DNA-condensing and chromatin-condensing properties of rat testes hla and hit compared to those of rat-liver hlbdec - hlt is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

Immediate hypersensitivity to Parthenium hysterophorus. I. Association of HLA antigens and Parthenium rhinitis

Lymphocytes collected from rhinitis subjects with strong positive skin reactions to the pollen allergens of Parthenium hysterophorus (American feverfew) having moderate to high titres of Parthenium-specific serum IgE were analysed for association of HLA-antigens covering 13 specificities of HLA-A, 17 specificities of HLA-B and eight specificities of HLA-DR loci by the NIH two-stage microlymphocytotoxicity assay. Comparison of the phenotypic frequencies of HLA-A and B antigens between Parthenium rhinitis subjects (n= 22) and control subjects (n= 137) did not suggest any significant association when tested for these antigen specificities. A significant correlation in the association of HLA-DR3 antigen with a relative risk of 11·33, however, was observed in Parthenium rhinitis subjects (n= 30) when compared to controls (n= 50).

HLA-B*57 and Gender Influence the Occurrence of Tuberculosis in HIV Infected People of South India

Background. Substantial evidence exists for HLA and other host genetic factors being determinants of susceptibility or resistance to infectious diseases. However, very little information is available on the role of host genetic factors in HIV-TB coinfection. Hence, a longitudinal study was undertaken to investigate HLA associations in a cohort of HIV seropositive individuals with and without TB in Bangalore, South India. Methods. A cohort of 238 HIV seropositive subjects were typed for HLA-A, B, and DR by PCR-SSP and followed up for 5 years or till manifestation of Tuberculosis. HLA data of 682 HIV Negative healthy renal donors was used as control. Results. The ratio of males and females in HIV cohort was comparable (50.4% and 49.6%). But the incidence of TB was markedly lower in females (12.6%,) than males (25.6%). Further, HLA-B* 57 frequency in HIV cohort was significantly higher among females without TB (21.6%, 19/88) than males (1.7%, 1/59); P = 0.0046; OR = 38. CD4 counts also were higher among females in this cohort. Conclusion. This study suggests that HIV positive women with HLA-B* 57 have less occurrence of TB as compared to males.

Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-beta and TNF-alpha production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E) from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP). In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-alpha as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-alpha and IFN-beta as well as the dsRNA analog, poly (I:C). Both IFN-beta and TNF-alpha further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.

Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NF kappa B and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NF kappa B dependent since it was blocked by inhibitors of NF kappa B activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NF kappa B inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-alpha was also inhibited by ShRNA mediated knockdown of NF kappa B. TNF-alpha treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NF kappa B-mediated induction of HLA-F following JEV infection and TNF-alpha exposure is being suggested for the first time. (C) 2014 Elsevier Inc. All rights reserved.

Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells

Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-gamma treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. It was also found that sHLA-E could inhibit IL-2 induced H-3]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit NK cell responses. Hence JEV-induced shedding of sHLA-E needs further investigation to better understand immune responses in JEV infections since it may have a role in viral evasion of NK cell responses. (C) 2014 Elsevier B.V. All rights reserved.

Deciphering complex patterns of class-I HLA-peptide cross-reactivity via hierarchical grouping

T-cell responses in humans are initiated by the binding of a peptide antigen to a human leukocyte antigen (HLA) molecule. The peptide-HLA complex then recruits an appropriate T cell, leading to cell-mediated immunity. More than 2000 HLA class-I alleles are known in humans, and they vary only in their peptide-binding grooves. The polymorphism they exhibit enables them to bind a wide range of peptide antigens from diverse sources. HLA molecules and peptides present a complex molecular recognition pattern, as many peptides bind to a given allele and a given peptide can be recognized by many alleles. A powerful grouping scheme that not only provides an insightful classification, but is also capable of dissecting the physicochemical basis of recognition specificity is necessary to address this complexity. We present a hierarchical classification of 2010 class-I alleles by using a systematic divisive clustering method. All-pair distances of alleles were obtained by comparing binding pockets in the structural models. By varying the similarity thresholds, a multilevel classification was obtained, with 7 supergroups, each further subclassifying to yield 72 groups. An independent clustering performed based only on similarities in their epitope pools correlated highly with pocket-based clustering. Physicochemical feature combinations that best explain the basis of clustering are identified. Mutual information calculated for the set of peptide ligands enables identification of binding site residues contributing to peptide specificity. The grouping of HLA molecules achieved here will be useful for rational vaccine design, understanding disease susceptibilities and predicting risk of organ transplants.

Estudo da frequência dos alelos de HLA-DRB1 em pacientes brasileiros com artrite reumatóide

Os alelos HLA-DRB1, que codificam uma sequência de aminoácidos (QKRAA/QRRAA/RRRAA) nas posições 70 a 74 da terceira região hipervariável da cadeia 1 do gene DRB1, denominada epítopo compartilhado (EC), estão associados com maior susceptibilidade e gravidade para artrite reumatóide (AR) em diversas populações. Uma nova classificação proposta por Du Montcel et al tem sido desenvolvida para apurar a associação entre HLA-DRB1 e AR. Este estudo foi desenhado com o objetivo de determinar a frequência dos alelos HLA-DRB1 em pacientes brasileiros com AR, e sua associação com o fator reumatoide (FR), anticorpos antipeptídeos citrulinados (ACPA) e lesão radiográfica articular e óssea. Quatrocentos e doze pacientes com AR e 215 controles foram incluídos. A tipificação HLA-DRB1 foi realizada pela reação em cadeia de polimerase (PCR) usando primers específicos e hibridação com oligonucleotídeos de sequência específica (SSOP). A pesquisa de ACPA foi determinada pela técnica de ELISA e a do FR por nefelometria, a avaliação radiográfica realizada pelo método do índice de Sharp modificado de Van Der Heijde. Para análises estatísticas foram utilizados os testes do qui-quadrado, t de Student e a regressão logística. Nos pacientes com AR alelos HLA-DRB1*04:01, *04:04, *04:05 se associaram com AR (p<0,05), embora o amplo intervalo de confiança, vale a pena ressaltar a associação observada com o alelo DRB1*09:01 e a doença (p<0,05). Alelos HLA-DRB1 EC+ foram observados em 62,8% dos pacientes e em 31,1% do grupo controle (OR 3,62; p <0,001) e estiveram associados com ACPA (OR 2,03; p<0,001). Alelos DRB1 DERAA mostraram efeito protetor para a AR (OR 0,42; p<0,001). A análise da nova classificação de HLA-DRB1 mostra que S2 e S3P se associaram a AR (p<0,05). Alelos S2 e/ou S3P esteve presente em 65% dos pacientes e 32% do grupo controle (OR 3,86; p<0,001) e estiveram associados a ACPA (OR.2,11; p=0,001). Alelos S3D, S1, X mostraram efeito protetor para a AR. Os resultados obtidos neste estudo demonstram que pacientes brasileiros com AR de etnia majoritariamente mestiça, alelos HLA-DRB1 avaliados segundo a hipótese do EC e a classificação proposta por Du Montcel estiveram associados à suscetibilidade à doença e à presença de ACPA.

Monitorização dos anticorpos anti-hla após transplante renal e sua correlação com episódios de rejeição aguda

Introdução: A associação entre a presença de anticorpo anti-HLA doador específico (DSA), em pacientes com prova cruzada negativa por citotoxicidade dependente de complemento (CDC), e a ocorrência de episódios de rejeição mediada por anticorpos (RMA) e menor sobrevida do enxerto já foi demonstrada por diversos autores. Entretanto,estimar a relevância clínica da presença desses anticorpos, em um determinado receptor, é um grande desafio e portanto novas estratégias de monitorização imunológicas são necessárias. Objetivo: O objetivo desse estudo foi monitorar a presença de DSA, bem como a variação dos seus títulos durante o primeiro ano após o transplante renal e correlacionar com episódios de rejeição aguda e função do enxerto ao final desse período. Metodologia: Foram analisados 389 soros de 71 pacientes incluídos no estudo. A pesquisa de DSA foi realizada utilizando os testes LABScreen single antigenbeads nas amostras correspondentes aos tempos: pré-transplante, 14, 30, 90, 180 e 365 dias após o transplante. Episódios de rejeição aguda comprovados por biópsia foram analisados de acordo com a classificação de Banff 2007. A taxa de filtração glomerular (TFG) ao final do primeiro ano foi estimada utilizando a fórmula Modificationof Diet in Renal Disease (MDRD). Os pacientes foram inicialmente separados em 3 grupos de diferentes riscos imunológicos (pré-transplante): A) DSA-, B) DSA+ com MFI >1000 e < 5000 e C) DSA+ com MFI > 5000. Num segundo momento, foram novamente agrupados de acordo com o perfil de mudança nos valores de MFI (intensidade de fluorescência média) ao longo do primeiro ano. Resultados: DSA estavam presentes pré-transplante em 15 pacientes. RMA foi mais frequente no grupo C (p = 0,02). De acordo com a variação dos títulos de DSA pós-transplante os pacientes foram novamente agrupados: grupo I) permaneceu DSA- durante todo acompanhamento = 50 pacientes, II) diminuiu ou manteve títulos de DSA em relação ao tempo zero = 13 pacientes e III) aumentou títulos em relação ao tempo zero = 8 pacientes (6 foram DSA de novo). Três pacientes dos grupos I e um paciente do grupo II apresentaram episódios de rejeição aguda celular. Não foi observada oscilação significativa nos títulos de anticorpos durante esses eventos. Nenhum paciente desse grupo apresentou episódio de RMA. Episódio de RMA ocorreu em dois pacientes do grupo III. Em ambos os pacientes foi detectado aumento significativo nos valores de MFI dos DSA em relação ao tempo zero. Não foi observada diferença significativa na TFG entre os grupos analisados nesse estudo. Entretanto, observou-se uma diferença estatisticamente significativa na TFG entre os pacientes que apresentaram episódio de rejeição aguda em relação aos que não tiveram, sendo menor nos primeiros (p = 0,04). Conclusão: A monitorização prospectiva dos anticorpos pode ajudar a identificar pacientes em maior risco para ocorrência de RMA e o aumento nos valores de MFI DSA deve ser interpretado como um sinal de alerta, sobretudo em pacientes previamente sensibilizados.

大蹼铃蟾抗菌肽22 对膀胱癌细胞株HLA-ABC以及DR表达影响的对照实验研究

　目的　探讨大蹼铃蟾抗菌肽22 (Maximin22) 对膀胱癌 细胞株HLA2DR 及HLA2ABC 表达的影响, 并与TNF2α、 IFN2α进行比较。方法　采用肿瘤细胞培养方法,流式细胞 仪检测各实验样品对膀胱癌细胞株T24 、BIU287 、SCaBER 的HLA2DR 及HLA2ABC 表达的影响。结果　Maximin22 对 不同的膀胱癌细胞株在小剂量下即有抑制作用,并呈剂量相 关性,各实验样品未见对各膀胱癌细胞株的HLA2DR 表达 有影响,Maximin22 、TNF2α对HLA2ABC 的表达均无影响; IFN2α则对HLA2ABC 的表达有上调作用。结论　Maximin2 2 、TNF2α抗癌机制与提高肿瘤细胞HLA2DR、ABC 的表达无 关, IFN2α可通过提高肿瘤细胞HLA2ABC 的表达提高T 细 胞对膀胱肿瘤的识别杀伤能力。

HLA-DRB1等位基因多态性与食管癌遗传易感性研究

目的 :从基因水平探讨湖北地区汉族人食管癌HLA -DRB1等位基因的遗传易感性。方法 :运用序列特异性引物聚合酶链反应结合基因序列分析等技术 ,检测无亲缘关系湖北汉族健康人 136例、食管癌组 4 2例患者的HLA -DRB1等位基因。SAS统计软件数据处理。结果 :湖北地区汉族人食管癌患者与正常人比较 ,HLA -DRB1 0 90 1基因频率显著增高 (0 2 5 0 0vs 0 1397,P =0 0 2 8,OR =2 0 5 3,病因分数 =0 12 82 ) ;两者间其余HLA -DRB1等

HLA-DRB1、-DQB1基因多态性与食管鳞癌遗传关联性

目的　从基因水平探讨食管鳞癌HLA DRB1 , DQB1等位基因的遗传易感性 ,以阐述其免疫遗传学特征。方法　运用序列特异性引物聚合酶链反应技术 ,检测无亲缘关系湖北汉族健康人 1 36例、食管鳞癌患者 42例的HLA DRB1 , DQB1等位基因。结果　湖北汉族人食管鳞癌患者与正常人比较 ,HLA DRB1 0 90 1等位基因分布频率显著增高 (0 .2 50 0比 0 .1 397,P =0 .0 2 8,OR =2 .0 53 ,病因分数 =0 .1 2 82 ) ,HLA DQB1 0 30