Eleni Dotsika

Asymptomatic canine leishmaniasis in Greater Athens area, Greece.

Leishmania(L. infantum is the etiological agent of human and canine visceral leishmaniasis in the Mediterranean subregion. Domestic dogs are the main reservoir of the parasite in most urban areas. A survey of 1638 asymptomatic dogs registered in Greater Athens area was carried out in the Hellenic Pasteur Institute during the period 1986–1994 to investigate the prevalence of canine visceral leishmaniasis in apparently healthy dogs. Dog sera was tested using the indirect fluorescent antibody technique (IFAT). Of the 1638 dogs, 366 (22.4%) had anti-Leishmania infantum antibodies at titre greater than or equal to 1/200 which were considered positive; 53 (3.2%) had antibody titres of 1/100 and were considered uncertain; and 1219 (74.4%) dogs were seronegative. From the 366 seropositive dogs, 212 were positive at 1/1600 serum dilution, 57 at 1/800, 38 at 1/400 and 59 at 1/200. The results were plotted according the site of residence, breed and age. The rate of asymptomatic infections with L. infantum dogs in Greater Athens area appears to be significantly high. Although there is an apparent lack of clinical symptoms in these dogs, asymptomatic animals harbor a chronic L. infantum infection and as such consist a ‘dangerous’ reservoir with regard to the spread of the disease.

CD8(+) T cells with parasite-specific cytotoxic activity and a Tc1 profile of cytokine and chemokine secretion develop in experimental visceral leishmaniasis.

Recently, a prominent role for CD8+ T cells in immunity against pathogens has emerged. The mode of action of CD8+ T cells in murine visceral leishmaniasis and their contribution to the clearance of the parasite has been addressed in the present study. We showed that during the course of experimental infection cytotoxic clones specific for Leishmania infantum antigens developed in the spleen of susceptible BALB/c mice, showed an activated phenotype and became susceptible to apoptotic cell death late in the course of the disease. CD8+ T cells exhibited considerable cytotoxic activity against cells expressing Leishmania antigens. This activity was mediated by both the perforin and the Fas/FasL pathway, as judged from in vitro and in vivo assays. The CD8+ T cells also up‐regulated mRNAs for cytokines (IFN‐γ and TNF‐α) and C‐C chemokines (RANTES and MIP‐1α), which have a major role in immunity against the pathogen. CD8+ T‐cells thus displayed a Tc1 pattern of differentiation. In conclusion, CD8+ T cells appear to play multiple roles in an experimental model of visceral leishmaniasis comprising both cytotoxic activity and secretion of cytokines and chemokines.

ISCOMs vaccine against experimental leishmaniasis

The major surface glycoprotein (gp63) of Leishmania major incorporated into the immunostimulating complexes (ISCOMs) was used to protect Balb/c mice against experimental infection. Two intraperitoneal vaccinations with low doses of gp63 into ISCOMs (gp63-ISCOMs) induced protective immunity in vaccinated mice as indicated by reduced inflammation and suppressed lesions after experimental challenge. An augmented IgG-specific secretion and a specific switching towards the IgG2a isotype was observed in the serum of vaccinated mice. Gp63-ISCOMs primed spleen cells restimulated in vitro with soluble Leishmania antigen (SLA) or live parasites displayed strong gp63-specific proliferative responses and secreted high levels of interleukin-2, interferon gamma and interleukin-10 but not interleukin-4. No delayed type hypersensitivity response to either SLA or LV39 was detected. These data indicate that gp63-ISCOMs induced a protective immunity in the susceptible Balb/c mice against Leishmania challenge, modulating the immune response towards a Th1 rather than Th2 type.

Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins.

Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4(+) and CD8(+) T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania infantum proteins, cysteine peptidase A (CPA), histone H1, KMP-11, and Leishmania eukaryotic initiation factor (LeIF) were analyzed for the prediction of binding epitopes to H2(d) MHC class I and II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in complete Freunds adjuvant/incomplete Freunds adjuvant. CPA_p2, CPA_p3, H1_p1, and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3, and LeIF_p6 induced IFN-γ-producing CD4(+) T cells indicating a TH1-type response. In addition, CPA_p2, CPA_p3, and H1_p1 induced also the induction of CD8(+) T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3, and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.

IMMUNOGLOBULIN AND CYTOKINE PROFILE IN MURINE SECONDARY HYDATIDOSIS

We have investigated specific immune responses in BALB/c mice with experimentally induced secondary hydatidosis. Following intraperitoneal inoculation of brood capsules containing Echinococcus granulosus‐protoscoleces, the course of the infection was followed for 513 days. The sera of the mice were screened for the presence of a number of cytokines, and for specific antibodies. During the first 129 days of infection, high levels of cytokines TNFα, IL‐α, IFNγ, IL‐6, andIL‐10 and specific IgGl andIgG3 isotypes were detected, as compared to uninfected controls. The levels oflgM and IgG2a were slightly increased following infection, and remained elevated throughout the period of observation. The levels of IL‐lα and specific immunoglobulin of all isotypes except IgM and IgG2α, were significantly decreased 103 days post infection fp.i.), whereas TNFα was sharply decreased 129 days p.i. During the period of 129 to 209 days of infection there was an increase in secreted IL‐10, and a slow decrease in the levels of IL‐6 andlFN‐γ. Levels of IgM, IgG, IgGl, and IgG2a plateaud during this period, whereas IgG3 and TNFα showed a peak at day 190 p.i. These data suggest the induction of Th2 antibody‐mediated immunity with a parallel expansion of Thl‐mediated inflammatory responses as important mechanism of host defence against the metacestode.

Generation of TNF alpha, IFN gamma, IL-6, IL-4 and IL-10 in mouse serum from trichinellosis: effect of the anti-inflammatory compound 4-deoxypyridoxine (4-DPD)

Infections caused by the nematode Trichinella spiralis is characterized in the host by an inflammatory response with cytokine production. In these studies we have detected TNF alpha, IL-6, IFN gamma, IL-4 and IL-10 in the serum of 10 mice infected with T. spiralis. Moreover, we detected, for the first time, these cytokines in the serum of mice treated with 4-DPD, a potent antagonist of vitamin B6 coenzyme which has anti-inflammatory properties. 4-DPD was used at 100, 400, 800 micrograms/bolus for 20 days, starting one day before the infection. After 15 days of T. spiralis infection, TNF alpha reached a maximum level, while IL-6 was maximal after 7 days, IFN gamma at 20 days and IL-4 at 14 days. IL-10 was not affected by the T. spiralis infection. When the animals were treated with 4-DPD at the reported dosages and infected with T. spiralis the inhibition of TNF alpha and IL-6, were dose-dependent in the first 7 days while IL-4 was reduced only at 400-800 micrograms/bolus. 4-DPD-treated mice did not statistically (P > 0.05) affect the generation of IFN gamma. In healthy animals the production of cytokines were not measurable, just as it was in non-infected animals treated with 4-DPD. The increase of cytokines such as, TNF alpha and IL-6 may be related to the severity of the disease, boosting the hosts resistance to the pathogen and inhibiting parasite survival. In addition, the augmentation of IL-4 production enhances T and B cells and macrophage responses and may stimulate T-cell antibody-mediated response to the pathogen. 4-DPD, an inhibitor of IL-1 and inflammatory reactions, proved to be most effective on TNF alpha and IL-6, which are mainly produced by macrophages.

Leishmanicidal activity assessment of olive tree extracts.

Leishmaniasis, a protozoan parasitic disease that remains a major worldwide health problem with high endemicity in developing countries, is prevalent around the Mediterranean basin. High cost, systemic toxicity, and diminished efficacy due to development of parasite resistance are the serious drawbacks of current treatment options. Thus, identifying new, effective, and safer anti-leishmanial drug(s) is of paramount importance. Here we tested the anti-promastigote and anti-amastigote activity of five natural products, including oleuropein and hydroxytyrosol, present in olive tree leaves and olive mill wastewater. These products are recognized as low-cost starting materials rich in bioactive compounds, particularly biophenols. Oleuropein and hydroxytyrosol exhibited the best inhibitory effect among the natural products tested in both stationary and middle logarithmic phase promastigotes of L. infantum, L. donovani, and L. major. Similarly, oleuropein and hydroxytyrosol demonstrated the highest selectivity index ratio against L. donovani amastigotes that parasitize J774A.1 macrophages. Moreover, oleuropein was tested in vivo in an experimental visceral leishmaniasis model. L. donovani-infected BALB/c mice received intraperitoneal oleuropein a total of 14 times at intervals of every other day. Three days after treatment termination, the spleen parasitic burden was reduced >80%. Of interest, this effect of oleuropein persisted and was even enhanced 6 weeks after the termination of the treatment, as determined by parasite depletion of >95% in liver and spleen. These findings contribute to the potential development of natural products as effective drugs against parasites of the Leishmania genus, with low cost and diminished cytotoxicity.

The Impact of a Successful Anti-Myxosporean Treatment on the Phagocyte Functions of Juvenile and Adult Sparus Aurata L

The aim of the present study was to investigate the impact of a successful anti-myxosporean medication on the innate immune system of fish intensively cultured in the Mediterranean basin. For this purpose, juvenile and adult gilthead seabream (S. aurata L.) naturally infected with Polysporoplasma sparis in the kidney were used in a small-scale field trial. The infected fish were treated orally with the combination of salinomycin and amprolium, two drugs well known for their anti-coccidial effect in other animals. Drug efficacy and safety was evaluated in terms of changes observed in histopathology, mortality and P. sparis intensity and prevalence rate. Phagocytic functions of head-kidney leucocytes were also investigated at the end as well as one month post the medication. Salinomycin with amprolium exhibited a significant reduction in intensity and prevalence rate in both juvenile and adult fish, and no histopathological evidence for toxic side effects was observed. In addition, the successful treatment was closely correlated with a complete restoration of the diminished phagocytic ability and capacity as well as NO, and lysozyme secretion in a time dependent manner. This data suggests that salilomycin with amprolium can be an alternative treatment for myxosporean infections in warm-water fish, possibly exhibiting their action through the enhancement of host innate functions.

Dendritic cells pulsed with peptides of gp63 induce differential protection against experimental cutaneous leishmaniasis.

The need for a vaccine against Leishmania spp., a major cause of worldwide morbidity and mortality, is urgent. We tested the efficacy of an experimental vaccination in murine models of cutaneous leishmaniasis, using dendritic cells (DCs) pulsed with synthetic or native parasite antigens. DCs pulsed with peptide 154–169aa of gp63 or soluble promastigote lysate (SPL) triggered antigen-specific immune responses and efficiently reduced lesion formation and parasite load of genetically susceptible BALB/c mice infected with Leishmania major. This effect was accompanied by a modulation of the cellular immune response towards a Th1 profile. Vaccination of genetically resistant CBA mice with DCs pulsed with peptide 154–169aa or SPL did not affect the course of the disease, whereas pulsing with the epitope 467–482aa of gp63 resulted in disease exacerbation, accompanied by a switch to a Th2 profile. In view of our continuously growing knowledge about the immunobiology of DCs, these findings suggest that vaccination with DCs pulsed with defined peptides could be a strategy against infectious diseases. Peptide selection is a prerequisite as they can differentially regulate the type of immune response in susceptible or resistant hosts.

Mannan-conjugated myelin peptides prime non-pathogenic Th1 and Th17 cells and ameliorate experimental autoimmune encephalomyelitis

Antigen presenting cells (APC) are critical for regulating immune responses. We tested mannan-peptide conjugates for targeting myelin peptides to APC to induce T cell tolerance and resistance to experimental autoimmune encephalomyelitis (EAE). Myelin peptides conjugated to mannan in oxidized (OM) or reduced (RM) forms protected mice against EAE in prophylactic and therapeutic protocols, with OM-conjugated peptides giving best results. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation, but not alterations in Th1, Th17 and Treg cell differentiation or T cell apoptosis compared to EAE controls. Bone marrow-derived dendritic cells (DC) loaded with OM-MOG showed up-regulated expression of co-stimulatory molecules, reduced PD-L1 expression and enhanced CD40-inducible IL-12 and IL-23 production compared to MOG DC, features consistent with immunogenic DC. OM-MOG induced active T cell tolerance because i.d. administration or passive transfer of OM-MOG DC suppressed ongoing EAE, while OM-MOG-vaccinated mice did not reduce the proliferation of transferred MOG-specific T cells. As in vivo, MOG-specific T cells cultured with OM-MOG DC showed reduced proliferation and equal Th1 and Th17 cell differentiation compared to those with MOG DC, but surprisingly cytokine production was unresponsive to CD40 engagement. Impaired effector T cell function was further evidenced in spinal cord sections from OM-MOG-vaccinated EAE mice, where markedly reduced numbers of CD3(+) T cells were present, restricted to leptomeninges and exceptional parenchymal lesions. Our results show that mannan-conjugated myelin peptides protect mice against EAE through the expansion of antigen-specific Th1 and Th17 cells with impaired proliferation responses and APC-induced co-stimulatory signals that are required for licensing them to become fully pathogenic T cells.