Sam Noppen

Hemozoin Induces Lung Inflammation and Correlates with Malaria-Associated Acute Respiratory Distress Syndrome

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1β, IL-6, IL-10, TNF, and transforming growth factor-β), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

Sulfated Escherichia coli K5 polysaccharide derivatives inhibit dengue virus infection of human microvascular endothelial cells by interacting with the viral envelope protein E domain III.

Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes cytokine-mediated alterations in the barrier function of the microvascular endothelium, leading to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We observed that DENV (serotype 2) productively infects primary (HMVEC-d) and immortalized (HMEC-1) human dermal microvascular endothelial cells, despite the absence of well-described DENV receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the mannose receptor on the cell surface. However, heparan sulfate proteoglycans (HSPGs) were highly expressed on these cells and pre-treatment of HMEC-1 cells with heparinase II or with glycosaminoglycans reduced DENV infectivity up to 90%, suggesting that DENV uses HSPGs as attachment receptor on microvascular endothelial cells. Sulfated Escherichia coli K5 derivatives, which are structurally similar to heparin/heparan sulfate but lack anticoagulant activity, were able to block DENV infection of HMEC-1 and HMVEC-d cells in the nanomolar range. The highly sulfated K5-OS(H) and K5-N,OS(H) inhibited virus attachment and subsequent entry into microvascular endothelial cells by interacting with the viral envelope (E) protein, as shown by surface plasmon resonance (SPR) analysis using the receptor-binding domain III of the E protein.

Nucleoside-catabolizing enzymes in mycoplasma-infected tumor cell cultures compromise the cytostatic activity of the anticancer drug gemcitabine.

Background: Gemcitabine is used to treat solid tumors. Some mycoplasmas preferentially colonize tumors in patients. Results: Mycoplasma-encoded cytidine deaminase and pyrimidine nucleoside phosphorylase compromise the cytostatic/antitumor activity of gemcitabine in mycoplasma-infected tumor cell cultures and xenografts in mice. Conclusion: Tumor-associated mycoplasmas may decrease the therapeutic efficiency of gemcitabine. Significance: Current treatment of mycoplasma-infected tumors with gemcitabine may be suboptimal. The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2′,2′-difluoro-2′-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2′,2′-difluoro-2′-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2′-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

Basic chemokine-derived glycosaminoglycan binding peptides exert antiviral properties against dengue virus serotype 2, herpes simplex virus-1 and respiratory syncytial virus

Chemokines attract leukocytes to sites of infection in a G protein-coupled receptor (GPCR) and glycosaminoglycan (GAG) dependent manner. Therefore, chemokines are crucial molecules for proper functioning of our antimicrobial defense mechanisms. In addition, some chemokines have GPCR-independent defensin-like antimicrobial activities against bacteria and fungi. Recently, high affinity for GAGs has been reported for the positively charged COOH-terminal region of the chemokine CXCL9. In addition to CXCL9, also CXCL12γ has such a positively charged COOH-terminal region with about 50% positively charged amino acids. In this report, we compared the affinity of COOH-terminal peptides of CXCL9 and CXCL12γ for GAGs and KD values in the low nM range were detected. Several enveloped viruses such as herpesviruses, hepatitis viruses, human immunodeficiency virus (HIV), dengue virus (DENV), etc. are known to bind to GAGs such as the negatively charged heparan sulfate (HS). In this way GAGs are important for the initial contacts between viruses and host cells and for the infection of the cell. Thus, inhibiting the virus-cell interactions, by blocking GAG-binding sites on the host cell, might be a way to target multiple virus families and resistant strains. This article reports that the COOH-terminal peptides of CXCL9 and CXCL12γ have antiviral activity against DENV serotype 2, clinical and laboratory strains of herpes simplex virus (HSV)-1 and respiratory syncytial virus (RSV). Moreover, we show that CXCL9(74-103) competes with DENV envelope protein domain III for binding to heparin. These short chemokine-derived peptides may be lead molecules for the development of novel antiviral agents.

Tryptophan dendrimers that inhibit HIV replication, prevent virus entry and bind to the HIV envelope glycoproteins gp120 and gp41

Dendrimers containing from 9 to 18 tryptophan residues at the peryphery have been efficiently synthesized and tested against HIV replication. These compounds inhibit an early step of the replicative cycle of HIV, presumably virus entry into its target cell. Our data suggest that HIV inhibition can be achieved by the preferred interaction of the compounds herein described with glycoproteins gp120 and gp41 of the HIV envelope preventing interaction between HIV and the (co)receptors present on the host cells. The results obtained so far indicate that 9 tryptophan residues on the periphery are sufficient for efficient gp120/gp41 binding and anti-HIV activity.

Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation

A small chemical drug CADA specifically binds to the signal peptide of the membrane pre-protein CD4, disturbing its synthesis, impeding the routing to and expression on the cell surface.

High mannose-specific lectin Msl mediates key interactions of the vaginal Lactobacillus plantarum isolate CMPG5300

To characterize the interaction potential of the human vaginal isolate Lactobacillus plantarum CMPG5300, its genome was mined for genes encoding lectin-like proteins. cmpg5300.05_29 was identified as the gene encoding a putative mannose-binding lectin. Phenotypic analysis of a gene knock-out mutant of cmpg5300.05_29 showed that expression of this gene is important for auto-aggregation, adhesion to the vaginal epithelial cells, biofilm formation and binding to mannosylated glycans. Purification of the predicted lectin domain of Cmpg5300.05_29 and characterization of its sugar binding capacity confirmed the specificity of the lectin for high- mannose glycans. Therefore, we renamed Cmpg5300.05_29 as a mannose-specific lectin (Msl). The purified lectin domain of Msl could efficiently bind to HIV-1 glycoprotein gp120 and Candida albicans, and showed an inhibitory activity against biofilm formation of uropathogenic Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. Thus, using a combination of molecular lectin characterization and functional assays, we could show that lectin-sugar interactions play a key role in host and pathogen interactions of a prototype isolate of the vaginal Lactobacillus microbiota.

Screening Platform toward New Anti-HIV Aptamers Set on Molecular Docking and Fluorescence Quenching Techniques

By using a new rapid screening platform set on molecular docking simulations and fluorescence quenching techniques, three new anti-HIV aptamers targeting the viral surface glycoprotein 120 (gp120) were selected, synthesized, and assayed. The use of the short synthetic fluorescent peptide V35-Fluo mimicking the V3 loop of gp120, as the molecular target for fluorescence-quenching binding affinity studies, allowed one to measure the binding affinities of the new aptamers for the HIV-1 gp120 without the need to obtain and purify the full recombinant gp120 protein. The almost perfect correspondence between the calculated Kd and the experimental EC50 on HIV-infected cells confirmed the reliability of the platform as an alternative to the existing methods for aptamer selection and measuring of aptamer-protein equilibria.

Antiviral activity of synthetic aminopyrrolic carbohydrate binding agents: targeting the glycans of viral gp120 to inhibit HIV entry

The binding abilities of a set of structurally related aminopyrrolic synthetic receptors for mannosides, endowed with antimycotic activity against yeast and yeast-like pathogens bearing mannoproteins on their cell surface, have been investigated towards the highly mannosylated gp120 and gp41 glycoproteins of the HIV envelope. A pronounced binding interaction with both glycoproteins was observed by SPR for most of the investigated compounds. Comparison of their binding properties towards the glycoproteins with their binding affinities toward mannosides revealed a direct correlation, supporting their role as carbohydrate binding agents (CBAs). Cytostatic activity studies revealed antiproliferative activity dependent on the nature and the structure of compounds. Antiviral activity studies against a broad panel of DNA and RNA viruses showed inhibitory effect against HIV infection of the T-lymphocyte CEM cell line for two compounds, suggesting antiviral activity similar to other CBAs, such as the nonpeptidic pradimicin antibiotics.

Inhibition of infection and transmission of HIV-1 and lack of significant impact on the vaginal commensal lactobacilli by carbohydrate-binding agents

OBJECTIVES A selection of carbohydrate-binding agents (CBAs) with different glycan specificities were evaluated for their inhibitory effect against HIV infection and transmission, and their interaction with vaginal commensal bacteria. METHODS Several assays were used for the antiviral evaluation: (i) cell-free virus infection of human CD4+ T lymphocyte C8166 cells; (ii) syncytium formation in co-cultures of persistently HIV-1-infected HUT-78/HIV-1 and non-infected CD4+ SupT1 cells; (iii) DC-SIGN-directed capture of HIV-1 particles; and (iv) transmission of DC-SIGN-captured HIV-1 particles to uninfected CD4+ C8166 cells. CBAs were also examined for their interaction with vaginal commensal lactobacilli using several viability, proliferation and adhesion assays. RESULTS The CBAs showed efficient inhibitory activity in the nanomolar to low-micromolar range against four events that play a crucial role in HIV-1 infection and transmission: cell-free virus infection, fusion between HIV-1-infected and non-infected cells, HIV-1 capture by DC-SIGN and transmission of DC-SIGN-captured virus to T cells. As candidate microbicides should not interfere with the normal human microbiota, we examined the effect of CBAs against Lactobacillus strains, including a variety of vaginal strains, a gastrointestinal strain and several non-human isolates. None of the CBAs included in our studies inhibited the growth of these bacteria in several media, affected their viability or had any significant impact on their adhesion to HeLa cell monolayers. CONCLUSIONS The CBAs in this study were inhibitory to HIV-1 in several in vitro infection and transmission models, and may therefore qualify as potential microbicide candidates. The lack of significant impact on commensal vaginal lactobacilli is an important property of these CBAs in view of their potential microbicidal use.