Biomedical subjects

D L Sacks

Publications and source records attributed to D L Sacks.

At least 37 records · Page 2Linked to original sources

PubMed1998

Analysis of cytokine production by inflammatory mouse macrophages at the single-cell level: selective impairment of IL-12 induction in Leishmania-infected cells.

Intracellular staining for cytokines and parasites, combined with two-color flow cytometric analyses, were used to examine the frequencies of IL-12-, TNF-alpha- and IL-6-producing macrophages in response to Leishmania major infection and/or activation with IFN-gamma/lipopolysaccharide (LPS). Inflammatory macrophages were obtained from nonimmune granulomas, initiated by the injection of polyacrylamide microbeads (Bio-gel P-100) into subcutaneous pouches of different mouse strains. Infection of inflammatory macrophages in vitro using metacyclic promastigotes produced identical effects on cytokine responses regardless of whether cells from genetically resistant or susceptible mouse strains were used: IL-12 was not produced in response to infection itself, virtually every infected cell lost its ability to produce IL-12 in response to IFN-gamma/LPS, and the IL-6 response was partially inhibited, while the TNF-alpha response of infected cells was unimpaired. Low-multiplicity infection of inflammatory macrophages in vivo using either metacyclic promastigotes or tissue amastigotes also resulted in the complete and selective inhibition of IL-12 responses in infected cells. These data establish the physiologic relevance of prior observations regarding the selective impairment of IL-12 induction pathways in infected macrophages, and suggest a mechanisms for the delayed onset of cell-mediated control mechanisms that is typical of even self-limiting forms of leishmanial disease.

Animals↗

PubMed1998

Vaccine requirements for sustained cellular immunity to an intracellular parasitic infection.

The humoral immunity induced by many viral and bacterial vaccines mediates protection that is maintained over a long period of time. In contrast, for other intracellular infections (such as with Leishmania major or Mycobacterium tuberculosis) for which cell-mediated immunity is required for protection, the mechanisms for developing durable responses after vaccination have not been well defined. Here we demonstrate that vaccination with plasmid DNA encoding a specific leishmanial antigen is more effective than leishmanial protein plus recombinant IL-12 in eliciting long-term immunity capable of controlling L. major infection. We also show that leishmanial protein plus IL-12 DNA produces an immunity that lasts much longer than does immunity elicited by leishmanial protein plus IL-12 protein, indicating that the persistence of IL-12 may be the essential determinant in maintaining durable cell-mediated immune responses for an intracellular parasitic infection.

Adjuvants, Immunologic↗

PubMed1998

Targeted gene deletion of Leishmania major genes encoding developmental stage-specific leishmanolysin (GP63).

The major surface glycoprotein of Leishmania major is a zinc metalloproteinase of 63 kDa referred to as leishmanolysin or GP63, which is encoded by a family of seven genes. Targeted gene replacement was used to delete gp63 genes 1-6 encoding the highly expressed promastigote and constitutively expressed GP63. In the L. major homozygous mutants deficient in gp63 genes 1-6, there was no expression of GP63 as detected by reverse transcription-polymerase chain reaction (RT-PCR) or fluorescent staining in promastigotes from the procyclic stage (logarithmic growth phase). The remaining L. major gP63 gene 7 was shown to be developmentally regulated, as it was expressed exclusively in infectious metacyclic stage (late stationary growth phase) promastigotes and in lesion amastigotes. The gp63 genes 1-6-deficient mutants showed increased sensitivity to complement-mediated lysis. The sensitivity to lysis was greater in procyclics than in metacyclics when compared with the equivalent wild-type stages. Increased resistance of the mutant metacyclic promastigotes correlated with the expression of gp63 gene 7 and was restored to the same levels as wild-type promastigotes by transfection with gp63 gene 1. Thus, expression of GP63 is clearly involved in conferring resistance to complement-mediated lysis. The L. major GP63 1-6 mutants were capable of infecting mouse macrophages and differentiating into amastigotes. Similar levels of infection and subsequent intracellular survival were observed when mouse macrophages were infected in vitro with wild type, GP63 1-6 mutants and mutants transfected with gp63 gene 1. The GP63 1-6 mutants were capable of lesion formation in BALB/c mice and, thus, gp63 genes 1-6 do not play a role in the survival of the parasite within mouse macrophages. The role of gp63 genes 1-6 in parasite development within the sandfly vector was studied. GP63 1-6 mutants grew normally in the blood-engorged midgut of both Phlebotomus argentipes and P. papatasi However, both wild-type and mutant promastigotes were lost after 2 days' growth in P. papatasi. The complete developmental pathway in P. argentipes was observed for wild-type promastigotes, GP63 1-6 mutants and mutants transfected with gp63 gene 1. Normal stage differentiation from amastigotes to procyclics, to nectomonads, to haptomonads and to infectious metacyclics was observed. Thus, the highly expressed promastigote forms of GP63, encoded by gp63 genes 1-6, do not appear to be required for nutrient utilization in the bloodmeal during the early stages of development in the sandfly or for midgut attachment and further development. gp63 1-6 genes do, however, play a major protective role against complement-mediated lysis when promastigotes are introduced into the mammalian host.

Animals↗

PubMed1998

Splenic cytokine responses in Indian kala-azar before and after treatment.

Cytokine mRNA levels were measured in serial splenic aspirates from 27 patients with visceral leishmaniasis during monotherapy with interferon-gamma (IFN-gamma; n = 9), sodium antimony gluconate (SAG; n = 8), or amphotericin B lipid complex (ABLC; n = 10). At baseline, mRNA for IFN-gamma was detected in 18 (86%) of 21 patients, and mRNA for interleukin (IL)-10 and IL-4 was detected in 21 (100%) and 10 (48%) of 21 patients, respectively. With IFN-gamma treatment alone, levels of IFN-gamma mRNA decreased by day 10 and then returned to baseline levels; IL-10 mRNA levels were high throughout treatment. In the SAG and ABLC groups, levels of IFN-gamma and IL-10 mRNA decreased significantly. Polarized Th2 cell type responses do not appear to develop in Indian kala-azar; instead, there is an initial mixed Th1-Th2 cell picture. With successful treatment and resolution of infection, both components of the immune response appear to involute.

Adolescent↗

PubMed1997

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 of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic protection was achieved by LACK DNA vaccination. The control of disease progression and parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon-gamma (IFN-gamma) production. Moreover, both the enhancement of IFN-gamma production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8(+) T cells at the time of vaccination or infection also abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8(+) T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.

Animals↗

PubMed1997

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-treated flies within the first 4 h after feeding. Substantial early killing was also observed in control flies, suggesting that the lack of PM exacerbates lethal conditions which normally exist in the blood-fed midgut. Early parasite mortality was reversed by soybean trypsin inhibitor. Allosamadin, a specific inhibitor of chitinase, led to a thickening of the PM, and also prevented the early parasite mortality seen in infected flies. Susceptibility to gut proteases was extremely high in transitional-stage parasites, while amastigotes and fully transformed promastigotes were relatively resistant. A novel role for the PM in promoting parasite survival is suggested, in which the PM creates a barrier to the rapid diffusion of digestive enzymes, and limits the exposure of parasites to these enzymes during the time when they are especially vulnerable to proteolytic damage.

Acetylglucosamine↗

PubMed1996

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 excretion of the digested bloodmeal. Biochemical characterization of Spock LPG revealed its structural similarity to the LPG of Leishmania donovani, a species whose inability to bind to and maintain infections in P. papatasi midguts has been strongly correlated with the expression of a surface LPG lacking galactose-terminated oligosaccharide side chains. An in vitro galactosyltransferase assay using wild-type or Spock membranes was used to determine that the defect in Spock LPG biosynthesis is a result of defective beta1,3-galactosyltransferase activity as opposed to a modification of LPG, which would prevent it from serving as a competent substrate for galactose addition. The results of these experiments show that Spock lacks the beta1, 3-galactosyltransferase for side chain addition and that the LPG side chains are required for L. major to bind to and to produce transmissible infection in P. papatasi.

Animals↗

PubMed1996

Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice.

Leishmania major promastigotes were found to avoid activation of mouse bone marrow-derived macrophages (BMM0) in vitro for production of cytokines that are typically induced during infection with other intracellular pathogens. Coexposure of BMM0 to the parasite and other microbial stimuli resulted in complete inhibition of interleukin (IL) 12 (p40) mRNA induction and IL-12 release. In contrast, mRNA and protein levels for IL-1(alpha), IL-1(beta), tumor necrosis factor (TNF) alpha, and inducible NO synthase (iNOS) were only partially reduced, and signals for IL-10 and monocyte chemoattractant protein (MCP-1/JE) were enhanced. The parasite could provide a detectable trigger for TNF-alpha and iNOS in BMM0 primed with interferon (IFN) gamma, but still failed to induce IL-12. Thus IL-12 induction is selectively impaired after infection, whereas activation pathways for other monokine responses remain relatively intact. Selective and complete inhibition of IL-12(p40) induction was observed using BMM0 from either genetically susceptible or resistant mouse strains, as well as IL-10 knockout mice, and was obtained using promastigotes from cutaneous, visceral, and lipophosphoglycan-deficient strains of Leishmania. The impaired production of the major physiological inducer of IFN-gamma is suggested to underlie the relatively prolonged interval of parasite intracellular survival and replication that is typically associate with leishmanial infections, including those producing self-limiting disease.

Animals↗

PubMed1996

Leishmania major: infection of human monocytes induces expression of IL-8 and MCAF.

Leishmania major, the causative agent of cutaneous leishmaniasis, is an intracellular parasite of monocytic cells. Leishmania lesions are characterized by recruitment of neutrophils, eosinophils, and blood monocytes. To clarify the mechanism of this cellular influx we examined the expression of IL-8 and MCAF, cytokines chemotactic for neutrophils and monocytes, respectively, in human monocytes infected by L. major. Supernatants of monocytes infected with L. major had chemotactic activity for neutrophils and monocytes that was neutralized by anti-IL-8 and anti-MCAF antibodies. Both IL-8 and MCAF mRNAs were induced within 1 hr of infection. Furthermore we showed that infection of monocytes with L. major induces IL-8 and MCAF but not the proinflammatory cytokines IL-1 beta and TNF alpha. These data suggest that the expression of chemokines may contribute to the cellular recruitment in Leishmania lesions.

Animals↗

PubMed1995

Indian kala-azar caused by Leishmania tropica.

Kala-azar, or visceral leishmaniasis, in India is generally assumed to be a result of infection with Leishmania donovani. 15 parasite isolates collected over the past 10 years from patients with classical disease were typed by monoclonal antibodies, isoenzymes, and kDNA analysis. 4 were shown to be L tropica, a species historically associated with cutaneous disease and more recently a mild "visceralising" disease from the Desert Storm experience. The results confirm that L tropica is a co-endemic agent of visceral leishmaniasis in India, and may shed light on the rising frequency of therapeutic unresponsiveness to sodium antimony gluconate, which complicates treatment of this lethal disease.

Adolescent↗

PubMed1995

Stage-specific binding of Leishmania donovani to the sand fly vector midgut is regulated by conformational changes in the abundant surface lipophosphoglycan.

The life cycle of Leishmania parasites within the sand fly vector includes the development of extracellular promastigotes from a noninfective, procyclic stage into an infective, metacyclic stage that is uniquely adapted for transmission by the fly and survival in the vertebrate host. These adaptations were explored in the context of the structure and function of the abundant surface lipophosphoglycan (LPG) on Leishmania donovani promastigotes. During metacyclogenesis, the salient structural feature of L. donovani LPG is conserved, involving expression of a phosphoglycan chain made up of unsubstituted disaccharide-phosphate repeats. Two important developmental modifications were also observed. First, the size of the molecule is substantially increased because of a twofold increase in the number of phosphorylated disaccharide repeat units expressed. Second, there is a concomitant decrease in the presentation of terminally exposed sugars. This later property was indicated by the reduced accessibility of terminal galactose residues to galactose oxidase and the loss of binding by the lectins, peanut agglutinin, and concanavalin A, to metacyclic LPG in vivo and in vitro. The loss of lectin binding was not due to downregulation of the capping oligosaccharides as the same beta-linked galactose or alpha-linked mannose-terminating oligosaccharides were present in both procyclic and metacyclic promastigotes. The capping sugars on procyclic LPG were found to mediate procyclic attachment to the sand fly midgut, whereas these same sugars on metacyclic LPG failed to mediate metacyclic binding. And whereas intact metacyclic LPG did not inhibit procyclic attachment, depolymerized LPG inhibited as well as procyclic LPG, demonstrating that the ligands are normally buried. The masking of the terminal sugars is attributed to folding and clustering of the extended phosphoglycan chains, which form densely distributed particulate structures visible on fracture-flip preparations of the metacyclic surface. The exposure and subsequent masking of the terminal capping sugars explains the stage specificity of promastigote attachment to and release from the vector midgut, which are key events in the development of transmissible infections in the fly.

Animals↗

PubMed1995

Changes in lipophosphoglycan and gene expression associated with the development of Leishmania major in Phlebotomus papatasi.

Stage-specific molecular and morphogenic markers were used to follow the kinetics of appearance, number, and position of metacyclic promastigotes developing during the course of L. major infection in a natural vector, Phlebotomus papatasi. Expression of surface lipophosphoglycan (LPG) on transformed promastigotes was delayed until the appearance of nectomonad forms on day 3, and continued to be abundantly expressed by all promastigotes thereafter. An epitope associate with arabinose substitution of LPG side-chain oligosaccharides, identified by its differential expression by metacyclics in vitro, was detected on the surface of a low proportion of midgut promastigotes beginning on day 5, and on up to 60% of promastigotes on days 10 and 15. In contrast 100% of the parasites egested from the mouthparts during forced feeding of 15 day infected flies stained strongly for this epitope. At each time-point, the surface expression of the modified LPG was restricted to morphologically distinguished metacyclic forms. Ultrastructural study of the metacyclic surface revealed an approximate 2-fold increase in the thickness of the surface coat compared to nectomonad forms, suggesting elongation of LPG as occurs during metacyclogenesis in vitro. A metacyclic-associated transcript (MAT-1), another marker identified by its differential expression in vitro, also showed selective expression by promastigotes in the fly, and was used in in situ hybridization studies to demonstrate the positioning of metacyclics in the anterior gut.

Animals↗

PubMed1994

Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmania is controlled by structural polymorphisms in the surface lipophosphoglycan.

Phlebotomine vectors can in some instances transmit only certain species of Leishmania. Comparison of a large number of vector/parasite pairs revealed that species-specific differences in vectorial competence were in every case directly correlated with the ability of promastigotes to attach to the sand-fly midgut, the variable outcomes of which were controlled by structural polymorphisms in the surface lipophosphoglycan (LPG) of the parasite. The ability of Phlebotomus papatasi to transmit only Leishmania major could be attributed to the unique, highly substituted nature of L. major LPG that provides for multiple terminally exposed beta-linked galactose residues for binding. While the relatively unsubstituted LPGs of other Leishmania species were unable to mediate promastigote attachment to P. papatasi, they could mediate binding to midguts of Phlebotomus argentipes, which was found to be a potentially competent vector for every Leishmania species examined. The data suggest that at least some phlebotomine vectors differ with respect to the parasite recognition sites which they express and that midgut adhesion is a sufficiently critical component of vectorial competence as to provide the evolutionary drive for LPG structural polymorphisms.

Animals↗

PubMed1994

Leishmania major: association of the differentially expressed gene B protein and the surface lipophosphoglycan as revealed by membrane capping.

The lipophosphoglycan (LPG) of Leishmania promastigotes forms a dense glycocalyx which effectively covers the entire surface of the cell, and which undergoes structural modifications during the differentiation of promastigotes to the infective or metacyclic stage. Recently, the first protein marker for metacyclic promastigotes of Leishmania major has been characterized. This protein, termed gene B protein, is located on the cell surface, yet it lacks any hydrophobic sequence for membrane attachment. It does contain an unusual amino acid repeat that is related to the peptidoglycan binding domain of protein A from Staphylococcus aureus, suggesting that the protein might interact with metacyclic LPG via this domain for attachment to the cell. We have studied the distribution of LPG, gene B protein, and the major surface protease, gp63, by labeling them with immunogold or immunofluorescence prior to and during capping events. Thin sections of double-labeled parasites revealed that the gene B protein-gold particles were colocalized with the LPG-gold particles in the LPG capping structures at the extremities of the cell. Cocapping of LPG and gene B protein was also observed with two-color fluorescence. No similar redistribution was seen in gp63 or with integral membrane proteins. In contrast to the gene B protein, gp63 could only be immunogold labeled on the metacyclic surface after capping and shedding of the LPG, providing further that it and other membrane-associated proteins are normally buried under the LPG coat. The unusual surface exposure of the gene B protein is consistent with its hydrophilic and LPG binding properties, which allow it to become incorporated into the cell coat and to localize to the most external aspects of the cell.

Animals↗

PubMed1994

Epitope mapping of monoclonal antibodies directed against lipophosphoglycan of Leishmania major promastigotes.

Monoclonal antibodies (MAbs) were generated against Leishmania major promastigote lipophosphoglycan (LPG) to use as tools in defining functional epitopes of this major cell surface glycoconjugate. Epitope mapping of four MAbs, designated 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2, revealed that the phosphorylated oligosaccharide repeat unit PO4-6[Gal(beta 1-3)]Gal(beta 1-4)Man alpha 1-, P3, is a highly immunogenic epitope which has previously been demonstrated, by chemical analyses, to be a repeat unit specific to L. major. Two antibodies, 4A2-A2 and 5E10-F2, also recognised the repeat unit PO4-6[Ara(beta 1-2)Gal(beta 1-3)]Gal(beta 1-4)Man alpha 1-, 4Pa, with less affinity than P3, while 2G11-A3 recognised P4a with greater affinity than for P3. The L. major metacyclic-specific antibody 3F12 only recognised repeat units terminating with arabinose residues. In particular, 3F12 recognised P4a, which is upregulated in metacyclic LPG compared to the procyclic form of the molecule. The oligosaccharides P3, P4a and P5a are specific to L. major LPG. The epitopes of 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2 were found on the cell surface and in the flagellar pocket of both procyclic and metacyclic V121 promastigotes, but were only detected at very low levels on amastigotes. The repeat unit P3 is able to inhibit attachment of procyclic promastigotes to the midgut of the sandfly vector, but neither Fab fragments of the four antibodies nor purified P3 could inhibit attachment of metacyclic promastigotes to the macrophage cell line J774. It was also shown that human sera from patients with cutaneous leishmaniasis recognised purified P3. The data suggests that while P3 is an immunogen in the natural course of infection of the human host, P3 plays no role in attachment and internalisation of promastigotes into the macrophages of the mammalian host.

Animals↗

PubMed1994

The role of the lipophosphoglycan of Leishmania in vector competence.

The surface lipophosphoglycans (LPG) of Leishmania promastigotes express stage- and species-specific polymorphisms that are defined by variations in the type and number of phosphorylated oligosaccharide repeats. We have studied how these polymorphic structures control the development of transmissible infections in the sandfly vector as well as the species-specificity of vectorial competence. Procyclic promastigotes displayed an inherent capacity to bind to midgut epithelial cells of a competent vector. This capacity was lost during their transformation of metacyclic promastigotes, permitting the selective release and anterior migration of infective-stage parasites for subsequent transmission by bite. Midgut attachment and release were found to be controlled by developmental modifications in terminally exposed saccharides on LPG, which, depending on the species of Leishmania, involved either substitution or capping of terminal side-chain sugars, loss of terminal side-chain sugars, substitution or loss of neutral capping sugars. The stage-specific terminal sugars involved in midgut adhesion are, in some cases, also species-specific, and the extent to which these differences affect midgut attachment, forcefully predicted vectorial competence.

Animals↗

PubMed1994

T-cell responses to infected autologous monocytes in patients with cutaneous and mucocutaneous leishmaniasis.

Although there is strong evidence that the control and resolution of human leishmanial infections depend primarily on activation of parasite-infected macrophages mediated by lymphokines derived from T cells, less is known about the nature of the responding cell type(s) which is protective or the antigen(s) (Ag[s]) that elicits these cells to respond. Studies using preparations of whole soluble Ag ("dead Ag") show that patients respond to a wide range of leishmanial Ags. The objective of the present study was to characterize the response of T cells from patients with healing or healed cutaneous or mucosal infections to Ag expressed by or derived from actively infected autologous monocytes ("live Ag"). Unfractionated T cells proliferated and produced gamma interferon in response to both live and dead Ags. Depletion of CD4+ T cells resulted in the loss of proliferative and gamma interferon responses to both live and dead Ags. The effect of CD8 depletion, although variable and not limited to the cells stimulated by infected monocytes, was clear for some patients. Expansion of T cells specific for live Ags by using amastigote-infected cells followed by restimulation with fast-protein liquid chromatography-fractionated soluble Ags revealed that a diversity of Ags are associated with infected monocytes. There may, however, be quantitative differences in the expression of certain Ags since prestimulation with live Ag induced higher responses to restimulation in mucocutaneous leishmaniasis patients than in localized cutaneous leishmaniasis patients. Prestimulation with dead Ag induced similar secondary responses in both patient groups.

Animals↗

PubMed1993

In vivo cytokine profiles in patients with kala-azar. Marked elevation of both interleukin-10 and interferon-gamma.

The immunological mechanisms underlying the susceptibility to disseminated visceral parasitism of mononuclear phagocytes in patients with kala-azar remain undefined. Resistance and susceptibility are correlated with distinct patterns of cytokine production in murine models of disseminated leishmanial disease. To assess lesional cytokine profiles in patients with kala-azar, bone marrow aspirates were analyzed using a quantitative reverse transcriptase PCR technique to amplify specific mRNA sequences of multiple Th1-, Th2-, and/or macrophage-associated cytokines. Transcript levels of IL-10 as well as IFN-gamma were significantly elevated in patients with active visceral leishmaniasis; IL-10 levels decreased markedly with resolution of disease. These findings suggest that IL-10, a potent, pleiotropic suppressor of all known microbicidal effector functions of macrophages, may contribute to the pathogenesis of kala-azar by inhibiting the cytokine-mediated activation of host macrophages that is necessary for the control of leishmanial infection.

Adolescent↗