Camel nanobodies: Promising molecular tools against leishmaniasis.

AIM: To characterize several anti-Leishmania tropica nanobodies and to investigate their effect on Leishmania infection. METHODS: Several immunological tests were implied to characterize five different (as confirmed by sequencing) anti-L tropica nanobodies (NbLt05, NbLt06, NbLt14, NbLt24 and NbLt36) against parasite lysates or intact cells from different stages, promastigotes and amastigotes. Direct inhibitory effect of these nanobodies on parasite infection cycle on macrophages was tested in cell culture. RESULTS: All the five nanobodies (with distinguished characteristics) were more specific to L tropica than to L major, but could equally recognize the lysate and the outer surface of the intact cells from the two main stages of the parasite. Nanobodies recognized several leishmania antigens (majorly between 75 and 63 kDa), and their proteinaceous nature was confirmed. Because of its role in leishmania life cycle, gp63 was considered a potential antigen candidate for nanobodies, and bioinformatics predicted such interaction. All nanobodies have a negative effect on the infectivity of L tropica, as they decreased the number of infected macrophages and the amastigotes inside those macrophages. CONCLUSION: Such anti-leishmania nanobodies, with outstanding characteristics and important target, can be of great use in the development of promising treatment strategies against leishmaniasis.

Heterogeneity of humoral immune response to Leishmania tropica in an experimental model.

Humoral (antibody) response is an important part of immunity against pathogens. Despite the clear role of cell-mediated immune response in protection against leishmaniasis, the role of humoral responses is challenging. There is very limited data regarding humoral immune response against Leishmania tropica which is the causative agent of human cutaneous leishmaniasis in many parts of the world. Here, we have compared pathogenicity and antibody response against six Iranian Leishmania tropica isolates in BALB/c mice. A Leishmania major isolate was used for comparison. The parasites were injected into the mice followed by the evaluation of the lesion development, parasite load, and antibody responses (IgG1 and IgG2a). Our findings showed that some isolates caused the large lesions and high parasite load in the spleen and lymph node, while other isolates led to no lesion, no splenic parasitism, and low parasite load in the lymph node. The more pathogenic isolates induced higher antibody responses (IgG1 and IgG2a). Our results indicated that there is substantial heterogeneity among various Leishmania tropica isolates regarding the humoral immune response as well as the pathogenicity.

Leishmania tropica: suggestive evidences for the effect of infectious dose on pathogenicity and immunogenicity in an experimental model.

Leishmania (L.) tropica is a causative agent of cutaneous and occasionally visceral or viscerotropic leishmaniasis in humans. The dose of parasites influences the course and outcome of disease in some Leishmania species. The effect of parasite dose on L. tropica infection in an experimental model was studied in the current paper. High and low doses of L. tropica were used for ear infection of BALB/c mice and lesion development, parasite load, and cytokine responses were assessed. L. major infection was used for comparison. Pre-infected mice were challenged in the footpad by a fixed high dose of L. tropica, and immune response and protection level were evaluated. High dose L. tropica infection in comparison to low dose results in higher lesion diameters, higher load of parasite in draining lymph node, higher levels of interferon-γ and interleukin-10, dissemination of parasite to spleen, and induction of protection against further L. tropica challenge. Comparison of L. tropica with L. major showed that L. tropica results in lower lesion diameters, more potential for growth in lymph nodes at early phases of infection, parasite dissemination to spleen, lower levels of IL-10, and a permanent lower cytokine response against low parasite dose in comparison to high dose. Our findings suggest that for L. tropica infection, only the high dose results in visceralization of the parasite and protection against further challenge of L. tropica. Therefore, the parasite dose may be an important factor in pathogenesis and immunity in L. tropica infection.

Seroprevalence of Leishmania infection and molecular detection of Leishmania tropica and Leishmania infantum in stray cats of Izmir, Turkey.

Leishmaniasis caused by more than 20 species of genus Leishmania is transmitted by the bite of infected phlebotomine sand flies. The studies on Leishmania infection in cats is very few in Turkey and therefore we aimed to screen stray cats living in city of Izmir located in western Turkey using nested PCR targeting kinetoplast DNA and serological techniques (ELISA and IFA). Leishmania DNA positive samples were also studied by ITS1 real time PCR. Whole blood and serum samples were obtained from stray cats (n: 1101) living in different counties of Izmir. In serological assays, a serum sample was considered positive in 1:40 dilution in IFA and for ELISA a serum sample was accepted positive when the absorbance value (AV) exceeded the mean AV + Standard Deviation (SD) of the negative control serum samples. According to the results, the seropositivity rates were 10.8% (119/1101) and 15.2% (167/1101) by in house ELISA and IFA, respectively. Among serology coherent samples, the seropositivity rate was 11.1% (116/1047) as detected by both assays after discordant samples (n: 54) were discarded. Of the 1101 stray cats, six (0.54%) were positive by nested PCR while only one of these six samples was positive by ITS1 real time PCR. During PCR, three controls designated as Leishmania infantum, Leishmania tropica, and Leishmania major were used for species identification. According to nested PCR results, L. tropica was identified in two cats (no.76 and 95). In another cat (no. 269), there were two bands in which one of them was well-matched with L. infantum and the other band had ∼850 bp size which does not match with any controls. Remaining three cats (no. 86, 514, and 622) also had the ∼850 bp atypical band size. ITS1 real time PCR detected L. tropica in only one cat (no. 622) which showed an atypical band size in nested PCR. These results indicated that three cats with only one atypical band (no. 86, 514, and 622) and the cat with mixed infection (no. 269) were infected with L. tropica. Altogether, L. tropica was detected in all six DNA positive cats and L. infantum was detected in one cat with mixed infection. In conclusion, although the reservoir role of cats in nature is still unclear the high seroprevalence rate against Leishmania parasites and detecting parasite DNA in stray cats in Izmir indicates that the stray cats are frequently bitten by infected sand flies. Further research activities are required to reveal the frequency of leishmaniasis in cats in different regions of Turkey where Leishmania species are endemic.

Specific K39 antibody response and its persistence after treatment in patients with imported leishmaniasis.

The sensitivity of a K39 ELISA (Leishmania IgG, Virion/Serion) for the detection of antibodies in patients with imported leishmaniasis was compared with an immunofluorescence assay (IFA), which was applied as "golden standard". The retrospective study comprised 93 IFA-positive or borderline sera from 42 patients with visceral (n = 16) or cutaneous (n = 26) leishmaniasis. Patients had acquired infection predominately in the Mediterranean area or the Middle East. The Leishmania species (Leishmania donovani/infantum, Leishmania tropica, Leishmania major) were identified by real-time PCR. The majority (94%) of first samples from patients with visceral leishmaniasis (VL) tested positive by K39 ELISA. Antibody levels ranged from low to very high (33.19-1990.00 U/ml; median 596.66 U/ml) but did not correlate with the respective IFA titers. High K39 ELISA values correlated with acute infection in immunocompetent individuals. K39 antibodies declined in all individuals after clinically successful therapy, but time to seronegativity varied considerably (51 weeks to >6 years). In patients with cutaneous leishmaniasis (CL), the sensitivity of the K39 ELISA was low (23%) compared to IFA (92% positive). Antibody levels ranged from low to medium (10.85-524.77 U/ml; median 19.77 U/ml). The highest antibody concentrations were seen in L. infantum-infected individuals. Summarizing, a high K39 ELISA value indicates active VL. The assay is, like IFA, not a measure for effective therapy but may support post-treatment monitoring. Low level positivity can indicate subclinical, previous or clinically cured VL or even CL. The K39 ELISA can supplement highly sensitive screening tests in the diagnosis and follow-up of imported leishmaniasis.

The immune microenvironment in cutaneous leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis is an infection that has spread to non-endemic regions, stimulating recent interest for the enhanced understanding of this disease. Downregulation of the CD1a receptor on Langerhans cells has been described in various cutaneous infections. OBJECTIVE: In this study, the immune response across different Ridley patterns and parasitic indices is outlined in a case series of cutaneous leishmaniasis. METHODS: Skin punch biopsies from the interface of normal and lesional cutaneous leishmaniasis were collected from 33 patients with molecularly confirmed Leishmania tropica or L. major infection. Ridley patterns (2-5) were assessed for various clinicopathological features including age, gender, disease duration, parasitic index and constituents of the inflammatory infiltrate. CD1a, CD68, CD3, CD4, CD8, CD20 and CD138 stains were performed on normal skin tissue, cutaneous leishmaniasis biopsies and cytospin/cell block cytology preparations of cultured leishmania promastigotes. CD1a was quantified per mm2 in the epidermis and dermis. The remaining stains were graded according to a 4-tiered grading system [0 (0-4%); 1 (5-24%); 2 (25-49%); 3 (50-74%) and 4 (75-100%). RESULTS: Total CD1a expression significantly decreased (14-fold) from parasitic indices (0-2) to (5-6); (ρ < 0.001). CD1a expression in the epidermis was at least 5-fold lower than normal skin (58 vs. 400 cells/mm2), inversely correlating with the parasitic index. There was an increase in dermal CD1a Langerhans cells (33 vs. 0 cells/mm² in the dermis). CD1a and CD68 staining of amastigotes was strong and diffuse, whereas promastigotes were negative. The major inflammatory infiltrate, in all Ridley patterns, consisted of macrophages and double-negative CD3(+) CD4(-) CD8(-) T lymphocytes. The double-negative CD3 T cells formed a ring around the parasitic laden macrophages. Apart from CD1a, there was no significant difference in inflammatory markers between the various Ridley patterns and parasitic indices. Disease duration did not correlate with Ridley pattern. CONCLUSION: The significant decrease in CD1a expression is postulated by two mechanisms; either via direct CD1a receptor uptake by leishmania amastigotes and/or negative feedback inhibition of CD1a Langerhans cells by double-negative CD3 T-regulatory cells. Modulation of the immune microenvironment in cutaneous leishmaniasis represents a potential therapeutic and prophylactic target.

Detection of high levels of anti-α-galactosyl antibodies in sera of patients with Old World cutaneous leishmaniasis: a possible tool for diagnosis and biomarker for cure in an elimination setting.

In the Kingdom of Saudi Arabia (KSA), Old World cutaneous leishmaniasis (CL) is mainly caused by Leishmania major and Leishmania tropica parasites. Diagnosis of CL is predominately made by clinicians, who at times fail to detect the disease and are unable to identify parasite species. Here, we report the development of a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to measure the levels of anti-α-galactosyl antibodies in human sera. Using this assay, we have found that individuals infected with either Leishmania spp. had significantly elevated levels (up to 9-fold higher) of anti-α-Gal IgG compared to healthy control individuals. The assay sensitivity was 96% for L. major (95% CI; 94-98%) and 91% for L. tropica (95% CI; 86-98%) infections and therefore equivalent to restriction fragment length polymorphism-polymerase chain reaction analysis of parasite ITS1 gene. In addition, the assay had higher sensitivity than microscopy analysis, which only detected 68 and 45% of the L. major and L. tropica infections, respectively. Interestingly, up to 2 years following confirmed CL cure individuals had 28-fold higher levels of anti-α-Gal IgG compared to healthy volunteers. Monitoring levels of anti-α-Gal antibodies can be exploited as both a diagnostic tool and as a biomarker of cure of Old World CL in disease elimination settings.

Intralesional gene expression profile of JAK-STAT signaling pathway and associated cytokines in Leishmania tropica-infected patients.

Background: The JAK-STAT signaling pathway is a central cascade of signal transduction for the myriad of cytokines in which dysregulation has been implicated in progression of inflammatory and infectious diseases. However, the involvement of this pathway in human cutaneous leishmaniasis (CL) due to Leishmania (L.) tropica warrants further investigation. Methods: This study sought to investigate differential gene expression of several cytokines and their associated jak-stat genes in the lesions of L. tropica-infected patients byquantitative Real-Time PCR. Further, the expression of five inhibitory immune checkpoint genes was evaluated. Results: Results showed that the gene expression levelsof both Th1 (ifng, il12, il23) and Th2 (il4, il10) types cytokines were increased in the lesion of studied patients. Further, elevated expression levels of il35, il21, il27 and il24 genes were detected in the lesions of CL patients. Notably, the expression of the majority of genes involved in JAK/STAT signaling pathway as well as checkpoint genes including pdl1, ctla4 and their corresponding receptors was increased. Conclusion: Our finding revealed dysregulation of cytokines and related jak-stat genes in the lesion of CL patients. These results highlight the need for further exploration of the functional importance of these genes in the pathogenesis of, and immunity to, CL.

Analysis of localized immune responses reveals presence of Th17 and Treg cells in cutaneous leishmaniasis due to Leishmania tropica.

PURPOSE: The interaction between the Leishmania parasite and the host cell involves complex, multifaceted processes. The disease severity in cutaneous leishmaniasis (CL) is largely dependent on the causative species. Most of the information on immune responses in human CL is available with respect to L. major infection and is lacking for L. tropica species. In this study, we employed cytokine/chemokine/receptor membrane cDNA array to capture comprehensive picture of immuno-determinants in localized human tissue during L. tropica infection. Expression of selected molecules was evaluated by real time PCR in dermal lesion tissues at pre- and post treatment stages. Plasma IL-17 level was estimated by sandwich ELISA. RESULTS: The cDNA array analysis identified several immuno-determinants in tissue lesions of Indian CL including cytokines (IFN-γ, TNF-α, IL-1ß, IL-10, IL-13), chemokines (IL-8, CCL2, CCL3, CCL4) and apoptotic molecules (Fas, TRAIL, IRF-1). Elevated mRNA levels of Th17 (IL-17, IL-23 and RORγt) and Treg (CD25, CTLA-4 and Foxp3) markers were observed in lesion tissues of CL patients compared to the control group, which subsided post treatment. Plasma IL-17 levels were found to be significantly higher in CL samples compared to controls. CONCLUSIONS: In addition to defining comprehensive immunological responses inside lesion tissues of CL patients, our study demonstrated the presence of Th17 and Treg cells in CL caused by L. tropica.

The route of Leishmania tropica infection determines disease outcome and protection against Leishmania major in BALB/c mice.

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

Human chorionic gonadotrophin (hCG) enhances immunity against L. tropica by stimulating human macrophage functions.

During pregnancy, there are important changes in hormone levels such as the huge production of human chorionic gonadotropin (hCG), which is supposed to influence the immune system. The aim of this study was to investigate the effect of hCG on immune response against Leishmania, through the evaluation of the functions of human macrophages infected with L. tropica. This study demonstrated that hCG significantly increased the NO production by rHu-IFNγ-primed macrophages then infected with L. tropica, which was correlated with decrease in the number of infected macrophages as well as the number of amastigotes per macrophage in a dose-dependent manner; however, the greatest effect was shown with the 250 U/mL concentration. The addition of the same concentration of hCG to rHu-IFNγ-primed macrophages caused also a major increase in both IL-6 and IL-12p40 production. In conclusion, hCG enhances different macrophage functions involved in immunity against L. tropica.

Protective immunity against Leishmania major induced by Leishmania tropica infection of BALB/c mice.

Leishmania (L.) tropica is a causative agent of human cutaneous and viscerotropic leishmaniasis. Immune response to L. tropica in humans and experimental animals are not well understood. We previously established that L. tropica infection induces partial protective immunity against subsequent challenge infection with Leishmania major in BALB/c mice. Aim of the present study was to study immunologic mechanisms of protective immunity induced by L. tropica infection, as a live parasite vaccine, in BALB/c mouse model. Mice were infected by L. tropica, and after establishment of the infection, they were challenged by L. major. Our findings shows that L. tropica infection resulted in protection against L. major challenge in BALB/c mice and this protective immunity is associated with: (1) a DTH response, (2) higher IFN-γ and lower IL-10 response at one week post-challenge, (3) lower percentage of CD4(+) lymphocyte at one month post-challenge, and (4) the source of IFN-γ and IL-10 were mainly CD4(-) lymphocyte up to one month post-challenge suggesting that CD4(-) lymphocytes may be responsible for protection induced by L. tropica infection in the studied intervals.

Evaluation of localized and systemic immune responses in cutaneous leishmaniasis caused by Leishmania tropica: interleukin-8, monocyte chemotactic protein-1 and nitric oxide are major regulatory factors.

We have established Leishmania tropica as the causative agent of cutaneous leishmaniasis (CL) in the region of India where the disease is endemic. The association between localized and circulating levels of immune-determinants in CL patients was evaluated. Reverse transcription-polymerase chain reaction analysis revealed up-regulation of interferon-gamma (IFN-gamma), interleukin (IL)-1beta, IL-8, tumour necrosis factor-alpha (TNF-alpha), IL-10 and IL-4 in dermal lesions at the pretreatment stage (n = 31) compared with healthy controls (P < 0.001) and a significant down-regulation after treatment (n = 14, P < 0.05). The results indicated that an unfavourable clinical outcome in CL was not related to an inadequate T helper 1 (Th1) cell response, but rather to impairment in multiple immune functions. Comparative assessment of treatment regimes with rifampicin (RFM) or sodium antimony gluconate (SAG) revealed tissue cytokine levels to be significantly reduced after treatment with RFM (P < 0.005), while no significant decrease was evident in the levels of IFN-gamma, TNF-alpha and IL-10 (P > 0.05) as a result of treatment with SAG. Increased transcripts of monocyte chemoattractant protein-1 (MCP-1) (P < 0.001) and inducible nitric oxide synthase (iNOS) (P < 0.05) were evident before treatment in tissue lesions and remained high after treatment. Immunohistochemistry demonstrated strong expression of myeloperoxidase (MPO) and IL-8, and moderate expression of iNOS in dermal lesions. The expression levels of IL-8, MCP-1 and nitric oxide (NO) were high in patient sera before treatment, as determined using cytokine bead array and enzyme-linked immunosorbent assay (ELISA). At the post-treatment stage, the serum IL-8 levels had decreased; however, the levels of MCP-1 and NO remained high. These data suggest that IL-8 is an effector immune-determinant in the progression of CL, whereas NO facilitates the parasite killing by macrophages via MCP-1-mediated stimulation.

Identification of Leishmania genes encoding proteins containing tandemly repeating peptides.

A genomic Leishmania major DNA expression library was screened using antibodies raised against L. major membranes. Two different clones were identified that encoded proteins containing regions of tandemly repeated peptides. Clone 20 encodes a repetitive peptide of 14 amino acids, while clone 39 encodes a repetitive peptide of 10 amino acids. DNA from clone 20 hybridized with two RNA species of 9,500 and 5,200 nucleotides in length, while DNA from clone 39 hybridized to a single RNA species of 7,500 nucleotides. Antibodies against clone 20 fusion protein recognized a series of L. major proteins of apparent mol wt 250,000. Regions of repetitive peptides is a characteristic shared by many malarial protein antigens and this feature has been implicated in immune evasion. Intracellular parasites such as Leishmania and Plasmodia, therefore, may have evolved similar mechanisms consisting of the expression of proteins containing tandemly repeating peptides that are involved in immune evasion.

Leishmania promastigotes are recognized by the macrophage receptor for advanced glycosylation endproducts.

In this paper we demonstrate the involvement of the macrophage receptor for advanced glycosylation endproducts (AGE) in the phagocytosis of Leishmania major promastigotes. Blocking of this receptor with the ligand, AGE-BSA, leads to a 50% decrease in phagocytosis relative to controls, and a comparable decrease in the respiratory burst. The inhibition of phagocytosis by AGE-BSA was specific to leishmania. The binding of zymosan or C3bi-RBC and the phagocytosis of IgG-RBC or latex beads was not affected by the presence of AGE-BSA. Blocking of both the AGE receptor and CR3 decreases leishmania binding by nearly 90%, and reduces the respiratory burst by 80%, indicating that the two receptors account for the bulk of L. tropica promastigote recognition and uptake by the macrophage.

Complement binding by two developmental stages of Leishmania major promastigotes varying in expression of a surface lipophosphoglycan.

The binding of complement by two developmentally distinct stages of Leishmania major has been studied. Noninfective log phase growth (LOG) promastigotes (serum sensitive) activate complement with deposition of covalently bound C3b onto the surface of the parasite. Infective, peanut agglutinin (PNA-) metacyclic stage promastigotes (serum resistant) also bear mainly C3b after incubation in serum, but a major portion of deposited C3 is present as a 110 X 10(3) mol wt C3 fragment. Whereas deposition of C3b on LOG promastigotes is mediated through the alternative pathway. PNA- parasites are unable to activate the alternative pathway in nonimmune serum. C3 is released from the parasite surface by proteolytic cleavage, at a rate which is nearly threefold greater for LOG than for PNA- promastigotes. Immunoprecipitation experiments show that the developmentally regulated lipophosphoglycan is a major C3 acceptor on both LOG and PNA- parasites. These experiments, which are the first to compare the form and processing of complement on infective and noninfective promastigotes of Leishmania, provide a framework for further definition of the differential C3 receptor-dependent uptake and survival of these parasites within mononuclear phagocytes.

Leishmania major-specific, CD4+, major histocompatibility complex class II-restricted T cells derived in vitro from lymphoid tissues of naive mice.

Several studies indicate that the outcome of experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm) is determined by immunological events occurring shortly after infection. These events lead to outgrowth of either protective CD4+ T cells in the C57BL/6 mouse, which cures, or exacerbative cells in the BALB/c mouse, which succumbs to disease. Potential factors influencing the outgrowth of protective or exacerbative T cells include antigen-presenting cells (APC), cytokines, and parasite antigens. An in vitro system, in which one could precisely control the factors shaping early events in the T cell response to Lm, would be very useful. To this end, we have examined the in vitro response of naive lymphocytes to Lm promastigotes. The data presented here show that Lm-specific CD4+ T cell receptor alpha/beta + T cells can be generated in vitro from spleen and lymph node cell populations of naive mice. Furthermore, they can be obtained from the CD44low (unprimed) population of T lymphocytes, indicating that in vitro priming occurs. The ability to generate these T cells is dependent on the presence of live parasites and is not due to a parasite-derived nonspecific T cell mitogen. Restimulation, as assayed by proliferation, requires APC bearing syngeneic I-A. Optimal restimulation of the in vitro derived T cells is achieved only when live promastigotes are used. The T cells do not proliferate in response to a frozen-and-thawed lysate of promastigotes, yet they exhibit mild reactivity to lysates prepared from heat-shocked promastigotes. Furthermore, they do not recognize two predominant antigens on the promastigote surface, lipophosphoglycan and gp63. T cells derived in vitro with Lm show crossreactivity with live L. donovani, less crossreactivity with live L. mexicana, and no crossreactivity with live Bacillus-Calmette-Guerin or live Brugia malayi microfilariae. Finally, these early T cells, whether derived from healing C57BL/6 or nonhealing BALB/c mice, produce interleukin 2 (IL-2), IL-4, and interferon gamma.

Antileishmanial defense in macrophages triggered by tumor necrosis factor expressed on CD4+ T lymphocyte plasma membrane.

In our studies of host defense to the intracellular protozoan Leishmania major, we uncovered a novel mechanism of antileishmanial defense that involves direct cell contact between effector CD4+ lymphocytes and Leishmania-infected macrophages. The mechanism is distinctive because it does not involve lymphokine secretion and induces no cytotoxic effects in the host cells; its expression is antigen-specific and genetically restricted. We now demonstrate that these effector CD4+ cells display tumor necrosis factor (TNF) on their surface and provide evidence that the membrane-associated TNF is involved in the activation of the antileishmanial defense. Using a Leishmania-specific cloned T-T cell hybridoma line (1B6; CD4+, T helper type 1) that activates antileishmanial defense in macrophages through cell contact and does not secrete TNF, we noted that only cells bearing surface TNF (TNF+), but not ones lacking surface TNF (TNF-), exerted these effects. Moreover, the antileishmanial effects excreted by TNF+ 1B6 cells as well as by lymph node CD4+ TNF+ lymphocytes could be blocked with anti-TNF antibody. We propose that membrane-associated TNF on CD4+ T cells may provide a mechanism of targeting activation signals to macrophages in an antigen-specific and genetically restricted manner.

Immunoregulation of cutaneous leishmaniasis. T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens.

BALB/c mice can be protected against a normally fatal Leishmania major infection by immunization with a partially purified, soluble subfraction of the parasite (fraction 9). In this study, we demonstrate that a T cell line established against fraction 9, designated line 9, transfers protection equivalent to that obtained by active immunization. In contrast, T cell lines (lines 1 and 9.2) responsive to a nonprotective soluble fraction (fraction 1) not only failed to protect BALB/c mice against L. major, but exacerbated the infection. Most importantly, in addition to differing in their antigen specificity, protective and exacerbative T cells lines could be distinguished on the basis of the lymphokines produced, a characteristic previously used to separate murine Th cells into two subsets, designated Th1 and Th2. We found that the protective cell line, line 9, displayed the Th1 property of secreting IL-2 and IFN-gamma, while the exacerbating lines secreted IL-4 and IL-5, a characteristic of Th2 cells. Our results demonstrate that Th1 and Th2 cells may have dramatically different effects on the outcome of an infection, and suggest that susceptibility and resistance in experimental leishmaniasis may depend upon a balance between the Th subsets induced.

Complement receptor type 3 (CR3) binds to an Arg-Gly-Asp-containing region of the major surface glycoprotein, gp63, of Leishmania promastigotes.

The major surface glycoprotein of Leishmania promastigotes, gp63, was isolated and reconstituted into a lipid membrane immobilized on the surface of 5-micron-diameter silica beads. These beads bound to the macrophage (MO), and the extent of binding correlated with the density of gp63 on the bead. The bead thus facilitated analysis of the binding specificity of a single ligand, gp63, without contribution from other molecules present on the surface of intact promastigotes. Plating of MO onto substrates coated with antibodies directed against several cell surface receptors indicated that the complement receptor CR3 was necessary for binding gp63. CR3 recognizes a portion of C3 that contains the sequence R G D. Since gp63 also contains such a sequence, we tested the ability of a synthetic peptide based on the R G D-containing region of gp63 to inhibit the binding of gp63 beads. The R G D-containing peptide from gp63 inhibited the binding of both gp63 beads and EC3bi to MO. Similarly, peptides previously shown to inhibit the binding of C3bi also inhibited the attachment of gp63 beads. The synthetic peptide from the R G D region of gp63 also reduced the binding of intact promastigotes to MO. These results indicate that gp63 binds directly to CR3.