The Relationship Between Fetal Weight with Sequestration of Infected Erythrocyte, Monocyte Infiltration, and Malaria Pigment Deposition in Placenta of Mother Giving Birth Suffering from Plasmodium Vivax Infection.

BACKGROUND: Malaria in pregnancy can cause fatal complications by parasite sequestration mechanism, which can cause monocyte infiltration in the intervillous space. P. vivax infection was significantly associated with malaria pigment in the placenta, indicating past subclinical infections. OBJECTIVE: This study aimed to determine the mechanism of P. vivax in the pathogenesis of placental malaria and its relationship with LBW. METHODS: This study was observational analytic with a cross-sectional approach. Placental tissue samples were obtained from pregnant women with LBW babies during delivery in Maumere, Nusa Tenggara Timur. The samples used in this study were confirmed by a polymerase chain reaction and consisted of 25 samples with 12 positive and 13 negative samples. Placental tissue samples were made with Hematoxylin-Eosin staining and observed under 1000x magnification at 100 fields using a light microscope. Parasite density, monocyte infiltration, and parasite pigments deposition were calculated. RESULTS: Microscopic observation revealed that there was a significant difference in infected erythrocytes sequestration between groups. Interestingly, monocyte and malaria pigments accumulation were found in malaria-positive and -negative groups, and no significant difference between groups. The correlation test showed no significant relationship between monocyte infiltration and LBW in the malaria-positive and -negative group and between parasite pigments and LBW in both groups. Moreover, there was no significant correlation between parasite density and LBW in the positive and negative groups. CONCLUSION: P. vivax infection causes acute, sub-acute, and chronic placental malaria in subclinical infected pregnant women in Maumere, Nusa Tenggara Timur that might cause an LBW baby.

BEWO trophoblast cells and Toxoplasma gondii infection modulate cell death mechanisms in THP-1 monocyte cells by interference in the expression of death receptor and intracellular proteins.

Crosstalk between trophoblast and monocytes is essential for gestational success, and it can be compromised in congenital toxoplasmosis. Cell death is one of the mechanisms involved in the maintenance of pregnancy, and this study aimed to evaluate the role of trophoblast in the modulation of monocyte cell death in the presence or absence of Toxoplasma gondii infection. THP-1 cells were stimulated with supernatants of BeWo cells and then infected or not with T. gondii. The supernatants were collected and analyzed for the secretion of human Fas ligand, and cells were used to determine cell death and apoptosis, cell death receptor, and intracellular proteins expression. Cell death and apoptosis index were higher in uninfected THP-1 cells stimulated with supernatants of BeWo cells; however, apoptosis index was reduced by T. gondii infection. Macrophage migration inhibitory factor (MIF) and transforming growth factor (TGF)-ß1, secreted by BeWo cells, altered the cell death and apoptosis rates in THP-1 cells. In infected THP-1 cells, the expression of Fas/CD95 and secretion of FasL was significantly higher; however, caspase 3 and phosphorylated extracellular-signal-regulated kinase (ERK1/2) were downregulated. Results suggest that soluble factors secreted by BeWo cells induce cell death and apoptosis in THP-1 cells, and Fas/CD95 can be involved in this process. On the other hand, T. gondii interferes in the mechanism of cell death and inhibits THP-1 cell apoptosis, which can be associated with active caspase 3 and phosphorylated ERK1/2. In conclusion, our results showed that human BeWo trophoblast cells and T. gondii infection modulate cell death in human THP-1 monocyte cells.

Interleukin-11 receptor expression on monocytes is dispensable for their recruitment and pathogen uptake during Leishmania major infection.

Interleukin-11 (IL-11) is an important member of the IL-6 family of cytokines. IL-11 activates its target cells via binding to a non-signaling α-receptor (IL-11R), which results in recruitment and activation of a gp130 homodimer. The cytokine was initially described as an anti-inflammatory protein, but has recently gained attention as a potent driver in certain types of cancer and different fibrotic conditions. Leishmania spp. are a group of eukaryotic parasites that cause the disease leishmaniasis. They infect phagocytes of their hosts, especially monocytes recruited to the site of infection, and are able to replicate within this rather harsh environment, often resulting in chronic infections of the patient. However, the molecular mechanisms underlying parasite and host cell interactions and factors of the immune cells that are crucial for Leishmania uptake are so far largely unspecified. Recently, increased IL-11 expression in the lesions of patients with cutaneous leishmaniasis has been reported, but the functional relevance is unknown. In this study, we show that monocytes express IL-11R on their cell surface. Furthermore, using an adoptive transfer model of IL-11R-/- monocytes, we analyze the contribution of IL-11 signaling on monocyte recruitment and monocyte infection in a mouse model of cutaneous leishmaniasis and find that IL-11 signaling is dispensable for monocyte recruitment and pathogen uptake during Leishmania major infection.

New Therapeutic Tools to Shape Monocyte Functional Phenotypes in Leishmaniasis.

In the innate immunity to Leishmania infection tissue-resident macrophages and inflammatory monocytes accumulate host-cell, effector, and efferocytosis functions. In addition, neutrophils, as host, effector, and apoptotic cells, as well as tissue-resident and monocyte-derived dendritic cells (DCs) imprint innate and adaptive immunity to Leishmania parasites. Macrophages develop phenotypes ranging from antimicrobial M1 to parasite-permissive M2, depending on mouse strain, Leishmania species, and T-cell cytokines. The Th1 (IFN-γ) and Th2 (IL-4) cytokines, which induce classically-activated (M1) or alternatively-activated (M2) macrophages, underlie resistance versus susceptibility to leishmaniasis. While macrophage phenotypes have been well discussed, new developments addressed the monocyte functional phenotypes in Leishmania infection. Here, we will emphasize the role of inflammatory monocytes to access how potential host-directed therapies for leishmaniasis, such as all-trans-retinoic acid (ATRA) and the ligand of Receptor Activator of Nuclear Factor-Kappa B (RANKL) might modulate immunity to Leishmania infection, by directly targeting monocytes to develop M1 or M2 phenotypes.

Ly6G deficiency alters the dynamics of neutrophil recruitment and pathogen capture during Leishmania major skin infection.

Neutrophils represent one of the first immune cell types recruited to sites of infection, where they can control pathogens by phagocytosis and cytotoxic mechanisms. Intracellular pathogens such as Leishmania major can hijack neutrophils to establish an efficient infection. However the dynamic interactions of neutrophils with the pathogen and other cells at the site of the infection are incompletely understood. Here, we have investigated the role of Ly6G, a homolog of the human CD177 protein, which has been shown to interact with cell adhesion molecules, and serves as a bona fide marker for neutrophils in mice. We show that Ly6G deficiency decreases the initial infection rate of neutrophils recruited to the site of infection. Although the uptake of L. major by subsequently recruited monocytes was tightly linked with the concomitant uptake of neutrophil material, this process was not altered by Ly6G deficiency of the neutrophils. Instead, we observed by intravital 2-photon microscopy that Ly6G-deficient neutrophils entered the site of infection with delayed initial recruitment kinetics. Thus, we conclude that by promoting neutrophils' ability to efficiently enter the site of infection, Ly6G contributes to the early engagement of intracellular pathogens by the immune system.

Individuals co-exposed to sand fly saliva and filarial parasites exhibit altered monocyte function.

BACKGROUND: In Mali, cutaneous leishmaniasis (CL) and filariasis are co-endemic. Previous studies in animal models of infection have shown that sand fly saliva enhance infectivity of Leishmania parasites in naïve hosts while saliva-specific adaptive immune responses may protect against cutaneous and visceral leishmaniasis. In contrast, the human immune response to Phlebotomus duboscqi (Pd) saliva, the principal sand fly vector in Mali, was found to be dichotomously polarized with some individuals having a Th1-dominated response and others having a Th2-biased response. We hypothesized that co-infection with filarial parasites may be an underlying factor that modulates the immune response to Pd saliva in endemic regions. METHODOLOGY/PRINCIPAL FINDINGS: To understand which cell types may be responsible for polarizing human responses to sand fly saliva, we investigated the effect of salivary glands (SG) of Pd on human monocytes. To this end, elutriated monocytes were cultured in vitro, alone, or with SG, microfilariae antigen (MF ag) of Brugia malayi, or LPS, a positive control. The mRNA expression of genes involved in inflammatory or regulatory responses was then measured as were cytokines and chemokines associated with these responses. Monocytes of individuals who were not exposed to sand fly bites (mainly North American controls) significantly upregulated the production of IL-6 and CCL4; cytokines that enhance leishmania parasite establishment, in response to SG from Pd or other vector species. This selective inflammatory response was lost in individuals that were exposed to sand fly bites which was not changed by co-infection with filarial parasites. Furthermore, infection with filarial parasites resulted in upregulation of CCL22, a type-2 associated chemokine, both at the mRNA levels and by its observed effect on the frequency of recruited monocytes. CONCLUSIONS/SIGNIFICANCE: Together, our data suggest that SG or recombinant salivary proteins from Pd alter human monocyte function by upregulating selective inflammatory cytokines.

Inhibition of gamma-secretase activity without interfering in Notch signalling decreases inflammatory response in patients with cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) patients present an exacerbated inflammatory response associated with tissue damage and ulcer development. Increasing numbers of patients have exhibited treatment failure, which remains not well understood. We hypothesized that adjuvant anti-inflammatory therapy would benefit CL patients. The aim of the present study was to investigate the contribution of Notch signalling and gamma-secretase activity to the inflammatory response observed in CL patients. Notch signalling is a molecular signalling pathway conserved among animal species. Gamma-secretase forms a complex of proteins that, among other pathways, modulates Notch signalling and immune response. We found that Notch 1 cell receptor signalling protects against the pathologic inflammatory response, and JLK6, a gamma-secretase inhibitor that does not interfere with Notch signalling, was shown to decrease the in-vitro inflammatory response in CL. Our data suggest that JLK6 may serve as an adjuvant treatment for CL patients.

Leishmania donovani Metacyclic Promastigotes Impair Phagosome Properties in Inflammatory Monocytes.

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells.

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

Epidemiological link between canine monocytic ehrlichiosis caused by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto in Argentina.

In this work, we analyze data that support an epidemiological link between cases of canine monocytic ehrlichiosis (CME) by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto as vector in an endemic area for this tick in Argentina. In a blood sample of a 1-year-old toy poodle with CME compatible clinical signs, which showed CME typical morulae in monocytes in Giemsa-stained blood smear, DNA of E. canis was detected by PCR. Further, DNA of E. canis was also detected in a female of R. sanguineus s.s. collected on the infected dog. Rhipicephalus sanguineus s.s. is the only member of the R. sanguineus group that prevails in the study area. The results of this study suggest that R. sanguineus s.s. may play a more important role in the transmission of E. canis than it was assumed so far. The epidemiological link between CME cases and R. sanguineus s.s. as vector in temperate areas of Argentina described in this work contrast previous studies which found that R. sanguineus sensu lato "tropical lineage" (which is absent in the study area) is competent to transmit E. canis but not R. sanguineus s.s.

Status of IL-4 and IL-10 driven markers in experimental models of Visceral Leishmaniasis.

AIM: Leishmania donovani, the causative agent for visceral leishmaniasis (VL), modulates host monocytes/macrophages to ensure its survival. However, knowledge regarding the host-parasite interactions underpinning the disease remains limited. As disease progression is associated with polarization of monocytes/macrophages towards M2, which is regulated by cytokines IL-4/IL-13 and IL-10, this study evaluated the status of key IL-4- and IL-10 driven markers in experimental models of VL, as also evaluated their correlation, if any, with parasite load. METHODS: In liver and splenic tissues from L donovani-infected hamsters and BALB/c mice, the parasite burden was determined along with mRNA expression of IL-4-driven markers, that is CD206, Arginase-I, CCL17, CCL22, PPAR-γ, STAT6, KLF4, FIZZ1 and YM1 along with IL-10-driven markers, CXCL13, IL-10, TGF-ß, VDR, CCR2 and CYP27A1. RESULTS: The mRNA expression of IL-4- and IL-10-driven markers was enhanced in both models, but only in the hamster model, the splenic tissues demonstrated a positive correlation between all the IL-10-driven markers and parasite load. CONCLUSIONS: Contrary to human VL, both models demonstrated an increased expression of IL-4- and IL-10-driven markers.

Cytokines in the immunity and immunopathogenesis in leishmaniases.

Cytokines are key mediators of immune responses to autoantigens, tumor antigens and foreign antigens including pathogens and transplant antigens. The cytokines are produced by a variety of immune and non-immune cells and are dynamically regulated. Remarkably, during toxic and septic shock syndromes, anaphylactic shock and in certain viral infections supra-physiologic levels of cytokine storms are produced culminating in multi-organ failure and death. However, Leishmania infection is a chronic parasitic infection with alternate outcomes- healing or non-healing. Leishmania invades macrophages and inflicts the complex of diseases called Leishmaniases. Depending on the species of Leishmania and the organs affected, the diseases are categorized into Cutaneous Leishmaniasis (CL), Muco-cutaneous Leishmaniasis (MCL) and Visceral Leishmaniasis (VL). After successful chemotherapy of VL, a dermal manifestation- termed post-kalazar dermal leishmaniasis (PKDL)- of the same infection occurs in some patients. The operational frameworks for different cytokines have been laid to discuss how these immune mediators control each of these forms of leishmaniases. One of these frameworks is the regulation of monocytopoiesis including the role of macrophages subsets and thrombopoiesis in leishmaniases. Macrophage metabolism is linked to different cytokines and is thereby associated with the manifestation of the resistance or susceptibility to Leishmania infection and of drug resistance. The chemokine-regulated immune cell movements present the landscape of infection and pathogenesis. T cells subsets- the IFN-γ-secreting Ly6C + T cells and the regulatory T cell subsets- provide the initial skewing of Th cell subset and regulation of effector Th subsets, respectively, eventually deciding the outcome of infection.

A further investigation of the leishmaniosis outbreak in Madrid (Spain): low-infectivity phenotype of the Leishmania infantum BOS1FL1 isolate to establish infection in canine cells.

Human leishmaniosis caused by Leishmania infantum is a zoonotic disease, with dogs as the main reservoir in Mediterranean Basin countries. The largest European outbreak of human leishmaniosis declared in the southwestern Madrid region (Spain) is characterized by unusual epidemiological and clinical features, such as the emergence of new wild reservoirs (hares and rabbits), whereas the seroprevalence, infection, and severity of canine leishmaniosis have not substantially changed since the first studies conducted in Madrid before the outbreak. Previous studies reported that L. infantum isolates from the Madrid leishmaniosis focus displayed elevated virulence in in vivo models of infection and increased infectivity in murine target cells. With the aim of studying whether changes in the host-parasite interaction and virulence profile have developed, we first assessed the behaviour of one circulating isolate of the outbreak, IPER/ES/2012/BOS1FL1 (BOS1FL1), compared to that of a well-characterized strain from canine leishmaniosis, MCAN/ES/1996/BCN150 (BCN150), in terms of infection capacity (percentage of infected cells, representing infectivity, and number of amastigotes per infected cell, representing the intensity of infection) in canine monocytes and macrophages. BCN150 displayed significantly higher infectivity (76.82 ±â€¯4.40 vs 38.58 ±â€¯2.19; P <  0.0001) and intensity of infection (3.64 ± 0.13 vs 1.83 ±â€¯0.12; P <  0.0001) than BOS1FL1 when interacting with canine cells. Our ROS induction results did not differ significantly between the two isolates or with the responses previously described for other L. infantum isolates. Paradoxically, increased resilience to hydrogen peroxide exposure was observed for BOS1FL1 (% viability 40.62 ±â€¯5.54 vs 26.37 ±â€¯2.93; P = 0.039). Finally, we demonstrated that a decreased intracellular load of BOS1FL1 was associated with increased IFN-γ (261.21 ±â€¯26.29 vs 69.80 ±â€¯9.02; P = 0.0151) and decreased IL-10 production (165.06 ±â€¯23.87 vs 264.41 ±â€¯30.58; P = 0.0002). In this study, we provide the first detailed insight into the differences between the isolate BOS1FL1 from the outbreak in Madrid and the well-characterized strain BCN150 MON-1 obtained from a dog in their response to interacting with canine cells. However, further studies are necessary to shed light on the immune mechanisms resulting in BOS1FL1 exhibiting less virulent behaviour in canine cells than in cells derived from other host species.

Characterization of ovine monocyte activity when cultured with Haemonchus contortus larvae in vitro.

AIMS: The objective of this study was to identify and characterize cell populations within ovine peripheral blood mononuclear cells (PBMCs) associated with Haemonchus contortus (Hc) larval morbidity and impairment in vitro. METHODS AND RESULTS: Monocytes and lymphocytes were separated from PBMC from parasite-resistant St. Croix (STC) sheep and parasite-susceptible Suffolk (SUF) sheep. Cells were cultured with Hc third stage larvae (L3) for 9 h. Larval morbidity was assessed using ATP concentration. Activation status was determined through gene expression analysis and enzyme inhibition. Enzymes arginase-1 (Arg1) and inducible nitric oxide synthase (iNOS) were inhibited using BEC (S-(2-boronoethyl)-I-cysteine) and 1400W (N-(3-(aminomethyl)benzyl)acetamidine), respectively. Larval ATP was lower when cultured with STC-derived monocytes (0.015 µmol/L ATP) compared to SUF-derived monocytes (0.067 µmol/L ATP) (P < .001), or lymphocytes from either breed (STC: 0.085 µmol/L, SUF: 0.112 µmol/L ATP) (P < .001). SUF-derived monocytes displayed higher expression of M1 genes, whereas STC-derived monocytes displayed M2 genes continuously. Inhibition of Arg1 decreased monocyte function in both breeds, whereas iNOS inhibition restored SUF-derived monocyte function. CONCLUSIONS: Together, these data indicate STC-derived monocytes favour M2 phenotype when exposed to L3, where SUF-derived monocyte function resembled M1 phenotype and described potential for improving Suffolk sheep through modulating inflammatory responses.

Role of growth hormone signaling pathways in the development of atherosclerosis.

OBJECTIVE: The direct actions of growth hormone (GH) in the development of atherosclerosis are unclear. The goal of this study was to characterize GH-induced changes in expression of signaling pathway elements and other proteins that may be related to atherosclerosis. METHODS: Human umbilical vein endothelial cells (HUVEC) and THP-1, a human acute monocytic leukemia cell line, were stimulated by exposure to 10-9 M or 10-8 M human GH with or without pretreatment with a mitogen-activated protein kinase kinase (MEK) 1 inhibitor. Levels of transcripts encoding vascular cell adhesion molecule (VCAM) -1, E-selectin, monocyte chemotactic protein (MCP-1), interleukin (IL) -6, and IL-8 were investigated by reverse transcription (RT) -PCR. For the quantitative adhesion assay, THP-1 cells or human primary monocytes were fluorescently labeled with 3'-O-acetyl-2',7'-bis(carboxyethyl) -4 diacetoxymethyl ester (BCECF/AM). HUVEC treated with human GH were co-incubated with BCECF-labeled THP-1 cells. One hour later, the number of BCECF-labeled THP-1 cells was assessed. An equivalent experiment was performed using BCECF-labeled primary monocytes, and the number of monocytes adhering to HUVEC was counted. RESULTS: Treatment with hGH increased the levels of E-selectin- and VCAM-1-encoding mRNAs in HUVEC. This effect was attenuated by pretreatment with a MEK1 inhibitor. Furthermore, hGH treatment increased adhesion of BCECF-labeled THP-1 cells or primary monocytes to HUVEC, and this effect was attenuated by pretreatment with a MEK1 inhibitor. CONCLUSIONS: VCAM-1 and E-selectin expression was stimulated by GH via the mitogen-activated protein kinase pathway, resulting in augmented adhesion of THP-1 cells and monocytes to HUVEC. These data suggested that GH directly stimulates the development of atherosclerosis.

An ex vivo multiparametric flow cytometry assay using human whole blood to simultaneously measure cytotoxicity and leishmanicidal activities.

Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.

Purification of Native Fasciola hepatica Fatty Acid-Binding Protein and Induction of Alternative Activation of Human Macrophages.

This chapter presents the different techniques to purify the native forms of Fasciola hepatica fatty acid-binding protein (Fh12) using size exclusion chromatography and isoelectric focusing (IEF). Also, it presents the procedure to study the immunological effect of the purified protein Fh12 using monocyte-derived macrophages (MDM) obtained from healthy human donors. For this purpose, I present the procedure to isolate and culture peripheral blood mononuclear cells (PBMCs) to generate alternatively activated macrophages (AAMΦ) by in vitro exposure to Fh12.

Possible Role for Toll-Like Receptors in Interaction of Fasciola hepatica Excretory-Secretory Products with Human Monocyte Cell Line.

This chapter presents a proteomic approach to purify and identify native excretory-secretory products (ESPs) in the range of >10-30 kDa proteins capable of interacting with toll-like receptors (TLRs). Here we present a protocol to fractionate the total ESPs using an ultrafiltration system to recover ESP proteins >10-30 kDa. The fraction of the proteins >10-30 kDa is purified by ion exchange chromatography (IEC) using a mono Q-column in a fast protein liquid chromatography system (FPLC) to separate its components based on charge. Finally, a screening system is presented using THP1-Blue CD14 cells to investigate whether TLRs could also be targeted by Fasciola hepatica ESPs and the interaction with TLR4 using HEK293 Blue-TLR4 cells.

Epigenetic regulation of defense genes by histone deacetylase1 in human cell line-derived macrophages promotes intracellular survival of Leishmania donovani.

Leishmania donovani, an intracellular protozoan parasite upon infection, encounters a range of antimicrobial factors within the host cells. Consequently, the parasite has evolved mechanisms to evade this hostile defense system through inhibition of macrophage activation that, in turn, enables parasite replication and survival. There is growing evidence that epigenetic down-regulation of the host genome by intracellular pathogens leads to acute infection. Epigenetic modification is mediated by chromatin remodeling, histone modifications, or DNA methylation. Histone deacetylases (HDACs) removes acetyl groups from lysine residues on histones, thereby leading to chromatin remodeling and gene silencing. Here, using L. donovani infected macrophages differentiated from THP-1 human monocytic cells, we report a link between host chromatin modifications, transcription of defense genes and intracellular infection with L. donovani. Infection with L. donovani led to the silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) transcript levels, protein expression, and enzyme activity showed a significant increase upon infection. HDAC1 occupancy at the promoters of the defense genes significantly increased upon infection, which in turn resulted in decreased histone H3 acetylation in infected cells, resulting in the down-regulation of mRNA expression of host defense genes. Small molecule mediated inhibition and siRNA mediated down-regulation of HDAC1 increased the expression levels of host defense genes. Interestingly, in this study, we demonstrate that the silencing of HDAC1 by both siRNA and pharmacological inhibitors resulted in decreased intracellular parasite survival. The present data not only demonstrate that up-regulation of HDAC1 and epigenetic silencing of host cell defense genes is essential for L. donovani infection but also provides novel therapeutic strategies against leishmaniasis.

Comparative Assessment of DNA Targets and Amplification Methods for Leishmania (Viannia) Detection in Human Samples.

Multiple polymerase chain reaction (PCR)-based approaches have been developed for Leishmania detection in clinical and laboratory samples, and this diversity limits inter-study comparisons, meta-analyses, and generalization of findings. Towards harmonization of a molecular tool for detection of Leishmania (Viannia) for research purposes, we evaluated the concordance of 18SrDNA quantitative polymerase chain reaction (qPCR) and minicircle kinetoplastid DNA (mkDNA) PCR followed by Southern blot (PCR-SB) in in vitro infection systems and in lesion and mucosal swab samples from Colombian patients with cutaneous leishmaniasis caused by L. (Viannia). The lower limit of parasite detection of 18SrDNA qPCR and mkDNA PCR-SB was 10-1 promastigotes and one intracellular amastigote per reaction. From cutaneous lesions (n = 63), an almost perfect concordance was found between the methods (κ = 0.92, 95% CI: 0.82-1.00). Despite equal limits of detection, mkDNA PCR-SB was more efficient for parasite detection in mucosal samples than 18SrDNA qPCR or 18SrDNA digital droplet PCR. The high concordance, sensitivity, scaling potential, and feasibility of implementation of the 18SrDNA qPCR, support its selection as the L. (Viannia) in research laboratories, as a first step towards harmonization of research protocols in the region.