Influence de l'anhydrobiose sur l'infestation de Biomphalaria glabrata par un miracidium de Schistosoma mansoni

Sur 410 B. glabrata infestées par 1 miracidium de S. mansoni, 300 ont été placées dans des boîtes aérées, sur de la terre humide, et soumises à 6 semaines de dessiccation progressive. Au terme de l'expérience, il y avait 71 survivantes (23.66%), dont 9 positives. Les 110 autres planorbes ont cosntitué le lot témein, avec 106 survivantes (96.36%) à la première semaine d'apparition des cercaires. L'étude hebdomadaire des émissions cercariennes a montré des variations périodiques pour les deux sexes, une plus grande production de certaines femelles chez les témoins, mais une production de cercaires mâles ou femelles semblable chez les mollusques ayant subi l'anhydrobiose. Le faible nombre de ces dernies n'a pas permis une étude comparée significative de la survie des porteurs de formes larvaires mâles et femelles. La durée du développement du parasite chez son hôte ne semble pas modifiée si l'on tient compte de la phase d'estivation.

Human leptospirosis in a slum area in the city of Rio de Janeiro, Brazil: a serological and epidemiological study

A serologic survey was carried out on slum dwellers in the city of Rio de Janeiro. A total of 259 serum samples from male and female individuals of different age groups were tested for the presence of antileptospire antibodies by microagglutination. Prevalence data were analyzed in relation to the major risk factors present at the site, mainly represented by the presence of carrier animals and the occurence of frequent floods. Of the samples tested, 25% reacted with antigens of different serogroups at titres ranging from 1:100 to 1:6400, with a predominance of titres <= 1:400; 35% of positive sera reacted with leptospirae of the Icterohaemorrhagiae serogroup. Reactions with Djasiman, Panama, Javanica, Canicola, Pyrogenes, Australis, Ballum, Sejroe, Bataviae, Grippotyphosa, Autumnalis and Cynopteri were also detected, though at lower frequencies. There was no statistically significant difference between sexes, but higher prevalence rates were found to be associated with increasing age. A focus of infection was characterized, in which social and economic factors contribute to the persistance of leptospirae by favoring the proliferation of the main reservoir.

Skeletal Muscle Mitochondrial and Lipid Droplet Volume Density: Validity of Electron Microscopy Point-counting Measurements

Mitochondrial (M) and lipid droplet (L) volume density (vd) are often used in exercise research. Vd is the volume of muscle occupied by M and L. The means of calculating these percents are accomplished by applying a grid to a 2D image taken with transmission electron microscopy; however, it is not known which grid best predicts these values. PURPOSE: To determine the grid with the least variability of Mvd and Lvd in human skeletal muscle. METHODS: Muscle biopsies were taken from vastus lateralis of 10 healthy adults, trained (N=6) and untrained (N=4). Samples of 5-10mg were fixed in 2.5% glutaraldehyde and embedded in EPON. Longitudinal sections of 60 nm were cut and 20 images were taken at random at 33,000x magnification. Vd was calculated as the number of times M or L touched two intersecting grid lines (called a point) divided by the total number of points using 3 different sizes of grids with squares of 1000x1000nm sides (corresponding to 1µm2), 500x500nm (0.25µm2) and 250x250nm (0.0625µm2). Statistics included coefficient of variation (CV), 1 way-BS ANOVA and spearman correlations. RESULTS: Mean age was 67 ± 4 yo, mean VO2peak 2.29 ± 0.70 L/min and mean BMI 25.1 ± 3.7 kg/m2. Mean Mvd was 6.39% ± 0.71 for the 1000nm squares, 6.01% ± 0.70 for the 500nm and 6.37% ± 0.80 for the 250nm. Lvd was 1.28% ± 0.03 for the 1000nm, 1.41% ± 0.02 for the 500nm and 1.38% ± 0.02 for the 250nm. The mean CV of the three grids was 6.65% ±1.15 for Mvd with no significant differences between grids (P>0.05). Mean CV for Lvd was 13.83% ± 3.51, with a significant difference between the 1000nm squares and the two other grids (P<0.05). The 500nm squares grid showed the least variability between subjects. Mvd showed a positive correlation with VO2peak (r = 0.89, p < 0.05) but not with weight, height, or age. No correlations were found with Lvd. CONCLUSION: Different size grids have different variability in assessing skeletal muscle Mvd and Lvd. The grid size of 500x500nm (240 points) was more reliable than 1000x1000nm (56 points). 250x250nm (1023 points) did not show better reliability compared with the 500x500nm, but was more time consuming. Thus, choosing a grid with square size of 500x500nm seems the best option. This is particularly relevant as most grids used in the literature are either 100 points or 400 points without clear information on their square size.

Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal stem cells

SUMMARY : Ewing's sarcoma is a member of Ewing's family tumors (ESPY) and the second most common solid bone and soft tissue malignancy of children and young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generates fusion of the 5' segment of the EWSR1 gene with the 3' segment of the ETS family gene FLI-1. The EWSR1-FLI-1 fusion protein behaves as an aberrant transcriptional activator and is believed to contribute to ESFT development. However, EWSR1-FLI-1 induces growth arrest and apoptosis in normal fibroblasts, and primary cells that are pemissive for its putative oncogenic properties have not been discovered, hampering basic understanding of ESFT biology. Here, we show that EWSR1-FLI-1 alone can transform mouse primary bone marrow-derived mesenchymal progenitor cells and generate tumors that display hallmarks of Ewing's sarcoma, including a small round cell phenotype, expression of ESFT-associated markers, insulin like growth factor-I dependence, and induction or repression of numerous EWSR1-FLI-1 target genes. Consistent with this finding, we tested the possibility that human mesenchymal stem cells (hMSC) might also provide a permissive cellular environment for EWSR1-FLI-1, and could represent the first adequate primary human cellular background for the oncogenic properties of the fusion protein. Indeed, expression of EWSR1-FLI-1 in human mesenchymal stem cells (hMSC) was not only stably maintained without inhibiting proliferation, but induced a gene expression profile bearing striking similarity to that of ESFT, including genes that are among the highest ESFT discriminators. Expression of EWSR1-FLI-1 in hMSCs may recapitulate the initial steps of Ewing's sarcoma development, allowing identification of genes that play an important role early in its pathogenesis. Among relevant candidate transcripts induced by EWSR1-FL/-1 in hMSC we found the polycomb group gene EZH2 which we show to play a critical role in Ewing's sarcoma growth. These observations provide the first identification of candidate primary cells from which ESFTs originate and suggest that EWSR1-FLI-1 expression may constitute the initiating event in ESFT pathogenesis. Le sarcome d' Ewing est un membre de la famille des tumeurs Ewing (ESFT) et représente la deuxième tumeur maligne solide de l'os et des tissus mous chez les enfants et les jeunes adultes. Cette tumeur est associée dans 85% des cas avec la translocation chromosomique t(11;22)(g24:g12), qui génère la fusion entre le segment 5' du gène EWSR1 avec le segment 3' du gène FLI-1, appartenant à la famille des facteurs de transcription ETS. La protéine de fusion EWSR1-FLI-1 qui en dérive joue le rSle d'un facteur de transcription aberrant, et est supposée contribuer de manière décisive au processus de développement des ESFTs. Néanmoins, l'expression de EWSR1-FLI-1 dans des fibroblastes normaux induit un arrêt de croissance et leur apoptose, et les cellules primaires permissives pour les propriétés oncogéniques attribuées à la translocation n'ont pas encore été identifiées, empêchant la compréhension de la biologie de base du sarcome d'Ewing. Dans ce travail on montre que l'expression de EWSR1-FLI-1 uniquement est capable de transformer des cellules souches mésenchymateuses dérivées de la moelle osseuse de la souris, pour générer des tumeurs qui présentent les caractéristiques du sarcome d' Ewing humain, et notamment une morphologie de petites cellules bleues et rondes, l'expression de marqueurs associés aux ESFTs, une dépendance du facteur de croissance IGF-1, et l'induction ou la répression de nombreux gènes cibles connus de EWSR1-FLI-1. Sur la base de ces observations, on a testé la possibilité que les cellules souches mésenchymateuses humaines (hMSCs) puissent aussi fournir un environnement cellulaire permissif pour EWSR1-FLI-1 ; et représenter le premier background cellulaire humain adéquat pour la manifestation du pouvoir oncogénique de la protéine de fusion. En effet, l'expression de EWSR1-FLI-1 dans des cellules souches mésenchymateuses humaines s'est révélée non seulement maintenue, mais elle a induit un profil d'expression génétique étonnamment similaire à celui des ESFTs humains, incluant les gènes qui ont été rapportés comme étant les plus discriminatifs pour ces tumeurs. L'expression de EWSR1-FLI-1 dans les hMSCs pourrait récapituler les étapes initiales du développement du sarcome d' Ewing, et de ce fait consentir à identifier les gènes qui jouent un rôle crucial dans sa pathogenèse précoce. Parmi les transcrits relevant indults par EWSR1-FL/-9 dans les hMSCs nous avons découvert le gène du groupe des polycomb EZH2, que nous avons par la suite démontré jouer un rôle essentiel dans la croissance du sarcome de Ewing. Ces observations apportent pour la première fois l'identification d'une cellule primaire candidate pour représenter la cellule d'origine des ESFTs, et en même temps suggèrent que l'expression de EWSR1-FLI-1 peut constituer l'événement initial dans la pathogenèse du sarcome d' Ewing.

Some biological properties of the human amniotic membrane interferon

Human amniotic interferon was investigated to define the species specificity of its antiviral action and compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cell in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.

The role of the Estrogen-Responsive B box protein in skin morphogenesis, wound repair and skin disease

SUMMARY: EBBP is a poorly characterized member of the RBCC/TRIM family (RING finger B-box coiled-coilltripartite motif). It is ubiquitously expressed, but particularly high levels are found in keratinocytes. There is evidence that EBBP is involved in inflammatory processes, since it can interact with pro-interleukin-1 ß (prolL-1 ß) in human macrophages and keratinocytes, and its downregulation results in reduced secretion of IL-1 ß. IL-1ß activation and secretion requires the proteolytic cleavage of prolL-1ß by caspase-1, which in turn is actìvated by a protein complex called the inflammasome. As it has been demonstrated that EBBP can bind two different proteins of the inflammasome (NALP-1 and caspase 1), we assumed that EBBP plays a role in the regulation of inflammation and that the inflammasome, which has as yet only been described in ínflammatory cells, may also exist in keratinocytes. Indeed, I could show in my thesis that the inflammasome components are expressed in human keratinócytes at the RNA and protein level and also in vivo in human epidermis. After irradiation with a physiological dose of UVB, keratinocytes activated prolL-1ß and secreted prolL-1 a, IL-1 ß, prolL-18 and inflammasome proteins, although all these proteins lack a classical signal peptide. The secretion was dependent on caspase-1 activity, but not on de novo protein synthesis. Knock-down of NALP1 and -3, caspase-1 and -5, EBBP and Asc strongly reduced the secretion of IL-1 ß, demonstrating that also in keratinocytes inflammasome proteins are directly involved in maturation of this cytokine. These results demonstrate for the first time the presence of an active inflammasome in non-professional immune cells. Moreover, they show that UV irradiation is a stimulus for inflammasome activation in keratinocytes. For the analysis of the ín vivo functions of EBBP, transgenic mice overexpressing EBBP in the epidermis were generated. To examine the influence of EBBP overexpression on inflammatory processes, we subjected the mice to different challenges, which induce inflammation. Wound-healing, UVB irradiation and delayed hypersensitivity were tested, but we did not observe any phenotype in the K14-EBBP mice. Besides, a conditional ebbp knockout mouse has been obtained, which will allow to determine the effects of EBBP gene deletion in different tissues and organs. RESUME: EBBP est un membre encore mal connu de la famille des RBCC/TRIM (RING finger B-box coiled-coil/tripartite motif). Il est exprimé de manière ubiquitaire, et en particulier dans les kératinocytes. EBBP étant capable d'interagir avec la prointerleukine-1 ß (prolL-1 ß) dans les macrophages et les kératinocytes humains et de réguler la sécrétion de l'IL-1 ß, il est très probable que cette protéine est impliquée dans l'inflammation. L'activation et la sécrétion de l'IL-1 ß requièrent le clivage protéolytique de son précurseur prolL-1ß par la caspase-1, qui est elle-même activée par un complexe protéique appelé l'inflammasome. Comme il a été démontré qu'EBBP peut lier deux protéines de l'inflammasome (NALP-1 et caspase-1), nous avons émis l'hypothèse qu'EBBP joue un rôle dans la régulation de l'inflammation et que l'inflammasome, jusqu'ici décrit exclusivement dans des cellules inflammatoires, existe dans les kératinocytes. En effet, j'ai pu montrer dans ma thèse que les composants de l'inflammasome sont exprimés dans les kératinocytes humains ainsi que in vivo dans l'épiderme humain. Après irradiation avec une dose, physiologique d'UVB, les kératinocytes activent la prolL-1 ß et sécrètent la prolL-1a, l'IL-1 ß, la prolL-18 et des protéines de l'inflammasome, bien que toutes ces protéines soient dépourvues de peptide signal. La sécrétion dépend de la caspase-1 mais pas de la synthèse protéique de novo. Le knock-down de NALP-1 et -3, des caspase-1 et -5, d'EBBP et d'Asc réduit de manière marquée la sécrétion d'IL-1 ß, démontrant que dans les kératinocytes également, les protéines de l'inflammasome sont impliquées directement dans la maturation de cette cytokine. Ces résultats démontrent pour la première fois la présence d'un inflammasome actif dans des cellules immunitaires non professionnelles. De plus, ils montrent que l'irradiation aux UV est un stimulus pour l'activation de l'inflammasome dans les kératinocytes. Pour l'analyse des fonctions d'EBBP in vivo, nous avons généré des souris transgéniques qui surexpriment EBBP dans l'épiderme. En vue d'examiner l'influence de la surexpression d'EBBP sur le processus inflammatoire, nous avons soumis ces souris à differents modèles d'inflammation. Nous avons testé cicatrisation, UVB et hypersensibilité retardée, mais n'avons pas observé de phénotype chez les souris transgéniques. En parallèle, nous avons également généré des souris knock-out pour ebbp qui devraient nous permettre de déterminer les effets de la suppression d'EBBP dans différents tissus et organes.

Risk Factors for Human T Cell Lymphotropic Virus Type I among Injecting Drug Users in Northeast Brazil: Possibly Greater Efficiency of Male to Female Transmission

It was observed in the city of Salvador, State of Bahia, the highest seroprevalence of human T cell lymphotropic virus type 1 (HTLV-I) infection in Brazil as demonstrated by national wide blood bank surveys. In this paper, we report results of an investigation of drug use and sexual behavior associated with HTLV-I infection among male and female injecting drug users (IDUs) in Salvador. A cross sectional study was conducted in the Historical District of Salvador from 1994-1996 (Projeto Brasil-Salvador) and 216 asymptomatic IDUs were selected using the snowball contact technique. Blood samples were collected for serological assays. Sera were screened for human immunodeficiency virus (HIV-1/2) and HTLV-I/II antibodies by ELISA and confirmed by Western blot. The overall prevalence of HTLV-I/II was 35.2% (76/216). The seroprevalence of HTLV-I, HTLV-II and HIV-1 was for males 22%, 11.3% and 44.1% and for females 46.2%, 10.3% and 74.4% respectively. HTLV-I was identified in 72.4% of HTLV positive IDUs. Variables which were significantly associated with HTLV-I infection among males included needle sharing practices, duration of injecting drug use, HIV-1 seropositivity and syphilis. Among women, duration of injecting drug use and syphilis were strongly associated with HTLV-I infection. Multivariate analysis did not change the direction of these associations. Sexual intercourse might play a more important role in HTLV-I infection among women than in men.

Methicillin-resistant Staphylococcus aureus in human milk

We collected and analyzed 500 samples of human milk, from five Brazilian cities (100 from each) to detect methicillin-resistant strains of Staphylococcus aureus (MRSA) producing enterotoxins. We found 57 strains of MRSA, and the mecA gene, responsible for resistance, was detected in all of them using a specific molecular probe. We examined 40 strains for the presence of four enterotoxins, after selecting a subset that included all strains from each region, except for the largest sample, from which 10 were randomly selected. Among these two presented enterotoxin B, and growth in human colostrum and trypicase soy broth. After 5 h of incubation at 37°C, population sizes were already higher than 9.4 x 105 UFC/ml and enterotoxin was released into culture medium and colostrum. Our results stress the importance of hygiene, sanitary measures, and appropriate preservation conditions to avoid the proliferation of S. aureus in human milk.

Human immunodeficiency virus/acquired immunodeficiency syndrome and tropical diseases: a Brazilian perspective

The paper summarizes recent findings on the epidemiology and pathogenesis of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/Aids), highlighting the role of co-infections with major tropical diseases. Such co-infections have been studied in the Brazilian context since the beginning of the Aids epidemic and are expected to be more frequent and relevant as the Aids epidemic in Brazil proceeds towards smaller municipalities and the countryside, where tropical diseases are endemic. Unlike opportunistic diseases that affect basically the immunocompromised host, most tropical diseases, as well as tuberculosis, are pathogenic on their own, and can affect subjects with mild or no immunossuppression. In the era of highly active anti-retroviral therapies (HAART), opportunistic diseases seem to be on decrease in Brazil, where such medicines are fully available. Benefiting from HAART in terms of restoration of the immune function, putative milder clinical courses are expected in the future for most co-infections, including tropical diseases. On the other hand, from an ecological perspective, the progressive geographic diffusion of Aids makes tropical diseases and tuberculosis a renewed challenge for Brazilian researchers and practitioners dealing with HIV/Aids in the coming years.

Prevalence of human immunodeficiency virus infection in alcoholics

To verify the prevalence of infection by human immunodeficiency virus (HIV) in alcoholics we studied 131 alcoholic patients (119 males and 12 females) with a mean age of 44.3 ± 10.8 years. Serum samples were collected from this group and analysed, by ELISA, for antibodies against HIV as well as for serological markers for hepatitis B virus (HBV) and hepatitis C virus (HCV). As we have previously described, we found a high prevalence of HBV (26.4%) and HCV (4.2%) markers as compared to the prevalence of these markers in samples of normal blood donors from Uberlândia's Hemocentro Regional, which are 4% and 0.4%, respectively. Of the 131 patients, four (3%) had antibodies against HIV, three (75%) of which were injecting drug users (IDU). In the HIV-negative group, only one patient was an IDU. The prevalence of HIV in our population, according to data from the city's Health Secretary, varies from 3.1% to 6.2%. We conclude that, at least for the moment, alcoholism per se, did not constitute an important risk factor for HIV infection. However, acquired immunodeficiency syndrome is a rather recent disease as compared to hepatitis B and C and, as the transmission routes are similar for HIV and hepatitis viruses, an increase in the incidence of HIV infection in alcoholics may be just a question of time.

The origin and dispersion of human parasitic diseases in the Old World (Africa, Europe and Madagascar)

The ancestors of present-day man (Homo sapiens sapiens) appeared in East Africa some three and a half million years ago (Australopithecs), and then migrated to Europe, Asia, and later to the Americas, thus beginning the differentiation process. The passage from nomadic to sedentary life took place in the Middle East in around 8000 BC. Wars, spontaneous migrations and forced migrations (slave trade) led to enormous mixtures of populations in Europe and Africa and favoured the spread of numerous parasitic diseases with specific strains according to geographic area. The three human plasmodia (Plasmodium falciparum, P. vivax, and P. malariae) were imported from Africa into the Mediterranean region with the first human migrations, but it was the Neolithic revolution (sedentarisation, irrigation, population increase) which brought about actual foci for malaria. The reservoir for Leishmania infantum and L. donovani - the dog - has been domesticated for thousands of years. Wild rodents as reservoirs of L. major have also long been in contact with man and probably were imported from tropical Africa across the Sahara. L. tropica, by contrast, followed the migrations of man, its only reservoir. L. infantum and L. donovani spread with man and his dogs from West Africa. Likewise, for thousands of years, the dog has played an important role in the spread and the endemic character of hydatidosis through sheep (in Europe and North Africa) and dromadary (in the Sahara and North Africa). Schistosoma haematobium and S. mansoni have existed since prehistoric times in populations living in or passing through the Sahara. These populations then transported them to countries of Northern Africa where the specific, intermediary hosts were already present. Madagascar was inhabited by populations of Indonesian origin who imported lymphatic filariosis across the Indian Ocean (possibly of African origin since the Indonesian sailors had spent time on the African coast before reaching Madagascar). Migrants coming from Africa and Arabia brought with them the two African forms of bilharziosis: S. haematobium and S. mansoni.

Clinical and immunological consequences of human T cell leukemia virus type-I and Schistosoma mansoni co-infection

Human T cell leukemia virus type-I (HTLV-I) infection is associated with spontaneous T cell activation and uncontrolled lymphocyte proliferation. An exacerbated type-1 immune response with production of pro-inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) is significantly higher in patients with myelopathy associated to HTLV-I than in HTLV-I asymptomatic carriers. In contrast with HTLV-I, a chronic Schistosoma mansoni infection is associated with a type-2 immune response with high levels of interleukin (IL-4, IL-5, and IL-10) and low levels of IFN-gamma. In this study, clinical and immunological consequences of the HTLV-I and S. mansoni infection were evaluated. The immune response in patients with schistosomiasis co-infected with HTLV-I showed low levels of IL-5 (p < 0.05) in peripheral blood mononuclear cells cultures stimulated with S. mansoni antigen (SWAP) and decreased SWAP-specific IgE levels when compared with patients with only schistosomiasis (p < 0.05). Liver fibrosis was mild in all HTLV-I co-infected patients. Immunological response was also compared in individuals who had only HTLV-I infection with those who were co-infected with HTLV-I and helminths (S. mansoni and Strongyloides stercoralis). In patients HTLV-I positive co-infected with helminths the IFN-gamma levels were lower than in individuals who had only HTLV-I. Moreover, there were fewer cells expressing IFN-gamma and more cells expressing IL-10 in individuals co-infected with HTLV-I and helminths. These dates indicate that HTLV-I infection decrease type 2-response and IgE synthesis and are inversely associated with the development of liver fibrosis. Moreover, helminths may protect HTLV-I infected patients to produce large quantities of pro-inflammatory cytokines such as IFN-gamma.