muramidase and Disease-Models--Animal

As the interactions of phage with mammalian innate and adaptive immune systems are better delineated and with our ability to recognize and eliminate toxins and other potentially harmful phage gene products, the potential of phage therapies is now being realized. Early efforts to use phage therapeutically were hampered by inadequate phage purification and limited knowledge of phage-bacterial and phage-human relations. However, although use of phage as an antibacterial therapy in countries that require controlled clinical studies has been hampered by the high costs of patient trials, their use as vaccines and the use of phage components such as lysolytic enzymes or lysozymes has progressed to the point of commercial applications. Recent studies concerning the intimate associations between mammalian hosts and bacterial and phage microbiomes should hasten this progress.

Topics: Animals; Anti-Bacterial Agents; Bacterial Infections; Bacteriophages; Disease Models, Animal; Humans; Muramidase; Vaccines

The airway provides numerous defense mechanisms to prevent microbial colonization by the large numbers of bacteria and viruses present in ambient air. An important component of this defense is the antimicrobial peptides and proteins present in the airway surface fluid (ASF), the mucin-rich fluid covering the respiratory epithelium. These include larger proteins such as lysozyme and lactoferrin, as well as the cationic defensin and cathelicidin peptides. While some of these peptides, such as human beta-defensin (hBD)-1, are present constitutively, others, including hBD2 and -3 are inducible in response to bacterial recognition by Toll-like receptor-mediated pathways. These peptides can act as microbicides in the ASF, but also exhibit other activities, including potent chemotactic activity for cells of the innate and adaptive immune systems, suggesting they play a complex role in the host defense of the airway. Inhibition of antimicrobial peptide activity or gene expression can result in increased susceptibility to infections. This has been observed with cystic fibrosis (CF), where the CF phenotype leads to reduced antimicrobial capacity of peptides in the airway. Pathogenic virulence factors can inhibit defensin gene expression, as can environmental factors such as air pollution. Such an interference can result in infections by airway-specific pathogens including Bordetella bronchiseptica, Mycobacterium tuberculosis, and influenza virus. Research into the modulation of peptide gene expression in animal models, as well as the optimization of peptide-based therapeutics shows promise for the treatment and prevention of airway infectious diseases.

Topics: Animals; Antimicrobial Cationic Peptides; Cathelicidins; Cystic Fibrosis; Defensins; Disease Models, Animal; Humans; Lactoferrin; Muramidase; Proteinase Inhibitory Proteins, Secretory; Proteins; Respiratory System; Toll-Like Receptors; Tuberculosis, Pulmonary; Virus Diseases

Topics: Animals; Antibodies, Neoplasm; Antibody Formation; Antigens, Neoplasm; BCG Vaccine; Cell Migration Inhibition; Dinitrochlorobenzene; Disease Models, Animal; Fibroblasts; Humans; Hybrid Cells; Hypersensitivity, Delayed; Immunity, Cellular; Immunization; Immunotherapy; Lectins; Lung Neoplasms; Lymphocyte Activation; Mice; Muramidase; Rosette Formation; Tissue Extracts; Vitamin A

Topics: Animals; Anti-Bacterial Agents; Clinical Trials as Topic; Dapsone; Disease Models, Animal; Drug Resistance, Microbial; Foot; gamma-Globulins; Glucocorticoids; Humans; Immunosuppressive Agents; Immunotherapy; Leprosy; Mice; Muramidase; Mycobacterium leprae; Phenazines; Sulfonamides; Sulfones; Thalidomide; Thioacetazone

Topics: Animals; Anti-Bacterial Agents; Bacteria; Cell Division; Cell Wall; Chemical Phenomena; Chemistry; Disease Models, Animal; Humans; Infections; L Forms; Lysostaphin; Muramidase; Osmosis; Protoplasts; Terminology as Topic; Virulence

Topics: Animals; Anti-Bacterial Agents; Clinical Trials as Topic; Dapsone; Disease Models, Animal; Drug Resistance, Microbial; Foot; gamma-Globulins; Glucocorticoids; Humans; Immunosuppressive Agents; Immunotherapy; Leprosy; Mice; Muramidase; Mycobacterium leprae; Phenazines; Sulfonamides; Sulfones; Thalidomide; Thioacetazone

Streptococcus mutans, a major pathogen of dental caries, is also known as a causative agent of cardiovascular disease. A 120 kDa collagen-binding protein (Cnm) of S. mutans is an important contributor to the pathogenicity of cardiovascular disease. Although dead bacteria have been detected in cardiovascular specimens by molecular biological methods, the pathogenicity of the bacteria remains unknown. Here, we analyzed the pathogenicity of killed S. mutans by focusing on collagen-binding ability and the effects on silkworms. In live S. mutans, Cnm-positive S. mutans had high collagen-binding activity, while Cnm-negative S. mutans had no such activity. After treatment with killed Cnm-positive S. mutans, amoxicillin-treated bacteria still had collagen-binding ability, while lysozyme-treated bacteria lost this ability. When live and amoxicillin-treated S. mutans strains were administered to silkworms, the survival rates of the silkworms were reduced; this reduction was more pronounced in Cnm-positive S. mutans infection than in Cnm-negative S. mutans infection. However, the administration of any of the lysozyme-treated bacteria did not reduce the survival rate of the silkworms. These results suggest that amoxicillin-killed Cnm-positive S. mutans strains maintain collagen-binding properties and pathogenicity in the silkworm model, and are possibly associated with pathogenicity in cardiovascular diseases.

Topics: Adhesins, Bacterial; Amoxicillin; Animals; Bombyx; Cardiovascular Diseases; Carrier Proteins; Collagen; Dental Caries; Disease Models, Animal; Humans; Muramidase; Saliva; Streptococcal Infections; Streptococcus mutans; Virulence

Heart failure (HF) ensuing myocardial infarction (MI) is characterized by the initiation of a systemic inflammatory response. We aimed to elucidate the impact of myelomonocytic cells and their activation by angiotensin II on vascular endothelial function in a mouse model of HF after MI.. HF was induced in male C57BL/6J mice by permanent ligation of the left anterior descending coronary artery. Compared to sham, HF mice had significantly impaired endothelial function accompanied by enhanced mobilization of Sca-1+c-Kit+ haematopoietic stem cells and Sca-1-c-Kit+ common myeloid and granulocyte-macrophage progenitors in the bone marrow as well as increased vascular infiltration of CD11b+Ly6G-Ly6Chigh monocytes and accumulation of CD11b+ F4/80+ macrophages, assessed by flow cytometry. Using mice with Cre-inducible expression of diphtheria toxin receptor in myeloid cells, we selectively depleted lysozyme M+ myelomonocytic cells for 10 days starting 28 days after MI. While the cardiac phenotype remained unaltered until 38 days post-MI, myeloid cell depletion attenuated vascular accumulation of Nox2+CD45+ cells, endothelial dysfunction, oxidative stress, and vascular expression of adhesion molecules and angiotensin II receptor type 1 (AT1R). Pharmacological blockade of this receptor for 4 weeks did not significantly alter cardiac function, but mimicked the effects of myeloid cell depletion: telmisartan (20 mg/kg/day, fed to C57BL/6J mice) diminished bone marrow myelopoesis and myeloid reactive oxygen species production, attenuated endothelial leucocyte rolling and vascular accumulation of CD11b+Ly6G-Ly6Chigh monocytes and macrophages, resulting in improved vascular function with less abundance of Nox2+CD45+ cells.. Endothelial dysfunction in HF ensuing MI is mediated by inflammatory Nox2+ myeloid cells infiltrating the vessel wall that can be targeted by AT1R blockade.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Animals, Genetically Modified; Disease Models, Animal; Endothelial Cells; Heart Failure; Leukocyte Rolling; Macrophages; Male; Mice, Inbred C57BL; Monocytes; Muramidase; Myeloid Cells; Myocardial Infarction; NADPH Oxidase 2; Oxidative Stress; Receptor, Angiotensin, Type 1; Signal Transduction; Telmisartan; Vasculitis

Individuals with postnatal growth retardation (PGR) are prone to developing chronic diseases. Abnormal development in small intestine is casually implicated in impaired growth. However, the exact mechanism is still implausible. In this present study, PGR piglets (aged 42 days) were employed as a good model to analyze developmental changes in intestinal mucosal barrier function. Our data demonstrated that PGR piglets exhibited impaired jejunal and ileal epithelial villous morphology and permeability, accompanied by decreased cell proliferation ability and increased apoptosis rate. In addition, the expression of tight junction proteins (ZO-1, claudin 1, and occludin) and E-cadherin was markedly inhibited by PGR. The expression of P-glycoprotein was significantly reduced in PGR piglets, as well as decreased activity of lysozyme. Moreover, the mRNA abundance and content of inflammatory cytokines were significantly increased in the intestinal mucosa and plasma of PGR piglets, respectively. PGR also contributed to lower level of sIgA, and higher level of CD68-positive rate, β-defensins, and protein expression involved p38 MAPK/NF-κB pathway. Furthermore, PGR altered the intestinal microbial community such as decreased genus Alloprevotella and Oscillospira abundances, and led to lower microbial-derived butyrate production, which may be potential targets for treatment. Collectively, our findings indicated that the intestinal mucosal barrier function of PGR piglets could develop the nutritional intervention strategies in prevention and treatment of the intestinal mucosal barrier dysfunction in piglets and humans.

Topics: Animals; Animals, Newborn; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bacteria; Butyrates; Cell Proliferation; Cytokines; Disease Models, Animal; Gastrointestinal Microbiome; Growth Disorders; Inflammation Mediators; Intestinal Mucosa; Intestine, Small; Muramidase; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Permeability; Sus scrofa; Tight Junction Proteins

Vitamin D exerts a regulatory role over mucosal immunity via the vitamin D receptor (VDR). Although Paneth cells and their products are known to regulate the commensal and pathogenic microbiota, the role that VDRs in Paneth cells play in these responses is unknown.. We identified the decreased intestinal VDR significantly correlated with reduction of an inflammatory bowel disease risk gene ATG16L1 and Paneth cell lysozymes in patients with Crohn's disease. We generated Paneth cell-specific VDR knockout (VDR. Lysozymes in the Paneth cells were significantly decreased in the VDR. We provide new insights into the tissue-specific functions of VDRs in maintaining Paneth cell alertness to pathogens in intestinal disorders. Targeting the VDR affects multiple downstream events within Paneth cells that inhibit intestinal inflammation and establish host defense against enteropathogens.

Topics: Animals; Autophagy; Autophagy-Related Proteins; Biopsy; Colon; Crohn Disease; Dextran Sulfate; Disease Models, Animal; Female; Humans; Ileum; Immunity, Mucosal; Male; Mice; Mice, Knockout; Microbiota; Muramidase; Paneth Cells; Receptors, Calcitriol; Vitamin D

Accumulating evidence has suggested a correlation of tumor infiltrating B cells (TiBcs) and a good prognosis of cancer diseases. In some cases, TiBcs appear to have experienced antigen stimulation since they have undergone class-switching and somatic hypermutation and formed tertiary lymphoid structures around tumors together with T cells. Assuming TiBcs include those that recognize some tumor antigens, we sought to investigate their possible usefulness for cell-mediated immunotherapies. To expand usually small number of TiBcs in vitro, we modified our B cell culture system: we transduced B cells with ERT2-Bach2 so that they grow unlimitedly provided with tamoxifen, IL-21 and our original feeder cells. Such cells differentiate into plasma cells and produce antibodies upon withdrawal of tamoxifen, and further by addition of a Bach2-inhibitor in vitro. As a preliminary experiment, thus expanded splenic B cells expressing a transgenic antigen receptor/antibody against hen egg lysozyme were intravenously injected into mice pre-implanted with B16 melanoma cells expressing membrane-bound HEL in the skin, which resulted in suppression of the growth of B16 tumors and prolonged survival of the recipient mice. To test the usefulness of TiBcs for the immunotherapy, we next used APCmin/+ mice as a model that spontaneously develop intestinal tumors. We cultured TiBcs separated from the tumors of APCmin/+ mice as above and confirmed that the antibodies they produce recognize the APCmin/+ tumor. Repeated injection of such TiBcs into adult APCmin/+ mice resulted in suppression of intestinal tumor growth and elongation of the survival of the recipient mice. Serum antibody from the TiBc-recipient mice selectively bound to an antigen expressed in the tumor of APCmin/+ mice. These data suggest a possibility of the novel individualized cancer immunotherapy, in which TiBcs from surgically excised tumor tissues are expanded and infused into the donor patients.

Topics: Animals; Antigens, Neoplasm; B-Lymphocytes; Basic-Leucine Zipper Transcription Factors; Cells, Cultured; Disease Models, Animal; Disease Progression; Immunohistochemistry; Immunotherapy; Interleukins; Intestinal Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Muramidase; Tamoxifen

Acute graft-versus-host disease (aGVHD) is a lethal complication after allogeneic hematopoietic stem cell transplantation. The mechanism involves the recognition of host antigens by donor-derived T cells which induces augmented response of alloreactive T cells. In this study, we characterized the role of a previously identified novel classical secretory protein with antitumor function-LYG1 (Lysozyme G-like 1), in aGVHD. LYG1 deficiency reduced the activation of CD4

Topics: Allografts; Animals; Cell Line, Tumor; Cell Polarity; Disease Models, Animal; Graft vs Host Disease; Graft vs Tumor Effect; Hematopoietic Stem Cell Transplantation; Humans; Interferon-gamma; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Muramidase; Recombinant Proteins; Signal Transduction; T-Lymphocytes, Regulatory; Transplantation, Homologous

It is a desirable and powerful strategy to precisely fabricate functional soft matter through self-assembly of molecular building blocks across a range of length scales. Proteins, nucleic acids, and polyphenols are the self-assemblers ubiquitous in nature. Assembly of proteins into flexible biocolloids, amyloid fibrils with high aspect ratio, has emerged as an unchallenged templating strategy for high-end technological materials and bio-nanotechnologies. We demonstrate the ability of these fibrils to support the deposition and self-assembly of polyphenols into hybrid nanofilaments and functional macroscopic hydrogels made thereof. The length scale of the substance that amyloid fibrils can attach with acting as the building templates was extended from nanometer down to sub-nanometer. Significantly increased loading capacities of polyphenols (up to 4.0 wt %) compared to that of other delivery systems and improved stability were realized. After oral administration, the hydrogels could transport from the stomach to the small intestine and finally to the gut (cecum, colon, rectum), with a long retention time in the colon. Oral administration of the hydrogels significantly ameliorated colitis in a mouse model, promoted intestinal barrier function, suppressed the pro-inflammatory mRNA expression, and very significantly (

Topics: Amyloid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Colitis; Dextran Sulfate; Disease Models, Animal; Dysbiosis; Gastrointestinal Microbiome; Male; Mice; Mice, Inbred C57BL; Muramidase; Nanoparticles; Polyphenols

Many conflicting reports about the involvement of serum amyloid P component (SAP) in amyloid diseases have been presented over the years; SAP is known to be a universal component of amyloid aggregates but it has been suggested that it can both induce and suppress amyloid formation. By using our Drosophila model of systemic lysozyme amyloidosis, SAP has previously been shown to reduce the toxicity induced by the expression of the disease-associated lysozyme variant, F57I, in the Drosophila central nervous system. This study further investigates the involvement of SAP in modulating lysozyme toxicity using histochemistry and spectral analyses on the double transgenic WT and F57I lysozyme flies to probe; i) formation of aggregates, ii) morphological differences of the aggregated lysozyme species formed in the presence or absence of SAP, iii) location of lysozyme and iv) co-localisation of lysozyme and SAP in the fly brain. We found that SAP can counteract the toxicity (measured by the reduction in the median survival time) induced by F57I lysozyme by converting toxic F57I species into less toxic amyloid-like structures, as reflected by the spectral changes that p-FTAA undergoes when bound to lysozyme deposits in F57I-F57I-SAP flies as compared to F57I-F57I flies. Indeed, when SAP was introduced to in vitro lysozyme fibril formation, the endpoint fibrils had enhanced ThT fluorescence intensity as compared to lysozyme fibrils alone. This suggests that a general mechanism for SAP's role in amyloid diseases may be to promote the formation of stable, amyloid-like fibrils, thus decreasing the impact of toxic species formed along the aggregation pathway.

Topics: Amyloid; Amyloidosis; Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Humans; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Serum Amyloid P-Component

In situ regeneration of the enamel-like structure of hydroxyapatite (HAp) crystals under oral conditions is significant for dental caries treatment. However, it is still a challenge for dentists to duplicate the elegant and well-aligned apatite structure bonding to the surface of demineralized enamel. A biocompatible amelogenin-inspired matrix, a phase-transited lysozyme (PTL) film mimicking an N-terminal amelogenin with central domain (N-Ame) combined with synthetic peptide (C-AMG) based on the functional domains of C-terminal telopeptide (C-Ame) is shown here, which is formed by amyloid-like lysozyme aggregation at the enamel interface through a rapid one-step aqueous coating process. In the PTL/C-AMG matrix, C-AMG facilitated the oriented arrangement of amorphous calcium phosphate (ACP) nanoparticles and their transformation to ordered enamel-like HAp crystals, while PTL served as a strong interfacial anchor to immobilize the C-AMG peptide and PTL/C-AMG matrix on versatile substrate surfaces. PTL/C-AMG film-coated enamel induced both of the in vivo and in vitro synthesis of HAp crystals, facilitated epitaxial growth of HAp crystals and recovered the highly oriented structure and mechanical properties to levels nearly identical to those of natural enamel. This work underlines the importance of amyloid-like protein aggregates in the biomineralization of enamel, providing a promising strategy for treating dental caries.

Topics: Amelogenin; Animals; Calcium Phosphates; Coated Materials, Biocompatible; Dental Caries; Dental Enamel; Disease Models, Animal; Elastic Modulus; Mice; Microscopy, Atomic Force; Muramidase; Nanoparticles; Peptides; Tooth Remineralization

The role of neuronal Toll-like receptor 4 (TLR4) in nerve injury is being pursued actively. However, the endogenous activation of neuronal TLR4 during neuroinflammation, in absence of the participation of glial TLR4, remains elusive. Here, we identified lysozyme as an endogenous activator of neuronal TLR4 signaling during nerve injury. Upon nerve injury, enhanced expression of lysozyme promoted neuronal hyperexcitability and neuropathic pain. Injections of lysozyme in healthy rats increased their mechanical and thermal pain sensitivity. Likewise, infusion of spinal cord slices with lysozyme increased neuronal excitability typical of neuropathic pain. Our results also showed that lysozyme activated excitability of both Aδ- and C-fibers. Thus, in addition to the discovery of lysozyme as an endogenous ligand for regulating neuronal TLR4 signaling, this study also lays the foundation of our understanding of its role in nervous system pathologies, providing multiple avenues for treating neuroinflammation.

Topics: Animals; Annexin A2; Cell Membrane; Disease Models, Animal; Female; Ganglia, Spinal; Humans; Injections; Kinetics; Muramidase; Nerve Tissue; Neuralgia; Neurons; Nociceptors; Protein Binding; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Toll-Like Receptor 4; Up-Regulation

Type II alveolar epithelial cells (AEC2s) play a crucial role in the regeneration of type I AECs after acute lung injury. The mechanisms underlying the regeneration of AEC2s are not fully understood. To address this issue, here, we investigated a murine model of acute lung injury using mice expressing human Diphtheria Toxin Receptor (DTR) under the control of Lysozyme M promoter (LysM-DTR). DT injection induced the depletion of AEC2s, alveolar macrophages, and bone marrow (BM)-derived myeloid cells in LysM-DTR mice, and the mice died within 6 days after DT injection. Apoptotic AEC2s and bronchiolar epithelial cells appeared at 24 hr, whereas Ki67-positive proliferating cells appeared in the alveoli and bronchioles in the lung of LysM-DTR mice at 72-96 hr after DT injection. Transfer of wild-type BM cells into LysM-DTR mice accelerated the regeneration of AEC2s along with the up-regulation of several growth factors. Moreover, several metabolites were significantly decreased in the sera of LysM-DTR mice compared with WT mice after DT injection, suggesting that these metabolites might be biomarkers to predict AEC2s injury. Together, LysM-DTR mice might be useful to identify growth factors to promote lung repair and the metabolites to predict the severity of lung injury.

Topics: Acute Lung Injury; Alveolar Epithelial Cells; Animals; Biomarkers; Bone Marrow Transplantation; Diphtheria Toxin; Disease Models, Animal; Female; Gene Expression Profiling; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Male; Metabolome; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Promoter Regions, Genetic; Wound Healing

Myelomonocytic cells are critical in injury and healing post-myocardial infarction (MI). Mechanisms of regulation, however, are incompletely understood. The aim of the study was to elucidate the role of interferon gamma (IFN-γ) in the orchestrated inflammatory response in a murine model of MI.. MI was induced in 8- to 12-week-old male mice (C57BL/6 background) by permanent ligation of the left anterior descending (LAD) coronary artery. Lysozyme M (LysM)+ cell-depleted LysMiDTR transgenic mice displayed a reduced influx of CD45.2+/CD3-/CD11b+/Gr-1high neutrophils into infarcted myocardium 1 day post-MI compared with infarcted controls, paralleled by decreased cardiac mRNA levels of IFN-γ and tumour necrosis factor alpha (TNF-α). Mortality after MI was significantly increased in LysM+ cell-depleted mice within 28 days post-MI. To more specifically address the role of neutrophils, we depleted C57BL/6 mice with a monoclonal anti-Gr-1 antibody and found increased mortality, deteriorated cardiac function as well as decreased cardiac IFN-γ mRNA expression early after MI. Ccl2, Cxcl1, Cx3cl1, and Il12b mRNA were reduced 3 days after MI, as was the amount of CD11b+/Ly-6G-/Ly-6Chigh inflammatory monocytes. LAD-ligated Cramp-/- mice lacking cathelicidin important in neutrophil-dependent monocyte chemotaxis as well as IFNγ-/- and TNFα-/- mice phenocopied Gr-1+ cell-depleted mice, supporting a regulatory role of IFN-γ impacting on both the sequence of inflammatory cell invasion and cardiac outcome early after MI. The use of conditional IFN-γ receptor deficient mice indicated a direct effect of IFN-γ on LysM+ cells in cardiac injury post-MI. Using IFN-γ reporter mice and flow cytometry, we identified cardiac lymphoid cells (CD4+ and CD8+ T cells and natural killer cells) as primary source of this cytokine in the cardiac inflammatory response post-MI.. IFN-γ directs a sequential chemotactic cellular immune response and determines survival and cardiac function post-MI.

Topics: Animals; Antimicrobial Cationic Peptides; Cathelicidins; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Chemotaxis, Leukocyte; Disease Models, Animal; Immunity, Cellular; Interferon gamma Receptor; Interferon-gamma; Killer Cells, Natural; Lymphocytes; Male; Mice, Inbred C57BL; Mice, Knockout; Muramidase; Myocardial Infarction; Myocardium; Receptors, Interferon; Signal Transduction; Time Factors; Tumor Necrosis Factor-alpha

In this study we compared polarized mouse T-helper (Th) lymphocytes of four populations, sensitized against an ocular antigen, for their patterns of migration and induction of inflammatory processes in recipient mouse eyes expressing the target antigen. Th1, Th2, Th9 and Th17 cells transgenically expressing T-cell receptor (TCR) specific against hen egg lysozyme (HEL) were adoptively transferred to recipient mice expressing HEL in their eyes. Recipient eyes collected 4 or 7 days post injection were analyzed for histopathological changes. Th1 and Th17 cells induced moderate to severe intraocular inflammation in the recipient mouse eyes, but essentially did not migrate into the conjunctiva. In contrast, Th2 and Th9 cells invaded minimally the intraocular space of recipient eyes, but accumulated in the limbus and migrated into the conjunctiva of the recipient mice and initiated allergy-like inflammatory responses, as indicated by remarkable eosinophil involvement. These data thus shed new light on the differences between the migration patterns and ocular pathogenic processes mediated by Th1/Th17 and by Th2/Th9 populations.

Topics: Animals; Cell Movement; Conjunctiva; Disease Models, Animal; Eosinophilia; Lens, Crystalline; Limbus Corneae; Mice; Muramidase; T-Lymphocytes, Helper-Inducer; Th1 Cells; Th17 Cells; Th2 Cells

Parenteral nutrition (PN) is associated with increased infectious risks due to impaired intestinal immunity. Although glucagon-like peptide-2 (GLP-2) enhances the gut barrier function, it is uncertain whether it improves mucosal immunologic barrier function. We hypothesized that injecting the PN mouse model with GLP-2 improved innate and acquired immunity, and prevented bacterial translocation. Forty-eight hours after venous cannulation, male Institute of Cancer Research mice were randomly divided into 3 groups based on their diet: chow with saline (n = 10), PN (n = 9), or PN + GLP-2 (30 μg bid per mouse, n = 10) provided for 5 days. Compared with chow, PN reduced interleukin (IL)-4 and IL-13 levels (P < .05, respectively), whereas, compared with PN alone, GLP-2 injection increased IL-4 and IL-13 levels (P < .05, respectively). Compared with chow, PN considerably suppressed, whereas GLP-2 improved, secretory phospholipase A2 and cryptdin-4 expression. PN, compared with chow, considerably decreased lysozyme and polymeric immunoglobulin receptor levels, whereas, compared with PN, GLP-2 significantly increased these protein levels (P < .01, respectively). In tissue and luminal samples, compared with chow, PN reduced secretory immunoglobulin A levels (P < .05), whereas, compared with PN alone, GLP-2 increased secretory immunoglobulin A levels (P < .05). Functionally, more bacterial translocation was observed in the PN group compared with the chow group (P < .001), and GLP-2 injection decreased bacterial translocation to chow levels (P < .05). In summary, GLP-2 treatment may improve intestinal innate and acquired immunity, and prevent bacterial translocation in mice on total parenteral nutrition.

Topics: Adaptive Immunity; Animals; Bacterial Translocation; Disease Models, Animal; Glucagon-Like Peptide 2; Immunity, Innate; Immunoglobulin A, Secretory; Interleukin-13; Interleukin-4; Intestinal Mucosa; Male; Mice, Inbred ICR; Muramidase; Parenteral Nutrition; Phospholipases A2; Random Allocation; Receptors, Polymeric Immunoglobulin

Streptococcus suis is a highly invasive pathogen that can cause sepsis and meningitis in pigs and humans. However, we have limited understanding of the mechanisms S. suis uses to evade innate immunity. To investigate the involvement of the two-component signal transduction system of S. suis in host immune defense, we examined the expression of 15 response regulators of S. suis following stimulation with polymorphonuclear leukocytes (PMNs). We found that several response regulators were significantly up-regulated including vraR. Thus, we constructed an isogenic deletion mutant of vraSR genes in S. suis and demonstrated VraSR promotes both bacterial survival in human blood and resistance to human PMN-mediated killing. The VraSR mutant was more susceptible to phagocytosis by human PMNs and had greater sensitivity to oxidant and lysozyme than wild-type S. suis. Furthermore, in vitro findings and in vivo evidence from a mouse infection model together strongly demonstrate that ΔvraSR had greatly attenuated virulence compared with wild-type S. suis. Collectively, our data reveal that VraSR is a critical regulatory system that contributes to the survival of S. suis and its ability to defend against host innate immunity.

Topics: Animals; Bacterial Proteins; Blood Bactericidal Activity; Cells, Cultured; Disease Models, Animal; Gene Deletion; Humans; Immune Evasion; Immunity, Innate; Mice; Microbial Viability; Muramidase; Neutrophils; Oxidants; Phagocytosis; Signal Transduction; Streptococcal Infections; Streptococcus suis; Virulence; Virulence Factors

Organ-specific transgenic membrane expression of hen egg lysozyme (HEL) as a "neo-self antigen" has been used in several models to study immunological tolerance. In this study we report the changes which occur in the B10.BR mouse retina when membrane-bound HEL is expressed in photoreceptors under the control of the promoter for interphotoreceptor retinoid binding protein (IRBP, RBP3). On direct clinical examination of the single transgenic (sTg-IRBP:HEL) mouse fundus, a low-level increase in retinal degeneration compared to non-transgenic controls was observed, presenting as drusenoid deposits and occasional small patches of atrophy. On histological examination, there was an overall shortening of outer segments and loss of photoreceptor nuclei in sTg-IRBP:HEL mice, which was more pronounced in the retinal periphery, particularly inferiorly. The fundoscopically observed lesions did not correlate with the photoreceptor shortening/loss but appeared to be located at the level of the retinal pigment epithelium/choriocapillaris layer and were an exaggeration in size and number of similar age-related changes found in wild type (WT) mice. In addition, neither the atrophic lesions nor the photoreceptor shortening were associated with common retinal degeneration genes, nor were they caused by exposure to light damage since mice housed at both high and low ambient light levels had similar degrees of retinal degeneration. Instead, sTg-IRBP:HEL mice expressed reduced levels of soluble retinal IRBP compared to WT mice which were present from postnatal day16 (P16) and preceded development of photoreceptor shortening (onset P21). We propose that insertion of the HEL transgene in the photoreceptor membrane disrupted normal photoreceptor function and led to reduced levels of soluble IRBP and retinal thinning. A similar phenotype has been observed in IRBP deficient mice. Despite the retinal thinning, the amount of HEL expressed in the retina was sufficient to act as an autoantigenic target when the mice were crossed to the HEL T cell receptor Tg mouse, since double transgenic (dTg-IRBP:HEL) mice spontaneously developed a severe uveoretinitis with onset at weaning. We suggest that, although membrane expression of foreign transgene products is likely to modify the structure and function of tissues and cells, the technology provides useful models to investigate mechanisms of antigen-specific immunological tolerance.

Topics: Animals; Blotting, Western; Disease Models, Animal; Eye Proteins; Immunohistochemistry; Mice; Mice, Transgenic; Muramidase; Photoreceptor Cells, Vertebrate; Polymerase Chain Reaction; Retinal Degeneration; Retinol-Binding Proteins; Transgenes

In this study we have reported an efficient antibacterial hybrid fabricated through surface functionalization of lysozyme capped gold nanoclusters (AUNC-L) with β-lactam antibiotic ampicillin (AUNC-L-Amp). The prepared hybrid not only reverted the MRSA resistance towards ampicillin but also demonstrated enhanced antibacterial activity against non-resistant bacterial strains. Most importantly, upon awakening through cis-2-decenoic acid (cis-DA) exposure, the MRSA persister got inhibited by the AUNC-L-Amp treatment. Intraperitoneal administration of this hybrid eliminates the systemic MRSA infection in a murine animal model. Topical application of this nano conjugate eradicated MRSA infection from difficult to treat diabetic wound of rat and accelerated the healing process. Due to inherent bio-safe nature of gold, AUNC-L alone or in the construct (AUNC-L-Amp) demonstrated excellent biocompatibility and did not indicate any deleterious effects in in vivo settings. We postulate that AUNC-L-Amp overcomes the elevated levels of β-lactamase at the site of MRSA antibiotic interaction with subsequent multivalent binding to the bacterial surface and enhanced permeation. Coordinated action of AUNC-L-Amp components precludes MRSA to attain resistance against the hybrid. We proposed that the inhibitory effect of AUNC-L-Amp against MRSA and its persister form is due to increased Amp concentration at the site of action, multivalent presentation and enhanced permeation of Amp through lysozyme-mediated cell wall lysis.

Topics: Ampicillin; Animals; Anti-Bacterial Agents; Disease Models, Animal; Gold; Injections, Intraperitoneal; Male; Metal Nanoparticles; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Muramidase; Rats; Staphylococcal Infections; Treatment Outcome

Activation of transforming growth factor-β-activated kinase 1 (TAK1) occurs after stroke and leads to an exacerbation of brain injury. TAK1 is involved in innate and adaptive immune responses, but it has divergent inflammatory effects that are dependent on the cell type in which it is activated. There is a robust infiltration of myeloid cells after stroke; however, the contribution of myeloid TAK1 to cerebral ischemia is currently unknown. We hypothesized that myeloid-specific deletion of TAK1 would protect against ischemic brain injury.. Myeloid TAK1. Infarcts were significantly smaller in TAK1. Our results showed that deletion of myeloid TAK1 resulted in smaller infarcts and improved functional outcomes at the peak of inflammation (day 3) and a reduction in brain-infiltrating immune cells that were primarily monocytes. Myeloid TAK1 deletion was also protective at 7 days post MCAo, reflecting a detrimental role of myeloid TAK1 in the progression of ischemic injury.

Topics: Animals; Antigens, CD; Cerebrovascular Circulation; Disease Models, Animal; Flow Cytometry; Infarction, Middle Cerebral Artery; Male; MAP Kinase Kinase Kinases; Mice; Mice, Inbred C57BL; Mice, Transgenic; Monocytes; Muramidase; Myeloid Cells; Neutrophil Infiltration; Neutrophils; Recovery of Function

To test the synergistic effect of Cpl-711 endolysin and antibiotics for antipneumococcal activity.. A combination of Cpl-711 and different antibiotics (amoxicillin, cefotaxime, levofloxacin and vancomycin) was tested in a checkerboard assay against several multidrug-resistant Streptococcus pneumoniae strains. Mouse and zebrafish models of pneumococcal sepsis were used to confirm the in vitro data.. The activity of Cpl-711 combined with amoxicillin or cefotaxime was synergistic in the bactericidal effect against a serotype 23F multiresistant clinical isolate of S. pneumoniae. Synergy between Cpl-711 and cefotaxime was validated using both mouse and zebrafish models.. Combination of Cpl-711 and cefotaxime may help in the treatment of diseases caused by multiresistant pneumococcal strains.

Topics: Animals; Anti-Bacterial Agents; Cefotaxime; Disease Models, Animal; Drug Resistance, Multiple, Bacterial; Drug Synergism; Female; Mice; Mice, Inbred BALB C; Muramidase; Pneumococcal Infections; Recombinant Fusion Proteins; Sepsis; Streptococcus Phages; Streptococcus pneumoniae; Zebrafish

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality in premature infants. Human and animal studies suggest a role for Paneth cells in NEC pathogenesis. Paneth cells play critical roles in host-microbial interactions and epithelial homeostasis. The ramifications of eliminating Paneth cell function on the immature host-microbial axis remains incomplete. Paneth cell function was depleted in the immature murine intestine using chemical and genetic models, which resulted in intestinal injury consistent with NEC. Paneth cell depletion was confirmed using histology, electron microscopy, flow cytometry, and real time RT-PCR. Cecal samples were analyzed at various time points to determine the effects of Paneth cell depletion with and without Klebsiella gavage on the microbiome. Deficient Paneth cell function induced significant compositional changes in the cecal microbiome with a significant increase in Enterobacteriacae species. Further, the bloom of Enterobacteriaceae species that occurs is phenotypically similar to what is seen in human NEC. This further strengthens our understanding of the importance of Paneth cells to intestinal homeostasis in the immature intestine.

Topics: Animals; Animals, Newborn; Autophagosomes; Cecum; Cytokines; Diphtheria Toxin; Disease Models, Animal; Dithizone; Enterobacteriaceae; Enterocolitis, Necrotizing; Gastrointestinal Microbiome; Klebsiella pneumoniae; Mice; Mice, Inbred C57BL; Muramidase; Paneth Cells

Neonatal brain injury is increasingly understood to be linked to inflammatory processes that involve specialised CNS and peripheral immune interactions. However, the role of peripheral myeloid cells in neonatal hypoxic-ischemic (HI) brain injury remains to be fully investigated.. We employed the Lys-EGFP-ki mouse that allows enhanced green fluorescent protein (EGFP)-positive mature myeloid cells of peripheral origin to be easily identified in the CNS. Using both flow cytometry and confocal microscopy, we investigated the accumulation of total EGFP. We demonstrate a temporally biphasic pattern of inflammatory monocyte and granulocyte infiltration, characterised by peak infiltration at 1 day and 7 days after hypoxia-ischemia. This occurs against a backdrop of continuous low-level resident monocyte infiltration. Antibody-mediated depletion of circulating myeloid cells reduced immune cell accumulation in the brain and reduced neuronal loss in male but not female mice.. This study offers new insight into sex-dependent central-peripheral immune communication following neonatal brain injury and merits renewed interest in the roles of granulocytes and monocytes in lesion development.

Topics: Animals; Animals, Newborn; Antibodies; Antigens, Ly; Calcium-Binding Proteins; Cytokinins; Disease Models, Animal; Functional Laterality; Green Fluorescent Proteins; Hypoxia-Ischemia, Brain; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Monocytes; Muramidase; Myeloid Cells; Nerve Tissue Proteins; Neutrophils

Malnutrition remains a leading contributor to the morbidity and mortality of children under the age of 5 years and can weaken the immune system and increase the severity of concurrent infections. Livestock milk with the protective properties of human milk is a potential therapeutic to modulate intestinal microbiota and improve outcomes. The aim of this study was to develop an infection model of childhood malnutrition in the pig to investigate the clinical, intestinal and microbiota changes associated with malnutrition and enterotoxigenic Escherichia coli (ETEC) infection and to test the ability of goat milk and milk from genetically engineered goats expressing the antimicrobial human lysozyme (hLZ) milk to mitigate these effects. Pigs were weaned onto a protein-energy-restricted diet and after 3 weeks were supplemented daily with goat, hLZ or no milk for a further 2 weeks and then challenged with ETEC. The restricted diet enriched faecal microbiota in Proteobacteria as seen in stunted children. Before infection, hLZ milk supplementation improved barrier function and villous height to a greater extent than goat milk. Both goat and hLZ milk enriched for taxa (Ruminococcaceae) associated with weight gain. Post-ETEC infection, pigs supplemented with hLZ milk weighed more, had improved Z-scores, longer villi and showed more stable bacterial populations during ETEC challenge than both the goat and no milk groups. This model of childhood disease was developed to test the confounding effects of malnutrition and infection and demonstrated the potential use of hLZ goat milk to mitigate the impacts of malnutrition and infection.

Topics: Animal Feed; Animals; Animals, Genetically Modified; Body Weight; Diet; Dietary Supplements; Disease Models, Animal; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Feces; Female; Gastrointestinal Microbiome; Genotype; Goats; Intestinal Diseases; Intestines; Male; Malnutrition; Milk; Muramidase; Organ Size; Permeability; Swine; Weaning

The present study aimed to investigate the effects of intestinal endotoxemia (IETM) in a rat model of aluminum neurotoxicity established by D-galactose and aluminum trichloride (AlCl3). Adult Wistar rats were administered D‑galactose and AlCl3 to create the aluminum neurotoxicity model. The learning and memory abilities of the rats were subsequently observed using a Morris water maze test and the serum levels of lipopolysaccharide (LPS), tumor necrosis factor (TNF)‑α, interleukin (IL)‑1, diamine oxidase (DAO), glutamine (Gln) and glutaminase were measured. The expression of S‑100β in the serum was detected using an enzyme‑linked immunosorbent assay. The expression levels of the amyloid β‑protein (Aβ) precursor (APP), presenilin 1 (PS1), β‑site APP‑cleaving enzyme (BACE), zona occludens protein (ZO)‑1 and Aβ 1‑40 in the brain of rats were detected via reverse‑transcription polymerase chain reaction, western blotting and immunohistochemistry. The levels of LPS, TNF‑α, IL‑1, DAO, Gln and S‑100β in serum and the mRNA and protein expression levels of APP, PS1, BACE and Aβ1‑40 in the brain were markedly increased in the model rats compared with controls. The level of glutaminase in the serum and the expression of ZO‑1 in the brain were decreased in the model rats compared with controls. IETM was present in the rat model of aluminum neurotoxicity established by D‑galactose and AlCl3 and may be important in the development of this neurotoxicity.

Topics: Aluminum; Aluminum Chloride; Aluminum Compounds; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Blood-Brain Barrier; Chlorides; Disease Models, Animal; Endotoxemia; Galactose; Interleukin-1; Intestinal Mucosa; Intestines; Lipopolysaccharides; Male; Memory; Muramidase; Neurotoxins; Peptide Fragments; Presenilin-1; Rats, Wistar; RNA, Messenger; Tumor Necrosis Factor-alpha; Zonula Occludens-1 Protein

Staphylococcus aureus (S. aureus) is one of the most common pathogen causing septic arthritis. To colonize the joints and establish septic arthritis this bacterium needs to resist the host innate immune responses. Lysozyme secreted by neutrophils and macrophages is an important defense protein present in the joint synovial fluids. S. aureus is known to be resistant to lysozyme due to its peptidoglycan modification by O-acetylation of N-acetyl muramic acid. In this study we have investigated the role of O-acetylated peptidoglycan in septic arthritis. Using mouse models for both local and hematogenous S. aureus arthritis we compared the onset and progress of the disease induced by O-acetyl transferase mutant and the parenteral wild type SA113 strain. The disease progression was assessed by observing the clinical parameters including body weight, arthritis, and functionality of the affected limbs. Further X-ray and histopathological examinations were performed to monitor the synovitis and bone damage. In local S. aureus arthritis model, mice inoculated with the ΔoatA strain developed milder disease (in terms of knee swelling, motor and movement functionality) compared to mice inoculated with the wild type SA113 strain. X-ray and histopathological data revealed that ΔoatA infected mice knee joints had significantly lesser joint destruction, which was accompanied by reduced bacterial load in knee joints. Similarly, in hematogenous S. aureus arthritis model, ΔoatA mutant strain induced significantly less severe clinical septic arthritis compared to its parental strain, which is in accordance with radiological findings. Our data indicate that peptidoglycan O-acetylation plays an important role in S. aureus mediated septic arthritis.

Topics: Acetylation; Acetyltransferases; Animals; Arthritis, Infectious; Cell Wall; Disease Models, Animal; Female; Knee Joint; Locomotion; Mice; Mice, Inbred BALB C; Muramic Acids; Muramidase; Mutation; Peptidoglycan; Single-Blind Method; Staphylococcal Infections; Staphylococcus aureus

A lysozyme-chitosan conjugate preparation (LYZOX), produced from egg white lysozyme and chitosan by Maillard reaction, is a commercial product developed as a cosmetic ingredient or food additive. Effects of LYZOX on in vitro growth of Candida albicans were examined. C. albicans cells were treated with LYZOX for 3 hrs, and then washed and cultured for an additional 16 hrs in modified RPMI1640 medium. Mycelial growth of C. albicans was clearly inhibited by more than 100 μg/ml of LYZOX in a concentration-dependent manner. On the other hand, corresponding concentration of chitosan or lysozyme or their mixture only scarcely showed clear inhibitory effect. Similarly, anti-Candida activity of the combination of LYZOX and decanoic acid, a middle-chain fatty acid, was also examined. Inhibitory activity of this combination against mycelial growth of C. albicans was very potent and appeared synergistic, since fractionated inhibitory concentration (FIC) index for 70% growth inhibition was calculated to be 0.20. Oral application of this combination improved the symptoms of Candida-infected-tongue in an experimental murine candidiasis model. On the basis of these results, the possible application of LYZOX as a new functional product with anti-candida activity was discussed.

Topics: Animals; Antifungal Agents; Candida albicans; Candidiasis, Oral; Chitosan; Decanoic Acids; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Combinations; Drug Resistance, Fungal; Drug Synergism; Drug Therapy, Combination; Mice, Inbred ICR; Muramidase; Treatment Outcome

Topics: Animal Feed; Animals; Animals, Genetically Modified; Animals, Newborn; Bacteroidetes; Diet; Disease Models, Animal; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Feces; Gastrointestinal Microbiome; Goats; Intestinal Diseases; Intestines; Milk; Muramidase; Swine; Swine Diseases

Alzheimer's disease (AD) is a chronic degenerative disease characterized by the presence of amyloid plaques and neurofibrillary tangles (NFTs), which results into memory and learning impairments. In the present study, we showed that the aggregates formed by a protein that has no link with Alzheimer's disease, namely the hen egg white lysozyme (HEWL), were cytotoxic and decreased spatial learning and memory in rats. The effect of Ag-nano particles (Ag-NPs) was investigated on disruption of amyloid aggregation and preservation of cognitive behavior of rats. Twenty-four male Wistar rats were divided into 4 groups including a control group, and injected with either scopolamine, lysozyme or aggregates pre-incubated with Ag-NPs. Rats' behavior was monitored using Morris water maze (MWM) twenty days after injections. HEWL aggregation in the presence and absence of the Ag-NPs was assayed by Thioflavin T binding, atomic force microscopy and cell-based cytotoxicity assay. Ag-NPs were capable to directly disrupt HEWL oligomerization and the resulting aggregates were non-toxic. We also showed that rats of the Ag-NPs group found MWM test platform in less time and with less distance traveled, in comparison with lysozyme group. Ag-NPs also increased the percentage of time elapsed and the distance swum in the target quadrant in the rat model of AD, in probe test. These observations suggest that Ag-NPs improved spatial learning and memory by inhibiting amyloid fibril-induced neurotoxicity. Furthermore, we suggest using model proteins as a valid tool to investigate the pathogenesis of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Disease Models, Animal; Male; Muramidase; Nanoparticles; Peptide Fragments; Rats; Rats, Wistar; Scopolamine; Silver; Spatial Learning; Spatial Memory

Th cells sensitized against autoantigens acquire pathogenicity following two sequential events, namely activation by their target Ag and a process named "licensing." In this study, we analyzed these processes in a transgenic mouse system in which TCR-transgenic Th cells specific to hen egg lysozyme (HEL) are adoptively transferred to recipients and induce inflammation in eyes expressing HEL. Our data show that the notion that the lung is the organ where "licensing" for pathogenicity takes place is based on biased data collected with cells injected i.v., a route in which most transferred cells enter via the lung. Thus, we found that when donor cells were activated in vitro and injected intraperitoneally, or were activated in vivo, they migrated simultaneously to the lung, spleen, and other tested organs. In all, tested organs donor cells undergo "licensing" for pathogenicity, consisting of vigorous increase in number and changes in expression levels of inflammation-related genes, monitored by both flow cytometry and microarray analysis. After reaching peak numbers, around day 3, the "licensed" donor cells migrate to the circulation and initiate inflammation in the HEL-expressing recipient eyes. Importantly, the kinetics of increase in number and of changes in gene expression by the donor cells were similar in lung, spleen, and other tested organs of the recipient mice. Furthermore, the total numbers of donor cells in the spleen at their peaks were 10- to 100-fold larger in the spleen than in the lung, contradicting the notion that the lung is the organ where "licensing" takes place.

Topics: Adoptive Transfer; Animals; Autoantigens; Autoimmune Diseases; Autoimmunity; Disease Models, Animal; Flow Cytometry; Lung; Lymphocyte Activation; Mice; Mice, Transgenic; Muramidase; Spleen; T-Lymphocytes, Helper-Inducer

Tumor-associated macrophages (TAMs) are often associated with a poor prognosis in cancer. To gain a better understanding of cellular recruitment and dynamics of TAM biology during cancer progression, we established a novel transgenic mouse model for in vivo imaging of luciferase-expressing macrophages.. In vivo imaging of LysM-LG mice showed luciferase activity was generated by macrophages. Clodronate liposome-mediated depletion of macrophages lowered overall bioluminescence while lipopolysaccharide injection increased macrophage bioluminescence in both the B16 and ID8 models. Tracking macrophages weekly in tumor-bearing animals after intraperitoneal (i.p.) or intraovarian (i.o.) injection resulted in distinct, dynamic patterns of macrophage activity. Animals with metastatic ovarian cancer after i.p. injection exhibited significantly higher peritoneal macrophage activity compared to animals after i.o. injection.. The LysM-LG model allows tracking of macrophage recruitment and activation during disease initiation and progression in a noninvasive manner. This model provides a tool to visualize and monitor the benefit of pharmacological interventions targeting macrophages in preclinical models.

Topics: Animals; Disease Models, Animal; Female; Genes, Reporter; Luminescent Measurements; Macrophage Activation; Macrophages; Melanoma; Mice, Transgenic; Molecular Imaging; Muramidase; Ovarian Neoplasms; Promoter Regions, Genetic

Topics: Animals; Anti-Bacterial Agents; beta-Lactams; Caenorhabditis elegans; Cell Wall; Disease Models, Animal; DNA Transposable Elements; Gene Knockout Techniques; Genetic Complementation Test; Genetic Testing; Mice, Inbred C57BL; Microbial Viability; Muramidase; Mutagenesis, Insertional; Pseudomonas aeruginosa; Pseudomonas Infections; Vancomycin; Virulence

Resident immune cells (e.g., macrophages [MΦs]) and airway mucus clearance both contribute to a healthy lung environment. To investigate interactions between pulmonary MΦ function and defective mucus clearance, a genetic model of lysozyme M (LysM) promoter-mediated MΦ depletion was generated, characterized, and crossed with the sodium channel β subunit transgenic (Scnn1b-Tg) mouse model of defective mucus clearance. Diphtheria toxin A-mediated depletion of LysM(+) pulmonary MΦs in wild-type mice with normal mucus clearance resulted in lethal pneumonia in 24% of neonates. The pneumonias were dominated by Pasteurella pneumotropica and accompanied by emaciation, neutrophilic inflammation, and elevated Th1 cytokines. The incidence of emaciation and pneumonia reached 51% when LysM(+) MΦ depletion was superimposed on the airway mucus clearance defect of Scnn1b-Tg mice. In LysM(+) MΦ-depleted Scnn1b-Tg mice, pneumonias were associated with a broader spectrum of bacterial species and a significant reduction in airway mucus plugging. Bacterial burden (CFUs) was comparable between Scnn1b-Tg and nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice. However, the nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice exhibited increased airway inflammation, the presence of neutrophilic infiltration, and increased levels of inflammatory cytokines in bronchoalveolar lavage fluid compared with Scnn1b-Tg mice. Collectively, these data identify key MΦ-mucus clearance interactions with respect to both infectious and inflammatory components of muco-obstructive lung disease.

Topics: Animals; Animals, Newborn; Cytokines; Diphtheria Toxin; Disease Models, Animal; Epithelial Sodium Channels; Genetic Predisposition to Disease; Inflammation Mediators; Luminescent Proteins; Lung; Macrophages; Mice, Inbred C57BL; Mice, Transgenic; Mucociliary Clearance; Muramidase; Pasteurella Infections; Pasteurella pneumotropica; Peptide Fragments; Phenotype; Pneumonia, Bacterial; Promoter Regions, Genetic

We have applied a serologic proteomic workflow involving three complementary MS approaches to a tissue-specific Kras(G12D) -knockin mouse model of pancreatic cancer that consistently forms precancerous lesions by 4 months of age. The three proteomics applications were highly complementary and allowed us to survey the entire range of low to high molecular weight serologic proteins. Combined, we identified 121 (49↓, 72↑) unique and statistically relevant serologic biomarkers with 88% previously reported to be associated with cancer and 38% specifically correlated with pancreatic cancer. Four markers, lysozyme C2, cytokeratin 19, Serpina1A and Pcf11, were further verified by Western blotting. When applying systems analysis, the top-associated gene ontology functions were tied to wound healing, RXR signaling, growth, differentiation and innate immune activation through the JAK/STAT pathway. Upon further investigation of the apparent immune response using a multiplex cytokine screen, we found that IFN-γ, VEGF and GM-CSF were significantly increased in serum from the Kras(G12D) animals compared to littermate controls. By combining three complementary MS applications, we were able to survey the native intact peptidome and the global proteome in parallel, unveiling pathways that may be biologically relevant to promotion of pancreatic cancer progression and serologic markers of noninvasive early-stage neoplasia.

Topics: alpha 1-Antitrypsin; Animals; Biomarkers, Tumor; Disease Models, Animal; Disease Progression; Gene Expression Regulation, Neoplastic; Gene Knock-In Techniques; Granulocyte-Macrophage Colony-Stimulating Factor; Interferon-gamma; Keratin-19; Mice; Mice, Inbred C57BL; Mice, Transgenic; mRNA Cleavage and Polyadenylation Factors; Muramidase; Pancreatic Neoplasms; Proteome; Proto-Oncogene Proteins p21(ras); Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Vascular Endothelial Growth Factor A

Corneal suturing is a surgical procedure used in patients with corneal trauma or transplants. It was reported that endogenous neutrophils are brightly labelled in gene-targeted mice expressing enhanced green fluorescent protein (eGFP) under the control of the endogenous lysozyme M promoter (LysM-eGFP mice).. We applied intravital imaging methods to analyse in vivo the dynamics of LysM-positive granulocytes (neutrophils) in LysM-eGFP mice with corneal sutures and examined their role in the elicitation of neutrophil infiltration.. We found that in the presuturing state, neutrophils strongly positive for LysM were located in the periphery of the corneal stromal layer; none were present in the centre of the cornea. After introducing a corneal suture, neutrophils accumulated in limbal vessels and then migrated to the corneal side and the conjunctival side, suggesting that they derived from limbal vessels. Thereafter they accumulated towards the central corneal area, arriving at the suture about 7 h after its placement. Although corneal sutures may elicit the continuous infiltration of neutrophils, their number was markedly decreased by day 1 after suture removal and continued to decrease thereafter.. Our results showed that corneal sutures may elicit the continuous infiltration of neutrophils.

Topics: Animals; Cell Movement; Cornea; Disease Models, Animal; Gene Transfer Techniques; Green Fluorescent Proteins; Keratitis; Mice; Mice, Transgenic; Microscopy, Fluorescence, Multiphoton; Muramidase; Neutrophils; Sutures

Food allergies are believed to be on the rise, and currently, management relies on the avoidance of the food. Hen's egg allergy is after cow's milk allergy the most common food allergy; eggs are used in many food products and thus difficult to avoid. A technological process using a combination of enzymatic hydrolysis and heat treatment was designed to produce modified hen's egg with reduced allergenic potential. Biochemical (SDS-PAGE, Size exclusion chromatography and LC-MS/MS) and immunological (ELISA, immunoblot, RBL-assays, animal model) analysis showed a clear decrease in intact proteins as well as a strong decrease of allergenicity. In a clinical study, 22 of the 24 patients with a confirmed egg allergy who underwent a double-blind food challenge with the hydrolysed egg remained completely free of symptoms. Hydrolysed egg products may be beneficial as low-allergenic foods for egg-allergic patients to extent their diet.

Topics: Allergens; Animals; Antibody Specificity; Chickens; Child, Preschool; Disease Models, Animal; Egg Hypersensitivity; Egg Proteins; Eggs; Female; Humans; Hydrolysis; Immune Tolerance; Immunoglobulin E; Infant; Male; Muramidase; Rats

Clostridium difficile (also known as Peptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibiotic-associated diarrhea throughout the world. Colonization of the intestinal tract is necessary for C. difficile to cause disease. Host-produced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yet C. difficile is able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition of d-alanine to teichoic acids, is important for C. difficile resistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that a dlt null mutant is severely attenuated for growth in lysozyme and that expression of the dltDABC operon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor σ(V) does not induce dlt expression in response to lysozyme, indicating that σ(V) is required for regulation of lysozyme-dependent d-alanylation of the cell wall. Using reporter gene fusions and 5' RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independent dlt expression. In addition, we observed that both a sigV mutant and a dlt mutant are more virulent in a hamster model of infection. These findings demonstrate that cell wall d-alanylation in C. difficile is induced by lysozyme in a σ(V)-dependent manner and that this pathway impacts virulence in vivo.

Topics: Alanine; Animals; Bacterial Proteins; Carrier Proteins; Cell Wall; Clostridioides difficile; Cricetulus; Disease Models, Animal; Enterocolitis, Pseudomembranous; Female; Gene Expression Regulation, Bacterial; Host-Pathogen Interactions; Muramidase; Mutation; Operon; Promoter Regions, Genetic; Protein Isoforms; Sigma Factor; Signal Transduction; Stereoisomerism; Teichoic Acids; Virulence

Resident microglia and infiltrating myeloid cells play important roles in the onset, propagation, and resolution of inflammation in central nervous system (CNS) injury and disease. Identifying cell type-specific mechanisms will help to appropriately target interventions for tissue repair. Arginase-1 (Arg-1) is a well characterised modulator of tissue repair and its expression correlates with recovery after CNS injury. Here we assessed the cellular localisation of Arg-1 in two models of CNS damage. Using microglia specific antibodies, P2ry12 and Fc receptor-like S (FCRLS), we show the LysM-EGFP reporter mouse is an excellent model to distinguish infiltrating myeloid cells from resident microglia. We show that Arg-1 is expressed exclusively in infiltrating myeloid cells but not microglia in models of spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). Our in vitro studies suggest that factors in the CNS environment prevent expression of Arg-1 in microglia in vivo. This work suggests different functional roles for these cells in CNS injury and repair and shows that such repair pathways can be switched on in infiltrating myeloid cells in pro-inflammatory environments.

Topics: Animals; Arginase; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Green Fluorescent Proteins; Inflammation; Mice; Mice, Inbred C57BL; Microglia; Muramidase; Myeloid Cells; Spinal Cord Injuries

Haemophilus parasuis is an opportunistic pathogen that causes Glässer's disease in swine, with polyserositis, meningitis, and arthritis. The high-temperature requirement A (HtrA)-like protease, which is involved in protein quality control, has been reported to be a virulence factor in many pathogens. In this study, we showed that HtrA of H. parasuis (HpHtrA) exhibited both chaperone and protease activities. Finally, nickel import ATP-binding protein (NikE), periplasmic dipeptide transport protein (DppA), and outer membrane protein A (OmpA) were identified as proteolytic substrates for HpHtrA. The protease activity reached its maximum at 40°C in a time-dependent manner. Disruption of the htrA gene from strain SC1401 affected tolerance to temperature stress and resistance to complement-mediated killing. Furthermore, increased autoagglutination and biofilm formation were detected in the htrA mutant. In addition, the htrA mutant was significantly attenuated in virulence in the murine model of infection. Together, these data demonstrate that HpHtrA plays an important role in the virulence of H. parasuis.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Biofilms; Complement Activation; Disease Models, Animal; Genetic Complementation Test; Haemophilus Infections; Haemophilus parasuis; Mice; Molecular Chaperones; Muramidase; Mutation; Proteolysis; Recombinant Fusion Proteins; Stress, Physiological; Substrate Specificity; Virulence; Virulence Factors

Hypohidrotic ectodermal dysplasia (HED) results from mutation of the EDA, EDAR or EDARADD genes and is characterized by reduced or absent eccrine sweat glands, hair follicles and teeth, and defective formation of salivary, mammary and craniofacial glands. Mouse models with HED also carry Eda, Edar or Edaradd mutations and have defects that map to the same structures. Patients with HED have ear, nose and throat disease, but this has not been investigated in mice bearing comparable genetic mutations. We report that otitis media, rhinitis and nasopharyngitis occur at high frequency in Eda and Edar mutant mice and explore the pathogenic mechanisms related to glandular function, microbial and immune parameters in these lines. Nasopharynx auditory tube glands fail to develop in HED mutant mice and the functional implications include loss of lysozyme secretion, reduced mucociliary clearance and overgrowth of nasal commensal bacteria accompanied by neutrophil exudation. Heavy nasopharynx foreign body load and loss of gland protection alters the auditory tube gating function and the auditory tubes can become pathologically dilated. Accumulation of large foreign body particles in the bulla stimulates granuloma formation. Analysis of immune cell populations and myeloid cell function shows no evidence of overt immune deficiency in HED mutant mice. Our findings using HED mutant mice as a model for the human condition support the idea that ear and nose pathology in HED patients arises as a result of nasal and nasopharyngeal gland deficits, reduced mucociliary clearance and impaired auditory tube gating function underlies the pathological sequelae in the bulla.

Topics: Animals; Disease Models, Animal; Ear, Middle; Ectodermal Dysplasia 1, Anhidrotic; Ectodysplasins; Edar Receptor; Edar-Associated Death Domain Protein; Genetic Predisposition to Disease; Humans; Mice; Muramidase; Mutation; NF-kappa B; Nose; Phenotype

Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP). SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies' neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies' lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures.

Topics: Amyloidosis; Animals; Animals, Genetically Modified; Apoptosis; Central Nervous System; Disease Models, Animal; Drosophila melanogaster; Gene Expression; Humans; Longevity; Muramidase; Mutation; Neurons; Plaque, Amyloid; Protective Factors; Protein Aggregation, Pathological; Serum Amyloid P-Component; Transgenes

Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer's disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome-wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aβ

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Disease Models, Animal; Drosophila melanogaster; Eye; Female; Humans; Male; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Mutation; Peptide Fragments; RNA, Messenger

Experimental autoimmune uveoretinitis (EAU) is a widely used experimental animal model of human endogenous posterior uveoretinitis. In the present study, we performed in vivo imaging of the retina in transgenic reporter mice to investigate dynamic changes in exogenous inflammatory cells and endogenous immune cells during the disease process.. Transgenic mice (C57Bl/6 J Cx 3 cr1 (GFP/+) , C57Bl/6 N CD11c-eYFP, and C57Bl/6 J LysM-eGFP) were used to visualize the dynamic changes of myeloid-derived cells, putative dendritic cells and neutrophils during EAU. Transgenic mice were monitored with multi-modal fundus imaging camera over five time points following disease induction with the retinal auto-antigen, interphotoreceptor retinoid binding protein (IRBP1-20). Disease severity was quantified with both clinical and histopathological grading.. In the normal C57Bl/6 J Cx 3 cr1 (GFP/+) mouse Cx3cr1-expressing microglia were evenly distributed in the retina. In C57Bl/6 N CD11c-eYFP mice clusters of CD11c-expressing cells were noted in the retina and in C57Bl/6 J LysM-eGFP mice very low numbers of LysM-expressing neutrophils were observed in the fundus. Following immunization with IRBP1-20, fundus examination revealed accumulations of Cx3cr1-GFP(+) myeloid cells, CD11c-eYFP(+) cells and LysM-eGFP(+) myelomonocytic cells around the optic nerve head and along retinal vessels as early as day 14 post-immunization. CD11c-eYFP(+) cells appear to resolve marginally earlier (day 21 post-immunization) than Cx3cr1-GFP(+) and LysM-eGFP(+) cells. The clinical grading of EAU in transgenic mice correlated closely with histopathological grading.. These results illustrate that in vivo fundus imaging of transgenic reporter mice allows direct visualization of various exogenously and endogenously derived leukocyte types during EAU progression. This approach acts as a valuable adjunct to other methods of studying the clinical course of EAU.

Topics: Animals; Autoimmune Diseases; CD11c Antigen; CX3C Chemokine Receptor 1; Disease Models, Animal; Disease Progression; Eye Proteins; Freund's Adjuvant; Luminescent Proteins; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multimodal Imaging; Muramidase; Peptide Fragments; Receptors, Chemokine; Retinal Vessels; Retinitis; Retinol-Binding Proteins; Time Factors; Uveitis

Autoimmune diseases result from a break in immune tolerance leading to an attack on self-antigens. Autoantibody levels serve as a predictive tool for the early diagnosis of many autoimmune diseases, including type 1 diabetes. We find that a genetic locus on mouse chromosome 12 influences the affinity maturation of antibodies as well as autoantibody production. Thus, we generated a NOD.H2(k) congenic strain bearing B10 alleles at the locus comprised within the D12Mit184 and D12Mit12 markers, which we named NOD.H2(k)-Chr12. We determined the biological relevance of the Chr12 locus on the autoimmune process using an antigen-specific TCR transgenic autoimmune mouse model. Specifically, the 3A9 TCR transgene, which recognizes a peptide from hen egg lysozyme (HEL) in the context of I-A(k), and the HEL transgene, which is expressed under the rat-insulin promoter (iHEL), were bred into the NOD.H2(k)-Chr12 congenic strain. In the resulting 3A9 TCR:iHEL NOD.H2(k)-Chr12 mice, we observed a significant decrease in diabetes incidence as well as a decrease in both the quantity and affinity of HEL-specific IgG autoantibodies relative to 3A9 TCR:iHEL NOD.H2(k) mice. Notably, the decrease in autoantibodies due to the Chr12 locus was not restricted to the TCR transgenic model, as it was also observed in the non-transgenic NOD.H2(k) setting. Of importance, antibody affinity maturation upon immunization and re-challenge was also impeded in NOD.H2(k)-Chr12 congenic mice relative to NOD.H2(k) mice. Together, these results demonstrate that a genetic variant(s) present within the Chr12 locus plays a global role in modulating antibody affinity maturation.

Topics: Animals; Antibody Affinity; Autoantibodies; Autoantigens; Autoimmunity; Chromosomes, Mammalian; Diabetes Mellitus, Type 1; Disease Models, Animal; Genetic Loci; Genetic Variation; Humans; Insulin; Mice; Mice, Congenic; Mice, Inbred NOD; Mice, Transgenic; Muramidase; Rats

In septic shock (SS), dysfunction of many organ systems develops during the course of the illness, although the mechanisms are not clear. In earlier studies, we reported that lysozyme-c (Lzm-S), a protein that is released from leukocytes and macrophages, was a mediator of the myocardial depression and vasodilation that develop in a canine model of Pseudomonas aeruginosa SS. Whereas both of these effects of Lzm-S are dependent on its ability to intrinsically generate hydrogen peroxide, we subsequently showed that Lzm-S can also deposit within the vascular smooth muscle layer of the systemic arteries in this model. In the present study, we extend our previous findings. We used a canine carotid artery organ bath preparation to study the time course and dose dependence of Lzm-S deposition within the vascular smooth muscle layer. We used a human aortic vascular smooth muscle cell preparation to determine whether Lzm-S can persistently inhibit contraction in this preparation. We also used a canine P. aeruginosa model to determine whether Lzm-S deposition might occur in other organs such as the kidney, liver, and small intestine. The results showed that, in the carotid artery organ bath preparation, Lzm-S deposition occurred within minutes of instillation and there was a dose-response effect. In the human aortic vascular smooth muscle cell preparation, Lzm-S inhibited contraction during a 4-day period. In the in vivo model, Lzm-S accumulated in the kidney and the superior mesenteric artery. In a canine renal epithelial preparation, we further showed that Lzm-S can be taken up by the renal tubules to activate inflammatory pathways. We conclude that Lzm-S can deposit in the systemic vasculature and kidneys in SS, where this deposition could lead to acute organ dysfunction.

Topics: Animals; Aorta; Carotid Arteries; Cells, Cultured; Disease Models, Animal; Dogs; Humans; Intestine, Small; Kidney Tubules, Distal; Macrophages; Mesenteric Arteries; Muramidase; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Organ Culture Techniques; Pseudomonas aeruginosa; Pseudomonas Infections; Sepsis

Production of protein therapeutics often involves in vitro refolding from bacterial inclusion bodies and subsequent PEGylation to improve protein stability and plasma half-life. Here, we devised a novel strategy for one-step production of site-specific mono-PEGylated proteins with good bioactivity and improved biostability by integrating PEGylation and protein refolding (IPPR). Using lysozyme and recombinant human fibroblast growth factor 21 (rhFGF21) as model proteins, we showed that both PEGylation and refolding of denatured proteins have been simultaneously accomplished by IPPR with high efficiency of refolding yield and bioconjugation. PEGylated rhFGF21 by IPPR has a similar capacity as the native rhFGF21 to stimulate glucose uptake in 3T3-L1 cells, but exhibits prolonged blood glucose and triglyceride lowering activity levels in the ob/ob diabetic mouse model. Hence, IPPR will significantly facilitate the generation of protein therapeutics.

Topics: 3T3-L1 Cells; Animals; Cells, Cultured; Disease Models, Animal; Fibroblast Growth Factors; Humans; Mice; Mice, Obese; Muramidase; Polyethylene Glycols; Protein Refolding; Recombinant Proteins

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that is responsible for almost 1.5 million deaths per year. Sensing of mycobacteria by the host's immune system relies on different families of receptors present on innate immune cells. Amongst them, several members of the TLR family are involved in the activation of immune cells by mycobacteria, yet the in vivo contribution of individual TLRs to the protective immune response remains controversial. On the contrary, MyD88, the adaptor molecule for most TLRs, plays a non-redundant role in the protection against tuberculosis and mice with a complete germline deletion of MyD88 succumb very early to infection. MyD88 is expressed in both immune and non-immune cells, but it is not clear whether control of mycobacteria requires ubiquitous or cell-type specific MyD88 expression. Therefore, using novel conditional switch-on mouse models, we aimed to investigate the importance of MyD88 signalling in DCs and macrophages for the induction of protective effector mechanisms against mycobacterial infection. We conclude that specific reactivation of MyD88 signalling in CD11c- or lysozyme M-expressing myeloid cells during Mycobacterium bovis Bacille Calmette-Guerin infection is sufficient to restore systemic and local inflammatory cytokine production and to control pathogen burden.

Topics: Animals; CD11c Antigen; Chronic Disease; Cytokines; Dendritic Cells; Disease Models, Animal; Gene Deletion; Humans; Macrophages; Mice; Mice, Knockout; Muramidase; Mycobacterium bovis; Myeloid Differentiation Factor 88; Signal Transduction; Tuberculosis

Early diagnosis of neurological disorders would greatly improve their management and treatment. A major hurdle is that inflammatory products of cerebral disease are not easily detected in blood. Inflammation in multiple organs and heterogeneity in disease present additional challenges in distinguishing the extent to which a blood-based marker reflects disease in brain or other afflicted organs. Murine models of the monogenetic disorder Niemann-Pick Type C present aggressive forms of cerebral and liver inflammatory disease. Microarray analyses previously revealed age-dependent changes in innate immunity transcripts in the mouse brain. We have now validated four putative secretory inflammatory markers that are also elevated in mouse liver. We include limited, first time analysis of human Niemann-Pick Type C liver and cerebellum. Furthermore, we utilized 2-hydroxypropyl-β-cyclodextrin (HPβCD, an emerging therapeutic) administered intraperitoneally in mice, which abrogates inflammatory pathology in the liver but has limited effect on the brain. By analyzing the corresponding effects on inflammatory plasma proteins, we identified cathepsin S as a lead indicator of liver disease. In contrast, lysozyme was a marker of both brain and liver disease. 2-Hydroxypropyl-β-cyclodextrin had no effect on transcripts of neuron-specific 24-hydroxylase, and its product 24(S)-hydroxycholesterol was not a useful indicator in mouse plasma. Our data suggest that dual analysis of levels of the inflammatory markers lysozyme and cathepsin S may enable detection of multiple distinct states of neurodegeneration in plasma.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; beta-Cyclodextrins; Brain; Cathepsins; Disease Models, Animal; Female; Gene Deletion; Humans; Inflammation; Intracellular Signaling Peptides and Proteins; Liver; Male; Mice; Mice, Inbred BALB C; Muramidase; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C; Proteins

Clostridium difficile is a clinically important pathogen and the most common cause of hospital-acquired infectious diarrhea. Expression of the C. difficile gene csfV, which encodes σ(V), an extracytoplasmic function σ factor, is induced by lysozyme, which damages the peptidoglycan of bacteria. Here we show that σ(V) is required for lysozyme resistance in C. difficile. Using microarray analysis, we identified the C. difficile genes whose expression is dependent upon σ(V) and is induced by lysozyme. Although the peptidoglycan of wild-type C. difficile is intrinsically highly deacetylated, we have found that exposure to lysozyme leads to additional peptidoglycan deacetylation. This lysozyme-induced deacetylation is dependent upon σ(V). Expression of pdaV, which encodes a putative peptidoglycan deacetylase, was able to increase lysozyme resistance of a csfV mutant. The csfV mutant strain is severely attenuated compared to wild-type C. difficile in a hamster model of C. difficile-associated disease. We conclude that the σ(V) signal transduction system, which senses the host innate immune defense enzyme lysozyme, is required for lysozyme resistance and is necessary during C. difficile infection.

Topics: Animals; Anti-Bacterial Agents; Clostridioides difficile; Cricetinae; Disease Models, Animal; DNA, Bacterial; Enterocolitis, Pseudomembranous; Microarray Analysis; Microbial Sensitivity Tests; Muramidase; Sigma Factor; Virulence

The aim of this study was to explore the possible interactions between vitelline membrane outer layer 1 homolog (VMO1) and other tear proteins and to determine the function of VMO1 in tear fluid.. Interactions between recombinant human VMO1 and several abundant tear proteins were determined by dot blot, His pull-down, immunoprecipitation, and Western blot assays, as well as by computer-assisted prediction and modeling of molecular interactions. Kirby-Bauer antibiotic testing was performed to determine whether VMO1 possesses antimicrobial activity. Tear samples were collected from dry eye patients and from healthy controls. The role of VMO1 in maintaining the stability of tear film was investigated by measurement of contact angles on Teflon, tear break-up time (TBUT) and the time-dependent reduction in tear film integrity in mice.. Vitelline membrane outer layer 1 homolog showed an interaction with lysozyme C (LYSC) in the dot-blot, His pull-down, and immunoprecipitation assays. Vitelline membrane outer layer 1 homolog revealed no zones of growth inhibition of standard strains of Staphylococcus aureus and Escherichia coli. Tears presented smaller contact angles on Teflon surfaces after the addition of VMO1 (P<0.05). Vitelline membrane outer layer 1 homolog-treated mice showed longer TBUTs (P<0.05). Tear films from VMO1-treated mice maintained their integrity for longer periods of time than tear films from the control group, and this effect was dose-dependent.. Vitelline membrane outer layer 1 homolog interacts with LYSC and has positive effects on the stabilization of tear film.

Topics: Animals; Blotting, Western; Disease Models, Animal; Dry Eye Syndromes; Egg Proteins; Humans; Immunoprecipitation; Mice; Mice, Inbred C57BL; Muramidase; Tears

To study the intestinal expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in acute liver failure (ALF) using rat models, and to determine the relation of these expressions to intestinal bacterial translocation.. Forty-eight healthy male Sprague-Dawley rats were divided into a control group (n=8) and a model group (n=40; intraperitoneal injection of 10% D-galactosamine). The model group was further divided into five subgroups according to the time lapse after model establishment (8, 16, 24, 48, and 72 hours). At the end of the experiments, homogenates of mesenteric lymph nodes, liver and spleen were cultured in agar for bacterial outgrowth.Hematoxylin-eosin stained sections of liver and terminal ileum were examined under an optical microscope to assess pathological changes. mRNA expression of RD-5, sPLA2 and lysozyme in the terminal ileum was determined by reverse transcription-polymerase reaction (RT-PCR), and protein expression of sPLA2 and lysozyme from the same anatomic location was determined by western blotting and immunohistochemistry. Means between groups were compared with one-way analysis of variance.. ALF was successfully induced in the D-galactosamine injected rats. No bacteria grew in the organ cultures from the control group, while 8.3%, 37.5% and 58.3% of the rats in the 24-, 48-and 72-hour model groups showed positive cultures. Despite this, the structure of the terminal ileum from the rats in the 72-hour model group was nearly intact, without obvious necrosis of mucosal epithelial cells. Expression of RD-5 and sPLA2 mRNA in the model groups gradually increased at early time points and peaked at 16 hours after induction of ALF (1.291+/-0.153 and 1.131+/-0.128), which was significantly higher than that detected in the control group (0.725+/-0.116 and 0.722+/-0.112, t=69.25, 95.71, all P<0.01). After that, the expression of RD-5 and sPLA2 mRNA progressively decreased, and by 72 hours after the induction of ALF, the expression (0.415+/-0.104 and 0.425+/-0.076) was significantly lower than that of the control group (t=31.55 and 44.98, all P<0.01). Lysozyme mRNA expression in the model group peaked at 8 hours after ALF induction (1.211+/-0.107), which was higher than that of the control group at this time point (0.853+/-0.093), and by 72 hours after ALF induction it declined to 0.704+/-0.103, which was significantly lower than that of the control group (t=9.224; all P=0.009). In addition, at 72 hours after ALF induction the protein expression of both lysozyme and sPLA2 was significantly lower in the model group (0.327+/-0.086 and 0.382+/-0.057) than in the control group (0.583+/-0.121 and 0.650+/-0.093, t=12.28 and 15.83, P=0.004 and 0.001). Similar results were obtained with immunohistochemical staining.. The function of the ileal mucosal immune barrier in the rat model of acute liver failure decreased, along with decreases in expression of RD-5, sPLA2 and lysozyme in the Paneth cells.At the same time, the rate of organ bacterial translocation increased without obvious injury to the intestinal mucosa structure.

Topics: Animals; Bacterial Translocation; Defensins; Disease Models, Animal; Galactosamine; Injections, Intraperitoneal; Intestines; Liver Failure, Acute; Male; Muramidase; Phospholipases A2; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger

Ouraim was to study influence of acute somatic pain on lysozyme activity of rats of different age: newborn, rats after eye opening, rats at the age of a month, adult and old rats.. Lysozyme activity was checked before pain irritation and 2, 30, 60, 120, 180min afterwards using Dorofeychuk's method in our modification. Pain effect was modeling by electrical stimulation.. activity of lysozyme was 0. 434±0. 01 units in intact newborn rats, It was higher than in adult rats - 0.260 ±0.01 units (p <0.001) and it was unchanged during the experiment. We found low lysozyme activity in rats after eye opening - 0.015±0.003 units and it was stable during the experiment. Rats at the age ofa month had diphasic reaction: lysozyme activity was 0.191±0.01 units in intact rats, it increased up to 0.378±0.01 units (p <0.001) in 2 min after painful irritation and it decreased up to 0.113±0.02 units (p <0.001) in 30 min. Lysozyme activity was 0.260±0.01 units. Single-phase reaction was determined after acute painful irritation: increase of lysozyme activity after acute somatic pain up to 0,450±0,014 units (p <0.001). Lysozyme activity was 0.246±0.02 units in blood plasma of old rats. It decreased up to 0.1701±0.01 units (p <0.01) after painful irritation and it was 0.387±0.01 (p <0.001) in the end of the experiment.. Response on pain irritation has differences in different groups. The common vector of pain response was the increase of lysozyme activity in rats at the age of a month, adult rats, rats after eye opening and old rats. Reaction of increasing lysozyme activity was strongly detected in adult rats. The results demonstrate preventive lasozyme resistance to potential tissue damage or contamination.

Topics: Acute Pain; Age Factors; Animals; Disease Models, Animal; Male; Muramidase; Nociceptive Pain; Rats

Neutrophils are considered responsible for the pathophysiologic changes during hepatic ischemia-reperfusion (I/R) injury; however, few studies have examined real-time intravital neutrophil recruitment. Here, we show a method for imaging the neutrophil recruitment in hepatic I/R injury using two-photon laser scanning microscopy (TPLSM).. LysM-eGFP mice were subjected to 45 min of partial warm hepatic ischemia followed by reperfusion. Mice received an intravenous injection of tetramethylrhodamine isothiocyanate-labeled albumin to visualize the microvasculature. Using time-lapse TPLSM technique, we directly observed the behavior of neutrophils in I/R injury.. At low magnification, four to six hepatic lobules could be visualized. The number of adherent neutrophils continued to increase for 4 hr after reperfusion, whereas their crawling velocity reached a maximum of 2 hr after reperfusion and then decreased gradually. High-magnification images revealed the presence or absence of blood circulation in sinusoids. Six hours after control operation or reperfusion, circulation was maintained in all sinusoids in the control group, whereas spotty nonperfused areas accompanied by neutrophil infiltration could be observed in the I/R group. Adherent neutrophils in perfused areas in the I/R group had more elongated shapes and moved more quickly than those in nonperfused areas and in the control group. Some hepatocytes affected by I/R injury showed the changes in their size and fluorescent intensity, which could attract neutrophils.. TPLSM was successfully used for intravital imaging of hepatic I/R injury in mice and has potential for a wide range of applications to investigate the mechanism of I/R injury.

Topics: Animals; Cell Death; Chemokine CXCL1; Chemokine CXCL2; Disease Models, Animal; Fluorescent Dyes; Green Fluorescent Proteins; Immunity, Innate; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Microscopy, Fluorescence, Multiphoton; Muramidase; Neutrophil Infiltration; Neutrophils; Promoter Regions, Genetic; Reperfusion Injury; Rhodamines; Time Factors; Warm Ischemia

The role of hematogenous (hMΦ) and microglial (mMΦ) macrophages following spinal cord injury (SCI) remains unclear as they are not distinguished easily from each other in the lesion area. We have recently described the temporal and spatial response to SCI of each MΦ population using the lys-EGFP-ki mouse that enables EGFP(+) hMΦ to be distinguished from EGFP(-) mMΦ at the lesion site. In the present study, we characterized the response of monocyte and hMΦ subsets and mMΦ to SCI. We describe, for the first time, the responses of circulating classical (pro-inflammatory) and non-classical monocyte subsets to SCI. Additionally, we show the presence of classical and non-classical hMΦ at the SCI lesion. Importantly, we demonstrate that the 'classical pro-inflammatory' hMΦ respond in the acute (1d, 3d) stages of SCI while the 'non-classical' hMΦ respond in the sub-acute (7d, 14d) phase of SCI. At later time points (6weeks post injury) classical hMΦ return to the injury site. Our study offers new insight into the cellular inflammatory response that occurs after SCI and suggests that the timing and targets of anti-inflammatory therapies may be crucial to maximize neuroprotection at the acute and more chronic stages of SCI.

Topics: Animals; Disease Models, Animal; Female; Flow Cytometry; Green Fluorescent Proteins; Macrophages; Male; Mice; Mice, Transgenic; Microglia; Monocytes; Muramidase; Spinal Cord Injuries; Time Factors

Social stress is associated with altered immunity and higher incidence of anxiety-related disorders. Repeated social defeat (RSD) is a murine stressor that primes peripheral myeloid cells, activates microglia, and induces anxiety-like behavior. Here we show that RSD-induced anxiety-like behavior corresponded with an exposure-dependent increase in circulating monocytes (CD11b(+)/SSC(lo)/Ly6C(hi)) and brain macrophages (CD11b(+)/SSC(lo)/CD45(hi)). Moreover, RSD-induced anxiety-like behavior corresponded with brain region-dependent cytokine and chemokine responses involved with myeloid cell recruitment. Next, LysM-GFP(+) and GFP(+) bone marrow (BM)-chimeric mice were used to determine the neuroanatomical distribution of peripheral myeloid cells recruited to the brain during RSD. LysM-GFP(+) mice showed that RSD increased recruitment of GFP(+) macrophages to the brain and increased their presence within the perivascular space (PVS). In addition, RSD promoted recruitment of GFP(+) macrophages into the PVS and parenchyma of the prefrontal cortex, amygdala, and hippocampus of GFP(+) BM-chimeric mice. Furthermore, mice deficient in chemokine receptors associated with monocyte trafficking [chemokine receptor-2 knockout (CCR2(KO)) or fractalkine receptor knockout (CX3CR1(KO))] failed to recruit macrophages to the brain and did not develop anxiety-like behavior following RSD. Last, RSD-induced macrophage trafficking was prevented in BM-chimeric mice generated with CCR2(KO) or CX3CR1(KO) donor cells. These findings indicate that monocyte recruitment to the brain in response to social stress represents a novel cellular mechanism that contributes to the development of anxiety.

Topics: Animals; Antigens, CD; Anxiety Disorders; Bone Marrow; Brain; Calcium-Binding Proteins; Cell Movement; Cytokines; Disease Models, Animal; Exploratory Behavior; Gene Expression Regulation; Green Fluorescent Proteins; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Microglia; Monocytes; Muramidase; Receptors, CCR2; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Stress, Psychological; Time Factors

Sickness behaviors, such as fatigue, mood alterations, and cognitive dysfunction, which result from changes in central neurotransmission, are prevalent in systemic inflammatory diseases and greatly impact patient quality of life. Although, microglia (resident cerebral immune cells) and cytokines (e.g., TNFα) are associated with changes in central neurotransmission, the link between peripheral organ inflammation, circulating cytokine signaling, and microglial activation remains poorly understood. Here we demonstrate, using cerebral intravital microscopy, that in response to liver inflammation, there is increased monocyte specific rolling and adhesion along cerebral endothelial cells (CECs). Peripheral TNFα-TNFR1 signaling and the adhesion molecule P-selectin are central mediators of these monocyte-CEC adhesive interactions which were found to be closely associated with microglial activation, decreased central neural excitability and sickness behavior development. Similar monocyte-CEC adhesive interactions were also observed in another mouse model of peripheral organ inflammation (i.e., 2,4-dinitrobenzene sulfonic acid-induced colitis). Our observations provide a clear link between peripheral organ inflammation and cerebral changes that impact behavior, which can potentially allow for novel therapeutic interventions in patients with systemic inflammatory diseases.

Topics: Alanine Transaminase; Animals; Cell Adhesion; Cerebral Cortex; Cholestasis; Colitis; Cytokines; Dinitrofluorobenzene; Disease Models, Animal; Endothelial Cells; Female; Hippocampus; Illness Behavior; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Monocytes; Muramidase; P-Selectin; Pentylenetetrazole

Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease.. Transgenic sickle mice, which express human α and β(S) globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffy(b) ) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array.. Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals.. In sum, homozygous β(S) expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.

Topics: Anemia, Sickle Cell; Animals; Disease Models, Animal; Erythrocyte Transfusion; Erythrocytes; Humans; Immunization; Isoantibodies; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Muramidase

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.

Topics: Amyloidosis; Animals; Animals, Genetically Modified; Disease Models, Animal; DNA-Binding Proteins; Drosophila melanogaster; Drosophila Proteins; Endoplasmic Reticulum Stress; Eye Abnormalities; Female; Green Fluorescent Proteins; Hemolymph; Humans; Male; Metamorphosis, Biological; Microscopy, Electron, Scanning; Muramidase; Photoreceptor Cells, Invertebrate; Solubility; Unfolded Protein Response

Peyer's patches (PPs) of the small intestine are antigen sampling and inductive sites that help establish mucosal immunity. Luminal antigens are transported from the mucosal surface of PPs to the subepithelial dome (SED), through the specialized epithelial M cells of the follicle-associated epithelium. Among the SED resident dendritic cells (DCs), which are situated ideally for taking up these antigens, some express high levels of lysozyme (LysoDC) and have strong phagocytic activity. We investigated the mechanisms by which LysoDCs capture luminal antigens in vivo.. We performed 2-photon microscopy on explants of PPs from mice in which the enhanced green fluorescent protein gene was inserted into the lysozyme M locus (lys-EGFP mice), allowing fluorescence detection of LysoDC.. LysoDC extended dendrites through M-cell-specific transcellular pores to the gut lumen. The M-cell adhesion molecules junctional adhesion molecule-A and epithelial cell adhesion molecule were recruited to sites of transcellular migration. Transcellular dendrites scanned the M-cell apical surface and the gut luminal content; they were able to take pathogenic bacteria and inert particles in the lumen before retracting back to the SED.. We describe an antigen sampling mechanism that occurs in PPs and involves cooperation between M cells of the follicle-associated epithelium and DCs of the subepithelial dome. This process might be developed to target vaccines to the mucosa.

Topics: Animals; Antigens; Antigens, Neoplasm; Cell Adhesion Molecules; Cell Communication; Cell Movement; Dendritic Cells; Disease Models, Animal; Epithelial Cell Adhesion Molecule; Fluorescent Antibody Technique; Green Fluorescent Proteins; Immunity, Mucosal; Intestinal Mucosa; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Muramidase; Permeability; Peyer's Patches; Receptors, Cell Surface; Salmonella Infections; Salmonella typhimurium; Time Factors

Genetic polymorphisms of autophagy-related genes have been associated with an increased risk to develop inflammatory bowel disease (IBD). Autophagy is an elementary process participating in several cellular events such as cellular clearance and nonapoptotic programmed cell death. Furthermore, autophagy may be involved in intestinal immune homeostasis due to its participation in the digestion of intracellular pathogens and in antigen presentation. In the present study, the role of autophagy in the intestinal epithelial layer was investigated. The intestinal epithelium is essential to maintain gut homeostasis, and defects within this barrier have been associated with the pathogenesis of IBD. Therefore, mice with intestinal epithelial deletion of Atg7 were generated and investigated in different mouse models. Knockout mice showed reduced size of granules and decreased levels of lysozyme in Paneth cells. However, this was dispensable for gut immune homeostasis and had no effect on susceptibility in mouse models of experimentally induced colitis.

Topics: Animals; Autophagy; Autophagy-Related Protein 7; Biomarkers; Colitis; Cytoplasmic Granules; Disease Models, Animal; Gene Knockout Techniques; Homeostasis; Immunity, Innate; Immunohistochemistry; Intestinal Mucosa; Mice; Mice, Knockout; Microtubule-Associated Proteins; Muramidase; Paneth Cells

The acute inflammatory response that follows spinal cord injury (SCI) contributes to secondary injury that results in the expansion of the lesion and further loss of neurologic function. A cascade of receptor-mediated signaling events after SCI leads to activation of innate immune responses including the migration of microglia and active recruitment of circulating leukocytes. Because conventional techniques do not always distinguish macrophages derived from CNS-resident microglia from blood-derived monocytes, the role that each macrophage type performs cannot be assessed unambiguously in these processes. We demonstrate that, in the normal and spinal cord-injured lys-EGFP-ki transgenic mouse, enhanced green fluorescent protein (EGFP) is expressed only in mature hematopoietic granulomyelomonocytic cells and not in microglia. This allowed us to assess the temporal and spatial relationships between microglia-derived and hematogenous macrophages as well as neutrophils during a period of 6 weeks after clip compression SCI. Within the lesion, EGFP-positive monocyte-derived macrophages were found at the epicenter surrounded by EGFP-negative-activated microglia and microglia-derived macrophages. Neutrophils were not present when EGFP-positive monocyte-derived macrophages were depleted, indicating that neutrophil persistence in the lesion depended on the presence of these monocytes. Thus, these 2 distinct macrophage populations can be independently identified and tracked, thereby allowing their roles in acute and chronic stages of SCI-associated inflammation to be defined.

Topics: Animals; Antigens, CD1; Antigens, Differentiation; Antigens, Ly; Clodronic Acid; Disease Models, Animal; Female; Flow Cytometry; Gene Expression Regulation; Green Fluorescent Proteins; Liposomes; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Muramidase; Neutrophils; Peroxidase; Spinal Cord Injuries; Time Factors

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl(-) and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca(2+) potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.

Topics: Amiloride; Animals; Biomarkers; Calcium; Cells, Cultured; Chlorides; Cyclic AMP; Cystic Fibrosis; Diffusion Chambers, Culture; Disease Models, Animal; Electric Impedance; Epithelial Cells; Exocrine Glands; Humans; Ion Transport; Lactoferrin; Methacholine Chloride; Mucin-5B; Muramidase; Swine; Tight Junctions; Trachea

C57Bl/6 mice were intranasally infected with influenza virus A/H5N1 A/goose/Krasnoozerskoye/627/05. The mortality rate of animals reached 70% on day 14 of the disease. The lungs of animals were characterized by necroses, destruction of vessels, hemorrhagic and thrombotic complications, edematous syndrome, and early fibrosis of the interstitium. On days 6-10 after infection, fibrosis was found in the zones of postnecrotic inflammatory infiltration. The expression of lysozyme and myeloperoxidase by pulmonary macrophages was initially increased, but decreased on day 10 of the study. The number of cathepsin D-expressing macrophages was elevated up to the 10th day of examination.

Topics: Animals; Cathepsin D; Disease Models, Animal; Fas-Associated Death Domain Protein; Influenza A Virus, H5N1 Subtype; Interleukin-6; Lung; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Muramidase; Nitric Oxide Synthase Type II; Orthomyxoviridae Infections; Peroxidase; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

The prominent host muramidase lysozyme cleaves bacterial peptidoglycan (PG), and the enzyme is abundant in mucosal secretions. The lytic enzyme susceptibility of Gram-negative bacteria and mechanisms they use to thwart lytic enzyme activity are poorly studied. We previously characterized a Helicobacter pylori PG modification enzyme, an N-deacetylase (PgdA) involved in lysozyme resistance. In this study, another PG modification enzyme, a putative PG O-acetyltransferase (PatA), was identified. Mass spectral analysis of the purified PG demonstrated that a patA strain contained a greatly reduced amount of acetylated muropeptides, indicating a role for PatA in H. pylori PG O-acetylation. The PG modification mutant strains (pgdA, patA, or pgdA patA) were more susceptible to lysozyme killing than the parent, but this assay required high lysozyme levels (up to 50 mg/ml). However, addition of host lactoferrin conferred lysozyme sensitivity to H. pylori, at physiologically relevant concentrations of both host components (3 mg/ml lactoferrin plus 0.3 mg/ml lysozyme). The pgdA patA double mutant strain was far more susceptible to lysozyme/lactoferrin killing than the parent. Peptidoglycan purified from a pgdA patA mutant was five times more sensitive to lysozyme than PG from the parent strain, while PG from both single mutants displayed intermediate sensitivity. Both sensitivity assays for whole cells and for purified PGs indicated that the modifications mediated by PgdA and PatA have a synergistic effect, conferring lysozyme tolerance. In a mouse infection model, significant colonization deficiency was observed for the double mutant at 3 weeks postinoculation. The results show that PG modifications affect the survival of a Gram-negative pathogen.. Pathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to peptidoglycan (PG, the site of action of lysozyme) are a known mechanism used by Gram-positive bacteria to protect against host lysozyme attack. However, Gram-negative bacteria contain a thin layer of PG and a recalcitrant outer membrane permeability barrier to resist lysis, so molecular modifications to cell wall structure in order to combat lysis remain largely unstudied. Here we show that two Helicobacter pylori PG modification enzymes (PgdA and PatA) confer a clear protective advantage to a Gram-negative bacterium. They protect the bacterium from lytic enzyme degradation, albeit via different PG modification activities. Many pathogens are Gram negative, so some would be expected to have a similar cell wall-modifying strategy. Understanding such strategies may be useful for combating pathogen growth.

Topics: Acetyltransferases; Animals; Disease Models, Animal; Helicobacter Infections; Helicobacter pylori; Host-Pathogen Interactions; Mass Spectrometry; Mice; Microbial Viability; Muramidase; Peptidoglycan; Virulence

Shigella flexneri is the causative agent of bacillary dysentery and generates a significant global disease burden. The aim of this study was to analyze the pathogenesis and host immune response, at both the physiological and molecular level, using the model organism Caenorhabditis elegans, in response to S. flexneri. C. elegans is a nematode that responds to infection with a simple innate immune system, key aspects of which have been shown to be conserved.. S. flexneri-mediated infection of C. elegans was performed in both solid and liquid assays. The expression and subsequent regulation of host candidate antimicrobial genes such as lysozymes, C-type lectins and pathogen virulence genes were kinetically analyzed in the S. flexneri-exposed nematode.. In solid assays, worms fed with S. flexneri showed complete killing at 153 ± 9 h. The kinetic studies showed that S. flexneri killed the worms upon continuous exposure at 41 ± 1.7 h. However, short-time exposure of the host to S. flexneri indicated that 14 h of exposure resulted in a loss of progeny, and death occurred after 46 h. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that mRNA levels of host candidate antimicrobial genes and pathogen virulence genes varied significantly at the time of early infection.. The killing of C. elegans requires live bacteria, and a minimal exposure time is sufficient for S. flexneri to have a lethal effect. The candidate antimicrobial genes and virulence genes are kinetically regulated within C. elegans during S. flexneri-mediated infections, thereby exhibiting their role and contribution in the host innate immune system.

Topics: Animals; Caenorhabditis elegans; Disease Models, Animal; Gene Expression Profiling; Host-Pathogen Interactions; Humans; Lectins; Muramidase; Shigella flexneri; Virulence Factors

Previous studies from this laboratory have shown that aerosolized recombinant human lysozyme (rhLZ) mitigates Pseudomonas aeruginosa (PA)-induced pneumonia. In the current investigation, our laboratory tested the hypothesis that aerosolized rhLZ can potentiate the effects of tobramycin (TBMN), thereby reducing the effective dose of this agent in the treatment of PA-induced pneumonia. Syrian hamsters were instilled intratracheally with PA, then exposed to an aerosol containing either 1% rhLZ, 3 μg TBMN, or a combination of both agents. In contrast to the initial studies with rhLZ, which involved 3 separate aerosol exposures, only a single treatment was used in the current investigation. Twenty-four hours after completion of the aerosol regimen, the following parameters were measured: (1) whole-lung colony-forming units (CFU), (2) total bronchoalveolar lavage fluid (BALF) CFU, (3) lung histopathology, and (4) total BALF neutrophils. The combination of rhLZ and TBMN significantly reduced whole-lung and BALF CFU, as well as the inflammatory index, compared to TBMN alone. Similar results were seen in vitro with regard to bactericidal activity. These findings provide a rationale for clinical testing of rhLZ as an adjunct to commercial antibiotic treatment.

Topics: Administration, Inhalation; Animals; Anti-Bacterial Agents; Bronchoalveolar Lavage Fluid; Cricetinae; Disease Models, Animal; Drug Synergism; Female; Humans; Lung; Mesocricetus; Muramidase; Neutrophils; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Recombinant Proteins; Tobramycin

Intravital microscopy is a useful tool for studying leukocyte trafficking in atherosclerosis. However, distinction between various subclasses of leukocytes using this technology is lacking. Therefore, we generated ApoE(-/-)/Lysozyme M(EGFP/EGFP) mice and investigated whether targeted cell types could be visualized by in vivo microscopy and whether absence of lysozyme M will influence atherosclerosis.. We crossed male ApoE(-/-) mice with mice homozygous for a knock-in mutation of enhanced green fluorescent protein (EGFP) in the lysozyme M locus (Lys(EGFP/EGFP)) creating ApoE(-/-)/Lys(EGFP/EGFP) mice. Mice were sacrificed at the age of 26 weeks. Blood was collected for serum lipid analysis, differential white blood cell count and flow cytometry. Lesion area was determined on en face mounted aortas and sections from aortic roots were stained for immunohistochemistry. Atherosclerotic lesions were also studied by confocal- and intravital microscopy.. Basic parameters, such as white blood cell count, cholesterol profile, lesion area and plaque composition was unaltered in ApoE(-/-)/Lys(EGFP/EGFP) mice compared to ApoE(-/-) mice. Fluorescent neutrophils and monocytes were clearly visualized by intravital fluorescence and confocal microscopy. Fluorescent cells were distributed primarily in the periphery of atherosclerotic lesions indicating a preference for recruitment in these areas.. ApoE(-/-)/Lys(EGFP/EGFP) mice will serve as a useful model to study leukocyte trafficking in atherosclerosis and how different subsets of leukocytes influence atherogenesis.

Topics: Animals; Aorta; Apolipoproteins E; Atherosclerosis; Disease Models, Animal; Green Fluorescent Proteins; Homozygote; Leukocytes; Male; Mice; Mice, Inbred C57BL; Monocytes; Muramidase; Neutrophils; Reactive Oxygen Species

Community-acquired pneumonia is a very common infectious disease associated with significant morbidity and mortality. Streptococcus pneumoniae is the predominant pathogen in this disease, and pneumococcal resistance to multiple antibiotics is increasing. The recently purified bacteriophage endolysin Cpl-1 rapidly and specifically kills pneumococci on contact. The aim of this study was to determine the therapeutic potential of Cpl-1 in a mouse model of severe pneumococcal pneumonia.. Controlled, in vivo laboratory study.. Female C57/Bl6 mice, 8-12 weeks old.. Mice were transnasally infected with pneumococci and therapeutically treated with Cpl-1 or amoxicillin by intraperitoneal injections starting 24 or 48 hours after infection.. Judged from clinical appearance, decreased body weight, reduced dynamic lung compliance and Pao2/Fio2 ratio, and morphologic changes in the lungs, mice suffered from severe pneumonia at the onset of therapy. When treatment was commenced 24 hours after infection, 100% Cpl-1-treated and 86% amoxicillin-treated mice survived otherwise fatal pneumonia and showed rapid recovery. When treatment was started 48 hours after infection, mice had developed bacteremia, and three of seven (42%) Cpl-1-treated and five of seven (71%) amoxicillin-treated animals survived. Cpl-1 dramatically reduced pulmonary bacterial counts, and prevented bacteremia, systemic hypotension, and lactate increase when treatment commenced at 24 hours. In vivo, treatment with Cpl-1 or amoxicillin effectively reduced counts of penicillin-susceptible pneumococci. The inflammatory response in Cpl-1-and amoxicillin-treated mice was lower than in untreated mice, as determined by multiplex cytokine assay of lung and blood samples. In human epithelial cell cultures, lysed bacteria evoked less proinflammatory cytokine release and cell death, as compared with viable bacteria.. Cpl-1 may provide a new therapeutic option in the treatment of pneumococcal pneumonia.

Topics: Amoxicillin; Animals; Disease Models, Animal; Female; Mice; Mice, Inbred C57BL; Muramidase; Pneumonia, Pneumococcal

Inflammatory bowel disease (IBD) is a chronic and recurring inflammation of the gastrointestinal tract, associated with a dysregulation of the mucosal immune system. There is an increasing prevalence of IBD; however, current pharmaceutical treatments are only moderately effective and have been associated with potential long-term toxicity. Lysozyme, a well-known antimicrobial protein found in large quantities in hen egg white, is a promising alternative for the treatment of IBD. A porcine model of dextran sodium sulfate (DSS)-induced colitis was used to examine the effect of hen egg lysozyme (HEL) supplementation on intestinal inflammation. Treatment with DSS resulted in weight loss, severe mucosal and submucosal inflammation, colonic crypt distortion, muscle wall thickening, down-regulation of mucin gene expression, and increased gastric permeability, but these symptoms were attenuated following supplementation with HEL and restored to basal levels observed in untreated control animals. Treatment with HEL also significantly reduced the local expression of pro-inflammatory cytokines TNF-alpha, IL-6, IFN-gamma, IL-8, and IL-17 while increasing the expression of the anti-inflammatory mediators IL-4 and TGF-beta, indicating that HEL may function as a potent anti-inflammatory and immunomodulator. Furthermore, the concomitant increases in TGF-beta and Foxp3 levels suggest that HEL may aid in restoring gut homeostasis by activating regulatory T cells, which are important in the regulation of the mucosal immune system. These results suggest that HEL is a promising novel therapeutic for the treatment of IBD.

Topics: Animals; Anti-Inflammatory Agents; Colitis; Cytokines; Dextran Sulfate; Disease Models, Animal; Gene Expression; Intestinal Mucosa; Muramidase; Swine

The progression of kidney disease to renal failure correlates with infiltration of mononuclear immune cells into the tubulointerstitium. These infiltrates contain macrophages, DCs, and T cells, but the role of each cell type in disease progression is unclear. To investigate the underlying immune mechanisms, we generated transgenic mice that selectively expressed the model antigens ovalbumin and hen egg lysozyme in glomerular podocytes (NOH mice). Coinjection of ovalbumin-specific transgenic CD8+ CTLs and CD4+ Th cells into NOH mice resulted in periglomerular mononuclear infiltrates and inflammation of parietal epithelial cells, similar to lesions frequently observed in human chronic glomerulonephritis. Repetitive T cell injections aggravated infiltration and caused progression to structural and functional kidney damage after 4 weeks. Mechanistic analysis revealed that DCs in renal lymph nodes constitutively cross-presented ovalbumin and activated CTLs. These CTLs released further ovalbumin for CTL activation in the lymph nodes and for simultaneous presentation to Th cells by distinct DC subsets residing in the kidney tubulointerstitium. Crosstalk between tubulointerstitial DCs and Th cells resulted in intrarenal cytokine and chemokine production and in recruitment of more CTLs, monocyte-derived DCs, and macrophages. The importance of DCs was established by the fact that DC depletion rapidly resolved established kidney immunopathology. These findings demonstrate that glomerular antigen-specific CTLs and Th cells can jointly induce renal immunopathology and identify kidney DCs as a mechanistic link between glomerular injury and the progression of kidney disease.

Topics: Animals; Antigen Presentation; Autoimmune Diseases; Cell Movement; Dendritic Cells; Disease Models, Animal; Glomerulonephritis; Kidney; Leukocytes, Mononuclear; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Muramidase; Ovalbumin; Podocytes; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer

Synthetic oligodeoxynucleotides (ODN) expressing 'suppressive' TTAGGG motifs down-regulate a variety of proinflammatory and T helper type 1 (Th1)-mediated pathological immune responses. The ability of the archetypal suppressive ODN A151 to inhibit ocular inflammation was examined in two murine models: experimental autoimmune uveitis, induced by immunization with a retinal antigen, interphotoreceptor retinoid-binding protein (IRBP) and adoptively transferred ocular inflammation, induced by transferring Th1 cells specific to hen egg lysozyme (HEL) into recipient mice that express HEL in their eyes. A151 treatment suppressed the inflammation in both models. In addition, A151 inhibited IRBP-specific cytokine production and lymphocyte proliferation in mice immunized with IRBP. These findings suggest that suppressive ODN affects both afferent and efferent limbs of the immunopathogenic process and may be of use in the treatment of autoimmune ocular inflammation.

Topics: Adoptive Transfer; Animals; Autoimmune Diseases; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Eye Proteins; Female; Immunosuppressive Agents; Inflammation Mediators; Lymphocyte Activation; Mice; Mice, Inbred Strains; Muramidase; Oligodeoxyribonucleotides; Retinol-Binding Proteins; Th1 Cells; Uveitis

To study an expanded time course of surgically induced choroidal neovascularization (CNV) in a porcine model applying fluorescence angiography and immunohistology.. Twenty-two porcine eyes underwent vitrectomy, a retinal bleb was raised and the detached retina perforated using endodiathermy. Bruch's membrane was perforated with a retinal perforator at a site where the overlying neuroretina was normal. Eyes were enucleated in a time interval between 30 min and 42 days after the perforation, and the pigs were subsequently killed. Immediately prior to enucleation, fundus photographs and fluorescein angiograms were obtained. Sections of paraffin-embedded eyes were immunohistochemically stained.. On fluorescein angiography, membranes aged 14 days or less exhibited leakage in 10/11 cases while the remaining showed persistent staining. The propensity to leak diminished with time and only 1/3 of the oldest membranes leaked. In eyes enucleated immediately after surgery, neuroretinas overlying the induced lesions were intact without apparent atrophy of cells. At day 3, macrophages and myofibroblasts formed membrane-like structures in the subretinal space. At day 7, the outer surface of the membrane was covered by retinal pigment epithelium (RPE) cells and the neuroretinas had suffered damage in the form of outer segment loss. In the time period 14-42 days, the CNV membrane became completely enveloped by RPE cells. The degree of membrane vascularization increased with time and was at its maximum after 42 days. Intact outer segments were identified over the oldest membranes.. The formation of surgical CNV membranes followed the normal reparatory pathway and the degree of vascularization of CNV membranes continued to increase during the 42 days. However, propensity to leak diminished with time. We believe that this was because of the fact that RPE cells completely enveloped older membrane and thus prevented leakage from the newly formed vessels. Photoreceptor outer segments, which had atrophied after 7 days, were able to regenerate over CNV membranes and could be identified again after 42 days.

Topics: Actins; Animals; Blood-Retinal Barrier; Bruch Membrane; Capillary Permeability; Choroidal Neovascularization; Collagen; Disease Models, Animal; Female; Fluorescein Angiography; Glial Fibrillary Acidic Protein; Immunoenzyme Techniques; Macrophages; Muramidase; Retinal Pigment Epithelium; Swine; Vitrectomy; von Willebrand Factor

Antimicrobial peptides are intrinsic to the innate immune system in many organ systems, but little is known about their expression in the central nervous system. We examined cerebrospinal fluid (CSF) and serum from patients with active bacterial meningitis to assess antimicrobial peptides and possible bactericidal properties of the CSF. We found antimicrobial peptides (human cathelicidin LL-37) in the CSF of patients with bacterial meningitis but not in control CSF. We next characterized the expression, secretion, and bactericidal properties of rat cathelin-related antimicrobial peptide, the homologue of the human LL-37, in rat astrocytes and microglia after incubation with different bacterial components. Using real-time polymerase chain reaction and Western blotting, we determined that supernatants from both astrocytes and microglia incubated with bacterial component supernatants had antimicrobial activity. The expression of rat cathelin-related antimicrobial peptide in rat glial cells involved different signal transduction pathways and was induced by the inflammatory cytokines interleukin 1beta and tumor necrosis factor. In an experimental model of meningitis, infant rats were intracisternally infected with Streptococcus pneumoniae, and rat cathelin-related antimicrobial peptide was localized in glia, choroid plexus, and ependymal cells by immunohistochemistry. Together, these results suggest that cathelicidins produced by glia and other cells play an important part in the innate immune response against pathogens in central nervous system bacterial infections.

Topics: Adolescent; Adult; Aged; Animals; Animals, Newborn; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Brain; Cathelicidins; Cells, Cultured; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Humans; Male; Meningitis, Pneumococcal; Middle Aged; Muramidase; Neuroglia; Nitrites; Rats; Rats, Wistar; RNA, Messenger; Time Factors; Young Adult

Activation of tubular epithelial cells by transforming growth factor-beta (TGF-beta) plays an important role in the pathogenesis of renal tubulointerstitial fibrosis. We developed a renally accumulating conjugate of a TGF-beta type-I receptor kinase inhibitor (TKI) and evaluated its efficacy in vitro and in vivo.. TKI was conjugated to the protein Lysozyme (LZM) via a platinum-based linker. TKI-LZM was evaluated in human tubular cells (HK-2) for its anti-fibrotic activity. Plasma, kidney and urine drug levels after a single intravenous dose of TKI-LZM in rats were determined by HPLC or immunodetection. Anti-fibrotic effects of TKI-LZM were examined in the unilateral ureteral obstruction (UUO) model.. TKI-LZM conjugate was successfully synthesized at an 1:1 drug/carrier ratio, and inhibited TGF-beta1-induced procollagen-1alpha1 gene expression in HK-2 cells. In vivo, TKI-LZM accumulated rapidly in tubular cells and provided a local depot for 3 days. Interestingly, a single dose of TKI-LZM inhibited the activation of tubular cells and fibroblasts in UUO rats and reduced renal inflammation. In contrast, free TKI at an equimolar (low) dosage exhibited little effects.. Inhibition of TGF-beta signaling by local drug delivery is a promising antifibrotic strategy, and demonstrated the important role of tubular activation in renal fibrosis.

Topics: Animals; Cell Line; Collagen Type I; Collagen Type I, alpha 1 Chain; Disease Models, Animal; Drug Carriers; Epithelial Cells; Fibroblasts; Fibrosis; Humans; Injections, Intravenous; Kidney Diseases; Kidney Tubules, Proximal; Male; Muramidase; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Rats; Rats, Wistar; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Transforming Growth Factor beta1; Ureteral Obstruction

Microbial products stimulate the immune system by interacting with Toll-like receptors (TLR) on antigen-presenting cells. This study examined the hypothesis that microbial products, which function as TLR ligands, are playing a major role in triggering pathogenic autoimmunity.. An experimental system was developed in which microbial TLR ligands were tested in vivo for their capacity to stimulate naïve CD4 cells specific against hen egg lysozyme (HEL) to become effector cells capable of inducing inflammation in eyes in which HEL is expressed. The ligands' mode of action was analyzed by determining their effects on the proliferation, acquisition of tissue-invading capacity, i.e. elevated CD49d and decreased CD62L expression, and production of interferon-gamma by the HEL-specific cells.. All the 7 tested TLR ligands triggered ocular inflammation in the experimental system used here, with pertussis toxin surpassing all other ligands in its activities. A correlation was found between the capacity of the ligands to trigger pathogenic immunity and to stimulate the proliferation, modification of cell surface and interferon-gamma production by T cells.. This study provides direct evidence to support the notion that microbial products are capable of triggering pathogenic autoimmunity.

Topics: Animals; Autoantigens; Autoimmune Diseases; Bacteria; Bacterial Toxins; CD4-Positive T-Lymphocytes; Disease Models, Animal; Integrin alpha2; Interferon-gamma; L-Selectin; Ligands; Mice; Mice, Transgenic; Muramidase; Polysaccharides, Bacterial; Toll-Like Receptors; Uveitis

The effects of the novel proline-containing nootropic and neuroprotective dipeptide, noopept (GVS-111, N-phenylacetyl-L-prolylglycine ethyl ester) were investigated in NMRI mice following olfactory bulbectomy. We have shown previously that these animals developed Alzheimer's disease (AD)-like behaviour, morphology and biochemistry including impairment of spatial memory, regional neuronal degeneration and elevated Abeta peptide brain levels. In the current investigation, spatial memory was assessed using the Morris water maze and serum antibodies to in vitro morphologically characterized amyloid structures of both Abeta((25-35)) peptide and equine lysozyme, as well as to neurotrophic glial factor S100b, were analyzed by enzyme-linked immunosorbent assay (ELISA). Noopept (administered at a dose of 0.01 mg/kg for a period of 21 days and during a further 5 days training) restored spatial memory and increased serum antibody levels to oligomers of Abeta((25-35)) peptide but not to equine lysozyme amyloid or S100b protein in bulbectomized animals. The positive immunotropic effect of noopept to Abeta((25-35)) peptide prefibrillar aggregates was more marked in sham-operated compared to the bulbectomized subjects which were characterized by an overall suppression of immunoreactivity. Enhancement of the immune response to Abeta((25-35)) peptide prefibrils caused by noopept may attenuate the neurotoxic consequences of amyloid fibrillization and also be associated with an improvement in spatial memory in bulbectomized mice. These actions of noopept, combined with its previously reported neuroprotective and cholinomimetic properties, suggests that this dipeptide may well be useful for improving cognitive deficits induced by neurodegenerative diseases.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Autoantibodies; Behavior, Animal; Dipeptides; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Male; Memory; Mice; Microscopy, Atomic Force; Muramidase; Nerve Growth Factors; Neuroprotective Agents; Nootropic Agents; Olfactory Bulb; Peptide Fragments; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Space Perception; Time Factors

Topics: Acute Disease; Administration, Intranasal; Animals; Disease Models, Animal; Female; Humans; Influenza, Human; Mice; Mice, Inbred BALB C; Muramidase; Orthomyxoviridae; Otitis Media; Pneumococcal Infections; Streptococcus pneumoniae

Photoreceptor degeneration in human photoreceptor dystrophies and in the relevant animal models has been thought to be executed by one common mechanism -- caspase-mediated apoptosis. However, recent experiments have challenged this concept. In previous experiments, analyzing gene expression in the degenerating rd/rd mouse retina, we have suggested that the gene defect leads to oxidative stress and altered metabolism, which may induce caspase-dependent and caspase-independent cell death mechanisms such as the activation of cystein-proteases, lysosomal proteases, autophagy and complement-mediated lysis. In this study we asked two questions. First, whether a temporal analysis of these different mechanisms during the course of degeneration would enable us to establish a causal relationship between these events; and second, whether photoreceptor degeneration in different models of photoreceptor dystrophies occurs by activating the same mechanisms. Three models of photoreceptor degeneration were chosen in which photoreceptor degeneration is caused by different events: the rd/rd mouse (calcium overload); the rds/rds mouse (structural defect); and light-damage (LD; oxidative stress). Marker genes were selected for the identified processes. PCR-analysis on laser capture microdissection samples was used to verify the expression of these genes in the rod photoreceptor layer. A temporal relationship between the processes was established at the mRNA level, using quantitative RT-PCR. The time course of gene expression was compared to that of cell loss (loss of rows of photoreceptor nuclei) and apoptosis (TUNEL labeling). Apoptosis and autophagy was analyzed using enzymatic assays. The time course of apoptosis and TUNEL labeling coincide in all three models. Complement-activated lysis was found to either parallel (rd/rd and rds/rds) or precede (LD) the development of TUNEL-positive cells. Autophagy was determined to parallel (rd/rd and LD) or lag (rds/rds) behind the development of TUNEL-positive cells. In all three models, glucose metabolism was found to be increased significantly prior to the onset of cell death, but then dropped in parallel with the loss of cells. The presence of the marker genes was verified by laser capture microdissection, and apoptosis (caspase activity) and autophagy (lysozyme and cathepsin activity) were verified in retina extracts. These results provide evidence that irrespective of whether photoreceptor degeneration is triggered by gene defec

Topics: Animals; Apoptosis; Autophagy; Caspase 1; Cell Death; Complement Activation; Disease Models, Animal; Gene Expression Regulation; Genetic Markers; In Situ Nick-End Labeling; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Muramidase; Oxidative Stress; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Time Factors

Because no biomarker that reflects small bowel allograft rejection is available, we applied surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to develop noninvasive markers required for routine diagnosis.. Heterotopic small bowel transplantation (SBT) was performed in rats, and they were divided into four experimental groups: sham-operated rats (sham), syngeneic transplants (syngeneic), allogeneic transplants (allogeneic), allogeneic transplants received FK506 (allo+FK). Plasma samples were analyzed with SELDI ProteinChip arrays to detect peaks that predominated in the allogeneic model. Possible biomarkers were identified in combination with SELDI retentate chromatography mass spectrometry (RCMS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The identified protein was further analyzed by immunohistochemistry.. An increase in the level of a 14.8-kDa protein, identified as lysozyme, was observed specifically in the plasma of the allogeneic group; the levels of this protein remained unchanged in the plasma of the other groups. On the other hand, the levels of a 10.1-kDa and a 13.0-kDa protein, identified as migration inhibitory factor-related proteins (MRP), MRP-8 and MRP-14, respectively, began to increase from an early stage of acute rejection. We also observed that lysozyme-positive macrophages had strongly infiltrated the lamina propria during acute rejection.. We identified three plasma proteins-MRP-8, MRP-14, and lysozyme-that increased during small bowel allograft rejection. The identified proteins appeared to be markers for inflammation associated with allograft rejection. This proteomic approach will be useful for the identification of candidate biomarkers.

Topics: Amino Acid Sequence; Animals; Biomarkers; Blood Proteins; Disease Models, Animal; Graft Rejection; Immunohistochemistry; Intestine, Small; Male; Molecular Sequence Data; Muramidase; Protein Array Analysis; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors

To evaluate the effects of an interleukin 1 receptor antagonist (IL-1RA) on the development of immune-mediated ocular inflammation in mice.. Recombinant, human, nonglycosylated IL-1RA (anakinra [kineret]) was tested for its inhibitory effects in 2 systems: (1) experimental autoimmune uveitis induced by interphotoreceptor retinoid-binding protein in B10.A mice using routine procedures and evaluated by clinical and histological examination, and (2) ocular inflammation in mice induced by transfer of hen egg lysozyme-specific T cells to hen egg lysozyme-transgenic mice. Treatment with IL-1RA included daily subcutaneous injections of the drug, at 300 and 500 mg/kg, or phosphate-buffered saline as control.. Mean +/- SE experimental autoimmune uveitis scores of histological ocular changes of the mice at day 14 postimmunization with interphotoreceptor retinoid-binding protein were 1.5 +/- 0.3 in control mice; 1.0 +/- 0.4 in 300-mg/kg anakinra-treated mice; and 0.5 +/- 0.2 in 500- mg/kg anakinra-treated mice (P = .004). There was a corresponding decrease in the cellular immune response and cytokine production of immune cells in treated mice. Suppression of ocular inflammation by anakinra in the transfer system was also observed (P = .04).. Human IL-1RA suppresses immune-mediated ocular inflammation in mice, affecting both the afferent and efferent components of the pathogenic immune response.Clinical Relevance Systemic administration of IL-1RA may have clinical application in the management of patients with uveitis.

Topics: Adoptive Transfer; Animals; Autoimmune Diseases; Cytokines; Disease Models, Animal; Eye Proteins; Female; Immunity, Cellular; Immunosuppression Therapy; Immunotherapy, Adoptive; Injections, Subcutaneous; Interleukin 1 Receptor Antagonist Protein; Lymphocyte Activation; Mice; Mice, Transgenic; Muramidase; Recombinant Proteins; Retinol-Binding Proteins; Sialoglycoproteins; Th1 Cells; Uveitis

Intraperitoneal adhesions are an important cause of postoperative intestinal obstruction, abdominal discomfort and infertility. In the present study we hypothesized that a combination of polypeptides with different surface properties, resulting in fine disperse low-soluble complexes, could be of benefit in the prevention of abdominal adhesions.. Various polypeptides including lysozyme, polyglutamate, polylysine and combinations of all three were evaluated as compared to hyaluronic acid. A standard wound on the parietal peritoneum in mice was used and the evaluated agents were administered immediately postoperatively. The extent of peritoneal adhesions to the injured area was measured and expressed as a percentage of the wound length as evaluated after 7 days. Flow cytometry was performed to evaluate the effect on peritoneal macrophage survival and phagocytic function and the Pick test was used to determine peroxide production in order to estimate toxicity and potential impairment of macrophage function caused by the chemicals.. Significant differences were seen among the treatment groups (p<0.001). Both polyglutamate and lysozyme, and polyglutamate together with polylysine significantly decreased adhesion formation as compared to hyaluronic acid. The polylysine-polyglutamate combination was still visible macroscopically on the peritoneal surface after 1 week, though not after 1 month. The polyglutamate-lysozyme mixture was less effective than these individual components alone. The chemicals did not show any toxic effects or altered function in macrophage cell culture.. Lysozyme, polyglutamate and, most effectively, a polyglutamate-polylysine combination significantly decreased experimental abdominal adhesion formation. A strong mechanical connection to the wound and prolonged attendance in the surface were noted. Peritoneal phagocyte function did not seem to be influenced by the chemicals.

Topics: Adjuvants, Immunologic; Animals; Anti-Infective Agents; Disease Models, Animal; Female; Flow Cytometry; Follow-Up Studies; Hyaluronic Acid; Macrophages, Peritoneal; Mice; Mice, Inbred Strains; Muramidase; Peritoneal Diseases; Polyglutamic Acid; Polylysine; Postoperative Complications; Tissue Adhesions; Wound Healing

Infection with Salmonella typhimurium can produce multiple organ dysfunctions. However, document concerning with gastric hemorrhagic ulcers occur in this infectious disease is lacking. The aim was to study modulation of gastric hemorrhagic ulcer by oxidative stress and mast cell histamine in S. typhimurium-infected rats. Additionally, the protective effects of drugs, such as ofloxacin, lysozyme chloride, ketotifen, ranitidine, and several antioxidants, including exogenous glutathione (GSH), allopurinol and dimethylsulfoxide (DMSO) were evaluated. Male Wistar rats were injected intrajejunally with a live culture of S. typhimurium (1 x 10(10) colony-forming units/rat) and followed by deprivation of food for 36 h. Age-matched control rats received sterilized vehicle only. Rat stomachs were irrigated for 3 h with either normal saline or a simulated gastric juice containing 100 mM HCl, 17.4 mM pepsin and 54 mM NaCl. S. typhimurium caused aggravation of offensive factors, including enhancing gastric acid back-diffusion, mucosal lipid peroxide generation, histamine release, microvascular permeability and hemorrhagic ulcer, as well as an attenuation of defensive substances, such as mucosal GSH and mucus level. Intragastric irrigation of gastric juice caused further aggravation of these gastric biochemical parameters. This exacerbation of ulcerogenic factors was abolished by pretreatment of ofloxacin and lysozyme chloride. Antioxidants, such as reduced GSH, allopurinol and DMSO also produced significant (P < 0.05) amelioration of gastric damage in S. typhimurium infected rats. In conclusion, gastric oxidative stress and histamine play pivotal roles in the formation of hemorrhagic ulcers that were effectively ameliorated by ofloxacin, lysozyme chloride, ketotifen, ranitidine, diamine oxidase and various antioxidants in S. typhimurium-infected rats.

Topics: Allopurinol; Animals; Antioxidants; Dimethyl Sulfoxide; Disease Models, Animal; Gastric Juice; Gastric Mucosa; Gastrointestinal Hemorrhage; Glutathione; Histamine Release; Indomethacin; Lipid Peroxides; Male; Mast Cells; Muramidase; Ofloxacin; Oxidative Stress; Rats; Rats, Wistar; Salmonella Infections, Animal; Sodium Chloride; Stomach Ulcer; Therapeutic Irrigation

Reversible myocardial depression in sepsis has been ascribed to the release of inflammatory mediators. We recently found that lysozyme c (Lzm-S), consistent with that originating from the spleen, was a mediator of myocardial depression in an Escherichia coli model of septic shock in dogs. We further showed in a right ventricular trabecular (RVT) preparation that Lzm-S's depressant activity could be blocked by N,N',N" triacetylglucosamine (TAC), a competitive inhibitor of Lzm-S. We hypothesized that Lzm-S binds to or cleaves a cardiac membrane glycoprotein, thereby interfering with myocardial contraction in sepsis. In the present study, we examined whether TAC could prevent myocardial depression in an in vivo preparation and whether other related N-acetylglucosamine (NAG) structures could also inhibit Lzm-S's effect in RVT.. Randomized experimental study.. University laboratory.. Anesthetized, mechanically ventilated dogs.. We produced sepsis by infusion of E. coli over an approximately 6-hr period.. We examined the effect of TAC on stroke work, our primary index of myocardial function, when treatment was administered before sepsis (pretreatment) and after 1.5 hrs (early treatment study) and 3.5 hrs of sepsis (late treatment study; LTS). In the pretreatment study and early treatment study, myocardial depression would have not yet occurred but would have already been present in the late treatment study. In RVT, we assessed the effect of other NAG oligosaccharides and variants to the NAG structure on Lzm-S's depressant activity. In pretreatment and the early treatment study, TAC prevented the reduction in stroke work observed in nontreated septic groups but did not reverse the reduction found in the late treatment study. In RVT, of the compounds tested, only N,N'-diacetylglucosamine showed an inhibitory effect.. We found that TAC, a competitive inhibitor of Lzm-S, prevented myocardial depression in experimental sepsis. Only specific NAG structures are inhibitory to Lzm-S's depressant activity. TAC may be useful in attenuating cardiovascular collapse in sepsis.

Topics: Acetylglucosaminidase; Animals; Cardiac Output; Disease Models, Animal; Dogs; Escherichia coli Infections; Female; Male; Muramidase; Myocardial Contraction; Myocardial Depressant Factor; Probability; Random Allocation; Reference Values; Sensitivity and Specificity; Shock, Septic; Stroke Volume; Ventricular Dysfunction, Left

Topics: Acetylglucosaminidase; Animals; Disease Models, Animal; Dogs; Escherichia coli Infections; Muramidase; Myocardial Contraction; Risk Assessment; Sensitivity and Specificity; Shock, Septic

Receptor-mediated endocytosis plays an important role in accumulation of aminoglycosides in renal proximal tubule. To prevent aminoglycoside-induced nephrotoxicity following concentrated accumulation of gentamicin in the kidney, effect of cationic proteins and their peptide fragments, which could inhibit gentamicin binding to its binding receptor(s), was investigated. Among several substrates for megalin, an endocytic receptor responsible for renal accumulation of aminoglycosides, cytochrome c potently inhibited gentamicin accumulation in renal cortex. Concentration-dependent inhibition by cytochrome c on gentamicin uptake was also observed in OK kidney epithelial cells expressing megalin. In addition, gentamicin-induced increase in urinary excretion of N-acetyl-beta-d-glucosaminidase (NAG), a marker of renal tubular damage, was significantly reduced by cytochrome c. We next attempted to find a peptide fragment with lower molecular size showing inhibitory effect on gentamicin uptake. Cyto79-88 inhibited gentamicin uptake in OK cells, but had little effect on renal accumulation of gentamicin in mice in vivo. On one hand, a peptide fragment of neural Wiskott-Aldrich syndrome protein (N-WASP), which interacts with acidic phospholipids like aminoglycosides, inhibited gentamicin accumulation not only in OK cells but also in mouse kidney. These results show that substrates and/or their peptide fragments for aminoglycoside binding receptor such as megalin might be useful for preventing aminoglycoside-induced nephrotoxicity.

Topics: Acetylglucosaminidase; Aminoglycosides; Animals; Aprotinin; Binding Sites; Cells, Cultured; Cytochromes c; Dehydration; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Delivery Systems; Drug Evaluation, Preclinical; Drug Therapy, Combination; Endocytosis; Gentamicins; Japan; Kidney Cortex; Kidney Tubules, Proximal; Low Density Lipoprotein Receptor-Related Protein-2; Male; Mice; Mice, Inbred Strains; Muramidase; Nerve Tissue Proteins; Peptide Fragments; Peptides; Rats; Rats, Wistar; Receptors, Drug; Species Specificity; Tissue Distribution; Tritium

We established a mouse model in which fatal pneumonia was induced by pneumococcal superinfection following influenza virus infection in chronic Pseudomonas aeruginosa infected mice. In this mouse model, influenza virus infection caused a significant increase in inflammatory cells, cytokines and severe tissue damage in the lungs of these P. aeruginosa infected mice, before pneumococcal infection. Intrapulmonary virus titres were significantly increased in mice with chronic P. aeruginosa infection, compared with control mice. Neutrophil function analysis showed significant reduction of myeloperoxidase (MPO) activity and lysozyme secretion by influenza virus infection in these mice. Our results suggest that influenza virus infection may play an important role in inducing pneumococcal pneumonia in chronic P. aeruginosa infected mice. Our results suggested that our mouse model is useful for investigating the pathogenesis of influenza virus infection in patients with chronic lung infection.

Topics: Acute Disease; Animals; Chronic Disease; Colony Count, Microbial; Cytokines; Disease Models, Animal; Disease Susceptibility; Lung; Lung Diseases, Parasitic; Male; Mice; Mice, Inbred Strains; Muramidase; Orthomyxoviridae Infections; Peroxidase; Pneumococcal Infections; Pneumonia, Pneumococcal; Pseudomonas Infections; Superinfection

The loss of spleen may increase the incidence of overwhelming sepsis. To prevent this, splenic autotransplantation has been performed in humans and experimental animals. However, there is still controversy about the effectiveness of regenerated splenic tissue in preventing infection. This study explored the effectiveness of splenic tissue autotransplantation in restoring host defense.. Rabbits were divided into three groups: splenic autotransplantation, sham operation, and total splenectomy. Histomorphology, T-lymphocyte count, serum lysozyme levels, hemolysin titers, and pneumococcal clearance were observed as read-out parameters over 24 weeks.. Histological study showed that the white pulp was poorly developed and central arterioles were missing in the regenerated splenic tissue of the autotransplanted rabbits. The weight of regenerated spleens recovered 6 months later in the splenic autotransplantation group was 11% of that in the sham operation group and was significantly less than the weight at implantation. There was no significant difference in the number of T lymphocytes or level of serum lysozyme between the three groups. A poor antibody response by the rabbits in the splenic autotransplantation and total splenectomy groups was noted after the primary intravenous administration of sheep red blood cells compared to those of sham operation group. After the challenge with type 3 pneumococci intravenously, pneumococcal clearance from the bloodstream in the splenic autotransplantation group did not differ significantly from that in the total splenectomy group, but was markedly delayed compared with that in the sham operation group.. The low quantity and poor quality of the regenerated splenic tissue contribute to the inferior immunoprotective ability of animals autotransplanted with one-third of the original spleen. This suggests that the regenerated spleen cannot compensate for the immunological function of the original one, especially host resistance to infection.

Topics: Animals; Disease Models, Animal; Immunity; Immunohistochemistry; Lymphocyte Count; Muramidase; Organ Transplantation; Pneumococcal Infections; Rabbits; Random Allocation; Reference Values; Regeneration; Sensitivity and Specificity; Sepsis; Spleen; Splenectomy; Survival Rate; Transplantation, Autologous; Treatment Outcome

Type 1 diabetes in NOD mice can be prevented through autoantigen vaccination by shifting lymphocyte differentiation toward a T-helper 2 (Th(2)) response. However, in other models of autoimmunity, this approach may be accompanied by unexpected triggering of Th(2)-dependent anaphylactic shock. To test the safety of vaccination therapy in the NOD mouse model, we evaluated the effects of immunization with a wide battery of antigens in NOD, BALB/c, and C57BL/6 mice. Surprisingly, a nondiabetogenic antigen, hen egg white lysozyme, induced severe shock exclusively in NOD mice (shock in 11 of 11 mice, lethal in 3 mice). Shock severity was further increased by a more pronounced Th(2) setting generated by 1alpha,25(OH)(2)D(3) administration (17 of 17 mice, lethal in 14 mice, P < 0.0001). Pretreatment with dexamethasone resulted in full rescue, indicating an immune-mediated mechanism. Serum IgE levels and Th(1)/Th(2) cytokine profile analysis showed that the shock phenomenon was paralleled by a Th(2) response. mRNA expression of platelet-activating factor receptor (PAF-R) was significantly higher in NOD mice (P < 0.01) and was further increased by 1alpha,25(OH)(2)D(3). Pretreatment with WEB2086 (PAF-R antagonist) again protected all mice from lethal shock, indicating PAF as an anaphylaxis effector. In conclusion, in NOD mice, vaccination leading to a Th(2) immune shift can result in a lethal anaphylactic reaction.

Topics: Animals; Autoantigens; Calcitriol; Chickens; Dexamethasone; Diabetes Mellitus, Type 1; Disease Models, Animal; DNA Primers; Immunoglobulin E; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Muramidase; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Shock; T-Lymphocytes, Helper-Inducer; Th2 Cells; Vaccines

Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are badly needed. We report the use of Cpl-1, the lytic enzyme of a pneumococcal bacteriophage, as an intravenous therapy for pneumococcal bacteremia in a mouse model. A 2000- microg dose of Cpl-1 reduced pneumococcal titers from a median of log(10) 4.70 CFU/ml to undetectable levels (

Topics: Animals; Anti-Bacterial Agents; Bacteremia; Disease Models, Animal; Drug Resistance, Bacterial; Enzyme Stability; Female; Hydrogen-Ion Concentration; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Muramidase; Nasopharynx; Pneumococcal Infections; Streptococcus Phages; Streptococcus pneumoniae

Alloreactive T lymphocytes can be primed through direct presentation of donor MHC:peptide complexes on graft cells and through indirect presentation of donor-derived determinants expressed by recipient APCs. The large numbers of determinants on an allograft and the high frequency of the alloreactive repertoire has further led to speculation that exposure to environmental Ags may prime T cells that cross-react with alloantigens. We sought to develop a model in which to test this hypothesis. We found that CD4(+) T cells obtained from C57BL/6 (B6) mice that clinically resolved Leishmania major infection exhibited statistically significant cross-reactivity toward P/J (H-2(p)) Ags compared with the response to other haplotypes. B6 animals that were previously infected with L. major specifically rejected P/J skin grafts with second set kinetics compared with naive animals. Although donor-specific transfusion combined with costimulatory blockade (anti-CD40 ligand Ab) induced prolonged graft survival in naive animals, the same treatment was ineffective in mice previously infected with L. major. The studies demonstrate that cross-reactive priming of alloreactive T cells can occur and provide direct evidence that such T cells can have a significant impact on the outcome of an allograft. The results have important implications for human transplant recipients whose immune repertoires may contain cross-reactively primed allospecific T cells.

Topics: Amino Acid Sequence; Animals; Antigens; Chickens; Cross Reactions; Disease Models, Animal; Female; Graft Rejection; Isoantigens; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Molecular Sequence Data; Muramidase; Ovalbumin; Rabbits; Skin Transplantation; T-Lymphocyte Subsets; Vaccination

The pathogenesis of gastric antral hemorrhage and ulceration is unclear. This paper first proposes that antral hemorrhagic ulcers produced in rats are associated with attenuation of defensive parameters (such as mucosal glutathione levels and histamine release, as well as aggravation of aggressive factors) including gastric acid back-diffusion and oxyradical generation. The protective effects of lysozyme chloride and antioxidants on this ulcer model were also evaluated. After being deprived of food for 24 hours followed by refeeding for 1 hour, rats were injected with 1.0 mol HCl/L intragastrically under potent analgesia of diethylether-anesthesia to induce antral ulcer. Control rats received a normal saline solution only. Rats were then given free access to water and food for 4 days. Before the experiment began, rats were again deprived of food for 24 hours. Following anesthetization, their stomachs were irrigated for 3 hours with either normal saline or a physiological acid solution containing 100 mmol HCl/L and 54 mmol NaCl/L. Aggravation of various aggressive and defensive parameters in antral mucosa was observed in refed rats that had received 1.0 mol HCl/L. A high relationship of mucosal glutathione level (r = -0.8754, P <.05) or lipid peroxides generation (r = 0.8198) to antral ulceration was obtained in those ulcerated rats. Intragastric lysozyme chloride (50-200 mg/kg) injected three times daily produced a dose-dependent attenuation of various gastric parameters in the acid-irrigated stomachs of antral ulcer rats. Intraperitoneal injections of various antioxidants, including exogenous glutathione, allopurinol, or dimethylsulfoxide also attenuated antral ulcer. In conclusion, the imbalance of aggressive factors, such as acid back-diffusion and oxyradicals-as well as defensive factors including glutathione and histamine-is important in modulating antral hemorrhagic ulcers that can be ameliorated by lysozyme chloride or antioxidants in rats.

Topics: Animals; Antioxidants; Diffusion; Disease Models, Animal; Dose-Response Relationship, Drug; Gastric Acid; Gastric Mucosa; Gastrointestinal Hemorrhage; Glutathione; Histamine; Hydrochloric Acid; Lipid Peroxides; Male; Muramidase; Pyloric Antrum; Rats; Rats, Wistar; Specific Pathogen-Free Organisms; Stomach Ulcer

Many glucosamine residues of the pneumococcal peptidoglycan (PG) are not acetylated, which makes the PG resistant to lysozyme. A capsular type III mutant with an inactivated pgdA gene (encoding the peptidoglycan N-acetylglucosamine deacetylase A) became hypersensitive to exogenous lysozyme and showed reduced virulence in the intraperitoneal mouse model.

Topics: Amidohydrolases; Animals; Bacterial Proteins; Disease Models, Animal; Humans; Mice; Muramidase; Mutation; Pneumococcal Infections; Streptococcus pneumoniae; Virulence

Nasal instillation is an effective method for inducing antigen-specific immune tolerance. However, it is not clear how a tolerization scheme established in one mouse strain will perform when used in a mouse of a different haplotype.. To compare the antigen-specific recall responses in four mouse strains--BALB/c, C57BL/6, NOD, and B10.PL--that were pretreated nasally with 50 micrograms of hen egg-white lysozyme prior to parenteral immunization with homologous antigen.. Mice were nasally treated with a prototype antigen, HEL, and then immunized with the same antigen emulsified in complete Freund's adjuvant. Spleens and lymph nodes were assayed for T cell proliferation measured by tritiated thymidine incorporation. Cytokine production was measured using ELISPOT assay. Serum antibody response to HEL was measured using an enzyme-linked immunosorbent assay.. Proliferative recall responses to HEL in B10.PL, C57BL/6, and BALB/c were greatly reduced compared to control mice, but non-obese diabetic mice were resistant to the tolerization regime. Despite their susceptibility to nasally induced suppression, the mechanisms responsible for tolerance induction differed in BALB/c and C57BL/6 mice.. Our findings demonstrate that while mucosal contacts with specific antigen consistently affect the outcome of subsequent exposure to the same antigen, the observed response will vary non-predictably, depending on the genetic background of the animal.

Topics: Animals; Autoimmune Diseases; Disease Models, Animal; Drug Evaluation, Preclinical; Epitopes; Haplotypes; Immune Tolerance; Immunity, Mucosal; Immunization; Instillation, Drug; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Muramidase; Nasal Mucosa; T-Lymphocytes

Cross beta-sheet structure formation and abnormal aggregation of proteins are thought to be pathological characteristics of some neurodegenerative disorders. To investigate the novel structural transformation and aggregation, the solid-state secondary structures of some proteins and peptides associated in thin films were determined by circular dichroism spectroscopy. Insulin, lysozyme, DsbA protein, luciferase, and ovalbumin peptide fall into one group; they show no or slight structural rearrangement from solution to the solid state. Another group, including bovine serum albumin, ovalbumin, alpha-synuclein, and plasminogen activator inhibitor-1 (PAIRC) peptide, undergo structural transformation with an increase of beta-sheet structure in the solid state. The beta-sheet formation of PAIRC peptide may reflect the structural transformation of the serpin reactive center that is relevant to the inhibitor activity. The beta-sheet structure of alpha-synuclein in the solid state may correspond to the amyloid-like aggregates, which are implicated in the pathogenesis of some neurodegenerative diseases.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Cattle; Circular Dichroism; Disease Models, Animal; Insulin; Luciferases; Models, Molecular; Muramidase; Nerve Tissue Proteins; Ovalbumin; Plasminogen Activator Inhibitor 1; Protein Conformation; Protein Disulfide-Isomerases; Protein Structure, Secondary; Serum Albumin, Bovine; Spectrum Analysis; Structure-Activity Relationship; Synucleins

Recent research has identified endogenous cationic antimicrobial peptides as important factors in the innate immunity of many organisms, including fish. It is known that antimicrobial activity, as well as lysozyme activity, can be induced in coho salmon (Oncorhynchus kisutch) mucus after exposure of the fish to infectious agents. Since lysozyme alone does not have antimicrobial activity against Vibrio anguillarum and Aeromonas salmonicida, a four-step protein purification protocol was used to isolate and identify antibacterial fractions from bacterially challenged coho salmon mucus and blood. The purification consisted of extraction with hot acetic acid, extraction and concentration on a C(18) cartridge, gel filtration, and reverse-phase chromatography on a C(18) column. N-terminal amino acid sequence analyses revealed that both the blood and the mucus antimicrobial fractions demonstrated identity with the N terminus of trout H1 histone. Mass spectroscopic analysis indicated the presence of the entire histone, as well as fragments thereof, including a 26-amino-acid N-terminal segment. These fractions inhibited the growth of antibiotic-supersuscptible Salmonella enterica serovar Typhimurium, as well as A. salmonicida and V. anguillarum. Synthetic peptides identical to the N-terminally acetylated or C-terminally amidated 26-amino-acid fragment were inactive in antimicrobial assays, but they potentiated the antimicrobial activities of the flounder peptide pleurocidin, lysozyme, and crude lysozyme-containing extracts from coho salmon. The peptides bound specifically to anionic lipid monolayers. However, synergy with pleurocidin did not appear to occur at the cell membrane level. The synergistic activities of inducible histone peptides indicate that they play an important role in the first line of salmon defenses against infectious pathogens and that while some histone fragments may have direct antimicrobial effects, others improve existing defenses.

Topics: Aeromonas; Animals; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Fish Diseases; Fish Proteins; Flounder; Gram-Negative Bacterial Infections; Histones; Mucus; Muramidase; Oncorhynchus kisutch; Peptides; Proteins; Vibrio; Vibrio Infections

Class A scavenger receptors (SR-A) have several proposed functions that could impact atherosclerosis and inflammatory processes. To define the function of SR-A in vivo, we created C57BL/6 transgenic mice that expressed bovine SR-A under the control of the restricted macrophage promoter, lysozyme (lyso-bSR-A). bSR-A mRNA was present in cultured peritoneal macrophages of transgenic mice and tissues that contain significant macrophages including spleen, lung, and ileum. Functional overexpression of SR-A was demonstrated in peritoneal macrophages both by augmented cholesterol ester deposition in response to AcLDL and enhanced adhesion in transgenic mice compared with nontransgenic littermates. To determine whether macrophage-specific expression of bSR-A regulated inflammatory responses, granulomas were generated by subcutaneous injection of carrageenan. Granuloma size was significantly increased in lyso-bSR-A transgenic mice compared with wild-type littermates [421 +/- 51 mg (n = 11) vs. 127 +/- 22 mg (n = 10), P < 0.001]. However, the larger granulomas in lyso-bSR-A transgenic mice were only associated with an increase in unesterified cholesterol, and not cholesterol esters. Furthermore, granulomas from transgenic mice had an increase in the number of macrophages within the tissue.Therefore, macrophage expression of bSR-A increased presence of this cell type in granulomas without enhancing the deposition of cholesterol esters, consistent with a role of the adhesive property of the protein.

Topics: Animals; Carrageenan; Cholesterol; Disease Models, Animal; Gene Expression; Granuloma; Inflammation; Lipoproteins, LDL; Macrophages, Peritoneal; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Promoter Regions, Genetic; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class A; Scavenger Receptors, Class B

The thyroid hormones T(3) (tri-iodothyronine) and T(4) (thyroxine) are disseminated throughout the body via the circulation and are maintained across a range of physiological concentrations under the control of thyroid-stimulating hormone (TSH). T(3) (and T(4) after conversion to T(3)) influences many biological activities, including gene expression and protein synthesis, though little is known about the nature of pituitary-thyroid immune interactions. In the present study we show that serum T(3) and T(4) levels are sharply but transiently reduced during the first 24 h of systemic antigen exposure and that this is followed by suppressed levels of free T(4), after which there is rapid recovery to normal levels. Splenic dendritic cells, depending upon the stage of maturation/activation, were found to be a rich source of TSH, and CD11c(+) cells with dendritic cell morphology were present in the thyroid 1-3 days after antigen exposure. Moreover, antigen priming of hypophysectomized mice that are unable to make pituitary-derived TSH resulted in significant increases in circulating T(4), implying that compensation in the drop in thyroid hormones can be regulated from extrapituitary sources. These findings thus identify a novel set of immune-endocrine interactions that transpire during the early phase of antigen exposure, and they suggest that under appropriate conditions the immune system directly participates in the process of maintaining physiological homeostasis by contributing to the regulatory control of thyroid hormone activity.

Topics: Animals; Cell Movement; Chickens; Dendritic Cells; Disease Models, Animal; Euthyroid Sick Syndromes; Female; Hypophysectomy; Isoantigens; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Neuroimmunomodulation; Receptors, Antigen, T-Cell, alpha-beta; Secretory Rate; Spleen; Thyroid Gland; Thyrotropin; Thyroxine; Triiodothyronine

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; Celiac Disease; Chick Embryo; Cyclooxygenase Inhibitors; Dinoprostone; Disease Models, Animal; Egg White; Humans; Indomethacin; Mice; Mice, Inbred Strains; Mice, Transgenic; Muramidase; Receptors, Antigen, T-Cell

Live mycobacteria secrete a number of unique proteins early in their multiplication which are important for both the pathogenesis and the stimulation of specific host responses. We have investigated the mechanisms by which the host mounts immune response against tuberculosis after vaccination with secretory proteins (SP) of a vaccine candidate Mycobacterium habana TMC 5135. Mice vaccinated with SP of 10th day growth of M. habana, either alone or emulsified in Freund's incomplete adjuvant (FIA) possessed antituberculous resistance and cellular immune responses against M. tuberculosis H37Rv. These proteins induced a significant cutaneous delayed type hypersensitivity response in guinea pigs vaccinated with heat killed M. tuberculosis H37Rv, which was equivalent to that observed with a standard purified protein derivative (PPD). The splenocytes of these guinea pigs have shown higher proliferative response after stimulation with SP than with PPD. The SP + FIA immunization has been found to exert maximum prophylactic effect by potentiating both the oxygen dependent arms and enzymatic activities of macrophages. Macrophages from mice vaccinated with SP of M. habana produced enhanced levels of interleukin(IL)-2, interleukin-12 and interferon(IFN)-gamma. The protective as well as cell mediated immune responses were upregulated in SP immunized animals when compared to whole cell (M. habana) vaccinated animals. SDS-PAGE of SP from M. habana showed the prominent bands of 60, 32, 31 and 30 kDa. Furthermore, the western analysis of SP with pulmonary tuberculosis patient's serum has revealed the presence of immunoreactive antigens of 36, 35, 33/32 kDa. Overall study demonstrated that the secretory antigens released by actively growing M. habana bacilli could activate different arms of effective immune response.

Topics: Acid Phosphatase; Animals; Bacterial Proteins; Blotting, Western; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Glucuronidase; Interferon-gamma; Interleukin-12; Interleukin-2; Mice; Mice, Inbred AKR; Muramidase; Mycobacterium; Reactive Oxygen Species; Tuberculosis

Abrogation of peripheral tolerance in transgenic mice that express a uniform B-cell receptor may create a powerful tool to examine the molecular mechanisms that underlie the autoimmune response in B cells. Here we report that processes that induce a systemic lupus erythematosus-like syndrome in normal mice, namely chronic graft vs host reaction, trigger systemic autoimmunity in a well-established transgenic mice model of B cell receptor peripheral tolerance. The induction of graft vs host reaction in mice that carry both a rearranged B cell Ag receptors specific for hen egg lysozyme and expressing chronically circulating hen egg lysozyme Ag resulted in induction of high and sustained levels of circulating anti-hen egg lysozyme autoantibodies and glomerulonephritis with proteinuria. This was associated with marked changes in expression of cell-surface proteins, such as CD23 and complement receptor 2. B cells from the graft vs host-induced mice could proliferate in vitro in response to self-Ag, and upon stimulation with anti-IgD demonstrated rapid phosphotyrosine phosphorylation of specific proteins, which could not be induced in the anergic double transgenic B cells. Conversely, loss of tolerance was not associated with a higher induction in the level of Syk kinase phosphorylation following stimulation with anti-IgD. Taken collectively, these data establish that 1) processes that induce a systemic lupus erythematosus-like syndrome in normal mice can abrogate peripheral tolerance in transgenic mice expressing self-tolerized B cells, and that 2) loss of tolerance in this model is associated with marked changes in surface expression of B cell coreceptors as well as with selective changes in IgD-induced signaling by discrete tyrosine-phosphoproteins, but not Syk kinase.

Topics: Animals; Autoantibodies; Autoantigens; B-Lymphocytes; Disease Models, Animal; Enzyme Precursors; Graft vs Host Reaction; Immune Tolerance; Immunoglobulin D; Immunoglobulin M; Intracellular Signaling Peptides and Proteins; Lupus Erythematosus, Systemic; Lupus Nephritis; Lymphocyte Activation; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Phosphoproteins; Phosphotyrosine; Protein-Tyrosine Kinases; Receptors, Antigen, B-Cell; Self Tolerance; Syk Kinase

Insulin-dependent diabetes is heavily influenced by genes encoded within the major histocompatibility complex (MHC), positively by some class II alleles and negatively by others. We have explored the mechanism of MHC class II-mediated protection from diabetes using a mouse model carrying the rearranged T cell receptor (TCR) transgenes from a diabetogenic T cell clone derived from a nonobese diabetic mouse. BDC2.5 TCR transgenics with C57Bl/6 background genes and two doses of the H-2(g7) allele exhibited strong insulitis at approximately 3 wk of age and most developed diabetes a few weeks later. When one of the H-2(g7) alleles was replaced by H-2(b), insulitis was still severe and only slightly delayed, but diabetes was markedly inhibited in both its penetrance and time of onset. The protective effect was mediated by the Abetab gene, and did not merely reflect haplozygosity of the Abetag7 gene. The only differences we observed in the T cell compartments of g7/g7 and g7/b mice were a decrease in CD4(+) cells displaying the transgene-encoded TCR and an increase in cells expressing endogenously encoded TCR alpha-chains. When the synthesis of endogenously encoded alpha-chains was prevented, the g7/b animals were no longer protected from diabetes. g7/b mice did not have a general defect in the production of Ag7-restricted T cells, and antigen-presenting cells from g7/b animals were as effective as those from g7/g7 mice in stimulating Ag7-restricted T cell hybridomas. These results argue against mechanisms of protection involving clonal deletion or anergization of diabetogenic T cells, or one depending on capture of potentially pathogenic Ag7-restricted epitopes by Ab molecules. Rather, they support a mechanism based on MHC class II-mediated positive selection of T cells expressing additional specificities.

Topics: Animals; Antigen-Presenting Cells; Antigens, CD; Diabetes Mellitus, Type 1; Disease Models, Animal; Genes, MHC Class II; Haplotypes; Hemocyanins; Hybridomas; Interleukin-2; Islets of Langerhans; Mice; Mice, Inbred NOD; Mice, Transgenic; Muramidase; Receptors, Antigen, T-Cell; T-Lymphocytes; Transgenes

Stress has been implicated as a factor in the pathogenesis of autoimmune disorders. In order to determine the effect of adrenergic stress on immune responses in vivo, C57BL/6 (B6; H-2b) mice, which respond weakly to hen-egg lysozyme (HEL), were immunized on day 0 with HEL (50-200 microg s.q.) and subsequently injected with epinephrine (EPI; 0.1-0.5 mg/kg s.q.) daily for up to 10 days. Controls included A/J mice (H-2k) which respond strongly to HEL. In some experiments, B6 mice were depleted of CD4+ or CD8+ lymphocytes by monoclonal antibody treatment in vivo, prior to immunization with HEL, and injection with EPI. On day 10, single cell suspensions of draining lymph nodes (LN) and spleen were examined for immune phenotype, proliferative responses to HEL, and lymphokine production. Minimal specific proliferative responses were detected in B6 mice compared to A/J mice. However, lymphocyte proliferation increased in HEL-immunized EPI-treated B6 mice but not in the A/J mice. IL-2-mediated proliferation and IL-2 secretion were both increased in the HEL-immunized EPI-treated B6 mice. The depletion of CD8+ but not CD4+ lymphocytes in vivo abrogated the effects of EPI, whereas adoptive transfer of naive CD8+ splenocytes to the CD8-depleted mice restored specific responses in the HEL-immunized EPI-treated animals. We conclude that EPI augments antigen-specific T-cell responses to HEL in B6 mice by a CD8+ T-cell-dependent mechanism.

Topics: Adoptive Transfer; Animals; Antigens; Autoimmune Diseases; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Chickens; Disease Models, Animal; Epinephrine; Female; Immunization; Interleukin-2; Lymphocyte Activation; Lymphocyte Depletion; Lymphokines; Mice; Mice, Inbred A; Mice, Inbred C57BL; Muramidase; Stress, Physiological

Endotoxemia is considered to be associated with the high mortality of gram-negative septic patients. Increasing evidence shows that beta-lactam antibiotics have a propensity to induce endotoxin release from the bacterial outer membrane while killing bacteria. We have recently found that egg white lysozyme (EW-LZM) shows strong inhibition of beta-lactam induced bacteriolysis and lipopolysaccharide (LPS) release from Escherichia coli O111, resulting in reduction of the LPS-initiated inflammatory response. In this study, we compared the effect of EW-LZM on E. coli J5, which possesses rough-type LPS (RaLPS), in order to demonstrate the effect of O-antigenic polysaccharide on endotoxin neutralizing activity of EW-LZM and on inhibition of beta-lactam induced lysis by LZM. Both of the beta-lactam induced bacterial lysis and subsequent LPS release were almost completely inhibited by EW-LZM. The effect was more potent than that of wild-type LPS as assessed by released LPS concentration and LPS induced cytokine syntheses. In addition, EW-LZM was effective against lethal infection of E. coli J5 in cyclophosphamide induced leukopenic mice. These facts strongly suggested that O-antigenic polysaccharide negatively modulates LPS neutralizing activity of EW-LZM.

Topics: Animals; Anti-Bacterial Agents; Bacteriolysis; Carrageenan; Cell Line; Cyclophosphamide; Disease Models, Animal; Endotoxemia; Endotoxins; Escherichia coli; Escherichia coli Infections; Galactose; Lactams; Leukopenia; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred ICR; Monokines; Muramidase; O Antigens

Cadmium represents a major aquatic pollutant in many parts of the world. Yet, despite the fact that cadmium accumulates in high concentrations in fish tissues, is found in polluted aquatic environments, and is carcinogenic and immunotoxic in a variety of mammalian species, the effects of cadmium on the immune responses of directly exposed aquatic species have not been clearly defined. This study was designed to assess the effects of in vivo cadmium exposure, at a concentration found in contaminated aquatic environments, on the immune defense mechanisms of fish. In this study, no effects were observed upon body weight, lysozyme activity, of cell viability, despite the high concentration of accumulated cadmium in the gills and liver. Furthermore, in the absence of any clinical manifestations or overt toxicity, exposure of rainbow trout to waterborne cadmium at 2 ppb altered macrophage-mediated immune functions, including phagocytosis and free radical production, in a time-dependent manner. Similar immunotoxic effects of cadmium have also been observed in mammals. Although interspecies comparisons between mammalian and fish immune responses are extremely complicated and need to be approached with caution, results from this study suggest the applicability of fish as an additional/alternative animal model for immunotoxicological studies.

Topics: Animals; Body Burden; Cadmium; Disease Models, Animal; Macrophages; Muramidase; Oncorhynchus mykiss; Phagocytosis; Reactive Oxygen Species

Although prolonged Gram-negative sepsis with high permeability alveolar edema, a well documented cause of adult respiratory distress syndrome, has been shown to result in surfactant alterations, the effects of acute endotoxemia on the lung surfactant system are largely unknown. In this study, lethal endotoxemia (> 80% mortality at 24 h) resulting in severe, rapid leukopenia with progressive thrombocytopenia was achieved through intraperitoneal injection of adult Fischer 344 rats with 3.5 mg of Escherichia coli endotoxin/kg. After assessment of pulmonary mechanics under general anesthesia, endotoxin-injected rats and appropriate controls were killed at 4, 8, and 12 h for morphological and biochemical analyses. Morphometric estimation of surfactant membrane subtypes in bronchoalveolar lavage fluid revealed prominent alterations including significant decrease (45%) in tubular myelin 12 h post-endotoxin, with a threefold increase in lamellar body-like forms at 8 and 12 h. Acute endotoxicosis resulted in decrease of total dynamic compliance, whereas pulmonary resistance remained unchanged. These changes were associated with margination of polymorphonuclear leukocytes in lung microcirculation, multifocal septal edema, and decrease in lamellar body lysozyme specific activity at 12 h. Alveolar edema, as determined by measurement of total protein in cell-free bronchoalveolar lavage fluid, was absent in both controls and endotoxin-injected rats. The results indicate that bloodborne lung injury induced by lethal endotoxicosis initiates acute perturbation of secreted surfactant membranes with pulmonary dysfunction in the absence of high protein alveolar edema.

Topics: Acute Disease; Animals; Biological Products; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Endotoxins; Lung; Male; Muramidase; Myelin Sheath; Proteins; Pulmonary Surfactants; Rats; Rats, Inbred F344; Respiratory Distress Syndrome; Toxemia; Tumor Necrosis Factor-alpha

We tried to develop a new delayed healing model of an open wound made on the dorsal skin of a rat by inducing malnutrition with restriction of food intake. We determined the effects of a newly introduced ointment gauze containing 5% lysozyme hydrochloride (M-1011G) on this model. Malnutrition characterized by a decrease in body weight and serum protein was produced by restricting the daily intake of commercially available food to 6 g for 2 weeks without liver disturbance and thereafter maintained with food intake of 12 g/day. Under these conditions, healing of the open wound made on the dorsal skin was prolonged, as compared with that of the wound made on a well-nourished animal. In this model, statistical studies showed that M-1011G was the most effective in accelerating the reduction of the wound area and shortening of the time required for the complete healing, among the following treatments: sterilized gauze alone, ointment gauze alone and 5% lysozyme hydrochloride-containing emulsified ointment. Histological findings showed that M-1011G greatly accelerated the granulation of this tissue which is essential for wound healing, probably due to stimulation of epidermization and regenerated granulation in the wound.

Topics: Animals; Bandages; Disease Models, Animal; Male; Muramidase; Nutrition Disorders; Ointments; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Wound Healing

Pentoxifylline was evaluated for its ability to enhance inactivation of group B streptococci in lungs of prematurely born rabbits. Mechanisms associated with intrapulmonary streptococcal clearance and the pharmacodynamics of pentoxifylline were also investigated.. Randomized, controlled animal trial.. University research laboratory.. A total of 123 New Zealand rabbits were delivered prematurely by cesarean section and were used for clearance studies. Twenty-three preterm pups were additionally utilized to study the pharmacodynamics of pentoxifylline.. Preterm rabbits were infected with group B streptococcal aerosols and given intraperitoneal injections of either pentoxifylline (25, 12.5, and 12.5 mg/kg) or placebo at 0, 6, and 12 hrs after infection.. At 0, 4, and 24 hrs, the numbers of streptococci were determined in the left lung, while the right lung underwent bronchoalveolar lavage to quantify intra-alveolar leukocytes, phagocytosis of inhaled bacteria, and concentrations of lysozyme and tumor necrosis factor. In a separate experiment, blood and bronchoalveolar fluid from infected animals were analyzed for pentoxifylline content. Streptococcal proliferation was less in pentoxifylline-treated animals than in controls at 24 hrs (p < .01). Pulmonary macrophages and polymorphonuclear leukocytes recovered from bronchoalveolar lavage fluid did not differ in numbers or phagocytic activity. Pentoxifylline-treated animals had lower levels of lysozyme (p < .02) and tumor necrosis factor (p < .005) in bronchoalveolar lavage fluid compared with placebo-treated pups. Therapeutic levels of pentoxifylline were achieved in blood and bronchoalveolar lavage fluid.. Despite lowering lysozyme and tumor necrosis factor content in epithelial lining fluid, pentoxifylline improves the inactivation of group B streptococci in preterm rabbit lungs. These findings suggest that increased group B streptococcal clearance was coincident with an anti-inflammatory effect due to pentoxifylline. We conclude pentoxifylline may be clinically useful as an adjunctive therapy for group B streptococcal pneumonia in newborns.

Topics: Animals; Animals, Newborn; Bronchoalveolar Lavage Fluid; Colony Count, Microbial; Disease Models, Animal; Drug Evaluation, Preclinical; Fetus; Leukocyte Count; Macrophages, Alveolar; Muramidase; Neutrophils; Pentoxifylline; Phagocytosis; Pneumonia; Rabbits; Random Allocation; Streptococcal Infections; Streptococcus agalactiae; Tissue Distribution; Tumor Necrosis Factor-alpha

Brucella abortus injected into CBA mice replicated primarily in the spleen and liver, reaching a peak bacterial count in both organs about 7 days postinfection. The organism was eliminated from the liver but declined to a chronic phase in the spleen. The infection caused hepatosplenomegaly. An influx of macrophages into the two organs was monitored by quantitative Northern (RNA blot) analysis of the macrophage-specific marker lysozyme mRNA. Lysozyme mRNA was detectable in spleen and increased three- to fourfold during infection. In liver, lysozyme mRNA was initially undetectable, but at about the peak of infection it reached a level comparable to that in the spleen. Macrophage colony-stimulating factor 1 (CSF-1) has been reported to be elevated in the circulation of animals infected with B. abortus and is known to stimulate monocytopoiesis. To investigate the role of CSF-1 in pathogenesis, we studied the effect of further increasing the CSF-1 concentration by administration of recombinant human CSF-1. Since the infection is characterized by several distinct phases, recombinant human CSF-1 was administered at defined times relative to these phases. Pronounced effects were observed only when CSF-1 administration was begun during the developing acute phase. The consequences were decreased bacterial numbers in the spleen but an increase in the liver, reduced antibody generation, and increased hepatosplenomegaly. A feature of many chronic intracellular infections is immunosuppression. B. abortus caused a substantial diminution of responsiveness of spleen cells to T-cell mitogens, particularly concanavalin A. This action was mimicked by CSF-1 treatment of the animals prior to spleen cell isolation. The results suggest that CSF-1 plays a role in macrophage recruitment in brucellosis and that recruited macrophages contribute to the immunopathology and immunosuppression.

Topics: Animals; Antibody Formation; Blotting, Northern; Brucellosis; Colony-Forming Units Assay; Disease Models, Animal; Dose-Response Relationship, Immunologic; Hepatomegaly; Hypersensitivity, Delayed; Liver; Lung; Lymphocyte Activation; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Mice, Inbred CBA; Muramidase; Organ Size; Recombinant Proteins; RNA, Messenger; Spleen; Splenomegaly; Time Factors

The hydroxyl radical scavengers dimethylthiourea (DMTU), sodium benzoate, and dimethylsulfoxide (DMSO) were administered to rats before doxorubicin hydrochloride (ADR) (5 mg/kg, IV) to probe the role of free radicals in mediating proteinuria in doxorubicin hydrochloride nephrosis (AN). Because ADR stimulates free radical production, the role of renal glutathione was also evaluated; glutathione metabolism is involved in tissue detoxification processes. DMTU administration to rats with AN caused a significant (p less than 0.01) reduction in their proteinuria after 7 days (52.84 +/- 13.21 mg/24 hours) when they were compared with ADR controls (155.81 +/- 20.16 mg/24 hours). In similar fashion, their urine albumin excretion was also significantly reduced when compared with that of ADR controls (11.13 +/- 2.75 mg/24 hours vs 32.08 +/- 4.14 mg/24 hours; p less than 0.01). DMTU-treated rats also had significantly (p less than 0.001) reduced urinary protein and albumin excretion at 14 days when compared with rats that received ADR alone. The urinary excretion of lysozyme and N-acetyl-glucosaminidase, markers of renal tubular injury, were significantly increased after 7 or 14 days in rats with AN, despite DMTU treatment. Creatinine clearance was significantly reduced (p less than 0.05) in rats receiving ADR alone (0.223 +/- 0.011 ml/min/100 gm) when compared with that in normal controls (0.331 +/- 0.027 ml/min/100 gm) or DMTU-treated rats (0.289 +/- 0.035 ml/min/100 gm). Unlike DMTU, neither sodium benzoate nor DMSO reduced proteinuria in rats with AN.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylglucosaminidase; Albuminuria; Animals; Benzoates; Benzoic Acid; Creatine; Dimethyl Sulfoxide; Disease Models, Animal; Doxorubicin; Free Radical Scavengers; Glomerular Filtration Rate; Glutathione; Hydroxides; Hydroxyl Radical; Injections, Intravenous; Kidney Cortex; Male; Muramidase; Nephrosis; Proteinuria; Rats; Rats, Inbred Strains; Thiourea

The aims of the present study were to define, under in vivo conditions, factors governing antigen binding and persistence in the rat joint and to establish a chronic arthritis model by means of a natural polycation. The influence of size as well as charge on antigen handling was examined using a range of chemically cationized proteins and natural polycations. Arthritis was induced by intraarticular challenge in preimmunized rats. Immunofluorescence studies revealed that not only pI, which must exceed pH 8-9, but also molecular size was a decisive parameter: only antigens of more than 40 kD were able to persist for significant periods in joint structures. All existing models of antigen induced chronic arthritis in rodents utilize chemically cationized proteins. We extended this system to natural polycations by showing that lysozyme (pI 11.3; MW 14 kD) in tetrameric, charge conserved form (MW 56 kD) as a model-antigen was able to induce chronic arthritis in the rat. After intraarticular challenge of preimmunized animals the course of inflammation was assessed both by 99mTechnetium-pertechnetate (99mTc) scintigram and from the histology. In contrast to monomeric lysozyme, which evoked only a transient inflammatory response (less than two weeks), tetrameric lysozyme induced a chronic arthritis, which still persisted at day 90. Our results show that the ability of cationic antigens to trigger chronic arthritis is vitally size dependent. This is also the first report of a natural polycation acting as an arthritogen, thus providing an experimental basis justifying the search for cationic microbial antigens in human post infectious reactive arthritis.

Topics: Animals; Antigens; Arthritis; Cations; Disease Models, Animal; Dose-Response Relationship, Drug; Knee Joint; Male; Molecular Weight; Muramidase; Proteins; Rats; Rats, Inbred Strains

Diabetes in the non-obese diabetic (NOD) mouse is a multigenic autoimmune disease and is possibly controlled by three recessive loci, including one that is linked to the major histocompatibility complex (MHC). The first external domain of the Class II MHC I-A beta chain in these mice is unique and has been suggested as being responsible for autoimmunity. The I-A alpha chain in these mice is I-A alpha d, and they lack the expression of I-E molecules. We have investigated immune responses to various Ir gene control antigens in NOD mice to determine the influence of the NOD Ia and particularly the I-A beta chain. We find that sheep insulin is highly immunogenic while other insulins are weakly immunogenic in these mice. Hen egg lysozyme, pigeon cytochrome C and the synthetic polypeptide Poly 18, Poly EYK(EYA)5 antigen produce good antibody responses. Apart from H-2d, NOD are the only mice where Poly 18 antigen is immunogenic. In these mice Poly 18 induced good T-cell proliferative response, which was inhibited by anti-Ia antibody, and the mice were able to respond to tyrosine-containing polypeptide Poly EYA but not to the phenylalanine-containing antigen Poly EFA. We also found that synthetic peptide 48-60 of the NOD I-A beta chain is highly immunogenic in syngeneic NOD mice both for T cells and B cells. Using an I-A beta chain-specific monoclonal antibody, we are able to prevent induction of diabetes when the antibody was administrated in prediabetic, young mice. Our results suggest that the immune response to various antigens and autoimmune diabetes in NOD mice is directly influenced by the I-A beta chain.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Formation; Autoimmune Diseases; Chickens; Columbidae; Cytochrome c Group; Diabetes Mellitus, Experimental; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Histocompatibility Antigens Class II; Immunization; Insulin; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Mice; Mice, Mutant Strains; Molecular Sequence Data; Muramidase; Peptide Fragments; T-Lymphocytes

The composition of the obligate anaerobic intestinal flora of patients with rheumatoid arthritis (RA) differed from that of healthy subjects (HS). Total numbers of aerobes as well as anaerobic coccoid rods were found elevated when compared with HS. Eubacterium species were found in all stool samples of both groups; Bifidobacterium species were present in seven (RA) and eight (HS) out of 10 subjects. From the flora of two RA patients and two HS Eubacterium species were isolated and identified. Cell wall fragments from four E. aerofaciens strains (two from RA, two from HS) were tested for arthritis induction in rats. All four strains induced chronic arthritis which was histologically confirmed. We concluded that in the normal intestinal flora of RA patients Eubacterium species are present in high numbers (i.e. greater than 10(9)/g faeces); cell walls from isolated E. aerofaciens strains had arthropathic properties.

Topics: Animals; Arthritis, Infectious; Arthritis, Rheumatoid; Cell Wall; Disease Models, Animal; Eubacterium; Feces; Female; Foot; Humans; Intestines; Joints; Muramidase; Rats; Rats, Inbred Lew; Rhamnose

Previous studies have shown that the decreased neutrophil migratory responsiveness seen in burned patients correlates with the extent of thermal injury and the extent of the neutrophil-specific granule deficiency. To understand better the relationship between the neutrophil dysfunction, degranulation, and thermal injury, a rabbit model was studied. Eighteen rabbits were burned over 20% of their surface area. Assay of peripheral blood heterophils disclosed decreased migratory activity compared with preburn levels and decreased lysozyme content vs preburn levels, but no change in the beta-glucuronidase content. The specific binding of tritiated formyl-methionyl-leucyl-phenylalanine to peripheral blood heterophils was increased fivefold over that of control cells. These studies indicate that, following thermal injury, there is a selective decrease of specific granule contents and an increase in chemoattractant binding to the cell and also suggest an abnormality in chemoattractant receptor processing. The rabbit provides a convenient model for the study of compromised host defenses following thermal injury.

Topics: Animals; Body Weight; Burns; Cell Migration Inhibition; Chemotaxis, Leukocyte; Disease Models, Animal; Endotoxins; Escherichia coli Infections; Muramidase; Neutrophils; Oligopeptides; Rabbits; Receptors, Formyl Peptide; Receptors, Immunologic; Staphylococcal Infections; Time Factors

Bacterial cell wall induced arthritis is an experimental model of chronic erosive synovitis in which arthritis is induced in rats by a single injection of an aqueous suspension of cell wall fragments from selected Gram-positive bacteria. To understand better the Gram-positive bacterial cell wall characteristics necessary for the induction of chronic arthritis we tested the arthritogenicity of five Gram-positive bacteria which were (1) lysozyme resistant and contained a polyrhamnose peptidoglycan side chain moiety, (2) lysozyme resistant, but had little or no rhamnose in the peptidoglycan, polysaccharide, or (3) neither lysozyme resistant, nor contained rhamnose in their peptidoglycan, polysaccharide. All of the lysozyme resistant cell walls tested induced acute arthritis, but only those cell walls which were both lysozyme resistant and contained rhamnose in their polysaccharide side chain were able to induce chronic arthritis. Cell walls which were neither lysozyme resistant nor contained rhamnose were not arthritogenic. These data suggest that both lysozyme resistance and the rhamnose moiety in the peptidoglycan, polysaccharide side chain play an important role in the induction of chronic arthritis by Gram-positive bacterial cell walls in aqueous suspension.

Topics: Animals; Arthritis; Cell Wall; Disease Models, Animal; Female; Lacticaseibacillus casei; Muramidase; Polysaccharides, Bacterial; Rats; Rats, Inbred Lew; Rhamnose; Structure-Activity Relationship

A wide variety of tests for the detection of circulating immune complexes (IC) has been proposed by different authors, but there is very little to no information concerning the performance of IC screening assays in samples known to contain in vivo-formed IC. The purpose of our investigation was to compare the behavior of a non-specific assay, the PEG-IgG screening test for IC, with an antigen-specific assay in serum samples sequentially obtained from rabbits to which we induced acute serum sickness. Five animals were used in the study; we were able to detect an increase of IC constituted by the heterologous antigen (human serum albumin) and corresponding antibodies in all, and in 4 animals the results of the PEG-IgG assay closely correlated with the results of the antigen-specific assay (rho values between 0.975 and 1.00). The 4 animals in which IC showed a definite peak by both assays developed proteinuria and IC deposits at the glomerular level, while the animal that failed to develop IC detectable by the PEG-IgG test remained normal throughout the study. These results demonstrate the ability of the PEG-IgG test to detect in vivo-formed IC and suggest that the IC detected by this test have pathogenic potential.

Topics: Acute Disease; Albuminuria; Animals; Antigen-Antibody Complex; Autoimmune Diseases; Disease Models, Animal; Immunoglobulin G; Longitudinal Studies; Muramidase; Polyethylene Glycols; Proteinuria; Rabbits; Serum Sickness

A model of allergic alveolitis was developed in inbred mice. Antigen suspension prepared from Thermoactinomyces vulgaris was applicated intranasally in C3H/He mice. Lung histology, serum IgG and IgA antibodies and serum lysozyme were followed during the course of sensitization. The lung histology showed an increased number of intra-alveolar macrophages and leucocytes in sensitized mice. Interstitial peribronchiolar and perivascular infiltration of mononuclear inflammatory cells and a few granulomas were also detected. An increase in the prevalence as well as titers of IgG and IgA antibodies was recorded during sensitization. An elevation in serum lysozyme level was also observed in sensitized mice. The lung histology of control animals was essentially normal and no increase in antibody titers or lysozyme levels was detected. Intranasal application of antigen prepared from Thermoactinomyces vulgaris seems to produce in mice allergic alveolitis resembling human farmer's lung.

Topics: Administration, Intranasal; Alveolitis, Extrinsic Allergic; Animals; Antibodies, Bacterial; Antigens, Bacterial; Disease Models, Animal; Female; Immunization; Immunoglobulin A; Immunoglobulin G; Leukocytes; Lung; Macrophages; Male; Mice; Mice, Inbred C3H; Micromonosporaceae; Muramidase; Pulmonary Alveoli; Time Factors

The proximal nephron of C57 beige mice with a genetic defect analagous to the Chédiak-Higashi syndrome (CHS) has been compared with that of normal C57 black mice. The concanavalin A-horseradish peroxidase (Con A-HRP) technique stained the brush border of the proximal straight tubule heavily in black mice and weakly in beige mice. In beige mice this method stained the brush border of the proximal convoluted tubules weakly and the brush border of the proximal straight tubules only negligibly. Periodic acid-Schiff staining showed no such difference between beige and black mice but revealed an increase distally in the size of the CHS inclusions in the proximal straight tubule of beige mice. Immunostaining visualized abundant lysozyme in the first portion of the proximal nephron but none in the more distal segments of beige and black mice alike. At the ultrastructural level, the proximal convoluted tubules of black mice contained two morphologic types of heterophagosomes, which apparently differed in accord with the stage of their development. Proximal straight tubules contained morphologically different heterophagic bodies. The mature stages of these heterophagosomes were greatly enlarged in CHS mice. With the periodic acid-thiocarbohydrazide-silver proteinate (PA-T-SP) method for localizing glycoprotein ultrastructurally, the microvillar brush border, apical invaginations of the plasmalemma, Golgi cisternae, and lysosomal inclusions stained selectively in the proximal nephron in both strains. The proximal straight nephron of beige mice after staining with the PA-T-SP method appeared depleted of the strongly reactive apical invaginations in some areas, particularly where large heterophagosomes bordered the apical plasmalemma. The enlarged secondary lysosomes of heterophagic origin in beige mice varied in showing both diffuse and focal PA-T-SP reactivity. Lysosomal acid phosphatase activity appeared decreased, and peroxisomes were normal in size but increased in number in the proximal nephron of beige mice.

Topics: Animals; Chediak-Higashi Syndrome; Disease Models, Animal; Immunoenzyme Techniques; Inclusion Bodies; Kidney Tubules, Proximal; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Microscopy, Electron; Muramidase; Phagocytes

The tissue damage induced by murine cytomegalovirus (MCMV) in mice with the beige mutation (bg/bg) and in their normal littermates (bg/ +) was investigated. The beige mutation in mice is a homolog of the Chédiak-Higashi syndrome in man, and various dysfunctions of phagocytes and decreased activity of natural killer cells have been demonstrated in these animals. Tissue damage, especially in the liver and spleen, was more conspicuous in bg/bg than in bg/ + mice and was associated with frequent intranuclear inclusions and a higher titer of virus. However, the mutation did not appear to alter the organ distribution of tissue damage induced by MCMV. The inflammatory response in the liver, which is presumed to contribute to host resistance, appeared under certain circumstances to be delayed and deficient in bg/bg mice.

Topics: Animals; Aspartate Aminotransferases; Chediak-Higashi Syndrome; Cytomegalovirus Infections; Disease Models, Animal; Disease Susceptibility; Female; Hematologic Tests; Kidney; Liver; Lymph Nodes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Muramidase; Mutation; Myocardium; Spleen; Thymus Gland

A single intraperitoneal injection of sodium oxalate was used to induce intrarenal tubular precipitation of calcium oxalate in rats. This experimental model was used to screen the efficacy of hydrochlorothiazide, orthophosphate, methylene blue, trypan blue, retinal folic acid, neuraminidase, and lysozyme in retarding intratubular calcium oxalate precipitation. Orthophosphate caused a 53 per cent reduction in calcium oxalate precipitation relative to the control animals.

Topics: Animals; Calcium; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Folic Acid; Hydrochlorothiazide; Methylene Blue; Muramidase; Neuraminidase; Oxalates; Phosphates; Rats; Trypan Blue; Urinary Calculi; Vitamin A

Topics: Animals; Body Temperature; Copper; Disease Models, Animal; Lung; Macrophages; Male; Mice; Muramidase; Organ Size; Orosomucoid; Q Fever; Respiratory Tract Infections; Spleen; Time Factors; Zinc

To correct a genetic defect, it would not be enough, say, to inject the missing enzyme, since the body's immune defenses would rapidly destroy it. A "Trojan horse" is needed to evade immune surveillance. The evolution of such an approach is described, as well as how it has been used to "cure" Tay-Sachs disease in culture, utilizing immunoglobulin-disguised liposomes to bring hexosaminidase A to deficient cells.

Topics: Animals; Cells, Cultured; Cytoplasm; Disease Models, Animal; Enzyme Therapy; Genetic Engineering; Hexosaminidases; Humans; Immunoglobulins; Leukocytes; Lipidoses; Liposomes; Lysosomes; Metabolism, Inborn Errors; Muramidase; Peroxidases; Phagocytes; Sharks

A transplantable myelogenous leukemia of an inbred Wistar/Furth rat has been established in tissue culture and cloned. The resulting transplantable leukemia line demonstrates in vitro doubling time of 20 hr, colony-forming efficiency of 5% in liquid and methylcellulos-containing medium, and a saturation density of 3.0 x 106 cells/sq cm in liquid medium. Following intraperitoneal inoculation, newborn rats developed solid tumors, ascities, and leukemia with ld50 of5 x 103 cells and mean latency of 60 days. The tumor cell morphology was consistent with that of acute myelogenous leukemia. Histochemical staining for myeloid enzymes revealed no evidence of myeloperoxidase, esterase, or leukocyte alkaline phosphatase; however, fluorescent antibody staining for lysozyme was markedly positive. Serum, urine, and ascitic fluid from rats with transplanted leukemia also contained elevated levels of lysozyme. There was no detectable type-CRNA virus production by this cell line after as long as 100 days in vitro. This inbred rat myelogenous leukemia should provide a useful model for studies of chemotherapy and immunoltherapy of human acute myelogenous leukemia.

Topics: Animals; Animals, Newborn; Ascitic Fluid; Cell Division; Cell Line; Culture Media; Culture Techniques; Disease Models, Animal; DNA; Female; Fluorescent Antibody Technique; Histocytochemistry; Leukemia, Experimental; Leukemia, Myeloid, Acute; Leukocytes; Muramidase; Pregnancy; Rats; Retroviridae; RNA-Directed DNA Polymerase; Staining and Labeling; Strontium Radioisotopes; Urine

Topics: Animals; Disease Models, Animal; Humans; Hypokalemia; Leukemia, Myeloid, Acute; Muramidase; Rats

Factors determining thrombus formation on a foreign surface were studied with the use of plastic flow chambers introduced into extracorporeal shunts. Silicone rubber shunts, joining the carotid artery and jugular vein, were implanted in dogs and remained patent for several weeks. The flow chamber geometry consisted of a 4.8 mm diameter straight tube having a 3.2 X 3.2 mm circumferential cavity in the wall. Chambers were introduced sequentially into the shunts for exposure times of 10 to 30 minutes and regulated blood flow rates of 100 to 400 ml/min. The dry weight of thrombus accumulated in the chamber (5 to 50 mg) was found to increase with exposure time up to 20 minutes and to decrease with increasing flow rate. Various components of the process of thrombus formation were altered by the administration of acetylsalicylic acid, heparin and lysozyme, used alone and in pairs. Heparin was found to be the most effective antithrombotic agent, dry weights of accumulated thrombus being on the order of 50 percent lower when compared to control values. The efficacy of heparin was found to be unaffected by the presence of aspirin and lysozyme, which themselves were not effective antithrombotic agents under the conditions of these experiments. The technique described here may provide a useful animal model for studying the influence of blood flow and different biomaterials on thrombus formation.

Topics: Animals; Aspirin; Blood Coagulation; Blood Flow Velocity; Disease Models, Animal; Dogs; Drug Synergism; Extracorporeal Circulation; Foreign-Body Reaction; Heparin; Muramidase; Platelet Aggregation; Rheology; Silicone Elastomers; Thrombosis; Time Factors

Topics: Alkaline Phosphatase; Animals; Arthritis; Blood Glucose; Diabetes Mellitus, Experimental; Diphenylamine; Disease Models, Animal; Indazoles; Male; Muramidase; Penicillamine; Phenylbutazone; Pyrazoles; Rats; Sulfanilamides; Sulfhydryl Compounds

The authors have analysed the influence of experimental infection of rabbits with Salmonella agona on lysozyme activity in the blood serum of examined animals. 37 rabbits were used in the experiments; experimental salmonellosis was developed in 22 out of this number, the remaining ones (15 rabbits) served as controls A statistically significant increase of lysozyme activity has been found in the serum of sick animals in the acute stage of the disease. A significantly high activity of this enzyme was observed in the diluted blood serum of experimental animals. No correlation was founds between the number of leucocytes and the percentage of granulocytes, and the lysozyme activity.

Topics: Animals; Disease Models, Animal; Leukocyte Count; Muramidase; Neutrophils; Rabbits; Salmonella Infections

Topics: Animals; Antibody Formation; Blood; Blood Bactericidal Activity; Cell Movement; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Estradiol; Female; Hemagglutination Tests; Injections, Intramuscular; Leukocytes; Liver; Monocytes; Muramidase; Phagocytosis; Pyelonephritis; Rats; Spleen

Topics: Animals; Arteriosclerosis; Cartilage; Disease Models, Animal; Female; Muramidase; Rats

Topics: Animals; Disease Models, Animal; Hypokalemia; Kidney; Kidney Tubules; Leukemia, Myeloid; Muramidase; Neoplasm Transplantation; Potassium; Rats

Topics: Animals; Aorta; Blood Cells; Calcification, Physiologic; Calcinosis; Calcium; Disease Models, Animal; Epiphyses; Lysosomes; Male; Muramidase; Phosphates; Rats; Rickets; Vitamin D

The induction of sterile unilateral pyelonephritis in rats with heat-killed Proteus mirabilis cells is described. The lesions were identical to those produced with viable bacteria. Lysozyme levels in both kidneys of rats developing unilateral sterile pyelonephritis underwent biphasic elevations similar to those produced with viable bacteria. In the injected kidney, the first elevation, associated with the trauma of injection, could be produced by injecting sterile saline. The second elevation was associated with the onset of chronicity in the injected kidney. The nonmanipulated, contralateral kidney showed a similar biphasic elevation, of equal duration but of greater magnitude.

Topics: Animals; Chronic Disease; Disease Models, Animal; Hot Temperature; Kidney; Muramidase; Proteus mirabilis; Pyelonephritis; Rats; Rats, Inbred Strains; Time Factors; Urease

Topics: Animals; Anti-Inflammatory Agents; Antibodies; Arthritis; Aspirin; Body Weight; Cyclophosphamide; Disease Models, Animal; Freund's Adjuvant; Hindlimb; Immunosuppressive Agents; Indomethacin; Male; Methotrexate; Muramidase; Phenylbutazone; Prednisolone; Rats

Spontaneous amyloidosis was noted in a significant number of germfree mice in comparison with their conventional contemporaries. The adjusted prevalence of this disease was increased in both groups by whole-body exposure at 6 weeks of age to 700 rad of ionizing radiation. The germfree groups demonstrated persistent hypogammaglobulinemia throughout their lifespans and no evidence of significant inflammatory processes at necropsy. The possible interpretation of these observations is discussed and it is concluded that defective or deficient immunoglobulin production may be the essential prerequisite for the development of amyloidosis.

Topics: Agammaglobulinemia; Age Factors; Amyloid; Amyloidosis; Animals; Aspartate Aminotransferases; Blood Chemical Analysis; Blood Proteins; Blood Urea Nitrogen; Cobalt Isotopes; Disease Models, Animal; Female; Germ-Free Life; Immunosuppression Therapy; Kidney; L-Lactate Dehydrogenase; Lymphoma, Non-Hodgkin; Mice; Muramidase; Radiation Effects; Serum Albumin; Serum Globulins; Thymus Neoplasms

Topics: Animals; Arthritis, Infectious; Arthritis, Rheumatoid; Body Temperature; Body Weight; Disease Models, Animal; Edema; Extremities; Leukocytosis; Male; Muramidase; Physical Exertion; Rats; Time Factors

Topics: Animals; Disease Models, Animal; Drug Synergism; Encephalomyocarditis virus; Female; Injections, Intraperitoneal; Injections, Intravenous; Leukocyte Count; Male; Mice; Muramidase; Orthomyxoviridae; Orthomyxoviridae Infections; Phagocytosis; Phenanthrenes; Poliomyelitis; Salmonella Infections, Animal; Salmonella typhimurium; Staphylococcal Infections; Staphylococcus