muramidase and Diabetes-Mellitus--Type-1

We discuss three areas of antigen presentation and macrophage biology being investigated in the laboratory. Using hen egg-white lysozyme as a protein antigen, all the segments of the molecules selected by the class II histocompatibility molecule I-A(k) were identified and characterized. The display of each family of peptides was explained biochemically and quantitated. Conformational isomers of a peptide-major histocompatibility complex (MHC) complex were identified. The relationship between the amounts of peptide-MHC displayed by the antigen-presenting cells and two biologic responses, central thymic selection and T-cell responses after immunization in adjuvant, were examined. The class II MHC molecule of the nonobese diabetic I-Ag7 is being examined for its properties of peptide selection. The objective is to identify the diabetogenic peptides, as well as the repertoire of protein antigens from beta-cells that trigger autoantibodies. The I-Ag7 molecule selects peptides that show very distinctive sequence motifs: one or more acidic residues at the carboxy terminus that interact at the P9 pocket of the binding groove. Finally, the investigations in listeriosis examined the early events in immune induction. More important, we found that Listeria causes marked apoptosis of lymphocytes around infective foci resulting from the apoptogenic properties of the pore-forming molecule Listeriolysin O.

Topics: Amino Acid Sequence; Animals; Antigen Presentation; Antigens; Apoptosis; Autoantibodies; Chickens; Diabetes Mellitus, Type 1; Epitopes; Listeriosis; Mice; Mice, Inbred NOD; Mice, Transgenic; Models, Immunological; Muramidase; Protein Conformation; T-Lymphocyte Subsets

The underlying cellular and molecular mechanism for the development of Type 1 diabetes is still to be fully revealed. We have previously demonstrated that the NOD mouse, a model for Type 1 diabetes, display a prolonged and enhanced immune response to both self and non-self-antigens. The molecular explanation for this defect however, has not been determined. In this study we immunized NOD and C57BL/6 (B6) with the conventional antigen i.e. hen egg lysozyme (HEL) and analyzed B cell activation, germinal center reaction and antibody clearance. Corroborating our previous observations NOD mice responded robustly to a single immunization of HEL. Immunofluorescence analysis of the spleen revealed an increased number of germinal centers in unimmunized NOD compared to B6. However, post immunization germinal center numbers were similar in NOD and B6. NOD mice showed lower response to BCR stimulation with anti-IgM, in particular at lower concentrations of anti-IgM. Antibody clearance in vivo did not differ between the strains. To determine the cell type that is responsible for the prolonged and enhance immune response, we reconstituted NOD-RAGs with cells from primed donors in different combinations. NOD B cells were required to reproduce the phenotype; however the non-lymphoid compartment of NOD origin also played a role. Based on our results we propose that preexisting GCs in the NOD promote the robust response and alteration in the BCR signaling could promote survival of stimulated cells. Overall, this mechanism could in turn also contribute to the activation and maintenance of autoreactive B cells in the NOD mouse.

Topics: Adoptive Transfer; Animals; Antibodies, Anti-Idiotypic; Autoimmunity; B-Lymphocytes; Chickens; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Progression; Gene Expression; Germinal Center; Humans; Immunization; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Muramidase; Receptors, Antigen, B-Cell; Spleen

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 an accompanying paper, we find specific localization of diabetogenic T cells only to islets of Langerhans bearing the specific antigen. Instrumental in the specific localization was the presence of intraislet dendritic cells bearing the β-cell-peptide-MHC complex. Here, we report that the entry of diabetogenic CD4 T cells very rapidly triggered inflammatory gene expression changes in islets and vessels by up-regulating chemokines and adhesion molecules. Vascular cell adhesion molecule-1 (VCAM-1) expression was notable in blood vessels, as was intercellular adhesion molecule-1 (ICAM-1). ICAM-1 was also found on β-cells. These expression changes induced the entry of nonspecific T cells that otherwise did not localize to the islets. In contrast to the entry of diabetogenic CD4 T cells, the entrance of nonspecific T cells required a chemokine response and VCAM-1 expression by the islets. IFN-γ was important for the early gene expression changes in the islets. By microarray analysis, we detected up-regulation of a group of IFN-inducible genes as early as 8 h post-T-cell transfer. These studies establish that entry of diabetogenic T cells induces a state of receptivity of islets to subsequent immunological insults.

Topics: Adoptive Transfer; Animals; Blood Vessels; CD4-Positive T-Lymphocytes; Dendritic Cells; Diabetes Mellitus, Type 1; Flow Cytometry; Gene Amplification; Gene Expression Profiling; Homeodomain Proteins; Intercellular Adhesion Molecule-1; Interferon gamma Receptor; Interferon-gamma; Islets of Langerhans; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, Transgenic; Muramidase; Oligonucleotide Array Sequence Analysis; Receptors, Interferon; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Vascular Cell Adhesion Molecule-1

Understanding the entry of autoreactive T cells to their target organ is important in autoimmunity because this entry initiates the inflammatory process. Here, the events that lead to specific localization of diabetogenic CD4 T cells into islets of Langerhans resulting in diabetes were examined. This was evaluated in two models, one in which T cells specific for a hen-egg white lysozyme (HEL) peptide were injected into mice expressing HEL on β cells and the other using T cells in the nonobese diabetic mouse strain, which develops spontaneous diabetes. Only T cells specific for β-cell antigens localized in islets within the first hours after their injection and were found adherent to intraislet dendritic cells (DCs). DCs surrounded blood vessels with dendrites reaching into the vessels. Localization of antigen-specific T cells did not require chemokine receptor signaling but involved class II histocompatibility and intercellular adhesion molecule 1 molecules. We found no evidence for nonspecific localization of CD4 T cells into normal noninflamed islets. Thus, the anatomy of the islet of Langerhans permits the specific localization of diabetogenic T cells at a time when there is no inflammation in the islets.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; CD4-Positive T-Lymphocytes; Dendritic Cells; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Histocompatibility Antigens Class II; Intercellular Adhesion Molecule-1; Islets of Langerhans; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Mice, Transgenic; Microscopy, Fluorescence, Multiphoton; Muramidase

One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs) have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns-independent component analysis-to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739), previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1) is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178), which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644) was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the mechanisms linking genome-wide association loci to disease.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Celiac Disease; Diabetes Mellitus, Type 1; Female; Gene Expression Profiling; Gene Expression Regulation; Genetic Predisposition to Disease; Genetic Variation; Genome-Wide Association Study; Genome, Human; Humans; Hypertension; Intracellular Signaling Peptides and Proteins; Male; Middle Aged; Monocytes; Muramidase; Polymorphism, Single Nucleotide; Proteins; Quantitative Trait Loci; Ribosomal Proteins; Transcription Factors

Antibacterial immunity in diabetes is impaired, which increases the risk of general and local infections. The aim of the study was to evaluate non-specific local antibacterial immunity based on lactoferrin and lysozyme concentration in tears in children with diabetes type 1.. Children at the age of 10-18 years old were studied. Group 1. consisted of children without diabetes, group 2. included patients with new onset of diabetes and group 3. consisted of children with decade-long diabetes. Among all patients tears were collected from inferior coniunctival fornix with hematocrit glass capillaries in purpose to measure lactoferrin and lysozyme concentration. ELISA method was used in laboratory testing.. Level of lactoferrin did not differ significantly among all groups. Concentration of lysozyme was statistically lower in group with decade-long diabetes (group 3.) compared to patients without diabetes. Mild correlation between lactoferrin and lysozyme levels was seen in individual patients in whole group of probands together.. Diabetes type 1 in children is associated with significant changes in concentration of tear proteins, which contribute to antibacterial immunity.

Topics: Adolescent; Child; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Diagnostic Techniques, Ophthalmological; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Female; Health Status Indicators; Humans; Lactoferrin; Male; Muramidase

Self-reactive B lymphocytes contribute to type 1 diabetes pathogenesis as APC and auto-Ab producers in NOD mice and humans. To shed light on the mechanisms responsible for the breakdown in B-lymphocyte self-tolerance to β-cell Ag, we utilised a model whereby hen-egg lysozyme (HEL)-specific Ig Tg (IgHEL-Tg)-Tg B lymphocytes were allowed to develop in or were transferred into mice expressing the HEL Tg under an insulin promoter (insHEL-Tg). IgHEL-Tg B lymphocytes enhanced type 1 diabetes susceptibility of insHEL-Tg NOD mice. A comparison of the tolerogenic activity of IgHEL-Tg B lymphocytes with NOD and non-autoimmune-prone C57BL/6 genetic backgrounds showed that both were rendered anergic in the presence of insHEL when competing with polyclonal B lymphocytes. Nevertheless, NOD IgHEL-Tg B lymphocytes transferred into insHEL-Tg mice were more readily susceptible to rescue from anergy than their C57BL/6 counterparts, following provision of in vivo T-cell help. The different tolerogenic outcomes were an intrinsic property of B lymphocytes rather than being related to the quality of T-cell help, with the defective response being at least partially controlled by genes mapping to insulin-dependent diabetes (Idd) susceptibility loci on Chromosome 1 (Idd5) and 4 (Idd9/11).

Topics: Adoptive Transfer; Animals; Autoantigens; B-Lymphocytes; Cytokines; Diabetes Mellitus, Type 1; Genetic Predisposition to Disease; Immunoglobulins; Insulin; Insulin-Secreting Cells; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Transgenic; Muramidase; Promoter Regions, Genetic; Self Tolerance; T-Lymphocytes, Helper-Inducer; Transgenes; Transplantation Chimera

Antigen-specific immunotherapy, an approach to selectively block autoimmune diabetes, generally declines in nonobese diabetic (NOD) mice as disease progresses. To define the parameters influencing the efficacy of antigen-specific immunotherapy once diabetes is established, plasmid DNA (pDNA) vaccination was used to suppress autoimmune-mediated destruction of syngeneic islet grafts in diabetic NOD recipients. pDNAs encoding a glutamic acid decarboxylase 65 (GAD65)-Ig molecule (pGAD65), interleukin (IL)-4 (pIL4), and IL-10 (pIL10) significantly delayed the onset of recurrent diabetes compared with pGAD65+pIL10-vaccinated recipients. Despite differences in efficacy, a similar frequency of GAD65-specific CD4(+) T-cells secreting IL-4, IL-10, or interferon-gamma were detected in mice treated with pGAD65+pIL4+pIL10 and pGAD65+pIL10. However, the frequency of FoxP3-expressing CD4(+)CD25(+)CD62L(hi) T-cells was increased in the renal and pancreatic lymph nodes of diabetic recipients vaccinated with pGAD65+pIL4+pIL10. These immunoregulatory CD4(+)CD25(+) T-cells (CD4(+)CD25(+) Treg) exhibited enhanced in vivo and in vitro suppressor activity that partially was transforming growth factor-beta dependent. Furthermore, duration of islet graft protection in pGAD65+pIL4+pIL10-vaccinated diabetic recipients correlated with the persistence of CD4(+)CD25(+) Treg. These data demonstrate that the frequency and maintenance of FoxP3-expressing CD4(+)CD25(+) Treg influence antigen-induced suppression of ongoing beta-cell autoimmunity in diabetic recipients.

Topics: Animals; Antigens; CD4 Lymphocyte Count; CD4-Positive T-Lymphocytes; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glutamate Decarboxylase; Immunotherapy; Insulin-Secreting Cells; Interleukin-2 Receptor alpha Subunit; Interleukin-4; Isoenzymes; Mice; Mice, Inbred NOD; Muramidase; Polymerase Chain Reaction; Transforming Growth Factor beta

We describe a novel TCR-transgenic mouse line, TCR7, where MHC class II-restricted, CD4+ T cells are specific for the subdominant H-2b epitope (HEL74-88) of hen egg lysozyme (HEL), and displayed an increased frequency in the thymus and in peripheral lymphoid compartments over that seen in non-transgenic littermate controls. CD4+ T cells responded vigorously to HEL or HEL74-88 epitope presented on APC and could develop into Th1 or Th2 cells under appropriate conditions. Adoptive transfer of TCR7 Ly5.1 T cells into Ly5.2 rat insulin promoter (RIP)-HEL transgenic recipient hosts did not lead to expansion of these cells or result in islet infiltration, although these TCR7 cells could expand upon transfer into mice expressing high levels of HEL in the serum. Islet cell infiltration only occurred when the TCR7 cells had been polarized to either a Th1 or Th2 phenotype prior to transfer, which led to insulitis. Progression from insulitis to autoimmune diabetes only occurred in these recipients when Th1 but not Th2 TCR7 cells were transferred and CTLA-4 signaling was simultaneously blocked. These findings show that regulatory pathways such as CTLA-4 can hold in check already differentiated autoreactive effector Th1 cells, to inhibit the transition from tolerance to autoimmune diabetes.

Topics: Adoptive Transfer; Animals; Antigens, CD; Antigens, Differentiation; CD4-Positive T-Lymphocytes; Cell Differentiation; CTLA-4 Antigen; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Insulin; Islets of Langerhans; Lymphocyte Activation; Mice; Mice, Transgenic; Muramidase; Peptides; Promoter Regions, Genetic; Receptors, Antigen, T-Cell; Th1 Cells; Th2 Cells

Two novel transgenic (Tg) strains were created expressing hen egg-white lysozyme (HEL) in a pancreas-specific fashion. RmHP.111 mice had levels of HEL per cell similar to that of the established ILK-3 strain, while RmHP.117 mice had 10-fold lower levels (50,000 molecules per cell). When bred to 3A9 TCR Tg mice, negative selection occurred equally in all three double-Tg combinations, yet only ILK-3 x 3A9 and RmHP.111 x 3A9 mice became diabetic. Additionally, activated 3A9 cells readily transferred diabetes into ILK-3 or RmHP.111 mice, but only marginally into the RmHP.117 strain. In the peripancreatic lymph node, division of naive 3A9 cells was similar between RmHP.111 and RmHP.117 strains, but pancreatic APCs from RmHP.111 x 3A9 mice stimulated HEL-reactive cells to a much greater degree than those from RmHP.117 x 3A9 mice. In this model, diabetes was dependent upon both initial priming in the peripancreatic lymph node and subsequent presentation in the pancreas, with disease incidence predicted by the beta cell level of autoantigen.

Topics: Animals; Antibodies; Autoantigens; Cell Line; Clonal Deletion; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Insulin; Islets of Langerhans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Predictive Value of Tests; Promoter Regions, Genetic; Rats; Receptors, Antigen, T-Cell

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

CTLA-4 is critical to the regulation of CD4 T cell homeostasis in vivo. However, whether CTLA-4 regulates responses to both self and foreign proteins is not clear. We have directly compared the role of CTLA-4 in controlling T cell responses to the same protein presented as an endogenous tissue Ag vs a foreign immunizing Ag. We show that CTLA-4 only modestly reduces responses to Ag administered with adjuvant, but dramatically inhibits responses to the same Ag expressed transgenically as a tissue self protein. The critical consequence of CTLA-4 engagement is to inhibit T cell accumulation in the local lymph node draining the Ag-bearing tissue, and failure of this control leads to the onset of autoimmune tissue destruction. Thus, CTLA-4 may preferentially dampen pathologic immune responses to self proteins while permitting protective immunity to foreign agents.

Topics: Abatacept; Adoptive Transfer; Animals; Antigen Presentation; Antigens; Antigens, CD; Antigens, Differentiation; Autoantigens; Autoimmunity; Chickens; CTLA-4 Antigen; Diabetes Mellitus, Type 1; Immunization; Immunoconjugates; Kinetics; Lymphocyte Activation; Mice; Mice, Knockout; Mice, Transgenic; Muramidase; Proteins; Rats; Recombinant Proteins; T-Lymphocytes

B lymphocytes partially contribute to autoimmune type 1 diabetes (T1D) as a subset of APC with a preferential ability to trigger pathogenic CD4 T cells. We hypothesized that this resulted from the unique ability of B lymphocytes to take up pancreatic beta cell proteins through Ig mediated capture. T1D was significantly delayed, but not prevented, in a NOD stock in which the B lymphocyte Ig repertoire was strongly restricted because of the allelic exclusion induced by transgenic Ig molecules specific for the disease irrelevant hen egg lysozyme (HEL) protein (NOD.IgHEL mice). However, introducing the Ig(mu)null mutation to eliminate the small residual numbers of non-transgenic B lymphocytes in the NOD.IgHEL stock strongly suppressed T1D to the same low levels that characterize B lymphocyte deficient NOD.Ig(mu)null mice. In contrast to standard NOD mice, both the NOD.IgHEL.Ig(mu)null and NOD.Ig(mu)null stocks were unable to generate T cell responses against the candidate diabetes autoantigen, glutamic acid decarboxylase. These results indicate that Ig-mediated capture of beta cell autoantigens accounts for why B lymphocytes have a greater capacity than other APC subtypes to trigger diabetogenic T cells. Hence, defects in B lymphocyte, as well as T lymphocyte, tolerance induction mechanisms may contribute to T1D in NOD mice.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; Autoantigens; B-Lymphocytes; Chickens; Diabetes Mellitus, Type 1; Female; Glutamate Decarboxylase; Immune Tolerance; Lymph Nodes; Male; Mice; Mice, Inbred NOD; Mice, Transgenic; Muramidase; Receptors, Immunologic; Spleen

We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus.

Topics: Adenovirus E2 Proteins; Amino Acid Sequence; Amino Acid Substitution; Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens, CD; Autoantigens; Diabetes Mellitus, Type 1; Histocompatibility Antigens Class II; Lysosomal Membrane Proteins; Lysosomes; Membrane Glycoproteins; Mice; Molecular Sequence Data; Muramidase; Peptide Fragments; Protein Binding; Tacrolimus Binding Proteins; Tumor Cells, Cultured

I-A(g7) is a unique class II MHC molecule that is clearly associated with autoimmune diabetes in non-obese diabetic (NOD) mice. To determine if I-A(g7) is defective in its ability to deliver tolerogenic signals in vivo, H-2(g7) mice were nasally pretreated with antigen, prior to immunization, to induce antigen-specific regulation. Nasally pretreated NOR (H-2(g7)) and (NON).NOD (H-2(g7)) congenic mice showed responses similar to those of NON (H-2(nb1)), BALB/c (H-2(d)) and B10.PL (H-2(u)) mice-a reduced recall response and a deviated T(h) cytokine profile. However, we found that NOD (H-2(g7)) mice are comparatively resistant to immunological tolerance induced by nasal pretreatment, such that at the usually effective dose no significant reduction was seen in the proliferative recall responses to nominal antigen after immunization. (NOD x BALB/c)F(1) (H-2(g7/d)) and (NOD x NOR)F(1) (H-2(g7)) mice were similarly resistant to nasal-induced tolerance, although significantly higher nasal doses of antigen were able to overcome the resistance in NOD and F(1) mice. Interestingly, activated NOD T cells were resistant to cell death induced by re-stimulation with plate-bound anti-CD3. These results demonstrate that activated T cells in NOD mice are defective in their ability to respond to regulatory signals delivered in vivo or in vitro. Furthermore, NOD T cells have an increased resistance to tolerance induced by I-A(g7)-dependent (antigen) or I-A(g7)-independent (anti-CD3) mechanisms. Thus, while I-A(g7) may contribute to insulin-dependent diabetes mellitus by selecting a particular repertoire of self-reactive T cell clones, additional defects in the peripheral T cells themselves are required to allow the expansion of diabetogenic clones and the development of autoimmune disease.

Topics: Administration, Intranasal; Animals; Clone Cells; Crosses, Genetic; Diabetes Mellitus, Type 1; Female; H-2 Antigens; Hematopoietic Stem Cells; Histocompatibility Antigens Class II; Immune Tolerance; Mice; Mice, Inbred NOD; Muramidase; Receptors, Antigen, T-Cell; Species Specificity; T-Lymphocytes

We report a mouse model for the spontaneous development of autoimmune diabetes: the 3A9 T cell receptor (TCR) transgenic mouse, which contains T cells that recognize the 52 - 61 family of hen egg-white lysozyme (HEL) peptides in the context of MHC class II I-A(k) molecules, was bred to the ILK3 mouse, that expresses HEL protein via the rat insulin promoter (RIP). Despite partial tolerance of 3A9 T cells in ILK3 mice, spontaneous diabetes developed in 64 % of 3A9xILK3 mice by 20 weeks of age. We provide evidence that APC from peri-pancreatic nodes have a large content of peptide-MHC complex and stimulate 3A9 T cells. We also report that cross presentation of HEL from beta cells to APC is 26-fold more efficient than presentation of soluble HEL. We previously reported on a biochemical margin of safety, based on the observation that activation of naive 3A9 T cells required 100-fold more peptide-MHC complexes than required for deletion of 3A9 thymocytes. We speculate that the high local density of autologous peptide-MHC complexes can be a determining factor that leads to the activation of autoreactive CD4 T cells and, consequently, to the development of autoimmunity.

Topics: Animals; Antigen Presentation; Diabetes Mellitus, Type 1; Disease Susceptibility; Histocompatibility Antigens; Islets of Langerhans; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Transgenic; Muramidase; T-Lymphocytes

Lysozyme (LZ), a host-defense protein, contains an 18 amino-acid domain with high affinity binding for sugar-derived proteins or lipids, called advanced glycation endproducts (AGE), that are implicated in diabetes- and age-dependent complications (DC).. A) The effects of LZ on AGE- removal were tested in vivo. LZ was injected (200 ug/day, i.p., X2 weeks) in non-obese diabetic (NOD), db/db (+/+) mice, and non-diabetic, AGE-infused Sprague-Dawley rats. B) LZ: AGE interactions with macrophage-like T1B-183 cells (Mf) and mesangial cells (MC) were tested in vitro.. A) In NOD mice, LZ reduced the elevated basal serum AGE (sAGE) (p < 0.05), enhanced urinary AGE (uAGE) excretion by approximately 2-fold (p < 0.01), while it reduced albuminuria (UA), p < 0.005. In db/db mice, LZ infusion also reduced the elevated sAGE (p < 0.05), doubled uAGE excretion (p < 0.05), and decreased UA (p < 0.01). In addition, LZ maintained normal sAGE in normal rats infused with AGE-BSA, as it doubled the urinary AGE (uAGE) clearance (p < 0.01). B) LZ stimulated the uptake and degradation of (125) I-labeled AGE-BSA and (25) I-human serum AGE by Mf, while suppressing AGE-induced TNFalpha and IGF-I production. In MC, LZ suppressed the AGE-promoted PDGF-B, alpha1 type IV collagen, and tenascin mRNA levels, and restored the AGE-suppressed expression and activity of MMP-9, but not MMP-2.. LZ may act to: a) accelerate renal in-vivo AGE clearance, b) suppress macrophage and mesangial cell- specific gene activation in vitro, and c) improve albuminuria due to diabetes. These data suggest that LZ by sequestering AGEs may protect against diabetic renal damage.

Topics: Animals; Anti-Infective Agents; Chickens; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Glomerular Mesangium; Glycation End Products, Advanced; Humans; In Vitro Techniques; Kidney; Macrophages; Male; Mice; Mice, Inbred NOD; Middle Aged; Muramidase; Rats

We have determined the crystal structure of I-Ag7, an integral component in murine type I diabetes development. Several features distinguish I-Ag7 from other non-autoimmune-associated MHC class II molecules, including novel peptide and heterodimer pairing interactions. The binding groove of I-Ag7 is unusual at both terminal ends, with a potentially solvent-exposed channel at the base of the P1 pocket and a widened entrance to the P9 pocket. Peptide binding studies with variants of the hen egg lysozyme I-Ag7 epitope HEL(11-25) support a comprehensive structure-based I-Ag7 binding motif. Residues critical for T cell recognition were investigated with a panel of HEL(11-25)-restricted clones, which uncovered P1 anchor-dependent structural variations. These results establish a framework for future experiments directed at understanding the role of I-Ag7 in autoimmunity.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Antigen Presentation; Chickens; Crystallography, X-Ray; Diabetes Mellitus, Type 1; Dimerization; Epitope Mapping; Epitopes, T-Lymphocyte; Histocompatibility Antigens Class II; Mice; Mice, Inbred NOD; Models, Molecular; Molecular Sequence Data; Muramidase; Peptide Fragments; Peptides; Protein Binding; T-Lymphocytes

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

Autoantigen-based immunotherapeutics have been shown to activate regulatory responses capable of inhibiting T cell-mediated autoimmune disease in animal models. However, their efficacy generally declines, as treatment occurs later in the disease process, and their mechanism of action is a matter of intense debate. Here, we report that the early administration of beta cell autoantigens (betaCAAs) to nonobese diabetic (NOD) mice broadly diverts the natural development of potentially pathogenic Thl-biased autoimmune responses toward the Th2 phenotype through Th2 spreading. With disease progression, there was a steady decline in the ability of betaCAA treatment to promote Th2-type cellular and humoral autoimmunity. Late in the disease process, some betaCAAs were still able to induce Th2 responses and Th2 spreading (although to a much lesser extent), while other autoantigens were not. This attenuation of inducible Th2 immunity with disease progression is likely to reflect a reduction in the availability of uncommitted autoantigen-reactive precursor T cells. These findings suggest that there are inherent differences in the frequency of betaCAA-reactive T cells and that, in advanced stages of autoimmune disease, regulatory responses may be best elicited with target tissue Ags against which large uncommitted T cell pools are still available. Since individuals presenting the first signs of autoimmune disease are likely to already have an advanced disease process, these findings may be useful for the rational design of Ag-based immunotherapeutics.

Topics: Animals; Autoantibodies; Autoantigens; beta-Galactosidase; Cell Movement; Chaperonin 60; Diabetes Mellitus, Type 1; Disease Progression; Glutamate Decarboxylase; Heat-Shock Proteins; Immunity, Cellular; Injections, Intraperitoneal; Islets of Langerhans; Mice; Mice, Inbred NOD; Muramidase; Peptide Fragments; Th2 Cells

One hundred and thirty-one patients with diabetes mellitus type 1 (IDDM) and 20 healthy controls were checked for the presence of periodontal diseases and for some oral microbiological parameters. Results demonstrated that IDDM patients, who were well compensated from both the metabolic and clinical point of view, showed a prevalence for periodontopathies, which only differed slightly from controls. Only the presence of gingivitis was significantly higher in IDDM patients than in healthy subjects. Both anaerobic and aerobic microbial flora did not show substantial differences for either group. Among the salivary antibacterial factors studied, lysozyme was significantly decreased in diabetic patients compared to controls. It is concluded that IDDM patients undergo periodontal complications with a frequency quite close to that of non-diabetic healthy subjects, when the disease is under strict metabolic and clinical control.

Topics: Acetylglucosaminidase; Adolescent; Adult; Aged; Anti-Bacterial Agents; Bacteria; Child; Child, Preschool; Colony Count, Microbial; Diabetes Mellitus, Type 1; Female; Humans; Immunoglobulin A, Secretory; Italy; Male; Middle Aged; Mouth Mucosa; Muramidase; Oral Hygiene; Periodontal Diseases; Prevalence; Saliva; Severity of Illness Index

Soluble bovine or ovine insulin given intravenously to female NOD mice shortly after weaning had a downregulating effect on several autoimmune parameters associated with insulin-dependent diabetes. The titer of spontaneous anti-insulin antibodies was reduced, insulitis was delayed and less severe, and only 25% of treated mice were diabetic at 30 weeks compared with 70% of untreated mice. An interesting paradox occurred in that bovine insulin, although poorly immunogenic in NOD mice and ineffective as a tolerogen for complete Freund's adjuvant-induced cellular and humoral responses to ovine insulin, was nearly as effective as immunogenic ovine insulin in protecting against diabetes and better than ovine insulin at downregulating spontaneous autoantibodies to insulin. Bovine and ovine insulins differ by only one amino acid on the A-chain loop, but whereas modulation of the induced response to ovine insulin appeared to be sheep-specific, modulation of the induced and spontaneous autoimmunity was achieved almost equally well by bovine or ovine insulin. We suggest therefore that modulation of the induced and spontaneous responses are dependent on different T-cell epitopes and that modulation of spontaneous autoimmunity appears to be governed by an epitope common to both insulins.

Topics: Amino Acid Sequence; Animals; Cattle; Diabetes Mellitus, Type 1; Female; Freund's Adjuvant; Injections, Intravenous; Insulin; Insulin Antibodies; Islets of Langerhans; Mice; Mice, Inbred NOD; Molecular Sequence Data; Muramidase; Sheep; Species Specificity; Swine; T-Lymphocytes

Malignant externa otitis is a potentially fatal disease in diabetic and other immunocompromised patients. Cerumen contains defense properties that protect the patient against infection. We tested the hypothesis that patients with diabetes mellitus have abnormalities in their cerumen that affect the environment of their external auditory canals and may predispose them to malignant externa otitis.

Topics: Aged; Cerumen; Diabetes Mellitus, Type 1; Fatty Acids; Fatty Acids, Unsaturated; Female; Humans; Hydrogen-Ion Concentration; Male; Middle Aged; Muramidase; Otitis Externa; Sex Characteristics

Serum lysozyme activity and nitroblue tetrazolium (NBT) reduction by blood phagocytes were studied in 24 dogs with controlled insulin-dependent diabetes mellitus and in 16 healthy controls aged from 6 to 12 years. In dogs with diabetes mellitus serum lysozyme activity was significantly (p less than 0.001) lowered. The NBT reducing value of circulating phagocytes in diabetic dogs was increased (p less than 0.05), whereas these cells increase in NBT reduction following stimulation (stimulation index) was decreased (p less than 0.05). It is suggested that in diabetic dogs changes in activity of circulating and tissue phagocytes may occur.

Topics: Animals; Diabetes Mellitus, Type 1; Dog Diseases; Dogs; Muramidase; Nitroblue Tetrazolium; Oxidation-Reduction; Phagocytosis

Renal tubular function was investigated in 98 non-insulin-dependent and 18 insulin-dependent diabetics under conditions of standard glycemic control. Mean urinary excretion of lysozyme, beta 2-microglobulin and N-acetyl-beta-D-glucosaminidase (NAG) in both Albustix-negative and positive patients were significantly elevated above the control range. The increased excretion of lysozyme, beta 2-microglobulin and NAG was found in 21, 55 and 62% of the normoalbuminuric patients, and in 40, 57 and 74% of the microalbuminuric patients, respectively. Besides the parameters cited above, urinary acid-soluble glycoprotein (ASP) was measured to assess its potential as an indicator of early renal dysfunction. Mean urinary ASP excretion was also elevated in both Albustix-negative and positive patients. The albumin/ASP ratio increased as nephropathy advanced. Such a mode of excretion was similar to those of low-molecular-weight proteins (lysozyme and beta 2-microglobulin). The results of multiple regression analysis showed that serum creatinine most highly correlated with the excretion of the urinary proteins except for NAG.

Topics: Acetylglucosaminidase; Adult; Albuminuria; beta 2-Microglobulin; Biomarkers; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Humans; Kidney Tubules; Male; Middle Aged; Muramidase; Proteinuria; Regression Analysis

We measured the excretion rates of six urinary enzymes that either originate from the proximal renal tubule, like alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), and N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), or that are typical low-molecular-mass proteins, like lysozyme (EC 3.2.1.17) and pancreatic ribonuclease (EC 3.1.27.5). These rates were compared with those of total protein and albumin in urine of 36 insulin-dependent diabetic men and 30 healthy men. Seventeen of the diabetics had "clinical proteinuria," defined as excretion of more than 7.5 g of protein per mole of urinary creatinine (group B). Group A comprised the 19 diabetics without proteinuria. Except for gamma-glutamyltransferase, the excretions of enzymes and proteins were significantly higher in diabetics than in controls and were greater in group B than in group A. N-Acetyl-beta-D-glucosaminidase was the analyte most often increased in group A (89%), followed by albumin and alkaline phosphatase (each 32%). All patients in group B showed increased excretion of N-acetyl-beta-D-glucosaminidase. We conclude from the comparative data that this enzyme may be useful as an early predictor of diabetic nephropathy.

Topics: Acetylglucosaminidase; Adult; Albuminuria; Alkaline Phosphatase; Aminopeptidases; CD13 Antigens; Diabetes Mellitus, Type 1; Diabetic Nephropathies; gamma-Glutamyltransferase; Humans; Male; Muramidase; Proteinuria; Ribonuclease, Pancreatic

Topics: Adult; Aged; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Enzyme Activation; Humans; Middle Aged; Muramidase; Urinary Tract Infections

Polymorphonuclear neutrophils' chemotaxis, surface charge, superoxide anions generation, NBT (nitro blue tetrazolium) reduction and intracellular lysozyme, and beta-glucuronidase content were estimated in patients with type I diabetes mellitus in a similar state of metabolic control. The chemotaxis of diabetic cells toward bacterial chemotactic factors was similar to controls, whereas migration toward complement-derived chemoattractants was significantly reduced. Polymorphonuclear neutrophils isolated from diabetic patients, when unstimulated, produced significantly greater amounts of superoxide anions and reduced NBT more efficiently. They also revealed reduced surface charge and lower intracellular content of lysozyme, whereas beta-glucuronidase content was similar to controls. The results obtained seem to indicate that neutrophils in patients with insulin-dependent diabetes manifest signs of being in the activated state. The possible mechanisms of such stimulation are discussed.

Topics: Adolescent; Adult; Chemotaxis, Leukocyte; Diabetes Mellitus, Type 1; Electrochemistry; Female; Glucuronidase; Humans; Male; Middle Aged; Muramidase; Neutrophils; Nitroblue Tetrazolium; Oxidation-Reduction; Superoxides; Surface Properties

Seeking to study whether measurement of lysozyme (EC 3.2.1.17) in urine by a reliable radioimmunoassay can provide a suitable index of renal tubular function and how lysozymuria develops in temporal relation to proteinuria in diabetic nephropathy, we have compared the urinary excretion of lysozyme and beta 2-microglobulin with the 15-min excretion rate of phenolsulfonphthalein in 39 patients with Type 2 (non-insulin-dependent) diabetes and investigated the temporal relation between the onset of lysozymuria and proteinuria in 15 patients with Type 1 (insulin-dependent) diabetes. The concentrations of lysozyme and beta 2-microglobulin in urine increased in proportion to the decrease in the rate of excretion of phenolsulfonphthalein in these patients. The coefficient of correlation between lysozyme concentration and the 15-min excretion rate of phenolsulfonphthalein (r = -0.70) was higher than that between beta 2-microglobulin concentration and the 15-min excretion rate of phenolsulfonphthalein (r = -0.46). Abnormally high lysozymuria, suggesting the existence of tubular dysfunction, was demonstrated in six of the patients with Type 1 diabetes who showed no proteinuria or only a slight increase in urinary protein excretion. Lysozymuria may thus be added to a list of the indicators for diabetic nephropathy.

Topics: Adolescent; Adult; Aged; beta 2-Microglobulin; Child; Child, Preschool; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Humans; Kidney Tubules; Middle Aged; Muramidase; Phenolsulfonphthalein; Proteinuria

The activity of non-specific protection factors (complement titer, lysozyme level, completed phagocytosis index and serum immunoglobulin (IG) concentration) was studied in 100 children, aged 3 to 14 years: 70 with diabetes mellitus and 30 normal subjects. The results obtained indicate a decreased reactivity of the child organism in diabetes mellitus, the reduction degree being directly proportional to the main process severity and decompensation extent. Serum IG level in children, suffering from diabetes mellitus, is disturbed, i.e. high IgA concentration is often seen in the presence of normal IgM level and normal or decreased IgG content. Disimmunoglobulinemia is intensified within decompensation phase, depending on the pathological process duration and severity. The reduced IgG level in patients with severe diabetes apart from lower indices of non-specific immunity, stipulates pred disposition of children with diabetes mellitus to the secondary infection development. The indices studied may be used not only for characterizing the body immunobiological reactivity and protective mechanisms, but also for determining the disease severity and form.

Topics: Adolescent; Child; Child, Preschool; Complement System Proteins; Diabetes Mellitus, Type 1; Dysgammaglobulinemia; Female; Humans; Hypergammaglobulinemia; IgG Deficiency; Immunoglobulin A; Male; Muramidase; Phagocytosis