Specific Identification of Biomphalaria tenagophila and Biomphalaria occidentalis Populations by the Low Stringency Polymerase Chain Reaction

Although Biomphalaria occidentalis and B. tenagophila are indistinguishable on the basis of shell morphology and the majority of their genital organs, only the latter is susceptible to infection with Schistosoma mansoni. Thus, the identification of these species is fundamental to epidemiological studies of schistosomiasis. Here we describe a simple and rapid method for differentiating B. tenagophila from B. occidentalis based on low stringency polymerase chain reaction and using a pair of primers specific for the amplification of the 18S rRNA gene. Analysis of the low stringency product profiles of populations of these snails from different geographical regions confirmed this approach as being applicable to the identification of B. tenagophila and B. occidentalis in cases where classical morphology is inconclusive

Use of a renal-specific oral supplement by haemodialysis patients with low protein intake does not increase the need for phosphate binders and may prevent a decline in nutritional status and quality of life.

BACKGROUND: Protein-energy wasting is a frequent and debilitating condition in maintenance dialysis. We randomly tested if an energy-dense, phosphate-restricted, renal-specific oral supplement could maintain adequate nutritional intake and prevent malnutrition in maintenance haemodialysis patients with insufficient intake. METHODS: Eighty-six patients were assigned to a standard care (CTRL) group or were prescribed two 125-ml packs of Renilon 7.5(R) daily for 3 months (SUPP). Dietary intake, serum (S) albumin, prealbumin, protein nitrogen appearance (nPNA), C-reactive protein, subjective global assessment (SGA) and quality of life (QOL) were recorded at baseline and after 3 months. RESULTS: While intention to treat analysis (ITT) did not reveal strong statistically significant changes in dietary intake between groups, per protocol (PP) analysis showed that the SUPP group increased protein (P < 0.01) and energy (P < 0.01) intakes. In contrast, protein and energy intakes further deteriorated in the CTRL group (PP). Although there was no difference in serum albumin and prealbumin changes between groups, in the total population serum albumin and prealbumin changes were positively associated with the increment in protein intake (r = 0.29, P = 0.01 and r = 0.27, P = 0.02, respectively). The SUPP group did not increase phosphate intake, phosphataemia remained unaffected, and the use of phosphate binders remained stable or decreased. The SUPP group exhibited improved SGA and QOL (P < 0.05). CONCLUSION: This study shows that providing maintenance haemodialysis patients with insufficient intake with a renal-specific oral supplement may prevent deterioration in nutritional indices and QOL without increasing the need for phosphate binders.

On Leishmania enriettii and Other Enigmatic Leishmania Species of the Neotropics

There are 20 named species of the genus Leishmania at present recognized in the New World, of which 14 are known to infect man. The present paper discusses the biological, biochemical and ecological features, where known, of six species which have not till now been found to cause human leishmaniasis; namely, Leishmania (Leishmania) enriettii, L. (L.) hertigi, L. (L.) deanei, L. (L.) aristidesi, L. (L.) forattinii and L. (Viannia) equatorensis. A protocol is suggested for attempts to discover the natural mammalian host(s) and sandfly vector of L. (L.) enriettii. Doubt is cast on the validity of the species L. herreri, described in Costa Rican sloths. Following the concensus of opinion that modern trypanosomatids derive from monogenetic intestinal flagellates of arthropods, phlebotomine sandflies are best regarded as the primary hosts of Leishmania species, with mammals acting as secondary hosts providing a source of parasites for these insects. There are probably natural barriers limiting the life-cycle of most leishmanial parasites to specific sandfly vectors

Cloning and Characterization of a Schistosoma mansoni cDNA Clone with a Specific Antigenic Expression during Development in a Vertebrate Host

Approximately 2.0 x 10 cDNA clones of an Schistosoma mansoni lgt11 cDNA library were screened in duplicate with serum from infected mice corresponding to distinct phases of infection. A cDNA clone (7/1) was isolated and recognized only by seven week serum. The clone was subcloned in pGEX-2T and Western-blot studies showed a specific antigenic expression confirming that only serum from the chronic phase is capable of recognizing this antigen. Dot-hybridization with RNA from different developmental phases of the parasite showed that the corresponding 7/1 RNA is expressed in all phases of parasite development in vertebrate hosts

Optimum in vitro expansion of human antigen-specific CD8 T cells for adoptive transfer therapy.

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

Two monoclonal rat antibodies with specificity for the beta-chain variable region V beta 6 of the murine T-cell receptor.

Two rat monoclonal antibodies (mAbs), 44-22-1 and 46-6B5, which recognize an alloreactive cytotoxic clone, 3F9, have been further tested on a panel of T hybridomas and cytotoxic T-cell clones for binding and functional activities. The mAbs recognized only those cells sharing the expression of the T-cell receptor beta-chain variable region gene V beta 6 with 3F9. All V beta 6+ cells were activated by these mAbs under cross-linking conditions and their antigen-specific activation was blocked by soluble mAb. Furthermore, depletion of 46-6B5+ normal lymph node T cells eliminated all cells expressing the epitope recognized by 44-22-1 and V beta 6 mRNA.

Inhibition of antigen-induced T-cell clone proliferation by antigen-specific antibodies.

Attempts to inhibit the recognition of soluble antigens by T lymphocytes using antibodies specific for the antigen in question have been uniformally unsuccessful, in contrast to the observed specific inhibition of antibody generation by B cells. One exception is the unique situation whereby anti-hapten antisera inhibit the T-cell proliferative responses observed when hapten-specific T lymphocytes or clones are cultured with hapten-derivatized cells or proteins. The inability to inhibit T-cell functions by antigen-specific antibodies has been interpreted in several ways: (1) T cells possess a different repertoire from B cells; (2) the antibodies tested recognize epitopes present on the native antigen, whereas T cells recognize non-native (processed) structures; (3) the antigenic determinant(s) recognized by T cells on the surface of antigen presenting cells are either not accessible to antibodies, or are present in low amounts. The development of antigen-specific T-cell clones and monoclonal antibodies both specific for the same antigenic determinants now allows this question to be investigated definitively. Here, we report for the first time the specific inhibition of antigen-induced T-cell clone proliferation by a monoclonal antibody directed against the relevant soluble protein antigen.

A Redescription of Riggia paranensis Szidat, 1948 (Isopoda, Cymothoidae) Based on Thirty-two Specimens from Curimatid Fish of Rio de Janeiro, Brazil, with an Emendation of the Genus

Riggia paranensis Szidat, 1948 is redescribed on the basis of 30 female and 2 male specimens collected from the pericardial cavities of the curimatid fish Cyphocarax (= Curimata) gilberti (Quoy & Gaimard). The fishes were caught in the Itabapoana River, State of Rio de Janeiro, southeastern Brazil. The presence of" dwarf" males, as reported by Szidat, was verified. The fusion of the pleonites and pleotelson in adult females was also confirmed. The generic diagnosis was emended to include details of the mouthparts and pleopods

Detection of Toxoplasma gondii-Specific Antibodies in Dogs. A Comparative Study of Immunoenzymatic, Immunofluorescent and Haemagglutination Titers

We evaluated the titers of anti-T. gondii antibodies by various serological tests in 40 serum samples from dogs exhibiting clinical signs of infectious diseases. Indirect immunofluorescence (IgG-IFI), indirect haemagglutination (IHA and M-Toxo) and immunoenzymatic (ELISA and PA-ELISA) tests were carried out. Titers ³ 64 were considered as positive. Anti-Toxoplasma antibodies were found in 9 (22.5%), 14 (35%), 14 (35%) and 12 (30%) samples, respectively for IHA, IgG-IFI, ELISA and PA-ELISA. The results showed that 57% were negative in all tests and 43% of the dogs presented antibodies to T. gondii; from these, 20% were positive in all three tests with high titers of antibodies and 23% were positive in only one or two tests with low titers of antibodies and mainly related to the IFI and ELISA tests. We observed 5 (12.5%) and 1 (2.5%) reactive samples, respectively, by M-Toxo and IHA with or without 2-mercapthoethanol, in the attempt to detect specific IgM. We can conclude that serodiagnosis of toxoplasmosis in dog have to be based on the combination of serological tests (IFI and ELISA) and with emphasis at the determination of the titers and the classes of the specific antibodies

Age-specific Prevalence of Antibodies to Hepatitis A in Children and Adolescents from Rio de Janeiro, Brazil, 1978 and 1995: Relationship of Prevalence to Environmental Factors

The age-specific prevalence of antibodies to hepatitis A virus (anti-HAV) was determined in two different population groups with low socio-economic status from Rio de Janeiro city, Brazil, whose serum samples were collected 17 years apart (Population 1, 1978; Population 2, 1995). In Population 2, analysis of the anti-HAV prevalence was also carried out with respect to environmental factors. Population 1 was composed of 520 stored sera collected from the umbilical cord of term neonates and children aged 1 month to 6 years. In population 2, 720 serum samples were collected from children and adolescents with ages ranging from 1 to 23 years. The overall prevalence rate of anti-HAV in Population 1 and Population 2 was 65.6% and 32.1%, respectively. In Population 1, the anti-HAV prevalence reached 88% at the age of 3, while in Population 2, it increased from 4.5% in children under the age of 3 to 66% in the group of adolescents over the age of 14. The low exposure to HAV infection in younger children from Population 2 could be a result of improved environmental hygiene and sanitation, as demonstrated by the presence of piped water, waste and sewage disposal systems in most houses from this population group. These findings indicate a possible change in the prevalence of hepatitis A in Rio de Janeiro

Enhancement of autophagic flux after neonatal cerebral hypoxia-ischemia and its region-specific relationship to apoptotic mechanisms.

The multiplicity of cell death mechanisms induced by neonatal hypoxia-ischemia makes neuroprotective treatment against neonatal asphyxia more difficult to achieve. Whereas the roles of apoptosis and necrosis in such conditions have been studied intensively, the implication of autophagic cell death has only recently been considered. Here, we used the most clinically relevant rodent model of perinatal asphyxia to investigate the involvement of autophagy in hypoxic-ischemic brain injury. Seven-day-old rats underwent permanent ligation of the right common carotid artery, followed by 2 hours of hypoxia. This condition not only increased autophagosomal abundance (increase in microtubule-associated protein 1 light chain 3-11 level and punctuate labeling) but also lysosomal activities (cathepsin D, acid phosphatase, and beta-N-acetylhexosaminidase) in cortical and hippocampal CA3-damaged neurons at 6 and 24 hours, demonstrating an increase in the autophagic flux. In the cortex, this enhanced autophagy may be related to apoptosis since some neurons presenting a high level of autophagy also expressed apoptotic features, including cleaved caspase-3. On the other hand, enhanced autophagy in CA3 was associated with a more purely autophagic cell death phenotype. In striking contrast to CA3 neurons, those in CA1 presented only a minimal increase in autophagy but strong apoptotic characteristics. These results suggest a role of enhanced autophagy in delayed neuronal death after severe hypoxia-ischemia that is differentially linked to apoptosis according to the cerebral region.

Exploration of immune competencies of viral-specific CD8+T cells in MS patients using tetramers and functional CTL assays

Introduction: Epstein-Barr Virus(EBV) has been repeatedly associatedwith multiple sclerosis (MS). Wehave previously shown that there is ahigh peripheral as well as intrathecalactivation of EBV-, but not cytomegalovirus(CMV)-specific CD8+ Tcells, early in the course of MS,strengthening the link between EBVand MS. However, the trigger of thisincreased EBV-specific CD8+ T cellresponse remains obscure. It could resultfrom a higher EBV viral load. Alternatively,it could be due to an intrinsicallydeficient EBV-specificCTL response, cytotoxic granulesmediated.Thus, we performed anin-depth study of the phenotype of exvivo EBV- and CMV-specific CD8+T cells in MS patients and control patients,assessing their cytotoxic activity.Methods:We analyzed the profileof cytotoxic granules in EBV- andCMV-specific CD8+ T cells in a cohortof 13 early MS patients, 20 lateMS, 30 other neurological diseases(OND) patients and 7 healthy controlsubjects. Ex vivo analysis of EBV- orCMV-specific CD8+ T cells was performedusing HLA class I/tetramercomplexes coupled to CCR7 andCD57 markers in conjunction withperforin, granzymes A, BandKstaining.In a sub-cohort of MS patientsand controls, cytotoxic activity ofEBV- and CMV-specific CD8+ Tcells was investigated using a functionalCFSE CTL assay. Results: UsingHLA Class I tetramers for EBVand CMV, we found that the frequencyof EBV- or CMV-specificCD8+ T cells were similar in all studysubjects. Most of EBV- and CMVspecificCD8+Tcells were highly differentiated(CCR7-) and a variousproportion expressed the exhaustionmarker CD57. MS and OND patientshad increased perforin expression inEBV-specific CD8+ T cells. Most importantly,we found that MS patientswith longer disease duration tended tohave lower CTL cytotoxicity as comparedto earlyMSpatients or controls.Conclusions: Effector EBV-specificCD8+ T cells are increased in earlyMS, however their cytotoxic granuleprofile does not seem to be fully alteredand the cytotoxic activity ofthese cells is normal. However, thecytotoxic activity of CTL decreasedin late MS patients suggesting an exhaustionof EBV-specific CD8+ Tcells possibly due to EBV reactivation.This work was supported by theSwiss National Foundation PP00B3-124893, the Swiss Society for MS,and the Biaggi Foundation to RADP.

Preliminary Evaluation of the Genetic Relatedness of Three Species of the Subgenus Dendromyia Theobald and Other Species of the Genus Wyeomyia Theobald (Diptera: Culicidae)

An eletrophoretic analysis of three species of the subgenus Dendromyia (Wyeomyia luteoventralis, Wy. ypsipola and Wy. testei) and three species belonging to different groups in the genus Wyeomyia (Wy. negrensis, Wy. mystes and Wy.confusa) was performed. Eight enzyme loci were analyzed. High values of genetic identity were detected among the species of the subgenus Dendromyia: Wy. luteoventralis, Wy. ypsipola and Wy. testei (mean value 0.63). On the other hand low values of genetic identity were observed among Wy. negrensis, Wy. mystes and Wy. confusa (mean value 0.23), suggesting that they belong, at least, to distinct subgenera within the Genus Wyeomyia. The UPGMA phenogram revealed the grouping of the Dendromyia species, while the others clustered at lower identity levels.

HIV Specific Humoral Immune Response in Rio de Janeiro, Brazil

Efforts to characterize HIV-1 polymorphism and anti-HIV immune response are being made in areas where anti-HIV/AIDS vaccines are to be employed. Anti-HIV-1 humoral immune response is being studied in infected individuals resident in Rio de Janeiro, in distinct cohorts involving recent seroconvertors, pregnant women or intravenous drug users (IDU). Comparative analyses of specificity of antibody response towards epitopes important for anti-HIV-1 immune response indicate quantitative differences between cohorts, with an exceptionally strong response in IDUs and weakest response in pregnant women. However, a comparative analysis between pregnant women cohorts from Rio de Janeiro and Rio Grande do Sul indicated an even lower response (with exception of the anti-V3-C clade peptide recognition) for the southern cohort. Studies analysing the immune function of the humoral response indicate a quite elevated occurrence of antibodies capable of neutralizing heterologous primary HIV-1 isolates from Rio de Janeiro. Attempts to correlate seroreactivity with HIV-1 neutralization with respect to HIV-1 polymorphism were not very successfull: while the Brazilian B clade B" variant could be recognized by binding assays, no significant distinction of HIV-1 clades/variants was observed in viral neutralization assays.