Publications

reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase.	nitric oxide (no) synthase (inos) is required for the resolution of acute cutaneous leishmaniasis in resistant c57bl/6 mice. as is the case in several other infections, the clinically cured host organism still harbors small amounts of live leishmania major parasites. here, we demonstrate lifelong expression of inos at the site of the original skin lesion and in the draining lymph node of long-term-infected c57bl/6 mice. inos activity in the lymph node was dependent on cd4+, but not on the cd8+ t ...	1996	8666908
protective role of the pefloxacin-ifn-gamma association in leishmania major-infected mice.	four groups of female balb/c mice were inoculated in the left hind footpad with 30 microliters of rpmi 1640 medium containing 10(7) leishmania major amastigotes/ml. one group was injected sc with 200 microliters of rpmi 1640 containing 180 micrograms of pefloxacin for 20 days, a second group with the same amount of medium containing 100 units of recombinant murine interferon gamma (rmifn-gamma). the third group was treated with the association, while the fourth group received plain medium in an ...	1996	8673851
leishmania major-human macrophage interactions: cooperation between mac-1 (cd11b/cd18) and complement receptor type 1 (cd35) in promastigote adhesion.	it has been suggested that the developmental maturation of leishmania major promastigotes can affect their interaction with human complement receptors. to study this, we measured the adhesion of metacyclic and logarithmic-phase l. major promastigotes to complement receptors expressed on primary macrophages, to recombinant receptors expressed on transfected cells, or to purified complement receptors in a cell-free system. we demonstrate that complement-opsonized promastigotes can bind to both mac ...	1996	8675328
treatment of cutaneous leishmaniasis with itraconazole. randomized double-blind study.	to compare the efficacy of itraconazole with placebo in the treatment of cutaneous leishmaniasis caused by leishmania major.	1996	8678570
experimental infection and transmission of leishmania major by laboratory-reared phlebotomus bergeroti parrot.	the ability of colony-reared phlebotomus bergeroti parrot to successfully acquire and transmit leishmania major (strain ipap/eg 89/si-177) was demonstrated in the laboratory. female p. bergeroti were fed naturally on infected mice and artificially on infected blood suspension using a chick-skin membrane apparatus. groups of sand flies, either infected on mice or by membrane feeding, were dissected and examined using light microscopy at 2-6, 8, 10, and 11 days postfeeding. heavy promastigote infe ...	1996	8686785
biosynthesis of lipophosphoglycan from leishmania major: solubilization and characterization of a (beta 1-3)-galactosyltransferase.	lipophosphoglycan (lpg), is the major cell surface molecule of promastigotes of all leishmania species. it is comprised of three domains: a conserved glycosylphosphatidylinositol anchor linked to a repeating phosphorylated disaccharide (p2; po4-6gal beta 1-4man alpha 1-) backbone and capped with a neutral oligosaccharide. in leishmania major the backbone is substituted at the c(o)3 of the galp residue with side chains containing galp, glcp and arap residues whereas in leishmania donovani the bac ...	1996	8694771
deficiency in beta1,3-galactosyltransferase of a leishmania major lipophosphoglycan mutant adversely influences the leishmania-sand fly interaction.	to study the function of side chain oligosaccharides of the cell-surface lipophosphoglycan (lpg), mutagenized leishmania major defective in side chain biosynthesis were negatively selected by agglutination with the monoclonal antibody wic79.3, which recognizes the galactose-containing side chains of l. major lpg. one such mutant, called spock, lacked the ability to bind significantly to midguts of the natural l. major vector, phlebotomus papatasi, and to maintain infection in the sand fly after ...	1996	8702802
purification and structural characterization of a filamentous, mucin-like proteophosphoglycan secreted by leishmania parasites.	parasitic protozoa of the genus leishmania secrete a filamentous macromolecule that forms networks and appears to be associated with cell aggregation. we report here the purification of this parasite antigen from leishmania major culture supernatant and its compositional (75.6% carbohydrate, 20% phosphate, 4.4% amino acids, w/w), structural, and ultrastructural characterization as a highly unusual proteophosphoglycan (ppg). mild acid hydrolysis, which cleaves preferentially hexose 1-phosphate bo ...	1996	8702946
towards a strategy of universally efficacious vaccination against pathogens uniquely susceptible to cell-mediated attack.	infection by some intracellular parasites is contained only by cell-mediated immunity, and yet antibody is produced at the expense of the cell-mediated response upon natural infection, leading to chronic or fatal disease. effective vaccination must therefore generate an immunological imprint ensuring a strong and stable cell-mediated response upon infection. such diseases include leprosy, tuberculosis, the leishmaniasis and aids (kaplan and cohn (1986) int. rev. exp. pathol. 28, 45-78; surcel et ...	1996	8717379
mice lacking the tnf receptor p55 fail to resolve lesions caused by infection with leishmania major, but control parasite replication.	tnf is involved in host resistance to several pathogens. recently it was found that mice lacking the p55 receptor for tnf (tnfrp55 -/-) do not control growth of the intracellular bacteria, listeria monocytogenes and mycobacterium tuberculosis. here we report that the course of infection in tnfrp55 -/- mice with another intracellular pathogen, the protozoan parasite leishmania major, is also quite different from normal mice. tnfrp55 -/- mice developed larger lesions than control mice and failed t ...	1996	8752935
psammomys obesus cretzschmar, 1828 and zoonotic cutaneous leishmaniasis in sinai peninsula, egypt.	in the middle east, the fat sand rat psammomys obesus is the most important reservoir host of zoonotic cutaneous leishmamiasis (zcl). it is the most incriminated host in outbreaks. two p. obesus caught in wadi el gedeiret (al arish, north sinai) were found naturally infected with leishmania major as indicated by enzyme electrophoresis. in egypt, the already known reservoir hosts are gerbillus pyramidum i. geoffroy st. hilaire, 1825; meriones crassus sundevall, 1984 and meriones sacramenti thomas ...	1996	8754646
two distinct stimulus-dependent pathways lead to production of soluble murine interleukin-4 receptor.	the il-4r exists in two forms, either membrane bound or as a soluble (s) molecule. since the sil-4r binds to its ligand with high affinity, thereby acting as an immunoregulatory molecule, we were interested in the processes leading to its release. first, the release of sil-4r in the model of murine leishmaniosis was analyzed. infection of mice with leishmania major resulted in up-regulation of sil-4r production by ag-stimulated cd4+ t cells, with a maximum around 7 days after infection. to clari ...	1996	8757301
evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to leishmania major: a comparison with direct microscopy of smears and sections from lesions.	we have compared the sensitivity of the polymerase chain reaction (pcr) as a diagnostic tool against conventional microscopical diagnostic techniques in patients with cutaneous leishmaniasis from the sudan. twenty-eight patients were diagnosed according to clinical criteria followed by microscopical examination of histological sections and slit or impression smears. the pcr had a sensitivity of 86% when used alone, and 93% when combined with southern blotting. in contrast, microscopy of histolog ...	1996	8761571
genetically resistant mice lacking interleukin-12 are susceptible to infection with leishmania major and mount a polarized th2 cell response.	mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (il-12) and derived from a strain genetically resistant to infection with leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional cd4+ t cell subsets in vivo. wild-type 129/sv/ev mice are resistant to infection with l. major showing only small lesions which resolve spontaneously within a few weeks an ...	1996	8766560
use of the green fluorescent protein as a marker in transfected leishmania.	we have tested the suitability of the green fluorescent protein (gfp) of aequorea victoria as a marker for studies of gene expression and protein targeting in the trypanosomatid parasite leishmania. leishmania promastigotes expressing gfp from episomal pxg vectors showed a bright green fluorescence distributed throughout the cell, readily distinguishable from control parasites. transfection of a modified gfp gene containing gc-rich synonymous codons and the s65t mutation (gfp+) yielded a much hi ...	1996	8784772
visceral leishmaniasis in somalia. significance of igg subclasses and of ige response.	we have determined the levels of igg subclasses and ige as well as specific antibodies of these isotypes in sera from 22 patients with clinical visceral leishmaniasis (vl) from somalia. the results are compared with those obtained from 30 somali and 23 swedish controls. we found markedly increased concentrations of igg1 in the vl sera, indicating that the pronounced increase in igg in vl which is generally considered to be due to polyclonal b-cell activation is mainly restricted to this subclass ...	1996	8793564
susceptibility to leishmania major in il-4 transgenic mice is not correlated with the lack of a th1 immune response.	il-4 transgenic mice of c3h genetic background expressing il-4 under the control of an mhc class i promoter were infected with leishmania major and the immune response was assessed. in contrast to littermate control mice, the transgenic mice were unable to restrict the growth of the parasites as shown by the strong increase in footpad swelling and parasite numbers in the spleen. the observed susceptibility was markedly less severe than that observed in balb/c mice. restimulation of lymph node ce ...	1996	8799723
broad spectrum antibiotic activity of the skin-pyy.	neuropeptide y (npy) and polypeptide yy (pyy) are two ubiquitous neuropeptides, found in brain and intestines, respectively, where they exert important regulatory functions. in this study, a new member of the yy family recently isolated from amphibian skin, skin-pyy (spyy), is reported to inhibit irreversibly the proliferation of a broad spectrum of pathogenic microorganisms. npy and pyy are shown to be endowed with the same activity. their potency is similar to that of other antibacterial pepti ...	1996	8601432
successful chemotherapy in experimental leishmaniasis is influenced by the polarity of the t cell response before treatment.	little is known about the influence of host factors on successful chemotherapy in leishmaniasis. although successfully treated patients will convert from a delayed-type hypersensitivity (dth)-negative to a dth-positive state, the importance of the immune status of the host before treatment remains largely unexplored. in experimental murine cutaneous leishmaniasis, it was found that increased polarization towards a th2 cytokine profile before the onset of drug therapy leads to an increased freque ...	1996	8603980
ex vivo evidence for pge2 and ltb4 involvement in cutaneous leishmaniasis: relation with infection status and cytokine production.	ex vivo culture of spleen cells from balb/c mice infected with 2 x 10(6) leishmania major (l. major) promastigotes were cultured with concanavalina (cona) or leishmanial antigen (l. ag) and tested for prostaglandin e2 (pge2) and for leukotriene b4 (ltb4), in order to study their involvement in the evolution of cutaneous leishmaniasis and the connexion with lymphokine-mediated responses. the data were compared with those obtained in balb/c mice protected against l. major by sublethal irradiation ...	1996	8587796
leishmania susceptibility puzzle gets another twist.		1996	8584928
genetic susceptibility to leishmania: il-12 responsiveness in th1 cell development.	the genetic background of t lymphocytes influences development of the t helper (th) phenotype, resulting in either resistance or susceptibility of certain mouse strains to pathogens such as leishmania major. with an in vitro model system, a difference in maintenance of responsiveness of t cells to interleukin-12 (il-12) was detected between balb/c and b10.d2 mice. although naive t cells from both strains initially responded to il-12, balb/c t cells lost il-12 responsiveness after stimulation wit ...	1996	8584935
susceptibility to leishmania major infection in interleukin-4-deficient mice.	interleukin-4 (il-4), a pleiotropic cytokine, is a major regulator of the immune system and is considered crucial for the development of t helper cell type 2 (th2) responses. the susceptibility of balb/c mice to infection with leishmania major has been associated with a polarized th2 response and an inability to down-modulate il-4 production. the role of il-4 in vivo was examined directly by disrupting the il-4 gene in balb/c embryonic stem cells. despite the absence of il-4, the genetically pur ...	1996	8584936
characterization of the immunological memory state generated in mice susceptible to leishmania major following exposure to low doses of l. major and resulting in resistance to a normally pathogenic challenge.	balb/c mice are susceptible to a high-dose infection of the protozoan leishmania major, which induces a parasite-specific antibody, th2-like response, exclusive of a significant and protective cell-mediated th1 component. we have shown, in contrast, that infection with a low number of parasites induces cell-mediated immunity exclusive of antibody production, and results in resistance to substantial subsequent high-dose infection. low-dose exposure thus constitutes effective vaccination. in the p ...	1996	8566074
pregnancy impairs resistance of c57bl/6 mice to leishmania major infection and causes decreased antigen-specific ifn-gamma response and increased production of t helper 2 cytokines.	resolution of cutaneous leishmaniasis in infected mice is associated with a polarized th1 immune response by the host, whereas maternal immune responses during pregnancy appear to be biased toward humoral (th2) and away from cell-mediated (th1) responses. the objective of this study was to evaluate whether the putative th2 bias in pregnant c57bl/6 mice would impair their normal ability to mount a curative th1 response against leishmania major infection. pregnant c57bl/6 mice developed larger cut ...	1996	8543816
t helper 1 response against leishmania major in pregnant c57bl/6 mice increases implantation failure and fetal resorptions. correlation with increased ifn-gamma and tnf and reduced il-10 production by placental cells.	maternal immune responses can influence fetal survival and several cytokines have harmful or protective effects on pregnancy. the th1 cytokines ifn-gamma and il-2 can cause fetal loss, whereas the th2 cytokine il-10 is protective. however, infections such as leishmaniasis show the opposite pattern: resistance is associated with the preferential mounting of a th1 response, whereas a th2 response exacerbates the disease. we therefore asked whether the curative th1 response against leishmania major ...	1996	8543817
resistance to leishmania major in mice.		1996	17772041
response: resistance to leishmania major in mice.		1996	17772042
modulating ongoing th2-cell responses in experimental leishmaniasis.	dramatically polarized t helper (th)-cell responses are seen in experimental murine infections with leishmania major. resistant mice develop a th1-cell type response and heal the primary lesion, while susceptible mice develop non-protective th2-cell responses, and the disease eventually proves fatal. deservedly, much effort has gone into determining factors that influence the development of these t-cell subsets early in infection; however, little is known about how the polarity of established re ...	1997	15275127
a simple method for quantifying leishmania in tissues of infected animals.	in experimental animals infected with leishmania major, the size of cutaneous lesions of the parasite often does not correlate with the number of parasites within the lesion. indeed, cutaneous lesions can heal, but still contain parasites. thus, the ability to determine parasite burden in infected animals becomes important, especially when assessing vaccines that are intended to induce sterilizing immunity. here, hermenio lima, julie bleyenberg and richard titus describe a simple technique for e ...	1997	15275128
vaccine efficacy of salmonella strains expressing glycoprotein 63 with different promoters.	the development of salmonella vaccine vectors has been hindered by both the requirement for multiple doses to induce immune responses and a lack of plasmid stability. direct comparisons of different promoter systems with the same antigen are necessary to address these important issues. we have previously described an aroa- arod- deletion mutant of salmonella typhimurium (gid101) which expresses the gene encoding the leishmania major promastigote surface glycoprotein gp63 (gid101). while this con ...	1997	8975908
effects of crude antigenic fractions of leishmania major on natural killer cell cytotoxicity, interferon-gamma and interleukin-4 secretion from peripheric blood lymphocytes of unexposed individuals.	the crude antigenic fraction (caf) isolated from leishmania major was fractionated into three subfractions by sodium dodecyl sulphate-polyacrylamide gel (sds-page). the effects of caf and its subfractions on nk cell cytotoxicity is investigated by chromium release assay. these subfractions designated as fractions 1, 2 and 3 correspond to 97.4-66 kd, 66-45 kd and 29 kd and below respectively. although both caf and its subfractions have inhibited the cytotoxicity of natural killer (nk) cells, the ...	1997	9143943
use of leishmania major derived leishmanin for skin test surveys of visceral leishmaniasis in ethiopia.	the potential use of leishmania major derived leishmanin in surveys of visceral leishmaniasis (vl) was examined. the study was conducted in konso sub-district, southwest ethiopia involving 51 vl patients, 18 vl contacts, four localised cutaneous leishmaniasis (lcl) cases and 15 healthy controls. sixty three percent of treated vl patients, 44.4% of vl contacts, all of lcl cases and none of untreated vl patients were positive to the test. leishmanin skin test (lst) induration sizes of 41 treated v ...	1997	9145577
human leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years.	the current interest in leishmaniasis stems from the importance of this disease with respect to travel medicine, veterans of operation desert storm, humanitarian concerns, and infection with human immunodeficiency virus. herein, i review aspects of leishmaniasis that are of practical value to practitioners, including presentation, diagnosis, and chemotherapy; i will emphasize advances in chemotherapy over the last 10 years. amphotericin b and its new lipid formulations are now competitive with p ...	1997	9145744
multiple lesions of sporotrichoid leishmaniasis in a filipino expatriate.	sporotrichoid cutaneous leishmaniasis (cl) was diagnosed in a filipino man who had worked in saudi arabia for 2 years. two primary lesions-one on the forearm and one on the abdomen-were characterized by "satellite" papules and subcutaneous nodules extending proximally in a sporotrichoid pattern. leishmania organisms were found in both primary lesions and a subcutaneous nodule. there was no evidence of systemic involvement. recognition that old world cl may disseminate through lymphatic channels ...	1997	9146565
combined immunomagnetic cell sorting and elispot assay for the phenotypic characterization of specific antibody-forming cells.	a combination of immunomagnetic cell sorting and elispot techniques has been evaluated to permit enrichment and characterization of antibody-secreting cells (asc). cell suspensions containing putative asc were first incubated with magnetic microbeads coated with antibodies specific for a given cell surface marker. after separation of bead-cell clusters and free cells, the resulting cell populations were examined for the presence of asc by an elispot assay. as a model system, the expression of se ...	1997	9149813
resistance to leishmania major is linked to the h2 region on chromosome 17 and to chromosome 9.	in leishmaniasis, as in many infectious diseases, clinical manifestations are determined by the interaction between the genetics of the host and of the parasite. here we describe studies mapping two loci controlling resistance to murine cutaneous leishmaniasis. mice infected with l. major show marked genetic differences in disease manifestations: balb/c mice are susceptible, exhibiting enlarging lesions that progress to systemic disease and death, whereas c57bl/6 are resistant, developing small, ...	1997	9151907
the roles of pteridine reductase 1 and dihydrofolate reductase-thymidylate synthase in pteridine metabolism in the protozoan parasite leishmania major.	trypanosomatid protozoans depend upon exogenous sources of pteridines (pterins or folates) for growth. a broad spectrum pteridine reductase (ptr1) was recently identified in leishmania major, whose sequence places it in the short chain alcohol dehydrogenase protein family although its enzymatic activities resemble dihydrofolate reductases. the properties of ptr1 suggested a role in essential pteridine salvage as well as in antifolate resistance. to prove this, we have characterized further the p ...	1997	9153248
host-cell cyclophilin is important for the intracellular replication of leishmania major.	the antiparasitic effects of cyclosporin a were examined in leishmanial infection by analysing the role of csa-binding proteins (cyclophilins) in the host-parasite interaction. we hypothesized that the leishmanicidal effects of csa on leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. two cyclophilins (20 and 22 kda) were purified from l. major parasites and n-terminally sequenced. although enzyme activity of these cyclophilins ...	1997	9159527
role of cd4+ t cells in pathogenesis associated with leishmania amazonensis infection.	most inbred strains of mice are susceptible to leishmania amazonensis infection. we have examined the mechanism(s) underlying this generalized susceptibility using mice deficient in t cell development or in the expression of either mhc class i or class ii. in contrast to wild-type c57bl/6 (b6) mice that uniformly developed large ulcerating lesions, mice lacking functional cd4+ t cells (due to targeted disruption of genes for either mhc class ii trans-activator or i-a beta) showed no signs of les ...	1997	9164958
efficient immunity against leishmania major in the absence of interleukin-6.	mice genetically deficient in the pleiotropic cytokine interleukin-6 (il-6) exhibit immunodeficiency against viral, bacterial, and fungal pathogens. when challenged with the protozoan parasite leishmania major, il-6-deficient mice controlled infection and mounted strong th1 responses as efficiently as il-6-sufficient littermates. thus, the successful activation of parasitized macrophages and class ii-restricted t cells does not require a full inflammatory repertoire.	1997	9169788
two new antiprotozoal 5-methylcoumarins from vernonia brachycalyx.	two new isomeric 5-methylcoumarins, 2'-epicycloisobrachycoumarinone epoxide (1) and cycloisobrachycoumarinone epoxide (2), have been isolated from the roots of vernonia brachycalyx by means of bioactivity-guided fractionation. the structures were elucidated by ms and nmr spectroscopic methods. compounds 1 and 2 showed in vitro activity against leishmania major promastigotes and against plasmodium falciparum schizonts and demonstrated an inhibition on the proliferation of human lymphocytes, which ...	1997	9170288
physical mapping across the dihydrofolate reductase-thymidylate synthase chromosome of leishmania major.	we have used a chromosome-specific approach to generate a 300 kb long 'contig' across leishmania major 500 kb chromosome. clones from a 13-hit genomic library served as templates to generate end-specific probes that were used in hybridization to a high density array of the library. the 'contig' generated contained 12 markers uniformly spaced. three restriction endonucleases were mapped within the map extending its resolution. map extension indicated a peculiar feature of sequence organization in ...	1997	9172423
il-4 rapidly produced by v beta 4 v alpha 8 cd4+ t cells instructs th2 development and susceptibility to leishmania major in balb/c mice.	balb/c mice develop aberrant t helper 2 (th2) responses and suffer progressive disease after infection with leishmania major. these outcomes depend on the production of interleukin-4 (il-4) early after infection. here we demonstrate that the burst of il-4 mrna, peaking in draining lymph nodes of balb/c mice 16 hr after infection, occurs within cd4+ t cells that express v beta 4 v alpha 8 t cell receptors. in contrast to control and v beta 6-deficient balb/c mice, v beta 4-deficient balb/c mice w ...	1997	9175832
serial backcross mapping of multiple loci associated with resistance to leishmania major in mice.	resistance or susceptibility of inbred mouse strains to the parasite leishmania major correlates with cd4+ t cell responses of the th1 or th2 subsets, respectively. to evaluate the genetic basis for this difference, resistant b10.d2 mice were backcrossed onto susceptible balb/c mice for five generations with selection for resistance. candidate resistance loci were identified by high frequency of heterozygosity in resistant n5 backcross mice. loci on chromosomes 6, 7, 10, 11, 15, and 16 were asso ...	1997	9175833
impaired th2 subset development in the absence of cd4.	prior studies in cd4-deficient mice established the capacity of t helper (th) lineage cells to mature into th1 cells. unexpectedly, challenge of these mice with nippostrongylus brasiliensis, a th2-inducing stimulus, failed to result in the development of th2 cells. additional studies were performed using cd4+ or cd4-cd8- (double-negative) t cell receptor (tcr) transgenic t cells reactive to lack antigen of leishmania major. double-negative t cells were unable to develop into th2 cells in vivo, a ...	1997	9175834
trans-kingdom transposition of the drosophila element mariner within the protozoan leishmania.	transposable elements of the mariner/tc1 family are postulated to have spread by horizontal transfer and be relatively independent of host-specific factors. this was tested by introducing the drosophila mauritiana element mariner into the human parasite leishmania major, a trypanosomatid protozoan belonging to one of the most ancient eukaryotic lineages. transposition in leishmania was efficient, occurring in more than 20 percent of random transfectants, and proceeded by the same mechanism as in ...	1997	9180085
altered immune responses in interleukin 10 transgenic mice.	interleukin (il)-10 is a pleiotropic cytokine which inhibits a broad array of immune parameters including t helper cell type 1 (th1) cytokine production, antigen presentation, and antigen-specific t cell proliferation. to understand the consequences of altered expression of il-10 in immune models of autoimmune disease, the response to infectious agents, and the response to tumors, we developed transgenic mice expressing il-10 under the control of the il-2 promoter. upon in vitro stimulation, spl ...	1997	9182682
cellulase activity of leishmania major in the sandfly vector and in culture.	the secretion of cellulose-degrading enzymes by leishmania promastigotes in culture and in the sandfly vector was demonstrated. two types of activity of cellulase enzyme-complexes were measured: endoglucanases, which randomly cleave cellulose chains and cellobioydrolases, which remove cellobiose from the nonreducing end of the molecule. the assays demonstrated that enzymes with these activities were secreted into the culture medium by leishmania major, l. donovani, and l. braziliensis. these act ...	1997	9183709
disruption of the trypanothione reductase gene of leishmania decreases its ability to survive oxidative stress in macrophages.	parasitic protozoa belonging to the order kinetoplastida contain trypanothione as their major thiol. trypanothione reductase (tr), the enzyme responsible for maintaining trypanothione in its reduced form, is thought to be central to the redox defence systems of trypanosomatids. to investigate further the physiological role of tr in leishmania, we attempted to create tr-knockout mutants by gene disruption in l. donovani and l. major strains using the selectable markers neomycin and hygromycin pho ...	1997	9184206
a monoclonal antibody directed to terminal residue of beta-galactofuranose of a glycolipid antigen isolated from paracoccidioides brasiliensis: cross-reactivity with leishmania major and trypanosoma cruzi.	a mouse monoclonal antibody, mest-1, was produced against band 1 glycolipid antigen of paracoccidioides brasiliensis. the glycan structure of band 1 antigen was recently elucidated and the monosaccharides sequence was defined as: galf beta 1-->6(manp alpha 1-->3)manp beta 1-->2ins. the reactivity of mest-1 mab was determined by solid-phase radioimmunoassay and high performance thin layer chromatography immunostaining. selective oxidation of galactofuranose residues and inhibition assays with dif ...	1997	9184826
carbohydrate-binding specificities and physico-chemical properties of lectins in various tissue of phlebotominae sandflies.	physico-chemical properties and carbohydrate-binding specificity of hemagglutination activity (ha) were compared in tissue lysates and haemolymph of unfed and bloodfed females of five sandfly species. sandfly gut lectins were found to be heat-labile, sensitive to dithiotreitol treatment, freezing/thawing procedures and were affected by divalent cations. the ph optimum of ha ranged between 7.0-7.5. specificity of gut ha of all species studied was directed towards aminosugars and some glycoconjuga ...	1997	9188176
parasite genome analysis. progress in the leishmania genome project.	genome projects have been established for 7 major groups of human parasitic infections: malaria, leishmaniasis, african trypanosomiasis, american trypanosomiasis, toxoplasmosis, schistosomiasis and filariasis. all except malaria and toxoplasmosis have come under the umbrella of the world health organization's strategic committee on parasite genome analysis. the focus of this meeting of the society was to review progress made in the leishmania and african trypanosome genome projects. this paper i ...	1997	9196742
parasite genome analysis. a global map of the leishmania major genome: prelude to genomic sequencing.	in 1994, the world health organization (tdr) launched a new strategic initiative in parasite genome analysis, establishing international genome networks for filariae, schistosoma, leishmania, trypanosoma brucei and t. cruzi. for leishmania, a number of different but complementary approaches have been adopted by members of the leishmania genome network. our laboratory has been using cosmid clone fingerprinting to produce a physical map of the genome. progress towards the completion of an integrat ...	1997	9196743
an in vitro model for infection with leishmania major that mimics the immune response in mice.	by using a primary in vitro response specific for leishmania major, normal t cells from resistant cba/cah-t6j and susceptible balb/c mice commit to a th1 and a th2 response, respectively. since commitment occurred, we measured the production of gamma interferon (ifn-gamma), interleukin-1 (il-1), il-2, il-4, il-5, il-10, and il-12, prostaglandin e2 (pge2), transforming growth factor beta (tgf-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for th1 ...	1997	9199457
diversity in repeat-containing surface proteins of leishmania major.	the gene b protein (gbp) is one of the products of the lmcdna16 gene family, a cluster of related but non-identical genes that are differentially-expressed during the leishmania life cycle. this protein, which is found on the surface of infective stage parasites, contains an extensive region of proline-rich amino acid repeats, constituting 45% of the total protein. the structure and stability of these repeats have been investigated in a number of l. major strains by polymerase chain reaction (pc ...	1997	9200128
the effects of honey on leishmania parasites: an in vitro study.	the activities of honey dilutions were investigated against three species of leishmania. the results were compared with the effects of the same concentrations of sugar. honey and sugar both have anti-leishmanial effects in vitro, but our results indicated that honey is superior to sugar.	1997	9204723
mariner sails into leishmania.		1997	9206830
a role for cd4 in peripheral t cell differentiation.	naive cd4+ t helper cells (th) differentiate into one of two well-defined cell types during immune responses. mature th1 and th2 cells regulate the type of response as a consequence of the unique cytokines that they secrete. cd4 serves a prominent role in potentiating antigen recognition by helper t cells. we have examined the role of cd4 in peripheral t cell differentiation by studying helper t cells from mice with a congenital defect in cd4 expression. after protein immunization or infection w ...	1997	9207001
interferon regulatory factor-1 is required for a t helper 1 immune response in vivo.	the transcription factor interferon regulatory factor-1 (irf-1) mediates the effects of ifn. no information exists on its role in lymphokine production. protection against the intracellular pathogen leishmania major depends on a th1 response. here, we show that cd4+ t cells from leishmania-infected mice lacking one (+/-) or both (-/-) alleles of the irf-1 gene developed a profound, gene dose-dependent decrease in ifngamma production. irf-1(-/-) mice showed dramatically exacerbated leishmaniasis. ...	1997	9208841
evaluation of leishmania antigens preparation and storage for use in enzyme immunoassays.	the influence of time and temperature on the storage of an alkaline antigen of l. major-like and l.(v.) braziliensis promastigotes added or not of a proteases inhibitor (pmsf) was evaluated by means of an igg-elisa. antibodies in assays using l. major-like antigen stored at -20 degrees c for 6 months had a statistically lower geometric mean titer (gmt) and different 95% confidence interval limits (cl) than antigens stored otherwise, as assessed by the "t" statistic. the pmsfl. major-like antigen ...	1997	9219443
transcription of the leishmania major hsp70-i gene locus does not proceed through the noncoding region.	primary transcripts in kinetoplastid protozoa are generally assumed to be multicistronic. we have analyzed the transcription in the gene locus which encodes the 70-kda heat shock protein by using nuclear run-on analysis. we find that rna synthesis in the hsp70-i gene locus either is terminated or pauses within the intergenic region approximately 250 nt downstream of the polyadenylation site. we therefore propose a discontinuous mode of transcription in the hsp70 genes of leishmania major.	1997	9225771
detrimental effect of nitric oxide on trypanosoma cruzi and leishmania major like cells.	the production of nitric oxide (no) by activated macrophages has been reported to be a non-specific immune-effect mechanism against several parasites. in this work we investigate whether the no has a detrimental effect on the intracellular parasites of the genus leishmania and as well as trypanosoma cruzi. this was assessed by co-cultivating infective leishmania promastigotes and t. cruzi trypomastigotes and non-infective t. cruzi epimastigotes forms of the parasites in the presence of the no re ...	1997	9227803
development of different leishmania major strains in the vector sandflies phlebotomus papatasi and p. duboscqi.	five lines of four leishmania major strains, which differ in geographical origin and virulence for mice, were used for experimental infections of phlebotomus papatasi and p. duboscqi. differences between the lines, which became evident 6 and 9 days after the infective feed, were more pronounced in p. papatasi. the highest infection rates were found for the more virulent line of strain lv561, while the lowest rates were recorded for strains l119 (low-virulence for mice) and neal (avirulent for mi ...	1997	9229020
description of a novel eukaryotic deoxyuridine 5'-triphosphate nucleotidohydrolase in leishmania major.	a leishmania major full-length cdna encoding a functional dutp nucleotidohydrolase (dutpase; ec 3.6.1.23) was isolated from a cdna expression library by genetic complementation of dutpase deficiency in escherichia coli. the cdna contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kda. although eukaryotic dutpases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dutpase e ...	1997	9230126
in vivo blocking of l-selectin rescues balb/c mice from fatal leishmania major infection.	susceptibility and resistance to experimental leishmania major (l. major) infection in mice are associated with a th2- or th1-type response, respectively. we have previously shown that immunological events occurring within the first 24 h after infection in the lymph node (ln) draining the site of parasite challenge are critical for the development of either type of t-cell responses. in the present study we manipulated these events by preventing the entry of naive lymphocytes into the draining ln ...	1997	9232431
successful therapy of chronic, nonhealing murine cutaneous leishmaniasis with sodium stibogluconate and gamma interferon depends on continued interleukin-12 production.	treatment of nonhealing forms of human leishmaniasis with antimonial drugs in combination with gamma interferon (ifn-gamma) may promote healing more effectively than conventional drug therapy. although the natures of immune responses in patients prior to treatment are often unclear, it is generally assumed that such therapy also promotes a switch from a th2-type response to a dominant th1-type response. we have examined the efficacy of ifn-gamma therapy, in combination with drug therapy, to prom ...	1997	9234779
synthesis, stability, and subcellular distribution of major histocompatibility complex class ii molecules in langerhans cells infected with leishmania major.	protozoan parasites of the genus leishmania exist as obligatory intracellular amastigotes and invade macrophages and langerhans cells, the dendritic cells of the skin. langerhans cells are much more efficient in presenting leishmania major antigen to t cells than macrophages are and have the unique ability to retain parasite antigen in immunogenic form for prolonged periods. to analyze the mechanisms that are responsible for this potency, we defined the synthesis, turnover, conformation, and loc ...	1997	9234810
synthesis and antiparasitic activities of amidinic azolated derivatives.	a set of heterocyclic n-acetamidinium hydrochlorides were prepared from the corresponding n-acetonitriles. the antiparasitic screening showed that, while all amidines are practically inactive, some nitriles present leishmanicide properties.	1997	9241827
structure-activity relationships for the antileishmanial and antitrypanosomal activities of 1'-substituted 9-anilinoacridines.	members of the class of 9-anilinoacridine topoisomerase ii inhibitors bearing lipophilic electron-donating 1'-anilino substituents are active against both the promastigote and amastigote forms of the parasite leishmania major. a series of analogues of the known 1'-nhhexyl lead compound were prepared and evaluated against l. major in macrophage culture to further develop structure-activity relationships (sar). toxicity toward mammalian cells was measured in a human leukemia cell line, and the rat ...	1997	9258370
production of tnf alpha and interleukin 6 by differentiated u937 cells infected with leishmania major.	several cytokines play a crucial role in the defense of the host against protozoa belonging to the genus leishmania. however, the role of tumor necrosis factor alpha (tnf alpha) and interleukin 6 (il-6) in human leishmaniasis is still controversial. the aim of this work was to study, in an "in vitro" model, the interactions of human phagocytes with l. major. the u937 human monocytic cell line, differentiated with phorbol myristate acetate (pma) or a combination of 1 alpha,25 dihydroxyvitamin d3 ...	1997	9258943
mitochondrial haplotypes and phylogeography of phlebotomus vectors of leishmania major.	haplotypes of eight phlebotomine species were characterized by cycle sequencing a mitochondrial (mt) dna fragment (cytochrome b to nadh1) amplified from single sandflies by pcr. phlebotomus (phlebotomus) papatasi displayed little variation throughout its large geographical range. we conclude that this vector of leishmania major suffered a population bottleneck late in the pleistocene and then radiated out from the eastern mediterranean subregion. there was no support for a recent domestic lineag ...	1997	9272439
non-mhc-linked th2 cell development induced by soluble protein administration predicts susceptibility to leishmania major infection.	continuous administration of soluble protein ag followed by immunization with the same ag in adjuvant results in the selective development of ag-specific cd4+ th2 cells in both normal and beta2-microglobulin-deficient balb/c mice. in addition to chronic administration by mini-osmotic pump, single bolus i.p., but not i.v., injection of protein ag induces th2 cell expansion. strong th2 cell priming depends on a non-mhc-linked genetic polymorphism. it is observed in all congenic strains on balb bac ...	1997	9278301
dendritic cells induce immunity to cutaneous leishmaniasis in mice.		1997	9286417
leishmania major: a clone with low virulence for balb/c mice elicits a th1 type response and protects against infection with a highly virulent clone.	balb/c mice are highly susceptible to infection with leishmania major and generally develop a severe, nonhealing form of disease following parasite inoculation. as opposed to protective th1 type immune responses which develop in resistant strains of mice, balb/c mice develop predominant th2 type responses characterized by the production of high levels of il-4, but only low levels of ifn-gamma. however, balb/c mice will develop resistance and th1 type responses following the inoculation of very l ...	1997	9287957
early steps in glycosylphosphatidylinositol biosynthesis in leishmania major.	a cell-free system based on washed leishmania major membranes was labelled with gdp-[3h]man in the presence of synthetic glucosaminyl-phosphatidylinositol (glcn-pi) and n-acetylglucosaminyl-phosphatidylinositol (glcnac-pi). in both cases, the major radiolabelled products were man alpha 1-4glcn alpha 1-6myo-inositol1-hpo4- (sn-1, 2-dipalmitoylglycerol) and man alpha 1-4glcn alpha 1-6myo-inositol1-hpo4- (sn-1-palmitoyl-2-lyso-glycerol), to which an additional d-mannose residue was added when a cha ...	1997	9291110
ultrastructural changes in parasites induced by nanoparticle-bound pentamidine in a leishmania major/mouse model.	drug targeting enhances drug efficacy. this principle was tested in the treatment of an experimental visceral leishmaniasis. using transmission electron microscopy (tem) we localized pentamidine-loaded polymethocrylate nanoparticles in the liver of mice infected with leishmania major and compared the ultrastructural changes in the parasites of these mice when they were treated with bound versus free pentamidine. between days 13 and 17 after infection, loaded nanoparticles treated group were inje ...	1997	9296058
metacyclogenesis modulates the ability of leishmania promastigotes to induce il-12 production in human mononuclear cells.	immunity against the intracellular protozoan leishmania species is highly dependent on th1 development. we have previously shown that il-12 is a powerful and probably obligatory factor for th1 cell generation and proliferation. we also observed that the experimental infection of c3h and balb/c mice with leishmania major is associated with il-12 production in vivo. now we demonstrate that metacyclic l. major promastigotes are poor inducers of il-12 in vitro in fresh human pbmc and monocytes. in a ...	1997	9300708
comparison of some molecular-genetic techniques for identification of leishmania circulating in natural foci of zoonotic cutaneous leishmaniasis in the central asia region.	different molecular-genetic methods were used to identify a cohort of leishmania strains from natural foci of zoonotic cutaneous leishmaniasis located in central asia, on the former ussr territory. the results obtained using isoenzymes, pcr, restriction fragment length polymorphisms of kdna and molecular hybridization techniques are discussed in terms of their applicability, discrimination power and feasibility for answering questions related to molecular epidemiological research and for detecti ...	1997	9302420
the role of chemokines and accessory cells in the immunoregulation of cutaneous leishmaniasis.	the course of infection with leishmania parasites is determined by the type of the developing cd4+ t cell immune response. macrophages and langerhans cells/dendritic cells play a decisive role in the interaction between the parasites and the host's immune system because they serve as host cells, as accessory cells that present parasite antigen, deliver costimulatory signals and secrete cytokines modulating the t cell activity and as effector cells eliminating the microorganisms. therefore, we pu ...	1997	9303204
vaccination with dna encoding the immunodominant lack parasite antigen confers protective immunity to mice infected with leishmania major.	to determine whether dna immunization could elicit protective immunity to leishmania major in susceptible balb/c mice, cdna for the cloned leishmania antigen lack was inserted into a euykaryotic expression vector downstream to the cytomegalovirus promoter. susceptible balb/c mice were then vaccinated subcutaneously with lack dna and challenged with l. major promastigotes. we compared the protective efficacy of lack dna vaccination with that of recombinant lack protein in the presence or absence ...	1997	9314562
leishmania major hsp100 is required chiefly in the mammalian stage of the parasite.	in leishmania major a 100-kda heat shock protein, hsp100, is abundant in the intracellular amastigote stage which persists in the mammalian host. a replacement of both clpb alleles which encode hsp100 does not affect promastigote viability under standard culture conditions but impairs thermotolerance in vitro. in experimental infections of balb/c inbred mice, the lack of hsp100 in the gene replacement mutants results in a markedly delayed lesion development compared with that in infections with ...	1997	9315657
structure activity studies of the cytokine macrophage migration inhibitory factor (mif) reveal a critical role for its carboxy terminus.	carboxy-truncated mutants of human mif (mif(1-104) and mif(1-109)) were used in structure activity studies. cd spectroscopy revealed an overall structural similarity between the mutants and mif. denaturant-induced unfolding demonstrated that the c-terminus contributed significantly to the conformational stability of mif. this appears to be due to the formation of two c-terminal beta-strands. the mutants were enzymatically active, exhibiting half of the enzymatic redox activity of mif. however, i ...	1997	9315691
rapid identification of causative species in patients with old world leishmaniasis.	conventional methods for the identification of species of leishmania parasite causing infections have limitations. by using a dna-based alternative, the present study tries to develop a new tool for this purpose. thirty-three patients living in marseilles (in the south of france) were suffering from visceral or cutaneous leishmaniasis. dna of the parasite in clinical samples (bone marrow, peripheral blood, or skin) from these patients were amplified by pcr and were directly sequenced. the sequen ...	1997	9316906
characterization of t cell-mediated responses in nonhealing and healing leishmania major infections in the absence of endogenous il-4.	il-4 drives polarized th2 responses, and differentiating th2 cells down-regulate their sensitivity to il-12. therefore, the failure of balb/c mice to heal leishmania major infection could be due to an il-4-dependent biased th2 response or to a reduced capacity of leishmania-specific th cells to respond to il-12. we examined the ability of cd4+ th cells from l. major-infected wild-type and il-4-deficient balb/c mice to respond to il-12. we show that the inability of normal and il-4-deficient balb ...	1997	9317142
using 3' untranslated sequences to identify differentially expressed genes in leishmania.	a more sensitive screen for leishmania major genes differentially expressed as the insect stage develops into an infectious form (metacyclogenesis) has been devised. the screen expolits the observation that in kinetoplastid protozoa differentially expressed genes are often associated with unique 3' untranslated regions (utrs). to obtain probes encoding this region, cdna is synthesised using an oligo-dt primer containing the universal vectorette sequence in the first strand reaction and an oligon ...	1997	9322753
immunological tolerance to a pancreatic antigen as a result of local expression of tnfalpha by islet beta cells.	recent experiments have suggested that tumor necrosis factor alpha (tnfalpha) can down-regulate islet-specific t cells and prevent the development of autoimmune diabetes. here we demonstrate that transgenic mice expressing both tnfalpha and the leishmania major lack antigen in the pancreas (rip-tnfalpha/rip-lack) exhibit an impaired ability to mount a cd4+ t cell response against lack. in addition, peripheral cd4+ t cells from tcr transgenic mice (tcr-lack/rip-tnfalpha/rip-lack) produced reduced ...	1997	9324360
leishmania spp.: mechanisms of toxicity of nitrogen oxidation products.	intracellular killing of leishmania parasites within activated murine macrophages is thought to result from the toxic activities of nitrogen oxidation products (referred to as no) released by the activated cells. in order to determine possible mechanisms of no toxicity for these microorganisms, promastigotes of leishmania major and leishmania enriettii were exposed to no generated chemically from acidified nitrite, s-nitrosocysteine, diethylamine nonoate, or nitroprusside. treatment with these a ...	1997	9326885
the role of il-12 and il-4 in leishmania major infection.		1997	9329218
interferon (ifn) consensus sequence-binding protein, a transcription factor of the ifn regulatory factor family, regulates immune responses in vivo through control of interleukin 12 expression.	mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (icsbp) develop a chronic myelogenous leukemia-like syndrome and mount impaired responses to certain viral and bacterial infections. to gain a mechanistic understanding of the contributions of icsbp to humoral and cellular immunity, we characterized the responses of control and icsbp-/- mice to infection with influenza a (flu) and leishmania major (l. major). mice of both genotypes survived infections wi ...	1997	9348311
interleukin-12 is indispensable for protective immunity against leishmania major.	interleukin-12 (il-12)-deficient mice derived from a strain genetically resistant to infection with leishmania major were recently shown to be susceptible toward this parasite, developing a strong th2 response after injection of a large number of parasites. we further investigated the role of il-12 in l. major infection by studying the responses of mutant mice against smaller numbers of parasites. il-12-deficient mice infected with only small numbers of parasites showed the progressive lesion de ...	1997	9353008
cytokines and nitric oxide as effector molecules against parasitic infections.	nitric oxide (no) derived from l-arginine by the catalytic action of inducible no synthase (inos) plays an important role in killing parasites. many cell types express high levels of inos when activated by a number of immunological stimuli which include interferon-gamma (ifn-gamma), tumour necrosis factor alpha, and lipopolysaccharide. ifn-gamma is typically produced by the th1 subject of cd4+ t cells, whose differentiation depends on interleukin-12 (il-12) produced by macrophages. mice with a d ...	1997	9355122
immunogenetics of leishmanial and mycobacterial infections: the belem family study.	in the 1970s and 1980s, analysis of recombinant inbred, congenic and recombinant haplotype mouse strains permitted us to effectively 'scan' the murine genome for genes controlling resistance and susceptibility to leishmanial infections. five major regions of the genome were implicated in the control of infections caused by different leishmania species which, because they show conserved synteny with regions of the human genome, immediately provides candidate gene regions for human disease suscept ...	1997	9355125
antiprotozoal compounds from asparagus africanus.	two antiprotozoal compounds have been isolated from the roots of asparagus africanus lam. (liliaceae), a new sapogenin, 2 beta, 12 alpha-dihydroxy-(25r)-spirosta-4,7-dien-3-one (1), which was named muzanzagenin, and the lignan (+)-nyasol (2), (z)-(+)-4,4'-(3-ethenyl-1-propene-1,3-diyl)-bisphenol. the structure of the sapogenin was elucidated by ms and by 1d and 2d nmr methods and established by a single crystal x-ray analysis. (+)-nyasol potently inhibits the growth of leishmania major promastig ...	1997	9358645
parasite and mammalian gpi biosynthetic pathways can be distinguished using synthetic substrate analogues.	glycosylphosphatidylinositol (gpi) structures are attached to many cell surface glycoproteins in lower and higher eukaryotes. gpi structures are particularly abundant in trypanosomatid parasites where they can be found attached to complex phosphosaccharides, as well as to glycoproteins, and as mature surface glycolipids. the high density of gpi structures at all life-cycle stages of african trypanosomes and leishmania suggests that the gpi biosynthetic pathway might be a reasonable target for th ...	1997	9362481
a novel role for the peritrophic matrix in protecting leishmania from the hydrolytic activities of the sand fly midgut.	the role of the peritrophic matrix (pm) in the development of leishmania major infections in a natural vector, phlebotomus papatasi, was investigated by addition of exogenous chitinase to the bloodmeal, which completely blocked pm formation. surprisingly, the absence of the pm was associated with the loss of midgut infections. the chitinase was not directly toxic to the parasite, nor were midgut infections lost due to premature expulsion of the bloodmeal. most parasites were killed in chitinase- ...	1997	9364562
molecular characterization of the heat-inducible lmsti1 protein of leishmania major.	we have recently isolated a cdna encoding the leishmania major homologue of the yeast stress-inducible protein sti1. southern blot analyses indicate that this protein is encoded by a single copy gene in l. major and that this gene is highly conserved throughout the leishmania genus. the sti1 gene is constitutively expressed in both l. major promastigotes and amastigotes however, sti1 transcript levels can be upregulated in promastigotes by a shift in culture temperature from 26 to 37 degrees c. ...	1997	9364964
leishmania major-infected c3h mice treated with anti-il-12 mab develop but do not maintain a th2 response.	leishmania major-infected c3h mice develop a th1 response, but studies have shown that treatment of c3h mice with anti-il-12 or anti-ifn-gamma mab promotes the development of a th2 response and susceptibility. however, we discovered that c3h mice treated for 3 wk with either anti-il-12 or anti-ifn-gamma mab eventually resolved their lesions and switched from a th2 to a th1 response. no significant differences in il-4, il-10, or ifn-gamma levels or in the parasite burden could be detected between ...	1997	9366430
biochemical analysis and immunogenicity of leishmania major amastigote fractions in cutaneous leishmaniasis.	soluble leishmania antigen (sla) from both developmental stages of l. major (l. major mrho/ir/75/er) were prepared. three and five subfractions of sla from amastigote and promastigote were obtained by fast protein liquid chromatography (fplc), respectively. biochemical analyses and comparison of amastigote and promastigote sla were done. the biochemical analyses revealed that the first fraction of l. major amastigote possesses a distinct band on its electrophoretic mobility pattern corresponding ...	1997	9367403
a leishmania protein that modulates interleukin (il)-12, il-10 and tumor necrosis factor-alpha production and expression of b7-1 in human monocyte-derived antigen-presenting cells.	leif, a gene homologue of the eukaryotic initiation factor 4a was first described as a leishmanial antigen that induced a th1-type t cell response in peripheral blood mononuclear cells (pbmc) from leishmaniasis patients. moreover, the interferon (ifn)-gamma production by pbmc was found to be interleukin (il)-12 dependent. herein, we characterize the effects of leif on cytokine production and expression of surface molecules by normal human monocytes as well as by monocyte-derived macrophages and ...	1997	9368620