Sequencing of E2 and NS5A regions of HCV genotype 3a in Brazilian patients with chronic hepatitis

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.

A molecular comparison of T lymphocyte populations infiltrating the liver and circulating in the blood of patients with chronic hepatitis B: Evidence for antigen-driven selection of a public complementarity-determining region 3 (CDR3) motif

Despite a large number of T cells infiltrating the liver of patients with chronic hepatitis B, little is known about their complexity or specificity. To characterize the composition of these T cells involved with the pathogenesis of chronic hepatitis B (CHB), we have studied the clonality of V beta T cell receptor (TCR)-bearing populations in liver tissue by size spectratyping the complementarity-determining region (CDR3) lengths of TCR transcripts. We have also compared the CDR3 profiles of the lymphocytes infiltrating the liver with those circulating in the blood to see whether identical clonotypes may be detected that would indicate a virus-induced expansion in both compartments. Our studies show that in most of the patients examined, the T cell composition of liver infiltrating lymphocytes is highly restricted, with evidence of clonotypic expansions in 4 to 9 TCR V beta subfamilies. In contrast, the blood compartment contains an average of 1 to 3 expansions. This pattern is seen irrespective of the patient's viral load or degree of liver pathology. Although the TCR repertoire profiles between the 2 compartments are generally distinct, there is evidence of some T cell subsets being equally distributed between the blood and the liver. Finally, we provide evidence for a putative public binding motif within the CDR3 region with the sequence G-X-S, which may be involved with hepatitis B virus recognition.

A single amino acid substitution in the mRNA capping enzyme λ2 of a mammalian orthoreovirus mutant increases interferon sensitivity.

In the last few years, the development of a plasmid-based reverse genetics system for mammalian reovirus has allowed the production and characterization of mutant viruses. This could be especially significant in the optimization of reovirus strains for virotherapeutic applications, either as gene vectors or oncolytic viruses. The genome of a mutant virus exhibiting increased sensitivity to interferon was completely sequenced and compared with its parental virus. Viruses corresponding to either the parental or mutant viruses were then rescued by reverse genetics and shown to exhibit the expected phenotypes. Systematic rescue of different viruses harboring either of the four parental genes in a mutant virus backbone, or reciprocally, indicated that a single amino acid substitution in one of λ2 methyltransferase domains is the major determinant of the difference in interferon sensitivity between these two viruses.

Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica

Salmonella enterica isolates (n = 182) were examined for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in parC that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the gyrB quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Len. Novel mutations were also found in parE encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both gyrA and parC were more susceptible to ciprofloxacin than were isolates with a mutation in gyrA alone.

The Irish paradigm on the natural progression of hepatitis C virus infection: an investigation in a homogeneous patient population infected with HCV 1b (Review)

The aetiological agent of chronic hepatitis C is the hepatitis C virus. The hepatitis C virus is spread by parenteral transmission of body fluids, primarily blood or blood products. In 1989, after more than a decade of research, HCV was isolated and characterised. The hepatitis C viral genome is a positive-sense, single-stranded RNA molecule approximately 9.4 kb in length, which encodes a polyprotein of about 3100 amino acids. There are 6 main genotypes of HCV, each further stratified by subtype. In 1994, a cohort of women was identified in Ireland as having been iatrogenically exposed to the hepatitis C virus. The women were all young and exposed as a consequence of the receipt of HCV 1b contaminated anti-D immunoglobulin. The source of the infection was identified as an acutely infected female. As part of a voluntary serological screening programme involving 62,667 people, 704 individuals were identified as seropositive for exposure to the hepatitis C virus; 55.4% were found to be positive for the viral genome 17 years after exposure. Of these women 98% had evidence of inflammation, but suprisingly, a remarkable 49% showed no evidence of fibrosis. Clinicopathology and virological analysis has identified associations between viral load and the histological activity index for inflammation, and, between inflammation and levels of the liver enzyme alanine aminotransferase. Infection at a younger age appears to protect individuals from progression to advanced liver disease. Molecular analyses of host immunogenetic elements shows that particular class II human leukocyte associated antigen alleles are associated with clearance of the hepatitis C virus. Additional class II alleles have been identified that are associated with stable viraemia over an extended period of patient follow-up. Although, investigation of large untreated homogeneous cohorts is likely to become more difficult, as the efficacy of anti-viral therapy improves, further investigation of host and viral factors that influence disease progression will help provide an evidence based approach were realistic expectations regarding patient prognosis can be ascertained.

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Accumulation of human heat shock protein 60-reactive T cells in the gingival tissues of periodontitis patients

Heat shock protein 60s (hsp60) are remarkably immunogenic, and both T-cell and antibody responses to hsp60 have been reported in various inflammatory conditions. To clarify the role of hsp60 in T-cell responses in periodontitis, we examined the proliferative response of peripheral blood mononuclear cells (PBMC), as well as the cytokine profile and T-cell clonality, for periodontitis patients and controls following stimulation with recombinant human hsp60 and Porphyromonas gingivalis GroEL. To confirm the infiltration of hsp60-reactive T-cell clones into periodontitis lesions, nucleotide sequences within complementarity-determining region 3 of the T-cell receptor (TCR) beta-chain were compared between hsp60-reactive peripheral blood T cells and periodontitis lesion-infiltrating T cells. Periodontitis patients demonstrated significantly higher proliferative responses of PBMC to human hsp60, but not to P. gingivalis GroEL, than control subjects. The response was inhibited by anti-major histocompatibility complex class 11 antibodies. Analysis of the nucleotide sequences of the TCR demonstrated that human hsp60-reactive T-cell clones and periodontitis lesion-infiltrating T cells have the same receptors, suggesting that hsp60-reactive T cells accumulate in periodontitis lesions. Analysis of the cytokine profile demonstrated that hsp60-reactive PBMC produced significant levels of gamma interferon (IFN-gamma) in periodontitis patients, whereas P. gingivalis GroEL did not induce any, skewing toward a type1 or type2 cytokine profile. In control subjects no significant expression of IFN-gamma or interleukin 4 was induced. These results suggest that periodontitis patients have human hsp60-reactive T cells with a type I cytokine profile in their peripheral blood T-cell pools.

Genotyping of two Neisseria gonorrhoeae fluroquinolone-resistant strains in the Brazilian Amazon Region

We report two ciprofloxacin and ofloxacin-resistant Neisseria gonorrhoeae strains that were isolated from the urethral discharge of male patients at the sexually transmitted diseases outpatient clinic of the Alfredo da Matta Foundation (Manaus, state of Amazonas, Brazil). The gonococci displayed minimal inhibitory concentrations (> 32.00 µg/mL) and three mutations in the quinolone resistance-determining region (S91F and D95G in GyrA and S87R in ParC). Both isolates were genotyped using N. gonorrhoeae multi-antigen sequence typing and the analysis showed that the ST225 which represented an emerging widespread multi-resistant clone that has also been associated with reduced susceptibility to ceftriaxone. We recommend continued surveillance of this pathogen to assess the efficacy of anti-gonococcal antibiotics in Brazil.

Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

Charakterisierung und Analyse der geschlechtsbestimmenden Region von Chironomus riparius

Die durch eine männchenspezifisch auftretende heterochromatische Bande und ein hemizygotes Cla-Element-Cluster gekennzeichnete geschlechtsbestimmende Region („sex determining region“: SDR) auf Chromosom III von C. riparius stellt ein frühes Stadium in der Evolution von Geschlechtschromosomen dar. Diese eindeutig lokalisierte chromosomale Region, die den molekular noch unbekannten männchen¬bestimmenden Faktor M enthalten muss, ist im Vergleich zu den Y-Chromosomen anderer Dipterenarten wie unter anderem M. domestica, die ebenfalls einen dominanten Männchenbestimmer besitzen, relativ klein. Aus diesem Grund bietet die SDR von C. riparius eine Möglichkeit, den männchenbestimmenden Faktor einzugrenzen und zu identifizieren. In der vorliegenden Arbeit konnte ein Bereich einer Größe von ca. 200 kb aus der SDR von C. riparius charakterisiert und analysiert werden. Durch bioinformatische Sequenzanalysen konnten an 20 Stellen der SDR mögliche Genstrukturen nachgewiesen werden. Von den gefundenen möglichen Genen ist bisher die Funktion in C. riparius unbekannt. Bei den Genen mit vermuteter Funktion deutet nichts eindeutig auf eine Beteiligung an der Geschlechtsbestimmung von C. riparius hin. Da allerdings davon auszugehen ist, dass für die Funktion des Männchenbestimmers M ein Gen rekrutiert wurde, welches zur Interaktion mit dem nachgeschalteten Gen der Geschlechts¬bestimmungskaskade fähig ist, muss die geschlechtsbestimmende Funktion des Gens M nicht unbedingt offensichtlich sein. Aus geschlechtsbestimmenden Genkaskaden anderer Dipteren bekannte Gene wie transformer und doublesex konnten im analysierten Bereich nicht nachgewiesen werden, obwohl zumindest zu doublesex homologe Gene im Genom von C. riparius vorkommen. Um möglicherweise proto-X- und proto-Y-Chromosom miteinander vergleichen zu können und einen Hinweis auf die chromosomale Herkunft der analysierten Sequenzen aus der SDR zu erlangen, wurden Sequenzen von 31 teilweise parallel liegenden BAC-Klonen aus der untersuchten Region verglichen. Dabei zeigte sich, dass die Klone zwei Gruppen bilden, deren Sequenzen sich durch 500 SNPs und 110 Indels unterschiedlicher Größe (1-800 Bp) unterscheiden, was für eine Herkunft von zwei sich erst seit kurzer Zeit unterscheidenden Geschlechtschromosomen spricht. Die zwölf größten dieser Indels wurden auf geschlechtsspezifische Unterschiede hin untersucht. Dabei zeigte sich, dass die Unterschiede zwischen den beiden Klongruppen zwar im 30 Jahre alten Laborstamm, der auch für die Konstruktion der durchsuchten BAC-Bibliotheken verwendet wurde, tatsächlich geschlechtsspezifisch sind, in zwei Wildfangpopulationen jedoch keine derartige Geschlechtsspezifität aufweisen. Somit kann keine Aussage zur Herkunft der untersuchten Klone aus der SDR von C. riparius getroffen werden, und es bleibt unklar, ob die analysierten Sequenzen vom proto-X oder vom proto-Y-Chromosom stammen.

Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution

We have developed an extremely sensitive technique, termed immuno-detection amplified by T7 RNA polymerase (IDAT) that is capable of monitoring proteins, lipids, and metabolites and their modifications at the single-cell level. A double-stranded oligonucleotide containing the T7 promoter is conjugated to an antibody (Ab), and then T7 RNA polymerase is used to amplify RNA from the double-stranded oligonucleotides coupled to the Ab in the Ab-antigen complex. By using this technique, we are able to detect the p185her2/neu receptor from the crude lysate of T6–17 cells at 10−13 dilution, which is 109-fold more sensitive than the conventional ELISA method. Single-chain Fv fragments or complementarity determining region peptides of the Ab also can be substituted for the Ab in IDAT. In a modified protocol, the oligonucleotide has been coupled to an Ab against a common epitope to create a universal detector species. With the linear amplification ability of T7 RNA polymerase, IDAT represents a significant improvement over immuno-PCR in terms of sensitivity and has the potential to provide a robotic platform for proteomics.

Loss of sequences 3' to the testis-determining gene, SRY, including the Y pseudoautosomal boundary associated with partial testicular determination.

The condition termed 46,XY complete gonadal dysgenesis is characterized by a completely female phenotype and streak gonads. In contrast, subjects with 46,XY partial gonadal dysgenesis and those with embryonic testicular regression sequence usually present ambiguous genitalia and a mix of Müllerian and Wolffian structures. In 46,XY partial gonadal dysgenesis gonadal histology shows evidence of incomplete testis determination. In 46,XY embryonic testicular regression sequence there is lack of gonadal tissue on both sides. Various lines of evidence suggest that embryonic testicular regression sequence is a variant form of 46,XY gonadal dysgenesis. The sex-determining region Y chromosome gene (SRY) encodes sequences for the testis-determining factor. To date germ-line mutations in SRY have been reported in approximately 20% of subjects with 46,XY complete gonadal dysgenesis. However, no germ-line mutations of SRY have been reported in subjects with the partial forms. We studied 20 subjects who presented either 46,XY partial gonadal dysgenesis or 46,XY embryonic testicular regression sequence. We examined the SRY gene and the minimum region of Y-specific DNA known to confer a male phenotype. The SRY-open reading frame (ORF) was normal in all subjects. However a de novo interstitial deletion 3' to the SRY-ORF was found in one subject. Although it is possible that the deletion was unrelated to the subject's phenotype, we propose that the deletion was responsible for the abnormal gonadal development by diminishing expression of SRY. We suggest that the deletion resulted either in the loss of sequences necessary for normal SRY expression or in a position effect that altered SRY expression. This case provides further evidence that deletions of the Y chromosome outside the SRY-ORF can result in either complete or incomplete sex reversal.

Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1.

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.

Pluripotent Reprogramming of Fibroblasts by Lentiviralmediated Insertion of SOX2, C-MYC, and TCL-1A

Reprogramming of somatic cells to pluripotency promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used 4 transcription factors, octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and v-myc myelocytomatosis viral oncogene homolog (C-MYC), can induce pluripotency in mouse and human fibroblasts. Here, we report that forced expression of a new combination of transcription factors (T-cell leukemia/lymphoma protein 1A [TCL-1A], C-MYC, and SOX2) is sufficient to promote the reprogramming of human fibroblasts into pluripotent cells. These 3-factor pluripotent cells are similar to human embryonic stem cells in morphology, in the ability to differentiate into cells of the 3 embryonic layers, and at the level of global gene expression. Induced pluripotent human cells generated by a combination of other factors will be of great help for the understanding of reprogramming pathways. This, in turn, will allow us to better control cell-fate and apply this knowledge to cell therapy.

The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

Background: In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results: The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions: These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state ( which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage ( in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.