Multiplex Patient-Based Drug Response Assay in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an extremely lethal malignancy arising from the pancreas. The treatment of PDA is complicated by ineffective treatments and a lack of biomarkers predictive of treatment success. We have designed a patient-derived organoid (PDO) based high-throughput drug screening assay to model treatment response to a variety of conventional and investigational treatments for PDA. Consecutive patients undergoing endoscopic ultrasound-guided fine-needle biopsy for tissue diagnosis of PDA at Rush University Medical Center were offered to participate in the study. Biopsies were immediately processed to develop organoids. Fifteen PDOs were screened for sensitivity to 18 compounds, including conventional PDA chemotherapies and FDA-approved investigational targeted therapies in cancer using Cell-titer GLO 3D (Promega) cell viability assay. The area under the curve (AUC) was calculated and normalized to the maximum area under the curve to generate a normalized AUC between 0 and 1. Molecular profiling of PDOs was conducted using RNA-seq. Human PDA transcriptomic was extracted from The Cancer Genome Atlas (TCGA). The drug response curves were reproducible. We observed variation in response to conventional therapies overall as well as among individual patients. There were distinct transcriptome signatures associated with response to the conventional chemotherapeutics in PDA. The transcriptomic profile of overall resistance to conventional therapies in our study was associated with poor survival in PDA patients in TCGA. Our pathway analysis for targeted drugs revealed a number of predictors of response associated with the mechanism of action of the tested drug. The multiplex organoid-based drug assay could be used in preclinical to inform patient stratification and therapeutic selection in PDA. When combined with omics data, ex vivo response to treatment could help identify gene signatures associated with response to novel therapies.

Organisational factors affecting performance in delivering influenza vaccination to staff in NHS Acute Hospital Trusts in England: A qualitative study.

Health care workers are a priority group for seasonal influenza vaccination, which is recommended by the World Health Organisation. There is a wide variation in uptake between and within countries. England has achieved 69.5% of health care workers vaccinated overall in 2017/18 across NHS acute and community health care settings, but it varies between Trusts from 50% to over 92.3%. While attitudinal factors have been well researched, there is limited evidence on organisational factors associated with high uptake. In England, most NHS Trusts are now implementing a similar range of interventions as part of their flu programme, and it remains unclear why performance remains so variable. This qualitative study is the first to explore reasons for this variation and provide recommendations for lower performing Trusts on how to improve. Fifty-seven interviews of managers and vaccinators were conducted in nine hospitals with flu vaccination uptake ranging from just over 55% to above 90%. Our study found that while Trusts deployed a wide range of both demand generating and supply interventions to increase uptake, there were marked differences in the organisational and delivery models utilised. Our study suggests that organisational culture was possibly the most important ingredient when trying to differentiate between high and low performing Trusts. We found that a positive culture aimed at fostering continuous improvement and favouring non-coercion on balance yielded more adherence from staff. Where influenza vaccination was embedded in the organisation wellbeing strategy, rather than executed as a siloed seasonal programme, this tended to foster good performance. Improving performance of influenza vaccination in health care workers will involve not only deploying the right interventions, and following "best practices". It will require the adaptation of flu progamme delivery strategies to the organisation context, and embedding vaccination into the organisational culture, thus supporting the normalisation of yearly vaccination.

Apoptosis and Compensatory Proliferation Signaling Are Coupled by CrkI-Containing Microvesicles.

Apoptosis has been implicated in compensatory proliferation signaling (CPS), whereby dying cells induce proliferation in neighboring cells as a means to restore homeostasis. The nature of signaling between apoptotic cells and their neighboring cells remains largely unknown. Here we show that a fraction of apoptotic cells produce and release CrkI-containing microvesicles (distinct from exosomes and apoptotic bodies), which induce proliferation in neighboring cells upon contact. We provide visual evidence of CPS by videomicroscopy. We show that purified vesicles in vitro and in vivo are sufficient to stimulate proliferation in other cells. Our data demonstrate that CrkI inactivation by ExoT bacterial toxin or by mutagenesis blocks vesicle formation in apoptotic cells and inhibits CPS, thus uncoupling apoptosis from CPS. We further show that c-Jun amino-terminal kinase (JNK) plays a pivotal role in mediating vesicle-induced CPS in recipient cells. CPS could have important ramifications in diseases that involve apoptotic cell death.

Glycogen Levels in Undiluted Genital Fluid and Their Relationship to Vaginal pH, Estrogen, and Progesterone.

BACKGROUND: Colonization of the female lower genital tract with Lactobacillus provides protection against STIs and adverse pregnancy outcomes. Growth of genital Lactobacillus is postulated to depend on epithelial cell-produced glycogen. However, the amount of cell-free glycogen in genital fluid available for utilization by Lactobacillus is not known. METHODS: Eighty-five genital fluid samples from 7 pre-menopausal women taken over 4-6 weeks were obtained using the Instead SoftCup® (EvoFem, Inc., San Diego, CA, USA) by consented donors. Cell-free glycogen and glucose in genital fluids and estrogen and progesterone in blood were quantified. FINDINGS: Glycogen ranged from 0.1-32 µg/µl. There were significant differences between women in glycogen over the observation period. There was a strong negative correlation between glycogen and vaginal pH (r = -0.542, p<0.0001). In multivariable analysis, free glycogen levels were significantly negatively associated with both vaginal pH and progesterone (p < 0.001 and p = 0.004, respectively). Estrogen, glucose, age, sexual intercourse 24 hours prior to visit, and days after the initial visit were not significantly associated with free glycogen levels. CONCLUSION: Cell-free glycogen concentrations can be very high, up to 3% of genital fluid, and are strongly associated with acidic vaginal pH. However, the fluctuations in glycogen levels in individuals and differences between individuals do not appear to be associated with estrogen.

Implementing an integrated in-situ coaching, observational audit, and story-telling intervention to support safe surgery.

This article describes an intervention that combined in-situ coaching, observational audits and story-telling to educate theatre teams at University College London Hospitals about the Five steps to safer surgery (NPSA 2010). Our philosophy was to educate theatre teams about 'what goes right' (good catches, exemplary leadership etc) as well as 'what could be improved'. Results showed improvements on 'behavioural reliability' metrics, a 68% increase in near miss reporting and a reduction in surgical harm incidents.

The vaginal microbiota over an 8- to 10-year period in a cohort of HIV-infected and HIV-uninfected women.

BACKGROUND: We identified predominant vaginal microbiota communities, changes over time, and how this varied by HIV status and other factors in a cohort of 64 women. METHODS: Bacterial DNA was extracted from reposited cervicovaginal lavage samples collected annually over an 8-10 year period from Chicago Women's Interagency HIV Study participants: 22 HIV-negative, 22 HIV-positive with stable infection, 20 HIV-positive with progressive infection. The vaginal microbiota was defined by pyrosequencing of the V1/V2 region of the 16S rRNA gene. Scheduled visits included Bacterial vaginsosis (BV) screening; clinically detected cases were referred for treatment. Hierarchical clustering identified bacterial community state types (CST). Multinomial mixed effects modeling determined trends over time in CST, by HIV status and other factors. RESULTS: The median follow-up time was 8.1 years (range 5.5-15.3). Six CSTs were identified. The mean relative abundance (RA) of Lactobacillus spp. by CST (with median number of bacterial taxa) was: CST-1-25.7% (10), CST-2-27.1% (11), CST-3-34.6% (9), CST-4-46.8% (9), CST-5-57.9% (4), CST-6-69.4% (2). The two CSTs representing the highest RA of Lactobacillus and lowest diversity increased with each additional year of follow-up (CST-5, adjusted odds ratio (aOR) = 1.62 [95% CI: 1.34-1.94]; CST-6, aOR = 1.57 [95 CI: 1.31-1.89]), while the two CSTs representing lowest RA of Lactobacillus and higher diversity decreased with each additional year (CST-1, aOR = 0.89 [95% CI: 0.80-1.00]; CST-2, aOR = 0.86 [95% CI: 0.75-0.99]). There was no association between HIV status and CST at baseline or over time. CSTs representing lower RA of Lactobacillus were associated with current cigarette smoking. CONCLUSIONS: The vaginal microbial community significantly improved over time in this cohort of women with HIV and at high risk for HIV who had regular detection and treatment referral for BV.

Exploratory comparison of vaginal glycogen and Lactobacillus levels in premenopausal and postmenopausal women.

OBJECTIVE: Previous studies have suggested that glycogen expression in the vaginal epithelium decreases during menopause, resulting in reduced levels of lactobacilli. However, free glycogen in genital fluids and its relationship with Lactobacillus levels have not been compared in premenopausal and postmenopausal women. METHODS: Eighty-two cervicovaginal lavage samples were collected at different phases of the menstrual cycle from 11 premenopausal (4 HIV-uninfected and 7 HIV-infected) and 12 postmenopausal (7 HIV-uninfected and 5 HIV-infected) women during a 1- to 3-month period. Free glycogen was quantified in genital fluids. Lactobacillus levels were quantified by real-time polymerase chain reaction. Estrogen and progesterone levels in blood were determined by enzyme-linked immunosorbent assay. RESULTS: Free glycogen was detected in both premenopausal and postmenopausal women. Across all samples, those from postmenopausal women had significantly lower levels of free glycogen than those from premenopausal women (median, 0.002 vs 0.065 µg/µL, respectively; P = 0.03). Lactobacillus levels correlated positively with free glycogen in both premenopausal (Spearman r = 0.68, P < 0.0001) and postmenopausal (r = 0.60, P < 0.002) women. Samples from premenopausal women had higher Lactobacillus levels and lower vaginal pH (median log, 8.1; median pH, 4) than those from postmenopausal women (median log, 7.1; median pH, 4.6), although these differences were not significant. HIV status had no significant effect on these relationships. CONCLUSIONS: Free glycogen is detected in both premenopausal and postmenopausal women and correlates with Lactobacillus in both groups. These results point to the complexity of the relationship between menopause and vaginal microbiota and indicate that more careful studies of the role of glycogen are warranted.

Free glycogen in vaginal fluids is associated with Lactobacillus colonization and low vaginal pH.

OBJECTIVE: Lactobacillus dominates the lower genital tract microbiota of many women, producing a low vaginal pH, and is important for healthy pregnancy outcomes and protection against several sexually transmitted pathogens. Yet, factors that promote Lactobacillus remain poorly understood. We hypothesized that the amount of free glycogen in the lumen of the lower genital tract is an important determinant of Lactobacillus colonization and a low vaginal pH. METHODS: Free glycogen in lavage samples was quantified. Pyrosequencing of the 16S rRNA gene was used to identify microbiota from 21 African American women collected over 8-11 years. RESULTS: Free glycogen levels varied greatly between women and even in the same woman. Samples with the highest free glycogen had a corresponding median genital pH that was significantly lower (pH 4.4) than those with low glycogen (pH 5.8; p<0.001). The fraction of the microbiota consisting of Lactobacillus was highest in samples with high glycogen versus those with low glycogen (median = 0.97 vs. 0.05, p<0.001). In multivariable analysis, having 1 vs. 0 male sexual partner in the past 6 months was negatively associated, while BMI ≥30 was positively associated with glycogen. High concentrations of glycogen corresponded to higher levels of L. crispatus and L. jensenii, but not L. iners. CONCLUSION: These findings show that free glycogen in genital fluid is associated with a genital microbiota dominated by Lactobacillus, suggesting glycogen is important for maintaining genital health. Treatments aimed at increasing genital free glycogen might impact Lactobacillus colonization.

Comparison of lower genital tract microbiota in HIV-infected and uninfected women from Rwanda and the US.

INTRODUCTION: Previous studies have shown that alterations of the bacterial microbiota in the lower female genital tract influence susceptibility to HIV infection and shedding. We assessed geographic differences in types of genital microbiota between HIV-infected and uninfected women from Rwanda and the United States. METHODS: Genera of lower genital tract bacterial microbiota were identified by high-throughput pyrosequencing of the 16S rRNA gene from 46 US women (36 HIV-infected, 10 HIV-uninfected) and 40 Rwandan women (18 HIV-infected, 22 HIV-uninfected) with similar proportions of low (0-3) Nugent scores. Species of Lactobacillus were identified by assembling sequences along with reference sequences into phylogenetic trees. Prevalence of genera and Lactobacillus species were compared using Fisher's exact tests. RESULTS: Overall the seven most prevalent genera were Lactobacillus (74%), Prevotella (56%), Gardnerella (55%), Atopobium (42%), Sneathia (37%), Megasphaera (30%), and Parvimonas (26%), observed at similar prevalences comparing Rwandan to US women, except for Megasphaera (20% vs. 39%, p = 0.06). Additionally, Rwandan women had higher frequencies of Mycoplasma (23% vs. 7%, p = 0.06) and Eggerthella (13% vs. 0%, p = 0.02), and lower frequencies of Lachnobacterium (8% vs. 35%, p<0.01) and Allisonella (5% vs. 30%, p<0.01), compared with US women. The prevalence of Mycoplasma was highest (p<0.05) in HIV-infected Rwandan women (39%), compared to HIV-infected US women (6%), HIV-uninfected Rwandan (9%) and US (10%) women. The most prevalent lactobacillus species in both Rwandan and US women was L. iners (58% vs. 76%, p = 0.11), followed by L. crispatus (28% vs. 30%, p = 0.82), L. jensenii (20% vs. 24%, p = 0.80), L. gasseri (20% vs. 11%, p = 0.37) and L. vaginalis (20% vs. 7%, p = 0.10). DISCUSSION: We found similar prevalence of most major bacterial genera and Lactobacillus species in Rwandan and US women. Further work will be needed to establish whether observed differences differentially impact lower genital tract health or susceptibility to genital infections.

Human α-amylase present in lower-genital-tract mucosal fluid processes glycogen to support vaginal colonization by Lactobacillus.

Lactobacillus colonization of the lower female genital tract provides protection from the acquisition of sexually transmitted diseases, including human immunodeficiency virus, and from adverse pregnancy outcomes. While glycogen in vaginal epithelium is thought to support Lactobacillus colonization in vivo, many Lactobacillus isolates cannot utilize glycogen in vitro. This study investigated how glycogen could be utilized by vaginal lactobacilli in the genital tract. Several Lactobacillus isolates were confirmed to not grow in glycogen, but did grow in glycogen-breakdown products, including maltose, maltotriose, maltopentaose, maltodextrins, and glycogen treated with salivary α-amylase. A temperature-dependent glycogen-degrading activity was detected in genital fluids that correlated with levels of α-amylase. Treatment of glycogen with genital fluids resulted in production of maltose, maltotriose, and maltotetraose, the major products of α-amylase digestion. These studies show that human α-amylase is present in the female lower genital tract and elucidates how epithelial glycogen can support Lactobacillus colonization in the genital tract.

Longitudinal assessment of pigtailed macaque lower genital tract microbiota by pyrosequencing reveals dissimilarity to the genital microbiota of healthy humans.

Vaginal bacterial communities play an important role in human health and have been shown to influence HIV infection. Pigtailed macaques (Macaca nemestrina) are used as an animal model of HIV vaginal infection of women. Since the bacterial microbiota could influence retrovirus infection of pigtailed macaques, the genital microbiota in 10 cycling macaques was determined by pyrosequencing. The microbiota of all macaques was polymicrobial with a median of 13 distinct genera. Strikingly, the genera Sneathia and Fusobacterium, both in the phylum Fusobacteria, accounted for 18.9% and 13.3% of sequences while the next most frequent were Prevotella (5.6%), Porphyromonas (4.1%), Atopobium (3.6%), and Parvimonas (2.6%). Sequences corresponding to Lactobacillus comprised only 2.2% of sequences on average and were essentially all L. amylovorus. Longitudinal sampling of the 10 macaques over an 8-week period, which spanned at least one full ovulatory cycle, showed a generally stable presence of the major types of bacteria with some exceptions. These studies show that the microbiota of the pigtailed macaques is substantially dissimilar to that found in most healthy humans, where the genital microbiota is usually dominated by Lactobacillus sp. The polymicrobial makeup of the macaque bacterial populations, the paucity of lactobacilli, and the specific types of bacteria present suggest that the pigtailed macaque microbiota could influence vaginal retrovirus infection.

A comparison of lower genital tract glycogen and lactic acid levels in women and macaques: implications for HIV and SIV susceptibility.

Understanding factors that affect heterosexual transmission of HIV in women is of great importance. Lactobacilli in the lower genital tract of women utilize glycogen in vaginal epithelial cells as an energy source and produce lactic acid. The resultant vaginal acidity is believed to provide protection against HIV infection. Conversely, bacterial vaginosis (BV) is characterized by less lactic acid and a higher pH, and is associated with increased susceptibility to HIV infection. Because vaginal infection of macaques with simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) is used as a model to study HIV sexual transmission, and because previous studies have shown a paucity of lactobacilli in rhesus macaques' lower genital tract, we compared lactic acid and glycogen levels in the genital fluid of rhesus and pigtail macaques with levels found in humans. The levels of lactic acid were lower in both rhesus (median=1.2 mol lactate/mg protein) and pigtail macaques (median=0.7 mol/mg) compared to women with healthy genital microbiota (median=4.2 mol/mg). Glycogen levels were significantly lower in both rhesus (median=0.004 µg glycogen/µg protein) and pigtail macaques (median=0 µg/µg) than in women (median=0.2 µg/µg). No significant differences in glycogen or lactate levels were observed comparing longitudinally collected samples from cycling pigtail macaques. These data show that the previously reported scarcity of lactobacilli in macaques correlates with low glycogen and lactic acid levels. These findings have important implications for studies of vaginal infection of macaques with SIV or SHIV and further our understanding of how the bacterial microbiota influences HIV infection.

Short-chain fatty acids induce pro-inflammatory cytokine production alone and in combination with toll-like receptor ligands.

PROBLEM: Short-chain fatty acids (SCFAs), produced at relatively high levels by anaerobic bacteria in bacterial vaginosis (BV), are believed to be anti-inflammatory. BV, a common alteration in the genital microbiota associated with increased susceptibility to HIV infection, is characterized by increased levels of both pro-inflammatory cytokines and SCFAs. We investigated how SCFAs alone or together with Toll-like receptor (TLR) ligands affected pro-inflammatory cytokine secretion. METHOD OF STUDY: Cytokines were measured by ELISA. Flow was used for phenotyping and reactive oxygen species (ROS) measurement. RESULTS: Short-chain fatty acids, at 20 mM, induced interleukin (IL)-8, IL-6, and IL-1ß release, while lower levels (0.02-2 mM) did not induce cytokine secretion. Levels >20 mM were toxic to cells. Interestingly, lower levels of SCFAs significantly enhanced TLR2 ligand- and TLR7 ligand-induced production of IL-8 and TNFα in a time- and dose-dependent manner, but had little effect on lipopolysaccharide-induced cytokine release. SCFAs mediated their effects on pro-inflammatory cytokine production at least in part by inducing the generation of ROS. CONCLUSION: Our data suggest that SCFAs, especially when combined with specific TLR ligands, contribute to a pro-inflammatory milieu in the lower genital tract and help further our understanding of how BV affects susceptibility to microbial infections.

The effects of commensal bacteria on innate immune responses in the female genital tract.

The innate and adaptive immune systems are important mechanisms for resistance to pathogens in the female lower genital tract. Lactobacilli at this site help maintain a healthy vagina by producing several factors including lactic acid. Indeed, bacterial vaginosis, a condition in which the genital microbiota is altered, is strongly associated with increased rates of a number of infections including HIV. However, the precise factors that contribute to increased rates of microbial and viral infections in bacterial vaginosis remain to be elucidated. We have studied the effects of bacterial microbiota in the lower genital tract on innate immunity and have found that Toll-like receptor ligands and short chain fatty acids, produced by bacterial microbiota, have dramatic effects on immune function. In this review, we will discuss these results, in addition to some recent articles that we believe will enhance our understanding of how microbes might interact with the immune system.

Pyrosequencing of the genital microbiotas of HIV-seropositive and -seronegative women reveals Lactobacillus iners as the predominant Lactobacillus Species.

The species of vaginal lactobacilli in HIV-seropositive and -seronegative women were determined by 16S gene pyrosequencing. Lactobacillus iners sequences were the predominant lactobacillus sequences in 66% of HIV(+) women and 90% of HIV(-) women. This has implications for resistance of HIV(+) and HIV(-) women to genital colonization by pathogenic organisms.

Identification of rhesus macaque genital microbiota by 16S pyrosequencing shows similarities to human bacterial vaginosis: implications for use as an animal model for HIV vaginal infection.

The composition of the lower genital tract microbiota in women is believed to affect the risk of sexually acquiring HIV. Since macaque genital microbiota could similarly impact vaginal infection with SIV we identified microbiota in 11 rhesus macaques using multitag pyrosequencing of the 16S rRNA gene. The microbiota was polymicrobial with a median of nine distinct bacterial taxa per macaque (range 3-16 taxa, each constituting 1% or more of the sequences). Taxa frequently found included Peptoniphilus, Sneathia, Porphyromonas, Mobiluncus, Atopobacter, Dialister, Thioreductor, Prevotella, and Streptococcus, many of which are also frequently found in women with bacterial vaginosis. Lactobacillus sequences (mostly L. johnsonii) were found in only four macaques but were not predominant in any (median of 0% of sequences, range 0-39%). All macaques were resampled 6 months after the first time point to determine the stability of the microbiota. The microbiota remained polymicrobial with a median of 10 taxa (range 6-18). Microbial patterns remained similar for six of the macaques, changed substantially in two, and had a mixed pattern in three. Significant sialidase enzyme activity, a marker of bacteria vaginosis in women, was detected in genital fluid from 9/11 and 8/11 macaques from the first and second time points, respectively. These results show that the macaque lower genital microbiota resembled a bacteria vaginosis-type microbiota in women and suggest that the microbiota of macaques in captivity promote rather than protect against vaginal infection with SIV. These results also suggest macaques could be used as an animal model to study some aspects of bacterial vaginosis.