Hypoxia inducible NOD2 interacts with 3-O-sulfogalactoceramide and regulates vesicular homeostasis.

BACKGROUND: Oxygen sensing in mammalian cells is a conserved signaling pathway regulated by hypoxia inducible factor type 1 (HIF-1). Inadequate oxygen supply (hypoxia) is common to many pathological disorders where autophagy plays an import role. The aim of this study was the identification and characterization of novel HIF-1 target genes that promote autophagy during hypoxia. METHODS: Whole genome Chromatin Immune Precipitation from hypoxic HeLa cells was used to identify novel HIF-1 target genes. Hypoxia induced expression and transcription regulation was studied in wild type and HIF-deficient cells. siRNA silencing of candidate genes was used to establish their role during autophagy. Recombinant protein was used for screening immobilized glycosylated lipids to identify potential ligands. RESULTS: We identified the Nucleotide Oligomerization Domain 2 (NOD2/CARD15) as a novel HIF-1 target and 3-O-sulfo-galactoceramide (sulfatide) and Mycobacterium sp. specific sulfolipid-1 as the first NOD2 ligands that both compete for binding to NOD2. Loss of NOD2 function impaired autophagy upstream of the autophagy inhibitor chloroquine by reducing the number of acidic vesicles. Inhibition of sulfatide synthesis elicited defects in autophagy similar to the NOD2 loss of function but did not influence NOD2-mediated NF-kB signaling. CONCLUSIONS: Our findings suggest that the interaction of NOD2 with sulfatide may mediate the balance between autophagy and inflammation in hypoxic cells. GENERAL SIGNIFICANCE: These findings may lead to a better understanding of complex inflammatory pathologies like Crohn's disease and tuberculosis where both NOD2 and hypoxia are implicated.

The rate of epidemiological and virological changes during the transition from nascent to concentrated HIV epidemic stage in the former Soviet Union countries.

The rate of processes accompanying the transition of the HIV-1 epidemic from nascent stage to concentrated one in the Former Soviet Union (FSU) during intravenous drug user (IDU)-associated HIV infection outbreaks in 1994-1999 has not been analyzed. To define the rates, we studied susceptible populations and circulating viruses before, during, and after the outbreaks. Our findings included the following: (1) the pattern of high HIV-1 genetic diversity characteristic of the nascent epidemic changed to a concentrated one within 1 year in St. Petersburg and in Moscow; (2) different FSU regions were at different stages of the HIV-1 epidemic in 1994-1996; (3) the change of serotypic patterns characteristic of different stages of the HIV/AIDS epidemic for the non-IDU risk group occurred within 1 year in Moscow, suggesting an extremely high rate of IDU-associated epidemic pattern distributions in regions and susceptible populations in the FSU.

Sport- and sample-specific features of trace elements in adolescent female field hockey players and fencers.

Active physical exercises and growth are associated with mineral imbalances in young athletes. The purpose of this study was to examine the impact of sport-related factors on tissue mineral status in adolescent female athletes. Saliva and hair samples were used for the analysis of immediate and more permanent tissue mineral status, respectively. Samples taken from a control non-athletic female group and two groups of female athletes (field hockey and fencing) were analyzed for seven essential minerals: calcium, chromium, iron, potassium, magnesium, selenium and zinc. Inductively-coupled plasma mass spectrometry was used for the quantification of elements having very low concentration range in samples (Se, Cr and Zn) whereas inductively coupled plasma optical emission spectrometry was used for quantification of more ubiquitous elements (Mg, К, Са, Fe). The obtained results for athletic groups were compared with control. Female athletes had increased levels of selenium in both saliva and hair as well as chromium in saliva. Field hockey players had the higher level of zinc in hair whereas fencers had the lower levels of salivary calcium. Strong negative correlation between potassium levels in saliva and hair was identified. Iron and magnesium did not differ between the studied groups. In conclusion, novel sport-specific features of chromium tissue levels in female athletes were found. The studied sport disciplines have different impact on the distribution of osteoporosis-related minerals (calcium and zinc). Our finding can help in the development of osteoporosis preventive trainings and in the proper nutrient supplementation to correct mineral imbalances in female athletes.

Interaction of HIV-1 with dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-expressing cells is influenced by gp120 envelope modifications associated with disease progression.

Dendritic cells can enhance the replication of HIV-1 in CD4(+) lymphocytes through the interaction of the gp120 envelope protein with such molecules as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin. The variable loops of gp120 have previously been shown to modulate the interaction of HIV-1 with its principal receptor CD4 and its various coreceptors, namely CCR5 and CXCR4. Here, we utilized a panel of molecular cloned viruses to identify whether gp120 modifications can influence the virus interaction with immature dendritic cells or a cell line expressing dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (Raji-DC-SIGN). The viruses encompass the R5, R5X4 and X4 phenotypes, and are based upon V1V2 and V3 sequences from a patient with disease progression. We found that dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin enhancement of virus replication can be modulated by the V1V2 length, the overall V3 charge and N-linked glycosylation patterns; similar results were observed with immature dendritic cells. Viruses with higher V3 charges are more readily transferred to CD4(+) lymphocytes when the V1V2 region is longer and contains an additional N-linked glycosylation site, whereas transfer of viruses with lower V3 charges is greater when the V1V2 region is shorter. Viruses differing in the V1V2 and V3 regions also demonstrated differential capture by Raji-DC-SIGN cells in the presence of mannan. These results indicate that the interaction between HIV-1 and immature dendritic cells via such molecules as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin may have a role in selecting viruses undergoing transmission and evolution during disease progression.

The vesicle-associated function of NOD2 as a link between Crohn's disease and mycobacterial infection.

Although Crohn's disease (CD) etiology remains unclear, a growing body of evidence suggests that CD may include an infectious component, with Mycobacterium avium subsp. paratuberculosis (MAP) being the most likely candidate for this role. However, the molecular mechanism of the MAP involvement in CD pathogenesis remains unclear. The polymorphism of the NOD2 gene, coding for an intracellular pattern recognition receptor, is a factor of predisposition to mycobacterial infections and CD. Recent findings on NOD2 interactions and functions provide the missing pieces in the puzzle of a NOD2-mediated mechanism common for mycobacterial infections and CD. Implications of these new findings for the development of a better understanding and treatments of CD and mycobacterial infections are discussed.

The DC-SIGN-CD56 interaction inhibits the anti-dendritic cell cytotoxicity of CD56 expressing cells.

BACKGROUND: This study aimed to clarify interactions of the pattern-recognition receptor DC-SIGN with cells from the HIV-infected peripheral blood lymphocyte cultures. METHODS: Cells from control and HIV-infected peripheral blood lymphocyte cultures were tested for the surface expression of DC-SIGN ligands. The DC-SIGN ligand expressing cells were analyzed for the role of DC-SIGN-ligand interaction in their functionality. RESULTS: In the vast majority of experiments HIV-infected lymphocytes did not express detectable DC-SIGN ligands on their cell surfaces. In contrast, non-infected cells, carrying NK-specific marker CD56, expressed cell surface DC-SIGN ligands. The weakly polysialylated CD56 was identified as a novel DC-SIGN ligand. The treatment of DC-SIGN expressing dendritic cells with anti-DC-SIGN antibodies increased the anti-dendritic cell cytotoxicity of CD56(pos) cells. The treatment of CD56(pos) cells with a peptide, blocking the weakly polysialylated CD56-specifc trans-homophilic interactions, inhibited their anti-dendritic cells cytotoxicity. CONCLUSIONS: The interaction between DC-SIGN and CD56 inhibits homotypic intercellular interactions of CD56(pos) cells and protects DC-SIGN expressing dendritic cells against CD56(pos) cell-mediated cytotoxicity. This finding can have an impact on the development of approaches to HIV infection and cancer therapy as well as in transplantation medicine.

Simultaneous introduction of HIV type 1 subtype A and B viruses into injecting drug users in southern Ukraine at the beginning of the epidemic in the former Soviet Union.

The vast majority of HIV-1 strains from the epidemic in the former Soviet Union (FSU) belong to subtype A (IDU-A) and CRF03_AB (IDU-A/B), for which IDU-A is one of parental strains; no epidemic by another parental virus, belonging to subtype B (IDU-B), has yet been identified. To characterize viruses present during the early stage of the epidemic in southern Ukraine, where the first outbreaks in the FSU were registered, we obtained partial env and pol sequences from IDUs from Odessa and Nikolaev and compared them with viruses from other outbreaks. All viruses from Odessa belonged to the IDU-A type, which is in accord with previous studies. At the same time, we found that the outbreak in Nikolaev was caused by IDU-B viruses, indicating that this outbreak is the result of an independent virus introduction. Phylogenetic analysis of viruses from the FSU supported the epidemiological data suggesting that the HIV-1 epidemic in the FSU started in southern Ukraine.

MUC1 in human milk blocks transmission of human immunodeficiency virus from dendritic cells to T cells.

Mother-to-child transmission of human immunodeficiency virus-1 (HIV-1) occurs frequently via breast-feeding. HIV-1 targets DC-SIGN+ dendritic cells (DCs) in mucosal areas that allow efficient transmission of the virus to T cells. Here, we demonstrate that the epithelial mucin MUC1, abundant in milk, efficiently bound to DC-SIGN on DC. The O-linked glycans within the mucin domain contained Lewis X structures, that were specifically recognized by the receptor. Interestingly, MUC1 prevented DC-SIGN-mediated transmission of HIV-1 from DCs to CD4+ T cells. We hypothesize that repetitive units of Lewis X, within the mucin domain, play an important role in inhibiting transmission of HIV-1 from mother to child.

C-type lectin Mermaid inhibits dendritic cell mediated HIV-1 transmission to CD4+ T cells.

Dendritic cells (DCs) are important in HIV-1 transmission; DCs capture invading HIV-1 through the interaction of the gp120 oligosaccharides with the C-type lectin DC-SIGN and migrate to the lymphoid tissues where HIV-1 is transmitted to T cells. Thus, the HIV-1 envelope glycoprotein gp120 is an attractive target to prevent interactions with DCs and subsequent viral transmission. Here, we have investigated whether the structural homologue of DC-SIGN, the nematode C-type lectin Mermaid can be used to prevent HIV-1 transmission by DCs. Our data demonstrate that Mermaid interacts with high mannose structures present on HIV-1 gp120 and thereby inhibits HIV-1 binding to DC-SIGN on DCs. Moreover, Mermaid inhibits DC-SIGN-mediated HIV-1 transmission from DC to T cells. We have identified Mermaid as a non-cytotoxic agent that shares the glycan specificity with DC-SIGN and inhibits DC-SIGN-gp120 interaction. The results are important for the anti-HIV-1 microbicide development directed at preventing DC-HIV-1 interactions.

Only five of 10 strictly conserved disulfide bonds are essential for folding and eight for function of the HIV-1 envelope glycoprotein.

Protein folding in the endoplasmic reticulum goes hand in hand with disulfide bond formation, and disulfide bonds are considered key structural elements for a protein's folding and function. We used the HIV-1 Envelope glycoprotein to examine in detail the importance of its 10 completely conserved disulfide bonds. We systematically mutated the cysteines in its ectodomain, assayed the mutants for oxidative folding, transport, and incorporation into the virus, and tested fitness of mutant viruses. We found that the protein was remarkably tolerant toward manipulation of its disulfide-bonded structure. Five of 10 disulfide bonds were dispensable for folding. Two of these were even expendable for viral replication in cell culture, indicating that the relevance of these disulfide bonds becomes manifest only during natural infection. Our findings refine old paradigms on the importance of disulfide bonds for proteins.

Efficient capture of antibody neutralized HIV-1 by cells expressing DC-SIGN and transfer to CD4+ T lymphocytes.

Infection of CD4+ T lymphocytes is enhanced by the capture and subsequent transfer of HIV-1 by dendritic cells (DCs) via the interaction with C-type lectins such as the DC-specific ICAM-grabbing nonintegrin (DC-SIGN). Numerous HIV-1 envelope-directed neutralizing Abs have been shown to successfully block the infection of CD4(+) T lymphocytes. In this study, we find that HIV-1-neutralized with the mAb 2F5 is more efficiently captured by immature monocyte-derived DCs (iMDDCs) and DC-SIGN-expressing Raji cells (Raji-DC-SIGN). Furthermore, a 2F5-neutralized virus captured by these cells was able to subsequently infect CD4+ T lymphocytes upon the release of HIV-1 from iMDDCs, thereby enhancing infection. We show that upon transfer via DC-SIGN-expressing cells, HIV-1 is released from immune-complexes with the Abs 2F5 and 4E10 (gp41-directed) and 2G12, 4.8D, and 1.7b (gp120-directed). The nonneutralizing V3-21 (V3 region of the gp120-directed) Ab enhanced HIV-1 infection upon capture and transfer via Raji-DC-SIGN cells, whereas no infection was observed with the neutralizing b12 Ab (gp120-directed), indicating that different Abs have variant effects on inhibiting HIV-1 transfer to CD4+ T lymphocytes. The increased capture of the 2F5-neutralized virus by iMDDCs was negated upon blocking the Fc receptors. Blocking DC-SIGN on iMDDCs resulted in a 70-75% inhibition of HIV-1 capture at 37 degrees C, whereas at 4 degrees C a full block was observed, showing that the observed transfer is mediated via DC-SIGN. Taken together, we propose that DC-SIGN-mediated capture of neutralized HIV-1 by iMDDCs has the potential to induce immune evasion from the neutralization effects of HIV-1 Abs, with implications for HIV-1 pathogenesis and vaccine development.

Statins disrupt CCR5 and RANTES expression levels in CD4(+) T lymphocytes in vitro and preferentially decrease infection of R5 versus X4 HIV-1.

BACKGROUND: Statins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1 (HIV-1) through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1 incorporation into viral particles. PRINCIPLE FINDINGS: Here we demonstrate that treatment of an enriched CD4(+) lymphocyte population with lovastatin (Lov), mevastatin (Mev) and simvastatin (activated and non-activated, Sim(A) and Sim(N), respectively) can reduce the cell surface expression of the CC-chemokine receptor CCR5 (P<0.01 for Sim(A) and Lov). The lowered CCR5 expression was associated with down-regulation of CCR5 mRNA expression. The CC-chemokine RANTES protein and mRNA expression levels were slightly increased in CD4(+) enriched lymphocytes treated with statins. Both R5 and X4 HIV-1 were reduced for their infection of statin-treated cells; however, in cultures where statins were removed and where a decrease in CCR5 expression was observed, there was a preferential inhibition of infection with an R5 versus X4 virus. CONCLUSIONS: The results indicate that the modulation of CC-chemokine receptor (CCR5) and CC-chemokine (RANTES) expression levels should be considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1 activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment could be considered as a way to modulate immune induction such as during vaccination protocols.

Intrapatient alterations in the human immunodeficiency virus type 1 gp120 V1V2 and V3 regions differentially modulate coreceptor usage, virus inhibition by CC/CXC chemokines, soluble CD4, and the b12 and 2G12 monoclonal antibodies.

We studied human immunodeficiency virus type 1 (HIV-1) chimeric viruses altering in their gp120 V1V2 and V3 envelope regions to better map which genetic alterations are associated with specific virus phenotypes associated with HIV-1 disease progression. The V1V2 and V3 regions studied were based on viruses isolated from an individual with progressing HIV-1 disease. Higher V3 charges were linked with CXCR4 usage, but only when considered within a specific V1V2 and V3 N-linked glycosylation context. When the virus gained R5X4 dual tropism, irrespective of its V3 charge, it became highly resistant to inhibition by RANTES and highly sensitive to inhibition by SDF-1alpha. R5 viruses with higher positive V3 charges were more sensitive to inhibition by RANTES, while R5X4 dualtropic viruses with higher positive V3 charges were more resistant to inhibition by SDF-1alpha. Loss of the V3 N-linked glycosylation event rendered the virus more resistant to inhibition by SDF-1alpha. The same alterations in the V1V2 and V3 regions influenced the extent to which the viruses were neutralized with soluble CD4, as well as monoclonal antibodies b12 and 2G12, but not monoclonal antibody 2F5. These results further identify a complex set of alterations within the V1V2 and V3 regions of HIV-1 that can be selected in the host via alterations of coreceptor usage, CC/CXC chemokine inhibition, CD4 binding, and antibody neutralization.