Adherent Population Research Articles

Critical areas for <i>Brettanomyces bruxellensis</i> contamination and biofilm formation in the cellar: on the origin of wine spoilage

The cellar environment harbours a consortium of microorganisms on the material surfaces and in the air. Among these microorganisms, the spoilage yeast Brettanomyces bruxellensis can colonise surfaces due to its specific bioadhesive properties. In this study, air and surface samples were collected in several wineries during the winter period. B. bruxellensis was detected in the cellar environment either in the air or on the surfaces of various materials, including in tartaric acid precipitates. Difficult-to-clean tank equipment (taps, wall angles, valves) were identified as critical areas where B. bruxellensis was frequently detected. To confirm that surfaces contaminated by B. bruxellensis could be involved in wine contamination, yeast growth and volatile phenol production were monitored in wine in contact with stainless steel harbouring biofilms. The presence of bioadherent cells and biofilms in contact with the wine resulted in significant cell release into the wine, leading to population growth and the production of volatile phenols at concentrations above the olfactory detection threshold. This study demonstrates the possibility of wine spoilage by resident and adherent populations of B. bruxellensis and confirms the need to pay special attention to the hygiene of hard-to-reach areas such as valves.

Daily Use Water Bottles as a Hub for Microbial Population: A Comparative Study of PET vs. Stainless Steel Water Bottles and Outcome of Washing Strategy Intervention

ABSTRACT Water bottles for everyday usage are a typical addition to people’s life and offer a practical way to stay hydrated. Even though safe and clean water is preferred for consumption, the water bottle or container used to drink water was never considered to be cleaned. Here, we examined the adhering microbial populations in water bottles composed of stainless steel (SS) and polyethylene terephthalate (PET). A total of 30 water bottles—15 PET and 15 SS—were gathered from different users. To identify and quantify the adhering microbial populations, microbial swabs from the inside surface of the bottles were collected and later cultivated on certain growth media. Overall the microbial load of PET is significantly higher than the SS water bottles of 68.8 + 19.1 cfu/ml and 35.4 + 8 cfu/ml respectively at initial sampling (P = 0.0027). We also evaluated the efficiency of various cleaning procedures in eliminating adherent bacteria populations. The cleaning strategy significantly reduced the microbial load (P<0.0001). The mean load observed was 11.2 + 2.3 cfu/ml post-wash. This comparison study offers important new information about the adherent microbial populations found in SS and PET water bottles used every day, in the end. This finding emphasizes the necessity of routine cleaning and upkeep of these bottles to reduce the possibility of microbial contamination and the accompanying health risks.

Abstract 3200: Assessing the utility of cell membrane lipid-extracted nanoliposomes (CLENs) as a targeted drug delivery system for pancreatic cancer

Abstract Background: Pancreatic cancer is a challenging type of cancer to treat as evidenced by a 9% 5-year survival and a dismal rate of mortality. Nanoparticles represent a revolutionary treatment method, primarily due to characteristics including cellular absorption, boosting therapeutic effectiveness, and mitigating side effects through localized actions. For nanomedicine, optimizing liposome formulations is crucial for achieving the highest level of specificity in drug targeting. This study aims to evaluate cellular interaction of pancreatic cancer lipid-extract modified nanoliposomes when compared to unmodified varieties in vitro. Method: Cell membrane lipid-extracted nanoliposomes (CLENs) were prepared via the thin film evaporation technique. The composition included various compositions and ratios of DOPC, DOTAP, Cholesterol, DPPE-PEG-5000, and lipid extracts derived from MS1-VEGF target cells. MS1-VEGF was the cellular model of pancreatic cancer used for this study. Cellular binding studies were performed using cellular suspension and adherent cell populations. Studies were performed in 48-well plates under different experimental conditions including alterations in time of incubation, and periods of cellular exposure to dual humidified and oscillation environments in the presence of lipid-extracted (LE) nanoliposomal preparations and conventional controls. Results: The particle size for nanoliposomes employed for this study was approximately 115 ± 0.7 nm for control and 128 ± 2.7 nm for experimental (lipid extract-modified) nanoliposomes. Following cellular exposure to different lipid extract concentrations (typically 0 to 100 nmoles per ml of growth medium), the fluorescence values demonstrated increased cellular uptake with greater LE content for floating and adhered MS1-VEGF cell populations. With the floating cells, LE-modified nanoliposomes demonstrated significantly higher fluorescence intensity values compared to conventional nanoliposomes. Similarly, with MS1-VEGF adherent cells, LE-modified nanoliposomes targeted cells to a greater extent compared to unmodified controls. Conclusions: The increased cellular uptake of CLENs designed for targeting pancreatic tumor cells (both floating and adherent cells) demonstrated an improvement over the use of conventional nanoliposomes under similar experimental conditions. Further research is needed to fully explore the potential use of CLENs for targeting and treatment of pancreatic cancer. Citation Format: Khaznah Alshammari, Sergio Verdery, Sujin George, Robert B Campbell. Assessing the utility of cell membrane lipid-extracted nanoliposomes (CLENs) as a targeted drug delivery system for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3200.

Maternal worry of children contracting COVID-19 predicts vaccine uptake in young children in Vermont

BackgroundYoung children, 6 months to 4 years of age, were the last population for whom the COVID-19 vaccines became available and remain the group with the lowest uptake. Scarce information exists on drivers of vaccine uptake in young children. The relationship between parental perceived risk of infection and child COVID-19 vaccination is unknown. ObjectiveExamine the association between perceived risk of infection and early childhood vaccination. MethodsIn June 2020, perinatal women in Vermont were enrolled in a longitudinal survey study assessing life changes during the pandemic. Surveys included worry of infection, Parents Attitude about Childhood Vaccines (PACV), intention to vaccinate children with the COVID-19 vaccine, and masking behaviors. Once vaccines became available, surveys also included child vaccination status. A demographic-adjusted stepwise logistic regression was conducted to assess the relationship of worry of infection and vaccine hesitancy on child vaccination status. Results85 women completed baseline and follow up surveys. Participants were highly adherent to adult COVID vaccines with 93 % at least partially vaccinated and only 3 % demonstrated general childhood vaccine hesitancy on the PACV. However, in this highly adherent population, only 61 % of young children are vaccinated. In univariate [OR = 7.35 95 % CI (2.70 to 20.02)] and demographic adjusted [OR = 6.03 95 % CI (1.30 to 27.90)] models, current worry about child infection was associated with higher odds of child vaccination. Previously established predictors, vaccine hesitancy [OR = 0.91 95 % CI (0.84 to 0.99)] and younger maternal age [OR = 1.33 95 % CI (1.07 to 1.65)] were again associated lower odds of child vaccination. ConclusionAmong vaccine adherent parents, worry of infection was a strong predictor of vaccine uptake. Continued efforts to articulate the potential morbidity of COVID in young children, along with established methods to address general vaccine hesitancy may aid in COVID vaccine uptake in this population.

Impact of Medication Adherence on Bone Mineral Density and Fracture Risk in Patients With Osteoporosis: A Systematic Review.

Osteoporosis is a chronic, prevalent disease marked by decreased bone mass and changes in bone anatomy associated with significant morbidity. The management of osteoporosis necessitates long-term therapy for which patient adherence is of vital importance. In the present review, we aim to collect all potential evidence from relevant studies that reported the impact of medication adherence on bone mineral density and fracture risk in patients with osteoporosis. We have conducted both electronic and manual search strategies within the potential databases and included articles and reviews to find relevant studies. We have assessed the effects of osteoporotic medication adherence on fracture rates and bone mineral density. The study participants were divided into two groups, adherent and non-adherent. Studies from the year 2010-2023 were included. Final inclusion consisted of 14 studies that showed variation in adherence rates with only three studies reporting optimal adherence followed by two studies with nearly half adherent population while the rest of the studies reported low medication adherence. The highest adherence rate reported was 82% while the lowest was 8%. Among the included studies the fracture rates varied significantly. Decreased rates of fracture were observedin the adherent population however two of the included studies were contrary to these findings. Additionally, only three studies discussed the effect of adherence on bone mineral density. Lack of medication adherence is linked to an increased risk of fracture, and low bone mineral density, further associated with more severe complications as per the evidence from the literature. However, variation in the fracture rates as observed in our findings advocates the need for further research for the generalizability of results.

Photo-Inactivation of Staphylococcus aureus by Diaryl-Porphyrins.

Photodynamic Antimicrobial Chemotherapy (PACT) has received great attention in recent years since it is an effective and promising modality for the treatment of human oral and skin infections with the advantage of bypassing pathogens' resistance to antimicrobials. Moreover, PACT applications demonstrated a certain activity in the inhibition and eradication of biofilms, overcoming the well-known tolerance of sessile communities to antimicrobial agents. In this study, 13 diaryl-porphyrins (mono-, di-cationic, and non-ionic) P1-P13 were investigated for their potential as photosensitizer anti-Staphylococcus aureus. The efficacy of the diaryl-porphyrins was evaluated through photo-inactivation tests. Crystal-violet staining combined with viable count techniques were aimed at assaying their anti-biofilm activity. Among the tested compounds, the neutral photosensitizer P4 was better than the cationic ones, irrespective of their corresponding binding rates. In particular, P4 was active in inhibiting the biofilm formation and in impairing the viability of the adherent and planktonic populations of a 24 h old biofilm. The inhibitory activity was also efficient against a methicillin resistant S. aureus strain. In conclusion, the diaryl-porphyrin family represents a reservoir of promising compounds for photodynamic applications against the pathogen S. aureus and in preventing the formation of biofilms that cause many infections to become chronic.

Ten Seconds to Implosion: The Magna Carta Lawful Rebellion

The Magna Carta Lawful Rebellion [MCLR] is an example of a “legal cult,” a social group organized around key guru leaders who obtain their elevated status and hold followers via alleged special knowledge of law. The MCLR’s two gurus, David Robinson and Jacquie Phoenix (legal name Jacqueline Robinson), claimed the true binding law for residents of the Commonwealth is a hidden secret law, called “pseudolaw.” Pseudolaw is a highly conserved set of false legal concepts nested in a conspiratorial narrative. Pseudolaw originated in the US, but post-2000 has spread world-wide. The MCLR pseudolaw variant is that the 1215 Magna Carta remains in effect and operates as a supraconstitutional authority. Robinson and Phoenix claim that individuals may defeat conventional legal and state authority by swearing an oath of allegiance to Lord Craigmyle of Invernesshire, a “New Rebel Baron.” Special paperwork then permits extraordinary authority, including the right to execute government authorities and other enemies. However, MCLR adherents instead employed these concepts in petty domestic circumstances, and without any success. The MCLR’s leadership and adherent population appear similar: low-income, uneducated, and marginal persons with conspiratorial anti-authority beliefs. This “cult of peers” largely operates online. The MCLR was a minor and comparatively unsuccessful branch of UK pseudolaw culture until 2019, when Phoenix attracted numerous followers, and led a “Redress” movement that promised defeat of New World Order oppressors via execution of “traitors and quislings.” The 2021 failure of the “Redress” effort now threatens Phoenix’s status, and the long-term viability of the MCLR and its concepts.

Impact of Medication Nonadherence in a Clinical Trial of Dual Antiplatelet Therapy

BackgroundNonadherence to antiplatelet therapy after percutaneous coronary intervention (PCI) is common, even in clinical trials. ObjectivesThe purpose of this study was to investigate the impact of nonadherence to study protocol regimens in the MASTER DAPT (Management of High Bleeding Risk Patients Post Bioresorbable Polymer Coated Stent Implantation With an Abbreviated Versus Prolonged DAPT Regimen) trial. MethodsAt 1-month after PCI, 4,579 high bleeding risk patients were randomized to single antiplatelet therapy (SAPT) for 11 months (or 5 months in patients on oral anticoagulation [OAC]) or dual antiplatelet therapy (DAPT) for ≥2 months followed by SAPT. Coprimary outcomes included net adverse clinical events (NACE), major adverse cardiac and cerebral events (MACE), and major or clinically relevant nonmajor bleeding (MCB) at 335 days. Inverse probability-of-censoring weights were used to correct for nonadherence Academic Research Consortium type 2 or 3. ResultsIn total, 464 (20.2%) patients in the abbreviated-treatment and 214 (9.4%) in the standard-treatment groups incurred nonadherence Academic Research Consortium type 2 or 3. At inverse probability-of-censoring weights analyses, NACE (HR: 1.01; 95% CI: 0.88-1.27) or MACE (HR: 1.07; 95% CI: 0.83-1.40) did not differ, and MCB was lower with abbreviated compared with standard treatment (HR: 0.51; 95% CI: 0.60-0.73) consistently across OAC subgroups; among OAC patients, SAPT discontinuation 6 months after PCI was associated with similar MACE and lower MCB (HR: 0.47; 95% CI: 0.22-0.99) compared with SAPT continuation. ConclusionsIn the MASTER DAPT adherent population, 1-month compared with ≥3-month DAPT was associated with similar NACE or MACE and lower MCB. Among OAC patients, SAPT discontinuation after 6 months was associated with similar MACE and lower MCB than SAPT continuation (Management of High Bleeding Risk Patients Post Bioresorbable Polymer Coated Stent Implantation With an Abbreviated Versus Prolonged DAPT Regimen [MASTER DAPT]; NCT03023020)

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

Facile, fluorine-free fabrication of bacterial antifouling titanium alloy Ti6Al4V surfaces for surgically implanted devices

The demand for hip arthroplasties and other joint replacement orthopedic surgeries is on the rise. Combined with the emergence of antibiotic drug resistant strains of pathogenic bacteria, there is an increasing need for the development of bacterial antifouling surface treatment technologies which can be applied to titanium alloy medical devices. Herein is reported the development of a durable, fluorine-free superhydrophobic surface treatment for Ti6Al4V, fabricated through facile means without the use of HF or fluoropolymers, utilizing safe reagents under mild reaction conditions. Nanoscale texturing was created with an alkaline hydrothermal process. An alkyl surface chemistry was imparted via the deposition of an alkyl self-assembled monolayer to achieve superhydrophobic, non-wetting behavior. This superhydrophobic surface is capable of imposing 1.55 ± 0.13 and 1.72 ± 0.23 log10 reductions in the adherent populations of Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa) bacteria, respectively. This represents a safe route to a significant potential mitigation in the costs and dangers arising from medical device associated infections.

Autophagy reprogramming stem cell pluripotency and multiple-lineage differentiation.

The cellular process responsible for the degradation of cytosolic proteins and subcellular organelles in lysosomes was termed "autophagy." This process occurs at a basal level in most tissues as part of tissue homeostasis that redounds to the regular turnover of components inside cytoplasm. The breakthrough in the autophagy field is the identification of key players in the autophagy pathway, compounded under the name "autophagy-related genes" (ATG) encoding for autophagy effector proteins. Generally, the function of autophagy can be classified into two divisions: intracellular clearance of defective macromolecules and organelles and generation of degradation products. Therapeutic strategies using stem cell-based approach come as a promising therapy and develop rapidly recently as stem cells have high self-renewability and differentiation capability as known as mesenchymal stem cells (MSCs). They are defined as adherent fibroblast-like population with the abilities to self-renew and multi-lineage differentiate into osteogenic, adipogenic, and chondrogenic lineage cells. To date, they are the most extensively applied adult stem cells in clinical trials. The properties of MSCs, such as immunomodulation, neuroprotection, and tissue repair pertaining to cell differentiation, processes to replace lost, or damaged cells, for aiding cell repair and revival. Autophagy has been viewed as a remarkable mechanism for maintaining homeostasis, ensuring the adequate function and survival of long-lived stem cells. In addition, authophagy also plays a remarkable role in protecting stem cells against cellular stress when the stem cell regenerative capacity is harmed in aging and cellular degeneration. Understanding the under-explored mechanisms of MSC actions and expanding the spectrum of their clinical applications may improve the utility of the MSC-based therapeutic approach in the future.

Determining the acceptability of point-of-care urine tenofovir testing and its performance in predicting HIV RNA suppression.

The detection of tenofovir (TFV) metabolites by point-of-care (POC) urine lateral flow immunoassays (LFIA) indicates adherence to tenofovir-containing HIV pre-exposure prophylaxis. However, the association between urine TFV metabolites as detected by LFIA and HIV viral load suppression in people receiving TFV-based antiretroviral therapy (ART) is unknown as is patient and clinician acceptability of POC urine LFIA testing in clinical practice in low- and middle-income country settings. We enrolled 409 people living with HIV from two HIV clinics in Lesotho and investigated the performance of POC urine LFIA TFV testing in predicting viral suppression. We interviewed 12 study participants and conducted a focus-group discussion with 5 clinicians to gather opinions on POC urine TFV testing. Using a viral load threshold of 1000copies/mL, 398 (98%) participants were virologically suppressed, and 8 were viremic. Tenofovir was detected in the urine of 405 (99%) participants. The sensitivity of the POC urine LFIA test in detecting TFV in participants with viral suppression was 99.3% (95% CI: 97.8-99.8); the specificity was 12.5% (95% CI: 0.3-52.6). The positive and negative predictive values were 98.3% and 25%, respectively. Point-of-care urine TFV testing was viewed favorably by both participants and clinicians. However, clinicians stated that the 2-3-days detection window of the assay studied limits adherence categorization. In our study cohort, urine POC TFV testing demonstrated high sensitivity in predicting viral suppression, but low specificity and negative predictive value. Urine POC TFV testing was highly acceptable to participants and clinicians; however, clinicians expressed concern about its clinical utility because of its limitations. While further research is needed to assess performance in less adherent populations, this test may support adherence counseling in some clinical settings.

Prevalence And Impact of Medical Comorbidities in A Real‐World Lung Cancer Screening Population

BackgroundLung cancer screening trials generally enroll motivated, relatively healthy, and adherent populations. We therefore evaluated the prevalence and effects of comorbidities in a real-world population undergoing low-dose computed tomography (LDCT) scans. Patients and MethodsWe calculated the Charlson Comorbidity Index (CCI) of patients for whom an initial low-dose computed tomography (LDCT) for lung cancer screening was ordered between February 2017 and February 2019 in an integrated safety-net healthcare system. We examined the association between CCI and initial LDCT completion using multivariable logistic regression, assessed the association between specific medical comorbidity and LDCT completion using Chi-square test or Fisher's exact test as appropriate, and examined the association between CCI and LDCT Lung-RADS results using Fisher's exact test. ResultsA total of 1358 patients were included in the analysis. Mean age was 63 years, 57% were women, and 50% were Black. Patients had moderate comorbidity burden (median CCI 3) with chronic pulmonary disease the most common comorbidity. Overall, 943 LDCT (70%) were completed. There was no difference in 30-day, 90-day, or 1-year completion rates of initial LDCT according to CCI. However, 30-day LDCT completion rates did increase over time (P < .001). Lung-RADS scores were not associated with CCI. ConclusionIn a real-world setting, patients undergoing lung cancer screening have moderate comorbidity burden. The degree and type of medical comorbidity are not associated with initial screening completion or results. Timeliness of LDCT completion may improve as program experience increases.

Adherence to the healthy lifestyle guideline in relation to the metabolic syndrome: Analyses from the 2013 and 2018 Indonesian national health surveys

In this study, we aimed to investigate differences in lifestyle factors and prevalence of metabolic syndrome (MetS) in the Indonesian population between 2013 and 2018. In addition, we investigated whether adherence to the 2015-released national healthy lifestyle guideline (‘GERMAS’) is associated with MetS in different sex, age, urban/rural, and BMI categories. We performed cross-sectional analyses in individuals aged >15 of the 2013 (n = 34,274) and 2018 (n = 33,786) Indonesian National Health Surveys. A stratified, multi-stage, systematic random sampling design and the probability proportional to size method were used to select households in the 34 provinces across the country. MetS was defined according to the Joint Interim Statement Criteria, and adherence to ‘GERMAS’ guideline was defined as fulfilling the national healthy lifestyle recommendations of ≥150 min/week physical activity (PA), ≥5 portions/day fruit and vegetable (FV), no smoking (NS), and no alcohol consumption (NA). We examined the associations of each lifestyle factor with MetS using logistic regression categorised by sex, age groups, urban/rural, and BMI, and adjusted for sociodemographic factors. We observed that men who adhered to the guideline had lower odds ratio of MetS [OR(95%CI) associated with PA: 0.85(0.75–0.97); NA: 0.75(0.56–1.00)] than non-adherent men. Middle-aged adults who adhered to the guideline had lower OR of MetS [PA: 0.85(0.72–1.01); FV: 0.78(0.62–0.99); NA: 0.66(0.46–0.93)] than non-adherent adults <45 years. The adherent urban population had lower OR of MetS [FV: 0.85(0.67–1.07); NA: 0.74(0.52–1.07)] than the non-adherent urban population. Those with overweight or obesity who adhered to the guideline had relatively lower odds of MetS than those who did not. In conclusion, in this nationally representative study, adherence to the ‘GERMAS’ guideline may confer cardiometabolic health benefits to several groups of the Indonesian population, particularly men, middle-aged, those with overweight and obesity, and potentially urban population.

Assessing the Impact of Diet on the Mucosa-Adhered Microbiome in Piglets Using Comparative Analysis of Rectal Swabs and Colon Content.

Previously, we demonstrated that rectal swabs provide a legitimate alternative to faecal sampling for the assessment of the intestinal microbiota in young piglets. However, we also reported that mucosa-adhered microbial populations were more represented in rectal swabs compared to faecal samples, albeit to a degree that varied per swab-sample. Here, we explored the possibility to exploit this variable enrichment of adhered populations in the rectal swabs to assess the impact of diet on mucosa-adhered microbiota in pre-weaning piglets. Paired samples of rectal swabs and colon luminal contents were collected from piglets just before weaning during two independent but similarly designed animal experiments [n = 28 piglets (experiment 1); n = 16 piglets (experiment 2)], with an early feeding treatment (EF) group that had access to customised fibrous feed in addition to sow’s milk and a control (CON) group exclusively reared on sow’s milk. The intestinal microbiome composition in rectal swabs and colon samples collected at 29 days of age were subjected to metataxonomic analysis. The results identified the genera Escherichia-Shigella, Anaerococcus, Peptostreptococcus, Enterococcus, Trueperella, Actinomyces, and Peptoniphilus as discriminative taxa enriched in rectal swabs compared to colon. Apart from Escherichia-Shigella (10–11% average relative abundance), most of these mucosa-adhered microbial genera display relatively low abundance. Rectal swab microbiota was found to be more variable, which is likely due to variable enrichment of mucosa-adhered microbes. Although almost exclusively driven by one of the experiments, the post-weaning diarrhoea-associated taxa Escherichia-Shigella, was enriched in CON compared to the EF group, suggesting that early life feeding may suppress post-weaning-diarrhoea-related problems in piglets. Our findings demonstrate that rectal swabs allow the investigation of the mucosa-adhered microbial populations as a function of dietary treatment in piglets. This offers opportunities to further study dietary approaches that suppress the abundance of the post-weaning diarrhoea associated adherent microbes like Escherichia-Shigella. Furthermore, we demonstrate that the paired swab-colon microbiota information (obtained from a subset of animals) can predict the mucosa-adhered populations or “mucosity factor” in rectal swab samples, facilitating the analysis of the adhered microbiota in large animal cohort studies using readily obtainable rectal swabs.

Estimating the treatment effect for adherers using multiple imputation

Randomized controlled trials are considered the gold standard to evaluate the treatment effect (estimand) for efficacy and safety. According to the recent International Council on Harmonization (ICH)-E9 addendum (R1), intercurrent events (ICEs) need to be considered when defining an estimand, and principal stratum is one of the five strategies to handle ICEs. Qu et al. (2020, Statistics in Biopharmaceutical Research 12:1-18) proposed estimators for the adherer average causal effect (AdACE) for estimating the treatment difference for those who adhere to one or both treatments based on the causal-inference framework, and demonstrated the consistency of those estimators; however, this method requires complex custom programming related to high-dimensional numeric integrations. In this article, we implemented the AdACE estimators using multiple imputation (MI) and constructed confidence intervals (CIs) through bootstrapping. A simulation study showed that the MI-based estimators provided consistent estimators with the nominal coverage probabilities of CIs for the treatment difference for the adherent populations of interest. As an illustrative example, the new method was applied to data from a real clinical trial comparing two types of basal insulin for patients with type 1 diabetes.

1393. Loss to Follow-up Rate in the Treatment of Latent Tuberculosis by Region of Origin

BackgroundAdherence in the treatment of latent tuberculosis infection (LTBI) is closely related to reactivation and infection control in the population. However, there has been little research on which populations are at higher risk of loss to follow-up. The aim of this study is to investigate how the adherence of LTBI patients in the United States (US) differs by region of origin.MethodsA retrospective, observational study was conducted from 2001 to 2020. LTBI patients were identified from the Cuyahoga County Tuberculosis Clinic in Cleveland, Ohio. Only patients who were informed of the diagnosis of LTBI were included. Patients were discharged from the Tuberculosis outpatient clinic upon completion of treatment or when the physician decided to discontinue treatment. We defined loss to follow-up as a case where LTBI was diagnosed but the patient was not formally discharged. Patients whose treatment was interrupted due to side effects were not considered loss to follow-up. Odds ratios were calculated using a multivariable regression model with patients from North America as the reference group.ResultsOf 4018 LTBI patients, 1171 (28.7%) were lost to follow-up, of which 950/2314 (41.0%) were from North America. Compared with LTBI patients from North America, significantly lower loss to follow-up rates were observed for those from Middle East and North Africa 30/170 (17.7% OR 0.52, 95% Confidence Interval (CI) 0.31-0.89), South Asia 60/692 (8.7% OR 0.41, 95% CI 0.21-0.78), and Sub-Saharan Africa 69/526 (13.1% OR 0.22, 95% CI 0.14-0.36). ConclusionThe analysis showed that a high loss to follow-up rate was observed in the patient groups from North America, Europe and Central Asia, and Latin America & the Caribbean. LTBI patients from North America had a significantly higher loss to follow-up rate than those from Middle East and North Africa, South Asia, and Sub-Saharan Africa, respectively. Further research is needed to determine how to intervene in the poorly adherent patient population, such as LTBI patients from North America, Europe and Central Asia, and Latin America & the Caribbean.Disclosures All Authors: No reported disclosures

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 °C. Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

CD14, A toll-like receptor co-factor, influences osteoclast differentiation and activity, but does not alter osteoblastic potential of bone-marrow preursors in mice

Purpose: Changes in subchondral bone structure occur in osteoarthritis (OA), and are visible by imaging as subchondral sclerosis on radiographs, and bone marrow lesions (BMLs) by MRI. These changes are the result of active bone remodeling that occurs in OA. BMLs in particular are correlated with pain in OA patients. We previously demonstrated that bone remodeling was significantly reduced after joint injury in mice deficient in CD14. CD14 enhances Toll-like receptor (TLR) mediated inflammation by acting as a co-receptor for multiple TLRs, increasing TLR sensitivity to damage associated molecular patterns (DAMPS) to activate proinflammatory signaling pathways. Soluble CD14, TLRs, and DAMPs (TLR ligands) are all elevated in OA patients, and sCD14 has been implicated as a biomarker of symptom and structural severity in OA. Increasing evidence suggestions CD14/TLR signaling influences bone remodeling as well, through activation of osteoclast precursor cells. Here, we investigate the potential mechanism by which CD14 influences bone remodeling through in vitro analysis of osteoclastogenic and osteoblastic potential and function. Methods: Bone marrow (BM) hematopoietic and mesenchymal precursors were isolated from long bones of 12-week-old male wild-type C57BL/6 (WT) and CD14-deficient (CD14-/-) mice. Briefly, BM was flushed from the femurs and tibia, and cells were cultured for 24 hours to separate non-adherent from adherent population. For osteoclastic differentiation, the non-adherent cells were seeded at a density of 26,000 cells per cm2 and were cultured in presence of monocyte/macrophage colony-stimulating factor (M-CSF; 35 ng/ml) and receptor activator of nuclear factor kβ ligand (RANKL; 100 ng/ml) for 9 days. Osteoblastic differentiation was accomplished by seeding the adherent BM cells at 30,000 cells per cm2 and culturing in the presence of 10 mM β-glycerophosphate ,50 μM ascorbic acid, and 100 nM Dexamethasone for up to 19 days. Osteoclasts were identified as multinucleated cells with positive TRAP staining. The resorptive ability of osteoclasts was assayed by quantifying resorption pits formed on bone slices. For osteoblast differentiation, Alizarin red was used to detect mineralized bone matrix. At the completion of each assay RNA was isolated from cultures and Real-time quantitative PCR analysis was performed to detect changes in mRNA levels of osteoclast (i.e. calcitonin receptor/CTR and cathepsin K/Ctsk) and osteoblast (i.e. osteopontin/OPN and osteocalcin/BLAP) differentiation markers. Results: Cells from CD14-/- mice demonstrated decreased percentages of TRAP positive cells and multi-nucleated cells (p value = 0.01; Fig. 1 A-D) and reduced Ctsk mRNA (p value = 0.03; Fig. 1 E&F), compared to WT controls. CD14-/- cells showed a 50% reduction in resorption pits and area compared to WT (Fig. 1 G&I). No significant differences were observed between strains in osteoblastic potential measured by mineralization and gene expression of osteoblastic markers (Fig. 2). Conclusions: CD14-/- BM precursor cells have reduced capacity to differentiate into functional osteoclasts, while osteoblastic potential is retained. This suggests that the reduced capacity of CD14-deficient osteoclastic cells may lead to the reduced bone remodeling in response to joint injury seen in the DMM model, by disrupting the balance between resorption and formation. Future experiments will determine the influence of DAMPs on osteoclast differentiation and activity, and the density of osteoclasts in SCB after DMM injury. These experiments will lead to a better understanding of the role of CD14 and TLR pathways on bone remodeling in response to joint injury and OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

PH and light-responsive polycaprolactone/curcumin@zif-8 composite films with enhanced antibacterial activity.

Food packaging materials, especially biodegradable polymer composites incorporated with natural antimicrobial agents with excellent antibacterial activities, are in high demand and attracted immense attention. Herein, a polycaprolactone/curcumin@zeolitic imidazolate framework-8 (PCL/Cur@ZIF-8) composite film with enhanced antibacterial activity was developed. Curcumin, a natural photosensitizer, was loaded in the highly porous nanocrystals ZIF-8 to improve its poor water solubility and stability. The integral structure of Cur@ZIF-8 was maintained well in the PCL matrix even at the highest loading of 35% (w/w), and all composite films had good light transmittance at 420-430nm. The PCL/Cur@ZIF-8 composite films responded to the acidic growth environment of bacteria by releasing zinc ions and curcumin molecules. Furthermore, upon blue light irradiation (420-430nm, 2.2mW/cm2 ), curcumin molecules generated singlet oxygen. With the synergistic effects of zinc ions and singlet oxygen, the composite films exhibited a 99.9% reduction of Escherichia coli and Staphylococcus aureus strains when the amount of Cur@ZIF-8 loading was more than 15% (w/w), as well as a strong anti-adhesion effect on bacteria. Moreover, bacterial resuscitation tests indicated that the composite films exhibited 99.9% reduction in the adhered bacteria population through treatment with photodynamic sterilization. This is the first study presenting that the incorporated curcumin ZIF-8 nanoparticles in the matrix of polymer are pH and light responsive for anti-adhesion of bacteria, which is of great potential application as antibacterial packaging material for the food industry. PRACTICAL APPLICATION: A novel, biodegradable, pH, and light-responsive composite film was developed for antibacterial activity. Natural photosensitizer curcumin was encapsulated in ZIF-8 nanocrystals (Cur@ZIF-8) as the antimicrobial agent. With the synergistic effects of Zn2+ and singlet oxygen, the composite film exhibited a 99.9% reduction of Escherichia coli and Staphylococcus aureus strains, and a strong anti-adhesion property toward bacteria. This composite film is of great potential application as an antibacterial packaging material that enhances the shelf life of fruits, meat, and so on.