RESUMO
The aim of this study is to evaluate some inflammatory parameter changes in septic shock patients and their possible correlation with clinical outcome, in particular when continuous veno-venous hemofiltration (CVVH) treatment is required. Considering the objective difficulty in enrolling this kind of patient, a preliminary study was initiated on seventeen septic shock patients admitted to a medical and surgical ICU. The mRNA expression of Toll-like receptor (TLR)-1, TLR-2, TLR-4, TLR-5, TLR-9, TNFα, IL-8 and IL-1ß was assessed, the plasmatic concentrations of IL-18, IL-2, IL-10 and TNFα were measured on the day of sepsis diagnosis and after 72 h. In those patients who developed acute renal failure unresponsive to medical treatment and who underwent CVVH treatment the same parameters were measured every 24 h during CVVH and after completion of the treatment. On sepsis diagnosis, gene expression of TLRs was up-regulated compared to the housekeeping gene in all the patients. After 72 h, in 35% of the patients a down-regulation of these genes was found compared to day 1, but it was not associated with a reduction of cytokine serum levels or improved clinical signs, better outcome or reduced mortality. After high volume hemofiltration treatment, cytokine serum levels and TLR expression were not significantly modified. In conclusion, considering the not numerous number of cases, from our preliminary study, we cannot certainly correlate TLR over-expression in septic shock patients with severity or outcome scores.
Assuntos
Choque Séptico/imunologia , Receptores Toll-Like/sangue , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/terapia , Adolescente , Idoso , Idoso de 80 Anos ou mais , Citocinas/sangue , Feminino , Regulação da Expressão Gênica , Hemofiltração , Humanos , Mediadores da Inflamação/sangue , Unidades de Terapia Intensiva , Itália , Cinética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/sangue , Índice de Gravidade de Doença , Choque Séptico/diagnóstico , Choque Séptico/genética , Choque Séptico/terapia , Receptores Toll-Like/genética , Adulto JovemRESUMO
The effect of Acanthamoeba on human platelets and erythrocytes has not been fully elucidated. This paper reports that cell-free supernatants prepared from A. castellanii can activate human platelets, causing both a significant increase in the cytosolic free-calcium concentration and platelet aggregation. In addition, we demonstrated that platelet activation depends on the activity of ADP constitutively secreted into the medium by trophozoites. This study also showed that A. castellanii can affect human red blood cells, causing hemolysis, and provided evidence that hemolysis occurs in both contact-dependent and contact-independent ways; there are differences in kinetics, hemolytic activity, and calcium dependency between the contact-dependent and contact-independent mechanisms. Partial characterization of contact-independent hemolysis indicated that ADP does not affect the plasma membrane permeability of erythrocytes and that heat treatment of amoebic cell-free supernatant abolishes its hemolytic activity. These findings suggest that some heat-labile molecules released by A. castellanii trophozoites are involved in this phenomenon. Finally, our data suggest that human platelets and erythrocytes may be potential cell targets during Acanthamoeba infection.
Assuntos
Acanthamoeba castellanii/fisiologia , Plaquetas/parasitologia , Eritrócitos/parasitologia , Animais , Cálcio/metabolismo , Sistema Livre de Células , Células Cultivadas , Humanos , Fatores de TempoRESUMO
Surface proteins of mucosal microbial pathogens play multiple and essential roles in initiating and sustaining the colonization of the heavily defended mucosa. The protist Trichomonas vaginalis is one of the most common human sexually transmitted pathogens that colonize the urogenital mucosa. However, little is known about its surface proteins. The recently completed draft genome sequence of T. vaginalis provides an invaluable resource to guide molecular and cellular characterization of surface proteins and to investigate their role in pathogenicity. Here, we review the existing data on T. vaginalis surface proteins and summarize some of the main findings from the recent in silico characterization of its candidate surface proteins.
Assuntos
Genoma de Protozoário , Proteínas de Membrana/genética , Proteínas de Protozoários/genética , Trichomonas vaginalis/genética , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Proteoma/genética , Alinhamento de Sequência , Trichomonas vaginalis/metabolismoRESUMO
Cell cultures obtained from the germinal layer of hydatid cysts of the parasitic tapeworm Echinococcus granulosus were characterized with respect to their microtubule and microfilament systems. These were stained using monospecific antibodies against tubulin from sea urchin spermatozoa or sheep brain and against Dictyostelium discoideum actin as well as rhodamine conjugated phalloidin. The results show that the distribution of microtubules nad actin containing fibres of these cells is remarkably similar to that of mammalian cells both during interphase and mitosis. Hydatid cells, however, could not be stained with a specific antivimentin antibody. Indirect immunofluorescence with antitubulin antibodies of inhibitor treated cells shows that hydatid cell microtubules are sensitive to several antimicrotubular drugs including benzimidazole derivatives, colchicine, vinblastine, and griseofulvin.
Assuntos
Citoesqueleto/química , Equinococose/tratamento farmacológico , Actinas/química , Animais , Anticorpos/metabolismo , Antifúngicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Encéfalo/metabolismo , Células Cultivadas , Colchicina/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Supressores da Gota/farmacologia , Griseofulvina/farmacologia , Humanos , Masculino , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Ouriços-do-Mar , Ovinos , Espermatozoides/metabolismo , Tubulina (Proteína)/química , Vimblastina/farmacologiaRESUMO
Our knowledge concerning cytopathogenicity of Trichomonas vaginalis has been enriched in the past by numerous findings. In this paper, we review the latest advances in the field and discuss the different mechanisms and molecules responsible for the parasite's virulence.
Assuntos
Trichomonas vaginalis/patogenicidade , Actinas/química , Actinas/imunologia , Animais , Sítios de Ligação , Citoesqueleto/química , Citoesqueleto/parasitologia , Endopeptidases/análise , Eritrócitos/química , Eritrócitos/parasitologia , Células Eucarióticas/citologia , Células Eucarióticas/parasitologia , Feminino , Hemólise , Humanos , Proteínas de Protozoários/análise , Espectrina/análise , Tricomoníase/parasitologia , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/química , Trichomonas vaginalis/citologia , VirulênciaRESUMO
We propose a new immunization method to stimulate a strong immune response against weak or diluted antigens. This technique is based on stimulation with polyclonal activators before exposure to the antigens. We also discuss the efficiency of various types of mitogen with particular regard to their capacity to produce monoclonal antibodies and serum antibodies. A specific immune response against soluble antigens is increased by pretreating mice with PPD. This preactivation permitted us to obtain monoclonal antibodies against weak antigens in a few days. No monoclonal antibodies were obtained by inoculating weak antigens or the activators by themselves.
Assuntos
Adjuvantes Imunológicos , Anticorpos Monoclonais/imunologia , Hibridomas/imunologia , Imunização/métodos , Ativação Linfocitária , Tuberculina/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Arginase/imunologia , Conalbumina/imunologia , Citoesqueleto/imunologia , Esquemas de Imunização , Camundongos , Feromônios/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Colágeno , Solubilidade , Trichomonas vaginalis/imunologiaRESUMO
We have investigated the mechanisms used by Trichomonas vaginalis to damage cellular membranes, using human erythrocytes as target cells. Haemolysis is a contact- and temperature-dependent phenomenon, and is inhibited in 4 mM EGTA. Osmotic protection experiments using carbohydrates with different molecular diameters as protectants demonstrated that the cytolytic activity of T. vaginalis is inhibited in 75 mM stachyose. On the basis of our data, we hypothesize a cytopathic mechanism mediated by the formation of functional pores into the target membrane. Some of the Trichomonas protein involved in haemolysis have been immunologically characterized.
Assuntos
Hemólise/fisiologia , Trichomonas vaginalis/patogenicidade , Animais , Carboidratos/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/parasitologia , Membrana Eritrocítica/ultraestrutura , Feminino , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Modelos Biológicos , Peso Molecular , Osmose , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Vaginite por Trichomonas/etiologia , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/isolamento & purificação , Trichomonas vaginalis/fisiologiaRESUMO
We studied a pili-independent adhesion mechanism to HEp-2 cells present in mucoid uropathogenic strains of Pseudomonas aeruginosa. Our data demonstrate that bacterial adhesion to HEp-2 cell surfaces is time dependent and that the phenotypes involved are influenced by bacterial growth conditions. Sonicated bacterial extracts competitively inhibit the adherence of homologous strains to HEp-2 cells. Adhesins that are heat and trypsin sensitive are located on the surface of the bacterial outer membrane. Immunogenic 55 kDa surface protein is required for the adherence to HEp-2 cell surfaces of non-piliated mucosal uropathogenic P. aeruginosa strains.
Assuntos
Aderência Bacteriana/fisiologia , Pseudomonas aeruginosa/fisiologia , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/imunologia , Proteínas de Bactérias/metabolismo , Extratos Celulares/farmacologia , Células Cultivadas , Humanos , Inibidores de Proteases/farmacologia , Ligação Proteica , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Tripsina/farmacologia , Infecções Urinárias/microbiologiaRESUMO
Diarrheagenic E. coli comprise a diverse group of microorganisms responsible for gastrointestinal diseases in humans. On the basis of their virulence traits they are distinguished from the non-pathogenic E. coli and classified in several categories. Molecular methods represent the most reliable techniques for distinguishing pathogenic from non-pathogenic E. coli and characterising their pathogenic features. In this paper we report the development of a set of three multiplex PCR assays for the simultaneous and rapid identification of diarrheagenic E. coli belonging to ETEC, EPEC, EHEC and EIEC groups. Assay 1 utilizes primer pairs specific for genes coding for ST and LT toxins of ETEC, and for the E. coli beta-glucuronidase (uidA); assay 2 detects the presence of the eae and bfpA genes of EPEC, and assay 3 recognizes stx1 and stx2 of EHEC, and ial of EIEC. This technique has been validated on 190 E. coli isolated in Angola, Italy and Mozambique from feces of children with diarrhea. Results obtained with the set of multiplex PCR demonstrated 100% accordance with those obtained for the same isolates by PCR on single target genes. The proposed set of multiplex PCRs is the first reported assay that allows the simultaneous characterization of the four categories of diarrheagenic E. coli.
Assuntos
Diarreia/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/patogenicidade , Reação em Cadeia da Polimerase/métodos , Angola , Criança , Primers do DNA/química , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Escherichia coli/classificação , Escherichia coli/genética , Fezes/microbiologia , Humanos , Itália , Moçambique , VirulênciaAssuntos
Anticorpos Antiprotozoários/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Trichomonas vaginalis/imunologia , Animais , Reações Cruzadas , Reações Falso-Positivas , Feminino , Anticorpos Anti-HIV/sangue , Infecções por HIV/diagnóstico , HIV-1/imunologia , Humanos , Coelhos , Vaginite por Trichomonas/imunologia , Vaginite por Trichomonas/parasitologiaAssuntos
Microtúbulos/ultraestrutura , Psoríase/patologia , Sarcoma de Kaposi/ultraestrutura , Neoplasias Cutâneas/ultraestrutura , Pele/ultraestrutura , Tubulina (Proteína)/imunologia , Síndrome de Werner/patologia , Animais , Anticorpos Heterófilos/imunologia , Células Cultivadas , Feminino , Fibroblastos/ultraestrutura , Imunofluorescência , Humanos , Pessoa de Meia-Idade , CoelhosAssuntos
Granuloma Inguinal/diagnóstico , Adulto , Calymmatobacterium/isolamento & purificação , Infecções por Chlamydia/complicações , Condiloma Acuminado/complicações , Granuloma Inguinal/complicações , Granuloma Inguinal/microbiologia , Humanos , Itália , Masculino , Neoplasias Penianas/complicações , Portugal/etnologiaRESUMO
This work was undertaken to determine whether Acanthamoeba could play a role in the survival and transmission of coxsackieviruses and focused on in vitro interactions between Acanthamoeba castellanii and coxsackie B3 viruses (CVB-3). Residual virus titer evaluations and immunofluorescence experiments revealed a remarkable CVB-3 adsorption on amoeba surfaces and accumulation inside cells. The survival of viruses was independent of the dynamics of amoeba replication and encystment. In addition, our results indicated that virus-infected amoebas can release infectious viruses during interaction with human macrophages. On the basis of these data, Acanthamoeba appears to be a potential promoter of the survival of coxsackieviruses and their transmission to human hosts.
Assuntos
Acanthamoeba castellanii/fisiologia , Acanthamoeba castellanii/virologia , Vetores de Doenças , Enterovirus/fisiologia , Receptores Virais/fisiologia , Replicação Viral , Acanthamoeba castellanii/crescimento & desenvolvimento , Animais , Membrana Celular/virologia , Imunofluorescência , Humanos , Macrófagos/ultraestrutura , Macrófagos/virologia , Viabilidade Microbiana , Fatores de TempoRESUMO
The cell cytoskeleton (microtubules, microfilaments and intermediate filaments) plays an important role in many cell functions, such as maintenance of cell locomotion, movement and compartimentalization of intracellular organelles and cell-to-cell interaction. Therefore, cytoskeleton alterations may result in sever impairment of cell functions. The aim of this paper was to study the in vitro effects of Pseudomonas aeruginosa on the cytoskeleton of cultivated Hep-2 cells. Cytoskeleton modifications were evidenced by immunofluorescence using monoclonal and polyclonal antibodies against tubulin and vimentin, and rhodamine conjugated phalloidin, that specifically binds to actin microfilaments. We report here that P. aeruginosa has a strong cytopathic effect on monolayers within a few hours of contact with the cells, and influences the organization of microfilaments, but has no discernable effect on microtubules or intermediate filaments.
Assuntos
Citoesqueleto/ultraestrutura , Pseudomonas aeruginosa/fisiologia , Citoesqueleto de Actina/ultraestrutura , Linhagem Celular , Imunofluorescência , Humanos , Filamentos Intermediários/ultraestrutura , Microscopia Eletrônica de Varredura , Microtúbulos/ultraestruturaRESUMO
Polypeptide mating pheromones Er-1 and Er-2, purified from the supernatant of Euplotes raikovi cultures of mating type I and mating type II, respectively, were used to immunize mice and obtain monoclonal antibodies. Five hybridoma clones producing antibodies specific to the mating pheromones were selected. They were analyzed for immunospecificity by immunoperoxidase assay, immunoblotting, and for their efficacy in inhibition of mating pheromone activity. Monoclonal antibodies from two hybridoma clones recognized only the mating pheromone used as antigen: those from the other three clones reacted, to comparable extents, with both mating pheromones. On the basis of these results it was assumed that two immunogenic sites exist in Er-1 and Er-2, one specific and the other common to both mating pheromones.
Assuntos
Cilióforos/imunologia , Epitopos/imunologia , Feromônios/imunologia , Atrativos Sexuais/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Especificidade de Anticorpos , Hibridomas , Técnicas Imunoenzimáticas , Isotipos de Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Atrativos Sexuais/antagonistas & inibidoresRESUMO
We report an outbreak of laboratory-acquired Brucella abortus infection originating in the accidental breakage of a centrifuge tube. A total of 12 laboratory workers were infected (attack rate of 31%), with an incubation time ranging from 6 weeks to 5 months. Antibody titers were evaluated weekly in all personnel exposed, allowing the diagnosis of the infection in most cases before the onset of clinical symptoms, so that specific therapy could be administrated.
Assuntos
Brucella abortus , Brucelose/epidemiologia , Surtos de Doenças , Pessoal de Laboratório Médico , Acidentes de Trabalho , Anticorpos Antibacterianos/sangue , Brucelose/diagnóstico , Brucelose/imunologia , Humanos , Itália/epidemiologia , Microbiologia , Fatores de TempoRESUMO
Adhesion of Trichomonas vaginalis is believed to be dependent on four adhesion proteins, which are thought to bind to vaginal epithelial cells in a specific manner with a ligand-receptor type of interaction. However, the specific receptors on the host cell have not yet been identified. In this work, the ability of the T. vaginalis adhesins to bind to cells of different histologic derivations and from different species has been studied. HeLa, CHO, and Vero cell lines; erythrocytes from different species; and a prokaryote without a cell wall, Mycoplasma hominis, were employed in order to investigate the cell specificity of the T. vaginalis adhesins. We observed that the T. vaginalis adhesins are able to bind to the different cell types to the same extent, suggesting that the host and tissue specificity of T. vaginalis adhesion should not be due to specificity of the parasite adhesins. Our results suggest that the data published to date on the subject are probably artifactual and that the experiments reported in the literature are not appropriate for identification of protozoan adhesins.
Assuntos
Proteínas de Protozoários/fisiologia , Trichomonas vaginalis/fisiologia , Trichomonas vaginalis/patogenicidade , Animais , Células CHO , Adesão Celular/fisiologia , Chlorocebus aethiops , Cricetinae , Eritrócitos/parasitologia , Feminino , Células HeLa , Humanos , Mycoplasma , Ligação Proteica , Células VeroRESUMO
The avidin-biotin-peroxidase technique is proposed for the immunological localization of beta-endorphin in the rat spinal cord. A rabbit specific antibody anti-human beta-endorphin was first obtained and then identified by immunoblotting and incubated with a quick-frozen section of young rat spinal cord. The use of a specific antibody with the immunoperoxidase reaction gave a morphological visualization of the beta-endorphin in the histological sections of the rat spinal cord.