Post-transplant recurrent hepatitis C: immunohistochemical detection of hepatitis C virus core antigen and possible pathogenic implications

Introduction: The mechanisms by which severe cholestatic hepatitis develops after liver transplantation are not fully understood. Reports on immunohistochemical distribution of hepatitis C virus (HCV) antigens are still scarce, but recently, HCV immunostaining was suggested for early diagnosis of cholestatic forms of recurrent hepatitis C in liver grafts. After purification, Rb246 pab anticore (aa1-68) yielded specific, granular cytoplasmic staining in hepatocytes. Signal amplification through the Envision-Alkaline Phosphatase System avoided endogenous biotin and peroxidase. Aims/Methods: Rb246 was applied to liver samples of explants of 12 transplant recipients, six with the most severe form of post-transplantation recurrence, severe cholestatic hepatitis (group 1) and six with mild recurrence (group 2). We also assessed immuno-reactivity at two time-points post-transplantation (median 4 and 22 months) in both groups. HCV-core Ag was semiquantified from 0 to 3+ in each time point. Serum HCV-RNA was also measured on the different time points by branched DNA. Results: In the early post-transplant time point, one patient had a mild staining (1+), two patients had a moderate staining (2+) and the other three had no staining in group 1, compared with five patients with no staining (0) and one patient with mild staining (1+) in group 2. Late post-transplant liver samples were available in nine patients, and two out of four samples in group 1 showed a mild staining, compared with no staining patients in five patients in group 2. Strikingly, on the explant samples, HCV immunostaining was strongly positive in group 1, and mildly positive in group 2. Two out of five samples showed 3+ staining, and three samples showed 2+ staining in group 1; two out of five samples showed no staining, two samples showed 1+ staining and one sample showed 2+ staining in group 2. Serum HCV-RNA was significantly higher in group 1, on both time-points post-transplantation. HCV-core Ag was not directly associated with serum HCV-RNA on the different time points. Conclusion: These preliminary results suggest that strong HCV immunostaining in the explant is predictive of more severe disease recurrence.

APOE polymorphism is associated with lipid profile, but not with arterial stiffness in the general population

Background: Cardiovascular diseases (CVD) are the main cause of death and disability in developed countries. In most cases, the progress of CVD is influenced by environmental factors and multifactorial inheritance. The purpose of this study was to investigate the association between APOE genotypes, cardiovascular risk factors, and a noninvasive measure of arterial stiffness in the Brazilian population. Methods: A total of 1493 urban Brazilian individuals were randomly selected from the general population of the Vitoria City Metropolitan area. Genetic analysis of the APOE polymorphism was conducted by PCR-RFLP and pulse wave velocity analyzed with a noninvasive automatic device. Results: Age, gender, body mass index, triglycerides, creatinine, uric acid, blood glucose, blood pressure phenotypes were no different between epsilon 2, epsilon 3 and epsilon 4 alleles. The epsilon 4 allele was associated with higher total-cholesterol (p < 0.001), LDL-C (p < 0.001), total-cholesterol/HDL-C ratio (p < 0.001), LDL/HDL-C ratio (p < 0.001), lower HDL-C values (p < 0.001) and higher risk to obesity (OR = 1.358, 95% CI = 1.019-1.811) and hyperuricemia (OR = 1.748, 95% CI = 1.170-2.611). Nevertheless, pulse wave velocity (p = 0.66) measures were no different between genotypes. The significant association between APOE genotypes and lipid levels persisted after a 5-year follow-up interval, but no interaction between time and genotype was observed for lipids longitudinal behavior. Conclusion: The epsilon 4 allele of the APOE gene is associated with a worse lipid profile in the Brazilian urban population. In our relatively young sample, the observed effect of APOE genotype on lipid levels was not translated into significant effects in arterial wall stiffness.

Genetic association and cellular function of MC1R variant alleles in human pigmentation

We have examined MC1R variant allele frequencies in the general population of South East Queensland and in a collection of adolescent dizygotic and monozygotic twins and family members to define statistical associations with hair and skin color, freckling, and mole count. Results of these studies are consistent with a linear recessive allelic model with multiplicative penetrance in the inheritance of red hair. Four alleles, D84E, R151C, R160W, and D294H, are strongly associated with red hair and fair skin with multinomial regression analysis showing odds ratios of 63, 118, 50, and 94, respectively. An additional three low-penetrance alleles V60L, V92M, and R163Q have odds ratios 6, 5, and 2 relative to the wild-type allele. To address the cellular effects of MC1R variant alleles in signal transduction, we expressed these receptors in permanently transfected HEK293 cells. Measurement of receptor activity via induction of a cAMP-responsive luciferase reporter gene found that the R151C and R160W receptors were active in the presence of NDP-MSH ligand, but at much reduced levels compared with that seen with the wild-type receptor. The ability to stimulate phosphorylation of the cAMP response element binding protein (CREB) transcription factor was also apparent in all stimulated MC1R variant allele-expressing HEK293 cell extracts as assessed by immunoblotting. In contrast, human melanoma cell lines showed wide variation in the their ability to undergo cAMP-mediated CREB phosphorylation. Culture of human melanocytes of known MC1R genotype may provide the best experimental approach to examine the functional consequences for each MC1R variant allele. With this objective, we have established more than 300 melanocyte cell strains of defined MC1R genotype.

Spondylocostal Dysostosis Associated with Methylmalonic Aciduria

Spondylocostal dysostosis (SCD) is a genetic disorder characterized by vertebral segmentation and formation defects associated with changes of the ribs. Autosomal dominant and recessive modes of inheritance have been reported. Methylmalonic aciduria (MMA) is an inborn error of propionate or cobalamin metabolism. It is an autosomal recessive disorder and one of the most frequent forms of branched-chain organic acidurias. Here we report on a case of a Brazilian boy with both diseases. As we know, it is the first case in the literature with the occurrence of both SCD and MMA-the first a skeletal disease and the latter an inborn error of metabolism.

Identification of novel mutations of the WFS1 gene in Brazilian patients with Wolfram syndrome

Objective: Wolfram syndrome (WS) is a rare, progressive, neurodegenerative disorder with an autosomal recessive pattern of inheritance. The gene for WS, WFS1, was identified on chromosome 4p16 and most WS patients carry mutations in this gene. However. some studies have provided evidence for genetic heterogeneity and the genotype-phenotype relationships are not clear. Our aim was to ascertain the spectrum of WFS1 mutations in Brazilian patients with WS and to examine the phenotype-genotype relationships in these patients. Design and methods: Clinical characterization and analyses of the WFS1. gene were performed in 27 Brazilian patients with WS from 19 families. Results: We identified 15 different mutations in the WFS1 gene in 26 patients, among which nine are novel. All mutations occurred in exon 8, except for one missense mutation which was located in exon 5. Although we did not find any clear phenotype-genotype relationship in patients with mutations in exon 8, the homozygous missense mutation in exon 5 was associated with a mild phenotype: onset of diabetes mellitus and optic atrophy during adulthood with good metabolic control being achieved with low doses of sulfonylurea Conclusions: Our data show that WFS1 is the major gene involved in WS in Brazilian patients and most mutations are concentrated in exon 8. Also, our study increases the spectrum of WFS1 mutations. Although no clear phenotype-genotype relationship was found for mutations in exon 8, a mild phenotype was associated with a homozygous missense mutation in exon 5.

HLA-DR in Brazilian patients with polyarteritis nodosa (PAN) and microscopic polyangiitis (MPA)

The aim of this study was to evaluate the frequency and clinical associations of HLA-DR alleles in Brazilian Caucasian patients with polyarteritis nodosa (PAN) or microscopic polyangiitis (MPA). We evaluated 29 Caucasian patients with vasculitis classified as PAN or MPA according to the American College of Rheumatology (ACR) 1990 Criteria, Chapel Hill Consensus Conference (CHCC) nomenclature for vasculitis and EULAR recommendations for conducting clinical studies in systemic vasculitis. HLA-DR alleles were typed using polymerase chain reaction-amplified DNA, hybridized with sequence-specific low resolution primers. DNA obtained from 59 Caucasian healthy blood donors were used as control. In order to evaluate if a specific HLA may have influence on the clinical profile of those diseases, we also divided the patients according to Birmingham vasculitis score (BVAS) and Five-Factors Score (FFS) at the time of diagnosis. Increased frequency of HLA-DRB1*16 (p = 0.023) and DRB4*01 (p = 0.048) was found in patients with higher disease activity at the time of diagnosis (BVAS >= 22). Patients with less severe disease (FFS = 0) had a higher frequency of HLA-DRB1*03 (p = 0.011). Patients with gastrointestinal tract involvement had significantly increased frequency of HLA-DRB1*11 or B1*12 (p = 0.046), B1*13 (p = 0.021) and B3 (p = 0.008). In contrast, patients with renal disease, had higher frequency of DRB1*15 or DRB1*16 (p = 0.035) and B5 (p = 0.035). In the subgroup of patients with MPA, increased frequency of HLA-DRB1*15 was found in patients with BVAS >= 22 (p = 0.038) and FFS >= 1 (p = 0.039) suggesting that this allele is associated with more aggressive disease. Antineutrophil cytoplasmic antibodies (ANCA) negative MPA patients had significantly increased frequency of HLA-DRB1*11 or DRB1*12 when compared to ANCA positive patients (p = 0.023). Our results suggest that HLA-DR alleles may influence PAN and MPA clinical expression and outcome and that in MPA they participate in the mechanisms involved in the development to ANCA.

Functional association of human Ki-1/57 with pre-mRNA splicing events

The cytoplasmic and nuclear protein Ki- 1 / 57 was first identified in malignant cells from Hodgkin`s lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki- 1 / 57 in human cells remains to be determined. Here, we investigated the relationship of Ki- 1 / 57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki- 1 / 57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki- 1 / 57 was able to bind to a poly- U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki- 1 / 57 can modify the splicing site selection of the adenoviral E1A minigene in a dose- dependent manner. Further confocal and. uorescence microscopy analysis revealed the localization of enhanced green. uorescent protein - Ki- 1 / 57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N- terminal region. In summary, our findings suggest that Ki- 1 / 57 is probably involved in cellular events related to RNA functions, such as pre- mRNA splicing.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Myosin Va phosphorylated on ser(1650) is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcription

Nuclear actin and nuclear myosins have been implicated in the regulation of geneexpression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser(1650) MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine(1650) and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser(1650) MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser(1650) MVa to nucleoli, as well as separating a fraction of phosphoser(1650) MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation.

P2X(7) receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1 beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1 beta into mature IL-1 beta, which is then secreted. Because both LPS (in vivo) and IL-1 beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1 beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco`s modified Eagle`s medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1 beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1 beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-L-arginine methyl ester or N-([3-(aminomethyl) phenyl] methyl) ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1 beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1 beta induced iNOS protein expression. Thus, activation of P2X 7 receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1 beta-mediated release of nitric oxide by iNOS.

A splice variant of the Neurospora crassa hex-1 transcript, which encodes the major protein of the Woronin body, is modulated by extracellular phosphate and pH changes

The Woronin body, a septal pore-associated organelle specific to filamentous ascomycetes, is crucial for preventing cytoplasmic bleeding after hyphal injury. In this study, we show that T1hex-1 transcript and a variant splicing T2hex-1 transcript are up-regulated at alkaline pH. We also show that both hex-1 transcripts are overexpressed in the preg(c), nuc-1(RIP), and pacC(ko) mutant strains of Neurospora crassa grown under conditions of phosphate shortage at alkaline pH, suggesting that hex-1 transcription may be coregulated by these genes. In addition, we present evidence that N. crassa PacC also has metabolic functions at acidic pH. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Angiogenic non-Hodgkin T/natural killer (NK)-cell lymphoma: Report of three cases

Angiogenic T/natural killer (NK)-cell lymphoma is a non-Hodgkin lymphoma characterized by necrosis and vascular destruction that is strongly associated with Epstein-Barr virus and AIDS. Early diagnosis is essential to improve the chances of patient survival, but severe local inflammatory infiltrate impairs histologic diagnosis by obscuring neoplastic cells. The most common markers are CD2, CD56, cytoplasmic CD3, and CD43 EBV We describe 3 cases of angiogenic T/NK-cell lymphoma that show the diverse Presentation of the same disease. Patient I was HIV positive and had nasal obstruction, facial edema, and ulceration of the nasal mucosa. Patient 2 had fever, a sore throat, and weight loss. Patient 3 had facial edema, fever, proptosis, and rapid development Of neurologic alterations. Several biopsies were needed for histologic confirmation in these patients, despite positivity for the CD3 and CD56 markers.

Transferability and characterization of nine microsatellite markers for the tropical tree species Tabebuia roseo-alba

Microsatellite loci that were previously developed in the tropical tree Tabebuia aurea were used for the genetic analysis of Tabebuia roseo-alba populations. Nine of 10 simple sequence repeat markers were amplified, and the polymorphism was assessed in 58 individuals sampled from two stands in southeastern Brazil. All loci were polymorphic with Mendelian inheritance. The allele numbers were high, ranging from 5 to 13 in population I and 3 to 7 in population II, with means of 8.9 and 5.5, respectively. We conclude that these markers can be efficiently used for parentage and gene-flow studies.

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta Aims: Thoracic ascending aortic aneurysms (TAA) are characterized by elastic fibre breakdown and cystic medial degeneration within the aortic media, associated with progressive smooth muscle cell (SMC) rarefaction. The transforming growth factor (TGF)-beta/Smad2 signalling pathway is involved in this process. Because the pericellular fibrinolytic system activation is able to degrade adhesive proteins, activate matrix metalloproteinase (MMP), induce SMC disappearance and increase the bioavailability of TGF-beta, the aim was to investigate the plasminergic system in TAA. Methods and results: Ascending aortas [21 controls and 19 TAAs (of three different aetiologies)] were analysed. Immunohistochemistry showed accumulation of t-PA, u-PA and plasmin in TAAs, associated with residual SMCs. Overexpression of t-PA and u-PA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and zymography on TAA extracts and culture medium conditioned by TAA. Plasminogen was present on the SMC surface and inside cytoplasmic vesicles, but plasminogen mRNA was undetectable in the TAA medial layer. Plasmin-antiplasmin complexes were detected in TAA-conditioned medium and activation of the fibrinolytic system was associated with increased fibronectin turnover. Fibronectin-related material was detected immunohistochamically in dense clumps around SMCs and colocalized with latent TGF-beta binding protein-1. Conclusions: The fibrinolytic pathway could play a critical role in TAA progression, via direct or indirect impact on ECM and consecutive modulation of TGF-beta bioavailability.

Light and scanning electron microcopy study of the tongue in Rhea americana

Morphological characteristics of the tongue were studied in adult rhea (Rhea americana). The lingual surface and the surface of epithelium-connective tissue interface of rhea tongue were examined macroscopically and by light and scanning electron microscopy. The rhea tongue revealed a triangular aspect, without adjustment of the inferior bill formation, occupying approximately of the length of the oral cavity. Lingual papilla-like structures were not observed over the lingual surface. The tongue mucosa was composed of a thick non-keratinized stratified squamous epithelium in the dorsal and ventral part, supported by a connective tissue core. The submucosa contained numerous glands with cytoplasmic granules, and luminal secretion was positive for histochemical reaction to Alcian Blue in pH 2.5 and PAS, and negative to Alcian Blue in pH 0.5. Despite the rudimentary characteristic of the tongue in rhea, our results suggest an important role of tongue secretions in food lubrication and humidification during the swallowing process, based on the enormous quantity of lingual glands in the submucosa and the histochemical characteristics of their secretions.