Genomic characterization of Brazilian hepatitis C virus genotypes 1a and 1b

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5' NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1%). The sequence of another isolate presented a three-nucleotide deletion at codon 329

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3%) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24%) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

Differential in vitro pathogenicity of predatory fungi of the genus Monacrosporium for phytonematodes, free-living nematodes and parasitic nematodes of cattle

In vitro tests were carried out on the pathogenicity of nine isolates of the predatory fungi of the genus Monacrosporium (5 M. sinense isolates, 3 M. appendiculatum and 1 M. thaumasium isolate) for a phytonematode (second stage juveniles from Meloidogyne incognita, race 3), a free-living nematode (Panagrellus spp), and two gastrointestinal parasitic nematodes of cattle (infective larvae of Cooperia punctata and Haemonchus placei). A suspension containing 2,000 nematodes from each species was added to Petri dishes containing fungi and grown on 2% water-agar medium at 25oC in the dark for up to 7 days. The dishes were examined every other day for 7 days and predation-free nematodes were counted. The results showed that the free-living nematodes, Panagrellus spp, were the most susceptible (P<0.05), followed by the phytonematode M. incognita, while the controls were ³98.5% viable. However, a variable susceptibility of the nematodes to different fungi was observed. This indicates that the use of predatory fungi for the environmental control of nematodes will be limited by the multiplicity of nematodes in the environment and their differential susceptibility to fungal isolates of the same genus.

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ß-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ß-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ß-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ß-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ß-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line

The LISP-I human colorectal adenocarcinoma cell line was isolated from a hepatic metastasis at the Ludwig Institute, São Paulo, SP, Brazil. The objective of the present study was to isolate morphologically different subpopulations within the LISP-I cell line, and characterize some of their behavioral aspects such as adhesion to and migration towards extracellular matrix components, expression of intercellular adhesion molecules and tumorigenicity in vitro. Once isolated, the subpopulations were submitted to adhesion and migration assays on laminin and fibronectin (crucial proteins to invasion and metastasis), as well as to anchorage-independent growth. Two morphologically different subpopulations were isolated: LISP-A10 and LISP-E11. LISP-A10 presents a differentiated epithelial pattern, and LISP-E11 is fibroblastoid, suggesting a poorly differentiated pattern. LISP-A10 expressed the two intercellular adhesion molecules tested, carcinoembryonic antigen (CEA) and desmoglein, while LISP-E11 expressed only low amounts of CEA. On the other hand, adhesion to laminin and fibronectin as well as migration towards these extracellular matrix proteins were higher in LISP-E11, as expected from its poorly differentiated phenotype. Both subpopulations showed anchorage-independent growth on a semi-solid substrate. These results raise the possibility that the heterogeneity found in the LISP-I cell line, which might have contributed to its ability to metastasize, was due to at least two different subpopulations herein identified.

Microencapsulation and tissue engineering as an alternative treatment of diabetes

In the 70's, pancreatic islet transplantation arose as an attractive alternative to restore normoglycemia; however, the scarcity of donors and difficulties with allotransplants, even under immunosuppressive treatment, greatly hampered the use of this alternative. Several materials and devices have been developed to circumvent the problem of islet rejection by the recipient, but, so far, none has proved to be totally effective. A major barrier to transpose is the highly organized islet architecture and its physical and chemical setting in the pancreatic parenchyma. In order to tackle this problem, we assembled a multidisciplinary team that has been working towards setting up the Human Pancreatic Islets Unit at the Chemistry Institute of the University of São Paulo, to collect and process pancreas from human donors, upon consent, in order to produce purified, viable and functional islets to be used in transplants. Collaboration with the private enterprise has allowed access to the latest developed biomaterials for islet encapsulation and immunoisolation. Reasoning that the natural islet microenvironment should be mimicked for optimum viability and function, we set out to isolate extracellular matrix components from human pancreas, not only for analytical purposes, but also to be used as supplementary components of encapsulating materials. A protocol was designed to routinely culture different pancreatic tissues (islets, parenchyma and ducts) in the presence of several pancreatic extracellular matrix components and peptide growth factors to enrich the beta cell population in vitro before transplantation into patients. In addition to representing a therapeutic promise, this initiative is an example of productive partnership between the medical and scientific sectors of the university and private enterprises.

Production and characterization of a monoclonal antibody against an Ascaris suum allergenic component

Ascaris suum allergenic components (PIII) separated by gel filtration chromatography of an adult worm extract were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells using polyethylene glycol (MW 1450) as fusogen. The hybridomas were cultured in HAT-containing medium and cloned at limiting dilutions. Supernatants from the growing hybrids were screened by ELISA using plates coated with PIII or the A. suum crude extract. The monoclonal antibody obtained, named MAC-3 (mouse anti-A. suum allergenic component), is an IgG1 kappa mouse immunoglobulin that specifically recognizes a 29,000 molecular weight protein (called allergenic protein) with an affinity constant of 1.7 x 10(9) M-1. The A. suum components recognized by MAC-3 induce specific IgE antibody production in immunized BALB/c mice. Ascitic fluid induced in Swiss mice by injecting ip the hybridoma cells and incomplete Freund's adjuvant was purified by affinity chromatography using a protein A-Sepharose column. The purified monoclonal antibody was then coupled to activated Sepharose beads in order to isolate the A. suum allergenic component from the whole extract by affinity chromatography.

Factors associated with penicillin-nonsusceptible pneumococcal infections in Brazil

Resistance of Streptococcus pneumoniae is a worldwide, growing problem. Studies of factors associated with resistance to penicillin have not been conducted in Brazil. The objective of the present study was to evaluate factors associated with infection by S. pneumoniae not susceptible to penicillin. A prevalence study was conducted including all patients with a positive culture for S. pneumoniae in a hospital from July 1991 to December 1992 and the year 1994. Of 165 patients identified, 139 were considered to have clinically relevant infections and 88% of them had invasive infections. All infections were community acquired and consisted of pneumonia (44%) and of central nervous system (19%), pelvic or abdominal (12%), upper airway or ocular (12%), primary bloodstream (9%) and skin and soft tissue (5%) infections. Mortality was 25%. Susceptibility to penicillin was present in 77.6% of the isolates; 21.8% were relatively resistant, and one isolate was resistant (minimal inhibitory concentration = 4 µg/ml). Multivariate analysis showed that age below 4 years (odds ratio (OR): 3.53, 95% confidence interval (95%CI): 1.39-8.96) and renal failure (OR: 5.50, 95%CI: 1.07-28.36) were associated with lack of susceptibility to penicillin. Bacteremia occurred significantly less frequently in penicillin-nonsusceptible infections (OR: 0.34, 95%CI: 0.14-0.84), possibly suggesting that lack of penicillin susceptibility is associated with lower virulence in S. pneumoniae.

Production and characterization of monoclonal antibodies to a Brazilian bovine herpesvirus type 5

Antigens of a bovine herpesvirus type 5 (BHV-5), isolated from a cow with a neurological infection in Rio Grande do Sul State, Brazil, were used to immunize BALB/c mice to produce monoclonal antibodies (mAbs). Eleven hybridomas secreting mAbs directed at BHV-5 antigens were obtained after two fusions and screening of 356 hypoxanthine-aminopterin-thymidine-resistant clones. The mAbs reacted at dilutions up to 1:500 (hybridoma culture supernatant) and up to >1:10,000 (ascitic fluid) in an indirect fluorescent antibody assay (IFA) and in immunoperoxidase staining of BHV-5-infected cells. Four mAbs (1D12, 2E2, 2G10 and 4E4) showed virus-neutralizing activity against the parental BHV-5 isolate. Five mAbs (1F3, 2A6, 2F9, 2G10 and HB24L) reacted in Western immunoblotting with a protein of approximately 90 kDa. Three other mAbs (2E2, 3D6 and 4E4) reacted in IFA with antigens of a BHV-1 mutant glycoprotein C- negative strain, demonstrating that they are directed at a viral antigen other than glycoprotein C. The eleven mAbs tested reacted with 20 BHV-5 field isolates and nine mAbs reacted with 10 BHV-1 isolates. Two mAbs (1F3 and 2F9) failed to react with BHV-1 field isolates, although they displayed a weak and nonreproducible reaction with the BHV-1 reference strain Los Angeles. These mAbs may be very useful in distinguishing between BHV-1 and BHV-5 infections since most of the traditional reagents and techniques are unable to do so. One mAb (2F9) was shown to bind to viral antigens by immunohistochemistry of histological sections of the brain of a BHV-5-infected calf. These results demonstrate that the mAbs produced here are suitable for use in a variety of immunological techniques and therefore may be useful for diagnostic and research purposes.

BCG lymphadenopathy detected in a BCG-vaccinated infant

Large-scale vaccination with BCG, the live attenuated strain of Mycobacterium bovis, is being adopted around the world, although sporadic complications have occurred after the procedure. Lymphadenopathy is not uncommon especially in babies under one year (0.73% of vaccinated infants), but the swelling subsides within 2 months in most cases, with no medical or surgical treatment. Brazil adopted BCG vaccination program earlier in the seventies and by 1995 more than 96% of the infant population received this immunization. We report here the occurrence of lymphadenopathy in a two-year-old child vaccinated with the Brazilian BCG strain. The diagnosis was made using a lymph node biopsy and intestinal aspirates that yielded a positive mycobacterial culture. The isolate was resistant to isoniazid, rifampicin, pyrazinamide and thiophen-2-carbonic acid hydrazide, sensitive to streptomycin, ethambutol, and p-nitrobenzoic acid, and reacted positively to cyclo-serine and negatively to niacin. The pncA gene involved in bacterial activation of pyrazinamide contains in M. bovis a point mutation that renders pyrazinamidase unable to catalyze drug activation. Therefore, this polymorphism is a good option for developing methods to differentiate M. bovis and M. tuberculosis. Taking advantage of this difference we further analyzed the isolates by single-stranded conformation polymorphism electrophoresis of DNA following PCR of the pncA gene. The isolate identity was confirmed by RFLP electrophoretic analysis of the amplified fragment following Eco065I digestion, which selectively cleaves M. tuberculosis DNA. From this result it is proposed that RFLP of pncA gene represents an alternative for differential diagnosis of M. bovis.

Evaluation by blue native polyacrylamide electrophoresis colorimetric staining of the effects of physical exercise on the activities of mitochondrial complexes in rat muscle

Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 ± 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 ± 0.65, P < 0.05) and soleus (9.8 ± 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.

Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil

In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 µg/ml. This isolate was sent to a reference laboratory (Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

L1 sequence of a new human papillomavirus type-58 variant associated with cervical intraepithelial neoplasia

The present study on molecular characterization of a human papillomavirus (HPV) isolated in Central Brazil describes the L1 gene sequence from a new variant of HPV-58, the isolate Bsb-02. The sample was from a smear obtained from a woman with cervical intraepithelial neoplasia grade II. The whole L1 gene from isolate Bsb-02 was sequenced automatically, showing 99.1% nucleotide identity with the gene from the HPV-58 reference. The clustering between Bsb-02 and HPV-58 reference sequence was also supported by phylogenetic analysis. Fourteen nucleotide substitutions were observed: eight were synonymous and six were associated with amino acid substitutions. A10V and V144I have not been previously described. At GenBank, the only complete L1 sequence from HPV-58 in addition to the HPV-58 reference one is that of Bsb-02. These data provide information that may be relevant to HPV diagnosis and to rational vaccine strategies. HPV variants may also be associated with host immune responses and with the risk of cervical neoplasia.

A multiplex PCR assay able to simultaneously detect Torque teno virus isolates from phylogenetic groups 1 to 5

Torque teno virus (TTV) is a circular, single-stranded DNA virus that chronically infects healthy individuals of all ages worldwide. TTV has an extreme genetic heterogeneity which is reflected in its current classification into five main phylogenetic groups (1-5). Using specific PCR assays, it has been shown that many individuals are co-infected with TTV isolates belonging to different phylogenetic groups. Here, a multiplex PCR assay was developed, using five recombinant plasmids. Each plasmid carried an insert of different size issued from a TTV isolate belonging to a different group. The assay was able to simultaneously amplify DNAs of TTV isolates belonging to all five phylogenetic groups. Multiplex PCR was then tested satisfactorily on DNAs extracted from 55 serum samples (47 health care workers and 8 AIDS patients). All individuals but nine were infected with at least one TTV isolate. Co-infection with multiple isolates was found in 29/47 (62%) health care workers and in 8/8 (100%) AIDS patients. A number of discrepancies were observed when results obtained with three thermostable DNA polymerases were compared. For example, four TTV phylogenetic groups were detected in a particular serum sample by using one of the three DNA polymerases, whereas the other two enzymes were able to detect only three TTV groups. However, none of the three enzymes used could be broadly considered to be more efficient than the others. Despite its limitations, the assay described here constitutes a suitable tool to visualize the degree of co-infection of a given population, avoiding time-consuming experiments.

HIV-1 binding and neutralizing antibodies of injecting drug users

Previous studies have demonstrated a stronger seroreactivity against some synthetic peptides responsible for inducing neutralizing antibodies in injecting drug users (IDU) compared to that of individuals sexually infected with HIV-1 (S), but the effectiveness in terms of the neutralizing ability of these antibodies has not been evaluated. Our objective was to study the humoral immune response of IDU by determining the specificity of their antibodies and the presence of neutralizing antibodies. The neutralization capacity against the HIV-1 isolate MN (genotype B), the primary HIV-1 isolate 95BRRJ021 (genotype F), and the seroreactivity with peptides known to induce neutralizing antibodies, from the V2 and V3 loops of different HIV-1 subtypes, were analyzed. Seroreactivity indicates that IDU plasma are more likely to recognize a broader range of peptides than S plasma, with significantly higher titers, especially of V3 peptides. Similar neutralization frequencies of the MN isolate were observed in plasma of the IDU (16/47) and S (20/60) groups in the 1:10 dilution. The neutralization of the 95BRRJ021 isolate was more frequently observed for plasma from the S group (15/23) than from the IDU group (15/47, P = 0.0108). No correlation between neutralization and seroreactivity with the peptides tested was observed. These results suggest that an important factor responsible for the extensive and broad humoral immune response observed in IDU is their infection route. There was very little difference in neutralizing antibody response between the IDU and S groups despite their differences in seroreactivity and health status.