Leishmaniasis: prevention, parasite detection and treatment.

Leishmaniasis remains a public health problem worldwide, affecting approximately 12 million people in 88 countries; 50 000 die of it each year. The disease is caused by Leishmania, obligate intracellular vector-borne parasites. In spite of its huge health impact on the populations in vast areas, leishmaniasis is one of the most neglected diseases. No safe and effective vaccine currently exists against any form of human leishmaniasis. The spectrum and efficacy of available antileishmanial drugs are also limited. First part of this review discusses the approaches used for the vaccination against leishmaniasis that are based on the pathogen and includes virulent or attenuated parasites, parasites of related nonpathogenic species, whole killed parasites, parasites' subunits, DNA vaccines, and vaccines based on the saliva or saliva components of transmitting phlebotomine vector. Second part describes parasite detection and quantification using microscopy assays, cell cultures, immunodetection, and DNA-based methods, and shows a progress in the development and application of these techniques. In the third part, first-line and alternative drugs used to treat leishmaniasis are characterized, and pre-clinical research of a range of natural and synthetic compounds studied for the leishmanicidal activity is described. The review also suggests that the application of novel strategies based on advances in genetics, genomics, advanced delivery systems, and high throughput screenings for leishmanicidal compounds would lead to improvement of prevention and treatment of this disease.

Specificity of anti-saliva immune response in mice repeatedly bitten by Phlebotomus sergenti.

Sand flies are bloodsucking insects transmitting parasites of genus Leishmania, the causative agents of diseases in humans and dogs. Experimental hosts repeatedly exposed to sand fly saliva can control Leishmania infection. Cell-mediated anti-saliva immune response is most likely responsible for this protective effect; however, there is no study so far concerning its antigenic specificity towards different sand fly vectors. In this study, splenocytes from BALB/c mice repeatedly exposed to the bites of Phlebotomus sergenti were challenged ex vivo with salivary gland homogenates from three different sand fly vectors -P. sergenti, P. papatasi, or P. arabicus. Mice bitten by P. sergenti had higher proliferative response to homologous antigen than splenocytes from naive mice. Splenocytes from P. sergenti bitten mice as well as anti-P. sergenti antibodies partially cross-reacted with P. papatasi saliva. In contrast, no cross-reactivity was found with P. arabicus saliva. Our data indicate that both arms of the immune system, cellular and humoral, react in a species-specific manner. Therefore, the presence of antibodies against salivary components of a certain species indicates the specificity of cell-mediated immune response as well. The data suggest that unique transmission-blocking vaccine would be required for each vector -Leishmania combination.

Genetics of susceptibility to leishmaniasis in mice: four novel loci and functional heterogeneity of gene effects.

Symptoms of human leishmaniasis range from subclinical to extensive systemic disease with splenomegaly, hepatomegaly, skin lesions, anemia and hyperglobulinemia, but the basis of this variation is unknown. Association of progression of the disease with Th2 lymphocyte response was reported in mice but not in humans. As most genetic studies in Leishmania major (L. major)-infected mice were restricted to skin lesions, we analyzed the symptomatology of leishmaniasis in mice by monitoring skin lesions, hepatomegaly, splenomegaly and seven immunological parameters. We detected and mapped 17 Leishmania major response (Lmr) gene loci that control the symptoms of infection. Surprisingly, the individual Lmr loci control 13 different combinations of pathological and immunological symptoms. Seven loci control both pathological and immunological parameters, 10 influence immunological parameters only. Moreover, the genetics of clinical symptoms is also very heterogeneous: loci Lmr13 and Lmr4 determine skin lesions only, Lmr5 and Lmr10 skin lesions and splenomegaly, Lmr14 and Lmr3 splenomegaly and hepatomegaly, Lmr3 (weakly) skin lesions, and Lmr15 hepatomegaly only. Only two immunological parameters, IgE and interferon-gamma serum levels, correlate partly with clinical manifestations. These findings extend the paradigm for the genetics of host response to infection to include numerous genes, each controlling a different set of organ-specific and systemic effects.

Modulation of murine cellular immune response and cytokine production by salivary gland lysate of three sand fly species.

Saliva of sand flies (Diptera: Phlebotominae) plays an important role in transmission of Leishmania parasites by modulating host immune response. However, because of the different protein compositions of saliva, the immunomodulatory effects may vary among sand fly species. We have therefore analysed and compared the immunomodulation effects of salivary gland lysate (SGL) of three different sand flies. Spleen cells from BALB/c mice were incubated with SGL of Phlebotomus papatasi, P. sergenti or Lutzomyia longipalpis. Concanavalin A-stimulated lymphocyte proliferation was significantly suppressed with SGLs of all three sand fly species and all SGL doses tested. This result indicates that saliva from different sand fly species is able to suppress host proliferative response even to the potent mitogen. In parallel experiments, we analysed the effect of SGL on IFN-gamma, IL-2, and IL-4 production; in mitogen-stimulated cells SGLs markedly inhibited IFN-gamma production in all intervals tested (reduced up to 31%) and to a lesser degree impaired production of the other two cytokines as well. Despite some species-specific differences in the intensity of immunomodulatory effects, saliva of all sand fly species modulated cell proliferation as well as cytokine production in a similar way.

Separation and mapping of multiple genes that control IgE level in Leishmania major infected mice.

The strain BALB/cHeA (BALB/c) is a high producer, and STS/A (STS) a low producer of IgE after Leishmania major infection. We analyzed this strain difference using 20 recombinant congenic (RC) BALB/c-c-STS/Dem (CcS/Dem) strains that carry different random subsets of 12.5% of genes of the strain STS on the BALB/c background. Strains CcS-16 and -20 exhibit a high and a low IgE level, respectively. In their F(2) hybrids with BALB/c we mapped nine Leishmania major response (Lmr) loci. Two of them we previously found to influence IgE level in CcS-5. IgE production in CcS-16 is controlled by loci on chromosomes 2, 10, 16 and 18 and in CcS-20 by loci on chromosomes 1, 3, 4, 5 and 8. The STS alleles of loci on chromosomes 1, 4, 5, 8 and 10 were associated with a low, whereas the STS alleles on chromosomes 16 and 18 with a high IgE production. The loci on chromosomes 2 and 3 have no apparent individual effect, but interact with the loci on chromosomes 10 and 1, respectively. The loci on chromosomes 10 and 18 were mapped in the regions homologous with the human regions containing genes that control total serum IgE and intensity of infection by Schistosoma mansoni, suggesting that some Lmr loci may participate in the pathways influencing atopic reactions and responses to several parasites. The definition of genes controlling anti-parasite responses will permit a better understanding of pathways and genetic diversity underlying the disease phenotypes.

Susceptibility to Leishmania major infection in mice: multiple loci and heterogeneity of immunopathological phenotypes.

Susceptibility as opposed to resistance of mouse strains (e.g., BALB/c vs C57BL/6) to Leishmania major has been attributed to a defective Th1 and a predominant Th2-response, resulting in increased IL-4 and IgE production, and decreased interferon gamma (IFN gamma) production, macrophage activation and elimination of parasites. Here we report dissection of genetic and functional aspects of susceptibility to leishmaniasis using two contrasting inbred strains BALB/cHeA (susceptible) and STS/A (resistant) and a resistant Recombinant Congenic (RC) Strain, CcS-5/Dem, which carries a random set of 12.5% of genes from the strain STS and 87.5% genes from the susceptible strain BALB/c. Linkage analysis of F2 hybrids between the resistant RC strain CcS-5 and the susceptible strain BALB/c revealed five loci affecting the response to the infection, each apparently associated with a different combination of pathological symptoms and immunological reactions. The correlation between Th2-type immune reactions and the disease in the F2 mice was either absent, or it was limited to mice with specific genotypes at loci on chromosomes 10 and 17. This suggests that the resistance vs susceptibility is influenced by mechanisms additional to the postulated antagonistic effects of Th1 and Th2 responses, and that the host's genotype affects the development of leishmaniasis in a complex way.

A new type of genetic regulation of allogeneic response. A novel locus on mouse chromosome 4, Alan2 controls MLC reactivity to three different alloantigens: C57BL/10, BALB/c and CBA.

The intensity of the mixed lymphocyte response (MLR) depends on the genetic disparity between the donors of responding and stimulating cells. Differences in the major histocompatibility complex (MHC) and Mls1 antigens induce the strongest responses. However, even with comparable incompatibilities in MHC and Mls antigens, some strains of genetically defined mice respond remarkably better than other strains. Apparently other, so far undefined, genetic factors contribute to the magnitude of the MLR. The strain OcB-9 (H2pz) has 87.5% genes from the strain O20/A (O20) and 12.5% genes from strain B10.O20 (both H2pz). In spite of the overal similarity of their genomes, OcB-9 mice differed from O20 mice in response to three different alloantigens C57BL/10 (H2b), BALB/c (H2d) and CBA (H2k). As both O20 and OcB-9 strains carry identical haplotype H2pz, their differences in alloantigen response depend only on non-MHC genes. We analyzed the genetic basis of these strain differences using (OcB-9 x O20)F2 hybrids, and we mapped a novel locus Alan2 (Alloantigen response 2) on chromosome 4 near D4Mit72 that influences the response to all alloantigens tested. This linkage was significant for C57BL/10 and for BALB/c alloantigens (corrected P values 0.0475 and 0.0158, respectively) and highly suggestive for CBA (corrected P = 0.0661). The response to DBA/1 (H2q) alloantigens exhibited a similar pattern but the linkage was not significant. As MLR reflects the recognition phase of transplantation reaction, identification of human counterparts of the Alan genes and a better understanding of the regulation of alloresponsiveness might lead to a better prediction of patients' reactions to allografts and to a more individualized measures to prevent rejection.

T-cell proliferative response is controlled by loci Tria4 and Tria5 on mouse chromosomes 7 and 9.

Lymphocytes of mouse strains BALB/cHeA (BALB/c) and STS/A (STS) differ in their response to CD3 antibody (anti-CD3). We analyzed the genetic basis of this strain difference, using the Recombinant Congenic Strains (RCS) of the BALB/c-c-STS/Dem (CcS/Dem) series. Each of the 20 CcS/Dem strains carries a different, random combination of 12.5% genes from the nonresponding strain STS and 87. 5% genes of the intermediate responder strain BALB/c. Differences in the magnitude of anti-CD3-induced response among CcS/Dem strains indicated that in addition to Fcgamma receptor 2 (Fcgr2) other genes are involved in the control of this response as well, and we have already mapped loci Tria1 (T cell receptor-induced activation 1), Tria2, and Tria3. In order to map additional Tria genes, we tested F2 hybrids between the high responder RC strain CcS-9 and the low responder strain CcS-11. Proliferation in complete RPMI medium without anti-CD3 is controlled by locus Sprol1 (spontaneous proliferation 1) linked to the marker D4Mit23 on Chr 4. At concentration 0.375 microg/ml anti-CD3 mAb, the response was controlled by a locus Tria4, which maps to the marker D7Mit32 on Chr 7. The response to the higher concentration of mAb, 3 microg/ml, was controlled by Tria5, which mapped to the marker D9Mit15 on Chr 9. Anti-CD3 is being used for modulation of lymphocyte functions in transplantation reactions and in cancer treatment. Study of mechanisms of action of different Tria loci could lead to better understanding of genetic regulation of these reactions.

The production of two Th2 cytokines, interleukin-4 and interleukin-10, is controlled independently by locus Cypr1 and by loci Cypr2 and Cypr3, respectively.

The strains BALB/cHeA (BALB/c) and STS/A (STS) differ in production of IL-4 and IL-10, two Th2 cytokines, after stimulation of spleen cells with Concanavalin A, STS being a low and BALB/c a high producer. We analyzed the genetic basis of this strain difference using the recombinant congenic (RC) strains of the BALB/c-c-STS/Dem (CcS/Dem) series. This series comprises 20 homozygous strains. Each CcS/Dem strain contains a different, random set of approximately 12. 5% genes of the "donor" strain STS and approximately 87.5% of the "background" strain BALB/c. We selected for further analysis the RC strain production intermediate between BALB/c and STS. In (CcS-20xBALB/c)F2 hybrids we found that different loci control expression of IL-4 and IL-10. Cypr1 (cytokine production 1) on chromosome 16 near D16Mit15 controls IL-4 production, whereas the production of IL-10 is influenced by loci Cypr2 near D1Mit14 and D1Mit227 on chromosome 1 and Cypr3 marked by D5Mit20 on chromosome 5. In addition, the relationship between the level of these two cytokines depends on the genotype of the F2 hybrids at a locus cora1 (correlation 1) on chromosome 5. This differential genetic regulation may be relevant for the understanding of biological effects of T-helper cells in mice of different genotypes.

IL-2-induced proliferative response is controlled by loci Cinda1 and Cinda2 on mouse chromosomes 11 and 12: a distinct control of the response induced by different IL-2 concentrations.

Lymphocytes of mouse strains BALB/cHeA (BALB/c) and STS/A (STS) differ in the IL-2-induced proliferative response, STS being a high and BALB/c a low responder in the range of concentrations 125-2000 IE/ml. We analyzed the genetic basis of this strain difference using the recombinant congenic (RC) strains of the BALB/c-c-STS/Dem (CcS/Dem) series. This series comprises 20 homozygous strains all derived from two parental inbred strains: the "background" strain BALB/c and the "donor" strain STS. Each CcS/Dem strain contains a different, random set of approximately 12.5% genes of the donor strain STS and approximately 87.5% genes of the background strain BALB/c. In this way, the STS genes controlling the IL-2-induced response became separated into individual CcS/Dem strains, as indicated by differences in the magnitude of the IL-2-induced response among CcS/Dem strains (M. Lipoldová et al., 1995, Immunogenetics 41: 301-311). To map some of these genes, we tested F2 hybrids between one of the high-responder RC strains, CcS-4, and the low-responder parental strain BALB/c. We found that the response to high IL-2 concentrations is controlled by a locus, Cinda1 (cytokine-induced activation 1), on chromosome 11 near the marker D11Mit4. The response to a lower dose of IL-2 tested on lymphocytes of the same mice was found to be controlled by another locus, Cinda2, in the centromeric part of chromosome 12, the higher response being linked to the STS allele of the marker D12Mit37. Understanding the action of genetic factors, such as Cinda1 and Cinda2, that control T cell function is expected to contribute to the efficient analysis of the genetic control of susceptibility to infections and autoimmune diseases.

Identical genetic control of MLC reactivity to different MHC incompatibilities, independent of production of and response to IL-2.

The inbred strain STS/A exhibits a higher proliferative response in the mixed lymphocyte culture (MLC) to stimulator cells of all 11 tested inbred mouse strains with 10 different major histocompatibility complex (MHC) haplotypes, as well as to stimulation with IL-2 than does the strain BALB/cHeA. However, alloantigen-stimulated BALB/c cells produce more IL-2 than STS/A cells. To study the genetic basis of these differences, we used 20 recombinant congenic strains (RCS) of the CcS/Dem series. Each of these CcS/Dem RC strains contains a different subset of about 12.5% of genes from the STS/A strain and the remaining approximately 87.5% of BALB/c origin genes. As a result the multiple non-linked genes responsible for phenotypic differences between BALB/c and STS/A became separated into different CcS/Dem strains. The strain distribution pattern (SD) of high or low MLC response of individual CcS/Dem strains to stimulator cells of four different strains was almost identical, indicating that differences in responsiveness, rather than the alloantigenic difference itself, determine the magnitude of the response, and that the responsiveness to different alloantigens is largely controlled by the same genes. The SDP of IL-2 stimulation was different from that of MLC responsiveness. The differences in the proliferative responses observed among individual CcS/Dem strains were not due to differences in numbers of CD3+, CD4+ or CD8+ cells or to the observed differences in IL-2 production, and hence they likely reflect genetically determined intrinsic properties of T cells. These results show that a set of non-linked genes controls proliferative responses in MLC irrespective of the MHC haplotype of the stimulator cells, and that stimulation with IL-2 and production of IL-2 are controlled by different subsets of genes. Since the genomes of all RCS are extensively characterized by microsatellite markers, they can be used to map the genes controlling proliferative responsiveness to stimulation with alloantigens and IL-2.

Separation of multiple genes controlling the T-cell proliferative response to IL-2 and anti-CD3 using recombinant congenic strains.

T lymphocytes of the strain BALB/cHeA exhibit a low proliferative response to IL-2 and a high response to the anti-CD3 monoclonal antibodies, while the strain STS/A lymphocyte response to these stimuli is the opposite. We analyzed the genetic basis of this strain difference, using a novel genetic tool: the recombinant congenic strains (RCS). Twenty BALB/c-c-STS/Dem (CcS/Dem) RCS were used, each containing a different random set of approximately 12.5% of the genes from STS and the remainder from BALB/c. Consequently, the genes participating in the multigenic control of a phenotypic difference between BALB/c and STS become separated into different CcS strains where they can be studied individually. The strain distribution patterns of the proliferative responses to IL-2 and anti-CD3 in the CcS strains are different, showing that different genes are involved. The large differences between individual CcS strains in response to IL-2 or anti-CD3 indicate that both reactions are controlled by a limited number of genes with a relatively large effect. The high proliferative response to IL-2 is a dominant characteristic. It is not caused by a larger major cell subset size, nor by a higher level of IL-2R expression. The response to anti-CD3 is known to be controlled by polymorphism in Fc gamma receptor 2 (Fcgr2) and the CcS strains carrying the low responder Fcgr2 allele indeed responded weakly. However, as these strains do respond to immobilized anti-CD3, while the STS strain does not, and as some CcS strains with the BALB/c allele of Fcgr2 are also low responders, additional gene(s) of the STS strain strongly depress the anti-CD3 response. In a backcross between the high responder and the low responder strains CcS-9 and CcS-11, one of these unknown genes was mapped to the chromosome 10 near D10Mit14. The CcS mouse strains which carry the STS alleles of genes controlling the proliferative response to IL-2 and anti-CD3 allow the future mapping, cloning, and functional analysis of these genes and the study of their biological effects in vivo.

Interleukin-1 and interferon-alpha augment interleukin-2 (IL-2) production by distinct mechanisms at the IL-2 mRNA level.

The production of interleukin-2 (IL-2) by phytohemaglutinin (PHA)-stimulated cells of human leukemia T cell line MOLT-16 can be significantly increased by interleukin-1 (IL-1) or interferon-alpha (IFN-alpha). The enhancing effect of IL-1 and IFN-alpha on IL-2 production was studied at the IL-2 mRNA level. We show that IL-1 enhances considerably and transiently, with the maximum level between 1 and 2 hr after stimulation, the expression of IL-2 mRNA in the PHA-stimulated cells. The level of IL-2 mRNA declined rapidly within 4 to 6 hr after stimulation in both PHA- and PHA plus IL-1-stimulated cell cultures. On the contrary, IFN-alpha does not elevate the level of IL-2 mRNA above the level in PHA-stimulated cultures, but maintains an enhanced level of IL-2 mRNA in the activated cells for more than 6 hr after stimulation. These observations correlate well with the kinetics of IL-2 protein production into the culture media. The results thus suggest that IL-1 and IFN-alpha may exert an enhancing effect on IL-2 production by distinct mechanisms. In addition, none of the five other lymphokines tested (i.e., IL-2, IL-3, IL-4, IL-5, and IL-6) had any significant effect on IL-2 mRNA expression in the activated MOLT-16 cells.