cord-factors and Disease-Models--Animal

Tuberculosis remains a major threat as drug resistance continues to increase. Pulmonary tuberculosis in adults is responsible for 80% of clinical cases and nearly 100% of transmission of infection. Unfortunately, since we have no animal models of adult type pulmonary tuberculosis, the most important type of disease remains largely out of reach of modern science and many fundamental questions remain unanswered. This paper reviews research dating back to the 1950's providing compelling evidence that cord factor (trehalose 6,6 dimycolate [TDM]) is essential for understanding tuberculosis. However, the original papers by Bloch and Noll were too far ahead of their time to have immediate impact. We can now recognize that the physical and biologic properties of cord factor are unprecedented in science, especially its ability to switch between two sets of biologic activities with changes in conformation. While TDM remains on organisms, it protects them from killing within macrophages, reduces antibiotic effectiveness and inhibits the stimulation of protective immune responses. If it comes off organisms and associates with lipid, TDM becomes a driver of tissue damage and necrosis. Studies emanating from cord factor research have produced (1) a rationale for improving vaccines, (2) an approach to new drugs that overcome natural resistance to antibiotics, (3) models of caseating granulomas that reproduce multiple manifestations of human tuberculosis. (4) evidence that TDM is a key T cell antigen in destructive lesions of tuberculosis, and (5) a new understanding of the pathology and pathogenesis of postprimary tuberculosis that can guide more informative studies of long standing mysteries of tuberculosis.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Disease Models, Animal; Humans; Macrophages; Mice; Mycobacterium tuberculosis; Tuberculosis; Tuberculosis Vaccines

Tuberculosis, once thought to have been controlled, is now resurgent in many parts of the world. Many gaps exist in understanding the pathogenesis of tuberculosis, especially secondary and cavitary disease. Evidence presented here suggests that cord factor (trehalose 6,6'-dimycolate, TDM) is a key driver of these processes. It is the most abundant lipid released by virulent M. tuberculosis (MTB) and can switch between two sets of activities. On organisms, TDM is non-toxic and protects them from killing by macrophages. On lipid surfaces, it becomes antigenic and highly toxic. Caseating granulomas, the hallmark of primary tuberculosis, develop from interaction of TDM with lipid within granulomas. New evidence indicates that secondary tuberculosis begins as a lipid pneumonia that accumulates mycobacterial antigens and host lipids in alveoli before developing conditions for activation of the toxicity and antigenicity of TDM. This rapidly produces caseation necrosis that leads to cavities. Finally, virulent MTB release large amounts of TDM during growth as a pellicle within cavities. We propose that such growth results in activation of the toxicity and antigenicity of TDM at the air interface and that presence of the activated TDM perpetuates the cavity.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Disease Models, Animal; Granuloma; Humans; Macrophages; Mice; Mycobacterium tuberculosis; Necrosis; Tuberculosis

Intravesical Bovis bacillus Calmette-Guérin (BCG) therapy is the most effective immunotherapy for bladder cancer, but it sometime causes serious side effects because of its inclusion of live bacteria. It is necessary to develop a more active but less toxic immunotherapeutic agent. Trehalose 6,6'-dimycolate (TDM), the most abundant hydrophobic glycolipid of the BCG cell wall, has been reported to show various immunostimulatory activities such as granulomagenesis and adjuvant activity. Here, we developed cationic liposomes incorporating TDM purified from Mycobacterium bovis BCG Connaught, and we investigated the antitumor effect of the cationic liposome TDM (Lip-TDM). Lip-TDM exerted an antitumor effect in bladder cancer, colon cancer, and melanoma-bearing mouse models that was comparable or even superior to that of BCG, with no body weight loss or granuloma formation. The antitumor effect of Lip-TDM disappeared in two types of mice: those with depletion of CD8

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents, Immunological; CD8-Positive T-Lymphocytes; Chemical Fractionation; Cord Factors; Cytokines; Dendritic Cells; Disease Models, Animal; Female; Humans; Immunologic Factors; Immunophenotyping; Infusions, Parenteral; Liposomes; Lymphocyte Activation; Mice; Molecular Structure; Mycobacterium bovis; Solvents; Treatment Outcome; Xenograft Model Antitumor Assays

Mycobacterium tuberculosis (MTB) is a pathogen that infects and kills millions yearly. The mycobacterium's cell wall glycolipid trehalose 6,6'-dimycolate (TDM) has been used historically to model MTB induced inflammation and granuloma formation. Alterations to the model can significantly influence the induced pathology. One such method incorporates intraperitoneal pre-exposure, after which the intravenous injection of TDM generates pathological damage effectively mimicking the hypercoagulation, thrombus formation, and tissue remodeling apparent in lungs of infected individuals. The purpose of these experiments is to examine the histological inflammation involved in the TDM mouse model that induces development of the hemorrhagic response. TDM induced lungs of C57BL/6 mice to undergo granulomatous inflammation. Further histological examination of the peak response demonstrated tissue remodeling consistent with hypercoagulation. The observed vascular occlusion indicates that obstruction likely occurs due to subendothelial localized activity leading to restriction of blood vessel lumens. Trichrome staining revealed that associated damage in the hypercoagulation model is consistent with intra endothelial cell accumulation of innate cells, bordered by collagen deposition in the underlying parenchyma. Overall, the hypercoagulation model represents a comparative pathological instrument for understanding mechanisms underlying development of hemorrhage and vascular occlusion seen during MTB infection.

Topics: Animals; Blood Coagulation; Cord Factors; Disease Models, Animal; Endothelium, Vascular; Female; Granuloma, Respiratory Tract; Lung; Mice, Inbred C57BL; Mycobacterium tuberculosis; Pneumonia; Tuberculosis, Pulmonary; Vascular Remodeling

Mycobacterial infection leads to the formation of characteristic immune aggregates called granulomas, a process accompanied by dramatic remodeling of the host vasculature. As granuloma angiogenesis favors the infecting mycobacteria, it may be actively promoted by bacterial determinants during infection. Using Mycobacterium marinum-infected zebrafish as a model, we identify the enzyme proximal cyclopropane synthase of alpha-mycolates (PcaA) as an important bacterial determinant of granuloma-associated angiogenesis. cis-Cyclopropanation of mycobacterial mycolic acids by pcaA drives the activation of host Vegf signaling within granuloma macrophages. Cyclopropanation of the mycobacterial cell wall glycolipid trehalose dimycolate is both required and sufficient to induce robust host angiogenesis. Inducible genetic inhibition of angiogenesis and Vegf signaling during granuloma formation results in bacterial growth deficits. Together, these data reveal a mechanism by which PcaA-mediated cis-cyclopropanation of mycolic acids promotes bacterial growth and dissemination in vivo by eliciting granuloma vascularization and suggest potential approaches for host-directed therapies.

Topics: Angiogenesis Inhibitors; Animals; Bacterial Proteins; Cord Factors; Disease Models, Animal; Humans; Indazoles; Macrophages; Methyltransferases; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Mycolic Acids; Neovascularization, Pathologic; Pyrimidines; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Sulfonamides; Tuberculoma; Zebrafish

Tuberculosis (TB) remains a global health concern. Trehalose 6'6-dimycolate (TDM) activates innate inflammation and likely also stimulates chronic inflammation observed during disease progression. Noninfectious models using purified TDM oil/water emulsions elicit pathologic findings observed in patients with TB. We introduce a new TDM model that promotes inflammatory lung pathologic findings and vascular occlusion and hemorrhage. C57BL/6 and BALB/c mice were injected with 10 μg of i.p. TDM in light mineral oil (TDM-IP). At day 7, another injection of 10 μg of i.v. TDM in oil/water emulsion was given (TDM-IV). The i.p./i.v. TDM (TDM-IVIP) group was compared with mice injected once with i.v. or i.p. TDM. The responses to TDM-IP, TDM-IV, or TDM-IPIV were consistent between mouse strains. Mice that received TDM-IV and TDM-IPIV had inflammatory pathologic findings with increases in inflammatory and T-cell cytokines, and the TDM-IPIV group had further enhancement of IL-10 and granulocyte-macrophage colony-stimulating factor. The TDM-IPIV group had increased CD4(+) T cells in lung tissue, significantly increased coagulation, decreased clot formation time, and increased maximum clot firmness. Masson's trichrome staining revealed increased deposition of collagen in the occluded vasculature. TDM-IPIV promotes a hypercoagulopathy state, independent of inflammation. This new model argues that TDM is sufficient to generate the hypercoagulopathy observed in patients with TB.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD; Collagen; Cord Factors; Cytokines; Disease Models, Animal; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Immunity, Innate; Lung; Lymphocytes; Macrophages; Mice, Inbred BALB C; Mice, Inbred C57BL; Mycobacterium tuberculosis; Neutrophils; Pneumonia; Pulmonary Veno-Occlusive Disease; Thrombelastography; Thrombophilia

A combination therapy for combined injury (CI) using a non-specific immunomodulator, synthetic trehalose dicorynomycolate and monophosphoryl lipid A (STDCM-MPL), was evaluated to augment oral antimicrobial agents, levofloxacin (LVX) and amoxicillin (AMX), to eliminate endogenous sepsis and modulate cytokine production.. Female B6D2F(1)/J mice received 9.75 Gy cobalt-60 gamma-radiation and wound. Bacteria were isolated and identified in three tissues. Incidence of bacteria and cytokines were compared between treatment groups.. Results demonstrated that the lethal dose for 50% at 30 days (LD(50/30)) of B6D2F(1)/J mice was 9.42 Gy. Antimicrobial therapy increased survival in radiation-injured (RI) mice. Combination therapy increased survival after RI and extended survival time but did not increase survival after CI. Sepsis began five days earlier in CI mice than RI mice with Gram-negative species predominating early and Gram-positive species increasing later. LVX plus AMX eliminated sepsis in CI and RI mice. STDCM-MPL eliminated Gram-positive bacteria in CI and most RI mice but not Gram-negative. Treatments significantly modulated 12 cytokines tested, which pertain to wound healing or elimination of infection.. Combination therapy eliminates infection and prolongs survival time but does not assure CI mouse survival, suggesting that additional treatment for proliferative-cell recovery is required.

Topics: Amoxicillin; Animals; Anti-Infective Agents; Bacterial Infections; Chemokines; Cord Factors; Cytokines; Disease Models, Animal; Drug Therapy, Combination; Female; Gamma Rays; Growth Substances; Immunologic Factors; Levofloxacin; Lipid A; Mice; Radiation Injuries, Experimental; Sepsis; Skin; Wound Infection

With the paucity of new drugs and HIV co-infection, vaccination remains an unmet research priority to combat visceral leishmaniasis (VL) requiring strong cellular immunity. Protein vaccination often suffers from low immunogenicity and poor generation of memory T cells for long-lasting protection. Cysteine proteases (CPs) are immunogenic proteins and key mediators of cellular functions in Leishmania. Here, we evaluated the vaccine efficacies of CPs against VL, using cationic liposomes with Toll like receptor agonists for stimulating host immunity against L. donovani in a hamster model.. Recombinant CPs type I (cpb), II (cpa) and III (cpc) of L. donovani were tested singly and in combination as a triple antigen cocktail for antileishmanial vaccination in hamsters. We found the antigens to be highly immunoreactive and persistent anti-CPA, anti-CPB and anti-CPC antibodies were detected in VL patients even after cure. The liposome-entrapped CPs with monophosphoryl lipid A-Trehalose dicorynomycolate (MPL-TDM) induced significantly high nitric oxide (up to 4 fold higher than controls) mediated antileishmanial activity in vitro, and resulted in strong in vivo protection. Among the three CPs, CPC emerged as the most potent vaccine candidate in combating the disease. Interestingly, a synergistic increase in protection was observed with liposomal CPA, CPB and CPC antigenic cocktail which reduced the organ parasite burden by 1013-1016 folds, and increased the disease-free survival of >80% animals at least up to 6 months post infection. Robust secretion of IFN-γ and IL-12, along with concomitant downregulation of Th2 cytokines, was observed in cocktail vaccinates, even after 3 months post infection.. The present study is the first report of a comparative efficacy of leishmanial CPs and their cocktail using liposomal formulation with MPL-TDM against L. donovani. The level of protection attained has not been reported for any other subcutaneous single or polyprotein vaccination against VL.

Topics: Animals; Antigens, Protozoan; Cations; Cord Factors; Cricetinae; Cysteine Proteases; Disease Models, Animal; Drug Delivery Systems; Leishmania donovani; Leishmaniasis; Lipid A; Liposomes; Mesocricetus; Protozoan Vaccines

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This 'genetic phenotype' was further confirmed by a 'chemical phenotype', whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.

Topics: Animals; Antitubercular Agents; Bacterial Proteins; Bridged Bicyclo Compounds, Heterocyclic; Chromatography, Thin Layer; Cord Factors; Disease Models, Animal; Dogs; Drug Resistance, Bacterial; Genotype; Hep G2 Cells; Humans; Kinetics; Mice; Microbial Sensitivity Tests; Microbial Viability; Mutation; Mycobacterium tuberculosis; Pyrazoles; Rats; Spiro Compounds; Treatment Outcome; Tuberculosis

Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6'-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB.

Topics: Adjuvants, Immunologic; Animals; Antigens, Bacterial; Bacterial Load; Cord Factors; Disease Models, Animal; Drug Carriers; Female; Humans; Lung; Mice; Mice, Inbred BALB C; Microspheres; Mycobacterium tuberculosis; Tuberculosis Vaccines; Tuberculosis, Pulmonary; Vaccines, DNA

Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice.. Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo.. Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated in a controlled release system against experimental VL.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Cytokines; Disease Models, Animal; Female; Humans; Injections, Subcutaneous; Leishmania donovani; Leishmaniasis Vaccines; Leishmaniasis, Visceral; Lipid A; Liposomes; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Nitric Oxide; Phosphatidylcholines; Th1 Cells; Vaccination; Vaccines, Synthetic

The immune system responds to tuberculosis (TB) infection by forming granulomas. However, subsequent immune-mediated destruction of lung tissue is a cause of significant morbidity and contributes to disease transmission. Lactoferrin, an iron-binding glycoprotein, has demonstrated immunomodulatory properties that decrease tissue destruction and promote T(H)1 immune responses, both of which are essential for controlling TB infection. The cord factor trehalose 6,6'-dimycolate (TDM) model of granuloma formation mimics many aspects of TB infection with a similar histopathology accompanied by proinflammatory cytokine production. C57BL/6 mice were injected intravenously with TDM. A subset of mice was given 1 mg of bovine lactoferrin 24 h post-TDM challenge. Lung tissue was analyzed for histological response and for the production of proinflammatory mediators. C57BL/6 mice demonstrated a granuloma formation that correlated with an increased production of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α,) IL-12p40, interferon-gamma (IFN-γ), and IL-10 protein. Mice treated with lactoferrin postchallenge had significantly fewer and smaller granulomas compared with those given TDM alone. Proinflammatory and T(H)1 cytokines essential to the control of mycobacterial infections, such as TNF-α and IFN-γ, were not significantly different in mice treated with lactoferrin. Furthermore, the anti-inflammatory cytokines IL-10 and transforming growth factor-β were increased. A potential mechanism for decreased tissue damage observed in the lactoferrin-treated mice is proposed. Because of its influence to modulate immune responses, lactoferrin may be a useful adjunct in the treatment of granulomatous inflammation occurring during mycobacterial infection.

Topics: Animals; Cord Factors; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Granuloma; Interleukin-10; Lactoferrin; Lung; Lung Diseases; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Protein Biosynthesis; Transforming Growth Factor beta; Tuberculosis

Tuberculosis has a staggering influence on world health, resulting in nearly 2 million deaths per year. The influence of glucocorticoids during Mycobacterium tuberculosis infection has been under investigation for decades; however, the identity of mycobacterial factors and the mechanism by which glucocorticoids are tissue specifically regulated to influence immune function during acute granuloma formation are unknown.. One factor implicated in initiating immunopathology during M. tuberculosis infection is trehalose-6,6'-dimycolate (TDM), a glycolipid component of the mycobacterial cell wall. Intravenous administration of TDM causes inflammatory responses in lungs of mice similar to M. tuberculosis infection and has been used as a successful model to examine proinflammatory regulation and early events involved in the manifestation of pathology.. IL-6, IL-1alpha and TNF-alpha mRNA and protein peaked during the initiation of granuloma formation. Pulmonary corticosterone levels were elevated when the proinflammatory response was greatest, dropping to half of that upon the establishment of granuloma pathology on day 7. It is hypothesized that once corticosterone reaches the site of inflammation, the enzymes 11beta-hydroxysteroid dehydrogenases (11betaHSDs) can influence bioavailability by interconverting corticosterone and the inert metabolite 11-dehydrocorticosterone. RT-PCR demonstrated that pulmonary 11betaHSD type 1 mRNA decreased 4-fold and 11betaHSD type 2 (11betaHSD2) mRNA expression increased 2.5-fold on day 3 after injection, suggesting that corticosterone regulation in the lung, specifically the reduction of active corticosterone by 11betaHSD2, may influence the progression of granuloma formation in response to the mycobacterial glycolipid.

Topics: 11-beta-Hydroxysteroid Dehydrogenase Type 1; 11-beta-Hydroxysteroid Dehydrogenase Type 2; Animals; Cord Factors; Corticosterone; Cytokines; Disease Models, Animal; Down-Regulation; Female; Granuloma, Respiratory Tract; Immune Tolerance; Lung; Mice; Mice, Inbred BALB C; Mycobacterium tuberculosis; RNA, Messenger; Tuberculosis, Pulmonary; Up-Regulation

M. tuberculosis (MTB) lose virulence during prolonged culture on artificial media. This loss of virulence is associated with a change in colony morphology. Several studies suggested that trehalose 6,6' dimycolate (TDM or cord factor), contributes to colony morphology.. To investigate the role of TDM in colony morphology of MTB using clinical isolates selected to have colony morphology typical of virulent or attenuated organisms.. Use immunohistochemical and physical chemical methods to assess the presence and distribution of TDM in rapidly growing pellicles of MTB.. TDM forms an insoluble crystalline monolayer at the air-water interfaces that is more rigid than that formed by any other biologic amphiphile and is strong enough to support a spreading pellicle of MTB. The surface of young pellicles of the isolate with virulent morphology displayed the regular linear pattern characteristic of monolayers of TDM. TDM was also identified in the open spaces of pellicles of MTB by immunohistochemistry. MTB with morphology of attenuated organisms had neither of these properties.. These data suggest that the characteristic morphology of colonies of virulent MTB is due to TDM released from the surface of the organisms.

Topics: Animals; Cord Factors; Disease Models, Animal; Mycobacterium tuberculosis; Rabbits; Virulence

Granulomatous colitis is a major entity of human intestinal diseases. We previously reported that intravenous injection of mycobacterial cord factor (CF), a potent macrophage activator, induced pulmonary granulomas in mice with enhanced production of Th1 cytokines and chemokines. In this study we made a murine model of granulomatous colitis by intramural injection of CF. A single dose of 300 microg CF was injected into the wall of the rat and mouse colon in the form of liposomes. After 1 week granulomas developed at the injection site, extending from the subserosa to the lamina propria, and persisted for longer than 6 weeks. They were composed mainly of ED1-positive macrophages, which often underwent apoptosis, and CD4(+) and CD8(+) lymphocytes, which preferentially infiltrated around the macrophage accumulation. Myofibroblast proliferation was not prominent, and no appreciable fibrosis resulted after the decline of granulomas. Although the intestinal epithelium was involved in inflammation, tissue injuries such as mucosal erosion or ulceration were not induced. When granulomas were formed near the Peyer's patches, they invaded deeply into the lymphoid tissue, producing many small islands. The mesenteric lymph nodes also had many granulomatous islands in the cortex and medulla, but the liver and spleen displayed no granulomatous changes, suggesting that liposomal CF spreads via the lymphatic vessels from the injection site. The CF-induced colonic granulomas associated with mesenteric lymphadenitis will be useful for investigating human granulomatous colitis.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD; Colon; Cord Factors; Crohn Disease; Disease Models, Animal; Immunoenzyme Techniques; Liposomes; Macrophages; Male; Mesenteric Lymphadenitis; Mycobacterium; Organ Size; Rats; Rats, Wistar; Specific Pathogen-Free Organisms; T-Lymphocyte Subsets

C57BL/6 mice were vaccinated with a bacterially expressed and purified polyhistidine-tagged full-length version of the microneme protein NcMIC3 (recNcMIC3) emulsified in Ribi Adjuvant System (RAS). Subsequently, they were challenged by intraperitoneal inoculation of 2 x 10(6) live Neospora caninum tachyzoites. As controls, groups of mice received phosphate-buffered saline (PBS)-RAS alone (adjuvant control) or were treated with PBS before infection (infection control). The protective effect of vaccination was assessed by Neospora-specific polymerase chain reaction (PCR), immunohistochemical investigation of brain tissue, and serological means (enzyme-linked immunosorbent assay). Assessment by PCR performed on DNA from different organs revealed that in all treatment groups parasite DNA could only be detected in brain tissue. According to the PCR results. the recNcMIC3 vaccine conferred protection to 75% of mice (n = 16 in 2 independent experiments), whereas application of PBS-RAS and of PBS alone resulted in protection of 12.5% and 0% of mice, respectively (n = 16 as above). Mice in the PBS-treated infection control group were affected by evident clinical signs of neosporosis starting on day 6 postinfection (p.i.). Conversely, none of the animals treated with either PBS-RAS or recNcMIC3 exhibited any symptoms until day 21 p.i. Immunohistochemical staining of paraffin-embedded brain tissue sections confirmed the protective effect of recNcMIC3 vaccination. Quantitative Neospora-specific real-time PCR revealed that infection intensities were lower in the brain tissues of recNcMIC3-vaccinated mice compared with PBS-RAS-treated adjuvant control mice. Serological analysis showed that the protective effect observed in recNcMIC3-vaccinated mice was associated with a Th2-type IgG1 antibody response directed against native NcMIC3 and a mixed IgG1-IgG2a antibody response directed against the recombinant antigen itself. Taken together, these results demonstrated that recombinant NcMIC3 vaccine confers a significant protectivity against experimentally induced cerebral neosporosis in mice.

Topics: Adhesins, Bacterial; Animals; Antibodies, Protozoan; Antigens, Protozoan; Brain; Brain Diseases; Carrier Proteins; Cell Wall Skeleton; Coccidiosis; Cord Factors; Disease Models, Animal; DNA, Protozoan; Female; Immunoglobulin G; Lipid A; Mice; Mice, Inbred C57BL; Neospora; Polymerase Chain Reaction; Protozoan Proteins; Protozoan Vaccines; Recombinant Proteins; Vaccination; Vaccines, Synthetic

Development of an effective vaccine against Leishmania infection is a priority of tropical disease research. We have recently demonstrated protection against Leishmania major in the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using the L. major challenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.

Topics: Adjuvants, Immunologic; Animals; Antigens, Protozoan; Antioxidants; Cell Wall Skeleton; Cord Factors; Disease Models, Animal; Female; Heat-Shock Proteins; Leishmania major; Leishmania mexicana; Leishmaniasis, Cutaneous; Lipid A; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Chaperones; Peptide Initiation Factors; Peroxidases; Peroxiredoxins; Polyproteins; Protozoan Proteins; Protozoan Vaccines; T-Lymphocytes; Vaccination; Vaccines, Synthetic

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Cytokines; Disease Models, Animal; Female; Gene Expression; Granuloma; Inflammation; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium; RNA, Messenger

Trehalose 6,6'-dimycolate (TDM), an immunomodulator, potentiates non-specific resistance in mice to influenza virus infection. When mice were injected intravenously with TDM, the striking proliferation of a minority of T-lymphocytes bearing gamma/delta T-cell receptors (gamma delta T-cells) that accumulated in granulomatous lungs was thought to be associated with the maintenance of acquired resistance to lethal influenza virus infection. To clarify the cellular basis of the defence against influenza virus, mice were depleted of gamma delta T-cells, alpha/beta (alpha beta) T-cells, or natural killer (NK) cells by in vivo administration of corresponding antibodies prior to influenza virus infection. The depletion of gamma delta T-cells significantly abrogated the augmented resistance of TDM-treated mice to infection, as did depletion of either alpha beta T-cells or NK cells. To gain insight into the functional ability of gamma delta T-cells, we evaluated the cytotoxic activity of this T-cell subset against a panel of target cell lines that were stably transfected with the influenza virus haemagglutinin (HA) gene from A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2) strains. The gamma delta T-cells from TDM-treated mice showed profound cytotoxicity against the target cells expressing HA of either the H1 or H3 subtype, in a non-major histocompatibility complex-restricted manner. Taken together, these results indicate that gamma delta T-cells play a non-specific role, in conjunction with alpha beta T-cells and NK cells, in protecting mice against influenza virus infection, and that the recognition and destruction of HA-expressing target cells by the activated gamma delta T-cells is one of the steps involved in this anti-influenza virus immunosurveillance.

Topics: Adjuvants, Immunologic; Animals; Cells, Cultured; Chick Embryo; Cord Factors; Cytotoxicity Tests, Immunologic; Disease Models, Animal; Female; Hemagglutinin Glycoproteins, Influenza Virus; Humans; Immunity, Innate; Influenza A virus; Influenza, Human; Killer Cells, Natural; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes

Preventive intraperitoneal trehalose dimycolate (TDM) treatment of mice, inoculated with encephalomyocarditis (EMC) virus by the same route, caused restriction of virus growth in the peritoneum, which was correlated to IFN production in peritoneal fluids prior to infection. Peritoneal macrophages from TDM-treated mice (TDM-PM) spontaneously secreted IFN-alpha/beta in large amounts. By their supernatants, TDM-PM could transfer an antiviral state against EMC virus to permissive resident peritoneal macrophages from control mice. IFN-alpha/beta produced by TDM-PM was found to be involved in this transfer activity. TDM-PM also exerted a strong antiviral effect on EMC virus-infected L-929 cells, which increased with time and the macrophage-target cell ratio. This activity also occurred by an IFN-alpha/beta-dependent mechanism. These data point to the role of IFN-alpha/beta production prior to EMC virus infection in the antiviral activities of TDM-PM and, more generally, in the outcome of viral infection.

Topics: Animals; Antiviral Agents; Ascitic Fluid; Cardiovirus Infections; Cell Line; Cells, Cultured; Cord Factors; Disease Models, Animal; Encephalomyocarditis virus; Female; Interferon-alpha; Interferon-beta; Macrophages, Peritoneal; Mice

Trehalose dimycolate (TDM) is a glycolipid contained in the cell walls of Mycobacteria, Nocardia and Corynebacteria. An intraperitoneal injection of TDM into mice has been known to produce hemorrhagic pneumonia without affecting any other organs. Thus, it provides a unique experimental model for studies of the mechanisms of alveolar hemorrhagic syndrome, including idiopathic pulmonary hemosiderosis. It has been reported that T lymphocytes are essential for the production of TDM-induced hemorrhagic pneumonia, however, the overall cellular mechanism is not yet clear. The purpose of this study is to re-examine and clarify the role of T lymphocytes in the pathogenesis of TDM-induced hemorrhagic pneumonia. To achieve it we considered 1) the dynamics of infiltrating lymphocytes to find out if there is a certain T lymphocyte subpopulation infiltrating predominantly into the lung, 2) the effect of in vivo depletion of T lymphocyte subpopulation by monoclonal antibodies, and 3) the effect of transfer of T lymphocytes into nude mice. The analysis of the dynamic change of the number of lymphocytes showed that the number of L3T4+ cells as well as Lyt2+ cells decreased on day 2 or 3 after TDM injection, thereafter increased, however, neither subpopulation infiltrated predominantly into the lung. Alveolar hemorrhages occurred in L3T4+ cell-depleted and/or Lyt2+ cell-depleted mice, and hemorrhages were enhanced in Lyt2+ cell-deplete mice. Alveolar hemorrhages occurred even in nude mice, and the intensity of hemorrhages or the cell numbers in the lung did not differ from those in T lymphocyte-reconstituted nude mice, however, hemorrhages were enhanced in L3T4+ cell-reconstituted nude mice. These results suggested that T lymphocytes are not primarily involved in the cellular mechanisms of the pathogenesis of TDM-induced hemorrhagic pneumonia, however, L3T4+ cells modify the process of the production of hemorrhagic pneumonia secondarily and enhance it.

Topics: Animals; Cord Factors; Disease Models, Animal; Female; Glycolipids; Hemorrhage; Male; Mice; Pneumonia; T-Lymphocytes

Trehalose dimycolate (TDM) treated and untreated mice were infected with Trypanosoma musculi. Compared to untreated mice, treated mice exhibited a five fold reduced number of circulating parasites. Untreated infected mice had a splenomegaly but only a slight increase of spleen weight of treated mice was observed. The role of trehalose dimycolate on T. musculi infection, especially via the macrophage is discussed.

Topics: Animals; Cord Factors; Disease Models, Animal; Female; Glycolipids; Mice; Splenomegaly; Trypanosomiasis