Dynamics of stress protein expression in leishmania-infected macrophages: evidence for selective induction and recognition of leishmania heat shock protein 60

Heat shock protein (HSP) expression was examined in murine bone marrow-derived macrophages infected with stationary phase promastigotes of Leishmania donovani. Immunoblotting with a rabbit polyclonal antiserum raised against HSP60 from Heliothis virescens (moth), revealed the de novo appearance of proteins with subunit sizes of M r 65,000 and 67,000 in leishmania infected macrophages. Expression of the novel M r 65,000 and 67,000 proteins in infected cells was coordinately regulated and at 24 hr of infection reached maximal levels of 52-100% increases above initial levels at 3 hr. Proteins with identical electrophoretic mobilities and which were similarly regulated in response to heat were also detected in leishmania promastigotes. The appearance of these proteins in macrophages was specific to leishmania infection in that neither protein was detected in non-infected cells either in the basal state or following several treatments including: (i) infection with Y. pseudotuberculosis, (ii) phagocytosis of S. aureus, (iii) NaAsC>2, or (iv) heat shock. A monoclonal antibody that recognizes both mammalian HSP70 and HSP70 from Plasmodia detected single isoforms of both leishmania and murine HSP70 in infected cells and neither protein changed quantitatively during infection. Levels of host heat shock proteins 60, 70 and 90 did not change in response to infection, however, marked induction of the mammalian stress protein heme oxygenase-1 was observed under these conditions. Further evidence for selective expression of the M r 65,000 and 67,000 heat-regulated leishmania proteins was provided by the finding that as their concentration was increasing, the abundance of the leishmania surface protease gp63 in infected cells was noted to decrease. The prominence of leishmania HSP60 within infected macrophages suggested the possibility that it might be a target of the host response. To facilitate further studies concerned with the role of this protein in the host response and in the pathogenesis of the leishmaniases, the HSP60 gene of Leishmania major was cloned, sequenced and expressed. A XEMBL-3 L. major genomic library was screened with a PCR-generated DNA probe derived from a highly conserved region of the leishmania HSP60 gene. A single clone that hybridized strongly was selected and characterized. Sequence analysis revealed the presence of an open reading frame of 1770 bp encoding a putative polypeptide of 589 amino acids with a predicted size of M r 64,790 and with the highest degree of amino acid sequence similarity (56%) to HSP60 from T. cruzi. Less extensive amino acid sequence similarity (48%) was observed between the leishmania HSP60 and the corresponding human protein. Notably, regions of significant sequence dissimilarity between the leishmania and human proteins were identified and these were concentrated principally within the carboxy-terminal regions of the proteins. The entire coding region of the leishmania HSP60 gene was amplified by PCR, subcloned into the pET-3a vector and expressed in E. coli. Purified recombinant protein was used to examine sera from patients with tegumentary leishmaniasis from South America for the presence of antibodies to HSP60. Unlike sera from healthy, uninfected controls, sera from patients reacted strongly with recombinant leishmania HSP60. In contrast, these same sera showed little or no reactivity with recombinant mycobacterial HSP65. To examine the protective potential of LHSP60, immunization studies were done in susceptible Balb/c mice. Recombinant leishmania HSP60 alone appeared to confer early (within the first 5 weeks) resistance against infection with L. major as determined by reduced lesion sizes in HSP60 immunized mice. In contrast, long-lasting protection did not develop in rLHSP60 immunized mice as lesion sizes and parasite numbers in these animals at 7 weeks of infection did not differ from those observed in control animals. These findings provide evidence for the selective induction of leishmania HSP60 in infected macrophages and for the recognition of this protein in humans with leishmaniasis. Further studies of this protein should clarify its role in the host response to Leishmania and in disease pathogenesis.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Characterization of Murine Dendritic Cell Line JAWS II and Primary Bone Marrow-Derived Dendritic Cells in Chlamydia muridarum Antigen Presentation and Induction of Protective Immunity▿

Dendritic cells (DCs) appear to orchestrate much of the immunobiology of Chlamydia infection, but most studies of Chlamydia-DC interaction have been limited by the availability and heterogeneity of primary bone marrow-derived DCs (BMDCs). We therefore evaluated the immunobiology of Chlamydia muridarum infection in an immortal DC line termed JAWS II derived from BMDCs of a C57BL/6 p53-knockout mouse. JAWS II cells were permissive to the developmental cycle of Chlamydia. Infection-induced cell death was 50 to 80% less in JAWS II cells than in BMDCs. Chlamydia infected JAWS II cells and yielded infectious progeny 10-fold greater than that with primary BMDCs. JAWS II cells showed an expression pattern of cell activation markers and cytokine secretion following Chlamydia infection similar to that of primary BMDCs by up-regulating the expression of CD86, CD40, and major histocompatibility complex class II and secreting significant amounts of interleukin-12 (IL-12) but not IL-10. JAWS II cells pulsed with Chlamydia stimulated immune CD4+ T cells to secrete gamma interferon. Adoptive transfer of ex vivo Chlamydia-pulsed JAWS II cells conferred levels of immunity on C57BL/6 mice similar to those conferred by primary BMDCs. Taken together, the data show that JAWS II cells exhibit immunobiological characteristics and functions similar to those of primary BMDCs in terms of Chlamydia antigen presentation in vitro and antigen delivery in vivo. We conclude that the JAWS II cell line can substitute for primary BMDCs in Chlamydia immunobiological studies

Evaluación de Trichoderma sobre hongos contaminantes de semillas de palma híbrida interespecífica OxG (Elaeis oleifera x Elaeis guineensis)

The germination process of hybrid palm interspecific OxG (E. oleifera x E guineensis.) seeds in the company Indupalma ltda has filed a decrease in production, which is reflected in economic losses due to the existence of filamentous fungi that affect seeds. Previous studies have shown the potential controller Trichoderma sp. on these fungi. Commercial and native strains of T. harzianum and T. viride were evaluated against prevalent fungal contaminants of interspecific hybrid palm seeds. They were isolated and identified eleven strains of pathogenic fungi from interspecific hybrid palm seeds that were removed from the different stages of germination. Dual plate tests were conducted at prevalent fungi and the percent inhibition of radial growth (PICO) and mycoparasitism were determined. These fungi were inhibited in growth by the antagonistic effect of T. harzianum and T. viride. Similarly, T.viride showed a great potential as a biocontrol agent to inhibit over 60% seven out of eleven strains of contaminating fungi.O processo de germinação de sementes de palma hibrida interespecífica OxG (E. oleifera x E. guineensis) da empresa Indupalma Ltda., tem apresentado uma diminuição na produção, que se reflete em perdas económicas, devido à existência de fungos filamentosos que afetam as sementes. Estudos prévios evidenciaram o potencial controlador de Trichoderma sp. sobre estes fungos. Nesta pesquisa cepas nativas e comerciais de T. harzianum e T. viride foram avaliadas frente a fungos contaminantes prevalentes de sementes de palma hibrida interespecífica. Foram isolados e identificados onze fungos patógenos prevalentes a partir de sementes de palma hibrida interespecífica que foram eliminadas das diferentes etapas do processo de germinação. Foram realizados tests de placa dupla aos fungos contaminantes selecionados e se determinou a porcentagem de inibição de crescimento radial (PICR) e micoparasitismo. Estes fungos foram inibidos em seu crescimento pelo efeito antagonista de T. harzianum e T. viride. Da mesma forma T. viride comercial evidenciou grande potencial biocontrolador, inhibindo a mais do 60% das onze cepas fùngicas contaminantes.El proceso de germinación de semillas de palma hibrida interespecífica OxG (E. oleifera x E. guineensis) de la empresa Indupalma ltda ha presentado una disminución en la producción, lo que se ve reflejado en pérdidas económicas, debido a la existencia de hongos filamentosos que afectan las semillas. Estudios previos evidenciaron el potencial controlador de Trichoderma sp. sobre estos hongos. En esta investigación se evaluaron cepas nativas y comerciales de T. harzianum y T. viride frente a hongos contaminantes prevalentes de semillas de palma hibrida interespecífica. Se aislaron e identificaron once hongos patógenos prevalentes a partir de semillas de palma hibrida interespecífica que fueron eliminadas de las diferentes etapas del proceso de germinación. Se llevaron a cabo pruebas de plato dual a los hongos contaminantes seleccionados y se determinó el porcentaje de inhibición de crecimiento radial (PICR) y micoparasitismo. Estos hongos fueron inhibidos en su crecimiento por el efecto antagonista de T. harzianum y T. viride. De igual manera T. viride comercial evidenció gran potencial biocontrolador al inhibir por encima del 60% a siete de las once cepas de hongos contaminantes

Schistosomiasis in the People's Republic of China: the Era of the Three Gorges Dam

Summary: The potential impact of the Three Gorges Dam (TGD) on schistosomiasis transmission in China has invoked considerable global concern. The TGD will result in changes in the water level and silt deposition downstream, favoring the reproduction of Oncomelania snails. Combined with blockages of the Yangtze River's tributaries, these changes will increase the schistosomiasis transmission season within the marshlands along the middle and lower reaches of the Yangtze River. The changing schistosome transmission dynamics necessitate a comprehensive strategy to control schistosomiasis. This review discusses aspects of the epidemiology and transmission of Schistosoma japonicum in China and considers the pathology, clinical outcomes, diagnosis, treatment, immunobiology, and genetics of schistosomiasis japonica together with an overview of current progress in vaccine development, all of which will have an impact on future control efforts. The use of synchronous praziquantel (PZQ) chemotherapy for humans and domestic animals is only temporarily effective, as schistosome reinfection occurs rapidly. Drug delivery requires a substantial infrastructure to regularly cover all parts of an area of endemicity. This makes chemotherapy expensive and, as compliance is often low, a less than satisfactory control option. There is increasing disquiet about the possibility that PZQ-resistant schistosomes will develop. Consequently, as mathematical modeling predicts, vaccine strategies represent an essential component in the future control of schistosomiasis in China. With the inclusion of focal mollusciciding, improvements in sanitation, and health education into the control scenario, China's target of reducing the level of schistosome infection to less than 1% by 2015 may be achievable