Infection Of Mononuclear Cells Research Articles

HIV-Infected Hepatic Stellate Cells or HCV-Infected Hepatocytes Are Unable to Promote Latency Reversal among HIV-Infected Mononuclear Cells.

Due to a common mode of transmission through infected human blood, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) co-infection is relatively prevalent. In alignment with this, HCV co-infection is associated with an increased size of the HIV reservoir in highly active antiretroviral therapy (HAART)-treated individuals. Hence, it is crucial to comprehend the physiological mechanisms governing the latency and reactivation of HIV in reservoirs. Consequently, our study delves into the interplay between HCV/HIV co-infection in liver cells and its impact on the modulation of HIV latency. We utilized the latently infected monocytic cell line (U1) and the latently infected T-cell line (J-Lat) and found that mediators produced by the infection of hepatic stellate cells and hepatocytes with HIV and HCV, respectively, were incapable of inducing latency reversal under the studied conditions. This may favor the maintenance of the HIV reservoir size among latently infected mononuclear cells in the liver. Further investigations are essential to elucidate the role of the interaction between liver cells in regulating HIV latency and/or reactivation, providing a physiologically relevant model for comprehending reservoir microenvironments in vivo.

Effect of hydro-alcoholic extract of Panax ginseng and Ampicillin treatment in an animal model of Listeria monocytogenes-induced endocarditis.

Endocarditis is a rare but serious infection caused by Listeria monocytogenes. Panax ginseng demonstrated multiple immunomodulatory effects in earlier studies. Ampicillin is known as an effective antibiotic in the treatment of this disease. Therefore, this study aimed to evaluate the effect of hydro-alcoholic extract of P. ginseng and ampicillin treatment in an animal model of Listeria monocytogenes-induced endocarditis. Thirty mice, 5-7 weeks old, were randomly divided into five groups (n=6) including Healthy Control, Infected, Ampicillin (20 mg/kg, subcutaneous) treatment, Ginseng (0.025 mg/kg, intraperitoneal) treatment, and Ginseng (0.025 mg/kg, intraperitoneal) +Ampicillin (15 mg/kg, subcutaneous) treatment groups. The concentration of cytokines in heart tissue, such as IL-1 (interleukine-1), IL-6, IL-8, and TNF-α (Tumor Necrosis Factor-α), was measured. Histopathological changes were evaluated in heart tissues. The levels of cytokines were significantly decreased in the Ampicillin+Ginseng treated group compared to the other experimental groups. Microscopically, pathologic changes in heart tissue were concomitant with biochemical findings, which in the infected group, neutrophils and mononuclear cells infiltration in endocardial tissue, myocardial cell necrosis, and edema were detectable. The Ampicillin+Ginseng group showed no significant changes compared to the normal control group. This study showed that ginseng hydro-alcoholic extract plus ampicillin has better efficacy than the extract or antibiotic alone against experimental endocarditis caused by Listeriosis.

Association between proliferation status of infected and non-infected mononuclear cells with tissue lesions in acute bovine theileriosis.

Tropical or Mediterranean theileriosis in dairy cattle is widely distributed in many tropical regions of the world. The purpose of this study was to evaluate the proliferation status of mononuclear cells infected with Theileria annulata schizonts in different tissues and its relationship with the pathogenesis of the parasite in cattle by histopathology, immuno-histochemistry and polymerase chain reaction (PCR). Blood and tissue samples of eight Holstein cattle that had been lost due to theileriosis and eight healthy slaughtered cattle of the same breed were collected as a control group after necropsy. The piroplasms in the blood smears and the schizonts in the cytoplasm of the lymphocytes and macrophages of the lymph nodes were microscopically detected. Histopathologically, the proliferation of macrophages, lymphocytes, and plasma cells in lymph nodes and the heart, congestion, and bleeding in the red pulp of the spleen, portal tracts of the liver, interstitial tissue of the kidneys, multifocal necrosis and ulceration in the abomasum together with hyperemia and hemorrhages and lymphoblastic infiltration in the submucosa and lamina propria adjacent to these lesions and emphysema with ecchymotic hemorrhage in the lungs were evident. Immunohistochemistry identified the proliferated cells as mostly Cluster of Differentiation 3- Positive T lymphocytes and macrophage marker antibody 387- positive macrophages. Positive results of PCR for the Tams1 30.00 kDa gene were observed in lymph nodes, liver, lung and abomasum. It was concluded that the pathological changes were the result of schizont-infected macrophage proliferation leading to severe uncontrolled proliferation of uninfected T lymphocytes.

HTLV-1 Carriers with Circulating Dominant Clones Harbor ATL-like Mutational and Transcriptomic Profiles Which Identify an Ultra-High Risk Group for Disease Transformation

Background: Identifying carriers of human T cell leukaemia virus type -1 (HTLV-1) at high risk of developing the aggressive T-cell malignancy adult T-cell leukaemia/lymphoma (ATL) is important as it will provide the opportunity for therapeutic intervention before disease transformation, which typically results in dismal outcomes. This further provides a rationale for broadening HTLV-1 screening. The pre-transformation processes take decades but remain elusive, with the main established risk factor being a high proviral load (proportion of infected mononuclear cells >4%) but this lacks specificity. We recently reported that ATL-like clonal T cell expansions can be identified by flow-cytometry based on T cell receptor (TCR)Vβ subunits in a subset of high-load HTLV-1 carriers ('oligoclonality-flow, OCI-flow') and that a high degree of oligoclonality (OCI flow-score >0.770), usually with a single dominant clone, predicts transformation risk. In this study, we characterize the mutational and transcriptomic profiles in these dominant clones in otherwise healthy carriers. Methods: A cohort of 19 high proviral load HTLV-1 infected individuals was selected, of whom 9 had a high OCI-flow consistent with ATL-like clonal expansion, and 10 had low OCI-flow (<0.770). Flow-sorting was used to isolate hyper-expanded ATL-like clones based on the immunophenotype CD3+CD4+CCR4+CD26-TCRVβX+ (representing the major clone) as well sorting fractions CD3+CD4+CCR4+CD26-TCRVβOther (infected polyclonal cells), CD3+CD4+CCR4-/CCR4+CD26+ cells (uninfected populations- termed 'other CD4+') and CD33/19+ (germline) cells. RNA was extracted from all flow-sorted populations for sequencing (RNA-Seq). DNA was extracted from CD33+/19+ cells for whole exome sequencing (WES). TCRα and -β complementarity-determining region 3 (CDR3) sequences were extracted and analysed with MiXCR software. Variant calling was performed using Mutect2 using paired RNA ('tumor') and DNA from CD33/CD19+ cells ('normal') samples. Differential gene expression was performed using DeSeq2. Results: Presence of a hyper-expanded major clone in high OCI samples was confirmed by analysis of CDR3 sequence diversity. Two of the nine subjects with a major clone also had an additional 'minor’ hyper-expanded T cell clone within the HTLV-1 infected polyclonal cell population. Eight of nine major clones (89%) harbored at least one mutation in a gene reported to be frequently mutated in ATL (VAF > 0.1, median 1 mutation, range 0-4). Both minor clones also harbored mutations in ATL driver genes. In total, sixteen mutations in genes that are frequently mutated in ATL were identified (Figure 1). These included mutations in CSNK2B (n =2), PRKCB (n =2), and CIC (n = 2) and CCR4 (n =1). Three subjects had mutations in a gene in the T-cell receptor/nuclear factor kappa B (TCR/NFkB) pathway, and all three subsequently transformed to ATL (median time since first detection of mutation(s) = 5.2 months). No other subject has yet transformed to ATL. There were no mutations detected in genes that are frequently mutated in ATL in any subject with low OCI-flow. Differential expression analysis demonstrated similarity in patterns of gene expression between major clones and ATL cells, and were distinct from patterns observed in infected polyclonal populations. Most differentially expressed genes in major clones were downregulated relative to uninfected CD4+ cells, and the majority (58%) of downregulated genes were downregulated in major clones compared to other HTLV-1 infected cells. Gene ontology analysis of differential gene expression in major clones showed enrichment of ATL-related pathways as well as suppression of pathways in lymphocyte differentiation and immune receptor activity. Conclusions: High proviral load HTLV-1 carriers with high OCI-flow cytometry scores carry hyper-expanded T cell clones with mutational and transcriptomic profiles that resemble ATL prior to transformation. TCR/NFkB pathway mutations were detected in cases who subsequently developed ATL and may represent a later stage in the transformation process. This offers the potential to identify ultra high-risk HTLV-1 carriers for novel pre-emptive, therapeutic intervention. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Future approaches for Brucellosis vaccines and therapies development based on molecular host-pathogen interactio

This review discusses Brucella-host interactions on molecular base and brucellosis immunobiological response. Also, the review handles pathogenesis-informed rationales to prevent or treat brucellosis. Brucella species is an animal pathogen that may cause incidental human brucellosis as a zoonotic disease. Brucellosis results in worldwide economic losses, human morbidity, and poverty. Despite Brucella species infect humans as an incidental host, 500,000 new human brucellosis occur annually, and no approved human vaccines or patient-friendly therapies are available. Brucella has strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that hinders its exposure to innate and adaptive immune responses, sequesters the pathogen from antibiotics effect and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion system(T4SS) dependent Brucella-containing vacuoles with lysosomal compartments, ii) inhibition of apoptosis of infected mononuclear cells, and iii) prevention of DC maturation, antigen presentation, and activation of naive T cells, pathogenesis lessons that may be informative for other intracellular pathogens. Data sets of NGS of Brucella and host time-series global expression fused with proteomics and metabolomics data from in vitro and in vivo experiments now approved human vaccines inform interactive cellular pathways and gene regulatory networks enabling full-scale systems biology analysis. The newly identified effector proteins of Brucella may represent novel targets for safer and more effective brucellosis vaccines and treatment.

Distribution of Pseudorabies Virus Antigen in Hunting Dogs with Concurrent Paragonimus westermani Infection

Following isolation of pseudorabies virus (PRV) from two hunting dogs in Oita prefecture, Japan, we investigated the PRV antigen distribution in the tissues of the infected animals. At necropsy, PRV-associated lesions included facial oedema, tonsillar and meningeal congestion, blotchy haemorrhages on the pericardium and mitral valves, and incomplete splenic contraction in one dog, with less prominent findings in the other dog. Multiple pulmonary nodules were seen in both cases, caused by the diploid form of Paragonimus westermani lung flukes, as confirmed by PCR-restriction fragment length polymorphism and gene sequencing analyses. Histological examination revealed that the PRV infection was associated with lesions of non-suppurative encephalitis in the brainstem. PRV antigen was detected in the cerebrum, cerebellum, brainstem, submucosal and myenteric plexuses, and mononuclear cells, mainly in the bone marrow, lymph nodes, tonsils and spleen tissues. There was evidence of PRV dissemination to the brain via the trigeminal or olfactory routes, in addition to possible spread to lymphoid organs via infected mononuclear cells.

Association of circulating fibroblast growth factor-2 with progression of HIV-chronic kidney diseases in children.

Children living with HIV frequently show high plasma levels of fibroblast growth factor-2 (FGF-2/bFGF). FGF-2 accelerates the progression of several experimental kidney diseases; however, the role of circulating FGF-2 in childhood HIV-chronic kidney diseases (HIV-CKDs) is unknown. We carried out this study to determine whether high plasma FGF-2 levels were associated with the development of HIV-CKDs in children. The plasma and urine FGF-2 levels were measured in 84 children (< 12 years of age) living with HIV during the pre-modern antiretroviral era, and followed for at least 3 years to determine the prevalence of proteinuria and HIV-CKDs. We also assessed the distribution of the kidney FGF-2 binding sites by autoradiography and Alcian blue staining, and explored potential mechanisms by which circulating FGF-2 may precipitate HIV-CKDs in cultured kidney epithelial and mononuclear cells derived from children with HIV-CKDs. High plasma FGF-2 levels were associated with a high viral load. Thirteen children (~ 15%) developed HIV-CKDs and showed a large reservoir of FGF-2 low-affinity binding sites in the kidney, which can facilitate the recruitment of circulating FGF-2. Children with high plasma and urine FGF-2 levels had 73-fold increased odds (95% CI 9-791) of having HIV-CKDs relative to those with normal FGF-2 values. FGF-2 induced the proliferation and decreased the expression of APOL-1 mRNA in podocytes, and increased the attachment and survival of infected mononuclear cells cultured from children with HIV-CKDs. High plasma FGF-2 levels appear to be an additional risk factor for developing progressive childhood HIV-CKDs.

In vitro dual (anticancer and antiviral) activity of the carotenoids produced by haloalkaliphilic archaeon Natrialba sp. M6

Halophilic archaea are a promising natural source of carotenoids. However, little information is available about the biological impacts of these archaeal metabolites. Here, carotenoids of Natrialba sp. M6, which was isolated from Wadi El-Natrun, were produced, purified and identified by Raman spectroscopy, GC-mass spectrometry, and Fourier transform infrared spectroscopy, LC–mass spectrometry and Nuclear magnetic resonance spectroscopy. The C50 carotenoid bacterioruberin was found to be the predominant compound. Because cancer and viral hepatitis are serious diseases, the anticancer, anti-HCV and anti-HBV potentials of these extracted carotenoids (pigments) were examined for the first time. In vitro results indicated that the caspase-mediated apoptotic anticancer effect of this pigment and its inhibitory efficacy against matrix metalloprotease 9 were significantly higher than those of 5-fluorouracil. Furthermore, the extracted pigment exhibited significantly stronger activity for eliminating HCV and HBV in infected human blood mononuclear cells than currently used drugs. This antiviral activity may be attributed to its inhibitory potential against HCV RNA and HBV DNA polymerases, which thereby suppresses HCV and HBV replication, as indicated by a high viral clearance % in the treated cells. These novel findings suggest that the C50 carotenoid of Natrialba sp. M6 can be used as an alternative source of natural metabolites that confer potent anticancer and antiviral activities.

Identification of Mycobacterium avium subsp. hominissuis secreted proteins using an in vitro system mimicking the phagosomal environment.

Background Mycobacterium avium subsp. hominissuis is a common intracellular pathogen that infects patients with HIV/AIDS and cause lung infection in patients with underlying lung pathology. M.avium preferably infects macrophages and uses diverse mechanisms to alter phagosome maturation. Once in the macrophage, the pathogen can alter the host cellular defenses by secreting proteins into the cytosol of host cells, but despite considerable research, only a few secreted effector proteins have been identified. We hypothesized that the environmental cues inside the phagosome can trigger bacterial protein secretion. To identify M. avium secretome within the phagosome, we utilized a previously established in vitro system that mimics the metal ion concentrations and pH of the M. avium phagosome.Results M. avium was exposed to phagosome metal concentrations for different time points and exported proteins were profiled and analyzed against bacterial proteins secreted in the culture medium. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 46 were unique to bacteria incubated in the metal mixture. Ten of potential effectors were selected and secretion of these proteins was monitored within M. avium infected mononuclear phagocytic cells using the beta-lactamase FRET-based reporter system. In addition, pull-down assay was performed for secreted calmodulin-like protein MAV_1356 protein to evaluate for eukaryotic target. All examined M. avium proteins were secreted into the macrophage cytosol, and gene expression analysis suggested that the metal environment likely stimulates secretion of pre-made proteins. Further investigation of bacterial secreted MAV_1356 protein, lead to the observation that the MAV_1356 interacts with the host proteins Annexin A1 and Protein S100-A8.ConclusionsWe established an in vitro system for the study if proteins secreted intracellularly, and revealed that the metal mixture mimicking the concentration of metals in the phagosome environment, triggers protein secretion.

Bacillary replication and macrophage necrosis are determinants of neutrophil recruitment in tuberculosis

We previously determined that burst size necrosis is the chief mode of mononuclear cell death in the lungs of mice with tuberculosis. The present study explored the link between infection-induced necrosis of mononuclear phagocytes and neutrophil accumulation in the lungs of mice challenged with one of four Mycobacterium tuberculosis strains of increasing virulence (RvΔphoPR mutant, H37Ra, H37Rv and Erdman). At all time points studied, Erdman produced the highest bacterial load and the highest proportion and number of M. tuberculosis-infected neutrophils. These parameters, and the proportion of TUNEL-positive cells, tracked with virulence across all strains tested. Differences in neutrophil infection were not reflected by levels of chemoattractant cytokines in bronchoalveolar lavage fluid, while interferon-γ (reported to suppress neutrophil trafficking to the lung in tuberculosis) was highest in Erdman-infected mice. Treating Erdman-infected mice with ethambutol decreased the proportion of mononuclear phagocytes with high bacterial burden and the ratio of infected neutrophils to infected mononuclear cells in a dose-dependent manner. We propose that faster replicating M. tuberculosis strains cause more necrosis which in turn promotes neutrophil recruitment. Neutrophils infected with M. tuberculosis constitute a biomarker for poorly controlled bacterial replication, infection-induced mononuclear cell death, and increased severity of immune pathology in tuberculosis.

Immunobiological activity and antiviral regulation efforts of Chinese goose (Anser cygnoides) CD8α during NGVEV and GPV infection

The CD8 molecule is a cell membrane glycoprotein expressed on cytotoxic T lymphocytes, which are involved in the clearance of viruses. However, the functional characterization of goose CD8α is still unclear. The immunobiological characterization of goose CD8α in goose spleen mononuclear cells (MNCs) was examined by real-time quantitative PCR (qPCR). It was shown that CD8α mRNA levels were significantly up-regulated by in vitro treatment of MNCs with phytohemagglutinin (PHA), concanavalin A (ConA), and polyinosinic-polycytidylic acid (poly I:C) in a dose-dependent way, but lipopolysaccharides (LPSs) did not have this same effect. Moreover, the time-course effect of CD8α expression in response to mitogens (PHA, ConA, and poly I:C) was evaluated in MNCs. A significant increase in the transcriptional levels of CD8α was detected in new type gosling viral enteritis virus (NGVEV)–infected goose MNCs at 48 h postinfection (PI) and in goose parvovirus (GPV)–infected MNCs at 72 h PI. Also, the number of CD8α+ cells was significantly increased during viral infection from 72 h on. The seminal changes in mRNA profiles of antiviral cytokines (IFN-α, IFN-γ, and IL-18) were observed and were significantly increased during late phases of NGVEV and GPV infection. Accordingly, our data not only contribute to the understanding of the immune characteristics of goose CD8α, but they also provide new insight into the innate antiviral immunity of geese.

Killed but Metabolically Active Mycobacterium bovis bacillus Calmette-Guérin Retains the Antitumor Ability of Live bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin is the most effective treatment for high risk noninvasive bladder cancer. Although bacillus Calmette-Guérin immunotherapy clearly decreases recurrence and progression rates, side effects are common and infection with the bacillus has been described. For these reasons it is necessary to find safer alternatives to the live bacillus. We explored the possibility of using killed but metabolically active bacillus Calmette-Guérin. T24, J82 and RT4 bladder tumor cell lines were cultured with live and irradiation or heat treated bacillus Calmette-Guérin Connaught. We measured the inhibition of cell proliferation and the production of cytokines in cell culture supernatants. Peripheral mononuclear blood cells were also infected and the production of different cytokines in cell culture supernatants was analyzed. Peripheral blood mononuclear cell and cell culture supernatants activated by mycobacteria were then cultured with T24 cells to analyze whether they showed cytotoxic activity. Compared to the other bacillus Calmette-Guérin treatments, γ irradiated bacillus Calmette-Guérin showed activity similar to that of the live bacillus for inhibiting tumor growth and inducing cytokine production. Irradiated bacillus Calmette-Guérin showed metabolic activity and, thus, was considered killed but metabolically active. This is the treatment that most accurately preserved the mycobacterial structure. Killed but metabolically active bacillus Calmette-Guérin induced cytokine production by infected peripheral mononuclear blood cells. Mycobacteria activated peripheral blood mononuclear cell and cell supernatants showed cytotoxic activity against tumor cells, retaining the antitumor capacity of the live bacillus. Our results suggest that killed but metabolically active bacillus Calmette-Guérin could be considered a safer immunotherapy alternative to treatment with the live bacillus.

Efficiency of Cell-Free and Cell-Associated Virus in Mucosal Transmission of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

Effective strategies are needed to block mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Here, we address a crucial question in HIV-1 pathogenesis: whether infected donor mononuclear cells or cell-free virus plays the more important role in initiating mucosal infection by HIV-1. This distinction is critical, as effective strategies for blocking cell-free and cell-associated virus transmission may be different. We describe a novel ex vivo model system that utilizes sealed human colonic mucosa explants and demonstrate in both the ex vivo model and in vivo using the rectal challenge model in rhesus monkeys that HIV-1-infected lymphocytes can transmit infection across the mucosa more efficiently than cell-free virus. These findings may have significant implications for our understanding of the pathogenesis of mucosal transmission of HIV-1 and for the development of strategies to prevent HIV-1 transmission.

PHARMACOLOGICAL STUDIES ON BUPARVAQUONE IN CATTLE

This study was planned to study the effect of buparvaquone on clinical signs, blood film, haematology and some liver and kidney function parameters of both normal and thielerial diseased cattle. Thirty Holstien cattle (2-4 years old) with average body weight 450-650 kg in a special farm at Dakahlia Governorate were used in this study. Ten of them showed clinical signs of theileriosis and confirmed by a thin blood film. They received buparvaquone once (2.5 mg/kg body weight). While the other 20 animals displayed no clinical symptoms,10 of them were kept as a control non treated group while, The last 10 animals showed no clinical signs and received the drug (2.5 mg/kg body weight) and kept as treated group. The obtained results revealed that buparvaquone (2.5 mg/kg body weight) given by intramuscular injection at once resulted in a complete disappearance of all clinical signs of the disease after 4 days post injection. Moreover, the present study showed abnormal erythrocyte morphology with occurrence of theileria schizoints in infected mononuclear cells and erythrocytes. The infected erythrocytes appeared as echinocytes thorn-like protrusions on the surface on blood smears which prepared directly from ear vein of all diseased animals. . Our haematological findings showed a significant decrease in the total erythrocyticand leukocyticcounts, haemoglobin concentration, and PCV% in the infected treated group when compared with control and non infected treated groups. The results also reflected that the diseased treated group showed a significant decrease in total protein, albumen, globulins, while a significant increase in Alanine Transaminase (ALT), Aspartate Transaminase (AST) and Alkaline Phosphatase (ALP) with a significant increase in serum bilirubin compared with other two groups.

Increased Production of Interleukin-4, Interleukin-10, and Granulocyte-Macrophage Colony-Stimulating Factor by Type 2 Diabetes’ Mononuclear Cells Infected with Dengue Virus, but Not Increased Intracellular Viral Multiplication

It has been reported that diabetes mellitus (DM) was an epidemiologically identified risk factor for development of dengue hemorrhagic fever (DHF)/severe dengue in dengue virus (DENV) affected patients, and T helper 2 (Th2) cytokines such as interleukin-4 (IL-4) and IL-10 each plays an important role in the immunopathogenesis of DHF in studies involving general population. To better understand the relationship between these epidemiological and immunological findings, we performed an in vitro study evaluating the sequential immunological reactions and viral load in the DENV infected mononuclear cells of adults with type 2 DM (T2DM group, n = 33) and normal adults (control group, n = 29). We found in the T2DM group significantly higher IL-4 level on the first (P = 0.049) and the third (P = 0.022) postinfection days, while higher IL-10 (P = 0.042) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P = 0.009) were detected on the third postinfection day. No significant difference in DENV viral load between the cultured mononuclear cells from both groups was found on the first and third post-infection days. These data immunologically suggest that patients with T2DM are at higher risk for development of DHF/severe dengue and strengthen the previously epidemiologically identified role of DM being a predictive risk factor for progressing into DHF/severe dengue in DENV-affected patients.

Expression of late viral proteins is restricted in nasal mucosal leucocytes but not in epithelial cells during early-stage equine herpes virus-1 infection

Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.

Efectos de la infección in vitro de células monocítico-macrofágicas por Chlamydia y su relación con los valores de lípidos

Introduction The mononuclear-fagocitic cellular type is a key element during the atherogenic process. The monocyte takes a transcendental role in the inflammatory process that is relevant at the onset and subsequent development of atheroma plaque. Otherwise, the cellular infiltration is a key feature to consider during the inflammatory process that we can found related with the atherosclerosis/infection association. According with the infectious explanation of atherosclerosis, microorganism more frequent associated with vascular pathology has been: Citomegalovirus, Helicobacter and Chlamydia. In the present paper we are interested to evaluate the characteristics and cellular behaviour of cultures of cells related with atherogenic process that has been previously infected with Chlamydia. Material and methods We have analized established human endothelial cells (Hep-2 cells), established mononuclear cells (THP-1 cells) and primary cultures of monocytes from blood health volunteers. Results All of cells types has been easily infected by Chlamydia. Nevertheless, the effects are different according with the different cells types. When we have compared the effect of normo or hyperlipidemic serum specimens over the cultures of infected mononuclear cells it is possible to show that the effect of infection is amortiguated by the presence of high serum levels of lipids.

Role of specific antibodies in Coxiella burnetii infection of macrophages

Q fever is a zoonosis with worldwide distribution caused by Coxiella burnetii, an obligate intracellular bacterium. Q fever is commonly divided into acute and chronic forms. Acute Q fever manifestations consist of self-limited febrile illness, pneumonia and granulomatous hepatitis as well as neurological disorders and miscellaneous manifestations [1]. The manifestations of chronic Q fever are endocarditis and, less frequently, vascular aneurysm and prosthesis infections. These usually occur in patients with previous vascular or valvular disease or in a context of immunosuppression. The chronic disease is characterised by impaired immune response, defective microbicidal activity of monocytes, and production of antibodies (Abs) directed against C. burnetii. Here, we examined the role of specific antibodies in the infection of human macrophages by C. burnetii. Immunoglobulins G (IgG) from three patients with chronic Q fever and three healthy controls were purified using protein A-sepharose columns (Pharmacia, Uppsala, Sweden). Their ability to opsonise C. burnetii was investigated by flow cytometry. About 85% of phase I organisms were opsonised by specific Abs (1 ⁄ 200 dilution) whereas irrelevant IgG were unable to opsonise bacteria. The intracellular fate of opsonised organisms within human monocyte-derived macrophages (MDM) was studied by real-time PCR as previously described [2]. Opsonised organisms intensively replicated within MDM whereas unopsonised organisms survived but poorly replicated: after 9 days, the bacterial load was 10 times higher in MDM infected with opsonised bacteria (98 400 vs. 10 200 C. burnetii DNA copies for 10 MDM). In addition, electron microscopy revealed that the replication of opsonised organisms was associated with the formation of spacious parasitophore vacuoles containing large amounts of bacteria (Fig. 1a), which were absent in MDM infected with unopsonised bacteria (Fig. 1b). At day 9 post-infection, about 30% of MDM infected with opsonised bacteria presented large vacuoles (Fig. 1c) whereas only 3% of MDM displayed large vacuoles when infected with unopsonised bacteria (Fig. 1c). Note that the large parasitophore vacuoles presented an electron-dense membrane and seemed unable to fuse with lysosomes (Fig. 1a). This prompted us to characterise the nature of the large vacuoles by scanning fluorescence confocal microscopy using cathepsin D as a marker of phagolysosomes [3]. It has been previously shown that C. burnetii prevents the phagosome conversion and survives in monocytes ⁄macrophages within late phagosomes, in contrast to avirulent and inactivated organisms, which are eliminated within phagolysosomes [3,4]. At day 9 post-infection, opsonised organisms did not co-localise with cathepsin D (Fig. 1d), demonstrating that the replicative large vacuoles are not phagolysosomes. Classically, it is believed that Abs provide protection against pathogens through different mechanisms, including opsonisation. We demonstrated here that the high levels of specific Abs found in chronic Q fever, which are likely to be related to the host inability to cure the infection, favoured C. burnetii replication within human MDM. In patients with C. burnetii endocarditis, the major manifestation of chronic Q fever, C. burnetii was found in infected cardiac valves as voluminous intracytoplasmic masses within infected mononuclear cells, and not extracellularly as for other aetiological agents of infective endocarditis [1]. We can hypothesise that C. burnetii-specific Abs produced during chronic Q fever are responsible for C. burnetii infection of cardiac valves. We also demonstrated that C. burnetii prevented the phagosome conversion because large parasitophore vacuoles containing organisms did not express cathepsin D, a specific Corresponding author and reprint requests: E. Ghigo, URMITE, Faculte de Medecine, 27 Bld. Jean Moulin, 13385 Marseille Cedex 05, France E-mail: eric.ghigo@univmed.fr

Atoxoplasmosis in Canary Fledglings: Severe Lymphocytic Enteritis with Preferential Parasitism of B Lymphocytes

All fledgling canaries (Serinum canarius) in a small private aviary died from atoxoplasmosis during a single breeding season. The birds were clinically normal when removed from their parents at 2 mo of age, but by 3 mo of age all had died following an illness characterized by progressive lethargy, anorexia, and debilitation. Cachexia, splenomegaly, and pale foci in the liver were evident at necropsy. Microscopically, there was striking infiltration of the intestinal lamina propria by mononuclear cells that contained intracytoplasmic protozoa. Protozoa were also observed in mononuclear cells in splenic and hepatic sinusoids and in vascular or perivascular spaces of other organs, but were much less numerous. Ultrastructural features of infected enteric mononuclear cells were suggestive of lymphocytes, and the majority of parasitized cells in paraffin sections of intestine were positive for CD-79 antigen, consistent with B lymphocytes. CD-3 staining was minimal, suggesting little or no T-cell infection. The following year, after egg-laying was completed, adults were treated with sulfadimethoxine, and no further fledgling losses occurred.

Mycolactone Diffuses from Mycobacterium ulcerans–Infected Tissues and Targets Mononuclear Cells in Peripheral Blood and Lymphoid Organs

BackgroundBuruli ulcer (BU) is a progressive disease of subcutaneous tissues caused by Mycobacterium ulcerans. The pathology of BU lesions is associated with the local production of a diffusible substance, mycolactone, with cytocidal and immunosuppressive properties. The defective inflammatory responses in BU lesions reflect these biological properties of the toxin. However, whether mycolactone diffuses from infected tissues and suppresses IFN-γ responses in BU patients remains unclear.Methodology/Principal FindingsHere we have investigated the pharmacodistribution of mycolactone following injection in animal models by tracing a radiolabeled form of the toxin, and by directly quantifying mycolactone in lipid extracts from internal organs and cell subpopulations. We show that subcutaneously delivered mycolactone diffused into mouse peripheral blood and accumulated in internal organs with a particular tropism for the spleen. When mice were infected subcutaneously with M. ulcerans, this led to a comparable pattern of distribution of mycolactone. No evidence that mycolactone circulated in blood serum during infection could be demonstrated. However, structurally intact toxin was identified in the mononuclear cells of blood, lymph nodes and spleen several weeks before ulcerative lesions appear. Importantly, diffusion of mycolactone into the blood of M. ulcerans–infected mice coincided with alterations in the functions of circulating lymphocytes.ConclusionIn addition to providing the first evidence that mycolactone diffuses beyond the site of M. ulcerans infection, our results support the hypothesis that the toxin exerts immunosuppressive effects at the systemic level. Furthermore, they suggest that assays based on mycolactone detection in circulating blood cells may be considered for diagnostic tests of early disease.