Riemerella anatipestifer UvrC is required for iron utilization, biofilm formation and virulence.

UvrC is a subunit of excinuclease ABC, which mediates nucleotide excision repair (NER) in bacteria. Our previous studies showed that transposon Tn4531 insertion in the UvrC encoding gene Riean_1413 results in reduced biofilm formation by Riemerella anatipestifer strain CH3 and attenuates virulence of strain YZb1. In this study, whether R. anatipestifer UvrC has some biological functions other than NER was investigated. Firstly, the uvrC of R. anatipestifer strain Yb2 was in-frame deleted by homologous recombination, generating deletion mutant ΔuvrC, and its complemented strain cΔuvrC was constructed based on Escherichia coli - R. anatipestifer shuttle plasmid pRES. Compared to the wild-type (WT) R. anatipestifer strain Yb2, uvrC deleted mutant ΔuvrC significantly reduced biofilm formation, tolerance to H2O2- and HOCl-induced oxidative stress, iron utilization, and adhesion to and invasion of duck embryonic hepatocytes, but not its growth curve and proteolytic activity. In addition, animal experiments showed that the LD50 value of ΔuvrC in ducklings was about 13-fold higher than that of the WT, and the bacterial loads in ΔuvrC infected ducklings were significantly lower than those in Yb2-infected ducklings, indicating uvrC deletion in R. anatipestifer attenuated virulence. Taken together, the results of this study indicate that R. anatipestifer UvrC is required for iron utilization, biofilm formation, oxidative stress tolerance and virulence of strain Yb2, demonstrating multiple functions of UvrC.RESEARCH HIGHLIGHTSDeletion of uvrC in R. anatipestfer Yb2 significantly reduced its biofilm formation.uvrC deletion led to reduced tolerance to H2O2- and HOCl-induced oxidative stress.The iron utilization of uvrC deleted mutant was significantly reduced.The uvrC deletion in R. anatipestifer Yb2 attenuated its virulence.

Inhibitory receptor CD47 binding to plasma TSP1 suppresses NK-cell IFN-γ production via activating the JAK/STAT3 pathway during HIV infection.

BACKGROUND: Natural killer (NK) cells play an important first-line role against tumour and viral infections and are regulated by inhibitory receptor expression. Among these inhibitory receptors, the expression, function, and mechanism of cluster of differentiation 47 (CD47) on NK cells during human immunodeficiency virus (HIV) infection remain unclear. METHODS: Fresh peripheral blood mononuclear cells (PBMCs) were collected from people living with HIV (PLWH) and HIV negative controls (NC) subjects. Soluble ligand expression levels of CD47 were measured using ELISA. HIV viral proteins or Toll-like receptor 7/8 (TLR7/8) agonist was used to investigate the mechanisms underlying the upregulation of CD47 expression. The effect of CD47 on NK cell activation, proliferation, and function were evaluated by flow cytometry. RNA-seq was used to identify downstream pathways for CD47 and its ligand interactions. A small molecule inhibitor was used to restore the inhibition of NK cell function by CD47 signalling. RESULTS: CD47 expression was highly upregulated on the NK cells from PLWH, which could be due to activation of the Toll-like receptor 7/8 (TLR7/8) pathway. Compared with NC subjects, PLWH subjects exhibited elevated levels of CD47 ligands, thrombospondin-1 (TSP1), and counter ligand signal regulatory protein-α (SIRPα). The TSP1-CD47 axis drives the suppression of interferon gamma (IFN-γ) production and the activation of the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway in NK cells. After treatment with a STAT3 inhibitor, the NK cells from PLWH showed significantly improved IFN-γ production. CONCLUSIONS: The current data indicate that the binding of the inhibitory receptor CD47 to plasma TSP1 suppresses NK cell IFN-γ production by activating the JAK/STAT3 pathway during HIV infection. Our results suggest that CD47 and its related signalling pathways could be targets for improving NK cell function in people living with HIV.

Double spiral chip-embedded micro-trapezoid filters (SMT filters) for the sensitive isolation of CTCs of prostate cancer by spectral detection.

Circulating tumor cells (CTCs) are cancer cells that are released from the original tumor and circulate in the blood vessels, carrying greatly similar constituents as the original tumor. Therefore, CTCs have a significant value in cancer prognosis, early diagnosis, and anti-cancer therapy. However, their rarity and heterogeneity make the isolation of CTCs an arduous task. In the present research, we propose a double spiral chip-embedded micro-trapezoid filter (SMT filter) for the sensitive isolation of the CTCs of prostate cancer by spectral detection. SMT filters were elongated to effectively capture CTCs and this distinctive design was conducive to their isolation and enrichment. The SMT filters were verified with tumor cells and artificial patient blood with a capture efficiency as high as 94% at a flow rate of 1.5 mL h-1. As a further validation, the SMT filters were validated in isolating CTCs from 10 prostate cancers and other cancers in 4 mL blood samples. Also, the CTCs tested positive for each patient blood sample, ranging from 83-114 CTCs. Significantly, we advanced hyperspectral imaging to detect the characteristic spectrum of CTCs both captured in situ on SMT filters and enriched after isolation. The CTCs could be positively identified by hyperspectral imaging with complete integrity of the cell morphology and an improved characteristic spectrum. This represents a breakthrough in the conventional surface-enhanced Raman scattering (SERS) spectroscopy of nanoparticles. Also, the characteristic spectrum of the CTCs would be highly beneficial for distinguishing the cancer type and accurate for enumerating tumor cells with varied intensities. Furthermore, a novel integrated flower-shaped microfilter was presented with all these aforementioned merits. The success of both the SMT filters and characteristic spectral detection indicated their feasibility for further clinical analysis, the evaluation of cancer therapy, and for potential application.

CD38+CD39+ NK cells associate with HIV disease progression and negatively regulate T cell proliferation.

The ectonucleotidases CD38 and CD39 have a critical regulatory effect on tumors and viral infections via the adenosine axis. Natural killer (NK) cells produce cytokines, induce cytotoxic responses against viral infection, and acquire immunoregulatory properties. However, the roles of CD38 and CD39 expressed NK cells in HIV disease require elucidation. Our study showed that the proportions of CD38+CD39+ NK cells in HIV-infected individuals were positively associated with HIV viral loads and negatively associated with the CD4+ T cell count. Furthermore, CD38+CD39+ NK cells expressed additional inhibitory receptors, TIM-3 and LAG-3, and produced more TGF-ß. Moreover, autologous NK cells suppressed the proliferation of CD8+ T and CD4+ T cells of HIV-infected individuals, and inhibiting CD38 and CD39 on NK cells restored CD8+ T and CD4+ T cell proliferation in vitro. In conclusion, these data support a critical role for CD38 and CD39 on NK cells in HIV infection and targeting CD38 and CD39 on NK cells may be a potential therapeutic strategy against HIV infection.

CD160 Promotes NK Cell Functions by Upregulating Glucose Metabolism and Negatively Correlates With HIV Disease Progression.

Natural killer (NK) cells are crucial for immune responses to viral infections. CD160 is an important NK cell activating receptor, with unknown function in HIV infection. Here, we found that CD160 expression was reduced on NK cells from HIV-infected individuals and its expression was negatively correlated with HIV disease progression. Further, GLUT1 expression and glucose uptake were higher in CD160+ NK cells, and the results of RNA-seq and flow cytometry demonstrated that CD160 positively regulated glucose metabolism through the PI3K/AKT/mTOR/s6k signaling pathway, thereby enhancing NK cell function. Moreover, we determined that reduced CD160 expression on NK cells could be attributed to the higher plasma levels of TGF-ß1 in HIV-infected individuals. Overall, these results highlight the vital role of CD160 in HIV disease progression and regulation of glucose metabolism, indicating a potential target for HIV immunotherapy.

Patterns of Sexually Transmitted Co-infections and Associated Factors Among Men Who Have Sex With Men: A Cross-Sectional Study in Shenyang, China.

Background: Men who have sex with men (MSM) are disproportionately affected by sexually transmitted infections (STIs). We sought to describe patterns of sexually transmitted co-infections and explore factors associated with increased acquisition of STIs among MSM. Methods: We enrolled MSM in Shenyang, China, between July and December 2020 to test for four STIs, including human papillomavirus (HPV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Treponema pallidum (TP). Data regarding demographic and behavioral characteristics of participants were collected through a self-administered digital questionnaire. We adopted the ordinal logistic regression model to identify factors associated with acquiring more STIs. Results: Overall, 177 participants with completed test results for all four STIs were analyzed. These participants had a median age of 29.0 (interquartile range: 23.0-38.0) years. The prevalence of STI co-infections was 23.7% [42/177; 95% confidence interval (CI), 17.8%-30.8%], among which HPV/CT (47.1%) and HPV/CT/NG (50.0%) co-infection were the predominant types among participants with dual and multiple infections, respectively. Participants who had a higher educational background [adjusted odds ratio (aOR), 0.46; 95% CI, 0.24-0.85; P = 0.014] and had a history of STIs (aOR, 2.53; 95% CI, 1.24-5.18; P = 0.011) were positively associated with acquiring more STIs. Conclusions: MSM in Shenyang suffer a substantial burden of sexually transmitted co-infections. An optimized multi-STI integration strategy targeting prevention, surveillance, screening, and treatment is warranted to reduce the prevalence of sexually transmitted co-infections, especially in less-educated MSM.

High Human Papillomavirus Vaccine Acceptability and Cost-Effectiveness of the Chinese 2-Valent Vaccine Among Men Who Have Sex With Men: A Cross-Sectional Study in Shenyang, China.

Background: Despite the insupportable burden caused by the human papillomavirus (HPV) and high vaccine acceptability, vaccination programs are not currently available for men who have sex with men (MSM). We aimed to assess HPV infection by examining the willingness for vaccination among MSM and cost-effectiveness of the Chinese 2-valent HPV vaccine. Methods: We recruited MSM in Shenyang, China between July and December 2020 to conduct anal HPV testing and an online survey regarding HPV-related knowledge and vaccine acceptability. We performed a cost-effectiveness analysis to evaluate the incremental cost-effectiveness ratios (ICERs) of the Chinese 2-valent HPV vaccine. Results: A total of 234 participants completed the online survey; of those, 203 were successfully tested for HPV. The median age was 30 years [interquartile range (IQR): 23-38 years]. Most participants had at least undergraduate education (136/234, 58.1%). The acceptability rate for the free HPV vaccine was 57.7% (135/234). The prevalence of HPV types 16 and 18 was 14.9% (18/121) and 26.8% (22/82) in the willing and unwilling to vaccinate groups, respectively (P > 0.05). The prevalence of high-risk HPV among participants aged <30 and ≥50 years was 48.6 and 38.9%, respectively. Using the Chinese per capita gross domestic product (GDP) as a threshold, the Chinese 2-valent HPV vaccine would be a "very cost-effective" strategy, with an ICER value of USD 4,411. This evidence showed that the Chinese 2-valent HPV vaccine was more cost-effective than other imported vaccines. Conclusions: Targeted strategies should be utilized in MSM with different rates of vaccine acceptability. A pilot HPV vaccination program based on the Chinese 2-valent HPV vaccine for MSM is urgently warranted to reduce the burden of HPV and anal cancer.

In-hospital Mortality and Causes of Death in People Diagnosed With HIV in a General Hospital in Shenyang, China: A Cross-Sectional Study.

Background: Acquired immune deficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV) infection, is a serious public health issue. This study investigated the correlated factors and possible changing trend of in-hospital death in patients diagnosed with HIV in the past decade in our hospital. Methods: We retrospectively collected data of firstly hospitalized patients with HIV in the Department of Infectious Disease in the First Affiliated Hospital of China Medical University from January 1, 2010 to December 31, 2019, and compared various factors that correlated with in-hospital death, including age, sex, opportunistic infections, and antiretroviral therapy (ART) status. Cox regression analysis was used to identify the risk factors for death. Results: In total, 711 patients were recruited for this study, and 62 patients died in the hospital. The in-hospital mortality rate was 8.72%. Tuberculosis (TB), malignancies, and thrombocytopenia were associated with mortality. Antiviral treatment before admission was found to be a protective factor. There was a declining trend in in-hospital mortality from 19.2% in 2010 to 6.3% in 2019 (linear-by-linear association test, p < 0.001), partly due to intensified medical care strategy. Conclusions: Till date, AIDS-defining illnesses remain the major cause of hospital admission and in-hospital mortality. TB and malignancies were correlated risk factors for in-hospital mortality. ART before admission was found to be beneficial, and considering the decreasing rate of in-hospital mortality, the implementation of intensified medical care strategy requires further effort.

Expression of the Inhibitory Receptor TIGIT Is Up-Regulated Specifically on NK Cells With CD226 Activating Receptor From HIV-Infected Individuals.

Natural killer (NK) cells are important for maintenance of innate immune system stability and serve as a first line of defense against tumors and virus infections; they can act either directly or indirectly and are regulated via co-operation between inhibitory and stimulatory surface receptors. The recently reported inhibitory receptor, TIGIT, can be expressed on the NK cell surface; however, the expression level and function of TIGIT on NK cells during HIV infection is unknown. In this study, for the first time, we investigated the expression and function of TIGIT in NK cells from HIV-infected individuals. Our data demonstrate that the level of TIGIT is higher on NK cells from patients infected with human immunodeficiency virus (HIV) compared with HIV-negative healthy controls. TIGIT expression is inversely correlated with CD4+ T cell counts and positively correlated with plasma viral loads. Additionally, levels of the TIGIT ligand, CD155, were higher on CD4+ T cells from HIV-infected individuals compared with those from healthy controls; however, there was no difference in the level of the activating receptor, CD226, which recognizes the same ligands as TIGIT. Furthermore, TIGIT was found to specifically up-regulated on CD226+ NK cells in HIV-infected individuals, and either rIL-10, or rIL-12 + rIL-15, could induce TIGIT expression on these cells. In addition, high TIGIT expression inhibited the production of interferon-gamma (IFN-γ) by NK cells, while TIGIT inhibition restored IFN-γ production. Overall, these results highlight the important role of TIGIT in NK cell function and suggest a potential new avenue for the development of therapeutic strategies toward a functional cure for HIV.

DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif.

Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PT-modifying enzyme DndEi, which has an additional domain compared with canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as a DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e. the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strand separation and intense ATP hydrolysis.

Roles of the TonB1 and TonB2 proteins in haemin iron acquisition and virulence in Riemerella anatipestifer.

Two TonB systems in Riemerella anatipestifer were found and characterized as ExbB1-ExbD1-TonB1 and ExbB2-ExbD2-ExbD2'-TonB2, but the significance of two sets of TonB complexes in R. anatipestifer is not clear. In this study, by deleting the tonB1 or tonB2 gene of R. anatipestifer strain CH3, we investigated the roles of the TonB1 and TonB2 proteins in iron acquisition and virulence. The results showed that strain CH3 could utilize haemin as the sole iron source in the presence of l-cysteine, but haemin iron acquisition was defective in the CH3ΔtonB1 mutant, and the deletion of either tonB1 or tonB2 significantly reduced adhesion to and invasion of Vero cells. Animal experiments indicated that the LD50 of the CH3ΔtonB1 and CH3ΔtonB2 mutants in ducklings was ∼224- and ∼87-fold, respectively, higher than that of the WT CH3 strain. Additional analysis indicated that blood bacterial loading of ducklings infected with CH3ΔtonB1 or CH3ΔtonB2 decreased significantly compared with that found for WT CH3-infected ducklings. Thus, our results indicated that the TonB1, but not TonB2 protein, is involved in haemin iron acquisition and that both TonB proteins are necessary for optimal bacterial virulence.

The siderophore-interacting protein is involved in iron acquisition and virulence of Riemerella anatipestifer strain CH3.

Riemerella anatipestifer causes epizootic infectious disease in poultry and serious economic losses especially to the duck industry. However, little is known regarding the molecular basis of its pathogenesis. The ability to acquire iron under low-iron conditions is related to the virulence of a variety of bacterial pathogens. In this study, a sip (Riean_1281) deletion mutant CH3Δsip was constructed and characterized for iron-limited growth, biofilm formation, and pathogenicity to ducklings. Results showed that siderophore-interacting protein (SIP) was involved in iron utilization and the sip deletion significantly reduced biofilm formation and adherence to and invasion of Vero cells. In addition, the sip gene was absent in 1 of 24 (4.17%) virulent strains and 2 of 3 (66.7%) avirulent strains of R. anatipestifer, and the sip gene from six R. anatipestifer strains, which belong to serotypes 1, 2, and 10, respectively, shared 100% amino acid identities to those of R. anatipestifer strains DSM15868 and RA-GD. These results suggested that siderophore-mediated iron acquisition may be an important iron-uptake pathway in R. anatipestifer. Animal experiments indicated that the median lethal dose of the CH3Δsip mutant in ducklings was about 35-fold higher than that of the wild-type CH3 strain. Thus, our results demonstrated that R. anatipestifer SIP was involved in iron acquisition and necessary for its optimal virulence.

The role of TonB-dependent receptor TbdR1 in Riemerella anatipestifer in iron acquisition and virulence.

Riemerella anatipestifer is an important duck pathogen and causes serious economic losses to the duck industry worldwide. To date, four full R. anatipestifer genomic sequences have been submitted to the GenBank database and 31 TonB-dependent outer membrane receptors, which may play critical roles in host-bacteria interactions, were predicted for R. anatipestifer strain GSM15868. In our previous study, we reported that the TonB-dependent receptor TbdR1 was a cross immunogenic antigen among R. anatipestifer serotypes 1, 2, and 10. However, the biological functions of TbdR1 in R. anatipestifer remain unclear. In the present study, a tbdR1 (Riean_1607) deletion mutant CH3ΔtbdR1 of R. anatipestifer strain CH3 was constructed and characterized for iron-limited growth, biofilm formation, and pathogenicity to ducklings. Our results showed that TbdR1 was involved in hemin iron acquisition and the tbdR1 deletion significantly reduced biofilm formation and adhesion to and invasion of Vero cells. Animal experiments indicated that the median lethal dose of the CH3ΔtbdR1 mutant in ducklings was about 45-fold higher than that of the wild-type CH3 strain. Additional analysis indicated that bacterial loads in blood, liver, and brain tissues in CH3ΔtbdR1-infected ducklings were decreased significantly compared to those in wild-type CH3-infected ducklings. Thus, our results demonstrated that TbdR1 was involved in hemin iron acquisition and necessary for optimal bacterial virulence.

KIR3DS1/L1 and HLA-Bw4-80I are associated with HIV disease progression among HIV typical progressors and long-term nonprogressors.

BACKGROUND: Natural killer (NK) cells have emerged as pivotal players in innate immunity, especially in the defense against viral infections and tumors. Killer immunoglobulin-like receptors (KIRs)--an important recognition receptor expressed on the surface of NK cells--regulate the inhibition and/or activation of NK cells after interacting with human leukocyte antigen (HLA) class I ligands. Various KIR genes might impact the prognosis of many different diseases. The implications of KIR-HLA interaction in HIV disease progression remains poorly understood. METHODS: Here, we studied KIR genotypes, mRNA levels, HLA genotypes, CD4+ T cell counts and viral loads in our cohort of Human Immunodeficiency Virus (HIV)-infected individuals, a group that includes HIV long-term nonprogressors (LTNPs) and typical progressors (TPs). RESULTS: We found that the frequency of KIR3DS1/L1 heterozygotes with HLA-Bw4-80I gene was much higher in LTNPs than in TPs (P = 0.001) and that the KIR3DL1 homozygotes without HLA-Bw4-80I gene had higher viral loads and lower CD4+ T cell counts (P = 0.014 and P = 0.021, respectively). Our study also confirmed that homozygosity for the HLA-Bw6 allele was associated with rapid disease progression. In addition to the aforementioned results on the DNA level, we observed that higher level expression of KIR3DS1 mRNA was in LTNP group, and that higher level expression of KIR3DL1 mRNA was in TP group. CONCLUSIONS: Our data suggest that different KIR-HLA genotypes and different levels of transcripts associate with HIV disease progression.

Higher NK cell IFN-γ production is associated with delayed HIV disease progression in LTNPs.

Natural killer (NK) cells are important effectors of the innate immune system that help control viral infections and tumorigenesis. However, the relationship between NK cell function and HIV disease progression remains poorly defined. In this study, we examined the function of NK cells in Chinese patients who were HIV-infected but treatment-naïve. These individuals include primary HIV-infected patients (PHIs), typical progressors (TPs), and long-term nonprogressors (LTNPs). We observed an increase of CD56(dim) NK cells in PHIs, but the production of interferon-gamma (IFN-γ) and CD107a expression in PHIs were not altered compared with normal control subjects (NCs). However, the NK cells from LTNPs exhibited increased activities in IFN-γ production, CD107a expression and granzyme B change after K562 stimulation compared with NCs. Furthermore, the percentage of IFN-γ(+)CD107a(-) NK cells in LTNPs was higher than that in TPs, PHIs and NCs; levels of IFN-γ production in LTNP NK cells exhibited an inverse correlation with viral loads. Similar correlations, however, were not observed in the PHI and TP groups. Taken together, these data demonstrate that enhanced NK cell function may contribute to the control of HIV infection, and increased IFN-γ secretion may be associated with delayed disease progression.

Barriers to acceptance of provider-initiated testing and counseling among men who have sex with men in Shenyang, China: a cross-sectional study.

A high prevalence of HIV infection is present among men who have sex with men (MSM) in China, but many people living with HIV or AIDS (PLWHs) are unaware of their HIV infection status. Provider-initiated HIV testing and counseling (PITC) is a streamlined model that can significantly enhance HIV detection and detect infections earlier. However, PITC has not yet been widely applied, and no studies have been conducted on MSM's attitudes towards PITC in China. In this study, a total of 438 MSM were recruited in Shenyang city. A multivariate logistic regression model showed that certain conditions made MSM more accepting of PITC: those who had attended VCT (voluntary counseling and testing) more than three times (odds ratio [OR]: 2.95, 95% CI: 1.36-6.37), those who considered PITC beneficial for family and friends (OR: 1.91, 95% CI: 1.25-2.92), those who obtained HIV/AIDS knowledge from brochures (OR: 2.52, 95% CI: 1.64-3.87), those who obtained HIV/AIDS knowledge from the Internet (OR: 1.66, 95% CI: 1.07-2.58), and those who were highly aware of their own risk of being infected with HIV (OR: 2.84, 95% CI: 1.37-5.91). To improve acceptance of PITC among MSM in China, stronger efforts are needed to lower the psychosocial barriers to receiving PITC, to promote HIV/AIDS awareness, and to encourage the extension of HIV testing.

[Cloning and expression of luxS and pfs and in vitro biosynthesis autoinducer 2 of avian pathogenic Escherichia coli from Anhui Province].

OBJECTIVE: The LuxS/AI-2 quorum sensing (QS) system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). In this study, the method of biosynthesis of AI-2 was established using recombinant LuxS and Pfs of avian pathogenic Escherichia coli (APEC), which will be benefit for future study of the role of AI-2 in APEC. METHODS: We investigated AI-2 production in APEC by vibrio harveyi BB170 (BB170). Furthermore, APEC LuxS and Pfs were expressed, purified and used to investigate the production of AI-2 in vitro. LuxS and Pfs were incubated with S-ribosylhomocysteine (SAH), the reaction was detected for the production of luminescence of BB170. RESULTS: APEC can produce AI-2 by BB170 bioassay. Purified LuxS and Pfs enzymes incubated with SAH and produced 300 micromol/L AI-2 in the reaction products. CONCLUSION: The results demonstrated that recombinant Pfs and LuxS synthesize AI-2 in vitro from SAH. These findings will be of benefit to future studies of the role of AI-2 in APEC.

R-848 triggers the expression of TLR7/8 and suppresses HIV replication in monocytes.

BACKGROUND: Toll-like receptors (TLR) 7 and 8 are important in single-stranded viral RNA recognition and may play a role in HIV infection and disease progression. We analyzed TLR7/8 expression and signaling in monocytes from HIV-infected and uninfected subjects to investigate a pathway with new potential for the suppression of HIV replication. METHODS: Eighty-one HIV-infected and uninfected subjects from Liaoning and Henan provinces in China participated in this study. Monocytes were isolated from subjects' peripheral blood mononuclear cells by magnetic bead selection. TLR7 and TLR8 mRNA was measured using quantitative real-time reverse transcriptase PCR. R-848 (resiquimod) was used as a ligand for TLR7 and TLR8 in order to 1) assess TLR7/8-mediated monocyte responsiveness as indicated by IL-12 p40 and TNF-α secretion and 2) to examine HIV replication in cultured monocytes in the presence of R-848. RESULTS: We found that expression of TLR7/8 mRNA in peripheral blood monocytes decreased with disease progression. TLR7 expression was decreased with stimulation with the TLR7/8 agonist, R-848, in vitro, whereas TLR8 expression was unaffected. Following R-848 stimulation, monocytes from HIV-infected subjects produced significantly less TNF-α than those from uninfected subjects, but trended towards greater production of IL-12 than stimulated monocytes from uninfected subjects. R-848 stimulation also suppressed HIV replication in cultured monocytes. CONCLUSIONS: Our study provides evidence that the TLR7 and TLR8 triggering can suppress HIV replication in monocytes and lead to postpone HIV disease progression, thereby offering novel targets for immunomodulatory therapy.

SOCS1 silencing can break high-dose dendritic cell immunotherapy-induced immune tolerance.

Dendritic cells (DCs) play a pivotal role in T cell-mediated immunity and have been shown to induce strong anti-tumor immune responses. As of yet, only a limited number of objective tumor regressions have been observed in clinical studies using a DC vaccine. Suppressor of cytokine signaling-1 (SOCS1) is a key negative regulator of the JAK/STAT signal pathway and plays an essential role in suppressing systemic autoimmunity that is mediated by DCs. The aim of this study was to investigate whether SOCS1-silenced DCs can break the vaccine-induced immune tolerance stimulated by high-dose DC, thereby enhancing anti-tumor activity. In the mouse melanoma model, we found that a 2x106 TRP2-pulsed DC vaccine was able to induce immune tolerance, while a 2x106 SOCS1-silenced DC/TRP2 vaccine prevented immune tolerance. Further experiments revealed that activation-induced T cell death (AICD) through the Fas/Fas-L pathway may play a crucial role in immune tolerance induced by 2x106 TRP2-pulsed DC. SOCS1-silencing in DCs could prevent immune tolerance by inhibiting Fas and Fas-L expression, induced by an increase in IL-12p70 and IL-6 production. In addition, in 2x106 SOCS1-silenced DC/TRP2 immunized mice, higher levels of IL-12p70 and IFN-γ and lower IL-17 production may inhibit tumor angiogenesis and therefore assist in breaking immune tolerance. In conclusion, high-doses of DCs can inhibit the vaccine-induced AICD of T cells and cytokine regulation in tumor angiogenesis. These results indicate that SOCS1-silenced DC vaccines may greatly enhance anti-tumor activity by breaking self-tolerance.