Longitudinal Analysis of Local Recurrence and Survival After Transanal Abdominal Transanal Radical Proctosigmoidectomy for Low Rectal Cancer Treated With Neoadjuvant Chemoradiation Therapy.

BACKGROUND: The transanal abdominal transanal radical proctosigmoidectomy was developed in 1984 as a sphincter preservation surgery in patients with low rectal cancers after preoperative radiation therapy. While serving as a catalyst for disruptive sphincter preservation surgery, it continues to be used and evolve. With the controversy over safety and local recurrence in other sphincter-preserving surgery, review of transanal abdominal transanal radical proctosigmoidectomy long-term oncologic outcomes is warranted. OBJECTIVE: To assess local recurrence and survival after transanal abdominal transanal radical proctosigmoidectomy after neoadjuvant chemoradiation therapy. DESIGN: Retrospective cohort study of a prospectively maintained database. SETTINGS: Tertiary rectal cancer referral center. PATIENTS: Patients with low adenocarcinoma (≤5 cm anorectal ring) receiving neoadjuvant chemoradiation therapy and then transanal abdominal transanal radical proctosigmoidectomy for curative resection between 1998 and 2021. MAIN OUTCOME MEASURES: Local recurrence rates and overall survival rates. RESULTS: Of 255 included patients, 67.8% were men (n = 173); the mean age was 58.7 years (SD 11.5) and the mean BMI was 27.1 (SD 5.4), with 50.2% (n = 128) having ASA class II and 49.8% (n = 127) having ASA class III/IV. The mean tumor size was 4.8 cm (SD 1.9), the majority of patients had clinical T3 disease (81.8%; n = 184), and 52.1% had nodal disease (n = 100). The median radiation dose was 5400 cGy, with 73.7% (n = 149) achieving good response and 90.2% (n = 230) receiving minimally invasive surgery. The complete total mesorectal excision rate was 94.3%, and 100% of patients (n = 255) had negative distal margins. The mean number of examined lymph nodes were 13.9 (SD 10.7). After a median follow-up of 55.4 months, 5.1% of patients (n = 13) developed local recurrence at a median time of 29.6 months. The 5-year overall survival was 84.1% (95% CI, 78.8-89.4). LIMITATIONS: Retrospective review with risk of bias and lack of generalizability. CONCLUSIONS: In this longitudinal study, the transanal abdominal transanal radical proctosigmoidectomy demonstrated excellent long-term locoregional control and survival in very low rectal cancers. The superior transanal abdominal transanal radical proctosigmoidectomy outcomes are durable over time, warranting expansion of the sphincter-preserving surgery technique. See Video Abstract . ANLISIS LONGITUDINAL DE LA RECURRENCIA LOCAL Y LA SUPERVIVENCIA DESPUS DE LA PROCTOSIGMOIDECTOMA RADICAL TRANSANAL ABDOMINAL TATA PARA EL CNCER DE RECTO BAJO TRATADO CON QUIMIORRADIACIN NEOADYUVANTE: ANTECEDENTES:La proctosigmoidectomía radical transanal abdominal se desarrolló en 1984 como una cirugía de preservación del esfínter en cánceres de recto bajo después de la radiación preoperatoria. Si bien sirve como catalizador para la cirugía disruptiva de preservación del esfínter, continúa utilizándose y evolucionando. Con la controversia sobre la seguridad y la recurrencia local en otras cirugías que preservan el esfínter, se justifica la revisión de los resultados oncológicos a largo plazo de la proctosigmoidectomía radical transanal abdominal.OBJETIVO:Evaluar localmente después de Proctosigmoidectomía Radical Transanal Abdominal Transanal después de quimiorradiación neoadyuvante.DISEÑO:Estudio de cohorte retrospectivo de una base de datos mantenida de forma prospectiva.AJUSTES:Centro terciario de referencia para el cáncer de recto.PACIENTES:Adenocarcinoma bajo (=/

Perioperative outcomes for single-port robotic versus single-incision laparoscopic surgery: a comparative analysis in colorectal cancer surgery.

BACKGROUND: Single-incision laparoscopic surgery (SILS) may offer improved cosmesis, reduced postoperative pain and faster recovery than conventional platforms, but widespread implementation was limited by technical demands. A single-port robotic platform was recently introduced, with components that further enhance SILS benefits without the technical challenges. No study to date has compared the two platforms to validate benefits. Our goal was to compare outcomes of SP robotics and SILS in colorectal cancer (CRC). METHODS: A prospective cancer registry was reviewed for CRC patients undergoing curative resection through a SILS or SP robotic approach from 2010 to 2022. Patient and cancer demographics, intraoperative, and postoperative outcomes were compared in a 1:1 propensity score-matched cohort, adjusting for baseline characteristics. The main outcome measures were complications, operative time, and oncologic quality measures. RESULTS: Matching resulted 50 SP robotic and 50 SILS patients. Cohorts were well matched in all demographics, but SP robotic rectal cancer cases were significantly closer to the anorectal ring than SILS (1.8 cm vs. 3.4 cm, p = 0.018). SP robotic and SILS platforms had similar operative times. Intraoperative conversions was comparable, but more SILS cases required additional ports to be placed (p = 0.040). The intraoperative complications rate, complete total mesorectal excision rates, and lymph node yield were not statistically significantly different. There were no positive margins in either group. Postoperatively, groups had analogous day of return of bowel function, comparable morbidity, and discharge destination. There was no mortality in either group. The length of stay was significantly shorter with SP robotics than SILS (mean 4.135 vs. 5.282 days, median 4 (2-8) vs. 5 (2-14) days; p = 0.045). CONCLUSIONS: Single-port robotics provided high quality oncologic surgery, adding the technical benefits of robotics to clinical and cosmetic benefits of single-port surgery. There were comparable operative time, complication rates, and oncologic outcomes in CRC cases, with shorter hospital stays with SP robotics. This early data is encouraging for expansion SP robotic technology.

Comparing advanced platforms for local excision of rectal lesions.

BACKGROUND: Transanal surgery facilitates organ preservation in select patients with benign and early malignant rectal lesions to avoid the functional consequences of radical surgery. The transanal endoscopic microsurgery (TEM) platform created a standard for local excision with lower margin positivity and recurrence rates than traditional transanal excision. The single-port robot (SP r) presents a promising alternative transanal platform. The goal of this study was to compare perioperative and pathologic outcomes of TEM and SP r for excision of rectal lesions. METHODS: A review of consecutive patients who underwent local excision of rectal lesions at a tertiary referral center from 1/2001 to 5/2022 was performed. Cases were stratified into TEM or SP rTAMIS in a 1:1 propensity score-matched cohort, adjusting for all baseline characteristics. Clinical, tumor-specific, and perioperative outcomes were compared using χ2, and Mann-Whitney U-tests. The main outcomes were oncologic quality measures, complications, and operative time. RESULTS: Matching resulted 50 patients in each cohort. Groups had similar age, gender, body mass index, comorbidity, diagnosis, lesion characteristics, and neoadjuvant chemoradiation rates. There were no intraoperative complications in either cohort. Three SP rTAMIS cases were converted intraoperatively; there were no conversions in TEM. SP rTAMIS had significantly shorter operative times than TEM (mean 104 vs. 245, p = 0.027). The rates of positive distal margins (2% TEM, 0% SP rTAMIS) and piecemeal resection (4% TEM, 0% SP rTAMIS) were similar. SP rTAMIS had significantly lower postoperative morbidity rates than TEM (9% vs. 20%, p = 0.031). There was no mortality in either cohort. CONCLUSIONS: SP robotics provided high-quality outcomes similar to TEM for local excision of rectal lesions. SP robotics had faster operative time with comparable clinical and oncologic outcomes to TEM. These early data are promising for expanding use of SP robotic platforms.

HMGB1 Is Involved in IFN-α Production and TRAIL Expression by HIV-1-Exposed Plasmacytoid Dendritic Cells: Impact of the Crosstalk with NK Cells.

Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover, Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands, such as HIV and CpG respectively, turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions, and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection, but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here, we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α, TNF-α, IFN-γ and IL-12, and CCR5-interacting chemokines (MIP-1α and MIP-1ß) in NK-pDCs co-cultures. At high HIV-1BaL concentrations, the addition of NK cells did not promote the release of these mediators, suggesting that once efficiently triggered by the virus, pDCs could not integrate new activating signals delivered by NK cells. However, high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly, we identified the alarmin HMGB1, released at pDC-NK cell synapse, as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover, HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1, HMGB1-specific antibodies, sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether, these findings identify HMGB1 as a trigger for IFN-α-mediated TRAIL expression at the surface of pDCs and NK cells, and they suggest a novel mechanism of innate control of HIV-1 infection.

Superior Efficacy of a Human Immunodeficiency Virus Vaccine Combined with Antiretroviral Prevention in Simian-Human Immunodeficiency Virus-Challenged Nonhuman Primates.

UNLABELLED: Although vaccines and antiretroviral (ARV) prevention have demonstrated partial success against human immunodeficiency virus (HIV) infection in clinical trials, their combined introduction could provide more potent protection. Furthermore, combination approaches could ameliorate the potential increased risk of infection following vaccination in the absence of protective immunity. We used a nonhuman primate model to determine potential interactions of combining a partially effective ARV microbicide with an envelope-based vaccine. The vaccine alone provided no protection from infection following 12 consecutive low-dose intravaginal challenges with simian-HIV strain SF162P3, with more animals infected compared to naive controls. The microbicide alone provided a 68% reduction in the risk of infection relative to that of the vaccine group and a 45% reduction relative to that of naive controls. The vaccine-microbicide combination provided an 88% reduction in the per-exposure risk of infection relative to the vaccine alone and a 79% reduction relative to that of the controls. Protected animals in the vaccine-microbicide group were challenged a further 12 times in the absence of microbicide and demonstrated a 98% reduction in the risk of infection. A total risk reduction of 91% was observed in this group over 24 exposures (P = 0.004). These important findings suggest that combined implementation of new biomedical prevention strategies may provide significant gains in HIV prevention. IMPORTANCE: There is a pressing need to maximize the impact of new biomedical prevention tools in the face of the 2 million HIV infections that occur each year. Combined implementation of complementary biomedical approaches could create additive or synergistic effects that drive improved reduction of HIV incidence. Therefore, we assessed a combination of an untested vaccine with an ARV-based microbicide in a nonhuman primate vaginal challenge model. The vaccine alone provided no protection (and may have increased susceptibility to a simian-HIV vaginal challenge), while the microbicide reduced the infection risk compared to that of vaccinated and naive animals. Importantly, the combined interventions provided the greatest level of protection, which was sustained following withdrawal of the microbicide. The data suggest that provision of ARV prophylaxis during vaccination reduces the potential for unexpected increased risks of infection following immunization and augments vaccine efficacy. These findings are important for the potential adoption of ARV prophylaxis as the baseline intervention for future HIV/AIDS vaccines.

Single-stranded DNA oligonucleotides inhibit TLR3-mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques.

TLR3 is a key receptor for recognition of double-stranded RNA and initiation of immune responses against viral infections. However, hyperactive responses can have adverse effects, such as virus-induced asthma. Strategies to prevent TLR3-mediated pathology are therefore desired. We investigated the effect of single-stranded DNA oligonucleotides (ssDNA-ODNs) on TLR3 activation. Human monocyte-derived dendritic cells up-regulate maturation markers and secrete proinflammatory cytokines on treatment with the synthetic TLR3 ligand polyinosine-polycytidylic acid (poly I:C). These events were inhibited in cultures with ssDNA-ODNs. Poly I:C activation of nonhematopoietic cells was also inhibited by ssDNA-ODNs. The uptake of poly I:C into cells was reduced in the presence of ssDNA-ODNs, preventing TLR3 engagement from occurring. To confirm this inhibition in vivo, we administered ssDNA-ODNs and poly I:C, alone or in combination, via the intranasal route in cynomolgus macaques. Proinflammatory cytokines were detected in nasal secretions in the poly I:C group, while the levels were reduced in the groups receiving ssDNA-ODNs or both substances. Our results demonstrate that TLR3-triggered immune activation can be modulated by ssDNA-ODNs and provide evidence of dampening proinflammatory cytokine release in the airways of cynomolgus macaques. These findings may open novel perspectives for clinical strategies to prevent or treat inflammatory conditions exacerbated by TLR3 signaling.

Escape of HIV-1-infected dendritic cells from TRAIL-mediated NK cell cytotoxicity during NK-DC cross-talk--a pivotal role of HMGB1.

Early stages of Human Immunodeficiency Virus-1 (HIV-1) infection are associated with local recruitment and activation of important effectors of innate immunity, i.e. natural killer (NK) cells and dendritic cells (DCs). Immature DCs (iDCs) capture HIV-1 through specific receptors and can disseminate the infection to lymphoid tissues following their migration, which is associated to a maturation process. This process is dependent on NK cells, whose role is to keep in check the quality and the quantity of DCs undergoing maturation. If DC maturation is inappropriate, NK cells will kill them ("editing process") at sites of tissue inflammation, thus optimizing the adaptive immunity. In the context of a viral infection, NK-dependent killing of infected-DCs is a crucial event required for early elimination of infected target cells. Here, we report that NK-mediated editing of iDCs is impaired if DCs are infected with HIV-1. We first addressed the question of the mechanisms involved in iDC editing, and we show that cognate NK-iDC interaction triggers apoptosis via the TNF-related apoptosis-inducing ligand (TRAIL)-Death Receptor 4 (DR4) pathway and not via the perforin pathway. Nevertheless, once infected with HIV-1, DC(HIV) become resistant to NK-induced TRAIL-mediated apoptosis. This resistance occurs despite normal amounts of TRAIL released by NK cells and comparable DR4 expression on DC(HIV). The escape of DC(HIV) from NK killing is due to the upregulation of two anti-apoptotic molecules, the cellular-Flice like inhibitory protein (c-FLIP) and the cellular inhibitor of apoptosis 2 (c-IAP2), induced by NK-DC(HIV) cognate interaction. High-mobility group box 1 (HMGB1), an alarmin and a key mediator of NK-DC cross-talk, was found to play a pivotal role in NK-dependent upregulation of c-FLIP and c-IAP2 in DC(HIV). Finally, we demonstrate that restoration of DC(HIV) susceptibility to NK-induced TRAIL killing can be obtained either by silencing c-FLIP and c-IAP2 by specific siRNA, or by inhibiting HMGB1 with blocking antibodies or glycyrrhizin, arguing for a key role of HMGB1 in TRAIL resistance and DC(HIV) survival. These findings provide evidence for a new strategy developed by HIV to escape immune attack, they challenge the question of the involvement of HMGB1 in the establishment of viral reservoirs in DCs, and they identify potential therapeutic targets to eliminate infected DCs.

Partial inactivation of CCR5- and CXCR4- tropic HIV-1 by human urine.

Human urine has been poorly investigated with regard to infection with human immunodeficiency virus (HIV). Here, we have studied the anti-infective functional properties of human urine against HIV. The effect of fresh urine pools on CCR5- and CXCR4-tropic HIV-1 was evaluated by using four in vitro mucosal models: reduction of infectivity of urine-treated HIV-1 particles, HIV-1 attachment to immature monocyte-derived dendritic cells (iMDDC), transfer of HIV-1 particles from iMDDC to autologous CD4 T cells, and HIV-1 transcytosis through epithelial cells. Human urine partially disrupted both CCR5- and CXCR4-tropic HIV-1 particles, moderately decreased the adsorption of HIV-1 on dendritic cells, and partially decreased the transfer of HIV-1 particles from dendritic cells to autologous T cells. These findings demonstrate partial inactivation of HIV infectivity and suggest that voiding urine after coitus could play a potential role in reducing the risk of HIV infection by both mechanically flushing out and neutralizing the infectivity of HIV-1 particles present in the genital tract.

Differential activity of candidate microbicides against early steps of HIV-1 infection upon complement virus opsonization.

BACKGROUND: HIV-1 in genital secretions may be opsonized by several molecules including complement components. Opsonized HIV-1 by complement enhances the infection of various mucosal target cells, such as dendritic cells (DC) and epithelial cells. RESULTS: We herein evaluated the effect of HIV-1 complement opsonization on microbicide candidates' activity, by using three in vitro mucosal models: CCR5-tropic HIV-1JR-CSF transcytosis through epithelial cells, HIV-1JR-CSF attachment on immature monocyte-derived dendritic cells (iMDDC), and infectivity of iMDDC by CCR5-tropic HIV-1BaL and CXCR4-tropic HIV-1NDK. A panel of 10 microbicide candidates [T20, CADA, lectines HHA & GNA, PVAS, human lactoferrin, and monoclonal antibodies IgG1B12, 12G5, 2G12 and 2F5], were investigated using cell-free unopsonized or opsonized HIV-1 by complements. Only HHA and PVAS were able to inhibit HIV trancytosis. Upon opsonization, transcytosis was affected only by HHA, HIV-1 adsorption on iMDDC by four molecules (lactoferrin, IgG1B12, IgG2G5, IgG2G12), and replication in iMDDC of HIV-1BaL by five molecules (lactoferrin, CADA, T20, IgG1B12, IgG2F5) and of HIV-1NDK by two molecules (lactoferrin, IgG12G5). CONCLUSION: These observations demonstrate that HIV-1 opsonization by complements may modulate in vitro the efficiency of candidate microbicides to inhibit HIV-1 infection of mucosal target cells, as well as its crossing through mucosa.

SUGT1 controls susceptibility to HIV-1 infection by stabilizing microtubule plus-ends.

Understanding the viral-host cell interface during HIV-1 infection is a prerequisite for the development of innovative antiviral therapies. Here we show that the suppressor of G2 allele of skp1 (SUGT1) is a permissive factor for human immunodeficiency virus (HIV)-1 infection. Expression of SUGT1 increases in infected cells on human brain sections and in permissive host cells. We found that SUGT1 determines the permissiveness to infection of lymphocytes and macrophages by modulating the nuclear import of the viral genome. More importantly, SUGT1 stabilizes the microtubule plus-ends (+MTs) of host cells (through the modulation of microtubule acetylation and the formation of end-binding protein 1 (EB1) comets). This effect on microtubules favors HIV-1 retrograde trafficking and replication. SUGT1 depletion impairs the replication of HIV-1 patient primary isolates and mutant virus that is resistant to raltegravir antiretroviral agent. Altogether our results identify SUGT1 as a cellular factor involved in the post-entry steps of HIV-1 infection that may be targeted for new therapeutic approaches.

In vitro synergistic activity against CCR5-tropic HIV-1 with combinations of potential candidate microbicide molecules HHA, KRV2110 and enfuvirtide (T20).

OBJECTIVES: To block the different mechanisms of HIV mucosal transmission, it is likely that use of several microbicide molecules will lead to the best protection against HIV transmission. Indeed, the combination of microbicides with complementary mechanisms of action is expected to increase the antiviral potency of the formulation. METHODS: The gp120-interacting plant lectin HHA ('Hippeastrum hybrid agglutinin'), the non-nucleoside reverse transcriptase inhibitor KRV2110 and the fusion inhibitor enfuvirtide (T20) were combined in 12 drug associations by using the Ray combination design method. Their activity against HIV-1(BaL) was assessed by the lymphocyte infectivity reduction assay and by the single-cycle BaL pseudovirus (PV) assay. In addition, their cell tolerance was evaluated for HEC-1 and HeLa epithelial cell lines, both originating from genital tissue. RESULTS: All evaluated combinations showed synergistic activity in both lymphocyte infectivity reduction and single-cycle BaL PV assays. The combination HHA + KRV2110 resulted in the highest cell viability, whereas the combinations including T20 exhibited a dose-dependent decrease in cell viability, demonstrating the differential tolerance of epithelial cell lines to the combinations. CONCLUSIONS: These observations provide a rational basis for in vitro testing of microbicide candidate molecule combinations, including anti-HIV-1 and cytotoxic cellular assays.

HIV-1 Envelope Overcomes NLRP3-Mediated Inhibition of F-Actin Polymerization for Viral Entry.

Purinergic receptors and nucleotide-binding domain leucine-rich repeat containing (NLR) proteins have been shown to control viral infection. Here, we show that the NLR family member NLRP3 and the purinergic receptor P2Y2 constitutively interact and regulate susceptibility to HIV-1 infection. We found that NLRP3 acts as an inhibitory factor of viral entry that represses F-actin remodeling. The binding of the HIV-1 envelope to its host cell receptors (CD4, CXCR4, and/or CCR5) overcomes this restriction by stimulating P2Y2. Once activated, P2Y2 enhances its interaction with NLRP3 and stimulates the recruitment of the E3 ubiquitin ligase CBL to NLRP3, ultimately leading to NLRP3 degradation. NLRP3 degradation is permissive for PYK2 phosphorylation (PYK2Y402∗) and subsequent F-actin polymerization, which is required for the entry of HIV-1 into host cells. Taken together, our results uncover a mechanism by which HIV-1 overcomes NLRP3 restriction that appears essential for the accomplishment of the early steps of HIV-1 entry.

Inhibition of HIV-1 transmission in trans from dendritic cells to CD4+ T lymphocytes by natural antibodies to the CRD domain of DC-SIGN purified from breast milk and intravenous immunoglobulins.

The present study demonstrates that human breast milk and normal human polyclonal immunoglobulins purified from plasma [intravenous immunoglobulins (IVIg)] contain functional natural immunoglobulin A (IgA) and IgG antibodies directed against the carbohydrate recognition domain (CRD) domain of the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) molecule, which is involved in the binding of human immunodeficiency virus (HIV)-1 to dendritic cells (DCs). Antibodies to DC-SIGN CRD were affinity-purified on a matrix to which a synthetic peptide corresponding to the N-terminal CRD domain (amino-acid 342-amino-acid 371) had been coupled. The affinity-purified antibodies bound to the DC-SIGN peptide and to the native DC-SIGN molecule expressed by HeLa DC-SIGN+ cells and immature monocyte-derived dendritic cells (iMDDCs), in a specific and dose-dependent manner. At an optimal dose of 200 microg/ml, natural antibodies to DC-SIGN CRD peptide purified from breast milk and IVIg stained 25 and 20% of HeLa DC-SIGN+ cells and 32 and 12% of iMDDCs, respectively. Anti-DC-SIGN CRD peptide antibodies inhibited the attachment of virus to HeLa DC-SIGN by up to 78% and the attachment to iMDDCs by only 20%. Both breast milk- and IVIg-derived natural antibodies to the CRD peptide inhibited 60% of the transmission in trans of HIV-1(JRCSF), an R5-tropic strain, from iMDDCs to CD4+ T lymphocytes. Taken together, these observations suggest that the attachment of HIV to DCs and transmission in trans to autologous CD4+ T lymphocytes occur through two independent mechanisms. Our data support a role of natural antibodies to DC-SIGN in the modulation of postnatal HIV transmission through breast-feeding and in the natural host defence against HIV-1 in infected individuals.

Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1beta and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1beta and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal-Wallis analysis of variance with Dunn's multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences ( P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1beta determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.

IFN-gamma-activated monocytes weakly produce HIV-1 but induce the recruitment of HIV-sensitive T cells and enhance the viral production by these recruited T cells.

The ability of macrophages to adapt to changing cytokine environments results in the dominance of a particular functional phenotype of macrophages, which would play a significant role in HIV pathogenesis. In comparison with untreated macrophages (M0), we examined the role of macrophages derived from IFN-gamma-activated monocytes (M1) in the HIV spread. We show that M0 and M1 bind with the same efficiency HIV-1 with a predominant role of C-type lectins in the R5-HIV attachment and of the heparan sulfate proteoglycans in the X4-HIV attachment. Despite similar levels of R5- and X4-HIV DNA, M1 replicates and weakly transmits the virus to activated T cells by releasing CXCR4- and CCR5-interacting chemokines. The blockade of dendritic cell-specific ICAM-3-grabbing nonintegrin expressed on M1 by mAb does not interfere with the viral transfer. Uninfected M1 recruits HIV-sensitive T cells efficiently and releases soluble factors, enhancing the viral production by these recruited cells. This study highlights the role of IFN-gamma to induce a population of macrophages that archive HIV-1 within a latent stage and cause the persistence of the virus by favoring the recruitment of T cells or enhancing the viral replication in infected CD4(+) T cells.

Pre-clinical development as microbicide of zinc tetra-ascorbo-camphorate, a novel terpenoid derivative: potent in vitro inhibitory activity against both R5- and X4-tropic HIV-1 strains without significant in vivo mucosal toxicity.

BACKGROUND: Terpenoid derivatives originating from many plants species, are interesting compounds with numerous biological effects, such as anti-HIV-1 activity. The zinc tetra-ascorbo-camphorate complex (or "C14"), a new monoterpenoid derivative was evaluated in vitro for its anti-HIV-1 activity on both R5- and X4-HIV-1 infection of primary target cells (macrophages, dendritic cells and T cells) and on HIV-1 transfer from dendritic cells to T cells. RESULTS: The toxicity study was carried out in vitro and also with the New Zealand White rabbit vaginal irritation model. C14 was found to be no cytotoxic at high concentrations (CC50 > 10 muM) and showed to be a potential HIV-1 inhibitor of infection of all the primary cells tested (EC50 = 1 muM). No significant changes could be observed in cervicovaginal tissue of rabbit exposed during 10 consecutive days to formulations containing up to 20 muM of C14. CONCLUSION: Overall, these preclinical studies suggest that zinc tetra-ascorbo-camphorate derivative is suitable for further testing as a candidate microbicide to prevent male-to-female heterosexual acquisition of HIV-1.

Differential in vitro inhibitory activity against HIV-1 of alpha-(1-3)- and alpha-(1-6)-D-mannose specific plant lectins: implication for microbicide development.

BACKGROUND: Plant lectins such as Galanthus nivalis agglutinin (GNA) and Hippeastrum hybrid agglutinin (HHA) are natural proteins able to link mannose residues, and therefore inhibit HIV-target cell interactions. Plant lectins are candidate for microbicide development. OBJECTIVE: To evaluate the activity against HIV of the mannose-specific plant lectins HHA and GNA at the cellular membrane level of epithelial cells and monocyte-derived dendritic cells (MDDC), two potential target cells of HIV at the genital mucosal level. METHODS: The inhibitory effects of HHA and GNA were evaluated on HIV adsorption to genital epithelial HEC-1A cell line, on HIV transcytosis throughout a monolayer of polarized epithelial HEC-1A cells, on HIV adsorption to MDDC and on transfer of HIV from MDDC to autologous T lymphocytes. RESULTS: HHA faintly inhibited attachment to HEC-1A cells of the R5-tropic HIV-1Ba-L strain, in a dose-dependent manner, whereas GNA moderately inhibited HIV adsorption in the same context, but only at high drug doses. Only HHA, but not GNA, inhibited HIV-1JR-CSF transcytosis in a dose-dependent manner. By confocal microscopy, HHA, but not GNA, was adsorbed at the epithelial cell surface, suggesting that HHA interacts specifically with receptors mediating HIV-1 transcytosis. Both plant lectins partially inhibited HIV attachment to MDDC. HHA inhibited more efficiently the transfer of HIV from MDDC to T cell, than GNA. Both HHA and GNA lacked toxicity below 200 microg/ml irrespective the cellular system used and do not disturb the monolayer integrity of epithelial cells. CONCLUSION: These observations demonstrate higher inhibitory activities of the lectin plant HHA by comparison to GNA, on HIV adsorption to HEC-1A cell line, HIV transcytosis through HEC-1A cell line monolayer, HIV adsorption to MDDC and HIV transfer from MDDC to T cells, highlighting the potential interest of HHA as effective microbicide against HIV.

HMGB1/anti-HMGB1 antibodies define a molecular signature of early stages of HIV-Associated Neurocognitive Isorders (HAND).

BACKGROUND: HIV-associated neurocognitive disorders (HAND) persist in the post-HAART era, characterized by asymptomatic neurocognitive impairment (ANI) and mild neurocognitive disorders (MND). High mobility group box 1 (HMGB1) is a non-histone chromosomal protein widely expressed in the nucleus of all eukaryotic cells, including brain cells, which acts as a potent proinflammatory cytokine when actively secreted from immune cells. Recent reports suggested that HMGB1 acts on microglial cells to promote neuroinflammation. In this study, our aim was to determine whether HMGB1 is involved in HAND, but also to identify early new markers of neurological impairment in HIV-infected patients. METHODS: CSF and serum were collected from 103 HIV-1-infected patients enrolled in Neuradapt, a prospective study of the prevalence of HAND in HIV-1 infected patients at Nice University Hospital. Stored fluids were assessed for immunological, virological, and brain metabolite parameters. In addition to HIV RNA and DNA measurements, expression of T-cell surface markers of activation (CD38 and HLA-DR) was analyzed on whole blood. Concentration of 27 cytokines and chemokines was measured using multiplex bead assays on serum and CSF. Concentration of HMGB1 and anti-HMGB1 IgG autoantibodies were also measured on the same samples. Changes in cerebral metabolites N-acetyl aspartate (NAA), Choline (Cho) and creatinine (Cr) were assessed by magnetic resonance microscopy (MRS). RESULTS: Clinical, virological and immunological characteristics were comparable between HAND (n = 30) and no HAND (n = 73) patients, except the absolute numbers of CD8+ T cells, which were higher in patients with HAND. Among the 29 molecules tested, only 4 of them were significantly upregulated in the CSF from HAND patients as compared to healthy donors i.e. HMGB1, anti-HMGB1 IgG antibodies, IP-10 and MCP1. CSF HMGB1 levels were positively correlated with HIV-1 DNA in aviremic HAND patients, suggesting a positive impact of HMGB1 on HIV reservoirs. Moreover, in contrast to NAA/Cr and Cho/NAA ratios, circulating anti-HMGB1 IgG antibody levels could discriminate patients with no HAND from patients with no HAND and a single deficit (average ROC-AUC = 0.744, p = 0.03 for viremic patients), thus enabling the identification of a very early stage of neurocognitive impairment. CONCLUSION: We report that brain injury in chronically HIV-infected patients on stable HAART is strongly associated with persistent CNS inflammation, which is correlated with increased levels of HMGB1 and anti-HMGB1 IgG in the CSF. Moreover, we identified circulating anti-HMGB1 IgG as a very early biomarker of neurological impairment in patients without HAND. These results might have important implication for the identification of patients who are at high risk of developing neurological disorders.

Erratum to 'HMGB1/anti-HMGB1 antibodies define a molecular signature of early stages of HIV-Associated Neurocognitive Disorders (HAND)'.

[This corrects the article DOI: 10.1016/j.heliyon.2017.e00245.].