[Polymorphic locus rs1061624 of the ТNFR2 gene is associated with the development of arterial hypertension in males].

Aim To study the involvement of cytokine polymorphous loci in development of arterial hypertension (AH) in men from the Central Black Earth region of Russia.Materials and methods 821 men were evaluated, including 564 patients with AH and 257 individuals of the control group. Analysis of 8 cytokine mononucleotide polymorphisms (MNP) was performed using the real-time polymerase chain reaction with TagMan probes. Statistical analysis was performed with the STATISTICA (v.10.0) and PLINK (v.1.06) software. The regulatory potential of MNP was analyzed with the HaploReg (v.4.1) service (http://archive.broadinstitute.org).Results The rs1061624 ТNFR2 polymorphous locus was associated with development of AH in men in recessive (odd ratio (OR), 0.33; 95 % confidence interval (CI): 0.18-0.61, рperm=0.0004) and additive (OR, 0.50, 95 % CI: 0.34-0.74, рperm=0.0006) genetic models and exerted a protective effect in development of AH. The rs1061624 MNP of the ТNFR2 gene has a regulatory significance; it is located in the DNA sites hypersensitive to the action of DNAase 1 and in binding sites for transcriptional factors and histones that mark enhancers and promoters in different organs and tissues.Conclusion The rs1061624 ТNFR2 gene polymorphism is involved in the development of AH in men of the Central Black Earth region of Russia.

[Study of associations of polymorphism of matrix metalloproteinases genes with the development of arterial hypertension in men].

The aim of research. To study the association of polymorphic loci of matrix metalloproteinases with the development of essential hypertension (EH) in men of the Central Chernozem Region of Russia. Materials and methods. A study of 564 men with EH and 257 control men was performed. Analysis of the polymorphic loci of metalloproteinases rs11568818 MMР7, rs1320632 MMР8, rs11225395 MMР8, rs1799750 MMР1, rs3025058 MMР3 was performed using real-time PCR. The study of associations of SNPs and their haplotypes with the development of arterial hypertension was carried out using logistic regression analysis in the PLINK software (v. 2.050). The regulatory potential of polymorphic loci was analyzed in the HaploReg software (v. 4.1) (http://archive.broadinstitute.org). The effect of SNP on gene expression was studied using the data of the Genotype-Tissue Expression project (http://www.gtexportal.org/). Results. Haplotype including rs11568818 MMP7, rs1320632 MMP8, rs11225395 MMP8 and rs1799750 MMP1 associated with a high risk of disease in men (OR=2,58, pperm=0,04). These polymorphisms located in region of promoter and enhancer histone marks and in the region of hypersensitivity to DNAse-1. They located in sites of proteins bound (TBP, CJUN, CFOS and GATA2) and they associated with the level of gene expression ММР7, ММР27 and RP11-817J15.3 (in peripheral blood, skeletal muscles, nervous tissue and other). Сonclusion. Haplotype G-A-C-1G for polymorphisms rs11568818 MMP7, rs1320632 MMP8, rs11225395 MMP8, rs1799750 MMP1 are associated with the development of essential hypertension in men in the Central Chernozem Region of Russia.

[The role of stress factors and genetic predisposition in the development of stroke in patients with essential hypertension]. / Rol' stressovogo faktora v realizatsii geneticheskoi predraspolozhennosti k razvitiiu insul'ta na fone gipertonicheskoi bolezni.

To study the interaction of polymorphic markers of matrix metalloproteinases (MMP) and chronic stress in the development of stroke associated with hypertension. MATERIAL AND METHODS: A total of 830 patients, including 303 patients with ischemic stroke associated with essential hypertension (EH) and 527 patients with EH without stroke, were examined. The study of metalloproteinases SNP was carried out using real-time PCR. The functional significance and influence of polymorphic loci on gene expression was studied using of HaploReg (v4.1) (http://archive.broadinstitute.org) and GTEx-portal (http://www.gtexportal.org). RESULTS AND CONCLUSION: An association of the genotype GG (rs11568818) of MMP7 with a high risk of stroke in patients exposed to regular stress (OR=1.71) was observed. It was found that allele 5A and genotype 5A/5A (rs3025058) of MMP3 had a protective effect on the development of stroke in patients without regular stress in the anamnesis (OR=0.73 and OR=0.60, respectively). Those SNPs are localized in the region of histone proteins H3K4me1 and H3K4me3, in the region of hypersensitivity to DNase-1, in the region of binding of regulatory proteins and transcription factors. The polymorphic locus rs11568818 is associated with the expression level of MMP7.

[Age-specific characteristics of acute myeloid leukemia karyotype].

AIM: To study distribution of some karyotype variants among patients of different age with acute myeloid leukemia (AML). MATERIAL AND METHODS: Distribution of balanced, normal, unbalanced, complex and monosomic karyotype among 244 patients with de novo AML in age groups 16-20, 21-30, 31-40, 41-50, 51-60, 61 and older was analysed. RESULTS: There is difference in frequency of balanced and complex karyotype in patients under and over 60 years. Number of AML patients with balanced aberrations including favourable variants t(8;21), t(15;17) and inv(16) falls after 60 years of age (6.7% versus 15.0% in patients aged 16-20 years; p < 0.001), while a complex karyotype occurs more frequently in AML patients at the age of 61 and older (56.8% versus 2.7% in the group 16-20 years; p < 0.001). With age, more frequently detected is the most unfavourable monosomic karyotype with aberrations similar to those in myelodysplastic syndrome (57.1% in patients aged 16-60 years and in 80.0% in the group of 61 years of age and over). CONCLUSION: Age-specific karyotype features detected may be explained by different biological mechanisms involved in leukosogenesis in young and elderly AML patients.

[Analysis of polymorphism of bone metabolism genes and evaluation of the risk of osteopenia in cosmonauts].

Densitometry of cosmonauts following long-duration missions shows reduction of bone mineral density (BMD). On the average, post-flight BMD remains within the normal range and the broad variability of individual BMD values sometimes is qualified as local osteopenia. Individual reactions are typed by similarity of amount and rate of BMD loss. At present, analysis of functionally significant polymorphism of bone metabolism genes is the most effective instrument for diagnostics of susceptibility to osteopenia and osteoporosis. The investigation was aimed to analyze polymorphism of genes of vitamin-D and (VDR) and calcitonin (CALCR) receptors, and of collagen-1 alpha-1-chain (Col1a-1) in candidate cosmonauts and cosmonauts returned from 5 to 7-mo. missions. According to the results of analysis, in the majority of cosmonauts rapid BMD loss correlated with TT genotype by VDR gene but not with genotypes Tt and tt and associated with carriage of incomplete s-allele in the Col1a1 gene. Yet, in several instances high BMD loss rates were personified with carriers of VDR gene alleles (homo- and heterozygote states--tt and Tt) and heterozygote by Col1a1 gene (Ss).

[Investigation of FLT3-ITD and NPM1 mutations in patients with myelodysplastic syndrome and mixed myeloid diseases].

Two FLT3-ITD mutations, one FLT3-TKD) and five NPM1 mutations were detected in 7 patients with de novo myelodysplastic syndrome (MDS) out of 44 cases of MDS and MDS/mixed myeloid diseases. Expression of one of the three investigated mutations was identified: 4 in gene NPM1 (9.1%) and 2--FLT3-ITD (4.5%); simultaneous FLT3-ITD and NPM1 mutation--1 (2.3%); no progression in NPM1 within 9-20 months--3, although with chromosome 7 damage--2. It was suggested that NPM1 mutation without complex karyotype may serve as marker of relatively favorable course.

[FLT3 and NPM1 gene mutations in patients with acute myeloid leukemias and the impact of FLT3-ITD mutations on the survival of patients with a normal karyotype].

AIM: To estimate the extent of FLT3 and NPM1 gene mutations and the impact of mutations of FLT3-ITD on the survival of patients with acute myeloid leukemias (AML). MATERIALS AND METHODS: The nucleus-containing cells of bone marrow and blood were studied in 43 patients with AML. Polymerase chain reaction analysis of total genomic DNA was applied. RESULTS: Mutations of FLT3-ITD, FLT3-TDK, and the NPM1 gene were found in 16 (37.2%) patients. A total of 19 mutations were revealed. There were 8 mutations of FLT3-ITD, 5 of FLT3-TKD, and 6 in the NPM1 gene. Single damages to genes were detected in 13 patients: FLT3-ITD in 6 (13.9%), FLT3-TKD in 4 (9.3%), and NPM1 in 3 (7%). Three (7%) patients exhibited 2 mutations simultaneously: in the NPM1 and FLT3-ITD in 2 (4.7%) and in the NPM1 gene and FLT3-TKD in 1 (2.3%). In AML patients with a normal karyotype and the FLT3-ITD-/NPM1 and FLT3-ITD+/ NPM-T genotypes, median overall survival was 17.3 versus 8 months (p = 0.069); and event-free survival (EFS) was 11 versus 5 months (p = 0.026). Univariate analysis established the negative impact of FLT3-1TD mutation on EFS. CONCLUSION: The findings allow AML patients with a normal karyotype and the FLT3-ITD-/NPM-genotypes to be identified as a poor prognosis group.

Genetic determinants of the efficiency of climatotherapy in patients with chronic heart failure.

We studied the dependence of climatotherapy effectiveness in patients with chronic heart failure (functional classes 0-II) on Ca(2+)-ATPase, phospholamban, beta1-adrenoceptor, and insulin-like growth factor 1 gene polymorphisms and possible interaction of these genes during the realization of the effect of climatotherapy. The effectiveness of climatotherapy depended on polymorphism of the studied genes; the maximum effect was attained in patients with the GG polymorphism of the Ca(2+)-ATPase gene, GT polymorphism of the phospholamban gene, ArgGly polymorphism of the beta1-adrenoceptor gene, and 19/19 polymorphism of the insulin-like growth factor 1 gene. We demonstrated additive interaction of Ca(2+)-ATPase and beta1-adrenoceptor genes during the realization of the cardiotonic effect of climatotherapy.

[A pathogenetic model of cerebral palsy in children born to mothers with antiphospholipid syndrome].

A pathogenetic model of infantile cerebral palsy caused by maternal antiphospholipid (APS) syndrome has been elaborated. Thirty-two children with cerebral palsy born to mothers with clinical signs of APS have been studied. The basic clinical feature of cerebral palsy in children was the prevalence of the double hemiplegic form, the absence of severe cognitive disorders, global muscular hypotrophy, rapid contracture formation and a tendency to frequent respiratory diseases. A seropositive APS variant was found in 42% of mothers examined, the seronegative one--in 58%. Such factors as (1) fetoplacental insufficiency and hypoxia caused by vascular infarctions of the placenta; (2) transplacental passage of antiphospholipid antibodies from a mother to a child; (3) intracerebral hemorrhages and periventricular leucomalacia in infants play the key role in the pathogenesis of cerebral palsy in children born to mothers with APS.

[Strokes and their causes in children]. / Insul'ty u detei i ikh prichiny.

Based on the literature and their own observations of 44 children aged 5-15 years (27 girls, 17 boys) with prior strokes, the authors characterize the types of strokes and principle causes of the disease as follows: (1) intracerebral and subarachnoidal hemorrhages (key etiology--arteriovenous malformations, blood disorders, coagulopathy, thrombocytopenias, thrombocytopathy, etc.) and (2) ischemic strokes i.e. (i) thrombotic (congenital and acquired vascular aplasias, angiitis, antiphospholipoid and viral vasculopathy, blood system coagulate activation, etc.); (ii) embolic (cardiogenic, septic, placental, etc.) and (iii) hemodynamic as a consequence of severe cardiomyopathy and disrupted total hemodynamics. The definitions of metabolic stroke as a complication of mitochondrial encephalopathy, homocystinuria and non-differentiated stroke caused more often by emergence of pathologic weariness of connective tissues were suggested. To elicit the stroke causes and types in children, the authors propose a reliable algorithm for clinico-instrumental diagnostic screening in acute period of stroke.

[Analysis of association of Col1a1 gene alleles with the development of osteoporosis]. / Analiz assotsiatsii allelei gena Col1a1 s razvitiem osteoporoza.

Allele frequencies of the G-->T polymorphism at the regulatory region of the Collal gene in the population of the northwestern Russia (control group) and in osteoporotic patients were estimated by the RFLP method based on PCR-mediated site-directed mutagenesis. Three patient groups with radiologically confirmed osteoporosis were examined. Group 1 consisted of 64 patients with severe osteoporosis complicated by fractures (SO); group 2 included 15 children with idiopathic osteoporosis (IO); group 3 consisted of 98 women with postmenopausal osteoporosis developed at the background of estradiol-deficiency state (PMO). The frequency of functionally defective allele s in the control group was 16.7%. It was statistically different from that in the SO patients (48.4%) (P < 0.01) and in the IO children (40%) (P < 0.01). The frequency of allele s in the PMO patients constituted 23.0% and it was similar to that in the control group (P > 0.05). Analysis of the Collal alleles provides early detection of the individuals with hereditary predisposition to osteoporosis and prophylaxis of the disease at the presymptomatic stage.

T-cell killing of heterogenous tumor or viral targets with bispecific chimeric immune receptors.

We have previously described several novel chimeric immune receptors (CIRs) that redirect human T cells to kill malignant or HIV-infected cells. These CIRs comprise a cancer- or virus-specific ligand or single-chain antibody fused to the signaling domain of the T-cell receptor CD3-zeta subunit. Binding of the ligand- or antibody-based CIR to the target antigen (Ag) triggers T-cell-mediated cytolysis of the tumor- or virus-infected cell independent of target cell major histocompatibility complex class I expression. A new type of CIR was developed to mediate the lysis of cells that expressed one or more distinct viral or tumor Ags; three bispecific CIRs (BCIRs) were generated that recognized the carcinoembryonic Ag (CEA) and TAG-72 tumor Ags or, alternatively, distinct epitopes in the HIV envelope (HIVenv). T cells expressing the antitumoral Ag BCIR lysed both CEA- and TAG-72-expressing targets and did not kill Ag-negative targets or target cells expressing other members of the CEA family. Similarly, T cells expressing the anti-HIVenv BCIR lysed target cells expressing both the wild-type HIVenv and a mutant HIVenv that lacked the epitopes recognized by the monospecific CIRs. This approach permits the generation of T cells with a broader spectrum of activity capable of killing virus-infected cells and malignant cells and reduces the potential of progression of disease due to Ag loss variants.

The p53-independent tumoricidal activity of an adenoviral vector encoding a p27-p16 fusion tumor suppressor gene.

We describe here that DE1-adenovirus vectors (AV) expressing a p27-p16 fusion molecule, termed W9, induce tumor cell apoptosis when overexpressed in a wide range of tumor cell types. However, in primary human cells derived from a variety of normal tissues, AV-W9 induced minimal apoptosis. In tumor cells AV-W9 demonstrated 5- to 50-fold greater tumoricidal activity than either of the parental molecules p16 and p27. In these studies, AV-W9 elicited apoptosis independent of the p53 and Rb status of the tumor cells. In several murine tumor models AV-W9 demonstrated p53-independent antitumor activity. It completely prevented tumor formation in two ex vivo models, whereas the parental molecules resulted in partial protection. Furthermore, AV-W9 induced tumor regression or suppressed tumor growth when introduced intratumorally into preestablished tumors in mice. This effect may be mediated through tumor cell apoptosis or antiangiogenic activity of AV-W9. Thus, this novel chimeric molecule is more potent and capable of killing a broader spectrum of tumors than the parental p16 and p27 molecules independent of the tumor cell p53 and phenotype and represents a powerful new therapeutic agent for cancer gene therapy.

Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure.

Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAV-hFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.

Anti-Tumor CC49-zeta CD4 T cells possess both cytolytic and helper functions.

The authors report that the nature of the T-cell-receptor--derived signal in normal CD4+ T cells can induce interleukin-2 (IL-2) secretion or perforin-mediated cytolytic activity. Normal human T cells were genetically modified to express the tumor antigen specific chimeric immune receptor, CC49-zeta. The CC49-zeta chimeric immune receptor is comprised of the intracellular signaling domains of the TCR CD3zeta protein fused to the single chain scFv of the humanized CC49 antibody, which binds the pan-adenocarcinoma tumor antigen TAG-72. Patient-specific T cells genetically modified to express the CC49-zeta receptor have been used in patients with colon cancer. The authors report that both CD4 and CD8 T cells expressing the CC49-zeta receptor mediated the major histocompatibility complex-unrestricted lysis of TAG-72--expressing tumor cells with comparable efficiency. However, although the CC49-zeta receptor mediated target cell lysis, it did not support the production of IL-2, even in the presence of CD28 stimulation. Robust IL-2 secretion and T-cell proliferation were observed when the same CD4 CC49-zeta T cells were stimulated through the CD28 receptor and endogenous T-cell receptor. These results indicate that CD4 T lymphocytes possess the capacity to act as both cytolytic and helper T cells and that this difference in effector function is controlled by the nature of the T-Cell receptor--derived signals.

Impact of chimeric immune receptor extracellular protein domains on T cell function.

Chimeric immune receptors (CIR) encompass tumor- or virus-specific ligands or antibodies fused to the signaling domains of either the T cell receptor or Fc receptor. T cells expressing these receptors recapitulate the cytopathic effects mediated by the T cell receptor and allow the targeting of tumor or virus infected cells in an MHC-independent manner. With this technology, large numbers of T cells with redirected target specificity can be generated. To define the structural features of recombinant CIRs required for optimal function, a panel of five closely related CIRs with identical target specificity were generated. These receptors recognized HIVenv through the single chain Fv (scFv) of an anti-gp 120 antibody. These scFv-zeta receptors were constructed to include alternative extracellular spacer and transmembrane protein domains derived from members of the immunoglobulin supergene family. The effect of these alternative extracellular protein domains on receptor stability, antigen affinity and T cell activity was assessed. We demonstrate that modifying the extracellular protein domains of the anti-HIVenv CIRs significantly impacted receptor stability and substrate binding affinity and that these effects, and not simply the level of cell surface expression, correlated most strongly with changes in CIR-mediated killing. These studies will aid in the rationale design of recombinant CIRs for the immunotherapy of viral infections, cancer and other diseases.

T cell tolerance is influenced by concomitant T cell recognition of cross-reactive self-peptides.

Although the current dogma of T cell recognition stresses its exquisite specificity, T cell clones selected for a given peptide can recognize other sequentially or structurally related peptides. Here, we have examined the immunogenicity and tolerogenicity of various self-peptides derived from region 61-80 of different MHC class I proteins co-expressed in the same mouse. Following immunization of B10.A mice (K(k), A(k), E(k), L(d), D(d)) with self-L(d) 61-80 peptide, vigorous MHC class II-restricted T cell proliferation was elicited after restimulation with either the immunogen or with self-K(k) 61-80 but not with self-D(d) 61-80. Furthermore, adult B10.A mice, tolerized with L(d) 61-80 prior to immunization with L(d) 61-80 did not respond to challenge with L(d) 61-80 and the cross-reactive K(k) 61-80. However, following K(k) 61-80 immunization, L(d) 61-80-tolerized mice responded to K(k) 61-80 but not to L(d) 61-80. Thus, tolerance induction to L(d) 61-80 resulted in the elimination/inactivation of L(d) 61-80-reactive T cells including the subpopulation that cross-reacted with K(k) 61-80. However, T cells that recognized K(k) 61-80 exclusively were preserved. Moreover, we showed that immunization with K(k) 61-80 resulted in tolerance breakdown to the cross-reactive, dominant self-peptide D(b) 61-80 in B10.A(4R) mice (K(k), A(k), L(d),D(b)). Together, these results show that the autoimmune T cell repertoire is influenced by the concomitant recognition of different cross-reactive self-peptides within the same individual.

[The immunomodulating action of interferon preparations. 3. Changes in the phagocytic activity of human blood neutrophilic granulocytes under the influence of interferon preparations]. / K immunomoduliruiushchemu deistviiu preparatov interferona. Soobshchenie 3. Izmenenie fagotsitarnoi aktivnosti neitrofil'nykh granulotsitov krovi cheloveka pod vliianiem preparatov interferona.

In vitro studies of phagocytosis-stimulating effects of interferon preparations differing in their origin and mode of purification were carried out with blood cells of dermatosis patients and healthy donors. Native preparations and preparations purified by sparing methods (alpha-interferon for intranasal administration and alpha-interferon for injections II) were found the most active: they induced a significant elevation of the activities of individual cells and of the count of actively phagocytizing cells. Phagocytosis-stimulating effect of well purified interferon preparations is essentially reduced. Recombinant alpha 2-interferon preparation has shown no such activity. A dose dependence of the effect of interferon preparations on blood neutrophil phagocytic activity has been revealed. The highest phagocytosis-stimulating effect of the above interferon preparations was observed in patients whose blood polymorphonuclear leukocyte phagocytic activity was essentially reduced.