In-situ proliferation contributes to the accumulation of myeloid cells in the spleen during progressive experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is characterized by expansion of myeloid cells in the liver and spleen, which leads to a severe splenomegaly associated with higher risk of mortality. This increased cellularity is thought to be a consequence of recruitment of cells to the viscera. We studied whether the local proliferation of splenic myeloid cells contributes to increased splenic cellularity. We found that a monocyte-like population of adherent splenic cells from Leishmania donovani-infected hamsters had enhanced replicative capacity ex vivo and in vivo (BrdU incorporation, p<0.0001). In vitro assays demonstrated that proliferation was more pronounced in the proinflammatory M1 environment and that intracellular infection prevented proliferation. Secondary analysis of the published splenic transcriptome in the hamster model of progressive VL revealed a gene expression signature that included division of tumoral cells (Z = 2.0), cell cycle progression (Z = 2.3), hematopoiesis (Z = 2.8), proliferation of stem cells (Z = 2.5) and overexpression of proto-oncogenes. Regulators of myeloid cell proliferation were predicted in-silico (CSF2, TLR4, IFNG, IL-6, IL-4, RTK signaling, and STAT3). The in-silico prediction was confirmed with chemical inhibitors of PI3K/AKT, MAPK and STAT3 which decreased splenic myeloid cell division ex vivo. Hamsters infected with L. donovani treated with a STAT3 inhibitor had reduced in situ splenic myeloid proliferation (p = 0.03) and parasite burden. We conclude that monocyte-like myeloid cells have increased STAT3-dependent proliferation in the spleen of hamsters with visceral leishmaniasis and that inhibition of STAT3 reduces myeloid cell proliferation and parasite burden.

Artemisinin-resistant Leishmania parasite modulates host cell defense mechanism and exhibits altered expression of unfolded protein response genes.

Artemisinin, extracted from a medicinal herb Artemisia annua, is widely used to treat malaria and has shown potent anticancer activity. Artemisinin has been found to be effective against experimental visceral and cutaneous leishmaniasis. Despite extensive research to understand the complex mechanism of resistance to artemisinin, several questions remain unanswered. The artesunate (ART)-resistant line of Leishmania donovani was selected and cellular mechanisms associated with resistance to artemisinin were investigated. ART-resistant (AS-R) parasites showed reduced susceptibility towards ART both at promastigote and amastigote stage compared with ART sensitive (WT) parasites. WT and AS-R parasites were both more susceptible to ART at the early log phase of growth compared with late log phase. AS-R parasites were more infective to the host macrophages (p < 0.05). Evaluation of parasites' tolerance towards host microbicidal mechanisms revealed that AS-R parasites were more tolerant to complement-mediated lysis and nitrosative stress. ROS levels were modulated in presence of ART in AS-R parasites infected macrophages. Interestingly, infection of macrophages by AS-R parasites led to modulated levels of host interleukins, IL-2 and IL-10, in addition to nitric oxide. Additionally, AS-R parasites showed upregulated expression of genes of unfolded protein response pathway including methyltransferase domain-containing protein (HSP40) and flagellar attachment zone protein (prefoldin), that are reported to be associated with ART resistance in Plasmodium falciparum malaria. This study presents in vitro model of artemisinin-resistant Leishmania parasite and cellular mechanisms associated with ART resistance in Leishmania.

Visceral Leishmaniasis in Hospitalized HIV-Infected Patients in Pernambuco, Brazil.

Common in four continents, visceral leishmaniasis (VL) is an important but neglected disease. Human immunodeficiency virus (HIV) infection increases the risk of developing VL in people from leishmaniasis-endemic areas, with worse prognosis when there is coinfection. We conducted a cross-sectional study to determine the prevalence of HIV/VL coinfection in patients admitted in three referral hospitals for HIV/acquired immunodeficiency syndrome (AIDS) in Pernambuco, Brazil, and to compare epidemiological, clinical, and laboratory characteristics among HIV/VL coinfected and HIV mono-infected individuals. The sample consisted of HIV patients aged 18 years or more, in a period of data collection of 6 months. We performed four Leishmania tests-polymerase chain reaction (PCR), direct agglutination test, rK39, and latex agglutination test-and individuals with at least one positive test were considered coinfected. The HIV/VL coinfection prevalence we found was 16.9%. We observed large variation in prevalence according to the Leishmania test used, with low coincidence of positive tests. The most frequent symptoms found were weight loss (75.6%), fever (67.6%), and cough (55.3%). When we compared HIV/VL coinfected and HIV mono-infected groups we did not observe statistically significant differences. Low educational level (P = 0.004) and pallor (P = 0.009) were more frequent in the coinfected group. Serum albumin level was higher in coinfected individuals (P = 0.009). It is important to follow-up these individuals to understand the dynamics of VL in people living with HIV. New tests are necessary, ideally differentiating active from latent infection. Testing for VL in people with HIV is important and should be considered as part of the initial investigation in these individuals.

Leishmania donovani induced Unfolded Protein Response delays host cell apoptosis in PERK dependent manner.

BACKGROUND: Endoplasmic reticulum (ER) stress generated unfolded stress response (UPR) is a basic survival mechanism which protects cell under unfavourable conditions. Leishmania parasite modulates host macrophages in various ways to ensure its survival. Modulation of PI3K-Akt pathway in delayed apoptotic induction of host; enables parasite to stabilize the infection for further propagation. METHODOLOGY: Infected RAW macrophages were exposed to campothecin or thagsigargin and phosphorylation status of PERK, Akt, BAD and Cyt-C was determined through western blotting using phospho specific antibody. Expression at transcriptional level for cIAP1 &2, ATF4, CHOP, ATF3, HO-1 and sXBP1 was determined using real time PCR. For inhibition studies, RAW macrophages were pre-treated with PERK inhibitor GSK2606414 before infection. FINDINGS: Our studies in RAW macrophages showed that induction of host UPR against L.donovani infection activates Akt mediated pathway which delays apoptotic induction of the host. Moreover, Leishmania infection results in phosphorylation and activation of host PERK enzyme and increased transcription of genes of inhibitor of apoptosis gene family (cIAP) mRNA. In our inhibition studies, we found that inhibition of infection induced PERK phosphorylation under apoptotic inducers reduces the Akt phosphorylation and fails to activate further downstream molecules involved in protection against apoptosis. Also, inhibition of PERK phosphorylation under oxidative exposure leads to increased Nitric Oxide production. Simultaneously, decreased transcription of cIAP mRNA upon PERK phosphorylation fates the host cell towards apoptosis hence decreased infection rate. CONCLUSION: Overall the findings from the study suggests that Leishmania modulated host UPR and PERK phosphorylation delays apoptotic induction in host macrophage, hence supports parasite invasion at early stages of infection.

Countervailing, time-dependent effects on host autophagy promotes intracellular survival of Leishmania.

Autophagy is essential for cell survival under stress and has also been implicated in host defense. Here, we investigated the interactions between Leishmania donovani, the main etiological agent of visceral leishmaniasis, and the autophagic machinery of human macrophages. Our results revealed that during early infection-and via activation of the Akt pathway-Leishmania actively inhibits the induction of autophagy. However, by 24 h, Leishmania switched from being an inhibitor to an overall inducer of autophagy. These findings of a dynamic, biphasic response were based on the accumulation of lipidated light chain 3 (LC3), an autophagosome marker, by Western blotting and confocal fluorescence microscopy. We also present evidence that Leishmania induces delayed host cell autophagy via a mechanism independent of reduced activity of the mechanistic target of rapamycin (mTOR). Notably, Leishmania actively inhibited mTOR-regulated autophagy even at later stages of infection, whereas there was a clear induction of autophagy via some other mechanism. In this context, we examined host inositol monophosphatase (IMPase), reduced levels of which have been implicated in mTOR-independent autophagy, and we found that IMPase activity is significantly decreased in infected cells. These findings indicate that Leishmania uses an alternative pathway to mTOR to induce autophagy in host macrophages. Finally, RNAi-mediated down-regulation of host autophagy protein 5 (ATG5) or autophagy protein 9A (ATG9A) decreased parasite loads, demonstrating that autophagy is essential for Leishmania survival. We conclude that Leishmania uses an alternative pathway to induce host autophagy while simultaneously inhibiting mTOR-regulated autophagy to fine-tune the timing and magnitude of this process and to optimize parasite survival.

Epidemiological profile of patients co-infected with visceral leishmaniasis and HIV/AIDS in Northeast, Brazil

Abstract INTRODUCTION: Visceral leishmaniasis (VL) and human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) co-infection has been a research topic of interest worldwide. In Brazil, it has been observed that there is a relative underreporting and failure in the understanding and management of this important association. The aim of this study was to analyze epidemiological and clinical aspects of patients with VL with and without HIV/AIDS. METHODS: We conducted an observational and analytical study of patients with VL followed in a Reference Service in the State of Maranhão, Brazil from 2007-2013. RESULTS: In total 126 patients were enrolled, of which 61 (48.4%) were co-infected with HIV/AIDS. There were more males among those with HIV/AIDS (85.2%, P>0.05) or with VL only (81.5%, P>0.05). These findings significantly differed based on age group (P<0.003); the majority of patients were aged 31-40 years (41.0%) and 21-30 years (32.3%) among those with and without HIV/AIDS co-infection, respectively. The incidence of diarrhea and splenomegaly significantly differed between the two groups (P=0.0014 and P=0.019, respectively). The myelogram parasitic examination was used most frequently among those with HIV/AIDS (91.8%), followed by those with VL only (69.2%). VL recurrences and mortality were significantly higher in the HIV/AIDS co-infected patients (P<0.0001 and P=0.012, respectively). CONCLUSIONS: Patients with VL with or without HIV/AIDS co-infection were mostly adult men. Diarrhea was more frequent in HIV/AIDS co-infected patients, whereas splenomegaly was more common in patients with VL only. In the group of HIV/AIDS co-infected patients, there was a higher rate of VL recurrence and mortality.

Visceral leishmaniasis and leishmaniasis-HIV coinfection: comparative study

Abstract INTRODUCTION: This study aimed to draw clinical and epidemiological comparisons between visceral leishmaniasis (VL) and VL associated with human immunodeficiency virus (HIV) infection. METHOD: Retrospective study. RESULTS: Of 473 cases of VL, 5.5% were coinfected with HIV. The highest proportion of cases of both VL and VL/HIV were found among men. A higher proportion of VL cases was seen in children aged 0-10 years, whereas coinfection was more common in those aged 18-50 years. CONCLUSIONS: VL/HIV coinfected patients presented slightly differently to and had a higher mortality rate than those with VL only.

TNF signalling drives expansion of bone marrow CD4+ T cells responsible for HSC exhaustion in experimental visceral leishmaniasis.

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These findings provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously.

Disease severity in patients with visceral leishmaniasis is not altered by co-infection with intestinal parasites.

Visceral leishmaniasis (VL) is a neglected tropical disease that affects the poorest communities and can cause substantial morbidity and mortality. Visceral leishmaniasis is characterized by the presence of Leishmania parasites in the spleen, liver and bone marrow, hepatosplenomegaly, pancytopenia, prolonged fever, systemic inflammation and low body mass index (BMI). The factors impacting on the severity of VL are poorly characterized. Here we performed a cross-sectional study to assess whether co-infection of VL patients with intestinal parasites influences disease severity, assessed with clinical and haematological data, inflammation, cytokine profiles and BMI. Data from VL patients was similar to VL patients co-infected with intestinal parasites, suggesting that co-infection of VL patients with intestinal parasites does not alter disease severity.

Disease Severity Prediction by Spirometry in Adults with Visceral Leishmaniasis from Minas Gerais, Brazil.

Visceral leishmaniasis (VL) is associated with interstitial pneumonitis according to histology and radiology reports. However, studies to address the functional impact on respiratory function in patients are lacking. We assessed pulmonary function using noninvasive spirometry in a cross-sectional study of hospitalized adult VL patients from Minas Gerais, Brazil, without unrelated lung conditions or acute infections. Lung conditions were graded as normal, restrictive, obstructive, or mixed patterns, according to Brazilian consensus standards for spirometry. To control for regional patterns of lung function, we compared spirometry of patients with regional paired controls. Spirometry detected abnormal lung function in most VL patients (70%, 14/20), usually showing a restrictive pattern, in contrast to regional controls and the standards for normal tests. Alterations in spirometry measurements correlated with hypoalbuminemia, the only laboratory value indicative of severity of parasitic disease. Abnormalities did not correlate with unrelated factors such as smoking or occupation. Clinical data including pulmonary symptoms and duration of therapy were also unrelated to abnormal spirometry findings. We conclude that the severity of VL is correlated with a restrictive pattern of lung function according to spirometry, suggesting that there may be interstitial lung involvement in VL. Further studies should address whether spirometry could serve as an index of disease severity in the management of VL.

[Visceral leishmaniasis concurrent with splenic marginal zone B-cell lymphoma].

Splenic marginal zone B-cell lymphoma (SMZBCL) is a rare non-Hodgkin B-cell lymphoma that presents with morphologically mature lymphoid cells corresponding in their immunological characteristics to secondary follicular marginal zone lymphocytes. It is clinically characterized by splenomegaly, moderate lymphocytosis, usually focal bone marrow lesion, sometimes moderate of monoclonal immunoglobulin in the serum (generally IgM or IgG) and/or urea, and a relatively benign course. Leishmaniasis is a transmissible natural focal infectious endemic disease that has a great diversity of clinical manifestations. The authors describe Russia's first case of SMZBCL concurrent with visceral leishmaniasis in a 52-year-old female patient admitted to a hematology hospital with weakness, splenomegaly, and lymphadenopathy. The simultaneous detection of lymphoma and leishmaniasis in the same biopsy specimen is extremely rare. Visceral leishmaniasis should be borne in mind as an opportunistic infection in patients with malignancies, particularly in immunocompromised persons who live or have stayed in the endemic areas.

Avaliação dos indicadores de lesão miocárdica em cães com leishmaniose visceral / Changes in myocardial injury indicators in naturally infected dogs with visceral leishmaniasis

O objetivo deste estudo foi avaliar os indicadores laboratoriais, eletrocardiográficos e histológicos de lesão cardíaca em diferentes grupos clínicos de cães com leishmaniose visceral. Foram analisados marcadores séricos, traçado eletrocardiográfico e fragmentos de tecido cardíaco de 41 cães naturalmente infectados, distribuídos em três grupos: assintomático, oligossintomático e sintomático. Todos os animais apresentaram aumento na atividade sérica da enzima creatina quinase fração MB. No traçado eletrocardiográfico, o complexo de baixa voltagem foi o distúrbio de condução mais frequente (8/10). Na análise histológica, 75,6% dos cães apresentaram reação inflamatória com predomínio de infiltrados linfo-histiocítico (13/31) de intensidade discreta a moderada e distribuição multifocal. As alterações microscópicas identificadas no miocárdio foram independentes dos achados laboratoriais, eletrocardiográficos e do quadro clínico apresentado pelos animais estudados. A ausência de associação entre alterações histopatológicas e os parâmetros investigados alerta para a dificuldade de identificação de cardiopatia em cães com leishmaniose visceral e ressalta a importância de incluir a leishmaniose visceral no diagnóstico de patologias cardíacas principalmente em regiões endêmicas para o agente.

The aim of this study was to evaluate the laboratory indicators, electrocardiographic and cardiac histological lesions in different clinical groups of dogs with visceral leishmaniasis. Serum markers were analyzed in conjunction with the electrocardiographic tracing and heart tissue fragments of 41 naturally infected dogs which were divided into three groups: asymptomatic, oligosymptomatic and symptomatic. All animals showed increased activity in serum creatine kinase MB fraction. In the electrocardiographic tracing, low voltage complex was the most frequent conduction disorder (8/12). In the histological analysis, 75.6% of the dogs showed inflammatory reaction with predominance of linfohistiocítico infiltrates (13/31) of mild to moderate intensity and multifocal distribution. Microscopic changes identified in the myocardium were independent laboratory findings, an electrocardiographic and clinical picture presented by the studied animals. The lack of association between histopathological changes and the parameters investigated indicate the difficulty in disease identification in dogs with visceral leishmaniasis and highlights the importance of including visceral leishmaniasis in the diagnosis of heart disease especially in endemic regions to the agent.

Novel markers of inflammatory response and hepatic dysfunction in canine leishmaniasis.

Dogs are the main host of Leishmania infantum, and the clinical presentation may range from asymptomatic to systemic manifestations. The immune mechanisms in infected, but clinically healthy dogs, prevails Th1 response mediated by cytokines. In this sense, adenosine deaminase (ADA) and butyrylcholinesterase (BChE) are considered as key enzymes in several physiological processes, including the modulation of inflammatory process. Considering the variable immune response against Leishmania and the known participation of ADA and BChE, the aim of this study was to assess the relation between these two enzymes with the inflammatory response as well as hepatic function in dogs naturally infected with L. infantum. For this purpose, the activity of ADA and BChE was assessed in sera of 24 dogs naturally infected with L. infantum, plus 17 healthy dogs. The naturally infected dogs had clinical signs compatible with leishmaniasis and sera activities of ADA (P<0.01) and BChE (P<0.05) decreased, when compared to the healthy group. The reduction of ADA activity probably represented an effect on inflammatory response, especially due to the decreased hydrolysis of extracellular adenosine, might in order to protect against tissue damage and, also, setting a down-regulation on pro-inflammatory cytokines. BChE enzyme had no effect on modulating the immune response in leishmaniasis, but it decreased, a fact may related to deficiency of synthesis in the liver. Therefore, ADA and BChE activities reduced probably in order to protect against extra tissue damage and due failure in synthesis, respectively.

Efficacy of intravenous cyclosporine in a case of cytophagic histiocytic panniculitis complicated by haemophagocytic syndrome after visceral leishmania infection.

Cytophagic histiocytic panniculitis (CHP) is a rare panniculitis characterized by systemic features, due to histiocytic infiltration along with haemophagocytosis, which may also appear in bone marrow, spleen, lymph nodes, and liver. Haemophagocytic lymphohistiocytosis (HLH) is a group of autoinflammatory disorders, which include macrophage activation syndrome, sometimes observed in the course of systemic autoimmune diseases, such as juvenile chronic polyarthritis, systemic lupus erythematosus or vasculitis, and infection-associated haemophagocytic syndrome; if not promptly recognised and treated, HLH can be fatal. Visceral leishmaniasis (VL) is a systemic disease caused by different forms of Leishmania spp., an intracellular protozoa. VL is endemic in tropical countries such as in the Middle East and the Mediterranean. The typical clinical and laboratory features are fever, hepato-splenomegaly, hypergammaglobulinaemia and pancytopenia. The features of VL may mimic some haematologic diseases. We report a case of cytophagic histiocytic panniculitis and HLH, triggered by a previous visceral leishmania infection. Cyclosporine was quickly effective in this case, after failure of high-dose glucocorticoids, anakinra and etoposide.

Clinicopathological and Immunological Changes in Indian Post Kala-Azar Dermal Leishmaniasis (PKDL) Cases in relation to Treatment: A Retrospective Study.

Post-kala-azar dermal leishmaniasis (PKDL) is an important factor in kala-azar transmission; hence its early detection and assessment of effective treatment is very important for disease control. In present study on 60 PKDL cases presented with macular, mixed papulonodular, or erythematous lesions, Leishmania parasites were demonstrated microscopically in 91% of papulonodular and 40% of macular lesions. Cellular infiltrates in skin biopsy imprint smears from lesions were mononuclear cells, 25-300/OIF (oil immersion field), predominantly histiocytes with vacuolation, many lymphocytes, some plasma cells, and Leishmania amastigotes 0-20/OIF. Cases with no demonstrable parasites were diagnosed on the basis of past history of VL, lesion's distribution, cytopathological changes, and positive DAT (86.83%). Following antileishmanial treatment with SAG, papulonodular forms of PKDL lesions disappeared clinically but microscopically the mononuclear cells (20-200/OIF) persisted in the dermal lesions. Response observed in macular PKDL lesions was poor which persisted both clinically and cytopathologically. Follow-up of PKDL will assess the effectivity of treatment as either disappearance of lesions or any relapse. Studies on involvement of immunological factors, that is, certain cytokines (IL-10, TGF-ß, etc.) and chemokines (macrophage inflammatory protein, MIP 1-α, etc.) in PKDL, may provide insight for any role in the treatment response.

Transcription of innate immunity genes and cytokine secretion by canine macrophages resistant or susceptible to intracellular survival of Leishmania infantum.

In this study we assessed the basal transcription of genes associated with innate immunity (i.e. Nramp1, NOD1, NOD2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9) in canine monocyte-derived macrophages from Leishmania-free dogs. Additionally, secretion of cytokines (IL-10, IL-12, TNF-α and IFN-γ) and nitric oxide in culture supernatants of macrophages with higher or lower resistance to intracellular survival of Leishmania infantum was also measured. Constitutive transcription of TLR9 and NOD2 were negligible; NOD1, TLR1, and TLR7 had low levels of transcription, whereas Nramp1 and TLR2, 3, 4, 5, and 6 had higher levels of constitutive transcription in canine monocyte-derived macrophages. There were no significant differences in transcription between macrophages with higher or lower resistance to intracellular survival of L. infantum. Secretion of TNF-α was higher in more resistant macrophages (designated as resistant) at 24h after infection when compared to less resistant macrophages (designated as susceptible), as well as the secretion of IFN-γ at 72 h post infection. Secretion of IL-10 was lower in resistant macrophages at 24h after infection. No detectable production of nitric oxide was observed. Interestingly, there was a negative correlation between NOD2 transcript levels and intracellular survival of L. infantum in resistant macrophages. This study demonstrated that decreased intracellular survival of L. infantum in canine macrophages was associated with increased production of TNF-α and IFN-γ and decreased production of IL-10; and that constitutive transcription of Nramp1, TLR and NLR does not interfere in intracellular survival of L. infantum.

[VISCERAL LEISHMANIASIS IN A THREE-YEAR-OLD CHILD].

The paper describes a clinical case of visceral leishmaniasis in a three-year-old child who Was born and is a permanent resident in Penm. Its clinical symptomatology; laboratory and instrumental findings; stages of a diagnostic search for fever of unknown origin concurrent with hepatosplenomegaly and pancytopenia; differential diagnosis with hemoblastosis; and an epidemiological history are detailed. Visceral leishmaniasis was diagnosed at examination of bone marrow specimens. The paper presents the positive results of combination etiopathogenetic treatment, which are confirmed by the time course of clinical changes and laboratory findings.

Leishmania donovani infection enhances lateral mobility of macrophage membrane protein which is reversed by liposomal cholesterol.

BACKGROUND: The protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment. METHODOLOGY/PRINCIPAL FINDINGS: We determined the lateral mobility of a membrane protein in normal, LD infected and liposome treated LD infected cells using GFP-tagged PLCδ1 as a probe. The mobility of PLCδ1 was computationally analyzed from the time lapse experiment using boundary distance plot and radial profile movement. Our results showed that the lateral mobility of the membrane protein, which is increased in infection, is restored to normal upon liposomal cholesterol treatment. The results of FRAP experiment lent further credence to the above notion. The membrane proteins are intimately linked with cellular actin and alteration of cellular actin may influence lateral mobility. We found that F-actin is decreased in infection but is restored to normal upon liposomal cholesterol treatment as evident from phalloidin staining and also from biochemical analysis by immunoblotting. CONCLUSIONS/SIGNIFICANCES: To our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.