RESUMO
The guinea pig Kurloff cell (KC) is an estradiol-dependent circulating mononuclear cell that had natural killer cytotoxic activity in vitro. We studied the variation of KC number during the development of transplanted leukemia in inbred strain 2 (S2) leukemia-sensitive guinea pigs. Grafts of leukemic cells (L2C) produced a significant increase in the number of KC. Leukemia occurred in 35.5% of non-estrogenized animals and in 18.3% of estrogenized guinea pigs. The increased number of KC seems to have antileukemia activity in vivo. This could be part of the general phenomenon of cancer resistance in guinea pigs.
Assuntos
Cobaias/imunologia , Células Matadoras Naturais/imunologia , Leucemia Experimental/imunologia , Animais , Estradiol/farmacologia , Imunidade Celular , Endogamia , Masculino , Transplante de NeoplasiasRESUMO
We examined the in vivo role of Kurloff cells (KC), guinea pig natural killer cells, during the development of L2C leukemia, by analysing changes in the arylsulfatases (Asases) in the lysosomal Kurloff body. The Kurloff body is rich in acid phosphatase, esterase and proteoglycans, as are large granular lymphocyte granules. Moreover, the Kurloff body contains lysosomal Asase B, with unusual anionic isoforms. Injection of L2C cells elicited a three-fold increase in KC Asase activity on day 6. The increase in KC Asase activity was correlated with the number of circulating L2C cells. The basic Asase form (pl 8) was lost, and a concomitant increase in anionic isoforms (pl 5-6) was observed on day 6. The role of the latter in cytolysis was investigated by examining their capacity to lyse L2C target cells. We conclude that Asase participates in cytolysis when lysis is mediated by the complete assembly of cytolytic proteins. Changing and increasing KC Asase activity during leukemia development may be a marker for activated KC in vivo. These findings suggest that the cytolytic activation of KC occurs during the preleukemic period.
Assuntos
Condro-4-Sulfatase/metabolismo , Isoenzimas/metabolismo , Células Matadoras Naturais/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Animais , Ânions , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Cobaias , Ativação Linfocitária , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Pré-Leucemia/sangue , Pré-Leucemia/enzimologiaRESUMO
The response of the intracellular pathogen Rhodococcus equi to acid shock, a stress potentially encountered after phagocytosis by macrophages, was analyzed. The wild-type and its avirulent plasmid-cured strain acquired increased acid tolerance during the exponential growth phase upon exposure to sublethal acid stress, a response referred to as the acid tolerance response. Maximal adaptation was observed when cells were pretreated for 90 min at pH 5.0 before exposure to the pH challenge. Search for plasmid-encoded proteins regulated by an acidic pH was performed using two-dimensional gel electrophoresis, and enabled us to detect several membrane and cytoplasmic proteins with altered expression during the adaptation phase, but none of them were plasmid-encoded. However, using a strategy based on plasmid-encoded gene expression, we showed that two operons located on the virulence plasmid of strain 85F were upregulated by acid pHs with a maximal induction at pH 5.0. One operon, containing vapA, was monocistronic whereas the other was polycistronic composed of vapD and an unknown open reading frame. Our combined results suggest that these genes may play an important role in the pathogenicity of R. equi.
Assuntos
Infecções por Actinomycetales/microbiologia , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA , Regulação Bacteriana da Expressão Gênica , Lipoproteínas/genética , Rhodococcus equi/genética , Fatores de Virulência , Infecções por Actinomycetales/genética , Infecções por Actinomycetales/patologia , Adaptação Fisiológica , Animais , Proteínas de Bactérias/fisiologia , Humanos , Ácido Clorídrico/farmacologia , Concentração de Íons de Hidrogênio , Lipoproteínas/fisiologia , Glicoproteínas de Membrana , Plasmídeos/genética , Rhodococcus equi/patogenicidade , Rhodococcus equi/fisiologia , Regulação para Cima , Virulência/genéticaRESUMO
Rhodococcus equi is an important gram-positive intracellular facultative pathogen in foals of less than 3 months of age, that causes suppurative bronchopneumonia, lymphadenitis and/or enteritis. The disease in young foals mainly occurs in spring and summer when weather conditions are favorable for survival and multiplication of the bacteria in the environment. R. equi is widespread in the environment of horsebreeding farms: it has been isolated from the soil of paddocks and from the feces of adult horses and foals. Aerosol infection via dust of paddocks seems to be the major route of foal infections. The molecular mechanisms associated with the pathogenesis are not well understood and little is known about the markers or factors associated with virulence of R. equi. However, the discovery of a large plasmid in virulent strains and its association with virulence in mice and in young foals was reported. In this report, we studied the acid resistance of virulent R. equi in comparison with its avirulent plasmid-free isogene.
Assuntos
Rhodococcus equi/fisiologia , Concentração de Íons de Hidrogênio , Plasmídeos , Rhodococcus equi/genética , Rhodococcus equi/patogenicidade , VirulênciaRESUMO
The authors report two cases of adenocarcinoma arising from Brünner's glands. The diagnosis was made on histological, histochemical and lectin-histochemical grounds. Brünner's glands carcinoma cells were alike and located very close to normal Brünner's glands. Brünner's glands carcinoma cells contained neutral glycoproteins and were positive for Concanavalin A.
Assuntos
Adenocarcinoma/diagnóstico , Glândulas Duodenais , Neoplasias Duodenais/diagnóstico , Idoso , Concanavalina A , Glicoproteínas/análise , Histocitoquímica , Humanos , MasculinoRESUMO
The major alpha 2-6 sialoglycoproteins in detergent-extracts of Kurloff cells were purified by anion-exchange and Sambucus nigra agglutinin-affinity chromatographies. The similar ultrastructural localisations of (1) S. nigra agglutinin-gold conjugates and (2) acid phosphatase activities on the Kurloff body and particularly on its myelin figures indicated that the major alpha 2-6 sialoglycoproteins of the Kurloff cell had acid phosphatase activity. Two-dimensional electrophoresis showed that these tartrate-sensitive phosphatases corresponded to 2 acidic (pI 3.4-3.7) polypeptides of 36 and 34 kDa. Hydrolysis with peptide-N-glycosidases F gave a 33 kDa apoprotein rich in alanine, glutamic acid, tyrosine and lysin. A lectin-affinity study demonstrated that they contained hybrid type bisected and fucosylated N-linked oligosaccharides. Cytotoxic properties were previously attributed to Kurloff cells and other studies suggested that not only acid phosphatases but also alpha 2-6-linked sialic acid residues themselves may participate in natural killer activity.
Assuntos
Fosfatase Ácida/metabolismo , Sialoglicoproteínas/isolamento & purificação , Linfócitos T Citotóxicos/metabolismo , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Glicosilação , Lectinas/metabolismo , Peso Molecular , Testes de Precipitina , Sialoglicoproteínas/metabolismoRESUMO
Following the previous ultrastructural demonstration of the presence of arylsulphatase (Asase) activities in Kurloff cells (KC) and of their quasi-exclusive localization in the Kurloff body (KB), this work investigates their biochemical and zymographic properties after extraction from purified KC suspensions. Using the discriminative inhibitory conditions of both the Baum or Lee-Vaupel and Conzelmann methods, nitrocatechol sulphate hydrolyzing enzymes of the KC were assumed to belong to the B class of the type II Asase alone. After electrophoretic separation under non-denaturing conditions in a 4-23% polyacrylamide gel, they were characterized by 55 kDa and 62 kDa zymographic bands. After isoelectric focusing, 'classical' cationic isoforms (pI 8.5) and two anionic isoforms (pI 4.4 and 4.6) were observed on zymograms. As expected for class B Asase, the different zymographic forms of KC Asase were only recovered in the unadsorbed fraction after anion-exchange chromatography on DEAE-cellulose column equilibrated with high ionic strength buffer. Their Km (2.1 mM), their optimum pH (5.8) and their inhibitions by sulfite, phosphate, sulphate and ascorbic acid as well as their slight stimulation by AgNO3 were also characteristic of this class of Asase. Finally, chondroitin-4-sulphate was shown to potentially be a physiological substrate for these lysosomal enzymes.
Assuntos
Arilsulfatases/metabolismo , Isoenzimas/metabolismo , Células Matadoras Naturais/enzimologia , Lisossomos/enzimologia , Organelas/enzimologia , Animais , Arilsulfatases/análise , Arilsulfatases/isolamento & purificação , Cromatografia DEAE-Celulose , Estradiol/farmacologia , Feminino , Cobaias , Focalização Isoelétrica , Isoenzimas/análise , Isoenzimas/isolamento & purificação , Células Matadoras Naturais/ultraestrutura , Cinética , Lisossomos/ultraestrutura , Masculino , Microscopia Eletrônica , Peso Molecular , Organelas/ultraestruturaRESUMO
This study deals with the acid phosphatase (AcPase) of the Kurloff body (KB), a large (10 microns diameter) periodic acid-Schiff-positive lysosomal inclusion body present in Kurloff cells (KC). KC AcPase were extracted, with similar yields, either with non-ionic detergent solution or after Dounce homogenization in low ionic strength buffer suggesting that they mainly correspond to hydrosoluble AcPase. After DEAE-cellulose chromatography of such crude Dounce-extracts, 97% of KC AcPase activity was recovered in the unbound glycoprotein fraction (peak I)1). The main protein content consisted of, as testified by SDS-PAGE analysis, major KC glycoproteins of 30-35 kDa. Thus, KC AcPase, and particularly sialoAcPase, may be assumed to correspond to these N-glycosylproteins among which the presence of alpha (2,6) sialoglycoproteins was previously established. Following electrophoresis on a 4-15% gradient native polyacrylamide gel or isoelectric focusing, the two size populations (200 kDa and 500 kDa) and up to 20 isoforms of KC AcPase were respectively detected by zymography in peak I. After Clostridium-derived sialidase digestion of peak I, the highly active bands observed at pH 3.5-5.2 disappeared. These zymographic patterns were similar to those obtained with crude extracts. After Concanavalin A (ConA)-Sepharose chromatography of peak I, the single ConA-bound glucosamine-labelled peak, eluted at 200 methyl-alpha-D-mannopyranose, contained the AcPase activity while the ConA-unbound peak was devoid of any acid phosphatase activity. After SDS-PAGE analysis, the ConA-bound fraction appeared to correspond only to a single broad protein band in the 30-35 kDa zone.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fosfatase Ácida/química , Glicoproteínas/química , Tecido Linfoide/citologia , Fosfatase Ácida/metabolismo , Animais , Cromatografia de Afinidade , Feminino , Cobaias , Tecido Linfoide/enzimologia , Masculino , Peso Molecular , Ácidos Siálicos/metabolismo , Frações Subcelulares/enzimologiaRESUMO
Treatment with a combination of erythromycin and rifampin has considerably improved survival rates of foals and immunocompromised patients suffering from severe pneumonia caused by Rhodococcus equi. Frequently, because of monotherapy, emergence of rifampin-resistant strains has been responsible for treatment failure. Using consensus oligonucleotides, we have amplified and sequenced the rifampin resistance (Rif(r))-determining regions of 12 rifampin-resistant R. equi strains isolated from three foals and of mutants selected in vitro from R. equi ATCC 3701, a rifampin-susceptible strain. The deduced amino acid sequences compared to those of four rifampin-susceptible R. equi strains showed several types of mutations. In 3 of the 10 strains isolated from one foal, His526Asn (Escherichia coli numbering) and Asp516Val mutations were associated with low-level resistance (rifampin MIC, 2 to 8 microg/ml), whereas His526Asp conferred high-level resistance (rifampin MIC, 128 microg/ml) in the 7 remaining strains. In strains from the two other foals, His526Asp and Ser531Leu mutations were found to be associated with high-level and low-level resistance, respectively. The in vitro mutants, highly resistant to rifampin, harbored His526Tyr and His526Arg substitutions. As described in other bacterial genera, His526, Ser531, and Asp516 are critical residues for rifampin resistance in R. equi, and the resistance levels are dependent on both the location and the nature of the substitution.
Assuntos
Infecções por Actinomycetales/veterinária , Antibióticos Antituberculose/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Doenças dos Cavalos/microbiologia , Mutação , Rhodococcus equi/efeitos dos fármacos , Rifampina/farmacologia , Infecções por Actinomycetales/microbiologia , Animais , Farmacorresistência Bacteriana/genética , Cavalos , Testes de Sensibilidade Microbiana , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/veterinária , Rhodococcus equi/genética , Análise de Sequência de DNARESUMO
Equine herpesviruses type 1 and 4 (EHV-1 and EHV-4) are ubiquitous in the equine population. One of their main properties is their ability to establish life-long latent infections in their hosts even in those with natural or vaccine-induced immunity. However, effect of vaccination status on prevalence and tissue tropism was not established. In this study, EHV-1 and EHV-4 were detected by polymerase chain reaction and by classical virus isolation from neural, epithelial and lymphoid tissues collected from unvaccinated (33) or vaccinated (23) horses. The percentage of EHV-1- and EHV-4-positive horses between vaccinates and unvaccinates was similar. Both viruses were detected in all tissues of both groups; in particular, lymph nodes draining the respiratory tract, nasal epithelium and nervous ganglia [i.e. trigeminal ganglia (TG)], which represent the main positive sites for EHV-1 and EHV-4. In vaccinated animals, the nervous ganglia (i.e. TG) were less frequently positive than in unvaccinated animals. Detection of positive TG was strongly correlated to the presence of EHV-1 in nasal epithelium.