Correlation of single nucleotide polymorphisms in the promoter region of the ANXA5 (annexin A5) gene with recurrent miscarriages in women of Greek origin.

Background: Recent findings show that a number of single nucleotide polymorphisms (SNPs) within the promoter region of the annexin A5-gene (ANXA5) reduce the expression of the reporter gene and so they display a significant association with recurrent pregnancy loss (RPL).Objective: The objective of the present study aimed to address the contribution of ANXA5 M2 haplotype consisting of four minor alleles: (SNP1: (-)467G > A, SNP2: (-)448A > C, SNP3: (-)422T > C, and SNP4: (-)373G > A) in the occurrence of recurrent pregnancy losses in the Greek population, and the role of further two minor alleles: SNP5: (-)302 T > G and SNP6: (-)1C > T as independent risk factors for RPL.Methods: A 752-bp genomic region of ANXA5 promoter was amplified by PCR using specific primers. Genotypic analysis by Sanger sequencing was performed for these six SNPs (minor alleles) in the promoter region of ANXA5 gene, in 100 (100) Greek women with recurrent miscarriages (median =3) and 70 (70) fertile controls. Statistical analysis was done using the SAS 9.3 for Windows (SAS Institute Inc, NC, USA) and SPSS packages for Windows (C.DiMaggio 2013, SAS Institute 2014).Results: This case-control study revealed that there is no significantly increased risk of RPL among the M2/ANXA5 haplotype carriers in the Greek population, as there were no statistical differences between the patients with recurrent pregnancy losses and the fertile controls (11.5% in RPL cases versus 9.29% in controls, p-value: .6364). There was no difference in SNP5 and SNP6 minor carriership between the two groups. In particular, carriers of SNP5 and SNP6 had an increased risk for RPL state with odds ratio: 1.2472 and 1.3846 respectively, however without statistically significant importance.Conclusion: The M2/ANXA5 haplotype does not differ between RPL patients and controls in the Greek population. Also, it is the first time that SNP5 and SNP6 minor alleles were evaluated extensively in women of European origin with recurrent pregnancy losses (RPL), and they do not seem to be independent risk factors in the occurrence of RPL in the Greek population. Though, this has to be confirmed in further and larger clinical trials with women of European origin.

Effect of the histone deacetylase inhibitor trichostatin a in human peripheral blood lymphocytes as a function of donor age.

The histone deacetylase inhibitor trichostatin A (TSA) is a promising agent for the treatment of certain types of cancers alone or in synergistic combination with other anticancer agents. One of the advantages of the use of histone deacetylase inhibitors, such as TSA, is that its effects have been found to be more potent toward cancer cells compared to normal cells. The effect of anticancer agents on the immune system, and on lymphocytes in particular, is of major importance to the success of anticancer regimens. In this respect, information documenting the effect of such agents on normal lymphocytes compared to malignant cells may be of significant value for the successful designing of clinical protocols. Moreover, the parameter of age may be a factor in the differential effects of such protocols. Histone deacetylase inhibitors lead to the accumulation of acetylated histones and, depending on the cell type, may induce either apoptosis, cell cycle arrest, or differentiation. Previous work from our lab has shown that TSA induces the accumulation of histone H4 acetylation and apoptosis in human peripheral blood lymphocytes. In light of the above, we have extended our investigation of the effects of TSA on human lymphocytes to include the parameter of age, which has not been previously studied. Our results show that TSA induces apoptosis of lymphocytes from donors of all age groups, but no age-related changes in the levels of apoptosis are observed.

Association of heart failure in homozygous beta-thalassemia with the major histocompatibility complex.

BACKGROUND: In beta-thalassemia major, heart failure primarily affecting left ventricular systolic function is the most common complication and cause of death. Apart from iron deposition, it has been recently reported that myocarditis might be another contributing factor in the pathogenesis of acute or chronic heart failure, acting possibly through an autoimmune mechanism. In an attempt to assess the role of immunogenetic factors in the development of heart failure associated with beta-thalassemia major, we studied the frequency of major histocompatibility antigens/alleles A, B, DR, and DQ in homozygous beta-thalassemic patients with and without heart failure primarily affecting the left ventricle. METHODS AND RESULTS: Forty-five consecutive unrelated Greek patients with homozygous beta-thalassemia and left-sided chronic heart failure were studied. Fifty-eight unrelated Greek patients with homozygous beta-thalassemia without heart failure and 130 unrelated Greek healthy controls were also studied. In all subjects, class I HLA-A and -B typing was performed by the complement-mediated lymphocytotoxicity assay, whereas class II HLA-DR and -DQ typing was performed by polymerase chain reaction. HLA-DRB1*1401 allele frequency was significantly increased in patients with beta-thalassemia major without left-sided heart failure compared with those with heart failure (corrected P [P(c)]=0. 02, odds ratio 0.1) and healthy controls (P(c)=0.001). HLA-DQA1*0501 allele frequency was increased in patients with heart failure compared with patients without heart failure (P(c)=0.04, odds ratio 14) and healthy controls (P(c)=0.004). CONCLUSIONS: Differences exist in the immunogenetic profile between homozygous beta-thalassemic patients with and without left-sided heart failure, raising the possibility that genetically defined immune mechanisms may play an important role in the pathogenesis of heart failure in beta-thalassemia.

Lack of the appropriate natural killer cell inhibitory receptors in women with spontaneous abortion.

Previous studies have revealed that women with unexplained recurrent spontaneous abortions have a limited repertoire of inhibitory KI receptors (inhKIRs) and that the inhKIRs they possess do not have specificity for the human leukocyte antigen (HLA)-Cw molecules that would be expressed on trophoblast. We sought to confirm these findings by direct definition of maternal inhKIR and trophoblastic HLA-Cw allotypes on the placental material of spontaneously missed pregnancies. The study included 30 women undergoing vacuum uterine curettage for first-trimester missed pregnancy (group A; n = 15) or for elective termination of normal pregnancy (group C, n = 15). DNA extracted from isolated decidual and trophoblastic cells was used for molecular detection of maternal inhKIRs (2DL1, 2DL2, 2DL3) and fetal HLA-Cw alleles, respectively. The results revealed that in the group of women who experienced abortion, 60% did not have the full repertoire of three inhKIRs (group A vs group C; p = 0.006); that in five of 15 patients (none in the controls), no epitope matching existed between maternal inhKIRs and trophoblastic HLA-Cw alleles (group A vs group C; p = 0.01); and that more cases were found with limited epitope matching (less than three inhKIRs with specificity for fetal HLA-Cw alleles). The results provide additional evidence that in some cases of spontaneous abortions, the women lack the appropriate inhKIRs to interact with the HLA-Cw molecules on trophoblasts and to deliver signals to inhibit natural killer cell activation and protect the embryo.

Investigation of the biomechanical integrity of decellularized rat abdominal aorta.

OBJECTIVES: The loss or damage of an organ or tissue is one of the most common and devastating problems in healthcare today. Tissue engineering applies the principles of engineering and biology toward the development of functional biological replacements that are able to maintain, improve, or restore the function of pathological tissues. The aim of the overall project is to study an already existing method for the decellularization of homograft vascular grafts for use in vascular surgery. MATERIALS AND METHODS: The biomechanical integrity of native and decellularized rat aortas was assessed under uniaxial tension tests. For this purpose, 36 male rats (12 Wistar and 24 Dark Agouti [DA]) were used to excise their abdominal aortas. Twelve of the aortas were tested fresh (Wistar and DA rats), within 24 hours from euthanasia, and the rest were decellularized using a modified protocol (DA rats only). Fresh and decellularized samples (n = 12) were subjected to uniaxial tensile loading to failure, and the recorded stress-strain behaviour of each specimen was assessed in terms of 6 biomechanical parameters. RESULTS: No statistically significant differences were found in any of the biomechanical parameters studied between the decellularized DA rat aorta group and both the native DA and Wistar rat aorta groups (P > .05). Also, no significant difference was shown between the native DA and native Wistar rat aorta groups. CONCLUSIONS: The results from this study have shown that the decellularization protocol did not affect the mechanical properties of the native rat aorta. In addition to this, both native Wistar and native/decellularized DA rat aorta groups shared similar mechanical properties.

Epitope analysis of HLA class I donor specific antibodies in sensitized renal transplant recipients.

OBJECTIVE: The goal of this study was to evaluate the epitope specificity of donor-specific HLA class I antibodies detected in the serum of alloimmunized from a previous renal graft patients. METHOD: A total of 410 serum samples from 87 patients who had lost a previous graft, were collected every 4 months during a 2-year follow-up period. All recipients and donors were typed for class I HLA-antigens by a standard lymphocytotoxicity technique. To define the specificities of the HLA class I antibodies, two techniques were used in parallel: the antihuman globulin augmented CDC (AHG-CDC) technique and an ELISA technique. The mismatched HLA-antigens and the detected HLA class I antibodies were categorized as intra-cross-reactive group mismatches (intra-CREGs-MMs) and other-CREG-MMs. For each sensitized patient actual and at risk epitope specificities were defined. RESULTS: Thirty-eight patients (43.7%) had developed IgG HLA class I-specific antibodies with stable specificities against mismatched alloantigens from the previous graft. A total of 60 antibody reactivity patterns and 82 specificities against private and public epitope were recognized. Patients with only intra-CREGs-MMs produced HLA class I-specific antibodies less frequently than patients with only other-CREG-MMs, although the difference was nearly statistically significant (P=0.053). All HLA class I donor-specific antibodies were considered to have specificities against the private epitopes of the mismatched graft HLA-antigens. In the cases where HLA class I alloreactivity was spreading to more than one donor antigens, we considered that the detected antibodies had specificities against the private and the shared between the alloantigens epitope(s). No epitope-specific antibodies were detected against shared epitopes between the mismatched alloantigens and the HLA-antigens of the patients. In 11/38 cases (28.9%) HLA class I alloreactivity spreading to non-graft antigens was detected. These antibodies were directed against HLA-antigens that share epitope(s) and have strong serological reactivity with the immunogenic alloantigens. CONCLUSION: Our data show that a small number of private and public alloepitopes seem to be responsible for antibody production in patients sensitized from a previous graft. A detailed description of these HLA-epitopes, in the context of clinical graft complications, may lead to an improved organ allocation strategy.

Assignment of HLA-antigens in CREGs facilitates the detection of acceptable mismatches in highly sensitized patients.

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 > 60%), panel reactive antibodies (PRA) value and the specificity of class I anti-HLA-antibodies were detected by two techniques in parallel: An anti-human 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 HLA-antigens 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 HLA-phenotypes 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.

A limited number of HLA epitopes are recognized from HLA class I-specific antibodies detected in the serum of sensitized patients.

The goal of this study was to evaluate the epitope specificity of HLA class I-specific antibodies detected in the serum of sensitized patients awaiting retransplantation. The study group consisted of 22 sensitized from previous graft patients, who produced stable IgG HLA class I-specific antibodies. A total of 60 serum samples were screened and analyzed by two techniques in parallel: the antihuman globulin augmented CDC (AHG-CDC) technique and an ELISA technique. All recipients and donors were typed for class I HLA antigens by a standard lymphocytotoxicity technique. The epitope identification was based on class I HLA antigens sequencing, where the multiple immunogenic epitopes are differentially shared among various HLA antigens. The unique epitope configuration on one HLA antigen represents the private epitope of the specific HLA antigen while epitopes shared by more than one HLA antigen represent public determinants. In some HLA antigens (HLA-A1), more than one private epitope has been defined, while in others (HLA-B35, -B51), the private epitopes are not yet known. In a total of 36 antibody reactivity patterns, the majority of the definable IgG HLA class I-specific antibodies corresponded to the A-locus (75%), and only 25% had specificities against the B-locus antigens, although the number of incompatibilities concerning both loci were almost identical (29 for the HLA antigens of the A-locus and 26 for those of B-locus). All patients produced HLA class I-specific antibodies with specificities against the private epitopes of the immunogenic mismatched HLA antigen(s). In 6/21 cases (28.6%), HLA class I alloreactivity spreading to nongraft HLA antigens was detected and 9 public (shared) immunogenic alloepitopes were recognized. In conclusion, appling the epitope analysis of HLA class I-specific antibodies produced by sensitized from previous graft patients, we were able to define the immunogenic alloepitopes. We consider that the immunogenic alloepitopes, during transplantation course, are mainly private epitopes of mismatched HLA antigens and, in certain cases, shared epitopes between the donor alloantigens and other HLA antigens. This knowledge may offer the potential of transplanting sensitized patients through improved donor selection.

HLA associations of anti-beta2 glycoprotein I response in a Greek cohort with antiphospholipid syndrome and meta-analysis of four ethnic groups.

Using molecular typing, we evaluated the strength of class II HLA associations in 67 Greek patients with antiphospholipid syndrome (APS), 54 of whom had antibodies against beta2-glycoprotein I (beta2GPI), as compared to 246 controls. To further clarify and delineate HLA associations of the beta2GPI response, we combined these data with individual patient data from three other ethnic groups including an additional 74 patients with beta2GPI response and 403 ethnically matched controls of white, African-American, and Mexican-American origin in a formal meta-analysis. The major alleles associated with anti-beta2GPI response are HLA-DQA1*03 (in particular *0301) and the HLA-DRB1*1302-DQB1*0604 haplotype, while protection against developing an anti-beta2GPI response is related primarily to the HLA-DRB1*0101-DQA1*0101 haplotype and the HLA-DRB1*1101 allele. These effects are not significantly heterogeneous across ethnic groups. The previously observed association with HLA-DQB1*0302 may simply reflect linkage disequilibrium with HLA-DQA1*0301 and the previously reported HLA-DQB1*06 effect is limited to HLA-DQB1*0604/0605, while HLA-DQB1*0602 is unlikely to be important. The meta-analysis clearly documents that the anti-beta2GPI response is determined by a few specific class II alleles and haplotypes.

MHC class I and class II phenotype, gene, and haplotype frequencies in Greeks using molecular typing data.

In the present study, DNA typing for HLA-A, C, B, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, and DPB1 was performed for 246 healthy, unrelated Greek volunteers of 20-59 years of age. Phenotype, genotype frequencies, Hardy-Weinberg equilibrium fit, and 3-locus haplotype frequencies for HLA-A, C, B, HLA-A, B, DRB1, HLA-DRB1, DQA1, DQB1, and HLA-DRB1, DQB1, DPB1 were calculated. Furthermore, linkage disequilibrium, deltas, relative deltas and p-values for significance of the deltas were defined. The population studied is in Hardy-Weinberg equilibrium, and many MHC haplotypes are in linkage disequilibrium. The most frequent specificities were HLA-A*02 (phenotype frequency = 44.3%) followed by HLA-A*24 (27.2%), HLA-B*51 (28.5%), HLA-B*18 (26.8%) and HLA-B*35 (26.4%) and HLA-Cw*04 (30.1%) and HLA-Cw*12 (26.8%). The most frequent MHC class II alleles were HLA-DRB1*1104 (34.1%), HLA-DQB1*0301 (54.5%) and HLA-DPB1*0401 with a phenotype frequency of 59.8%. The most prominent HLA-A, C, B haplotypes were HLA-A*24, Cw*04, B*35, and HLA-A*02, Cw*04, B*35, each of them observed in 21/246 individuals. The most frequent HLA-A, B, DRB1 haplotype was HLA-A*02, B*18, DRB1*1104 seen in 20/246 individuals, while the haplotype HLA-DRB1*1104, DQB1*0301, DPB1*0401 was found in 49/246 individuals. Finally, the haplotype DRB1*1104, DQA1*0501, DQB1*0301 was observed in 83/246 individuals. These results can be used for the estimation of the probability of finding a suitable haplotypically identical related or unrelated stem cell donor for patients of Greek ancestry. In addition, they can be used for HLA and disease association studies, genetic distance studies in the Balkan and Mediterranean area, paternity cases, and matching probability calculations for the optimal allocation of kidneys in Greece.

Humoral sensitization against rejected grafts: specific antibodies to graft immunogenic amino acid triplets.

Humoral sensitization against immunogenic amino acid (aa) triplets expressed on a rejected graft was analyzed in 83 retransplant candidates. All patients had lost a graft with HLA-A,-B mismatches. The alloantibodies were detected by a complement-dependent cytotoxicity (CDC) technique and an ELISA method in parallel; they were classified as HLA graft-specific (GS) and non-GS antibodies. The aa triplet specificity of the antibodies was assessed using the HLAMatchmaker algorithm. HLA class I antibodies were detected in 74 of 78 (94%) cases, including GS reactivity in 55 (74.3%) and non-GS in 72 (97.2%), either alone (n = 19) or in parallel with GS antibodies (n = 53). For all HLA-GS-antibody-reactive patients, we defined the specificity against immunogenic aa triplets on the previous graft. Moreover, antibodies specific to graft aa triplets were observed within the non-GS antibodies among 19 of 19 and 28 of 53 cases, respectively. Therefore, aa triplet-specific antibodies against the rejected graft were present in all 74 cases with HLA class I antibodies. Antibodies against aa triplets expressed on all HLA class I-mismatched graft antigens were present in 73% of cases. The high extent of humoral alloreactivity against a rejected graft supports the decision to avoid repeated exposure to immunogenic aa triplet mismatches on a second graft. An accurate analysis for performed antibodies in these cases may be beneficial to select the most suitable second donor.

Minor histocompatibility antigen HA-1 and HPA-5 polymorphisms in HLA-identical related bone marrow transplantation.

The minor histocompatibility antigens (mHags), HA-1 and HPA-5, are immunogenic alloantigens shown to be responsible for graft-versus-host disease (GVHD) in HLA-identical bone marrow transplantation. Both antigens have two known alleles each, resulting in a single amino acid polymorphism. The HA-1H allele encodes histidine, whereas the HA-1R allele encodes arginine. The HPA-5b (Br(a)) allele encodes lysine, whereas the HPA-5a (Br(b)) encodes glutamic acid. In this study, 49 bone marrow transplant recipients and their genetically related HLA-identical donors were evaluated for the presence of HA-1, whereas 39 recipients, different from the abovementioned ones, and their HLA-identical siblings were analyzed for the presence of HPA-5. The frequencies of the two alleles of HA-1 in the recipient population were HA-1R = 0.663 and HA-1H = 0.336. In the donor population, the respective frequencies were 0.704 and 0.296. Seven donors (14.5%) were mismatched with the recipients for HA-1H. In contrast, the frequencies of the two alleles of HPA-5 in the recipient population were HPA-5a = 0.859 and HPA-5b = 0.141; whereas, among donors, they were 0.820 and 0.180, respectively. Five donors (12.8%) were found to be mismatched with their recipients for HPA-5. These results provide insight into the polymorphism of mH antigens based on the study of their frequencies in bone marrow transplant recipients and their genetically HLA-identical siblings, an endeavor that is essential to investigate the presence of HA-1 and HPA-5 mHags.

HLA class I donor-specific triplet antibodies detected after renal transplantation.

The purpose of this study was to investigate whether IgG, non-donor-specific anti-HLA class I antibodies (HLAabI) detected after renal transplantation recognize immunogenic amino acid triplets expressed on the foreign graft. In addition, we sought to evaluate the effect of these antibodies as well as other posttransplant HLAabI on graft outcome. Posttransplant sera from 264 renal recipients were tested for the presence of IgG HLAabI and HLA class II-specific alloantibodies (HLAabII) by ELISA. The HLAMatchmaker computer algorithm was used to define the HLA class I non-donor-specific antibodies, which seem to recognize immunogenic amino acid triplets. Donor-specific triplet antibodies (DSTRab) were detected in 16 of 22 (72.7%) recipients based on at least one HLA-A or -B mismatched antigen with the donor. DSTRab were found either without (n = 7) or with (n = 9) HLA donor-specific antibodies (HLA-DSA). The presence of DSTRab alone in the periphery was associated with acute rejection, whereas the presence of both DSTRab and HLA-DSA was associated with chronic rejection and graft failure.

Non-indexed medical journals in the Web: new perspectives in the medical literature.

Many medical journals, publishing in national languages, meet serious financial problems and difficulties when they attempt to become indexed in the international indices. Obviously, this not only affects the scientific quality of non-indexed medical journals (NIMJs) but also affects the awareness of the scientific community of topics with apparently local but potentially broader scientific significance. This is a reality for over 100 Greek medical journals, none of which has a life longer than 30 years or more than 2000 subscribers. Among them, the 'Archives of Hellenic Medicine' (AHM) is published and sponsored by the Athens Medical Society (the oldest medical society in Greece founded in 1835). This peer-reviewed Journal is being published for 13 years, bimonthly, in Greek. Attempting to overcome the above mentioned problems and to be involved in the process of discovering the most effective way of scientific 'skywriting', 2 years ago, the AHM entered full-text in the Web and it was decided that up to 500% of its volume should be covered by English-language papers. As a result, the AHM are now included in the main Web lists of medical journals and their home page is linked in many academic pages having approximately 500 hits/month. Furthermore, 45 retrievals of AHM's English-language papers or English abstracts of Greek-language articles were reported by e-mail response from abroad. Considered apart from the paper-publishing, the expenses of the digital publishing of the AHM are about half of those of paper-publishing, as they were before the appearance of the Journal in the Web. Up to now, about 40% of the Journal's digital publishing cost is covered by advertisements included in its pages and by a modification of its paper-publishing policy. It is concluded that the international scientific community is not indifferent for information published in NIMJs. Medical national minorities working abroad express special interest for this type of information. The Web makes the NIMJs accessible to these potential readers, who would never have the chance to acquire them in their printed form.

Evaluation of decellularization in umbilical cord artery.

Major achievements in creating decellularized whole tissue scaffolds have drawn considerable attention to decellularization as a promising approach for tissue engineering. Developing a tissue-engineered small-diameter (≤2 mm) vascular graft, using decellularized human umbilical arteries (hUAs), for reconstructive surgery is a challenging task. Polymers used in the past, proved unsuitable due to serious adverse effects and autologous vessels are available only in 40% of patients. In this study, histological and proteomic analysis was performed to evaluate the efficiency of two decellularization protocols. In decellularization protocol A, hUAs were incubated in 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) followed by incubation in alpha minimal essential medium (α-MEM) with foetal bovine serum (FBS) while in decellularization protocol B the hUAs were incubated in Hypotonic Tris and SDS followed by incubation in nuclease solution. Histological analysis of decelullarised hUA with both protocols revealed good preservation of extracellular cell matrix (ECM) proteins and immunofluorescent staining detected collagen I and fibronectin. The DNA content within the hUAs after decellularization with protocol A was 6.2% and with protocol B 17.3%. Proteomic analysis identified cytoplasmic enzymes such as, dehydrogenase X, α-enolase and peptidyl-prolyl cis-trans isomerase A only in native samples, while, cytoskeletal proteins such as a-actin, filamin and ECM proteins like collagens were found both in native and decellularised hUA. In conclusion, both decellularization protocols effectively removed the cellular material while the ECM remained intact. Future studies are warranted to elucidate the specific effects of altered structure-function relationships on the overall fate of decellularized hUAs.