An immunoresponsive three-dimensional urine-tolerant human urothelial model to study urinary tract infection.

Introduction: Murine models of urinary tract infection (UTI) have improved our understanding of host-pathogen interactions. However, given differences between rodent and human bladders which may modulate host and bacterial response, including certain biomarkers, urothelial thickness and the concentration of urine, the development of new human-based models is important to complement mouse studies and to provide a more complete picture of UTI in patients. Methods: We originally developed a human urothelial three-dimensional (3D) model which was urine tolerant and demonstrated several urothelial biomarkers, but it only achieved human thickness in heterogenous, multi-layered zones and did not demonstrate the comprehensive differentiation status needed to achieve barrier function. We optimised this model by altering a variety of conditions and validated it with microscopy, flow cytometry, transepithelial electrical resistance and FITC-dextran permeability assays to confirm tissue architecture, barrier integrity and response to bacterial infection. Results: We achieved an improved 3D urine-tolerant human urothelial model (3D-UHU), which after 18-20 days of growth, stratified uniformly to 7-8 layers comprised of the three expected, distinct human cell types. The apical surface differentiated into large, CD227+ umbrella-like cells expressing uroplakin-1A, II, III, and cytokeratin 20, all of which are important terminal differentiation markers, and a glycosaminoglycan layer. Below this layer, several layers of intermediate cells were present, with a single underlying layer of CD271+ basal cells. The apical surface also expressed E-cadherin, ZO-1, claudin-1 and -3, and the model possessed good barrier function. Infection with both Gram-negative and Gram-positive bacterial classes elicited elevated levels of pro-inflammatory cytokines and chemokines characteristic of urinary tract infection in humans and caused a decrease in barrier function. Discussion: Taken together, 3D-UHU holds promise for studying host-pathogen interactions and host urothelial immune response.

The urothelium: a multi-faceted barrier against a harsh environment.

All mucosal surfaces must deal with the challenge of exposure to the outside world. The urothelium is a highly specialized layer of stratified epithelial cells lining the inner surface of the urinary bladder, a gruelling environment involving significant stretch forces, osmotic and hydrostatic pressures, toxic substances, and microbial invasion. The urinary bladder plays an important barrier role and allows the accommodation and expulsion of large volumes of urine without permitting urine components to diffuse across. The urothelium is made up of three cell types, basal, intermediate, and umbrella cells, whose specialized functions aid in the bladder's mission. In this review, we summarize the recent insights into urothelial structure, function, development, regeneration, and in particular the role of umbrella cells in barrier formation and maintenance. We briefly review diseases which involve the bladder and discuss current human urothelial in vitro models as a complement to traditional animal studies.

Severe Acute Respiratory Syndrome Type 2-Causing Coronavirus: Variants and Preventive Strategies.

COVID-19 vaccines have constituted a substantial scientific leap in countering severe acute respiratory syndrome type 2-causing coronavirus (SARS-CoV-2), and worldwide implementation of vaccination programs has significantly contributed to the global pandemic effort by saving many lives. However, the continuous evolution of the SARS-CoV-2 viral genome has resulted in different variants with a diverse range of mutations, some with enhanced virulence compared with previous lineages. Such variants are still a great concern as they have the potential to reduce vaccine efficacy and increase the viral transmission rate. This review summarizes the significant variants of SARS-CoV-2 encountered to date (December 2021) and discusses a spectrum of possible preventive strategies, with an emphasis on physical and materials science.

Novel antibiotic-loaded particles conferring eradication of deep tissue bacterial reservoirs for the treatment of chronic urinary tract infection.

A significant proportion of urinary tract infection (UTI) patients experience recurrent episodes, due to deep tissue infection and treatment-resistant bacterial reservoirs. Direct bladder instillation of antibiotics has proved disappointing in treating UTI, likely due to the failure of infused antibiotics to penetrate the bladder epithelium and accumulate to high enough levels to kill intracellular bacteria. This work investigates the use of nitrofurantoin loaded poly(lactic-co-glycolic acid) (PLGA) particles to improve delivery to intracellular targets for the treatment of chronic UTI. Using electrohydrodynamic atomisation, we produced particles with an average diameter of 2.8 µm. In broth culture experiments, the biodegradable particles were effective against a number of UTI-relevant bacterial strains. Dye-loaded particles demonstrated that intracellular delivery was achieved in all cells in 2D cultures of a human bladder epithelial progenitor cell line in a dose-dependent manner, achieving far higher efficiency and concentration than equivalent quantities of free drug. Time-lapse video microscopy confirmed that delivery occurred within 30 min of administration, to 100% of cells. Moreover, the particles were able to deliver the drug to cells through multiple layers of a 3D human bladder organoid model causing minimal cell toxicity, displaying superior killing of bacterial reservoirs harboured within bladder cells compared with unencapsulated drug. The particles were also able to kill bacterial biofilms more effectively than the free drug. These results illustrate the potential for using antibiotic-loaded microparticles to effectively treat chronic UTIs. Such a delivery method could be extrapolated to other clinical indications where robust intracellular delivery is required, such as oncology and gene therapy.

Effect of Environment on the Evolutionary Trajectories and Growth Characteristics of Antibiotic-Resistant Escherichia coli Mutants.

The fitness cost to bacteria of acquisition of resistance determinants is critically under-investigated, and the identification and exploitation of these fitness costs may lead to novel therapeutic strategies that prevent the emergence of antimicrobial resistance. Here we used Escherichia coli and amoxicillin-clavulanic acid (AMC) resistance as a model to understand how the artificial environments utilized in studies of bacterial fitness could affect the emergence of resistance and associated fitness costs. Further, we explored the predictive value of this data when strains were grown in the more physiologically relevant environments of urine and urothelial organoids. Resistant E. coli isolates were selected for following 24-h exposure to sub-inhibitory concentrations of AMC in either M9, ISO, or LB, followed by growth on LB agar containing AMC. No resistant colonies emerged following growth in M9, whereas resistant isolates were detected from cultures grown in ISO and LB. We observed both within and between media-type variability in the levels of resistance and fitness of the resistant mutants grown in LB. MICs and fitness of these resistant strains in different media (M9, ISO, LB, human urine, and urothelial organoids) showed considerable variation. Media can therefore have a direct effect on the isolation of mutants that confer resistance to AMC and these mutants can exhibit unpredictable MIC and fitness profiles under different growth conditions. This preliminary study highlights the risks in relying on a single culture protocol as a model system to predict the behavior and treatment response of bacteria in vivo and highlights the importance of developing comprehensive experimental designs to ensure effective translation of diagnostic procedures to successful clinical outcomes.

Chronic Rejection of Cardiac Allografts Is Associated With Increased Lymphatic Flow and Cellular Trafficking.

BACKGROUND: Cardiac transplantation is an excellent treatment for end-stage heart disease. However, rejection of the donor graft, in particular, by chronic rejection leading to cardiac allograft vasculopathy, remains a major cause of graft loss. The lymphatic system plays a crucial role in the alloimmune response, facilitating trafficking of antigen-presenting cells to draining lymph nodes. The encounter of antigen-presenting cells with T lymphocytes in secondary lymphoid organs is essential for the initiation of alloimmunity. Donor lymphatic vessels are not anastomosed to that of the recipient during transplantation. The pathophysiology of lymphatic disruption is unknown, and whether this disruption enhances or hinders the alloimmune responses is unclear. Although histological analysis of lymphatic vessels in donor grafts can yield information on the structure of the lymphatics, the function following cardiac transplantation is poorly understood. METHODS: Using single-photon emission computed tomography/computed tomography lymphoscintigraphy, we quantified the lymphatic flow index following heterotrophic cardiac transplantation in a murine model of chronic rejection. RESULTS: Ten weeks following transplantation of a minor antigen (HY) sex-mismatched heart graft, the lymphatic flow index was significantly increased in comparison with sex-matched controls. Furthermore, the enhanced lymphatic flow index correlated with an increase in donor cells in the mediastinal draining lymph nodes; increased lymphatic vessel area; and graft infiltration of CD4+, CD8+ T cells, and CD68+ macrophages. CONCLUSIONS: Chronic rejection results in increased lymphatic flow from the donor graft to draining lymph nodes, which may be a factor in promoting cellular trafficking, alloimmunity, and cardiac allograft vasculopathy.

Evaluation of Appropriateness Admission and Hospital Stay at Educational Hospital.

BACKGROUND: Appropriate admissions and patients' length of stay are two of the most important indicators of efficient health care delivery in hospitals. Paying due attention to these indicators may lead to optimal use of hospital resources as well as provision of ambulatory services to a larger population of patients. The purpose of the current study is to quantify the rate of inappropriate hospital admissions and days of hospital stay to identify factors affecting them. METHODS: Data were collected regarding admissions and length of stay of 1815 patients admitted to an educational hospital in Tehran, Iran, with a total 12,629 days of hospitalization using the Appropriateness Evaluation Protocol. A qualitative study was conducted using content analysis method by analyzing data from interviews with the hospital personnel about the factors affecting patients' length of stay. RESULTS: The results indicated that the average length of stay in medical and surgical wards was 9.4 to 6.3 days, and 8.5% of admissions and 3.4% of stays were inappropriate. The necessity to receive nursing care and/or to receive medical services and/or the patients' conditions accounted for 57.6% of total hospitalization days, followed by the need to receive nursing care alone (36.6%). Planning/Procedures/ Personnel factors were responsible for 77.3% of inappropriate stays. The qualitative study revealed that in addition to sound in house policy setting, abundance of suitable equipment and facilities at the hospital site had positively affected the appropriateness of hospital stay while incoordination of health care delivery groups, rotation of residents and other wards personnel and lack of a proper complementary patient follow-up system, had a negative impact on the same indicator. CONCLUSION: Inappropriate admissions and inappropriate stays are influenced by numerous factors, both inside and outside of the hospitals; the results of the current study indicate that structural factors such as techniques adopted in the studied hospital, contributed significantly to decreasing inappropriate stays. Improving and upgrading these techniques will make optimal use of hospital beds possible.

Caring for Acutely Ill Patients in General Wards: A Qualitative Study.

INTRODUCTION: The number of acutely ill patients has risen in general wards due to the aging population, more advanced and complicated therapeutic methods, economic changes in the health system, therapeutic choices and shortage of intensive care unit beds. This may lead to adverse events and outcomes with catastrophic results. The purpose of this study was to describe the conditions of acutely ill patients, from the perspective of caregivers. METHODS: The study was conducted in Tehran University of Medical Sciences and its two affiliated general teaching hospitals. Ten nurses and physicians participated in interviews, which were analyzed using qualitative content analysis methods. RESULTS: Four main categories of difficulties in caring for acutely ill patients in general wards were described: problems in identifying acutely ill patients, problems in clinical management of acutely ill patients, inappropriate use of Intensive Care Unit (ICU) beds, and poor structure for mortality control. The staff do not appropriately diagnose the signs of deterioration. There are problems with the appropriate management of acutely ill patients, even if they are considered to be acutely ill and in need of special attention in general wards. CONCLUSION: Many shortcomings exist caring for acutely ill patients, ranging from identification to clinical management; there are also structural and contextual problems. An immediate plan is necessary to circumvent the challenges and to improve the care for acutely ill patients. These challenges highlight the need for changes in current levels of care for acutely ill patients, as well as the need for appropriate support systems.

Evaluation of a critical care outreach service in a middle-income country: A stepped wedge cluster randomized trial and nested qualitative study.

PURPOSE: This trial evaluates implementation of critical care outreach in a middle-income country. MATERIALS AND METHODS: Critical care outreach delivered by a team of intensive care nurses was implemented across general hospital wards in an Iranian university hospital. The order of implementation was randomized with wards stratified by predicted mortality rates. Effectiveness was evaluated using a stepped wedge cluster randomized controlled trial design, comparing outcomes between patients admitted before and after implementation. The primary outcomes were inhospital mortality and cardiopulmonary resuscitation. A nested qualitative study explored challenges to implementation and contextualized the trial outcomes. RESULTS: Between July 2010 and December 2011, 13 wards were sequentially randomized to implement the critical care outreach: 7802 patients were admitted before implementation and 10 880 after implementation. There were 370 deaths (4.74%) among patients admitted before implementation and 384 deaths (3.53%) after implementation. Adjusting for clustering and temporal trends, the odds ratio for mortality was 1.03 (95% confidence interval, 0.68-1.53). Results for other outcomes were broadly similar. Focus groups revealed a lack of endorsement of the intervention by management and ward nurses. CONCLUSIONS: This pragmatic evaluation of critical care outreach in a middle-income country did not show a reduction in mortality or other outcomes.

Clostridium difficile-mediated effects on human intestinal epithelia: Modelling host-pathogen interactions in a vertical diffusion chamber.

Clostridium difficile infection is one of the leading causes of healthcare associated diarrhoea in the developed world. Although the contribution of C. difficile toxins to disease pathogenesis is now well understood, many facets of host-pathogen interactions between the human intestinal epithelia and the C. difficile bacterium that may contribute to asymptomatic carriage and/or clinical disease remain less clear. Herein, we tested the hypothesis that C. difficile strains mediate intestinal epithelial cell (IEC) antimicrobial immunity via toxin dependent and independent means and that the 'anaerobic' environment has a significant impact on bacterial-IEC interactions. Crosstalk between three C. difficile PCR ribotypes (RT) [RT027 (strain R20291), RT012 (strain 630) and RT017 (strains M68 and CF5)] and IEC cell-lines were investigated. All RTs showed significant engagement with human Toll-like receptors (TLR)-5, TLR2-CD14 and TLR2/6 as measured by IL-8 release from TLR-transfected HEK cells. Co-culture studies indicated minimal impact of R20291 and 630 TcdA and TcdB on bacterial adherence to Caco-2 cells. An apical anaerobic environment had a major effect on C. difficile-T84 crosstalk as significantly greater cytokine immunity and trans-epithelial electrical resistance (TEER) dysfunction was recorded when co-cultures were performed in an Ussing chamber system compared to standard 5% CO2 conditions. Overall, this study suggests that anaerobic C. difficile engagement with human IECs is a complex interplay that involves bacterial and toxin-mediated cellular events.

Host immunity to Clostridium difficile PCR ribotype 017 strains.

Clostridium difficile is an important nosocomial pathogen and the leading cause of antibiotic-associated diarrhea. Multilocus sequence typing indicates that C. difficile strains belong to five distinct genetic clades encompassing several PCR ribotypes (RT). Since their emergence in 2003, hypervirulent RT027 strains have been a major focus of research; in contrast, our current understanding of RT017-mediated disease pathogenesis lags far behind. In this study, we aimed to characterize host immunity to CF5 and M68, two genetically well-defined RT017 strains. Both strains engaged with host Toll-like receptor 2/6 (TLR2/6), TLR2-CD14, and TLR5 to similar extents in a model cell line. Despite this, CF5 mediated significantly greater dendritic cell (DC) interleukin-12 (IL-12), IL-27, and IL-10 immunity than M68. Both strains elicited similar IL-1ß mRNA levels, and yet only M68 caused a marked increase in secretory IL-1ß. A CF5 cocultured-DC cytokine milieu drove an equipotent Th1 and Th17 response, while M68 promoted greater Th17 immunity. Human gastrointestinal ex vivo cytokine responses to both strains were characterized. Taken together, our data suggest that C. difficile strains mediate overlapping and yet distinct mucosal and DC/T cell immunity. Finally, toxin-driven IL-1ß release supports the hypothesis that this cytokine axis is a likely target for therapeutic intervention for C. difficile infection.

Clostridium difficile modulates host innate immunity via toxin-independent and dependent mechanism(s).

Clostridium difficile infection (CDI) is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea and currently represents a significant health burden. Although the role and contribution of C. difficile toxins to disease pathogenesis is being increasingly understood, at present other facets of C. difficile-host interactions, in particular, bacterial-driven effects on host immunity remain less studied. Using an ex-vivo model of infection, we report that the human gastrointestinal mucosa elicits a rapid and significant cytokine response to C. difficile. Marked increase in IFN-γ with modest increase in IL-22 and IL-17A was noted. Significant increase in IL-8 suggested potential for neutrophil influx while presence of IL-12, IL-23, IL-1ß and IL-6 was indicative of a cytokine milieu that may modulate subsequent T cell immunity. Majority of C. difficile-driven effects on murine bone-marrow-derived dendritic cell (BMDC) activation were toxin-independent; the toxins were however responsible for BMDC inflammasome activation. In contrast, human monocyte-derived DCs (mDCs) released IL-1ß even in the absence of toxins suggesting host-specific mediation. Infected DC-T cell crosstalk revealed the ability of R20291 and 630 WT strains to elicit a differential DC IL-12 family cytokine milieu which culminated in significantly greater Th1 immunity in response to R20291. Interestingly, both strains induced a similar Th17 response. Elicitation of mucosal IFN-γ/IL-17A and Th1/Th17 immunity to C. difficile indicates a central role for this dual cytokine axis in establishing antimicrobial immunity to CDI.

Association of adiponectin with dietary factors and cardiovascular risk factors in type 2 diabetes mellitus patients.

BACKGROUND AND AIM: Adiponectin, is an adipose tissue-specific adipokine, that circulates in human plasma at high levels, although lower levels are noted with insulin resistance and atherosclerosis. We investigated the relationship of adiponectin concentrations with dietary factors and some of the cardiovascular risk factors in patients with T2DM. METHODS AND RESULTS: Totally 107 patients with T2DM were recruited from the out patients clinic of Shariati Hospital, Tehran, Iran. Patients were evaluated for laboratory and anthropometric measurements including serum adiponectin, fasting insulin, FPG, OGTT, HbA1c, HOMA-IR, hsCRP, weight, height, BMI and WHR. Nutrients intakes were obtained via 24-h recall from each patient in three successive days. Nutrients and data analysis were done using FPII and SPSS version 13 softwares. The mean of log10-transformed serum adiponectin concentration was 0.79±0.27µg/ml. The univariate linear regression analysis could not show any significant relation between the log of serum adiponectin and dietary factors. In multivariate linear regression after multiple adjustment, the log of serum adiponectin was independently associated with WHR (P=0.02, t=-2.33), HDL-C (P=0.050, t=2.03) and markedly but not significantly with age of patients (P=0.058, t=1.92). CONCLUSIONS: Our findings showed that WHR, one of the important cardiovascular risk factors, can modulate independently adiponectin levels of T2DM patients in inverse manner. Also, the age of patients and HDL-C levels have marked positive effect on circulating levels of this adipocytokine. Thus, adiponectin might be a useful biomarker to prevent developing CVD in type 2 diabetes.

Human intestinal epithelial response(s) to Clostridium difficile.

Clostridium difficile is a gram-positive, spore-forming, toxin-producing anaerobic bacillus that is being increasingly implicated as the leading cause of diarrhea and colitis, particularly in hospitalized, elderly patients. Studies to date suggest that C. difficile toxins A and B play a major role in the observed colonic inflammation and associated disease pathogenesis; however, the role of other potential bacterial factors at present remains unknown. Early effects of C. difficile on host intestinal epithelia include modest induction of innate immune responses with progressive loss of intestinal epithelial cell barrier function and cell death.