Human Immunology

The aim of this meta-analysis was to evaluate whether specific maternal HLA alleles and HLA sharing of couples are associated with the occurrence of recurrent miscarriage (RM). A systematic literature search was performed for studies that evaluated the association between HLA alleles, HLA sharing and RM. RM was defined as three or more consecutive unexplained miscarriages and a control group was included of women with at least one live birth and no miscarriages in their history. Meta-analyses were performed and the pooled odds ratio (OR) was calculated. We included 41 studies. Selection bias was present in 40 studies and information bias in all studies. Meta-analyses showed an increased risk of RM in mothers carrying a HLA-DRB1*4 (OR 1.41, 95% CI 1.05-1.90), HLA-DRB1*15 (OR 1.57, 95% CI 1.15-2.14), or a HLA-E*01:01 allele (OR 1.47, 95% CI 0.20-1.81), and a decreased risk with HLA-DRB1*13 (OR 0.63, 95% CI 0.45-0.89) or HLA-DRB1*14 (OR 0.54, 95% CI 0.31-0.94). Pooling results for HLA ...

Sequencing based typing (SBT) has become the gold standard in HLA-typing. In recent years various strategies have been developed. However the most time consuming and error prone part of SBT is still dataanalysis including reporting and archiving of the generated data. In addition, the ever vast increasing number of HLA alleles, increase the number of ambiguous results which require a special approach to resolve. In recent IMGT/HLA database release the total number has succeeded even 6000 different HLA-alleles. Methods: We have improved our software package SBTengine® significantly; e.g allowing now a reduced number of crucial position to check manually, as the software is using a quality scoring per nucleotide. Moreover also the CWD list and NMDP allele frequency list are exploited elegantly such that the attention of the reviewer is directed to most important crucial positions. Results: SBTengine® combines all sequences of a single sample, either homozygous or heterozygous to a conclusive allele assignment. The software guides the user easily through the typing process. In case of an ambiguity, the DART system of SBTengine® suggests a consecutive action, either to sequence an additional region or to apply a Group Specific Sequencing Primer to separate the HLA alleles. Furthermore, almost all known null-alleles are excluded or confirmed automatically. SBTengine® is an open software package and accepts any HLA sequencing data generated independently of the reagents used. Conclusions: We will show new and unique features of the latest version of SBTengine®. The versatility of SBTengine® will enable laboratories to easily implement SBT Typing into their daily routine laboratory activities and to obtain high resolution and accurate HLA typing in a straightforward manner.

The evidence of association between HLA-B ⁄ 1502 and anticonvulsant induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) from the Indian population is scant. Patients with a history of SJS/TEN secondary to carbamazepine or phenytoin were enrolled. The control group comprised of patients who had received carbamazepine/phenytoin for P6 months without any adverse cutaneous event. Low-resolution DNA typing for HLA-B and high resolution HLA-B ⁄ 15 typing was performed. Seventeen patients with history of SJS/TEN secondary to carbamazepine (9) or phenytoin (8) and 50 tolerant controls (carbamazepine-37; phenytoin-13) were enrolled. The mean age of patients and controls was 33.9 ± 11.6 and 28.1 ± 9.9 years, respectively. HLA-B ⁄ 1502 was observed in 2/9 (22.2%) carbamazepine-SJS/TEN patients and none of the 37 carbamazepine tolerant controls (p = 0.035). HLA-B ⁄ 1502 was not observed in any of the 8 phenytoin-SJS/TEN patients or the 13 phenytoin tolerant controls. Our data suggests that HLA-B ⁄ 1502 is a risk factor for carbamazepine induced SJS/TEN. Therefore, HLA-B ⁄ 1502 testing should be performed prior to initiating carbamazepine in North Indian population.

To examine the genetic diversity in Morocco, the polymorphism at the HLA-DRB1 locus was investigated in two populations: the Metalsa group consisting of Berbers from north Morocco (who speak the Tarifit language and live in the Nador area), and the Chaouya group who are Arabic-speaking people from west Morocco (Atlantic coast) living in the Settat area. The DRB1 alleles of 197 healthy unrelated individuals were identified by direct DNA sequencing of exon 2 using fluorescently-labeled primers. A total of 28 and 29 alleles at DRB1 locus were identified in the Metalsa and Chaouya groups, respectively. The most frequent alleles in the Metalsa group are DRB1*03011 (20.2%), DRB1*0701 (12.12%), and DRB1*1302 (11.11%). In the Chaouya group, DRB1*0701 (16.33%), DRB1*15011 (12.76%), and DRB1*03011 (11.73%) are most common. Each population exhibits some specific variants and some uncommon alleles. The frequency of the DRB1*03011 allele differs significantly between the two populations (p ϭ 0.0311). The DRB1 frequency distributions in the two groups suggest the effects of balancing selection. The interpopulation analysis highlighted a strong relatedness, based on genetic distances, between the two Moroccan groups and the other north Africans (the Moroccans from El Jadida area, Moroccan Souss Berbers, Algerians, and Tunisians), and to a lesser extent with the Iberians, French, and Ethiopians.

Diversity is characteristic of the HLA system. In this study 4727 potential BMT donors from 5 ethnic groups were typed for DRB1/3/4/5 loci by SBT and for DQB1 by RLS. Even though the panel analyzed here may be skewed by the occurrence of rare patient's HLA genotypes important information about linkage disequilibrium patterns may be gained. Haplotype assignments were done by examining previously known associations and by applying the EM algorithm; both procedures yielded identical results. We observed 71 DRB1 alleles Ϫ46 were typed in at least 5 subjects accounting for 99.5 percent of the alleles. Analysis of the 3 loci haplotypes showed 157 haplotypes that were found in at least two instances each (99.1 percent); 25 haplotypes had unique alleles and 59 corresponded to unique arrays of alleles. In spite of the finding of conserved haplotype blocks we could identify at least 2 haplotypes for each DRB1 allele that was observed more than 17 times. The strength of specific associations varied in different ethnicities. DRB5*01011 associates almost absolutely with DRB1*15011 in all ethnic groups and with DRB1*1503 in African subjects however we observed this allele associated with various DRB1*15/16 alleles in Asians. We observed almost absolute associations between DRB1*13021 and DRB3*03011, and DRB1*13011 with DRB3* 0101 or 0202 in all populations with exception of Africans in which DRB1-DRB3 exceptions were more frequent (10 percent). The present study demonstrates that less than 60 DRB1 alleles account for more than 99 percent of the gene pool of all populations combined; in spite of this conservation we observed multiple DRB-DQ haplotypes for almost all common DRB1 alleles.

Dendritic cells (DCs) exposed to tumor antigens followed by treatment with T h 1-polarizing differentiation signals have paved the way for the development of DC-based cancer vaccines. Critical parameters for assessment of the optimal functional state of DCs and prediction of the vaccine potency of activated DCs have in the past been based on measurements of differentiation surface markers like HLA-DR, CD80, CD83, CD86, and CCR7 and the level of secreted cytokines like interleukin-12p70. However, the level of these markers does not provide a complete picture of the DC phenotype and may be insufficient for prediction of clinical outcome for DC-based therapy. We therefore looked for additional biomarkers by investigating the differential expression of microRNAs (miRNAs) in mature DCs relative to immature DCs. A microarray-based screening revealed that 12 miRNAs were differentially expressed in the two DC phenotypes. Of these, four miRNAs, hsa-miR-155, hsa-miR-146a, hsa-miR-125a-5p, and hsa-miR-29a, were validated by real-time polymerase chain reaction and northern blotting. The matured DCs from 12 individual donors were divided into two groups of highly and less differentiated DCs, respectively. A pronounced difference at the level of miRNA induction between these two groups was observed, suggesting that quantitative evaluation of selected miRNAs potentially can predict the immunogenicity of DC vaccines. ᭧

We have been utilizing the LabScreen methodology for assessing PRA levels & antibody (Ab) specificity for several years. As a result of Luminex's increased sensitivity we are able to more accurately identify anti-HLA Abs and better assist our transplant team. As with any Ab screening method, not all patient sera are successfully assessed with this method. We occasionally come across pts with non-specific high background (bkgrd) that makes Ab assessment difficult (due to control values outside our acceptable range [very high neg ctrls or very low pos:Neg ctrls]. The object of this study was to determine if Adsorb Out could reduce nonspecific high bkgrd in our problem sera without affecting normal bkgrd while still allowing the detection of "true" anti-HLA Abs.

It is generally accepted that human and experimental tumor cells can lose major histocompatibility complex (MHC) class I molecules. These human leukocyte antigen (HLA) losses are detected when the primary tumor breaks the basal membrane, invades the surrounding tissues, and starts to metastasize. These altered HLA class I phenotypes probably constitute the major tumor escape mechanism facing anti-tumor T-cell mediated responses. Thus, it is important to characterize these phenotypes in clinical tumor samples, analyze the mechanism(s) responsible for them, and counsel patients before and during peptide anti-cancer immunotherapy. The present paper summarizes the most relevant altered HLA class I phenotypes found in human tumor samples, indicates their frequency, and outlines the mechanisms implicated. This review also points out that the natural killer (NK) escape mechanism of HLA class I deficient cancer cells is yet to be defined. Knowledge accumulated to date reveals that HLA class I molecules are an important crossroad in tumor immunology. Human Immunology 61, 65-73 (2000).

The interleukin-18 (IL-18) gene on chromosome 11q22 has been suggested as a susceptibility factor for allergies. To test for a possible role of IL-18 polymorphisms in Czech population, case-control study including 958 subjects (633 allergic patients and 325 healthy controls) was performed. An allele-specific polymerase chain reaction was used to analyze variants at positions Ϫ607 C/A (rs1946518) and Ϫ137 G/C (rs187238) in the promoter region together with the polymerase chain reaction-restriction fragment length polymorphism method for the detection of polymorphism at position Ϫ140 C/G (previously Ϫ133 C/G, rs360721) in intron 1 of the IL-18 gene. The Ϫ1297 C/T (rs360719) polymorphism was genotyped by real-time-polymerase chain reaction, using a predevelopment TaqMan allele discrimination assay. There were no significant differences in distribution of alleles or genotypes in any of four single nucleotide polymorphisms in the IL-18 gene between controls and patients. However, subsequent analysis revealed a significant difference in haplotype frequencies between the allergic patients and healthy subjects (p Ͻ 0.01). Haplotype formed by Ϫ1297 C/Ϫ607 A/Ϫ137 C/Ϫ140 C alleles occurred significantly more frequently in patients than controls (0.0433 vs 0.0129; p Ͻ 0.0003; p corr Ͻ 0.01, OR ϭ 3.37; 95% CI ϭ 1.59 -7.14). In contrast, there was no relationship among the IL-18 variants and total serum IgE level. Our results indicate that promoter polymorphisms in the IL-18 gene act in interaction and could play a role in allergic disorders. ᭧

This population from Buenos Aires, Argentina, Latitude 34.3S, Longitude 58.2W, consisted of 157 Caucasian individuals. The individuals were drawn from an urban environment and alleles were determined using sequence specific oligonucleotide probes.

Celiac disease (CD) is a rare example of multifactorial disorder in which a genetic test is of great clinical relevance, as the disease rarely develops in the absence of specific HLA alleles. We typed DR-DQ genes in 437 Italian children with celiac disease, 834 first-degree relatives, and 551 controls. Of patients, 91% carried DQ2 and/or DQ8 heterodimers, 6% only had ␤2 chain, 2% was ␣5 positive, and four were DQ2/DQ8/␤2/␣5 negative. Only the presence of ␣5 resulted negatively associated to disease (p ϭ 2 ϫ 10 Ϫ4 ), whereas we confirmed the effect of the ␤ half of DQ2 dimer on CD predisposition (p ϭ 4 ϫ 10 Ϫ12 ). Considering 1:100 disease prevalence, we obtained a risk gradient ranging from 1:7 for DQ2 and DQ8 individuals down to 1:2518 for subjects lacking all predisposing factors. The DQB1*02 and DQB1*0302 concurrence (p ϭ 9 ϫ 10 Ϫ4 ), besides the DQB1*02/*02 homozygosity, had an additional role in disease genetic determination. The CD prevalence rose to 17.6% in sisters, 10.8% in brothers, and 3.4% in parents. In the three groups, the subjects carrying high-risk HLA molecules were 57%, 71%, and 58%; among them, 29%, 15%, and 6% respectively had CD. Those siblings and parents with no susceptible factors were not affected. These findings indicate the impact of the HLA test for CD in clinical practice.

Substances occurring naturally in the sera of patients can interfere with Luminex ᭨ antibody assays, causing increased background and changes in antibody specificity. We present data on the effectiveness of hypotonic dialysis (HD) or dithiothreitol (DTT) treatment in eliminating this interference. HD significantly increased reaction strength of positive control beads and reduced reaction strength of negative control beads. HD also improved specificity identification, determination of donor-specific antibody (DSA) strength, and crossmatch predictability compared with values in untreated serum. DTT also increased the reaction strength of positive control beads, but in most cases, further increased reactivity of negative control beads. DTT improved crossmatch predictability but to a lesser extent than did HD and may differ with specificities defined in other assays. Because interference is frequently observed in sera from highly sensitized patients, it is important to recognize and eliminate interference in Luminex ᭨ antibody assays for accurate and meaningful test interpretation. ᭧ Numbers shown below HLA alleles are MFI values. For serum number 1, the A2 alleles were among the 10 lowest reacting beads for the untreated serum.

The presence of complement fixing anti-human leukocyte antigen (HLA) antibodies in the circulation of organ transplant recipients may result in heart allograft rejection. Here, we assessed the clinical impact of pre-and post-transplantation allosensitization on long-term survival of heart allografts. Sequential samples of sera from heart allograft recipients were screened pretransplantation for panel reactive antibodies using the complement-dependent cytotoxicity test. Patients were monitored post-transplantation for donor specific anti-HLA class I and class II antibodies. Kaplan-Meier graft survival plots were generated to analyze the effect of anti-HLA antibodies on transplantation outcomes. Statistical analysis showed that the post-transplantation development of alloantibodies was a significant risk factor that was associated with low long-term survival rates; in contrast, recipients' gender, age, previous transplantations, and degree of HLA matching with the donor had no effect on long-term survival. The presence in pretransplantation sera of antibodies against more than 10% of the HLA reference panel (PRA Ͼ10%) was associated with AMR and with a relatively lower rate of graft survival after 1 year but did not affect 10-year survival. The present data underline the importance of monitoring the development of anti-HLA antibodies as a tool for early diagnosis and treatment of AMR. ᭧

For over 50 years, the International HLA and Immunogenetics Workshops (IHIW) have advanced the fields of histocompatibility and immunogenetics (H&I) via community sharing of technology, experience and reagents, and the establishment of ongoing collaborative projects. In the fall of 2017, the 17th IHIW will focus on the application of next generation sequencing (NGS) technologies for clinical and research goals in the H&I fields. NGS technologies have the potential to allow dramatic insights and advances in these fields, but the scope and sheer quantity of data associated with NGS raise challenges for their analysis, collection, exchange and storage. The 17th IHIW has adopted a centralized approach to these issues, and we have been developing the tools, services and systems to create an effective system for capturing and managing these NGS data. We have worked with NGS platform and software developers to define a set of distinct but equivalent NGS typing reports that record NGS data ...

All animals develop in association with complex microbial communities. It is now well established that commensal microbiota is essential for the correct functionality of each organ in the host. Particularly, the commensal gastro-intestinal microbiota (CGIM) is a key factor for development, immunity and nutrient conversion, rendering them bio-available for various uses. Thus, nutritional inputs generate a positive loop in maintaining host health and are essential in shaping the composition of the CGIM communities. Probiotics, which are live exogenous microorganisms, selectively provided to the host, are a promising concept for manipulating the microbiota and thus for increasing the host health status. Nevertheless, most mechanisms induced by probiotics to fortify the immune system are still a matter of debate. Alternatively, prebiotics, which are non-digestible food ingredients, can favor the growth of specific target groups of CGIM. Several metabolites are produced by the CGIM, one of the most important are the short-chain fatty acids (SCFAs), which emerge from the fermentation of complex carbohydrates. SCFAs have been recognized as key players in triggering beneficial effects elicited by simple diffusion and by specific receptors present thus far only in epithelial cells of higher vertebrates at different GI locations. However, both strategies have shown to provide resistance against pathogens during periods of high stress. In fish, knowledge about the action of pro- and prebiotics and SCFAs is still limited. Thus, in this review, we briefly summarize the mechanisms described on this topic for higher vertebrates and discuss why many of them may operate in the fish gut representing a model for different mucosal tissues.

The main function of major histocompatibility complex (MHC) class II molecules is to present processed antigens, which are derived primarily from exogenous sources, to CD4 ϩ T-lymphocytes. MHC class II molecules thereby are critical for the initiation of the antigen-specific immune response. Besides antigen presentation, growing evidence is showing that ligation of MHC class II molecules also activates intracellular signaling pathways, frequently leading to apoptosis. Constitutive expression of MHC class II molecules is confined to professional antigen-presenting cells (APC) of the immune system, and in nonprofessional APCs MHC class II molecules can be induced by a variety of immune regulators. Interestingly, activated T cells from many species, with the exception of mice, synthesize and express MHC class II molecules at their cell surface. In this review, we discuss our current knowledge on the transcriptional regulation of MHC class II expression in activated human and mouse T cells, and the contribution of DNA methylation of the T-cell employed class II transactivator promoter III to the MHC class II deficiency of mouse T cells. We also discuss the proposed functions of the activated T cell synthesized and expressed MHC class II molecules, including antigen presentation, T-T cell interactions, and MHC class II-mediated intracellular signaling. Human Immunology 65, 282-290 (2004).

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient

The major histocompatibility complex (MHC) is highly polymorphic and more than 1500 human MHC alleles are known to date. These alleles do not bind to a given peptide with identical affinity. Although MHC alleles are functionally related, it is difficult to quantify the functional variation between them. Threedimensional structures of known MHC-peptide (MHCp) complexes suggest that specific peptide residues bind selectively to functional pockets in the binding groove. From a set of known MHCp structures we identified 21 critical polymorphic functional residue positions (CPFRP) that significantly reduced functional pocket variability to just 189 among 212 HLA-A alleles. Interestingly 101 HLA-A alleles clustered into 29 clusters such that the six functional pockets formed by the CPFRPs are identical within the cluster. Human Immunology 64, 718 -728 (2003).

The National Kidney Matching Scheme (NKMS) allows matching and allocation of donor organs throughout Australia. Sera from potential recipients are distributed to each interstate tissue typing laboratory on a monthly basis for crossmatching in the event of a cadaver donor. Therefore, it is essential there is consensus for results between these laboratories in order for donated organs to be allocated appropriately. A quality control program conducted under the auspices of ASEATTA was undertaken for (1) panel reactive antibody characterization; (2) routine T lymphocyte crossmatching; and (3) characterization of antibody isotype by DTT treatment. These aims were achieved by distribution of (1) six sera for the determination of PRA activity; (2) 20 scrambled trays including replicate dilutions of a strongly positive lymphocytotoxic serum, high titer monoclonal antibody and negative sera and; (3) 20 trays containing sera with IgG and/or IgM antibodies. These were then evaluated by each laboratory on a panel of T cells. There was concordance between laboratories for PRA levels and antibody characterization. However, there was considerable variation in crossmatch sensitivity and reproducibility, several laboratories had carryover and others could not detect weak IgM antibodies. These results demonstrate the utility and need for ongoing crossmatch exchange programs, particularly for laboratories participating in organ exchange programs.

The Lacandon of the Mayanse branch belong to the same linguistic family of the Guatemalan Mayans. They are a cultural preserved group of 700 individuals culturally and geographically isolated, that was never reached by the conquerors. They lived in small clans dispersed throughout the vast jungle. They survived avoiding contact with foreign diseases during a long period of time. By 1940 a strong influenza epidemics devastated the population and only 48 natives survived, resulting in the nowadays inbred population.We published previously their class II profile, and herein class I was completed in 99 individuals, since class I molecular typing adds a very valuable information for the MHC characterization of populations. Methods: DNA typing was performed using a reverse dot-blot technique. Since it was not possible to select unrelated individuals because of high inbreeding, allele frequency (AF%) was calculated by direct counting from 66 less related individuals. Genetic Distances were calculated and dendrograms were constructed using allele frequency of DRB1 high resolution typing of 39 Amerindians, Europeans and Asians. The Mexican Mestizos groups previously published by us were also included. The UPGMA tree construction from allele frequency data was used. Scope: The MHC characterization of unique populations that have lived isolated is a valuable tool for analysis of the epidemiological impact that the genes may have on the disease prevalence of natives. Ancestral origins can also be tracked back. Conclusions: The results at the two digit level showed 6 A* alleles, 8 B* and 6 Cw* types; 11 DRB1* and 5 DQB1 4 digit alleles were present being the most frequent *0411 (35.3%) followed by *1402, *0403, *0407, *0404,*1406, *1602. Admixture was evident, due to the Whites that came to evangelize the group since 1940. The % of Caucasian/Mediterranean admixture found was 3.04% for A* and B* loci; 3.04; 4.56% for DRB1 & 1.52% for DQB1* locus. These alleles are an example of admixture:

Aim: SLE is a multi-systemic and polygenic chronic inflammatory disease, in which the MHC plays a central role in susceptibility to disease and disease subtype expression. Most important is the autoantibody synthesis that has been shown to be linked to different class II alleles, indicating that there are class II-Ir genes that control autoab production. SLE is prevalent in females (18 -50 years). It is characterized by the presence of antinuclear antibodies (ANA) in 97% of the patients. These are directed against different nuclear components: double stranded anti-DNA (anti-DNA dc); or anti-Smith (Sm), that are present in Ͻ1% of healthy people. Several DR alleles and particular sequences in the DQ groove are relevant to the auto-ab synthesis. The aim of this study was to investigate which sequences are responsible of autoantibody synthesis in mexican patients with SLE.

The immunogenetics of celiac disease demonstrates a highly significant association with the HLA class II alleles DQAI*0501 DQBI*0201 encoded in either a cis-or trans-configuration. In Northern Europe, these alleles are found in linkage disequilibrium with DRBI*0301 while in Southern Europe an additional secondary association through linkage disequilibrium is seen with the combination DRB 1"1101/0701. This study examines 34 Ashkenazi Jews with celiac disease and 36 ethnically matched controls to determine alleles at the DRB, DQA1, DQB1, and DPB1 loci using SSO probes in conjunction with gene amplification by the PCR. The results confirm a highly significant association with the DQA 1"0501 DQB 1"0201 allelic combination (71% ce-liac vs 8% control individuals; p = 0.00005; )~2 = 21.4). Of celiac subjects, 29% were negative for the proposed DQ susceptibility alleles, the majority of whom were DRB 1"0402 positive (20% overall celiac group). No additional susceptibility was associated at the DRB3 and DPB loci. This study confirms that the MHC-linked celiac disease susceptibility among Ashkenazi Jews is closely associated with the presence of the combination of alleles DQAI*0501 DQBI*0201. However, within this population of relatively high-prevalence celiac disease, 30% of celiac patients do not carry these alleles and are therefore not covered by a single "unifying" hypothesis. Human Immunology 38. 270-276 (1993) ABBREVIATIONS EF etiologic fraction MHC major histocompatibility complex PCR polymerase chain reaction SSO RR sequence-specific oligonucleotide relative risk From The Rayne Institute (M. R. T., P. J. CJ and Molecular lmmunogenetics IM. A. H., J.

Three new allelic forms of the HLA-G DNA sequence (HLA-G*II, HLA-G*III, and HLA-G*IV) have been identified. With the HLA-G*I sequence (previously designated HLA 6.0) as a reference, HLA-G*II shows a silent (G → A) mutation at the third base of codon 57, HLA-G*III bears a non-synonymous (A → T), but conservative, (Thr → Ser) substitution at the first base of codon 31, and HLA-G*IV shows two silent substitutions: (A → T) at the third base of codon 107 and (G → A) at the third base of codon 57. A rapid method of singling out each allele on genomic DNA has been developed by using polymerase chain reaction amplification followed by restriction endonuclease treatment. Also, more or less strong linkage disequilibria has been found between most HLA-A alleles and either HLA-G*I or *II, both being the most prevalent alleles in the population, with a genotypic frequency of 0.55 and 0.38, respectively; HLA-G*III is very rare and HLA-G*IV has a genotypic frequency of 0.07. An evolutive classification of HLA-A alleles results according to their association with either HLA-G*I or HLA-G*II, which does not correlate with the classical serological cross-reacting groups classification. The finding of a strong and selective A/G linkage disequilibria with most HLA-A alleles, together with the existence of less frequent random A/G associations, may suggest that there exist in different haplotypes true and varied A/G genetic distances (and not a recombinational hotspot). It may be inferred from preliminary data that in primates HLA-A/G haplotypes bearing G*II may have appeared later than those bearing G*I.

The human leukocyte antigen (HLA) class III region, located on chromosome 6p21, has been regarded as one of the susceptible loci for type 1 diabetes. Because it contains many genes related to inflammatory and immune responses, including tumor necrosis factor (TNF), lymphotoxin-␣ (LT-␣), and allograft inflammatory factor 1 (AIF-1) genes, it is unclear which gene within the class III region is responsible for the susceptibility to the disease. We sequenced the AIF-1 gene region and detected three novel polymorphisms, all of which were diallelic and localized at introns. Then, we investigated AIF-1, TNF, and LT-␣ gene polymorphisms in 165 patients with type 1 diabetes, consisting of 90 patients with young-onset type 1 diabetes, 75 patients with adult-onset type 1 diabetes, and 200 control patients. We also analyzed TNF receptors type 1 (TNFR1) and type 2 (TNFR2) gene polymorphisms, located on chromosome 12p13 and 1p36, respectively. Although there were significant differences between type 1 diabetes patients and controls in the distributions of TNF promoter poly-morphisms at position Ϫ1031 and Ϫ857, and LT-␣ gene NcoI polymorphism, none of them was independently associated with the disease after two-locus analysis with HLA class II alleles. We detected the significantly increased frequency of the Ϫ383C allele, located in the TNFR-1 promoter region, in both young-onset and adult-onset diabetes patients compared with controls. In addition, the Ϫ383C allele was found to be associated with higher expression of the TNFR1 gene than that of Ϫ383A allele in in vitro expression. These results suggest that the TNFR1 gene region might be a susceptible locus to type 1 diabetes in Japanese.

Block matching is a valuable tool for selecting donors for bone marrow transplantation. Identical, electrophoretic profiles of unrelated bone marrow donor-recipient pairs have been shown to be associated with long-term survival and a reduction of graft versus host disease (GVHD). This study was undertaken to determine the sequences of the PCR products which are generated. PCR products obtained with beta-block

Aim: Dried Blood Spots (DBS) on FTA cards serve as a good resource for HLA and other SNP genotyping studies. Isolation of gDNA from small punches of DBS for genetic testing, such as HLA typing, is cumbersome and inefficient. Herein, we describe a quick and inexpensive protocol for the preparation of DNA extract from DBS that shows promise for HLA typing based on results from LIFECODES HLA-A SSO typing kit. Methods: Small 3 mm discs of DBS were washed with FTA washing buffer to remove possible PCR inhib-490 Abstracts / Human Immunology 74 (2013) 483-499

The purpose of this study was to investigate whether in highly sensitized patients (HSPs) the acceptable HLA-A and -B mismatches (AMs) can be predicted on the basis of patients' HLA-phenotype. To this affect, 1000 historical serum samples obtained from 50 HSPs (PRA Ͼ 60%), panel reactive antibodies (PRA) value and the specificity of class I anti-HLA-antibodies were detected by two techniques in parallel: An antihuman globulin augmented cytotoxicity (AHG-CDC) and an Elisa technique. Thereafter, class I HLA-antigens of the nonreactive cells in the screening panel and class I HLA-antigens of the patients were assigned to CREGs. The AMs for each one of the patients were detected using a separate cell panel, which was prepared in a way to include almost all the HLA-antigens belonging to the CREGs of the patients as well as to those of the nonreactive cells in the screening panel. It was found that the AMs in HSPs, detected with this protocol were more, compared to those we usually detect using only the HLAantigens of the nonreactive cells in the screening panel (up to 8 versus 2-5). Both, the definitively detected AMs, and the HLA-specificities of the nonreactive cells of the screening panel belonged to the same CREGs. These CREGs were equivalent to the CREGs of class I HLAphenotypes of each patient. The data presented in this paper introduce a new, rapid and easier way for the detection of AMs in HSPs. According to this proposed protocol, the assignment of patients' standard class I private HLA-phenotypes in CREGs, not only greatly facilitates the detection of AMs, but the detected AMs are also in fact significantly more than those determined by the conventional methodology. We have also confirmed that the majority of antibodies induced by HLA alloimmunization are directed against mismatched shared or public group epitopes CREGs. Moreover, we have confirmed that prospective matching for major CREGs would be feasible on a national level and would not significantly prolong waiting time, which could result in a significant augmentation of the potential donor pool. Human Immunology 59, 587-596 (1998).

Familial Mediterranean fever (FMF) inflammatory attacks are often triggered by metabolic or physical stress. mTOR signaling and autophagy modulate cellular responses to metabolic danger signals. In this study, we investigated the implication of mTOR inhibition and autophagy in FMF pathophysiology. mTOR inhibition induced MEFV gene expression in polymorphonuclear cells (PMNs) from healthy individuals, whereas it had no effect on PMNs from attack-free FMF patients. A significant reduction in pyrin levels in PMNs from FMF patients after mTOR inhibition was also observed. Pyrin levels in control PMNs remained unaffected. Moreover, the basal autophagic status in PMNs from FMF patients was reduced, as indicated by the lower LC3B-II/I ratio and ATG mRNA expression levels. However, mTOR inhibition had similar effects on the induction of autophagy in the two groups. The differential pyrin expression after metabolic stress induction and the impaired basal autophagy suggest a potential role in the triggering of FMF attacks.

Vogt-Koyanagi-Harada’s syndrome (VKH) is an autoimmune disease prevalent in Mongoloids with evident participation of HLA. The aim of this study was to identify the class II DNA sequences involved in the etiopathogenesis of VKH in Mexican Mestizos. This study included 46 VKH patients and 170 controls. 75% were females (mean age at onset of 33.5 years). The disease evolved to

Interactions between inhibitory killer cell immunoglobulin-like receptors (iKIR) and HLA class I molecules regulate natural killer (NK) cell responses to eliminate infected and transformed cells while maintaining tolerance to healthy cells. Unlinked polymorphic gene families encode KIR and HLA, and their independent segregation results in a variable number and type of iKIRϩHLA pairs inherited in individuals. The aim of study is to test if coexistence of a minimum of one iKIR and its specific HLA class I is necessary to maintain self-tolerance and enable NK cell competency. Methods: KIR and HLA genotyping was performed in 759 unrelated individuals. The diversity in the coinheritance of iKIRϩHLA pairs and activating KIR (aKIR) genes were analyzed in four ethnic populations. Scope: The results showed that every individual studied inherited a minimum of one iKIRϩHLA pair suggesting that MHC class I-dependent inhibitory signaling is essential for human NK cells. In contrast, 13.4% of the study group lacked all aKIR genes. Twenty percent of the study group carried only one of the four iKIRϩHLA pairs. Interestingly, 3% of the study group carrying only KIR2DL3ϩHLA-C1 as an iKIRϩHLA pair lacked aKIR genes. These data suggest that a single iKIR can maintain self-tolerance and constitute the minimal KIR repertoire for human NK cells. Genotypes carrying an equal number of iKIRϩHLA pairs and aKIR genes were represented in 20% of our study group. The remaining individuals had either a dominant inhibitory KIR genotype (iKIRϩHLAϾaKIR) or a dominant activating KIR genotype (iKIRϩHLAϽaKIR). Conclusions: Genotypes encoding imbalanced inhibitory and activating interactions may contribute to the susceptibility or resistance to human diseases.

Aim: Complement-fixing (CF) HLA antibodies (Ab) play an important role in transplantation because they can initiate inflammatory processes within the graft that lead to rejection. The Luminex Ig-binding assay (LUM-Ig) is now the principal method for HLA Ab testing. The C1q-binding assasy (LUM-C1q) can distinguish between CF and non-CF Abs. We show here evidence that the differential reactivity of CF HLA Ab depends on the structural composition of epitope (Ep)-carrying alleles (Al). Methods: Human cytotoxic class I Ep-specific monoclonal antibodies (mAb) were tested in LUM-Ig and LUM-C1q (OneLambda). Results were compared to CDC reactivity with informative cells from the 13th International Workshop. Results: Two reactivity patterns were observed. For some mAb, Ep-carrying Al reacted similarly in all assays. But certain mAb reacted with more Al in LUM-Ig than in LUM-C1q and CDC. Reactivity was analyzed with HLAMatchmaker, incorporating the concept that eplets are essential parts of structural Eps which can contact the 6 Complementarity Determining Regions (CDRs) of Ab. The data show that LUM-Ig mAb are specific for eplets but that LUM-C1q and CDC reactivity require an additional polymorphic amino acid configuration on eplet-carrying Al. Conclusions: The findings must be viewed in context of formation of an antigen-Ab complex resulting in release of free energy necessary to stabilize binding and to induce conformational change in the Fc part of the Ab structure to expose the C1q receptor, the first step of Complement activation. The CF properties of HLA Ab require not only specific recognition of an eplet but also depend on interactions of other CDRs with critical amino acid configurations within the structural Ep. Eplet-carrying Al that lack such critical configurations will bind with Ab but the amount of free energy is insufficient to expose the C1q-binding site and there is no CF.

In Saudi the chance of finding matched sibling is around 70% which is higher than many other countries, due to large family size. For the remaining 30% of the patients, the probability of finding a match in the international unrelated bone marrow registries is slim to null. The emergence of the cord blood transplantation that allows less than perfect HLA matching between patient and cord blood unit (CBU), gave a chance to more patients to be transplanted. In this study we analyzed the chance of finding a suitable CBU, the level of HLA ABDR matching and the cost effectiveness of the cord blood bank at King Faisal Specialist Hospital and Research Center. Methods: A total of 266 consecutive searches (216 for pediatrics and 50 for adult patients) were evaluated within the 3440 CBU inventory at KFSH & RC cord blood bank that resulted in 69 transplants between Oct-2008 to Dec-2011. The CBU was typed at the low resolution level for HLA-A, HLA-B and at the high resolution level for HLA-DRB1 alleles. Results: 89.1% of the patients found at least one CBU equal or greater than 4/6 HLA match with total nucleated (TNC) cell P3.7 Â 10 7 /kg. 50.2 of the patients had 4/6 HLA match, 42.2% of the patients had 5/6 HLA match and 7.6% had 6/6 HLA match CBU. 10.9% of the patients did not find 4/6 or better match. As the numbers of CBU is adding up, the need to imported CBU is decreased. In 2009, 44% of the donor CBU were imported, in 2010 16.6% and in 2011 only 9% were imported. In addition to less time from search until the unit ready for transplant, the cost of the CBU from our cord blood bank is only 21% of the cost of the imported CBU. Conclusions: This study clearly indicates the cost effectiveness of the institution cord blood bank and the needs to increase the inventory size to improve the probability of finding 5/6 or full house 6/6 CBU.

Nuclear factor κ B (NFκB) is an important transcription factor that together with its inhibitor (IκB) participates in the activation of genes involved in immune responses. We examined the CA repeat polymorphism of the NFKB1 gene (encoding for NFκB) and A/G point variation in the 3′UTR region of the nuclear factor kappa B inhibitor alpha (NFKBIA) gene (encoding for IκB) in Czech and German patients with type 2 diabetes. The sample consisted of 211 patients, both with and without kidney complications, and 159 controls. Additionally, 152 patients with systemic lupus erythematosus (SLE) were genotyped for NFKBIA polymorphism. We observed a significant increase in the homozygous AA genotype of the NFKBIA gene when compared with the control group (the highest value was in diabetics without diabetic nephropathy [pc* = 0.0015, odds ratio = 3.59]). No differences were seen between the SLE and control groups. With regard to the polymorphism of the NFKB1 gene, we did not observe any significant differences between the groups. Since the AA genotype of the NFKBIA gene presents a risk for type 2 diabetes development but not for diabetic nephropathy alone, we believe that the NFκB gene polymorphism can influence the pathogenesis of diabetes mellitus and affect its complications. Negative findings relative to other inflammatory autoimmune diseases, such as SLE, suggest a specific relationship between NFκB and type 2 diabetes mellitus.

Aim: Many sequencing strategies for HLA-DRB1 utilize tailed amplification primers targeting binding sites within the exon resulting loss of meaningful antigen binding site polymorphism. Tailed amplicon may also yield increased levels of baseline noise due to anomalous PCR fragment formation. We describe a new strategy for HLA-DRB1 sequence based typing (SBT) that uses intronic primers for amplification and sequencing of complete exon 2 and exon 3 to increase resolution and ensure detection of novel alleles. Our design produces uniform fragment lengths for each exon resulting in robust amplification and uses nested sequencing primers to produce high quality, low background data.

Increased production of matrix metalloproteinases (MMPs) plays an important role in tissue damage in inflammatory bowel disease (IBD). Genetically encoded variation between individuals in MMP production may therefore contribute to disease onset, type, or severity. We undertook an extensive candidate gene single nucleotide polymorphism (SNP) study of MMP-1,-2,-3,-7,-8,-9,-10,-12,-13, and-14 and tissue inhibitor of metalloproteinases (TIMPs)-1,-3, and-4 in ulcerative colitis (UC). We identified tagging SNPs across these genes, and genotyped these SNPs in a Caucasian New Zealand dataset consisting of 419 UC patients and 907 controls. SNPs in a number of MMP genes were associated with UC. After correcting for multiple testing SNPs in MMP-3, MMP-8, MMP-10, and MMP-14 remained significant in their associations with UC. In a second study, using samples from a Dutch cohort, most of the significant findings in the New Zealand cohort were not replicated. However, data from an international meta-analysis provide some support for the initial findings. In conclusion, this study provides preliminary evidence to suggest that genetic variation in the MMPs may play a role in interindividual differences in UC susceptibility and clinical outcome. Further studies are needed in other cohorts to determine the robustness of these observations in different populations.

The HLA allele frequency distribution of the Mayans from Guatemala was studied and compared with those of other First American Natives and worldwide populations (a total of 12,364 chromosomes and 6182 individuals from 60 different populations). The main conclusions were (1): the closest Amerindian group to Mayans is the Arhuacs, who were the first recorded Caribbean Islands' inhabitants (2). Mayans are not so close to Mesoamerican Zapotec, Mixe and Mixtec Amerindians, who genetically cluster together. Mixe had been related to Mayans only on linguistic bases (3). DRB1*0407 and DRB1*0802 alleles are found in 50% of Mayans; these alleles are also found in other Amerindians, but the Mayans' high frequencies may be showing a founder effect for this Mesoamerican-Caribbean population (4). Extended Mayan specific HLA haplotypes are described for the first time (5).

Rituximab (RIT), a murine/human chimeric monoclonal antibody directed against human CD20 is under investigation for its role in transplantation. RIT causes B-cell crossmatches to appear positive. Pronase, a proteolytic enzyme that targets F c receptors removes CD20 from B cells. After CD20 is removed, RIT should not bind, making it possible to detect class I or class II antibodies on treated B cells. In this study, we incubated RIT with normal human serum (NHS, negative control) or pooled sera from highly sensitized (Ͼ50% panel reactive antibody, HLAϩ) subjects awaiting renal transplantation (positive control) and then performed B-cell flow cytometric crossmatches using untreated or pronase treated B cells as targets. We observed that untreated B cells incubated with RIT-spiked NHS displayed a signif-icant increase in surface fluorescence compared with NHS without RIT, similar to the fluorescence that occurs with a positive crossmatch. In contrast, when CD20 was cleaved from the B cells with pronase, B cells displayed a negative crossmatch with the RIT-spiked NHS. In addition, there was no change in the crossmatches of pooled high panel reactive antibody (PRA) sera after pronase treatment. RIT could be used without worry about losing the ability to perform transplant immunologic monitoring.