Toll-like receptor 4 promotes control of Leishmania infantum infection through inducement of leishmanicidal activity in host macrophages: role of mitogen activated kinases.

Establishment of Leishmania infection inside macrophages requires deactivation of various signaling pathways that are dispensable for effective immune responses against the parasite. In the present study, we provide evidence that Leishmania infantum promastigotes attachment on the surface of peritoneal macrophages, internalization and transformation to amastigotes abrogated the activation of extracellular signal-regulated protein kinases (ERK) 1/2, p38 mitogen activated protein kinases (MAPK) and c-Jun NH2-terminal kinases (JNK) and the production of pro-inflammatory cytokines IL-12 and TNFalpha. Subsequent macrophage stimulation with lipopolysaccharide (LPS) during the first hours of exposure to parasite or infection resulted in restoration of MAPK phosphorylation. However, LPS-mediated MAPK activation required parasite internalization (uptake) since cytochalasin-D pretreated macrophages did not responded to LPS stimulation. IL-12, TNFalpha, and NO production was positively regulated with MAPK phosphorylation in contrast to nuclear factor-kappaB (NF-kB) which was MAPK independent. Specifically, inhibition of MAPK activation with specific inhibitors revealed that IL-12 production required p38 MAPK activation, whereas TNFalpha and NO production required all three MAPK. The restoration of NO production resulted in decrease of infection rates. Hence, these results suggest that in contrast to phagocytosis of L. infantum promastigotes, establishment of infection does not desensitize macrophages to subsequent stimulation with LPS, resulting in parasite elimination through MAPK and NF-κB activation and partial restoration of IL-12, TNFalpha and NO synthesis.

KPC-producing, multidrug-resistant Klebsiella pneumoniae sequence type 258 as a typical opportunistic pathogen.

The virulence of a KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) strain representing those circulating in Greece was assessed in a mouse septicemia model. The strain was virtually avirulent (50% lethal dose, >10(8) and 5 × 10(7) CFU for immunocompetent and neutropenic animals, respectively). Also, it was highly susceptible to serum killing, rapidly phagocytosed in vitro, and classified as K41, which is not among the virulent capsular types. The findings indirectly support the notion that high ST258-associated mortality is largely due to inefficient antimicrobial treatment.

Modulation of NF-kappaΒ signalling pathways by parasites.

NF-kappaB is implicated in lymphocyte development, maturation, proliferation and survival. This inducible transcription factor is widely expressed by virtually all cell types. In mammals, the genes rela, relb, crel, nfkappaΒ1, and nfkappaB encode the five NF-kB protein family members RelA (p65), RelB, c-Rel, p50, and p52, respectively, which form homo- and heterodimeric DNA-binding complexes capable of regulating target gene transcription of specific biological responses differentially. NF-kappaB regulates the expression of a wide variety of genes that play critical roles in innate and adaptive immune responses, is strongly linked to the inhibition of apoptosis, and contributes to tumor growth, metastasis, and chemoresistance. Parasites have targeted several parts of the NF-kappaB pathway, allowing them to interfere with the transcription of immune response genes. The biology of different parasites is critical in influencing the patterns and kinetics of NF-kappaB activity and thereby the development of subsequent immune responses.

Microarray analysis of NF-kappaB signaling pathways in PBMC of mice infected by Trichinella spiralis.

The NF-kappaΒ pathway gene expression profiles were compared between 10, 20 and 39 days after Trichinella spiralis experimental infection in BALB/c mice. Out of 128 genes, 19 (14.8%) genes were present in non-infected and post-infected mice. The expression of 7 (36.8%) genes was downregulated 10 and 20 days post-infection while 3 (15.8%) genes were upregulated 39 days post-infection. The present study lists the candidate genes of the NF-kappaB signaling pathway that were commonly and differentially expressed between the specific points of T. spiralis infection, thus suggesting that these genes need to be further investigated to reveal the mechanism of the T. spiralis modulation of the NF-kappaB signaling pathways.

Vaccination with Trichinella spirallis antigens increases CD8+ peripheral T cells and enhances the Th2 immune response in Leishmania infantum challenged mice.

In this study we investigate the effect of Trichinella spiralis vaccination on immune responses elicited in BALB/c mice challenged subcutaneously with 0.5 x 10 6 of Leishmania infantum promastigotes. Secretion of specific anti-L. infantum antibodies and changes in the number of CD4+, CD8+ T cell and CD19+ B cells in the peripheral blood were tested for the evaluation of immune responses. Immunization with low amounts of T. spiralis antigens induced depression in anti-Leishmania specific antibodies of the IgG1 isotype, while no changes in the number of CD4+ and CD8+ T cell subpopulations or CD19+ B cells were observed. In contrast, high amounts of T. spiralis antigens induced an enhancement in anti-Leishmania specific antibodies of total IgG and IgG1 isotype, increase of CD8+ T cell number and activation of CD19+ B cells, indicated by the co-expression of CD69 marker. Our results suggest that immunization with a certain dose of T. spiralis antigens in experimentally challenged mice with L. infantum leads to an increase of peripheral CD8+ T cells which are responsible for the control of L. infantum infection, although a simultaneous enhancement in Th2-type of immune response is also observed.

Antiparasitic and immunomodulatory effect of innovative treatments against Myxobolus sp. infection in Diplodus puntazzo.

The potential antiparasitic and immunomodulatory effect of three treatments against myxosporean parasites on the innate immune system of sharpsnout sea bream (Diplodus puntazzo) was investigated. Fish naturally infected with Myxobolus sp. (Bivalvulida/Platysporina), a histozoic parasite mainly affecting the renal interstitial tissue, were treated by oral administration of a combination of salinomycin with amprolium, Origanum essential oil or fumagillin in a small-scale field trial. Various leucocyte functions influenced by myxosporean infection were examined in order to determine treatment effects on leucocyte immunocompetence of treated fish. One month post treatment all drugs caused a significant decrease in prevalence and intensity of infection in comparison to untreated, infected fish. The effect was most prominent in salinomycin with amprolium treated fish, which 1-month post treatment contained either no cysts at all or a few spores free in melanomacrophage centres revealing almost total elimination of the parasite and the antiparasitic action of the treatment. There was no histopathological evidence of drug toxicity. Antiparasitic action was accompanied by a significant enhancement of phagocytic activity demonstrated by ingestion of large numbers of latex beads and the secretion of high levels of reactive nitrogen intermediates by phagocytes in vitro. Complete restoration of the diminished mitogenic responses and serum lysozyme secretion was also detected in salinomycin with amprolium-treated fish compared to untreated, infected fish. These data suggest that salilomycin with amprolium may be a promising treatment for myxosporean infections in intensively cultured warm-water fish, exhibiting action partially via the enhancement of host, innate immune functions and leading to parasite elimination.

Inhibition of MCP-1 and MIP-2 chemokines in murine trichinellosis: effect of the anti-inflammatory compound L-mimosine.

Mimosine is a plant amino-acid which has been reported to block DNA replication in mammalian cells and to arrest cell reversibly towards the end of the G1 phase or at the beginning of the S phase. In this study, 42 mice were infected with T. spiralis a nematode parasite, and treated with the anti-inflammatory compound L-mimosine, to determine if any alteration in the chronic inflammatory state occurred, by investigating the hosts immunological response. MCP-1, a C-C chemokine and MIP-2, a C-X-C chemokine were tested and calculated in the sera of infected animals, after 1, 10, 20, 30, 40, 50 and 60 days post infection, by ELISA method. The diaphragm and the masseters of the infected mice, were tested for inflammatory response. Here we found, that MCP-1 was partially inhibited by L-mimosine, while MIP-2 was totally inhibited. Moreover in sections of the diaphragm and masseters, the infiltration of inflammatory cells, such as macrophages, lymphocytes and eosinophils were more intense in untreated animals compared to those treated with L-mimosine. These findings show, that L-mimosine may have an inhibitory effect on MCP-1 and MIP-2 serum levels in Trichinellosis and may influence the recruitment of inflammatory cells and the intensity of the inflammatory reaction in this parasitic disease.

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 tropical 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.

T cell help is required to induce idiotypic-anti-idiotypic autoantibody network after immunization with complementary epitope 289-308aa of La/SSB autoantigen in non-autoimmune mice.

Immunotherapies against autoimmune diseases have been of limited success. Preventive vaccines could be developed on the basis to abrogate unwanted immune responses to defined autodeterminants. In this study it is shown that immunization of BALB/c mice with two linear T and B cell epitopes of the human La/SSB autoantigen (spanning the regions 289-308aa and 349-364aa) and their complementary forms specified by the complementary mRNA, results in characteristic B and T cell responses. Mice immunized with the 289-308aa epitope or its complementary peptide elicited specific antibodies against both epitopes. In contrast, mice immunized with the 349-364aa epitope or its complementary peptide mounted antibody titres against the immunizing peptide only. According to these data, the 289-308aa epitope and its complementary form were capable to generate an idiotypic-anti-idiotypic response, which were cross-regulated. Peptide-specific T cell proliferation and cytokine production in vitro revealed the induction of a two-stage T helper response (Th1-->Th2 type) after immunization with either the epitope 289-308 or its complementary peptide. IgG1 was the predominant subclass after immunization with the two forms of epitopes 289-308 and 349-364, while a response of the IgG2b > IgG2a was obtained after the immunization with the complementary form of 349-364 epitope reflecting the TH2/TH1 polarization, respectively. Our data suggest that the complementary peptides of two immunodominant epitopes of human LaSSB can mimic the autoantibodies against these epitopes and establish an active idiotypic-anti-idiotypic network.

Efficacy and toxicity of orally administrated anti-coccidial drugs for innovative treatments of Myxobolus sp. infection in Puntazzo puntazzo.

This study tested drugs and therapeutic compounds to determine effective commercial treatment for fishes infected with myxosporeans. Two series of shore-based experiments and 1 field trial were performed. For the shore-based experiments we used Puntazzo puntazzo (ca. 20 g weight) with kidneys infected with Myxobolus sp. Initially, 6 different doses of Fumagillin, 2 doses of Toltrazuril, and 1 dose of Amprolium, ESB3 and Salinomycin were tested. In the second shore-based experiment, infected fish were treated with Origanum essential oils, Toltrazuril with propylene glycol, Amprolium, and a combination of Salinomycin 12% + Amprolium (SA). In the field trial, P. puntazzo (ca. 165 g) infected with the parasite were treated with SA, Origanum essential oils and Fumagillin. In all trials, the drugs were added to the feed and administered according to the selected regimen. Their efficacy was evaluated in terms of mortality (acceptable level was <3%), pathology and prevalence rate of Myxobolus sp. Lesions were observed only in fish treated with Fumagillin and Toltrazuril. Pathology due to treatment with Fumagillin was observed only at doses > 6 mg kg(-1) body wt for 6 wk in the interstitial renal tissue, where slight inflammation arose. The highest dose tested (25 mg kg(-1)) also produced necrosis in the interstitial tissue, degeneration of the epithelial cells of the tubules and a reduction in melanomacrophage centre numbers. The SA combination proved the most effective treatment for Myxobolus sp. infection of P. puntazzo as (1) the therapeutic regimen and commercial product was not toxic and (2) a significant reduction occurred in the prevalence rate.

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.

IgG, IgG1 and IgM response in Trichinella spiralis-infected mice treated with 4-deoxypirydoxine or fed a Vitamin B6-deficient diet.

The aim of this study was to investigate the effect of pyridoxine (Vitamin B6) deficiency on the immunological response of BALB/c mice infected with the parasite T. spiralis. Specific anti-parasite IgM and IgG immunoglobulins were detected by ELISA method in the serum of treated animals at different periods for 60 days post infection. Vitamin B6-deficiency was induced in two separate groups of mice by either (1) maintaining the mice on a Vitamin B6-deficient synthetic pellet diet for 40 days before infection, or (2) by daily intraperitoneal injection of 8 x 10(5) M/100 microl of 4-Deoxypyridoxine (4-DPD), a potent antagonist of Vitamin B6 for 20 days prior to infection. These two groups of mice were then injected with 100 larvae (L1-T. spiralis) per os. Parasite burdens in the mice were observed by light microscopy. Cysts were present in the diaphragms of the mice after 60 days post-infection. Parasite specific IgG, as well as IgG1 levels were determined in the sera of infected mice fed a normal diet. These levels were found to be lower in the 4-DPD-treated mice compared to the untreated mice. The inhibition started from the 10th day and continued to the 60th day, and in the 4-DPD-treated group the inhibition initiated after 24 h to 60 days. IgM level also was depressed by 4-DPD, starting from 24 h after injection of the compound. In mice fed Vitamin B6-deficient diets the levels of IgG were lower than in mice fed normal diets. These results show that BALB/c mice infected with T. spiralis and fed either a Vitamin B6-deficient diet or a diet which included the Vitamin B6-antagonist, 4-DPD, both influence the course of IgG, IgG1 and IgM production.

A simple method for the serodiagnosis of human hydatid disease based on a protein A/colloidal dye conjugate.

We have developed a dot immunobinding assay (CM) for the serodiagnosis of the human hydatidosis. The assay employs Samaron blue colloidal dye particles conjugated to hydatid antigen (HA) or protein A, both of which serve as visualizing agents. The test is simple and can be completed in 1 h. It requires few reagents, a nitrocellulose membrane (NC) strip onto which the HA has been bound, and the colored conjugates. It is sensitive and specific for the detection of anti-HA antibodies and generally agrees closely with the data from the enzyme-linked immunosorbent assay (ELISA).

Interleukin-1beta and interleukin-1alpha may affect the implantation rate of patients undergoing in vitro fertilization-embryo transfer.

OBJECTIVE: To investigate whether interleukin-1beta (IL-1beta) and interleukin-1alpha (IL-1alpha) affect the implantation rate of patients undergoing IVF-ET. DESIGN: Follicular fluid and serum were obtained on the day of hCG administration, the day of oocyte retrieval, and the day of embryo transfer. SETTING: Cellular immunology laboratory in a research institute, a high technology IVF unit in a medical center, and a university hospital. PATIENT(S): Thirty-three women who were undergoing IVF-ET. MAIN OUTCOME MEASURE(S): IL-1beta and IL-1alpha were measured by specific ELISA and their levels were correlated with the implantation rate. RESULT(S): Classification of IVF-ET patients according to their implantation rate revealed significantly higher amounts of follicular fluid IL-1beta in the implantation versus nonimplantation cycles (68.5+/-24.6 pg/mL versus 20.5+/-13.4 pg/mL); The difference between the level of IL-1alpha in the two groups was not statistically significant(11.6+/-5.1 pg/mL versus 7.3+/-1.9 pg/mL). In parallel, systemic FSH/hMG-dependent IL-1beta and IL-1alpha production was observed in implantation cycles but not in nonimplantation cycles. Statistically significant IL-1beta and IL-1alpha production was observed after administration of hCG. CONCLUSION(S): Gonadotropins used during IVF-ET induce local and systemic production of IL-1beta and IL-1alpha. In addition, the implantation rate for IVF-ET patients who have detectable serum concentrations of IL-1beta and IL-1beta on the day of hCG administration could be higher than the rate for IVF-ET patients who do not have detectable concentrations of these cytokines.

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.

Influence of Quillaja saponaria triterpenoid content on the immunomodulatory capacity of Epstein-Barr virus iscoms.

The immune responses to immunostimulating complexes (iscoms) containing recombinant Epstein-Barr virus (EBV) gp340 envelope protein was evaluated in BALB/c (H-2(d)) and CBA (H-2(k)) mice. Gp340-iscoms were used either with a low content of Quillaja triterpenoid adjuvant (L-iscoms) or supplemented with additional Quillaja adjuvant in the form of iscomatrix (S-iscoms). Class and subclass distribution of anti-gp340 antibodies, EBV-neutralizing antibodies, antigen-specific T cell proliferation and cytokine production were determined and these results compared to those obtained by immunization with non-adjuvated gp340. The H-2(d) and H-2(k) mice were characterized as low or high responders in respect to the level of specific anti-gp340 antibodies, secretion of IgG2a isotype, antigen-specific lymphoproliferative capacity, interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) production in the basic immunizations with gp340. While presentation of the antigen in iscom formulations with low levels of Quillaja triterpenoids induces a moderate enhancement of the immune responses in the low responder H-2(d) mice, supplementation with high levels of iscomatrix immunomodulator was required to enhance the immune responses in the high responder H-2(k) mice. In both mouse strains subcutaneous immunization with S-iscoms resulted in a significant increase of IgG1- and IgG2a-specific antibodies, as well as in strong antigen-specific proliferative response confirmed by the simultaneous cytokine production. The enhanced antigen-specific secretion of IL-2 and IFN-gamma together with the abrogation of IL-10 and the absence of IL-4 indicates that the responses were driven towards a Th1-type rather than Th2-type immune response. The S-iscom formulations minimized the differences in immune responses between the two mouse strains, but the capacity of immune sera to neutralize EBV transformation in vitro remained completely strain-dependent. These data indicate that immune responses generated by iscoms can be manipulated by altering the triterpenoid composition of the iscoms and that the levels of triterpenoids can determine whether or not a Th1-type response is made.