Membrane Permeabilization by Trypanosome Lytic Factor, a Cytolytic Human High Density Lipoprotein

Trypanosome lytic factor (TLF) is a subclass of human high density lipoprotein (HDL) that mediates an innate immune killing of certain mammalian trypanosomes, most notably Trypanosoma brucei brucei, the causative agent of a wasting disease in cattle. Mechanistically, killing is initiated in the lysosome of the target trypanosome where the acidic pH facilitates a membrane-disrupting activity by TLF. Here we utilize a model liposome system to characterize the membrane binding and permeabilizing activity of TLF and its protein constituents, haptoglobin-related protein (Hpr), apolipoprotein L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas non-lytic human HDL exhibits inefficient permeabilizing activity. Purified, delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic membranes. Analysis of the relative particle sizes of susceptible liposomes reveals distinctly different membrane-active behavior for native TLF and the delipidated protein components. We propose that lysosomal membrane damage in TLF-susceptible trypanosomes is initiated by the stable association of the TLF particle with the lysosomal membrane and that this is a property unique to this subclass of human HDL.High density lipoproteins (HDL)² are complex yet ordered macromolecules consisting of characteristic proteins embedded in a phospholipid monolayer that surrounds a hydrophobic core of esterified cholesterol and triglycerides. A subclass of HDL is responsible for an innate immune killing of the African blood stream parasite Trypanosoma brucei brucei (1–3), and very recently, has been shown to be cytotoxic to intracellular Leishmania promastigotes (4). The trypanolytic HDL particle, termed trypanosome lytic factor (TLF), is characterized by the presence of two proteins, apolipoprotein L-1 (apoL-1) and haptoglobin-related protein (Hpr), as well as the HDL ubiquitous apolipoprotein A-1 (apoA-1) (1, 5–7). Killing of the susceptible parasite involves high affinity binding to a cell-surface receptor, endocytosis, and trafficking of the TLF particle to the lysosome (8–12). The acidic lysosomal environment facilitates a membrane-disrupting activity by the TLF particle and subsequent cell death (9, 13). It has been shown that purified, delipidated apoL-1 or Hpr are sufficient for trypanosome killing. When these proteins are incorporated into the same lipoprotein particle, a several hundredfold increase in killing activity is exhibited (5). In addition, Molina-Portela et al. (14) show that maximal protection against T. b. brucei in a transgenic mouse model requires the expression of human Hpr, apoL-1, and apoA-1, supporting a synergistic mode of action.Haptoglobin-related protein evolved during primate evolution and is restricted to apes, old world monkeys, and humans (15). Haptoglobin-related protein is highly similar (92%) to the acute phase serum protein haptoglobin (Hp) (16). All mammals use Hp as a scavenger of hemoglobin (Hb) released during hemolysis associated with infection or trauma. Haptoglobin binds cell-free Hb with high affinity and facilitates its removal from the circulation through a receptor-mediated process in the liver (17). Like Hp, Hpr binds free Hb, yet this Hpr·Hb complex is not recognized by the requisite receptors in mammals and is thus not removed from the circulation (18). TLF uptake by susceptible trypanosomes requires specific binding to an Hpr·Hb complex that facilitates trafficking of the TLF particle to the lysosome (10). It has been proposed that once inside the lysosomal compartment, Hpr·Hb contributes directly to membrane disruption through the generation of oxygen radicals with the bound Hb providing the iron necessary for Fenton chemistry (7, 10, 19).Apolipoprotein L-1 is a unique member of the apolipoprotein L protein family in that, unlike the remaining apoL proteins, it possesses an N-terminal signal sequence and is thus secreted from cells. As is the case for Hpr, apoL-1 appeared during primate evolution (20–22). Within the circulation of primates, apoL-1 is exclusively associated with HDL, and the majority of the protein is in the TLF subclass (5). The apoL family members are all predicted to adopt amphipathic α-helical conformations, suggesting that their physiological role involves membrane interaction (20). Apolipoprotein L-1 shares limited homology with channel-forming colicins and, consistent with this observation, has been shown to function as an ion channel when incorporated into lipid bilayers (23).The ultimate fate of TLF-targeted lysosomal membranes is not firmly established. Several studies employing both in vivo cellular analysis and artificial membrane systems address this point with conflicting results. Electron microscopy studies with gold-conjugated TLF revealed accumulation of TLF in intracellular vesicles and subsequent vesicle membrane breakdown and appearance of gold particles in the cytoplasm (9). Widener et al. (10) observed efflux of lysosomally localized large molecular mass dextrans (500 kDa) in TLF-treated T. b. brucei. These data suggest that the lysosomal membrane experiences large scale disruption. In contrast, Perez-Morga et al. (23) and Vanhollebeke et al. (24) report uncontrollable lysosomal swelling in susceptible trypanosomes treated with normal human serum, suggesting stability of the lamellar structure of the lysosomal membrane after TLF attack. Swelling is attributed to apoL-1-mediated influx of Cl^– ions and concomitant osmotic flow of water into the lysosome. However, Molina-Portela et al. (25) observed the formation of cation-selective pores in TLF-treated planar lipid bilayers composed of trypanosome lipids. The diversity of activities reported for TLF and normal human serum may reflect the packaging of multiple toxins within the same complex that can act synergistically to provide optimal killing activity (5, 14).Here we utilize model liposomes to monitor the membrane activity of TLF and its protein constituents. We describe the effects of TLF, delipidated Hpr, apoL-1, and apoA-1 on the permeability of unilamellar liposomes. Additionally, we show that TLF, apoL-1, and apoA-1 exhibit lipid specificity and that Hpr, apoL-1, and apoA-1 induce large scale changes in the geometry of liposomes. These results provide a molecular basis for the recognition of lysosomal membranes by this toxic HDL and support a multicomponent mechanism for trypanosome killing.     

Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1^- and lpg5A^-/lpg5B^- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania.     

Association between Serum C-Peptide as a Risk Factor for Cardiovascular Disease and High-Density Lipoprotein Cholesterol Levels in Nondiabetic Individuals

Objective

Objective: Although serum C-peptide has increasingly received attention as a new and important risk factor for cardiovascular disease (CVD), the potential mechanisms remain unclear. This study aimed to investigate the association between serum C-peptide as a risk factor for CVD and high-density lipoprotein cholesterol (HDL-C) levels.

Methods

The present study included 13,185 participants aged ≥20 years. Serum C-peptide and HDL-C levels were measured according to a standard protocol. Stratified analysis of covariance was used to compare serum HDL-C levels between different quartiles of serum C-peptide levels. Logistic regression analysis was used to determine the association between serum C-peptide and HDL-C levels. Cox proportional hazard regression analysis was conducted to determine the hazard ratio of serum HDL-C for CVD-related mortality.

Results

The results of the ANCOVA analysis showed a significant linear trend between the mean serum HDL-C level and the different quartiles of serum C-peptide. Compared to the first quartile (25th percentile), the second, third, and fourth quartiles had gradual reduction in serum HDL-C levels. Logistic regression analyses showed a strong negative association between serum C-peptide levels and HDL-C levels; the p value for the linear trend was <0.001. In men, compared with the lowest quartile of the serum C-peptide level, the relative risk was 1.75, 2.79, and 3.07 for the upper three quartiles of the serum C-peptide level. The relative risk was 1.60, 2.61, and 3.67 for women. The results of the survival analysis showed that serum HDL-C levels were negatively associated with CVD-related death in both men and women.

Conclusion

Serum C-peptide as a risk factor for CVD was significantly and negatively associated with serum HDL-C levels in individuals without diabetes. These findings suggest that serum C-peptide levels association with CVD death can be caused, at least in part, by the low serum HDL-C level.     

Platelet-Activating Factor (PAF) Modulates Peritoneal Mouse Macrophage Infection by Leishmania amazonensis

The effects of platelet-activating factor (PAF) on the infection of peritoneal mouse macrophages by Leishmania amazonensis were investigated. Prior to the infection, the parasites and/or the macrophages were treated with PAF and/or one of the following modulators: WEB 2086 (PAF antagonist), and the modulators of protein kinase C, phorbol-12-myristate-13-acetate (PMA), and sphingosine. The infection was inhibited when the macrophages or both the parasites and the macrophages were treated with PAF, but stimulated by PAF-treated parasites. WEB 2086 abrogated PAF effects in both systems. The infection was stimulated when the macrophages were treated with sphingosine plus PAF, but inhibited when the macrophages were treated with sphingosine and the parasites with sphingosine plus PAF. The infection was inhibited by sphingosine-treated parasites, either in the presence or in the absence of PAF. Leishmania amazonensis–macrophage infection was inhibited by PMA in all systems tested. Received: 27 September 2000 / Accepted: 15 December 2000     

Enteric YaiW Is a Surface-Exposed Outer Membrane Lipoprotein That Affects Sensitivity to an Antimicrobial Peptide

yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions.     

Latent Infection with Leishmania donovani in Highly Endemic Villages in Bihar,India

Introduction

Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4–10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.

Methods

We selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.

Results

We enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30). Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years).

Discussion

Although infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.     

Dolabelladienetriol, a Compound from Dictyota pfaffii Algae, Inhibits the Infection by Leishmania amazonensis

Background

Chemotherapy for leishmaniasis, a disease caused by Leishmania parasites, is expensive and causes side effects. Furthermore, parasite resistance constitutes an increasing problem, and new drugs against this disease are needed. In this study, we examine the effect of the compound 8,10,18-trihydroxy-2,6-dolabelladiene (Dolabelladienetriol), on Leishmania growth in macrophages. The ability of this compound to modulate macrophage function is also described.

Methodology/Principal Findings

Leishmania-infected macrophages were treated with Dolabelladienetriol, and parasite growth was measured using an infectivity index. Nitric oxide (NO), TNF-α and TGF-β production were assayed in macrophages using specific assays. NF-kB nuclear translocation was analyzed by western blot. Dolabelladienetriol inhibited Leishmania in a dose-dependent manner; the IC₅₀ was 44 µM. Dolabelladienetriol diminished NO, TNF-α and TGF-β production in uninfected and Leishmania-infected macrophages and reduced NF-kB nuclear translocation. Dolabelladienetriol inhibited Leishmania infection even when the parasite growth was exacerbated by either IL-10 or TGF-β. In addition, Dolabelladienetriol inhibited Leishmania growth in HIV-1-co-infected human macrophages.

Conclusion

Our results indicate that Dolabelladienetriol significantly inhibits Leishmania in macrophages even in the presence of factors that exacerbate parasite growth, such as IL-10, TGF-β and HIV-1 co-infection. Our results suggest that Dolabelladienetriol is a promising candidate for future studies regarding treatment of leishmaniasis, associated or not with HIV-1 infection.     

Antimicrobial Peptide-induced Apoptotic Death of Leishmania Results from Calcium-de pend ent, Caspase-independent Mitochondrial Toxicity

α- and θ-defensin-, magainin-, and cathelicidin-type antimicrobial peptides (AMPs) can kill the pathogenic protozoan Leishmania. Comparative studies of a panel of AMPs have defined two distinct groups: those that induce nonapoptotic (Class I) and apoptotic (Class II) parasite killing based on their differential ability to induce phosphatidyl serine exposure, loss of mitochondrial membrane potential and decreased ATP production, induction of caspase-3/7 and -12 activity, and DNA degradation. Class II AMPs cause rapid influx of the vital stain SYTOX and an increase in intracellular Ca²⁺, whereas Class I AMPs cause a slow accumulation of SYTOX and do not affect intracellular Ca²⁺ levels. Inhibitors of cysteine or caspase proteases diminished fast influx of SYTOX through the surface membrane and DNA degradation but do not ablate the annexin V staining or the induction of apoptosis by Class II AMPs. This suggests that the changes in surface permeability in AMP-mediated apoptosis are related to the downstream events of intracellular cysteine/caspase activation or the loss of ATP. The activation of caspase-12-like activity was Ca²⁺-dependent, and inhibitors of voltage-gated and nonspecific Ca²⁺ channels diminished this activity. Flufenamic acid, a nonspecific Ca²⁺ inhibitor, completely ablated AMP-induced mitochondrial dysfunction and cell death, indicating the importance of dysregulation of Ca²⁺ in antimicrobial peptide-induced apoptosis.Leishmania are vector-borne protozoa that parasitize host macrophage phagolysosomes. Activated macrophages kill intracellular parasites in part by induction of apoptosis by nitric oxide in a caspase- and cysteine protease-independent manner (1, 2). Limited apoptosis of Leishmania within the sandfly vector, prior to infection of host, may be important to the establishment of infection (3, 4). In vitro Leishmania can undergo apoptosis by exposure to many different compounds that lead to alteration of surface the cell membrane causing enhanced annexin V staining, activation of caspase-like proteases, DNA laddering, and mitochondrial membrane depolarization. Our work has recently shown that antimicrobial peptide exposure of Leishmania can induce apoptosis, which occurs concomitant with activation of both caspase and cysteine protease activities (5).Antimicrobial peptides (AMPs)2 are structurally diverse highly cationic proteins between 10 and 50 amino acids and are components of the innate immune systems of organisms within all kingdoms. AMPs have a myriad of functions and are known to interact with and disrupt microbial surface membranes leading to cell death. Magainins are α-helical AMPs expressed in the skin of frogs, whereas cathelicidins are mammalian β-pleated sheet peptides, both of which can kill Leishmania at physiological concentrations (5). We have documented that magainins are quite apt at causing AMP-induced apoptosis of surface metalloprotease null mutants of L. major and that these exhibited features of apoptotic Leishmania induced by different stimuli (5). These two different structurally diverse classes of AMPs are known to interact with membranes in distinct ways (6), yet it is unknown whether we they can kill parasites via disparate mechanisms.Apoptosis of Leishmania induced by antimony, camptothecin, and hydrogen peroxide all induce significant increases in cytosolic calcium, which is toxic to mitochondrial membrane potential (7–9). Calcium-related cell death and mitochondrial toxicity are related to the activity of nonspecific calcium channels (7, 8, 10), which may be attributable to their location in the mitochondrial membrane (11, 12). Apoptosis of Leishmania is also associated with increases in caspase-like and cysteine protease activity (5, 7, 9, 13, 14). Caspase-3/7-like activation can be induced by a number of stimuli despite the absence of genes encoding caspases within the L. major genome (15). In addition, the ancestral metacaspase expressed by Leishmania donovani is a trypsin-like protease not inhibitible with caspase inhibitors (16). We and others have found that cysteine protease activity may be important for certain features of Leishmania apoptosis such as DNA degradation (5). It is quite clear that Leishmania can undergo apoptosis in caspase-dependent and caspase-independent pathways (17), yet we do not know what mode of apoptosis occurs upon AMP exposure.In the work presented here we show that two structurally diverse AMPs kill Leishmania differentially inducing either nonapoptotic or apoptotic cell death. We have characterized AMP-induced apoptotic cell death and found that although it is associated with the activation of caspase-3/7- and -12-like activities, these activities are not essential to apoptosis. Conversely we find that a dramatic increase in cytosolic Ca²⁺ correlates with the mitochondrial membrane dysfunction and the decline in ATP production and cell death and that this is completely inhibited by blockade of nonspecific calcium channels. Blockade of caspase and cysteine proteases affect surface membrane permeability changes and DNA degradation but do not prevent cell death, suggesting that AMP-induced apoptosis may be a caspase-independent process. These data provide a framework to understand the critical events in AMP-mediated apoptosis and may be helpful in the rational design of chemotherapeutic strategies for manipulation of cell death pathways in Leishmania.     

Development of an HIV-1 Microbicide Based on Caulobacter crescentus: Blocking Infection by High-Density Display of Virus Entry Inhibitors

The HIV/AIDS pandemic remains an enormous global health concern. Despite effective prevention options, 2.6 million new infections occur annually, with women in developing countries accounting for more than half of these infections. New prevention strategies that can be used by women are urgently needed. Topical microbicides specific for HIV-1 represent a promising prevention strategy. Conceptually, using harmless bacteria to display peptides or proteins capable of blocking entry provides an inexpensive approach to microbicide development. To avoid the potential pitfalls of engineering commensal bacteria, our strategy is to genetically display infection inhibitors on a non-native bacterium and rely on topical application of stabilized bacteria before potential virus exposure. Due to the high density cell-surface display capabilities and the inherent low toxicity of the bacterium, the S-layer mediated protein display capabilities of the non-pathogenic bacterium Caulobacter crescentus has been exploited for this approach. We have demonstrated that C. crescentus displaying MIP1α or CD4 interfered with the virus entry pathway and provided significant protection from HIV-1 pseudovirus representing clade B in a standard single cycle infection assay. Here we have expanded our C. crescentus based microbicide approach with additional and diverse classes of natural and synthetic inhibitors of the HIV-1 entry pathway. All display constructs provided variable but significant protection from HIV-1 infection; some with protection as high as 70%. Further, we describe protection from infection with additional viral clades. These findings indicate the significant potential for engineering C. crescentus to be an effective and readily adaptable HIV-1 microbicide platform.     

Inflammatory and Antimicrobial Responses to Methicillin-Resistant Staphylococcus aureus in an In Vitro Wound Infection Model

Treatment of patients with burn wound infections may become complicated by the presence of antibiotic resistant bacteria and biofilms. Herein, we demonstrate an in vitro thermal wound infection model using human skin equivalents (HSE) and biofilm-forming methicillin-resistant Staphylococcus aureus (MRSA) for the testing of agents to combat such infections. Application of a liquid nitrogen-cooled metal device on HSE produced reproducible wounds characterized by keratinocyte death, detachment of the epidermal layer from the dermis, and re-epithelialization. Thermal wounding was accompanied by up-regulation of markers for keratinocyte activation, inflammation, and antimicrobial responses. Exposure of thermal wounded HSEs to MRSA resulted in significant numbers of adherent MRSA/HSE after 1 hour, and multiplication of these bacteria over 24-48 hours. Exposure to MRSA enhanced expression of inflammatory mediators such as TLR2 (but not TLR3), IL-6 and IL-8, and antimicrobial proteins human β-defensin-2, -3 and RNAse7 by thermal wounded as compared to control HSEs. Moreover, locally applied mupirocin effectively reduced MRSA counts on (thermal wounded) HSEs by more than 99.9% within 24 hours. Together, these data indicate that this thermal wound infection model is a promising tool to study the initial phase of wound colonization and infection, and to assess local effects of candidate antimicrobial agents.     

Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis,but Not by Leishmania (Viannia) guyanensis

Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.     

Immunostimulatory Properties of Dendritic Cells after Leishmania donovani Infection Using an In Vitro Model of Liver Microenvironment

Background

Recent advances demonstrated that liver dendritic cells (DCs) promote immunologic hyporesponsiveness that may contribute to hepatic tolerance. Although there has been significant work on the phenotypic and functional roles of such DCs, the impact of liver microenvironment on the immune properties of infected DC is still poorly explored, probably because of the limitations of modelization.

Methodology/Principal Findings

Here, we hypothesized that DC tolerogenic properties have an impact on the antimicrobial response, particularly during the infection by the protozoan parasite Leishmania donovani. Indeed, a lymphocytic Th2 environment was reported to favour the growth and proliferation of L. donovani. We first modelized an adequate monocyte-differentiated DC model, either in rat liver epithelial cell- or in a human hepatic non-parenchymal cell-conditioned medium in order to infect them further. We established that DCs differentiated in a hepatic microenvironment displayed a CD14+/CD16+/CD123+ phenotype, secreted low IL-12p70 and had an impaired capacity to stimulate allogeneic T lymphocyte proliferation and IFNγ secretion. We then infected DCs with L. donovani in the in vitro-defined hepatic microenvironment. The infection of hepatic DCs restored their capacity to stimulate allogeneic T-cell proliferation and to induce lymphocytic secretion of IFNγ. Such characteristics were recently shown to favour granuloma formation in mice liver.

Conclusions/Significance

Our results suggest that the specific immunostimulatory properties of infected hepatic DCs might amplify the granuloma maturation, which warrants the effective control of infection in the liver during visceral leishmaniasis.     

Leishmania major and Leishmania tropica: I the in vitro effects of an immunomodulator, S2-Complex

This study was undertaken to try to determine the possible anti-leishmanial activity of S2-Complex, an organic complex of copper chloride, ascorbic acid, and nicotinamide. The promastigotes, axenic amastigotes, and intracellular amastigotes of both Leishmania major and Leishmania tropica were incubated with different concentrations of S2-Complex. The EC50 for each form was calculated. Results show that all forms of the parasites were dose dependently inhibited by S2-Complex. The promastigotes of both parasites were the most resistant with highest EC50 followed by axenic amastigotes. While intracellular amastigotes were the most sensitive with the lowest EC50.These results indicate that S2-Complex has a direct anti-leishmanial effect. When mice were treated with S2-Complex or BCG for four days before harvesting the macrophages, and the macrophages infected with both L. major and L. tropica, they showed increased phagocytosis and increased parasite killing. The results of S2-Complex were not statistically different from the immunomodulating agent BCG. These results indicate that S2-Complex has an immunomodulating effect in addition to the direct anti-leishmanial effect.     

Expression of a Peroral Infection Factor Determines Pathogenicity and Population Structure in an Insect Virus

A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus is being studied as a possible biological insecticide. This virus exists as a mixture of complete and deletion genotypes; the latter depend on the former for the production of an essential per os transmission factor (pif1) in coinfected cells. We hypothesized that the virus population was structured to account for the prevalence of pif1 defector genotypes, so that increasing the abundance of pif1 produced by a cooperator genotype in infected cells would favor an increased prevalence of the defector genotype. We tested this hypothesis using recombinant viruses with pif1 expression reprogrammed at its native locus using two exogenous promoters (egt, p10) in the pif2/pif1 intergenic region. Reprogrammed viruses killed their hosts markedly faster than the wild-type and rescue viruses, possibly due to an earlier onset of systemic infection. Group success (transmission) depended on expression of pif1, but overexpression was prejudicial to group-specific transmissibility, both in terms of reduced pathogenicity and reduced production of virus progeny from each infected insect. The presence of pif1-overproducing genotypes in the population was predicted to favor a shift in the prevalence of defector genotypes lacking pif1-expressing capabilities, to compensate for the modification in pif1 availability at the population level. As a result, defectors increased the overall pathogenicity of the virus population by diluting pif1 produced by overexpressing genotypes. These results offer a new and unexpected perspective on cooperative behavior between viral genomes in response to the abundance of an essential public good that is detrimental in excess.     

Imipramine Is an Orally Active Drug against Both Antimony Sensitive and Resistant Leishmania donovani Clinical Isolates in Experimental Infection

Background

In an endeavor to find an orally active and affordable antileishmanial drug, we tested the efficacy of a cationic amphiphilic drug, imipramine, commonly used for the treatment of depression in humans. The only available orally active antileishmanial drug is miltefosine with long half life and teratogenic potential limits patient compliance. Thus there is a genuine need for an orally active antileishmanial drug. Previously it was shown that imipramine, a tricyclic antidepressant alters the protonmotive force in promastigotes, but its in vivo efficacy was not reported.

Methodology/Principal Findings

Here we show that the drug is highly active against antimony sensitive and resistant Leishmania donovani in both promastigotes and intracellular amastigotes and in LD infected hamster model. The drug was found to decrease the mitochondrial transmembrane potential of Leishmania donovani (LD) promastigotes and purified amastigotes after 8 h of treatment, whereas miltefosine effected only a marginal change even after 24 h. The drug restores defective antigen presenting ability of the parasitized macrophages. The status of the host protective factors TNF α, IFN γ and iNOS activity increased with the concomitant decrease in IL 10 and TGF β level in imipramine treated infected hamsters and evolution of matured sterile hepatic granuloma. The 10-day therapeutic window as a monotherapy, showing about 90% clearance of organ parasites in infected hamsters regardless of their SSG sensitivity.

Conclusions

This study showed that imipramine possibly qualifies for a new use of an old drug and can be used as an effective orally active drug for the treatment of Kala-azar.