Identification of unique proteomic signatures in allergic and non-allergic skin disease.

BACKGROUND: Atopic dermatitis (AD), psoriasis (PS), and contact dermatitis (CD) are common skin diseases, characterized by barrier disruption and systemic inflammation, with unique epidermal signatures and common inflammatory pathways identified by transcriptomic profiling. This study profiled proteomic signatures in serum from subjects with AD, PS, and CD compared with healthy controls (HC). OBJECTIVE: Identify unique proteomic signatures to distinguish between inflammatory diseases with similar epidermal disruption and overlapping epithelial inflammation. METHODS: Sera from 20 subjects with moderate to severe AD, 10 subjects with CD, 12 subjects with moderate to severe PS, 10 subjects with both AD and CD, and 10 HC with no history of skin disease was analysed using high-throughput proteomic analysis that detects expression of 1129 protein targets. Protein expression was compared between disease and HC, and across diseases for statistical significance (fold change≥1.5 and false discovery rate≤0.05), to identify unique proteomic signatures for each disease. RESULTS: Complement C5A anaphylatoxin (C5A), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), ILT-4, C-C motif ligand 18 (PARC), and sialic acid-binding Ig-like lectin 14 (SIG14) were significantly modulated in all three diseases compared with HC. We identified unique signatures for AD (Immunoglobulin E (IgE), thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC)), CD (10 proteins), and PS (kynureninase (KYNU)). Proteomic profiling in subjects with both AD and CD identified additional dysregulated proteins compared with subjects with either condition alone, indicating an exacerbated inflammation reaction. CONCLUSIONS AND CLINICAL RELEVANCE: Unique sera proteomic signatures may distinguish between inflammatory skin diseases despite similar epidermal barrier disruption and epithelial inflammation. This may provide insight into disease pathogenesis, diagnosis, and therapeutic intervention in difficult-to-treat subjects.

How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy.

Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 because of predominant skipping of exon 7 during pre-mRNA splicing. With the recent US Food and Drug Administration approval of nusinersen (Spinraza), the potential for correction of SMN2 exon 7 splicing as an SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here, we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions.

Vitamin D in clinically isolated syndrome: evidence for possible neuroprotection.

BACKGROUND AND PURPOSE: Vitamin D status has been associated with inflammatory activity in multiple sclerosis (MS), but it is not known if it is associated with gray matter volume, the loss of which predicts long-term disability in MS. The association of vitamin D levels with brain volume measures and inflammatory activity in patients with clinically isolated syndrome (CIS) was investigated. METHODS: In the phase 2 CIS trial of atorvastatin, 25-hydroxyvitamin D levels were evaluated for their age-adjusted associations with normalized gray matter and brain parenchymal volumes on brain magnetic resonance imaging (MRI). The relationships between 25-hydroxyvitamin D levels and clinical and MRI measures of inflammatory activity were also assessed. RESULTS: In 65 patients in this substudy, each 25 nmol/l higher 25-hydroxyvitamin D level was associated with 7.8 ml higher gray matter volume (95% confidence interval 1.0, 14.6, P = 0.025). There was a tendency for an inverse association of average 25-hydroxyvitamin D levels and the composite end-point of ≥3 new brain T2 lesions or ≥1 relapse within a year (odds ratio per 25 nmol/l higher 25-hydroxyvitamin D level 0.66, 95% confidence interval 0.41, 1.08, P = 0.096). CONCLUSIONS: Vitamin D status may impact neurodegeneration after CIS, although these results should be replicated in a second study. If confirmed in clinical trials, vitamin D supplementation may reduce long-term disability.

Past, present, and future for biologic intervention in atopic dermatitis.

Atopic dermatitis (AD) is a debilitating disease that significantly alters the quality of life for one in four children and one in 10 adults. Current management of AD utilizes combinations of treatments to symptomatically alleviate disease by suppressing the inflammatory response and restoring barrier function in the skin, reducing disease exacerbation and flare, and preventing secondary skin infections. Resolution is temporary and long-term usage of these treatments can be associated with significant side-effects. Antibody therapies previously approved for inflammatory diseases have been opportunistically evaluated in patients with atopic dermatitis; however, they often failed to demonstrate a significant clinical benefit. Monoclonal antibodies currently in development offer hope to those individuals suffering from the disease by specifically targeting immune and molecular pathways important for the pathogenesis of atopic dermatitis. Here, we review the underlying biological pathways and the state of the art in therapeutics in AD.

Lectican proteoglycans, their cleaving metalloproteinases, and plasticity in the central nervous system extracellular microenvironment.

The extracellular matrix (ECM) in the central nervous system actively orchestrates and modulates changes in neural structure and function in response to experience, after injury, during disease, and with changes in neuronal activity. A component of the multi-protein, ECM aggregate in brain, the chondroitin sulfate (CS)-bearing proteoglycans (PGs) known as lecticans, inhibit neurite outgrowth, alter dendritic spine shape, elicit closure of critical period plasticity, and block target reinnervation and functional recovery after injury as the major component of a glial scar. While removal of the CS chains from lecticans with chondroitinase ABC improves plasticity, proteolytic cleavage of the lectican core protein may change the conformation of the matrix aggregate and also modulate neural plasticity. This review centers on the roles of the lecticans and the endogenous metalloproteinase families that proteolytically cleave lectican core proteins, the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), in neural plasticity. These extracellular metalloproteinases modulate structural neural plasticity-including changes in neurite outgrowth and dendritic spine remodeling-and synaptic plasticity. Some of these actions have been demonstrated to occur via cleavage of the PG core protein. Other actions of the proteases include cleavage of non-matrix substrate proteins, whereas still other actions may occur directly at the cell surface without proteolytic cleavage. The data convincingly demonstrate that metalloproteinases modulate physiological and pathophysiological neural plasticity.

The association of lactate and vasopressor need for mortality prediction in survivors of cardiac arrest.

BACKGROUND: Currently there are few tools available for clinicians to predict outcomes in cardiac arrest survivors. Our objective was to determine if the combination of simple clinical parameters (initial blood lactate and vasopressor use) can predict outcome in post-cardiac arrest patients. METHODS: The design was a retrospective medical record review. The study was carried on in two urban, tertiary-care, university teaching hospitals. As for patients, inclusion criteria were: 1) age ≥18 years; 2) non-traumatic out-of-hospital cardiac arrest with return of spontaneous circulation; 3) lactic acid measured within one hour of return of circulation. No interventions was performed. RESULTS: Patients were divided into groups based on two variables: 1) vasopressor status (receipt of vasopressors vs. no vasopressors); and 2) initial blood lactate (categories defined as lactate <5 mmol/L, lactate 5 to 10 mmol/L, lactate ≥10 mmol/L); 128 out-of-hospital cardiac arrest patients met study inclusion criteria. Overall mortality was 71% (95%CI 63-79%). Patients who received vasopressors had significantly higher mortality rates compared to patients who did not receive vasopressors (80% vs. 52%; P=0.002). A stepwise increase in mortality is associated with increasing lactate levels (39% lactate <5 mmol/L, 67% lactate 5 mmol/L to10 mmol/L, and 92% lactate ≥10 mmol/L; P<0.001). The AUC for our model was 0.82. CONCLUSION: The combination of two clinical parameters, vasopressor need and lactic acid levels, is an accurate severity of illness classification system and can predict mortality in patients following out-of-hospital cardiac arrest. Prospective validation of these variables in post-cardiac arrest is needed.

Human factors in resuscitation: Lessons learned from simulator studies.

Medical algorithms, technical skills, and repeated training are the classical cornerstones for successful cardiopulmonary resuscitation (CPR). Increasing evidence suggests that human factors, including team interaction, communication, and leadership, also influence the performance of CPR. Guidelines, however, do not yet include these human factors, partly because of the difficulties of their measurement in real-life cardiac arrest. Recently, clinical studies of cardiac arrest scenarios with high-fidelity video-assisted simulations have provided opportunities to better delineate the influence of human factors on resuscitation team performance. This review focuses on evidence from simulator studies that focus on human factors and their influence on the performance of resuscitation teams. Similar to studies in real patients, simulated cardiac arrest scenarios revealed many unnecessary interruptions of CPR as well as significant delays in defibrillation. These studies also showed that human factors play a major role in these shortcomings and that the medical performance depends on the quality of leadership and team-structuring. Moreover, simulated video-taped medical emergencies revealed that a substantial part of information transfer during communication is erroneous. Understanding the impact of human factors on the performance of a complex medical intervention like resuscitation requires detailed, second-by-second, analysis of factors involving the patient, resuscitative equipment such as the defibrillator, and all team members. Thus, high-fidelity simulator studies provide an important research method in this challenging field.

Congestive heart failure and outpatient risk of venous thromboembolism: a retrospective, case-control study.

Although CHF has been considered a risk factor for venous thromboembolism, this has not been directly studied. We hypothesized that congestive heart failure would increase the risk of venous thromboembolism in an outpatient population, and that this risk would increase as patients' ventricular function worsened. We conducted a case-control study to examine whether CHF due to left ventricular dysfunction was an independent risk factor for acute venous thromboembolism in outpatients, once established risk factors such as recent surgery and prior venous thromboembolism are taken into account. We reviewed 106 cases of DVT and 603 controls, admitted for diabetes mellitus or infection, matched for month of admission at a VA hospital. Assignment of a diagnosis of venous thromboembolism required a definitive test, as did classification as CHF. In a logistic regression model CHF was an independent predictor of venous thromboembolism. A second logistic regression model showed that the risk of venous thromboembolism increased as the ejection fraction (EF) decreased, with an EF < 20 associated with a venous thromboembolism OR of 38.3 (95% CI 9.6, 152.5). CHF is an independent risk factor for venous thromboembolism, and the risk increases markedly as the EF decreases. These results support the use of anticoagulation in selected patients with CHF.

Soluble tumor necrosis factor receptor type I enhances tumor development and persistence in vivo.

Secretion of human soluble tumor necrosis factor receptor type I (sTNFRI) by the mouse fibrosarcoma cell line, L929, previously has been demonstrated to confer resistance to in vitro lysis by TNF and to LAK- and CTL-mediated cytolysis. These findings suggest that, in vivo, sTNFRI contributes to tumor survival by inhibiting these immunologic mechanisms. To evaluate this hypothesis, we compared the growth of sTNFRI-secreting L929 cells with that of the unmodified parental fibrosarcoma in an in vivo mouse transplantation model. Secretion of sTNFRI by L929 cells markedly enhanced their tumorigenicity and persistence in syngeneic recipients. This benefit was abrogated by sTNFRI-neutralizing antibodies induced by immunization prior to tumor challenge. These data demonstrate that sTNFRI directly influences tumor formation and persistence in vivo and suggest the selective removal and/or inactivation of sTNFRI as a promising new avenue for cancer immunotherapy.

Comparison of a maedi-visna virus CA-TM fusion protein ELISA with other assays for detecting sheep infected with North American ovine lentivirus strains.

A maedi-visna virus CA-TM fusion protein ELISA (MVV ELISA) was evaluated for the detection of antibody in sheep infected with North American ovine lentivirus (OvLV). The results of the MVV ELISA were compared with other assays for OvLV antibody and with viral infection in an intensively studied group of 38 sheep with a high prevalence of OvLV infection and disease. The sensitivity, specificity, and concordance of assays for OvLV antibody (MVV ELISA, indirect ELISA, Western blot, and AGID), virus (virus isolation, PCR, antigen ELISA), and OvLV-induced disease in each animal were compared with OvLV infection status as defined by a positive result in two or more of the assays. Five sheep met the criteria for absence of OvLV infection. The sensitivity of the MVV ELISA in detecting OvLV infected sheep was 64%, whereas the sensitivity of the other three tests for antibody ranged from 85 to 94%. All the antibody assays were 100% specific in this group of animals. Of the assays for virus, the PCR test had the highest sensitivity and the best concordance with OvLV infection, but it also had the lowest specificity of any of the virus or antibody assays. Among the antibody tests, the concordance of the MVV ELISA compared most favorably with the AGID test for detecting OvLV-infected sheep. Analysis of serum samples from 28 lambs experimentally-infected with one of three North American strains of OvLV suggested that there were no significant strain differences detectable by antibody assay. Twenty virus-inoculated lambs were positive by both the MVV ELISA and the AGID test, five lambs were MVV ELISA negative and AGID test positive, and three lambs were MVV ELISA positive and AGID test negative. No pre-inoculation samples were positive by either assay. In a longitudinal study involving seven lambs, antibodies to OvLV were detected by AGID 3-5 weeks post-inoculation, but were not detected by MVV ELISA until 5-10 weeks post-inoculation. Among 128 naturally and experimentally-infected sheep that were seropositive in the AGID test, the overall sensitivity of the MVV ELISA was higher in the naturally infected sheep (84%) than in the experimentally infected sheep (69%). The data indicated that the MVV ELISA represents a less sensitive, but specific alternative for the detection of OvLV antibodies.

Analysis of sheep T-cell receptor beta-chain heterogeneity.

We analyzed nucleotide and deduced amino acid sequence heterogeneity of sheep T-cell receptor beta-chain cDNAs isolated from an anchored-polymerase chain reaction library. Evaluation of 34 individual rearrangements has defined 18 new beta-chain variable region sequences which have been clustered into 13 families. Presumptive allelic polymorphisms of four of these variable regions have been defined, as well as ten distinct beta-chain joining region sequences. The present analysis indicates that sheep T-cell receptor beta-chains are composed of characteristic leader, variable, joining, and constant region sequences, and that imprecise joining and N-region addition contribute significantly to diversity in the third hypervariable region. Thus, it appears that sheep, like all other mammals studied to date, employ somatic rearrangement of multiple germline genes to create beta-chain heterogeneity. These findings have allowed us to estimate the diversity of the sheep T-cell receptor beta-chain variable region repertoire, and they provide information that will permit the evaluation of the role that specific T-cell populations play in naturally occurring and experimental diseases of sheep.

Multifaceted inhibition of anti-tumour immune mechanisms by soluble tumour necrosis factor receptor type I.

Soluble tumour necrosis factor receptor type I (sTNFRI) is a potent inhibitor of TNF with the potential to suppress a variety of effector mechanisms important in tumour immunity. That sTNFRI influences tumour survival in vivo is suggested by results from human clinical trials of Ultrapheresis, an experimental extracorporeal treatment for cancer. While the considerable clinical benefit provided by Ultrapheresis is correlated with the removal of plasma sTNFRI, there is no direct evidence that sTNFRI inhibits immune mechanisms which mediate tumour cell elimination. To evaluate formally the ability of sTNFRI to inhibit these mechanisms, we have engineered sTNFRI production into the TNF-sensitive murine fibrosarcoma cell line, L929. Soluble TNFRI-secreting L929 cells display increased resistance to direct lysis by TNF, and to lysis by syngeneic lymphokine-activated killer cells and cytotoxic T cells. These findings confirm the suggestion that sTNFRI inhibits immunological mechanisms important in tumour cell eradication, and further support a role for sTNFRI in tumour survival in vivo. In addition, these observations suggest the development of methods for more specific removal and/or inactivation of sTNFRI as promising new avenues for cancer immunotherapy.

Neonatal appendectomy impairs mucosal immunity in rabbits.

We compared the effects of neonatal appendectomy in rabbits on total Ig and antigen (Ag)-specific Ig levels in the serum and gut, and on plasma cell numbers in the small intestine in response to intraperitoneal (i.p.) and intraduodenal (i.d.) immunizations with ovalbumin (OVA). Animals were sacrificed after 9 weeks. Antibodies (Abs) in the duodenum were collected and quantified by enzyme-linked immunosorbent assay (ELISA) while plasma cells were quantified by double immunofluorescent staining. Appendectomy markedly reduced total intestinal IgA (P < 0.0006), IgM (P < 0.003), and IgG (P < 0.05) relative to controls, whereas total serum Ig levels were not lowered significantly. Moreover, appendectomy nearly ablated OVA-specific IgA (P < 0.007) in the gut and severely depleted OVA-specific IgG in the gut (P < 0.03) and serum (P < 0.007). The sharp decreases in total IgA and anti-OVA IgA were paralleled by decreases in total IgA+ plasma cells (P < 0.0005) and OVA-reactive IgA+ plasma cells (P < 0.05). These results support a major role of the rabbit appendix in seeding the intestinal lamina propria with plasma cell precursors, especially those producing IgA.

Homologies between T cell receptor junctional sequences unique to multiple sclerosis and T cells mediating experimental allergic encephalomyelitis.

The selection of T cell clones with mutations in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene has been used to isolate T cells reactive to myelin basic protein (MBP) in patients with multiple sclerosis (MS). These T cell clones are activated in vivo, and are not found in healthy individuals. The third complementarity determining regions (CDR3) of the T cell receptor (TCR) alpha and beta chains are the putative contact sites for peptide fragments of MBP bound in the groove of the HLA molecule. The TCR V gene usage and CDR3s of these MBP-reactive hprt-T cell clones are homologous to TCRs from other T cells relevant to MS, including T cells causing experimental allergic encephalomyelitis (EAE) and T cells found in brain lesions and in the cerebrospinal fluid (CSF) of MS patients. In vivo activated MBP-reactive T cells in MS patients may be critical in the pathogenesis of MS.

T cell receptors, immunoregulation, and autoimmunity.

T cell receptor (TCR) peptide vaccines have proven useful in the prevention and treatment of autoimmune disease in animal models. Prospects for developing TCR peptide vaccines for human autoimmune disease are only now being explored. Preliminary indications provide cause for optimism that immunization with TCR peptides eventually will be a viable treatment option for autoimmune pathologies in humans. In the long term, development of this technology may permit reliable manipulation of T cell immunity, leading to treatments for autoimmunity, T lymphoproliferative disorders, and, in the broadest interpretation, any pathogenesis mediated by oligoclonal T cell populations.

Immunoregulation of autoimmune disease by vaccination with T cell receptor peptides.

Restricted TCR gene usage in animal models of autoimmune disease has led to strategies for control of these diseases by targeting the idiotypic determinants within the TCR sequence. Rats can be rendered resistant to EAE by immunization with synthetic peptides representing sequences contained within the V beta, J alpha and VDJ beta regions of the TCR that are conserved among encephalitogenic T cells. We propose that the mechanism of immunoregulation thus produced results from the stimulation of an anticlonotypic response directed at endogenously synthesized TCR peptides presented by Class I MHC on the surface of the autoreactive T cell, and that this mechanism may be part of the natural immunoregulation of T cell responses. The experimental data demonstrate the utility of this therapeutic approach and its potential for treatment of any pathogenic condition mediated by specific, oligoclonal T cell populations.

Limited T-cell receptor beta-chain heterogeneity among interleukin 2 receptor-positive synovial T cells suggests a role for superantigen in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disease affecting the synovial membranes of articulating joints that is thought to result from T-cell-mediated autoimmune phenomena. T cells responsible for the pathogenesis of RA are likely present in that fraction of synovial T cells that expresses the interleukin 2 receptor (IL-2R), one marker of T-cell activation. We report herein an analysis of T-cell receptor (TCR) beta-chain gene expression by IL-2R-positive synovial T cells. These T cells were isolated from uncultured synovial tissue specimens by using IL-2R-specific monoclonal antibodies and magnetic beads, and TCR beta-chain transcription was analyzed by PCR-catalyzed amplification using a panel of primers specific for the human TCR beta-chain variable region (V beta). Multiple V beta gene families were found to be transcribed in these patients samples; however, three gene families, V beta 3, V beta 14, and V beta 17, were found in a majority of the five synovial samples analyzed, suggesting that T cells bearing these V beta s had been selectively retained in the synovial microenvironment. In many instances, the V beta 3, V beta 14, or V beta 17 repertoires amplified from an individual patient were dominated by a single rearrangement, indicative of clonal expansion in the synovium and supportive of a role for these T cells in RA. Of note is a high sequence similarity between V beta 3, V beta 14, and V beta 17 polypeptides, particularly in the fourth complementarity-determining region (CDR). Given that binding sites for superantigens have been mapped to the CDR4s of TCR beta chains, the synovial localization of T cells bearing V beta s with significant CDR4 homology indicates that V beta-specific T-cell activation by superantigen may play a role in RA.

A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis.

We have examined previously the peptide specificity of the T cell response to myelin basic protein (MBP) in patients with multiple sclerosis (MS) and healthy controls, and demonstrated that an epitope spanning amino acids 87-106 was frequently recognized. Because this region is encephalitogenic in some experimental animals, it has been postulated that the response to the epitope may have relevance to MS. In this study, the fine specificity of this response is studied using four well-characterized, monospecific T cell lines from three MS patients and an identical twin of a patient. Each of the lines recognized a peptide with the same core sequence, amino acids 89-99, although the responses were affected to various degrees by truncations at the COOH- or NH2 terminal ends of the 87-106 epitope. Importantly, the epitope was recognized in conjunction with four different HLA-DR molecules. Also, the T cell receptor beta chain usage was heterogeneous, and each line expressed a different VDJ sequence. The four HLA-DR molecules restricting the response to this epitope have been shown to be overrepresented in MS populations in various geographic areas, suggesting that the response to this region of the MBP molecule may be relevant to the pathogenesis of MS. These findings may have important implications in designing therapeutic strategies for the disease.

Vaccination against experimental allergic encephalomyelitis with T cell receptor peptides.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by CD4+ T cells reactive with myelin basic protein (MBP). Rats were rendered resistant to the induction of EAE by vaccination with synthetic peptides corresponding to idiotypic determinants of the beta chain VDJ region and J alpha regions of the T cell receptor (TCR) that are conserved among encephalitogenic T cells. These findings demonstrate the utility of TCR peptide vaccination for modulating the activity of autoreactive T cells and represent a general therapeutic approach for T cell-mediated pathogenesis.

An extended HLA-D region haplotype associated with celiac disease.

Celiac disease has one of the strongest associations with HLA (human leukocyte antigen) class II markers of the known HLA-linked diseases. This association is primarily with the class II serologic specificities HLA-DR3 and -DQw2. We previously described a restriction fragment length polymorphism (RFLP) characterized by the presence of a 4.0-kilobase Rsa I fragment derived from an HLA class II beta-chain gene, which distinguishes the class II HLA haplotype of celiac disease patients from those of many serologically matched controls. We now report the isolation of this beta-chain gene from a bacteriophage genomic library constructed from the DNA of a celiac disease patient. Based on restriction mapping and differential hybridization with class II cDNA and oligonucleotide probes, this gene was identified as one encoding an HLA-DP beta chain. This celiac disease-associated HLA-DP beta-chain gene was flanked by HLA-DP alpha-chain genes and, therefore, was probably in its normal chromosomal location. The HLA-DP alpha-chain genes of celiac disease patients also were studied by RFLP analysis; 84% of HLA-DR3, -DQw2 patients had a 16-kb Xba I fragment that was present in only 36% of HLA-DR3, -DQw2 controls. Moreover, 79% of these patients had both alpha- and beta-chain polymorphisms in contrast to 27% of controls. Thus, celiac disease is associated with a subset of HLA-DR3, -DQw2 haplotypes characterized by HLA-DP alpha- and beta-chain gene RFLPs. Within the celiac-disease patient population, the joint segregation of these HLA-DP genes with those encoding the serologic specificities HLA-DR3 and -DQw2 indicates: (i) that the class II HLA haplotype associated with celiac disease is extended throughout the entire HLA-D region, and (ii) that celiac-disease susceptibility genes may reside as far centromeric on this haplotype as the HLA-DP subregion.