Phenotypic characterization of Clostridium botulinum strains isolated from infant botulism cases in Argentina / Caracterización fenotípica de cepas de Clostridium botulinum aisladas de casos de botulismo del lactante en Argentina

Infant botulism is the most common form of human botulism; however, its transmission has not been completely explained yet. Some of the most recognized potential sources of Clostridium botulinum spores are the soil, dust, honey and medicinal herbs. In Argentina, 456 cases of infant botulism were reported between 1982 and 2007. C. botulinum type A was identified in 455 of these cases whereas type B was identified in just one case. However, in Argentina, types A, B, E, F, G, and Af have been isolated from environmental sources. It is not clearly known if strains isolated from infant botulism cases have different characteristics from strains isolated from other sources. During this study, 46 C. botulinum strains isolated from infant botulism cases and from environmental sources were typified according to phenotypic characteristics. Biochemical tests, antimicrobial activity, and haemagglutinin-negative botulinum neurotoxin production showed uniformity among all these strains. Despite the variability observed in the botulinum neurotoxin's binding to cellular receptors, no correlation was found between these patterns and the source of the botulinum neurotoxin. However, an apparent geographical clustering was observed, since strains isolated from Argentina had similar characteristics to those isolated from Italy and Japan, but different to those isolated from the United States.

El botulismo del lactante es la forma más común del botulismo humano; sin embargo, su forma de transmisión no ha sido totalmente explicada. El suelo, el polvo ambiental, la miel y algunas hierbas medicinales son potenciales fuentes de esporas de Clostridium botulinum. Entre 1982 y 2007 se informaron en Argentina 456 casos de botulismo del lactante, 455 casos debidos al serotipo A y uno al serotipo B. Sin embargo, los serotipos A, B, E, F, G y Af han sido aislados de suelos y otras fuentes en Argentina. No se conoce si las cepas aisladas de casos de botulismo del lactante poseen características diferentes de las cepas aisladas de otras fuentes. Durante este estudio se caracterizaron 46 cepas de C. botulinum. Las pruebas bioquímicas y de sensibilidad a los antimicrobianos y la producción de neurotoxina botulínica hemaglutinina-negativa mostraron uniformidad entre estas cepas. A pesar de la variabilidad observada respecto de la unión de la neurotoxina a receptores celulares, no se observó una correlación entre estos patrones de unión y la fuente de aislamiento. Sin embargo, se observó una aparente agrupación geográfica, ya que las cepas aisladas en Argentina tuvieron características similares a las observadas en las cepas aisladas en Italia y Japón, pero diferentes de las que se registraron en las cepas aisladas en los Estados Unidos.

Role of Leishmania (Leishmania) amazonensis amastigote glycosphingolipids in macrophage infectivity

The role of glycosphingolipids (GSLs) present in amastigote forms of Leishmania (Leishmania) amazonensis during infection of macrophages was analyzed, with particular emphasis on GSLs presenting the terminal Galpß1-3Galpa disaccharide. Macrophage invasion by L. (L.) amazonensis amastigotes was reduced by 37 percent when the disaccharide Galpß1-3Galp (1 mM) was added to the culture medium. The putative macrophage receptor/lectin for ß-Gal-globotriaosylceramide (Galpß1-3Galpa1-4Galpß1-4Glc pß1-1Cer) and other structurally related GSLs from L. (L.) amazonensis amastigotes were analyzed by micelles and parasite binding assay to peritoneal macrophage proteins fractionated by SDS-PAGE under nonreducing conditions. Micelles containing purified amastigote GSLs or a suspention of L. (L.) amazonensis amastigotes fixed with 2 percent formaldehyde were incubated with nitrocellulose membrane containing the macrophage proteins transferred by Western blotting. Binding of micelles containing purified GSLs from amastigote forms or fixed L. (L.) amazonensis amastigotes to nitrocellulose membrane was probed using monoclonal antibody ST-3, which recognizes the glycoepitope Galpß1-3Galpa1-R present either in the micelle preparation or on the amastigote surface. Macrophage protein with molecular mass ~30 kDa bound the amastigote GSL and appeared to be a doublet on electrophoresis. The specificity of this interaction was confirmed using fixed L. (L.) chagasi amastigotes, which do not express GSLs such as ß-Galp-globotriaosylceramides, and which do not bind to 30-kDa protein.

Expressão de glicoesfingolipídeos no carcinoma espinocelular do trato aerodigestivo superior / Glycosphingolipid expression in squamous cell carcinoma of the upper aerodigestive tract

Os glicoesfingolipídios (GSLs) são importantes componentes da membrana celular, organizados em microdomínios, relacionados a receptores de membrana e comportamento anti-social da célula neoplásica como crescimento descontrolado, invasão e ocorrência de metástases. OBJETIVO: Como a expressão de GSLs no carcinoma espinocelular (CEC) é tema pouquíssimo estudado decidiu-se realizar estudo prospectivo visando avaliar a expressão de GSLs no CEC do trato aerodigestivo superior. MÉTODO: Coletou-se 33 amostras de CEC e mucosa normal e GSLs extraídos e purificados por cromatografia de fase reversa em coluna de C-18 e hidrólise alcalina em metanol. Os GSLs foram quantificados por densitometria das placas de cromatografia de alta resolução em camada delgada coradas com orcinol. RESULTADOS: Observou-se aumento significativo de GSLs no CEC (3,57æg/mg) em comparação à mucosa normal (1,92æg/mg), principalmente do monosialogangliosídeo (GM3), trihexosilceramida (CTH), dihexosilceramida (CDH), globosídeo (Gb4). A expressão de monohexosilceramida (CMH) foi semelhante no CEC e na mucosa normal. O aumento do GM3 no CEC foi demonstrado por métodos imunoquímicos empregando-se MAb DH2 (anti-GM3). Analisando-se os carboidratos do CMH por cromatografia gasosa acoplado a espectrômetro de massa constatou-se que a mucosa normal expressa glucosilceramida e o CEC glucosilceramida e galactosilceramida. CONCLUSÃO: O aumento de GSLs no tecido tumoral pode representar alterações dos microdomínios da membrana celular resultantes do processo de transformação maligna, responsáveis por uma maior interação célula-célula e célula-matriz aumentando seu potencial de infiltração e metástase, possibilitando o emprego dos GSLs e de MAbs no diagnóstico e no tratamento do CEC, a exemplo do que ocorre no melanoma.

Glycosphingolipids are integral constituents of cellular membrane, arranged in rafts, and with neoplasic cell anti-social behavior, like uncontrolled cell growth, invasiveness, and metastatic potential. AIM: However, there are few studies about glycosphingolipids (GSL) expression in squamous cell carcinoma (SCC). Since GSL are known to be tumor-associated markers we decided to perform a prospective study on the GSL profiles of SCC. METHOD: Specimens of 33 SCC and normal mucosa were obtained and GSLs were extracted and purified by reverse-phase chromatography on C18 column and alkaline hydrolysis in methanol. GSLs were quantified using densitometry of orcinol-stained HPTLC plates. RESULT: A significant increase of GSLs in SCC (3.57æg/mg) was observed as compared to normal mucosa (1.92æg/mg). In SCC, an increase of 2 to 3 times in the amounts of CDH, CTH, Globoside, and GM3 was observed in comparison to normal mucosa. The identification of GM3 as well as its increased expression in SCC was confirmed unequivocally by HPTLC immunostaining and indirect immunofluorescence using MAb DH2 (anti-GM3). BY analyzing SCC and normal mucosa CMHs by GC/MS, normal mucosa expresses only glucosylceramide whereas SCC cells express both glucosylceramide and galactosylceramide. CONCLUSION: The increase in the amount of GSLs in tumor tissue may represent changes of cell membrane microdomains resulting from the malignant transformation process, which is responsible for greater cell-cell or cell-matrix interaction thereby increasing their potential for infiltration and metastasis.

Accidente cerebro-vascular en la enfermedad de Fabry: algo más que una simple estenosis / Stroke in Fabry disease: more than a simple stenosis

Se analiza la evidencia existente a la fecha sobre los mecanismos fisiopatológicos que puedem generar accidentes cerebrovasculares en la enfermedad de Fabry. Esta entidad es el resultado de la deficiencia de alfa-galactosidasa A, lo que resulta en depósito patológico de glicoesfingilípidos en distintas poblaciones celulares. Asociados a la insuficiencia renal y cardíaca, los accidentes cerebrovasculares pueden derivar en la muerte de los pacientes. Durante mucho tiempo el único mecanismo generador de daño vascular informado fue la oclusión vascular por depósito lipídico a nivel endotelial. En la actualidad se describen otros mecanismos. El advenimento de la terapia de reemplazo enzimático ha generado gran expectativa en cuanto la posibilidad de reversión de estos mecanismos. Si bien la evidencia es escasa y son necesarios más estudios a largo plazo, algunos informes demuestran que luego de meses, el tratamiento ha logrado revertir algunos de los mecanismos implicados

The objective is to analyze the updated evidence on the physiopathological mechanisms that can generate cerebrovascular damage in Fabry disease. Fabry disease is the result of the deficiency of alpha-galactosidasa A, which causes pathological storage of glycosphingolipids, in different cells. Associated to renal and cardiac insufficiency, cerebrovascular complications can derive in the death of the patients. During a long time the only reported mechanism was the vascular occlusion by deposit of lipids at endothelial level. At the present time, other mechanisms are postulated. The arrival of enzyme replacement therapy has generated great expectation on the possibility of reversion of these alterations. Although the evidence is scarce and more long-term studies are necessary, some reports demonstrate that after months, the treatment has managed to revert some of the mechanisms involved

Specific binding of Burkholderia pseudomallei cells and their cell-surface acid phosphatase to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2).

Specific binding between bacterial cells and host tissue is an early step of the pathogenesis of infection. Burkholderia pseudomallei cells, the causative micro-organisms of melioidosis, were demonstrated to bind specifically to tissue glycolipids (asialo GM1 and asialo GM2) by solid phase binding assay on thin layer chromatograms. The detection limit was around 400 pmol of the glycolipids. Acid phosphatase purified from the culture filtrate of B. pseudomallei was tested for such binding properties, and the same results were obtained. According to our previous studies, the enzyme is a glycoprotein located on the cell surface, and hydrolysed tyrosine phosphate most actively among the substrates so far tested. The mode of binding between the enzyme and the glycolipids was analyzed by comparison of binding levels among three samples different in protein content, sugar content and specific phosphatase activities per protein and sugar residue. The results suggested the possibility of a receptor-ligand relationship between the bacterial enzyme and the host-cell glycolipids (asialo GM).

Glycosphingolipid expression in solid tumours and transformed cell lines.

Glycosphingolipids are assumed to play a crucial role in cell-cell and cell-substrate interactions, including cell adhesion, proliferation, differentiation and apoptosis. Furthermore, cell surface glycolipid profile changes in the so called "social disorders", such as malignant transformation. To better investigate these modifications, the ganglioside composition in different solid tumours and in two transformed cell lines was analyzed. In some of these models we also tried to correlate the pattern of gangliosides to the key enzymes involved in their metabolism. The results we obtained can be summarized as follows:(1), meningiomas with or without chromosome 22 deletion: predominance of ganglioside GD3 in the former and of ganglioside GM3 in the latter. Correlation between GM3/GD3 ratio and SAT-2 activity; (2), mammary carcinomas developed in MMTV/c-neu transgenic mice: accumulation of GM3-derived species. The different ganglioside distribution seems to correlate with the tumour size; (3), Sarcoma Galliera-strain cells SGS/3A and normal syngenic murine fibroblasts FG: transformed cells exhibit a lower activity of sialyltransferases (SAT-1, SAT-2, SAT-4) compared to normal fibroblasts, suggesting a possible correlation with the ganglioside pattern. The neuraminidase activity seems to correlate to the glycoprotein sialic acid content; (4), 3T3 normal murine fibroblasts and SVT2 transformed cells: GM3 is absent in 3T3, while it accounts for the main ganglioside species in SVT2. On the contrary, GM2 present in a large amount in normal fibroblasts, is practically absent in transformed cells. No correlation has been observed between ganglioside profile and glycosyltransferase activities so far examined.