Immune stimulation of rainbow trout reveals divergent regulation of MH class II-associated invariant chain isoforms.

Major histocompatibility complex (MHC) class II-associated invariant chain is a chaperone responsible for targeting the MHC class II dimer to the endocytic pathway, thus enabling the loading of exogenous antigens onto the MHC class II receptor. In the current study, in vivo and in vitro methods were used to investigate the regulation of the rainbow trout invariant chain proteins S25-7 and INVX, upon immune system activation. Whole rainbow trout and the macrophage/monocyte-like cell line RTS11 were treated with PMA at concentrations shown to induce IL-1ß transcripts and homotypic aggregation of RTS11. S25-7 transcript levels remained unchanged in the gill, spleen, and liver and were found to be significantly decreased in head kidney beginning 24 h post-stimulation. Meanwhile, INVX transcript levels remained unchanged in all tissues studied. Both S25-7 and INVX proteins were produced in gill and spleen tissues but their expression was unaffected by immune system stimulation. Surprisingly, neither INVX nor S25-7 protein was detected in the secondary immune organ, the head kidney. Analysis of RTS11 cultures demonstrated that both INVX and S25-7 transcript levels significantly increased at 96 h and 120 h following PMA stimulation before returning to control levels at 168 h. Meanwhile, at the protein level in RTS11, S25-7 remained unchanged while INVX had a significant decrease at 168 h post-stimulation. These results indicate that neither INVX nor S25-7 is upregulated upon immune system activation; thus, teleosts have evolved a system of immune regulation that is different than that found in mammals.

Assessment of the genetic basis of rosacea by genome-wide association study.

Rosacea is a common, chronic skin disease that is currently incurable. Although environmental factors influence rosacea, the genetic basis of rosacea is not established. In this genome-wide association study, a discovery group of 22,952 individuals (2,618 rosacea cases and 20,334 controls) was analyzed, leading to identification of two significant single-nucleotide polymorphisms (SNPs) associated with rosacea, one of which replicated in a new group of 29,481 individuals (3,205 rosacea cases and 26,262 controls). The confirmed SNP, rs763035 (P=8.0 × 10(-11) discovery group; P=0.00031 replication group), is intergenic between HLA-DRA and BTNL2. Exploratory immunohistochemical analysis of HLA-DRA and BTNL2 expression in papulopustular rosacea lesions from six individuals, including one with the rs763035 variant, revealed staining in the perifollicular inflammatory infiltrate of rosacea for both proteins. In addition, three HLA alleles, all MHC class II proteins, were significantly associated with rosacea in the discovery group and confirmed in the replication group: HLA-DRB1*03:01 (P=1.0 × 10(-8) discovery group; P=4.4 × 10(-6) replication group), HLA-DQB1*02:01 (P=1.3 × 10(-8) discovery group; P=7.2 × 10(-6) replication group), and HLA-DQA1*05:01 (P=1.4 × 10(-8) discovery group; P=7.6 × 10(-6) replication group). Collectively, the gene variants identified in this study support the concept of a genetic component for rosacea, and provide candidate targets for future studies to better understand and treat rosacea.

Assessment of biodiversity in Chilean cattle using the distribution of major histocompatibility complex class II BoLA-DRB3 allele.

Bovine leukocyte antigens (BoLAs) are used extensively as markers for bovine disease and immunological traits. In this study, we estimated BoLA-DRB3 allele frequencies using 888 cattle from 10 groups, including seven cattle breeds and three crossbreeds: 99 Red Angus, 100 Black Angus, 81 Chilean Wagyu, 49 Hereford, 95 Hereford × Angus, 71 Hereford × Jersey, 20 Hereford × Overo Colorado, 113 Holstein, 136 Overo Colorado, and 124 Overo Negro cattle. Forty-six BoLA-DRB3 alleles were identified, and each group had between 12 and 29 different BoLA-DRB3 alleles. Overo Negro had the highest number of alleles (29); this breed is considered in Chile to be an 'Old type' European Holstein Friesian descendant. By contrast, we detected 21 alleles in Holstein cattle, which are considered to be a 'Present type' Holstein Friesian cattle. Chilean cattle groups and four Japanese breeds were compared by neighbor-joining trees and a principal component analysis (PCA). The phylogenetic tree showed that Red Angus and Black Angus cattle were in the same clade, crossbreeds were closely related to their parent breeds, and Holstein cattle from Chile were closely related to Holstein cattle in Japan. Overall, the tree provided a thorough description of breed history. It also showed that the Overo Negro breed was closely related to the Holstein breed, consistent with historical data indicating that Overo Negro is an 'Old type' Holstein Friesian cattle. This allelic information will be important for investigating the relationship between major histocompatibility complex (MHC) and disease.

Clinical application of pharmacogenomics: the example of HLA-based drug-induced toxicity.

Pharmacogenomics is gradually becoming more and more indispensable in modern medicine. In several cases, a pharmacogenomics test may alleviate serious drug-induced adverse reactions, if it precedes drug prescription. In this article, we provide an overview of the well-established HLA-based carbamazepine- and allopurinol-induced adverse reactions, as one of the most characteristic examples of the clinical application of pharmacogenomics, highlighting its regional impact in Southeast Asian populations in preventing adverse reactions of certain drug/allele pairs. This example provides useful insights towards evidence generation for policy implementation, including economic evaluation analysis, the implementation of pharmacogenomics testing procedures and monitoring of policy effectiveness, hence serving, per se or in the context of international collaborative efforts, as a model for similar cases in several national healthcare systems worldwide.

Significance of semiquantitative assessment of preformed donor-specific antibody using luminex single bead assay in living related liver transplantation.

AIM: To analyze the risks of preoperatively produced donor-specific antibody (DSA) in liver transplantation. METHODS: DSA was assessed using direct complement-dependent cytotoxicity (CDC) and anti-human globulin- (AHG-) CDC tests, as well as the Luminex Single Antigen assay. Among 616 patients undergoing blood type identical or compatible living donor liver transplantation (LDLT), 21 patients were positive for CDC or AHG-CDC tests, and the preserved serum from 18 patients was examined to determine targeted Class I and II antigens. The relationships between the mean fluorescence intensity (MFI) of DSA and the clinical outcomes were analyzed. RESULTS: Patients were divided into 3 groups according to the MFI of anti-Class I DSA: high (11 patients with MFI > 10,000), low (2 patients with MFI < 10,000), and negative (5 patients) MFI groups. Six of 11 patients with high Class-I DSA showed positive Class-II DSA. Hospital death occurred in 7 patients of the high MFI group. High MFI was a significant risk factor for mortality (P = 0.0155). Univariate analysis showed a significant correlation between MFI strength and C4d deposition (P = 0.0498). CONCLUSIONS: HLA Class I DSA with MFI > 10,000 had a significant negative effect on the clinical outcome of patients with preformed DSA in LDLT.

Polymorphisms in the F8 gene and MHC-II variants as risk factors for the development of inhibitory anti-factor VIII antibodies during the treatment of hemophilia a: a computational assessment.

The development of neutralizing anti-drug-antibodies to the Factor VIII protein-therapeutic is currently the most significant impediment to the effective management of hemophilia A. Common non-synonymous single nucleotide polymorphisms (ns-SNPs) in the F8 gene occur as six haplotypes in the human population (denoted H1 to H6) of which H3 and H4 have been associated with an increased risk of developing anti-drug antibodies. There is evidence that CD4+ T-cell response is essential for the development of anti-drug antibodies and such a response requires the presentation of the peptides by the MHC-class-II (MHC-II) molecules of the patient. We measured the binding and half-life of peptide-MHC-II complexes using synthetic peptides from regions of the Factor VIII protein where ns-SNPs occur and showed that these wild type peptides form stable complexes with six common MHC-II alleles, representing 46.5% of the North American population. Next, we compared the affinities computed by NetMHCIIpan, a neural network-based algorithm for MHC-II peptide binding prediction, to the experimentally measured values and concluded that these are in good agreement (area under the ROC-curve of 0.778 to 0.972 for the six MHC-II variants). Using a computational binding predictor, we were able to expand our analysis to (a) include all wild type peptides spanning each polymorphic position; and (b) consider more MHC-II variants, thus allowing for a better estimation of the risk for clinical manifestation of anti-drug antibodies in the entire population (or a specific sub-population). Analysis of these computational data confirmed that peptides which have the wild type sequence at positions where the polymorphisms associated with haplotypes H3, H4 and H5 occur bind MHC-II proteins significantly more than a negative control. Taken together, the experimental and computational results suggest that wild type peptides from polymorphic regions of FVIII constitute potential T-cell epitopes and thus could explain the increased incidence of anti-drug antibodies in hemophilia A patients with haplotypes H3 and H4.

Assessment of bioactivity of a recombinant chicken interferon-gamma expressed using a baculovirus expression system.

The full-length coding sequence of chicken interferon-γ (ChIFN-γ) was cloned into a baculovirus nonfusion vector, pFastBacDual, and expressed in Sf21 insect cells. Recombinant ChIFN-γ (rChIFN-γ) protein was found to be expressed both intracellularly as well as in the culture supernatants. The affinity-purified rChIFN-γ contained 14, 17, and 28 kDa proteins, possibly representing both glycosylated and nonglycosylated protein forms of ChIFN-γ. The bioactivity of rChIFN-γ was confirmed in vitro by production of nitric oxide in a chicken macrophage cell line (HD11) and antiviral activity against vesicular stomatitis virus in primary chicken embryonic fibroblast cells. Further, HD11 cells stimulated with rChIFN-γ showed significant upregulation of inducible nitric oxide synthases, IFN-γ, interleukin-1ß, interleukin-12p35, signal transducers and activators of transcription 1, class II, major histocompatibility complex, transactivator, and major histocompatibility complex II-ß chain (BL-B) transcripts. In conclusion, the present study provides information on the ability of functionally active rChIFN-γ expressed in a baculovirus system in inducing significant transcriptional upregulation of various immune system-related genes, including those that encode cytokines, antigen-presenting molecules, and transcription factors involved in the major histocompatibility complex and IFN-signaling pathway.

Temperature and length-dependent modulation of the MH class II beta gene expression in brook charr (Salvelinus fontinalis) by a cis-acting minisatellite.

It is widely recognized that the variation in gene regulation is an important factor from which evolutionary changes in diverse aspects of phenotype can be observed in all organisms. Distinctive elements with functional roles on gene regulation have been identified within the non-coding part of the genome, including repeated elements. Major histocompatibility complex (MHC) genes have been the subject of an abundant literature which made them unique candidates for studies of adaptation in natural populations. Yet, the vast majority of studies on MHC genes have dealt with patterns of polymorphism in sequence variation while very few paid attention to the possible implication of differential expression in adaptive responses. In this paper, we report the identification of a polymorphic minisatellite formed of a 32 nucleotides motif (38% G+C) involved in regulation of the major histocompatibility class II beta gene (MHII beta) of brook charr (Salvelinus fontinalis). Our main objectives were: to analyze the variability of this minisatellite found in the second intron of the MHII beta gene and to document its effect to the variation of expression level of this gene under different environmental conditions. Distinctive number of the minisatellite repeats were associated with each different MHII beta alleles identified from exon 2 sequences. Relative expression levels of specific alleles in heterozygous individuals were determined from fish lymphocytes in different genotypes. We found that alleles carrying the longest minisatellite showed a significant 1.67-2.56-fold reduction in the transcript expression relatively to the shortest one. Results obtained in three different genotypes also indicated that the repressive activity associated to the longest minisatellite was more effective at 18 degrees C compared to 6 degrees C. In contrast, no significant difference was observed in transcript levels between alleles with comparable minisatellite length at both temperatures. We also depicted a significant up-regulation of the total MHII beta transcript at 6 degrees C relative to 18 degrees C. These results reveal for the first time that a temperature-sensitive minisatellite could potentially play an important role in the gene regulation of the adaptive immune response in fishes.

Major histocompatibility class II genes in rainbow trout (Oncorhynchus mykiss) exhibit temperature dependent downregulation.

Major histocompatibility (MH) class II receptors are expressed on the surface of specialized antigen-presenting cells in vertebrate immune systems. Their function is to present peptides derived from exogenous pathogens to CD4+ T cells. Variation in the level of expression of these genes has been linked to pathogenesis in various diseases. Very little has been published on the function of MH class II receptors in teleost fish to date. In this study, we have produced polyclonal antibodies recognizing MH class II alpha and beta proteins of rainbow trout and employed them to characterize the expression pattern of these genes. Deglycosylation using N-glycosidase F and endoglycosidase H showed that MH class II alpha is glycosylated in rainbow trout. MH class II beta was also found to be glycosylated as reported previously. Results from Northern blotting revealed that the expression of these genes was not affected by exposure of rainbow trout to temperature of 5 degrees C. However, at 2 degrees C, downregulation of MH class II alpha and beta genes was evident at both the mRNA and protein levels as assessed by Northern and Western blotting, respectively. Because MH class II antigens play an important role in generating an immune response to bacterial and fungal pathogens, downregulation of these genes at low temperature could account for the susceptibility of fish to low temperature-related diseases such as bacterial cold-water disease and winter saprolegniosis.

Quantitative and qualitative assessment of circulating NY-ESO-1 specific CD4+ T cells in cancer-free individuals.

The germ cell antigen NY-ESO-1 is characterized by its frequent expression in patients bearing cancers of various histological types, that positively correlates with stage of disease, together with its frequent spontaneous immunogenicity in patients with advanced cancer. Because of these features, NY-ESO-1 is presently viewed as a prototype antigen for the development of cancer vaccines aimed at preventing disease progression. To gain a global view of the CD4+ T cell repertoire available for NY-ESO-1 in individuals of different genetic background, in this study, we have addressed the presence, frequency, and fine specificity of CD4+ T cells reactive against NY-ESO-1-derived sequences among circulating lymphocytes from healthy donors. NY-ESO-1 specific CD4+ T cells were present among circulating lymphocytes at a frequency between 0.5 and 5 precursors per million CD4+ T cells. In the majority of the cases, the reactivity of NY-ESO-1 specific CD4+ T cells was directed towards immunodominant regions located in the carboxyl-terminal half of the protein. Interestingly, immunodominant regions were confined to parts of the NY-ESO-1 protein containing hotspot sequences with predicted high binding for multiple frequently expressed MHC class II molecules. In contrast, no reactivity was found against the amino-terminal part of the protein, which was concomitant with the paucity, in this region, of sequences with predicted high binding to MHC class II molecules.

HLA-DRB1*, -DQB1* in Piramalai Kallars and Yadhavas, two Dravidian-speaking castes of Tamil Nadu, South India.

Two Dravidian-speaking castes of Tamil Nadu, Piramalai Kallars (PKs, n = 205) and Yadhavas (YDs, n = 239) and a random panel (84) were studied for HLA-DRB1* and -DQB1* polymorphisms by DNA-SSOP typing methods. XI and XII International Histocompatibility primers and non-radioactive-labelled oligo probes were employed to identify the alleles. Results revealed that PKs possessed >0.1 allele frequencies of HLA-DRB1*15011, 0301, -DQB1*0201, 0501 and 0601; YDs, HLA-DRB1*0301, 0401, 07 and -DQB1*0601; and the random panel, DRB1*15021, 0401, 07, -DQB1 0201, 0301, 0302 and 0501. The highest frequency of DRB1*1501 in the world (GF = 0.225) was found in PKs. The most frequent two-locus haplotype (>500/10,000) in all the study samples was DRB1*10-DQB1*0501, while 1501-0601 was frequent in PKs and YDs. Comparison of the HLA-DRB1* data with Eastern European and South-East Asian populations suggested migration as the prime cause of the observed diversity in DRB1* allele frequencies. Nonetheless, the heterozygocity test and Watterson's homozygosity test indicated that balancing selection still operates on HLA-DRB1* locus, in this endemic region of various infectious diseases. This and spatial autocorrelation analysis support the view that selection may be a cause of "generating" new variants and allelic diversity in different ancient settlements. The study suggested that South Indian, inbred, endogamous, sympatrically isolated castes or similar well-defined breeding isolates around the world, living under the same milieu-epidemiology, may be ideal models to test the immunogenetic basis of disease susceptibility.

Immunization with plasmid DNA encoding MHC class II binding peptide/CLIP-replaced invariant chain (Ii) induces specific helper T cells in vivo: the assessment of Ii p31 and p41 isoforms as vehicles for immunization.

A single helper T cell (Th) epitope-specific T cell subset was successfully induced in vivo by immunization with plasmid DNA encoding MHC class II binding peptide/class II-associated invariant chain peptide (CLIP)-replaced murine Ii molecules. Spleen cells from mice immunized by gene gun bombardment with plasmid DNA for Ii p31 and p41 molecules, whose CLIP regions were replaced with an I-A(d)-restricted Th epitope, ovalbumin (OVA) 323-336, showed the specific proliferation and interferon-gamma (IFN-gamma) production. A20-2J B cell lines having these plasmids were capable of stimulating spleen cells from the immunized mice and naïve DO10-transgenic mice bearing the epitope-specific T cell receptor (TCR) transgenes by examining the specific proliferative response and IFN-gamma production. Some mice immunized with the Ii p41-OVA323, but not with the Ii p31-OVA323 plasmid, produced the peptide-specific antibodies, suggesting the functional difference between Ii isoforms.

Comparative assessment of MHC antigen expression in bladder and testis tumour biopsies and established urological tumour cell lines: the relevance of cytokine and gene transfection for correction of defective MHC antigens.

The aim of this study using radio-binding (RB), peroxidase-anti-peroxidase (PAP) and immunoprecipitation (IP) techniques was to investigate the pattern of major histocompatibility complex (MHC) antigen expression in urological malignancies and to compare the results with those seen in established urological human tumour cell lines. The results showed that using PAP technique, the percent bladder cases showing complete loss or cases with greater than 90% of tumour cells negative with W6/32 (detects all class I antigens), HC10 (detects free heavy chain) and BMM.1 (detects beta2-mirogobulin) monoclonal antibodies (Mab) were 16%, 44% and 2% respectively. In a subgroup of 37 cases, the intensity of MHC class II antigen expression for strong, weak and negative cases were 9 (24%), 8 (22%) and 20 (54%) respectively. The expression for class I antigens on testis tumours was mainly negative and when positive, it was present in a small percent of tumour cells. This was also observed for class II antigens where only 8% of cases showed some degree of positivity. Using RB technique, 10 of 12 (85%) of tumour lines expressed class I antigens spontaneously and following interferon gamma (IFNgamma) stimulation, the 2 negative lines one testis (Tera I) and one bladder (Fen) remained negative and 2 lines (both testis lines Tera II and Ep2102) showed a significant class I up-regulation. None of the lines expressed class II antigens spontaneously and following IFNgamma stimulation, 8 of 12 (66%) were induced. The absence of class I and II antigens in the negative lines was confirmed using IP technique. In the case of one class I negative bladder cell line i.e. Fen, the biochemical analysis showed the absence of beta2-m gene product which could not be restored by IFNgamma stimulation. However, transfection of the cells with beta2-m gene resulted in the expression of fully assembled class I antigens, indicating that the loss of antigens was due to the absence of functional beta2-m gene. These results indicated the similarity between the pattern of expression of MHC antigens on tumour biopsies and established tumour cell lines. They also demonstrated that both cytokine stimulation and gene transfection could be used to correct defective class I antigens in tumour cell lines. These approaches might have important implications for pre-selection of bladder cancer patients for cytokine or gene therapies.

The extent of HLA class II allele level disparity in unrelated bone marrow transplantation: analysis of 1259 National Marrow Donor Program donor-recipient pairs.

A comprehensive analysis of the HLA-D region loci, DRB1, DRB3, DRB5, DQA1, DQB1, DPA1 and DPB1, was performed to determine allelic diversity and underlying HLA disparity in 1259 bone marrow recipients and their unrelated donors transplanted through the National Marrow Donor Program. Although 43.0% of DRB1 alleles known to exist at the beginning of the study were found in this predominantly Caucasian transplant population, a few alleles predominated at each locus. In recipients, 67.1% of DRB1 alleles identified were one or two of six common DRB1 alleles. Only 118 (9.4%) donor-recipient pairs were matched for all alleles of DRB1, DQA1, DQB1, DPA1 and DPB1. While 79.4% of the pairs were matched for DRB1, only 13.2% were matched for DPB1 alleles. Almost 66% of pairs differed by more than one allele mismatch and 59.0% differed at more than one HLA-D locus. DQB1 was matched in 85.9% of DRB1-matched pairs. In contrast, only 13.9% of the pairs matched for DRB1, DQA1 and DQB1 were also matched for DPA1 and DPB1. This database, highlighting the underlying HLA disparity within the pairs, forms the foundation of an ongoing study to establish the relationship between HLA matching and successful outcome in unrelated allogeneic stem cell transplant.

Effect of transforming growth factor-beta1 on microglial MHC-class II expression.

In the present report, the effects of IFN-gamma and transforming growth factor beta1 (TGF-beta1) on major histocompatibility complex class II (MHC-II) gene expression in isolated mouse brain microglial cells, in the MH-S macrophage cell line and in the primary mouse macrophage cultures were examined. IFN-gamma is a potent inducer of MHC-II gene and this induction was further elevated in microglia by TGF-beta1, while TGF-beta1 inhibited IFN-gamma, induction in macrophages. The enhancing effect of TGF-beta1 was also detected in microglia at the protein level. Transient transfection of microglia with 5' deletional mutants of the MHC-II IAalpha promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated that TGF-beta1 acts at the transcriptional level to enhance the MHC-II expression induced by IFN-gamma.

CAG repeat mutation in the mouse IL-2 gene enables concurrent assessment of T- and B-cell-specific gene expression by northern analysis.

The unique presence of CAG triplet repeats in the mouse IL-2 gene, lending the IL-2 cDNA a second specificity to MHC class II invariant chain (Ii) mRNA, coincides with another unusual genetic feature of the mouse, the lack of Ii gene upregulation in stimulated T cells. While the former anomaly allows simultaneous measurement of IL-2 and Ii mRNAs by Northern analysis, the latter condition ensures that Ii mRNA remains a T-cell-independent, specific marker of Ii positive, primarily B cell function. Thus, Northern analysis using CAG repeat containing IL-2 cDNA enables concurrent assessment of T and B cell activities in the spleen or in other mixtures of mouse lymphocytes.

Human immunodeficiency virus-1 reverse transcriptase immunodominant CD4+ T cell epitopes: a peptide-based multiparametric assessment in the mouse.

We previously identified an immunodominant CD4+ T cell determinant in the carboxy-terminal region of HIV-1 reverse transcriptase (RT528-543). The present study aimed at enumerating all the potential sites of HIV-1 RT recognized by Th cells in the BALB/c (H-2d) mouse model. To achieve this we used a panel of 62 overlapping 15-mer synthetic peptides covering the whole RT sequence to assay the following parameters: (i) immunogenicity in naive BALB/c mice injected either with peptides pools or individual peptides; (ii) antigenicity, as detected by their ability to restimulate in vitro T cells from BALB/c mice primed with native RT; (iii) MHC class II (Ad)-binding capacity as measured by the inhibition of the antigen-specific, Ad-restricted presentation of unfolded apamin (4-Acm) by fixed antigen-presenting cells to Ad/4-Acm-specific, interleukin-2-producing T hybridoma cells; and (iv) the presence of typical or degenerate consensus Ad-binding motifs. The results in this study permitted identification of three novel immunodominant RT mouse CD4+ T cell sites (RT276-290, RT375-389 and RT411-425) located in regions of limited polymorphism among RT from several HIV isolates. Some of these RT segments were found to be in the vicinity of B cell or H-2Kk- or HLA-A2-restricted cytotoxic T lymphocyte epitopes. Finally, the approach used in this study was found to be very efficient for enumerating most T cell recognition sites in a complex protein, a result that would have not been achieved by a single parameter-based analysis.

A simple and economical DRB1 typing procedure combining group-specific amplification, DNA heteroduplex and enzyme restriction analysis.

A new procedure for HLA-DRB1 typing is proposed. The method combines six group-specific amplifications with heteroduplex analysis and, in some cases, enzyme restriction analysis. This technique, which is as discriminative as oligotyping, is simple, economical and does not require probes. These characteristics make this approach a valid alternative to other HLA genomic typing procedures, especially in those cases such as donor-recipient pairs matching where a small number of samples has to be managed at once.

Accurate and rapid assessment of MHC antigen upregulation following cytokine stimulation.

Three human tumour cell lines, A431 (cervical), Scaber (bladder) and Fen (bladder), were studied using immunohistochemical staining (IC), radiobinding (RB), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA) and dot-blot (DB) techniques in order to assess major histocompatibility antigen (MHC) induction in response to interferon-gamma (IFN-gamma). Induction of class II antigens by IFN-gamma was observed on all three cell lines using all techniques. Monoclonal antibody (Mab) staining showed that both Scaber and A431 lines were positive for intact class I (Mab W6/32), class I free heavy chain (Mab HC10) and beta 2 microglobulin (beta 2-m) (Mab BBM.1), while Fen cells were positive only with HC10. The IP technique demonstrated the presence of a 45 kDa band on precipitation of the Fen lysate with HC10 Mab, whereas no such band was observed when W6/32 was used. The DB technique confirmed the negative reaction with W6/32 and BBM.1 Mabs, while HC10 showed positive staining which was upregulated by IFN-gamma. Transfection of the Fen cells with the beta 2-m gene resulted in the surface expression of fully assembled class I molecules. The DB technique showed upregulation of class I antigens following IFN-gamma stimulation, while RB detected no significant increase in cell surface expression (t test; p = 0.104). The binding values for transfected Fen cells before and after IFN-gamma stimulation were 2000 +/- 48 and 2161 +/- 156 cpm respectively. These results demonstrate that the DB technique facilitates an accurate assessment of cytokine induced antigens, corrected against a background of total cellular protein synthesis. The ease of execution, simplicity, non-radioactive nature and economy make it the method of choice for routine screening prior to the selection of suitable patients for cytokine therapy.

HLA matching of unrelated bone marrow transplant pairs: direct sequencing of in vitro amplified HLA-DRB1 and -DQB1 genes using magnetic beads as solid support.

Sequencing of HLA genes can offer complete information on the HLA class II genes relevant for the outcome of bone marrow transplantation (BMT). Genomic HLA matching of unrelated BMT patient/donor pairs is often based on PCR-SSO typing of HLA class II alleles. Typing a small number of samples by this approach is both expensive and time-consuming, due to the large number of SSO probes required to perform a complete class II typing. Moreover, only polymorphisms explicitly tested for will be found. We now provide the first report of the use of direct sequencing of HLA-DRB1 and -DQB1 genes, using PCR-amplified genomic DNA attached to magnetic beads, for clinical routine HLA matching. Sequencing ladders obtained by this procedure are easily readable, the patterns can be interpreted in HLA homozygous as well as heterozygous individuals, and sequence differences or similarities between the BMT donor and recipient can be directly identified. This genomic typing method is informative, relatively fast and therefore well-suited for the small number of samples usually analyzed in matching of BMT pairs. Furthermore, this technique has the potential for automation.