Magali Garcia

Distribution of Spontaneous gyrA Mutations in 97 Fluoroquinolone-Resistant Helicobacter pylori Isolates Collected in France

ABSTRACT We determined the prevalence of gyrA mutations conferring fluoroquinolone resistance in 97 Helicobacter pylori isolates collected in France from 2007 to 2010. Ninety-four harbored one or two mutations already found in the quinolone resistance determining region (QRDR) of gyrA (for T87I, n = 23; for N87K, n = 32; for D91N, n = 30; for D91G, n = 7; for D91Y, n = 6), 2 harbored a mutation never previously described (D91H and A88P), and one strain was resistant (ciprofloxacin MIC of 8 mg/liter) without a detected mutation conferring this resistance in gyrA or gyrB genes.

Chemokines and Antimicrobial Peptides Have a cag-Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

ABSTRACT Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. The cag pathogenicity island (cag PAI) of H. pylori allows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response to H. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori B128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models with H. pylori B128ΔcagM, a cag PAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells with H. pylori, inflammatory-mediator production was largely due to cag PAI substrate-independent virulence factors. Thus, H. pylori cag PAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation during H. pylori infection.

Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

ABSTRACT Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5′-terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5′ stem-loop I, which is an RNA secondary structure required for viral RNA replication. In this study, we assessed the consequences of the genomic deletions observed in vivo for coxsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5′ deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5′ deletions greatly reduced the synthesis of viral RNA in vitro, which was detected only for the 7- and 21-nucleotide deletions. Since 5′ stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA replication, we investigated the binding of the host cell protein PCBP2, as well as viral protein 3CDpro, to deleted positive-strand RNAs corresponding to the 5′ end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CDpro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA replication observed for the 5′-deleted viral genomes—a less stable ribonucleoprotein complex formed with proteins involved in viral RNA replication. IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one-third of idiopathic dilated cardiomyopathies. Terminal deletions at the 5′ end of the viral genome involving an RNA secondary structure required for RNA replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA replication. In this report, we demonstrate that transfected RNAs harboring 5′-terminal sequence deletions are able to direct the synthesis of viral proteins, but not genomic RNAs, in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral replication factors to viral RNA is conserved despite genomic deletions but that the impaired RNA synthesis associated with terminally deleted viruses could be due to destabilization of the ribonucleoprotein complexes formed.

Inflammatory signaling pathways induced by Helicobacter pylori in primary human gastric epithelial cells

Inflammatory signaling pathways induced by Helicobacter pylori remain unclear, having been studied mostly on cell-line models derived from gastric adenocarcinoma with potentially altered signaling pathways and nonfunctional receptors. Here, H. pylori-induced signaling pathways were investigated in primary human gastric epithelial cells. Inflammatory response was analyzed on chemokine mRNA expression and production after infection of gastric epithelial cells by H. pylori strains, B128 and B128ΔcagM, a cag type IV secretion system defective strain. Signaling pathway involvement was investigated using inhibitors of epidermal growth factor receptor (EGFR), MAPK, JAK and blocking Abs against TLR2 and TLR4. Inhibitors of EGFR, MAPK and JAK significantly reduced the chemokine mRNA expression and production induced by both H. pylori strains at 3 h and 24 h post-infection. JNK inhibitor reduced chemokine production at 24 h post-infection. Blocking Abs against TLR2 but not TLR4 showed significant reduction of chemokine secretion. Using primary culture of human gastric epithelial cells, our data suggest that H. pylori can be recognized by TLR2, leading to chemokine induction, and that EGFR, MAPK and the JAK/STAT signaling pathways play a key role in the H. pylori-induced CXCL1, CXCL5 and CXCL8 response in a cag pathogenicity island-independent manner.

Skin innate immune response to flaviviral infection

Skin is a complex organ and the largest interface of the human body exposed to numerous stress and pathogens. Skin is composed of different cell types that together perform essential functions such as pathogen sensing, barrier maintenance and immunity, at once providing the first line of defense against microbial infections and ensuring skin homeostasis. Being inoculated directly through the epidermis and the dermis during a vector blood meal, emerging Dengue, Zika andWest Nile mosquito-borne viruses lead to the initiation of the innate immune response in resident skin cells and to the activation of dendritic cells, which migrate to the draining lymph node to elicit an adaptive response. This literature review aims to describe the inflammatory response and the innate immune signalization pathways involved in human skin cells during Dengue, Zika and West Nile virus infections.

Manifestations cutanées inhabituelles au cours d’une leishmaniose viscérale associée au VIH

BACKGROUND Visceral leishmaniasis is not normally expressed in skin. Herein, we describe the case of an HIV-positive patient who developed two unusual skin manifestations during an episode of visceral leishmaniasis. PATIENTS AND METHODS A 48-year-old female patient consulted initially for infiltrated purpura of all four limbs. Skin biopsy revealed leukocytoclastic vasculitis with Leishman-Donovan bodies. Laboratory tests showed medullary, splenic, gastric and colic involvement, suggesting systemic disease, and enabling visceral leishmaniasis to be diagnosed. Two years later, despite prolonged treatment, the patient presented maculopapular exanthema, and histology revealed persistent Leishman-Donovan bodies. DISCUSSION We report herein an association of two rare skin manifestations in an HIV-positive patient with visceral leishmaniasis: infiltrated purpura and maculopapular exanthema. However, the underlying mechanisms remain hypothetical. The initial leukocytoclastic exanthema could be secondary to either polyclonal hypergammaglobulinaemia or to IgA deposits, or possibly to mechanical impairment of blood vessels by the actual parasite. The maculopapular exanthema occurring later raised the possibility of post-Kala-Azar leishmaniasis due to blood-borne dissemination in an anergic subject or perhaps even immune reconstitution inflammatory syndrome.

Anti-inflammatory activity of Crateva adansonii DC on keratinocytes infected by Staphylococcus aureus: From traditional practice to scientific approach using HPTLC-densitometry

ETHNOPHARMACOLIGICAL RELEVANCE Leaves of Crateva adansonii DC (Capparidaceae), a small bush found in Togo, are widely used in traditional medicine to cure infectious abscesses. Traditional healers of Lomé harvest only budding leaves early in the morning, in specific area in order to prepare their drugs. AIM OF THE STUDY The main goal was to validate the ancestral picking practices, and to assess the activity of C. adansonii medicine towards infectious abscesses. MATERIALS AND METHODS A phytochemical screening of various C. adansonii leaf samples was performed using an original HPTLC-densitometry protocol and major flavonoids were identified and quantified. C. adansonii samples were collected in different neighborhoods of Lomé, at different harvesting-times and at different ages. Radical scavenging capacity, using DPPH assay, was used to quickly screen all extracts. Extracts were tested for anti-Staphylococcus aureus activity and anti-inflammatory effect on human primary keratinocytes infected by S. aureus. IL6, IL8 and TNFα expression and production were assessed by RT-PCR and ELISA assays. RESULTS Using antioxidant activity as selection criteria, optimal extracts were obtained with budding leaves, collected at 5:00am in Djidjolé neighborhood. This extract showed the strongest anti-inflammatory effect on S. aureus-infected keratinocytes by reducing IL6, IL8 and TNFα expression and production. None of the extracts inhibited the growth of S. aureus. CONCLUSIONS Those results validate the traditional practices and the potential of C. adansonii as anti-inflammatory drug. Our findings suggest that traditional healers should add to C. adansonii leaves an antibacterial plant of Togo Pharmacopeia, in order to improve abscess healing.

In vitro culture and phenotypic and molecular characterization of gastric stem cells from human stomach.

Human gastric mucosa shows continuous self‐renewal via differentiation from stem cells that remain poorly characterized.

Respiratory viruses as a cause of sudden death

Adults and children are unevenly affected by sudden death. Children under 5 are the main targets of sudden death [1,2]. An infectious cause is detected on average in 50% (15–86% depending on the study) of sudden death in children and infection concerns mostly the respiratory system [3–5]. A viral etiology of respiratory infection is then identified in 20– 48% of cases during the postmortem investigations [1,2]. Viral respiratory infections are therefore one of the main etiologies of sudden deaths of children ahead of heart attacks and far ahead of the central nervous system involvement. The viruses then involved are identical to those responsible for high or low respiratory tract infections in the general population. The influenza virus A and B (FluA or FluB), respiratory syncytial virus (RSV), parainfluenza virus, metapneumovirus, adenovirus (ADV), and rhino-enterovirus (EV) are thus classically found (Table 1) [1,2,6–9]. The epidemiology of sudden deaths associated with respiratory virus responds to obvious seasonal changes and coincides with epidemic winter peaks of these viruses, with the notable exception of the EV group mainly circulating during summer and autumn. Some of these viruses have a specific clinical presentation; EV often associate heart and lung damage; ADV are also responsible for gastroenteritis; finally, the pandemic FluA is responsible for more deaths than seasonal virus due to increased virulence of the pathogen, its circulation within an immunologically naïve population leading to more readily systemic spread through the body [10]. Other viruses involved in common childhood diseases such as herpes simplex, Varicella Zoster virus (VZV), Epstein–Barr virus, cytomegalovirus, human herpes virus type 6, and Parvovirus B19 have also been detected in lungs of deceased infants, but their involvement in the death remained sometimes uncertain [11]. However, the epidemiology is probably still largely incomplete as it totally depends on the methodology used during the postmortem investigations. It is likely also biased since respiratory virus testing is more commonly performed in children than in adults leading to an underestimation in this second group of patients. Furthermore, the use of viral culture, a method known to be less sensitive than PCR and not isolating many pathogenic viruses, probably contributed to distort the epidemiology of sudden respiratory viral deaths. Its profile is thus gradually refined as we move from accumulated case reports using various diagnostic methodologies to standardized cohort studies where broad spectrum molecular biology tools are now conventionally used. Nevertheless, the epidemiological data are still missing for many respiratory viruses. Sudden death caused by respiratory viruses is due to septic shock, obstruction of bronchioles, and impaired breathing responsible for apnea and severe, potentially life-threatening hypoxemia [7,16]. It occurs in the context of an acute infection or an exacerbation of a chronic respiratory disease such as asthma or chronic obstructive pulmonary disease. The pathophysiology of sudden death is double (Figure 1). It combines an infectious part with an inflammatory part [17]. The infectious side itself is multiple. First, it is related to the own virus virulence. The most pathogenic and epidemic viruses like RSV or influenza viruses are also those commonly identified in sudden death [18]. Pandemic influenza viruses are responsible for a larger number of deaths than the seasonal influenza virus [10]. Second, infectious part is also based on host immunodeficiency that mainly affects children under 2 years at a period of their development where they lose the protection provided by maternal antibodies while still having an immature immune system. Third, pre-existing viral infections have a facilitating role on bacterial infections. Viral infections cause an increase in the number of bacteria receptors promoting adherence and colonization of the respiratory tract by a higher number and greater variety of bacterial species [19]. Moreover, lethal doses of bacterial endotoxins and exotoxins in animal model are reduced during mild or asymptomatic viral infection [20,21]. Inappropriate inflammatory response to viral infection remains, however, the main element likely to trigger sudden death [22]. Well described in children, this immunopathological component, known as cytokine storm hypothesis, is a combination of inadequate secretion in response to viral infection of pro-inflammatory cytokines (IFNγ, IL-1, IL-6, and TNFα) with a deficit in anti-inflammatory cytokines such as IL10 [23]. It causes microscopic inflammatory changes of the respiratory tract, frequently observed in sudden deaths, consisting in peribronchial inflammatory infiltrates, increase in IgM-producing cells in trachea, and mast cell degranulation [24,25]. The RSV, in particular, is known to cause a major and inappropriate inflammatory response, which explains its frequent detection in sudden death of the child [26]. Genetic factors are also involved since a recent study showed a single nucleotide polymorphism interferon gamma (IFNG) T + 874A genotype associated with higher IFNγ response that is EXPERT REVIEW OF ANTI-INFECTIVE THERAPY, 2016 VOL. 14, NO. 4, 359–363 http://dx.doi.org/10.1586/14787210.2016.1157470

Plasma exchange significantly affects darunavir exposure

Sir, Plasma exchange (PE) is a therapeutic procedure aimed at reducing the amount of abnormal or toxic substances in the blood, e.g. immunoglobulins. It consists in removing a large volume of plasma from a patient and replacing it with some form of replacement fluid. PE is non-selective and may also remove circulating medications from blood (both protein-bound and unbound drug). Darunavir is a synthetic non-peptidic HIV protease inhibitor (PI) metabolized mainly by CYP3A4 isoenzymes and 95% bound by plasma proteins, primarily a-1-acid glycoprotein. Increased clearance of atazanavir, another PI, during PE has already been reported, but the effect of PE on darunavir disposition is unknown. Since darunavir exhibits concentration–effect relationships, knowledge of this effect is critical for treatment efficacy. We report here the case of a white woman in her 50s receiving highly active antiretroviral therapy (HAART) including darunavir and requiring PE treatment. Consent for publication was obtained from the patient. She was diagnosed HIV-1 positive in 2001 and started HAART in 2005. Viral load (VL) and CD4 count were 720956 copies/mL and 13 cells/mm, respectively, when she was hospitalized for visceral leishmaniasis with polyclonal hypergammaglobulinaemia. She then started a new HAART combination consisting of darunavir/ritonavir (600/100 mg twice daily) with emtricitabine/tenofovir (200/245 mg once daily). Despite good virological response (VL ,20 copies/mL), immunological restoration was not achieved (CD4 count1⁄4150 cells/mm and CD4/CD8 ratio1⁄40.08) and hypergammaglobulinaemia increased. Because of hyperviscosity syndrome, she underwent PE sessions twice a week during which darunavir pharmacokinetics (PK) were explored. Several blood samples were drawn over the dosing interval, i.e. just before, during and immediately after a 2 h PE session, in order to estimate the elimination rate constant during PE (keon); a plasma sample collected during PE was also assayed in order to determine the amount of darunavir eliminated by PE. Three additional samples were drawn after PE in order to estimate the darunavir elimination rate constant after a PE session (keoff). Plasma darunavir concentrations were determined using a validated HPLC-UV method and ke values were estimated as the slope from the linear regression of log-concentrations on sample times. Corresponding t1 2 were estimated as loge2 divided by ke. In order to demonstrate the impact of PE on steady-state darunavir PK profiles, PK simulations were performed using Nonmem population PK software (Monte Carlo simulation, n1⁄41000 concentration –time profiles). Calculated keon and mean estimated PK parameters obtained from the literature (CL/F1⁄410.7 L/h, V/F1⁄4198 L and ka1⁄40.95 h) were used for that purpose. PK analysis was performed during three PE sessions. The mean+SD plasma volume and darunavir amount removed per session were 2.51+0.03 L and 9.3+8.3 mg, respectively. Actual darunavir concentrations measured before, during and after PE sessions were 8802+4835, 4505+2930 and 3314+2103 ng/mL, respectively. Estimated keoff and keon were 0.10+0.1 and 0.53+0.13 h, respectively. These data