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).

The dominant alopecia phenotypes Bareskin, Rex-denuded, and Reduced Coat 2 are caused by mutations in gasdermin 3.

Reduced Coat 2 (Rco2) is an ENU-induced mutation affecting hair follicle morphogenesis by an abnormal and protracted catagen. We describe chromosomal mapping and molecular identification of the autosomal dominant Rco2 mutation. The Rco2 critical region on mouse chromosome 11 encompasses the alopecia loci, Bareskin (Bsk), Rex-denuded (Re(den)), Recombination induced mutation 3 (Rim3), and Defolliculated (Dfl). Recently, the gasdermin (Gsdm) gene was described as predominantly expressed in skin and gastric tissues. We provide evidence for a murine-specific gene cluster consisting of Gsdm and two closely related genes which we designate as Gsdm2 and Gsdm3. We show that Gsdm3 reflects a mutation hotspot and that Gsdm3 mutations cause alopecia in Rco2, Re(den), and Bsk mice. We infer a role of Gsdm3 during the catagen to telogen transition at the end of hair follicle morphogenesis and the formation of hair follicle-associated sebaceous glands.

Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS.

The authors report mutation screening of the p150 subunit of dynactin (DCTN1) and the cytoplasmic dynein heavy chain (DNCHC1) genes in 250 patients with ALS and 150 unrelated control subjects. Heterozygous missense mutations of the DCTN1 gene were detected in one apparently sporadic case of ALS (T1249I), one individual with familial ALS (M571T), two patients with familial ALS, and two unaffected relatives in the same kindred (R785W). The allelic variants of the DCTN1 gene may represent a previously unknown genomic risk factor for ALS.

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.

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.

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.

Amphetamines induce apoptosis and regulation of bcl-x splice variants in neocortical neurons.

Amphetamineanalogs have emerged as popular recreational drugs of abuse. The number of reports of these substances producing severe acute toxicity and death is increasing. In 'Ecstasy' -associated deaths, focal necrosis in the liver and individual myocytic necrosis has been reported. Furthermore, serotonergic and dopaminergic neuronal cell damage has been observed in experimental amphetamine intoxication in laboratory animals. Here we demonstrate that subchronic exposure to D-amphetamine, methamphetamine, methylenedioxyamphetamine, and methylenedioxymethamphetamine ('Ecstasy') results in significant neurotoxicity in rat neocortical neurons in vitro. This neuronal cell death is accompanied by endonucleosomal DNA cleavage and differential expression of anti- and proapoptotic bcl-xL/S splice variants. In addition, we observed pronounced induction of cell stress-associated transcription factor c-jun and translation initiation inhibitor p97 after amphetamine treatment. These data support that the neurotoxic effects of different amphetamines are extended to rat neocortical neurons and that apoptotic pathways are involved in amphetamine-induced neurotoxicity.

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.

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.

Genomic instability in colorectal carcinomas: comparison of different evaluation methods and their biological significance.

In order to demonstrate the relationship between microsatellite instability and other types of genomic instability, a series of 56 sporadic colorectal carcinomas was investigated by flow cytometrical ploidy analysis, oligonucleotide fingerprinting, and microsatellite polymerase chain reaction (PCR). Stabilization of the p53 gene product was analysed by immunohistochemistry and proliferative activity was determined flow cytometrically and by silver staining of nucleolar organizer regions (AgNORs). Of the 56 carcinomas, 11 (19 per cent) exhibited microsatellite instability; 33 of the cases were aneuploid (59 per cent) and 29 (52 per cent) showed alterations of the oligonucleotide fingerprints. There was a significant correlation of microsatellite instability with localization of these tumours proximal to the splenic flexure, diploid DNA content, and less frequent p53 stabilization. A solid growth pattern, mucinous differentiation, and a Crohn's-like lymphoid infiltrate were also characteristic for those tumours. The results demonstrate for the first time a significantly lower proliferative activity in tumours with microsatellite instability. Data obtained from DNA flow cytometry or from oligonucleotide fingerprinting did not correlate with such tumour characteristics. It is proposed that the use of microsatellite PCR facilitates specifically the detection of a group of colorectal cancers which may differ in pathogenesis and perhaps prognosis.

Concomitant overexpression of the EGFR and erbB-2 genes in renal cell carcinoma (RCC) is correlated with dedifferentiation and metastasis.

Gene amplification or structural alteration of different erbB genes exerts a transforming effect in a variety of human neoplasms. Overexpression of the EGF receptor is associated with tumor initiation and progression of renal cell carcinoma (RCC). However, the role of erbB-2 in these processes remains unknown. We investigated 34 renal cell carcinomas for gene amplification and expression of the EGFR and erbB-2 genes at the mRNA and protein level and their relationship to pathological and clinical parameters. No amplification of both genes has been observed. However, high expression of the EGF receptor protein and p185erbB2 was frequent in RCC and statistically significantly related to higher tumor grades. We could demonstrate a close correlation of p185erbB2 overexpression with high EGF receptor levels. Co-overexpression of both receptor types was significantly associated with metastatic disease. Our results suggest a synergistic involvement of both EGF receptor and p185erbB2 in the progression of RCC.

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.

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.

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.

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.

[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.

Amplification and differential expression of members of the erbB-gene family in human glioblastoma.

The objective of the present study was to determine the frequency of amplifications of three different members of the erbB gene family in human glioblastoma multiforme (GBM). We investigated 47 glial tumors (37 GBM WHO grade IV, 5 anaplastic astrocytomas WHO III and 5 astrocytomas WHO II) by Southern and Western analysis, and immunocytochemistry. Gene amplification of erbB genes in human malignant gliomas was restricted to the EGF receptor (EGFR) gene, erbB-1. We found amplification of the EGFR gene in 49% (18/37) of GBM but not in the astrocytomas WHO II/III. The erbB-2 and erbB-3 genes showed no amplification in the tumor specimens investigated in this study. At the protein level we found overexpression of the EGF receptor in 86% (32/37) by Western analysis and in 92% (34/37) by immunocytochemistry. Expression of the ERBB2 protein was present in 54% (20/37) but immunoreactivity was much weaker than for EGF receptor and in most cases barely detectable by Western analysis and immunocytochemistry. The ERBB3 protein was not expressed in the glial tumors investigated in this study. Of the three erbB genes only gene amplification and overexpression of the EGF receptor seems to have an impact on tumor progression of human gliomas. Our data from immunohistochemistry indicate that ERBB2 expression in GBM is closely correlated with EGF receptor levels and is therefore not useful as an independent prognostic parameter.

Amplification of the epidermal-growth-factor-receptor gene correlates with different growth behaviour in human glioblastoma.

The objective of our study was to determine the frequency of EGF-receptor-gene rearrangement in relation to tumour-growth behaviour in an unselected group of glioma patients. We investigated 73 glial tumours with different grades of malignancy (17 low-grade gliomas, 14 anaplastic variants, and 42 GBM) by Southern analysis, reverse transcriptase PCR (RT-PCR) amplification of mRNA, and Western analysis. An amplification of the EGF-receptor gene was present in 19/42 GBM but in only 1 anaplastic astrocytoma. By RT-PCR, 4/19 GBM with gene amplification showed a specific amino-terminal aberrant splice mutation of 801 bp in addition to undeleted mRNA. By Western analysis, 27/42 GBM showed expression of the EGF-receptor protein. Protein levels, however, varied among individual tumours. Four GBM containing an aberrant splice mutation exhibited an immunoreactive protein of 130 kDa MW in addition to the normal EGF-receptor protein p170. All GBM patients underwent surgery followed by a standard course of radiotherapy. Neuroradiological follow-up in 31/42 GBM patients consisted of bimonthly MRI examinations. There was a statistically significant difference in the mean latency period until tumour regrowth of patients suffering from GBM with and without EGF-receptor-gene amplification (9 weeks vs. 32 weeks). Our data indicate more rapid tumour regrowth kinetics of GBM with amplified EGF receptor genes in vivo.