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PubMedDate not supplied

The long-term efficacy of interferon alfa in chronic hepatitis C patients: a critical review.

With current therapeutic regimens, sustained responses occur in no more than 25% of patients with chronic hepatitis C who are treated with interferon. Relapses occur usually within 6 months from therapy suspension, but clinical and virologic recurrencies can be observed as late as after 3 years of follow up. The rate of long-term responses seems to depend on the dosage and the period of administration of interferon, but the best therapeutic protocol remains unknown. As a direct marker of permanent recovery is not available, indirect signs of disease resolution are: (i) continuously normal alanine aminotransferase levels; (ii) clearance of HCV-RNA; (iii) disappearance of anti-C100/NS4; and (iv) significant histological improvements assessed at least 2 years after therapy withdrawal. Known baseline predictive features of long-term response are the absence of cirrhosis, low viraemic levels and infection with HCV of type III or IV genotype (Okamoto's classification). According to recent reports, the lower the heterogeneity of the hypervariable region of the envelope 2 gene of HCV, the higher the chance of a sustained remission. There is not yet any consensus on the efficacy of a second therapeutic course of interferon in inducing a permanent response, and controlled trials are needed to clarify this issue.

Chronic Disease↗
PubMed1996

Hepatitis C transmission between spouses.

To examine inter-spouse transmission as one of the potential routes of infection for hepatitis C virus (HCV), 121 patients with HCV-related chronic liver disease who tested positive for antibodies to HCV (anti-HCV) and their spouses were studied. Of these, 21 (17.4%) patients had spouses with anti-HCV. In 12 couples, the HCV genotype matched (type II: 10 couples, type III: two couples). The genotype differed in six couples. One patient was positive for anti-HCV, but negative for HCV-RNA, while the spouse was positive for both. The remaining two couples were only positive for anti-HCV. Genetic heterogeneity in the hypervariable region 1 of HCV was analysed in 11 couples with matched genotypes. In two couples, no mutation was recognized in the putative E2/NS1 genes using the heteroduplex method. The present study provides evidence to verify household transmission of HCV between patients and their spouses. These results suggest that inter-spouse transmission may be a potential route of transmission of HCV infection. However, other environmental factors (e.g. the duration of the marriage) cannot be ignored.

Adult↗
PubMed2005

Outbreak of hepatitis C virus infection in an outpatient clinic.

BACKGROUND: From January through September 2001, seven patients were admitted to Fukaya Red Cross Hospital with typical clinical manifestations of acute hepatitis. Six were outpatients of the clinic, which is located near the hospital. An extensive survey of clinic outpatients conducted by the local health department revealed six more new acute hepatitis cases during this period. METHODS: A case control study was carried out to identify potential risk factors for infection. In total, 1946 outpatients with clinic records were scheduled to undergo hepatitis C virus (HCV)-antibody testing. For the HCV-Ab positive patients, HCV-RNA was subtyped and quantified, and sequences of HCV hypervariable region 1 were determined. RESULTS: Ultimately, 12 patients with acute hepatitis and two asymptomatic subjects were found to be a part of this outbreak. HCV isolates were divided into three major groups using phylogenetic tree analysis. Only a past history of visiting the clinic was significantly associated with acute hepatitis. The timing of the parenteral medical procedure at the clinic and the onset of acute hepatitis strongly suggested association of the two events. CONCLUSIONS: Our findings suggest that nosocomial HCV infection can occur in an outpatient clinic, even in countries where post-transfusion hepatitis has been almost entirely eliminated.

Adult↗
PubMed1995

HLA-DQB1 allele associates with idiopathic nephrotic syndrome in Japanese children.

HLA-DQA1 and -DQB1 genes were investigated in 30 Japanese children with idiopathic nephrotic syndrome (INS) using the polymerase chain reaction-restriction fragment length polymorphism method. Compared with control children, there was a significant increase in the HLA-DQA1*0501, DQB1*0301 and DQB1*0601 alleles, whereas the frequency of DQB1*0501 showed a significant decrease in the patients. The increase of DQA1*0501 can be explained as being a result of a linkage disequilibrium with DQB1*0301. The previous result demonstrating a positive association of DRw6 and DRw8 with INS is also assumed to be attributable to a linkage disequilibrium with DQB1*0301 and 0601. A total of 83% of patients compared with 37% of controls possessed DQB1*0301 and/or DQB1*0601 allele (P < 0.001, RR = 8.6). Only these alleles have alanine at residue 13 and tyrosine at residue 26 in the hypervariable region in the Beta 1 domain of DQB1 gene. These findings suggest that the unique amino acid residues in the DQB1 gene may contribute to disease susceptibility in Japanese children with INS.

Adolescent↗
PubMed2005

Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition.

Serial analysis of ribosomal sequence tags (SARST) is a novel technique for characterizing microbial community composition. The SARST method captures sequence information from concatemers of short 16S rDNA polymerase chain reaction (PCR) amplicons from complex populations of DNA. Here, we describe a similar method, serial analysis of V6 ribosomal sequence tags (SARST-V6), which targets the V6 hypervariable region of bacterial 16S rRNA genes. The SARST-V6 technique exploits internal primer sequences to generate compatible restriction digest overhangs, thereby improving upon the efficiency of SARST. Serial analysis of V6 ribosomal sequence tags of bacterial community composition in hydrothermal marine sediments from Guaymas Basin resembled results of cloning and sequencing of single, full-length PCR products from ribosomal RNA genes of the same microbial community. Both methods identified the same major bacterial groups, but only SARST-V6 recovered thermodesulfobacteria and gamma-proteobacteria sequences, while only full-length PCR product cloning recovered candidate division OP11 se-quences. There were differences in the relative frequencies of some phylotypes. The disparities reflect differences in the amplicon pool obtained during initial amplification that may result from different primer affinities or DNA degradation. These results demonstrate the utility of SARST-V6 in collecting taxonomically informative data for high-throughput analysis of microbial communities.

Bacteria↗
PubMed2006

Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition.

Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.

Bacteria↗
PubMedDate not supplied

A genetic analysis of type 2 (non-insulin-dependent) diabetes mellitus in Punjabi Sikhs and British Caucasoid patients.

A genetic analysis of diabetic and non-diabetic Punjabi Sikhs (n = 164) was made for markers of non-insulin-dependent diabetes mellitus using insulin receptor, insulin, and HLA-D alpha chain gene probes. Additionally British Caucasoids (n = 163) were studied using the insulin receptor probe. Insulin receptor gene restriction fragment length polymorphisms were defined using Southern blot techniques and the restriction enzyme Bgl II and BAm Hl. In Punjabi Sikhs and British Caucasoids neither of the restriction fragment length polymorphisms distinguished non-insulin-dependent diabetes mellitus subjects from controls. In the Sikhs no association with non-insulin-dependent diabetes mellitus was seen with the hypervariable region of the insulin gene or with HLA-DR/DQ/DX alpha chain restriction fragment length polymorphisms. We therefore conclude that despite the high prevalence of non-insulin-dependent diabetes mellitus in Asians we were unable to find any genetic markers for this disease using the available cloned gene probes.

Diabetes Mellitus, Type 2↗
PubMed1989

Coronary artery disease, HDL cholesterol, and insulin-gene flanking sequences.

The relationship between coronary artery disease, HDL cholesterol, and the hypervariable region flanking the human insulin gene was studied in Saudi Arabian non-diabetic subjects (n = 68) and in patients with Type 2 diabetes (n = 35). A locus of insulin-gene-linked DNA polymorphism with three average size classes of alleles was found: a small class 1, a rare medium size class 2, and a large class 3 allele. In the total group of subjects (n = 103), those with the class 3 allele had a lower plasma HDL cholesterol concentration than those without the class 3 allele (0.93 +/- 0.26 vs 1.12 +/- 0.30 mmol l-1, 2p less than 0.003). No difference in genotype and allelic frequency was found between patients with and without coronary artery disease in the combined group of subjects, with and without diabetes. Similarly the genotype distribution was not different between non-diabetic subjects and patients with Type 2 diabetes in the combined group of subjects, both with and without coronary artery disease. In conclusion, the study did not confirm a previous study showing an association between the class 3 allele and atherosclerosis in a Caucasian population. However, the class 3 allele was associated with a low plasma HDL cholesterol concentration.

Alleles↗
PubMed1996

Genetic diversity in the Iberian Peninsula determined from mitochondrial sequence analysis.

We have analysed 302 bp of the first hypervariable region of the mitochondrial D-loop in 271 individuals from different regions of the Iberian Peninsula and 85 individuals from Algeria. The Basque population is significantly different from neighbouring populations in terms of overall levels of diversity. This is because the majority of sequences in the Basques are restricted to the lineage group defined by the CRS (Cambridge Reference Sequence) and its derivatives although, like other Iberian populations, they showed a unimodal distribution of pairwise sequence differences. The timing of divergence of populations within Iberia points to a shared ancestry of all populations in the Upper Palaeolithic. Further genetic subdivision is apparent in Catalonia and Andalusia, with increased genetic diversity in the latter. Lineage diversity comparisons of Iberian populations with European (Tuscan) and North African (Algerian) populations shows the Iberian Peninsula to be more similar to other European populations, although a small number of Iberian lineages can be traced to North Africa.

Algeria↗
PubMed2005

The genetics of the pre-Roman Iberian Peninsula: a mtDNA study of ancient Iberians.

The Iberians developed a surprisingly sophisticated culture in the Mediterranean coast of the Iberian Peninsula from the 6th century BC until their conquest by the Romans in the 2nd century BC. They spoke and wrote a non-Indo-European language that still cannot be understood; their origins and relationships with other non-Indo-European peoples, like the Etruscans, are unclear, since their funerary practices were based on the cremation of bodies, and therefore anthropology has been unable to approach the study of this people. We have retrieved mitochondrial DNA (mtDNA) from a few of the scarce skeletal remains that have been preserved, some of them belonging to ritualistically executed individuals. The most stringent authentication criteria proposed for ancient DNA, such as independent replication, amino-acid analysis, quantitation of template molecules, multiple extractions and cloning of PCR products, have been followed to obtain reliable sequences from the mtDNA hypervariable region 1 (HVR1), as well as some haplogroup diagnostic SNPs. Phylogeographic analyses show that the haplogroup composition of the ancient Iberians was very similar to that found in modern Iberian Peninsula populations, suggesting a long-term genetic continuity since pre-Roman times. Nonetheless, there is less genetic diversity in the ancient Iberians than is found among modern populations, a fact that could reflect the small population size at the origin of the population sampled, and the heterogenic tribal structure of the Iberian society. Moreover, the Iberians were not especially closely related to the Etruscans, which points to considerable genetic heterogeneity in Pre-Roman Western Europe.

Adult↗
PubMedDate not supplied

Identification of Trypanosoma cruzi zymodemes by kinetoplast DNA probes.

Analysis of zymograms of extracts of Trypanosoma cruzi isolated from different hosts in Argentina allowed characterization of 12 zymodemes or "isozymic strains," only six of which were found in human patients. Two of these six zymodemes (Z1 and Z12) were widely distributed and found in more than 80% of human patients. These two "major natural clones" differed significantly in pathogenic activity. Because the groupings obtained by studying enzymes and kinetoplast DNA (kDNA) were similar, it is possible to identify the zymodeme by analyzing kDNA. A 290-bp fragment was amplified by PCR using primers for the sequences flanking the hypervariable regions of kDNA minicircles. Labeled probes for this fragment, prepared from Z1 and Z12 reference stocks, hybridized specifically with PCR-amplified kDNA from parasite stocks, allowing identification of zymodemes.

Adolescent↗
PubMed2002

Genetic complexity and serum reactivity of HVR1 quasispecies of hepatitis C virus in patients with cirrhosis.

OBJECTIVE: The RNA genome of hepatitis C virus varies considerably, especially within the hypervariable region 1 (HVR1), a domain located on the 5' end of the E2/NS1 envelope region. Our previous study has suggested there were greater numbers of quasispecies in the liver than in matched serum, independent of the viral load. However, the significance of this finding has not been examined extensively at genetic and serological levels. METHODS: By large scale cloning and sequencing, we studied the genetic complexity of HVR1 quasispecies in two selected patients with cirrhosis. The serum reactivity of peptides representing different HVR1 quasispecies isolated from these cases was also estimated by standard ELISA format. RESULTS: We found the same major (dominant and/or subdominant) viral quasispecies variants in serum and in the cirrhotic liver. Genetic analysis suggested that the evolutionary pressure on HVR1 was higher than on its flanking region in quasispecies derived from the liver, whereas this trend is attenuated in quasispecies from serum. The immunoreactivity to peptides representing different HVR1 quasispecies variants showed considerable cross-reactivity with heterologous sera, whereas the reactivity was strongest against the dominant HVR1 peptide over time in homologous sera. CONCLUSIONS: These findings indicate that the formation and selection of HVR1 quasispecies may not be driven solely by humoral immune pressure, at least in these two cases.

Amino Acid Sequence↗
PubMed1995

Development of a PCR-based technique for detection of Helicobacter pylori.

A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori, using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.

Adult↗
PubMed2000

Detection of sequence variation in PCR-amplified fragments of omp2 gene from three species of the family Chlamydiaceae using agarose gel electrophoresis containing bisbenzimide-PEG.

A simple technique providing a means for rapid genetic differentiation of chlamydial strains is described. The technique is based on a single-step sequence-specific separation of PCR-amplified DNA fragments by electrophoresis in an agarose gel containing a DNA ligand - bisbenzimide-PEG. A hypervariable region at the 5' end of the omp2 gene of Chlamydiaceae species encoding the 60-kDa cysteine-rich outer membrane protein was selected as a target for PCR. The appropriate fragments were amplified from strains of Chlamydia trachomatis, Chlamydophila pneumoniae, and Chlamydophila psittaci, and the PCR products originating from different species were electrophoretically separated in the presence of the DNA ligand. We therefore demonstrated that PCR with a single pair of primers followed by simple agarose gel electrophoresis with bisbenzimide-PEG can be applied to the differentiation of three members of the family Chlamydiaceae which are commonly recognized as human pathogens.

Animals↗
PubMed2002

Identification of enteric pathogens by heat shock protein 60 kDa (HSP60) gene sequences.

A highly specific and reproducible approach for the simultaneous detection of enteric pathogenic bacteria was developed using bacterial hsp60 gene and molecular biological tools. A single pair of universal primers was derived from the highly conserved sequence of hsp60 genes encompassing a 600-bp hypervariable region. PCR amplification followed by either dot blot hybridization or restriction enzyme digestion performed on 38 enteric bacteria indicated that this approach could differentiate not only different genera such as Campylobacter, Yersinia and Vibrio, but also species that are closely related genetically, such as between C. jejuni and C. coli, or between Salmonella and Shigella or Escherichia coli.

Amino Acid Sequence↗
PubMed2005

Natural history of hepatitis C virus-related liver fibrosis after renal transplantation.

The aim of our study was to assess hepatitis C virus (HCV) evolution and long-term liver histology outcome in anti-HCV(+)/RNA(+) renal transplant (RT) patients. A total of 51 anti-HCV(+)/RNA(+) RT patients underwent liver biopsies (LB) every 3-4 years after transplantation (two LBs, n = 51; three LBs, n = 42; four LBs, n = 9). The hypervariable region (HVR)-1 of the HCV genome from all patients was characterized over time. Overall, the rate of liver fibrosis progression was 0.09 +/- 0.03 Metavir units/year. We identified three groups of patients: those in whom liver fibrosis remained stable (n = 21), those with progressing liver fibrosis (n = 21) and those with a regression in liver fibrosis (n = 10). In the last two groups, the progression and the regression of liver fibrosis were gradual during follow-up. Ferritin levels and hepatosiderosis were significantly higher in fibrosers. Initial fibrosis stage and high diversification of the HVR-1 of HCV genome between transplantation and the first liver biopsy were independent factors associated with liver fibrosis regression. In conclusion, in the current study, more than 10 years after renal transplantation, HCV infection was not harmful upon liver histology in more than 50% of patients. The diversification of the HVR-1 might be used to predict liver fibrosis outcome.

Adult↗
PubMed2005

HCV infection of the transplanted liver: changing CD81 and HVR1 variants immediately after liver transplantation.

The second envelope protein at hypervariable region 1 (HVR1) has been implicated in contributing to hepatitis C virus (HCV)-host cell interactions and CD81 (a multifunctional protein) has been demonstrated to act as a cell surface receptor for HCV and may interact directly with HVR1. The purpose of the current study was to determine if certain HVR1 quasispecies variants more effectively associate with and infect allografts after liver transplantation than other HVR1 variants and whether CD81 receptor expression changes after transplantation. Blood and allograft samples were obtained from the peritransplant period in seven patients. Clones of RT-PCR product were directly sequenced to identify HVR1 quasispecies variants. Explanted liver and serial allograft biopsies in recipients with HCV were examined by immunohistochemistry (IHC) for CD81 expression. Examination of HVR1 sequences demonstrated that only a fraction of the quasispecies variants recovered from each patient's blood sampled immediately prior to transplantation associated with and infected the allografts. Genetic diversity at HVR1 decreased with reperfusion but did not significantly decrease with infection. Expression of CD81 varied during the immediate post-transplant period. In conclusion, HVR1 quasispecies variants differentially associate with, and infect allografts, after liver transplantation. Additionally, allografts express variable amounts of CD81 after transplantation.

Amino Acid Sequence↗
PubMed2005

Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion.

The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 degrees C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, approximately 25% of the allergen remained intact, approximately 50% corresponded to a large fragment of M(r) 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of approximately 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B- and T-cell epitopes.

2S Albumins, Plant↗