Virginia Iniesta

Arginase I induction in macrophages, triggered by Th2-type cytokines, supports the growth of intracellular Leishmania parasites

Leishmania spp. are intracellular protozoan parasites that invade and replicate within macrophages. In a previous report, we have demonstrated that the growth of intracellular amastigotes could be controlled by inhibition of arginase. This enzyme, induced in host cells by Th2 cytokines, synthesizes L‐ornithine which can be used by parasites to generate polyamines and proliferate. In this study, we have designed experiments to better analyse the dependence of parasite proliferation on arginase induction in infected macrophages. Treatment of Leishmania major‐infected BALB/c macrophages with interleukin (IL)‐4, IL‐10 or transforming growth factor‐β, which are all inducers of arginase I in murine macrophages, led to a proportional increase in the number of intracellular amastigotes. Moreover, parasite proliferation and arginase activity levels in macrophages from the susceptible BALB/c mice were significantly higher than those from infected C57BL/6 cells when treated with identical doses of these cytokines, indicating that a strong correlation exist between the permissibility of host cells to L. major infection and the induction of arginase I in macrophages. Specific inhibition of arginase by Nω‐hydroxy‐nor‐L‐arginine (nor‐LOHA) reverted growth, while L‐ornithine and putrescine promoted parasite proliferation, indicating that the parasite cell division depends critically on the level of L‐ornithine available in the host. Therefore, arginase induction in the context of a Th2 predominant response might be a contributor to susceptibility in leishmaniasis.

Arginase I Induction during Leishmania major Infection Mediates the Development of Disease

ABSTRACT In a previous work, we demonstrated that the induction of arginase I favored the replication of Leishmania inside macrophages. Now we have analyzed the differential expression of this enzyme in the mouse model of L. major infection. Ours results show that arginase I is induced in both susceptible and resistant mice during the development of the disease. However, in BALB/c-infected tissues, the induction of this protein parallels the time of infection, while in C57BL/6 mice, the enzyme is upregulated only during footpad swelling. The induction of the host arginase in both strains is mediated by the balance between interleukin-4 (IL-4) and IL-12 and opposite to nitric oxide synthase II expression. Moreover, inhibition of arginase reduces the number of parasites and delays disease outcome in BALB/c mice, while treatment with l-ornithine increases the susceptibility of C57BL/6 mice. Therefore, arginase I induction could be considered a marker of disease in leishmaniasis.

Immunohistological features of visceral leishmaniasis in BALB/c mice

It has been reported that the level of protection provided by vaccines against murine visceral leishmaniasis (VL) is low and that progress in research on VL may be due to the lack of appropriate models to study protective immunity. We have analysed the immunohistological features occurring in BALB/c mice after intravenous administration of 10 3 , 10 5 and 10 6 parasites of Leishmania infantum. Our results show that in all cases parasite administration leads to the establishment of infection and to the development of quantifiable immunohistological features which are dependent on the inoculum size. This study demonstrates that differences in the parasite challenge result in changes in the evolution of some of the parameters associated with the degree of the infection in the BALB/c model: level of anti‐Leishmania antibodies, up‐regulation of spleen arginase activity, balance between IFN‐γ and IL‐10, extent of lymphoid follicle depletion in the splenic white pulp and ineffective development of hepatic granulomas. Also, and depending on the initial infectious inoculum, the absence of parasites in the bone marrow and the number of mature and empty type granulomas were parameters associated with protection. We think that in this model a challenge of the order of 105 parasites should prove useful for vaccine studies against VL.

The Chimerical Multi-Component Q protein from Leishmania in the absence of adjuvant protects dogs against an experimental Leishmania infantum infection

The protective potential against Leishmania infection of the Leishmania chimerical Q protein administered as a single (Q) or double dose (Q+Q) without adjuvant was analyzed in a double-blind placebo controlled experiment in dogs. During vaccination the protein induced an intense early anti-Q response but no reactivity against total Leishmania infantum proteins was detected. Several end-points were taken into consideration. In the vaccinated animals the amount and intensity of clinical symptoms was lower than in the control group. Pathological signs of disease were observed in liver, kidney and spleen of all dogs from the control group in contrast to the normal appearance of the organs of the vaccinated animals. Vaccination was able to induce parasite clearance in most dogs. Only 1/7 dog was parasite DNA positive in skin in the Q group in contrast to 6/7 dogs in control and 4/7 in Q+Q. Significant anti-SLA clearance was observed in the vaccinated animals at the end of the study. Differences between control and vaccinated animals were also observed at the biochemical level, DTH and nitrite oxide production.

Detection of Leishmania infantum kinetoplast minicircle DNA by Real Time PCR in hair of dogs with leishmaniosis

It is known that hair can accumulate environmental toxics and excrete foreign chemical or biological substances. In this context, we hypothesized that foreign DNA could be found in the hair of an infected organism, and thus, be detected by Real Time PCR in the hair of Leishmania infantum naturally infected dogs. A population of 28 dogs living in Leishmania endemic areas was divided into two groups: A (13 Leishmania infected dogs) and B (15 healthy dogs). Blood, lymph node and ear hair samples from all of them were tested for the presence of parasite kinetoplast DNA (kDNA). For the same purpose, hair of several body areas and hair sections of two infected dogs were also analyzed. Epidermal keratinocytes from an infected animal were also analyzed for reactivity against Leishmania antigens by ELISA and for the presence of kDNA. Regarding to dogs from group A, parasite kDNA was detected in the 100% of lymph node samples. The sensitivity of Real Time PCR in ear hair was similar to that obtained in blood (9 positive out of 13 versus 8 positive out of 13, respectively). Moreover, the presence of L. infantum kDNA was also detected in the hair of all the analyzed body zones, in all hair sections and in epidermal keratinocytes. In infected dogs, parasite kDNA could be detected and quantified from just one single hair, whereas it was not detected in any of the samples of the healthy dogs. This work describes a new method for a reliable and non-invasive diagnosis of canine leishmaniosis using hair samples of infected animals. The data presented also provide some insights for the understanding of the physiology of keratinocytes and the role of hair as a specialized tissue in the kidnapping and removal of foreign DNA.

Experimental model for reproduction of canine visceral leishmaniosis by Leishmania infantum

In this report an experimental model of Leishmania infantum (L. infantum) infection in dogs is described. The data presented are derived from an overall and comparative analysis of the clinical outcomes of three groups of dogs intravenously infected with 500,000 promastigotes on different dates (2003, 2006 and 2008). The parasites used for challenge were isolated from a dog having a patent form of leishmaniosis, classified as MCAN/ES/1996/BCN150 zymodeme MON-1. Late-log-phase promastigote forms derived from cultured amastigotes obtained from the spleen of the heavily infected hamsters were used for infection. Only one single infective dose was administered to each dog. After challenge, the animals were monitored for 12 months. To analyze the disease outcome, several biopathological, immunological and parasitological end-points were considered. The analysis of the infected dogs indicated that the development of the clinical disease was very similar in the three experimental challenges, as shown by the immune response, the parasite load and the clinical and histopathological lesions detected at necropsy. A high similarity was also observed between the disease development after the experimental challenge and the one reported to occur in endemic natural infection areas, as various degrees of susceptibility to the disease and even resistance were observed in the experimentally infected animals. We believe that this challenge model faithfully reproduces and mimics the course of a natural infection and that it could be used as a suitable tool for analyzing the efficacy of anti-Leishmania drugs and vaccines.

First detection of Leishmania infantum kinetoplast DNA in hair of wild mammals: Application of qPCR method to determine potential parasite reservoirs

The data presented in this paper describe the application of a method for a reliable and non-invasive diagnosis of leishmaniosis in wild reservoirs, based on the detection of Leishmania infantum kinetoplast DNA (kDNA) in hair samples by Real Time PCR (qPCR). The study has been performed on 68 ear/leg hair samples from 5 different wild species (Vulpes vulpes, Canis lupus, Martes foina, Rattus norvegicus and Erinaceus europaeus) from several geographic areas of West and North Spain. The presence of Leishmania kDNA was detected in 14 of the 68 analyzed samples, being the highest quantity of DNA observed in foxes. This is the first report of the presence of Leishmania in a hedgehog. The kDNA remained stable under the exposure of hair to different environmental conditions (freezing or high temperature, ultraviolet rays or treatment with tanning salts). This detection method could constitute a suitable alternative for the search of the parasite in wild hosts, due to the numerous advantages that hair samples present for collection, transport and storage processes.

Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

Background Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Methodology/Principal Findings Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. Conclusion/Significance The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis.

Leishmania major infection in susceptible and resistant mice elicit a differential humoral response against a total soluble fraction and defined recombinant antigens of the parasite

In the present work, we analyzed the humoral response of Leishmania major experimentally infected BALB/c and C57BL/6 mice against three Leishmania antigens: total soluble antigen (soluble leishmania antigen(SLA)), a chimerical recombinant protein formed by the genetic fusion of four cytoplasmic proteins (PQ), and a kinetoplastic membrane protein (Kmp-11). We determined the correlation between the immune response against these proteins and the histopathological changes induced in the susceptible and resistant mice after infection. The data showed the existence of wide differences in the recognition of SLA, PQ, and Kmp-11 by the sera from both strains. The anti-SLA titer of BALB/c was 100 times higher than that of C57BL/6 mice. Antibodies against the recombinant Kmp-11 were detected only in infected BALB/c during the first stage of the infection. In contrast, the PQ protein was recognized by the sera from infected BALB/c mice but exclusively when they were in a late-lesion period. The data suggest that the response against the membrane Kmp-11 protein is transient and correlates with early developmental stages of the infection, whereas the response against cytoplasmic proteins as those present in PQ is sustained and could be considered as a marker of an advanced stage of the infection and disease.

Detection and chronology of parasitic kinetoplast DNA presence in hair of experimental Leishmania major infected BALB/c mice by Real Time PCR.

Hair can accumulate foreign chemical or biological substances. Recently, it has been reported that parasite DNA can also be detected in the hair of Leishmania infantum infected dogs. The aim of this work has been to find out whether parasite DNA incorporates in the hair of Leishmania major experimentally infected animals. For this purpose, a group of 4 BALB/c mice, intradermally inoculated in both ears with 1000 L. major V1 strain promastigote forms, was monitored for parameters associated to the infection during 35 days. Weekly, ear swelling was measured, and hair samples from ears and leg were collected. Blood samples were obtained before challenge and at day 35 post infection, when parasite load was measured in ear, lymph node and spleen by limit dilution. Ear swelling and other parameters observed in the infected mice were consistent with those described for this model. The presence of parasite kinetoplast DNA (kDNA) was detected by Real Time PCR in all ear and leg hair samples at the final timepoint. These data suggests that hair is a specialized tissue in the sequestration and removal of foreign DNA. Detection of DNA in hair could be, therefore, a useful tool to chronologically record the infection process during experimental mice assays.