Nuclear rDNA-based molecular clock of the evolution of triatominae (Hemiptera: Reduviidae), vectors of Chagas disease

The evolutionary history and times of divergence of triatomine bug lineages are estimated from molecular clocks inferred from nucleotide sequences of the small subunit SSU (18S) and the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA of these reduviids. The 18S rDNA molecular clock rate in Triatominae, and Prosorrhynchan Hemiptera in general, appears to be of 1.8% per 100 million years (my). The ITS-2 molecular clock rate in Triatominae is estimated to be around 0.4-1% per 1 my, indicating that ITS-2 evolves 23-55 times faster than 18S rDNA. Inferred chronological data about the evolution of Triatominae fit well with current hypotheses on their evolutionary histories, but suggest reconsideration of the current taxonomy of North American species complexes.

Borrelia-like spirochetes recovered from ticks and small mammals collected in the Atlantic Forest Reserve, Cotia county, State of São Paulo, Brazil

Forty-four marsupials, 77 rodents and 161 ticks were captured in an Atlantic Forest Reserve in Cotia county, State of São Paulo, where human cases of Lyme disease (LD) simile were reported. Twenty-one borrelia-like spirochete isolates were recovered from the mammals' blood and rodent livers or spleens, and triturated ticks inoculated into BSK II medium. Our results suggest that the reservoirs and ticks collected may harbor borrelia-like spirochetes, some of which have an antigenic similarity with the unknown causative agent of LD simile in Brazil, and/or with North American Borrelia burgdorferi s.s.

Lymphatic vessel contractile activity and intestinal inflammation

Edema is a consistent observation in inflamatory bowel disease (IBD), and immune responses are inevitable in inflammation. Because the lymphatic system is an integral part of both tissue fluid homeostasis and immune reactions, it is likely that lymphatics play a role in the complex etiology of IBD. Despite the consistent findings that the lymphatic system is altered during gastrointestinal inflammation, the majority of studies conducted on the disease only mention the lymphatic system in passing. The effects of inflammatory mediators on lymphatic vessel function also remain poorly defined, despite its essential role in immunity and prevention of tissue edema. Processes allowing effective lymph transport are altered during inflammation, however, the mode of alteration and reason why lymphatics are ineffective in inflammatory reactions need to be further investigated. In addition, these processes have not yet been examined in an appropriate animal model and little has been done using in vivo methods of investigation in any model of gastrointestinal inflammation. This paper reviews the role of the lymphatic system in intestinal inflammation, as well as the role of the inflammatory products in mediating lymphatic contractile function.

Biogeography and evolution of Amazonian triatomines (Heteroptera: Reduviidae): implications for Chagas disease surveillance in humid forest ecoregions

An ecological-evolutionary classification of Amazonian triatomines is proposed based on a revision of their main contemporary biogeographical patterns. Truly Amazonian triatomines include the Rhodniini, the Cavernicolini, and perhaps Eratyrus and some Bolboderini. The tribe Rhodniini comprises two major lineages (pictipes and robustus). The former gave rise to trans-Andean (pallescens) and Amazonian (pictipes) species groups, while the latter diversified within Amazonia (robustus group) and radiated to neighbouring ecoregions (Orinoco, Cerrado-Caatinga-Chaco, and Atlantic Forest). Three widely distributed Panstrongylus species probably occupied Amazonia secondarily, while a few Triatoma species include Amazonian populations that occur only in the fringes of the region. T. maculata probably represents a vicariant subset isolated from its parental lineage in the Caatinga-Cerrado system when moist forests closed a dry trans-Amazonian corridor. These diverse Amazonian triatomines display different degrees of synanthropism, defining a behavioural gradient from household invasion by adult triatomines to the stable colonisation of artificial structures. Anthropogenic ecological disturbance (driven by deforestation) is probably crucial in the onset of the process, but the fact that only a small fraction of species effectively colonises artificial environments suggests a role for evolution at the end of the gradient. Domestic infestation foci are restricted to drier subregions within Amazonia; thus, populations adapted to extremely humid rainforest microclimates may have limited chances of successfully colonising the slightly drier artificial microenvironments. These observations suggest several research avenues, from the use of climate data to map risk areas to the assessment of the synanthropic potential of individual vector species.

Urban transmission of Chagas disease in Cochabamba, Bolivia

Chagas disease is a major public health problem in Bolivia. In the city of Cochabamba, 58% of the population lives in peripheral urban districts ("popular zones") where the infection prevalence is extremely high. From 1995 to 1999, we studied the demographics of Chagas infections in children from five to 13 years old (n = 2218) from the South zone (SZ) and North zone (NZ) districts, which differ in social, environmental, and agricultural conditions. Information gathered from these districts demonstrates qualitative and quantitative evidence for the active transmission of Trypanosoma cruzi in urban Cochabamba. Seropositivity was high in both zones (25% in SZ and 19% in NZ). We observed a high risk of infection in children from five to nine years old in SZ, but in NZ, a higher risk occurred in children aged 10-13, with odds ratio for infection three times higher in NZ than in SZ. This difference was not due to triatomine density, since more than 1,000 Triatoma infestans were captured in both zones, but was possibly secondary to the vector infection rate (79% in SZ and 37% in NZ). Electrocardiogram abnormalities were found to be prevalent in children and pre-adolescents (SZ = 40%, NZ = 17%), indicating that under continuous exposure to infection and re-infection, a severe form of the disease may develop early in life. This work demonstrates that T. cruzi infection should also be considered an urban health problem and is not restricted to the rural areas and small villages of Bolivia.

Small intestinal inflammation following oral infection with Toxoplasma gondii does not occur exclusively in C57BL/6 mice: review of 70 reports from the literature

Small intestinal immunopathology following oral infection with tissue cysts of Toxoplasma gondii has been described in C57BL/6 mice. Seven days after infection, mice develop severe small intestinal necrosis and succumb to infection. The immunopathology is mediated by local overproduction of Th1-type cytokines, a so-called "cytokine storm". The immunopathogenesis of this pathology resembles that of inflammatory bowel disease in humans, i.e., Crohn's disease. In this review, we show that the development of intestinal pathology following oral ingestion of T. gondii is not limited to C57BL/6 mice, but frequently occurs in nature. Using a Pubmed search, we identified 70 publications that report the development of gastrointestinal inflammation following infection with T. gondii in 63 animal species. Of these publications, 53 reports are on accidental ingestion of T. gondii in 49 different animal species and 17 reports are on experimental infections in 19 different animal species. Thus, oral infection with T. gondii appears to cause immunopathology in a large number of animal species in addition to mice. This manuscript reviews the common features of small intestinal immunopathology in the animal kingdom and speculates on consequences of this immunopathology for humankind.

She’s a femme fatale: low-density larval development produces good disease vectors

Two hypotheses for how conditions for larval mosquitoes affect vectorial capacity make opposite predictions about the relationship of adult size and frequency of infection with vector-borne pathogens. Competition among larvae produces small adult females. The competition-susceptibility hypothesis postulates that small females are more susceptible to infection and predicts frequency of infection should decrease with size. The competition-longevity hypothesis postulates that small females have lower longevity and lower probability of becoming competent to transmit the pathogen and thus predicts frequency of infection should increase with size. We tested these hypotheses for Aedes aegypti in Rio de Janeiro, Brazil, during a dengue outbreak. In the laboratory, longevity increases with size, then decreases at the largest sizes. For field-collected females, generalised linear mixed model comparisons showed that a model with a linear increase of frequency of dengue with size produced the best Akaike’s information criterion with a correction for small sample sizes (AICc). Consensus prediction of three competing models indicated that frequency of infection increases monotonically with female size, consistent with the competition-longevity hypothesis. Site frequency of infection was not significantly related to site mean size of females. Thus, our data indicate that uncrowded, low competition conditions for larvae produce the females that are most likely to be important vectors of dengue. More generally, ecological conditions, particularly crowding and intraspecific competition among larvae, are likely to affect vector-borne pathogen transmission in nature, in this case via effects on longevity of resulting adults. Heterogeneity among individual vectors in likelihood of infection is a generally important outcome of ecological conditions impacting vectors as larvae.

Assessing the mitochondrial DNA diversity of the Chagas disease vector Triatoma sordida (Hemiptera: Reduviidae)

Triatoma sordida is a species that transmits Trypanosoma cruzi to humans. In Brazil, T. sordida currently deserves special attention because of its wide distribution, tendency to invade domestic environments and vectorial competence. For the planning and execution of control protocols to be effective against Triatominae, they must consider its population structure. In this context, this study aimed to characterise the genetic variability of T. sordida populations collected in areas with persistent infestations from Minas Gerais, Brazil. Levels of genetic variation and population structure were determined in peridomestic T. sordida by sequencing a polymorphic region of the mitochondrial cytochrome b gene. Low nucleotide and haplotype diversity were observed for all 14 sampled areas; π values ranged from 0.002-0.006. Most obtained haplotypes occurred at low frequencies, and some were exclusive to only one of the studied populations. Interpopulation genetic diversity analysis revealed strong genetic structuring. Furthermore, the genetic variability of Brazilian populations is small compared to that of Argentinean and Bolivian specimens. The possible factors related to the reduced genetic variability and strong genetic structuring obtained for studied populations are discussed in this paper.

Breeding, genetic and genomic of citrus for disease resistance

Although the citriculture is one of the most important economic activities in Brazil, it is based on a small number of varieties. This fact has contributed for the vulnerability of the culture regarding the phytosanitary problems. A higher number of varieties/genotypes with potential for commercial growing, either for the industry or fresh market, has been one of the main objectives of citrus breeding programs. The genetic breeding of citrus has improved, in the last decades, due to the possibility of an association between biotechnological tools and classical methods of breeding. The use of molecular markers for early selection of zygotic seedlings from controlled crosses resulted in the possibility of selection of a high number of new combination and, as a consequence, the establishment of a great number of hybrids in field experiments. The faster new tools are incorporated in the program, the faster is possibility to reach new genotypes that can be tested as a new variety. Good traits should be kept or incorporate, whereas bad traits have to be excluded or minimized in the new genotype. Scion and rootstock can not be considered separately, and graft compatibility, fruit quality and productivity are essential traits to be evaluated in the last stages of the program. The mapping of QTLs has favored breeding programs of several perennial species and in citrus it was possible to map several characteristics with qualitative and quantitative inheritance. The existence of linkage maps and QTLs already mapped, the development of EST and BAC library and the sequencing of the Citrus complete genome altogether make very demanding and urgent the exploration of such data to launch a wider genetic study of citrus. The rising of information on genome of several organisms has opened new approaches looking for integration between breeding, genetic and genome. Genome assisted selection (GAS) involves more than gene or complete genome sequencing and is becoming an import support in breeding programs of annual and perennial species. An huge information amount can be derivate from genome analysis. The use and benefit of such informations will depend on the genetic basis of the breeding program.

Disaccharidase levels in normal epithelium of the small intestine of rats with iron-deficiency anemia

Iron-deficiency anemia is the nutritional deficiency most frequently occurring throughout the world, which manifests as a complex systemic disease involving all cells, affecting enzyme activities and modifying protein synthesis. In view of these considerations, the objective of the present study was to determine the effects of iron-deficiency anemia on disaccharidases and on the epithelial morphokinetics of the jejunal mucosa. Newly weaned male Wistar rats were divided into 4 groups of 10 animals each: C6w received a standard ration containing 36 mg elemental iron per kg ration for 6 weeks; E6w received an iron-poor ration (5-8 mg/kg ration) for 6 weeks; C10w received an iron-rich ration (36 mg/kg ration) for 10 weeks; E10w received an iron-poor ration for 6 weeks and then an iron-rich ration (36 mg/kg) for an additional 4 weeks. Jejunal fragments were used to measure disaccharidase content and to study cell proliferation. The following results were obtained: 1) a significant reduction (P<0.001) of animal weight, hemoglobin (Hb), serum iron and total iron-binding capacity (TIBC) in group E6w as compared to C6w; reversal of the alterations in Hb, serum iron and TIBC with iron repletion (E10w = C10w); animal weights continued to be significantly different in groups E10w and C10w. 2) Sucrase and maltase levels were unchanged; total and specific lactase levels were significantly lower in group E6w and this reduction was reversed by iron repletion (E10w = C10w). 3) The cell proliferation parameters did not differ between groups. On the basis of these results, we conclude that lactase production was influenced by iron deficiency and that this fact was not related to changes in cell population and proliferation in the intestinal mucosa

Morphological changes of the jejunal mucosa in protracted diarrhea and their correlation with disease duration, weight loss and serum albumin levels

The pathogenesis of protracted diarrhea is multifactorial. In developing countries, intestinal infectious processes seem to play an important role in triggering the syndrome. Thirty-four children aged 1 to 14 months, mean 6.5 months, with protracted diarrhea were studied clinically and in terms of small intestinal mucosal morphology. Mild, moderate or severe hypotrophy of the jejunal mucosa was detected in 82% of cases, and mucosal atrophy was observed in 12%. The intensity of the morphological changes of the jejunal mucosa correlated negatively with serum albumin levels. No correlation was detected between mucosal grading and duration of diarrhea or between mucosal grading and weight reported as percentile. After nutritional support was instituted, serial jejunal biopsies were obtained from 12 patients: five patients submitted to parenteral nutrition for 7 to 38 days, mean 17 days, and 7 patients receiving a hypoallergenic oral diet (semi-elemental formula, 3; chicken formula, 3; human milk, 1). In seven cases (58%) a progressive increase in villus height and a decrease in the number of inflammatory cells were noted. Recovery of the morphologic pattern was accompanied by clinical improvement in all patients

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

Antagonist G-mediated targeting and cytotoxicity of liposomal doxorubicin in NCI-H82 variant small cell lung cancer

The aim of the present study was to characterize the interactions of antagonist G (H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH 2)-targeted sterically stabilized liposomes with the human variant small cell lung cancer (SCLC) H82 cell line and to evaluate the antiproliferative activity of encapsulated doxorubicin against this cell line. Variant SCLC tumors are known to be more resistant to chemotherapy than classic SCLC tumors. The cellular association of antagonist G-targeted (radiolabeled) liposomes was 20-30-fold higher than that of non-targeted liposomes. Our data suggest that a maximum of 12,000 antagonist G-targeted liposomes were internalized/cell during 1-h incubation at 37ºC. Confocal microscopy experiments using pyranine-containing liposomes further confirmed that receptor-mediated endocytosis occurred, specifically in the case of targeted liposomes. In any of the previously mentioned experiments, the binding and endocytosis of non-targeted liposomes have revealed to be negligible. The improved cellular association of antagonist G-targeted liposomes, relative to non-targeted liposomes, resulted in an enhanced nuclear delivery (evaluated by fluorimetry) and cytotoxicity of encapsulated doxorubicin for incubation periods as short as 2 h. For an incubation of 2 h, we report IC50 values for targeted and non-targeted liposomes containing doxorubicin of 5.7 ± 3.7 and higher than 200 µM doxorubicin, respectively. Based on the present data, we may infer that receptors for antagonist G were present in H82 tumor cells and could mediate the internalization of antagonist G-targeted liposomes and the intracellular delivery of their content. Antagonist G covalently coupled to liposomal drugs may be promising for the treatment of this aggressive and highly heterogeneous disease.

Neuropsychological rehabilitation of memory deficits and activities of daily living in patients with Alzheimer's disease: a pilot study

Patients with Alzheimer's disease (AD) gradually lose their cognitive competence, particularly memory, and the ability to perform daily life tasks. Neuropsychological rehabilitation is used to improve cognitive functions by facilitating memory performance through the use of external aids and internal strategies. The effect of neuropsychological rehabilitation through memory training - motor movements, verbal association, and categorization - and activities of daily living (ADL) training was tested in a sample of 5 elderly out-patients (mean age: 77.4 ± 2.88 years), with mild AD (Mini-Mental State Examination score: 22.20 ± 2.17) and their caregivers. All patients had been taking rivastigmine (6-12 mg/day) for at least 3 months before being assigned to the rehabilitation sessions, and they continued to take the medication during the whole program. Just before and after the 14-week neuropsychological rehabilitation program all patients were assessed by interviewers that did not participate in the cognitive training, using the Mini-Mental State Examination, Montgomery-Alsberg Depression Rating Scale, Hamilton Anxiety Scale, Interview to Determine Deterioration in Functioning in Dementia, Functional Test, Memory Questionnaire of Daily Living for patient and caregiver, Quality of Life Questionnaire for patient and caregiver, and a neuropsychological battery. The results showed a statistically significant improvement in ADL measured by Functional Test (P = 0.04), and only a small improvement in memory and psychiatric symptoms. Our results support the view that weekly stimulation of memory and training of ADL is believed to be of great value in AD treatment, not only delaying the progress of the disease, but also improving some cognitive functions and ADL, even though AD is a progressively degenerative disease.

In vitro modulation of Bcl-2 levels in small cell lung cancer cells: effects on cell viability

Small cell lung cancer (SCLC) is an aggressive disease, representing 15% of all cases of lung cancer, has high metastatic potential and low prognosis that urgently demands the development of novel therapeutic approaches. One of the proposed approaches has been the down-regulation of BCL2, with poorly clarified and controversial therapeutic value regarding SCLC. The use of anti-BCL2 small interfering RNA (siRNA) in SCLC has never been reported. The aim of the present study was to select and test the in vitro efficacy of anti-BCL2 siRNA sequences against the protein and mRNA levels of SCLC cells, and their effects on cytotoxicity and chemosensitization. Two anti-BCL2 siRNAs and the anti-BCL2 G3139 oligodeoxynucleotide (ODN) were evaluated in SCLC cells by the simultaneous determination of Bcl-2 and viability using a flow cytometry method recently developed by us in addition to Western blot, real-time reverse-transcription PCR, and cell growth after single and combined treatment with cisplatin. In contrast to previous reports about the use of ODN, a heterogeneous and up to 80% sequence-specific Bcl-2 protein knockdown was observed in the SW2, H2171 and H69 SCLC cell lines, although without significant sequence-specific reduction of cell viability, cell growth, or sensitization to cisplatin. Our results question previous data generated with antisense ODN and supporting the present concept of the therapeutic interest in BCL2 silencing per se in SCLC, and support the growing notion of the necessity of a multitargeting molecular approach for the treatment of cancer.