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1.
FEMS Immunol Med Microbiol ; 43(3): 413-7, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15708316

RESUMO

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.


Assuntos
Burkholderia mallei/genética , Burkholderia pseudomallei/genética , Burkholderia/genética , Animais , Sequência de Bases , Burkholderia/isolamento & purificação , Burkholderia mallei/isolamento & purificação , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/isolamento & purificação , Burkholderia pseudomallei/patogenicidade , Primers do DNA/genética , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade , Especificidade da Espécie
2.
Br J Haematol ; 126(6): 828-36, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15352987

RESUMO

The mixed lymphocyte reaction (MLR) has been used extensively to measure alloreactive T cells. In clinical practice, a negative MLR of recipient T cells responding to donor cells does not necessarily mean that a donor-specific tolerance has been established. This discrepancy indicates that the presently used methods fail to demonstrate the full repertoire of alloreactive T cells. This could be the result of the fact that some alloreactive T cells do not respond in vitro but will mount a response towards alloantigens in vivo, or that some alloreactive T cells do not respond during the MLR but will respond later if the alloantigen stimulation remains. We therefore examined the non-proliferating T-cell population in a mouse primary alloreactive response. Spleen and lymph node cells derived from C57BL/6J (H-2(b)) mice were stained with carboxy-fluorescein diacetate succinimidyl ester and injected intravenously into C.B-17 severe combined immunodeficient (SCID) mice (H-2(d)). The donor cells were recovered 5 d after the injection. The non-proliferating T cells were sorted and were non-reactive to alloantigen stimulation in vitro. Nevertheless, these non-proliferating T cells could proliferate and cause acute graft-versus-host disease after being adoptively transferred to secondary recipients of SCID mice. These results suggest that there exists an alloreactive T-cell population that does not respond to in vitro alloantigen stimulation but can mount a delayed alloresponse in vivo.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Isoantígenos/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Doença Aguda , Transferência Adotiva , Animais , Divisão Celular/imunologia , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID
3.
J Eukaryot Microbiol ; 50(1): 70-5, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12674482

RESUMO

After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11-day-old cultures of B. pythoni, 68.8% of the organisms (= 2.2 x 10(8) cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze-fracture images of the plasma membrane of non-cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non-cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle-rich small depressions and smooth small protrusions observed on the P face and E face of non-cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non-cyst cells.


Assuntos
Blastocystis/citologia , Blastocystis/ultraestrutura , Répteis/parasitologia , Animais , Blastocystis/química , Parede Celular/química , Parede Celular/ultraestrutura , Células Cultivadas , Técnica de Fratura por Congelamento , Histocitoquímica , Humanos , Dinâmica Populacional
4.
Int J Parasitol ; 32(7): 789-804, 2002 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12062550

RESUMO

Despite being discovered more than 80 years ago, progress in Blastocystis research has been gradual and challenging, due to the small number of laboratories currently working on this protozoan parasite. To date, the morphology of Blastocystis hominis has been extensively studied by light and electron microscopy but all other aspects of its biology remain little explored areas. However, the availability of numerous and varied molecular tools and their application to the study of Blastocystis has brought us closer to understanding its biology. The purpose of this review is to describe and discuss recent advances in B. hominis research, with particular focus on new, and sometimes controversial, information that has shed light on its genetic heterogeneity, taxonomic links, mode of transmission, in vitro culture and pathogenesis. We also discuss recent observations that B. hominis has the capacity to undergo programmed cell death; a phenomenon similarly reported for many other unicellular organisms. There are still many gaps in our knowledge of this parasite. Although there is a growing body of evidence suggesting that B. hominis can be pathogenic under specific conditions, there are also other studies that indicated otherwise. Indeed, more studies are warranted before this controversial issue can be resolved. There is an urgent need for the identification and/or development of an animal model so that questions on its pathogenesis can be better answered. Another area that requires attention is the development of methods for the transfection of foreign/altered genes into B. hominis in order to facilitate genetic experiments.


Assuntos
Infecções por Blastocystis/parasitologia , Blastocystis hominis/classificação , Animais , Infecções por Blastocystis/patologia , Infecções por Blastocystis/transmissão , Blastocystis hominis/genética , Blastocystis hominis/ultraestrutura , Humanos
5.
J Med Microbiol ; 51(1): 76-82, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11800476

RESUMO

A novel 10-bp repeat (5'-CGACGCAGGC-3')34 was identified in a strain of Burkholderia pseudomallei, the first repetitive element found in this species. A pair of primers, based on the flanking sequences of the repetitive region, was used in PCR and DNA sequence analysis to determine the genomic structure and distribution of the repetitive element in 76 arabinose- (ara-) and 7 ara+ B. pseudomallei isolates. DNA fragments of 400-700 bp were amplified in all ara- isolates. Ara+ isolates were characterised by a uniform fragment of 402 bp. Nucleotide sequence analysis of these fragments revealed broad heterogeneity of the variable-number tandem repeats with 26 distinct alleles ranging between (5'-CGACGCAGGC-3')13 and (5'-CGACGCAGGC-3')45 identified in the ara- isolates. In contrast, a novel non-repetitive sequence was identified in each of the ara+ isolates. This was confirmed by Southern blot analysis. Such biotype-specific variable-number tandem repeats may be useful as genetic markers for rapid strain differentiation of ara- isolates.


Assuntos
Arabinose/metabolismo , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/genética , DNA Bacteriano/análise , Sequências de Repetição em Tandem , Alelos , Sequência de Aminoácidos , Animais , Técnicas de Tipagem Bacteriana , Sequência de Bases , Southern Blotting , Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidade , DNA Bacteriano/química , Eletroforese em Gel de Ágar , Marcadores Genéticos , Humanos , Dados de Sequência Molecular , Peso Molecular , Técnicas de Amplificação de Ácido Nucleico , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Virulência
6.
Infect Immun ; 70(2): 504-11, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11796576

RESUMO

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with protean clinical manifestations. The major route of infection is thought to be through subcutaneous inoculation of contaminated soil and water, although ingestion and inhalation of contaminated aerosols are also possible. This study examines infection through the intranasal route in a murine model to mimic infection through inhalation. Two strains of mice, C57BL/6 and BALB/c, exhibit differential susceptibilities to the infection, with the C57BL/6 mice being considerably more resistant. To examine host factors that could contribute to this difference, bacterial loads and cytokine profiles in the two strains of mice were compared. We found that infected BALB/c mice exhibited higher bacterial loads in the lung and spleen and that they produced significantly higher levels of gamma interferon (IFN-gamma) in the serum than C57BL/6 mice. Although tumor necrosis factor alpha and interleukin-1 could be detected in the nasal washes and sera of both strains of mice, the production in serum was transient and much lower than that of IFN-gamma. C57BL/6 mice also exhibited memory responses to bacteria upon reinfection, with the production of serum immunoglobulin G (IgG) and mucosal IgA antibodies. Thus, it is possible that the production of systemic and mucosal antibodies is important for protection against disease in C57BL/6 mice.


Assuntos
Melioidose/imunologia , Mucosa Nasal/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Burkholderia pseudomallei/imunologia , Burkholderia pseudomallei/fisiologia , Citocinas/sangue , Citocinas/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Imunidade nas Mucosas , Interferon gama/genética , Fígado/imunologia , Pulmão/imunologia , Melioidose/sangue , Melioidose/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Líquido da Lavagem Nasal/imunologia , Especificidade da Espécie , Baço/imunologia
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