The Effect of Heparin and Other Exogenous Glycosaminoglycans (GAGs) in Reducing IL-1ß-Induced Pro-Inflammatory Cytokine IL-8 and IL-6 mRNA Expression and the Potential Role for Reducing Inflammation.

Glycosaminoglycans (GAGs) are long linear polysaccharides found in every mammalian tissue. Previously thought only to be involved in cellular structure or hydration, GAGs are now known to be involved in cell signaling and protein modulation in cellular adhesion, growth, proliferation, and anti-coagulation. In this study, we showed that GAGs have an inhibitory effect on the IL-1ß-stimulated mRNA expression of IL-6 and IL-8. Exogenous heparin (p < 0.0001), heparan (p < 0.0001), chondroitin (p < 0.049), dermatan (p < 0.0027), and hyaluronan (p < 0.0005) significantly reduced the IL-1ß-induced IL-8 mRNA expression in HeLa cells. Exogenous heparin (p < 0.0001), heparan (p < 0.0001), and dermatan (p < 0.0027) also significantly reduced IL-1ß-induced IL-6 mRNA expression in HeLa cells, but exogenous chondroitin and hyaluronan had no significant effect. The exogenous GAGs may reduce the transcription of these inflammatory cytokines through binding to TILRR, a co-receptor of IL-1R1, and block/reduce the interactions of TILRR with IL-1R1.

NFκB1 Polymorphisms Are Associated with Severe Influenza A (H1N1) Virus Infection in a Canadian Population.

Background: We examined associations between NFκB1 polymorphisms and influenza A (H1N1) clinical outcomes in Canadian. Methods: A total of thirty-six Caucasian patients admitted to the intensive care unit (ICU) in hospitals in Canada were recruited during the 2009 H1N1 pandemic. Genomic DNA was extracted from the whole blood samples. The NFkB1 gene was targeted for genotyping using next-generation sequencing technology­Roche 454. Results: A total of 136 single nucleotide polymorphisms (SNPs) were discovered within the NFκB1 gene. Among them, 63 SNPs were significantly enriched in patients admitted in the ICU (p < 0.05) compared with the British Caucasian population in the 1000 Genomes study. These enriched SNPs are mainly intron variants, and only two are exon SNPs from the non-transcribing portion of the NFκB1 gene. Conclusions: Genetic variations in the NFκB1 gene could influence clinical outcomes of pandemic H1N1 infections. Our findings showed that sequence variations of the NFκB1 gene might influence patient response to influenza infection.

High level of plasma TILRR protein is associated with faster HIV seroconversion.

BACKGROUND: TILRR (Toll-like Interleukin-1 Receptor Regulator) is a modulator of many genes in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. It promotes the production of inflammatory mediators and the migration of immune cells. Recently, we showed that TILRR protein circulates in human blood. Thus, it could influence systemic inflammation. Systemic and mucosal inflammations increase the susceptibility to HIV infection. In this study, we analyzed the TILRR protein levels of the archived plasma samples of women enrolled in the Pumwani cohort to determine whether the plasma TILRR protein levels before seroconversion are correlated with differential risk of HIV seroconversion. METHODS: TILRR protein of 941 archived HIV negative plasma samples from 390 women who were HIV negative at the cohort enrollment was quantified with an in-house developed multiplex bead array method. Proinflammatory cytokines/chemokines were measured using a 14-plex bead array method. Spearman rank correlation analysis was used to determine the correlation between plasma TILRR protein and proinflammatory cytokines/chemokines. Kaplan-Meier survival analysis was conducted to evaluate whether the median plasma TILRR protein levels correlate with increased risk of HIV seroconversion. FINDINGS: The level of plasma TILRR protein was positively correlated with plasma IL-1ß (rho: 0.2593, p<0.0001), MCP-1 (rho: 0.2377, p<0.0001), and IL-17A (rho: 0.1225, p=0.0216). Women with median plasma TILRR protein levels ≥100 ng/ml seroconverted significantly faster than women with plasma TILRR protein levels <100 ng/ml (log-rank= 100.124, p<0.0001; relative risk= 3.72 and odds ratio= 15.29). Furthermore, the factors causing genital inflammation, such as STIs (sexually transmitted infections), vaginal discharge, and genital ulcers were not statistically significantly different among women with different median plasma TILRR protein levels. INTERPRETATION: The high plasma TILRR protein levels are highly correlated with several plasma proinflammatory cytokines/chemokines. High median plasma TILRR protein (≥100 ng/ml) strongly predicted an increased risk of HIV seroconversion. Reducing plasma TILRR protein levels may reduce the risk of HIV acquisition. FUNDING: The study was funded by an operating grant from the Canadian Institutes of Health Research (CIHR), operating grant-PA: CHVI Vaccine Discovery and Social Research (http://www.cihr-irsc.gc.ca/e/193.html), and National Microbiology Laboratory of Canada.

High Level of Pre-Treatment HIV-1 Drug Resistance and Its Association with HLA Class I-Mediated Restriction in the Pumwani Sex Worker Cohort.

BACKGROUND: We analyzed the prevalence of pre-antiretroviral therapy (ART) drug resistance mutations (DRMs) in a Kenyan population. We also examined whether host HLA class I genes influence the development of pre-ART DRMs. METHODS: The HIV-1 proviral DNAs were amplified from blood samples of 266 ART-naïve women from the Pumwani Sex Worker cohort of Nairobi, Kenya using a nested PCR method. The amplified HIV genomes were sequenced using next-generation sequencing technology. The prevalence of pre-ART DRMs was investigated. Correlation studies were performed between HLA class I alleles and HIV-1 DRMs. RESULTS: Ninety-eight percent of participants had at least one DRM, while 38% had at least one WHO surveillance DRM. M184I was the most prevalent clinically important variant, seen in 37% of participants. The DRMs conferring resistance to one or more integrase strand transfer inhibitors were also found in up to 10% of participants. Eighteen potentially relevant (p < 0.05) positive correlations were found between HLA class 1 alleles and HIV drug-resistant variants. CONCLUSIONS: High levels of HIV drug resistance were found in all classes of antiretroviral drugs included in the current first-line ART regimens in Africa. The development of DRMs may be influenced by host HLA class I-restricted immunity.

The Potential Role of FREM1 and Its Isoform TILRR in HIV-1 Acquisition through Mediating Inflammation.

FREM1 (Fras-related extracellular matrix 1) and its splice variant TILRR (Toll-like interleukin-1 receptor regulator) have been identified as integral components of innate immune systems. The potential involvement of FREM1 in HIV-1 (human immunodeficiency virus 1) acquisition was suggested by a genome-wide SNP (single nucleotide polymorphism) analysis of HIV-1 resistant and susceptible sex workers enrolled in the Pumwani sex worker cohort (PSWC) in Nairobi, Kenya. The studies showed that the minor allele of a FREM1 SNP rs1552896 is highly enriched in the HIV-1 resistant female sex workers. Subsequent studies showed that FREM1 mRNA is highly expressed in tissues relevant to mucosal HIV-1 infection, including cervical epithelial tissues, and TILRR is a major modulator of many genes in the NF-κB signal transduction pathway. In this article, we review the role of FREM1 and TILRR in modulating inflammatory responses and inflammation, and how their influence on inflammatory responses of cervicovaginal tissue could enhance the risk of vaginal HIV-1 acquisition.

Assessment of the population coverage of an HIV-1 vaccine targeting sequences surrounding the viral protease cleavage sites in Gag, Pol, or all 12 protease cleavage sites.

Development of an effective HIV-1 vaccine has been a great challenge faced by the research community. Recently a novel strategy targeting the viral protease cleavage sites (PCSs) has been tested and shown promising results. This T cell-based vaccine strategy depends on individuals expressing certain HLA class I molecules and since each population has unique distributions of HLA class I alleles, population coverage analysis is required to assess feasibility. Utilizing the validated CD8 T cell epitope data from previous studies we conducted coverage analysis of an HIV-1 vaccine targeting the sequences surrounding all 12-PCSs, Gag-PCSs, and Pol-PCSs. The population coverage, average epitope hit, and minimum number of epitopes recognized by 90% of the population (PC90) was compiled for 66 countries and 16 geographical regions using the web tool provided by "Immune Epitope Database and Analysis Resource". Our analysis shows that the coverage for an HIV-1 vaccine targeting sequences surrounding all 12 PCSs, 5 PCSs in Gag or 6 PCSs in Pol can cover ~ 70% to ~ 100% of the populations analyzed. There was no statistical difference in population coverages for the majority of populations examined when comparing the CD8 T cell epitope sets (12-PCSs, Gag-PCSs, and Pol-PCSs). As expected, vaccines targeting more sequences will have more CD8 T cell epitopes, as the mean average epitope hit for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 9.45, 4.76, and 4.74, respectively, and across all geographical regions was 9.76, 4.99, and 4.92, respectively. The average PC90 for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 2.53, 1.31, and 1.41, respectively, and across all geographical regions was 2.24, 1.23, and 1.29, respectively. Thus, vaccines targeting sequences surrounding the HIV-1 PCSs can cover broad populations; however, whether targeting a subset of the PCSs is sufficient to prevent acquisition requires further preclinical investigation.

Genomic characterization and evolution of SARS-CoV-2 of a Canadian population.

COVID-19 has greatly affected public health and world economy. In this study, we analyzed 129 full-length genomes of SARS-CoV-2 viruses of a Canadian population during early phase of the pandemic. Phylogenetic analysis revealed three major paths of transmission of SARS-CoV-2 viruses into Canada. Twenty-one substitutions that have frequencies greater than 3% of viral population were identified. Analysis of these substitutions indicated that P1427I (ORF1b), Y1464C (ORF1b), and Q57H (ORF3a) might affect functions of the corresponding SARS-CoV-2 encoded proteins. Additionally, we found the evidence of positive selection on the ORF3a and codon 614 of Spike protein, suggesting the viral components responsible for host entry and activation of inflammation response were targeted by host immune responses. The study showed genomic variation and evolution of SARS-CoV-2 in a Canadian population. These information may help develop preventive strategies and be used for further study of SARS-CoV-2 pathogenesis and therapeutics development.

Vaccine targeting SIVmac251 protease cleavage sites protects macaques against vaginal infection.

After over 3 decades of research, an effective anti-HIV vaccine remains elusive. The recently halted HVTN702 clinical trial not only further stresses the challenge to develop an effective HIV vaccine but also emphasizes that unconventional and novel vaccine strategies are urgently needed. Here, we report that a vaccine focusing the immune response on the sequences surrounding the 12 viral protease cleavage sites (PCSs) provided greater than 80% protection to Mauritian cynomolgus macaques against repeated intravaginal SIVmac251 challenges. The PCS-specific T cell responses correlated with vaccine efficacy. The PCS vaccine did not induce immune activation or inflammation known to be associated with increased susceptibility to HIV infection. Machine learning analyses revealed that the immune microenvironment generated by the PCS vaccine was predictive of vaccine efficacy. Our study demonstrates, for the first time to our knowledge, that a vaccine which targets only viral maturation, but lacks full-length Env and Gag immunogens, can prevent intravaginal infection in a stringent macaque/SIV challenge model. Targeting HIV maturation thus offers a potentially novel approach to developing an effective HIV vaccine.

TILRR Promotes Migration of Immune Cells Through Induction of Soluble Inflammatory Mediators.

TILRR has been identified as an important modulator of inflammatory responses. It is associated with NF-κB activation, and inflammation. Our previous study showed that TILRR significantly increased the expression of many innate immune responsive genes and increased the production of several pro-inflammatory cytokines/chemokines by cervical epithelial cells. In this study, we evaluated the effect of TILRR-induced pro-inflammatory cytokines/chemokines on the migration of immune cells. The effect of culture supernatants of TILRR-overexpressed cervical epithelial cells on the migration of THP-1 monocytes and MOLT-4 T-lymphocytes was evaluated using Transwell assay and a novel microfluidic device. We showed that the culture supernatants of TILRR-overexpressed HeLa cells attracted significantly more THP-1 cells (11-40%, p = 0.0004-0.0373) and MOLT-4 cells (14-17%, p = 0.0010-0.0225) than that of controls. The microfluidic device-recorded image analysis showed that significantly higher amount with longer mean cell migration distance of THP-1 (p < 0.0001-0.0180) and MOLT-4 (p < 0.0001-0.0025) cells was observed toward the supernatants of TILRR-overexpressed cervical epithelial cells compared to that of the controls. Thus, the cytokines/chemokines secreted by the TILRR-overexpressed cervical epithelial cells attracted immune cells, such as monocytes and T cells, and may potentially influence immune cell infiltration in tissues.

A MiSeq-HyDRA platform for enhanced HIV drug resistance genotyping and surveillance.

Conventional HIV drug resistance (HIVDR) genotyping utilizes Sanger sequencing (SS) methods, which are limited by low data throughput and the inability of detecting low abundant drug resistant variants (LADRVs). Here we present a next generation sequencing (NGS)-based HIVDR typing platform that leverages the advantages of Illumina MiSeq and HyDRA Web. The platform consists of a fully validated sample processing protocol and HyDRA web, an open web portal that allows automated customizable NGS-based HIVDR data processing. This platform was characterized and validated using a panel of HIV-spiked plasma representing all major HIV-1 subtypes, pedigreed plasmids, HIVDR proficiency specimens and clinical specimens. All examined major HIV-1 subtypes were consistently amplified at viral loads of ≥1,000 copies/ml. The gross error rate of this platform was determined at 0.21%, and minor variations were reliably detected down to 0.50% in plasmid mixtures. All HIVDR mutations identifiable by SS were detected by the MiSeq-HyDRA protocol, while LADRVs at frequencies of 1~15% were detected by MiSeq-HyDRA only. As compared to SS approaches, the MiSeq-HyDRA platform has several notable advantages including reduced cost and labour, and increased sensitivity for LADRVs, making it suitable for routine HIVDR monitoring for both patient care and surveillance purposes.

Current advances in HIV vaccine preclinical studies using Macaque models.

The macaque simian or simian/human immunodeficiency virus (SIV/SHIV) challenge model has been widely used to inform and guide human vaccine trials. Substantial advances have been made recently in the application of repeated-low-dose challenge (RLD) approach to assess SIV/SHIV vaccine efficacies (VE). Some candidate HIV vaccines have shown protective effects in preclinical studies using the macaque SIV/SHIV model but the model's true predictive value for screening potential HIV vaccine candidates needs to be evaluated further. Here, we review key parameters used in the RLD approach and discuss their relevance for evaluating VE to improve preclinical studies of candidate HIV vaccines.

Toll-like Interleukin 1 Receptor Regulator Is an Important Modulator of Inflammation Responsive Genes.

TILRR (Toll-like interleukin-1 receptor regulator), a transcript variant of FREM1, is a novel regulatory component, which stimulates innate immune responses through binding to IL-1R1 (Interleukin-1 receptor, type 1) and TLR (Toll-like receptor) complex. However, it is not known whether TILRR expression influences other genes in the NFκB signal transduction and pro-inflammatory responses. Our previous study identified FREM1 as a novel candidate gene in HIV-1 resistance/susceptibility in the Pumwani Sex worker cohort. In this study, we investigated the effect of TILRR overexpression on expression of genes in the NFκB signaling pathway in vitro. The effect of TILRR on mRNA expression of 84 genes related to NFκB signal transduction pathway was investigated by qRT-PCR. Overexpression of TILRR on pro-inflammatory cytokine/chemokine(s) secretion in cell culture supernatants was analyzed using Bioplex multiplex bead assay. We found that TILRR overexpression significantly influenced expression of many genes in HeLa and VK2/E6E7 cells. Several cytokine/chemokine(s), including IL-6, IL-8 (CXCL8), IP-10, MCP-1, MIP-1ß, and RANTES (CCL5) were significantly increased in the cell culture supernatants following TILRR overexpression. Although how TILRR influences the expression of these genes needs to be further studied, we are the first to show the influence of TILRR on many genes in the NFκB inflammatory pathways. The NFκB inflammatory response pathways are extremely important in microbial infection and pathogenesis, including HIV-1 transmission. Further study of the role of TILRR may identify the novel intervention targets and strategies against HIV infection.

Transvaginal Natural Orifice Transluminal Endoscopic Surgery Sacrocolpopexy: Tips and Tricks.

STUDY OBJECTIVE: To demonstrate helpful tips and tricks for the successful use of transvaginal natural orifice transluminal endoscopic surgery (NOTES) for performing sacrocolpopexy and salpingo-oophorectomy surgery. Minimally invasive approaches for treating pelvic organ prolapse via sacrocolpopexy have traditionally included laparoscopy either with or without robotic assistance. Transvaginal NOTES is a novel minimally invasive approach that both avoids abdominal incisions and provides improved visualization; however, it can be technically challenging. DESIGN: Stepwise demonstration with narrated video footage (Canadian Task Force classification III). SETTING: An academic tertiary care hospital in Guangdong, China. PATIENT: A 61-year-old gravida 3, para 3 woman with 3 spontaneous vaginal deliveries and stage III uterine prolapse, stage III cystocele, and stage III rectocele. The preoperative vaginal length was 6 cm. INTERVENTION: After performing vaginal hysterectomy, we show the usefulness of NOTES for salpingo-oophorectomy. We also demonstrate useful techniques for transvaginal NOTES sacrocolpopexy including hydrodissection, division of the Y mesh, anchoring of the anterior mesh before reducing prolapse, retroperitoneal tunneling, and hand suturing of the mesh and vaginal cuff. MEASUREMENTS AND MAIN RESULTS: The procedure was successfully performed in approximately 190 minutes. The postoperative vaginal length was 5 cm. Postoperative pelvic organ prolapse quantification was stage 0. CONCLUSION: The transvaginal NOTES approach is feasible and efficient for sacrocolpopexy and salpingo-oophorectomy; additionally, it is a reasonable option for patients who desire a minimally invasive approach with excellent cosmetic results. Surgical techniques that aid in effectively performing transvaginal NOTES sacrocolpopexy include the use of hydrodissection, Y mesh division, anterior mesh anchoring before reducing prolapse, retroperitoneal tunneling, and hand suturing. Using the techniques presented here, we were able to insert the port only 1 time, which improves the efficiency and safety of this surgery.

Mucosal antibody responses to vaccines targeting SIV protease cleavage sites or full-length Gag and Env proteins in Mauritian cynomolgus macaques.

HIV mutates rapidly and infects CD4+ T cells, especially when they are activated. A vaccine targeting conserved, essential viral elements while limiting CD4+ T cell activation could be effective. Learning from natural immunity observed in a group of highly HIV-1 exposed seronegative Kenyan female sex workers, we are testing a novel candidate HIV vaccine targeting the 12 viral protease cleavage sites (PCSs) (the PCS vaccine), in comparison with a vaccine targeting full-length Gag and Env (the Gag/Env vaccine) in a Mauritian cynomolgus macaque/SIV model. In this study we evaluated these vaccines for induction of mucosal antibodies to SIV immunogens at the female genital tract. Bio-Plex and Western blot analyses of cervicovaginal lavage samples showed that both the PCS and Gag/Env vaccines can elicit mucosal IgG antibody responses to SIV immunogens. Significantly higher increase of anti-PCS antibodies was induced by the PCS vaccine than by the Gag/Env vaccine (p<0.0001). The effect of the mucosal antibody responses in protection from repeated low dose pathogenic SIVmac251 challenges is being evaluated.

Transvaginal natural orifice transluminal endoscopic surgery tubal reanastomosis: a novel route for tubal surgery.

OBJECTIVE: To demonstrate how a transvaginal natural orifice transluminal endoscopic surgery (NOTES) tubal reanastomosis is a novel route for tubal surgery. The surgical technique is a combination of traditional vaginal surgery with single-site surgical skills. DESIGN: The surgical technique is explained in a stepwise fashion with the use of surgical video footage. The video uses a surgical case to demonstrate the specific techniques necessary to perform a NOTES tubal reanastomosis. SETTING: Teaching university. PATIENT(S): A 42-year-old female G2P2 with a history of tubal ligation 11 years before presentation requesting a tubal recanalization. INTERVENTION(S): Transvaginal NOTES tubal reanastomosis was initiated with a posterior colpotomy. A single-site gelport was placed. The fallopian tubes were hydrodissected, the blocked portion of each tube was removed, an epidural catheter was threaded through each lumen, and the two remaining segments of each tube were sutured together in an end-to-end fashion using single-site suturing skills. MAIN OUTCOME MEASURE(S): Transvaginal NOTES tubal reanastomosis as an alternative route for tubal reanastomosis. RESULT(S): The bilateral fallopian tubes were recanalized with bilateral tubal patency. This was confirmed 8 weeks postoperatively with a three-dimensional sonohystogram, which showed patency of the bilateral fallopian tubes. CONCLUSION(S): The current preferred technique for reversal of a tubal sterilization is to perform a minimally invasive surgery with an end-to-end anastomosis. This gives the patient a 60%-90% intrauterine pregnancy rate postoperatively. NOTES has the benefits of a fast recovery, no abdominal incisional pain, and an extremely cosmetic outcome. Current research has shown a 0%-3.1% range for the risk of pelvic infection in transvaginal NOTES if prophylactic antibiotics are administered during the surgery. The NOTES tubal reanastomosis combines the traditional vaginal surgery technique of creating a posterior colpotomy with single-site surgical skills like suturing and knot tying. The surgery is completed through a single transvaginal port without an abdominal incision. In the hands of a skilled minimally invasive surgeon, transvaginal NOTES tubal reanastomosis is a feasible and alternative route for this procedure.

Hypothetical endogenous SIV-like antigens in Mauritian cynomolgus macaques.

Simian immunodeficiency virus (SIV) infection of Mauritian cynomolgus macaques (MCMs) is an increasingly important nonhuman primate model for HIV vaccine research. We previously reported that in MCMs anti-SIV antibodies can be naturally developed without exogenous infection or vaccination, and that a vaccine targeting SIV protease cleavage sites (PCS) can cross-induce antibodies to non-PCS SIV antigens. We speculate that this is potentially caused by the existence of endogenous SIV-like antigens. External stimuli (such as environmental factors and vaccination) may induce expression of endogenous SIV-like antigens to elicit these antibodies. Database and mass spectrometry analyses were conducted to search for such antigens. We identified endogenous SIV-like DNA sequences in cynomolgus macaque genome and non-PCS peptide homologous to SIV Env protein in PBMCs of a PCS-vaccinated monkey. Our preliminary insights suggest that endogenous SIV-like antigens may be one of the possible reasons for the natural and cross-inducible SIV antibodies in MCMs.

Identification and Characterization of Positively Selected Mutations in Nef of Four HIV-1 Major Subtypes from Los Alamos National Laboratory.

BACKGROUND: Human immunodeficiency virus-1 (HIV-1) mutates rapidly to escape host immune pressure. This results in the generation of positively selected mutations (PSM) throughout the viral genome. Escape mutations in Nef, one of the accessory proteins of HIV-1, which plays an important role in viral pathogenicity have previously been identified in several large cohort studies, but the evolution of PSMs overtime in various HIV-1 subtypes remains unknown. METHODS: 161 clade A1, 3093 clade B, 647 clade C and 115 clade D HIV-1 nef sequences were obtained from the HIV Database of Los Alamos National Laboratory and aligned using MEGA 6.0. The sequences from each clade were grouped based on the year of collection. Quasi analysis was used to identify PSMs and the number and locations of PSMs were compared among different subtypes. RESULTS: PSMs for all four subtypes were distributed across the sequence of Nef, and conserved residues F90, W113, PxxPxR (a.a 72-77) remain unaltered overtime. The frequency of PSMs was stable among subtype B sequences but increased overtime for other subtypes. Phylogenetic analysis shows that sequences containing PSMs tend to cluster together at both inter and intra- subtype levels. CONCLUSION: Identification of PSMs and their changes overtime within various subtypes of HIV-1 is important in defining global viral evolutionary patterns that can provide insights for designing therapeutic strategies.

Transvaginal Natural Orifice Transluminal Endoscopic Surgery Myomectomy: A Novel Route for Uterine Myoma Removal.

STUDY OBJECTIVE: Transvaginal surgery is the most minimally invasive surgery for a gynecologic procedure, but it has the limitation of lack of exposure and limited surgical space when using traditional vaginal surgical instrumentation, such as in a hysterectomy for a uterus without descent or for a myomectomy. Transvaginal natural orifice transluminal endoscopic surgery (NOTES) offers similar benefits of traditional vaginal surgery but also expands the horizon of transvaginal surgery by allowing the surgeon to perform procedures that are typically limited to an abdominal approach. The advantages of NOTES may include no incisional pain as well as a better cosmetic outcome. These benefits help outweigh the obstacle of learning this novel approach. Our objective is to demonstrate the transvaginal NOTES technique as a combination of traditional vaginal surgical skill with single-site surgical skill. DESIGN: Stepwise demonstration of the transvaginal NOTES technique for myomectomy with narrated video footage (Canadian Task Force classification III). SETTING: Academic tertiary care hospital. PATIENT: A 42-year-old woman. INTERVENTIONS: Transvaginal NOTES myomectomy with combined transvaginal surgical and single-site surgical skills. MEASUREMENTS AND MAIN RESULTS: A 42-year-old woman (gravida 2 para 2) with a preoperative transvaginal ultrasound diagnosis of a 6-cm left anterior myoma requested myoma removal with uterine preservation. She presented with a 2-year history of left pelvic pain and menorrhagia. The myoma was removed with minimal blood loss, and pathology revealed a necrotic myoma. The patient had resolution of her left-sided pelvic pain. CONCLUSIONS: Combined with traditional transvaginal anterior colpotomy, single-site surgical skills allow the surgeon to access the entire abdomen and perform myomectomy through a transvaginal single port. Transvaginal NOTES myomectomy is not only possible but allows myomectomy to be performed with no abdominal incision.

Natural and cross-inducible anti-SIV antibodies in Mauritian cynomolgus macaques.

Cynomolgus macaques are an increasingly important nonhuman primate model for HIV vaccine research. SIV-free animals without pre-existing anti-SIV immune responses are generally needed to evaluate the effect of vaccine-induced immune responses against the vaccine epitopes. Here, in order to select such animals for vaccine studies, we screened 108 naïve female Mauritian cynomolgus macaques for natural (baseline) antibodies to SIV antigens using a Bio-Plex multiplex system. The antigens included twelve 20mer peptides overlapping the twelve SIV protease cleavage sites (-10/+10), respectively (PCS peptides), and three non-PCS Gag or Env peptides. Natural antibodies to SIV antigens were detected in subsets of monkeys. The antibody reactivity to SIV was further confirmed by Western blot using purified recombinant SIV Gag and Env proteins. As expected, the immunization of monkeys with PCS antigens elicited anti-PCS antibodies. However, unexpectedly, antibodies to non-PCS peptides were also induced, as shown by both Bio-Plex and Western blot analyses, while the non-PCS peptides do not share sequence homology with PCS peptides. The presence of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques should be considered in animal selection, experimental design and result interpretation, for their best use in HIV vaccine research.