April 28-May 4, 2001
J Immunol Methods 2001 Jun
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(+)
PMID: 11334981 [PubMed - as supplied by publisher]
J Immunol Methods 2001 Jun 1;252(1-2):15-23
- Isolation and
characterization of rhesus blood dendritic cells using flow
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,
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
PMID: 11334961 [PubMed - as supplied by publisher]
Cell Immunol 2001 Mar 15;208(2):73-9
- Influence 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,
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
PMID: 11333139 [PubMed - in process]
Histol Histopathol 2001 Apr;16(2):655-67
- Towards a new generation of
vaccines: the cytokine IL-12 as an adjuvant to enhance
cellular immune responses to pathogens during prime-booster
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):255-256
- Will 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):128-30
- Persistence of Plasmodium
falciparum in the placenta after apparently effective
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
PMID: 11324359 [PubMed - in process]
Trends Parasitol 2001 May;17(5):242-9
- Targeting 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
PMID: 11323309 [PubMed - in process]
Trends Parasitol 2001 May;17(5):236-42
- Of mice and malaria mutants:
unravelling the genetics of drug resistance using rodent
Carlton JM, Hayton K, Cravo PV, Walliker D.
National Center for Biotechnology Research, National Library
of Medicine, National Institutes of Health, 20892, Bethesda,
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):219-23
- Pre-erythrocytic immunity to
Plasmodium falciparum: the case for an LSA-1 vaccine.
Kurtis JD, Hollingdale MR, Luty AJ, Lanar DE, Krzych U,
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):215-6
- Liver size a useful clinical
indicator in childhood malaria.
PMID: 11323296 [PubMed - in process]
Trends Parasitol 2001 May;17(5):215
- Malaria Immunology and
Pathogenesis Consortium (MIMPAC) formed.
PMID: 11323294 [PubMed - in process]
Trends Parasitol 2001 May;17(5):213
- Immunity to malaria increases
PMID: 11323290 [PubMed - in process]
Trends Parasitol 2001 May;17(5):209-11
- Plasmodium 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,
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):111-4
- Duffy-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,
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):2600-2608
- Pfnek-1, a NIMA-related
kinase from the human malaria parasite Plasmodium falciparum
Biochemical properties and possible involvement in MAPK
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.