Rapid screening for the detection of HLA-B57 and HLA-B58 in prevention of drug hypersensitivity.

HLA-B57 and HLA-B58 are major histocompatibility class (MHC)-I allotypes that are potentially predictive of important clinical immune phenotypes. HLA-B*5701 is strongly associated with hypersensitivity to the HIV drug abacavir, liver toxicity from the antibiotic flucloxacillin and is a marker for slow progression of HIV AIDS. HLA-B*5801 is associated with hypersensitivity to allopurinol used to treat hyperuricaemia and recurrent gout. Here we describe a monoclonal antibody (mAb) specific for HLA-B57 and HLA-B58 that provides an inexpensive and sensitive screen for these MHC-I allotypes. The usefulness of HLA-B57 screening for prediction of abacavir hypersensitivity was shown in three independent laboratories, including confirmation of the mAb sensitivity and specificity in a cohort of patients enrolled in the PREDICT-1 trial. Our data show that patients who test negative by mAb screening comprise 90%-95% of all individuals in most human populations and require no further human leukocyte antigen (HLA) typing. Patients who test positive by mAb screening should proceed to high-resolution typing to ascertain the presence of HLA-B*5701 or HLA-B*5801. Hence, mAb screening provides a low-cost alternative to high-resolution typing of all patients and lends itself to point-of-care diagnostics and rapid ascertainment of low-risk patients who can begin immediate therapy with abacavir, flucloxacillin or allopurinol.

Tumour necrosis factor-alpha single nucleotide polymorphisms are not independent of HLA class I in UK Caucasians with adult onset idiopathic inflammatory myopathies.

OBJECTIVE: To investigate haplotype tagging single nucleotide polymorphisms (SNPs) in the tumour necrosis factor alpha (TNF-alpha) gene, in UK Caucasian idiopathic inflammatory myopathy (IIM) patients. METHODS: A cross-sectional, case-control study of four TNF-alpha SNPs was undertaken, comparing cases of polymyositis (PM) (n = 121), dermatomyositis (DM) (n = 109) and myositis overlapping with other connective tissue diseases (CTD-overlap) (n = 73) with normal subjects (n = 177). Subgroup analyses were undertaken after stratifying for myositis specific/associated antibodies. RESULTS: The TNF-308A allele demonstrated a strong association with each myositis disease subgroup vs controls [PM, odds ratio (OR) 2.8, 95% confidence interval 1.9-4.3; DM, OR 2.5, 1.6-3.8; CTD-overlap, OR 3.3, 2.1-5.1]. The TNF-308GA/AA genotype frequency was significantly increased vs controls (PM, OR 3.7, 2.1-6.3; DM, OR 3.2, 1.8-5.5; CTD-overlap, OR 5.0, 2.6-9.6) suggesting a dominant model. The association was strongest in patients possessing anti-aminoacyl transfer RNA synthetase (anti-synthetase) (OR 5.1, 3.3-8.0) or -PM-Scl (OR 5.0, 2.7-8.9) antibodies. The -1031T allele was also a significant risk factor in DM (OR 2.2, 1.4-3.6), anti-synthetase (OR 2.9, 1.6-5.3) and -PM-Scl (OR 5.6, 1.9-6.4) antibody positive patients. The TNF-308A association was lost after adjusting for HLA-B*08, but remained independent of HLA-DQB1*02 (both are alleles forming part of the common ancestral haplotype). The HLA-B*08/TNF-308A/DRB1*03/DQA1*05/DQB1*02 haplotype was a risk factor in all myositis subgroups vs controls (OR 3.0, 1.8-5.3). CONCLUSIONS: TNF-308A and -1031T alleles are significant risk factors in the IIMs. In the IIMs, the TNF-308A allele is part of the common ancestral haplotype, but is not independent of HLA-B*08.

HLA expression and cancer--14th IHIWS immunohistochemistry quality control exercise exchange results.

An immunohistochemistry (IH) quality control exercise was conducted as part of the 14th International HLA (human leukocyte antigen) and Immunogenetics Workshop (IHIWS) HLA Expression and Cancer component. Six laboratories participated and the exercises involved performing IH using three monoclonal antibodies (HC-10, beta2m and SI00) on three sequential paraffin-embedded melanoma sections provided by one laboratory. High-resolution digital photographs of five IH-stained sections were also distributed for interpretation. While there was generally good agreement between laboratories, several differences in staining and interpretation of IH sections were identified and possible reasons given. Interpretation of the high-resolution digital photographs showed a high level of concordance between laboratories. It is suggested that further exercises are conducted as part of future collaborative activities in order to further characterise areas of variability between IH performance and interpretation of results.

Fourier transform infrared imaging as a method for detection of HLA class I expression in melanoma without the use of antibody.

Human leukocyte antigen (HLA) class I expression in melanoma is usually assessed using immunohistochemical staining. Here we report on the use of Fourier transform infrared (FTIR) hyperspectral imaging, a method widely used in two-dimensional analysis of chemical components, to study HLA class I expression in tissue. Two-dimensional cluster colour images derived from unsupervised hierarchical cluster analysis of FTIR hyperspectral data on melanoma sections were compared with consecutive sections that were immunohistochemically stained for class I expression. HLA-class-I-positive and -negative areas were differentiated by FTIR cluster images in all eight melanoma sections investigated without the need for antibody attachment. FTIR imaging enables the distinction of HLA-class-I-positive from class-I-negative areas in melanoma. This method is accurate, rapid and cost-effective.

Analysis of HLA class I alterations in tumors: choosing a strategy based on known patterns of underlying molecular mechanisms.

The application of peptide-based immunotherapy in the treatment of cancer has known limitations in patients with loss or downregulation of human leukocyte antigen (HLA) class I expression on tumor cells. These alterations diminish the ability of cancer cells to present tumor peptides to T cells and therefore lead to failure of peptide-based cancer vaccination. Abnormal expression of HLA class I molecules in malignant cells is a frequent event that ranges from total loss of class I molecules to partial loss of HLA-specific haplotypes or alleles. Different mechanisms underlie these alterations and might require different therapeutic approaches. A complete characterization of molecular defects may suggest strategies for the selection and follow-up of patients undergoing T-cell based immunotherapy. Moreover, a precise identification of the mechanism leading to HLA class I defects in patients with cancer will help develop new, personalized patient-tailored treatment protocols. Here, we describe several examples showing the necessity and feasibility of making detailed individual analysis of HLA alteration mechanisms based on previously described molecular patterns in different types of malignancy. We recommend using this approach, at least in some patients, to enhance the therapeutic benefit of cancer immunotherapy.

Donor methylenetetrahydrofolate reductase genotype is associated with graft-versus-host disease in hematopoietic stem cell transplant patients treated with methotrexate.

Methotrexate (MTX), used as a graft-versus-host disease (GvHD) prophylactic agent in hematopoietic stem cell transplantation (HSCT), exerts its effect via folate cycle inhibition. A critical enzyme involved in folate metabolism is 5,10-methylenetetrahydrofolate reductase (MTHFR). We examined the association of a single nucleotide polymorphism (SNP) at position 677 in the MTHFR gene on GvHD outcomes in allogeneic HSCT patients administered MTX. MTHFR genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 193 HSCT patients and donors. A total of 140 patients were transplanted with an HLA-matched related donor and 53 with an unrelated donor. GvHD outcomes were compared between genotypes by univariate and multivariate analysis. The combined donor 677CT and TT genotypes were associated with a decreased incidence of GvHD (acute and chronic combined) in HSCT recipients with an HLA-matched related donor (75% at 1 year in the CT and TT group compared with 91% in the wild type CC group, P=0.01), increased time to onset of first GvHD (P=0.001) and time to first GvHD treated with systemic therapy (P=0.022). Unrelated donor MTHFR genotype was not associated with outcome parameters and no associations of recipient genotype in either related or unrelated donor cohorts were observed.

4-Hydroxy-5,6-dihydropyrones as inhibitors of HIV protease: the effect of heterocyclic substituents at C-6 on antiviral potency and pharmacokinetic parameters.

Due largely to the emergence of multi-drug-resistant HIV strains, the development of new HIV protease inhibitors remains a high priority for the pharmaceutical industry. Toward this end, we previously identified a 4-hydroxy-5,6-dihydropyrone lead compound (CI-1029, 1) which possesses excellent activity against the protease enzyme, good antiviral efficacy in cellular assays, and promising bioavailability in several animal species. The search for a suitable back-up candidate centered on the replacement of the aniline moiety at C-6 with an appropriately substituted heterocyle. In general, this series of heterocyclic inhibitors displayed good activity (in both enzymatic and cellular tests) and low cellular toxicity; furthermore, several analogues exhibited improved pharmacokinetic parameters in animal models. The compound with the best combination of high potency, low toxicity, and favorable bioavailabilty was (S)-3-(2-tert-butyl-4-hydroxymethyl-5-methyl-phenylsulfanyl)-4-hydroxy-6-isopropyl-6-(2-thiophen-3-yl-ethyl)-5,6-dihydro-pyran-2-one (13-(S)). This thiophene derivative also exhibited excellent antiviral efficacy against mutant HIV protease and resistant HIV strains. For these reasons, compound 13-(S) was chosen for further preclinical evaluation.

HLA class I expression on human cancer cells. Implications for effective immunotherapy.

Early studies demonstrated the role of cytotoxic T cells as an immune defence mechanism against tumour cells. The demonstration of tumour antigen peptides and their presentation to T cells on major histocompatibility complex class I molecules highlighted the importance of these molecules in effective anti-tumour responses. It is well established that many tumours escape T cell recognition by loss or down regulation of class I molecule expression on the cell surface of tumour cells. Tumours which have lost class I expression are immunoselected and as a result have a propensity for growth and metastatic spread. With the development of cancer vaccine strategies for clinical use, there will be a future role for histocompatibility laboratories in determining class I expression on tumour cells in individual patients. These studies of expression will require not just the demonstration of total class I expression but the demonstration of locus and allele specific class I molecules involved in the relevant tumour peptide presentation. These studies will be pivotal in tailoring individual patient therapies. The identification of appropriate monoclonal antibody reagents for class I expression and techniques used on different kinds of tissue sections will be a component of the forthcoming 13th International Histocompatibility Workshop.

Nonpeptidic HIV protease inhibitors: 6-alkyl-5,6-dihydropyran-2-ones possessing a novel and achiral 3-(2-t-butyl-5-methyl-4-sulfamate)phenylthio moiety.

Dihydropyran-2-ones possessing a sulfamate moiety at the 4-position of the thiophenyl ring were designed to reach S3' pocket of the HIV protease. Synthetic routes for the preparation of thiotosylates possessing 3-(2-t-butyl-5-methyl-4-sulfamate) phenylthio moiety were established. SAR of various sulfamate analogs including HIV protease binding affinities, antiviral activities and therapeutic indices will be described.

Matching for HLA DPA1 and DPB1 alleles in unrelated bone marrow transplantation.

The impact of donor-recipient DPA1 and DPB1 matching was examined in 122 unrelated bone marrow transplant pairs. All pairs were serologically matched at the time of transplantation for HLA class I and II and a majority also DRB1 allele matched. Retrospective A, B, C, DRB1, DQA1, DQB1 in addition to DPA1 and DPB1 allele matching was performed by molecular techniques. The percentage of pairs that were allele matched was as follows; HLA-A = 91% (n = 80), HLA-B = 94% (n = 80), HLA-C = 78% (n = 80), HLA-DRB1 = 96% (n = 122), HLA-DQA1 = 99% (n = 80), HLA-DQB1 = 92% (n = 122). 92 recipient/donor pairs with informative clinical data were available for analysis. DPA1 identity (no incompatibility in either direction) was observed in 57% and DPA1 compatibility in 76% of pairs with no apparent beneficial effect of matching on patient survival or Graft Versus Host Disease (GVHD). DPB1 identity was observed in 11% and compatibility in 27% of pairs. A significant improvement in patient survival was observed in DPB1 matched compared to one DPB1 mismatch (p < 0.01) and combined one and two DPB1 mismatched transplants (p = 0.03). This beneficial effect remained when allele mismatches at HLA-A, B, C, DRB1, DQA1, DQB1 were excluded (p = 0.05, p = 0.03, respectively). There was a significant association of increased frequency of severe GVHD (grades III-IV) compared to mild GVHD (grades I-II) with DPB1 mismatched transplants compared to DPB1 matched transplants (p = 0.04). In DPB1 mismatched transplants an association between patient survival and matching for individual DPB1 polymorphic regions was not observed; however in the HLA-A, B, DRB1, DQA1, DQB1 allele matched transplants a non significant increase in the frequency of Grade IV GVHD was observed in recipients who were negative compared to those who were positive for DPB1 alleles coding for glutamic acid at position 69.

Coexistent rheumatoid arthritis and systemic lupus erythematosus: clinical, serological, and phenotypic features.

The clinical and serological features and HLA phenotypes are reported for 11 patients with coexistent features of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). All patients had a symmetrical small joint polyarthritis and features of SLE such as rash, photosensitivity, oral ulceration, serositis, cytopenia, and biopsy proved lupus nephritis. Eight had hypocomplementaemia. Autoantibodies were characteristic of the two diseases: all patients had rheumatoid factor and antibodies to double stranded DNA, eight (73%) had antibodies to collagen, and five (46%) had antibodies to Ro (SS-A). There was also an overlap of HLA phenotypes. Six patients were DR4 and seven were DR2 or DR3 positive, and of the five patients who were DR4 negative, four shared class I alleles often associated with DR4. If RA and SLE share a common autoimmune dysfunction, those patients who have the two diseases do so because they have genetic determinants of both.

Reactivity to human islets and fetal pig proislets by peripheral blood mononuclear cells from subjects with preclinical and clinical insulin-dependent diabetes.

A simple, direct assay for T-lymphocyte reactivity to islet antigen(s) in human insulin-dependent diabetes mellitus (IDDM) should facilitate preclinical diagnosis and the evaluation of intervention therapy to avert autoimmune-mediated beta-cell destruction. In subjects with preclinical or clinical IDDM, we measured the reactivity of peripheral blood mononuclear cells (PBMCs) incubated over 6 days with either adult human islets or fetal pig proislets, or other fetal pig tissues, and with human insulin. With islets, the stimulation index (SI) of [3H]thymidine uptake by PBMCs exceeded the mean + 2SD of control subjects in 6 of 6 preclinical subjects (SI 8.7 +/- 3.7), 7 of 11 clinical subjects (SI 5.2 +/- 3.4), and 1 of 12 control subjects (SI 2.7 +/- 1.7); with insulin, the responses were less in frequency and magnitude, being 4 of 6 (2.7 +/- 1.6), 3 of 11 (2.2 +/- 1.1), and 0 of 12 (1.20 +/- 0.55), respectively. The mean responses to islets of PBMCs from preclinical and clinical subjects differed significantly from control subjects (P less than 0.02 by 2-tailed Kruskal-Wallis test). Secretion of granulocyte macrophage colony-stimulating factor by PBMCs over 6 days was assayed in the preclinical group and generally paralleled the uptake of [3H]thymidine. PBMC reactivity to islets appeared to be at least as sensitive a marker of preclinical IDDM as autoantibodies to a 64,000-Mr protein, presumably the enzyme glutamic acid decarboxylase, in fetal pig proislets. In conclusion, islet-reactive T lymphocytes in subjects with preclinical and clinical IDDM can be identified in bulk culture of PBMCs.(ABSTRACT TRUNCATED AT 250 WORDS)

An immunologic and genetic study of asthma in workers in an aluminum smelter.

The cause or causes of asthma among employees in aluminum smelters is unknown. We attempted to ascertain whether such workers who developed asthma differed in respect to indices of immunological function and certain genetic markers. Data were collected from 33 asthmatic and 127 nonasthmatic potroom workers. Asthmatic workers had significantly lower mean serum levels of immunoglobulin (Ig)M; however, mean levels of IgG and IgA, median levels of IgE, the capacity for recall of delayed type hypersensitivity, levels of immune complexes, and frequency of antinuclear or other autoantibodies did not differ from values for nonasthmatic workers. Asthma was found to develop on a background of atopy in 21 workers (64%), whereas there were no features of atopy in 12 workers (36%). Cigarette smoking had independent effects on immunological function. In respect to genetic markers, there was a higher frequency among asthmatic workers of the alpha-1-anti-trypsin deficiency phenotype MS, but the frequency of blood groups, Gm allotypes, or human leucocyte antigen types was similar. The study established that the profile of immune function, or genetic markers tested, did not differ essentially for workers in an aluminum smelter who did or did not develop asthma; however, there was an indication of heterogeneity in causation, as judged by "atopy-related" and "non-atopy-related" groups in the asthma population.

Genetic markers in rheumatoid arthritis relationship to toxicity from D-penicillamine.

In a 3-centre study involving 144 patients with rheumatoid arthritis (RA), a relationship between side effects from D-penicillamine and HLA antigens, allotypic markers of the IgG heavy chain (Gm) and allotypes of complement components Bf, C4A and C4B was sought. There was a significant association between proteinuria induced by D-penicillamine and the antigens DR3 and B8. However, the presence of DR2 seemed to protect against the development of proteinuria. Thrombocytopenia from D-penicillamine was significantly associated with HLA-A1 and DR4; 15 of 23 patients who possessed both antigens developed thrombocytopenia (p less than 0.001 uncorrected, approximate relative risk (RR) = 5.5). A null complement allele located at the C4B locus (C4BQO) was also associated with thrombocytopenia from D-penicillamine (p less than 0.005, RR = 17.3). Our study confirms the findings from other series which indicate that there is a genetic predisposition for the development of proteinuria from D-penicillamine in RA and suggests that this may also be the case in D-penicillamine induced thrombocytopenia.

HLA-DR typing of fetal human spleen and liver lymphoblastoid cells transformed by Epstein-Barr virus.

Because few HLA-DR-positive cells are present in the fetal spleen and liver, full HLA typing cannot be performed. However, B lymphocyte precursors can be transformed with Epstein-Barr virus to produce lymphoblastoid cells which express HLA-A, B, and DR antigens. Successful transformation was achieved, usually with spleen and liver, in nine fetuses aged from 15 to 18 weeks, mostly within 7 to 14 days of initiation of the cultures. Spleen-derived lymphoblasts were more suitable for typing because of their greater homogeneity and higher viability. Tissues from two 13-week fetuses from prostaglandin-induced abortions and from a spontaneously aborted 22-week fetus could not be transformed. This is probably attributable to prolonged ischemia before the tissues were obtained but, in the 13-week fetuses, absence of B lymphocyte precursors was not excluded. HLA-DR typing may be useful in obtaining well matched donor-recipient pairs in fetal pancreatic islet transplantation.