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Sat Feb 18 11:22:33 BRST 2006

phlebotomine were done to the insect monitoring. The collection was done
 in an area of infection of American cutaneous leishmaniasis in the 
basin of the Araguari River in the municipality of Uberlandia (MG).The 
first collection was made in May (a cold, damp month), the second in 
June 2000 (a cold, dry month) the third in October 2000 (a hot, dry 
month) and the fourth in January 2001 (a hot, rainy month).CDC and 
Shannon light traps were used 6551 phlebotomne were captured and 
identified, 1990 male and 4562 female, comprised of two lines (Lutzomyia
 and Brumptomyia) and 8 species. Lutzomyia intermedia predominated with 
the largest number of specimens (6531), which accounted for 99,7% of the
 collected insects. In the four collections, it was observed that 
Lutzomyia intermedia manifested a preference for the month preceding the
 rainy season, with its high temperatures and humidity.

PMID: 15712555

TITLE: Diagnosis of cutaneous leishmaniasis by fine needle aspiration cytology:
a report of 66 cases.

AUTHORS: Masoom Kassi, Pashtoon Murtaza Kasi

REFERENCE: Trop Doct 2005 Jan 35(1):50-1

PMID: 15721387

TITLE: Tuftsin-bearing liposomes as antibiotic carriers in treatment of
macrophage infections.

AUTHORS: C M Gupta, W Haq

REFERENCE: Methods Enzymol 2005  391():291-304

Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg) that specifically binds 
monocytes, macrophages, and polymorphonuclear leukocytes and potentiates
 their natural killer activity against tumors and pathogens. The 
antimicrobial activity of this peptide is significantly increased by 
attaching at the C-terminus a fatty acyl residue through the 
ethylenediamine spacer arm. This activity is further augmented by 
incorporating the modified tuftsin in the liposomes. The tuftsin-bearing
 liposomes not only enhance the host's resistance against a variety of 
infections but also serve as useful vehicles for the site-specific 
delivery of drugs in a variety of macrophage-based infections, such as 
tuberculosis and leishmaniasis.

PMID: 15613100

TITLE: Soluble hemoglobin-haptoglobin scavenger receptor CD163 as a
lineage-specific marker in the reactive hemophagocytic syndrome.

AUTHORS: Dominik J Schaer, Boris Schleiffenbaum, Michael Kurrer, Alexander
Imhof, Esther BÃ¤chli, JÃ¶rg Fehr, Holger J Moller, Soren K Moestrup, Andreas
Schaffner

AFFILIATION: Department of Medicine, University Hospital, Zurich, Switzerland.
schaer_dominik at hotmail.com

REFERENCE: Eur J Haematol 2005 Jan 74(1):6-10

Reactive hemophagocytic syndrome (RHS) is a disease of overwhelming 
macrophage activity triggered by infection, malignancy or autoimmune 
disorders. Currently used laboratory markers for the quantitative 
assessment of monocyte/macrophage activation lack lineage-restricted 
expression patterns and thus specificity. Serum levels of the macrophage
 specific scavenger receptor CD163 were determined by enzyme-linked 
immunosorbent assay (ELISA) and were found to be highly increased in 
patients with RHS (median 39.0 mg/L). Significantly lower levels were 
determined in patients with sepsis (median 9.1 mg/L), acute 
mononucleosis (median 8.2 mg/L), Leishmania infection (median 6.7 mg/L) 
and healthy controls (median 1.8 mg/L). Follow-up of patients with a 
relapsing course of the disease revealed close correlations of sCD163 
with clinical disease activity, serum ferritin and other markers of 
macrophage activity. Large sinusoidal accumulations of CD163 expressing 
macrophages actively engaged in phagocytosis of blood cells were 
detected in spleen sections of RHS patients. Our data suggests sCD163 to
 be a macrophage-specific marker in patients with disorders of 
inappropriate macrophage activation.

PMID: 15715249

TITLE: Diagnostic value of rK39 dipstick in zoonotic visceral leishmaniasis in
Turkey.

AUTHORS: Seray Ozensoy Toz, Kwang-Poo Chang, Yusuf Ozbel, M Ziya Alkan

AFFILIATION: Department of Parasitology, Ege University Medical School, 35100
Bornova-Izmir, Turkey.

REFERENCE: J Parasitol 2004 Dec 90(6):1484-6

K39 is a repetitive immunodominant epitope in a kinesin-related protein 
expressed predominantly in the amastigotes of visceral Leishmania spp. 
Enzyme immunoassays of patient's sera with recombinant K39 (rK39) proved
 to be highly specific and sensitive for diagnosis of active visceral 
leishmaniasis (VL, kala-azar). The same assays in dipstick format were 
also found effective for diagnosis of both human VL (HVL) and canine VL
 (CanVL) in most endemic areas of these diseases. Fifty-eight human 
patients and 22 dogs, clinically suspected of kala-azar, were screened 
with rK39 dipstick in comparison with the conventional methods of 
diagnosis, i.e., microscopic examinations of bone marrow and lymph node 
aspirates and immunofluorescent antibody tests (IFAT), respectively. 
Sixteen patients and 12 dogs were found to be rK39 dipstick positive. 
The results were corroborated with those of parasitological examinations
, except 1, rK39-positive but smear-negative, case in each group. IFAT 
of the 2 discordant cases gave positive results. The rK39 dipstick is 
thus reliable for diagnosis of both HVL and canVL cases in Turkey.

PMID: 15720898

TITLE: Is hypertriglyceridaemia a new concept for visceral leishmaniasis?

AUTHORS: Omer Erdeve, Yildaz Dallar, Zeynep Siklar

AFFILIATION: Department of Pediatrics, Ankara Education and Research Hospital,
Ankara, Turkey.

REFERENCE: Ann Trop Paediatr 2004 Dec 24(4):369

PMID: 15356347

TITLE: Leashing Leishmania.Some old mice fight parasitic infection better than
young mice do.

AUTHORS: R John Davenport

REFERENCE: Sci Aging Knowledge Environ 2004 Sep 2004(36):nf82

PMID: 15533297

TITLE: Efficacy of Trypan: a diminazene based drug as antileishmanial agent.

AUTHORS: J C Macharia, A J Bourdichon, M M Gicheru

AFFILIATION: Department of Parasitology, Institute of Primate Research, National
Museums of Kenya, P.O. Box 24481, Karen, Nairobi, Kenya.

REFERENCE: Acta Trop 2004 Nov-Dec 92(3):267-72

Trypan, a diamidine based drug, was tested as an antileishmanial agent. 
Duplicate cultures of both Leishmania major and Leishmania donovani 
promastigotes in M199 medium and Trypan at various concentrations were 
tested. The cultures were incubated at 25 degrees C and parasites 
counted at 48 h interval, and the data generated was used to establish 
growth inhibition curves. Drug-free cultures were included to serve as 
control. In the in vivo study, a total of 40 BALB/c mice were divided 
into five groups of 8 mice each. They were infected with 2 x 10(6) 
promastogotes on the left footpad. Two groups were treated with 70 
microg/ml of Trypan, a total of 500 microl used immediately after 
infection, one group by topical application and the other administered 
intraperitoneally. The treatments were repeated for the two other groups
 10 weeks post infection, one by topical application and the other 
administered intraperitoneally. One group was not treated and thus 
served as control. Footpad sizes were measured using Vernier calliper 
every 2 weeks for 21 weeks. In the in vitro studies, Trypan inhibited 
growth of either L. major or L. donovani promastigotes in all the 
concentrations tested with more dramatic inhibition in high 
concentrations. Based on the in vivo studies, it was evident that Trypan
 had effect on L. major infected lesions when applied topically 
immediately after infection. However, there was no effect when treatment
 commenced after the lesions were established. The data is discussed.

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PMID: 15579454

TITLE: Distinct roles for lymphotoxin-alpha and tumor necrosis factor in the
control of Leishmania donovani infection.

AUTHORS: Christian R Engwerda, Manabu Ato, Simona StÃ¤ger, Clare E Alexander,
Amanda C Stanley, Paul M Kaye

AFFILIATION: Immunology and Infection Laboratory and Australian Centre for
International and Tropical Health and Nutrition, Queensland Institute of
Medical Research, Herston, Queensland, Australia 4029. chrise at qimr.edu.au

REFERENCE: Am J Pathol 2004 Dec 165(6):2123-33

Tumor necrosis factor (TNF) is critical for the control of visceral 
leishmaniasis caused by Leishmania donovani. However, the role of the 
related cytokine lymphotoxin (LT) alpha in this infection is unknown. 
Here we report that C57BL/6 mice deficient in TNF (B6.TNF(-/-)) or LT 
alpha (B6.LT alpha(-/-)) have increased susceptibility to hepatic L. 
donovani infection. Furthermore, the outcome of infection in bone marrow
 chimeric mice is dependent on donor hematopoietic cells, indicating 
that developmental defects in lymphoid organs were not responsible for 
increased susceptibility to L. donovani. Although both LT alpha and TNF 
regulated the migration of leukocytes into the sinusoidal area of the 
infected liver, their roles were distinct. LT alpha was essential for 
migration of leukocytes from periportal areas, an event consistent with 
LT alpha-dependent up-regulation of VCAM-1 on liver sinusoid lining 
cells, whereas TNF was essential for leukocyte recruitment to the liver
. During visceral leishmaniasis, both cytokines were produced by radio-
resistant cells and by CD4(+) T cells. LT alpha and TNF production by 
the former was required for granuloma assembly, while production of 
these cytokines by CD4(+) T cells was necessary to control parasite 
growth. The production of inducible nitric oxide synthase was also found
 to be deficient in TNF- and LT alpha-deficient infected mice. These 
results demonstrate that both LT alpha and TNF are required for control 
of L. donovani infection in noncompensatory ways.

PMID: 15305696

TITLE: [Nitric oxide and anti-protozoan chemotherapy]

AUTHORS: L Gradoni, P Ascenzi

AFFILIATION: Reparto di Malattie Trasmesse da Vettori e SanitÃ  Internazionale,
Dipartimento MIPI, Istituto Superiore di SanitÃ , Roma, Italy.

REFERENCE: Parassitologia 2004 Jun 46(1-2):101-3

Constitutive nitric oxide (NO) is generated by constitutively expressed 
types of NO-synthase enzymes (NOS-I and -III), being involved in 
physiological processes such as nervous transmission and vasodilatation
. Inducible NO, synthesized by the NO-synthase isoform NOS-II, is an 
anti-pathogen and tumoricidal agent. However, inducible NO production 
requires a tight control because of cytotoxic and immune-modulation 
activity. NO produced by human and canine macrophages has long been 
demonstrated to be involved in the intracellular killing of Leishmania. 
Mechanisms of parasite survival and persistence in the host have been 
throughly investigated, and include suppression of NOS-II and the 
parasite entry into NOS-II negative cells. Both intracellular and 
extracellular morphotypes of Trypanosoma cruzi are killed by NO in vitro
 and in vivo, although a role of NO in the pathogenesis of heart disease
 has been reported. Killing of extracellular protozoa such as 
Trichomonas vaginalis and Naegleria fowleri by activated macrophages is 
also mediated by NO. The main control of Plasmodium spp infection in 
human and murine hepatocytes, and in human monocytes is achieved by NO-
mediated mechanisms. Protection from severe malaria in African children 
has been found associated with polymorphisms of the NOS-II promoter; 
however, a pathogenic role of endogenous NO has been documented in 
cerebral malaria. Although several macromolecules are putative NO 
targets, recent experimental work has shown that NO-releasing compounds 
inhibit cysteine proteases (CP) of P. falciparum, T. cruzi and L. 
infantum in a dose-dependent manner. CPs are present in a wide range of 
parasitic protozoa and appear to be relevant in several aspects of the 
life cycle and of the parasite-host relationships. Comparative analysis 
of 3-D amino acid sequence models of CPs from a broad range of living 
organisms, from viruses to mammals, suggests that the Sy atom of the Cys
 catalytic residue undergoes NO-dependent chemical modification (S-
nytrosilation and disulfide bridge formation), with the concomitant loss
 of enzyme activity. The NO-donor S-nitroso-N-acetilpenicillamine (SNAP
) was shown to kill T. cruzi epimastigotes and L. infantum promastigotes
 in culture, while a combination of nitrite plus acid organic salts was 
highly effective against L. major amastigotes in mouse macrophages. A 
parasitostatic effect--with both encystation and excystation inhibition
--of S-nitrosoglutathione and spermine-NONOate was documented in 
trophozoite cultures of Giardia duodenalis. Recently, a novel 
formulation of metronidazole bearing a NO-releasing group was found to 
enhance significantly the in vitro killing of Entamoeba histolytica 
trophozoites, compared to metronidazole. So far, only two clinical 
studies were performed on human patients, suffering from cutaneous 
leishmaniasis. In one study, 16 Ecuadorean patients were treated with a 
SNAP cream administered on lesions for 10 days. All lesions were 
parasitologically cured and clinically healed by day 30. In the second 
study, a different NO-producing cream (basically nitrite in acidic 
environment) was employed to treat 40 Syrian patients. Only 28% of them 
showed improvement and 12% were cured by day 60. In conclusion, despite 
the wide evidence that NO can be regarded as a natural anti-protozoal 
weapon, little efforts have been made to develop and test NO-based drugs
 in human medicine. This is mainly due to the difficulty in designing 
suitable chemical carriers able to release the right amount of NO, in 
the right place and in the right time, to avoid toxic effects against 
non-target host cells.

PMID: 15187137

TITLE: Protection against progressive leishmaniasis by IFN-beta.

AUTHORS: Jochen Mattner, Alexandra Wandersee-SteinhÃ¤user, Andreas Pahl, Martin
RÃ¶llinghoff, Gerard R Majeau, Paula S Hochman, Christian Bogdan

AFFILIATION: Institute of Clinical Microbiology, Immunology and Hygiene,
University of Erlangen-Nuremberg, Erlangen, Germany.

REFERENCE: J Immunol 2004 Jun 172(12):7574-82

Type I IFNs (IFN-alphabeta) exert potent antiviral and immunoregulatory 
activities during viral infections, but their role in bacterial or 
protozoan infections is poorly understood. In this study, we demonstrate
 that the application of low, but not of high doses of IFN-beta protects
 60 or 100% of BALB/c mice from progressive cutaneous and fatal visceral
 disease after infection with a high (10(6)) or low (10(4)) number of 
Leishmania major parasites, respectively. IFN-beta treatment of BALB/c 
mice restored the NK cell cytotoxic activity, increased the lymphocyte 
proliferation, and augmented the production of IFN-gamma and IL-12 in 
the draining lymph node. Low, but not high doses of IFN-beta caused 
enhanced tyrosine phosphorylation of STAT1 and STAT4, suppressed the 
levels of suppressor of cytokine signaling-1, and up-regulated the 
expression of inducible NO synthase in vivo. The IFN-beta-induced 
increase of IFN-gamma production was dependent on STAT4. Protection by 
IFN-beta strictly required the presence of inducible NO synthase. In the
 absence of STAT4 or IL-12, IFN-beta led to an amelioration of the 
cutaneous and visceral disease, but was unable to prevent its 
progression. These results identify IFN-beta as a novel cytokine with a 
strong, dose-dependent protective effect against progressive cutaneous 
leishmaniasis that results from IL-12- and STAT4-dependent as well as -
independent events.

PMID: 15109961

TITLE: Cathepsin L is crucial for a Th1-type immune response during Leishmania
major infection.

AUTHORS: Kotaro Onishi, Yang Li, Kazunari Ishii, Hajime Hisaeda, Lijun Tang,
Xuefeng Duan, Teruki Dainichi, Yoichi Maekawa, Nobuhiko Katunuma, Kunisuke
Himeno

AFFILIATION: Department Microbiology and Immunology, Faculty of Medical
Sciences, Kyushu University, Fukuoka 812-8582, Japan. koonishi at jikei.ac.jp

REFERENCE: Microbes Infect 2004 Apr 6(5):468-74

Prior to the activation of CD4+ T cells, exogenous proteins are digested
 by endo/lysosomal enzymes in antigen-presenting cells (APCs) to produce
 antigenic peptides that are presented on MHC class II molecules. In the
 studies described here, the functional significance of cathepsin L for 
antigen processing and Th1/Th2 differentiation in experimental 
leishmaniasis was investigated. We first demonstrated that cathepsin L 
is one of the candidates for endo/lysosomal enzymes in the processing of
 soluble Leishmania antigen (SLA) by using CLIK148, a specific inhibitor
 of cathepsin L. Treatment of BALB/c or DBA/2 mice with CLIK148 
exacerbated the disease by enhancing an SLA-specific Th2-type response 
such as IL-4 production. CLIK148 did not exert any direct influence on 
Leishmania major promastigotes themselves or on the course of L. major 
infection in SCID mice. Taken together, these findings suggest that 
treatment of host mice with CLIK148 affects the processing of SLA in 
APCs, resulting in the potentiation of Th2-type immune responses and 
thus leading to exacerbation of the disease. Furthermore, endo/lysosomal
 cathepsin L was found to be functionally distinct from previously 
described cathepsins B and D.

PMID: 15039311

TITLE: Toll-like receptor 4 contributes to efficient control of infection with
the protozoan parasite Leishmania major.

AUTHORS: Pascale Kropf, Marina A Freudenberg, Manuel Modolell, Helen P Price,
Shanti Herath, Simone Antoniazi, Chris Galanos, Deborah F Smith, Ingrid
MÃ¼ller

AFFILIATION: Faculty of Medicine, Department of Immunology, Imperial College
London, London, United Kingdom.

REFERENCE: Infect Immun 2004 Apr 72(4):1920-8

The essential role of Toll-like receptors (TLR) in innate immune 
responses to bacterial pathogens is increasingly recognized, but very 
little is known about the role of TLRs in host defense against 
infections with eukaryotic pathogens. For the present study, we 
investigated whether TLRs contribute to the innate and acquired immune 
response to infection with the intracellular protozoan parasite 
Leishmania major. Our results show that TLR4 contributes to the control 
of parasite growth in both phases of the immune response. We also 
addressed the mechanism that results in killing or growth of the 
intracellular parasites. Control of parasite replication correlates with
 the early induction of inducible nitric oxide synthase in TLR4-
competent mice, whereas increased parasite survival in host cells from 
TLR4-deficient mice correlates with a higher activity of arginase, an 
enzyme known to promote parasite growth. This is the first study showing
 that TLR4 contributes to the effective control of Leishmania infection 
in vivo.

PMID: 14768057

TITLE: Control of Leishmania major in the absence of Tyk2 kinase.

AUTHORS: Ulrike Schleicher, Jochen Mattner, Martin Blos, Heike Schindler, Martin
RÃ¶llinghoff, Marina Karaghiosoff, Mathias MÃ¼ller, Gabriele Werner-Felmayer,
Christian Bogdan

AFFILIATION: Institute of Clinical Microbiology, Immunology and Hygiene,
University of Erlangen-Nuremberg, Erlangen, Germany.

REFERENCE: Eur J Immunol 2004 Feb 34(2):519-29

IL-12 is indispensable for the control of many intracellular pathogens, 
but the components of the signaling pathway that are essential for its 
function in vivo are incompletely understood. Here, we investigated in 
the Leishmania major mouse model whether Tyk2 kinase is required for the
 generation of a protective immune response. Unlike C57BL/6 controls, 
Tyk2(-/-)mice developed severe skin lesions after infection that 
frequently ulcerated, but ultimately healed. NK cell cytotoxicity was 
absent in infected Tyk2(-/-) mice, even after IL-12 pretreatment, which 
correlated with a STAT4 activation defect. IFN-alpha / beta, which was 
still able to activate STAT1 in Tyk2(-/-) NK cells, reconstituted their 
cytotoxic activity, but not their IL-12 responsiveness. The IL-12-
induced production of IFN-gamma by NK cells and CD8(+) T cells was 
strongly suppressed in Tyk2(-/-) mice at day 1 of infection, but partly 
regained during the late phase of infection. Tyk2(-/-) CD4(+) T cells 
developed into Th1 cells (although in a delayed fashion) and infected 
Tyk2(-/-) mice expressed normals levels of inducible NO synthase. Thus, 
Tyk2 is required for the IL-12 response of NK cells and CD8(+) T cells 
in L. major-infected mice, but not for the generation of Th1 cells and 
the ultimate control of the disease.

PMID: 12933876

TITLE: Both the Fas ligand and inducible nitric oxide synthase are needed for
control of parasite replication within lesions in mice infected with Leishmania
major whereas the contribution of tumor necrosis factor is minimal.

AUTHORS: Reza Chakour, Reto Guler, MÃ©lanie Bugnon, Cindy Allenbach, IrÃ¨ne
Garcia, Jacques MauÃ«l, Jacques Louis, Fabienne Tacchini-Cottier

AFFILIATION: The World Health Organization Immunology Research and Training
Center and Institute of Biochemistry, University of Lausanne, Epalinges,
Switzerland.

REFERENCE: Infect Immun 2003 Sep 71(9):5287-95

Following infection with the protozoan parasite Leishmania major, C57BL/
6 mice develop a small lesion that heals spontaneously. Resistance to 
infection is associated with the development of CD4(+) Th1 cells 
producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), 
which synergize in activating macrophages to their microbicidal state. 
We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (
gld TNF(-/-) mice) infected with L. major neither resolved their lesions
 nor controlled Leishmania replication despite the development of a 
strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) 
activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld 
mice, but only gld and gld TNF(-/-) mice failed to control parasite 
replication. Parasite numbers were high in gld mice and even more 
elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the
 Fas/FasL pathway is required for successful control of parasite 
replication and that TNF contributes only a small part to this process. 
Furthermore, FasL was shown to synergize with IFN-gamma for the 
induction of leishmanicidal activity within macrophages infected with L
. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion 
size throughout infection, despite being able to largely control 
parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential
 for the complete control of parasite replication, while the 
contribution of TNF is more important in controlling inflammation at the
 site of parasite inoculation.

PMID: 12884305

TITLE: TNF-alpha mediates the induction of nitric oxide synthase in macrophages
but not in neutrophils in experimental cutaneous leishmaniasis.

AUTHORS: Simone G Fonseca, Pedro R T RomÃ£o, FlorÃªncio Figueiredo, Ruth H
Morais, HermÃªnio C Lima, SÃ©rgio H Ferreira, Fernando Q Cunha

AFFILIATION: Department of Biochemistry and Immunology, School of Medicine of
RibeirÃ£o Preto, University of SÃ£o Paulo, SÃ£o Paulo, Brazil.

REFERENCE: Eur J Immunol 2003 Aug 33(8):2297-306

Leishmania major infection in C57BL/6 mice is controlled by the 
activation of a Th1 response and nitric oxide (NO) production by 
macrophages. TNF-alpha is considered one of the most important cytokines
 involved in this response. In the present study, we investigated the 
expression of nitric oxide synthase (iNOS) in the inflammatory cells 
present in the lesion and draining lymph nodes, and the cytokine 
production by lymph node cells in animals treated with anti-TNF-alpha. 
Our results demonstrated that mice treated with anti-TNF-alpha presented
 an increase in the number of parasites and the size of lesion, but they
 were able to control the infection. The increase in the lesion size 
correlated to the reduction of iNOS activity in the draining lymph nodes
. Furthermore, the anti-TNF-alpha treatment also reduced the expression 
of iNOS in the macrophages, but did not affect the iNOS expression in 
the neutrophils. The anti-TNF-alpha mAb did not reduce the iNOS 
expression in IFN-gamma-stimulated L. major infected neutrophils in 
vitro. Anti-TNF-alpha mAb treatment caused an increase in the production
 of IFN-gamma and IL-10 by the lymph node cells from infected mice. 
Consequently, these results suggest that neutrophils do not respond to 
anti-TNF-alpha treatment and might be a source of NO to control L. major
 infection under these experimental conditions.

PMID: 12874364

TITLE: Both interleukin-4 (IL-4) and IL-4 receptor alpha signaling contribute to
the development of hepatic granulomas with optimal antileishmanial activity.

AUTHORS: Simona StÃ¤ger, James Alexander, K Christine Carter, Frank Brombacher,
Paul M Kaye

AFFILIATION: Department of Infectious and Tropical Diseases, London School of
Hygiene and Tropical Medicine, London, United Kingdom.

REFERENCE: Infect Immun 2003 Aug 71(8):4804-7

The roles of interleukin-4 (IL-4) and IL-13 in the regulation of 
immunity to Leishmania donovani infection are still poorly understood. 
Here we show that the increased parasite load observed in IL-4(-/-) and 
IL-4 receptor alpha(-/-) mice correlates with retarded granuloma 
maturation and antileishmanial activity and that the increased parasite 
load observed in IL-4 receptor alpha(-/-) mice correlates with increased
 NOS2 expression and decreased serum gamma interferon levels. IL-4 and 
IL-13 appear to play little role in regulating collagen deposition in L
. donovani-induced granulomas.

PMID: 12870129

TITLE: Determinants of response to interleukin-10 receptor blockade
immunotherapy in experimental visceral leishmaniasis.

AUTHORS: Henry W Murray, Andre L Moreira, Cristina M Lu, Jennifer L DeVecchio,
Maki Matsuhashi, Xiaojing Ma, Frederick P Heinzel

AFFILIATION: Department of Medicine, Weill Medical College of Cornell
University, New York, USA. hwmurray at med.cornell.edu.

REFERENCE: J Infect Dis 2003 Aug 188(3):458-64

In established Leishmania donovani visceral infection in normal mice, 
anti-interleukin (IL)-10 receptor (IL-10R) monoclonal antibody (MAb) 
treatment induced intracellular parasite killing within liver 
macrophages. IL-10R blockade maintained IL-12 protein 40, markedly 
increased interferon (IFN)-gamma serum levels, and enhanced tissue 
inducible nitric oxide synthase (iNOS) expression and granuloma assembly
. Optimal MAb-induced killing, including synergism with antimony 
chemotherapy, required endogenous IL-12 and/or IFN-gamma and at least 
one IFN-gamma-regulated macrophage mechanism-iNOS or phagocyte oxidase. 
However, in IFN-gamma knockout mice, anti-IL-10R also induced both 
granuloma formation and leishmanistatic activity. As judged by IL-10R 
blockade, endogenous IL-10 primarily regulates killing in L. donovani 
infection by suppressing production of and responses to the Th1 cell-
type cytokines, IL-12, and IFN-gamma. However, because anti-IL-10R also 
released IFN-gamma-independent effects, IL-10 appears to act more 
broadly and suppresses multiple antileishmanial mechanisms.

PMID: 12778468

TITLE: Targeting of immunostimulatory DNA cures experimental visceral
leishmaniasis through nitric oxide up-regulation and T cell activation.

AUTHORS: Neeta Datta, Snigdha Mukherjee, Lopamudra Das, Pijush K Das

AFFILIATION: Molecular Cell Biology Laboratory, Indian Institute of Chemical
Biology, Calcutta, India.

REFERENCE: Eur J Immunol 2003 Jun 33(6):1508-18

Active targeting of CpG-containing oligodeoxynucleotide (CpG-ODN) to 
macrophages was studied by incorporating it in mannose-coated liposomes
, using visceral leishmaniasis as the model macrophage disease. 
Mannosylated liposomal CpG-ODN was more effective than liposomal or free
 CpG-ODN in inhibiting amastigote multiplication within macrophages. 
Moreover, in a 60-day mouse model of visceral leishmaniasis, complete 
elimination of spleen parasite burden was achieved by mannosylated 
liposomal CpG-ODN, compared to 62% and 81% parasite suppression by free 
and liposomal ODN, respectively, at a similar dose. Although in vitro 
exposure of CpG-ODN did not induce marked nitric oxide (NO) generation 
by macrophages, considerably enhanced amount of NO was generated by 
macrophages of CpG-ODN-treated animals. Their splenocytes secreted 
soluble factors required for the induction of NO generation, and the 
increased NO generation was paralleled by an increase in antileishmanial
 activity. Inducible NO generation was suppressed by treating splenocyte
 supernatants with anti-IFN-gamma or anti-IL-12 antibodies, whereas in 
vivo administration of these anti-cytokine Ab along with CpG-ODN 
reversed protection against infection. CpG-ODN treatment resulted in 
reduced levels of IL-4, but increased levels of IFN-gamma, IL-12 and 
inducible NO synthase in infected spleen cells, which was magnified by 
encapsulation in mannose-coated liposomes. This targeted treatment was 
not only curative, but it also imparted resistance to reinfection. These
 results represent a general approach for intracellular targeting of CpG
-ODN, which effectively enhances its therapeutic potential in 
redirecting curative Th1 responses in Th2-driven disorders.

PMID: 12780717

TITLE: Nitric oxide and cellular immunity in experimental cutaneous
leishmaniasis.

AUTHORS: N L DÃaz, M FernÃ¡ndez, E Figueira, R RamÃrez, I B Monsalve, F J
Tapia

AFFILIATION: Laboratorio de BiologÃa Molecular, Instituto de Biomedicina,
Universidad Central de Venezuela, Caracas, Venezuela.

REFERENCE: Clin Exp Dermatol 2003 May 28(3):288-93

We examined the local and systemic production of nitric oxide (NO) and 
the pattern of cytokine during the course of Leishmania mexicana 
infection in susceptible BALB/c and resistant C57BL/6 mice. NO 
derivatives were measured in serum, and the expression of inducible 
nitric oxide synthase (iNOS), interferon (IFN-gamma), interleukin (IL-4
) and epidermal Langerhans cells (LC) was measured in the lesions by 
immunohistology. Circulating NO concentrations, iNOS+ cell density, IFN-
gamma+ Th1 cells and CD205+ Langerhans cells were higher in early 
lesions of resistant C57BL/6 mice. In contrast, susceptible BALB/c mice 
developed chronic and progressive lesions with a predominance of IL-4+ 
Th2 cells. In both susceptible and resistant mice, lesion size and lymph
 node volume followed a similar course. The early local and systemic 
production of NO in resistant mice may be related with the premature 
production of IFN-gamma observed, contributing to the resolution of the 
lesion.

PMID: 12731047

TITLE: Organ-specific and stage-dependent control of Leishmania major infection
by inducible nitric oxide synthase and phagocyte NADPH oxidase.

AUTHORS: Martin Blos, Ulrike Schleicher, F Janaina Soares Rocha, Udo Meissner,
Martin RÃ¶llinghoff, Christian Bogdan

AFFILIATION: Institute of Clinical Microbiology, Immunology and Hygiene,
University of Erlangen-Nuremberg, Erlangen, Germany.

REFERENCE: Eur J Immunol 2003 May 33(5):1224-34

In the Leishmania major mouse model of cutaneous leishmaniasis inducible
 nitric oxide synthase (iNOS) is crucial for the killing of the parasite
 in the skin and draining lymph node. However, the effector mechanism 
operating against L. major in the spleen is unknown. As reactive oxygen 
intermediates might play a role, we analyzed macrophages and mice 
lacking the gp91phox subunit of the phagocyte NADPH oxidase (phox) for 
their ability to combat an infection with L. major. Macrophages from 
wild-type and gp91phox(-/-) mice had an equal capacity to kill L. major 
after activation by cytokines. Unlike iNOS, the activity of phox was 
dispensable for the resolution of the acute skin lesions and exerted 
only a limited effect on the containment of the parasites in the 
draining lymph node, but was essential for the clearance of L. major in 
the spleen. During the chronic phase of infection, parasites persisted 
at high levels in gp91phox(-/-) mice, and cutaneous lesions re-emerged 
in approximately 60% of these mice. gp91phox deficiency did not impair 
the expression of iNOS or the production of TNF and IFN-gamma. These 
results demonstrate that iNOS and phox are both required for the control
 of L. major in vivo and display unexpected organ- and stage-specific 
anti-leishmanial effects.

PMID: 12644314

TITLE: Infection pattern and immune response in the spleen and liver of BALB/c
mice intracardially infected with Leishmania donovani amastigotes.

AUTHORS: Piyali Mukherjee, Asish K Ghosh, Asoke C Ghose

AFFILIATION: Department of Microbiology, Bose Institute, P-1/12 CIT Scheme
VII-M, Kolkata, 700 054, India.

REFERENCE: Immunol Lett 2003 Apr 86(2):131-8

Intracardial inoculation of BALB/c mice with Leishmania donovani 
amastigotes induced progressive visceral leishmaniasis (VL) with 
increasing splenic parasite load when followed upto 4-month 
postinfection period. In contrast, the liver parasite load reached 
maximum around 2-month postinfection period following which it started 
declining. The infection pattern differed somewhat from the earlier 
reports on mouse model of VL induced by intravenous inoculation of 
parasites with respect to the duration as well as magnitude of parasite 
burden in the organs (liver and spleen) and associated 
hepatosplenomegaly. Immunosuppression in mice with progressive VL was 
manifested in the form of impairment of proliferative response of the 
splenic mononuclear cells (SPMC) to in vitro stimulation with 
leishmanial antigen or the mitogen concanavalin A (ConA), although ConA 
stimulated cells were found to be capable of IL-2 and IFN-gamma 
synthesis. Differential expression of activating (IL-2, IFN-gamma and 
TNF-alpha) as well as deactivating (IL-4 and TGF-beta) cytokines was 
demonstrable in the spleen and liver of animals during the course of 
infection. Further, the synthesis of inducible nitric oxide synthase (
iNOS) enzyme increased considerably in the liver as well as in the 
spleen of 4-month infected animal with parallel increase in the 
transcripts of the iNOS activating cytokines IFN-gamma and TNF-alpha. 
The temporal variation in the organ specific immune response could be 
related to the differential control of parasite burden in the liver and 
spleen of the infected host.

PMID: 12695487

TITLE: CD40 signaling is impaired in L. major-infected macrophages and is
rescued by a p38MAPK activator establishing a host-protective memory T cell
response.

AUTHORS: Amit Awasthi, Ramkumar Mathur, Aslam Khan, Bimba N Joshi, Nitya Jain,
Sangeeta Sawant, Ramanamurthy Boppana, Debashis Mitra, Bhaskar Saha

AFFILIATION: National Centre for Cell Science, Ganeshkhind, Pune 411007, India.

REFERENCE: J Exp Med 2003 Apr 197(8):1037-43

Leishmania, a protozoan parasite, lives and multiplies as amastigote 
within macrophages. It is proposed that the macrophage expressed CD40 
interacts with CD40 ligand on T cells to induce IFN-gamma, a Th1-type 
cytokine that restricts the amastigote growth. Here, we demonstrate that
 CD40 cross-linking early after infection resulted in inducible nitric 
oxide synthetase type-2 (iNOS2) induction and iNOS2-dependent amastigote
 elimination. Although CD40 expression remained unaltered on L. major-
infected macrophages, delay in the treatment of macrophages or of mice 
with anti-CD40 antibody resulted in significant reduction in iNOS2 
expression and leishmanicidal function suggesting impaired CD40 
signaling in Leishmania infection. The inhibition of CD40-induced iNOS2 
expression by SB203580, a p38-mitogen activated protein kinase (p38MAPK
)-specific inhibitor, and the reversal of the inhibition by anisomycin, 
a p38MAPK activator, suggested a crucial role of p38MAPK in CD40 
signaling. Indeed, the CD40-induced p38MAPK phosphorylation, iNOS2 
expression and anti-leishmanial function were impaired in Leishmania-
infected macrophages but were restored by anisomycin. Anisomycin's 
effects were reversed by SB203580 emphasizing the role of p38MAPK in 
CD40-induced iNOS2-dependent leishmanicidal function. Anisomycin 
administration in L. major-infected BALB/c mice resulted in significant 
reduction in the parasite load and established a host-protective Th1-
type memory response. Also implicated in these findings is a scientific 
rationale to define novel anti-parasite drug targets and to bypass the 
problem of drug resistance.

PMID: 12384497

TITLE: Leishmania EF-1alpha activates the Src homology 2 domain containing
tyrosine phosphatase SHP-1 leading to macrophage deactivation.

AUTHORS: Devki Nandan, Taolin Yi, Martin Lopez, Crystal Lai, Neil E Reiner

AFFILIATION: Department of Medicine, Division of Infectious Diseases, The
University of British Columbia, Research Institute of the Vancouver Hospital
and Health Sciences Center, Vancouver, British Columbia V5Z 3J5, Canada.
dnandan at interchange.ubc.ca

REFERENCE: J Biol Chem 2002 Dec 277(51):50190-7

The human leishmaniasis are persistent infections of macrophages caused 
by protozoa of the genus Leishmania. The chronic nature of these 
infections is in part related to induction of macrophage deactivation, 
linked to activation of the Src homology 2 domain containing tyrosine 
phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of
 SHP-1 activation, lysates of Leishmania donovani promastigotes were 
subjected to SHP-1 affinity chromatography and proteins bound to the 
matrix were sequenced by mass spectrometry. This resulted in the 
identification of Leishmania elongation factor-1alpha (EF-1alpha) as a 
SHP-1-binding protein. Purified Leishmania EF-1alpha, but not host cell 
EF-1alpha, bound directly to SHP-1 in vitro leading to its activation. 
Three independent lines of evidence indicated that Leishmania EF-1alpha 
may be exported from the phagosome thereby enabling targeting of host 
SHP-1. First, cytosolic fractions prepared from macrophages infected 
with [(35)S]methionine-labeled organisms contained Leishmania EF-1alpha
. Second, confocal, fluorescence microscopy using Leishmania-specific 
antisera detected Leishmania EF-1alpha in the cytosol of infected cells
. Third, co-immunoprecipitation showed that Leishmania EF-1alpha was 
associated with SHP-1 in vivo in infected cells. Finally, introduction 
of purified Leishmania EF-1alpha, but not the corresponding host protein
 into macrophages activated SHP-1 and blocked the induction of inducible
 nitric-oxide synthase expression in response to interferon-gamma. Thus
, Leishmania EF-1alpha is identified as a novel SHP-1-binding and 
activating protein that recapitulates the deactivated phenotype of 
infected macrophages.

PMID: 12555666

TITLE: Control of New World cutaneous leishmaniasis is IL-12 independent but
STAT4 dependent.

AUTHORS: Laurence U Buxbaum, Jude E Uzonna, Michael H Goldschmidt, Phillip
Scott

AFFILIATION: Department of Pathobiology, School of Veterinary Medicine,
University of Pennsylvania, Philadelphia, USA.

REFERENCE: Eur J Immunol 2002 Nov 32(11):3206-15

Leishmania mexicana, a New World protozoan parasite, induces small, 
chronic, but non-progressive lesions in C57BL/6 (B6) mice. In this study
 we investigated the role of IL-12, and subsequent Th1 factors, in 
controlling cutaneous L. mexicana infection. IL-12 treatment failed to 
promote disease resolution, suggesting that the inability of mice to 
heal is not related to a deficiency of endogenous IL-12 production. 
Surprisingly, L. mexicana-induced cutaneous lesions in wild-type and IL-
12p40-deficient mice were indistinguishable, with similar parasite 
burdens, immune responses, and lesion histopathology. In contrast, iNOS
, IFN-gamma, and STAT4-deficient mice developed progressive disease and 
uncontrolled parasite growth. These results differ dramatically from L. 
major infection, in which IL-12p40-deficient mice are highly susceptible
, with very rapid lesion growth, very large parasite burdens, and the 
development of a strong Th2 response. These data uncover the existence 
of an alternate IFN-gamma and iNOS pathway for control of Leishmania 
lesions, which is IL-12 independent, but which unexpectedly requires 
STAT4.

PMID: 12379707

TITLE: Interleukin-10 (IL-10) in experimental visceral leishmaniasis and IL-10
receptor blockade as immunotherapy.

AUTHORS: Henry W Murray, Christina M Lu, Smita Mauze, Sherry Freeman, Andre L
Moreira, Gilla Kaplan, Robert L Coffman

AFFILIATION: Department of Medicine, Weill Medical College of Cornell
University, New York, New York 10021, USA. hwmurry at med.cornell.edu

REFERENCE: Infect Immun 2002 Nov 70(11):6284-93

Interleukin-10 (IL-10) is thought to promote intracellular infection, 
including human visceral leishmaniasis, by disabling Th1 cell-type 
responses and/or deactivating parasitized tissue macrophages. To develop
 a rationale for IL-10 inhibition as treatment in visceral infection, 
Th1 cytokine-driven responses were characterized in Leishmania donovani-
infected BALB/c mice in which IL-10 was absent or overexpressed or its 
receptor (IL-10R) was blockaded. IL-10 knockout and normal mice treated 
prophylactically with anti-IL-10R demonstrated accelerated granuloma 
assembly and rapid parasite killing without untoward tissue inflammation
; IL-12 and gamma interferon mRNA expression, inducible nitric oxide 
synthase reactivity, and responsiveness to antimony chemotherapy were 
also enhanced in knockout mice. In IL-10 transgenic mice, parasite 
replication was unrestrained, and except for antimony responsiveness, 
measured Th1 cell-dependent events were all initially impaired. Despite 
subsequent granuloma assembly, high-level infection persisted, and 
antimony-treated transgenic mice also relapsed. In normal mice with 
established infection, anti-IL-10R treatment was remarkably active, 
inducing near-cure by itself and synergism with antimony. IL-10's 
deactivating effects regulate outcome in experimental visceral 
leishmaniasis, and IL-10R blockade represents a potential immuno- and/or
 immunochemotherapeutic approach in this infection.

PMID: 12443660

TITLE: Saponins, IL12 and BCG adjuvant in the FML-vaccine formulation against
murine visceral leishmaniasis.

AUTHORS: Wania Renata Santos, Valeria M F de Lima, Edilma Paraguai de Souza,
Robson Ronney Bernardo, Marcos Palatnik, Clarisa Beatriz Palatnik de Sousa

AFFILIATION: Instituto de Microbiologia, "Professor Paulo de GÃ³es" Universidade
Federal do Rio de Janeiro (UFRJ), CCS, Cidade UniversitÃ¡ria, Ilha do FundÃ£o,
Caixa Postal 68040, CEP 21941-590, RJ, Rio de Janeiro, Brazil.

REFERENCE: Vaccine 2002 Nov 21(1-2):30-43

The FML antigen of Leishmania donovani, in combination with either 
Riedel de HaÃ«n (R), QuilA, QS21 saponins, IL12 or BCG, was used in 
vaccination of an outbred murine model against visceral leishmaniasis (
VL). Significant and specific increases in anti-FML IgG and IgM 
responses were detected for all adjuvants, and in anti-FML IgG1, IgG2a 
and IgG2b and delayed type of hypersensitivity to L. donovani lysate (
DTH), only for all saponins and IL12. The QS21-FML and QuilA-FML groups 
achieved the highest IgG2a response. QuilA-FML developed the strongest 
DTH and QS21-FML animals showed the highest serum IFN-gamma 
concentrations. The reduction of parasitic load in the liver in response
 to each FML-vaccine formulation was: 52% (P<0.025) for BCG-FML, 73
% (P<0.005) for R-FML, 93% (P<0.005) for QuilA-FML and 79.2% (P&lt
;0.025) for QS21-FML treated animals, respectively. Protection was 
specific for R-FML and QS21-FML while the QuilA saponin treatment itself
 induced 69% of LDU reduction. The FML-saponin vaccines promote 
significant, specific and strong protective effects against murine 
visceral leishmaniasis. BCG-FML induced minor and non-specific 
protection while IL12-FML, although enhancing the specific antibody and 
IDR response, failed to reduce the parasitic load of infected animals.

PMID: 12471424

TITLE: B-cell infiltration and frequency of cytokine producing cells differ
between localized and disseminated human cutaneous leishmaniases.

AUTHORS: M G S Vieira, F Oliveira, S Arruda, A L Bittencourt, A A Barbosa, M
Barral-Netto, A Barral

AFFILIATION: Faculdade de Medicina, Universidade Federal da Bahias, Salvador,
BA, Brasil.

REFERENCE: Mem Inst Oswaldo Cruz 2002 Oct 97(7):979-83

Biopsies from human localized cutaneous lesions (LCL n = 7) or 
disseminated lesions (DL n = 8) cases were characterized according to 
cellular infiltration,frequency of cytokine (IFN-gamma, TNF-alpha) or 
iNOS enzyme producing cells. LCL, the most usual form of the disease 
with usually one or two lesions, exhibits extensive tissue damage. DL is
 a rare form with widespread lesions throughout the body; exhibiting 
poor parasite containment but less tissue damage. We demonstrated that 
LCL lesions exhibit higher frequency of B lymphocytes and a higher 
intensity of IFN-gamma expression. In both forms of the disease CD8+ 
were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha
 and iNOS producing cells, as well as the frequency of CD68+ macrophages
, did not differ between LCL and DL. Our findings reinforce the link 
between an efficient control of parasite and tissue damage, implicating 
higher frequency of IFN-gamma producing cells, as well as its possible 
counteraction by infiltrated B cells and hence possible humoral immune 
response in situ.

PMID: 12183555

TITLE: Endogenous interleukin-12 is critical for controlling the late but not
the early stage of Leishmania mexicana infection in C57BL/6 mice.

AUTHORS: Fabiola Aguilar Torrentera, Jon D Laman, Marjan Van Meurs, Luciano
Adorini, Eric Muraille, Y Carlier

AFFILIATION: Laboratory of Parasitology, Free University of Brussels (ULB),
Brussels, Belgium.

REFERENCE: Infect Immun 2002 Sep 70(9):5075-80

The role of interleukin-12 (IL-12) has been clearly established in the 
resistance of C57BL/6 (B6) mice to Leishmania major infection, but its 
involvement in the control of Leishmania mexicana infection remains to 
be determined. Here, we show the following. (i) L. mexicana, in contrast
 to L. major, induces the development of nonhealing lesions in B6 mice
. (ii) Cells expressing IL-12p40, gamma interferon (IFN-gamma), NOS2, 
and CD40L are numerous in the footpad lesion and/or the draining 
popliteal lymph node of animals infected for up to 14 weeks with L. 
mexicana. (iii) B6 mice, either IL-12p40 deficient or treated with IL-
12p40-neutralizing antibodies, display a dramatic enhancement of primary
 and secondary lesions leading to death 10 weeks after inoculation with 
L. mexicana. (iv) Splenocytes harvested 4 and 8 weeks after infection of
 IL-12p40(-/-) B6 mice with L. mexicana are unable to produce IFN-gamma
, but secrete IL-4, IL-10, and IL-18. Thus, the early control of L. 
mexicana infection by B6 mice is independent of IL-12, whereas IL-12 and
 Th1 responses play a key role in controlling the late stages of L. 
mexicana infection. However, they fail to resolve lesions, in contrast 
to L. major infection, emphasizing the different outcomes induced by 
these two Leishmania species in B6 mice.

PMID: 12117977

TITLE: Expression of inducible nitric oxide synthase in skin lesions of patients
with american cutaneous leishmaniasis.

AUTHORS: Muna Qadoumi, Inge Becker, Norbert Donhauser, Martin RÃ¶llinghoff,
Christian Bogdan

AFFILIATION: Institute of Clinical Microbiology, Immunology and Hygiene,
University of Erlangen, Germany.

REFERENCE: Infect Immun 2002 Aug 70(8):4638-42

Cytokine-inducible (or type 2) nitric oxide synthase (iNOS) is 
indispensable for the resolution of Leishmania major or Leishmania 
donovani infections in mice. In contrast, little is known about the 
expression and function of iNOS in human leishmaniasis. Here, we show by
 immunohistological analysis of skin biopsies from Mexican patients with
 local (LCL) or diffuse (DCL) cutaneous leishmaniasis that the 
expression of iNOS was most prominent in LCL lesions with small numbers 
of parasites whereas lesions with a high parasite burden (LCL or DCL) 
contained considerably fewer iNOS-positive cells. This is the first 
study to suggest an antileishmanial function of iNOS in human Leishmania
 infections in vivo.

PMID: 11895981

TITLE: Early enhanced Th1 response after Leishmania amazonensis infection of
C57BL/6 interleukin-10-deficient mice does not lead to resolution of
infection.

AUTHORS: Douglas E Jones, Mark R Ackermann, Ulrike Wille, Christopher A Hunter,
Phillip Scott

AFFILIATION: Department of Veterinary Pathology, College of Veterinary Medicine,
Iowa State University, Ames, Iowa 50011, USA. jonesdou at iastate.edu

REFERENCE: Infect Immun 2002 Apr 70(4):2151-8

C3H and C57BL/6 mice are resistant to Leishmania major but develop 
chronic lesions with persistent parasite loads when they are infected 
with Leishmania amazonensis. These lesions develop in the absence of 
interleukin-4 (IL-4), indicating that susceptibility to this parasite is
 not a result of development of a Th2 response. Expression of the 
cytokine IL-10 during infection could account for the lack of IL-12 
expression and poor cell-mediated immunity towards the parasite. 
Therefore, we tested the hypothesis that IL-10 plays a central role in 
downmodulating the Th1 response after L. amazonensis infection. 
Infection of C57BL/6 IL-10-deficient mice indicated that in the absence 
of IL-10 there was early enhancement of a Th1 response, which was 
downregulated during the more chronic stage of infection. In addition, 
although there were 1- to 2-log reductions in the parasite loads within 
the lesions, the parasites continued to persist, and they were 
associated with chronic lesions whose size was similar to that of the 
control lesions. These experiments indicated that L. amazonensis 
resistance to killing in vivo is only partially dependent on expression 
of host IL-10. However, IL-10-deficient mice had an enhanced delayed-
type hypersensitivity response during the chronic phase of infection, 
indicating that there were Th1 type effector cells in vivo at this late 
stage of infection. These results indicate that although IL-10 plays a 
role in limiting the Th1 response during the acute infection phase, 
other immunomodulatory factors are responsible for limiting the Th1 
response during the chronic phase.

PMID: 11985871

TITLE: Detection of iNOS gene expression in cutaneous leishmaniasis biopsy
tissue.

AUTHORS: Iracema Arevalo, Brian Ward, Greg Matlashewski

AFFILIATION: Department of Microbiology and Immunology, McGill University,
Montreal, Que., Canada.

REFERENCE: Mol Biochem Parasitol 2002 Apr 121(1):145-7

PMID: 11825771

TITLE: Leishmania (L.) amazonensis-induced inhibition of nitric oxide synthesis
in host macrophages.

AUTHORS: Filomena M Perrella Balestieri, Allan R Pires Queiroz, Cristoforo
Scavone, Vlaudia M Assis Costa, Manoel Barral-Netto, Ises de Almeida
Abrahamsohn

AFFILIATION: Departamento de Fisiologia e Patologia/LaboratÃ³rio de Tecnologia
FarmacÃªutica, UFPB, JoÃ£o Pessoa, CEP 58051-970, PB, Brazil.

REFERENCE: Microbes Infect 2002 Jan 4(1):23-9

Inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) 
production was demonstrated in J774-G8 macrophages infected with 
Leishmania (L.) amazonensis promastigotes. The downmodulation of NO 
production observed in infected and LPS-stimulated J774-G8 cells 
correlated with a reduction in inducible nitric oxide synthase (iNOS) 
activity. Reduction in iNOS activity was not paralleled by decreased 
iNOS mRNA expression, suggesting that the parasite affects post-
transcriptional events of NO synthesis. Supplementation with L-arginine 
or tetrahydrobiopterin did not increase NO production, suggesting that 
inhibition is not due to an insufficiency of substrate or co-factor. 
Treatment with anti-IL-10, anti-IL-4 or anti-TGF-beta neutralizing 
antibodies also failed to increase NO production, indicating that these 
cytokines are not involved in the observed parasite-induced inhibition 
of NO synthesis. However, treatment of the cultures with IFN-gamma 
resulted in a marked increase in NO production by infected LPS-
stimulated cells. These results show that although L.(L.) amazonensis 
infection inhibits iNOS activity and NO production by J774-G8 cells, 
activation by IFN-gamma is capable of overriding the suppression of NO 
synthesis.

PMID: 12078472

TITLE: Nitric oxide production by Leishmania-infected macrophages and modulation
by cytokines and prostaglandins.

AUTHORS: O Brandonisio, M A Panaro, M Sisto, A Acquafredda, L Fumarola, D
Leogrande, V Mitolo

AFFILIATION: Dipartimento di Clinica Medica, Immunologia e Malattie Infettive,
Sezione di Microbiologia ed Immunologia, FacoltÃ  di Medicina e Chirurgia,
UniversitÃ  di Bari, Policlinico, Piazza G. Cesare, 70124 Bari, Italy.
brandoni at cimedoc.uniba.it

REFERENCE: Parassitologia 2001 Dec 43 Suppl 1():1-6

Nitric oxide (NO) produced by an inducible nitric oxide synthase (iNOS 
or NOS2) plays a major microbicidal role in murine macrophages and its 
importance is now emerging also in the dog and human models. In dogs we 
demonstrated that macrophages in vitro infected with Leishmania infantum
 produced NO, after stimulation with cytokine-enriched peripheral blood 
mononuclear cell supernatants. In addition, parasite killing was reduced
 by the NOS inhibitor L-NG monomethylarginine. On the contrary, canine 
blood monocytes before macrophage differentiation did not release NO, 
and their leishmanicidal activity was instead correlated with superoxide
 anion and interferon (IFN)-gamma production. In human macrophage 
cultures, after infection with Leishmania infantum, we showed both iNOS 
expression by immunofluorescence and western blotting and NO release by 
the Griess reaction for nitrites. Various cytokines and prostaglandins 
can differently modulate NO synthesis. In our experiments, stimulation 
by recombinant human IFN-gamma and bacterial lipopolysaccharide greatly 
enhanced iNOS expression and NO production in human macrophages. In 
addition, the prostaglandin E2 increased NO release in activated, 
Leishmania-infected human macrophages. These results are interesting in 
the light of a possible immunological or pharmacological regulation of 
NO synthesis and microbicidal functions of macrophages.

PMID: 11592059

TITLE: IL-10 mediates susceptibility to Leishmania donovani infection.

AUTHORS: M L Murphy, U Wille, E N Villegas, C A Hunter, J P Farrell

AFFILIATION: Department of Pathobiology, School of Veterinary Medicine,
University of Pennsylvania, Philadelphia 19104, USA.

REFERENCE: Eur J Immunol 2001 Oct 31(10):2848-56

Human visceral leishmaniasis (VL) results in a severe and potentially 
fatal systemic disease, accompanied by cellular immune depression. The 
production of IL-10 correlates with ongoing disease and it has been 
suggested that the cellular immune depression that accompanies active 
disease may be due to a predominance of IL-10 production rather than a 
lack of IFN-gamma production, which is essential for optimal macrophage 
activation and parasite elimination. To examine the role of IL-10 in 
resistance during L. donovani infection (a causative agent of VL), the 
course of infection was examined in mice lacking the gene for IL-10. 
BALB/c IL-10-/-, as well as C57BL/6 IL-10-/- mice, were highly resistant
 to L. donovani infection, as evidenced by liver parasite burdens which 
were tenfold lower than those in control mice after 14 days of infection
. Enhanced resistance was accompanied by increased production of IFN-
gamma and nitric oxide in BALB/c IL-10-/- mice. Susceptibility to 
infection in BALB/c IL-10-/- mice was enhanced following in vivo 
treatment with a neutralizing antibody to IFN-gamma or IL-12. Together 
these studies demonstrate for the first time that IL-10 is a critical 
component of the immune response that inhibits resistance to L. donovani.

PMID: 11561959

TITLE: Inducible nitric oxide synthase expression in Leishmania-infected dog
macrophages.

AUTHORS: M Sisto, O Brandonisio, M A Panaro, A Acquafredda, D Leogrande, A
Fasanella, T Trotta, L Fumarola, V Mitolo

AFFILIATION: Dipartimento di Clinica Medica, Immunologia e Malattie Infettiva e
Sezione di Microbiologia e Immunologia, University of Bari, Italy.

REFERENCE: Comp Immunol Microbiol Infect Dis 2001 Oct 24(4):247-54

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2
) represents one of the main microbicidal mechanisms of murine 
macrophages, but its role in other animal models is poorly investigated
. Therefore, the aim of this work was to evaluate NOS2 expression in dog
 macrophages infected with Leishmania infantum. Macrophages obtained 
from peripheral blood of healthy dogs were activated with recombinant 
human interferon (rhIFN)-gamma and bacterial lipopolysaccharide (LPS) 
and then infected with L. infantum promastigotes. zymodeme MONI. For the
 immunofluorescence assay fixed macrophages were incubated with 
polyclonal rabbit anti-NOS2 and then with rhodamine F(ab')2 goat anti-
rabbit IgG. For immunoblotting, cell lysates were submitted to SDS-PAGE 
and blots were incubated with polyclonal rabbit anti-NOS2 and then with 
horseradish peroxidase-conjugated goat anti-rabbit IgG. Results 
demonstrated that L. infantum-infected cells, after stimulation with 
rhIFN-gamma and LPS, displayed high levels of fluorescence for the NOS2 
in their cytoplasm, unlike unstimulated uninfected macrophages. In 
western blotting, polyclonal anti-NOS2 reacted specifically with a 
protein band corresponding to 130 kDa. The signal produced in Leishmania
-infected cells stimulated with rhIFN-gamma and LPS was higher than that
 produced in Leishmania-infected unstimulated cells. No band was 
detected in cellular lysates from uninfected unstimulated cells. These 
results indicate that dog macrophages can express NOS2, and suggest a 
role for IFN-gamma and LPS in NOS2 induction also in this animal model.

PMID: 11447142

TITLE: Malnutrition alters the innate immune response and increases early
visceralization following Leishmania donovani infection.

AUTHORS: G M Anstead, B Chandrasekar, W Zhao, J Yang, L E Perez, P C Melby

AFFILIATION: Medical Service, Department of Veterans Affairs Medical Center,
South Texas Veterans Health Care System, University of Texas Health Science
Center at San Antonio, San Antonio, Texas 78229-3900, USA.

REFERENCE: Infect Immun 2001 Aug 69(8):4709-18

Malnutrition is a risk factor for the development of visceral 
leishmaniasis. However, the immunological basis for this susceptibility 
is unknown. We have developed a mouse model to study the effect of 
malnutrition on innate immunity and early visceralization following 
Leishmania donovani infection. Three deficient diets were studied, 
including 6, 3, or 1% protein; these diets were also deficient in iron, 
zinc, and calories. The control diet contained 17% protein, was zinc and
 iron sufficient, and was provided ab libitum. Three days after 
infection with L. donovani promastigotes, the total extradermal (lymph 
nodes, liver, and spleen) and skin parasite burdens were equivalent in 
the malnourished (3% protein) and control mice, but in the malnourished 
group, a greater percentage (39.8 and 4.0%, respectively; P = 0.009) of 
the extradermal parasite burden was contained in the spleen and liver. 
The comparable levels of parasites in the footpads in the two diet 
groups and the higher lymph node parasite burdens in the well-nourished 
mice indicated that the higher visceral parasite burdens in the 
malnourished mice were not due to a deficit in local parasite killing 
but to a failure of lymph node barrier function. Lymph node cells from 
the malnourished, infected mice produced increased levels of 
prostaglandin E(2) (PGE(2)) and decreased levels of interleukin-10. 
Inducible nitric oxide synthase activity was significantly lower in the 
spleen and liver of the malnourished mice. Thus, malnutrition causes a 
failure of lymph node barrier function after L. donovani infection, 
which may be related to excessive production of PGE(2) and decreased 
levels of IL-10 and nitric oxide.

PMID: 11441096

TITLE: Oxidative responses of human and murine macrophages during phagocytosis
of Leishmania chagasi.

AUTHORS: K R Gantt, T L Goldman, M L McCormick, M A Miller, S M Jeronimo, E T
Nascimento, B E Britigan, M E Wilson

AFFILIATION: Immunology Program, Department of Internal Medicine, University of
Iowa, Iowa City, IA 52242, USA.

REFERENCE: J Immunol 2001 Jul 167(2):893-901

Leishmania chagasi, the cause of South American visceral leishmaniasis, 
must survive antimicrobial responses of host macrophages to establish 
infection. Macrophage oxidative responses have been shown to diminish in
 the presence of intracellular leishmania. However, using electron spin 
resonance we demonstrated that murine and human macrophages produce O2-
during phagocytosis of opsonized promastigotes. Addition of the O2- 
scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures 
resulted in increased infection, suggesting that O2- enhances macrophage
 leishmanicidal activity. The importance of NO. produced by inducible NO
 synthase (iNOS) in controlling murine leishmaniasis is established, but
 its role in human macrophages has been debated. We detected NO. in 
supernatants from murine, but not human, macrophages infected with L. 
chagasi. Nonetheless, the iNOS inhibitor N(G)-monomethyl-L-arginine 
inhibited IFN-gamma-mediated intracellular killing by both murine and 
human macrophages. According to RNase protection assay and 
immunohistochemistry, iNOS mRNA and protein were expressed at higher 
levels in bone marrow of patients with visceral leishmaniasis than in 
controls. The iNOS protein also increased upon infection of human 
macrophages with L. chagasi promastigotes in vitro in the presence of 
IFN-gamma. These data suggest that O2- and NO. each contribute to 
intracellular killing of L. chagasi in human and murine macrophages.

PMID: 11378016

TITLE: Th1/Th2-regulated arginase availability modulates Leishmania infection.

AUTHORS: A Taylor-Robinson

REFERENCE: Trends Parasitol 2001 Jun 17(6):262

PMID: 11368921

TITLE: Role of peroxynitrite in macrophage microbicidal mechanisms in vivo
revealed by protein nitration and hydroxylation.

AUTHORS: E Linares, S Giorgio, R A Mortara, C X Santos, A T Yamada, O Augusto

AFFILIATION: Departamento de BioquÃmica, Instituto de QuÃmica, Universidade de
SÃ£o Paulo, SÃ£o Paulo, Brazil.

REFERENCE: Free Radic Biol Med 2001 Jun 30(11):1234-42

The cytotoxins produced by phagocytic cells lacking peroxidases such as 
macrophages remain elusive. To elucidate macrophage microbicidal 
mechanisms in vivo, we compared the lesion tissue responses of resistant
 (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis 
infection. This comparison demonstrated that parasite control relied on 
lesion macrophage activation with inducible nitric oxide synthase 
expression (iNOS), nitric oxide synthesis, and extensive nitration of 
parasites inside macrophage phagolysosomes at an early infection stage. 
Nitration and iNOS expression were monitored by confocal microscopy; 
nitric oxide synthesis was monitored by EPR. The main macrophage 
nitrating agent was shown to be peroxynitrite derived because parasite 
nitration occurred in the virtual absence of polymorphonuclear cells (
monitored as peroxidase activity) and was accompanied by protein 
hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies 
confirmed that peroxynitrite is cytotoxic to parasites whereas nitric 
oxide is cytostatic. The results indicate that peroxynitrite is likely 
to be produced close to the parasites and most of it reacts with carbon 
dioxide to produce carbonate radical anion and nitrogen dioxide whose 
concerted action leads to parasite nitration. In parallel, some 
peroxynitrite decomposition to the hydroxyl radical should occur due to 
the detection of hydroxylated proteins in the healing tissues. 
Consequently, peroxynitrite and derived radicals are likely to be 
important macrophage-derived cytotoxins.

PMID: 11298294

TITLE: Leishmania donovani-induced macrophages cyclooxygenase-2 and
prostaglandin E2 synthesis.

AUTHORS: C Matte, G Maion, W Mourad, M Olivier

AFFILIATION: Centre de Recherche en Infectiologie, UniversitÃ© Laval, Ste-Foy,
QuÃ©bec, Canada. Centre de Rhumatologie et Immunologie du CHUL, UniversitÃ©
Laval, Ste-Foy, QuÃ©bec, Canada.

REFERENCE: Parasite Immunol 2001 Apr 23(4):177-84

Prostaglandin E2 (PGE2) secretion during Leishmania infection has been 
reported. However, the signalling mechanisms mediating this response are
 not well understood. Since cyclooxygenase-2 (COX-2) and cytosolic 
phospholipase A2 (cPLA2) are involved in PGE2 synthesis in response to 
various stimuli, the implication of these enzymes was evaluated in 
Leishmania-infected phorbol myristate acetate-differentiated U937 human 
monocytic cell line. Time-course experiments showed that PGE2 synthesis 
increased significantly in parallel with COX-2 expression when cells 
were incubated in the presence of Leishmania donovani promastigotes or 
lipopolysaccharides (LPS). Increase in cPLA2 mRNA expression was only 
detected when cells were stimulated with LPS. Indomethacin, genistein, 
and H7, which are antagonists of COX-2, protein tyrosine kinase (PTK) 
and protein kinase C (PKC), respectively, inhibited PGE2 production 
induced by L. donovani and LPS. However, only H7 inhibited COX-2 mRNA 
synthesis, and there was a significant correlation between PGE2 
inhibition and reduced COX-2 expression. Collectively, our results 
indicate that infection of U937 by L. donovani leads to the generation 
of PGE2 in part through a PKC-dependent signalling pathway involving COX
-2 expression. They further reveal that PTK-dependent events are 
necessary for Leishmania-induced PGE2 generation, but not for COX-2 
expression. A better understanding of the mechanisms by which Leishmania
 can induce PGE2 production could provide insight into the 
pathophysiology of leishmaniasis and may help to improve therapeutic 
approaches.

PMID: 11257027

TITLE: Identification of genes induced by a macrophage activator, S-28463, using
gene expression array analysis.

AUTHORS: S Buates, G Matlashewski

AFFILIATION: Department of Microbiology and Immunology, McGill University,
Montreal, Canada H3A 2B4.

REFERENCE: Antimicrob Agents Chemother 2001 Apr 45(4):1137-42

S-28463 and imiquimod are imidazoquinoline compounds which stimulate 
microbicidal activity by inducing a local immune response at the site of
 application. Imiquimod-containing cream is an effective clinical 
treatment against cervical warts caused by human papillomavirus 
infection. Imiquimod also induces leishmanicidal activity both in vitro 
in macrophages and in vivo in a mouse model for cutaneous leishmaniasis
. The major target cells of S-28463 and imiquimod are macrophages. To 
explore the molecular basis in which imidazoquinolines generate 
macrophage microbicidal activity, a cDNA gene array analysis was 
undertaken to identify genes induced by S-28463. Out of 588 genes 
screened in this assay, only 13 genes were significantly induced by S-
28463. Remarkably, virtually all of the induced genes are involved in 
macrophage activation and inflammatory response. This experimental 
approach defines the mechanism of action of this clinically relevant 
compound in the induction of microbicidal activity in macrophages and 
also potentially identifies novel genes associated with microbicidal 
activity in this cell type.

PMID: 11238648

TITLE: Rapidly fatal leishmaniasis in resistant C57BL/6 mice lacking TNF.

AUTHORS: P Wilhelm, U Ritter, S Labbow, N Donhauser, M RÃ¶llinghoff, C Bogdan, H
KÃ¶rner

AFFILIATION: Institut fÃ¼r Klinische Mikrobiologie, Immunologie, UniversitÃ¤t
Erlangen-NÃ¼rnberg, Erlangen, Germany.

REFERENCE: J Immunol 2001 Mar 166(6):4012-9

The resolution of infections with the protozoan parasite Leishmania 
major in mice requires a Th1 response that is closely associated with 
the expression of IL-12, IFN-gamma, and inducible NO synthase. Previous 
Ab neutralization studies or the use of mice deficient for both TNF 
receptors suggested that TNF plays only a limited role in the control of
 parasite replication in vivo. In this study we demonstrate that 
resistant C57BL/6 (B6.WT) mice locally infected with L. major rapidly 
succumb to progressive visceral leishmaniasis after deletion of the TNF 
gene by homologous recombination. A reduction of the parasite inoculum 
to 3000 promastigotes did not prevent the fatal outcome of the disease. 
An influence of the altered morphology of secondary lymphoid organs in 
C57BL/6-TNF(-/-) (B6.TNF(-/-)) mice on the course of disease could be 
excluded by the generation of reciprocal bone marrow chimeras. Although 
infected B6.TNF(-/-) mice mounted an L. major-specific IFN-gamma 
response and expressed IL-12, the onset of the immune reaction was 
delayed. After in vitro stimulation, B6.TNF(-/-) inflammatory 
macrophages released 10-fold less NO in response to IFN-gamma than B6.WT
 cells. However, in the presence of a costimulus, e.g., L. major 
infection or LPS, the production of NO by B6.WT and B6.TNF(-/-) 
macrophages was comparable. In vivo, inducible NO synthase protein was 
readily detectable in skin lesions and draining lymph nodes of B6.TNF
(-/-) mice, but its expression was more disperse and less focal in the 
absence of TNF. These are the first data to demonstrate that TNF is 
essential for the in vivo control of L. major.

PMID: 11159954

TITLE: Pretreatment with recombinant Flt3 ligand partially protects against
progressive cutaneous leishmaniasis in susceptible BALB/c mice.

AUTHORS: I B Kremer, M P Gould, K D Cooper, F P Heinzel

AFFILIATION: Department of Dermatology, University Hospitals of Cleveland,
Cleveland, Ohio 44106, USA.

REFERENCE: Infect Immun 2001 Feb 69(2):673-80

Dendritic cells are potent antigen-presenting cells that also produce 
interleukin-12 (IL-12) during innate and adaptive cellular immune 
responses and that thereby promote the differentiation of gamma 
interferon (IFN-gamma)-producing Th1-type CD4(+) T lymphocytes. We 
hypothesized that expanded dendritic-cell populations in mice pretreated
 with the hematopoietic cytokine Flt3L would protect against cutaneous 
Leishmania major infection. Pretreatment of disease-susceptible BALB/c 
mice with 10 microg of recombinant Flt3L (rFlt3L) for 9 to 10 days 
before infection increased lymph node IL-12 p40 productive capacity 20-
fold compared to that of saline-injected controls. Furthermore, 9 of 22
 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous 
infections, whereas none of the 22 control BALB/c mice healed. Healed, 
rFlt3L-pretreated mice did not develop disease following reinfection. 
Flt3L pretreatment also reduced parasite numbers 1,000-fold in the 
cutaneous lesions at 2 weeks after infection relative to numbers in 
lesions of untreated controls. However, Flt3L pretreatment did not 
significantly alter L. major-induced IFN-gamma and IL-4 production in 
lymph node culture at 1, 2, and 4 weeks after infection. Despite the 
lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes 
expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2
) nitric oxide synthase mRNA at 7 days after infection. In contrast, 
treatment with rFlt3L after infection failed to protect against disease 
despite comparable expansions of dendritic cells and IL-12 p40 
productive capacity in both infected and uninfected BALB/c mice treated 
with rFlt3L. We conclude that rFlt3L pretreatment before infection with 
L. major reduces parasite load and promotes healing of cutaneous lesions
 without stable cytokine deviation towards a dominant Th1 cytokine 
phenotype.

PMID: 11160239

TITLE: The hamster as a model of human visceral leishmaniasis: progressive
disease and impaired generation of nitric oxide in the face of a prominent
Th1-like cytokine response.

AUTHORS: P C Melby, B Chandrasekar, W Zhao, J E Coe

AFFILIATION: Medical Service, Department of Veterans Affairs Medical Center,
South Texas Veterans Health Care System, San Antonio, TX 78284, USA.
melby at uthscsa.edu

REFERENCE: J Immunol 2001 Feb 166(3):1912-20

Active human visceral leishmaniasis (VL) is characterized by a 
progressive increase in visceral parasite burden, cachexia, massive 
splenomegaly, and hypergammaglobulinemia. In contrast, mice infected 
with Leishmania donovani, the most commonly studied model of VL, do not 
develop overt, progressive disease. Furthermore, mice control Leishmania
 infection through the generation of NO, an effector mechanism that does
 not have a clear role in human macrophage antimicrobial function. 
Remarkably, infection of the Syrian hamster (Mesocricetus auratus) with 
L. donovani reproduced the clinicopathological features of human VL, and
 investigation into the mechanisms of disease in the hamster revealed 
striking differences from the murine model. Uncontrolled parasite 
replication in the hamster liver, spleen, and bone marrow occurred 
despite a strong Th1-like cytokine (IL-2, IFN-gamma, and TNF/lymphotoxin
) response in these organs, suggesting impairment of macrophage effector
 function. Indeed, throughout the course of infection, inducible NO 
synthase (iNOS, NOS2) mRNA or enzyme activity in liver or spleen tissue 
was not detected. In contrast, NOS2 mRNA and enzyme activity was readily
 detected in the spleens of infected mice. The impaired hamster NOS2 
expression could not be explained by an absence of the NOS2 gene, 
overproduction of IL-4, defective TNF/lymphotoxin production (a potent 
second signal for NOS2 induction), or early dominant production of the 
deactivating cytokines IL-10 and TGF-beta. Thus, although a Th1-like 
cytokine response was prominent, the major antileishmanial effector 
mechanism that is responsible for control of infection in mice was 
absent throughout the course of progressive VL in the hamster.

PMID: 11421463

TITLE: Tannins and related compounds: killing of amastigotes of Leishmania
donovani and release of nitric oxide and tumour necrosis factor alpha in
macrophages in vitro.

AUTHORS: A F Kiderlen, O Kayser, D Ferreira, H Kolodziej

AFFILIATION: Robert Koch-Institut, Abteilung fÃ¼r Infektionskrankheiten, Berlin,
Germany.

REFERENCE: Z Naturforsch [C] 2001 May-Jun 56(5-6):444-54

The antileishmanial and immunomodulatory potencies of a series of 28 
polyphenols were evaluated in terms of extra- and intracellular 
leishmanicidal activity and macrophage activation for release of nitric 
oxide (NO), tumour necrosis factor (TNF) and interferon (IFN)-like 
properties. For this, several functional bioassays were employed 
including an in vitro model for leishmaniasis in which murine bone 
marrow-derived macrophages (BMMphi) were infected with the obligate 
intracellular parasite Leishmania donovani, an extracellular Leishmania 
proliferation assay, a fibroblast-lysis assay (TNF-activity), and a 
biochemical assay for NO. Except for gallic acid, its methyl ester, 
shikimic acid and catechin (EC50 25.8-67.9 nM) all polyphenols tested 
significantly inhibited the intracellular survival of L. donovani 
amastigotes (EC50 0.4-13.9 nM) when compared with the clinically used 
agent, sodium stibogluconate (EC50 10.6 nM). In contrast, none of the 
samples proved to be directly toxic for the extracellular promastigote 
form of the parasite. Noteworthy, the phenolic samples showed only 
moderate or no cytotoxicity against the murine host cells (EC50 10 to >
144 nM). Although NO is an important effector molecule in macrophage 
microbicidal activity, the inducing potential of the test compounds for 
its release was found to be very moderate ranging from 7-54 microM (IFN-
gamma/LPS 119 microM). On the other hand, inhibition of NO production 
had no apparent effect on intracellular leishmanicidal activity of 
polyphenols. Their in vitro TNF-inducing potential producing 50% lysis 
in murine L929 cells increased in the order of simple phenols and 
flavanols (34-48 U/ml) < A-type proanthocyanidins (53-80 U/ml) < B
-type proanthocyanidins (64-200 U/ml) < hydrolyzable tannins (287-350
 U/ml) at the host cell subtoxic concentration of 50 microg/ml. 
Furthermore, gallic acid and some hydrolyzable tannins showed 
appreciable IFN-like activities (14-23 U/ml) as reflected by inhibition 
of the cytopathic effect of encephalomyocarditis virus on fibroblast L 
929 cells. The results provide a rational basis for the recorded anti-
infectious efficacy of traditionally used herbal medicines containing 
tannins in vivo, in the light of both only moderate direct antimicrobial
 activities of distinct polyphenols in vitro and the limited knowledge 
on their uptake in humans.

PMID: 11399524

TITLE: Sustained parasite burden in the spleen of Leishmania infantum-infected
BALB/c mice is accompanied by expression of MCP-1 transcripts and lack of
protection against challenge.

AUTHORS: D Rousseau, S Demartino, F AnjuÃ¨re, B Ferrua, K Fragaki, Y Le Fichoux,
J Kubar

AFFILIATION: Groupe de Recherche en Immunopathologie de la Leishmaniose (EA
2675), Laboratoire de Parasitologie, FacultÃ© de MÃ©decine, avenue de
Valombrose, 06107 Nice Cedex 2, France.

REFERENCE: Eur Cytokine Netw 2001 Apr-Jun 12(2):340-7

We analyzed differential responses of spleen and liver, major organ 
targets for viscerotropic Leishmania species, to experimental infection 
and examined if resistance to challenge was organ-specific. In liver, 
parasites were spontaneously cleared and iNOS trancripts expression 
paralleled that of amastigote load. In the spleen, amastigote 
multiplication was only partly controlled, and iNOS transcripts 
expression was transient. Total numbers of spleen cells, B cells, and T 
cells were decreased, while F4/80(+) and Mac1(+) cells were conserved. 
Expression of splenic MCP-1 transcripts remained constant, indicating 
its possible contribution to immigration of Leishmania host cells and to
 sustained parasite load. Spleen cells produced both, Th1- and Th2-type 
cytokines and Th2-type response was dominant, compatible with the 
sustained MCP-1 expression. Challenge experiments showed that in 
contrast to the liver, where initial infection conferred a progressively
 established immunity, in the spleen there was no induced protection 
against reinfection. Organ-specific resistance against challenge could 
be important for designing antileishmanial vaccines.

PMID: 11169417

TITLE: The outcome of Leishmania major experimental infection in BALB/c mice can
be modulated by exogenously delivered iron.

AUTHORS: S Bisti, G Konidou, F Papageorgiou, G Milon, J R Boelaert, K
Soteriadou

AFFILIATION: Department of Biochemistry, Laboratory of Molecular Parasitology,
Hellenic Pasteur Institute, Athens, Greece.

REFERENCE: Eur J Immunol 2000 Dec 30(12):3732-40

We previously established that Leishmania promastigotes express a 
transferrin receptor and that iron chelators inhibit promastigote growth
 in vitro. Thus, we were interested in modulating the vertebrate host 
iron pool and to monitor whether such changes will affect the outcome of
 L. major infection in BALB / c mice, inoculated in the footpad with 106
 stationary phase promastigotes. Treatment of mice with desferrioxamine 
resulted in a slight delay of the development of cutaneous lesions. In 
contrast and unexpectedly, systemic iron delivery, at early time points 
of parasite delivery, significantly limited footpad pathology. 
Accordingly, parasite loads at the site of parasite delivery, the 
draining lymph node, liver and spleen were significantly reduced in iron
-loaded mice. Importantly, the "protective" effect of iron 
delivery correlated with the presence, at the site of inoculation, of 
lower levels of IL-4 and IL-10 transcripts while both IFN-gamma and 
inducible nitric oxide synthase transcripts were at higher levels. The 
presence of more type 1 cytokine transcripts was further supported by 
the increased levels of IgG2a in their sera. These data strongly suggest
 that susceptibility to L. major as assessed in the footpad model is 
modifiable by interventions that alter the iron status of the host at 
early time points of parasite delivery.

PMID: 11035745

TITLE: Essential role of platelet-activating factor in control of Leishmania
(Leishmania) amazonensis infection.

AUTHORS: M V Lonardoni, M Russo, S Jancar

AFFILIATION: Department of Clinical Analyses, State University of MaringÃ¡,
MaringÃ¡, ParanÃ¡, Brazil.

REFERENCE: Infect Immun 2000 Nov 68(11):6355-61

In the present study we investigated the role of platelet-activating 
factor (PAF) and prostaglandins in experimental Leishmania (Leishmania) 
amazonensis infection and the relationship between these mediators and 
nitric oxide (NO) production. Mouse peritoneal macrophages elicited with
 thioglicolate were infected with leishmania amastigotes, and the 
infection index determined 48 h later. The course of infection was 
monitored for 5 weeks in mice infected in the footpad with promastigotes
 by measuring the footpad swelling and parasite load in regional lymph 
nodes and spleen. The addition of PAF to C57BL/6 mouse macrophages 
significantly inhibited parasite growth and induced NO production. 
Treatment of macrophages with a selective PAF antagonist, WEB2086, 
increased the infection, indicating that endogenously produced PAF 
regulates macrophage ability to control leishmania infection. This 
effect of PAF was abolished by addition of the inhibitor of NO synthesis
, L-NAME, to the cultures. The addition of prostaglandin E(2) 
significantly increased the infection and NO production. Treatment with 
cyclo-oxygenase inhibitor, indomethacin, reduced the infection and PAF-
induced release of NO. Thus, the increased NO production induced by PAF 
seems to be mediated by prostaglandins. The more-selective inhibitors of
 cyclo-oxygenase 2, nimesulide and NS-398, had no significant effect. 
Thus, antileishmanial activity correlates better with the presence of 
PAF or absence of prostaglandins than with NO production. In vivo 
treatment with PAF antagonists significantly increased leishmania 
lesions, as well as the parasite load, in regional lymph nodes and 
spleens. These findings indicate that PAF is essential for the control 
of leishmania infection.

PMID: 11069076

TITLE: Endogenous IL-4 is necessary for effective drug therapy against visceral
leishmaniasis.

AUTHORS: J Alexander, K C Carter, N Al-Fasi, A Satoskar, F Brombacher

AFFILIATION: Department of Immunology, University of Strathclyde, Glasgow, GB.
j.alexander at strath.ac.uk

REFERENCE: Eur J Immunol 2000 Oct 30(10):2935-43

It is well established that a fully competent immune response is 
required for the successful drug treatment of visceral leishmaniasis. 
However, recent studies have cast some doubt as to which elements of the
 immune response synergize with chemotherapeutic treatment. The role of 
the Th2 response and IL-4 in particular during visceral leishmaniasis 
awaits clarification. We, therefore, examined the effectiveness of 
sodium stibogluconate treatment on Leishmania donovani infection in BALB
/c wild-type and IL-4-/- mice. Parasite burdens in L. donovani-infected 
IL-4+/- and IL-4-/-, as we have previously shown for B6/129 mice, were 
similar, despite an apparent type 1 antibody response in infected IL-4
-/- mice, demonstrated by increased levels of parasite-specific IgG2a 
and decreased IgG1. Unexpectedly IL-4-/- mice responded poorly to sodium
 stibogluconate treatment with increased parasite burdens in all tissues
 examined. Furthermore, drug therapy of IL-4-/- but not IL-4+/+ mice 
resulted in significant reductions in splenocyte IFN-gamma mRNA 
transcripts and in serum IFN-gamma levels. These results demonstrate 
that IL-4 has an important role in effective anti-leishmanial 
chemotherapy which seems to be related to modulation of IFN-gamma 
production.

PMID: 11009089

TITLE: Peroxovanadium-mediated protection against murine leishmaniasis: role of
the modulation of nitric oxide.

AUTHORS: C Matte, J F Marquis, J Blanchette, P Gros, R Faure, B I Posner, M
Olivier

AFFILIATION: Centre de Recherche en Infectiologie and DÃ©partement de Biologie
MÃ©dicale, Centre Hospitalier Universitaire de QuÃ©bec, FacultÃ© de MÃ©decine,
UniversitÃ© Laval, Ste-Foy, Canada.

REFERENCE: Eur J Immunol 2000 Sep 30(9):2555-64

The phosphotyrosine phosphatase inhibitor bpV(phen) has the ability to 
markedly decrease the progression of leishmaniasis in vivo. Here, we 
have identified the mechanisms that are responsible for this protective 
effect. We report that two potent peroxovanadium (pV) compounds, bpV(
phen) and bpV(pic), control progression of leishmaniasis in a similar 
manner by modulating NO-dependent microbicidal action. We observed that 
their injection can rapidly and transiently induce the expression of 
inducible NO synthase (iNOS) in livers of mice and enhance circulating 
nitrate levels. Treatment of mice with bpV(phen) or bpV(pic) completely 
controlled progression of leishmaniasis in an NO-dependent manner, since
 inhibition of iNOS with aminoguanidine completely reversed this pV-
mediated protection. This NO-dependent pV-mediated protection was 
further demonstrated by the incapacity of bpV(phen)-treated Nramp-/-, 
iNOS-/- mutant mice to control Leishmania major infection. Using an air 
pouch model, we showed that bpV(phen) can strongly modulate secretion of
 L. major-induced pro-inflammatory molecules and neutrophil recruitment
. In addition, we observed that bpV(phen) per se can strongly induce the
 expression of Th1 type cytokines over Th2 in spleens of animals. 
Overall, this study has allowed us to establish the in vivo functional 
and immunological events involved in pV-mediated protective mechanism 
against leishmaniasis and that NO plays a pivotal role in this process.

PMID: 10719664

TITLE: The role of nitric oxide in innate immunity.

AUTHORS: C Bogdan, M RÃ¶llinghoff, A Diefenbach

AFFILIATION: Institute of Clinical Microbiology, Immunology and Hygiene,
University of Erlangen, Germany. christian.bogdan at mikrobio.med.uni-erlangen.de

REFERENCE: Immunol Rev 2000 Feb 173():17-26

Type 2 nitric oxide synthase (iNOS or NOS2) was originally described as 
an enzyme that is expressed in activated macrophages, generates nitric 
oxide (NO) from the amino acid L-arginine, and thereby contributes to 
the control of replication or killing of intracellular microbial 
pathogens. Since interferon (IFN)-gamma is the key cytokine for the 
induction of NOS2 in macrophages and the prototypic product of type 1 T-
helper cells, high-level expression of NOS2 has been regarded to be 
mostly restricted to the adaptive phase of the immune response. In this 
review, we summarize data that demonstrate a prominent role of NOS2/NO 
also during innate immunity. During the early phase of infection with 
the intracellular pathogen Leishmania major, focally expressed NOS2/NO 
not only exerts antimicrobial activities but also controls the function 
of natural killer cells and the expression of cytokines such as IFN-
gamma or transforming growth factor-beta. Some of these effects result 
from the function of NOS2/NO as an indispensable co-factor for the 
activation of Tyk2 kinase and, thus, for interleukin-12 and IFN-alpha/
beta signaling in natural killer cells.

PMID: 10761741

TITLE: Adenosine, AMP, and protein phosphatase activity in sandfly saliva.

AUTHORS: O Katz, J N Waitumbi, R Zer, A Warburg

AFFILIATION: Department of Parasitology, The Kuvin Center for the Study of
Infectious and Tropical Diseases, The Hebrew University-Hadassah Medical
School, Ein Kerem, Jerusalem, Israel.

REFERENCE: Am J Trop Med Hyg 2000 Jan 62(1):145-50

As they probe the skin for blood, sand flies inject saliva that prevents
 hemostasis. Sand fly saliva also promotes leishmaniasis by suppressing 
immunologic functions of macrophages. Saliva of Phlebotomus papatasi, 
the vector of Old World cutaneous leishmaniasis, contains adenosine and 
AMP. We show that Ph. papatasi saliva as well as pure adenosine down-
regulate the expression of the inducible nitric oxide (NO) synthase gene
 in activated macrophages. In addition Ph. papatasi, but not Lutzomyia 
longipalpis, saliva inhibits the production of NO. Taken together, these
 data suggest that salivary adenosine is responsible for the down-
regulation of NO synthesis. Saliva of both genera Phlebotomus and 
Lutzomyia contains significant levels of endogenous protein phosphatase-
1/2A-like activity that is heat labile, inhibitable by okadaic acid and 
calyculine a, and does not require divalent cations.

PMID: 10647995

TITLE: Human monocytic U937 cells transfected with human hepatic inducible
nitric oxide synthase exhibit leishmanicidal activity.

AUTHORS: S Bertholet, J MauÃ«l

AFFILIATION: Institute of Biochemistry, University of Lausanne, Switzerland.
Sylvie.BertholetGirardin at ib.unil.ch

REFERENCE: J Leukoc Biol 2000 Jan 67(1):34-9

In mice, the high inducible synthesis of nitric oxide (NO) resulting 
from inducible NO synthase (iNOS, NOS2) expression by macrophages (Mphi
) is considered an essential component of the protective immune response
 against infection by intracellular pathogens. Conversely, in humans, 
the question of a role for NO as an antimicrobial defense mechanism has 
been the subject of much debate. Recently, however, iNOS expression by 
human Mphi and formation of NO or its derivatives have been reported 
both in vivo and in vitro, strongly suggesting that human Mphi are 
indeed capable of inducible NO synthesis. However, the conditions 
allowing NO production by human Mphi in culture remain poorly defined, 
rendering more difficult the study of the effector functions of NO in 
these cells. To alleviate this problem, cells of the U937 monocytoid 
line were engineered to express iNOS by transfection with human hepatic 
iNOS (DFGiNOS), leading to production of NO on supplementation with the 
cofactor tetrahydrobiopterin. We report that U937 cells, when 
differentiated with 1,25-dihydroxyvitamin D3 and retinoic acid, acquire 
a phenotype allowing infection by Leishmania parasites and maintain 
viable intracellular microorganisms up to 72 h post-infection. 
Leishmania survival in DFGiNOS cells is strongly decreased when the 
cells are treated with tetrahydrobiopterin. Intracellular killing is 
evident by 24 h and increases up to 72 h post-infection, and is 
inhibited by L-N5-(1-iminoethyl)ornithine, an inhibitor of NO synthesis
. In contrast, superoxide anion does not appear to play a role in the 
killing of Leishmania by DGFiNOS U937 cells. The relevance of this model
 to the study of the mechanisms of intracellular killing by human 
macrophages is discussed.

PMID: 10603400

TITLE: Roles of endogenous gamma interferon and macrophage microbicidal
mechanisms in host response to chemotherapy in experimental visceral
leishmaniasis.

AUTHORS: H W Murray, S Delph-Etienne

AFFILIATION: Department of Medicine, Weill Medical College of Cornell
University, New York, New York 10021, USA. hwmurray at mail.med.cornell.edu

REFERENCE: Infect Immun 2000 Jan 68(1):288-93

In experimental visceral leishmaniasis, in which the tissue macrophage 
is the target, in vivo responsiveness to conventional chemotherapy (
pentavalent antimony [Sb]) requires a T-cell-dependent mechanism. To 
determine if this mechanism involves gamma interferon (IFN-gamma)-
induced activation and/or specific IFN-gamma-regulated macrophage 
leishmanicidal mechanisms (generation of reactive nitrogen or oxygen 
intermediates, we treated gene-deficient mice infected with Leishmania 
donovani. In IFN-gamma gene knockout (GKO) mice, Sb inhibited but did 
not kill intracellular L. donovani (2% killing versus 76% in controls). 
Sb was active (>94% killing), however, in both inducible nitric oxide 
synthase (iNOS) knockout (KO) and respiratory burst (phagocyte oxidase)-
deficient chronic granulomatous disease (X-CGD) mice. Sb's efficacy was 
also maintained in doubly deficient animals (X-CGD mice treated with an 
iNOS inhibitor). In contrast to Sb, amphotericin B (AmB) induced high-
level killing in GKO mice; AmB was also fully active in iNOS KO and X-
CGD animals. Although resolution of L. donovani infection requires iNOS
, residual visceral infection remained largely suppressed in iNOS KO 
mice treated with Sb or AmB. These results indicate that endogenous IFN-
gamma regulates the leishmanicidal response to Sb and achieves this 
effect via a pathway unrelated to the macrophage's primary microbicidal 
mechanisms. The role of IFN-gamma is selective, since it is not a 
cofactor in the response to AmB. Treatment with either Sb or AmB permits
 an iNOS-independent mechanism to emerge and control residual 
intracellular L. donovani infection.

PMID: 10438370

TITLE: Synergistic effect of interferon-gamma and mannosylated
liposome-incorporated doxorubicin in the therapy of experimental visceral
leishmaniasis.

AUTHORS: L Kole, L Das, P K Das

AFFILIATION: Department of Molecular Cell Biology, Indian Institute of Chemical
Biology, Calcutta, India.

REFERENCE: J Infect Dis 1999 Sep 180(3):811-20

Active targeting of doxorubicin to macrophages was studied by 
incorporating it in mannose-coated liposomes by use of visceral 
leishmaniasis in BALB/c mice as the model macrophage disease. 
Mannosylated liposomal doxorubicin was more effective than liposomal 
doxorubicin or free doxorubicin. Because leishmaniasis is accompanied by
 immunosuppression, immunostimulation by interferon (IFN)-gamma was 
evaluated to act synergistically with mannosylated liposomal doxorubicin
 therapy. Combination chemotherapy with a suboptimal dose of IFN-gamma 
resulted in possibly complete elimination of spleen parasite burden. 
Analysis of mRNA levels of infected spleen cells suggested that targeted
 drug treatment together with IFN-gamma, in addition to greatly reducing
 parasite numbers, resulted in reduced levels of interleukin (IL)-4 but 
increased levels of IL-12 and inducible nitric oxide synthase. Such 
combination chemotherapy may provide a promising alternative for the 
cure of leishmaniasis, with a plausible conversion of antiparasitic T 
cell response from a Th2 to Th1 pattern indicative of long-term 
resistance.

PMID: 10487453

TITLE: A double-blind randomized clinical trial of a topical herbal extract
(Z-HE) vs. systemic meglumine antimoniate for the treatment of cutaneous
leishmaniasis in Iran.

AUTHORS: F Zerehsaz, R Salmanpour, F Handjani, S Ardehali, M R Panjehshahin, S Z
Tabei, H R Tabatabaee

AFFILIATION: Shiraz Clinic and Department of Dermatology, Shiraz University of
Medical Sciences, Iran.

REFERENCE: Int J Dermatol 1999 Aug 38(8):610-2

PMID: 10458764

TITLE: Effects of nitric oxide on the induction and differentiation of Th1
cells.

AUTHORS: W Niedbala, X Q Wei, D Piedrafita, D Xu, F Y Liew

AFFILIATION: Department of Immunology, University of Glasgow, Glasgow, GB.

REFERENCE: Eur J Immunol 1999 Aug 29(8):2498-505

We have previously shown that mice lacking inducible NO synthase are 
markedly more susceptible to Leishmania major infection but developed a 
significantly enhanced Th1 cell response compared with wild-type mice. 
Furthermore, at high concentrations, NO inhibited IL-12 synthesis by 
activated macrophages, thereby indirectly suppressing the expansion of 
Th1 cells. We report here that at low concentrations, NO selectively 
enhanced the induction of Th1 cells and had no effect on Th2 cells. NO 
exerted this effect in synergy with IL-12 during Th1 cell 
differentiation and had no effect on fully committed Th1 cells. NO 
appears to affect CD4(+) T cells directly and not at the antigen-
presenting cells. These results therefore provide an additional pathway 
by which NO modulates the immune response and contributes to the 
homeostasis of the immune system.

PMID: 10438949

TITLE: BCL-6-deficient mice reveal an IL-4-independent, STAT6-dependent pathway
that controls susceptibility to infection by Leishmania major.

AUTHORS: A L Dent, T M Doherty, W E Paul, A Sher, L M Staudt

AFFILIATION: Metabolism Branch, National Cancer Institute, Laboratory of
Parasitic Diseases, National Institutes of Health, Bethesda, MD, USA.

REFERENCE: J Immunol 1999 Aug 163(4):2098-103

The BCL-6 gene negatively regulates Th2 responses as shown by the 
finding that BCL-6-deficient (BCL-6-/-) mice develop a lethal Th2-type 
inflammatory disease. The response of inbred mouse strains to infection 
with Leishmania major is under genetic control; BALB/c mice are 
susceptible and develop a progressive parasite burden, whereas most 
other common laboratory strains of mice are resistant to infection. We 
found that BCL-6-/- mice on a resistant genetic background (C57BL/6 x 
129 intercrossed mice) were highly susceptible to L. major infection; 
they resembled BALB/c mice in terms of lesion size, parasite load, and 
the production of Th2 cytokines. BCL-6-/-IL-4-/- double-mutant mice were
 also susceptible to L. major infection and produced 10-fold higher 
levels of the Th2 cytokine IL-13 than IL-4-/- littermate controls. By 
contrast, BCL-6-/-STAT6-/- double-mutant mice were resistant to L. major
 infection despite also producing elevated levels of IL-13. These 
results show that STAT6 is required for susceptibility to L. major 
infection and suggest that IL-13 signaling through STAT6 may contribute 
to a nonhealing, exacerbated L. major infection.

PMID: 10320373

TITLE: Requirement for type 2 NO synthase for IL-12 signaling in innate
immunity.

AUTHORS: A Diefenbach, H Schindler, M RÃ¶llinghoff, W M Yokoyama, C Bogdan

AFFILIATION: Institut fÃ¼r Klinische Mikrobiologie, Immunologie und Hygiene,
UniversitÃ¤t Erlangen, Wasserturmstrasse 3, D-91054 Erlangen, Germany.

REFERENCE: Science 1999 May 284(5416):951-5

Interleukin-12 (IL-12) and type 2 NO synthase (NOS2) are crucial for 
defense against bacterial and parasitic pathogens, but their 
relationship in innate immunity is unknown. In the absence of NOS2 
activity, IL-12 was unable to prevent spreading of Leishmania parasites
, did not stimulate natural killer (NK) cells for cytotoxicity or 
interferon-gamma (IFN-gamma) release, and failed to activate Tyk2 kinase
 and to tyrosine phosphorylate Stat4 (the central signal transducer of 
IL-12) in NK cells. Activation of Tyk2 in NK cells by IFN-alpha/beta 
also required NOS2. Thus, NOS2-derived NO is a prerequisite for cytokine
 signaling and function in innate immunity.

PMID: 9989990

TITLE: Macrophage microbicidal mechanisms in vivo: reactive nitrogen versus
oxygen intermediates in the killing of intracellular visceral Leishmania
donovani.

AUTHORS: H W Murray, C F Nathan

AFFILIATION: Department of Medicine, Division of Hematology-Oncology, Cornell
University Medical College, New York 10021, USA. hwmurray at mail.med.cornell.edu

REFERENCE: J Exp Med 1999 Feb 189(4):741-6

To determine the relative contributions of respiratory burst-derived 
reactive oxygen intermediates (ROI) versus reactive nitrogen 
intermediates (RNI) to macrophage-mediated intracellular host defense, 
mice genetically deficient in these mechanisms were challenged with 
Leishmania donovani, a protozoan that selectively parasitizes visceral 
tissue macrophages. During the early stage of liver infection at wk 2, 
both respiratory burst-deficient gp91(phox)-/- (X-linked chronic 
granulomatous disease [X-CGD]) mice and inducible nitric oxide synthase
 (iNOS) knockout (KO) mice displayed comparably increased susceptibility
. Thereafter, infection was unrestrained in mice lacking iNOS but was 
fully controlled in X-CGD mice. Mononuclear cell influx into infected 
liver foci in X-CGD and iNOS KO mice was also overtly impaired at wk 2. 
However, granuloma assembly in parasitized tissue eventually developed 
in both hosts but with divergent effects: mature granulomas were 
functionally active (leishmanicidal) in X-CGD mice but inert in iNOS-
deficient animals. These results suggest that (a) ROI and RNI probably 
act together in the early stage of intracellular infection to regulate 
both tissue recruitment of mononuclear inflammatory cells and the 
initial extent of microbial replication, (b) RNI alone are necessary and
 sufficient for eventual control of visceral infection, and (c) although
 mature granulomas have traditionally been associated with control of 
such infections, these structures fail to limit intracellular parasite 
replication in the absence of iNOS.

PMID: 10592110

TITLE: Inducible nitric oxide synthase and nitric oxide production in Leishmania
infantum-infected human macrophages stimulated with interferon-gamma and
bacterial lipopolysaccharide.

AUTHORS: M A Panaro, A Acquafredda, S Lisi, D D Lofrumento, T Trotta, R
Satalino, M Saccia, V Mitolo, O Brandonisio

AFFILIATION: Istituto di Anatomia Umana Normale, University of Bari,
Policlinico, I-70124, Bari, Italy.

REFERENCE: Int J Clin Lab Res 1999  29(3):122-7

Nitric oxide produced by an inducible nitric oxide synthase constitutes 
one of the main microbicidal mechanisms of murine macrophages and its 
importance is now being recognized for human macrophages. In this study 
we evaluated inducible nitric oxide synthase expression, nitric oxide 
release, and parasitocidal ability of Leishmania infantum-infected 
monocyte-derived human macrophages. The inducible nitric oxide synthase 
was detected by immunofluorescence and western blotting and nitric oxide
 production was measured by the Griess reaction for nitrites. Parasite 
killing was microscopically evaluated by fluorescent dyes. Experiments 
were performed on macrophages with or without previous stimulation with 
recombinant human interferon-gamma and bacterial lipopolysaccharide. 
Inducible nitric oxide synthase expression and nitric oxide release were
 higher in Leishmania-infected stimulated macrophages than in uninfected
 cells or infected cells without previous stimulation. Nitric oxide 
production and parasitocidal activity against Leishmania infantum were 
reduced in macrophages treated with the nitric oxide synthase inhibitor 
L-N(G) monomethylarginine. These results suggest a microbicidal role for
 nitric oxide in human leishmaniasis, with the possible practical 
application of immunological or pharmacological regulation of nitric 
oxide synthesis in the treatment of this infection.

PMID: 9820534

TITLE: Phlebotomus papatasi sand fly salivary gland lysate down-regulates a Th1,
but up-regulates a Th2, response in mice infected with Leishmania major.

AUTHORS: M L Mbow, J A Bleyenberg, L R Hall, R G Titus

AFFILIATION: Department of Pathology, Colorado State University College of
Veterinary Medicine and Biomedical Sciences, Fort Collins 80523-1671, USA.

REFERENCE: J Immunol 1998 Nov 161(10):5571-7

A vertebrate host becomes infected with Leishmania major when the sand 
fly vector injects parasites into skin along with saliva. Previous 
studies showed that salivary gland lysate of the New World sand fly 
Lutzomyia longipalpis markedly enhanced L. major infection in CBA mice. 
However, L. major is an Old World parasite transmitted in nature by the 
Old World sand fly Phlebotomus papatasi. Here we examine the ability of 
P. papatasi salivary gland lysate to enhance infection (lesion size and 
parasite burden) by L. major. In addition, we examine the effects of 
salivary gland lysate on the immune response to L. major by monitoring 
the levels of cytokine mRNA from the lymph nodes draining cutaneous 
lesions. We found that P. papatasi salivary gland lysate dramatically 
exacerbated lesion development in disease-resistant CBA mice. This 
exacerbation of disease correlated with inhibition of the production of 
Thl cytokines and associated factors (IFN-gamma, IL-12, and inducible 
nitric oxide synthase), but with enhancement of the Th2 cytokine IL-4, 
whereas no changes in the levels of IL-10 and TGF-beta were noted. 
Importantly, salivary gland lysate directly up-regulated expression of 
IL-4 mRNA in mice in the absence of infection with L. major.

PMID: 9924547

TITLE: Lack of a nitric-oxide response during the course of Leishmania infantum
infection in the golden hamster (Mesocricetus auratus), with or without
treatment with liposomal amphotericin B.

AUTHORS: C Bories, C Coffin, D Mathieu, P N Bories, E Scherman, D Rivollet, M
Deniau

AFFILIATION: Service de Radiologie, Centre Hospitalier Universitaire Henri
Mondor, CrÃ©teil, France. Christian.Bories at cep.u-psud.fr

REFERENCE: Ann Trop Med Parasitol 1998 Sep 92(6):685-92

Liver and spleen volumes and serum concentrations of nitrate (the end-
product of NO in vivo), albumin, gamma-globulin, protein, creatine and 
urea were measured during the course of progressive infections with 
Leishmania infantum MON-1 (MHOM/PR/93/CRE29) in 10 Syrian golden 
hamsters. Each hamster was infected by intraperitoneal injection with 4 
x 10(7) promastigotes. Five of the infected animals were treated, with 6
 mg liposomal amphotericin B (L-AmB)/kg given by intracardiac injection
, on day 107 post-infection (p.i.). Compared with those in the 
uninfected hamsters used as controls, the liver volumes in the infected 
animals became significantly enlarged by day 40 p.i. (38% larger than 
the controls; P < 0.001) whereas significant enlargement of the 
spleen was first detected on day 72. Each infected animal had detectable
 serum levels of antileishmanial antibodies on day 72. There were 
significant elevations in gamma-globulin concentration as early as day 
40 (P < 0.05) but significant falls in albumin concentrations were 
only detected from day 107 (P < 0.001). Nitrate, creatinine and urea 
concentrations remained unchanged during the course of infection, even 
after L-AmB treatment. Serum nitrate levels were not enhanced by L. 
infantum infection nor by the L-AmB treatment which induced a 98.2% 
decrease in parasite burden. The lack of NO production in visceral 
leishmaniasis, with or without L-AmB treatment, points to the 
unresponsiveness of inducible nitric oxide synthase in this rodent model.

PMID: 9725203

TITLE: Switch of CD4+ T cell differentiation from Th2 to Th1 by treatment with
cathepsin B inhibitor in experimental leishmaniasis.

AUTHORS: Y Maekawa, K Himeno, H Ishikawa, H Hisaeda, T Sakai, T Dainichi, T
Asao, R A Good, N Katunuma

AFFILIATION: Department of Parasitology and Immunology, University of Tokushima
School of Medicine, Japan.

REFERENCE: J Immunol 1998 Sep 161(5):2120-7

When activated, CD4+ T helper cells differentiate functionally into one 
of two subsets, Th1 or Th2. Before the Th differentiation, Ags must be 
processed into peptide epitopes and presented to CD4+ T cells in 
association with MHC class II molecules. However, the proteases 
responsible for this Ag processing have not been well defined. When BALB
/c mice susceptible to infection with Leishmania major were treated with
 a specific inhibitor (CA074) of cathepsin B, a lysosomal cysteine 
protease that digests exogenous antigenic proteins, those mice acquired 
resistance against infection with L. major and showed the shift of 
immune responses from Th2 to Th1; that is, they produced specific IgG2a 
Ab and generated IFN-gamma in contrast to untreated and infected mice 
that produced IgG1 and IgE and generated IL-4. CA074 interfered with the
 digestion of L. major Ags with lysosomal enzymes in vivo as well as in 
vitro. However, this inhibitor did not show any direct influence on the 
growth of L. major and the functions of T cells stimulated with anti-CD3
 Ab. These findings indicate that cathepsin B inhibitor could switch CD4
+ T cell differentiation from Th2 to Th1, suggesting that the alteration
 in Ag processing modulates the polarity of Th differentiation.

PMID: 9605154

TITLE: Control of Leishmania major infection in mice lacking TNF receptors.

AUTHORS: M Nashleanas, S Kanaly, P Scott

AFFILIATION: University of Pennsylvania School of Veterinary Medicine,
Philadelphia 19104, USA.

REFERENCE: J Immunol 1998 Jun 160(11):5506-13

TNF participates in the induction of nitric oxide (NO) production and 
macrophage activation, leading to the elimination of intracellular 
pathogens. We previously found that TNF receptor p55-deficient mice (
TNFRp55-/-) control replication of Leishmania major in vivo but fail to 
resolve their lesions. Here we report that mice lacking the p75 receptor
 (TNFRp75-/-) or both receptors (TNFRp55p75-/-), also control parasite 
replication, albeit mice lacking the p55 receptor (either TNFRp55-/- or 
TNFRp55p75-/-) are delayed in their elimination of L. major compared 
with controls. All TNF receptor-deficient mice developed a Thl-type 
immune response and up-regulated inducible NO synthase (iNOS) mRNA gene 
expression in lesions during infection. Thus, neither TNF receptor 
appears to be absolutely required for NO production or elimination of L
. major in vivo. In vitro, however, while macrophages from naive TNFRp75
-/- mice could be activated to produce NO and kill L. major, we observed
 a defect in NO production and parasite killing by resident peritoneal 
macrophages from naive TNFRp55-/- or TNFRp55p75-/- mice. However, when 
macrophages were elicited with leishmanial Ag from 4-wk-infected TNFRp55
-/- or TNFRp55p75-/- mice, they produced NO and were leishmanicidal. 
These data suggest that the TNFRp75 plays no essential role in L. major 
infection in mice and that the p55 receptor may be required for optimal 
macrophage activation. However, the results also show that a mechanism 
exists by which macrophages can be primed in vivo during L. major 
infection to produce NO and kill L. major in the absence of signaling 
through either of the TNF receptors.

PMID: 9529078

TITLE: Phlebotomus papatasi saliva inhibits protein phosphatase activity and
nitric oxide production by murine macrophages.

AUTHORS: J Waitumbi, A Warburg

AFFILIATION: Department of Parasitology, The Kuvin Center for the Study of
Infectious and Tropical Diseases, Hebrew University-Hadassah Medical School,
Jerusalem, Israel.

REFERENCE: Infect Immun 1998 Apr 66(4):1534-7

Leishmania parasites, transmitted by phlebotomine sand flies, are 
obligate intracellular parasites of macrophages. The sand fly 
Phlebotomus papatasi is the vector of Leishmania major, a causative 
agent of cutaneous leishmaniasis in the Old World, and its saliva 
exacerbates parasite proliferation and lesion growth in experimental 
cutaneous leishmaniasis. Here we show that P. papatasi saliva contains a
 potent inhibitor of protein phosphatase 1 and protein phosphatase 2A of
 murine macrophages. We further demonstrate that P. papatasi saliva down
 regulates expression of the inducible nitric oxide synthase gene and 
reduces nitric oxide production in murine macrophages. Partial 
biochemical characterization of the protein phosphatase and nitric oxide
 inhibitor indicated that it is a small, ethanol-soluble molecule 
resistant to boiling, proteolysis, and DNase and RNase treatments. We 
suggest that the P. papatasi salivary protein phosphatase inhibitor 
interferes with the ability of activated macrophages to transmit signals
 to the nucleus, thereby preventing up regulation of the induced nitric 
oxide synthase gene and inhibiting the production of nitric oxide. Since
 nitric oxide is toxic to intracellular parasites, the salivary protein 
phosphatase inhibitor may be the mechanism by which P. papatasi saliva 
exacerbates cutaneous leishmaniasis.

PMID: 9570545

TITLE: Protective effect on Leishmania major infection of migration inhibitory
factor, TNF-alpha, and IFN-gamma administered orally via attenuated Salmonella
typhimurium.

AUTHORS: D Xu, S J McSorley, L Tetley, S Chatfield, G Dougan, W L Chan, A
Satoskar, J R David, F Y Liew

AFFILIATION: Department of Immunology, University of Glasgow, United Kingdom.

REFERENCE: J Immunol 1998 Feb 160(3):1285-9

The genes encoding murine macrophage migration inhibitory factor (MIF), 
IL-2, IFN-gamma or TNF-alpha were cloned individually into an expression
 plasmid under the control of the inducible promoter nirB and 
transfected into the aroA- aroD- deletion mutant strain of Salmonella 
typhimurium (BRD509). These S. typhimurium derivatives (henceforward 
called constructs and termed GIDMIF, GIDIL2, GIDIFN and GIDTNF) 
expressed their respective cytokines in vitro under anaerobic conditions
 and stably colonized BALB/c mice up to 14 days after oral 
administration. The highly susceptible BALB/c mice that had received the
 constructs orally and that had been subsequently infected via the 
footpad with Leishmania major, developed significantly reduced disease 
compared with control mice administered the untransfected Salmonella 
strain (BRD509). Importantly, a combination of GIDMIF, GIDIFN, and 
GIDTNF administered orally after L. major infection was able to 
significantly limit lesion development and reduced parasite loads by up 
to three orders of magnitude. Spleen and lymph node cells of mice 
administered this combination expressed markedly higher levels of 
inducible nitric oxide synthase (iNOS) compared with those from mice 
receiving an equivalent dose of the control strain of Salmonella (BRD509
). These data therefore demonstrate the feasibility of therapeutic 
treatment in an infectious disease model using cytokines delivered by 
attenuated Salmonella. The protective effect observed correlates with 
the induction of inducible nitric oxide synthase in vivo.

PMID: 9261952

TITLE: Haemolytic activities of plant saponins and adjuvants. Effect of
Periandra mediterranea saponin on the humoral response to the FML antigen of
Leishmania donovani.

AUTHORS: W R Santos, R R Bernardo, L M PeÃ§anha, M Palatnik, J P Parente, C B
Palatnik de Sousa

AFFILIATION: Instituto de Microbiologia, Universidade Federal do Rio de Janeiro
(UFRJ), CCS, Cidade UniversitÃ¡ria, Brazil.

REFERENCE: Vaccine 1997 Jun 15(9):1024-9

An 87.7% (P < 0.01) and 84% (P < 0.001) of protection against 
visceral leishmaniasis was achieved in CB hamsters and Balb/c mice, 
respectively, with saponin combined to the fucose-mannose ligand of 
Leishmania donovani (FML). However, an undesirable haemolytic effect was
 described for several saponins. Aiming to improve the formulation with 
FML/saponin, we comparatively analysed the haemolytic potential of 
recently characterized plant saponins and currently used adjuvants. The 
haemolytic activity of steroidic saponins from Agave sisalana; Smilax 
officinalis as well as commercial saponin (Riedel De HaÃ«n's), was 
higher than that of triterpenoid ones (Bredemeyera floribunda; Periandra
 mediterranea) and the Freund's complete adjuvant. The concentration 
resulting in 50% haemolysis was 500 micrograms ml-1 for aluminum 
hydroxide. The low haemolytic effect of P. mediterranea saponin was 
abolished by removal of its glycidic moiety and its sapogenin fraction 
as well as the Freund's Incomplete Adjuvant were non-haemolytic within 
this range. Furthermore, the adjuvant effect of three doses of P. 
mediterranea saponin injected with the FML antigen of L. donovani, was 
assayed in mice, either by the intraperitoneal (i.p.) or the 
subcutaneous (s.c.) route. The anti-FML IgG antibody levels increased 
and detectable levels were observed up to 3 months in the s.c. group. 
The response was expanded in both groups after an injection with a 
fourth vaccine dose. The IgG response showed increased levels of IgG2a 
only in the i.p. group, while IgG2b and IgG1 but not IgG3 antibodies 
were higher than controls in both groups. In conclusion, the results 
suggest that the recently described triterpenoid fractions of P. 
mediterranea can be safely used as adjuvant with low or non-haemolytic 
effect.

PMID: 8713607

TITLE: The FML (Fucose Mannose Ligand) of Leishmania donovani. a new tool in
diagnosis, prognosis, transfusional control and vaccination against human
kala-azar.

AUTHORS: C B Palatnik de Sousa, E M Gomes, E P de Souza, W R dos Santos, S R de
Macedo, L V de Medeiros, K Luz

AFFILIATION: Instituto de Microbiologia, Universidade Federal do Rio de
Janeiro.

REFERENCE: Rev Soc Bras Med Trop 1996 Mar-Apr 29(2):153-63

The Fucose-Mannose Ligand (FML) of Leishmania donovani is a complex 
glycoproteic fraction. Its potential use as a tool for diagnosis of 
human visceral leishmaniasis was tested with human sera from Natal, Rio 
Grande do Norte, Brazil. The FML-ELISA test, showed 100% sensitivity and
 96% specificity, identifying patients with overt kala-azar (p < 0.
001, when compared to normal sera), and subjects with subclinical 
infection. More than 20% apparently healthy subjects with positive 
reaction to FML developed overt kala-azar during the following 10 months
. In the screening of human blood donnors, a prevalence of 5% of 
sororeactive subjects was detected, attaining 17% in a single day. The 
GP36 glycoprotein of FHL is specifically reconized by human kala-azar 
sera. The immunoprotective effect of FML on experimental L. donovani 
infection was tested in swiss albino mice. The protection scheemes 
included three weekly doses of FML, supplemented or not with saponin by 
the subcutaneous or intraperitoneal routes and challenge with 2 x 10(7) 
amastigotes of Leishmania donovani. An enhancement of 80.0% in antibody 
response (p < 0.001) and reduction of 85.5% parasite liver burden (p
 < 0.001) was detected in animals immunized with FML saponin, 
unrespectively of the immunization route.

PMID: 3324215

TITLE: The discovery of urea stibamine.

AUTHORS: P D Marsden

REFERENCE: Rev Soc Bras Med Trop 1986 Apr-Jun 19(2):115

REQUEST: [ leishmania ]

(112 articles match this request. 69 articles matching other requests removed)

PMID: 15722039

TITLE: LdARL-1 His-tagged recombinant protein: purification by immobilized metal
affinity expanded bed adsorption.

AUTHORS: Annelise Sahin, Emmanuel Tetaud, Gilles Merlin, Xavier Santarelli

AFFILIATION: CNRS UMR 5162 GÃ©nomique Fonctionnelle des Trypanosomatides,
UniversitÃ© Victor Segalen Bordeaux 2, 146 rue LÃ©o Saignat, 33076 Bordeaux
cedex, France.

REFERENCE: J Chromatogr B Analyt Technol Biomed Life Sci 2005 Apr 818(1):19-22

Previously we have cloned three ADP-ribosylation factor-like (ARL) genes
 from the parasitic protozoan Leishmania donovani: LdARL-3A and 3B, 
LdARL-1. LdARL-3A was previously purified as an active native form, 
which was able to bind GTP in vitro. In this paper, we have performed 
the production and the purification of Histidine-tagged (His-tagged) 
LdARL-1 recombinant protein by immobilized metal affinity chromatography
 (IMAC) using expanded bed adsorption (EBA) technology. This protein was
 purified with more than 95% purity and could be successfully used for 
GTP-binding assay.

PMID: 15720561

TITLE: Leishmania salvage and remodelling of host sphingolipids in amastigote
survival and acidocalcisome biogenesis.

AUTHORS: Kai Zhang, Fong-Fu Hsu, David A Scott, Roberto Docampo, John Turk,
Stephen M Beverley

AFFILIATION: Department of Molecular Microbiology, Box 8230, Washington
University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.

REFERENCE: Mol Microbiol 2005 Mar 55(5):1566-78

Summary Sphingolipids (SLs) play essential roles in most eukaryotes, but
 in the trypanosomatid protozoan Leishmania major their functions differ
 significantly. Previously we showed that null mutants defective in de 
novo sphingoid base synthesis (spt2(-)) lacked SLs but grew well and 
retained lipid rafts while replicating as promastigotes in vitro. 
However, they experienced catastrophic defects in membrane trafficking 
on entry into stationary phase, and failed to differentiate to the 
infective metacyclic form. Here we showed this mutant retained the 
ability to enter macrophages silently and inhibit activation, although 
as expected most parasites were destroyed. However, in mouse infections
, after a delay rapidly progressive lesions appeared, and purified 
amastigotes were fully virulent to macrophages and mice. Mass 
spectrometry of spt2(-) amastigote lipids revealed the presence of high 
levels of parasite-specific inositol phosphorylceramides (IPCs) not 
synthesized by the mammalian hosts. Inhibitor studies showed that 
salvage occurs at the level of complex SLs, suggesting that parasites 
carry out 'headgroup' remodelling. Additionally, we describe a new 
defect of the spt2(-) promastigotes involving 'empty' acidocalcisomes (
ACs), which may point to the origin of this organelle from the lysosome-
related organelle/multivesicular body biogenesis pathway. However, ACs 
in spt2(-) amastigotes appeared quantitatively and morphologically 
normal. Thus salvage of SLs and other molecules by intracellular 
amastigotes play key roles in AC biogenesis and parasite survival in the
 host.

PMID: 15721837

TITLE: Leishmania donovani engages in regulatory interference by targeting
macrophage protein tyrosine phosphatase SHP-1.

AUTHORS: Devki Nandan, Neil E Reiner

AFFILIATION: Division of Infectious Diseases, Department of Medicine, Faculties
of Medicine and Science, Vancouver Coastal Health Research Institute (VCHRI),
The University of British Columbia, Vancouver, British Columbia, Canada.

REFERENCE: Clin Immunol 2005 Mar 114(3):266-77

Protozoan parasites of the genus leishmania are obligate intracellular 
parasites of monocytes and macrophages. These pathogens have evolved to 
invade the mammalian immune system and typically survive for long 
periods of time. Leishmania have developed a variety of remarkable 
strategies to prevent their elimination by both innate and acquired 
immune effector mechanisms. One particular strategy of interest involves
 manipulation of host cell regulatory pathways so as to prevent 
macrophage activation required for efficient microbicidal activity. 
These interference mechanisms are the main focus of this review. Several
 lines of evidence have been developed to show that the Src homology-2 
domain containing tyrosine phosphatase-1 (SHP-1) becomes activated in 
leishmania-infected cells and that this contributes to disease 
pathogenesis. Recent studies aimed at understanding the mechanism 
responsible for the change in activation state of SHP-1 led to the 
identification of leishmania EF-1alpha as an SHP-1 binding protein and 
SHP-1 activator. This was a surprising finding given that this 
ubiquitous and highly conserved protein plays an essential role in 
protein translation in both prokaryotic and eukaryotic cells. The role 
of leishmania EF-1alpha as an SHP-1 activator and its contribution to 
pathogenesis are reviewed with particular attention to the properties 
that distinguish it from host EF-1alpha.

PMID: 15721836

TITLE: Modulation of phagolysosome biogenesis by the lipophosphoglycan of
Leishmania.

AUTHORS: Robert Lodge, Albert Descoteaux

REFERENCE: Clin Immunol 2005 Mar 114(3):256-65

Promastigotes of the protozoan parasite Leishmania are inoculated into 
the mammalian host by an infected sandfly and are phagocytosed by 
macrophages. There, they differentiate into amastigotes, which replicate
 in phagolysosomes. A family of glycoconjugates, the phosphoglycans (PGs
), plays an important role in the ability of promastigotes to survive 
the potentially microbicidal consequences of phagocytosis. 
Lipophosphoglycan (LPG), an abundant promastigote surface glycolipid, 
has received considerable attention over the past several years. Of 
interest for this review, lipophosphoglycan confers upon Leishmania 
donovani promastigotes the ability to inhibit phagolysosome biogenesis. 
This inhibition correlates with an accumulation of periphagosomal F-
actin, which may potentially form a physical barrier that prevents L. 
donovani promastigote-harboring phagosomes from interacting with late 
endosomes and lysosomes. Thus, similar to several other pathogens, 
Leishmania promastigotes hijack the host cell's cytoskeleton early 
during the infection process. Here, we review this phenomenon and 
discuss the potential underlying mechanisms.

PMID: 15713445

TITLE: Strand asymmetry patterns in trypanosomatid parasites.

AUTHORS: Daniel Nilsson, BjÃ¶rn Andersson

AFFILIATION: Center for Genomics and Bioinformatics, Karolinska Institutet,
Berzeliusv. 35, SE-171 77 Stockholm, Sweden.

REFERENCE: Exp Parasitol 2005 Mar 109(3):143-9

The genome organization of kinetoplastid parasites is unusual, with 
chromosomes containing several long regions of polycistronically 
transcribed genes. The regions where the direction of transcription 
switches have been hypothesized to contain origins of replication and 
possibly also centromers and promoters. We report that overall strand 
asymmetry patterns can be observed in Trypanosoma cruzi and Trypanosoma 
brucei with optima on strand-switch regions. The base skews of T. cruzi 
and T. brucei divergent strand-switches show patterns analogous to those
 for bacterial origins of replication, but they differ from those of 
Leishmania major. Bias in codon usage and the trypanosomatid 
unidirectional gene clusters predict most of this skew, but fail to 
properly explain the same trend in intergenic regions, as does the 
current knowledge of regulatory sequences.

PMID: 15708793

TITLE: Water soluble cationic trans-platinum complexes which induce programmed
cell death in the protozoan parasite Leishmania infantum.

AUTHORS: Paul A Nguewa, Miguel A Fuertes, Salvador Iborra, Yousef Najajreh, Dani
Gibson, Enrique MartÃnez, Carlos Alonso, JosÃ© M PÃ©rez

AFFILIATION: Departamento de ParasitologÃa, Facultad de Farmacia, Universidad
de la Laguna, Tenerife, Spain.

REFERENCE: J Inorg Biochem 2005 Mar 99(3):727-36

We have evaluated the cytotoxic properties against the protozoan 
Leishmania infantum of four water soluble cationic trans-Pt(II)Cl(2) 
compounds containing as inert groups NH(3) and piperazine (1), 4-
picoline and piperazine (2), n-butylamine and piperazine (3), and NH(3) 
and 4-piperidino-piperidine (4). The leishmanicidal activity of 
compounds 3 and 4 against promastigotes of the parasite Leishmania 
infantum was 2.5- and 1.6-times higher than that of the cytotoxic drug 
cis-diamminedichloroplatinum(II), respectively. Interestingly, compounds
 3 and 4 produce in Leishmania infantum promastigotes a higher amount of
 programmed cell death than cisplatin, which is associated with cell 
cycle arrest in G2/M. In contrast to cis-diamminedichloroplatinum(II), 
binding of compounds 3 and 4 to calf thymus DNA induces conformational 
changes more similar to those of trans-diamminedichloroplatinum(II) that
 may be attributed to denaturation of the double helix. Similarly to cis
-diamminedichloroplatinum(II) and trans-diamminedichloroplatinum(II), 
the interaction of compounds 3 and 4 with ubiquitin results in an 
increase of the alpha-helix content of the protein as observed by 
circular dichroism spectroscopy. However, fluorescence studies indicate 
that compounds 3 and 4 produce a decrease in the fluorescence of the 
tyrosine 59 residue of ubiquitin higher than both cis-
diamminedichloroplatinum(II) and trans-diamminedichloroplatinum(II). 
Altogether, our results suggest that the biochemical mechanism of 
cytotoxic activity of compounds 3 and 4 against Leishmania infantum must
 be different from that of cis-diamminedichloroplatinum(II). To the best
 of our knowledge, compounds 3 and 4 are the first reported trans-
platinum complexes that show antiparasitic activity.

PMID: 15720564

TITLE: LmxMPK9, a mitogen-activated protein kinase homologue affects flagellar
length in Leishmania mexicana.

AUTHORS: Florian Bengs, Anne Scholz, Daniela Kuhn, Martin Wiese

AFFILIATION: Bernhard Nocht Institute for Tropical Medicine, Parasitology
Section, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany.

REFERENCE: Mol Microbiol 2005 Mar 55(5):1606-15

Summary Components of mitogen-activated signal transduction pathways 
have been shown to be involved in flagellum biogenesis and maintenance. 
A mitogen-activated protein kinase homologue, designated LmxMPK9 from 
Leishmania mexicana, has been recently identified in a homology screen 
and its mRNA found to be present in all life stages. Three different 
splice-addition sites were used for mRNA maturation in trans-splicing in
 the different life stages. However, here we show that LmxMPK9 protein 
is exclusively found in the promastigote stage. Recombinant expression 
of LmxMPK9 in Escherichia coli and kinase assays revealed a temperature 
optimum at 27 degrees C, the optimal growth temperature for L. mexicana 
promastigotes, and a preference for manganese to promote substrate 
phosphorylation of myelin basic protein. A deletion mutant for the 
single-copy gene revealed significantly elongated flagella, whereas 
overexpression led to a subpopulation with rather short to no flagella 
suggesting a role for LmxMPK9 in flagellar morphogenesis.

PMID: 15710544

TITLE: Effects of hyperbaric oxygen on Leishmania amazonensis promastigotes and
amastigotes.

AUTHORS: Wagner Weber Arrais-Silva, Marcelle Carolina Collhone, Diana Copi
Ayres, Paula Cristina de Souza Souto, Selma Giorgio

AFFILIATION: Department of Parasitology, Biology Institute, Universidade
Estadual de Campinas, Caixa Postal 6109, Cep 13083-970, Campinas, SÃ£o Paulo,
Brazil.

REFERENCE: Parasitol Int 2005 Mar 54(1):1-7

In the present study, we evaluated the effects of hyperbaric oxygen (HBO
) exposure in both Leishmania amazonensis life stages (promastigotes and
 amastigotes) and on macrophage cultures infected with the parasite. HBO
 treatment protocols, which can be tolerated by humans and animals, 
induced irreversible metabolic damage and affected parasite morphology, 
growth and ability to transform. The observation that the antioxidant N-
acetylcysteine (NAC) prevents some of these deleterious effects 
indicated an involvement of oxidative stress during parasite HBO 
exposure. In addition, HBO exposed L. amazonensis-infected macrophage 
cultures showed reduction of the percentage of infected cells and of the
 number of intracellular parasites per cell. Thus, the demonstration 
that HBO, a therapy used in the management of different diseases, is 
toxic for both L. amazonensis life stages and can alter macrophage 
susceptibility to the infection encourages further studies of this 
therapy in animal models of Leishmania infection.

PMID: 15657927

TITLE: Selective targeting of indel-inferred differences in spatial structures
of highly homologous proteins.

AUTHORS: Artem Cherkasov, Devki Nandan, Neil E Reiner

AFFILIATION: Department of Medicine (Division of Infectious Diseases),
University of British Columbia, Vancouver, British Columbia, Canada.

REFERENCE: Proteins 2005 Mar 58(4):950-4

Recent findings have shown that the protein elongation factor-1alpha (EF
-1alpha) from the eukaryotic pathogen Leishmania donovani possesses 
virulence properties. This was unexpected, since it has greater than 80
% sequence identity with its human homologue. Given that EF-1alpha is 
essential for cell survival, in principle, it can be considered an 
attractive drug target. However, the challenge is to be able to 
selectively target the protein so as not to affect function of the human
 homologue. While a limited number of discrete differences were 
scattered throughout the sequence, most of the difference between these 
2 homologues could be attributed to a 12-amino acid insert present in 
human EF-1alpha and absent from the leishmania sequence. In the present 
study, we modeled the spatial differences in structures of human and L. 
donovani EF-1alpha's inferred by this insertion-deletion (or "indel
"). The protein models were used to develop antibodies directed 
specifically toward the deletion region of the pathogen protein. The 
strategy described allowed successful selective targeting of this 
putative leishmania virulence factor while avoiding recognition of the 
highly similar human EF-1alpha homologue. These findings may establish a
 new strategy for the development of antagonists directed against 
certain pathogenic targets having close human homologues. Proteins 2005
 (c) 2005 Wiley-Liss, Inc.

PMID: 15722076

TITLE: Leishmania braziliensis: a novel mechanism in the lipophosphoglycan
regulation during metacyclogenesis.

AUTHORS: Rodrigo P P Soares, Tatiana L Cardoso, Tamara Barron, MÃ¡rcio S S
AraÃºjo, Paulo F P Pimenta, Salvatore J Turco

AFFILIATION: Department of Biochemistry, University of Kentucky Medical Center,
Lexington, KY 40536, USA.

REFERENCE: Int J Parasitol 2005 Mar 35(3):245-53

During metacyclogenesis of Leishmania in its sand fly vector, the 
parasite differentiates from a noninfective, procyclic form to an 
infective, metacyclic form, a process characterised by morphological 
changes of the parasite and also biochemical transformations in its 
major surface lipophosphoglycan (LPG). This lipid-anchored 
polysaccharide is polymorphic among species with variations in sugars 
that branch off the conserved Gal(beta1,4)Man(alpha1)-PO(4) backbone of 
repeat units and the oligosaccharide cap. Lipophosphoglycan has been 
implicated as an adhesion molecule that mediates the interaction with 
the midgut epithelium of the sand fly in the subgenus Leishmania. This 
paper describes the LPG structure for the first time in a species from 
the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from 
the procyclic form of L. braziliensis was found to lack side chain sugar
 substitutions. In contrast to other species from the subgenus 
Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1
-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate
 repeat units of LPG. Thus, this represents a novel mechanism in the 
regulation of LPG structure during metacyclogenesis.

PMID: 15691375

TITLE: Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in
intracellular parasite survival: studies by gene disruption and antisense mRNA
inhibition.

AUTHORS: Vasanthakrishna Mundodi, Ashwini S Kucknoor, Lashitew Gedamu

AFFILIATION: Department of Biological Sciences, University of Calgary, Calgary
AB T2N1N4, Canada. lgedamu at ucalgary.ca.

REFERENCE: BMC Mol Biol 2005 Feb 6(1):3

BACKGROUND: The parasitic protozoa belonging to Leishmania (L.) donovani
 complex possess abundant, developmentally regulated cathepsin L-like 
cysteine proteases. Previously, we have reported the isolation of 
cysteine protease gene, Ldccys2 from Leishmania (L.) chagasi. Here, we 
have further characterized this cysteine protease gene and demonstrated 
its role during infection and survival of Leishmania (L.) chagasi within
 the U937 macrophage cells. RESULTS: The amastigote specific Ldccys2 
genes of L. (L.) chagasi and L. (L.) donovani have identical gene 
organization, as determined by southern blots. In vivo expression 
analyses by Northern blots showed that Ldccys2 is amastigote specific. 
Western blot using anti-Ldccys2 antibody confirmed the amastigote 
specific protein expression. Recombinant expression of Ldccys2, a 30 kDA
 protein, was functionally active in a gelatin assay. Results from 
Ldccys2 heterozygous knockout mutants showed its role during macrophage 
infection and in intra-macrophage survival of the parasites. Since 
attempts to generate null mutants failed, we used antisense RNA 
inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the 
results from antisense studies further confirmed the results from 
heterozygous knockout mutants, reiterating the importance of amastigote 
specific cysteine proteases in Leishmania infection and pathogenesis. 
CONCLUSIONS: The study shows that Ldccys2 is a developmentally regulated
 gene and that Ldccys2 is expressed only in infectious amastigote stages
 of the parasite. The collective results from both the heterozygous 
knockout mutants and antisense mRNA inhibition studies shows that 
Ldccys2 helps in infection and survival of L. (L.) chagasi amastigotes 
within the macrophage cells. Finally, antisense RNA technique can be 
used as an alternate approach to gene knockout, for silencing gene 
expression in L. (L.) chagasi, especially in cases such as this, where a
 null mutant cannot be achieved by homologous recombination.

PMID: 15579464

TITLE: Hemoglobin receptor in leishmania is a hexokinase located in the
flagellar pocket.

AUTHORS: Ganga Krishnamurthy, Rajagopal Vikram, Sudha B Singh, Nitin Patel,
Shruti Agarwal, Gauranga Mukhopadhyay, Sandip K Basu, Amitabha Mukhopadhyay

AFFILIATION: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi
110067, India and Center for Molecular Medicine, Jawaharlal Nehru University,
New Delhi 110067, India.

REFERENCE: J Biol Chem 2005 Feb 280(7):5884-91

Hb endocytosis in Leishmania is mediated through a 46-kDa protein 
located in the flagellar pocket. To understand the nature of the Hb 
receptor (HbR), we have purified the 46-kDa protein to homogeneity from 
Leishmania promastigote membrane. Purified HbR specifically binds Hb. 
The gene for HbR was cloned, and sequence analysis of the full-length 
HbR gene indicates the presence of hexokinase (HK) signature sequences, 
ATP-binding domain, and PTS-II motif. Four lines of evidence indicate 
that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb
, and the Hb-binding domain resides in the N terminus of the protein; 2
) recombinant proteins and cell lysate prepared from HbR-overexpressing 
Leishmania promastigotes show enhanced HK activity in comparison with 
untransfected cells; 3) immunolocalization studies using antibodies 
against the N-terminal fragment (Ld-HbR-DeltaC) of Ld-HbR indicate that 
this protein is located in the flagellar pocket of Leishmania; and 4) 
binding and uptake of (125)I-Hb by Leishmania is significantly inhibited
 by anti-Ld-HbR-DeltaC antibody and Ld-HbR-DeltaC, respectively. Taken 
together, these results indicate that HK present in the flagellar pocket
 of Leishmania is involved in Hb endocytosis.

PMID: 15619607

TITLE: Correction of Translational Defects in Patient-derived Mutant
Mitochondria by Complex-mediated Import of a Cytoplasmic tRNA.

AUTHORS: Bidesh Mahata, Suvendra Nath Bhattacharyya, Saikat Mukherjee, Samit
Adhya

AFFILIATION: Genetic Engineering Laboratory, Indian Institute of Chemical
Biology, 4 Raja S. C. Mullick Road, Calcutta 7000032, India.

REFERENCE: J Biol Chem 2005 Feb 280(7):5141-4

A variety of clinical disorders result from mutations in mitochondrial 
tRNA genes, leading to translational defects. We show here that a 
protein complex from the kinetoplastid protozoon Leishmania induces 
specific, ATP-dependent import of human cytoplasmic tRNA(1)(Lys) into 
human mitochondria in vitro. The imported tRNA undergoes efficient 
aminoacylation within the organelle and supports organellar protein 
synthesis. Moreover, translation in mitochondria from patients with 
myclonic epilepsy with ragged red fibers (MERRF) and Kearns-Sayre 
syndrome (KSS), containing mutant tRNA(Lys) genes, is stimulated to near
-wild-type levels and the formation of aberrant polypeptides suppressed 
by complex-mediated import. These results suggest a novel way to 
introduce RNAs for the modulation of mitochondrial gene expression.

PMID: 15561707

TITLE: Molecular characterization, expression, and in vivo analysis of LmexCht1:
the chitinase of the human pathogen, Leishmania mexicana.

AUTHORS: Manju B Joshi, Matthew E Rogers, Alison M Shakarian, Mat Yamage, Saeed
A Al-Harthi, Paul A Bates, Dennis M Dwyer

AFFILIATION: Cell Biology Section, Laboratory of Parasitic Diseases, NIAID,
National Institutes of Health, Bethesda, Maryland 20892-0425, USA.

REFERENCE: J Biol Chem 2005 Feb 280(5):3847-61

Chitinases have been implicated to be of importance in the life cycle 
development and transmission of a variety of parasitic organisms. Using 
a molecular approach, we identified and characterized the structure of a
 single copy LmexCht1-chitinase gene from the primitive trypanosomatid 
pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an 
approximately 50 kDa protein, with well conserved substrate binding and 
catalytic domains characteristic of members of the chitinase-18 protein 
family. Further, we showed that LmexCht1 mRNA is constitutively 
expressed by both the insect vector (i.e. promastigote) and mammalian (i
.e. amastigote) life cycle developmental forms of this protozoan 
parasite. Interestingly, however, amastigotes were found to secrete/
release approximately >2-4-fold higher levels of chitinase activity 
during their growth in vitro than promastigotes. Moreover, a homologous 
episomal expression system was devised and used to express an epitope-
tagged LmexCht1 chimeric construct in these parasites. Expression of the
 LmexCht1 chimera was verified in these transfectants by reverse 
transcription-PCR, Western blots, and indirect immunofluorescence 
analyses. Further, results of coupled immunoprecipitation/enzyme 
activity experiments demonstrated that the LmexCht1 chimeric protein was
 secreted/released by these transfected L. mexicana parasites and that 
it possessed functional chitinase enzyme activity. Such transfectants 
were also evaluated for their infectivity both in human macrophages in 
vitro and in two different strains of mice. Results of those experiments
 demonstrated that the LmexCht1 transfectants survived significantly 
better in human macrophages and also produced significantly larger 
lesions in mice than control parasites. Taken together, our results 
indicate that the LmexCht1-chimera afforded a definitive survival 
advantage to the parasite within these mammalian hosts. Thus, the 
LmexCht1 could potentially represent a new virulence determinant in the 
mammalian phase of this important human pathogen.

PMID: 15715010

TITLE: Detection of Leishmania infantum in canine peripheral blood.

AUTHORS: V Foglia Manzillo, D Piantedosi, L Cortese

AFFILIATION: Dipartimento di Scienze Cliniche Veterinarie, Sezione Clinica
Medica, UniversitÃ  degli Studi di Napoli Federico II, via F. Delpino 1,80137
Naples, Italy.

REFERENCE: Vet Rec 2005 Jan 156(5):151-2

PMID: 15539112

TITLE: Rocking the curve.

AUTHORS: Marc Choisy, Mallorie Hide, Anne-Laure BaÃ±uls, Jean-FranÃ§ois GuÃ©gan

REFERENCE: Trends Microbiol 2004 Dec 12(12):534-6

PMID: 15711773

TITLE: Regulation of k-cl cotransport: from function to genes.

AUTHORS: N C Adragna, M Di Fulvio, P K Lauf

AFFILIATION: Departments of Pharmacology and Toxicology, Wright State
University, School of Medicine, Dayton, OH, 45435-0002, USA,
norma.adragna at wright.edu.

REFERENCE: J Membr Biol 2004 Oct 201(3):109-37

This review intends to summarize the vast literature on K-Cl cotransport
 (COT) regulation from a functional and genetic viewpoint. Special 
attention has been given to the signaling pathways involved in the 
transporter's regulation found in several tissues and cell types, and 
more specifically, in vascular smooth muscle cells (VSMCs). The number 
of publications on K-Cl COT has been steadily increasing since its 
discovery at the beginning of the 1980s, with red blood cells (RBCs) 
from different species (human, sheep, dog, rabbit, guinea pig, turkey, 
duck, frog, rat, mouse, fish, and lamprey) being the most studied model
. Other tissues/cell types under study are brain, kidney, epithelia, 
muscle/smooth muscle, tumor cells, heart, liver, insect cells, 
endothelial cells, bone, platelets, thymocytes and Leishmania donovani. 
One of the salient properties of K-Cl-COT is its activation by cell 
swelling and its participation in the recovery of cell volume, a process
 known as regulatory volume decrease (RVD). Activation by thiol 
modification with N-ethylmaleimide (NEM) has spawned investigations on 
the redox dependence of K-Cl COT, and is used as a positive control for 
the operation of the system in many tissues and cells. The most accepted
 model of K-Cl COT regulation proposes protein kinases and phosphatases 
linked in a chain of phosphorylation/dephosphorylation events. More 
recent studies include regulatory pathways involving the phosphatidyl 
inositol/protein kinase C (PKC)-mediated pathway for regulation by 
lithium (Li) in low-K sheep red blood cells (LK SRBCs), and the nitric 
oxide (NO)/cGMP/protein kinase G (PKG) pathway as well as the platelet-
derived growth factor (PDGF)-mediated mechanism in VSMCs. Studies on VSM
 transfected cells containing the PKG catalytic domain demonstrated the 
participation of this enzyme in K-Cl COT regulation. Commonly used 
vasodilators activate K-Cl COT in a dose-dependent manner through the NO
/cGMP/PKG pathway. Interaction between the cotransporter and the 
cytoskeleton appears to depend on the cellular origin and experimental 
conditions. Pathophysiologically, K-Cl COT is altered in sickle cell 
anemia and neuropathies, and it has also been proposed to play a role in
 blood pressure control. Four closely related human genes code for KCCs
 (KCC1-4). Although considerable information is accumulating on tissue 
distribution, function and pathologies associated with the different 
isoforms, little is known about the genetic regulation of the KCC genes 
in terms of transcriptional and post-transcriptional regulation. A few 
reports indicate that the NO/cGMP/PKG signaling pathway regulates KCC1 
and KCC3 mRNA expression in VSMCs at the post-transcriptional level. 
However, the detailed mechanisms of post-transcriptional regulation of 
KCC genes and of regulation of KCC2 and KCC4 mRNA expression are unknown
. The K-Cl COT field is expected to expand further over the next decades
, as new isoforms and/or regulatory pathways are discovered and its 
implication in health and disease is revealed.

PMID: 15601228

TITLE: The choice of animal model and reduction.

AUTHORS: Michael F W Festing

AFFILIATION: FRAME, Russell & Burch House, 96-98 North Sherwood Street,
Nottingham NG1 4EE, UK.

REFERENCE: Altern Lab Anim 2004 Sep 32 Suppl 2():59-64

Careful choice of the animal model is essential, if research is to be 
conducted efficiently, by using the minimum number of animals in order 
to provide the maximum amount of information. Inbred strains of rodents 
provide an excellent way of controlling and investigating genetic 
variation in characters of interest and in response to experimental 
treatments. Outbred stocks, in which genetic and non-genetic factors are
 inextricably mixed, are much less suitable, because random and 
uncontrolled genetic variation tends to obscure any treatment responses
. In some cases, the use of inbred strains has led to major advances in 
scientific understanding. The specific example given here is in the 
understanding of host-parasite relationships but, more generally, inbred
 strains have been of critical importance in research which has resulted
 in the award of at least 17 Nobel prizes. And yet, despite the 
extensive literature on the properties and scientific value of inbred 
strains, many scientists continue to use outbred stocks in the mistaken 
belief that the use of such animals will, in some mysterious way, make 
their research more applicable to humans. There is really no evidence 
that this is so, and there is much evidence that the use of inbred 
strains has been highly successful in many disciplines.

PMID: 15183913

TITLE: Piperazine-linked bisbenzamidines: a novel class of antileishmanial
agents.

AUTHORS: Annie Mayence, Jean Jacques Vanden Eynde, Louis LeCour, Larry A Walker,
Babu L Tekwani, Tien L Huang

AFFILIATION: Division of Basic Pharmaceutical Sciences, College of Pharmacy,
Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, USA.

REFERENCE: Eur J Med Chem 2004 Jun 39(6):547-53

A series of 13 1,4-diarylpiperazines has been prepared, evaluated for 
antileishmanial activity and their binding affinity to DNA was measured
. Among these compounds, 1,4-bis[4-(1H-benzimidazol-2-yl)phenyl]
piperazine (14) emerged as the most active compound with an IC(50) value
 of 0.41 microM which is about sevenfold more potent than pentamidine.

********************************************************************************************************************

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PMID: 15474490

TITLE: Toxoplasma gondii exposes phosphatidylserine inducing a TGF-beta1
autocrine effect orchestrating macrophage evasion.

AUTHORS: Sergio H Seabra, Wanderley de Souza, Renato A Damatta

AFFILIATION: LaboratÃ³rio de Biologia Celular e Tecidual, Centro de BiociÃªncias
e Biotecnologia, Universidade Estadual do Norte Fluminense, 28013-600 Campos dos
Goytacazes, RJ, Brazil.

REFERENCE: Biochem Biophys Res Commun 2004 Nov 324(2):744-52

Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii. 
Activated macrophages control T. gondii growth by nitric oxide (NO) 
production. However, T. gondii active invasion inhibits NO production, 
allowing parasite persistence. Here we show that the mechanism used by T
. gondii to inhibit NO production persisting in activated macrophages 
depends on phosphatidylserine (PS) exposure. Masking PS with annexin-V 
on parasites or activated macrophages abolished NO production inhibition
 and parasite persistence. NO production inhibition depended on a 
transforming growth factor-beta1 (TGF-beta1) autocrine effect confirmed 
by the expression of Smad 2 and 3 in infected macrophages. TGF-beta1 led
 to inducible nitric oxide synthase (iNOS) degradation, actin filament (
F-actin) depolymerization, and lack of nuclear factor-kappaB (NF-kappaB
) in the nucleus. All these features were reverted by TGF-beta1 
neutralizing antibody treatment. Thus, T. gondii mimics the evasion 
mechanism used by Leishmania amazonensis and also the anti-inflammatory 
response evoked by apoptotic cells.

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