Leishmania parasite detection and quantification using PCR-ELISA.

This protocol describes an improved and optimized PCR-ELISA method for detection and quantification of Leishmania parasites in host tissues. Unlike other DNA-based assays, this method uses digoxigenin- and biotin-labeled primers. This eliminates the need for a separate step of hybridization of the PCR product with labeled probes. The PCR product is detected using sandwich ELISA with antidigoxigenin-detecting antibodies. Primers are complementary to the kinetoplast minicircle conserved region of parasite DNA, allowing the detection of several Leishmania species. For measurement of a wide range of parasite concentrations, +/-25 cycles were optimal. The sensitivity of this technique is 0.3 fg of parasite DNA per reaction in 40-cycle PCR-ELISA, corresponding to 0.004 parasites. DNA preparation by a standard TRI reagent procedure takes about 4 h. When DNA is prepared, a single person can test a large number of samples (at least 150) in a maximum of 7 h. This method might also be suitable for detecting and quantifying other pathogens, especially for detecting small differences in pathogen numbers.

Loci controlling lymphocyte production of interferon c after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility.

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1­Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2pz) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2b) or BALB/ cHeA (H2d) mice, or by ConA. IFNγ production in MLCs of individual (O20 9 OcB-9)F2mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.

Novel loci controlling lymphocyte proliferative response to cytokines and their clustering with loci controlling autoimmune reactions, macrophage function and lung tumor susceptibility.

Novel genotyping and statistical tools have led to mapping of numerous QTL loci for multigenic traits that previously could not be detected. The relationships of these QTL families to other QTL families and the functional specialization of their members can now be studied. We have mapped a number of loci controlling activation of T lymphocytes by mitogens and cytokines and their capacity to produce cytokines. In (O20xOcB-9)F2 hybrids, we mapped 3 novel loci controlling proliferative T-cell response to cytokines IL-2 and IL-4 (Cinda3) or IL-4 only (Cinda4 and Cinda5). OcB-9 allele at Cinda3 controls a higher response than the O20 allele to both IL-2 and IL-4, and OcB-9 alleles of Cinda4 and Cinda5 control higher response to IL-4. These novel Cinda loci and the previously mapped Cinda1 locus seem to be located in genomic regions together with other QTL families: macrophage function loci Marif1 and Marif2, proteoglycan-induced arthritis loci Pgia4, Pgia7 and Pgia12 and lung tumor susceptibility loci Sluc1, Sluc4, Sluc6 and Sluc20. The possible relevance of these QTL associations in several different sites of the genome for the immune response, inflammation and tumorigenesis has to be elucidated.

Different genetic control of cutaneous and visceral disease after Leishmania major infection in mice.

The mouse strains BALB/cHeA (BALB/c) and STS/A (STS) are susceptible and resistant to Leishmania major-induced disease, respectively. We analyzed this difference using recombinant congenic (RC) BALB/c-c-STS/Dem (CcS/Dem) strains that carry different random subsets of 12.5% genes of the strain STS in a BALB/c background. Previously, testing the resistant strain CcS-5, we found five novel Lmr (Leishmania major response) loci, each associated with a different combination of pathological and immunological reactions. Here we analyze the response of RC strain CcS-16, which is even more susceptible to L. major than BALB/c. In the (CcS-16 x BALB/c)F(2) hybrids we mapped three novel loci that influence cutaneous or visceral pathology. Lmr14 (chromosome 2) controls splenomegaly and hepatomegaly. On the other hand Lmr15 (chromosome 11) determines hepatomegaly only, and Lmr13 (chromosome 18) determines skin lesions only. These data confirm the complex control of L. major-induced pathology, where cutaneous and visceral pathology are controlled by different combinations of genes. It indicates organ-specific control of antiparasite responses. The definition of genes controlling these responses will permit a better understanding of pathways and genetic diversity underlying the different disease phenotypes.

Mouse genetic model for clinical and immunological heterogeneity of leishmaniasis.

Systematic assessment of the role of host genes in clinico-pathological and immunological manifestations of Leishmania major-induced disease in mice was performed using 20 recombinant congenic (RC) strains. As the RC strains are homozygous and each carries a different, random set of 12.5% genes from the resistant strain, STS/A, and 87.5% genes from the susceptible strain, BALB/cHeA, they allowed us to study the pathological and immunological characteristics of infected hosts in 20 fixed different random combinations of BALB/c and STS genes. The 20 RC strains differ widely in expression of different symptoms of disease and in immunological characteristics. Disease or healing in different strains occurred in association with different components of immune response -- with the exception of a frequently occurring correlation between the disease and IgE levels. Moreover, some parameters of the immune response were highly correlated in some strains but not at all in others. This shows that several patterns of the immune response may be associated with the same clinical outcome, depending on the host genotype. Our data also suggest that despite the complexity of regulation, when a sufficient number of controlling loci is known, the prediction of a phenotype is possible. Combining functional and clinical information with multilocus genotyping may improve our ability to predict the progression of the disease and to optimize the treatment.