Lack of association between human leukocyte antigen polymorphisms rs9277535 and rs7453920 and chronic hepatitis B in a Brazilian population.

Hepatitis B virus (HBV) infection is a serious public health problem worldwide. The progression of the disease depends on several host and viral factors and may result in fulminant hepatitis (very rare), acute hepatitis with spontaneous clearance, and chronic hepatitis B infection. Previous studies demonstrated that variations in the human leukocyte antigen (HLA) class II (HLA-DPB1 and HLA-DQB2 genes) are related to the chronic HBV infection. This study aimed to investigate the association of two single nucleotide polymorphism (SNPs), one in the HLA-DPB1 (rs9277535) and one in the HLA-DQB2 (rs7453920), with chronic hepatitis B infection in a southern Brazilian sample. This case-control study included 260 HBV patients attended in a Specialized Center for Health in Caxias do Sul (Brazil) between 2014 and 2016. The same number of controls (matching for age, gender, and ethnicity) was obtained in a University Hospital in the same city and period. Blood samples were collected and genomic DNA was extracted. Genotyping were performed by real-time Taqman PCR method. Odds ratios with 95% confidence intervals and significance level of 5% (P < 0.05) were calculated. Allele frequencies in the SNP rs9277535 were 72.6% for A and 27.4% for G nucleotides in cases and 75.0% for A and 25.0% for G in controls. Allele frequencies in the SNP rs7453920 were of 25.7% for A and 74.3% for G in cases and 28.8% for A and 71.2% for G in controls. No statistically significant association was found between both SNPs and chronic hepatitis B (P > 0.05).

Comparative genomic in situ hybridization of colon carcinomas with replication error.

The aim of the present study was to detect complex genetic alterations in colorectal carcinomas with and without microsatellite instability (MIN) by comparative genomic in situ hybridization. MIN due to replication errors is the hallmark of hereditary nonpolyposis colon cancer. None of 6 MIN-positive tumors showed amplifications, and only 2 tumors displayed deletions of one chromosomal segment each. In contrast, different gains and losses were observed in 11 of 12 MIN-negative carcinomas. The most frequent gains affected chromosomes 7, 13, and 20q, whereas deletions were observed on chromosomes 17, 18, and 9p. These results demonstrate different mechanisms of genetic instability in subgroups of colorectal carcinomas and may, therefore, support the hypothesis of different etiologies in tumors with and without MIN.

Hairless, a Drosophila gene involved in neural development, encodes a novel, serine rich protein.

Hairless is a dominant loss of function mutation in Drosophila affecting the formation of adult sensory organs. In the mutants, neuronal precursor cells do not differentiate, suggesting that Hairless might be involved in specifying or realizing neuronal fate in the fly, similar to the 'pro-neural' genes of the achaete-scute complex. As highlighted by the manifold phenotypic interactions of Hairless with most of the neurogenic loci, the gene might play an important role in nervous system development. Therefore, we initiated a molecular analysis of the Hairless locus in order to elucidate the function of its gene product and gain insight into the biochemical nature of the observed genetic interactions in which it participates. Here, we report the molecular cloning of the Hairless locus, confirmed by breakpoint and transformation analysis. Unexpectedly, Hairless activity peaks during embryogenesis, where transcripts accumulate primarily in endo- and mesodermal cell layers, and is lowest during larval stages, the lethal phase of Hairless mutants. The putative Hairless protein deduced from DNA sequencing is extremely basic and highly enriched in serine residues. Hairless appears to encode a novel protein without compelling homology to other known proteins which function in specifying peripheral nervous system development in Drosophila.

Alopecia in a novel mouse model RCO3 is caused by mK6irs1 deficiency.

Reduced coat 3 (Rco3) is a new spontaneous autosomal recessive mutation with defects in hair structure and progressive alopecia. Here we describe chromosomal mapping and molecular identification of the Rco3 mutation. The murine Rco3 locus maps to a 2-Mb interval on chromosome 15 encompassing the keratin type II gene cluster. Recently, mK6irs1 was described as a type II keratin expressed in Henle's and Huxley's layer of the murine inner root sheath. Genomic sequencing revealed a 10-bp deletion in exon 1 of mK6irs1 resulting in a frameshift after 58 amino acid residues and, therefore, the absence of 422 carboxy-terminal amino acid residues containing the complete alpha-helical rod domain. Henle's and Huxley's layers show no immunoreactivity with mK6irs1-specific antibodies and the absence of intermediate filament formation in electron microscopic images. These results indicate that the expression of functional mK6irs1 is indispensable for intermediate filament formation in the inner root sheath and highlights the importance of the keratinization of the inner root sheath in the normal formation of the hair shaft.

Detection of complex genetic alterations in human glioblastoma multiforme using comparative genomic hybridization.

The aim of the present study was to detect complex genetic alterations in human glioblastoma multiforme (GBM) by comparative genomic in situ hybridization (CGH). Of the 24 GBM that were examined, increased fluorescence intensities indicating chromosomal polysomy of chromosome 7 and gene amplification at chromosome 7p were found in 42% of the tumors. In addition, signal enhancement of chromosome 19 was present in 29% and at 12q13-15 in 21% of the tumors. We also detected reduction of fluorescence intensities indicating gross deletions on chromosomes 10 (58%), 9p (46%), and 13 (29%). There was a close correlation of CGH results when compared with Southern analysis of the EGFR gene localized on chromosome 7 and loss of heterozygosity detection of chromosome 9 and 10 by microsatellite PCR. A close correlation was also observed between copy number changes of chromosome 7 and deletions of chromosome 10. Amplification of chromosome 12q and deletions of chromosomes 9p and 13 seemed to be complementary in the tumors investigated in the present study.

Differential transcription and translation of immediate early genes in the gerbil hippocampus after transient global ischemia.

Excitotoxic activation of glutamate receptors is thought to be a key event for the molecular pathogenesis of postischemic delayed neuronal death of CA-1 neurons in the gerbil hippocampus. Glutamate receptor stimulation also causes induction of transcription factors that belong to the class of immediate early genes. We examined the expression of six different immediate early genes in the gerbil hippocampus after transient global ischemia. Comparative analysis of c-fos and Krox-24 expression was carried out in the same animals at the transcriptional and translational level by in situ hybridization and immunocytochemistry. Postischemic synthesis of four additional immediate early gene (IEG)-encoded proteins (FOS-B, c-JUN, JUN-B, and JUN-D) was investigated by immunocytochemistry at recirculation intervals between 1 and 48 h. After 5 min of ischemia, transcription of c-fos and Krox-24 mRNA was induced in all hippocampal subpopulations with peak expression at 1 h after recirculation. In vulnerable CA-1 neurons, increased transcription of c-fos and Krox-24 was not followed by translation into protein. Induction of immediate early gene-encoded proteins was restricted to neuronal populations less vulnerable to brief ischemia and identified neurons that are targets of glutamate receptor-mediated neurotoxicity but that are destined to survive. Our data indicate an asynchronous synthesis and persistence of individual IEG-encoded proteins in these neurons. The staggered induction implies that combinatorial changes of transcription factors allow a differential postischemic regulation of target gene expression both spatially and over time.

Microsatellite instability: new aspects in the carcinogenesis of colorectal carcinoma.

Very recently a new molecular mechanism in the tumorigenesis of colorectal carcinoma has been described which is closely linked to hereditary non-polyposis colonic cancer (HNPCC). Ubiquitous changes in the length of simple repetitive DNA sequences between constitutional and tumour DNA occur in about 90% of cases of HNPCC and in about 15% of cases of non-familial, sporadic colorectal carcinoma. Such microsatellite instabilities have been shown to be the phenotypical marker of mutations in the human homologues of prokaryotic mismatch repair genes (MutS, MutL, MutH). These data provide crucial new tools in the detection of patients at high risk of developing colon cancer and other HNPCC-related carcinomas. In addition, these developments provide new insights into a new, presumably primary event in oncogenesis, i.e. the occurrence of mutations in genomic stability genes leading to an increased cellular mutation rate ("mutator phenotype") and thus to cancer.

Chemical carcinogenesis in the nervous system: past and future.

The model of experimental tumors of the nervous system has greatly contributed to our understanding of growth and management of intracranial tumors, but has been somewhat neglected in the last years, because a wealth of new data concerning oncogenic action came from viral oncogenesis. These new issues led to a much better insight into human tumor induction and promotion. Yet one example of the impact of oncogenic transformation stems from the "neurooncogenic" model: the discovery of the neu oncogene and its product as a putative differentiation receptor in the cell membrane of experimental Schwann cell derived tumors. In the light of this unique finding the history of the "neurooncogenic" model and the morphological and "clinical" result of tumors produced within the model are reviewed. There is a large open field for future investigation both in basic and applied science.

Early morphological findings in experimental high pressure neurological syndrome.

HPNS (high pressure neurological syndrome) is considered to be reversible condition of the nervous system caused by elevated (atmospheric) pressure. Clinical observations and experimental findings gave rise to the belief that this syndrome at least partly functions as a model of a dopamin dependent psychosis. Morphological alterations during or after HPNS in man and animals have not been reported so far. We treated rats for three hours with an increasing pressure of helium-oxygen mixture up to 61 ATA in a pressure chamber. This pressure was subsequently maintained for one hour and then released to zero within twenty seconds. The rats died within the first three seconds of pressure release due to complete deoxygenation. Brains were immediately removed and either cooled in liquid nitrogen or fixed in formalin. In both instances the central nervous tissue was excellently preserved. In paraffin embedded formalin fixed specimens, dark neurons in different brain regions were found, especially within parts of the dentate gyrus, the CA 4 subfield of the ammons horn, in dopaminergic brainstem nuclei and in some cortical pyramidal cells. In dopaminergic cells, tyrosine hydroxylase was found to be absent in cells transformed into dark neurons. These dark neurons which have long been recognized in neuropathology, probably represent reversibly damaged neurons transformed into the dark configuration by aldehyde fixation. They may correspond to early apoptosis or they may be the consequence of cytoskeletal disruption.

Quantitative DNA analysis of an intracerebrally transplanted brain tumour model after experimental chemotherapy with BCNU and CCNU.

In the present study we investigated the susceptibility of high passages of the rat glial transplantation tumour G-XIII to chemotherapy using nitrosourea compounds. We observed a significant increase in lifespan (ILS) of animals treated with BCNU (37%, p < 0.01) and CCNU (27%, p < 0.01). There were no difference in the efficiency between these two substances. Using a semi-quantitative score system no histopathological changes were observed which were associated with the response to therapy. The only predicative parameter in the present study was the quantitative DNA distribution pattern. There was a close correlation between treatment and the occurrence of unimodal DNA distribution patterns indicating clonal regrowth of recurrent tumours. Moreover, we also observed a correlation of the DNA distribution pattern of recurrent tumours with the result of experimental chemotherapy. Survival times of animals suffering from tumours containing unimodal DNA histogram was significantly longer than survival times of rats with multimodal DNA distribution, i.e. bimodal or broad DNA histograms. A unimodal, near-diploid stem line was only present in treated animals suggesting that these clones are more resistant against therapy using nitrosourea compounds. Our data indicate DNA cytophotometry as comprehensive tool for the monitoring of therapy response and the design of experimental chemotherapy using rat glial tumours.

Early and late morphological effects of experimental HPNS--animal model of psychosis?

The high pressure neurological syndrome (HPNS), a neurological condition during elevated pressure especially in deep diving, has been simulated with experimental animals. Rats were subjected to 61 bars with slow pressure increase and one or two hours constant high pressure; subsequently the pressure was released to sea level within 20 seconds--leading to immediate oxygen depletion and death of animals--or with slow decompression rates allowing survival. In all animals, brains and partly other organs were investigated morphologically. In animals sacrificed immediately, subtle changes in different brain regions were found: symmetrical occurrence of dark neurons in the hippocampus formation, cortex and brain stem, reduced expression of tyrosin hydroxylase in the substantia nigra and enhanced expression of Bax protein in some of these regions. The dark neurons were only observed after aldehyde fixation, otherwise the brains were unaltered despite ultrarapid decrease of highly elevated pressure. In animals that were allowed to survive for different time periods, some of these subtle changes were equally noted by light and electron microscopy. Furthermore, the ventricles were enlarged, the astrocytic reaction in the hippocampus increased and some signs of the destruction of the adrenal gland were visible. We conclude, that HPNS leads to minimal changes within the nervous system. The behaviour of animals during pressure was slightly altered, the weights after the experiments reduced, but no lasting sequelae were noted. Since both in human and experimental deep diving conditions signs of psychosis were reported, this HPNS model must be considered as a tentative animal model of human psychosis.

Differential gene expression of vascular smooth muscle cells. Detection by RNA arbitrarily primed polymerase chain reaction.

BACKGROUND: Vascular smooth muscle cells (VSMC) play an important role in the development of restenotic lesions. However, regulation of proliferation, migration, and matrix synthesis of these cells is still poorly understood. The aim of this study was to analyze gene expression of differently stimulated bovine VSMC. MATERIAL AND METHODS: RNA was isolated from stimulated bovine VSMC after different time periods. For stimulation we used growth factors (platelet-derived growth factors PDGF-AA, PDGF-BB, basic fibroblast growth factor) and a nitric oxide donating drug (sodium nitroprusside). Gene expression of stimulated and control cells was analyzed by non-radioactive RNA fingerprinting (RNA arbitrarily primed polymerase chain reaction, RAP-PCR) and standard gel electrophoresis. Polymorphic fragments were sequenced and further characterized. RESULTS: By RAP-PCR we detected changes in the RNA fingerprint pattern of stimulated cells compared with unstimulated cells. Sequences of five fragments out of 12 showed high homology to known human genes (serine-methyl-transferase, DUTT1, laminin B2, a newly cloned translational regulator (p97), and a human expressed sequence tag). For laminin B2 we could confirm an upregulation after stimulation with growth factors at 1 and 6 hours and after stimulation with SNP at 1 hour in comparison to controls. For p97 we could show a downregulation after stimulation with SNP, bFGF and PDGF-BB but not PDGF-AA. CONCLUSION: RAP-PCR is well suited for analysis of VSMC gene expression in vitro. The laminin B2 and p97 gene are differently expressed after growth factor stimulation in bovine VSMC.

[Structure-activity relationships applied to quinoxaline-1,4-dioxides]. / Struktur-Wirkungs-Beziehungen bei Chinoxalin-1,4-dioxiden.

Studies on structure-activity relationships applied to quinoxaline 1,4-dioxides were performed on the basis of Hansch analysis. It could be demonstrated that correlations exist between MIC values (Escherichia coli) and physicochemical parameters. On the other hand, correlations were also found between nutritive effects determined on chicken and structural parameters. The results obtained could be confirmed by means of Free-Wilson analysis.

Chromosome numbers and DNA-content in intracerebrally transplanted experimental gliomas.

Serially transplanted experimental tumors of the central and peripheral nervous system can be used as models to investigate open questions in human neurooncology. Altered susceptibility of higher passages to chemotherapy might be correlated with chromosome number and DNA-content variations which would be partly expressed as changes in proliferation behaviour. Karyotypes therefore were analysed in the 73rd to 90th generations of transplanted experimental gliomas. Wide variation of chromosome number was observed; 2 major types of distribution occurred, the one presenting with, the other without stemlines. Large chromosomes # 1 and # 4 were often monosomic, while small chromosomes of ## 8 to 20 were increased up to the fivefold. Lines with prominent and few markers were observed. On the whole, cells of the proliferating pool of the tumor had to be considered as hypotetraploid. Comparison of chromosome numbers and DNA content gave good correlation; differences between the 2 were explained by the fact that only the number of chromosomes was taken into account, regardless of whether small or large chromosomes were lacking or in excess. When intracerebrally transplanted tumors had been previously treated by administration of BCNU, the DNA content was altered, indicating an increased share of diploid cells in the proliferation pool. Results are at variance with earlier findings in tissue cultures of directly induced malignant gliomas and neurinomas in rats. The findings in transplanted tumors can be interpreted as a result of increased malignancy in transplantation tumors, documented by rapid growth in the animal and dedifferentiated histologic morphology.

Growth inhibition by dominant-negative mutations of the neu-encoded oncoprotein.

In the present study, kinase-deficient mutants of the neu gene were constructed in order to generate dominant-negative receptor molecules, which should abolish phosphorylation of receptor complexes. One construct carried a mutation of the putative ATP-binding site (K758M), while the other mutant was generated by deletion of the kinase domain (ID400). Neither receptor showed phosphorylation by in vitro kinase assay. When NIH3T3 fibroblasts were co-transfected by the oncogenic neu gene and one of either construct, the transforming effect could be partially reversed. Therefore, kinase-negative mutations of the neu-encoded receptor seemed to have a dominant-negative effect on the action of the activated protein. To test this hypothesis, rat neurinoma cell lines containing oncogenic neu genes were transfected with the constructs. Expression of the kinase-defective mutants and reduced phosphorylation could be detected in different clones derived from single transfected cells. Striking growth inhibition and reduction of colony formation in soft agar were observed in these cell lines when compared with untransfected cells. Thus, kinase-deficient mutants exert a dominant-negative effect on phosphorylation of receptor complexes, resulting in a reversion of the transformed phenotype.

[Detection of amplified DNA sequences by comparative genomic in situ hybridization with human glioma tumor DNA as probe]. / Nachweis amplifizierter DNA-Sequenzen mittels Komparativer Genomischer in situ Hybridisierung und Tumor-DNA humaner gliome als Probe.

Comparative genomic hybridization (CGH) provides a new possibility for the investigation of genetic alterations in tumour genomes. In our experiments CGH was carried out using genomic DNA from human glioblastoma multiforme (GBM) as a probe for chromosomal in situ suppression hybridization. Amplified DNA sequences contained in the tumour DNA showed specific signals, revealing the chromosomal positions of these sequences. Using this approach we detected amplifications of different chromosomal segments in individual GBM specimens. In accordance with the results from Southern analysis demonstrating amplification of the EGFR gene in 45% of human GBM, CGH signals in different GBM mapped to the region of this gene on chromosome 7p. Other signals detected by CGH involved chromosome 12q and 8q. Our data demonstrate CGH as a novel comprehensive and rapid approach for the analysis of complex genomic alterations in glial tumours.

Expression of cytokines and growth factors in human glomerulonephritides.

Numerous experimental studies point to the potential role of cytokines and growth factors in the pathogenesis of renal disease. However, from the various autocrine and paracrine mediators identified in vitro and in animal models, so far only a few have been demonstrated in selected human glomerulopathies. We examined two types of glomerulonephritis (GN): extracapillary GN with anti-neutrophil cytoplasmic autoantibodies (ANCA), an example of an acute form of GN, and mesangial IgA GN, usually a chronic form of GN, with immunocytochemistry, in situ hybridization and the polymerase chain reaction. Normal renal tissue from tumour nephrectomies served as a control. In ANCA-positive GN with active renal lesions (crescents, glomerular and vascular necrosis), infiltrating mononuclear cells in glomeruli and in the interstitium expressed interleukin (IL)-1 beta, tumour necrosis factor (TNF)-alpha, IL-2, interferon (IFN)-gamma, platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta. Cytokine expression was also observed in activated resident cells, including endothelial cells, capsular epithelial cells, smooth muscle cells of vessel walls, fibroblasts and some tubular epithelial cells. In addition, we noted an increase in the cytokine and growth factor receptors TNF-R, IL-1R type II, IL-2R, IFN-gamma R and PDGF beta-R. In contrast, in mesangial IgA-GN, IL-1 beta, TNF-alpha, IFN-gamma and IL-2 were usually absent in glomeruli. Mesangial expansion in this disorder was accompanied by an increased expression of PDGF, PDGF beta-R, TGF-beta and IL-6 in mesangial areas. In both conditions a good correlation was observed between cytokine expression at the mRNA (in situ hybridization) and protein level (immunocytochemistry).(ABSTRACT TRUNCATED AT 250 WORDS)

[Psychoanalysis and psychotherapy management in Austria]. / Psychoanalyse und psychotherapeutische Versorgung in Osterreich.

In a study on the psychotherapeutic provision in Austria the participation of the psychoanalysts was also researched. All over Austria there is a lack of psychotherapists in numbers and regionally. Long term psychotherapy except for a few special institutions are free of charge for the client. In private practice however the patients have to pay heavily. Non-medical psychotherapists are not eligible for refunds by the health insurance system. Almost 4/5 of all psychotherapists belong to a non-medical profession (e.g. psychologists). Only about 1/5 are medical doctors who work as psychotherapists on the basis of a therapeutic training. Psychoanalysts in Austria primarily work as psychoanalytic oriented psychotherapists and to a lesser extent as psychoanalysts.

Estrogen hormones reduce lipid peroxidation in cells and tissues of the central nervous system.

Effects of estrogen hormones on lipid peroxidation (LPO) were examined in rat brain homogenates (RBHs), hippocampal HT 22 cells, rat primary neocortical cultures, and human brain homogenates (HBHs). Dose-response curves indicated half-maximal effective concentrations (EC50) of 5.5 and 5.6 mM for iron-induced LPO in RBHs and HT 22 homogenates. Incubation of living rat primary neocortical cultures with iron resulted in an EC50 of 0.5 mM, whereas culture homogenates showed an EC50 of 1.2 mM. Estrogen hormones reduced LPO in all systems: In RBHs, estrone inhibited iron-induced LPO to 74.1 +/- 5.8% of control levels (17beta-estradiol: 71.3 +/- 0.1%) at a concentration of 10 microM. In hippocampal HT 22 cell homogenates, levels of LPO were reduced to 74.8 +/- 5.5% by estrone and to 47.8 +/- 6.2% by 17beta-estradiol. In living neocortical cultures, 17beta-estradiol decreased iron-induced LPO to 79.2 +/- 4.8% and increased the survival of cultured neuronal cells. Of the other steroid compounds tested (corticosterone, progesterone, testosterone), only progesterone decreased LPO in HT 22 cell homogenates. In HBHs, LPO was dose-dependently increased by iron concentrations from 2.7 to 6.0 mM. Incubation with estrogens resulted in a dose-dependent inhibition of LPO to 53.8 +/- 8.6% with 10 microM 17beta-estradiol, whereas estrone failed to affect iron-induced LPO to a significant extent. Nonestrogenic steroids, including hydrocortisol, did not show significant effects on LPO in HBHs.