Protein-DNA associations in a gender-specific gene of Schistosoma mansoni: characterization by UV cross-linking, DNase I footprinting and band shift assays

Protein extracts obtained from male and female shistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3'end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generatingrelatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of proteins occured at the 5'end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide the F-10 HRE, evidence proteins having MWS of 30,45 and 65 kDNA. These proteins are presumably involved in the regulation of transcription of the F-10 gene.

Cross-sectional and evolutive studies of schistosomiasis mansoni in untreated and mass treated endemic areas in the southeast and northeast of Brazil

Cross-sectional and evolutive studies on schistosomiasis mansoni were carried out before and after mass treatment in the endemic areas of Capitao Andrade and Padre Paraíso, state of Minas Gerais, Riachuelo, state of Sergipe, Alhandra, state of Paraíba, and Aliança, Alegre and Coroatá, lowland of the state of Maranhao, Brazil, in the last eighteen years. The studies included clinical and fecal examination by the Kato-Katz quantitative technique, skin testfor Schistosoma mansoni infection, evaluation of man-water contact and other epidemiological investigations such as infection rate and dynamic of the snail population. Results showed: (1) Higher prevalence of S. mansoni infection, greater egg load elimination and higher and earlier morbidity of the chronic froms of the disease in the southeast areas of Capitao Andrade and Padre Paraíso; (2) The incidence of hepatosplenic form is higher in some family clusters, in whites and mullattos in all the endemic areas but develop earlier in the southeast; (3) The prevalence and morbidity of schistosomiasis are decreasing both in the mass treated northeast and in the untreated southeast areas; (4) The mass treatment reduces rapidily the prevalence of the infection and the morbidity of the disease but can not control it because of the frequent reinfections due to the intensity of man-water contact.

Human schistosomiasis mansoni: studies on in vitro granuloma modulation

Infection with Schistosoma mansoni induces humoral and T cell mediated responses and leads to delayed hipersensitivity that results in granulomatous inflamatory disease around the parasite eggs. Regulation of these responses resulting in a reduction in this anti-egg inflamatory disease is appsrently determined by idiotypic repertoires of the patient, associated with genetic background and multiple external factors. We have previously reported on idiotype/anti-idiotype-receptor transactions in clinical human schistosomiasis. These findings support a hypothesis that anti-SEA cross-reactive idiotypes develop in some patients during the course of a chronic infection and participate in regulation of anti-SEA cellular immune responses. We repport here on experiments wich extend those observations to the regulation of granulomatous hypersensitivity measured by an in vitro granuloma model. T cells from chronic intestinal schistosomiasis patients were stimulated in vitro with anti-SEA idiotypes and assayed in an autologous in vitro granuloma assay for modulation of granuloma formation. These anti-SEA idiotype reactive T cells were capable of regulating autologous in vitro granuloma formation. This regulatory activity, initiated with stimulatory anti-SEA idiotypic antibodies, was antigenically specific and was dependent on the present of intact (F(ab')2 immunoglobulin molecules. The ability to elicit this regulatory activity appears to be dose dependent and is more easily demonstrated in chronically infected intestinal patients or SEA sensitized individuals. These data support the hypothesis that anti-SEA cross reactive idiotypes are important in regulating granulomatous hypersensitivy in chronic intestinal schistosomiasis patients and these cross-reactive idiotypes appear to play a major role in cell-cell interactions which result in the regulation of anti-SEA cellular immune responses.

Onchocerciasis in Ecuador: Prevalence of Infection on the Ecuador-Colombia Border in the Province of Esmeraldas

The prevalence of onchocerciasis infection was determined in communities on 7 rivers located in the northern area of the cantón San Lorenzo, province of Esmeraldas. Diagnosis of the infection was obtained by skin biopsies and recombinant-antigen based-serology. No evidence of infection was detected in 9 communities studied along the Río Mataje, which forms the frontier between Ecuador and Colombia, nor in 10 adjacent communities located on 5 interior rivers. Evidence for Onchocerca volvulus infection was found in 4 communities on the Río Tululví with the following prevalence: La Boca (3.5% by biopsy and 3.9% by serology), Guayabal (9.1% by both biopsy and serology), La Ceiva (51.5% by biopsy and 53% by serology), and Salidero (4% by biopsy and 7.7% by serology). A few individuals in these communities were seropositive for O. volvulus in the absence of detectable dermal microfilariae: these might harbor very light or prepatent infections. No clinical disease attributable to onchocerciasis was found. The infected communities will be included in the ivermectin-based National Control Program for the disease, with no evidence of the infection having extended north of the Ecuadorian-colombian border

The Influence of Self-fertilization performance and Copulation Behaviour in Reproduction by Cross-fertilization in Groups of Biomphalaria tenagophila (Mollusca, Planorbidae)

The following hypotheses were tested for groups of simultaneous hermaphrodites Biomphalaria tenagophila: (a) snails that have low reproductive success during the process of self-fertilization do not increase their reproductive success after the end of grouping; (b) the copulation behaviour and the presence of one snail whose eggs have a low viability rate influence the partner's reproductive success by cross-fertilization. Groups were constituted by a homozygous pigmented snail and two albinos: one with a viability rate higher than 70% ("good reproducers") and the other less than 10% ("bad reproducers"). All pigmented snails had viability rates higher than 70%. The "good" and "bad" reproducer albino snails had similar copulation behaviour. However, after the end of grouping, the "bad reproducers" continued to have viability rates less than 10% over 30 days. In 100% of the cases that pigmented snails copulated (performing either a male role or simultaneously male and female roles) exclusively with "good" reproducer albinos, they presented high reproductive success (producing, on average of 8.4 pigmented embryos/egg-mass). However, in 100% of the cases that pigmented snails copulated with both partners, the "good" reproducer albino snails produced none or very few embryos (the highest average was 2.2 pigmented embryos/egg-mass). Therefore, the production of viable embryos by cross-fertilization was more influenced by self-fertilization performance than by copulation behaviour. The presence of a snail whose eggs have a low viability rate could decrease their partners reproductive success

A dhfr-ts- Leishmania major Knockout Mutant Cross-protects against Leishmania amazonensis

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65% smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75% in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57% by SC and 49% by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization.

Epidemiology of hookworm infection in Itagua, Paraguay: a cross sectional study

A cross-sectional study in Itagua, Paraguay tested 192 people for the presence, intensity and species of hookworm infection. Fifty-nine percent of these individuals were found to be infected. Intensity of infection was determined on 92% of infected individuals by quantitative egg counts. The high intensity hookworm infections, which cause the greatest morbidity, were clustered between the ages of five and 14 years. No differences were seen between genders. The species of hookworm was determined for parasites reared from 72% of infected individuals. Both Necator americanus and Ancylostoma duodenale were identified, although the former species predominated. We conclude that hookworm infection continues to be a public health problem in Paraguay, particularly among children and adolescents who suffer from high intensity infections. A. duodenale continues to persist in the Western Hemisphere and has not been completely displaced by N. americanus.

Mexican Trypanosoma cruzi stocks: analysis of minicircle kDNA homologies by cross-hybridization

Homologies of minicircle kDNA of 27 Mexican stocks were studied by cross-hybridization with four kDNA probes derived from three reference stocks belonging to groups Trypanosoma cruzi I (SO34 cl4 and Silvio) and T. cruzi II (MN) and one Mexican stock. High homologies were only observed with Silvio (six stocks) and Mexican probes (11 stocks). After 30 min exposure (low homology) additional stocks were recognized with SO34 cl4 (three stocks) and Silvio (six stocks) probes; with the Mexican probe only five stocks remained non-reactive. All the stocks were typed by isoenzyme (16 loci) and Mexican stocks belonged to T. cruzi I. Hybridization patterns were not strictly correlated with the observed clustering and cross-hybridization of kDNA minicircles is not available to distinct Mexican stocks.

Cross-sectional study defines difference in malaria morbidity in two Yanomami communities on Amazonian boundary between Brazil and Venezuela

It is well established that immunity to malaria is short-lived and is maintained by the continuous contact with the parasite. We now show that the stable transmission of malaria in Yanomami Amerindian communities maintains a degree of immunity in the exposed population capable to reduce prevalence and morbidity of malaria. We examined 508 Yanomami Amerindians living along Orinoco (407) and Mucajaí (101) rivers, on the Venezuelan and Brazilian Amazon region, respectively. At Orinoco villages, malaria was hyperendemic and presented stable transmission, while at Mucajaí villages it was mesoendemic and showed unstable transmission. The frequency of Plasmodium vivax and P. falciparum was roughly comparable in Venezuelan and Brazilian communities. Malaria presented different profiles at Orinoco and Mucajaí villages. In the former communities, malaria showed a lower prevalence (16% x 40.6%), particularly among those over 10 years old (5.2% x 34.8%), a higher frequency of asymptomatic cases (38.5% x 4.9%), and a lower frequency of cases of severe malaria (9.2% x 36.5%). Orinoco villagers also showed a higher reactivity of the immune system, measured by the frequency of splenomegaly (72.4% x 29.7%) and by the splenic index (71.4% over level 1 x 28.6), and higher prevalence (91.1% x 72.1%) and mean titer (1243 x 62) of antiplasmodial IgG antibodies, as well as a higher prevalence (77.4% x 24.7%) and mean titer (120 x 35) of antiplasmodial IgM antibodies. Our findings show that in isolated Yanomami communities the stability of malaria transmission, and the consequent continuous activation of the immune system of the exposed population, leads to the reduction of malaria prevalence and morbidity.

Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis

We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.

Polymerase chain reaction genotyping of Epstein-Barr virus in scraping samples of the tongue lateral border in HIV-1 seropositive patients

The Epstein-Barr virus (EBV) is the etiological agent of oral hairy leukoplakia (OHL), an oral lesion with important diagnostic and prognostic value in acquired immunodeficiency disease syndrome. The two EBV genotypes, EBV-1 and EBV-2, can be distinguished by divergent gene sequences encoding the EBNA-2, 3A, 3B, and 3C proteins. The purpose of this study was to identify the EBV genotype prevalent in 53 samples of scrapings from the lateral border of the tongue of HIV-1 seropositive patients, with and without OHL, and to correlate the genotypes with presence of clinical or subclinical OHL with the clinic data collected. EBV-1 and EBV-2 were identified through PCR and Nested-PCR based on sequence differences of the EBNA-2 gene. EBV-1 was identified in the 31 samples (15 without OHL, 7 with clinical OHL and 9 with subclinical OHL), EBV-2 in 12 samples (10 without OHL, 1 with clinical and 1 subclinical OHL), and a mixed infection in 10 samples (2 without OHL, 3 with clinical and 5 with subclinical OHL). The presence of EBV-1 was higher in women, but a significant statistical result relating one the EBV genotypes to the development of OHL was not found. We conclude that the oral epithelium in HIV-1 seropositive patients can be infected by EBV-1, EBV-2 or by a mixed viral population.

Mixed-function oxidases and esterases associated with cross-r esistance between DDT and lambda-cyhalothrin in Anopheles darlingi Root 1926 populations from Colombia

In order to establish the insecticide susceptibility status for Anopheles darlingi in Colombia, and as part of the National Network on Insecticide Resistance Surveillance, five populations of insects from three Colombian states were evaluated. Standardised WHO and CDC bottle bioassays, in addition to microplate biochemical assays, were conducted. Populations with mortality rates below 80% in the bioassays were considered resistant. All field populations were susceptible to deltamethrin, permethrin, malathion and fenitrothion. Resistance to lambda-cyhalothrin and DDT was detected in the Amé-Beté population using both bioassay methods with mortality rates of 65-75%. Enzyme levels related to insecticide resistance, including mixed function oxidases (MFO), non-specific esterases (NSE), glutathione S-transferases and modified acetylcholinesterase were evaluated in all populations and compared with a susceptible natural strain. Only mosquitoes from Amé-Beté presented significantly increased levels of both MFO and NSE, consistent with the low mortalities found in this population. The continued use of lambda-cyhalothrin for An. darlingi control in this locality has resulted in a natural resistance to this insecticide. In addition, DDT resistance is still present in this population, although this insecticide has not been used in Colombia since 1992. Increased metabolism through MFO and NSE may be involved in cross-resistance between lambda-cyhalothrin and DDT, although kdr-type nerve insensitivity cannot be discarded as a possible hypothesis. Additional research, including development of a kdr specific assay for An. darlingi should be conducted in future studies. Our data demonstrates the urgent need to develop local insecticide resistance management and surveillance programs throughout Colombia.

Visceral leishmaniasis in border areas: clustered distribution of phlebotomine sand flies in Clorinda, Argentina

Three years after the first report of Lutzomyia longipalpis in Clorinda, Argentina, a border city near Asunción, Paraguay, the city was surveyed again. Lu. longipalpis was found clustered in the same neighbourhoods in 2007 as in 2004, even though the scattered distribution of canine visceral leishmaniasis was more related to the traffic of dogs through the border.