Viability of Ascaris lumbricoides eggs eliminated after anti-helminthic therapy

The viability of Ascaris lumbricoides eggs passed in the feces was evaluated after treatment of patients with one of the anti-helminthic drugs (thiabendazole, levamisole, cambendazole, pyrantel pamoate, mebendazole or praziquantel). For each drug, a group of 5 children was selected and their feces collected 24 h before treatment and 24, 48 and 72 h after drug administration, except for mebendazole, with the feces being collected throughout the period of treatment. After sedimentation, the total amount of eggs from each collection was transferred to tissue culture flasks containing 10 ml H2So4 O, 1N, with the addtion of 3 drops of a miconazol solution, and incubated at 28 graus centígrados, individually, for 80 days. The flasks wee maintained open and the culture were oxigenated daily by manual agitation. On the 80th day of culture, 20-days-old albino mice were inoculated with 3,200 embryonated eggs, per os. Larvae were recovered from their lungs and hearts, on the 8th day after infection, according to Baerman's method (Morais, 1948). Thiabendazole showed 100.0% ovicidal capacity as early as 48 after treatment. Inhibition of embrionary development was observed when thiabendazole was used. This drug also had an effect on the eggs infectivity when inoculated into normal mice. No significant effect on embrionary development was observed for the other drugs tested.

Diagnostic accuracy of G-CSF, IL-8, and IL-1ra in critically ill children with suspected infection.

OBJECTIVE: To elucidate the diagnostic accuracy of granulocyte colony-stimulating factor (G-CSF), interleukin-8 (IL-8), and interleukin-1 receptor antagonist (IL-1ra) in identifying patients with sepsis among critically ill pediatric patients with suspected infection. DESIGN AND SETTING: Nested case-control study in a multidisciplinary neonatal and pediatric intensive care unit (PICU) PATIENTS: PICU patients during a 12-month period with suspected infection, and plasma available from the time of clinical suspicion (254 episodes, 190 patients). MEASUREMENTS AND RESULTS: Plasma levels of G-CSF, IL-8, and IL-1ra. Episodes classified on the basis of clinical and bacteriological findings into: culture-confirmed sepsis, probable sepsis, localized infection, viral infection, and no infection. Plasma levels were significantly higher in episodes of culture-confirmed sepsis than in episodes with ruled-out infection. The area under the receiver operating characteristic curve was higher for IL-8 and G-CSF than for IL-1ra. Combining IL-8 and G-CSF improved the diagnostic performance, particularly as to the detection of Gram-negative sepsis. Sensitivity was low (<50%) in detecting Staphylococcus epidermidis bacteremia or localized infections. CONCLUSIONS: In this heterogeneous population of critically ill children with suspected infection, a model combining plasma levels of IL-8 and G-CSF identified patients with sepsis. Negative results do not rule out S. epidermidis bacteremia or locally confined infectious processes. The model requires validation in an independent data-set.

Trypanosoma cruzi: metacyclogenesis in vitro - I. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods

In this work we have studied the modifications in the biological properties of Trypanosoma cruzi when the parasite is maintained for a long time in axenic culture. The studies were done with a clone from an avirulent strain (Dm30L) and a non-cloned virulent strain (EP) of T. cruzi. Both parasiteswere maintained, for at least three years, by successive triatomine/mouse alternate passage (control condition), or by serial passage in axenic medium (culture condition), or only in the mouse (mouse condition). The comparison between parasites of culture and control condition showed that metacyclogenesis capacity was reduced in the former and that the resulting metacyclics displayed an attenuatedvirulence. In order to compare the virulence of metacyclics from the urine of the insect vector, Rhodnius prolixus were infected by artificial feeding with parasites of the control or culture condition. After three triatomine/triatomine passages, there was observed an almost identical biological behavior for these parasites, hence indicating that the maintenance of T. cruzi for a long time in axenic culture affects the differentiation capacity and the virulence of the parasite. Additionally, it was demonstrated that it is possible to maintain T. cruzi exclusively through passages in the invertebrate host.

Development of a coculture model of encapsulated cells.

In the whole animal, metabolic regulations are set by reciprocal interactions between various organs, via the blood circulation. At present, analyses of such interactions require numerous and uneasily controlled in vivo experiments. In a search for an alternative to in vivo experiments, our work aims at developing a coculture system in which different cell types are isolated in polymer capsules and grown in a common environment. The signals exchanged between cells from various origins are, thus, reproducing the in vivo intertissular communications. With this perspective, we evaluated a new encapsulation system as an artificial housing for liver cells on the one hand and adipocytes on the other hand. Murine hepatocytes were encapsulated with specially designed multicomponent capsules formed by polyelectrolyte complexation between sodium alginate, cellulose sulphate and poly(methylene-coguanidine) hydrochloride, of which the permeability has been characterized. We demonstrated the absence of cytotoxicity and the excellent biocompatibility of these capsules towards primary culture of murine hepatocytes. Encapsulated hepatocytes retain their specific functions--transaminase activity, urea synthesis, and protein secretion--during the first four days of culture in minimum medium. Mature adipocytes, isolated from mouse epidydimal fat, were embedded in alginate beads. Measurement of protein secretion shows an identical profile between free and embedded adipocytes. We finally assessed the properties of encapsulated hepatocytes, cryopreserved over a periods of up to four months. The perspective of using encapsulated cells in coculture are discussed, since this system may represent a promising tool for fundamental research, such as analyses of drug metabolism, intercellular regulations, and metabolic pathways, as well as for the establishment of a tissue bank for storage and supply of murine hepatocytes.

Down-modulation of lymphoproliferation and interferon-gamma production by beta-glucan derived from Saccharomyces cerevisiae

beta-glucan, one of the major cell wall components of Saccharomyces cerevisiae, has been found to enhance immune functions. This study investigated in vivo and in vitro effects of beta-glucan on lymphoproliferation and interferon-gamma (IFN-gamma) production by splenic cells from C57BL/6 female mice. All experiments were performed with particulate beta-glucan derived from S. cerevisiae. Data demonstrated that both, i.p administration of particulate beta-glucan (20 or 100 µg/animal) and in vitro stimulation of splenic cells (20 or 100 µg/ml of culture) decreased lymphoproliferation and IFN-gamma production induced by concanavalin A. These results suggest that beta-glucan can trigger a down-modulatory effect regulating a deleterious immune system hyperactivity in the presence of a strong stimulus.

Detection of Entamoeba histolytica/Entamoeba dispar in stool specimens by using enzyme-linked immunosorbent assay

Entamoeba histolytica actually comprises two genetically distinct but morphologically indistinguishable species. E. histolytica can cause invasive intestinal and extra intestinal disease, while E. dispar cannot. Identification and differentiation of E. dispar and E. histolytica in stool sample by microscopy is imprecise. Several weeks of culture and isoenzyme analysis are required to differentiate E. histolytica from E. dispar. In this study, we have used an enzyme-linked immunosorbent assay (ELISA) for detection of E. histolytica/E.dispar and compared it with microscopy. Eighty-eight samples were evaluated, trichrome staining was positive in 20.4% (18) and ELISA was positive in 29.5% (26). Both tests were positive in 14 (15.9%) samples, 4 (4.5%) only with direct microscopy, and 12 (13.6%) only with ELISA. Both tests were negative in 58 (65.9%) samples. Microscopy has low sensitivity and high specificity, low negative predictive value and high positive predictive value in comparison with ELISA. E. histolytica/E. dispar antigen detection ELISA tests are inexpensive compared to the specific tests, yield objective results and do not require experienced microscopists and can therefore be recommended for screening of stools worldwide and the results can be taken for treatment that are fitting with its clinic.

Phenotypic, functional, and quantitative characterization of canine peripheral blood monocyte-derived macrophages

The yield as well as phenotypic and functional parameters of canine peripheral blood monocyte-derived macrophages were analyzed. The cells that remained adherent to Teflon after 10 days of culture had high phagocytic activity when inoculated with Leishmania chagasi. Flow cytometric analysis demonstrated that more than 80% of cultured cells were positive for the monocyte/macrophage marker CD14.

Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell-mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigen-specific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4(+)CD25(-) T cells could be expanded 8-fold into alloantigen-specific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO(+)CCR7(-) memory cells, and had a CD4(high), CD25(+), Foxp3(+), and CD62L (L-selectin)(+) phenotype. Although these CD4(high)CD25(+)Foxp3(+) alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.

Personality profiles of cultures: Aggregate personality traits

Personality profiles of cultures can be operationalized as the mean trait levels of culture members. College students from 51 cultures rated an individual from their country whom they knew well (N 12,156). Aggregate scores on Revised NEO Personality Inventory (NEO-PI-R) scales generalized across age and sex groups, approximated the individual-level 5-factor model, and correlated with aggregate self-report personality scores and other culture-level variables. Results were not attributable to national differences in economic development or to acquiescence. Geographical differences in scale variances and mean levels were replicated, with Europeans and Americans generally scoring higher in Extraversion than Asians and Africans. Findings support the rough scalar equivalence of NEO-PI-R factors and facets across cultures and suggest that aggregate personality profiles provide insight into cultural differences.

Differentiation of postmitotic neuroblasts into substance P-immunoreactive sensory neurons in dissociated cultures of chick dorsal root ganglion.

Counts performed on dissociated cell cultures of E10 chick embryo dorsal root ganglia (DRG) showed after 4-6 days of culture a pronounced decline of the neuronal population in neuron-enriched cultures and a net gain in the number of ganglion cells in mixed DRG cell cultures (containing both neurons and nonneuronal cells). In the latter case, the increase in the number of neurons was found to depend on NGF and to average 119% in defined medium or 129% in horse serum-supplemented medium after 6 days of culture. The lack of [3H]thymidine incorporation into the neuronal population indicated that the newly formed ganglion cells were not generated by proliferation. On the contrary, the differentiation of postmitotic neuroblasts present in the nonneuronal cell compartment was supported by sequential microphotographs of selected fields taken every hour for 48-55 hr after 3 days of culture. Apparently nonneuronal flat dark cells exhibited morphological changes and gradually evolved into neuronal ovoid and refringent cell bodies with expanding neurites. The ultrastructural organization of these evolving cells corresponded to that of primitive or intermediate neuroblasts. The neuronal nature of these rounding up cell bodies was indeed confirmed by the progressive expression of various neuronal cell markers (150 and 200-kDa neurofilament triplets, neuron specific enolase, and D2/N-CAM). Besides a constant lack of immunoreactivity for tyrosine hydroxylase, somatostatin, parvalbumin, and calbindin-D 28K and a lack of cytoenzymatic activity for carbonic anhydrase, all the newly produced neurons expressed three main phenotypic characteristics: a small cell body, a strong immunoreactivity to MAG, and substance P. Hence, ganglion cells newly differentiated in culture would meet characteristics ascribed to small B sensory neurons and more specifically to a subpopulation of ganglion cells containing substance P-immunoreactive material.

Retrovirus-mediated gene transfer in polyclonal T cells results in lower apoptosis and enhanced ex vivo cell expansion of CMV-reactive CD8 T cells as compared with EBV-reactive CD8 T cells.

To modulate alloreactivity after hematopoietic stem cell transplantation, "suicide" gene-modified donor T cells (GMCs) have been administered with an allogeneic T-cell-depleted marrow graft. We previously demonstrated that such GMCs, generated after CD3 activation, retrovirus-mediated transduction, and G418 selection, had an impaired Epstein-Barr virus (EBV) reactivity, likely to result in an altered control of EBV-induced lymphoproliferative disease. To further characterize the antiviral potential of GMCs, we compared the frequencies of cytomegalovirus (CMV)-specific CD8+ T (CMV-T) cells and EBV-specific CD8+ T (EBV-T) cells within GMCs from CMV- and EBV-double seropositive donors. Unlike anti-EBV responses, the anti-CMV responses were not altered by GMC preparation. During the first days of culture, CMV-T cells exhibited a lower level of CD3-induced apoptosis than did EBV-T cells. In addition, the CMV-T cells escaping initial apoptosis subsequently underwent a higher expansion rate than EBV-T cells. The differential early sensitivity to apoptosis could be in relation to the "recent activation" phenotype of EBV-T cells as evidenced by a higher level of CD69 expression. Furthermore, EBV-T cells were found to have a CD45RA-CD27+CCR7- effector memory phenotype, whereas CMV-T cells had a CD45RA+CD27-CCR7- terminal effector phenotype. Such differences could be contributive, because bulk CD8+CD27- cells had a higher expansion than did bulk CD8+CD27+ cells. Overall, ex vivo T-cell culture differentially affects apoptosis, long-term proliferation, and overall survival of CMV-T and EBV-T cells. Such functional differences need to be taken into account when designing cell and/or gene therapy protocols involving ex vivo T-cell manipulation.

Production of human secretory component with dimeric IgA binding capacity using viral expression systems.

The cDNA encoding the NH2-terminal 589 amino acids of the extracellular domain of the human polymeric immunoglobulin receptor was inserted into transfer vectors to generate recombinant baculo- and vaccinia viruses. Following infection of insect and mammalian cells, respectively, the resulting truncated protein corresponding to human secretory component (hSC) was secreted with high efficiency into serum-free culture medium. The Sf9 insect cell/baculovirus system yielded as much as 50 mg of hSC/liter of culture, while the mammalian cells/vaccinia virus system produced up to 10 mg of protein/liter. The M(r) of recombinant hSC varied depending on the cell line in which it was expressed (70,000 in Sf9 cells and 85-95,000 in CV-1, TK- 143B and HeLa). These variations in M(r) resulted from different glycosylation patterns, as evidenced by endoglycosidase digestion. Efficient single-step purification of the recombinant protein was achieved either by concanavalin A affinity chromatography or by Ni(2+)-chelate affinity chromatography, when a 6xHis tag was engineered to the carboxyl terminus of hSC. Recombinant hSC retained the capacity to specifically reassociate with dimeric IgA purified from hybridoma cells.

Long-term ex vivo expansion of human fetal liver primitive haematopoietic progenitor cells in stroma-free cultures

Successful expansion of haematopoietic cells in ex vivo cultures will have important applications in transplantation, gene therapy, immunotherapy and potentially also in the production of non-haematopoietic cell types. Haematopoietic stem cells (HSC), with their capacity to both self-renew and differentiate into all blood lineages, represent the ideal target for expansion protocols. However, human HSC are rare, poorly characterized phenotypically and genotypically, and difficult to test functionally. Defining optimal culture parameters for ex vivo expansion has been a major challenge. We devised a simple and reproducible stroma-free liquid culture system enabling long-term expansion of putative haematopoietic progenitors contained within frozen human fetal liver (FL) crude cell suspensions. Starting from a small number of total nucleated cells, a massive haematopoietic cell expansion, reaching > 1013-fold the input cell number after approximately 300 d of culture, was consistently achieved. Cells with a primitive phenotype were present throughout the culture and also underwent a continuous expansion. Moreover, the capacity for multilineage lymphomyeloid differentiation, as well as the recloning capacity of primitive myeloid progenitors, was maintained in culture. With its better proliferative potential as compared with adult sources, FL represents a promising alternative source of HSC and the culture system described here should be useful for clinical applications.

Increased connexin43 expression in human sphenous veins in culture is associated with intimal hyperplasia

Résumé Objectif : L'hyperplasie intimale est un processus de remodelage vasculaire qui apparaît après une lésion vasculaire. Les mécanismes impliqués dans l'hyperplasie intimale sont la prolifération, la dédifférentiation et la migration des cellules musculaires lisses depuis la média vers l'espace sous-intimal. Nous avons émis l'hypothèse que les jonctions communicantes de type gap, qui coordonnent certains processus physiologiques tels que la croissance et la différentiation cellulaire, pouvaient participer au développement de l'hyperplasie intimale. Méthodes : Des segments de veines saphènes humaines prélevées chirurgicalement lors de pontages, ont été ouverts longitudinalement avec la surface luminale placée vers le haut et maintenus en culture pendant 14 jours. Des fragments veineux ont été préparés pour une évaluation histologique, pour des mesures de l'épaisseur de la néointima, et pour des analyses immunocytochimiques de l'ARN messager ainsi que des protéines. Résultats : Parmi les 4 connexines (Cxs 37, 40, 43 et 45) qui forment les jonctions communicantes dans les veines, nous avons focalisé notre étude sur l'expression des Cxs 43 et 40; nous avons démontré que la Cx43 est exprimée dans les cellules musculaires lisses et les cellules endothéliales alors que la Cx40 est uniquement présente dans l'endothélium. Après 14 jours en culture, des analyses histomorphométriques ont montré une augmentation significative de l'épaisseur de l'intima démontrant la présence d'hyperplasie intimale. Une analyse temporelle a révélé une augmentation progressive de la Cx43 jusqu'à une augmentation maximale de six à huit fois au niveau de l'ARN messager et des protéines après 14 jours en culture. Au contraire, l'expression de la Cx40 n'était pas modifiée. Des analyses par immunofluorescence ont montré également une augmentation de la Cx43 dans les membranes des cellules musculaires lisses de la média. Le développement de l'hyperplasie intimale in vitro est diminué en présence de fluvastatin et cette diminution est associée à une réduction de l'expression de la Cx43. Conclusions : Ces données démontrent que la Cx43 est augmentée in vitro pendant le processus d'hyperplasie intimale et que la fluvastatin prévient cette induction. Ces résultats suggèrent un rôle crucial joué par la communication intercellulaire impliquant la Cx43 dans la veine humaine durant le développement de l'hyperplasie intimale. Abstract Objective: Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. Methods: Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis. Results: Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. Afrer 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. Atime-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression. Conclusions: These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia. (J Vasc Surg 2005;41:1043-52.)

Secretion of an endogenous subtilisin by Pichia pastoris strains GS115 and KM71.

The methylotrophic yeast Pichia pastoris is widely used for the expression of heterologous enzymes. While the purity of the desired expression product is of major importance for many applications, we found that recombinant enzymes produced in methanol medium were contaminated by a 37-kDa endogenous yeast protease. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) but not by 1,10-phenanthroline, EDTA, and pepstatin A, suggesting the nature of a serine protease. Its secretion was abolished in P. pastoris strains GS115 and KM71 by specific mutagenesis of a subtilisin gene (SUB2) but not by inactivation of the gene encoding vacuolar proteinase B (PRB). Bioinformatic comparisons of Sub2 protein with subtilisins from other fungal genomes and phylogenetic analyses indicated that this enzyme is not an orthologue of the vacuolar protease cerevisin generally present in yeasts but is more closely related to another putative subtilisin found in a small number of yeast genomes. During growth of P. pastoris, Sub2 was produced as a secreted enzyme at a concentration of 10 microg/ml of culture supernatant after overexpression of the full-length SUB2 gene. During fermentative production of recombinant enzymes in methanol medium, 1 ml of P. pastoris culture supernatant was found to contain approximately 3 ng of Sub2, while the enzyme was not detected during growth in a medium containing glycerol as a carbon source. The mutant strain GS115-sub2 was subsequently used as a host for the production of recombinant proteases without endogenous subtilisin contamination.