HCV infection by cell-to-cell transmission: choice or necessity?

In vitro models of HCV infection have allowed for the clarifying of molecules and mechanisms involved in the main steps of virus cell-entry. HCV entry and neutralization appear to be closely related. Neutralizing antibodies inhibit the E2-CD81 binding, therefore CD81 is considered to be a major target of immune response. The tight-junction proteins are also implicated in E2-binding to CD81 and successive steps of virus entry, in cooperation with several co-receptors, whose involvement has still to be elucidated. Increasing evidence has emphasized the importance of cell-to-cell HCV-transmission in chronic infection. This route for infection could favour virus-escape from host-neutralization though its CD81-dependency is still debated. The main reasons which have delayed our understanding of HCV-infection are here critically reviewed, as are the challenges faced by investigators in the field. A deeper insight into the different pathways involved could help to elucidate some crucial features of HCV infection mechanisms and disclose important implications in its pathogenesis, which could help in suggesting new targets for successful immune-prophylactic/therapeutic strategies.

Strategies for successful vaccination against hepatocellular carcinoma.

Current therapies against hepatocellular carcinoma (HCC) are not curative in the majority of patients. In the past, immunotherapy approaches aimed to non-specifically stimulate immune response were quite ineffective. New treatments based on stimulation of specific anti-tumor immune response are currently proposed and appear more promising. Tumor-specific antigens identified in HCC demonstrated immunogenicity both in preclinical and clinical trials. Effectiveness in animal studies raised interest in the clinical applicability of non-specific adoptive immunotherapy that prevented disease recurrence after tumor resection. Dendritic cell (DC)-based tumor vaccines achieved encouraging results, and cellular vaccines based on DCs have already entered clinical trials. Preventive and therapeutic DNA vaccination have been proposed, all based on tumor-associated antigens (TAAs), either modified or not, an example being alpha-fetoprotein (AFP). The concomitant expression of co-stimulatory molecules and cytokines was used to increase tumor immunogenicity. Syngeneic or nude mice models indicated that immunotherapy for HCC could stimulate an anti-tumor T-cell response leading to clinical benefit devoid of significant toxicity. The use of DNA-based vaccination raises exciting possibilities in preventing HCC in high-risk individuals such as those with cirrhosis. Novel immunotherapy strategies may contribute in the future to prevention and treatment of HCC.

Susceptibility trends of Bacteroides fragilis group isolates from Buenos Aires, Argentina.

The aim of this study was to analyze the susceptibility trends to seven antibiotics of Bacteroides fragilis group isolates based on three survey studies performed by the Committee of Anaerobic Bacteria between 1989 and 2002. Fifty three, 82 and 65 B. fragilis group isolates were collected during each period. The antimicrobial agents included were: ampicillin, ampicillin-sulbactam (2:1), cefoxitin, piperacillin, imipenem, clindamycin, and metronidazole. Minimal inhibitory concentrations (MICs) were determined according to the reference agar dilution method described by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS). The most active antibiotics for B. fragilis and non-B. fragilis species throughout the three periods were: imipenem with 99.1 and 100% of activity, respectively, and metronidazole with 100% of activity. The susceptibility to ampicillin-sulbactam showed a decrease, from 100% to 90.3% and to 82.4 % in the last period, for both B. fragilis and non-B. fragilis species, respectively. The overall susceptibility rates for cefoxitin, piperacillin, and clindamycin were significantly different between B. fragilis and non-B. fragilis species (84.2% vs. 56.5%; 85.9% vs. 66.7% and 88.8% vs. 64.7%, respectively, p < 0.05). Cefoxitin was the antibiotic that showed more variations as regards periods and species. The susceptibility rates for clindamycin were low, about 60%, for non-B. fragilis species during the last two periods. The variations observed in the susceptibility patterns of the B. fragilis group isolates emphasize the need to continue monitoring the emergence of resistance in order to guide the election of the most appropriate antibiotic therapy scheme for anaerobic infections.

[Deoxyribonuclease activity detection in Clostridium chauvoei strains]. / Detección de la actividad de desoxiribonucleasa (DNasa) en cepas de Clostridium chauvoei.

Beta toxin of C. chauvoei has desoxiribonuclease (DNase) activity which is regarded as one of its virulence factors. The production of DNase was detected in strains isolated from bovines, using as controls C. chauvoei ATCC 10092, and C. perfringens Type A and C. septicum, both laboratory isolates. The enzyme activity was made evident on a DNA substrate observing the macroscopic degradation. A simple methodology was developed using a commercial medium for DNase test, with the incorporation of sterile horse serum. Each strain was streaked on the surface of the medium, incubated in anaerobic atmosphere at 37 degrees C for 48 hours. The plates were revealed with HCI 1 N. The appearance of a clear and transparent zone around and under the microbial growing was considered a positive reaction. Enzyme activity was detected in 10 of 12 strains and also in the controls. The serum addition to the commercial basal medium allows the optimum development of the microorganism showing the enzymatic digestion zone.

Detección de la actividad de desoxiribonucleasa (DNasa) en cepas de Clostridium chauvoei / [Deoxyribonuclease activity detection in Clostridium chauvoei strains]

Beta toxin of C. chauvoei has desoxiribonuclease (DNase) activity which is regarded as one of its virulence factors. The production of DNase was detected in strains isolated from bovines, using as controls C. chauvoei ATCC 10092, and C. perfringens Type A and C. septicum, both laboratory isolates. The enzyme activity was made evident on a DNA substrate observing the macroscopic degradation. A simple methodology was developed using a commercial medium for DNase test, with the incorporation of sterile horse serum. Each strain was streaked on the surface of the medium, incubated in anaerobic atmosphere at 37 degrees C for 48 hours. The plates were revealed with HCI 1 N. The appearance of a clear and transparent zone around and under the microbial growing was considered a positive reaction. Enzyme activity was detected in 10 of 12 strains and also in the controls. The serum addition to the commercial basal medium allows the optimum development of the microorganism showing the enzymatic digestion zone.

Detección de la actividad de desoxiribonucleasa (DNasa) en cepas de Clostridium chauvoei. / [Deoxyribonuclease activity detection in Clostridium chauvoei strains]

Beta toxin of C. chauvoei has desoxiribonuclease (DNase) activity which is regarded as one of its virulence factors. The production of DNase was detected in strains isolated from bovines, using as controls C. chauvoei ATCC 10092, and C. perfringens Type A and C. septicum, both laboratory isolates. The enzyme activity was made evident on a DNA substrate observing the macroscopic degradation. A simple methodology was developed using a commercial medium for DNase test, with the incorporation of sterile horse serum. Each strain was streaked on the surface of the medium, incubated in anaerobic atmosphere at 37 degrees C for 48 hours. The plates were revealed with HCI 1 N. The appearance of a clear and transparent zone around and under the microbial growing was considered a positive reaction. Enzyme activity was detected in 10 of 12 strains and also in the controls. The serum addition to the commercial basal medium allows the optimum development of the microorganism showing the enzymatic digestion zone.

["In vitro" activity of ten antimicrobial agents against anaerobic bacteria. A collaborative study, 1999-2002]. / Actividad "in vitro" de 10 antimicrobianos frente a bacterias anaerobias. Estudio multicéntrico, 1999-2002.

The antimicrobial activity of ampicillin, ampicillin-sulbactam, cefoxitin, ceftriaxone, imipenem, piperacillin, piperacillin-tazobactam, clindamycin, metronidazole, and azitromycin was assesed against 166 strains of anaerobic bacteria recovered from eight hospitals in Buenos Aires. The strains studied were Bacteroides fragilis group (65), Fusobacterium spp. (26), Prevotella spp. (21), Porphyromonas spp. (10), Clostridium difficile (10), other clostridia (12), and gram-positive cocci (22). The MICs were determined by the agar dilution method according to NCCLS document M11-A5. Metronidazole and piperacillin-tazobactam were the most active antimicrobial agents tested and exhibited MIC90 values of < or = 2 microg/ml and < or = 4 microg/ml against gram-negative organisms, and < or = 2 microg/ml, and < or = 8 microg/ml against gram-positive organisms, respectively. Among beta-lactams the activity against gram-negative rods was in the following order: imipenem > piperacillin > cefoxitin > ceftriaxone > ampicillin. Among the gram-positive bacteria the decreased activity was: piperacillin > imipenem > cefoxitin > ceftriaxone > ampicillin. The majority of the species studied showed different degrees of resistance to clindamycin and azitromycin. Nevertheless, 90% of Fusobacterium nucleatum and Porphyromonas spp. isolates were inhibited by 0.125 mg/ml of clindamycin and azitromycin, respectively.

Actividad “in vitro” de 10 antimicrobianos frente a bacterias anaerobias: Estudio multicéntrico, 1999-2002 / “In vitro” activity of ten antimicrobial agents against anaerobic bacteria. A collaborative study, 1999-2002

Se evaluó la actividad de ampicilina, ampicilina-sulbactama, cefoxitina, ceftriaxona, imipenem, piperacilina, piperacilina-tazobactama, clindamicina, metronidazol y azitromicina frente a 166 cepas de bacterias anaerobias aisladas en 8 hospitales de Buenos Aires. Se estudiaron: Bacteroides grupo fragilis (65), Fusobacterium spp. (26), Prevotella spp. (21), Porphyromonas spp. (10), Clostridium difficile (10), otros clostridios (12) y cocos gram-positivos (22). Las CIMs se determinaron usando el método patrón de dilución en agar recomendado por el NCCLS, documento M11-A5. Los antibióticos más activos fueron metronidazol y piperacilina-tazobactama que exhibieron valores de CIM90£ 2 µg/ml y £ 4 µg/ml frente a los microorganismos gram-negativos y £ 2 µg/ml y £ 8 µg/ml frente a los microorganismos gram-positivos, respectivamente. Entre los b-lactámicos el orden de actividad frente a bacilos gram-negativos fue: imipenem > piperacilina > cefoxitina > ceftriaxona > ampicilina. En gram-positivos la actividad decreciente fue: piperacilina> imipenem > cefoxitina > ceftriaxona > ampicilina. La mayoría de las especies estudiadas mostraron distintos niveles de resistencia con clindamicina y azitromicina. Sin embargo, el 90% de las cepas de Fusobacterium nucleatum y Por-phyromonas spp. fue inhibido por una concentración de 0,125 µg/ml de clindamicina y azitromicina, respectivamente.

The antimicrobial activity of ampicillin, ampicillin-sulbactam, cefoxitin, ceftriaxone, imipenem, piperacillin, piperacillin-tazobactam, clindamycin, metronidazole, and azitromycin was assesed against 166 strains of anaerobic bacteria recovered from eight hospitals in Buenos Aires. The strains studied were Bacteroidesfragilis group (65), Fusobacterium spp. (26), Prevotella spp. (21), Porphyromonas spp. (10), Clostridium difficile (10), other clostridia (12), and gram-positive cocci (22). The MICs were determined by the agar dilution method according to NCCLS document M11-A5. Metronidazole and piperacillin-tazobactam were the most active antimicrobial agents tested and exhibited MIC90values of £ 2 µg/ml and £ 4 µg/ml against gram-negative organisms, and £ 2 µg/ml, and £ 8 µg/ml against gram-positive organisms, respectively. Among b-lactams the activity against gram-negative rods was in the following order: imipenem> piperacillin > cefoxitin > ceftriaxone > ampicillin. Among the gram-positive bacteria the decreased activity was: piperacillin> imipenem> cefoxitin > ceftriaxone > ampicillin. The majority of the species studied showed different degrees of resistance to clindamycin and azitromycin. Nevertheless, 90% of Fusobacterium nucleatum and Porphyromonas spp. isolates were inhibited by 0.125 mg/ml of clindamycin and azitromycin, respectively.

Actividad [quot ]in vitro[quot ] de 10 antimicrobianos frente a bacterias anaerobias. Estudio multicéntrico, 1999-2002 / [[quot ]In vitro[quot ] activity of ten antimicrobial agents against anaerobic bacteria. A collaborative study, 1999-2002]

The antimicrobial activity of ampicillin, ampicillin-sulbactam, cefoxitin, ceftriaxone, imipenem, piperacillin, piperacillin-tazobactam, clindamycin, metronidazole, and azitromycin was assesed against 166 strains of anaerobic bacteria recovered from eight hospitals in Buenos Aires. The strains studied were Bacteroides fragilis group (65), Fusobacterium spp. (26), Prevotella spp. (21), Porphyromonas spp. (10), Clostridium difficile (10), other clostridia (12), and gram-positive cocci (22). The MICs were determined by the agar dilution method according to NCCLS document M11-A5. Metronidazole and piperacillin-tazobactam were the most active antimicrobial agents tested and exhibited MIC90 values of piperacillin > cefoxitin > ceftriaxone > ampicillin. Among the gram-positive bacteria the decreased activity was: piperacillin > imipenem > cefoxitin > ceftriaxone > ampicillin. The majority of the species studied showed different degrees of resistance to clindamycin and azitromycin. Nevertheless, 90

of Fusobacterium nucleatum and Porphyromonas spp. isolates were inhibited by 0.125 mg/ml of clindamycin and azitromycin, respectively.

Actividad [quot ]in vitro[quot ] de 10 antimicrobianos frente a bacterias anaerobias. Estudio multicéntrico, 1999-2002. / [[quot ]In vitro[quot ] activity of ten antimicrobial agents against anaerobic bacteria. A collaborative study, 1999-2002]

The antimicrobial activity of ampicillin, ampicillin-sulbactam, cefoxitin, ceftriaxone, imipenem, piperacillin, piperacillin-tazobactam, clindamycin, metronidazole, and azitromycin was assesed against 166 strains of anaerobic bacteria recovered from eight hospitals in Buenos Aires. The strains studied were Bacteroides fragilis group (65), Fusobacterium spp. (26), Prevotella spp. (21), Porphyromonas spp. (10), Clostridium difficile (10), other clostridia (12), and gram-positive cocci (22). The MICs were determined by the agar dilution method according to NCCLS document M11-A5. Metronidazole and piperacillin-tazobactam were the most active antimicrobial agents tested and exhibited MIC90 values of < or = 2 microg/ml and < or = 4 microg/ml against gram-negative organisms, and < or = 2 microg/ml, and < or = 8 microg/ml against gram-positive organisms, respectively. Among beta-lactams the activity against gram-negative rods was in the following order: imipenem > piperacillin > cefoxitin > ceftriaxone > ampicillin. Among the gram-positive bacteria the decreased activity was: piperacillin > imipenem > cefoxitin > ceftriaxone > ampicillin. The majority of the species studied showed different degrees of resistance to clindamycin and azitromycin. Nevertheless, 90

of Fusobacterium nucleatum and Porphyromonas spp. isolates were inhibited by 0.125 mg/ml of clindamycin and azitromycin, respectively.

PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues.

The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.

Identification of six putative novel human papillomaviruses (HPV) and characterization of candidate HPV type 87.

Six putative novel human papillomavirus (HPV) types were detected by using general primers for a conserved L1 HPV region in patients examined in gynecologic centers. One of the isolates, detected in samples from 4 patients with koilocytic atypia at cervical cytology (3 of whom were also infected with human immunodeficiency virus type 1), was completely sequenced, identified as a new HPV genotype, and designated candidate HPV87 (candHPV87) by the Reference Center for Human Papillomavirus. candHPV87 shows the classic HPV genome organization and the absence of a functional E5 coding region. Phylogenetic analysis documented that the candHPV87 genome clusters within the A3 group of HPVs, together with HPV61, HPV72, HPV83, HPV84 and candHPV86, which have been completely sequenced, and a number of other putative novel genotypes (two of which are described in this work), which have been partially characterized. To address the growth-enhancing potential of candHPV87, the E6 and E7 putative coding regions were cloned and expressed in tissue cultures. The data indicate that both proteins stimulate cell division in tissue cultures more than those of low-risk HPVs, though not as much as those of HPV16. Taken together, the clinical, molecular, and biological data suggest that the novel papillomavirus characterized in the present study is a low- to intermediate-risk HPV.

[Leydig cell tumor of the testicle. Clinical case]. / I tumori a cellule di Leydig del testicolo. Caso clinico.

The case of a young man affected by Leydig cell tumor of the right testis, without gynecomastia and feminization signs is reported. The plasmatic level of testosterone, estrogenic hormones, APF and Beta-HCG were normal. The diagnostic and therapeutical aspects are discussed and the role of the radical orchifuniculectomy in T1N0M0 stage is pointed out.

Morphological modifications induced by HCV infection in the TOFE human lymphoblastoid cell line.

In this study, we analysed by transmission electron microscopy (TEM), sequential details of morphological modifications that accompanied viral morphogenesis in the lymphoblastoid cell line (LCL) TOFE infected in vitro with hepatitis C virus (HCV). As previously reported, we observed virus-like particles (VLPs) in cytoplasmic vesicles mainly located in the perinuclear region of infected cells. In this area, the Golgi apparatus and the endoplasmic reticulum (ER) appeared hyperplastic, remarkably enriched in vesicles and lysosomal structures. Furthermore, only in this perinuclear region, cytopathic-effect(CPE)-like changes seemed to originate, consisting in enlarged cytoplasmic vacuoles filled with degenerative amorphous material containing VLPs. Finally, the complete filling-up of the cytoplasm with these degenerative vacuoles, in addition to cellular lysis displayed by some cells, appeared as the possible terminal pattern of the infectious process. Our data suggest that in vitro HCV-infected TOFE cells undergo typical CPE-like changes that may be connected with virus replication.

Detection of virus-like particles in liver biopsies from HCV-infected patients.

In order to directly ascertain the presence of HCV virus infection in livers of patients with HCV chronic hepatitis, we investigated, by transmission electron microscopy (TEM), liver biopsies from 2 adults and 4 children for the presence of virus-like particles (VLPs). The plasmas of these HCV-positive patients were HCV-RNA-positive, with high ALT values. In liver tissue samples examined, we were able to detect plus and minus strands of HCV RNA by strand-specific RT-PCR. Aggregates or single VLPs of about 45 nm in diameter were detectable in variable amounts in endoplasmic cisternae and in hepatocyte cytoplasms of infected patients. These results emphasize the relevance of performing TEM assays to confirm the diagnosis of HCV infection.

TOFE human-B-cell-line-based adsorption-inhibition assay to detect HCV neutralizing antibodies.

We previously demonstrated that the human lymphoblastoid B-cell line (LCL) TOFE, derived from normal human bone marrow, is permissive to HCV infection. In this report we developed an in vitro HCV adsorption-inhibition assay based on TOFE cells, to reveal the presence of neutralizing antibodies in sera from acutely infected patients.

Susceptibility of human lymphoblastoid B-cell line CE to HIV1 infection.

In this preliminary report, we provide evidence that the human B-lymphoblastoid cell line (LCL) CE, bone-marrow-derived, previously reported to be permissive to hepatitis C virus, is also permissive to HIV1 infection. HIV1 genomes were detectable in cell supernatants, virus RNA transcripts and proviral DNAs in cell extracts at different times post-infection. Therefore, we propose this LCL cell line as a tool for exploring the mutual interactions of the two viruses in double-infected cells.

Molecular characterization and dynamics of hepatitis C virus replication in human fetal hepatocytes infected in vitro.

The molecular features of hepatitis C virus (HCV) replication in human fetal hepatocytes (HFHs) were addressed in this study. Using a competitive reverse-transcription polymerase chain reaction (RT-PCR) assay for the quantitation of HCV-RNA molecules, the highest level of viral replication was detected 30 days' postinfection. At this time point, viral particles of 41 to 45 nm in diameter accumulated in the cell cytoplasm. Their density in cell extracts and culture medium was distributed between heavy (1.180-1.360 g/cm3) and light fractions (1.105-1.050 g/cm3) of a sucrose gradient, while, in the serum inoculum, they had a positive fraction at 1.180 g/cm3. In infected HFHs, minus-strand HCV RNA was observed in fractions displaying a sedimentation coefficient of 28 S to 18 S, while plus-strand HCV RNA showed a peak restricted to the 21 S fraction; the HCV RNA of serum inoculum had a sedimentation coefficient of 38 to 40 S, which revealed the presence of HCV RNA of unique positive polarity. The 21 S RNA fraction of cell extracts was resistant to 20 minutes of RNase I digestion, while the same incubation time totally inactivated a comparable amount of HCV RNA purified from the serum inoculum, revealing the presence of completely and/or partially double-stranded HCV-RNA molecules in the infected cells. Detection in HFHs of replicative forms and replicative intermediates suggests that the dynamic profile of HCV replication in these cells is similar to that described in other flaviviruses.

Quantitation of hepatitis C virus RNA production in two human bone marrow-derived B-cell lines infected in vitro.

The ability of hepatitis C virus (HCV) to replicate in two B-cell lines, CE and TOFE, derived from bone marrow of healthy subjects was compared using qualitative and quantitative molecular methods. The presence of intracellular negative-stranded HCV RNA (replicative intermediate) was investigated by nested polymerase chain reaction (PCR) in the infected cultures at different times after infection. The amounts of positive-stranded HCV RNA (genomic RNA copies) synthesized and released from cells one week after in vitro infection were determined by competitive PCR after reverse transcription of viral RNA for the 5' viral untranslated region. In both cell lines, HCV RNA replication took place, but the TOFE cell line appeared to be a more efficient virus producer than the CE cell line. The TOFE cell line could be a valuable and reliable tool for basic and clinical HCV studies.