Specific Immunoassays Confirm Association of Mycobacterium avium Subsp. paratuberculosis with Type-1 but Not Type-2 Diabetes Mellitus

Mycobacterium avium subspecies paratuberculosis (MAP) is a versatile pathogen with a broad host range. Its association with type-1 diabetes mellitus (T1DM) has been recently proposed. Rapid identification of infectious agents such as MAP in diabetic patients at the level of clinics might be helpful in deciphering the role of chronic bacterial infection in the development of autoimmune diseases such as T1DM.We describe use of an ELISA method to identify live circulating MAP through the detection of a cell envelope protein, MptD by a specific M13 phage--fMptD. We also used another ELISA format to detect immune response to MptD peptide. Both the methods were tested with blood plasma obtained from T1DM, type-2 diabetes (T2DM) patients and non-diabetic controls. Our results demonstrate MptD and fMptD ELISA assays to be accurate and sensitive to detect MAP bacilli in a large fraction (47.3%) of T1DM patients as compared to non-diabetic controls (12.6%) and those with confirmed T2DM (7.7%). Comparative analysis of ELISA assays performed here with 3 other MAP antigen preparations, namely HbHA, Gsd and whole cell MAP lysates confirmed comparable sensitivity of the MptD peptide and the fMptD based ELISA assays. Moreover, we were successful in demonstrating positive bacterial culture in two of the clinical specimen derived from T1DM patients.The MptD peptide/fMptD based ELISA or similar tests could be suggested as rapid and specific field level diagnostic tests for the identification of MAP in diabetic patients and for finding the explanations towards the occurrence of type-1 or type-2 diabetes in the light of an active infectious trigger

Islet-Specific CTL Cloned from a Type 1 Diabetes Patient Cause Beta-Cell Destruction after Engraftment into HLAA2 Transgenic NOD/SCID/IL2RG Null Mice

Despite increasing evidence that autoreactive CD8 T-cells are involved in both the initiation of type 1 diabetes (T1D) and the destruction of beta-cells, direct evidence for their destructive role in-vivo is lacking. To address a destructive role for autoreactive CD8 T-cells in human disease, we assessed the pathogenicity of a CD8 T-cell clone derived from a T1D donor and specific for an HLA-A2-restricted epitope of islet-specific glucose-6-phosphatase catalytic-subunit related protein (IGRP). HLA-A2/IGRP tetramer staining revealed a higher frequency of IGRP-specific CD8 T-cells in the peripheral blood of recent onset human individuals than of healthy donors. IGRP(265-273)-specific CD8 T-cells that were cloned from the peripheral blood of a recent onset T1D individual were shown to secrete IFNγ and Granzyme B after antigen-specific activation and lyse HLA-A2-expressing murine islets in-vitro. Lytic capacity was also demonstrated in-vivo by specific killing of peptide-pulsed target cells. Using the HLA-A2 NOD-scid IL2rγ(null) mouse model, HLA-A2-restricted IGRP-specific CD8 T-cells induced a destructive insulitis. Together, this is the first evidence that human HLA-restricted autoreactive CD8 T-cells target HLA-expressing beta-cells in-vivo, demonstrating the translational value of humanized mice to study mechanisms of disease and therapeutic intervention strategies

A Signature of Maternal Anti-Fetal Rejection in Spontaneous Preterm Birth: Chronic Chorioamnionitis, Anti-Human Leukocyte Antigen Antibodies, and C4d

Chronic chorioamnionitis is found in more than one-third of spontaneous preterm births. Chronic chorioamnionitis and villitis of unknown etiology represent maternal anti-fetal cellular rejection. Antibody-mediated rejection is another type of transplantation rejection. We investigated whether there was evidence for antibody-mediated rejection against the fetus in spontaneous preterm birth.This cross-sectional study included women with (1) normal pregnancy and term delivery (n = 140) and (2) spontaneous preterm delivery (n = 140). We analyzed maternal and fetal sera for panel-reactive anti-HLA class I and class II antibodies, and determined C4d deposition on umbilical vein endothelium by immunohistochemistry. Maternal anti-HLA class I seropositivity in spontaneous preterm births was higher than in normal term births (48.6% vs. 32.1%, p = 0.005). Chronic chorioamnionitis was associated with a higher maternal anti-HLA class I seropositivity (p<0.01), significant in preterm and term birth. Villitis of unknown etiology was associated with increased maternal and fetal anti-HLA class I and II seropositivity (p<0.05, for each). Fetal anti-HLA seropositivity was closely related to maternal anti-HLA seropositivity in both groups (p<0.01, for each). C4d deposition on umbilical vein endothelium was more frequent in preterm labor than term labor (77.1% vs. 11.4%, p<0.001). Logistic regression analysis revealed that chronic chorioamnionitis (OR = 6.10, 95% CI 1.29–28.83), maternal anti-HLA class I seropositivity (OR = 5.90, 95% CI 1.60–21.83), and C4d deposition on umbilical vein endothelium (OR = 36.19, 95% CI 11.42–114.66) were associated with preterm labor and delivery.A major subset of spontaneous preterm births has a signature of maternal anti-fetal cellular and antibody-mediated rejections with links to fetal graft-versus-host disease and alloimmune reactions

Nasal tolerance induces antigen-specific CD4+CD25- regulatory T cells that can transfer their regulatory capacity to naive CD4+ T cells.

The mucosal immune system is uniquely adapted to elicit immune responses against pathogens but also to induce tolerogenic responses to harmless antigens. In mice, nasal application of ovalbumin (OVA) leads to suppression of both T(h)1 and T(h)2 responses. This tolerance can be transferred to naive mice by CD4(+) T(r) cells from the spleen. Using the allotypic Ly5 system, we were able to demonstrate in vivo that T(r) cells not only suppress naive CD4(+) T cells, but also induce them to differentiate into T(r) cells. The effector function of these mucosal T(r) cells is not restricted by cytokine polarization, since T(r) cells from T(h)1-tolerant mice can suppress a T(h)2 response and vice versa. Transfer of splenic CD4(+)CD25(+) and CD4(+)CD25(-) T cell subsets from OVA-tolerized mice revealed that both subsets were equally able to suppress a delayed-type hypersensitivity response in acceptor mice. In contrast to the CD25(-) T cell subset, the CD25(+) cells were not specific for the antigen used for tolerization. Together, these findings demonstrate a role for CD4(+)CD25(-) T(r) cells in mucosal tolerance, which suppresses CD4(+) T cells in an antigen-specific fashion, irrespective of initial T(h)1/T(h)2 skewing of the immune response. This offers a major advantage in the manipulation of mucosal tolerance for the treatment of highly cytokine-polarized disorders such as asthma and autoimmune diseases

Proteases and Protease-activated receptors signalling: at the crossroads of acquired and innate immunity

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71652/1/j.1365-2222.2007.02738.x.pd