Identification of a novel HLA-B allele, HLA-B*08:177, in a Hungarian patient and her HLA identical sibling.

The newly detected HLA-B*08:177 differs from HLA-B*08:01:01:01 by 1 single nucleotide substitution at position 365 of exon 3.

The potential role of HLA-DRB1*11 in the development and outcome of haematopoietic stem cell transplantation-associated thrombotic microangiopathy.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a serious complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) with high mortality rate. We retrospectively studied the frequency, clinical and genetic associations and prognostic effect of TA-TMA, in a total of 425 consecutive adult patients, who underwent allo-HSCT for a malignant haematological condition between 2007 and 2013 at our single centre. TA-TMA developed in 19% of the patients. Unrelated donor type (P<0.001), acute GvHD grades II-IV (P<0.001), myeloablative conditioning regimens (P=0.003), tacrolimus-based GvHD prophylaxis (P=0.003), CMV infection (P=0.003) and carriership for HLA-DRB1*11 (P=0.034) were associated with the development of TA-TMA. Survival was adversely affected by the presence of TA-TMA (P<0.001). Among patients with TA-TMA, the outcome of HLA-DRB1*11 carriers was significantly better compared with non-carriers (P=0.003). As a new finding, our observations suggest that the presence of HLA-DRB1*11 antigen contributes to the development of TA-TMA and affects the outcome.

HLA genetic diversity in Hungarians and Hungarian Gypsies: complementary differentiation patterns and demographic signals revealed by HLA-A, -B and -DRB1 in Central Europe.

Systematic analyses of human leukocyte antigen (HLA) profiles in different populations may increase the efficiency of bone marrow donor selection and help reconstructing human peopling history. We typed HLA-A, -B, and -DRB1 allele groups in two bone marrow donor cohorts of 2402 Hungarians and 186 Hungarian Gypsies and compared them with several Central-European, Spanish Gypsy, and Indian populations. Our results indicate that different European Gypsy populations share a common origin but diverged genetically as a consequence of founder effect and rapid genetic drift, whereas other European populations are related genetically in relation to geography. This study also suggests that while HLA-A accurately depicts the effects of genetic drift, HLA-B, and -DRB1 conserve more signatures of ancient population relationships, as a result of balancing selection.

Low occurrence of the HLA-C*04:09N allele in a large Hungarian cohort.

The presence of null alleles may affect the outcome of stem cell transplantation. HLA-C*04:09N was defined as 'common' with a frequency of 2-5/1000 in Caucasians, and its presence is routinely tested as part of haplotypes HLA-A*02:01/A*23:01-B*44:03-DRB1*07:01-DQB1*02:01. We aimed to investigate HLA-C*04:09N in a representative Hungarian cohort. HLA-typing data of 7345 unrelated persons were analyzed. The presence of HLA-C*04:09N was excluded in 157 chromosomes with either serology typing or with an allele-specific polymerase chain reaction for HLA-C*04:09N. HLA-C*04:09N was identified in a single chromosome with HLA-A*02, B*44, C*04, DRB1*07 resulting in a HLA-C*04:09N allele frequency of 0.0068% (1/14,690). This is approximately a 10- to 40-fold lower frequency compared with the previous data. Our results emphasize the need of precise local population-specific HLA-data, allowing appropriate modifications of local HLA-typing protocols.

Decrease in cold ischemic times as a result of protocol changes of urgent immunogenetic testing during cadaveric kidney transplantation in Hungary.

BACKGROUND: Based on national ethics committee permission, the procedure of urgent immunogenetics testing prior to cadaveric kidney transplantation was changed in Hungary from January 1, 2011 allowing HLA typing of the donor and prospective crossmatching using peripheral blood samples from the donor prior to the definitive declaration of brain death. The aim of the current study was to compare key indicators of transplantation primarily cold ischemic time [CIT], between time periods with outcomes. METHODS: The following indicators were systematically collected prospectively and retrospectively for each deceased heart-beating donor transplantation between January 1, 2010 and October 31, 2010 (n = 114) versus January 1, 2011 and October 31, 2011 (n = 91): CIT for the first and second kidney; laboratory turnaround times (TAT), and time for final preparation of the selected recipient. RESULTS: As a result of the new procedure, the CIT for the first kidney decreased from 16.5 ± 3.5 to 12.4 ± 3.2 hours (P < .001). Similarly, for the second kidney the parameters were a 19.8 ± 3.4 versus 16.0 ± 3.8 hours (P < .001). As a consequence of more hands-on time in the laboratory, the TAT increased from 5.6 ± 0.8 hours to 7.2 ± 1.1 hours (TAT1) followed by an additional 4.2 ± 1.0 hours (TAT2). We also compared the times necessary for preparation of immunologically suitable recipients for transplantation, namely, 9.5 ± 2.3 hours in the earlier system, increasing to 15.5 ± 4.3 hours during the new procedure. CONCLUSION: As a consequence of the procedural change, the CIT parameter decreased significantly for both kidneys, which may have contributed to improved short-term outcomes of transplantation. The time available for final preparation of selected recipients was increased allowing improvements in CIT.

ATG16L1 and IL23 receptor (IL23R) genes are associated with disease susceptibility in Hungarian CD patients.

BACKGROUND: North American and European genome-wide association scans have identified ATG16L1 and IL23R as novel inflammatory bowel disease (IBD) susceptibility genes and subsequent reports confirmed these findings in large independent populations. The aims of this study were to investigate the association and examine genotype-phenotype relationships in a Hungarian IBD cohort. METHODS: 415 unrelated IBD patients (CD: 266, age: 35.2+/-12.1 years, duration: 8.7+/-7.5 years and UC: 149, age: 44.4+/-15.4 years, duration: 10.7+/-8.9 years) and 149 healthy subjects were investigated. IL23R Arg381Gln (R381Q, rs11209026) and ATG16L1 Thr300Ala (T300A, rs2241880) polymorphisms were tested using LightCycler allele discrimination method. Detailed clinical phenotypes were determined by reviewing the medical charts. RESULTS: The association between IL23R rs11209026, ATG16L1 rs2241880 and CD was confirmed (OR(IL23R381Q): 0.38, 95% CI: 0.16-0.87; OR(ATG16L1300AA): 1.86, 95% CI: 1.04-3.40). No difference was found between patients with UC and either controls or CD. In CD, IL23R 381Gln heterozygosity was associated with inflammatory disease (70% vs. 34%, p=0.037), while disease restricted to the colon was more prevalent in patients with the ATG16L1 300Ala/Ala homozygosity (33.3% vs. 21.1%, p=0.036). In addition, carriage of the variant alleles did not predict response to steroids, infliximab or need for surgery. CONCLUSIONS: We confirmed that ATG16L1 and IL23R are susceptibility loci for CD in Hungarian CD patients. Further studies are needed to confirm the reported phenotype-genotype associations found in this study.

A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity.

To establish a simple and rapid method for the screening of stable recombinant Chinese hamster ovary (CHO) cell lines, we have developed a cell surface labeling technique using fluorescently tagged antibodies that bind to secreted target proteins at low temperature. Using fluorescence intensity as the sole criterion for selection of cells, we are able to enrich populations of highly productive cells using preparative flow cytometry sorting. Reiterative sorting based on selection of cells having the highest fluorescence intensity of cell surface labeled protein results in dramatic increases in specific cellular productivity. Using lymphotoxin-beta receptor IgG fusion protein as a model system, we have demonstrated a greater than 20-fold increase in specific productivity (0.49-11.5 pg cell(-1) day(-1)) (pcd) without the use of methotrexate (MTX)-mediated selection or amplification. In addition, the flow cytometry used to enrich for and clone high producer cell lines has reduced development time by more than 50% and the number of screening assays by more than 10-fold. When a transfected population of CHO cells expressing a humanized version of the murine monoclonal antibody (mAb) AQC2 directed against human alpha 1 beta 1 integrin was subjected to the same treatment, a 25-fold improvement in specific productivity (0.3-8.0 pcd) was observed. Furthermore, similar application of this technique to MTX-amplified clones resulted in up to 120-fold overall improvement in specific productivity (up to 42 pcd). Greater than 20 examples are also presented to demonstrate the robustness and performance of this technique.

Co-expression of interleukin-2 and green fluorescent protein reporter in mycobacteria: in vivo application for monitoring antimycobacterial immunity.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing green fluorescent protein (rBCG-GFP), driven by the mycobacterial heat shock protein 70 (HSP 70) promoter from an autonomously replicating plasmid, was genetically engineered to co-express mouse interleukin-2 (IL-2) by introduction of an independent HSP 60 promoter. To monitor host antimycobacterial immunity, C57BL/6 mice were intravenously infected with IL-2 expressing and non-expressing GFP rBCGs. Both rBCGs were clearly imaged and easily quantified with ultraviolet microscopy of tissue sections and whole organ suspensions. Enhanced mycobacterial clearance from the spleens of mice infected with the rBCG-IL-2/GFP strain was apparent by both diminished bacterial counts and spleen weights during the first 6 weeks post-infection relative to rBCG-GFP. T helper type 1 (TH1) cytokine production and proliferative response to BCG restimulation was also elevated from in vitro splenocyte cultures taken from the rBCG-IL-2/GFP-infected group. Taken together, these results suggest that IL-2 expression from rBCG augmented host protective immunity to mycobacterial infection via an enhanced TH1 immune response. Mycobacterial expression vectors that allow simultaneous but independent production of reporter proteins and bioactive substances provide an ideal means for monitoring the in vivo fate of recombinant mycobacteria.

Role of IL-12 in the induction and potentiation of IFN-gamma in response to bacillus Calmette-Guérin.

Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been accepted as the most effective agent in clinical use against superficial bladder cancer, its mechanism of action remains incompletely understood. A kinetic analysis in assessing the potential role of cytokines from BCG-stimulated murine splenocytes showed that IL-12 expression preceded that of other cytokines. Experiments subtracting endogenous BCG-driven IL-12 using neutralizing Ab or augmenting its activity with supplemental rIL-12 revealed not only that IL-12 plays a dominant role in IFN-gamma induction but also that it is normally dose limiting. A striking increase in IFN-gamma production could be generated in both mouse and human immunocompetent cell culture by the addition of even a small amount of rIL-12. Moreover, this same synergistic effect could be replicated during in vivo administration of BCG plus rIL-12 into the mouse bladder and was observed in a patient receiving intravesical combination therapy. In costimulation cultures, this synergy appeared to partially rely on IL-18 and IL-2 and could be down-regulated by IL-10. This suggests that a dynamic interplay between Th1 and Th2 cytokines is responsible for net IFN-gamma production. The ability of supplemental exogenous IL-12 to strongly shift this balance toward Th1 provides an immunological basis for using it in conjunction with intravesical BCG for bladder cancer immunotherapy.

Pharmacological biocompatibility between intravesical preparations of BCG and interferon-alpha 2B.

PURPOSE: To determine if BCG and interferon alpha-2B are mutually compatible as mixed intravesical agents for clinical bladder cancer therapy. MATERIALS AND METHODS: Mutual compatibility was assessed by measuring IFN-alpha's effect on BCG metabolic activity, growth rate, and clumping tendency and conversely by observing BCG's effect on IFN-alpha's anti-viral activity. Optical density at 600 nm. (OD600) was used to estimate the number of colony forming units of BCG in suspension during 3 hours measurements of clumping and 8 days measurements of BCG proliferation. BCG viability was evaluated using a substrate marker, MTT, which correlates with BCG density and metabolic activity. The anti-viral activity of IFN-alpha was determined in a cytopathic protection bioassay using the encephalomyocarditis virus/FS-4 cell system. RESULTS: Continuous shaking of reconstituted BCG for 3 hours at 37C resulted in a marginal (11.3%) drop in OD600 which was minimally altered by inclusion of IFN-alpha at 2 million units (MU)/ml. (12.7% drop). Metabolic activity and growth rate of BCG alone or BCG with IFN-alpha were essentially identical. IFN-alpha's antiviral activity was not affected by incubation with BCG. CONCLUSIONS: The inclusion of IFN-alpha into the usual BCG formulation for intravesical administration has no apparent effect on BCG's viability or tendency to form clumps in suspension. Similarly, the physical mixing of IFN-alpha with BCG does not impair its biological activity. Thus, both agents are pharmacologically compatible for future clinical studies involving combination intravesical therapy.

Streptococcal preparation OK-432 is a potent inducer of IL-12 and a T helper cell 1 dominant state.

Streptococcal preparation OK-432 is a bacterial immunopotentiator extensively used in Japan for adjuvant cancer therapy. Using a C57BL/6 mouse model, OK-432 was found to induce multiple cytokines including the Th1 polarizing cytokine IL-12. Expression of IL-12 protein by murine splenocytes was restricted to macrophages and B cells and led to high levels of IFN-gamma production from both CD4+ and CD8+ T cells. Of the Th2 cytokines IL-4 and IL-10, only IL-10 protein was detected and originated primarily from the adherent cell population. Its expression was delayed relative to IL-12. A similar pattern of cytokine induction was observed from human PBMCs. OK-432-driven IFN-gamma production was inhibited by anti-IL-12 Ab, anti-IL-2 Ab, anti-TNF-alpha Ab, and anti-IL-2R alpha Ab, suggesting that IFN-gamma production from Th1 cells is induced by the cooperation action of these cytokines through the IL-2R alpha pathway. When compared with another widely used immunopotentiator bacillus Calmette-Guérin (BCG), OK-432 was a stronger IL-12 and IFN-gamma inducer. Furthermore, the mechanism of IFN-gamma induction by OK-432 differed from BCG in that coincident granulocyte-macrophage CSF and IL-1 expression played little to no role. These results suggest that OK-432 is a potent multicytokine inducer, specifically a strong inducer of IL-12, and that OK-432 may exert its antitumor effect by promoting a Th1-dominant state.

A novel method for monitoring Mycobacterium bovis BCG trafficking with recombinant BCG expressing green fluorescent protein.

To better understand intracellular and extracellular trafficking of Mycobacterium bovis bacillus Calmette-Guérin (BCG) when used as an intravesical agent in the treatment of transitional cell carcinoma (TCC) of the bladder, recombinant BCG (rBCG) expressing the jellyfish green fluorescent protein (GFP) was created. When the MB49.1 murine TCC cell line was incubated with GFP-expressing rBCG, internalization of the pathogen could be directly visualized by UV microscopy and quantitated by flow cytometry. The in vitro internalization of the GFP rBCG by the bladder tumor cells was temperature dependent, occurring most readily at 37 degrees C and being severely inhibited at 4 degrees C. Optimum internalization was achieved in vitro at a 10:1 BCG-to-tumor cell ratio over 24 h during which approximately 16% of the tumor cells became infected. Cytochalasin B, a phagocytosis inhibitor, abrogated the ingestion by almost 100% at a concentration of 200 micrograms/ml, indicating that contractile microfilaments likely played an important role in this process. By using mitomycin, a DNA cross-linking reagent, to inhibit proliferation of MB49.1 cells, clearance of about 40% of the green rBCG was achieved by 3 days postinfection. No significant difference between the GFP rBCG and wild-type BCG was observed in the ability to induce the expression of cell membrane proteins of major histocompatibility classes I and II, ICAM-I and -II, B7-1 and -2, of Fas from MB49.1 cells or cytokine production from mouse spleen cells. These results indicate that GFP rBCG may serve as a useful substitute for wild-type BCG in future studies of in vivo trafficking experimental and clinical immunotherapy.

Bacillus Calmette-Guérin plus interleukin-2 and/or granulocyte/macrophage-colony-stimulating factor enhances immunocompetent cell production of interferon-gamma, which inhibits B16F10 melanoma cell growth in vitro.

Although immunotherapy with bacillus Calmette Guérin (BCG) is an established adjuvant treatment for malignant melanoma, the mechanism of its role in this process is unclear. To investigate the possible contribution of tumor-inhibitory cytokines induced by BCG, B16F10 melanoma cell growth in culture was assessed in response to purified cytokines and conditioned media of BCG-stimulated splenocytes. Interferon-gamma (IFN gamma) was the most potent single agent (IC50 approximately 50 pg/ml). Tumor necrosis factor alpha was substantially weaker (IC50 > 10 ng/ml) but provided synergy with IFN gamma. None of the other cytokines such as interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-12, or granulocyte/macrophage-colony-stimulating factor had direct antitumor activity against B16F10 melanoma cells. However, when IL-2 and/or GM-CSF were combined with BCG either by exogenous addition or through endogenous production by novel cytokine-secreting recombinant BCG (rBCG), a substantial increase in INF gamma production by splenocytes was observed. Antitumor activity of this conditioned medium directly correlated with IFN gamma concentration and was completely blocked by neutralizing antibody to IFN gamma. These results suggest that BCG may exert part of its antitumor action on melanoma through the induction of IFN gamma, which can be greatly enhanced through the concomitant addition of IL-2 and/or GM-CSF. Furthermore, by utilizing rBCG that secrete these cytokines, it may be possible to potentiate the antitumor effect of BCG directly at the site of BCG inoculation.

Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes.

Mycobacterium bovis BCG was genetically engineered to express and secrete mouse interleukin-2 (IL-2) and rat IL-2. Genes encoding IL-2 were inserted into an Escherichia coli-BCG shuttle plasmid under the control of the BCG HSP60 promoter. To facilitate study of proteins produced in this system, the IL-2 gene product was expressed (i) alone, (ii) with the mycobacterial alpha-antigen secretion signal sequence at the amino terminus, (iii) with an influenza virus hemagglutinin epitope tag at the amino terminus, and (iv) with both the secretion signal sequence and the epitope tag. When expressed with the alpha-antigen signal sequence, biologically active IL-2 was secreted into the extracellular medium. Western blot (immunoblot) analysis of the intracellular IL-2 and extracellular IL-2 revealed that the secretion signal was appropriately cleaved from the recombinant lymphokine upon secretion. To assess the ability of recombinant BCG to stimulate cytokine production in a splenocyte population, mouse splenocytes were cultured together with wild-type or IL-2-producing BCG. IL-2-secreting BCG clones stimulated substantial increases in gamma interferon production, which could be reproduced by the addition of exogenous IL-2 to BCG. Levels of IL-6, IL-10, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor were not significantly changed, while IL-4 and IL-5 remained undetectable (less than 50 pg/ml). The enhanced production of gamma interferon in response to IL-2-secreting BCG was strain independent. Recombinant BCG expressing mammalian cytokines provides a novel means to deliver cytokines and may augment the immunostimulatory properties of BCG in immunization and cancer therapy.