An entomological and seroepidemiological study of the vectorial-transmission risk of Chagas disease in the coast of northern Chile.

Four species of triatomines are known from Chile: Triatoma infestans Klug, Mepraia spinolai Porter, M. gajardoi Frías, Henry & González, and M. parapatrica Frías (Hemiptera: Reduviidae), the last three are endemic. The geographical distribution of M. gajardoi includes the coastal areas in the north of Chile between 18° and 21°S, an area with both a resident workforce and summer-season visitors. A study was developed to assess the risk of vectorial transmission of Chagas disease by M. gajardoi in hut settlements on the coast of the Tarapacá Region, in particular in Caleta San Marcos and Caleta Río Seco. The study comprised fingerstick sampling of 95 persons, venous samples from 29 domestic dogs and capture of 52 triatomines, from both fishing coves. The samples were analysed by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) techniques. The results show that, of the total number of persons studied, 100% were negative for Trypanosoma cruzi Chagas (Trypanosomatida: Trypanosomatidae) antibodies, 10.34% of canids were positive for the antibody and 5.8% of M. gajardoi were infected to the PCR technique. The presence of this species in areas close to human settlements constitutes a risk to human populations established on the coast of northern Chile.

Involvement of KLF14 and egr-1 in the TGF-beta1 action on Leydig cell proliferation.

Transforming growth factor ß1 (TGF-ß1) is a pleiotropic cytokine that modulates cell homeostasis. In Leydig cells, TGF-ß1 exerts stimulatory and inhibitory effect depending on the type I receptor involved in the signaling pathway. The aim of the present work was to study the signaling mechanisms and the intermediates involved in the action of TGF-ß1 on TM3 Leydig cell proliferation in the presence or absence of progesterone. The MTT assay showed that the presence of progesterone in the culture media lead to a proliferative effect that was blocked by Ru 486, an inhibitor of progesterone receptor; and ALK-5 did not participate in this effect. TGF-ß1 (1 ng/ml) increased the expression of p15 (an inhibitor of cell cycle) in TM3 Leydig cells, and this effect was blocked by progesterone (1µM). The expression of PCNA presented a higher increase in the cell cultured with TGF-ß1 plus progesterone than in cells cultured only with TGF-ß1. Progesterone induced the gene expression of endoglin, a cofactor of TGF-ß1 receptor that leads to a stimulatory signaling pathway, despite of the absence of progesterone response element in endoglin gene. In addition, the presence of progesterone induced the gene expression of egr-1 and also KLF14, indicating that this steroid channels the signaling pathway into a non-canonical mechanism. In conclusion, these findings suggest that the proliferative action of TGF-ß1 involves endoglin. This co-receptor might be induced by KLF14 which is probably activated by progesterone.

Germ cell differentiation and proliferation in the developing testis of the South American plains viscacha, Lagostomus maximus (Mammalia, Rodentia).

Cell proliferation and cell death are essential processes in the physiology of the developing testis that strongly influence the normal adult spermatogenesis. We analysed in this study the morphometry, the expression of the proliferation cell nuclear antigen (PCNA), cell pluripotency marker OCT-4, germ cell marker VASA and apoptosis in the developing testes of Lagostomus maximus, a rodent in which female germ line develops through abolished apoptosis and unrestricted proliferation. Morphometry revealed an increment in the size of the seminiferous cords with increasing developmental age, arising from a significant increase of PCNA-positive germ cells and a stable proportion of PCNA-positive Sertoli cells. VASA showed a widespread cytoplasmic distribution in a great proportion of proliferating gonocytes that increased significantly at late development. In the somatic compartment, Leydig cells increased at mid-development, whereas peritubular cells showed a stable rate of proliferation. In contrast to other mammals, OCT-4 positive gonocytes increased throughout development reaching 90% of germ cells in late-developing testis, associated with a conspicuous increase in circulating FSH from mid- to late-gestation. TUNEL analysis was remarkable negative, and only a few positive cells were detected in the somatic compartment. These results show that the South American plains viscacha displays a distinctive pattern of testis development characterized by a sustained proliferation of germ cells throughout development, with no signs of apoptosis cell demise, in a peculiar endocrine in utero ambiance that seems to promote the increase of spermatogonial number as a primary direct effect of FSH.

Comments on the Classification of Neotropical Scionini (Tabanidae) with a Description of a New Genus.

The classification of the Tabanidae had remained stable over the last 60 years after Mackerras proposed a great revolution in the arrangement of the family. Recently, some new proposals based on molecular data have once again changed the classification of the family, mainly with a focus on the tribe Scionini. The present paper introduces a critical analysis based on the taxonomical view of the recent proposed classification of Scionini. Three genera are discussed: Lepmia Fairchild, Parosca Enderlein, and Pseudoscione Lutz. Lepmia atra (Philippi), L. grisea (Jaennicke), and L. leucothorax (Ricardo) are transferred to a new genus, Sixtomyiagen.n., based on its morphological differences from Lepmia. Other three species receive a new combination: Parosca subulipalpis (Enderlein) n. comb., Pseudoscione albifrons (Macquart) n. comb., Ps. hibernus (Wilkerson & Coscarón) n. comb. A key to species of Sixtomyia is provided and diagnostic characters are illustrated.

Regulation of visceral adipose tissue-derived serine protease inhibitor by nutritional status, metformin, gender and pituitary factors in rat white adipose tissue.

Visceral adipose tissue-derived serine protease inhibitor (vaspin) is a recently discovered adipocytokine mainly secreted from visceral adipose tissue, which plays a main role in insulin sensitivity. In this study, we have investigated the regulation of vaspin gene expression in rat white adipose tissue (WAT) in different physiological (nutritional status, pregnancy, age and gender) and pathophysiological (gonadectomy, thyroid status and growth hormone deficiency) settings known to be associated with energy homeostasis and alterations in insulin sensitivity. We have determined vaspin gene expression by real-time PCR. Vaspin was decreased after fasting and its levels were partially recovered after leptin treatment. Chronic treatment with metformin increased vaspin gene expression. Vaspin mRNA expression reached the highest peak at 45 days in both sexes after birth and its expression was higher in females than males, but its levels did not change throughout pregnancy. Finally, decreased levels of growth hormone and thyroid hormones suppressed vaspin expression. These findings suggest that WAT vaspin mRNA expression is regulated by nutritional status, and leptin seems to be the nutrient signal responsible for those changes. Vaspin is influenced by age and gender, and its expression is increased after treatment with insulin sensitizers. Finally, alterations in pituitary functions modify vaspin levels. Understanding the molecular mechanisms regulating vaspin will provide new insights into the pathogenesis of the metabolic syndrome.

Novel expression and direct effects of adiponectin in the rat testis.

Adiponectin is an adipocyte hormone, with relevant roles in lipid metabolism and glucose homeostasis, recently involved in the control of different endocrine organs, such as the placenta, pituitary and, likely, the ovary. However, whether as described previously for other adipokines, such as leptin and resistin, adiponectin is expressed and/or conducts biological actions in the male gonad remains unexplored. In this study, we provide compelling evidence for the expression, putative hormonal regulation, and direct effects of adiponectin in the rat testis. Testicular expression of adiponectin was demonstrated along postnatal development, with a distinctive pattern of RNA transcripts and discernible protein levels that appeared mostly located at interstitial Leydig cells. Testicular levels of adiponectin mRNA were marginally regulated by pituitary gonadotropins but overtly modulated by metabolic signals, such as glucocorticoids, thyroxine, and peroxisome proliferator-activated receptor-gamma, whose effects were partially different from those on circulating levels of adiponectin. In addition, expression of the genes encoding adiponectin receptor (AdipoR)-1 and AdipoR2 was detected in the rat testis, with developmental changes and gonadotropin regulation for AdipoR2 mRNA, and prominent levels of AdipoR1 in seminiferous tubules. Moreover, recombinant adiponectin significantly inhibited basal and human choriogonadotropin-stimulated testosterone secretion ex vivo, whereas it failed to change relative levels of several Sertoli cell-expressed mRNAs, such as stem cell factor and anti-Müllerian hormone. In summary, our data are the first to document the expression, regulation and functional role of adiponectin in the rat testis. Taken together with its recently reported expression in the ovary and its effects on LH secretion and ovarian steroidogenesis, these results further substantiate a multifaceted role of adiponectin in the control of the reproductive axis, which might operate as endocrine integrator linking metabolism and gonadal function.

Description of the Terrestrial Larva of Parosca latipalpis (Macquart) (Diptera: Tabanidae) from Southern Chile.

The terrestrial larva of the austral horsefly, Parosca latipalpis (Macquart), identified by molecular techniques, is described. The larva of P. latipalpis resembles Scaptia auriflua (Donovan), Copidapha vicina (Taylor), Myioscaptia muscula (English), and Osca lata (Guérin-Meneville) in many morphological characters, as well as in their terrestrial habitats. Some characters that are shared between these species are unique among Tabanidae and provide evidence of their monophyletic origin, suggesting a typical Gondwanaland group. Larvae of P. latipalpis were found 2-3 cm below of the soil surface and associated with larvae of Coleoptera, Lepidoptera, and Diptera in southern Chile.

Trypanosoma cruzi diversity in infected dogs from areas of the north coast of Chile.

As part of a multi-site research program on the eco-epidemiology and control of Chagas disease in northern Chile, we sought to identify the Trypanosoma cruzi discrete typing units (DTUs) infecting rural and peridomestic dogs, using direct methods without grown of the parasite in the laboratory and thus to assess the use of this species as a sentinel of the disease in well-defined endemic areas of T. cruzi in Chile. Infected dogs (35) from three villages were included in the study. The studied villages were Caleta Río Seco and Caleta San Marcos, both in the Tarapacá Region, and La Serena in the Coquimbo Region. These villages were selected based on previous evidence of Mepraia infection reports of the Chilean Ministry of Health. Amplicons from nested-PCR positive samples were used as targets to determine the infective T. cruzi DTUs circulating in blood using PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). Results of hybridization with dog samples from Caleta Rio Seco showed single infections in 2 out of 16 and mixed infections in 14 out of 16. TcVI was the most frequent DTU found in this area. A highlight is that for the first time the presence of TcIII is reported in this area. Samples from Caleta San Marcos showed single infections in 5 out of 9 and mixed infections in 4 out of 9. TcVI was the most frequent DTU found in this area. Samples from La Serena showed single infections in 5 out of 10 and mixed infections in 2 out of 10; we were unable to genotype the other 3 samples. Our results indicate that infection by T. cruzi DTUs in dogs is not homogeneously distributed but rather specific to each region of our country, as demonstrated by the differences in the T. cruzi DTU distribution in some localities.

Construction of CTB fusion proteins for screening of monoclonal antibodies against Salmonella typhi OmpC peptide loops.

Mice were immunized with resin-bound peptides whose sequences have been proposed to be part of exposed loops in Salmonella typhi outer membrane protein OmpC. To screen hybridomas for monoclonal antibodies against those epitopes, we designed fusion proteins where the candidate peptide sequence was attached to the amino end of cholera toxin B-subunit (CTB). The constructed fusion proteins allowed the efficient selection of positive clones by GM1-ELISA. Selected antibodies recognized purified OmpC and whole Salmonella bacteria. This suggests a native structure of our genetically attached peptides in agreement with immunological properties reported for previous CTB recombinant fusion proteins. In a more general context, CTB hybrids could be used to screen for antibodies towards immunogenic epitopes in other systems. This might turn out to be particularly useful when producing antibodies against peptide sequences in microorganisms whose handling is difficult or that pose inherent health risks.

Immune response to porins isolated from Salmonella typhi in different mouse strains.

Outer membrane proteins (OMPs) are able to induce protection against the challenge with S. typhi in a murine model. Both humoral and cellular immunity are involved in the protective mechanisms. In order to determine whether the responsiveness to S. typhi porins is genetically controlled in mice, different strains were immunized i.p. with 30 micrograms of OMPs isolated from S. typhi 9,12,Vi:d at days 0 and 7. On day 21, spleen cells were recovered and the lymphoproliferative response to porins was assessed. The highest responses were found in mice with H-2k and H-2a haplotypes (C3H/HeJ and A/J), intermediate responses were found in mice with H-2b haplotype (C57Bl/6) and the lowest responses in H-2d mice (Balb/c). These results demonstrate that the responsiveness to S. typhi porins is in part controlled by the major histocompatibility complex class II molecules and will help to further study the mechanisms of the immune response to these proteins.

Dasybasis elquiensis, a new species of horse fly from Northern Chile (Diptera: Tabanidae: Diachlorini).

The female of Dasybasis elquiensis, new species, is described and illustrated from specimens collected in Paso La Ternera, Elqui Province, north Chile. Its relationships to other Dasybasis species are discussed.

Dasybasis (Agelanius) cortesi, a new species of horse fly from Chile (Diptera:Tabanidae:Diachlorini).

The female and male of Dasybasis (Agelanius) cortesi, new species, is described and illustrated from specimens collected in the National Reserve of Río Clarillo, Cordillera Province, Central Chile. Its relationships to other Dasybasis species are discussed.

Simultaneous expression of CFA/I and CS3 colonization factor antigens of enterotoxigenic Escherichia coli by delta aroC, delta aroD Salmonella typhi vaccine strain CVD 908.

Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS3 (the common antigen in the CFA/II family of fimbrial antigens) are two of the most prevalent fimbrial antigens found in clinical isolates but are never expressed by the same wild-type strain. We manipulated the genetic determinants encoding CS3 and CFA/I fimbriae so that these two important colonization factors are expressed simultaneously in attenuated Salmonella typhi live oral vaccine strain CVD 908, including after growth in liquid medium (CFA/I is poorly expressed by wild-type ETEC in broth culture). The recombinant fimbrial structures produced by CVD 908 are morphologically indistinguishable from the CS3 fibrillae and CFA/I rod-like fimbriae produced by ETEC, and are recognized by monospecific CS3 and CFA/I antibodies. This prototype construct may prove useful in investigating the live vector approach to immunoprophylaxis of ETEC diarrheal disease.

Lymphocytic proliferative response to outer-membrane proteins isolated from Salmonella.

Porins isolated from Salmonella typhi have been demonstrated to protect against the challenge with this bacteria in mice. The mechanism has not been clarified, but could be associated with activation of both humoral and cellular immunity. In order to evaluate the induction of specific T cell responses, the lymphocytic proliferation to porins isolated from Salmonella typhimurium, Salmonella typhi and Escherichia coli was examined by 3H-thymidine incorporation assay in mice immunized with three different antigens: acetone-killed S. typhimurium, its porins, or outer-membrane proteins (OMPs) isolated from S. typhi. Higher proliferative responses were observed in mice immunized with porins and OMPs compared with those which received the acetone-killed bacteria. Although cross-reactivity was observed between porins, they were not mitogenic. Moreover, porins were able to activate T lymphocytes isolated from mice immunized with S. typhi OMPs. These results suggest that T cell activation, through the release of lymphokines, may play a role in the induction of protective immunity with porins.

Active protection of mice against Salmonella typhi by immunization with strain-specific porins.

NIH mice were immunized with between 2.5 and 30 micrograms of two highly purified porins, 34 kDa and 36 kDa, isolated from the virulent strain Salmonella typhi 9,12, Vi:d. Of mice immunized with 10 micrograms of porins, 90% were protected against a challenge with up to 500 LD50 (50% lethal doses) of S. typhi 9,12,Vi:d and only 30% protection was observed in mice immunized with the same dose of porins but challenged with the heterologous strain Salmonella typhimurium. These results demonstrate the utility of porins for the induction of a protective status against S. typhi in mice.

[Conjugate vaccines against bacterial infections: typhoid fever]. / Vacunas conjugadas contra infecciones bacterianas: fiebre tifoidea.

Capsular polysaccharides have been studied as possible vaccines against infectious diseases. However, they are capable to induce only short-run protection because of their T-independent properties and they would not be protective against infection in high-risk populations. The alternative to face this problem is to develop methods to join covalently the polysaccharide and proteins to both increase the immunogenicity of and to confer the property of T-dependence to this antigen. In order to obtain a conjugate vaccine against typhoid fever, in our laboratory we have tried to synthesize a conjugate immunogen between the Vi antigen and porins from Salmonella typhi.

On carbide dissolution in an as-cast ASTM F-75 alloy.

The solution treatment of an as-cast ASTM F-75 alloy was investigated. Microstructural evolution was followed during thermal processing, in particular with regard to the content and type of carbides formed. To evidence any probable carbide transformations occurring during the heating stage, as well as to clarify their effect on the carbide dissolution kinetics, three heating rates were studied. Image analysis and scanning electron microscopy techniques were used for microstructural characterization. For the identification of precipitates, these were electrolytically extracted from the matrix and then analyzed by X-ray diffraction. It was found that the precipitates in the as-cast alloy were constituted by both a M(23)C(6) carbide and a sigma intermetallic phase. The M(23)C(6) carbide was the only phase identified in solution-treated specimens, regardless of the heating rate employed, which indicated that this carbide dissolved directly into the matrix without being transformed first into an M(6)C carbide, as reported in the literature. It was found that the kinetics of dissolution for the M(23)C(6) carbide decreased progressively during the solution treatment, and that it was sensitive to the heating rate, decreasing whenever the latter was decreased. Because the M(23)C(6) carbide was not observed to suffer a phase transformation prior to its dissolution into the matrix, the effect of the heating rate was associated to the morphological change occurred as the specimens were heated. The occurrence of the observed phases was analyzed with the aid of phase diagrams computed for the system Co-Cr-Mo-C.