Porphyromonas gingivalisinduces CCR5-dependent transfer of infectious HIV-1 from oral keratinocytes to permissive cells
© Giacaman et al; licensee BioMed Central Ltd. 2008
Received: 18 September 2007
Accepted: 27 March 2008
Published: 27 March 2008
Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells.
Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced.
P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1.
Systemic infection after oral exposure to HIV-1 has been reported in breastfed infants from seropositive mothers . Whether HIV/AIDS is acquired through oral exposure to seminal fluid from HIV-positive individuals remains equivocal . Yet, experimental evidence points to the plausibility that exposure of the oral mucosal epithelium to HIV-1 results in primary infection of the oral tissues followed by systemic dissemination. For example, when simian immunodeficiency virus (SIV) is non-traumatically swabbed on the gingival and buccal mucosa of primates, oral epithelial infection is evident within one day [3, 4], while systemic infection occurs within a week . Consistent with these observations, human oral epithelial cells of HIV-infected patients contain integrated HIV-1 DNA, which may result from either primary infection or systemic dissemination of the virus . HIV-1 has also been suggested to infect human oral epithelial cells in vitro [7, 8]. Recent work from our laboratory shows that replication aborts after viral integration, while harbored virions are transmissible from oral keratinocytes to permissive cells . In vivo, however, human oral epithelium is generally not considered a target for primary infection by HIV-1 [10, 11].
Mucosal exposure is responsible for the vast majority of the current HIV infections worldwide  and R5-tropic HIV-1 accounts for most of primary infections [13–15]. In mucosal tissues such as in the gut, CCR5 has been proposed to act as a "gatekeeper", facilitating primary infection by R5-tropic while excluding X4-tropic HIV-1 [14, 16, 17]. Indeed, primary R5-tropic HIV-1 infection generally requires target cells that carry a specific receptor for gp120 such as CD4 and the chemokine coreceptor CCR5 . Interestingly, a homozygous defect in expression of the R5-tropic coreceptor CCR5 is associated with resistance to HIV-1 infection in frequently exposed individuals . On mucosal surfaces where epithelial cells predominate, the mechanism by which R5-tropic HIV-1 is specifically selected, and X4 HIV-1 is relatively excluded remains unclear. Many potential "gatekeeper" mechanisms have been proposed . More than relying on a single "gatekeeper", selective R5-HIV transmission seems to depend on the aggregate activity of cell and tissue specific restrictive barriers and facilitated uptake mechanisms encountered as HIV-1 passes from the mucosal surface to permissive cells in the organized lymphoid tissues .
Healthy squamous oral keratinocytes predominately express CXCR4 , but low to undetectable levels of CCR5 [19, 20] and there is no expression of the major HIV-1 receptor, CD4 [7, 11, 21, 22]. Given that oral keratinocytes can integrate HIV-1 DNA, alternative HIV-1 receptors have been proposed, including galactosyl ceramide (GalCer) [23, 24], heparan sulfate proteoglycans [11, 25], syndecans [26, 27], and mannose receptor [28, 29]. In concert with CXCR4 (X4-tropic HIV-1 specific) or CCR5 (R5-tropic HIV-1 specific) chemokine coreceptors, these alternative receptors have been suggested to take up infectious HIV-1 , which can then be transferred to permissive cells [27, 30–32].
Since oral epithelial cells express only CXCR4 [7, 19, 20, 22], and oral keratinocytes in vitro can internalize and transfer infectious HIV-1 , we sought to learn if CCR5 coreceptor regulation by co-infecting oral bacteria could result in increased uptake and transfer of R5-tropic HIV-1. Co-infecting viruses, such as human herpesvirus 6 (HHV-6) and HHV-7, down-regulate expression of the HIV-1 co-receptor, CXCR4 [33, 34]. Since HHV modulation does not affect CCR5, CXCR4 down-regulation may increase the relative expression of CCR5, enhancing the "gatekeeper". Our group has recently shown that Porphyromonas gingivalis, an endogenous periodontal pathogen, selectively up-regulates CCR5 in oral keratinocytes . These cells increase CCR5 expression when signaled through protease-activated receptors (PAR) and TLRs by the P. gingivalis putative virulence factors, gingipains (Rgp and Kgp) and LPS, respectively . We, therefore, hypothesized that P. gingivalis co-infection increases HIV-1 transfer of infectious R5-tropic HIV-1 from oral keratinocytes to permissive cells. In the absence of productive infection in oral keratinocytes, we showed that P. gingivalis caused a CCR5-dependent increase in transfer of R5-tropic HIV-1. As a consequence of primary non-productive infection, R5-tropic HIV-1 is suggested to disseminate selectively from oral mucosal epithelium in association with P. gingivalis infection in periodontitis.
P. gingivalis-induced release of infectious R5-specific HIV-1 from oral epithelial cells
Effect of P. gingivalison TERT-2 cell-associated HIV-1
To determine if P. gingivalis increased viral association with TERT-2 cells, RNA from infected TERT-2 cells was recovered between 7 and 54 hours post-inoculation and HIVgag RNA was quantified by real-time PCR. In the presence and absence of P. gingivalis, greater levels of HIVgag RNA were generally recovered from R5- (Fig. 2C) than X4-HIV-1 (Fig. 2D) infected TERT-2 cells.
P. gingivaliseffects on HIV-1 replication
P. gingivalisincreases harbored infectious HIV-1 in TERT-2 cells
Increase in TERT-2 cell-associated infectious R5-tropic HIV-1 blocked by anti-CCR5 antibodies
We next tested whether up-regulation of the CCR5 HIV-1 coreceptor on TERT-2 cells  by P. gingivalis could contribute to the infectivity of R5-tropic HIV-1. Oral keratinocytes were pre-incubated with P. gingivalis, then incubated with anti-CCR5 antibody, and inoculated with HIV Ba-L. At 18 h post-inoculation, spent culture supernatants and TERT-2 cell lysates were recovered and assayed for infectivity using TZM-bl reporter cells. Anti-CCR5 caused a dose-dependent reduction in infectious R5-tropic HIV-1 from both the culture supernatants and the intracellular compartment of TERT-2 cells (Fig. 5D). At the highest dose tested, anti-CCR5 blocked the increase in R5-tropic HIV-1 infectivity attributable to P. gingivalis (Fig. 5D).
P. gingivalis increases cell-to-cell transinfection of intracellular infectious HIV-1 from oral keratinocytes
We next sought to learn whether oral keratinocytes pre-incubated with P. gingivalis transferred more infectious intracellular R5-HIV to permissive cells in co-culture. After trypsinization and removal of extracellular virus, P. gingivalis pre-incubated TERT-2 cells were incubated with R5-HIV-1 or X4-HIV-1 for 6 h and cultured for 18 h post-inoculation. At 18 h, infected TERT-2 cells were harvested and co-cultured with TZM-bl reporter cells for an additional 24 h. TERT-2 cells trans infected TZM-bl cells with more infectious R5-tropic HIV-1 when pre-incubated in the presence of P. gingivalis than in the absence (Fig. 6C). In contrast, X4-HIV-1 trans infection from TERT-2 cells to TZM-bl cells was not affected by P. gingivalis and was lower than R5-HIV-1 (data not shown).
To determine whether trans infection of intracellular R5-HIV-1 was CCR5-dependent, TERT-2 cells pre-incubated with P. gingivalis were incubated with anti-CCR5 antibody and then inoculated with R5- or X4-HIV-1. TERT-2 cells incubated with CCR5 antibody and R5- or X4-HIV-1, in the absence of P. gingivalis served as negative control. Blocking the TERT-2 cell CCR5 receptor with antibodies significantly reduced trans infection of R5-HIV-1 (p < 0.001) (Fig. 6C) but not X4-HIV-1 (not shown) to TZM-bl cells. After pre-incubation with P. gingivalis, anti-CCR5 reduced trans infection of R5-tropic HIV-1 to levels similar to HIV-1 inoculated TERT-2 cells without P. gingivalis.
To confirm the role of CCR5, TERT-2 cells with or without pre-incubation with P. gingivalis were incubated with the CCR5 ligand, RANTES, at 30 and 300 ng/mL or with 10 or 100 nM of TAK-779. TAK-779 selectively blocks HIV gp120 interaction with CCR5 . Consistent with the CCR5 antibody data, cells incubated with RANTES (Fig. 6D) or TAK-799 (data not shown) before inoculation with HIV-1 showed statistically significant (p < 0.05) dose-dependent reductions in the increased transfer of R5-HIV-1 mediated by P. gingivalis. As expected, TERT-2 cells inoculated with X4 viruses in the presence or absence of P. gingivalis were not affected by RANTES or TAK-779 (data not shown). In the absence of HIV-1, TERT-2 cells incubated with either P. gingivalis, CCR5 antibody, RANTES or TAK-779, showed no false-positive transfer (staining by TZM-bl reporter) (data not shown).
At harvest, co-cultured TERT-2 cells, which had been washed and trypsinized to remove extracellular and plasma membrane-associated HIV-1, appeared to trans infect the TZM-bl reporter cells (Fig. 6E). In some fields, blue TZM-bl cells and TERT-2 cells appeared to grow independently (Fig. 6E, panel I). More commonly, blue TZM-bl cells and TERT-2 cells grew in direct contact (panels II, III). In comparison to co-culture with HIV-1 infected TERT-2 cells, TZM-bl-to-TZM-bl trans infection of R5-tropic HIV-1 resulted in 100-fold more infected reporter cells (data not shown), increased multinuclear cells with syncytia formation, and more intense staining (Fig. 6E, panel IV). Although the contact status with TZM-bl cells at the time of trans infection was not established, the images suggest that TERT-2 cells trans infect either released virus or directly transferred internalized HIV-1 mediated by cell-to-cell contacts. P. gingivalis-mediated increased transfer of intracellular HIV-1 was unrelated to reverse transcription. Indeed, AZT (500 μM) maintained continuously in TERT-2 cells cultures did not affect the increase in R5-tropic HIV-1 trans infection caused by P. gingivalis (data not shown).
Endogenous bacteria may modulate HIV-1 infection. For example, we have shown that the oral endogenous pathogen, P. gingivalis, can up-regulate CCR5 on oral keratinocytes . We next sought to learn whether CCR5 up-regulation by P. gingivalis could modulate dissemination of R5-tropic HIV-1 from oral keratinocytes. In this report, we show that P. gingivalis increases the transmissibility of infectious R5-tropic HIV-1 to proximal permissive cells in vitro without affecting the dissemination of X4-tropic HIV-1. To the best of our knowledge, this is the first report of interactions between septic oral epithelial cells and HIV-1.
HIV-1 infection never occurs in a sterile environment and the septic mucosal environment may affect susceptibility to HIV-1 infection. The oral mucosa and virtually all mucosal epithelial tissues are colonized by polymicrobial biofilms that may modify the acquisition of HIV-1 infection. Co-infecting microorganisms that affect the clinical course of HIV-AIDS or mechanisms of infection include Mycobacterium tuberculosis , human hepatitis C virus , hepatitis B (HBV)  herpes simplex virus-2 (HSV-2) , Neisseria gonorrhea , cytomegalovirus, Epstein-Barr virus, HHV-6, -7, and -8, and human papilloma virus . When HIV and Leishmania co-infect, the severity increases for both infections . Similarly, the malaria-causing protozoan Plasmodium is highly associated with the occurrence  and severity of HIV infections . Co-infection with Mycobacterium avium may directly increase the severity of infection by increasing HIV-1 replication .
P. gingivalis is a putative pathogen associated with periodontitis, a polymicrobial infection of the gingiva and tooth-supporting connective tissues, bone and ligament . When challenged with commensal and pathogenic bacteria, oral keratinocytes release cytokines and chemokines , which may function as chemoattractants for CD4-positive T cells . Infiltrating CD4+ T cells can then co-localize with keratinocytes, facilitating docking and transfer of infectious virions . In the gingiva, immature dendritic (Langerhans) cells  typically co-localize with keratinocytes and T cells . Indeed, we show that TERT-2 cells contact and apparently transfer virus to co-cultured CD4-positive TZM-bl cells (Fig. 6E panels II–III), whereas the virus does not appear to interact directly with P. gingivalis (Fig. 5). The data also show that virus released by TERT-2 cells can also be captured by TZM-bl cells (Fig. 6E panel I). TZM-bl cells were used to both assay infectivity of cell-free HIV-1 and serve as a permissive target for trans infection of HIV-1 from oral keratinocytes. The results in TZM-bl cells parallel data we have obtained using primary tonsil keratinocytes and several keratinocyte cell lines using peripheral blood mononuclear cells as permissive targets (Vacharaksa et al, unpublished data).
In the presence of P. gingivalis, TERT-2 (Fig. 6C, D) and primary tonsil epithelial cells (data not shown) selectively increase trans infection of R5-tropic HIV-1 to TZM-bl cells. The selective increase in infectious R5-tropic HIV-1 on the plasma membranes and within TERT-2 cells (Fig. 4), and release into the extracellular environment (Fig. 1A) is independent of new viral replication (Fig. 3). TERT-2 cells appear to take up and contain the same amount of HIV-1 over time in the presence and absence of P. gingivalis based on HIV-1gag RNA and p24 levels (Fig. 2). The reason for this striking increase in infectivity is not clear. The amount of recovered virus protein or RNA can be discordant with levels of infectious virions [26, 52]. Furthermore, non-permissive HIV-1 infection of oral keratinocytes occurs at low frequency, and small differences in the presence and absence of P. gingivalis may challenge the sensitivity and discrimination of the detection assays. To estimate viral infectivity, we show that counting 10 to 40 positive cells of the 1 × 104 TZM-bl cells per well is reproducible and reliable. Infectivity apparently discriminates better than detection or quantification of viral protein. It is clear, however, that the ability of keratinocytes to bind and capture HIV-1, as estimated by HIVgag RNA and p24 levels, does not reflect the persistence and transfer of infectious virions to target cells.
Two plausible mechanisms emerge to explain the P. gingivalis-mediated selective increase in infectious R5-tropic HIV-1. As a consequence of P. gingivalis, TERT-2 cells selectively harbor and protect infectious R5-tropic HIV-1, but not CXCR4-tropic virus. In addition, trans infection of R5-tropic HIV-1 to permissive TZM-bl cells also increases in a CCR5 up-regulation-dependent manner. Although both are dependent on pre-incubation with P. gingivalis, these mechanisms differ.
In response to P. gingivalis, infectious virions were consistently recovered from TERT-2 cell culture medium (Fig. 1A), cell surface (Fig. 4A) and within the cell (Fig. 4C). AZT treatment did not affect viral infectivity suggesting that transfer of intracellular R5-HIV-1 from oral keratinocytes was independent of intracellular viral uncoating or reverse transcription. Harbored HIV-1 remains infectious for up to two days (Figs. 1A, 4A, C). Dendritic cells show similar capability. For example, attachment of HIV-1 to DC-SIGN preserves infectious virus up to 4 days . When compared to cells that do not express DC-SIGN, preservation of viral infectivity results in an increase in trans infection to CD4+ permissive cells . The P. gingivalis-mediated selective increase in cell-associated R5-tropic HIV-1 suggests a novel protective activity is expressed in oral epithelial cells. This protective activity for harbored HIV-1 may be independent of the expression of CCR5. Protective activity may function directly on R5-tropic virus or by inhibiting HIV-1 inactivation mechanisms. P. gingivalis alters the gene expression profile in TERT-2 cells through lipopolysaccharide activation of Toll-like receptors and protease activation of protease-activated receptors . Therefore, modulation of innate immunity by P. gingivalis may enable keratinocytes to increase the infectivity of harbored R5-tropic HIV-1.
Perhaps in concert with protection of harbored virus, we also showed that P. gingivalis-mediated upregulation of CCR5 in TERT-2 cells  increases the effectiveness of trans infection to permissive TZM-bl cells (CD4+ CXCR4+ CCR5+). In TERT-2 cells, CCR5 appears to function primarily in trans, increasing the delivery of R5-tropic HIV-1 to CD4+ permissive cells. The trans function appears to be analogous to DC-SIGN on dendritic cells, which enables trans infection to permissive cells . Clearly, CCR5 blockade with specific antibodies, RANTES or a receptor antagonist inhibits the P. gingivalis-mediated increase in selective R5-tropic HIV-1 trans infection (Figs. 5, 6). The CCR5-dependent increase in trans infection of R5-tropic HIV-1 could reflect TERT-2 cell uptake, release or cell-to-cell transfer of virus. That P. gingivalis does not affect levels or kinetics of intracellular HIV-1gag RNA (Fig. 2C, D) and p24 (Fig. 6A, B) argues against a significant role for CCR5 in selective R5-tropic HIV-1 uptake in these cells. While the data suggest strongly that CCR5 is necessary for the specific trans infection of R5-tropic HIV-1, it is likely that internalization of the virus within the keratinocyte is CCR5-independent. Consistent with our findings, blocking CCR5 antibodies reduced transcytosis of R5-specific HIV-1 through primary genital epithelial cells, resulting in attenuated infection of CD4+ cells . Furthermore, up-regulation of CCR5 appears to be necessary, but may not be sufficient for trans infection. The net effect of the P. gingivalis-mediated up-regulation of CCR5 expression, however, appears to be an increase in the proportion of R5-tropic HIV-1 that successfully transits though the keratinocyte to trans infect permissive targets.
Interestingly, P. gingivalis proteases, particularly RgpA, inhibit gp120-mediated HIV-1 fusion with the highly permissive MT4 T-cell line and facilitate proteolysis of the CD4 receptor . In the present study, the CD4-negative oral keratinocytes [7, 11, 21, 22] show HIV internalization in the presence of P. gingivalis Rgp. Like dendritic cells (DCs) , HIV-1 appears to enter CD4- cells by endocytosis, involving clathrin-coated vesicles [55, 56] or macropinosomes . Like DCs [53, 58], infection of oral keratinocytes is non-productive (Vacharaksa et al, unpublished), but it remains to be learned whether, like DCs [12, 59], keratinocytes use synapse formation to trans infect. Indeed, recent evidence suggests that HIV-1 enters oral keratinocytes by an endocytic pathway within minutes (Dietrich et al, unpublished) without apparent reliance on gp120 and CD4-mediated membrane fusion [56, 57]. If P. gingivalis modifies interactions between receptors or co-receptors and HIV-1, candidate targets would be other than cell-fusion associated CD4, CXCR4 and CCR5. Clearly, the interactions between TERT-2 cells and P. gingivalis are complex and up-regulation of CCR5 provides only a partial explanation for the increase in trans infection of R5-tropic HIV-1. If these mechanisms simulate pathways in vivo, the oral keratinocyte would be placed in the circuit of transmission of HIV-1, capturing R5-tropic HIV-1 from the mucosal surface and transferring the infectious virus to permissive cells such as infiltrating CD4-positive T cells or specific intraepithelial dendritic cells (DCs). Since iDCs dock with CD4+ T cells, P. gingivalis-infected oral keratinocytes can contribute to the selective systemic dissemination of R5-tropic HIV-1.
Collectively, these data suggest that select mucosal sites in the oral cavity such as the periodontal tissues, where organisms like P. gingivalis are often abundant in the complex microflora , may contribute to the complex set of restrictions and enabling pathways that in aggregate serve as the mucosal gatekeeper system for primary R5-tropic HIV-1 clinical infection . A CCR5-dependent gatekeeper mechanism, or one that is regulated by an endogenous co-pathogen, P. gingivalis, has not been previously recognized in oral epithelia. Somewhat analogous to P. gingivalis in oral keratinocytes , the oral pathogen Actinobacillus actinomycetemcomitans increases expression of HIV-1 coreceptors in monocytes . Under conditions of inflammation or infection, polarized human endometrial cells also increase release of infectious HIV-1 to the extracellular compartment . Furthermore, in periodontitis, the epithelial barrier is disrupted , increasing the proximity of virus or virus-infected keratinocytes to target T-cells [32, 62], release of pro-inflammatory cytokines  and activation of TLR-dependent signaling pathways by bacteria . Inflammation in the gingiva and oral mucosa could enhance HIV-1 infection of the oral tissues. For example, certain bacterial pathogens  and E. coli LPS  increase HIV-1 promoter activity by signaling through TLR4. We find no evidence that P. gingivalis increase HIV-1 transcriptional activity in TERT-2 cells. In control experiments, we ruled out that P. gingivalis and its products in spent bacterial media could activate the LTR promoter in TZM-bl cells (data not shown). Since infectious HIV-1 can be harbored in oral keratinocytes, the squamous mucosal epithelium may also constitute a cryptic reservoir of infection in vivo, which is enhanced specifically for R5-tropic HIV-1 in the presence of P. gingivalis. Periodontal disease and other oral infections and inflammatory conditions may, therefore, affect the risk for systemic dissemination of HIV-1 from an oral focus. If this speculation is confirmed, novel therapeutic strategies could be developed to thwart HIV-1 at its point of entry.
Immortalized human oral keratinocytes OKF6/TERT-2 (TERT-2)  were provided by James Rheinwald of Harvard Medical School and cultured essentially as described previously . In brief, cells were grown in 5% CO2 at 37°C in keratinocyte serum-free (KSF-M; Gibco), supplemented with 0.3 mM CaCl2, 25 μg/mL bovine pituitary extract and 0.2 ng/mL epidermal growth factor (TERT-2 medium). Culture media was changed every two days and cells were subcultured at 60 to 70% confluency (about 5 days). Using an IRB approved protocol, palatine tonsil tissues were obtained from routine tonsillectomies performed at the Hennepin County Medical Center, Minneapolis, MN. Primary tonsil epithelial cells were isolated and cultured using a protocol modified from Oda and Watson  as described . In brief, tonsil cells were cultured and the medium was partially replaced (70%) every 2 days. Once the primary culture was established, cells were passaged after 4 days in culture at 70 to 80% confluence. Only cells growing in passage 3 or 4 were used for the experiments. Tonsil epithelial cells were at least 96% epithelial based on flow cytometric analysis using epithelial and fibroblast markers. To serve as a positive control and also as permissive targets for HIV-1 infection, TZM-bl cells (JC53)  were obtained from and cultured as recommended by the NIH AIDS Research and Reference Reagent Program, MD.
Viruses and bacteria
The X4- (IIIb) (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HTLV-IIIB/H9 from Dr. Robert Gallo, Cat. 398) and R5-tropic HIV-1 (Ba-L) (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1Ba-L from Dr. Suzanne Gartner, Dr. Mikulas Popovic and Dr. Robert Gallo, Cat. 510) strains were propagated in peripheral blood mononuclear cells (PBMCs) using the protocols of the NIH AIDS Research and Reference Reagent Program. To estimate the amount of infectious virus, the 50% infection endpoint method (TCID50) of Reed-Muench was used [71, 72]. TCID50 of virus stocks was determined in PHA-activated PBMCs. A multiplicity of HIV-1 infection (MOI) of 0.005 was used to infect the cells (TCID50 per cell).
P. gingivalis, strain ATCC 33277, was grown under anaerobic conditions in a Coy anaerobic chamber (85% N2, 5% CO2 and 10% H2) at 37°C on Todd-Hewitt agar plates (Difco) supplemented with 5% (v/v) defibrinated sheep blood or in Todd-Hewitt broth supplemented with 5 μg/mL hemin (Sigma) and 1 μg/mL menadione (Sigma). Bacteria were grown in 5 mL of broth for approximately 72 h to an OD620 nm of 0.9 to 1.1 (early stationary phase) and counted for determination of the bacterial MOI by the spiral plate method .
Infections with P. gingivalisand HIV-1
To serve as targets of HIV-1 infection, TERT-2 or tonsil epithelial cells (1 × 105) were seeded in 24-well plates and grown overnight. Culture medium was removed, replaced with fresh pre-warmed medium and freshly harvested P. gingivalis were added at a MOI of 100 for 3 h at 37°C in spent Todd-Hewitt broth to a final volume of 500 μL. After P. gingivalis incubation, cells were washed 5 times with Dulbecco's Phosphate-Buffered Saline (DPBS) (Gibco). Negative control cells (unexposed to P. gingivalis) were incubated with anaerobic Todd-Hewitt broth, and grown under identical conditions. After removing the bacteria at 3 h, HIV-1 IIIb or Ba-L in 500 μL of medium was inoculated at an MOI 0.005 onto keratinocyte cultures and incubated. At 6 h, cells were washed 5 times with DPBS to remove excess virus, fresh medium was added, and incubation continued for up to 54 h post-inoculation with P. gingivalis (48 h after washing to remove HIV-1). At each time point, three different samples were obtained: (i) released HIV-1 in 500 μL of spent cell culture supernatants; (ii) plasma membrane-associated HIV-1, and (iii) intracellular, trypsin-resistant HIV-1. To recover plasma membrane-associated virus (sample ii), cells were treated with 250 μL of 0.05% trypsin-EDTA (Gibco) for 5 min at 37°C. Trypsin was inactivated by addition of equal volumes of DMEM (Mediatech) supplemented with 10% FBS (TZM-bl medium). Cell suspensions were collected, centrifuged at 5,255 × g for 1 min and supernatants were recovered containing membrane-associated HIV that was released by trypsin. The resulting cell pellet containing intracellular HIV-1 (sample iii) was resuspended in 500 μL of TZM-bl medium, lysed by 3 cycles of freezing under liquid N2 and thawing at room temperature. Lysis was verified by light microscopy. The three samples were stored at -20°C for infectious virion assay (see below) or p24 ELISA. HIV p24 in cell samples was estimated by ELISA (Beckman-Coulter) as described by the manufacturer.
TZM-bl reporter assay for infectious HIV
To determine transfer of infectious HIV-1 from oral keratinocytes, TZM-bl cells were seeded at 1 × 104 cells per well and grown overnight in 96-well plates. The HIV-1 samples (100 μL of a 1:2 dilution) were incubated with TZM-bl reporter cells for 2 h at 37°C in 5% CO2, washed 3 times with TZM-bl medium, and incubated for 24 h. TZM-bl cells were then fixed with 100 μL of 0.05% glutaraldehyde for 5 min, and washed 3 times with DPBS. Cells were then covered with 50 μL of X-Gal solution (500 mM potassium ferrocyanide, 500 mM potassium ferricyanide, 0.1 M magnesium chloride and 20 mg/mL X-Gal; all from Sigma) and incubated for 2 h at 37°C in 5% CO2. After staining, blue positive cells were counted in each well under a light microscope. To assess specificity of the reporter cell to HIV infection, monolayers of TZM-bl cells were exposed to P. gingivalis-exposed and unexposed TERT-2 or TZM-bl supernatants without HIV-1 (negative control). At most, 1 or 2 blue-stained cells per well were detected as a false positive result.
Co-culture of infected TERT-2 cells with reporter TZM-bl cells
TERT-2 cells were pre-incubated with P. gingivalis for 3 h, washed 5 times, incubated with R5-HIV-1 (Ba-L) for 6 h, washed 5 times and incubated in TERT medium for 18 h post-inoculation. Extracellular HIV-1 was then removed by trypsin, cells were washed twice with TZM-bl medium and resuspended in TERT medium. TERT-2 cells (1 × 104) in suspension were added to monolayers of TZM-bl reporter cells per well in a mixture of equal volumes of TERT and TZM-bl media. Co-cultures were maintained for 24 h at 37°C in 5% CO2 and stained with X-Gal solution, as above.
Primer sequences and PCR conditions
50°C, 2 min, 95°C, 2 min, and 95°C, 15 s and 60°C, 30 s, for 50 cycles
50°C, 2 min, 95°C, 2 min, and 95°C, 15 s and 60°C, 30 s, for 50 cycles
95°C, 15 s, 55°C, 30 s, 72°C, 15 s for 30 cycles
95°C, 15 s, 60°C, 30 s, 72°C, 15 s for 30 cycles
TERT-2 cells (2 × 105) were seeded on cover slips in 12-well plates and cultured overnight at 37°C in 5% CO2. Cells were incubated for 3 h with P. gingivalis ATCC 33277 at MOI 100, washed 5 times with DPBS, inoculated with HIV Ba-L for 2 h at MOI 0.005 and washed 5 more times. TERT-2 cells were prepared for microscopic observation, as described . For some experiments, P. gingivalis and HIV Ba-L were co-cultured on glass slides without cells. Cells were incubated with a 1:100 dilution (v/v) of murine anti-HIV p24 IgG1 monoclonal antibody (Chemicon Int., Millipore), murine IgG1 isotype control antibody (BD Biosciences, Pharmingen), or rabbit polyclonal anti-P. gingivalis IgG. After antibody incubation, cells were washed 5 times and incubated with ALEXA 568-conjugated donkey anti-murine IgG antibody (Molecular Probes) diluted 1:2000 (v/v) and/or FITC-conjugated goat anti-rabbit IgG antibody (Sigma) (1:1000 (v/v) dilution) and DAPI (Molecular Probes) at a 1:3000 (v/v) dilution for 1 h. Samples were observed using a confocal microscope (Olympus FluoView 1000). From each field, 10 to 30 consecutive sections of 0.5 μm thickness were resolved at 1024 × 1024 pixels, analyzed and pseudo-colored. Co-localization of antigens was determined in single sections from the image stacks. Final images were generated using ImageJ 1.37V software (National Institutes of Health). Microscope settings were kept identical for all images captured.
CCR5 blocking experiments
CCR5 was blocked on TERT-2 cells using goat anti-human CCR5 polyclonal antibody (CKR-5; Santa Cruz), recombinant human RANTES (CCL5) (Peprotech) or TAK-779 (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH:TAK-779)  essentially as reported previously . In the presence of anti-human CCR5 or RANTES, TERT-2 cells were pre-incubated in the presence or absence of P. gingivalis. Cells were then inoculated with HIV-1 Ba-L or IIIb at a MOI 0.005, incubated for 6 h, washed 5 times with DPBS and cultured for 18 h post-inoculation. Culture supernatants or cell lysates were recovered, stored at -20°C and inoculated onto TZM-bl cells to report infectious HIV. In some experiments, TERT-2 cells were trypsinized at 18 h post-inoculation and co-cultured with TZM-bl cells, as described above. Infectious virus from the samples was assayed in TZM-bl cells as described above. Oral keratinocytes in the presence or absence of P. gingivalis were used as positive and negative controls, respectively.
Statistical analyses were performed by the Student's t test for paired values using GraphPad software. Differences were considered significant at a p < 0.05.
These studies were supported by NIH grants-in-aid DE015503 (to MCH), DE15506 (KFR), the Veterans Affairs Research Service, and the Mucosal and Vaccine Research Center. The expert technical support and advice of Claudine Fasching is greatly appreciated. This manuscript was submitted in partial fulfillment of the requirements for the PhD degree by RAG.
- Coovadia HM, Rollins NC, Bland RM, Little K, Coutsoudis A, Bennish ML, Newell ML: Mother-to-child transmission of HIV-1 infection during exclusive breastfeeding in the first 6 months of life: an intervention cohort study. Lancet. 2007, 369 (9567): 1107-1116. 10.1016/S0140-6736(07)60283-9.View ArticlePubMedGoogle Scholar
- Younai FS: Oral HIV transmission. J Calif Dent Assoc. 2001, 29 (2): 142-148.PubMedGoogle Scholar
- Milush JM, Kosub D, Marthas M, Schmidt K, Scott F, Wozniakowski A, Brown C, Westmoreland S, Sodora DL: Rapid dissemination of SIV following oral inoculation. Aids. 2004, 18 (18): 2371-2380.PubMedGoogle Scholar
- Milush JM, Stefano-Cole K, Schmidt K, Durudas A, Pandrea I, Sodora DL: Mucosal innate immune response associated with a timely humoral immune response and slower disease progression after oral transmission of simian immunodeficiency virus to rhesus macaques. J Virol. 2007, 81 (12): 6175-6186. 10.1128/JVI.00042-07.View ArticlePubMedGoogle Scholar
- Abel K, Pahar B, Van Rompay KK, Fritts L, Sin C, Schmidt K, Colon R, McChesney M, Marthas ML: Rapid virus dissemination in infant macaques after oral simian immunodeficiency virus exposure in the presence of local innate immune responses. J Virol. 2006, 80 (13): 6357-6367. 10.1128/JVI.02240-05.View ArticlePubMedGoogle Scholar
- Rodriguez-Inigo E, Jimenez E, Bartolome J, Ortiz-Movilla N, Bartolome Villar B, Jose Arrieta J, Manzarbeitia F, Carreno V: Detection of human immunodeficiency virus type 1 RNA by in situ hybridization in oral mucosa epithelial cells from anti-HIV-1 positive patients. J Med Virol. 2005, 77 (1): 17-22. 10.1002/jmv.20409.View ArticlePubMedGoogle Scholar
- Liu X, Zha J, Chen H, Nishitani J, Camargo P, Cole SW, Zack JA: Human immunodeficiency virus type 1 infection and replication in normal human oral keratinocytes. J Virol. 2003, 77 (6): 3470-3476. 10.1128/JVI.77.6.3470-3476.2003.View ArticlePubMedGoogle Scholar
- Moore JS, Rahemtulla F, Kent LW, Hall SD, Ikizler MR, Wright PF, Nguyen HH, Jackson S: Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro. Virology. 2003, 313 (2): 343-353. 10.1016/S0042-6822(03)00283-6.View ArticlePubMedGoogle Scholar
- Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, MacDonald ME, Stuhlmann H, Koup RA, Landau NR: Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell. 1996, 86 (3): 367-377. 10.1016/S0092-8674(00)80110-5.View ArticlePubMedGoogle Scholar
- Lu FX, Jacobson RS: Oral mucosal immunity and HIV/SIV infection. J Dent Res. 2007, 86 (3): 216-226.View ArticlePubMedGoogle Scholar
- Kumar RB, Maher DM, Herzberg MC, Southern PJ: Expression of HIV receptors, alternate receptors and co-receptors on tonsillar epithelium: implications for HIV binding and primary oral infection. Virol J. 2006, 3: 25-10.1186/1743-422X-3-25.View ArticlePubMedGoogle Scholar
- Wilkinson J, Cunningham AL: Mucosal transmission of HIV-1: first stop dendritic cells. Curr Drug Targets. 2006, 7 (12): 1563-1569. 10.2174/138945006779025482.View ArticlePubMedGoogle Scholar
- Meng G, Wei X, Wu X, Sellers MT, Decker JM, Moldoveanu Z, Orenstein JM, Graham MF, Kappes JC, Mestecky J, Shaw GM, Smith PD: Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells. Nat Med. 2002, 8 (2): 150-156. 10.1038/nm0202-150.View ArticlePubMedGoogle Scholar
- Moore JP, Kitchen SG, Pugach P, Zack JA: The CCR5 and CXCR4 coreceptors--central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection. AIDS Res Hum Retroviruses. 2004, 20 (1): 111-126. 10.1089/088922204322749567.View ArticlePubMedGoogle Scholar
- Reynes J, Portales P, Segondy M, Baillat V, Andre P, Avinens O, Picot MC, Clot J, Eliaou JF, Corbeau P: CD4 T cell surface CCR5 density as a host factor in HIV-1 disease progression. Aids. 2001, 15 (13): 1627-1634. 10.1097/00002030-200109070-00004.View ArticlePubMedGoogle Scholar
- Bomsel M, David V: Mucosal gatekeepers: selecting HIV viruses for early infection. Nat Med. 2002, 8 (2): 114-116. 10.1038/nm0202-114.View ArticlePubMedGoogle Scholar
- Margolis L, Shattock R: Selective transmission of CCR5-utilizing HIV-1: the 'gatekeeper' problem resolved?. Nat Rev Microbiol. 2006, 4 (4): 312-317.View ArticlePubMedGoogle Scholar
- Sullivan N, Sun Y, Sattentau Q, Thali M, Wu D, Denisova G, Gershoni J, Robinson J, Moore J, Sodroski J: CD4-Induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization. J Virol. 1998, 72 (6): 4694-4703.PubMedGoogle Scholar
- Cutler CW, Jotwani R: Oral mucosal expression of HIV-1 receptors, co-receptors, and alpha-defensins: tableau of resistance or susceptibility to HIV infection?. Adv Dent Res. 2006, 19 (1): 49-51.View ArticlePubMedGoogle Scholar
- Giacaman RA, Nobbs AH, Ross KF, Herzberg MC: Porphyromonas gingivalis Selectively Up-Regulates the HIV-1 Coreceptor CCR5 in Oral Keratinocytes. J Immunol. 2007, 179 (4): 2542-2550.View ArticlePubMedGoogle Scholar
- Quinones-Mateu ME, Lederman MM, Feng Z, Chakraborty B, Weber J, Rangel HR, Marotta ML, Mirza M, Jiang B, Kiser P, Medvik K, Sieg SF, Weinberg A: Human epithelial beta-defensins 2 and 3 inhibit HIV-1 replication. Aids. 2003, 17 (16): F39-48. 10.1097/00002030-200311070-00001.View ArticlePubMedGoogle Scholar
- Vacharaksa A, Asrani A, Gebhard K, Faschling C, Janoff E, Ross K, Herzberg M: Infection and Replication of HIV-1 in Immortalized Oral Keratinocytes. Submitted. 2007Google Scholar
- Kage A, Shoolian E, Rokos K, Ozel M, Nuck R, Reutter W, Kottgen E, Pauli G: Epithelial uptake and transport of cell-free human immunodeficiency virus type 1 and gp120-coated microparticles. J Virol. 1998, 72 (5): 4231-4236.PubMedGoogle Scholar
- Fantini J, Cook DG, Nathanson N, Spitalnik SL, Gonzalez-Scarano F: Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor. Proc Natl Acad Sci U S A. 1993, 90 (7): 2700-2704. 10.1073/pnas.90.7.2700.View ArticlePubMedGoogle Scholar
- Saidi H, Magri G, Nasreddine N, Requena M, Belec L: R5- and X4-HIV-1 use differentially the endometrial epithelial cells HEC-1A to ensure their own spread: implication for mechanisms of sexual transmission. Virology. 2007, 358 (1): 55-68. 10.1016/j.virol.2006.07.029.View ArticlePubMedGoogle Scholar
- Bobardt MD, Saphire AC, Hung HC, Yu X, Van der Schueren B, Zhang Z, David G, Gallay PA: Syndecan captures, protects, and transmits HIV to T lymphocytes. Immunity. 2003, 18 (1): 27-39. 10.1016/S1074-7613(02)00504-6.View ArticlePubMedGoogle Scholar
- Bobardt MD, Chatterji U, Selvarajah S, Van der Schueren B, David G, Kahn B, Gallay PA: Cell-free human immunodeficiency virus type 1 transcytosis through primary genital epithelial cells. J Virol. 2007, 81 (1): 395-405. 10.1128/JVI.01303-06.View ArticlePubMedGoogle Scholar
- Szolnoky G, Bata-Csorgo Z, Kenderessy AS, Kiss M, Pivarcsi A, Novak Z, Nagy Newman K, Michel G, Ruzicka T, Marodi L, Dobozy A, Kemeny L: A mannose-binding receptor is expressed on human keratinocytes and mediates killing of Candida albicans. J Invest Dermatol. 2001, 117 (2): 205-213. 10.1046/j.1523-1747.2001.14071.x.View ArticlePubMedGoogle Scholar
- Liu Y, Liu H, Kim BO, Gattone VH, Li J, Nath A, Blum J, He JJ: CD4-independent infection of astrocytes by human immunodeficiency virus type 1: requirement for the human mannose receptor. J Virol. 2004, 78 (8): 4120-4133. 10.1128/JVI.78.8.4120-4133.2004.View ArticlePubMedGoogle Scholar
- Delezay O, Koch N, Yahi N, Hammache D, Tourres C, Tamalet C, Fantini J: Co-expression of CXCR4/fusin and galactosylceramide in the human intestinal epithelial cell line HT-29. Aids. 1997, 11 (11): 1311-1318. 10.1097/00002030-199711000-00004.View ArticlePubMedGoogle Scholar
- Bomsel M: Transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier. Nat Med. 1997, 3 (1): 42-47. 10.1038/nm0197-42.View ArticlePubMedGoogle Scholar
- Carreno MP, Krieff C, Irinopoulou T, Kazatchkine MD, Belec L: Enhanced transcytosis of R5-tropic human immunodeficiency virus across tight monolayer of polarized human endometrial cells under pro-inflammatory conditions. Cytokine. 2002, 20 (6): 289-294. 10.1006/cyto.2002.2009.View ArticlePubMedGoogle Scholar
- Yasukawa M, Hasegawa A, Sakai I, Ohminami H, Arai J, Kaneko S, Yakushijin Y, Maeyama K, Nakashima H, Arakaki R, Fujita S: Down-regulation of CXCR4 by human herpesvirus 6 (HHV-6) and HHV-7. J Immunol. 1999, 162 (9): 5417-5422.PubMedGoogle Scholar
- Secchiero P, Zella D, Barabitskaja O, Reitz MS, Capitani S, Gallo RC, Zauli G: Progressive and persistent downregulation of surface CXCR4 in CD4(+) T cells infected with human herpesvirus 7. Blood. 1998, 92 (12): 4521-4528.PubMedGoogle Scholar
- Nisapakultorn K, Ross KF, Herzberg MC: Calprotectin expression in vitro by oral epithelial cells confers resistance to infection by Porphyromonas gingivalis. Infect Immun. 2001, 69 (7): 4242-4247. 10.1128/IAI.69.7.4242-4247.2001.View ArticlePubMedGoogle Scholar
- Baba M, Nishimura O, Kanzaki N, Okamoto M, Sawada H, Iizawa Y, Shiraishi M, Aramaki Y, Okonogi K, Ogawa Y, Meguro K, Fujino M: A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc Natl Acad Sci U S A. 1999, 96 (10): 5698-5703. 10.1073/pnas.96.10.5698.View ArticlePubMedGoogle Scholar
- Breen RA, Swaden L, Ballinger J, Lipman MC: Tuberculosis and HIV co-infection: a practical therapeutic approach. Drugs. 2006, 66 (18): 2299-2308. 10.2165/00003495-200666180-00003.View ArticlePubMedGoogle Scholar
- Parodi C, Belmonte L, Bare P, de Bracco MM, Ruibal-Ares B: Impact of human immune deficiency virus infection on hepatitis C virus infection and replication. Curr HIV Res. 2007, 5 (1): 55-67. 10.2174/157016207779316341.View ArticlePubMedGoogle Scholar
- Salmon-Ceron D, Lewden C, Morlat P, Bevilacqua S, Jougla E, Bonnet F, Heripret L, Costagliola D, May T, Chene G: Liver disease as a major cause of death among HIV infected patients: role of hepatitis C and B viruses and alcohol. J Hepatol. 2005, 42 (6): 799-805. 10.1016/j.jhep.2005.01.022.View ArticlePubMedGoogle Scholar
- Lingappa JR, Celum C: Clinical and therapeutic issues for herpes simplex virus-2 and HIV co-infection. Drugs. 2007, 67 (2): 155-174. 10.2165/00003495-200767020-00001.View ArticlePubMedGoogle Scholar
- Schwarcz SK, Kellogg TA, McFarland W, Louie B, Klausner J, Withum DG, Katz MH: Characterization of sexually transmitted disease clinic patients with recent human immunodeficiency virus infection. J Infect Dis. 2002, 186 (7): 1019-1022. 10.1086/342954.View ArticlePubMedGoogle Scholar
- Mbopi-Keou FX, Belec L, Teo CG, Scully C, Porter SR: Synergism between HIV and other viruses in the mouth. Lancet Infect Dis. 2002, 2 (7): 416-424. 10.1016/S1473-3099(02)00317-1.View ArticlePubMedGoogle Scholar
- Krisanaprakornkit S, Weinberg A, Perez CN, Dale BA: Expression of the peptide antibiotic human beta-defensin 1 in cultured gingival epithelial cells and gingival tissue. Infect Immun. 1998, 66 (9): 4222-4228.PubMedGoogle Scholar
- Abu-Raddad LJ, Patnaik P, Kublin JG: Dual infection with HIV and malaria fuels the spread of both diseases in sub-Saharan Africa. Science. 2006, 314 (5805): 1603-1606. 10.1126/science.1132338.View ArticlePubMedGoogle Scholar
- Idemyor V: Human immunodeficiency virus (HIV) and malaria interaction in sub-Saharan Africa: the collision of two Titans. HIV Clin Trials. 2007, 8 (4): 246-253. 10.1310/hct0804-246.View ArticlePubMedGoogle Scholar
- Guenthner PC, Secor WE, Dezzutti CS: Trichomonas vaginalis-induced epithelial monolayer disruption and human immunodeficiency virus type 1 (HIV-1) replication: implications for the sexual transmission of HIV-1. Infect Immun. 2005, 73 (7): 4155-4160. 10.1128/IAI.73.7.4155-4160.2005.View ArticlePubMedGoogle Scholar
- Nonnenmacher C, Dalpke A, Rochon J, Flores-de-Jacoby L, Mutters R, Heeg K: Real-time polymerase chain reaction for detection and quantification of bacteria in periodontal patients. J Periodontol. 2005, 76 (9): 1542-1549. 10.1902/jop.2005.76.9.1542.View ArticlePubMedGoogle Scholar
- Bodet C, Chandad F, Grenier D: Modulation of cytokine production by Porphyromonas gingivalis in a macrophage and epithelial cell co-culture model. Microbes Infect. 2005, 7 (3): 448-456. 10.1016/j.micinf.2004.11.021.View ArticlePubMedGoogle Scholar
- Qi X, Koya Y, Saitoh T, Saitoh Y, Shimizu S, Ohba K, Yamamoto N, Yamaoka S, Yamamoto N: Efficient induction of HIV-1 replication in latently infected cells through contact with CD4(+) T cells: Involvement of NF-kappaB activation. Virology. 2007, 361 (2): 325-334. 10.1016/j.virol.2006.11.014.View ArticlePubMedGoogle Scholar
- Allam JP, Novak N, Fuchs C, Asen S, Berge S, Appel T, Geiger E, Kochan JP, Bieber T: Characterization of dendritic cells from human oral mucosa: a new Langerhans' cell type with high constitutive FcepsilonRI expression. J Allergy Clin Immunol. 2003, 112 (1): 141-148. 10.1067/mai.2003.1607.View ArticlePubMedGoogle Scholar
- Hasseus B, Jontell M, Bergenholtz G, Dahlgren UI: Langerhans cells from human oral epithelium are more effective at stimulating allogeneic T cells in vitro than Langerhans cells from skin. Clin Exp Immunol. 2004, 136 (3): 483-489. 10.1111/j.1365-2249.2004.02469.x.View ArticlePubMedGoogle Scholar
- Saphire AC, Bobardt MD, Gallay PA: Cyclophilin a plays distinct roles in human immunodeficiency virus type 1 entry and postentry events, as revealed by spinoculation. J Virol. 2002, 76 (9): 4671-4677. 10.1128/JVI.76.9.4671-4677.2002.View ArticlePubMedGoogle Scholar
- Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, Cornelissen IL, Nottet HS, KewalRamani VN, Littman DR, Figdor CG, van Kooyk Y: DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell. 2000, 100 (5): 587-597. 10.1016/S0092-8674(00)80694-7.View ArticlePubMedGoogle Scholar
- Xie H, Belogortseva NI, Wu J, Lai WH, Chen CH: Inhibition of human immunodeficiency virus type 1 entry by a binding domain of Porphyromonas gingivalis gingipain. Antimicrob Agents Chemother. 2006, 50 (9): 3070-3074. 10.1128/AAC.01578-05.View ArticlePubMedGoogle Scholar
- Daecke J, Fackler OT, Dittmar MT, Krausslich HG: Involvement of clathrin-mediated endocytosis in human immunodeficiency virus type 1 entry. J Virol. 2005, 79 (3): 1581-1594. 10.1128/JVI.79.3.1581-1594.2005.View ArticlePubMedGoogle Scholar
- Marechal V, Clavel F, Heard JM, Schwartz O: Cytosolic Gag p24 as an index of productive entry of human immunodeficiency virus type 1. J Virol. 1998, 72 (3): 2208-2212.PubMedGoogle Scholar
- Marechal V, Prevost MC, Petit C, Perret E, Heard JM, Schwartz O: Human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis. J Virol. 2001, 75 (22): 11166-11177. 10.1128/JVI.75.22.11166-11177.2001.View ArticlePubMedGoogle Scholar
- Geijtenbeek TB, Torensma R, van Vliet SJ, van Duijnhoven GC, Adema GJ, van Kooyk Y, Figdor CG: Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell. 2000, 100 (5): 575-585. 10.1016/S0092-8674(00)80693-5.View ArticlePubMedGoogle Scholar
- Turville SG, Santos JJ, Frank I, Cameron PU, Wilkinson J, Miranda-Saksena M, Dable J, Stossel H, Romani N, Piatak M, Lifson JD, Pope M, Cunningham AL: Immunodeficiency virus uptake, turnover, and 2-phase transfer in human dendritic cells. Blood. 2004, 103 (6): 2170-2179. 10.1182/blood-2003-09-3129.View ArticlePubMedGoogle Scholar
- Marsh PD: Dental plaque: biological significance of a biofilm and community life-style. J Clin Periodontol. 2005, 32 Suppl 6: 7-15. 10.1111/j.1600-051X.2005.00790.x.View ArticlePubMedGoogle Scholar
- Lei J, Wu C, Wang X, Wang H: p38 MAPK-dependent and YY1-mediated chemokine receptors CCR5 and CXCR4 up-regulation in U937 cell line infected by Mycobacterium tuberculosis or Actinobacillus actinomycetemcomitans. Biochem Biophys Res Commun. 2005, 329 (2): 610-615. 10.1016/j.bbrc.2005.02.022.View ArticlePubMedGoogle Scholar
- Fichorova RN, Tucker LD, Anderson DJ: The molecular basis of nonoxynol-9-induced vaginal inflammation and its possible relevance to human immunodeficiency virus type 1 transmission. J Infect Dis. 2001, 184 (4): 418-428. 10.1086/322047.View ArticlePubMedGoogle Scholar
- Decrion AZ, Dichamp I, Varin A, Herbein G: HIV and inflammation. Curr HIV Res. 2005, 3 (3): 243-259. 10.2174/1570162054368057.View ArticlePubMedGoogle Scholar
- Bafica A, Scanga CA, Schito M, Chaussabel D, Sher A: Influence of coinfecting pathogens on HIV expression: evidence for a role of Toll-like receptors. J Immunol. 2004, 172 (12): 7229-7234.View ArticlePubMedGoogle Scholar
- Equils O, Faure E, Thomas L, Bulut Y, Trushin S, Arditi M: Bacterial lipopolysaccharide activates HIV long terminal repeat through Toll-like receptor 4. J Immunol. 2001, 166 (4): 2342-2347.View ArticlePubMedGoogle Scholar
- Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, Louis DN, Li FP, Rheinwald JG: Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol. 2000, 20 (4): 1436-1447. 10.1128/MCB.20.4.1436-1447.2000.View ArticlePubMedGoogle Scholar
- Oda D, Watson E: Human oral epithelial cell culture I. Improved conditions for reproducible culture in serum-free medium. In Vitro Cell Dev Biol. 1990, 26 (6): 589-595. 10.1007/BF02624208.View ArticlePubMedGoogle Scholar
- Wei X, Decker JM, Liu H, Zhang Z, Arani RB, Kilby JM, Saag MS, Wu X, Shaw GM, Kappes JC: Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother. 2002, 46 (6): 1896-1905. 10.1128/AAC.46.6.1896-1905.2002.View ArticlePubMedGoogle Scholar
- Popovic M, Read-Connole E, Gallo RC: T4 positive human neoplastic cell lines susceptible to and permissive for HTLV-III. Lancet. 1984, 2 (8417-18): 1472-1473. 10.1016/S0140-6736(84)91666-0.View ArticlePubMedGoogle Scholar
- Gartner S, Markovits P, Markovitz DM, Kaplan MH, Gallo RC, Popovic M: The role of mononuclear phagocytes in HTLV-III/LAV infection. Science. 1986, 233 (4760): 215-219. 10.1126/science.3014648.View ArticlePubMedGoogle Scholar
- Reed LJ: A simple method of estimating fifty per cent endpoint. Am J Hyg. 1938, 27: 493-497.Google Scholar
- Biacchesi S, Skiadopoulos MH, Yang L, Murphy BR, Collins PL, Buchholz UJ: Rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression. J Virol Methods. 2005, 128 (1-2): 192-197. 10.1016/j.jviromet.2005.05.005.View ArticlePubMedGoogle Scholar
- Gilchrist JE, Campbell JE, Donnelly CB, Peeler JT, Delaney JM: Spiral plate method for bacterial determination. Appl Microbiol. 1973, 25 (2): 244-252.PubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25 (4): 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.