Cross-immunity against SARS-COV-2 variants of concern in naturally infected critically ill COVID-19 patients.

Critically ill patients infected with SARS-CoV-2 display adaptive immunity, but it is unknown if they develop cross-reactivity to variants of concern (VOCs). We profiled cross-immunity against SARS-CoV-2 VOCs in naturally infected, non-vaccinated, critically ill COVID-19 patients. Wave-1 patients (wild-type infection) were similar in demographics to Wave-3 patients (wild-type/alpha infection), but Wave-3 patients had higher illness severity. Wave-1 patients developed increasing neutralizing antibodies to all variants, as did patients during Wave-3. Wave-3 patients, when compared to Wave-1, developed more robust antibody responses, particularly for wild-type, alpha, beta and delta variants. Within Wave-3, neutralizing antibodies were significantly less to beta and gamma VOCs, as compared to wild-type, alpha and delta. Patients previously diagnosed with cancer or chronic obstructive pulmonary disease had significantly fewer neutralizing antibodies. Naturally infected ICU patients developed adaptive responses to all VOCs, with greater responses in those patients more likely to be infected with the alpha variant, versus wild-type.

Elevated vascular transformation blood biomarkers in Long-COVID indicate angiogenesis as a key pathophysiological mechanism.

BACKGROUND: Long-COVID is characterized by prolonged, diffuse symptoms months after acute COVID-19. Accurate diagnosis and targeted therapies for Long-COVID are lacking. We investigated vascular transformation biomarkers in Long-COVID patients. METHODS: A case-control study utilizing Long-COVID patients, one to six months (median 98.5 days) post-infection, with multiplex immunoassay measurement of sixteen blood biomarkers of vascular transformation, including ANG-1, P-SEL, MMP-1, VE-Cad, Syn-1, Endoglin, PECAM-1, VEGF-A, ICAM-1, VLA-4, E-SEL, thrombomodulin, VEGF-R2, VEGF-R3, VCAM-1 and VEGF-D. RESULTS: Fourteen vasculature transformation blood biomarkers were significantly elevated in Long-COVID outpatients, versus acutely ill COVID-19 inpatients and healthy controls subjects (P < 0.05). A unique two biomarker profile consisting of ANG-1/P-SEL was developed with machine learning, providing a classification accuracy for Long-COVID status of 96%. Individually, ANG-1 and P-SEL had excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, P < 0.0001; validated in a secondary cohort). Specific to Long-COVID, ANG-1 levels were associated with female sex and a lack of disease interventions at follow-up (P < 0.05). CONCLUSIONS: Long-COVID patients suffer prolonged, diffuse symptoms and poorer health. Vascular transformation blood biomarkers were significantly elevated in Long-COVID, with angiogenesis markers (ANG-1/P-SEL) providing classification accuracy of 96%. Vascular transformation blood biomarkers hold potential for diagnostics, and modulators of angiogenesis may have therapeutic efficacy.

Nutritional, Microbial, and Allergenic Changes during the Fermentation of Cashew 'Cheese' Product Using a Quinoa-Based Rejuvelac Starter Culture.

Fermentation has been applied to a multitude of food types for preservation and product enhancing characteristics. Interest in the microbiome and healthy foods makes it important to understand the microbial processes involved in fermentation. This is particularly the case for products such as fermented cashew (Anacardium occidentale). We hereby describe the characterisation of cashew samples throughout an entire fermentation production process, starting at the quinoa starter inoculum (rejuvelac). The viable bacterial count was 108 -109 colony forming units/g. The nutritional composition changed marginally with regards to fats, carbohydrates, vitamins, and minerals. The rejuvelac starter culture was predominated by Pediococcus and Weissella genera. The 'brie' and 'blue' cashew products became dominated by Lactococcus, Pediococcus, and Weissella genera as the fermentation progressed. Cashew allergenicity was found to significantly decrease with fermentation of all the end-product types. For consumers concerned about allergic reactions to cashew nuts, these results suggested that a safer option is for products to be made by fermentation.

Effect of Oral Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the Vaginal Microbiota, Cytokines and Chemokines in Pregnant Women.

Spontaneous preterm birth is associated with vaginal microbial dysbiosis. As certain strains of lactobacilli help restore homeostasis in non-pregnant women, the goal was to determine the effect of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 administered orally, twice daily for 12 weeks on the vaginal microbiota, cytokines and chemokines of low-risk pregnant women. A double-blind, placebo-controlled, randomized trial comparing probiotic lactobacilli to placebo daily was performed in 86 asymptomatic pregnant women who had an Intermediate or Bacterial Vaginosis Nugent score at 13 weeks. After drop outs, 32 women receiving probiotics and 34 receiving placebo completed the study. The Nugent score returned to normal in 30% of the women in both groups at 28 weeks and was maintained until 35 weeks. The majority of subjects had normal pregnancy outcomes. Ninety-three bacterial species were detected at 13 weeks, with Lactobacillus iners, Lactobacillus crispatus, Gardnerella vaginalis and Atopobium vaginae being the most abundant across pregnancy. There was no difference in the Shannon diversity index between the probiotic and placebo groups at 13, 28 or 35 weeks. Almost all subjects consumed fermented foods and many of the organisms in the vagina are also known to be present in fermented foods. Interleukin-4 in the placebo group and Interleukin-10 in both probiotic and placebo groups increased slightly at 28 weeks but were not different at 35 weeks when compared to 13 weeks. In conclusion, this study showed no adverse issues resulting from 12 week use of probiotic Lactobacillus strains GR-1 and RC-14 during pregnancy in women at low risk for premature birth. The vaginal microbiota demonstrated flux irrespective of this oral probiotic administration.

Expired probiotics: what is really in your cabinet?

The popularity of using probiotics has surged, since they became widely accepted as safe and help improve general health. Inevitably, some of these products are used after expiration when microbial cell viability is below the recommended effective dose. Given that probiotics must be live microorganisms administered in adequate amounts, the aim of this study was to measure viability in expired products and assess how packaging and storage conditions impact efficacy, if at all. Thirty-three expired probiotic products were evaluated, of which 26 were stored in conditions recommended by the manufacturer. The viable microbial cells were enumerated and representative isolates identified by 16S and internally transcribed spacer rRNA gene sequencing. While the products had a mean past expiration time of 11.32 (1-22) years, 22 still had viable contents, and 5 were within or above the original product cell count claim. Product formulation and the number of species present did not appear to impact the stability of the products. However, overall packaging type, storage conditions and time since expiry were found to affect viability. All products with viable cells had the strain stipulated on the label. Despite some selected probiotic products retaining viability past their expiry date (indicating long-term storage is possible), the total counts were mostly well below that required for efficacious use as recommended by the manufacturer. Consuming expired probiotics may not yield the benefits for which they were designed.

Enterococcus faecalis persistence in pediatric patients treated with antibiotic prophylaxis for recurrent urinary tract infections.

AIM: Enterococcus faecalis is one of the most common causes of recurrent urinary tract infection (RUTI), yet enterococcal pathogenesis is poorly understood. Our aims were to identify the prevalence of enterococci in RUTI patients and characterize the enterococcal response to nitrofurantoin and trimethoprim-sulfamethoxazole. MATERIALS & METHODS: We studied pediatric patients receiving antibiotic prophylaxis and those only under clinical observation for 12 months (n = 39). We then assessed the response of uropathogenic E. faecalis to nitrofurantoin and trimethoprim-sulfamethoxazole. RESULTS: Enterococci were isolated from almost half of patients and exposure of Enterococcus to nitrofurantoin increased virulence properties; this did not correlate with increased expression of virulence factors. CONCLUSION: Our results demonstrate that antibiotic prophylaxis may not be suitable for treatment of enterococcal RUTI (NCT02357758).

Promising Prebiotic Candidate Established by Evaluation of Lactitol, Lactulose, Raffinose, and Oligofructose for Maintenance of a Lactobacillus-Dominated Vaginal Microbiota.

Perturbations to the vaginal microbiota can lead to dysbiosis, including bacterial vaginosis (BV), which affects a large portion of the female population. In a healthy state, the vaginal microbiota is characterized by low diversity and colonization by Lactobacillus spp., whereas in BV, these species are displaced by a highly diverse population of bacteria associated with adverse vaginal health outcomes. Since prebiotic ingestion has been a highly effective approach to invigorate lactobacilli for improved intestinal health, we hypothesized that these compounds could stimulate lactobacilli at the expense of BV organisms to maintain vaginal health. Monocultures of commensal Lactobacillus crispatus, Lactobacillus vaginalis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, and Lactobacillus iners, in addition to BV-associated organisms and Candida albicans, were tested for their ability to utilize a representative group of prebiotics consisting of lactitol, lactulose, raffinose, and oligofructose. The disaccharide lactulose was found to most broadly and specifically stimulate vaginal lactobacilli, including the strongly health-associated species L. crispatus, and importantly, not to stimulate BV organisms or C. albicans Using freshly collected vaginal samples, we showed that exposure to lactulose promoted commensal Lactobacillus growth and dominance and resulted in healthy acidity partially through lactic acid production. This provides support for further testing of lactulose to prevent dysbiosis and potentially to reduce the need for antimicrobial agents in managing vaginal health.IMPORTANCE Bacterial vaginosis (BV) and other dysbioses of the vaginal microbiota significantly affect the quality of life of millions of women. Antimicrobial therapy is often poorly effective, causes side effects, and does not prevent recurrences. We report one of very few studies that have evaluated how prebiotics-compounds that are selectively fermented by beneficial bacteria such as Lactobacillus spp.-can modulate the vaginal microbiota. We also report use of a novel in vitro polymicrobial model to study the impact of prebiotics on the vaginal microbiota. The identification of prebiotic lactulose as enhancing Lactobacillus growth but not that of BV organisms or Candida albicans has direct application for retention of homeostasis and prevention of vaginal dysbiosis and infection.

A Novel Millet-Based Probiotic Fermented Food for the Developing World.

Probiotic yogurt, comprised of a Fiti sachet containing Lactobacillus rhamnosus GR-1 and Streptococcus thermophilus C106, has been used in the developing world, notably Africa, to alleviate malnutrition and disease. In sub-Saharan African countries, fermentation of cereals such as millet, is culturally significant. The aim of this study was to investigate the fermentation capability of millet when one gram of the Fiti sachet consortium was added. An increase of 1.8 and 1.4 log CFU/mL was observed for S. thermophilus C106 and L. rhamnosus GR-1 when grown in 8% millet in water. Single cultures of L. rhamnosus GR-1 showed the highest µmax when grown in the presence of dextrose, galactose and fructose. Single cultures of S. thermophilus C106 showed the highest µmax when grown in the presence of sucrose and lactose. All tested recipes reached viable counts of the probiotic bacteria, with counts greater than 106 colony-forming units (CFU)/mL. Notably, a number of organic acids were quantified, in particular phytic acid, which was shown to decrease when fermentation time increased, thereby improving the bioavailability of specific micronutrients. Millet fermented in milk proved to be the most favorable, according to a sensory evaluation. In conclusion, this study has shown that sachets being provided to African communities to produce fermented milk, can also be used to produce fermented millet. This provides an option for when milk supplies are short, or if communities wish to utilize the nutrient-rich qualities of locally-grown millet.

Sitagliptin in patients with non-alcoholic steatohepatitis: A randomized, placebo-controlled trial.

AIM: To evaluate the effect of sitagliptin vs placebo on histologic and non-histologic parameters of non-alcoholic steatohepatitis (NASH). METHODS: Twelve patients with biopsy-proven NASH were randomized to sitagliptin (100 mg daily) (n = 6) or placebo (n = 6) for 24 wk. The primary outcome was improvement in liver fibrosis after 24 wk. Secondary outcomes included evaluation of changes in NAFLD activity score (NAS), individual components of NAS (hepatocyte ballooning, lobular inflammation, and steatosis), glycemic control and insulin resistance [including measurements of glycated hemoglobin (HbA1C) and adipocytokines], lipid profile including free fatty acids, adipose distribution measured using magnetic resonance imaging (MRI), and thrombosis markers (platelet aggregation and plasminogen activator inhibitor 1 levels). We also sought to determine the correlation between changes in hepatic fat fraction (%) [as measured using the Iterative Decomposition of water and fat with Echo Asymmetry and Least-squares estimation (IDEAL) MRI technique] and changes in hepatic steatosis on liver biopsy. RESULTS: Sitagliptin was not significantly better than placebo at reducing liver fibrosis score as measured on liver biopsy (mean difference between sitagliptin and placebo arms, 0.40, P = 0.82). There were no significant improvements evident with the use of sitagliptin vs placebo for the secondary histologic outcomes of NAS total score as well as for the individual components of NAS. Compared to baseline, those patients who received sitagliptin demonstrated improved HbA1C (6.7% ± 0.4% vs 7.9% ± 1.0%, P = 0.02), and trended towards improved adiponectin levels (4.7 ± 3.5 µg/mL vs 3.9 ± 2.7 µg/mL, P = 0.06) and triglyceride levels (1.26 ± 0.43 mmol/L vs 2.80 ± 1.64 mmol/L, P = 0.08). However, when compared with placebo, sitagliptin did not cause a statistically significant improvement in HbA1C (mean difference, -0.7%, P = 0.19) nor triglyceride levels (mean difference -1.10 mmol/L, P = 0.19) but did trend towards improved adiponectin levels only (mean difference, 0.60 µg/mL, P = 0.095). No significant changes in anthropometrics, liver enzymes, other adipocytokines, lipid profile, thrombosis parameters, or adipose distribution were demonstrated. The MRI IDEAL procedure correlated well with steatosis scores obtained on liver biopsy in both groups at baseline and post-treatment, and the Spearman correlation coefficients ranged from r = 0.819 (baseline) to r = 0.878 (post-treatment), P = 0.002. CONCLUSION: Sitagliptin does not improve fibrosis score or NAS after 24 wk of therapy. The MRI IDEAL technique may be useful for non-invasive measurement of hepatic steatosis.

Microbiota at Multiple Body Sites during Pregnancy in a Rural Tanzanian Population and Effects of Moringa-Supplemented Probiotic Yogurt.

The nutritional status of pregnant women is vital for healthy outcomes and is a concern for a large proportion of the world's population. The role of the microbiota in pregnancy and nutrition is a promising new area of study with potential health ramifications. In many African countries, maternal and infant death and morbidity are associated with malnutrition. Here, we assess the influence of probiotic yogurt containing Lactobacillus rhamnosus GR-1, supplemented with Moringa plant as a source of micronutrients, on the health and oral, gut, vaginal, and milk microbiotas of 56 pregnant women in Tanzania. In an open-label study design, 26 subjects received yogurt daily, and 30 were untreated during the last two trimesters and for 1 month after birth. Samples were analyzed using 16S rRNA gene sequencing, and dietary recalls were recorded. Women initially categorized as nourished or undernourished consumed similar calories and macronutrients, which may explain why there was no difference in the microbiota at any body site. Consumption of yogurt increased the relative abundance of Bifidobacterium and decreased Enterobacteriaceae in the newborn feces but had no effect on the mother's microbiota at any body site. The microbiota of the oral cavity and GI tract remained stable over pregnancy, but the vaginal microbiota showed a significant increase in diversity leading up to and after birth. In summary, daily micronutrient-supplemented probiotic yogurt provides a safe, affordable food for pregnant women in rural Tanzania, and the resultant improvement in the gut microbial profile of infants is worthy of further study.

A systems biology approach investigating the effect of probiotics on the vaginal microbiome and host responses in a double blind, placebo-controlled clinical trial of post-menopausal women.

UNLABELLED: A lactobacilli dominated microbiota in most pre and post-menopausal women is an indicator of vaginal health. The objective of this double blinded, placebo-controlled crossover study was to evaluate in 14 post-menopausal women with an intermediate Nugent score, the effect of 3 days of vaginal administration of probiotic L. rhamnosus GR-1 and L. reuteri RC-14 (2.5×109 CFU each) on the microbiota and host response. The probiotic treatment did not result in an improved Nugent score when compared to when placebo. Analysis using 16S rRNA sequencing and metabolomics profiling revealed that the relative abundance of Lactobacillus was increased following probiotic administration as compared to placebo, which was weakly associated with an increase in lactate levels. A decrease in Atopobium was also observed. Analysis of host responses by microarray showed the probiotics had an immune-modulatory response including effects on pattern recognition receptors such as TLR2 while also affecting epithelial barrier function. This is the first study to use an interactomic approach for the study of vaginal probiotic administration in post-menopausal women. It shows that in some cases multifaceted approaches are required to detect the subtle molecular changes induced by the host to instillation of probiotic strains. TRIAL REGISTRATION: ClinicalTrials.gov NCT02139839.

Correlations between changes in cytokines and clinical outcomes for early phase (proof of concept) trials in active diffuse systemic sclerosis using data from an imatinib study.

OBJECTIVE: Data from a small study testing imatinib to treat SSc were used to determine if cytokine changes were related to differences in clinical parameters to model future early phase trials pairing cytokine changes and clinical parameters. METHODS: Plasma and punch skin biopsy specimens collected at baseline and 6 months were analysed for levels of 26 fibrotic and inflammatory cytokines using multiplexed immunoassays and ELISA. Seven of nine patients on active treatment had paired data. Biopsies were biopulverized and standardized to protein levels in the tissue homogenate. Plasma was frozen at -80°C and analysed using multiplexed immunoassays or ELISAs standardized to CRP. Correlations between fold changes in cytokines and differences in clinical parameters (skin score, physician and patient global assessments and HAQ) were performed. P < 0.01 was considered significant. RESULTS: After 6 months of imatinib treatment, plasma levels of soluble vascular cell adhesion molecule 1 decreased significantly (P < 0.001), while tissue levels of soluble intercellular adhesion molecule 1 increased (P < 0.01). Some significant correlations between fold changes in certain plasma fibrotic and inflammatory cytokines and changes in clinical parameters after 6 months of treatment were found: patient global scores and IL-13 (r = 0.964, P < 0.0001); ESR and IL-12p70 (r = -0.903, P < 0.01); in tissue samples, patient global score and soluble E-selectin (r = 0.913, P < 0.01); and physician global score with sCD40L (r = -0.883, P < 0.01). CONCLUSION: Some serum and tissue cytokines may have a role in early phase clinical trials of SSc, correlating with changes in clinical parameters. Serum and tissue samples could be analysed in early phase trials to determine whether they support the clinical observations. TRIAL REGISTRATION: http://clinicaltrials.gov/show/NCT01545427.

The use of triclosan eluting stents effectively reduces ureteral stent symptoms: a prospective randomized trial.

UNLABELLED: What's known on the subject? and What does the study add? Infection, encrustation and ureteral-stent-related symptoms (USRS) including pain, urgency and frequency are all major problems associated with stent use. No current ureteral stent or exogenously applied therapy adequately deals with these problems and antibiotic use is ineffective once a bacterial biofilm forms on the device. Triclosan is a broad spectrum antibacterial agent widely used in numerous healthcare products and has been previously shown to reduce inflammation on the skin and in the oral cavity. This study tested a triclosan-impregnated ureteral stent for its ability to reduce infection, encrustation and USRS. This study shows that while a triclosan-impregnated ureteral stent cannot reduce infection rates alone compared with antibiotic use, the stent can reduce several USRS including pain during indwelling. This study suggests that the triclosan eluting stent may have a role in treating patients, perhaps in combination with standard antibiotic therapy. OBJECTIVE: To evaluate the capacity of triclosan-loaded ureteral stents to reduce stent-associated bacterial attachment, biofilm formation and encrustation, thereby potentially reducing infection development and other device-related sequelae. PATIENTS AND METHODS: Twenty subjects requiring short-term stenting (7-15 days) were randomized to receive either a Percuflex Plus(®) non-eluting stent (control) or a Triumph(®) triclosan eluting stent. Control-stented subjects received 3 days of levofloxacin prophylaxis (500 mg once daily) while Triumph(®)-stented subjects did not. All subjects were assessed for positive urine and stent cultures, stent biofilm development and encrustation. Following device removal, each subject completed an analogue-scale symptom assessment questionnaire. RESULTS: Ureteral stenting was performed after nine ureteroscopic and one extracorporeal shock wave lithotripsy procedure in the control group and eight ureteroscopic and two shock wave lithotripsy procedures in the triclosan group. No significant differences were observed for culture, biofilm and encrustation between the two groups. Subjects in the triclosan group reported significant reductions in lower flank pain scores during activity (58.1% reduction, P = 0.017) and urination (42.6%, P = 0.041), abdominal pain during activity (42.1%, P = 0.042) and urethral pain during urination (31.7%, P = 0.049). CONCLUSIONS: In this study, the use of the Triumph(®) triclosan eluting stent had no marked impact on biofilm formation, encrustation or infection development in short-term stented patients. The Triumph(®) device led to significant reductions in several common ureteral-stent-related symptoms, supporting its use in this patient population.

Imatinib in active diffuse cutaneous systemic sclerosis: Results of a six-month, randomized, double-blind, placebo-controlled, proof-of-concept pilot study at a single center.

OBJECTIVE: To better understand the feasibility of using imatinib, a tyrosine kinase inhibitor, to treat active diffuse cutaneous systemic sclerosis (dcSSc). METHODS: We performed a 6-month, randomized, double-blind, placebo-controlled, proof-of-concept pilot study of imatinib in patients with active dcSSc. Data on safety, modified Rodnan skin thickness scores (MRSS), Health Assessment Questionnaire (HAQ) scores, patient's and physician's global assessments (100-mm visual analog scale), and biomarkers in serum and skin biopsy samples were collected. We used a 4:1 randomization strategy (imatinib 200 mg administered twice a day versus placebo), stratifying according to current use of methotrexate. The plan was to enroll 20 dcSSc patients. RESULTS: After enrolling 10 patients (9 receiving active drug and 1 receiving placebo), we found poor tolerability and high rates of adverse events with imatinib, and study enrollment was discontinued. There was no significant difference in the mean MRSS in all patients who took imatinib (31.1 at baseline versus 29.4 at 6 months) or in only those who completed 6 months of imatinib (31.0 at baseline versus 30.3 at 6 months), and there was no difference in the C-reactive protein level, erythrocyte sedimentation rate, physician's global assessment, patient's global assessment, response to the Health Transition query, or the HAQ scores between those who did and those who did not complete 6 months of therapy. Side effects were edema, fluid retention, fatigue, nausea, cramps/myalgias, diarrhea, alopecia, and anemia. Most side effects occurred within the first week of treatment, and even when imatinib was reintroduced at a lower dosage (200 mg daily), it was poorly tolerated. Two patients were hospitalized because of side effects of the medication. In general, biomarker levels in plasma and skin did not change. CONCLUSION: Imatinib was poorly tolerated, and this could limit its application in SSc. The study was too small to form conclusions about the efficacy of imatinib in SSc.

Results of a Pilot Randomized Placebo-Controlled Trial in Primary and Secondary Raynaud's Phenomenon with St. John's Wort: Detecting Changes in Angiogenic Cytokines When RP Improves.

Objectives.To perform a 6-week double-blind RCT in Raynaud's phenomenon (RP) comparing the plant extract St. John's Wort (SJW) to placebo. Methods. RP patients having at least 7 attacks per week were stratified by primary and secondary RP and within secondary by systemic sclerosis or other connective tissue disease. Subjects completed a daily standardized diary recording all RP attacks (frequency, duration and severity). Serum levels of 18 inflammatory and angiogenic cytokines were measured pre- and post-treatment. Results. Eighteen patients completed the study; 8 received SJW and 10 placebo. The decrease in mean number of attacks per day was 0.75 with SJW and 1.01 with placebo, P = 0.06. Attack duration and severity were not different between groups. Cytokine analyses demonstrated no between-groups differences. Combining treatment groups, those with >50% improvement in frequency of attacks yielded a significant increase in E-selectin (P = 0.049), MMP-9 (P = 0.011), G-CSF (P = 0.02), and VEGF (P = 0.012) pre- versus post-treatment. A ≥50% improvement in severity of attacks corresponded to a significant increase in levels of sVCAM-1 (P = 0.003), sICAM-1 (P = 0.007), and MCP-1 (P = 0.004). Conclusions. There were no clinical or biomarker benefit of SJW versus placebo in RP. However, combining all patients, there were changes in some cytokines that may be further investigated.

Novel in vitro model for studying ureteric stent-induced cell injury.

OBJECTIVE: To develop a novel in vitro model for the study of bladder and kidney epithelial cell injury akin to stent movement, as ureteric stents are associated with urinary tract complications that can significantly add to patient morbidity. These sequelae may be linked to inflammation triggered by stent-mediated mechanical injury to the urinary tract. MATERIALS AND METHODS: T24 bladder and A498 kidney cell line monolayers were damaged mechanically by segments of either Percuflex Plus (PP) or Triumph (triclosan-eluting) stents (both from Boston Scientific Corporation Inc. Natick, MA, USA) and the resulting expression profiles of several pro-inflammatory cytokines and growth factors were analysed. RESULTS: After control injury using the PP stent, supernatants of both cell lines had significantly increased levels of interleukin (IL)-6, IL-8, basic fibroblast growth factor and platelet-derived growth factor BB, and A498 cells also had increased tumour necrosis factor alpha. In almost all cases, the presence of triclosan within the media abrogated the pro-inflammatory cytokine increases, while its effects on growth factors varied. CONCLUSION: This study suggests that stent-related symptoms in the bladder and kidney may be partially due to a local inflammatory response to epithelial damage caused by the presence and movement of the stent. Future stent design should take these inflammatory responses, with respect to physical injury, into consideration, using either more biocompatible materials or anti-inflammatory compounds such as triclosan.

Effect of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the ability of Candida albicans to infect cells and induce inflammation.

Vulvovaginal candidiasis, a high prevailing infection worldwide, is mainly caused by Candida albicans. Probiotic Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 have been previously shown to be useful as adjuvants in the treatment of women with VVC. In order to demonstrate and better understand the anti-Candida activity of the probiotic microorganisms in an in vitro model simulating vaginal candidiasis, a human vaginal epithelial cell line (VK2/E6E7) was infected with C.albicans 3153a and then challenged with probiotic L. rhamnosus GR-1 and/or L. reuteri RC-14 or their respective CFS (alone or in combination). At each time point (0, 6, 12 and 24 hr), numbers of yeast, lactobacilli and viable VK2/E6E7 cells were determined and, at 0, 6 and 12 hr, the supernatants were measured for cytokine levels. We found that C. albicans induced a significant increase in IL-1alpha and IL-8 production by VK2/E6E7 cells. After lactobacilli challenge, epithelial cells did not alter IL-6, IL-1alpha, RANTES and VEGF levels. However, CFS from the probiotic microorganisms up-regulated IL-8 and IP-10 levels secreted by VK2/E6E7 cells infected with C. albicans. At 24 hr of co-incubation, L. reuteri RC-14 alone and in combination with L. rhamnosus GR-1 decreased the yeast population recoverable from the cells. In conclusion, L. reuteri RC-14 alone and together with L. rhamnosus GR-1 have the potential to inhibit the yeast growth and their CFS may up-regulate IL-8 and IP-10 secretion by VK2/E6E7 cells, which could possibly have played an important role in helping to clear VVC in vivo.

Use of triclosan-eluting ureteral stents in patients with long-term stents.

BACKGROUND AND PURPOSE: Long-term use of ureteral stents is prevented by biofilm-related infection and encrustation mandating stent changes every few months. Triclosan is a broad-spectrum antimicrobial in numerous consumer and medical products and has been incorporated into a ureteral stent. We sought to determine the clinical effects of the triclosan-eluting stent in patients who needed long-term ureteral stenting. PATIENTS AND METHODS: Eight patients with long-term stents were enrolled prospectively. All received a control stent for 3 months along with preoperative and postoperative antibiotics. After 3 months, the control stent was removed, and a triclosan-eluting stent was placed for 3 months with no antibiotics administered. For both indwelling periods, urine cultures were obtained weekly and biweekly for the first and last 6 weeks, respectively, and antibiotics were prescribed when patients had both a positive urine culture and symptoms of urinary tract infection. On removal, stents were assessed for microorganisms and encrustation. RESULTS: Overall, similar microorganisms were isolated during each indwell period, although Staphylococcus and Enterococcus strains were isolated more frequently during control and triclosan stenting, respectively. Significantly fewer antibiotics were used during triclosan stenting, coinciding with a slightly higher number of positive urine cultures and significantly fewer symptomatic infections. No bacterial isolates developed antibiotic resistance during triclosan stent placement. CONCLUSIONS: Antibiotic use with control stents resulted in bacterial antibiotic resistance, which was not the case with the triclosan-eluting stents. Although triclosan-eluting stents did not show a clinical benefit in terms of urine and stent cultures or overall subject symptoms compared with controls, their use did result in decreased antibiotic usage and significantly fewer symptomatic infections. The triclosan-eluting stent alone is not sufficient to reduce device-associated infections in this difficult patient population.

Triclosan inhibits uropathogenic Escherichia coli-stimulated tumor necrosis factor-alpha secretion in T24 bladder cells in vitro.

BACKGROUND AND PURPOSE: Triclosan is an antimicrobial agent commonly used in consumer and medical products that inhibits bacterial fatty acid synthesis. In addition to its bactericidal effects, sublethal concentrations of triclosan reduce local inflammation, inhibit the growth of bacterial uropathogens, induce membrane stress, and inhibit P-fimbrial expression in uropathogenic Escherichia coli (UPEC). We tested whether sublethal concentrations of triclosan could reduce the adherence of UPEC to bladder and kidney cells and reduce the amount of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) produced by these cells during bacterial challenge in vitro. MATERIALS AND METHODS: Assays of bacterial growth, adhesion, and intracellularization were performed using UPEC GR12 incubated for 4 hours on monolayers of human T24 bladder cells or A498 kidney cells with various sublethal concentrations of triclosan. The expression profile of TNF-alpha from bladder cells was evaluated using ELISA. RESULTS: No significant decreases were observed in the adherence or invasion percentages of UPEC GR12 with either cell line when treated with sublethal amounts of triclosan. However, treatment with triclosan 0.5 microg/mL led to a significant decrease in the total number of UPEC GR12 recovered from T24 monolayers (P < 0.05). Importantly, a reduction in the expression of TNF-alpha by T24 cells was shown when UPEC GR12 was treated with triclosan (P < 0.05). CONCLUSIONS: Sublethal concentrations of triclosan did not inhibit the adhesion or intracellularization of UPEC into kidney or bladder cell lines but did significantly reduce the amount of TNF-alpha secreted by bladder cells. Therefore, the use of triclosan on ureteral stents may prove clinically beneficial, not only by inhibiting bacterial survival and growth within the urinary tract, but by reducing local inflammation as well.

Wnt expression is not correlated with beta-catenin dysregulation in Dupuytren's Disease.

BACKGROUND: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in finger flexion contractures. Increased cellular beta-catenin levels have been identified as characteristic of this disease. As Wnts are the most widely recognized upstream regulators of cellular beta-catenin accumulation, we have examined Wnt gene expression in surgical specimens and in DD-derived primary cell cultures grown in two-dimensional monolayer culture or in three-dimensional FPCL collagen lattice cultures. RESULTS: The Wnt expression profile of patient-matched DD and unaffected control palmar fascia tissue was determined by a variety of complimentary methods; Affymetrix Microarray analysis, specific Wnt and degenerative primer-based Reverse Transcriptase (RT)-PCR, and Real Time PCR. Microarray analysis identified 13 Wnts associated with DD and control tissues. Degenerate Wnt RT-PCR analysis identified Wnts 10b and 11, and to a lesser extent 5a and 9a, as the major Wnt family members expressed in our patient samples. Competitive RT-PCR analysis identified significant differences between the levels of expression of Wnts 9a, 10b and 11 in tissue samples and in primary cell cultures grown as monolayer or in FPCL, where the mRNA levels in tissue > FPCL cultures > monolayer cultures. Real Time PCR data confirmed the down-regulation of Wnt 11 mRNA in DD while Wnt 10b, the most frequently isolated Wnt in DD and control palmar fascia, displayed widely variable expression between the methods of analysis. CONCLUSION: These data indicate that changes in Wnt expression per se are unlikely to be the cause of the observed dysregulation of beta-catenin expression in DD.