Genetic evidence of functional ficolin-2 haplotype as susceptibility factor in cutaneous leishmaniasis

Background

Ficolin-2 coded by FCN2 gene is a soluble serum protein that plays an important role in innate immunity. In this study, we analyzed five functional polymorphisms of the FCN2 gene for their possible association with cutaneous leishmaniasis.

Methods

Initially we screened 40 Syrian Arabs for the entire FCN2 gene. We investigated the contribution of FCN2 functional variants in 226 patients with cutaneous leishmaniasis and 286 healthy controls from Syria. Polymorphisms in the promoter regions (−986G/A, −602G/A, −4A/G) of the FCN2 gene were assessed by TaqMan real time PCR, whereas polymorphisms in exon8 (+6359C/T and +6424G/T) were assessed by DNA sequencing. We also measured serum ficolin-2 levels in 70 control Syrian Arabs and correlated the serum concentrations to FCN2 genotypes and haplotypes respectively.

Results

Nine new FCN2 variants including two with non synonymous substitutions in exon6 and exon8 were observed. The homozygous genotypes +6424T/T were distributed more in controls and none in patients (P = 0.04). The AGACG haplotype were observed more in patients than in controls (OR = 2.0, 95%CI 1.2–3.4, P = 0.006). The serum ficolin-2 levels were significantly distributed among the reconstructed ficolin-2 haplotypes (P<0.008) and the haplotype AGACG was observed with higher ficolin-2 levels in 70 control individuals.

Conclusion

Our results demonstrate a significant association of FCN2 AGACG haplotype with cutaneous leishmaniasis in a Syrian Arab population. These first results provide a basis for a future study that could confirm or disprove possible relationships between FCN2 gene polymorphisms with cutaneous leishmaniasis.     

Antigen-stimulated lymphokines from patients with cutaneous leishmaniasis induce monocyte killing of Leishmania major intracellular amastigotes

The specific immune response of patients with cutaneous leishmaniasis including the ability of their lymphokines to enhance the monocytes' leishmaniacidal activity was studied. In 16 patients with cutaneous leishmaniasis, their concanavalin A-induced lymphocyte proliferative responses, interferon-gamma and interleukin 2 activities and the ability of their concanavalin A-induced lymphokines to kill monocyte intracellular amastigotes were not different from normal controls. Antigen-stimulated lymphocyte cultures showed that 13 of 13 patients had an increased lymphocyte proliferative response; 11 of 16 produced interleukin 2 and 12 of 13 produced interferon-gamma; in addition, 10 of 11 of these antigen-induced supernatants increased the monocytes' killing of Leishmania major amastigotes. Antibody levels to parasite membrane antigens determined by radioimmunoassay showed that 8 of 13 titers were greater than 10 and 4 of 13 titers were 2 to 10 times higher than control. Our findings demonstrate that patients with cutaneous leishmaniasis elicit a specific immune response to L. major antigens and part of this response is the production of lymphokine capable of promoting monocyte killing of intracellular amastigotes.     

Chemotactic response of macrophages to Acanthamoeba castellanii antigen and antibody-dependent macrophage-mediated killing of the parasite.

The chemotactic potential of antigens of Acanthamoeba castellanii for macrophages and the ability of naive and immune rat peritoneal macrophages to kill A. castellanii in vitro were assessed. The amoebolytic capacity of immune rat serum and complement was also examined. No parasite was killed in the presence of heat-inactivated naive rat serum. Low numbers of parasites were lysed in the presence of heat-inactivated immune rat serum, whereas significantly greater numbers of parasites were lysed in the presence of nonheat-inactivated naive and immune rat serum. Macrophages from naive rats were capable of lysing some parasites. However, the amoebolytic capability of these cells was significantly increased in the presence of serum from immune rats. Regardless of the source of serum used, macrophages from immune rats demonstrated about twice the amoebolytic proficiency of cells from naive rats. Macrophages from naive rats showed their highest capacity for lysing amoebae when incubated in the presence of gamma interferon and immune rat serum. The greatest overall proficiency in lysing parasites was displayed by cells from immune rats incubated with A. castellanii in the presence of gamma interferon and nonheat-inactivated serum from immune rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid peroxidation as a factor in gallstone pathogenesis

Phospholipid peroxidation markedly reduces the stability of mixed micellar systems composed of cholate, phosphatidylcholine and supersaturating levels of cholesterol. This suggests that lipid peroxidation is likely to play a significant role in the precipitation of cholesterol from gallbladder bile, thus in the pathogenesis of cholesterol gallstones. This conclusion is supported by studies of the nucleation time of cholesterol in gallbladder biles, which was significantly reduced by exposure to a stream of oxygen. This effect of phospholipid peroxidation on cholesterol solubility may occur in other biological fluids as well. In view of the increased lipid peroxidation in the elderly, it may explain the effect of age on the frequency of various diseases related to cholesterol precipitation.     

Immunotherapy as a treatment of American cutaneous leishmaniasis: preliminary studies in Brazil.

A prophylactic vaccine composed of killed promastigotes of five stocks of Leishmania was tested as an immunotherapeutic agent against American cutaneous leishmaniasis (ACL). The agent was administered by deep intramuscular injection daily for 10 days, followed by a 10-day interval. Out of 62 patients so treated, 47 (76%) were considered clinically cured; 41 required 2-10 treatment courses and the other six 11-19 courses. None of the patients treated by immunotherapy displayed adverse side-effects. Immunotherapy proved to be effective in the treatment of single cutaneous lesions, multiple cutaneous lesions and in cases of mucocutaneous leishmaniasis. In comparison with chemotherapy (Glucantime), immunotherapy is less efficient and more prolonged but can be safely used when antimonials are contra-indicated or are found to be ineffective. Consideration is given to the treatment of victims of ACL living in rural areas remote from a medical centre.     

Migratory history as a factor influencing dietary habit

A traditional concept of staple food is present in the dietary habits of Japanese, and the prominent role of rice as a staple food has changed since the end of World War II. The influence of migration on staple food patterns was studied using Hayashi's method of multivariate analysis for the quantification of qualitative data. The result of the study suggested that migration clearly influences the choice of a staple food, and that the influence of migration is more conspicuous in comparatively younger generations.     

Serum as a factor influencing adhesion of Enterococcus faecalis to glass and silicone

The purpose of this work was to analyze the effect of serum on the physicochemical surface properties and adhesion to glass and silicone of Enterococcus faecalis ATCC 29212 at 37 degrees C. As is presented using thermodynamics analysis, serum minimizes the interaction of cells with water, which correlates well with the increase in hydrophobicity and in bacterial adhesion to glass and silicone.     

Single gene control of resistance to cutaneous leishmaniasis in mice

A series of inbred, congenic resistant, and hybrid strains of mice were intradermally inoculated with 10⁶ promastigotes of Leishmania tropica. These mice were divided into susceptible and resistant groups using the criteria of lesion size, development of metastatic foci and skin-test reactivity. At 16 weeks of infection, resistant strains A/J, DBA/1J, AKR/J, CBA/J, C3H/HeJ, NZB/BINJ, C57BL/6J, C57BL/10Sn, B10.D2, B10.129(10M), and B10.CE(30NX) had completely resolved their lesions, while susceptible SWR/J and BALB/cJ mice demonstrated large, nonhealing cutaneous lesions. In addition, BALB/cJ developed metastatic lesions on the extremities which progressively increased in size. All BALB/cJ and SWR/J mice died by 7 1/2 months of infection. The BALB/cJ x C57BL/6JF₁ hybrid behaved in an intermediate fashion showing a slower expansion of cutaneous ulcers and a delayed development of metastatic foci, however, the infection ultimately proved fatal. The F₂ generation could be separated into three distinct groups: resistant, intermediate, and susceptible mice with a lesion size distribution pattern in conformity with a 1:2:1 ratio. Male/female susceptibility differences were not noted. These data indicated that development of acquired resistance may be under the control of a single, autosomal gene. The gene did not appear to be H-2-, Ir-2-, or H-11-linked as is seen with Leishmania donovani infections.     

Salivary β-glucosidase as a direct factor influencing the occurrence of halitosis

β-Glucosidases are enzymes present in all living organisms, playing a pivotal role in diverse biological processes. These enzymes cleave β-glycosidic bonds between carbohydrates, or between a carbohydrate and a non-carbohydrate moiety, which may result in the liberation of volatile aglycones. Released compounds execute diverse physiological roles, while the industry takes advantage of exogenously added β-glucosidases for aroma enrichment during food and beverage production. β-Glucosidase enzymatic activity has been reported in human saliva and given the fact that these enzymes are involved in aroma release, we investigated here the correlation between β-glucosidase activity in human saliva and the occurrence of halitosis. Measurement of salivary enzyme activity of 48 volunteers was performed using p-nitrophenyl-β-d-glucopyranoside as substrate. Each volunteer was clinically evaluated by a dental surgeon and clinical and laboratorial data were statistically analyzed. Gas-chromatography of saliva headspace allowed the analysis of the direct role of exogenous β-glucosidase on aromatic /volatile profile of saliva samples. The data demonstrated a positive correlation between halitosis and enzymatic activity, suggesting that the enzyme exerts a direct role in the occurrence of bad breath. Gas-chromatography analysis demonstrated that exogenously added enzyme led to the alteration of volatile organic content, confirming a direct contribution of β-glucosidase activity on saliva volatile compounds release. Although halitosis is a multifactorial condition, the complete understanding of all governing factors may allow the development of more effective treatment strategies. Such studies may pave the way to the use of β-glucosidase inhibitors for halitosis clinical management.     

Clay minerals as a factor influencing the biochemical activity of soil microorganisms

The results of the study confirm the significance of clay minerals as a factor influencing the biochemical activity of soil microorganisms. The soil microflora is influenced both by the direct effect of clays on the microbial cells and indirectly, by their effect on the environment. The direct effect is projected into fundamental processes of the cycle of biogenic elements, including humification processes. The character and mechanism of the effect depend on the species of microorganism, on the quality and quantity of the mineral sorbents present in the soil and on other ecological factors. A further study will be carried out to investigate different aspects of the influence of clay minerals on the incidence, growth and biochemical activity of soil microorganisms.     

Resistance of Histoplasma capsulatum to killing by human neutrophils

The basis for resistance of yeast form of Histoplasma capsulatum to antifungal activity of human neutrophils was studied. In limiting dilution assays and short term coculture assays human neutrophils were ineffective in killing H. capsulatum whereas Candida albicans was readily killed. By contrast, in a cell free hydrogen peroxide-peroxidase-halide system H. capsulatum was as sensitive to killing as C. albicans. Moreover, lysate of human neutrophils effectively substituted for horse-radish peroxidase in a cell free system for killing H. capsulatum. H. capsulatum elicited significant products of the oxidative burst in human neutrophils as detected by luminol-enhanced chemiluminescence. However, the response was two-fold less (p<0.05) than that induced by C. albicans. Transmission electron microscopy studies showed that phagosome-lysosome fusion took place when neutrophils phagocytosed C. albicans or H. capsulatum. Taken together, these findings indicate that, even though H. capsulatum elicits an oxidative burst and phagosome-lysosome fusion within the phagosome, it is capable of evading damage in short term assays.Abbreviations CFU colony forming units - PMN polymorphonuclear neutrophil - CTCM complete tissue culture medium - CL chemiluminescence - HPO horseradish peroxidase - P-L lysosomal peroxidase positive material     

Resistance to killing by tumor necrosis factor in an adipocyte cell line caused by a defect in arachidonic acid biosynthesis

We have found that TA1-R6, which are resistant to the cytotoxic effects of tumor necrosis factor (TNF) in the presence of cycloheximide (Reid, T. R., Torti, F., and Ringold, G. M. (1989) J. Biol. Chem. 264, 4583-4589), have reduced ability to release arachidonic acid (20:4) from membrane phospholipids in response to either TNF or the calcium ionophore A23187 treatment. However, no defect in the activity of phospholipase A2, the principal enzyme responsible for the release of 20:4 from phospholipids, was observed in these cells. Detailed biochemical characterization of these TNF-resistant cells has revealed that these cells are unable to synthesize 20:4 endogenously because of a defect in delta 6-desaturase, the rate-limiting enzyme of 20:4 biosynthesis. This deficiency leads to a marked decrease in the steady-state levels of 20:4 present in choline-containing phospholipid (PC) and ethanolamine-containing phospholipid (PE). The TA1-R6 cells, however, are capable of incorporating exogenous 20:4 into PC and PE, and when loaded in such manner they become significantly more sensitive to the cytotoxic effects of TNF in the presence of cycloheximide. Therefore, the release of arachidonic acid from phospholipids appears to be a critical element in the signaling pathway utilized by TNF and is essential to the rapid cytotoxic response elicited by TNF in the absence of protein synthesis in wild-type TA1 cells.     

Moisture as a factor influencing the distributions of two species of terrestrial salamanders

Summary Plethodon richmondi shenandoah occurs in at least three geographically isolated talus slopes in Shenandoah National Park, Virginia, U.S.A., each surrounded by a continuous population of Plethodon c. cinereus in the soil outside the talus. Distributions are contiguous but largely non-overlapping. The talus presents a much drier habitat than does the surrounding soil. Four experiments were designed to test the responses of the two species to moisture and substrate. Although shenandoah lives in a habitat generally drier than that of cinereus, both species choose the wet end of a moisture gradient and do not differ significantly in moisture preference. When given choices between a substrate of rock or soil, the two species respond similarly: neither expresses a preference when both substrates are moist and both choose soil over rock as the substrates dry, showing that substrate preference is based on moisture content and not texture. A third experiment demonstrates that cinereus suffers significantly greater mortality and loss of body water when subjected to a drying rock substrate than when subjected to a soil substrate, since the latter holds moisture longer. Thus the talus most likely presents a greater stress of dehydration to salamanders than does the soil. A fourth experiment shows that when forced to dehydrate, shenandoah survives longer, loses significantly less body water per hour, and withstands a greater loss of body water before death than does cinereus.The conclusions drawn are that cinereus inhabits areas of deep soil not due to a preference for that substrate but due to the requirement of a moist substrate, and it cannot enter the talus due to the dry conditions there. P. r. shenandoah, on the other hand, neither prefers the rocky nor the dry conditions of the talus and is probably excluded from the soil by the presence of cinereus. The survival of shenandoah in the talus is due, at least in part, to its ability to withstand the stress of dehydration for a longer period than can cinereus.