ELISPOT assay to measure antigen-specific murine CD8(+) T cell responses.Carvalho LH, Hafalla JC, Zavala F.
Department of Medical and Molecular Parasitology, New York University Medical Center, 341 East 25th Street, 10010, New York, NY, USA
The enzyme-linked immunospot technique (ELISPOT) relies on the visualization of cytokine secretion by individual T cells following in vitro stimulation with antigen. This assay has been developed and standardized for the quantitative detection of antigen-specific CD8(+) T cells in mice subjected to different immunization protocols [J. Immunol. Methods 181 (1995) 45]. We have identified important variables that affect the efficacy of the ELISPOT assay and in this protocol we describe this methodology in detail. As a model, we used the production of interferon-gamma by CD8(+) T cells from peripheral blood, spleen and liver of mice immunized with malaria sporozoites expressing the H-2K(d)-restricted SYVPSAEQI. This protocol has also been used successfully to detect Th1 and Th2 epitope specific CD4(+) T cells.
PMID: 11334981 [PubMed – as supplied by publisher]
J Immunol Methods 2001 Jun 1;252(1-2):15-23Isolation and characterization of rhesus blood dendritic cells using flow cytometry.
Pichyangkul S, Saengkrai P, Yongvanitchit K, Limsomwong C, Gettayacamin M, Walsh DS, Stewart VA, Ballou WR, Heppner DG.
Department of Immunology and Medicine, US Army Medical Component, US Armed Forces Research Institute of Medical Science (AFRIMS), 315/6 Rajvithi Road, 10400, Bangkok, Thailand
Recognition of dendritic cells (DCs) as initiators and modulators of immune responses and growing use of rhesus monkeys for the preclinical optimization of vaccine formulations prompted characterization of the phenotype and function of isolated rhesus peripheral blood DCs. We developed a flow cytometric method to directly identify and isolate DCs from rhesus peripheral blood whereby a T cell depleted population negative for CD3, CD14, CD16 and CD20 but positive for CD83 yielded a cell population with surface markers, morphology, and a cytokine profile similar to human myeloid DCs. Rhesus blood DCs were more effective than monocytes and B cells in mixed lymphocyte reactions and in the presentation of recombinant malaria blood stage antigen MSP-1((42)) to autologous T cells. The ability to isolate rhesus blood DC from peripheral blood should be a useful tool for immunological investigations.
PMID: 11334961 [PubMed – as supplied by publisher]
Cell Immunol 2001 Mar 15;208(2):73-9Influence of Tumor Necrosis Factor-alpha on the Ability of Monocytes and Lymphocytes to Destroy Intraerythrocytic Plasmodium falciparum in Vitro.
Muniz-Junqueira MI, dos Santos-Neto LL, Tosta CE.
Laboratorio de Imunologia Celular, Patologia, Clinica Medica, Faculdade de Medicina, Universidade de Brasilia, Brasilia, DF, 70.910-900, Brazil
It has been shown that administration of TNF-alpha causes an increase of survival of plasmodium-infected mice. However, this anti-parasitic effect cannot be reproduced in vitro upon direct incubation of the cytokine with the parasite. This suggests that TNF-alpha may act through modulation of some plasmodicidal mechanism not yet clarified. We evaluated the effect of exogenous TNF-alpha on the phagocytosis of Plasmodium falciparum-infected erythrocytes by monocytes and its influence on the ability of monocytes and lymphocytes to inhibit parasite growth. The capacity of endogenous TNF-alpha to influence the ability of monocytes to inhibit the parasite was also verified. We found that addition of 33 ng TNF-alpha/mL to cultures of human monocytes and P. falciparum-infected erythrocytes increased the phagocytic index from 3.8 to 7.8 in the presence of serum containing P. falciparum antibody. TNF-alpha increased the capacity of monocyte plus lymphocyte to inhibit parasite growth by about 3 times at 0.5 and 5 ng/mL. Sera from severely ill P. falciparum-infected individuals inhibited the parasite growth, but addition of anti-TNF-alpha antibody was unable to modify this inhibition. These data show that TNF-alpha can increase the phagocytic capacity. This was probably due to an increased expression of Fc receptors on monocytes or to the modulation of Fc receptor signaling pathways by signals originating from the binding of TNF-alpha to its receptors. TNF-alpha also acted on lymphocytes plus monocytes by increasing the inhibition of P. falciparum by a mechanism not related to phagocytosis. These findings suggest that TNF-alpha has a pleiotropic anti-malaria effect and that this protective effect depends on the interplay of different factors, such as monocytes/macrophages, lymphocytes, and antibodies, in addition to other cells and molecules. Copyright 2001 Academic Press.
PMID: 11333139 [PubMed – in process]
Histol Histopathol 2001 Apr;16(2):655-67Towards a new generation of vaccines: the cytokine IL-12 as an adjuvant to enhance cellular immune responses to pathogens during prime-booster vaccination regimens.
Gherardi MM, Ramirez JC, Esteban M.
Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologia, CSIC, Madrid, Spain.
A main goal of the industrialized world is the development of effective vaccines to control infectious diseases with major health and socio-economic impact. Current understanding of the immune response triggered during infection with pathogens causing malaria, hepatitis C and AIDS emphasizes the importance of cytotoxic T lymphocytes (CTLs) in combating these infections. This has led to the development of new vaccination strategies, some of which are in phase I/II clinical trials. Promising strategies of vaccination are based on highly attenuated viral vectors, such as Vaccinia virus (VV) in combination with heterologous like vectors naked DNA, referred to as priming/booster vaccination. While these immunization schedules increased the production of specific CTLs, there is a need to further expand the CD8+T cell population to control an infection. Among molecules that play a significant role in the modulation of the CTL response is the cytokine IL-12. Immunoregulation by IL-12 is of central importance in cell-mediated immunity (CMI) against those pathogens and tumors that are controlled by cell-mediated mechanisms, supported by Thl cells. The use of this cytokine in combination with highly immunogenic VV-derived vectors is a promising system for development of future vaccination schedules. In this review, we summarize recent data on the use of IL-12 in vaccination procedures, as well as undesired side-effects of the cytokine that can be overcome by accurate use of dose, route and time-window administration of IL-12 encoding vectors. Results described here indicate that VV IL-12-mediated enhancement of the specific CMI response against a model antigen HIV-1 env was time- and dose-dependent and that the antigen and the cytokine could be expresed from two different rVVs modulating the doses of the vectors and allowing for enhancement of a specific CMI response. Moreover, the use of IL-12 during DNA prime/VV boost regimens enhanced the specific anti-HIV-1 env cellular response 20 times compared to that generated after a single rVVenv inoculation. Variables such as: a) dose of the cytokine applied, b) time of its administration and c) routes of inoculation play a critical role in the final outcome of the response. The findings presented here can be extended to other antigens, suggesting that immunomodulatory cytokines can be useful in the development of the future vaccines against numerous infectious diseases and tumors.
PMID: 11332721 [PubMed – in process]
Br J Haematol 2001 Apr;113(1):255-256Will the high rates of post-treatment sexual stage parasitaemia seen in malaria-endemic areas make the optiMAL antigen test unreliable in predicting malaria treatment outcome?
Tjitra E, Anstey NM.
National Institute of Health Research and Development, Ministry of Health, Jakarta, Indonesia, and Tropical Medicine and International Health Unit, Menzies School of Health Research and Northern Territory Clinical School, Darwin, Australia. [email protected]
PMID: 11328311 [PubMed – as supplied by publisher]
J Perinatol 2001 Mar;21(2):128-30Persistence of Plasmodium falciparum in the placenta after apparently effective quinidine/clindamycin therapy.
Procop GW, Jessen R, Hyde SR, Scheck DN.
Section of Microbiology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
The persistence of Plasmodium falciparum in the placenta after apparently adequate therapy with quinine has been described. We describe this phenomenon in the placenta of a 19-year-old woman with falciparum malaria, who was treated with a combination of quinidine and clindamycin. Although this therapy was effective and diminished her peripheral blood parasitemia from 3% at presentation to almost undetectable at the time of delivery, vast numbers of P. falciparum-infected erythrocytes were present in the maternal sinusoids of the placenta. This sequestration of infected erythrocytes produced a local parasitemia in the placenta of 70% to 80%. Additionally, rare Plasmodium-infected erythrocytes were also seen in the fetal blood of the placenta. We review malaria in pregnancy, parasitic involvement of the placenta and emphasize that Plasmodium-infected erythrocytes may persist in the placenta even after clearance of parasites from the peripheral blood.
PMID: 11324359 [PubMed – in process]
Trends Parasitol 2001 May;17(5):242-9Targeting polyamines of parasitic protozoa in chemotherapy.
Muller S, Coombs GH, Walter RD.
Wellcome Trust Biocentre, University of Dundee, DD1 5EH, UK
All parasitic protozoa contain polyamines and in recent years they, and their associated enzymes, have attracted attention as drug targets because they might reveal novel antiparasite therapies. How justified is this approach to drug discovery? In this review, Sylke Muller, Graham Coombs and Rolf Walter summarize the current status of research into drugs that exploit polyamine metabolism of trypanosomatid and malaria parasites, and propose priorities for research into such drugs. This review was inspired by an Expert Meeting entitled ‘Polyamine Metabolism of Parasitic Protozoa as a Drug Target'(*).
PMID: 11323309 [PubMed – in process]
Trends Parasitol 2001 May;17(5):236-42Of mice and malaria mutants: unravelling the genetics of drug resistance using rodent malaria models.
Carlton JM, Hayton K, Cravo PV, Walliker D.
National Center for Biotechnology Research, National Library of Medicine, National Institutes of Health, 20892, Bethesda, MD, USA
It is well recognized that drug resistance is the most significant obstacle to gaining effective malaria control. Despite the enormous advances in the knowledge of the biochemistry and molecular biology of malaria parasites, only a few genes determining resistance to the commonly used drugs have been identified. The idea that rodent malaria parasites should be exploited more widely for such work, in view of the practical problems of studying this subject experimentally in human malaria, is presented.
PMID: 11323308 [PubMed – in process]
Trends Parasitol 2001 May;17(5):219-23Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine.
Kurtis JD, Hollingdale MR, Luty AJ, Lanar DE, Krzych U, Duffy PE.
Walter Reed Army Institute of Research, Dept of Immunology, 20910, Silver Spring, MD, USA
A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.
PMID: 11323304 [PubMed – in process]
Trends Parasitol 2001 May;17(5):215-6Liver size a useful clinical indicator in childhood malaria.
PMID: 11323296 [PubMed – in process]
Trends Parasitol 2001 May;17(5):215Malaria Immunology and Pathogenesis Consortium (MIMPAC) formed.
PMID: 11323294 [PubMed – in process]
Trends Parasitol 2001 May;17(5):213Immunity to malaria increases during puberty.
PMID: 11323290 [PubMed – in process]
Trends Parasitol 2001 May;17(5):209-11Plasmodium invasion of mosquito cells: hawk or dove?
Sinden RE, Billingsley PF.
Dept of Biology, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, SW7 2AZ, London, UK
In the past five years, there has been renewed interest in the early development of the malaria parasite in the mosquito. Numerous exciting studies have examined in more detail the cellular and molecular interactions of the ookinete with the peritrophic matrix, midgut epithelium and basal lamina of the mosquito midgut, and a plethora of new responses by the mosquito to this invasion process have been described.
PMID: 11323288 [PubMed – in process]
FEBS Lett 2001 Apr 20;495(1-2):111-4Duffy-null promoter heterozygosity reduces DARC expression and abrogates adhesion of the P. vivax ligand required for blood-stage infection.
Michon P, Woolley I, Wood EM, Kastens W, Zimmerman PA, Adams JH.
The Duffy blood group antigen is an essential receptor for Plasmodium vivax entry into erythrocytes in a process mediated by the parasite ligand, the Duffy binding protein (DBP). Recently, individuals living in a malaria endemic region of Papua New Guinea were identified as heterozygous for a new allele conferring Duffy negativity, which results in 50% less Duffy antigen on their erythrocytes. We demonstrate that DBP adherence to erythrocytes is significantly reduced for erythrocytes from heterozygous individuals who carry one Duffy antigen negativity allele. These data provide evidence that emergence of this new allelic form of Duffy negativity is correlated with resistance against vivax malaria.
PMID: 11322957 [PubMed – in process]
Eur J Biochem 2001 May;268(9):2600-2608Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum Biochemical properties and possible involvement in MAPK regulation.
Dorin D, Le Roch K, Sallicandro P, Alano P, Parzy D, Poullet P, Meijer L, Doerig C.
Institut National de la Sante et de la Recherche Medicale (INSERM), Paris, France; Istituto Superiore de Sanita, Roma, Italy; Institut de Medecine Tropicale du Service de Sante des Armees (IMTSSA), Marseille, France; The European Bioinformatics Insitute, EMBL Cambridge Outstation, Cambridge, UK. Station Biologique de Roscoff, CNRS, Roscoff, France.
We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials.
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