A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Isolation and characterization of pLS 1 plasmid mutants with increased copy numbers

Abstract Streptococcus pneumoniae genetic systems designed for isolation of plasmid mutants with copy-up phenotypes have been developed. The target plasmids have the pLS1 replicon, and two different strategies have been followed: (i) selection of clones exhibiting augmented resistance to antibiotics, or (ii) obligatory co-existence of incompatible plasmids. We have isolated 23 plasmid mutants exhibiting increased number of copies. All the mutations corresponded to four different alleles of the copG gene of plasmid pLS1. These strategies could be used with other plasmids.     

Characterization of SCAR markers of Eimeria spp. of domestic fowl and construction of a public relational database (The Eimeria SCARdb)

This study reports the development and characterization of 151 sequence characterized amplified region (SCAR) markers for the seven Eimeria species that infect the domestic fowl. From this set, 84 markers are species-specific and 67 present partial specificity. The complete nucleotide sequence was derived for all markers, revealing the presence of micro- and minisatellite repetitive units in 22 SCARs, with up to five distinct repeat units being observed per marker. Only 15 markers showed significant hits in similarity searches against public sequence databases, thus confirming their anonymous and non-coding character. Finally, a relational database of the markers (the Eimeria SCARdb) was developed and made available on the Internet, providing a valuable resource of SCAR markers that can be useful for molecular diagnosis, and also for epizootiological, genetic variability and genome mapping studies.     

Biology of the first generation of a laboratory colony of Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae)

The phlebotomine sand flies Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) are very close and may be involved in the transmission of Leishmania spp. Ross, 1903 in Brazil. The biology of the first laboratory-reared generations of these species, descended from insects captured in Além Paraíba (N. intermedia) and Corinto (N. neivai) in the Brazilian state of Minas Gerais, is described here. The captured females were fed on hamsters and maintained individually in rearing pots. Laboratory temperature and relative humidity were maintained at 25-26 masculineC and 80% respectively. The productivity of the first generation of N. intermedia was greater than that of N. neivai, and its development time clearly shorter, particularly for the second and third larval instars.     

Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney

Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a co-receptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis.     

Redescription of Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae)

The phlebotomine sand flies Nyssomyia intermedia and Nyssomyia neivai are the probable vectors of American tegumentary leishmaniasis in the Southern and Southeastern regions of Brazil. These species form a complex, being difficult to separate between either females or males of the two members based on recognized morphological characteristics. Both N. intermedia and N. neivai are redescribed here in the search for characters that facilitate their correct identification. It was possible to differentiate females by means of spermathecal characteristics. Males could be separated with confidence by the tips of the genital filaments, which have the form of a deep spoon, the angle of the concavity being well accentuated in N. intermedia and much shallower in N. neivai.     

T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia) braziliensis

Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL) patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4+ as compared to CD8+ Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-gamma) production in comparison to CCL patients. On the other hand, interleukin (IL-10) production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-gamma) and regulatory mechanisms (IL-10). The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10) and effector cytokines (IFN-gamma), leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease.