Evaluation of cellular immunological responses in mono- and polymorphic clinical forms of post-kala-azar dermal leishmaniasis in India.

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis (VL). The disease manifests into macular, papular and/or nodular clinical types with mono- or polymorphic presentations. Here, we investigated differences in immunological response between these two distinct clinical forms in Indian PKDL patients. Peripheral blood mononuclear cells of PKDL and naive individuals were exposed in vitro to total soluble Leishmania antigen (TSLA). The proliferation index was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based lymphoproliferative assay. Cytokines and granzyme B levels were determined by cytometric bead array. Parasite load in tissue biopsy samples of PKDL was quantified by quantitative polymerase chain reaction (qPCR). The proportion of different lymphoid subsets in peripheral blood and the activated T cell population were estimated using flow cytometry. The study demonstrated heightened cellular immune responses in the polymorphic PKDL group compared to the naive group. The polymorphic group showed significantly higher lymphoproliferation, increased cytokines and granzyme B levels upon TSLA stimulation, and a raised proportion of circulating natural killer (NK) T cells against naive controls. Furthermore, the polymorphic group showed a significantly elevated proportion of activated CD4(+) and CD8(+) T cells upon in-vitro TSLA stimulation. Thus, the polymorphic variants showed pronounced cellular immunity while the monomorphic form demonstrated a comparatively lower cellular response. Additionally, the elevated level of both activated CD4(+) and CD8(+) T cells, coupled with high granzyme B secretion upon in-vitro TSLA stimulation, indicated the role of cytotoxic cells in resistance to L. donovani infection in polymorphic PKDL.

Effective humoral and cellular immunoprotective responses in Li ESAp-MDP vaccinated protected dogs.

Cell-mediated and humoral immunity were explored in LiESAp-MDP vaccinated protected dogs versus susceptible placebo dogs 2 months and 8 months post-vaccination. As previously described, a strong and long-lasting cell-mediated immunity, critical for protection against Leishmania infantum was exclusively revealed in vaccinated dogs as confirmed by a positive response to the intradermal inoculation of leishmanin and by a significant higher anti-leishmanial activity of canine monocytes-derived macrophages. Moreover, our results support the view that cooperation of humoral antibody with cell-mediated immunity might be important in developing protective immunity in LiESAp-MDP vaccinated dogs. Anti-LiESAp serum samples were found functionally active in vitro, promoting (i) early killing of pretreated promastigotes and amastigotes, (ii) strong inhibitory effect on the in vitro growth of both parasitic developmental stages of L. infantum and (iii) most importantly, a significant inhibition of pretreated promastigotes in vitro infectivity in canine macrophages. However, anti-LiESAp antibody response was not implicated in the promastigotes-amastigotes differentiation process. In these experiments, we have added additional support to the concept that antibodies to Leishmania may be important in developing protective immunity.

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis.

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.

Detection of antibodies to Ureaplasma urealyticum in pregnant women by enzyme-linked immunosorbent assay using membrane antigen and investigation of the significance of the antibodies.

Optimal conditions of a microenzyme-linked immunosorbent assay using a group-specific membrane antigen of Ureaplasma urealyticum serotype 7 were established with rabbit antisera and applied for the evaluation of immunoglobulin M (IgM) and IgG antibodies in 139 serum specimens from pregnant women between 26 and 38 weeks of gestation, and the assay was compared with microorganism culture and investigated to determine the role of U. urealyticum in perinatal morbidity and mortality. U. urealyticum was isolated from 75 (54%) of 139 patients; 40 had a colonization greater than or equal to 10(6) cells per ml of swab (29%); 64 (85%) of 75 culture-positive patients had IgG antibodies (absorbance mean, 0.650), versus 4 (6%) of 64 culture-negative patients (absorbance mean, 0.103) (P less than 0.001). There was no cross-reactivity with Chlamydia trachomatis infection from patients from whom no mycoplasmas were isolated, but this cross-reactivity occurred in 24% of patients with other mycoplasma infections. There was a good correlation between quantitative evaluation of U. urealyticum colonization and antibody level (P less than 0.05). However, IgM antibody was found in 30% of culture-positive patients but also in 25% of the culture-negative group. Frequency of U. urealyticum colonization was greater in unmarried young women (less than 25 years old) with a history of genital infection, and a significantly greater frequency was detected in patients who smoked (P less than 0.01) and had a lower socioeconomic status (P less than 0.001). A lower infant birth weight was more associated with U. urealyticum colonization greater than or equal to 10(5) cells per ml. The enzyme-linked immunosorbent assay provided an additional means to diagnose and evaluate U. urealyticum infection in pregnant women.

Preparative electroelution of specific protein antigens from Mycoplasma pneumoniae: use in an enzyme-linked immunosorbent assay (ELISA).

A rapid, simple and preparative method is described for the recovery of the seven highest molecular weight proteins (HMWP) from Mycoplasma pneumoniae membrane. The yield of proteins obtained was approximately 90%. The method involved the separation of M. pneumoniae proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroelution of HMWP. These eluted antigens were used in an ELISA to measure IgG antibodies in sera from 9 blood donors and 9 patients with M. pneumoniae infection. The specificity of M. pneumoniae HMWP was examined by competition ELISA and immunoblotting with different mycoplasma species encountered in the respiratory tract.