Leishmaniosis
Manager: Jose María Requena Rolanía UAM-B
Leishmaniases are a series of diseases caused by different species of the genus Leishmania. Of the various clinical forms of the disease, visceral leishmaniasis is the severest and tends to prove fatal if not diagnosed and treated in time.
Leishmaniases are endemic in 88 countries across Africa, Asia, Europe, South and North America. A total of 12-14 million people are estimated to be currently affected by these parasitoses, with 1.5-2 million new cases every year. Furthermore, the data indicate that there is a rising trend in the incidence of these diseases owing to risk factors, such as HIV infection, malnutrition and mass migration. Currently, there is no effective prophylactic method and chemotherapeutic treatments do not have the desirable efficacy.
In Spain and other countries around the Mediterranean Basin, the predominant species is L. infantum, which mainly causes visceral leishmaniasis among those it infects. Apart from countries bordering the Mediterranean, this species is also to be found spread through West Africa, America, northern China and western Asia. Its main host is the dog, which acts as a reservoir for transmission to humans. Moreover, since the appearance and spread of AIDS, there has been a renewed outbreak of leishmaniasis incidence in humans, in view of the fact that L. infantum has been shown to be the main opportunistic parasite in these patients.
Along with the great amount of information being accumulated from knowledge of the genome sequence of several species of Leishmania, recent years have also witnessed the development of other molecular tools, which are enabling rapid advances to be made in understanding many unique aspects of the molecular and cellular biology of these organisms. Among these, mention should be made of proteomic techniques, which allow for large-scale analysis of proteins expressed at a specific moment in the life cycle of a given organism. Accordingly, the combination of bidimensional electrophoresis together with mass spectrometry has become a magnificent tool for ascertaining changes in the expression and rapid identification of proteins.
Consequently, the prospects of controlling such diseases will require the development of new research approaches aimed at furthering knowledge of this parasite's molecular biology, which will in turn enable new targets to be identified for diagnosis, treatment and development of vaccines.
Subprogram: Molecular Epidemiology, Diagnosis and Control
Manager: Jose María Requena Rolanía UAM-B
The taxonomic status of Leishmania species causing visceral leishmaniasis (LV) in East Africa is a controversial topic. Initially L. donovani, the cause of LV and post Kala-azar dermic leishmaniasis (PKDL), was fundamentally associated with an anthroponotic cycle, and L. infantum, the cause of cutaneous leishmaniasis (LC) and LV, was associated with a zoonotic cycle. Nevertheless, recent findings of L. donovani strains in dogs in the Sudan, along with new reviews of the philogeny of the donovani complex, make it necessary for these concepts to be revised. Control of the disease would vary substantially depending upon whether one was dealing with an anthroponosis or a zoonosis. Thus, aside from the L. donovani/L. infantum conflict, better knowledge of LV epidemiology on the Horn of Africa calls for markers that would enable more to be known about the strains circulating in the various epidemiological cycles, their geographical distribution/spread, the study of foci and outbreaks, as well as establishing which patients will develop PKDL.
Goals
1. Analysis of the genetic variability of isolates from the Sudan and Ethiopia of L. infantum/L. donovani.
2. Obtaining epidemiological markers for the study of outbreaks and identification of cycles
3. Obtaining markers of the progression of visceral leishmaniasis to post Kala-azar dermic leishmaniasis
4. Providing the WHO with a tool for control of leishmaniasis on the Horn of Africa
Genomic approach for the development of methods of diagnosis and control of leishmaniases
Head Researcher Jose María Requena UAM-B:
Despite the considerable incidence of leishmanioses among the human population world-wide, there is currently no vaccine against these parasitoses, and chemotherapeutic treatments lack the desirable efficacy. Furthermore, the appearance of strains resistant to the drugs in current use is beginning to be a serious problem. In the former Tropical Disease Co-operative Research Network (Red de Investigaciones de Enfermedades Tropicales-Ricet) genomic macroarrays of L. infantum were constructed, with these showing themselves to be very useful tools for analysing different aspects of this parasite's biology which can be exploited for the development of tools for the diagnosis and control of leishmaniases. In addition, the recent sequencing of the Leishmania genome has generated an immense amount of information, whch is a good complement for the genomic approach sought in this project.
Goals
1. The generation of genomic macroarrays of L. infantum in a sufficient amount for use by the different Groups.
2. Using genomic macroarrays to develop new diagnostic tools that enable the following to be determined: degree of virulence of strains, differences in parasite tropism in the host, and resistance to drugs.
3. Analysing: genes involved in folate metabolism as targets for the development of new drugs, changes in gene expression induced by treatment with new leishmanicidal substances, alterations in gene expression caused by leishmanicide peptides, effect on induced gene expression after insertion into Leishmania of L1Tc heterologous retrotransposon, and the implication of certain groups of genes in promastigote-amastigote differentiation in L. Infantum.
Applications of the experimental model of infection to control of canine leishmaniasis.
Head Researcher Javier Moreno, CIB-CSIC
The dog is the principal reservoir of L. infantum and plays a key role in transmission to humans, which is why effective control of zoonotic visceral leishmaniasis requires effective control of infection in the dog. Implementation of effective canine leishmaniasis control measures with impact on human incidence calls for the development of new diagnostic, therapeutic and prophylactic approaches and trials of these in the canine model.
Characterisation in the canine model of immune response against different recombinant proteins of L. infantum will enable their immunogenic capacity to be ascertained and their use for diagnosis, protection or immunomodulator role in L. infantum infection to be established.
Goals
1. Isolation and purification to homogeneity of Leishmania recombinant proteins.
2. Characterisation of humoral response against Leishmania infantum recombinant molecules. Usefulness for immunodiagnosis of the infection and its prognostic value in naturally- and experimentally-infected dogs.
3. Analysis of the cellular response induced by L. infantum recombinant molecules, by means of in vitro lymphoproliferation assay . Determination of the profile of cytokines induced by recombinant proteins.
4. Study of the immunomodulator capacity of L. infantum recombinant molecules. Effects on innate immunity
Subprogram: Leishmania treatment
Manager: Francisco Gamarro Conde IPBLN-C
Within the initiative of a co-ordinated project, the present proposal seeks to establish and characterise the mechanism of action of some of these compounds, characterise the mechanisms of resistance that Leishmania develops against the most promising leishmanicidal drugs and antibiotic peptides, and study epidemiological markers of tropism and virulence in leishmaniasis. All of this will lead to the design of new drugs and to the availability of drug-resistance, tropism and virulence markers which will ultimately facilitate control of this parasitic disease. Treatment of leishmaniasis with pentavalent antimony compounds is not only toxic, but is also increasingly meeting with the appearance of resistance. Alternative therapy with amphotericin B formulations is toxic and costly. The problem is becoming more acute due to a progressive increase in HIV coinfections, in which case the treatment ceases to be effective. Recently, India approved oral treatment with alkyl phospholipid miltefosine (hexadecylphosphocholine), which displays great efficacy and could be used on HIV-coinfected patients. Leishmania, like other organisms, may develop drug-resistance processes, due mainly to overexpression or mutation of the protein targeted for drug action, or reduction in drug accumulation owing to expression of ABC transporter proteins that act as efflux pumps. The study of the proteomic patterns of parasites subjected to the presence of new and potential leishmanicidal drugs or resistant lines, will enable identification of possible therapeutic targets as well as the mechanisms whereby such parasites can become resistant. Similarly, this analysis with enable factors of virulence and tropism to be selected on the basis of the proteomic study of L. infantum promastigotes belonging to dermotropic and viscerotropic zymodemes isolated from HIV-coinfected patients and canine leishmaniasis. This will lead in future to the rational design of new drugs, as well as the use of alternative treatment strategies in cases of appearance of resistance and control of the disease.
Goal
The project's main goal is to identify new therapeutic targets in the Leishmania parasite, ascertain the mechanisms of resistance to leishmanicidal drugs and new antibiotic peptides, and determine the factors of virulence and tropism by application of proteomics.
New alternatives in Leishmania chemotherapy
Head Researcher: Luis Rivas, CIB-CSIC
At present, Leishmania chemotherapy is the only means of treating this condition; it is, however, confronted by the high rate of cytotoxic effects inherent in the majority of leishmanicidal drugs, except liposomal amphotericin B and miltefosine, and a growing incidence of resistence. The final consequence is the need to develop new drugs that: i).- have a high therapeutic index; ii).-have the possibility of low-cost production; iii).-have a low induction of resistance; iv) rely on new targets; and v).-ideally, have additional activity on parasites other than Leishmania.
Within the context of RICET's co-operative project, possible alternatives to these problems will be addressed in accordance with: i).-availability of banks of natural products with untested activities; ii) synthetic capacity for development and/or modification of drugs, as well as the existence of a chemobank; iii).- development of eukaryotic antimicrobial peptides (EAPs) active in membrane. These peptides, derived from components of innate immunity, are highly cationic and have a capacity for adoption of amphipathic structures. As a new mechanism of action, they exploit the destructuring of the pathogen's plasma membrane, by stoichiometric preferential interaction with anionic phospholipids exposed on the external leaflet of the plasma membrane, a characteristic common to lower (protozoa, fungi) but not higher prokaryotes and eukaryotes, with dissipation of ionic gradients and death of the parasite. This mechanism renders induction of resistance improbable, on requiring a drastic change in the composition of the phospholipids that simultaneously affect all the enzyme and transport systems located therein.
In order to achieve the most effective screening possible of drugs and targets alike, we propose the development of a collection of Leishmania strains with altered gene expression for the limiting steps of metabolic pathways and signal transduction, which would allow for a general definition of the targets implicated for a given drug or, vice-versa, focus the obtaining of drugs for a given target.
Current availability of leishmanicidal lead molecules with tried and tested in vitro and in vivo action, and the obtaining of new design and synthesis agents will, moreover, allow for testing on other parasites targeted by other Network projects. Reciprocally, this project will benefit from the development of bioactive compounds developed in the Network for other pathogens, and for trypanosomatidae in particular. For instance, mention could be made of the fact that the original development of amphotericin B and miltefosine was as antifungicidal and antineoplastic agents respectively.
Goals
1.- Screening of new compounds active in Leishmania.
2.- Development of leishmanicidal antibiotic peptides active in membrane, both by design and from natural peptide sequences.
3.- Development of a collection of Leishmania parasites with altered, drug-targeted gene expression
Immunomodulators in leishmaniasis therapy.
Head Researcher Basilio Valladares, IUETSPC
Treatment of leishmaniasis is an as yet unresolved issue. Since the 1950s drugs have been -and indeed are still being- used, which require parenteral administration and prolonged treatments, and, in addition, give rise to a series of adverse reactions that hamper their use (cardiotoxicity with abnormal electrocardiograms, pancreatitis, etc.). There are active ingredients of plant and synthetic origin with promising leishmanicidal activity and low toxicity (peptides, synthetic organic molecules, etc.) but, until now, they have not, in themselves, been capable of totally eliminating the parasite. Furthermore, modulation of the immune system towards a type-TH1 cellular response is known to favour the death of these protozoa. Likewise, nitric oxide stimulation contributes to the death of the protozoa.
We are seeking to use active ingredients of proven leishmanicidal activity, which have been identified at various laboratories, in conjunction with proteins of Leishmania sp. which, in the form of recombinant proteins, have been shown to induce a type-TH1 immunological reponse. The sum of the two actions could eliminate the parasite, thereby bringing about the cure of the host.
Goals
1. Expression and purification of recombinant proteins which have been obtained from Leishmania sp. and have shown immunomodulation with induction of a TH1 response and production of nitric oxide.
2. The search for and isolation, purification and synthesis of molecules that have shown in vitro leishmanicidal activity.
3. Assessment of the leishmanicidal activity in experimental animals of joint administration of immunomodulators and chemical substances obtained above.
4. The search for and isolation, expression, purification and study of new molecules with immunomodulator activity.
Subprogram: Development of vaccine candidate molecules
Manager: Manuel Carlos López López IPBLN-A
Control of leishmaniasis will require induction of a multiple immune response that integrates cellular activation and production of antibodies with a certain balance towards a type 1 response, of differing intensity depending on the species of Leishmania and type of leishmaniasis generated. It is postulated that molecules capable of inducing protection against infection by this parasite ought to carry different antigenic components of the parasite, duly selected for their immune capacity, as well as immunomodulators capable of boosting and/or directing the host's immune response towards a specific protective profile. Hence, immunological characterisation and analysis of the protective capacity against Leishmania infection of certain antigenic proteins of the parasite or fragments thereof associated with immunomodulators in the context of DNA molecules and/or chimera recombinant proteins, will be essential in order to obtain molecules capable of combating and/or preventing the infection in question.
Goal:
we are seeking to design and obtain chimera molecules carrying antigens of the Leishmania parasite and, in murine experimentation systems (mice of different haplotype and susceptibility to visceral and cutaneous leishmaniasis in hamsters), to study their immunogenicity and capacity to induce protection against infection by this parasite.
