ovalbumin and Diabetes-Mellitus--Type-1

Mucosal immune response to an antigen leads to a state of attenuated systemic response to the same antigen, known as mucosal or oral tolerance. Thus, autoantigen administration via mucosal routes could be useful in the prevention of autoimmune diseases. Although in various models of autoimmune disease it is often effective, in some cases it is ineffective and in other cases even harmful. In these cases it is likely that, concomitantly with tolerance, a productive immune response is induced that exacerbates autoimmunity. Recent evidence suggests that induction of cytotoxic T lymphocytes capable of destroying pancreatic beta cells may be an unavoidable consequence of mucosal administration of pancreatic beta-cell associated autoantigen. To improve the safety and efficacy of mucosal tolerance induction in the prevention of type 1 diabetes, further means to control the induction of cytotoxic T lymphocytes and other potentially tissue-destructive immune effectors may be required.

Topics: Administration, Intranasal; Administration, Oral; Animals; Autoantigens; Autoimmune Diseases; Desensitization, Immunologic; Diabetes Mellitus, Type 1; Digestive System; Humans; Immune Tolerance; Immunization; Islets of Langerhans; Lymphoid Tissue; Mice; Mice, Inbred NOD; Mice, Transgenic; Mucous Membrane; Ovalbumin; Rats; Safety; T-Lymphocytes, Cytotoxic

The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice but diminished in monoclonal models specific to artificial or neoantigens. Rationally designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels and reduced T cell infiltration and proinflammatory cytokine expression in newly diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.

Topics: Alleles; Amino Acid Sequence; Animals; Autoantigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Crosses, Genetic; Cytokines; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Gene Deletion; Germ Cells; Humans; Inflammation Mediators; Lymph Nodes; Lymphocyte Activation; Male; Mice, Inbred C57BL; Mice, Inbred NOD; Ovalbumin; Pancreas; Peptides; Receptors, Antigen, T-Cell; Spleen; T-Lymphocytes; Trans-Activators; Transcription, Genetic

Adoptive cellular therapy (ACT) using T-cell receptor (TCR)-engineered lymphocytes holds promise for eradication of disseminated tumors but also an inherent risk of pathologic autoimmunity if targeted antigens or antigenic mimics are expressed by normal tissues. We evaluated whether modulating TCR affinity could allow CD8

Topics: Allergens; Animals; Antigens, Neoplasm; Autoantigens; CD8-Positive T-Lymphocytes; Cell Line; Diabetes Mellitus, Type 1; Female; Immunotherapy, Adoptive; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Ovarian Neoplasms; Receptors, Antigen, T-Cell

The cause of most hypertensive disease is unclear, but inflammation appears critical in disease progression. However, how elevated blood pressure initiates inflammation is unknown, as are the effects of high blood pressure on innate and adaptive immune responses. We now report that hypertensive mice have increased T cell responses to antigenic challenge and develop more severe T cell-mediated immunopathology. A root cause for this is hypertension-induced erythrocyte adenosine 5'-triphosphate (ATP) release, leading to an increase in plasma ATP levels, which begins soon after the onset of hypertension and stimulates P2X7 receptors on antigen-presenting cells (APCs), increasing APC expression of CD86. Hydrolyzing ATP or blocking the P2X7 receptor eliminated hypertension-induced T cell hyperactivation. In addition, pharmacologic or genetic blockade of P2X7 receptor activity suppressed the progression of hypertension. Consistent with the results in mice, we also found that untreated human hypertensive patients have significantly elevated plasma ATP levels compared with treated hypertensive patients or normotensive controls. Thus, a hypertension-induced increase in extracellular ATP triggers augmented APC and T cell function and contributes to the immune-mediated pathologic changes associated with hypertensive disease.

Topics: Adenosine Triphosphate; Adult; Aged; Animals; Antigens; B7-2 Antigen; Dendritic Cells; Diabetes Mellitus, Type 1; Female; Hepatitis; Humans; Hypertension; Male; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Ovalbumin; Receptors, Purinergic P2X7; T-Lymphocytes

D-mannose, a C-2 epimer of glucose, exists naturally in many plants and fruits, and is found in human blood at concentrations less than one-fiftieth of that of glucose. However, although the roles of glucose in T cell metabolism, diabetes and obesity are well characterized, the function of D-mannose in T cell immune responses remains unknown. Here we show that supraphysiological levels of D-mannose safely achievable by drinking-water supplementation suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation, and increased the proportion of Foxp3

Topics: Adoptive Transfer; Animals; Colitis; Colon; Diabetes Mellitus, Type 1; Dietary Supplements; Disease Models, Animal; Fatty Acids; Flow Cytometry; Forkhead Transcription Factors; Humans; In Vitro Techniques; Inflammation; Integrins; Lipid Metabolism; Lung; Lung Diseases; Mannose; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Ovalbumin; Oxidation-Reduction; Pancreas; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Respiratory Hypersensitivity; Reverse Transcriptase Polymerase Chain Reaction; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

Proinsulin is the first autoantigen in type 1 diabetes (T1D). We reasoned that coupling hematopoietic stem cells (HSCs) transplantation with ex vivo transduction of syngeneic HSCs with lentiviral vectors to express proinsulin II could prevent T1D in nonobese diabetic (NOD) mice.. Hematopoietic stem cells were isolated from 6- to 8-week-old NOD female mice and transduced in vitro with lentiviral vectors encoding proinsulin II. Preconditioned 3- to 4-week-old female NOD mice were transplanted with transduced or nontransduced HSCs and compared with age-matched unmanipulated control. The insulitis, T1D development, and immune reconstitution were assessed.. The mean (SD) insulitis score was significantly reduced (1.156 [0.575] vs 2.156 [0.892] or 3.043 [0.728], P = 0.009 or <0.001), and diabetes was nearly completely prevented (1/13 vs 5/12 or 4/9, P = 0.031 or 0.013) in recipients of transduced HSCs expressing proinsulin II as compared with recipients of nontransduced HSCs or unmanipulated control. Sialitis, reconstitution of peripheral blood leukocytes, and in vitro recall responses to ovalbumin were not different between 3 groups of mice.. Syngeneic transplantation of HSCs transduced ex vivo with lentiviral vectors to encode proinsulin II is a novel strategy to prevent T1D.

Topics: Animals; Cytokines; Diabetes Mellitus, Type 1; Enzyme-Linked Immunospot Assay; Female; HEK293 Cells; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Immune Tolerance; Mice, Inbred NOD; Ovalbumin; Pancreatitis; Proinsulin; Sialadenitis; T-Lymphocytes; Transfection

T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity.

Topics: Animals; Autoantigens; Autoimmunity; Binding, Competitive; Diabetes Mellitus, Type 1; Flow Cytometry; Insulin-Secreting Cells; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Protein Binding; Receptors, Antigen, T-Cell; Self Tolerance; T-Lymphocytes

During pregnancy there is an exchange of cells between the fetus and the mother including T lymphocytes that can persist after delivery. Previous studies have described an increased numbers of maternal cells in children with juvenile diabetes as compared to their unaffected siblings. Our objective was to assess the possibility for these chimeric T cells to trigger an anti-beta cell response.. We mated OT2 transgenic female mice having T cells specifically targeting ovalbumin to RIP-OVA males expressing ovalbumin in pancreatic β cells. This allowed us to examine RIP-OVA progeny from OT2 mothers to assess the consequences of maternal T cells acquired during gestation or lactation. We quantitatively analyzed the pancreas of RIP-OVA mice from OT2 mothers for islet infiltration and compared them to RIP-OVA mice not exposed to OT2 mothers or to wild-type mice from OT2 mothers.. RIP-OVA mice from OT2 mothers had significantly more peri-insulitis (p=0.0083) compared to wild-type littermates. Similarly RIP-OVA mice from OT2 mothers had more peri-insulitis as compared to RIP-OVA mice from RIP-OVA mothers (p=0.0073). Presence and specific anti-ovalbumin activity of maternal OT2 cells in the offsprings' peripheral lymphoid tissues was found in a separate group of mice. In animals presenting islet inflammation, CD3+ infiltrating cells in the pancreas were however derived from the offspring and not from OT2 mothers. In accordance, OT2 and RIP-OVA double transgenic mice with high levels of auto-reactive T cells had more peri-insulitis and sometimes intense insulitis when they were from OT2 mothers as compared to RIP-OVA mothers (p=0.046).. In highly specific fetal/maternal combinations, maternal T cells with activity against the offspring pancreatic beta cells, presumably chimeric in fetal organs, initiate islet inflammation and may therefore predispose to auto-immune diabetes.

Topics: Animals; Chimera; Diabetes Mellitus, Type 1; Female; Fetus; Insulin-Secreting Cells; Male; Maternal-Fetal Exchange; Mice; Ovalbumin; Pregnancy; T-Lymphocytes

Both CD4(+) Th17-cells and CD8(+) cytotoxic T lymphocytes (CTLs) are involved in type 1 diabetes and experimental autoimmune encephalomyelitis (EAE). However, their relationship in pathogenesis of these autoimmune diseases is still elusive. We generated ovalbumin (OVA)- or myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells expressing RORγt and IL-17 by in vitro co-culturing OVA-pulsed and MOG(35-55) peptide-pulsed dendritic cells (DC(OVA) and DC(MOG)) with CD4(+) T cells derived from transgenic OTII and MOG-T cell receptor mice, respectively. We found that these Th17 cells when transferred into C57BL/6 mice stimulated OVA- and MOG-specific CTL responses, respectively. To assess the above question, we adoptively transferred OVA-specific Th17 cells into transgenic rat insulin promoter (RIP)-mOVA mice or RIP-mOVA mice treated with anti-CD8 antibody to deplete Th17-stimulated CD8(+) T cells. We demonstrated that OVA-specific Th17-stimulated CTLs, but not Th17 cells themselves, induced diabetes in RIP-mOVA. We also transferred MOG-specific Th17 cells into C57BL/6 mice and H-2K(b-/-) mice lacking of the ability to generate Th17-stimulated CTLs. We further found that MOG-specific Th17 cells, but not Th17-activated CTLs induced EAE in C57BL/6 mice. Taken together, our data indicate a distinct role of Th17 cells and Th17-stimulated CTLs in the pathogenesis of TID and EAE, which may have great impact on the overall understanding of Th17 cells in the pathogenesis of autoimmune diseases.

Topics: Adoptive Transfer; Animals; CD4 Antigens; CD8 Antigens; Cells, Cultured; Dendritic Cells; Diabetes Mellitus, Type 1; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Interleukin-17; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin Proteins; Myelin-Oligodendrocyte Glycoprotein; Ovalbumin; Peptide Fragments; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes, Cytotoxic; Th17 Cells

The gels of Aloe species contain immunomodulatory components such as aloctin A and acemannan. Most studies on these gels were performed in in vitro cell culture systems. Although several studies examined their immunomodulatory activity in vivo, the route of administration was intraperitoneal or intramuscular. Here, we evaluated the in vivo immunomodulatory activity of processed Aloe vera gel (PAG) in mice. Oral administration of PAG significantly reduced the growth of C. albicans in the spleen and kidney following intravenous injection of C. albicans in normal mice. PAG administration also reduced the growth of C. albicans in streptozotocin-induced diabetic mice. PAG administration did not increase ovalbumin (OVA)-specific cytotoxic T lymphocyte (CTL) generation in normal mice, but did increase it in high-fat-diet induced diabetic mice. These findings provide the first clear evidence for the immunomodulatory activity of orally administered Aloe vera gel.

Topics: Administration, Oral; Aloe; Animals; Candida albicans; Candidiasis; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Gels; Immunologic Factors; Kidney; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Ovalbumin; Plant Preparations; Spleen; T-Lymphocytes, Cytotoxic; Time Factors

In APCs, the protein tyrosine kinase Syk is required for signaling of several immunoreceptors, including the BCR and FcR. We show that conditional ablation of the syk gene in dendritic cells (DCs) abrogates FcgammaR-mediated cross priming of diabetogenic T cells in RIP-mOVA mice, a situation phenocopied in wild-type RIP-mOVA mice treated with the selective Syk inhibitor R788. In addition to blocking FcgammaR-mediated events, R788 also blocked BCR-mediated Ag presentation, thus broadly interrupting the humoral contributions to T cell-driven autoimmunity. Indeed, oral administration of R788 significantly delayed spontaneous diabetes onset in NOD mice and successfully delayed progression of early-established diabetes even when treatment was initiated after the development of glucose intolerance. At the DC level, R788 treatment was associated with reduced insulin-specific CD8 priming and decreased DC numbers. At the B cell level, R788 reduced total B cell numbers and total Ig concentrations. Interestingly, R788 increased the number of IL-10-producing B cells, thus inducing a tolerogenic B cell population with immunomodulatory activity. Taken together, we show by genetic and pharmacologic approaches that Syk in APCs is an attractive target in T cell-mediated autoimmune diseases such as type 1 diabetes.

Topics: Aminopyridines; Animals; Cross-Priming; Dendritic Cells; Diabetes Mellitus, Type 1; Female; Gene Targeting; Immunoglobulin G; Immunoglobulin M; Insulin; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Mice, Transgenic; Morpholines; Ovalbumin; Oxazines; Protein-Tyrosine Kinases; Pyridines; Pyrimidines; Receptors, Antigen, B-Cell; Receptors, IgG; Syk Kinase

To investigate a new clinically relevant immunoregulatory strategy based on treatment with murine Thymoglobulin mATG Genzyme and CTLA4-Ig in NOD mice to prevent allo- and autoimmune activation using a stringent model of islet transplantation and diabetes reversal.. Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig.. BALB/c islets transplanted into hyperglycemic NOD mice under prolonged mATG+CTLA4-Ig treatment showed a pronounced delay in allograft rejection compared with untreated mice (mean survival time: 54 vs. 8 days, P < 0.0001). Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment. The striking effect on autoimmunity was confirmed by 100% diabetes reversal in newly hyperglycemic NOD mice and 100% indefinite survival of syngeneic islet transplantation (NOD.SCID into NOD mice).. The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.

Topics: Abatacept; Animals; Antibodies, Monoclonal; Antilymphocyte Serum; Autoimmunity; Cell Division; Cytokines; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Graft Rejection; Humans; Immunoconjugates; Immunosuppressive Agents; Islets of Langerhans Transplantation; Mice; Mice, Inbred NOD; Ovalbumin; Skin Transplantation; Transplantation, Homologous

Type 1 diabetes (T1D) results from the immune-mediated destruction of the insulin-producing β-islet cells in the pancreas. The genetic and environmental mechanisms promoting the development of this disease remain poorly understood. We have explored the cellular requirements for T1D development in DO11.10xRIPmOVA (DORmO) mice, which carry a TCR transgene specific for an MHC class II-restricted epitope from OVA and express membrane-bound OVA in the pancreas under the control of the rat insulin promoter. We found that DORmO.RAG2(-/-) mice do not develop insulitis and are completely protected from diabetes, demonstrating that endogenous lymphocyte receptor rearrangement is required for disease development. Diabetes in DORmO mice is preceded by the development of OVA-specific autoantibodies and is delayed in B cell-deficient DORmO.JhD(-/-) mice, demonstrating that B cells contribute to disease progression. In addition, transfer of CD8(+) T cells from diabetic animals into DORmO.RAG2(-/-) mice promoted insulitis by OVA-specific CD4(+) T cells. Finally, although diabetes develops in DORmO mice in the presence of a significant population of Foxp3(+) OVA-specific regulatory T cells, boosting regulatory T cell numbers by injecting IL-2 immune complexes dampens autoantibody production and prevents development of insulitis and overt diabetes. These results help define the events leading to diabetes in DORmO mice and provide new insights into the cellular interactions required for disease development in an Ag-specific model of T1D.

Topics: Adoptive Transfer; Animals; B-Lymphocytes; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Separation; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fluorescent Antibody Technique; Histocompatibility Antigens Class II; Immunohistochemistry; Insulin; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Transgenic; Ovalbumin; Promoter Regions, Genetic; Rats; T-Lymphocyte Subsets

The definitions of tolerogenic vs immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allogeneic Ags. However, we have previously reported that mature DC (mDC) prevented the onset of autoimmune diabetes, whereas immature DC (iDC) were therapeutically ineffective. In this study, islet-specific CD4(+) T cells from BDC2.5 TCR-transgenic mice were stimulated in the absence of exogenous cytokine with iDC or mDC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both iDC and mDC presenting low peptide doses induced weak TCR signaling via the Akt/mammalian target of rapamycin (mTOR) pathway, resulting in significant expansion of Foxp3(+) Treg. Furthermore, unpulsed mDC, but not iDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3(neg) Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-transgenic T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with Ag dose and inversely with Treg expansion. Studies with T cells or DC from IL-6(-/-) mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low Ag doses. These studies indicate that the strength of Akt/mTOR signaling, a critical T cell-intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production.

Topics: Animals; Antibodies; Antigens; Carrier Proteins; CD28 Antigens; CD3 Complex; CD4-Positive T-Lymphocytes; Coculture Techniques; Cytokines; Dendritic Cells; Diabetes Mellitus, Type 1; Dose-Response Relationship, Immunologic; Forkhead Transcription Factors; Interleukin-6; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Oncogene Protein v-akt; Ovalbumin; Peptides; Phosphotransferases (Alcohol Group Acceptor); Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases

The activity of regulatory T cells (Treg) is widely accepted to play a central role in preventing pathogenic immune responses against self-Ags. However, it is not clear why such regulation breaks down during the onset of autoimmunity. We have studied self-Ag-specific Treg during the induction of spontaneous diabetes. Our data reveal a shift in the balance between regulatory and pathogenic islet-reactive T cells in the pancreas-draining lymph nodes during disease onset. Treg function was not compromised during disease initiation, but instead conventional T cells showed reduced susceptibility to Treg-mediated suppression. Release from Treg suppression was associated with elevated levels of IL-21 in vivo, and provision of this cytokine abrogated Treg suppression in vitro and in vivo. These data suggest that immunological protection of a peripheral tissue by Treg can be subverted by IL-21, suggesting new strategies for intervention in autoimmunity.

Topics: Adoptive Transfer; Animals; Autoimmunity; Blood Glucose; Diabetes Mellitus, Type 1; Disease Progression; Immune Tolerance; Interleukins; Lymph Nodes; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Mice, Transgenic; Ovalbumin; Pancreas; T-Lymphocytes, Regulatory

Organ infiltration by T cells depends on the adhesion molecules expressed in these sites and on homing receptors expressed by the T cells. Here, we have studied which form of priming can enable T cells to home to pancreatic islets. To this end, we have used transgenic mice expressing the model autoantigen ovalbumin in pancreatic islets and transgenic ovalbumin-specific CD4 and CD8 T cells. We demonstrate that these T cells were imprinted with homing receptor patterns characteristic for the site of priming, such as alpha4beta7 integrin for mucosal antigen delivery or functionally active alpha4beta1 integrin for islet autoantigens. The adhesion molecules corresponding to these receptors were found to be constitutively expressed in islets, enabling T cells bearing these receptors to infiltrate the islets and to cause diabetes. Disease was prevented only by blockade of the endothelial adhesion molecule, ligand of homing receptors with which the T cells were imprinted. Thus, different priming locations induced different homing mechanisms, allowing T cells to target the islets. This may contribute to the susceptibility of islets to T-cell-mediated attack. Furthermore, it may pertain to the design of adhesion-modulating therapies alone or in combination with external autoantigen administration.

Topics: Animals; Antigen Presentation; Autoantigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Movement; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Insulin-Secreting Cells; Integrin alpha4beta1; Integrins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Receptors, Antigen, T-Cell; Receptors, Lymphocyte Homing

Peripheral tolerance is required to prevent autoimmune tissue destruction by self-reactive T cells that escape negative selection in the thymus. One mechanism of peripheral tolerance in CD8(+) T cells is their activation by resting dendritic cells (DC). In contrast, DC can be "licensed" by CD4(+) T cells to induce cytotoxic function in CD8(+) T cells. The question that then arises, whether CD4(+) T cell help could impair peripheral tolerance induction in self-reactive CD8(+) T cells, has not been addressed. In this study we show that CD4(+) T cell activation by resting DC results in helper function that transiently promotes the expansion and differentiation of cognate CD8(+) T cells. However, both the CD4(+) and CD8(+) T cell populations ultimately undergo partial deletion and acquire Ag unresponsiveness, disabling their ability to destroy OVA-expressing pancreatic beta cells and cause diabetes. Thus, effective peripheral tolerance can be induced by resting DC in the presence of CD4(+) and CD8(+) T cells with specificity for the same Ag.

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Clonal Deletion; Dendritic Cells; Diabetes Mellitus, Type 1; Gene Expression Regulation; Immune Tolerance; Insulin-Secreting Cells; Lymphocyte Activation; Mice; Mice, Transgenic; Ovalbumin

T cells causing autoimmunity must escape tolerance. We observed that CD8(+) T cells with high avidity for an antigen expressed in the pancreas, kidney, and thymic medulla were efficiently removed from a polyclonal repertoire by central and peripheral tolerance mechanisms. However, both mechanisms spared low-avidity T cells from elimination. Neither the introduction of activated, self-antigen-specific CD4(+) helper T cells nor a global inflammatory stimulus were sufficient to activate the low-avidity CD8(+) T cells and did not break tolerance. In contrast, challenge with a recombinant bacterium expressing the self antigen primed the low-avidity T cells, and the animals rapidly developed autoimmune diabetes. We suggest that whereas thymic and peripheral tolerance mechanisms remove cells that can be primed by endogenous amounts of self antigen, they do not guard against tissue destruction by low-avidity effector T cells, which have been primed by higher amounts of self antigen or by crossreactive antigens.

Topics: Animals; Antibody Affinity; Antigens; Autoimmunity; Cells, Cultured; Dendritic Cells; Diabetes Mellitus, Type 1; Immune Tolerance; Immunoglobulin Heavy Chains; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Mice; Mice, Transgenic; Organ Specificity; Ovalbumin; Rats; T-Lymphocytes

Bidens pilosa is claimed to be useful for immune or anti-inflammatory disorders; however, little scientific evidence has been published concerning its function. In this paper, immune disease mouse models were used to study the function of a butanol fraction of B.pilosa. We demonstrated treatment with the butanol fraction of B.pilosa ameliorated Th1 cell-mediated autoimmune diabetes in nonobese diabetic (NOD) mice but caused deterioration of Th2 cell-mediated airway inflammation induced by ovalbumin (OVA) in BALB/c mice. We next showed that Th2 cytokines (IL-4 and/or IL-5) increased but Th1 cytokine (IFN-gamma) decreased following injections with the butanol fraction of B.pilosa in both mouse strains. Accordingly, Th2 cytokine-regulated IgE production in mouse serum increased following treatment with this fraction. Finally, we found that the butanol fraction of B.pilosa inhibited Th1 cell differentiation but promoted Th2 cell differentiation. Taken together, the butanol fraction of B.pilosa has a dichotomous effect on helper T cell-mediated immune disorders, plausibly via modulation of T cell differentiation.

Topics: Animals; Bidens; Bronchoalveolar Lavage Fluid; Butanols; Cell Differentiation; Cytokines; Diabetes Mellitus, Type 1; Drugs, Chinese Herbal; Female; Immunoglobulin E; Inflammation; Lung; Mice; Mice, Inbred NOD; Ovalbumin; Th1 Cells; Th2 Cells

The NOD mouse has proved to be a relevant model of insulin-dependent diabetes mellitus, closely resembling the human disease. However, it is unknown whether this strain presents a general biastoward Th1-mediated autoimmunity or remains capable of mounting complete Th2-mediated responses. Here, we show that NOD mice have the capacity to develop a typical Th2-mediated disease, namely experimental allergic asthma. In contrast to what might have been expected, they even developed a stronger Th2-mediated pulmonary inflammatory response than BALB/c mice, a strain that shows a typical Th2 bias in this model. Thus, after allergen sensitization and intra-nasal challenge, the typical features of experimental asthma were exacerbated in NOD mice, including enhanced bronchopulmonary responsiveness, mucus production and eosinophilic inflammation in the lungs as well as specific IgE titers in serum. These hallmarks of allergic asthma were associated with increased IL-4, IL-5, IL-13 and eotaxin production in the lungs, as compared with BALB/c mice. Notwithstanding their quantitative and functional defect in NOD mice, CD1d-dependent NKT cells contribute to aggravate the disease, since in OVA-immunized CD1d(-/-) NOD mice, which are deficient in this particular T cell subset, airway eosinophilia was clearly diminished relative to NOD littermates. This is the first evidence that autoimmune diabetes-prone NOD mice can also give rise to enhanced Th2-mediated responses and might thus provide a useful model for the study of common genetic and cellular components, including NKT cells that contribute to both asthma and type 1 diabetes.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD1; Antigens, CD1d; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Chemokine CCL11; Chemokines, CC; Cytokines; Diabetes Mellitus, Type 1; Immunoglobulin E; Interleukin-5; Male; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Knockout; Mucus; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells

Adoptive transfer of ovalbumin (OVA)-specific T cells from the DO.11 TCR transgenic mouse on a Rag(-/-) background into mice expressing OVA in pancreatic islet cells induces acute insulitis and diabetes only if endogenous lymphocytes, including regulatory T cells, are removed. When wild-type OVA-specific/Rag(-/-) T cells, which are all CD25(-), are transferred into islet antigen-expressing mice, peripheral immunization with OVA in adjuvant is needed to induce diabetes. In contrast, naive CTLA-4(-/-)/Rag(-/-) OVA-specific T cells (also CD25(-)) develop into Th1 effectors and induce disease upon recognition of the self-antigen alone. These results suggest that CTLA-4 functions to increase the activation threshold of autoreactive T cells, because in its absence self-antigen is sufficient to trigger autoimmunity without peripheral immunization. Further, CTLA-4 and regulatory T cells act cooperatively to maintain tolerance, indicating that the function of CTLA-4 is independent of regulatory cells, and deficiency of both is required to induce pathologic immune responses against the islet self-antigen.

Topics: Animals; Antigens, CD; Antigens, Differentiation; Autoantigens; Autoimmunity; CTLA-4 Antigen; Diabetes Mellitus, Type 1; DNA-Binding Proteins; Immune Tolerance; Islets of Langerhans; Mice; Mice, Knockout; Mice, Transgenic; Ovalbumin; T-Lymphocytes

Here we show that Janus kinase (JAK) 3 is an important molecular target for treatment of autoimmune insulin-dependent (type 1) diabetes mellitus. The rationally designed JAK3 inhibitor JANEX-1 exhibited potent immunomodulatory activity and delayed the onset of diabetes in the NOD mouse model of autoimmune type 1 diabetes. Whereas 60% of vehicle-treated control NOD mice became diabetic by 25 weeks, the incidence of diabetes at 25 weeks was only 9% for NOD females treated with daily injections of JANEX-1 (100 mg/kg/day) from Week 10 through Week 25 (P = 0.007). Furthermore, JANEX-1 prevented the development of insulitis and diabetes in NOD-scid/scid females after adoptive transfer of splenocytes from diabetic NOD females. Chemical inhibitors such as JANEX-1 may provide the basis for effective treatment modalities against human type 1 diabetes. To our knowledge, this is the first report of the immunosuppressive activity of a JAK3 inhibitor in the context of an autoimmune disease.

Topics: Adoptive Transfer; Animals; Cytokines; Diabetes Mellitus, Type 1; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Interleukin-2; Janus Kinase 3; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Milk Proteins; Ovalbumin; Phosphorylation; Protein-Tyrosine Kinases; Quinazolines; STAT5 Transcription Factor; T-Lymphocytes; Trans-Activators; Tyrosine

Bone marrow or hematopoietic stem cell transplantation is a potential treatment for autoimmune disease. The clinical application of this approach is, however, limited by the risks associated with allogeneic transplantation. In contrast, syngeneic transplantation would be safe and have wide clinical application. Because T cell tolerance can be induced by presenting antigen on resting antigen-presenting cells (APCs), we reasoned that hematopoietic stem cells engineered to express autoantigen in resting APCs could be used to prevent autoimmune disease. Proinsulin is a major autoantigen associated with pancreatic beta cell destruction in humans with type 1 diabetes (T1D) and in autoimmune NOD mice. Here, we demonstrate that syngeneic transplantation of hematopoietic stem cells encoding proinsulin transgenically targeted to APCs totally prevents the development of spontaneous autoimmune diabetes in NOD mice. This antigen-specific immunotherapeutic strategy could be applied to prevent T1D and other autoimmune diseases in humans.

Topics: Animals; Autoantigens; Diabetes Mellitus, Type 1; Hematopoietic Stem Cell Transplantation; Humans; Immunization; Islets of Langerhans; Lymphocyte Subsets; Mice; Mice, Inbred NOD; Ovalbumin; Proinsulin; Spleen; T-Lymphocytes; Transplantation, Isogeneic

When conjugated to various proteins, the nontoxic B-chain of cholera toxin (CTB) significantly increases the ability of these proteins to induce immunological tolerance after oral administration. Here, we investigated if a nonconjugated form of CTB enhances the induction of immune tolerance after oral insulin administration. Induction of immunological tolerance was studied after oral administration of insulin preparations in three mouse models; an insulin/ovalbumin coimmunization model, a model of virus-induced diabetes in transgenic RIP-LCMV-NP mice and in nonobese diabetic (NOD) mice serving as a model of spontaneous diabetes. In the immunization model, we demonstrate that mixing with CTB increases the tolerogenic potential of insulin, approximately 10 fold. Titration of the CTB concentration in this system revealed that an insulin : CTB ratio of 100 : 1 was optimal for the induction of bystander suppression. Further studies revealed that this insulin : CTB ratio also was optimal for the prevention of diabetes in a virus-induced, transgenic diabetes model. In addition, the administration of this optimal insulin-CTB preparation significantly prevented the onset of diabetes in old NOD mice with established islet infiltration. The data presented here demonstrate that CTB, even in its unconjugated form, functions as a mucosal adjuvant, increasing the specific tolerogenic effect of oral insulin.

Topics: Adjuvants, Immunologic; Administration, Oral; Animals; Autoantigens; Bystander Effect; Cholera Toxin; Diabetes Mellitus, Type 1; Female; Humans; Immune Tolerance; Immunity, Mucosal; Immunization; Insulin; Islets of Langerhans; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Transgenic; Ovalbumin; Pharmaceutical Vehicles; Promoter Regions, Genetic; T-Lymphocytes, Cytotoxic

NOD mice have a relative deficiency of CD4+CD25+ regulatory T cells that could result in an inability to maintain peripheral tolerance. The aim of this study was to induce the generation of CD4+CD25+ regulatory T cells in response to autoantigens to prevent type 1 diabetes (T1D). We found that immunization of NOD mice with insulin B-chain peptide B:9-23 followed by 72 h in vitro culture with B:9-23 peptide induces generation of CD4+CD25+ regulatory T cells. Route of immunization has a critical role in the generation of these cells. Non-autoimmune mice BALB/c, C57BL/6 and NOR did not show up regulation of CD4+CD25+ regulatory T cells. These cells secreted large amounts of TGF-beta and TNF-alpha with little or no IFN-gamma and IL-10. Adoptive transfer of these CD4+CD25+ regulatory T cells into NOD-SCID mice completely prevented the adoptive transfer of disease by diabetogenic T cells. Although, non-self antigenic OVA (323-339) peptide immunization and in vitro culture with OVA (323-339) peptide does result in up regulation of CD4+CD25+ T cells, these cells did not prevent transfer of diabetes. Our study for the first time identified the generation of antigen-specific CD4+CD25+ regulatory T cells specifically in response to immunization with B:9-23 peptide in NOD mice that are capable of blocking adoptive transfer of diabetes. Our results suggest the possibility of using autoantigens to induce antigen-specific regulatory T cells to prevent and regulate autoimmune diabetes.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Antigen Presentation; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; CD3 Complex; CD4 Antigens; Coculture Techniques; Diabetes Mellitus, Type 1; Drug Administration Routes; Female; Glycosuria; Immune Tolerance; Insulin; Interferon-gamma; Interleukin-10; Islets of Langerhans; L-Selectin; Lectins, C-Type; Leukocyte Common Antigens; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Ovalbumin; Peptide Fragments; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Receptors, Interleukin-2; Spleen; T-Lymphocytes; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vaccination

Antigen administration via oral and other mucosal routes can suppress systemic immunity to the antigen and has been used to prevent experimental autoimmune disease. This approach may prove ineffective or even harmful if it leads to a concomitant induction of cytotoxic T lymphocytes (CTLs), and indeed, mucosal administration of the model antigen ovalbumin (OVA) has been shown to elicit CTL activation while simultaneously inducing oral tolerance. Here we show that induction by oral OVA of CTLs in wild-type mice, and of diabetes in mice expressing OVA transgenically in pancreatic beta cells, can be prevented by transiently blocking the CD40 ligand (CD40L). However, CD40L blockade did not diminish oral tolerance, as measured by suppression of systemic OVA-primed T cell proliferation, IFN-gamma secretion, and Ab production. Consistent with these findings, mice lacking CD40 expression could be orally tolerized to OVA. Transient CD40L blockade therefore dissociates pathogenic from protective immunity and should enhance the efficacy and safety of oral tolerance for preventing autoimmune disease.

Topics: Administration, Oral; Animals; CD40 Antigens; CD40 Ligand; Diabetes Mellitus, Type 1; Female; Humans; Immune Tolerance; Immunity, Mucosal; Interferon-gamma; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Signal Transduction; T-Lymphocytes, Cytotoxic

Type I diabetes is associated with a low incidence of asthma. We tested whether a decrease in sensory neuropeptide release is associated with an attenuated bronchoconstrictive response to field stimulation (FS; 100 stimuli, 20 V, 0.1 ms, 20 Hz) in streptozotocin (STZ)-induced diabetes. The organ fluid of the preparations were also tested for substance P, calcitonin gene-related peptide (CGRP), and somatostatin concentrations by RIA. Preparations were from either normal rats or those pretreated with 50 mg/kg STZ iv 8 wk before experiment. A group of STZ-treated animals was supplied with insulin delivery (4 IU/day sc) implants between 4 and 8 wk. A subgroup was formed to study the effect of capsaicin desensitization. The atropine-resistant contraction was attenuated by diabetes without capsaicin-sensitive relaxation response. Exogenous CGRP and substance P potentiated, whereas somatostatin inhibited (1 nM-10 microM) the FS-induced contractions in rings from either group. FS released somatostatin, CGRP, and substance P from 0.17 +/- 0.024, 0.15 +/- 0.022, and 1.65 +/- 0.093 to 0.58 +/- 0.032, 0.74 +/- 0.122, and 5.34 +/- 0.295 in preparations from normal, and from 0.19 +/- 0.016, 0.11 +/- 0.019, and 0.98 +/- 0.116 to 0.22 +/- 0.076, 0.34 +/- 0.099, and 1.84 +/- 0.316 fmol/mg wet wt in preparations from diabetic rats. Insulin supplementation restored neuropeptide release in rings from STZ-treated rats. The results show that the decreased FS-induced contractions occurred with a decrease in sensory neuropeptide release in STZ-diabetic rats.

Topics: Animals; Blood Glucose; Body Weight; Bronchial Hyperreactivity; Bronchoconstriction; Calcitonin Gene-Related Peptide; Capsaicin; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Guinea Pigs; Insulin; Male; Neural Conduction; Neurons, Afferent; Ovalbumin; Rats; Rats, Wistar; Somatostatin; Substance P; Trachea

Cows' milk proteins have been proposed to play a part in the pathogenesis of Type I (insulin-dependent) diabetes mellitus but both epidemiological and immunological studies have given conflicting results. Thus we aimed to study the immunological response to cows' milk proteins among diabetic and healthy children, focusing on the balance of Th1- and Th2-like lymphocytes.. Peripheral blood mononuclear cells from 30 Type I diabetic children (4 to 18 years old) were examined and compared with peripheral blood mononuclear cells from 18 healthy age-matched control children (7 to 15 years old). Expression of IFN-gamma and IL-4 mRNA were detected by realtime RT-PCR and as protein by ELISA after stimulation with BSA, the ABBOS-peptide (a. a. 152-169) and beta-lactoglobulin (betaLG) from cows' milk and ovalbumin from hens' egg. Phytohaemagglutinin and keyhole limpet haemocyanin were used as positive and negative controls, respectively.. Bovine serum albumin caused a weak Th2-like response in Type I diabetic children, whereas BSA antibodies decreased with age only among healthy children. Otherwise, cows' milk proteins (BSA, ABBOS and betaLG) caused increased expression for IFN-gamma and IL-4 mRNA in diabetic and healthy children. BetaLG caused the strongest immunological response, which decreased with age only among diabetic children. However, ovalbumin from egg caused a similar activation of the immune system and the immune response was similar in both diabetic and healthy children.. Proteins from cows' milk caused an equal Th1- and Th2-like immune response in diabetic and healthy children. Thus, our results do not support the hypothesis that cows' milk antigens are important for the immune process associated with Type I diabetes.

Topics: Adolescent; Animals; Antibodies; Antigens; C-Peptide; Cattle; Child; Child, Preschool; Diabetes Mellitus, Type 1; Dietary Proteins; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Hemocyanins; Humans; Insulin; Interferon-gamma; Interleukin-4; Lactoglobulins; Male; Milk Proteins; Ovalbumin; Peptide Fragments; Phytohemagglutinins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serum Albumin, Bovine; Th1 Cells; Th2 Cells

Insulin-dependent diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic beta cells with the primary mechanism being cell mediated. The BB rat develops insulitis and IDDM with many features analogous to the disease in man. In previous studies we reported that weekly administration of 2'-deoxycoformycin (dCF) for four months reduces significantly the incidence of IDDM in the BB rat by 70%, and that the animals remain free of diabetes for a minimum of two months after drug withdrawal. Since the diabetes-prone BB rat is lymphopenic, with a reduction of both CD4 and CD8 cells, the continuous failure of dCF treated animals to develop diabetes may have been due to generalized immunosuppression. To test this possibility, the ability of dCF treated diabetes-free BB rats to mount an immune response after challenge with Ovalbumin was examined five months after drug withdrawal. The results showed that the post-immunization levels of total IgG and specific IgG in these animals did not differ from those observed in non-dCF treated controls nor those of control diabetes-resistant non-lymphopenic BB rats. Moreover, FACS analysis indicated no change in the percentages of total numbers of CD4+ or CD8+ cells between the two groups of animals. Histological assessment of the pancreata of the post-dCF treated animals showed varying degrees of mononuclear cell infiltrates in the islets. These data demonstrate that treatment by dCF is not permanent, and may require intermittent or continuous administration to prevent development of diabetes. Further studies are needed to determine the mechanism of action of dCF in this model of IDDM.

Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Immunoglobulin G; Immunosuppressive Agents; Immunotherapy; Islets of Langerhans; Ovalbumin; Pentostatin; Prediabetic State; Rats; Rats, Inbred BB

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common alpha-chain with I-Ad but has a peculiar beta-chain. As with most beta-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position beta57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the beta-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Antigens, Differentiation, B-Lymphocyte; Cell Line; Crystallization; Diabetes Mellitus, Type 1; Drosophila melanogaster; Genetic Vectors; Glutamate Decarboxylase; Histocompatibility Antigens Class II; Isoenzymes; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Molecular Sequence Data; Ovalbumin; Peptide Fragments; Peptide Library; Protein Binding; Sequence Alignment

Autoantigens found on pancreatic islets can move to draining lymph nodes, where they are able to cause the activation and consequent deletion of autoreactive T cells by a mechanism termed cross-tolerance. This deletion depends on signalling through CD95 (also known as Fas), a member of the superfamily of tumour-necrosis-factor receptors. Here we describe a new mechanism that protects against autoimmunity: this mechanism involves another member of this superfamily, CD30, whose function was largely unknown. CD30-deficient islet-specific CD8-positive T cells are roughly 6,000-fold more autoaggressive than wild-type cells, with the transfer of as few as 160 CD30-deficient T cells leading to the complete destruction of pancreatic islets and the rapid onset of diabetes. We show that, in the absence of CD30 signalling, cells activated but not yet deleted by the CD95-dependent cross-tolerance mechanism gain the ability to proliferate extensively upon secondary encounter with antigen on parenchymal tissues, such as the pancreatic islets. Thus, CD30 signalling limits the proliferative potential of autoreactive CD8 effector T cells and protects the body against autoimmunity.

Topics: Adoptive Transfer; Animals; Autoimmunity; CD8-Positive T-Lymphocytes; Clonal Deletion; Diabetes Mellitus, Type 1; Immune Tolerance; Islets of Langerhans; Ki-1 Antigen; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Signal Transduction; Transplantation Chimera

The impact of exposure to lead on gut cytokine gene expression and oral tolerance was analyzed. Oral tolerization with ovalbumin (OVA) increased levels of IL-10 and TGF-beta in gut tissue while IFN-gamma mRNA levels remained unchanged in both autoimmune diabetes prone NOD and normal C57BL/6 mice. This shift towards Th2/Th3 type cytokine gene expression was completely abolished by concomitant treatment with PbCl2 (6 x 0.5 mg/kg) in NOD mice while the cytokine balance in C57BL/6 mice was unaffected. Suppression of Th2/Th3 type cytokine expression was associated with a dampened oral tolerance response to OVA as determined by T cell proliferation assays. We conclude that in autoimmunity prone NOD mice environmental toxicants may disturb immune homeostasis by targeting the gut immune system.

Topics: Administration, Oral; Animals; Antigens; Cytokines; Diabetes Mellitus, Type 1; Female; Gene Expression; Immune Tolerance; Intestine, Small; Lead; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Ovalbumin; RNA, Messenger; Spleen; T-Lymphocyte Subsets; Transforming Growth Factor beta

The non-obese diabetic (NOD) mouse develops insulin dependent diabetes mellitus (IDDM) spontaneously with a higher incidence in females than in males. There are many similarities to the human disease, making it an ideal model. Our group is examining the role that CD4(+) and CD8(+) T cells play in IDDM in the NOD mouse, as it is known that both T cell subsets are required for onset of disease. Although IDDM has an autoimmune etiology, the initial triggering event is unknown and the autoantigen involved has not been identified. This investigation focussed on one of the potential autoantigens involved, the enzyme glutamic acid decarboxylase (GAD). We raised GAD peptide-specific CD8(+) T cells by immunising NOD mice with the GAD peptide alongside an irrelevant peptide that induced a CD4(+) T cell response. In order to maintain these peptide specific T cells in vitro and generate clones, it was found that antibodies specific to CD4(+) and MHC class II molecules needed to be included in the culture medium. This paper outlines the methods we employed to generate and maintain these CD8(+) T cells in vitro.

Topics: Amino Acid Sequence; Animals; Autoantigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Culture Techniques; Clone Cells; Cytotoxicity Tests, Immunologic; Diabetes Mellitus, Type 1; Epitopes; Female; Glutamate Decarboxylase; Humans; Immunization; Male; Mice; Mice, Inbred NOD; Oligopeptides; Ovalbumin

To investigate the humoral immune response to common food antigens in Japanese children with IDDM.. IgG antibodies to cow's milk, beta-lactoglobulin, bovine serum albumin (BSA), alpha-lactalbumin, and hens egg ovalbumin were examined by enzyme-linked immunosorbent assay in the sera of 33 patients with IDDM, ages 11.8 +/- 3.4 years. The data were compared with that of 50 normal subjects, ages 10.3 +/- 5.1 years, who acted as control subjects. A positive antibody to a food antigen was defined as an antibody titer greater than the 95th percentile value in normal subjects.. Children with IDDM had significantly higher median titers of IgG antibodies to beta-lactoglobulin and ovalbumin (P = 0.03 and P = 0.0005 respectively). More children with IDDM than control subjects had positive IgG antibody to ovalbumin (21 vs. 6%, P = 0.04). Titers, as well as the number of positive antibodies to other food antigens, including BSA, did not differ between the two groups.. Japanese children with IDDM show an enhanced humoral immune response to beta-lactoglobulin and ovalbumin, a phenomenon that may be related to the pathogenesis of the disease.

Topics: Adolescent; Animals; Child; Diabetes Mellitus, Type 1; Food Hypersensitivity; Humans; Immunoglobulin G; Japan; Lactalbumin; Lactoglobulins; Milk; Ovalbumin; Serum Albumin, Bovine

Human epidemiological studies delineated early exposure to intact dietary protein (e.g., most infant formulas) as an environmental risk factor for the development of IDDM. The Trial to Reduce IDDM in the Genetically at Risk (TRIGR), an international IDDM prevention trial, has been designed to determine if avoidance of intact dairy protein in high-risk infants < or =6 months of age can reduce the subsequent diabetes incidence. We here studied the casein hydrolysate-based trial diet (Nutramigen) in NOD mice. When given either continuously or for 10 weeks after weaning, the test diet was highly effective in preventing autoimmune diabetes (32-week incidence: 4.6 vs. 58.8%) and in preserving pancreatic insulin levels, with little effect on islet inflammation. Spleen cells from protected NOD mice failed to adoptively transfer diabetes into irradiated syngeneic recipients. When co-transferred with splenocytes from diabetic donors, cells from diet-protected mice inhibited adoptive diabetes transfer (incidence 50 vs. 94%, P < 0.001). T-cell reactivity to the islet cell autoantigens ICA69 (islet cell antigen 69) and GAD65 developed only in diabetic recipients of spleen cell grafts, indicating that diabetes protection extends to more than one autoantigen. In protected mice, ICA69 T-cell reactivity was not detectable spontaneously nor after priming with this autoantigen; however, priming with the cross-reactive non-self-antigen bovine serum albumin recruited T-cells responsive to ICA69. Thus, diabetes prevention with the clinical trial diet is effective in NOD mice, where it affects some T-cell repertoires and allows development of regulatory cells that interfere with destructive autoimmunity.

Topics: Adoptive Transfer; Animals; Autoantigens; Caseins; Cattle; Cross Reactions; Diabetes Mellitus, Type 1; Diet; Disease Models, Animal; Female; Hydrolysis; Incidence; Insulin; Islets of Langerhans; Male; Mice; Mice, Inbred NOD; Ovalbumin; Pancreas; Pilot Projects; Serum Albumin; Spleen; T-Lymphocytes; Time Factors

Endogenous advanced glycation endproducts (AGEs) include chemically crosslinking species (glycotoxins) that contribute to the vascular and renal complications of diabetes mellitus (DM). Renal excretion of the catabolic products of endogenous AGEs is impaired in patients with diabetic or nondiabetic kidney disease (KD). The aim of this study was to examine the oral absorption and renal clearance kinetics of food AGEs in DM with KD and whether circulating diet-derived AGEs contain active glycotoxins. Thirty-eight diabetics (DM) with or without KD and five healthy subjects (NL) received a single meal of egg white (56 g protein), cooked with (AGE-diet) or without fructose (100 g) (CL-diet). Serum and urine samples, collected for 48 hr, were monitored for AGE immunoreactivity by ELISA and for AGE-specific crosslinking reactivity, based on complex formation with 125I-labeled fibronectin. The AGE-diet, but not the CL-diet, produced distinct elevations in serum AGE levels in direct proportion to amount ingested (r = 0.8, P < 0.05): the area under the curve for serum ( approximately 10% of ingested AGE) correlated directly with severity of KD; renal excretion of dietary AGE, although normally incomplete (only approximately 30% of amount absorbed), in DM it correlated inversely with degree of albuminuria, and directly with creatinine clearance (r = 0.8, P < 0.05), reduced to <5% in DM with renal failure. Post-AGE-meal serum exhibited increased AGE-crosslinking activity (two times above baseline serum AGE, three times above negative control), which was inhibited by aminoguanidine. In conclusion, (i) the renal excretion of orally absorbed AGEs is markedly suppressed in diabetic nephropathy patients, (ii) daily influx of dietary AGEs includes glycotoxins that may constitute an added chronic risk for renal-vascular injury in DM, and (iii) dietary restriction of AGE food intake may greatly reduce the burden of AGEs in diabetic patients and possibly improve prognosis.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Albuminuria; Creatinine; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Diet, Diabetic; Female; Food Analysis; Fructose; Glycation End Products, Advanced; Humans; Intestinal Absorption; Male; Middle Aged; Ovalbumin; Reference Values; Risk Factors

Oral administration of antigen leads to systemic immune unresponsiveness. Low dose oral tolerance generates regulatory cells which, when triggered in an antigen-specific manner, suppress inflammatory responses. We have previously shown that oral administration of an organ-specific antigen, porcine insulin, protects against diabetes development in the NOD mouse. In the present study we extend these observations to the B-chain of insulin, a 30-amino-acid peptide which has now been shown by others to contain the immunogenic epitope. Oral administration of the B-chain slowed diabetes development in a co-transfer model in which cells from B-chain-fed animals were co-transferred with diabetogenic cells (P=0.02). Further exposure to antigen via feeding of the co-transfer recipient animals not only slowed diabetes development but prevented diabetes in some animals (P=0.01). In vitro proliferation of popliteal lymph node cells from fed and immunized animals was suppressed in an antigen-specific manner when cells were restimulated with the fed antigen. When those cells were cultured and restimulated in vitro with the B-chain of insulin, we also observed a decrease in IFN-gamma expression and an increase in IL-4, TGF-beta and IL-10 expression. These results demonstrate that an orally protective epitope resides in the B-chain of insulin and that refeeding following adoptive transfer enhances protection. Finally, the orally administered antigen is associated with a decrease in Th1 responses and an increase in Th2 responses to the insulin B-chain.

Topics: Administration, Oral; Adoptive Transfer; Animals; Autoimmunity; Cytokines; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Immunodominant Epitopes; Immunosuppression Therapy; In Vitro Techniques; Insulin; Lymphocyte Activation; Male; Mice; Mice, Inbred NOD; Ovalbumin; Th1 Cells; Th2 Cells

Nonobese diabetic (NOD) mice develop spontaneous insulin-dependent diabetes mellitus (IDDM), and the pancreas-infiltrating T cells invariably show a Th1 phenotype. We demonstrated here that the interleukin (IL)-12 antagonist (p40)2 can deviate the default Th1 development of naive T cell receptor (TCR)-transgenic CD4+ cells to the Th2 pathway in vitro. Although (p40)2 does not modify the cytokine profile of polarized Th1 cells, it prevents further recruitment of CD4- cells into the Th1 subset. To study the involvement of Th1 and Th2 cells in the initiation and progression of IDDM, we targeted endogenous IL-12 by administration of (p40)2 in NOD mice. (p40)2 administration to NOD mice inhibits interferon-gamma but not IL-10 production in response to lipopolysaccharide (LPS) or to the putative autoantigen IA-2. Serum immunoglobulin isotypes determined after (p40)2 treatment indicate an increase in Th2 and a decrease in Th1 helper activity. Administration of (p40)2 from 3 weeks of age onwards, before the onset of insulitis, results in the deviation of pancreas-infiltrating CD4+ but not CD8+ cells to the Th2 phenotype as well as in the reduction of spontaneous and cyclophosphamide-accelerated IDDM. After treating NOD mice with (p40)2 from 9 weeks of age, when insulitis is well established, few Th2 and a reduced percentage of Th1 cells are found in the pancreas. This is associated with a slightly decreased incidence of spontaneous IDDM, but no protection from cyclophosphamide-accelerated IDDM. In conclusion, deviation of pancreas-infiltrating CD4+ cells to Th2 is associated with protection from IDDM. However, targeting IL-12 after the onset of insulitis, when the pancreas contains polarized Th1 cells, is not sufficient to induce an effective immune deviation able to significantly modify the course of disease.

Topics: Animals; Autoantigens; Cyclophosphamide; Diabetes Mellitus, Type 1; Dimerization; Immunoglobulin Isotypes; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-4; Islets of Langerhans; Lipopolysaccharides; Mice; Mice, Inbred NOD; Ovalbumin; Pancreas; T-Lymphocyte Subsets; Th1 Cells; Th2 Cells; Time Factors

Self-antigens expressed in extrathymic tissues such as the pancreas can be transported to draining lymph nodes and presented in a class I-restricted manner by bone marrow-derived antigen-presenting cells. Such cross-presentation of self-antigens leads to CD8+ T cell tolerance induction via deletion. In this report, we investigate the influence of CD4+ T cell help on this process. Small numbers of autoreactive OVA-specific CD8+ T cells were unable to cause diabetes when adoptively transferred into mice expressing ovalbumin in the pancreatic beta cells. Coinjection of OVA-specific CD4+ helper T cells, however, led to diabetes in a large proportion of mice (68%), suggesting that provision of help favored induction of autoimmunity. Analysis of the fate of CD8+ T cells indicated that CD4(+) T cell help impaired their deletion. These data indicate that control of such help is critical for the maintenance of CD8+ T cell tolerance induced by cross-presentation.

Topics: Adoptive Transfer; Animals; Autoantigens; Autoimmunity; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Diabetes Mellitus, Type 1; Immune Tolerance; Islets of Langerhans; Mice; Ovalbumin

Elevated levels of antibodies to cow's milk proteins, i.e., beta-lactoglobulin (BLG) and bovine serum albumin (BSA), have been associated with IDDM. We observed enhanced cellular immune response by a proliferation test of peripheral blood mononuclear cells to BLG in 22 of 40 (55%) patients with newly diagnosed IDDM compared with 7 of 32 healthy children (22%) (P = 0.004, chi 2 test). The median stimulation index to BLG was 3.3 in patients and 1.5 in healthy children (P = 0.003, Mann-Whitney U test). No difference was found in cellular reactivity to other cow's milk proteins, such as BSA or alpha-casein, or to a dietary immunogenic protein, ovalbumin. Cellular responsiveness to BLG was not associated with HLA-DQB1* risk alleles of IDDM, which suggests that immune response to the protein does not only reflect the accumulation of these HLA alleles in the patients with IDDM. We suggest that enhanced cellular immune response to dietary BLG may reflect a disturbance in the regulation of immune response to oral antigens in IDDM. This kind of defect may play a fundamental role in the development of beta-cell autoimmunity in IDDM.

Topics: Adolescent; Animals; Caseins; Cattle; Child; Child, Preschool; Diabetes Mellitus, Type 1; Humans; Infant; Lactoglobulins; Lymphocyte Activation; Milk Proteins; Ovalbumin

Topics: Administration, Oral; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line; Cells, Cultured; Cytokines; Diabetes Mellitus, Type 1; Immune Tolerance; Immunohistochemistry; Immunotherapy, Adoptive; Insulin; Lymphocyte Activation; Macromolecular Substances; Macrophages; Mice; Mice, Inbred Strains; Ovalbumin; T-Lymphocytes

CD28 ligation delivers a costimulatory signal important in T cell activation. This study demonstrates that the disruption of the CD28/B7 pathway early in the nonobese diabetic mouse strain, using CD28-/- and CTLA41g transgenic mice, promoted the development and progression of spontaneous autoimmune diabetes. Functional analyses of T cells isolated from CD28-deficient mice demonstrated that the GAD-specific T cells produced enhanced Th1-type cytokines (IL-2 and IFN gamma) and diminished Th2-type cytokine, IL-4. Moreover, there was a significant decrease in serum levels of anti-GAD antibodies of the IgG1 isotype consistent with a profound suppression of Th2-type responses in these animals. Thus, the early differentiation of naive diabetogenic T cells into the Th2 subset is dependent upon CD28 signaling and extends our understanding of the importance of Th1/Th2 balance in the regulation of this spontaneous autoimmune disease.

Topics: Abatacept; Animals; Antigens, CD; Antigens, Differentiation; Autoantigens; B7-1 Antigen; Base Sequence; CD28 Antigens; CTLA-4 Antigen; Diabetes Mellitus, Type 1; Glutamate Decarboxylase; Immunoconjugates; Immunoglobulin G; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Molecular Sequence Data; Ovalbumin; Th1 Cells; Th2 Cells

We investigated the association between serum antibodies to cow's milk proteins and insulin-dependent diabetes mellitus (IDDM) in Hungarian children. Forty-eight children 1.0-17.1 years of age with newly diagnosed IDDM and 74 control children 1.0-16.0 years of age were studied for serum IgG, IgA and IgM antibodies to cow's milk, beta-lactoglobulin, bovine serum albumin and ovalbumin by enzyme-linked immunosorbent assays. The specificity of IgM antibodies to beta-lactoglobulin and bovine serum albumin was controlled by Western blot. The levels of IgG and IgA antibodies to cow's milk proteins were similar in children with and without IDDM, with the exception of slightly increased levels of IgA antibodies to beta-lactoglobulin in diabetic children (P = 0.05). The levels of IgM antibodies to cow's milk were significantly higher in IDDM patients than in control children (P = 0.0002). Children with IDDM more often had IgM antibodies to beta-lactoglobulin (46.3% vs 18.8%; P = 0.002) and bovine serum albumin (87.8% vs 49.3%, P < 0.0001) than control children. Neither the levels of IgG or IgA antibodies to ovalbumin nor the frequency of IgM antibodies to ovalbumin differed between diabetic and control children.. In Hungarian children, clinical manifestation of IDDM is often associated with IgM antibody response to cow's milk protein and its fractions, beta-lactoglobulin and bovine serum albumin, indicating a loss of immunological tolerance to these proteins. IgG and IgA antibodies to cow's milk proteins, associated with an early introduction of cow's milk in diet, seem to play a minor role in the development of childhood IDDM in Hungary.

Topics: Adolescent; Child; Child, Preschool; Diabetes Mellitus, Type 1; Humans; Hungary; Immunoglobulin A; Immunoglobulin M; Infant; Lactoglobulins; Milk Proteins; Ovalbumin; Serum Albumin

Topics: Animals; Antigens; Autoimmune Diseases; Callithrix; CD8-Positive T-Lymphocytes; Cytokines; Diabetes Mellitus, Type 1; Encephalomyelitis, Autoimmune, Experimental; Humans; Immune Tolerance; Immunotherapy; Mice; Myelin Proteins; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; Ovalbumin; Th1 Cells; Th2 Cells

We previously reported that immunity to the p277 peptide of the human 60-kDa heat shock protein (hsp60) was a causal factor in the diabetes of non-obese diabetic (NOD) mice, which are genetically prone to develop spontaneous autoimmune diabetes. The present study was done to test whether immunization with the p277 peptide could cause diabetes in standard strains of mice. We now report that a single administration of the p277 peptide conjugated to carrier molecules such as bovine serum albumin or ovalbumin can induce diabetes in C57BL/6 mice and in other strains not genetically prone to develop diabetes. The diabetes was marked by hyperglycemia, insulitis, insulin autoantibodies, glucose intolerance and low blood levels of insulin. The diabetes could be transferred to naive recipients by anti-p277 T cell lines. Similar to other experimentally induced autoimmune diseases, the autoimmune diabetes remitted spontaneously. After recovery, the mice were found to have acquired resistance to a second induction of diabetes. Susceptibility to induced diabetes in C57BL/6 mice was influenced by sex (males were much more susceptible than were females) and by class II genes in the major histocompatibility complex (B6.H-2bm12 mice with a mutation in the MHC-II molecule were relatively resistant). Other strains of mice susceptible to induced diabetes were C57BL/KSJ, C3HeB/FeJ, and NON/Lt. BALB/c and C3H/HeJ strains were relatively resistant. Immunization to p277-carrier conjugates could also induce transient hyperglycemia in young NOD mice, but upon recovery from the induced diabetes, the NOD mice were found to have acquired resistance to later development of spontaneous diabetes. Thus, T cell immunity to the p277 peptide can suffice to induce diabetes in standard mice, and a short bout of induced diabetes can affect the chronic process that would otherwise lead to spontaneous diabetes in diabetes-prone NOD mice.

Topics: Amino Acid Sequence; Animals; Autoantibodies; Autoimmune Diseases; Blood Glucose; Cattle; Chaperonin 60; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Susceptibility; Female; Genes, MHC Class II; Glucose Tolerance Test; Humans; Immunization; Immunotherapy, Adoptive; Insulin; Islets of Langerhans; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred NOD; Molecular Sequence Data; Ovalbumin; Peptide Fragments; Receptors, Interleukin-1; Serum Albumin, Bovine; Species Specificity; T-Lymphocytes

To study the humoral immune response to bovine serum albumin (BSA) and ovalbumin (OA) in children with newly diagnosed insulin-dependent diabetes mellitus (IDDM).. We examined serum samples from 505 children 0.8-14.9 years of age with newly diagnosed IDDM for antibodies to BSA and OA by enzyme-linked immunosorbent assay (ELISA). We also had two control groups: 85 unrelated control children (0.8-7.1 years of age) and 395 nondiabetic siblings (3.0-14.9 years of age). The specificity of antibodies detected in ELISA was confirmed by immunoblotting in a subset of sera with varying levels of antibodies.. Diabetic children < 7 years of age had a significantly higher level of IgG (immunoglobulin) antibodies to BSA than did unrelated control children (P < 0.0001). The difference was greatest in the youngest group of children, 0.8-2.9 years of age. IgA antibodies to BSA were detected more frequently among diabetic than control children (P = 0.0009). Levels of IgG and IgA antibodies to ovalbumin did not differ between diabetic and control children. Diabetic children 3.0-14.9 years of age also had higher levels of IgG and IgA antibodies to BSA than did their age- and sex-matched nondiabetic siblings (P = 0.02 and P < 0.0001, respectively). Those siblings who contracted IDDM during the follow-up period (n = 15) had a measurable level of IgA antibodies to BSA more often than did those who remained nondiabetic (60 and 34%, respectively; P = 0.04). Neither before nor after diagnosis of IDDM was there any significant trend in antibody levels.. A high level of antibodies to BSA commonly associates with IDDM, whereas the humoral immune response to OA is similar in diabetic and nondiabetic children.

Topics: Adolescent; Antibodies; Antibody Formation; Child; Child, Preschool; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Finland; Humans; Immunoblotting; Immunoglobulin A; Immunoglobulin G; Infant; Infant, Newborn; Ovalbumin; Serum Albumin, Bovine

The non-obese diabetic (NOD) mouse is a model for the study of insulin-dependent diabetes mellitus (IDDM). Recently transgenic NOD mice have been derived (NOD-E) that express the major histocompatibility complex (MHC) class II I-E molecule. NOD-E do not become diabetic and show negligible pancreatic insulitis. The possibility pertained that NOD-E mice are protected from disease by a process of T-cell deletion or anergy. This paper describes our attempts to discover whether this was so, by comparing NOD and NOD-E mouse T-cell receptor V beta usage. Splenocytes and lymph node cells were therefore tested for their ability to proliferate in response to monoclonal anti-V beta antibodies. We were unable to show any consistent differences between NOD and NOD-E responses to the panel of antibodies used. Previously proposed V beta were shown to be unlikely candidates for deletion or anergy. T cells present at low frequency (V beta 5+) in both NOD and NOD-E mice were shown to be as capable of expansion in response to antigenic stimulation as were more frequently expressed V beta. Our data therefore do not support deletion or anergy as mechanisms which could account for the observed disease protection in NOD-E mice.

Topics: Animals; Cell Division; Diabetes Mellitus, Type 1; Gene Deletion; H-2 Antigens; Immune Tolerance; Lymph Nodes; Mice; Mice, Inbred CBA; Mice, Inbred NOD; Mice, Transgenic; Ovalbumin; Receptors, Antigen, T-Cell, alpha-beta; Spleen; T-Lymphocytes

There is compelling evidence from animal models that type I diabetes is a consequence of T cell-mediated destruction of islet beta-cells. The recent isolation of islet-specific T cell clones from nonobese diabetic mice provides a means of identification of the Ag on islet cells that are responsible for stimulation of autoreactive T cells. We describe an APC line constructed by fusion of spleen B cells obtained from nonobese diabetic mice to a B lymphoma that was transfected with the H and L chains of an IgM specific to the hapten TNP. Using this hybrid APC we have observed a dramatic increase in the efficiency of presentation of TNP-conjugated islet cell protein preparations compared to that seen with conventional APC. Our results illustrate the potential use of this APC line for isolation and characterization of islet Ag relevant to the T cell response.

Topics: Adenoma, Islet Cell; Animals; Antigen-Presenting Cells; Antigens, Neoplasm; Autoantigens; B-Lymphocytes; Cell Line; Diabetes Mellitus, Type 1; Haptens; Hybrid Cells; Interleukin-2; Islets of Langerhans; Lymphocyte Activation; Mice; Mice, Inbred NOD; Ovalbumin; Receptors, Antigen, B-Cell; T-Lymphocytes; Trinitrobenzenes

Topics: Adolescent; Adult; Albumins; Animals; Antibodies; Antigen-Antibody Reactions; Cardiovascular Diseases; Cattle; Child; Collagen Diseases; Communicable Diseases; Diabetes Mellitus; Diabetes Mellitus, Type 1; Female; gamma-Globulins; Gastrointestinal Diseases; Hematologic Diseases; Humans; Hypersensitivity; Infant; Infant, Newborn; Insulin; Iodine Isotopes; Lactalbumin; Maternal-Fetal Exchange; Milk; Nervous System Diseases; Ovalbumin; Pregnancy; Respiratory Tract Diseases; Serum Albumin; Serum Albumin, Bovine; Skin Tests; Viral Vaccines