Quantitative assessment of a novel flow-through porous microarray for the rapid analysis of gene expression profiles.

A novel microarray system that utilizes a porous aluminum-oxide substrate and flow-through incubation has been developed for rapid molecular biological testing. To assess its utility in gene expression analysis, we determined hybridization kinetics, variability, sensitivity and dynamic range of the system using amplified RNA. To show the feasibility with complex biological RNA, we subjected Jurkat cells to heat-shock treatment and analyzed the transcriptional regulation of 23 genes. We found that trends (regulation or no change) acquired on this platform are in good agreement with data obtained from real-time quantitative PCR and Affymetrix GeneChips. Additionally, the system demonstrates a linear dynamic range of 3 orders of magnitude and at least 10-fold decreased hybridization time compared to conventional microarrays. The minimum amount of transcript that could be detected in 20 microl volume is 2-5 amol, which enables the detection of 1 in 300,000 copies of a transcript in 1 microg of amplified RNA. Hybridization and subsequent analysis are completed within 2 h. Replicate hybridizations on 24 identical arrays with two complex biological samples revealed a mean coefficient of variation of 11.6%. This study shows the potential of flow-through porous microarrays for the rapid analysis of gene expression profiles in clinical applications.

Colorectal mucus binds DC-SIGN and inhibits HIV-1 trans-infection of CD4+ T-lymphocytes.

Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4+ T-lymphocytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1-3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1) as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.

HIV-1 autologous antibody neutralization associates with mother to child transmission.

The HIV-1 characteristics associated with mother to child transmission (MTCT) are still poorly understood and if known would indicate where intervention strategies should be targeted. In contrast to horizontally infected individuals, exposed infants possess inherited antibodies (Abs) from their mother with the potential to protect against infection. We investigated the HIV-1 gp160 envelope proteins from seven transmitting mothers (TM) whose children were infected either during gestation or soon after delivery and from four non-transmitting mothers (NTM) with similar viral loads and CD4 counts. Using pseudo-typed viruses we tested gp160 envelope glycoproteins for TZM-bl infectivity, CD4 and CCR5 interactions, DC-SIGN capture and transfer and neutralization with an array of common neutralizing Abs (NAbs) (2F5, 2G12, 4E10 and b12) as well as mother and infant plasma. We found no viral correlates associated with HIV-1 MTCT nor did we find differences in neutralization with the panel of NAbs. We did, however, find that TM possessed significantly higher plasma neutralization capacities than NTM (P = 0.002). Furthermore, we found that in utero (IU) TM had a higher neutralization capacity than mothers transmitting either peri - partum (PP) or via breastfeeding (BF) (P = 0.002). Plasma from children infected IU neutralized viruses carrying autologous gp160 viral envelopes as well as those from their corresponding mothers whilst plasma from children infected PP and/or BF demonstrated poor neutralizing capacity. Our results demonstrate heightened autologous NAb responses against gp120/gp41 can associate with a greater risk of HIV-1 MTCT and more specifically in those infants infected IU. Although the number of HIV-1 transmitting pairs is low our results indicate that autologous NAb responses in mothers and infants do not protect against MTCT and may in fact be detrimental when considering IU HIV-1 transmissions.

HIV-1 disease progression is associated with bile-salt stimulated lipase (BSSL) gene polymorphism.

BACKGROUND: DC-SIGN expressed by dendritic cells captures HIV-1 resulting in trans-infection of CD4(+) T-lymphocytes. However, BSSL (bile-salt stimulated lipase) binding to DC-SIGN interferes with HIV-1 capture. DC-SIGN binding properties of BSSL associate with the polymorphic repeated motif of BSSL exon 11. Furthermore, BSSL binds to HIV-1 co-receptor CXCR4. We hypothesized that BSSL modulates HIV-1 disease progression and emergence of CXCR4 using HIV-1 (X4) variants. RESULTS: The relation between BSSL genotype and HIV-1 disease progression and emergence of X4 variants was studied using Kaplan Meier and multivariate Cox proportional hazard analysis in a cohort of HIV-1 infected men having sex with men (n = 334, with n = 130 seroconverters). We analyzed the association of BSSL genotype with set-point viral load and CD4 cell count, both pre-infection and post-infection at viral set-point. The number of repeats in BSSL exon 11 were highly variable ranging from 10 to 18 in seropositive individuals and from 5-17 in HRSN with 16 repeats being dominant (>80% carry at least one allele with 16 repeats). We defined 16 to 18 repeats as high (H) and less than 16 repeats as low (L) repeat numbers. Homozygosity for the high (H) repeat number BSSL genotype (HH) correlated with high CD4 cell numbers prior to infection (p = 0.007). In HIV-1 patients, delayed disease progression was linked to the HH BSSL genotype (RH = 0.462 CI = 0.282-0.757, p = 0.002) as was delayed emergence of X4 variants (RH = 0.525, 95% CI = 0.290-0.953, p = 0.034). The LH BSSL genotype, previously found to be associated with enhanced DC-SIGN binding of human milk, was identified to correlate with accelerated disease progression in our cohort of HIV-1 infected MSM (RH = 0.517, 95% CI = 0.328-0.818, p = 0.005). CONCLUSION: We identify BSSL as a marker for HIV-1 disease progression and emergence of X4 variants. Additionally, we identified a relation between BSSL genotype and CD4 cell counts prior to infection.

Innate immune factors associated with HIV-1 transmission.

PURPOSE OF REVIEW: Relatively little is known with regards to the mechanisms of HIV-1 transmission across a mucosal surface and more specifically what effects host factors have on influencing infection and early viral dissemination. The purpose of this review is to summarize which factors of the innate immune response can influence mucosal transmission of HIV-1. RECENT FINDINGS: A large array of cell types reside at the mucosal surface ranging from Langerhans cells, dendritic cells, macrophages as well as CD4⁺ lymphocytes, all of which interact with the virus in a unique and different way and which can contribute to risk of HIV-1 transmission. Numerous factors present in bodily secretions as well as the carrier fluids of HIV-1 (breast milk, vaginal secretions, semen and intestinal mucus) can influence transmission and early virus replication. These range from cytokines, chemokines, small peptides, glycoproteins as well as an array of host intracellular molecules which can influence viral uncoating, reverse transcription as well as egress from the infected cell. SUMMARY: Better understanding the cellular mechanisms of HIV-1 transmission and how different host factor can influence infection will aide in the future development of vaccines, microbicides, and therapies.

Binding of human milk to pathogen receptor DC-SIGN varies with bile salt-stimulated lipase (BSSL) gene polymorphism.

OBJECTIVE: Dendritic cells bind an array of antigens and DC-SIGN has been postulated to act as a receptor for mucosal pathogen transmission. Bile salt-stimulated lipase (BSSL) from human milk potently binds DC-SIGN and blocks DC-SIGN mediated trans-infection of CD4(+) T-lymphocytes with HIV-1. Objective was to study variation in DC-SIGN binding properties and the relation between DC-SIGN binding capacity of milk and BSSL gene polymorphisms. STUDY DESIGN: ELISA and PCR were used to study DC-SIGN binding properties and BSSL exon 11 size variation for human milk derived from 269 different mothers distributed over 4 geographical regions. RESULTS: DC-SIGN binding properties were highly variable for milks derived from different mothers and between samplings from different geographical regions. Differences in DC-SIGN binding were correlated with a genetic polymorphism in BSSL which is related to the number of 11 amino acid repeats at the C-terminus of the protein. CONCLUSION: The observed variation in DC-SIGN binding properties among milk samples may have implications for the risk of mucosal transmission of pathogens during breastfeeding.

Mucin 6 in seminal plasma binds DC-SIGN and potently blocks dendritic cell mediated transfer of HIV-1 to CD4(+) T-lymphocytes.

Many viruses transmitted via the genital or oral mucosa have the potential to interact with dendritic cell-specific intercellular adhesion molecule-3 grabbing non integrin (DC-SIGN) expressed on immature dendritic cells (iDCs) that lie below the mucosal surface. These cells have been postulated to capture and disseminate human immunodeficiency virus type-1 (HIV-1) to CD4(+) lymphocytes, potentially through breaches in the mucosal lining. We have previously described that BSSL (bile salt-stimulated lipase) in human milk can bind DC-SIGN and block transfer. Here we demonstrate that seminal plasma has similar DC-SIGN blocking properties as BSSL in human milk. Using comparative SDS-PAGE and Western blotting combined with mass spectrometry we identified mucin 6 as the DC-SIGN binding component in seminal plasma. Additionally, we demonstrate that purified mucin 6 binds DC-SIGN and successfully inhibits viral transfer. Mucin 6 in seminal plasma may therefore interfere with the sexual transmission of HIV-1 and other DC-SIGN co-opting viruses.

Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells.

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.