The diabetic antigen glutamic acid decarboxylase (GAD 65) in the human peripheral blood.

BACKGROUND: Glutamic acid decarboxylase (GAD 65) is a diabetes-associated antigen which is generally considered to be strictly intracellular. In order to better understand autoimmunity, this study demonstrates the appearance of GAD 65 in the peripheral human blood and presents implications for the diagnosis and therapy of some autoimmune diseases. METHODS: The GAD 65 molecules are detected by their interaction with monoclonal antibodies labeled with dyes in an experimental setup with fluorescence correlation spectroscopy (FCS). These interactions result in changes in Brownian motion measured as fluorescence fluctuations. Sera from 153 patients with diabetes mellitus type 1 and controls were investigated. To enable the representation of the molecule as a model for further discussions, we present structural visualizations of its hydrophobic properties, leading to possible interactions with the cell membrane lipids and epitope locations. RESULTS: The GAD65 antigen could be measured with a sensitivity of 2.65 microg/ml in 'clean systems' resulting from spiking experiments and human sera. The GAD 65 antigen could be identified in 8 patient sera: 4 children with diabetes mellitus type 1 and 4 adults initially taken as controls but who retrospectively showed signs of autoimmunity. CONCLUSION: We conclude that these findings are of significance for the concept of autoimmunity, i.e. in an initial step the immune system is primed by its accessibility to GAD 65. Our experimental results may also be important for the therapy of diabetes mellitus type 1 and other autoimmune diseases by the passive administration of GAD 65 antibodies.

Expression of HLA-G in inflammatory bowel disease provides a potential way to distinguish between ulcerative colitis and Crohn's disease.

In addition to being involved in nutrient uptake, the epithelial mucosa constitute the first line of defense against microbial pathogens. A direct consequence of this physiological function is a very complex network of immunological interactions that lead to a strong control of the mucosal immune balance. The dysfunction of immunological tolerance is likely to be a cause of inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD). HLA-G is a non-classical major histocompatibility complex (HLA) class I molecule, which is highly expressed by human cytotrophoblast cells. These cells play a role in immune tolerance by protecting trophoblasts from being killed by uterine NK cells. Because of the deregulation of immune system activity in IBD, as well as the immunoregulatory role of HLA-G, we have analyzed the expression of HLA-G in intestinal biopsies of patients with UC and CD. Our study shows that the differential expression of HLA-G provides a potential way to distinguish between UC and CD. Although the reason for this differential expression is unclear, it might involve a different mechanism of immune regulation. In addition, we demonstrate that in the lamina propria of the colon of patients with UC, IL-10 is strongly expressed. In conclusion, the presence of HLA-G on the surface of intestinal epithelial cell in patients with UC lends support to the notion that this molecule may serve as a regulator of mucosal immune responses to antigens of undefined origin. Thus, this different pattern of HLA-G expression may help to differentiate between the immunopathogenesis of CD and UC.

HLA-G protein processing and transport to the cell surface.

Data are presented on the intracellular trafficking of HLA-G protein, taking the unique features of this non-classical molecule into consideration: the existence of seven isoforms resulting from alternative splicing (HLA-G1 to G7), and reduced tail length compared with HLA class I antigens. Biochemical studies and analysis of viral strategies for escaping the host immune system led to the demonstration that (i) both the membrane-bound (HLA-G1) and the soluble (HLA-G5) forms of the molecule require peptide association for cell surface expression, using TAP-dependent or TAP-independent pathways; (ii) peptide loading onto the HLA-G protein plays a critical role in controlling the quality of the molecule reaching the cell surface; (iii) surface expression of truncated HLA-G molecules is possible, and (iv) HLA-G expression may be restricted to soluble HLA-G5. These data reveal that HLA-G presents specific cell trafficking pathways and strongly support the contention that the primary function of HLA-G is as of an inhibitor ligand for immune-competent cells.

Expanded polyglutamines in Caenorhabditis elegans cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death.

Huntington's disease (HD) is a dominant neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). HD pathogenesis appears to involve the production of mutated N-terminal htt, cytoplasmic and nuclear aggregation of htt, and abnormal activity of htt interactor proteins essential to neuronal survival. Before cell death, neuronal dysfunction may be an important step of HD pathogenesis. To explore polyQ-mediated neuronal toxicity, we expressed the first 57 amino acids of human htt containing normal [19 Gln residues (Glns)] and expanded (88 or 128 Glns) polyQ fused to fluorescent marker proteins in the six touch receptor neurons of Caenorhabditis elegans. Expanded polyQ produced touch insensitivity in young adults. Noticeably, only 28 +/- 6% of animals with 128 Glns were touch sensitive in the tail, as mediated by the PLM neurons. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins with 19, 88, and 128 Glns were observed. In contrast, significant deposits and morphological abnormalities in PLM cell axons were observed with expanded polyQ (128 Glns) and partially correlated with touch insensitivity. PLM cell death was not detected in young or old adults. These animals indicate that significant neuronal dysfunction without cell death may be induced by expanded polyQ and may correlate with axonal insults, and not cell body aggregates. These animals also provide a suitable model to perform in vivo suppression of polyQ-mediated neuronal dysfunction.

Soluble HLA-G protein secreted by allo-specific CD4+ T cells suppresses the allo-proliferative response: a CD4+ T cell regulatory mechanism.

We recently reported that the nonclassical HLA class I molecule HLA-G was expressed in the endomyocardial biopsies and sera of 16% of heart transplant patients studied. The aim of the present report is to identify cells that may be responsible for HLA-G protein expression during the allogeneic reaction. Carrying out mixed lymphocyte cultures in which the responder cell population was depleted either in CD4(+) or CD8(+) T cells, we found that soluble HLA-G5 protein but not the membrane-bound HLA-G isoform was secreted by allo-specific CD4(+) T cells from the responder population, which suppressed the allogeneic proliferative T cell response. This inhibition may be reversed by adding the anti-HLA-G 87G antibody to a mixed lymphocyte culture. That may indicate a previously uncharacterized regulatory mechanism of CD4(+) T cell proliferative response.

HLA-G2, -G3, and -G4 isoforms expressed as nonmature cell surface glycoproteins inhibit NK and antigen-specific CTL cytolysis.

HLA-G is a nonclassical MHC class I molecule that plays a major role in maternal-fetal tolerance. Four membrane-bound (HLA-G1 to -G4) and two soluble (HLA-G5, and -G6) proteins are generated by alternative splicing. Only HLA-G1 has been extensively studied in terms of both expression and function. We provide evidence here that HLA-G2, -G3, and -G4 truncated isoforms reach the cell surface of transfected cells, as endoglycosidase H-sensitive glycoproteins, after a 2-h chase period. Moreover, cytotoxicity experiments show that these transfected cells are protected from the lytic activity of both innate (NK cells) and acquired (CTL) effectors. These findings highlight the immunomodulatory role that HLA-G2, -G3, and -G4 proteins will assume during physiologic or pathologic processes in which HLA-G1 expression is altered.

The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the protein huntingtin (htt). Pathogenesis in HD appears to involve the formation of ubiquitinated neuronal intranuclear inclusions containing N-terminal mutated htt, abnormal protein interactions, and the aggregate sequestration of a variety of proteins (noticeably, transcription factors). To identify novel htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library. We found a predicted WW domain protein (ZK1127.9) that interacts with N-terminal fragments of htt in two-hybrid tests. A human homologue of ZK1127.9 is CA150, a transcriptional coactivator with a N-terminal insertion that contains an imperfect (Gln-Ala)(38) tract encoded by a polymorphic repeat DNA. CA150 interacted in vitro with full-length htt from lymphoblastoid cells. The expression of CA150, measured immunohistochemically, was markedly increased in human HD brain tissue compared with normal age-matched human brain tissue, and CA150 showed aggregate formation with partial colocalization to ubiquitin-positive aggregates. In 432 HD patients, the CA150 repeat length explains a small, but statistically significant, amount of the variability in the onset age. Our data suggest that abnormal expression of CA150, mediated by interaction with polyglutamine-expanded htt, may alter transcription and have a role in HD pathogenesis.

Spontaneous retinopathy in HLA-A29 transgenic mice.

Humans who have inherited the class I major histocompatibility allele HLA-A29 have a markedly increased relative risk of developing the eye disease termed birdshot chorioretinopathy. This disease affecting adults is characterized by symmetrically scattered, small, cream-colored spots in the fundus associated with retinal vasculopathy and inflammatory signs causing damage to the ocular structures, leading regularly to visual loss. To investigate the role of HLA-A29 in this disease, we introduced the HLA-A29 gene into mice. Aging HLA-A29 transgenic mice spontaneously developed retinopathy, showing a striking resemblance to the HLA-A29-associated chorioretinopathy. These results strongly suggest that HLA-A29 is involved in the pathogenesis of this disease. Elucidation of the role of HLA-A29 should be assisted by this transgenic model.

HLA-G1 co-expression boosts the HLA class I-mediated NK lysis inhibition.

It is now acknowledged that the pattern of HLA-G expression is not restricted to extravillous cytotrophoblast cells, as several studies described HLA-G in HLA class I+ cells, such as thymic epithelial cells, cytokine-activated monocytes and some tumors. In these situations, HLA-G may provide an additional inhibitory signal to escape from NK cell-mediated cytotoxicity. Accordingly, the aim of this study was to define the behavior of HLA-G once it is co-expressed into an HLA-A, -B, -C and -E+ cell line. For this purpose, HLA-G1 cDNA was transfected into an HLA class I+ melanoma cell line which was used as a target towards freshly isolated peripheral blood NK cells. Cytotoxic experiments using either anti-HLA-G1 or anti-HLA-G1 inhibitory receptor mAb show that HLA-G1 boosts the HLA class I-mediated inhibition of polyclonal NK cells through interaction with ILT-2, which appears as the major HLA-G1 inhibitory receptor involved. Nevertheless, HLA-G1 is also able to inhibit the cytolytic activity of an ILT-2- NK clone which otherwise expresses another HLA-G1 inhibitory receptor belonging to the KIR103 gene family. In order to more precisely define the relative role exerted by HLA-G1 versus -E on polyclonal NK cells, antibody-blocking assays were carried out using either anti-HLA class I or anti-CD94/NKG2A. Results demonstrate that in the absence of HLA-G1, the naturally expressed HLA class I-mediated NK inhibition is predominantly exerted by HLA-E through binding with CD94/NKG2A. In contrast, once HLA-G1 is expressed, it becomes the major NK inhibitory ligand.

Hla-g hoy / HLA-G today

HLA-G es una molécula del complejo mayor de histocompatibilidad tipo I no clásica que se expresa de forma selectiva en los citotrofoblastos en la interfase feto-materna , los cuales pierden la expresión de moléculas tipo I clásicas como HLA-A y HLA-B. Esta molécula ejerce un papel protector frente a la actividad lítica llevada a cabo por las células Natural Killer (NK) de la decidua uterina, protegiendo al feto de ser rechazado por la madre y creando un estado de tolerancia. HLA-G exhibe una distribución tisular re stringida, un polimorfismo limitado. Se transcribe en 7 isoformas, de las cuales 4 aparecen ligadas a membrana (G1G4) y las otras 3 son formas solubles (G5-G7). Todas las isoformas de HLA-G inhiben la citolisis de células mononucleares de sangre periférica mediadas por células NK.HLA-G inhibe la función citotóxica de células T durante la reacción primaria alogénica. Las secuencias reguladoras implicadas en la expresión génica de HLA-I clásica y HLAII (particularmente la caja XI unida al complejo RFX) están también implicadas en el mecanismo de transcripción de HLA-G. En tumores, la expresión de HLA-G le confiere protección frente a la actividad lítica de células NK y T, pudiendo impedir la eliminación de las células tumorales por parte de las células efectoras. En trasplantes cardiacos, la presencia de HLA-G reduce de forma significativa los rechazos agudos y muestra una total ausencia de los rechazos crónicos. En definitiva, podemos considerar a HLA-G como una molécula implicada en la tolerancia inmunitaria (AU)

Altered HLA-G transcription in pre-eclampsia is associated with allele specific inheritance: possible role of the HLA-G gene in susceptibility to the disease.

Pre-eclampsia is a disorder of human pregnancy occurring in 5-10% of all births, and represents the leading cause of infant morbidity and mortality and maternal death. In pre-eclampsia, invasion of fetal trophoblasts into maternal arteries during early pregnancy is shallow or absent. Here we examined the hypothesis that HLA-G, a non-classical class I HLA expressed in cytotrophoblasts, may act as a key gene in pre-eclampsia. We analysed HLA-G at the level of transcription and genotyped a silent CAC-CAT polymorphism in exon 3 and a 14-bp insertion/deletion in the 3' untranslated region. A deficit in levels of the HLA-G3 transcript was observed in mild pre-eclampsia compared to normal placentas. The distribution of HLA-G polymorphisms was different between normal and pre-eclampsia samples. A correlation between the alteration in transcription of the HLA-G gene and certain HLA-G genotypes was also observed. Thus we provide the first evidence for a possible role of HLA-G in genetic susceptibility to, and pathogenesis of pre-eclampsia.

A specific interferon (IFN)-stimulated response element of the distal HLA-G promoter binds IFN-regulatory factor 1 and mediates enhancement of this nonclassical class I gene by IFN-beta.

Type I interferons display a broad range of immunomodulatory functions. Interferon beta increases gene expression at the transcriptional level through binding of factors to the interferon-stimulated response element (ISRE) within the promoters of interferon-inducible genes, such as HLA class I. Despite mutation of the class I ISRE sequence within the nonclassical HLA-G class I gene promoter, we show that interferon beta enhances both transcription and cell surface expression of HLA-G in trophoblasts and amniotic and thymic epithelial cells that selectively express it in vivo. Deletion and mutagenesis analysis of a putative interferon-regulatory factor (IRF)-1 binding site within the HLA-G promoter show that HLA-G transactivation is mediated through an ISRE sequence 746 base pairs upstream from ATG, which is distinct from the interferon-responsive element described within proximal classical class I gene promoters. Electrophoretic mobility shift analysis and supershift analysis further demonstrate that interferon-responsive transcription factors, including IRF-1, specifically bind to the HLA-G ISRE. Our results provide evidence that IRF-1 binding to a functional ISRE within the HLA-G promoter mediates interferon beta-induced expression of the HLA-G gene. These observations are of general interest considering the implication of HLA-G in mechanisms of immune escape involved in fetal-maternal tolerance and other immune privilege situations.

Heat shock and arsenite induce expression of the nonclassical class I histocompatibility HLA-G gene in tumor cell lines.

The nonclassical histocompatibility class I gene HLA-G has a tissue-restricted expression. To explore mechanisms involved in HLA-G transcriptional regulation, we have investigated the effect of stress, including heat shock and arsenite treatment, on HLA-G expression in tumor cell lines. We show that stress induces an increase of the level of the different HLA-G alternative transcripts without affecting other MHC class I HLA-A, -B, -E, and -F transcripts. A heat shock element (HSE) that binds to heat shock factor 1 (HSF1) on stress conditions was further identified within the HLA-G promoter. Considering the ability of HLA-G to modulate the function of immunocompetent cells, we hypothesize a new feature of HLA-G as a signal regulating the immune response to stress.

Implication of HLA-G molecule in heart-graft acceptance.

HLA-G found in five of 31 heart-transplant recipients was associated with a decrease of acute and chronic rejection episodes.

Human leukocyte antigen-G: immunotolerant major histocompatibility complex molecule in transplantation.

HLA-G is a nonclassical major histocompatibility complex class I molecule selectively expressed on cytotrophoblasts at the fetal-maternal interface, where it plays a role in maternofetal tolerance. In this review, attempts were made to summarize the current state of knowledge of the effects of HLA-G on both natural killer cell and T cell functions and their implications in transplantation.

Historic landmarks in clinical transplantation: conclusions from the consensus conference at the University of California, Los Angeles.

The transplantation of organs, cells, and tissues has burgeoned during the last quarter century, with the development of multiple new specialty fields. However, the basic principles that made this possible were established over a three-decade period, beginning during World War II and ending in 1974. At the historical consensus conference held at UCLA in March 1999, 11 early workers in the basic science or clinical practice of transplantation (or both) reached agreement on the most significant contributions of this era that ultimately made transplantation the robust clinical discipline it is today. These discoveries and achievements are summarized here in six tables and annotated with references.