Peripheral blood mononuclear cell responses to heat shock proteins and their derived synthetic peptides in juvenile idiopathic arthritis patients

Background Sequence homology and cross reactivity between microbial and human heat shock proteins (hsps) led to the concept that hsps might be involved in the etiopathogenesis of autoimmune diseases. Objective In our study we stimulated peripheral blood mononuclear cells (PBMC) of patients with juvenile idiopathic arthritis (JIA) and healthy controls with various hsp-derived peptides together with the whole molecules of corresponding hsp. Methods PBMC were cultured with recombinant human hsp60 (rh-hsp60), rh-hsp70, Mycobacterium bovis hsp65 (M.bovis hsp65), P562–571 human hsp60, P180–188 M. bovis hsp65, P450–463 human hsp70 and P545–554 cytokeratin derived synthetic peptides. Cell responses were measured after incorporation of ³H-thymidine and expressed as stimulation indices. Results and conclusion We found elevated proliferative response to rh-hsp60, M. bovis hsp65 and P562–571 human hsp60 derived peptide in patients with JIA polyarthritis. Significantly elevated proliferation to P180–188 M. bovis hsp65 was found in JIA lasting more than 2 years. None of the particular clinical characteristics (RF, ANA, HLA B27 and disease activity) seemed to be associated with hsp or hsp-derived synthetic peptide proliferative response in the JIA cohort. Received 26 September 2005; returned for revision 13 December 2005; accepted by G. Wallace 3 January 2006     

Immunosuppression in hamsters with progressive visceral leishmaniasis: an evaluation of the role of nitric oxide toward impairment of the lymphoproliferative response

The progressive visceral infection caused in golden hamsters by Leishmania donovani amastigotes led to gradual impairment of the proliferative response of their splenic (SPMC) or peripheral blood (PBMC) mononuclear cells to in vitro stimulation with leishmanial antigen, with mitogen (concanavalin A), and even with a combination of phorbol myristate acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals, however, almost completely restored their proliferative response to PMA + Io, thus ruling out the possibility of any intrinsic defect in the signal-transduction pathways of lymphocyte activation and proliferation. Subsequent studies demonstrated that the generation of soluble mediators such as nitric oxide by these adherent cells is responsible, albeit partially, for the down-regulation of the lymphoproliferative response in hamsters with visceral leishmaniasis. Received: 8 December 1998 / Accepted: 15 January 1999     

Measurement of Serum Antigen Concentration by Ultrasound-Enhanced Immunoassay and Correlation with Clinical Outcome in Meningococcal Disease

 The distribution of Neisseria meningitidis serogroup B and C polysaccharide antigen in blood and the prognostic significance of antigen concentration was examined by ultrasound-enhanced immunoagglutination of coated microparticles. Specimens (169 sera/plasma from 145 patients with confirmed meningococcal disease) were tested retrospectively. The ultrasonic immunoassay detected serum antigen in 136 samples from 112 patients. Titration of antigen-positive specimens allowed estimation of blood antigen concentration. The modal blood antigen titre was 1/16, corresponding to an estimated polysaccharide concentration of 0.85 μg/ml. The lowest mean blood antigen concentration found ultrasonically was 0.05 μg/ml; compared to the 1.98 μg/ml found by conventional latex agglutination, this represents an approximately 30-fold improvement in sensitivity. Three grades of outcome were correlated with the presenting antigen titre in 83 patients: (i) <2 weeks hospitalisation, (ii) ≥2 weeks hospitalisation and (iii) mortality. High polysaccharide concentrations correlated with mortality. Nine of 15 patients with a serum antigen titre of 1/64 or greater (≥3.4 μg/ml polysaccharide) died, whereas no patient with titres equal to or less than 1/4 (≤0.21 μg/ml) died, including those patients in whom antigen was undetectable by ultrasonic immunoassay. Increasing antigen concentration significantly correlated with severity of outcome (P<0.001). Ultrasound-enhanced agglutination provides a rapid prognostic indicator by sensitive measurement of serum antigen level.     

Stimulation of synovial fluid mononuclear cells with the human 65-kD heat shock protein or with live enterobacteria leads to preferential expansion of TCR-gamma delta+ lymphocytes.

T lymphocyte responses to heterologous or self 65-kD heat shock protein (hsp) have been implicated in the pathogenesis of various forms of arthritis. To delineate the relationship of 65-kD hsp to different synovial fluid (SF) T cell subsets, we stimulated synovial fluid (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with different inflammatory rheumatic diseases and from healthy controls with human or mycobacterial 65-kD hsp, tetanus toxoid (TT), heat-killed or live Yersinia enterocolitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR-gamma delta+ lymphocytes in the 65-kD hsp-stimulated SF-derived lines. This expansion of TCR-gamma delta+ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR-gamma delta+ cells was also shown after SFMC stimulation with live, but not with heat-killed Yersinia or with TT. We conclude that a common mechanism is involved in the selective expansion of TCR-gamma delta+ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65-kD hsp. Out of a panel of TCR-gamma delta+ T lymphocyte clones (TLC) derived from a human 65-kD hsp-stimulated line, only a minority of TLC proliferated weakly upon restimulation with this antigen in the presence of autologous monocytes, whereas TCR-alpha beta+ TLC responded vigorously to the human 65-kD hsp and in some cases also cross-recognized the mycobacterial hsp homologue and/or heat-killed Yersinia. This implies that additional factors or cells may be present in the milieu of SFMC cultures that propagate the expansion of TCR-gamma delta+ cells in response to 65-kD hsp or live bacteria.     

Effect of T-helper cytokine environment on specificity of T-cell responses to mycobacterial 65,000 MW heat-shock protein.

The purpose of this work was to determine if the fine specificity of T cells differed between mice immunized with an antigen in a T helper 1 (Th1) cytokine-dominated environment as compared with a T helper 2 (Th2) cytokine-dominated environment. It was found that splenic T cells from mice immunized with mycobacterial heat-shock protein (hsp 65) and interleukin-12 (IL-12) produced less interleukin-4 (IL-4) and more interferon-gamma (IFN-gamma) in response to stimulation with hsp 65 in vitro than did T cells from mice immunized with hsp 65 alone. The T-cell proliferative response to hsp 65 did not differ between the two groups of mice, although the responses were higher than those of T cells from non-immunized mice. Strikingly, T cells from mice given hsp 65 and IL-12 gave significantly higher responses to six peptides (corresponding to the sequence of hsp 65) to which T cells from mice immunized with hsp 65 alone did not respond. It is considered that different epitopes are presented to T cells (possibly owing to changes in antigen processing) if the environment is shifted, by IL-12, from Th2 towards Th1 cytokines.     

Soluble interleukin-2 receptor and soluble CD8 antigen in active rheumatoid arthritis

Concentrations of soluble interleukin-2 receptor (sIL-2R) and of soluble CD8 antigen (sCD8) in sera and in supernatants of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) derived from patients with active rheumatoid arthritis (RA) were studied. sIL-2R concentrations in sera derived from patients with RA (1484 +/- 382 U/ml) were significantly higher than in sera derived from healthy controls (380 +/- 110 U/ml; P less than 0.0005). In contrast, supernatants of PHA-stimulated PBMC derived from patients with RA contained similar amounts of sIL-2R (727 +/- 467 U/ml) as those derived from healthy control individuals (833 +/- 508 U/ml; P greater than 0.1). When investigated for the presence of sCD8 antigen, sera derived from patients with RA contained significantly lower amounts (30 +/- 28 U/ml) than sera derived from healthy controls (405 +/- 136 U/ml; P less than 0.0005). Similarly, PHA stimulation of PBMC derived from patients with RA resulted in a significantly lower production of sCD8 (35 +/- 46 U/ml) as compared to the one obtained by PHA stimulation of PBMC derived from healthy controls (177 +/- 59 U/ml; P less than 0.0005). This difference could not be explained by a lower proliferative response to PHA by PBMC derived from patients with RA (21,474 +/- 14,022 cpm) as compared to healthy controls (29,549 +/- 11,188 cpm; P greater than 0.05). Our data demonstrate that PBMC derived from patients with active RA differ from PBMC derived from healthy individuals concerning their ability to produce sIL-2R and sCD8.     

Soluble CD4 suppress the antigen driven proliferative response of certain T cell clones specific for mycobacteria and for peptides by mycobacterial heat shock proteins.

We have investigated the effect of soluble recombinant CD4 (sCD4) on the antigen specific (BCG, peptides of mycobacterial 65 kDa hsp) responses of T cell lines of T cell clones. The majority of the antigen specific clones could be suppressed in their antigen driven response by the addition of sCD4, while others, including the parental polyclonal T cell line, were not. The suppression of the specific T cell response was reversed by the addition of anti-CD3, did not affect the proliferative response to IL-2, and was independent of the amount of antigen. A decreased capacity to produce IFN-gamma in response to the antigen by the addition of sCD4 was seen only with those clones that were also inhibited in their specific proliferative response. This model may be used to delineate further the interaction between T cells and the antigen presenting cell, and the finding may limit the possible in vivo use of sCD4 in the therapy of human immunodeficiency virus (HIV) infections.     

Functional Properties of Mesenchymal Stem Cells Labeled with Magnetic Microparticles <Emphasis Type="Italic">in Vitro</Emphasis> and Analysis of Their Distribution after Transplantation

Mesenchymal stem cells enzymatically isolated from human placenta were labeled with magnetic fluorescent microparticles (d=0.96 μ). We showed that microparticles in high doses (>10 μl stock suspension per 1 ml culture medium) significantly inhibited cell proliferation in culture. In our work we determined the optimal concentration of particles not affecting physiological properties of mesenchymal stem cells: it does not change cell proliferation, does not induce apoptosis, and does not modulate their transdifferentiation into neuronal cells. In vivo experiments showed that the chosen particles allow easy visualization of transplanted cells ex vivo on sections of different tissues.     

Synovial Cells Responding to a 65-kDa Mycobacterial Heat Shock Protein have a High Proportion of a TcRγδ Subtype Uncommon in Peripheral Blood

We have analysed the ability of T cells from synovial fluid mononuclear cells (SFMC) and from peripheral blood mononuclear cells (PBMC) of inflammatory arthritic diseases to proliferate in response to mycobacterial antigens (65-kDa heat shock protein [hsp] of BCG, whole BCG) and to rat collagen type II. The SFMC demonstrated a significantly greater ability to respond to 65-kDa hsp of BCG, and to whole BCG, compared with PBMC from the same patients. With collagen type II, only a small proportion of the patients showed a proliferative response, although with this antigen also SFMC responded better than PBMC. There was no difference between SFMC and PBMC in the response to control antigen (tetanus toxoid), phytohaemagglutinin (PHA), or interleukin 2 (IL-2). A high proportion of cells in SFMC-derived short-term T-cell lines were of TcR gamma delta type, often exceeding the number of TcR gamma beta type. There was a significantly higher proportion of TcR gamma delta cells in the SFMC lines compared with the PBMC lines, and a large part of the TcR gamma delta cells in the SFMC cultures was CD8+. The SFMC lines had a high proportion of delta-TCS-1+ cells (V delta 1) among their TcR gamma delta cells, always exceeding the percentages of Ti gamma A+(V gamma 9) and BB3+ (V delta 2). In the PBMC lines, the distribution of TcR gamma delta subtypes was markedly different, with a Ti gamma A+/BB3+ population in the majority. These data argue for a different subpopulation distribution of TcR gamma delta cells in synovial fluid compared with peripheral blood of patients with inflammatory arthritic diseases.     

Differential rat T cell recognition of cathepsin D-released fragments of mycobacterial 65 kDa heat-shock protein after immunization with either the recombinant protein or whole mycobacteria

T cells specific for the mycobacterial 65 kDa heat-shock protein(hsp65) play a pivotal role in the development of adjuvant arthritis(AA) in Lewis rats. Upon adoptive transfer, CD4⁺ T cells recognizinga particular hsp65 epitope trigger the onset of disease. Activationof hsp65-reactlve T cells can be achieved by immunization withheat-killed mycobacteria in mineral oil—complete Freund'sadjuvant (CFA)—or with purified recombinant hsp65. Arthritis,however, will only develop after immunization with CFA. In fact,prelmmunlzatlon with hsp65 protects against any subsequent attemptto induce AA. In this study, we examined polyclonal lymph nodecell responses in Lewis rats, Immunized with either CFA or purifiedrecombinant hsp65 in incomplete Freund's adjuvant, to a setof hsp65 fragments generated by a mild digestion with cathepsinD. Prollferatlve responses to several hsp65 fragments variedwith the type of antigen used for immunization. A cathepsinD-released fragment, Identified as residues 376–408, preferentiallytriggered proliferation of rat T cells after hsp65 Immunization.Prelmmunlzatlon of Lewis rats with this peptlde delayed theonset and reduced the severity of AA. Prelmmunlzatlon with anotherfragment which was preferentially recognized after CFA immunization,representing residues 40–60, did not have such a protectiveeffect. Our findings suggest the presence of mycobacterial hsp65determinants that selectively trigger AA-regulatlng T cellsand illustrate that cathepsin D may be used as an experimentaltool to generate such determinants.     

Dietary microparticles implicated in Crohn’s disease can impair macrophage phagocytic activity and act as adjuvants in the presence of bacterial stimuli

Objective and design: Western diets regularly expose the gastrointestinal tract (GI) to large quantities ( > 10¹²/day) of man-made, submicron-sized, particles derived from food additives and excipients. These are taken up by M cells, accumulate in gut macrophages, and may influence the aetiology of inflammatory bowel diseases (IBD). Materials: We investigated the effects of common dietary microparticles on the function of macrophages from healthy donors or active Crohn’s disease (CD) patients. Methods: Macrophages were incubated for 24 h with microparticles before being assayed for cytokine production and phagocytic activity. Results: Microparticles alone were non-stimulatory but, in the presence of bacterial antigens such as LPS, they could act as adjuvants to induce potent cytokine responses. Uptake of high concentrations of microparticles also impaired macrophage phagocytic capacity – but not their ability – to take up 2μM fluorescent beads. Conclusions: While dietary microparticles alone have limited effects on basic macrophage functions, their ability to act as adjuvants could aggravate ongoing inflammatory responses towards bacterial antigens in the GI tract. Received 26 January 2007; returned for revision 27 March 2007; accepted by I. Ahnfelt-R?nne 19 April 2007     

Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat-shock protein 60

The hypothesis that T-cell responses to the 60 000 MW family of heat-shock proteins (hsp) may be related to the severity of rheumatoid arthritis (RA) was examined. Peripheral blood mononuclear cells (PBMC) from most normal individuals and both early and established RA patients proliferated in vitro in response to human hsp 60 and mycobacterial hsp 65 as well as tetanus toxoid (TT) and mycobacterial purified protein derivative (PPD). PBMC from some patients with established RA gave responses to hsp 60 that were above the normal range and/or peaked earlier than PBMC from normal individuals. The responses of PBMC from established RA to hsp 65, but not PPD or TT, were also higher than those from normal individuals, but the peak responses to all three antigens appeared delayed. Thus a selective increase in responsiveness to hsp 60 develops with disease duration in many RA patients. Six assessments of disease activity and severity were made but apart from rheumatoid factor titre, they were unrelated to the proliferative response. Similarly, disease activity and severity did not differ between those RA patients whose hsp 60 stimulated cells produced interferon-gamma and those who did not, although patients whose hsp 60-stimulated T cells produced interleukin-4 (IL-4) and/or IL-10, appeared to have less disease activity and severity than those who did not. Significant negative correlations were found between IL-10 production by hsp 60-stimulated cells and disease assessments. It is considered that RA is less severe in those patients whose hsp 60-stimulated cells produce T-helper 2 type cytokines.     

Evidence of a T helper type 2 activation in human schistosomiasis

The lymphocyte proliferative response and cytokine production to S. mansoni antigen in vitro were evaluated in 22 schistosomiasis patients living in an area endemic for this disease. The majority of patients (86%) showed no lymphocyte proliferative response and none of them showed interferon-γ (IFN-γ) production, following in vitro stimulation with soluble adult worm antigen preparation (SWAP). In contrast, interleukin (IL)-5 (2038 ± 1757 pg/ml) and IL-10 (867 ± 762 pg/ml) were detected in peripheral blood mononuclear cell (PBMC) cultures stimulated with SWAP. Moreover, mRNA for IL-4 was detected in SWAP-stimulated PBMC from 4 of 6 patients evaluated. Restoration of lymphoproliferative response was achieved in 4 of 6 patients by adding anti-IL-10 monoclonal antibody (mAb) to PBMC cultures [mean stimulation index (SI) in the presence of antigen = 2.7 ± 2.9; SI in the presence of antigen plus anti-IL-10, 21 ± 16]. Restoration of IFN-γ production by addition of anti-IL-10 mAb was achieved in 4 of 12 patients evaluated (248, 350, 687 and 710 pg/ml). Moreover, the addition of IL-10 to PBMC cultures of 3 schistosomiasis patients and 2 cured subjects who had high lymphoproliferative responses to SWAP resulted in the suppression of these responses by 90%, and completely suppressed IFN-γ production in one of the subjects, whose PBMC produced IFN-γ after stimulation with SWAP. The presence of IL-4 mRNA, high levels of IL-5, and the absence of IFN-γ in PBMC culture supernatants from infected patients, supports the conclusion that patients living in an endemic area of schistosomiasis express a predominant T helper type 2 response. The high levels of IL-10 and the ability of neutralizing anti-IL-10 mAb to restore T cell responses indicate that this cytokine plays an important role in the modulation of T cell responses in schistosomiasis.     

Opposite Effects of 5-Hydroxymethyluracil on Mitogenic Response of T Cells Stimulated through T-Cell Receptor or through T-Cell Receptor and CD28 Co-Receptor Molecule

The effect of a synthetic analog of “minor” pyrimidine base 5-hydroxymethyluracil (0.01- 10 μg/ml) on the mitogenic response of donor peripheral blood T cells was studied in vitro under conditions of lymphocyte stimulation through T-cell receptor (antiCD3 monoclonal antibodies) or through T-cell receptor and CD28 co-receptor molecule (antiCD3 and antiCD28 monoclonal antibodies). 5-Hydroxymethyluracil suppressed the mitogenic response of T cells stimulated with antiCD3 antibodies, which was paralleled by an increase in the count of silent cells and decrease in the count of dividing lymphocytes, but not by stimulation of apoptosis of activated cells. Under conditions of integration of stimuli (lymphocyte stimulation with antiCD3 and antiCD28), 5-hydroxymethyluracil stimulated the mitogenic response, which was paralleled by suppression of activation apoptosis and increase in proliferative potential of cells.     

Effect of Dynorphin A (1-17) on Proliferation and IL-2, IL-4, IFN-γ Production by Peripheral Blood Mononuclears

Dynorphin A (1-17) in concentrations of 10⁻⁸–10⁻⁹ M inhibits phytohemagglutinin (2.5 μg/ml)-induced proliferative response of mononuclear fraction lymphocytes. In mitogen-stimulated cultures, 10⁻⁸ M dynorphin A (1-17) stimulates the production of IL-4, inhibits the production of IL-2, and does not modify the production of IFN-γ. Nonselective κ-receptor antagonist naloxone and selective antagonist binaltorphimine hydrochloride abolish the inhibitory effects of both dynorphin A concentrations on the lymphocyte proliferative response. On the other hand, evaluation of the effect of κ-receptor blockade on the production of IL-2 and IL-4 showed that this effect depends on peptide concentration and antagonist type. Hence, the results attest to an important role of κ-receptors in modulation of functional activity of immune cells.     

Effect of tuftsin on the functional activity and intracellular pH of murine peritoneal macrophages

The effect of tuftsin and its tripeptide analog in various concentrations (from 0.001 to 10.0 μg/ml) on phagocytosis and on the intracellular pH is studied in murine peritoneal macrophages. Tuftsin causes a uniform dose-dependent increase of these two parameters in the cells. This effect is maximally pronounced at concentrations of the peptide close to its physiological level (about 0.3 μg/ml) and gradually decreases as its content in the incubation medium is lowered or raised. On the other hand, the tripeptide analog of tuftsin does not exhibit such an effect on the cells and under the same conditions suppresses phagocytosis and acidifies their intracellular medium. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 3, pp. 265–267, March 1994 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

FK506 (tacrolimus) improves lung injury through inhibition of Fas-mediated inflammation

Objective To investigate whether FK506 (tacrolimus) can inhibit Fas- or A23187-induced interleukin (IL)-8 expression and cell death in A549 human alveolar epithelial cells, plus Fas-mediated acute lung injury in vivo. Methods Assays for IL-8, cell death, and caspase-3 activity were performed. A549 cells were treated with 25 μmol A23187 or 0.2 μg/ml agonistic anti-Fas antibody plus 5 ng/ml interferon-gamma (IFN-γ). Tacrolimus was treated at 0.1–10 ng/ml. For in vivo experiment, agonistic anti-Fas antibody (Jo2) at 2.5 μg/g was intratracheally instilled into C57BL/6 mice. Neutrophils and protein contents in bronchoalveolar lavage (BAL) fluid were measured within 24 h of instillation. Mice were orally treated with 32 mg/kg of tacrolimus 24 h and 1 h prior to instillation. Results Both Fas and A23187 caused significant IL-8 expression and cell death in A549 cells. Tacrolimus inhibited A23187-induced IL-8 expression alone while it protected all Fas-mediated responses. Mice instilled intratracheally with Jo2 at 2.5 μg/g had significant increases in neutrophils, protein contents in BAL fluid and in expression of chemoattractants for neutrophils. These increases were reversed by tacrolimus. Conclusions Tacrolimus serves as a therapeutic option for improving lung injury through inhibition of Fas-mediated inflammation. Received 7 November 2005; returned for revision 28 December 2005; accepted by G. Wallace 2 February 2006     

Proliferative response of synovial fluid and peripheral blood mononuclear cells to arthritogenic and non-arthritogenic microbial antigens and to the 65-kDa mycobacterial heat-shock protein

Cellular immune responses to microbial antigens have been implicated in the pathogenesis of some forms of arthritis including reactive arthritis, Reiter's syndrome, ankylosing spondylitis and rheumatoid arthritis. We investigated the proliferative T cell responses of paired peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MC) to so-called arthritogenic bacteria (Yersinia entero-colitica and Salmonella typhimurium), to control antigens, such as Candida albicans, mumps virus and purified protein derivative, to the recombinant mycobacterial 65-kDa heat-shock protein (hsp 65) and the mitogen phytohemagglutinin (PHA) in 16 patients with different inflammatory rheumatic diseases. The [³H]thymidine uptake of unstimulated cells (medium control) as well as the proliferative response to the different antigens tested was markedly increased in SFMC irrespective of the underlying rheumatic disease. In contrast, mitogenic stimulation was decreased in SFMC. The proliferative response to the hsp 65 correlated significantly with the responses to Yersinia, Salmonella and Candida. These results may reflect an enhanced function of SF antigen-presenting cells, different functional properties and subset distributions of PB and SF T cells with a preferetial accumulation of helper-inducer/memory T cells or a maintenance of an ongoing immune response by T cells cross-recognizing self epitopes such as epitopes located on the hsp 65. Thus, care should be taken in the interpretation of SF T cell responses to microbial antigens as diagnostic tools in arthritis.     

Identification of mycobacteria of the MAIS complex and M. tuberculosis by restriction fragment length polymorphism analysis of hsp65 gene

Restriction fragment length polymorphism analysis of hsp65 gene was performed on museum strains of mycobacteria using Hin6I restrictase. Study of restriction profiles allowed us to distinguish mycobacterial species of the MAIS complex and several strains of nontuberculous mycobacteria. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 188–191, August, 2006     

Antileishmanial potential of a marine sponge, <Emphasis Type="Italic">Haliclona exigua</Emphasis> (Kirkpatrick) against experimental visceral leishmaniasis

In this study, we are reporting antileishmanial activity of a marine sponge Haliclona exigua, belonging to phylum Porifera. The crude methanol extract and its three fractions were tested both in vitro and in vivo. The crude extract exerted almost complete inhibition of promastigotes at 50 μg/ml and 76.4 ± 6.5% inhibition of intracellular amastigotes at 100 μg/ml concentration with IC₅₀ values of 18.6 μg/ml and 47.2 μg/ml, respectively. When administered to Leishmania donovani infected hamsters at a dose of 500 mg/kg × 5, p.o., it resulted in 72.2 ± 10.4% inhibition of intracellular amastigotes. At a lower dose (250 mg/kg), it exhibited 43.9 ± 5.1% inhibition. Among the fractions, highest antileishmanial activity both in vitro (>90%) and in vivo (60.9 ± 18.3%) was observed in n-butanol (soluble) fraction with IC₅₀ values of 8.2 μg/ml and 31.2 μg/ml against promastigotes and intracellular amastigotes, respectively. Hexane fraction also showed comparatively good activity against both the stages of parasites in vitro but was moderately active in leishmania-infected hamsters. Chloroform fraction resulted in 45 ± 10.2% inhibition in vivo at a dose of 500 mg/kg × 5, p.o., whereas it was inactive in vitro. n-Butanol (insoluble) fraction was inactive both in vitro and in vivo. Araguspongin C, an alkaloid isolated from n-butanol (soluble) fraction exhibited moderate inhibition of promastigotes and intracellular amastigotes at 100 μg/ml but showed weak antileishmanial action in vivo. Our findings indicate that this marine sponge has the potential to provide new lead toward development of an effective antileishmanial agent and, hence, calls for more exhaustive studies for exploiting the vast world of marine resources to combat the scourge of several parasitic diseases.