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http://www.elsevier.de/protisPublished online date 26 January 2009

& 2008 Elsevdoi:10.1016/j

160, 151—158, May 2009

Protist, Vol.

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Meeting Report IFoLeish-2008: Current Status andFuture Challenges in Leishmania Research andLeishmaniasis

Introduction

Despite progress in the diagnosis and treatment,leishmaniasis remains a major public healthproblem particularly in developing countries. Theimpact of leishmaniasis on human health has beengrossly underestimated for many years, and it hasnow been classified by the World Health Organi-zation (WHO) as one of the most neglectedtropical diseases. During the last decade, ende-mic areas have been spreading and a sharpincrease in the number of leishmaniasis caseshas been recorded. At present, an estimated12 million people are infected in 88 countries(72 developing and 16 developed). Two millionclinical cases of leishmaniasis are assumed tooccur annually with 1.5 million cases of self-healing but disfiguring cutaneous leishmaniasis(CL), and 500 000 cases of life-threatening visceralleishmaniasis (Kala-Azar; VL). At least 60 000people succumb to VL each year and a loss of2.4 million disability-adjusted life years (DALYs)has been calculated as the total disease burden ofleishmaniasis (Hotez et al. 2004).

The numerous medical problems caused byleishmaniasis are in sharp contrast to the com-prehensive understanding of the cellular andmolecular biology of Leishmania parasites and oftheir interactions with the hosts. A wealth of dataon the genomes, transcriptomes and proteomesof representative Leishmania species have beenaccumulated and methods have been developedthat allow the rapid generation of transgenicparasites for phenotypic and pathogenic analyses.A major challenge thus appears to be thetranslation of the results of basic science intofeasible and affordable strategies for a bettercontrol of leishmaniasis and a reduction ofthe disease burden. In order to foster such atranslational approach in Leishmania research,the Interdisciplinary Forum on Leishmaniasis —

ier GmbH. All rights reserved..protis.2008.12.001

IFoLeish-2008 — was held at the Institute forHygiene and Medical Microbiology of theUniversity of Heidelberg, Germany, from 3 to5 April 2008. Scientists and clinicians with long-standing expertise in leishmaniasis and tropicalmedicine fuelled an exchange of their views on thecurrent status and future prospects of researchdedicated to the prophylaxis and treatment ofLeishmania infections. The participants of themeeting concordantly agreed that continuousinterdisciplinary efforts are crucial to translateour detailed knowledge of the parasite, the vectorbiology and the pathogenesis of disease intoimproved control measures and the developmentof vaccines against one of the major parasiticdiseases of humans. The IFoLeish-2008 meetingwas organized by the German Society of Hygieneand Microbiology, section ‘Eukaryotic Pathogens’,in collaboration with the German Society forParasitology.

Diagnosis and Clinical Disease

The genus Leishmania comprises approximately20 different species that can cause humandisease. Although three major clinical forms canbe distinguished, i.e. cutaneous, mucocutaneousand visceral leishmaniasis, a broad spectrumof different clinical symptoms including severalrare manifestations can occur, thereby hinderingclinical diagnosis (Gerd Burchard, Bernhard NochtInstitute for Tropical Medicine, Hamburg,Germany). Fever, hepatosplenomegaly and pan-cytopenia are typical symptoms of overt VL, butgastrointestinal, renal and neurological disorderscan also occur. The situation is further compli-cated by a low manifestation index of VL ofapproximately 10% which allows reactivation of

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sub-clinical infections after immunosuppression,e.g. in HIV patients or transplant recipients.Alternatively, overt disease can result from anti-retroviral therapy (ART) in Leishmania/HIV-co-infected patients due to an Immune ReconstitutionInflammatory Syndrome (IRIS). The number ofsuch cases is low but may increase in the futurewith a broader access to ART in countries withlimited health resources. In contrast to VL,cutaneous leishmaniasis (CL) is normally a self-limiting skin disease, although visceral manifesta-tion due to L. major can occur. Skin lesions candiffer markedly in morphology and localization,and in HIV-co-infected patients, relapses andmulti-focal skin lesions that respond poorly totreatment are common. CL due to L. braziliensiscan further develop to destructive and life-threa-tening mucocutaneous leishmaniasis (MCL).Unfortunately, risk factors for developing MCLare largely unknown, but their identification wouldbe highly beneficial for therapeutic prevention ofMCL.

A chronic dermal complication that arises as asequel of VL due to L. donovani in India, EastAfrica and China is Post Kala-Azar DermalLeishmaniasis (PKDL). Importantly, the manifesta-tion rate depends on the parasite strain with5—15% or 55—60% of Indian or Sudanesepatients, respectively, developing PKDL aftersuccessful treatment of VL (Poonam Salotra,Institute of Pathology, Safdarjung Hospital Cam-pus, New Delhi, India). The disease manifests invarious clinical forms from hypopigmentedmacules to infiltrated nodules or ulceration thatis particularly common in Africa. Reactivation ofpersistent Leishmania infection rather then re-infection is considered to underlie PKDL develop-ment and is an important issue in the molecularbasis of manifestation. Transcriptome analyses ofL. donovani RNA isolated from VL and PKDLpatients identified 3% of differentially expressedgenes with gp63, PSA-2, amastin- and calpain-like protein being up-regulated during PKDL.Although their contribution remains to be func-tionally resolved, they represent candidate genesthat may affect parasite persistence and clinicalmanifestation during PKDL. Beside parasite fac-tors, the systemic and local immune responsesplay a crucial role. Indeed, TNF-a serum levels aswell as transcripts of IFN-g, TNF-a and IL-6 in theaffected tissue were specifically elevated in IndianPKDL but not VL patients and normalized follow-ing treatment. This indicates that expression of adistinct mixture of cytokines may contribute to thepathogenesis of PKDL although differences in

cytokine responses of Indian and Sudanesepatients have been identified.

As discussed above, the heterogeneity ofclinical manifestations after Leishmania infectionshampers clinical diagnosis. Furthermore, all Leish-mania species are morphologically identical andcan thus not be distinguished by microscopy. Aspointed out by Henk Schallig (Royal TropicalInstitute, Amsterdam, The Netherlands), speciesidentification is crucial for adequate treatment,since development of secondary drug resistancediffers between species. Isoenzyme analysis,microsatellite analysis, or PCR in combinationwith restriction fragment length polymorphism(RFLP) analysis can be used. These techniques,however, are often not applicable in the countrieswhere leishmaniasis is most prevalent. Nucleicacid sequence-based amplification (NASBA) ofthe 18S rRNA has been recently developed as anovel promising approach. Although it does notyet allow differentiation of Leishmania species, itnevertheless detects active Leishmania infectionswith high specificity and sensitivity. Since it isbased on the amplification of RNA and parasitescan also be quantitated (QT-NASBA), it is particu-larly useful to monitor disease progression or theoutcome of anti-leishmanial therapy. The isother-mal amplification does not require sophisticatedequipment and is therefore much easier applic-able in most endemic countries than conventionalPCR or RT-PCR. The detection of NASBA ampli-cons on an oligochromatography (OC) dipstickthrough hybridization with a gold-conjugatedprobe is a further significant step towards asensitive and specific diagnosis of leishmaniasisin developing countries and is now ready forevaluation in tropical areas.

Epidemiology

In order to determine the impact of differentgenotypes on the course of infection and/or thehost range, to identify genotypic traits of drug-resistant parasites, to analyse the dynamics ofpopulation structures and the origin of newgenotypes, and for surveillance purposes, thedifferentiation of Leishmania at the level of para-site strains is required. Multilocus enzyme electro-phoresis (MLEE) is the gold standard for taxonomyand strain typing, but has several theoretical andpractical drawbacks. Gabriele Schonian (Charite-University Medical Center, Berlin, Germany) sug-gested the analysis of multiple highly polymorphicand evolutionary neutral DNA repeats (multilocus

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microsatellite typing, MLMT) as a fast, reliable,and powerful alternative. Suitable markers arenow available for several Leishmania species.Analyses of 91 strains of the L. donovani complexfrom different endemic areas revealed 6 majorpopulations with different geographic distribu-tions. Importantly, the heterogeneity of thesepopulations differed considerably between geo-graphic areas, with the Mediterranean populationbeing the most diverse. Typing of 141 EuropeanL. infantum strains proved the high discriminationpotential of MLMT, since it recognized one non-MON-1 (MON: zymodemes typed by the Labor-atoire d’Ecologie Medicale et Pathologie Para-sitaire, Montpellier) and three distinct MON-1subpopulations. Although these populationsshowed predominantly clonal structures, geneflow even between non-MON-1 and MON-1populations became evident. Unfortunately, dis-tinct genotypes of L. infantum did not correlatewith host range or clinical outcome. In addition,identification of drug-resistant L. donovani strainsby MLMT also failed. This may be due to drugresistance rather relying on single point mutationsthat can only be identified by genome sequencingefforts but not by MLMT.

Since 15 years, the geographical distribution ofleishmaniasis has expanded significantly andcertain risk factors, including migration, urbaniza-tion, and increased susceptibility due to immuno-suppression and malnutrition, certainly haveplayed important roles. Another important issueis the impact of climate changes, including globalwarming, on the spreading of the sand fly vectorsand the emergence of autochthonous leishmania-sis in formerly non-endemic regions. Paul Ready(Natural History Museum, London, UK) pointedout that novel endemic foci of canine L. infantuminfections undoubtedly emerged in northern Italy.In addition, a survey among French veterinariansalso suggested spreading of canine leishmaniasisto northern France, although it remains unclearhow many of these cases are really due toautochthonous transmission. A high-resolutionspatial modelling identified the local habitat,urbanization, land use, temperature and otherclimatic parameters as the most critical factors forthe distribution of sand flies and endemic diseasein a model region in southwest France. Impor-tantly, a combination of environmental and cli-matic variables rather than a single factor as, forexample, increasing temperatures, determines therisk of spreading of endemic leishmaniasis intoformerly non-affected areas. The risk of a generalemergence of autochthonous L. infantum infec-

tions in central or northern Europe thus appears tobe limited. It should be mentioned, however, thatcases of leishmaniasis have been clearly docu-mented in humans and dogs from central Europewithout any history of travelling into endemicareas. In addition, clinicians from non-endemicareas should routinely consider Leishmania asetiological agent of diseases, mostly skin lesionsin travellers returning from tropical or temperateareas (August Stich, Medical Mission Institute,Wurzburg, Germany). For example, approximately100 cases are diagnosed and registered each yearin Germany. Since the manifestation index islow and many cases may remain unrecognized,the actual number, however, is considered to bemuch higher.

Pathogenomics and Postgenomics

During transmission from the invertebrate vectorto the mammalian host, Leishmania parasiteshave to adapt to distinct environmental condi-tions. However, Leishmania and other kinetoplas-tids are unusual in the developmental regulation ofgene expression. In these parasites, arrays ofopen reading frames are normally constitutivelytranscribed into polycistronic RNA precursors thatare further processed into individual mRNAs bytrans-splicing of a leader sequence bearing atrimethyl cap at the 50-end (Spliced Leader, SL)and polyadenylation at the 30-end. Regulation ofgene expression thus has to be achieved post-transcriptionally and RNA degradation has beenidentified to be crucial (Christine Clayton, Centerfor Molecular Biology, University of Heidelberg,Germany). This involves the sequential dead-enylation of the poly A-tail by the CAF1/NOTcomplex, removal of the 50-cap by a yet unidenti-fied decapping protein and subsequent RNAdegradation by a 30-exosome and 50—30 exonu-cleases. Alternatively, developmentally unwantedor misfolded mRNAs that are destabilized byregulatory factors are rapidly decapped anddegraded by the 50—30 exonuclease XRNA follow-ing exit from the nucleus. Importantly, the compo-sition and crystal structure of the CAF1/NOTdeadenylase complex and the Leishmania exo-some differ considerably from their mammalianhomologues. Therefore, and since post-transcrip-tional control of gene expression is so critical forregulated gene expression, the RNA degradationmachinery and its regulation may prove apromising drug target to combat Leishmania andrelated parasites.

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Besides differential expression due to RNAdegradation, developmental differentiation ofLeishmania also involves post-translational mod-ifications. Gerald Spath (Institut Pasteur, Paris,France) presented phospho-proteomic analysesof L. donovani promastigotes and amastigotes.Quantitative 2-D difference gel electrophoresis(DIGE) identified 94 distinct Leishmania phospho-proteins of known function that were predomi-nantly categorized to ubiquitin-dependent proteindegradation, protein refolding and translationinitiation. This suggests that protein synthesis,stability and degradation in Leishmania criticallydepend on phosphorylation-dependent regulatorynetworks. Remarkably, more than 35% of thephosphoproteins of known or unknown functionwere differentially regulated between promasti-gotes and amastigotes. Among them, chaper-ones, including components of a putative HSP90/foldosome complex, were particularly abundantand were shown to be differentially phosphory-lated in the amastigote stage. Together, these dataindicate that phosphorylation may be criticalduring developmental differentiation of Leishmaniaand must be regulated by stage-specific kinasesand/or phosphatases of chaperone substrates.However, chaperones may not only become clientproteins of kinases during promastigote-to-amas-tigote transition but can themselves also influencethe phospho-proteome of Leishmania as sug-gested by reduced protein phosphorylationfollowing pharmacological inhibition of HSP90(Joachim Clos, Bernhard Nocht Institute forTropical Medicine, Hamburg, Germany). Thus, acomplex network of phosphorylation/dephosphor-ylation events of and by chaperones may establisha regulatory pathway that — among other factors— governs developmental differentiation in Leish-mania.

Heat-shock proteins are stress-induced chaper-ones several of which are up-regulated upontransmission of Leishmania from the insect vectorto the warm-blooded mammalian host. They areable to disassemble complexes of denaturatedproteins that can subsequently be refolded.HSP100 is strongly up-regulated in the amastigotestage. Importantly, Leishmania mutants that lackHSP100 fail to proliferate inside macrophages andare essentially avirulent in mice but grow normallyas promastigotes or axenic amastigotes (JoachimClos). Although the exact function is not yetknown, HSP100 obviously stabilizes the amasti-gote stage inside macrophages and this mayagain be regulated in a phosphorylation-depen-dent manner.

The surfaces of Leishmania promastigotesand amastigotes are covered with various glyco-conjugates including lipophosphoglycan (LPG),glycoinositolphopholipids (GIPLs), glycosylpho-sphatidylinositol (GPI)-anchored proteins, andglycosphingolipids. Since they are at the interfaceof the parasite and its insect or mammalian hosts,they are considered to be of prime importance forthe host—parasite interplay. However, the impactof individual classes of glycoconjugates on thecourse of leishmaniasis is difficult to unravel.Galactofuranose is a monosaccharide that isabsent in vertebrates but present in L. majorLPG and GIPLs (Franc-oise Routier, HannoverMedical School, Germany). Deletion of glf, i.e.the gene encoding the UDP-galactopyranosemutase that catalyzes the formation of galactofur-anose, did not impair the viability of L. major,although LPG was absent and GIPLs weretruncated. In vivo, the virulence of the mutantwas nevertheless attenuated with a clearlydelayed onset of pathology. Interestingly, thelesion sizes did not differ between mutants andwild-type parasites indicating that galactofura-nose is critical for establishing the infection bypromastigotes but not for amastigote survival andreplication. This is reminiscent of LPG-deficientL. major, and shows that GIPLs of amastigotes(and possibly also promastigotes) are obviouslynot essential for parasite pathogenesis.

Immune Response and its Impact onClinical Outcome

Beside the Leishmania species, the host’s immuneresponse largely determines the course of infec-tion with predominant Th1-type reactivities beingable to control the pathogen. Dendritic cells (DC)are central for antigen presentation and priming ofnaıve T cells. Uwe Ritter (University of Regens-burg, Germany) dissected the role of DC subtypesin the initiation of an adaptive immune response ina mouse model of CL. Surprisingly, epidermalLangerhans cells appear to be dispensable forinitiating the adaptive immune response againstL. major and parasite control. They rather lead tothe initial priming and expansion of CD8+ T cellsthat — during prolonged infection — diminish theIFN-g response and dampen anti-parasitic effec-tor responses in the dermis. In contrast, dermalDCs are instrumental in the migration to skin-draining lymph nodes and the priming of CD4+ Tcells that subsequently activate dermal macro-phages to kill the parasite. It has to be stressed

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that depletion of Langerin-positive cells includingLangerhans cells nevertheless led to increasedparasite loads at the site of infection.

The cytokine milieu is critical for the develop-ment of Th1-type innate and adaptive immuneresponses that are instrumental in controllingLeishmania. Type-I interferons (IFN-a/b) haverecently been implicated in the regulation of Th1-type innate immune responses against Leishmaniathrough activation of NK cells. Christian Bogdan(University of Erlangen, Germany) clarified theregulation and function of type-I IFNs duringcutaneous and visceral murine leishmaniasis.Plasmacytoid DC (pDC) but not myeloid DC(mDC) secretes high levels of IFN-a/b in responseto Leishmania or Leishmania DNA in a TLR9-,MyD88- and IFN-a/b receptor (IFNAR)-dependentmanner whereas both DC subtypes secrete IL-12.Furthermore, expression of type-I interferons isup-regulated in vivo after infection of mice withL. major or L. infantum. Importantly, followingLeishmania infection, IFNAR-deficient mice arenevertheless able to activate NK cells for IFN-gproduction and to resolve cutaneous lesionscaused by L. major. Thus, while NK cell activitydoes not rely on IFNAR-mediated responses, itclearly depends on mDC-derived IL-12 and TLR9-mediated signalling during both visceral andcutaneous leishmaniasis.

It has long been recognized that DC-derivedcytokines also regulate the differentiation ofT-helper subsets which are decisive for theadaptive immunity during cutaneous leishmania-sis. IL-12 is the prototype of cytokines required fordevelopment of protective Th1 lymphocytes, but avariety of other cytokines emerged that alsoinstructs Th polarization in cutaneous leishmania-sis (Esther von Stebut-Borschitz, University ofMainz, Germany). DCs from infected BALB/c miceproduce less IL-1a/b and more IL-12p40 homo-dimers (IL-12p80) than those from infectedC57BL/6 mice. Administration of IL-1a protectsBALB/c mice from progressive CL and subse-quent death in an IL-12p70-dependent manner.Importantly, the beneficial influence of IL-1a/b isrestricted to the first days of infection, while thecytokine leads to the differentiation of Th2 ratherthan Th1 cells during prolonged infection. Incontrast, binding of IL-12p80 to the IL-12 receptordeteriorates the clinical outcome of CL by block-ing IL-12 receptor signalling and promoting aTh2-biased Th population. Another importantsource of IL-12 receptor-blocking IL-12p80 maybe BALB/c neutrophils (Franc-oise Tacchini-Cot-tier, World Health Organization (WHO) Immunol-

ogy Research and Training Center, Epalinges,Switzerland). Recently, the Th1/Th2 paradigmhas been modified by the detection of additionalTh subsets including the Th17 lineage. Esther vonStebut-Borschitz provided evidence of increasedIL-17 and IL-23p19 levels in BALB/c mice indica-tive for expansion of Th17 cells. Importantly,genetic deletion of IL-17A conferred partial pro-tection against L. major and progressive diseasein otherwise susceptible BALB/c mice. Protectionin IL-17A�/� mice correlated with reduced infiltra-tion of antigen-presenting cells and polymorpho-nuclear neutrophils (PMN) into the infected tissueindicating an immunomodulatory role of thesecells at the site of infection.

High numbers of persisting neutrophils haveindeed been described in CL lesions of suscep-tible BALB/c mice whereas they are cleared inresistant C57BL/6 mice (Franc-oise Tacchini-Cot-tier). Loss of PMNs in C57BL/6 mice is achievedby contact-dependent macrophage-inducedapoptosis. Importantly, such apoptotic cell deathis amplified by L. major and, therefore, occurspreferentially at the site of infection. It relies on theexpression of trans-membrane TNF on macro-phages thus indicating a role of surface TNF inthe early control of the inflammatory response toL. major. However, PMNs also fulfil critical roles inshaping the adaptive response and as effectorcells that help to control both L. major andL. donovani. Depletion of PMNs in L. donovani-infected mice leads to increased parasite burdenand exacerbation of disease due to a predominantTh2 response. Furthermore, resistance against CLis associated with expression of high levelsof TLR2, TLR7, TLR9, and signalling-competentIL-12, but low levels of inhibitory IL-12p80(see above) and TGF-b by murine neutrophils inresponse to L. major. Thus, neutrophils may fulfilboth beneficial and detrimental roles in controllingthe inflammatory and adaptive responses toLeishmania infection and this likely depends onthe time of infection and the PMN phenotype.

Eukaryotic parasites such as Leishmania haveevolved elaborate mechanisms to partially evadeimmune responses and to ensure growth andeventually persistence in the host. L. major andpossibly also other Leishmania species hijackPMNs to gain entry into appropriate host cells(Tamas Laskay, University of Lubeck, Germany).Phagocytic neutrophils are certainly well suited ashost cells for Leishmania early during infectionsince they rapidly colonize the infected tissue.However, Leishmania also face certain problemswithin these cells. First, PMNs are short-lived cells

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that rapidly die via apoptosis. In order to counter-act host cell death, L. major delay the apoptoticprogram of neutrophils. Second, the parasite hasto gain entry into macrophages in order todifferentiate into the amastigote stage and toallow sustained proliferation. To this end, the anti-apoptotic activity of L. major ceases duringprolonged infection and apoptotic PMNs contain-ing viable promastigotes are readily engulfed bymacrophages. Remarkably, uptake of apoptoticPMNs harboring a parasite cargo leads to auto-crine or paracrine TGF-b-mediated silencing ofmacrophages and a productive infection withLeishmania. In addition, apoptotic PMNs contain-ing Leishmania parasites also inhibit the inflam-matory response of and can be phagocytosedby neutrophils. These findings have led to theconcept of neutrophils as ‘Trojan horses’ forL. major. More recently, it has been shown thatTGF-b-mediated silencing of phagocytes is alsoconferred by stationary-phase promastigotesthemselves that — unexpectedly — contain ahigh proportion of parasites with an apoptotic-likephenotype. Importantly, depletion of the ‘apopto-tic’ parasites abrogate the pathogenic potential ofa virulent L. major population indicating that analtruistic ‘suicide’ of a subset of promastigotesfacilitate the intracellular survival of viable para-sites and promotes productive infection. Thus,apoptotic PMNs as ‘Trojan horses’ and promas-tigotes with an apoptotic-like death phenotypeboth dampen the anti-parasitic inflammatoryresponse at the site of L. major-infection.

Therapy and Prevention

A detailed map of the antigenicity of Leishmaniahas provided valuable insights into parasite—hostinteractions and the prospects of effective vac-cines and new sensitive immunodiagnosticdevices. Using a proteome-serological approachwith sera from VL patients from a highly endemicarea in India, Peter Walden (Charite — UniversityMedical Center, Berlin, Germany) identified 4500different antigens out of 1200 protein spots thatwere reproducibly silver-stained in 2-D gels.Importantly, serological responses to L. donovanidiffered extensively between patients and immu-nodominant antigens could not be identified.Furthermore, parasite isolates from different fociin a district of the Indian state of Bihar displayeddiverse antigenicities that could be partiallyattributed to antigenic drift towards parasitestrains that are non-immunogenic in a given VL

patient. Despite the large heterogeneity of serolo-gical responses and antigenicities, a number ofshared L. donovani antigens have been recog-nized that could be instrumental in the develop-ment of multi-subunit diagnostic reagents orvaccines. Nevertheless, due to this heterogeneityand the mostly intracellular localization of Leish-mania in the mammalian host, T-cell antigens maybe more promising vaccine candidates. Kineto-plastid membrane protein (kmp)-11 of L. donovaniwas proven to be such a candidate since it bears ahigh density of immunogenic CD8+ and CD4+

T-cell epitopes. In addition, it is processed andpresented by Leishmania-infected macrophagesin the context of different HLA class I allomorphswith different sequence preferences thus suggest-ing that it may induce CD8+ T-cell responses inpatients with different HLA specificities. It will beinteresting to see whether T-cell reactivities of anextended cohort of VL patients confirm thesuitability of this antigen as a potential T-cellvaccine.

The route of antigen delivery certainly has amajor impact on the efficacy of putative anti-Leishmania vaccines. Heidrun Moll (University ofWurzburg, Germany) presented promising data onexperimental vaccination of mice with Leishmaniaantigen-pulsed DC. Although several DC subpo-pulations, including Langerhans cells, mDC andpDC that have been pulsed ex vivo with Leishma-nia lysate or distinct antigens, are capable toinduce a robust and long-lasting immunity againstchallenge with L. major, the requirements forprotection clearly differ. For example, Leishmaniaantigen-pulsed mDC, but not pDC, only protect ifthey are co-activated ex vivo with the TLR9 ligandCpG. In addition, the protective effect of antigen-pulsed mDC depends on the capacity to induce apolarized Th1 response in the recipient micewhereas this is dispensable for protection bypDC. The protection conferred by pulsed mDCrelies on the activation of resident DC and thismay be achieved via the cross-activation of NKcells in the recipient in a cell contact-dependentmanner. Together, the results show that DC mayindeed be suitable to manipulate the host’simmune response by either therapeutic vaccina-tion with antigen-pulsed autologous DC or —more feasible — by in situ targeting of DC forimmunoprophylaxis. Although several criticalparameters have been unravelled, further workhas to be done to develop efficient strategies forDC targeting in tissues.

During the last 2—3 decades, the efficacy ofantimonial therapy of visceral leishmaniasis due to

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L. donovani decreased considerably. The alterna-tive drug, amphotericin B, also requires hospitali-zation of patients and is quite toxic. Therefore,novel drugs are urgently needed. An importantlandmark in the chemotherapy of leishmaniasiswas the development of oral miltefosine, analkylphosphocholine (Shyam Sundar, Institute ofMedical Sciences, Varanasi, India). Indeed, 100 mgoral miltefosine per day for 28 days (X25 kg bodyweight) led to long-term cure rates of 94% thusbeing comparable with parenterally administeredamphotericin B. Miltesosine is better toleratedthan amphotericin B, although limited vomiting ordiarrhea still occurs in 40% and 20% of miltefo-sine-treated adults, respectively. The drug can alsobe given to children (2.5 mg/kg for 28 days) withthe same efficacy and less adverse effects than inadults but is contraindicated during pregnancy.However, despite these promising data, thenecessity of a 4-week regimen, the uncontrolledavailability of the drug, relatively high costs, andthe discontinuation of therapy in 20—33% ofpatients largely increase the risk of rapid emer-gence of resistant L. donovani strains. Importantly,a recent phase II combination trial indicated ahigher cure rate of VL by the combination ofmiltefosine with liposomal amphotericin B (Ambi-some) for only 7 days as compared to mono-therapy with either Ambisome or Miltefosine for 28days. Consequently, a combination therapy includ-ing miltefosine may increase the compliance,thereby reducing the discontinuation rate and theemergence of resistant parasite strains.

Efficient vector control measures may reduce oreven prevent the transmission of Leishmaniaparasites by sand flies, thereby avoiding thenecessity of chemotherapy or vaccination. Dueto the terrestrial life style of phlebotomine larvaeand dispersed breeding places in domestic,peridomestic and sylvatic environments, controlis only feasible for the population of adult sandflies and the vector—host contact (Michele Maroli,Istituto Superiore di Sanita, Rome, Italy). Severalinsecticide residual spraying (IRS) campaigns inboth South America and the Mediterranean regionhave been implemented recently and may indeedreduce the sand fly population. However, theyare restricted to indoor IRS, depend on a suitableinfrastructure and trained personnel, and requireapplication of a detailed monitoring in order toevaluate their long-term efficacy and allow cost-benefit analyses. Due to the mostly nocturnalbiting activity of the phlebotomine females, insec-ticide-treated nets (ITNs) represent a suitable andcost-effective alternative for reducing indoor

Leishmania transmission. They are easy to use,their implementation requires only limitedresources, and — most importantly — severalfield studies using pyrethroid-treated nets haveproven their efficacy against sand flies and theincidence of cutaneous leishmaniasis. Chemicalrepellents and protective clothing may be the onlymeasures that prevent outdoor exposure to sandflies. Whereas diethyltoluamid (DEET) is the goldstandard and highly effective against haemato-phagous insects, Picaridin (KBR 3023) is apromising alternative with 100% protectionagainst biting by Phlebotomus sp. until 7 h afterapplication. Finally, control of canine leishmaniasisin Mediterranean countries may have beneficialeffects on VL in humans, since domestic dogs arethe principal reservoir of L. infantum. Indeed, theuse of different pyrethroid formulations on dogslargely prevents blood meals by and exerts toxiceffects on sand flies. Consequently, canine leish-maniasis is reduced by 50—90% and appears toalso reduce the incidence of VL in humans. It hasto be stressed, however, that this strategy isinappropriate for VL due to L. donovani that do notuse dogs as reservoir.

Future Directions and Conclusions

Despite considerable progress in our understand-ing of the biology of Leishmania parasites andphlebotomine vectors and the pathogenesis ofdisease during the genomic and post-genomiceras, a variety of open questions still remain to beresolved (Werner Solbach, University of Lubeck,Germany). For example, how does the recentlyidentified gene flow between distinct Leishmaniastrains affect the dynamics of the parasitepopulations and what is the consequence onclinical disease and the emergence of resistantstrains? What are the determinants of the differentclinical outcomes others than parasite speciesand hosts’ immune responses, e.g. populationgenetics of the host, epigenetic influences, reacti-vation versus re-infection, co-infections with otherpathogens, vector species and their transmissioncompetence, or subtle differences in immune cellsubpopulations and the cytokine milieu? Resol-ving these issues is crucial in order to define anarray of risk factors which are indicative fordeveloping overt VL, PKDL, mucocutaneouslesions, or reactivation of persistent parasites.In this context, the development of animal modelsothers than mice would certainly be helpful inorder to identify pathogenetic traits that more

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closely resemble the situation in humans.Although important progress has been made, weare still far away from a clinical diagnosis that issensitive and specific, easy and quick to perform,suitable and affordable in those areas whereleishmaniasis is most prevalent, and — impor-tantly — that also allows species identification. Inaddition, the development of novel drugs or drugregimens with a better compliance and lower sideeffects are of major concern. Finally, the develop-ment of immunoprophylactic measures is still atthe beginning although potentially suitable anti-gens and promising modes of delivery have beenidentified. While this list of issues to be resolved iscertainly not complete, it highlights the impor-tance of integrative efforts that combine clinicalexpertise and molecular and cellular scientificknowledge in order to translate basic Leishmaniaresearch into an improved control of a neglectedparasitic disease. The meeting participants con-curred that the IFoLeish-2008 represented anexcellent platform to strengthen such an inter-disciplinary exchange of ideas and to implementresearch collaborations.

Acknowledgements

The meeting was supported by the DeutscheForschungsgemeinschaft (DFG), the GermanSociety of Hygiene and Microbiology (DGHM),section ‘Eukaryotic pathogens’, the GermanSociety for Parasitology (DGP), the C.H.S.foundation Heidelberg, and by industrial sponsors.

References

Hotez PJ, Remme JHF, Buss P, Alleyne G, Morel C,Breman G (2004) Combating tropical infectious diseases:report of the disease control priorities in developing countriesproject. Clin Infect Dis 38: 871—878

Stefan Zimmermanna,1, Heidrun Mollb,Werner Solbachc, and Carsten G.K. Luderd,1

aInstitute for Hygiene and Medical Microbiology,University of Heidelberg, Im Neuenheimer Feld

324, 69120 Heidelberg, Germany

bInstitute for Molecular Infection Biology,University of Wurzburg, Rontgenring 11,

97070 Wurzburg, Germany

cInstitute for Medical Microbiology and Hygiene,University of Lubeck, Ratzeburger Allee 160,

23538 Lubeck, Germany

dInstitute for Medical Microbiology,University of Gottingen,

Kreuzbergring 57,37075 Gottingen, Germany

1Corresponding authors;fax +49 6221 56 4343 (S. Zimmermann),

fax +49 551 39 5861 (C.G.K. Luder)e-mail

stefan.zimmermann@med.uni-heidelberg.de(S. Zimmermann),

clueder@gwdg.de (C.G.K. Luder)