Tyrosine kinase inhibitors (TKIs) have been adopted in the treatment of a variety of malignancies. Despite their popularity, the underlying mechanism of the adverse effects seen with the use of TKIs is not completely understood. Acute liver injury is a known side effect of many of these drugs. Some papers have demonstrated that N-acetylcysteine may have a role in non-acetaminophen induced acute liver failure (NAI-ALF). There is little evidence supporting the use of N-acetylcysteine in the treatment of tyrosine kinase inhibitor-induced acute liver injury. This case report adds to the limited body of existing knowledge.We present a 67-year-old Caucasian female with a past medical history of anxiety, hyperlipidemia, in utero exposure to diethylstilbestrol (DES), and well-differentiated angiosarcoma of the right breast. She achieved remission for approximately six years after mastectomy with adjuvant chemotherapy and radiation. Subsequent surveillance imaging revealed new hepatic and cervical lesions. Further investigation with cutaneous biopsy near the occipital region confirmed recurrent metastatic angiosarcoma. The patient was started on high-dose pazopanib and initially tolerated the TKI without any adverse effects. However, after approximately two weeks of therapy, she began to experience dark colored urine, myalgias, and fatigue. These symptoms, along with significant elevations in liver enzymes , prompted admission for evaluation of acute liver injury. The etiology of the acute liver injury was suspected to be secondary to TKI therapy. Treatment with intravenous N-acetylcysteine was initiated for non-acetaminophen induced acute liver failure (NAI-ALF) and resulted in a dramatic improvement in transaminases before discharge.Evidence suggests that there is a beneficial role for N-acetylcysteine in the management of NAI-ALF. However, when it comes specifically to the management of TKI induced acute liver injury, there is limited evidence to support its use. This case report highlights a possible use of N-acetylcysteine in the management of TKI mediated acute liver injury. Additional studies should be conducted to determine the role N-acetylcysteine plays in the management of TKI mediated liver injury. The use of N-acetylcysteine (NAC) in the setting of acetaminophen-induced acute liver failure (AI-ALF) has been well studied and has become the standard of care in the management of this condition. There has been limited research regarding the use of NAC in the management of non-acetaminophen induced acute liver failure (NAI-ALF). NAC may produce an anti-inflammatory and antioxidant effect in the setting of NAI-ALF . AdditioThe use of tyrosine kinase inhibitors (TKI) in the management of malignancy has increased dramatically in the last decade. The underlying mechanism of the adverse effects seen with this class of novel drugs is still under investigation; however, acute liver injury has been reported with several TKIs -6. In thThe patient is a 67-year-old Caucasian female with a past medical history of anxiety, hyperlipidemia, in utero exposure to diethylstilbestrol (DES), and well-differentiated angiosarcoma of the right breast that was initially diagnosed via a core biopsy in 2011. She underwent right mastectomy with adjuvant radiotherapy and chemotherapy (gemcitabine-Taxotere). She achieved remission of her disease for approximately six years without any evidence of malignancy. However, subsequent surveillance computed tomography of the abdomen in 2017 revealed new scattered sub-centimeter enhancing hepatic lesions. Further evaluation of these hepatic lesions with magnetic resonance imaging (MRI) redemonstrated multiple sub-centimeter enhancing liver lesions  without any adverse effects. However, after approximately two weeks of therapy, she began to experience dark colored urine, myalgias, and fatigue. Subsequent evaluation at a local urgent care center revealed a new elevation in liver enzymes with alanine transaminase (ALT) of 522 units/L and aspartate transaminase (AST) of 456 units/L. These laboratory abnormalities prompted a referral to a tertiary care center for further evaluation, where repeat laboratory tests showed a significant increase in liver enzymes (ALT 1377 units/L and AST 1212 units/L). Additional testing revealed an international normalized ratio of 1.1, prothrombin time of 12.7 seconds, alkaline phosphatase 275 units/L, bilirubin of 1.5 mg/dL, and albumin of 4.3 g/dL. Serum acetaminophen levels were undetectable. An abdominal ultrasound of the right upper quadrant demonstrated unremarkable liver size, appearance, and echogenicity without intrahepatic ductal dilation or masses. Additionally, vascular ultrasound of the abdomen revealed no filling defects in the hepatic vein or main portal vein. Further studies, including serologies for herpes simplex virus, Ebstein Barr virus, cytomegalovirus, viral hepatitis, anti-nuclear antibody, and anti-smooth muscle antibody, were negative. Evaluation ultimately revealed no anatomic, infectious, or autoimmune etiologies for the patient's acute liver injury. The etiology of acute liver injury was, therefore, suspected to be secondary to the use of TKI (pazopanib).Prompt treatment with intravenous NAC for NAI-ALF was initiated on admission, and pazopanib was discontinued. The patient was monitored with serial complete metabolic panels and coagulation studies every eight hours. The acute liver injury rapidly improved during the hospital course, and the patient was ultimately discharged home 48 hours post-admission Figure . Close follow-up was arranged with the hepatology clinic, where repeat laboratory testing showed continued improvement in liver enzymes.A limited body of knowledge describes the use of NAC in the setting of NAI-ALF. The available literature is even more limited concerning the use of NAC in the context of TKI induced acute liver injury.One of the early mentions of the use of NAC in NAI-ALF was described in a prospective study of 91 patients studied over a seven-year period. In this study, the primary causes of NAI-ALF were from viral hepatitis  as well as drug-induced liver injury from tuberculosis drug therapy. However, none of the studied patients demonstrated NAI-ALF from TKI. A drawback of this study was a historical control group derived from chart review. Despite these limitations, the study showed that the use of NAC for NAI-ALF in a non-liver transplant center is a relatively safe intervention that significantly reduced mortality and length of hospitalization . The use of NAC in NAI-ALF is further supported by a prospective randomized case-control trial, conducted over two years, which assessed the effect of NAC therapy on mortality in patients with NAI-ALF. The trial enrolled 80 patients that were randomized to NAC versus placebo. Once again, the majority of the causes of NAI-ALF were secondary to acute viral hepatitis and tuberculosis drug therapy. There were no cases of NAI-ALF due to TKI therapy. Nevertheless, significant improvement in length of hospitalization and mortality with NAC was once again demonstrated, with reported 28% mortality in the NAC group compared to 53% mortality in the placebo group .The overall evidence points to a decrease in mortality and hospital length of stay when NAC is used in the treatment of NAI-ALF ,7. HowevThere is evidence that exists to suggest that there is a role for N-acetylcysteine in the management of non-acetaminophen induced acute liver failure. However, when it comes specifically to the management of tyrosine kinase inhibitor-induced acute liver injury, there is no large body of evidence to support its use. This case report highlights the possible use of NAC in the management of TKI mediated acute liver injury. Further case reports, case series, and prospective trials are needed to build the body of evidence in favor of this particular intervention.


Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are incurable, progressive and fatal neurodegenerative diseases with patients variably affected clinically by motor, behavior, and cognitive deficits. The accumulation of an RNA-binding protein, TDP-43, is the most significant pathological finding in approximately 95% of ALS cases and 50% of FTD cases, and discovery of this common pathological signature, together with an increasing understanding of the shared genetic basis of these disorders, has led to FTD and ALS being considered as part of a single disease continuum. Given the widespread aggregation and accumulation of TDP-43 in FTD-ALS spectrum disorder, TDP-43 may have potential as a biomarker in these diseases.We therefore conducted a systematic review and meta-analysis to evaluate the diagnostic utility of TDP-43 detected in the cerebrospinal fluid (CSF) of patients with FTD-ALS spectrum disorder.p = 0.02).From seven studies, our results demonstrate that patients with ALS have a statistically significantly higher level of TDP-43 in CSF (effect size 0.64, 95% CI: 0.1–1.19, These data suggest promise for the use of CSF TDP-43 as a biomarker for ALS. Frontotemporal dementia (FTD) is an umbrella term for a spectrum of neurodegenerative disorders. These disorders primarily affect behaviour, personality, executive function and language (and may include motor impairment). It is the second most common form of dementia after Alzheimer’s disease (AD) in people under 65 years old, with the average age of onset being between 45 and 65 years old, and the peak prevalence being around age 65 to 69 . PrognosFTD also affects the motor system and is now increasingly thought to be on a syndromic spectrum with amyotrophic lateral sclerosis . ALS isC9orf72, found to be present in the majority of cases of familial cases of FTD-ALS spectrum disorders The title and abstract of each paper were screened for relevance with respect to inclusion and exclusion criteria. For studies meeting our predefined criteria the full text was retrieved, imported to Excel and duplicate records were discarded. The quality of each paper was then evaluated by the QUADAS-2 quality score and relevant data were extracted.All studies that measure TDP-43 in the CSF of living patients with ALS or FTD, compared to controls.Inclusion criteriaNo control groupAnimal studiesPost mortem studiesReviewsLetters and commentsAbstractsNon-parametric results Exclusion criteriaStudy ID: (i) Author and (ii) YearIntervention: FTD , ALS or both (FTD-ALS)Medical diagnosis of comparison/controlsIntervention quantification method (western blot or ELISA)Age: mean age of patients and controlsSex: of patients and controlsDiagnosis method of patientsGenetic factorsSample size: of patients and controlsStudy characteristics to be extracted:Two similar searches were done, with the similar objectives. These were to look at the effect size of lymphocytic bound TDP-43 in FTD, ALS and FTD-ALS patients compared to controls, and the effect size of plasma TDP-43 in FTD, ALS and FTD-ALS compared to controls. However, neither of these searches produced enough papers in order to do a systematic review. AND (Lymphocyte [MeSH] OR White Cell [MeSH] or White Blood Cell [MeSH]) AND TDP [MeSH]. AND (Plasma [MeSH] OR Serum [MeSH] OR Blood [MeSH]) AND TDP [MeSH]2-values, and a funnel plot and Egger’s regression test was used to assess publication bias.An individual meta-analysis was carried out for each group of patients (i) FTD alone, (ii) ALS alone and (iii) FTD and ALS patients combined as an overall evaluation of FTD-ALS spectrum disorders. Additional subgroup analyses included: an assessment of heterogeneity and a quality score. Outcome measures were calculated for each of the studies identified and included on a forest plot. Given the variability of intervention quantification included in the analysis, outcome measures were recorded in standardised mean differences (SMD), to allow for meaningful comparisons between studies. SMD was compared using Hedges g-statistic, to account for bias from small sample sizes, using a random effects model. SMDs were reported as odds ratios with 95% confidence intervals. Heterogeneity was assessed for all outcome measures using Ip = 0.8029) or funnel plot. Systematic review, risk of bias and quality assessment revealed that only 3 of the 7 identified papers reported on measures to reduce bias, such as blinding  demonstrating a significant increase in detectable TDP-43 in the CSF of patients with FTD or ALS compared to controls. However, Chi2 was 20.57 and I2 was 76% showing significant heterogeneity in this dataset . There was a similarly significant heterogeneity with a Chi2 of 10.75 and an I2 of 81%. Meta-analysis of studies assessing TDP-43 in the CSF of patients with ALS alone showed an effect estimate of 0.64 (95% CI: 0.10–1.19). A Z-score of 2.3 (p = 0.02) showing a significant increase in detectable TDP-43 in the CSF of ALS patients. Chi2 was 11.80 and I2 was 66% showing moderate heterogeneity  FTD-ALS spectrum disorders (ALS and FTD combined), (ii) FTD alone, and (iii) ALS alone. Meta-analysis of FTD-ALS spectrum disorders versus controls using standard mean difference found an effect estimate of 0.55 (95% CI: 0.01–1.09) and a Z-score of 2.0  showed a significantly increased CSF TDP-43. We also detected substantial heterogeneity and relative risk of bias in these studies. This heterogeneity is likely due to the wide variety of methods used to analyse CSF TDP-43 concentrations, but could also reflect the disease heterogeneity  seen in these spectrum disorders and the heterogeneity of controls used for comparison. Two of the studies used normal healthy controls , 20 whilC9orf72 hexanucleotide expansion might provide an indication as to how early TDP-43 aggregation occurs, and potentially would allow us to diagnose FTD-ALS spectrum disorders pre-symptomatically in these patients [C9orf72) that might skew data. The study should be carried out longitudinally in order to map the progression of TDP-43 in both controls and patients. Diagnosis would be done using currently approved diagnostic tools, with post-mortem pathological verification of diagnosis. Both the sampler and the pathologist should be blinded to whether the CSF is from a control or a patient. Equally, analysis should be done with an ELISA and presented in empirical concentrations to improve transparency. As of yet, there are not enough studies into the TDP-43 in the blood, either in lymphocytes or in plasma. However, these are avenues for future meta-analyses (given more studies), as they provide less invasive diagnostic tests than lumbar puncture.Given the significant effect size shown in this meta-analysis, demonstrating an increase in the levels of detectable TDP-43 in patients with ALS, further research to refine the use of CSF TDP-43 as a diagnostic tool is warranted. Furthermore, a longitudinal study of CSF TDP-43 concentrations in patients with FTD-ALS spectrum disorders might show if CSF TDP-43 increases with disease progression. Similarly, a longitudinal study investigating carriers of the patients . This woOur data demonstrate a significant difference in TDP-43 levels in ALS patients compared to controls, implying that CSF TDP-43 levels show promise as a potential biomarker in ALS. It is probable that the statistically significant result observed in FTD-ALS spectrum disorder cases Fig. , could bOur systematic review and meta-analysis reveals early supportive data indicating that TDP-43 detected in the CSF of patients with FTD-ALS spectrum disorders in particular ALS patients could be a promising biomarker in these diseases. Given the current paucity of diagnostic and prognostic biomarkers in these disorders, this result clearly indicates that further studies in to its utility are warranted.


Aiming to improve the treatment outcomes of current daily tuberculosis (TB) chemotherapy over several months, we investigated whether nanoencapsulation of existing drugs would allow decreasing the treatment frequency to weekly, thereby ultimately improving patient compliance. Nanoencapsulation of three first-line anti-TB drugs was achieved by a unique, scalable spray-drying technology forming free-flowing powders in the nanometer range with encapsulation efficiencies of 82, 75, and 62% respectively for rifampicin, pyrazinamide, and isoniazid. In a pre-clinical study on TB infected mice, we demonstrate that the encapsulated drugs, administered once weekly for nine weeks, showed comparable efficacy to daily treatment with free drugs over the same experimental period. Both treatment approaches had equivalent outcomes for resolution of inflammation associated with the infection of lungs and spleens. These results demonstrate how scalable technology could be used to manufacture nanoencapsulated drugs. The formulations may be used to reduce the oral dose frequency from daily to once weekly in order to treat uncomplicated TB. M. tuberculosis (M.tb), has followed humanity for millennia [Tuberculosis (TB) is arguably the best established human infectious disease in existence. The disease, caused by infection, usually of the lungs, with the bacterium illennia . The Worillennia .The current drug treatment regimens are effective for drug-susceptible TB, but its duration over months and toxic side effects increase the risk of non-compliance with concomitant relapse and development of drug resistance . The incIn order to reduce the TB deaths by 90% by 2030 as promoted by WHO ,6,7. VarOne recent success of an orally delivered nanoencapsulated drug system was demonstrated by Horvati and colleagues (2015) who reported a Poly DL-lactic-co-glycolic acid polymer (PLGA) nanoparticle for the encapsulation of a pyridopyrimidine derivative showing improved anti-mycobacterial activity and low toxicity compared to the unencapsulated drug in an infected guinea pig model . Even a The entrapment of the therapeutic agents in biodegradable polymeric nano-systems designed to enhance targeted entry, slow-release, and retention of the antibiotics in the cells for longer periods should not only achieve an improved minimum inhibitory concentration, but also reduce dose frequency. Thus, it should improve patient compliance and reduce systemic side effects associated with conventional free anti-TB drugs. Previously, we have demonstrated the safety of orally administered uncoated PLGA nanoparticles in an in vivo mouse model. No pathological lesions or inflammation as assessed by histopathology assays were observed, compared to controls. It was also illustrated in the same study how the migration of these particles occurred from the gastrointestinal tract after oral administration to various organs of the body . When PEW: 45,000–75,000), partially hydrolysed (87–89%) Polyvinyl alcohol (PVA) MW 13,000–23,000, polyethylene glycol (PEG) MW 9 000, lactose monohydrate, Chitosan , glycerol, Tween 20, Pluronic F127™ and salts for the preparation of phosphate buffered saline were purchased from Sigma-Aldrich Chemical Co., . Difco Middlebrook 7H9 medium and OADC  were from Becton Dickinson and Company .Rifampicin (RIF), Pyrazinamide (PZA), Isoniazid (INH), PLGA 50:50  was used . BrieflyC57Bl/6 female mice were bred under specific pathogen-free conditions at the University of Cape Town and used between 8–12 weeks. Infected animals were maintained in individually ventilated cages under biosafety level 3 conditions. All experiments were approved by the Research Ethics Committee of the University of Cape Town, Cape Town, South Africa.M.tb H37Rv was grown in Difco Middlebrook 7H9 medium containing 0.5% glycerol and enriched with 10% OADC. Cultures were incubated at 37 °C and grown until log phase, aliquoted and maintained as frozen stocks at −70 °C. For infection, an aliquot of M.tb H37Rv was rapidly thawed at 37 °C, passed 30 times through a 29.5 G needle and diluted in sterile saline. Mice were infected using a Glas-Col Inhalation Exposure System Model A4224. The pulmonary infection dose of 50–100 cfu/lung was confirmed by 10 mice one day after infection and plating the homogenised lung tissue on Difco Middlebrook 7H10 agar plates in 10-fold serial dilutions. Plates were semi-sealed in plastic bags, incubated for 17–21 days at 37 °C, after which the number of mycobacterial colonies were counted, and the infection dose calculated.Isoniazid containing PLGA (INH-PLGA), rifampicin containing PLGA (RIF-PLGA) and pyrazinamide containing PLGA (PZA-PLGA) nanoparticles were prepared in 20% v/v Tween/Saline solution at concentrations based on the daily standard therapy doses prescribed for human adults. Empty PLGA particles were similarly prepared. For concentration calculations, the encapsulated drug weight and not the total particle weight was used. Encapsulated nanoparticles were therefore prepared at the following concentrations: INH-PLGA = 5 mg/kg/day, RIF-PLGA = 10 mg/kg/day, PZA-PLGA = 20 mg/kg/day.For preparation of chemotherapy in drinking water, it was assumed that a 20 g mouse would drink 2.5 mL–5 mL water/day. Chemotherapy in drinking water was therefore prepared at the following concentrations: INH = 40 mg/L, RIF = 80 mg/L, and PZA = 160 mg/L to provide a final therapeutic dose of INH = 5–10 mg/kg, RIF = 10–20 mg/kg, and PZA = 20–40 mg/kg.M.tb H37Rv at a dose of 50–100 cfu/lung, the infection was allowed to be established for three weeks and the bacilli burden determined. Thereafter, infected animals received therapy for durations of four weeks, six weeks, and nine weeks respectively in three separate experiments. Each experiment (n = 10) contained the following treatment groups namely (1) untreated (UT-control), (2) empty particles only (NP-control), (3) chemotherapy in water (CT-water), (4) encapsulated particles chemotherapy (CT-NP). Empty nanoparticles and encapsulated drug particles were administered in a volume of 200 µl by gavage every seven days to respective groups at doses 7x the daily dosage to ensure sufficient drug availability for the 7-day duration. CT-water was always available in the drinking water of the animals in that group for the treatment duration.Upon infection with At the end of treatment, all animals were sacrificed by halothane administration. The spleens and lungs were removed and weighed. The left lung lobes were fixed in 4% paraformaldehyde for sectioning and subsequent hemoxylin/eosin and Ziehl Neelson staining for acid-fast bacilli. The rest of the lungs were processed for bacilli burden determination.The data are expressed as the mean ± SEM. Statistical analysis was performed by ANOVA  using GraphPad Prism software (version 4.01). For all tests, significance was determined at p-value < 0.05.dK is a constant, nD is the orifice diameter of the nozzle, and fŋ and gŋ are kinematic viscosity of feed and gas, respectively. fF and gF are the flow rates of feed and gas whereas eW is the Weber dimensionless number correlating the gas pressure acting on the liquid to the liquid capillary pressure (surface tension) [pD is the diameter of the dried particle, dD is the diameter of the droplet before drying, sY is the solid concentration in the spray solution, dρ is the spray solution droplet density and pρ the density of the dried particle [Free-flowing powder of drug-loaded polymeric nanoparticles was obtained by a novel technique consisting of spray drying a multiple emulsion. It is extensively reported in literature that for a spray-drying process using a pneumatic or multi-fluid nozzle for atomisation of a liquid or suspension, the obtained droplet size is influenced by the feeding rate, surface tension and viscosity of the feed solution, the nozzle diameter as well as the pressure and density of the atomising fluid (air or inert gas such as nitrogen). Usually, the finest droplet size that can be achieved with the current nozzle configuration ranges between 10–100 μm. The average particle size D50 of a sample can be predicted by Lubanska’s equation as follows:tension) . Severalparticle . In ordeAll formulations yielded free-flowing powder after spray-drying. As depicted in Evaluation of the surface morphology of the particles was conducted using atomic force microscopy (AFM).All the formulations prepared for the study had a similar topography as presented by M.tb H37Rv in an aerosol inhalation challenge mouse model were investigated. After a 3-week infection period, all animals were treated for nine weeks thereafter. The treatment consisted of a daily oral dose of free INH/RIF/PZA (abbreviated CT-water) and a once-off weekly dose of the encapsulated drugs (abbreviated CT-NP). Untreated (UT) mice and empty nanoparticles (NP) served as controls. Each test group consisted of 10 mice.In this study, the bactericidal efficacy of INH-PLGA, RIF-PLGA, and PZA-PLGA nanoparticles against M.tb H37Rv infectious dose administered in all three experiments was verified to be between 50–100 cfu/lung by enumeration of colonies obtained after 18–24 h infection by serial dilution of homogenised lung tissue. After three weeks, the establishment of pulmonary infection was calculated to be between 1 × 105–1 × 106 cfu/lung and dissemination to the spleen averaged at 3 × 103 cfu/spleen. These were normal and well within published ranges. The overall kinetic changes in lung, spleen and body weights over the experimental period are represented in The It is well established that infection will induce inflammation and recruitment of cells to sites of infection. Here lung and spleen weights were used as surrogate markers to reflect increased inflammatory responses as measured in p > 0.05). Measurement of lung weights reflected significant differences (p< 0.01) between UT-control groups and CT-water control groups indicating the resolution of inflammation in the CT-water treated group. Similarly, a significant difference (p< 0.01) between NP-control treated mice and CT-NP treated mice were found indicating the resolution of inflammation in CT-NP treated mice. There was no significant difference between the UT-control and NP-control treated mice indicating that the treatment with the empty particles did not influence the resolution of inflammation. Therefore, the particles in the lungs appeared to be inert and did not contribute to enhanced inflammation. It was noted that the lung weights in the CT-water treated group was significantly lower (p< 0.01) than that of the CT-NP treated group at this time point. This indicated that resolution of inflammation in the CT-water treated group happened faster than that of the CT-NP treated animals.At six weeks after treatment i.e., nine weeks post-infection similar observations were noted with respect to lung and spleen weights as at four weeks, again with no significant differences in weights noted in the spleens of the different groups. However, the results differed in one respect, in that comparison of lung weights between the CT-water treated group and the CT-NP treated group no longer showed a significant difference after six weeks of treatment, indicating an equivalent resolution of inflammation at the longer treatment time point. These two groups also showed significant differences towards their control counterparts.p< 0.001) was noted in spleen weights between the UT-control group and CT-water group, indicating the resolution of inflammation in the spleen in the CT-water treated animals. Interestingly, significant spleen weight differences (p< 0.05) were also noted between the NP-control group and the CT-NP treated group, suggesting resolution of splenic inflammation in animals treated with drug encapsulated particles. Spleen weights of both the UT-water and NP-control groups were not significantly different, again indicating no enhancing effect by the empty nanoparticles on the induction of inflammation. Importantly, resolution of splenic inflammation in CT-water treated and CT-NP treated groups were found to be equivalent, indicating that both treatment approaches were effective for resolution of TB induced inflammation associated with the spleen after nine weeks of treatment.At nine weeks after treatment, i.e., 12 weeks post-infection, lung weights essentially reflected the same differences obtained at six weeks after treatment. However, for the first time a significant difference  between the UT-control group and the CT-water group was noted with bacilli burdens 10–100 times lower in the CT-water treated group for both the lungs and spleens, indicating effective inhibition of bacterial replication when drugs were administered free in drinking water. Moreover, significant lower bacilli burdens were also noted in the CT-NP treated group compared to the NP-control group (p< 0.001) or the UT-control group (p< 0.01). Although the mean of the CT-NP treated group was higher than that of the CT-water treated group, there was no significant difference between the two groups for either the lungs or the spleens. Similar differences were noted after six weeks of chemotherapeutic treatment. The overall bacilli burden for the lungs was however 10–100 times lower after six weeks treatment compared to four weeks treatment in all the groups that received chemotherapy either as encapsulated drugs or as free drugs in drinking water.ur weeks A, six weur weeks B, and niur weeks C of chemM.tb infected mice with free drugs administered in water resulted in clearance of bacilli in the lungs (undetectable levels <10 cfu) in 8/10 mice and complete clearance in the spleen. Pulmonary burdens in animals treated with encapsulated drugs ranged from 1 × 102–1 × 103 cfu whereas complete clearance in the spleen was noted for 4/9 mice.No detectable bacilli were noted in the spleens of animals which received chemotherapy as free drugs after six weeks of treatment (the detection limit is 10 cfu), suggesting complete clearance. In contrast, detectable burden levels, although low, were measured in CT-NP treated mice. The relative bactericidal activity after nine weeks of chemotherapeutic treatment in the lungs and spleens of Several positive outcomes of this study were found by making use of an easily scalable spray-drying technology to produce formulations in the nano-size range, namely the experiments reproducibly demonstrated that chemotherapy by nanoencapsulated anti-TB drugs matches the effect of administration of free drugs in respect of (a) controlling the inflammation, (b) the bactericidal properties, and (c) the non-toxic nature of the particles.Poor patient compliance represents a major challenge for drug-susceptible and drug-resistant TB treatment. Apart from severe side effects, another factor for poor patient compliance is the daily drug regimens that are extended over at least nine months of treatment. Although the latter is managed by directly observed treatment (DOTS) protocols, it is cumbersome to both the patients and clinical staff ,21. TherThree first-line anti-TB drugs, INH, RIF and PZA, were incorporated separately into the biodegradable PLGA particles through a unique spray-drying encapsulation technology. This resulted in free-flowing powders in the nano-size range, which could be easily dispersed into solution. Due to the varying physical properties of the drugs, their encapsulation efficiencies differed. Therefore, these compounds were encapsulated separately to rule out any bias on the dosing concentrations for the animals as compared to combined formulations where mean drug encapsulation efficiencies were used ,6.M.tb H37Rv infected mouse model was used to evaluate the efficacy of these nanoencapsulated drugs in a once-off weekly dose, compared to the conventional unencapsulated drugs that were administered daily.During the manufacturing process, these PLGA particles were coated with a PEG/chitosan solution in order to reduce protein binding and extend the circulation time of the particles in the blood . PreviouUpon infection, disease manifestation was allowed for three weeks whereafter the treatments were initiated for periods of four, six, and nine weeks. Lung and spleen weights were used as indicators to reflect any inflammatory responses ,23. The The rationale behind the increased dose that was used for the once a week encapsulated drugs compared to the daily dose was due to increasing reports in the literature on the limited uptake of polymeric nanoparticles after oral delivery, most probably due to the gastrointestinal mucus barriers. One such example was when samarium oxide- encapsulated PLGA nanoparticles, after oral delivery in rats, were shown to be distributed throughout most organs but the majority of particles remained within the intestines .M.tb replication. More specifically, there is a distinct benefit for longer treatment with encapsulated particles when comparing data of for weeks to that of six and nine weeks of treatment. Treatment with drug encapsulated nanoparticles also resulted in a rapid decline in bacilli burden, demonstrating effective delivery of drugs by nanoparticles. Although the rate of mycobacterial killing using free drugs was higher compared to the encapsulated drugs for the time period, it was promising to note that there was a tendency towards bacterial clearance and that the rate of bacterial killing remained consistent after four weeks when treating animals with drug encapsulated nanoparticles.The bactericidal efficacy of a once-off weekly dose was similar when compared to the conventional daily dose treatment. The data clearly indicated effective inhibition of bacilli replication with chemotherapy treatment delivered in the form of free drugs in drinking water. Importantly, the data showed that treatment with drugs encapsulated in PLGA and delivered once every seven days by oral gavage is equally effective to inhibit These observations are in accordance with previous studies done on polymeric particles where the potential to reduce the dosing frequency was shown without compromising the efficacy of the drugs ,27,28,29Apart from solving the shortfalls of free drug treatment, another advantage of nanoencapsulated anti-TB drugs to consider is their ease of manufacturing in terms of scalability and cost-effectiveness . The newReformulation of existing anti-TB chemotherapy by nanoencapsulation holds various advantages, clinical data, stretching over several decades, of the actual anti-TB drug gives guidance to the limitations that should be addressed by reformulation into nanoparticles. These nanoparticles could offer the advantage to introduce slow-release of the drug, leading to the opportunity of reducing the number of administrations and increasing patient compliance. In addition, slow-release provides the benefit in reducing the systemic exposure and thereby the toxic side effects of the drug itself. Encapsulation also protects the pharmaceutical active from the acidic gastric environment for oral delivery and is particularly relevant for molecules such as RIF that is degraded at such low pH values . A lot o


Background. The main purpose of this study was to explore the body-mass index and waist circumference associated with physical fitness by gender. Methods. In this cross-sectional study, we included 1036 adolescents (55.4% girls) from eight randomly selected secondary schools within the city of Zagreb (Croatia). Body-mass index and waist circumference were objectively measured. Physical fitness included three tests: (1) 1 min sit-ups, (2) standing long jump and (3) a sit-and-reach test. Associations were calculated using linear regression models. Results. Boys had higher body-mass index and waist circumference values, compared to girls (p < 0.001). They also performed better in 1 min sit-ups and the standing long jump tests (p < 0.001), while girls obtained higher values in the sit-and-reach test (p < 0.001). In boys, body-mass index and waist circumference were associated with 1 min sit-ups and the standing long jump. In girls, waist circumference was also associated with 1 min sit-ups and the standing long jump, while body-mass index was only associated with this standing long jump. Conclusions. Our study shows that anthropometric indices have non-linear associations with physical fitness tests in a large sample of Croatian adolescents. Screening for thinness and obesity to predict the level of physical fitness should be of a great interest. Obesity has become a major public health problem in the past three decades, especially in children and adolescents . SpecifiPhysical fitness represents a significant part of physical activity . It is dSeveral previous studies have examined the association between overweight/obesity (by using the body-mass index indicator) and physical fitness ,13,14,15Results of those studies were consistent with each other, showing a non-linear association between the body-mass index and several fitness tests; that is both thin and overweight/obese participants scored lower on the physical fitness tests. According to the aforementioned, more studies have been using the body-mass index as an indicator of nutritional status, yet the body-mass index cannot differ fat mass from free-fat mass, and may lead to a misclassification . Thus, sAfter an extensive literature review, there has been a lack of studies examining combined non-linear associations between body-mass index and waist circumference with physical fitness. Therefore, the main purpose of the study was to explore the body-mass index and the waist circumference associated with physical fitness in a large sample of Croatian adolescents aged 15–18 years by gender. We hypothesized that the association between all anthropometric indices and physical fitness would be non-linear with a parabolic shape.In this cross-sectional study, participants were secondary-school students. A detailed study protocol has been described elsewhere . In brie2) was calculated as weight (in kilograms) divided by the square of height (in meters). WC was measured for each participant remaining still in a standing position. We used anthropometric tape placed horizontally midway between the lower rib margin and the iliac crest at the end of normal expiration [p < 0.001).Body height was measured to the nearest millimeter in bare or stocking feet with the adolescent standing upright against a stadiometer . The result was given in meters. Body weight was measured to the nearest 0.1 kg, and the participant wore light clothes with no shoes . The result was given in kilograms. BMI  .Sit-ups in 1 min: Trunk strength was assessed as the maximum number of sit-ups achieved in one minute. Children were seated on the floor, backs straight, hands clasped behind their neck, knees bent at 90°, with their heels and feet flat on the mat. They then lay down on their backs, shoulders touching the mat, and returned to the sitting position with their elbows out in front to touch their knees, keeping the hands clasped behind their neck the whole time. The total amount of correctly performed sit-ups in 60 s was the score .Sit-and reach test: Sitting on the floor or upon a mat, legs straight under the angle of 90°, the person being tested reached forward with the arms (hands overlapping). The distance of reach was measured in centimeters using a measuring non-elastic tape attached to the floor .p ≤ 0.05, and it was two sided (2-sided). All of the analysis were performed in the Statistical Package for Social Sciences Software, ver. 22 .Data are presented as mean (SD). All variables were grouped according to sex (boys vs. girls). First, we calculated the differences between the sexes in variables which were analyzed using univariate analysis of variance. Previous studies have shown sex differences in perceived and estimated physical fitness . Therefop < 0.001). They also performed better in 1 min sit-ups (ES = 0.81) and standing long jump (ES = 1.69) tests (p < 0.001), while girls obtained higher values in the sit-and-reach test .Basic descriptive statistics of the study participants are presented in Quadratic associations between anthropometric indices and physical fitness tests and overall physical fitness for both boys and girls are presented in The main question of the study was as to whether body-mass index and waist circumference were associated with some aspects of physical fitness. This study shows that both body-mass index and waist circumference are associated with physical fitness tests, especially 1-min sit-ups and the standing long jump.Our results of the association between body-mass index and physical fitness are in line with previous studies conducted among a sample of children ,15,17 anOur study also shows a non-linear association between waist circumference and standing long jump and overall physical fitness score in boys, and overall physical fitness score in girls. Only a few previous studies have examined the aforementioned association ,12,14. HN = 1036). Second, we used both body-mass index and waist circumference as indicators of obesity. Third, we presented non-linear regression results, regarding anthropometric indices and some aspects of physical fitness.This study has several strengths. First, we conducted our study on a relatively large sample of secondary-school students (Our study has several limitations. First, by using a cross-sectional design, we cannot conclude the causality of the correlation. Second, to assess objectively estimated physical fitness, we only used musculoskeletal fitness. Previous studies have used aerobic capacity as the measure of physical fitness ,15,16,17Our study shows that anthropometric indices are associated with some aspects of physical fitness tests in a large sample of Croatian adolescents. Although overweight and obesity have become a major public health problem worldwide, our study also shows that thin adolescents might also be a group at risk of performing poorly on physical fitness tests. Therefore, screening for both thinness and obesity to predict the level of physical fitness should be of great interest for future longitudinal research, and those findings might serve as certain recommendations for optimal nutritional status and better physical fitness performance in adolescents.


The polyphenolic compounds in the pyrolytic lignins which are responsible for radical scavenging activity and antioxidant properties can be readily profiled with HPLC-MS/MS combined with untargeted metabolomics. The results also suggest that DPPH, TEAC, and TPC assays are suitable methods for the measurement of antioxidant activity in a variety of pyrolytic lignins. These data show that the pyrolytic lignins can be considered as promising sources of natural antioxidants and value-added chemicals.A challenge in recent years has been the rational use of forest and agriculture residues for the production of bio-fuel, biochemical, and other bioproducts. In this study, potentially useful compounds from pyrolytic lignins were identified by HPLC-MS/MS and untargeted metabolomics. The metabolites identified were 2--1-(4-hydroxy-3-methoxyphenyl)-1-propanol, benzyl benzoate, fisetinidol, phenyllactic acid, 2-phenylpropionic acid, 6,3′-dimethoxyflavone, and vanillin. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH), trolox equivalent antioxidant capacity (TEAC), and total phenolics content (TPC) per gram of pyrolytic lignin ranged from 14 to 503 mg ascorbic acid equivalents, 35 to 277 mg trolox equivalents, and 0.42 to 50 mg gallic acid equivalents, respectively. A very significant correlation was observed between the DPPH and TPC ( Renewable plant biomass holds an eco-friendly and promising future in the years ahead, since it is an effective alternative source of renewable energy in the current scenario of depleting oil reserves, global warming, and growing environmental concerns. The biomass can be converted to other valuable forms of energy by a number of processes such as biological, mechanical and chemical. However, thermal processes  can yield complex chemicals at a very high yield (75 wt %) .Pyrolysis has been applied for thousands of years for charcoal production and historical use of pyrolysis dates back to ancient Egyptian times, when they were using pyrolysis for the preparation of tar for caulking boats and as an embalming agent . In receFast pyrolysis is regarded as one of the most promising thermo-chemical conversion techniques for producing liquid biofuels . In addiPyrolysis is able to provide material that has a wide range of qualities for a variety of applications ,5. Bio-oPyrolytic lignin derived from fast pyrolysis is a very complex phenolic polymer that can be obtained from agriculture and forestry residues. In general, lignins are classified as three-dimensional amorphous polymers consisting of methoxylated phenylpropane structures . The impDespite the significance of lignin as a valuable bio-polymer, its real industrial potential has not been developed. In order to develop new practical applications from lignin, there is strong need to characterize its chemical reactivity and functional properties  that areLignins have antioxidant potential, which is their capacity to quench the production of free radicals, and their ability to stabilize free radicals induced by oxygen and its radical species. A better understanding of natural antioxidants from pyrolytic lignins will facilitate their use in the food industry and as feedstock as value-added chemicals to preserve food, colour, flavor, and active vitamin contents . The incp < 0.05) from the pyrolytic lignins. The significance of the heat map lies in its unique ability to identify clusters of samples with similar metabolic patterns and discriminating metabolites that can drive the sample clustering https://xcmsonline.scripps.edu) bioinformatics platform [m/z = 15 ppm, minimum peak width = 10 s and maximum peak width = 120 s); obiwarp settings for retention-time correction (prof Step = 1); and parameters for chromatogram alignment, including mzwid = 0.015, minfrac = 0.5 and bw = 5. The relative quantification of metabolite features was based on peak areas [Data were analyzed using multi-group method of XCMS online (platform . The HPLak areas .Antioxidant activities of the pyrolytic lignins were determined using three methods (i) 2,2-diphenyl-1-picrylhydrazyl scavenging effect (DPPH); (ii) trolox equivalent antioxidant capacity assay (TEAC); and (iii) total phenolics assay (TPC). TEAC and TPC are an electron transfer (ET) based assays, whereas DPPH is mainly ET based and has a hydrogen-atom abstraction mechanism (HAT) . Due to DPPH radical-scavenging activity was determined using by microplate assay with some modifications of Yu and coworkers . Briefly•+ radical scavenging activity of pyrolytic lignins were determined after Re and coworkers [•+, which is a blue-green chromophore and has a characteristic absorption at 734 nm. ABTS•+ solution was prepared, thereby mixing ABTS salt (7 mM) with potassium persulfate (2.45 mM). The solution was prepared 12–16 h before use and was stored in the dark at room temperature. The ABTS•+ solution was diluted with methanol to obtain an absorbance of 0.70 (±0.002) at 734 nm. In general, the addition of antioxidants to the preformed radical cation decolourizes the ABTS•+, which is then reduced to ABTS. Standards and samples (of appropriate dilution) were prepared in methanol at various concentrations and 300 µL of ABTS•+ was added to the reaction mixture. The reaction contents were incubated in TECAN plate reader at 25 °C and readings were obtained after 6 min. The results were expressed as mg trolox equivalents/g of pyrolytic lignin. Trolox  was used as a standard.The ABTSoworkers  with somTotal phenolics content (TPC) of the pyrolytic lignins was determined after Singleton and coworkers  with somp < 0.05. In addition, correlation analysis between DPPH, TEAC and TPC was performed using the Pearson product moment correlation method. CoStat  was used for the statistical analysis.All assays were performed in triplicates and the results obtained were analyzed using one-way analysis of variance (ANOVA) to compare the data (mean). Furthermore, means were separated using the Student-Newan-Keul’s test, where The current study provides a first report on the HPLC-MS/MS combined with untargeted metabolomic analysis of pyrolytic lignins. The untargeted metabolomics provided a useful tool for the rapid identification of metabolites from pyrolytic lignins. The HPLC-MS/MS and untargeted metabolomics revealed that pyrolytic lignins have high compound amounts and high antioxidant activity (as determined by antioxidant assays) in contrast to non-pyrolytic lignins. Over all, the autonomous untargeted metabolomics provided an efficient means of high-throughput metabolomic profiling of lignins derived from fluidized-bed fast pyrolysis. Pyrolytic lignins contain natural antioxidants that can be used as a source of value-added chemicals. The results also show that the pyrolytic lignins have a varying level of antioxidant activity, which is related to the presence of phenolic compounds. It is likely that novel compound from pyrolytic lignins may be extracted and can be used as natural antioxidants in the food, pharmaceutical and nutraceutical industry. Lignin-derived value-added chemicals can provide food additives that are natural, safe, and acceptable by consumers. Furthermore, TEAC, DPPH, and TPC assays are suitable methods for the analysis of antioxidant activity of pyrolytic lignins.


The exact route of iron through the kidney and its regulation during iron overload are not completely elucidated. Under physiologic conditions, non-transferrin and transferrin bound iron passes the glomerular filter and is reabsorbed through kidney epithelial cells, so that hardly any iron is found in the urine. To study the route of iron reabsorption through the kidney, we analyzed the location and regulation of iron metabolism related proteins in kidneys of mice with iron overload, elicited by iron dextran injections. Transferrin Receptor 1 was decreased as expected, following iron overload. In contrast, the multi-ligand hetero-dimeric receptor-complex megalin/cubilin, which also mediates the internalization of transferrin, was highly up-regulated. Moreover, with increasing iron, intracellular ferritin distribution shifted in renal epithelium from an apical location to a punctate distribution throughout the epithelial cells. In addition, in contrast to many other tissues, the iron exporter ferroportin was not reduced by iron overload in the kidney. Iron accumulated mainly in interstitial macrophages, and more prominently in the medulla than in the cortex. This suggests that despite the reduction of Transferrin Receptor 1, alternative pathways may effectively mediate re-absorption of iron that cycles through the kidney during parenterally induced iron-overload. The most iron consuming process of the body, erythropoiesis, is regulated by the renal erythropoietin producing cells in kidney interstitium. We propose, that the efficient re-absorption of iron by the kidney, also during iron overload enables these cells to sense systemic iron and regulate its usage based on the systemic iron state. The kidneys are extremely sensitive to heme and hemoglobin exposure during hemolytic anemias –4. In coMost iron transport proteins including TfR1 are regulated by iron to maintain cellular and systemic iron homeostasis. Iron uptake by TfR1 is post-transcriptionally down-regulated by the Iron Regulatory Protein (IRP)-system when the cellular iron is high . The twoAnother promising candidate receptor that permits renal Tf and iron reabsorption during iron overload is the multi-ligand receptor, cubilin . Cubilin2+) [2+ is transported by the divalent metal transporter 1 (DMT1), that is expressed apically in the Loop of Henle [Non-transferrin bound iron (NTBI) may be bound to albumin which is also taken up through the megalin-cubilin complex or it is bound to small molecules such as citrate and is taken up through their transporters or is reduced and reabsorbed as ferrous iron (Fe2+) –23. Fe2+of Henle  or throuof Henle . Thus, DOnce iron is absorbed into the epithelial cells, it is either used for cellular needs or basolaterally exported. Intermittently iron is stored in the iron storage protein ferritin which is also regulated by the IRPs but in the opposite direction of TfR1, as IRP binding to the two ferritin-subunit transcripts in low iron conditions inhibits ferritin translation. Thus, ferritin is highly expressed during iron overload, as well as in mice with targeted deletion of IRP2 .Ferroportin (FPN) is the only ferrous iron exporter known to date and is located basolaterally on kidney epithelium, where it mediates the exit of iron from the epithelial cell and thus completes re-absorption . FPN is In this research we studied proteins involved in iron re-absorption through the kidney during Parenterally induced systemic Iron Overload (PIO). PIO was elicited by iron dextran-injections, that mimic parenteral administration of iron supplements. We found that TfR1 is indeed down-regulated. In contrast, cubilin is highly up-regulated during PIO. We further show evidence that the cubilin-regulation by iron is post-transcriptional and may be mediated by an iron-dependent stabilization of cubilin through the transcriptional up-regulation of megalin. In epithelial cells, the intracellular ferritin distribution is shifted towards the basolateral membrane, close to the interstitium. In the interstitium, iron and ferritin accumulate mainly in macrophages, and more prominently in the medulla than in the cortex. This stands in contrast to iron absorbed through the diet, where iron accumulates mainly in the renal epithelium.Animal experiments were done according to protocols approved by the Technion Animal Ethics Committee, Haifa, Israel. All mice were on C57Bl/6J background. PIO was elicited in 3 month-old male mice by 5 daily intra-peritoneal injections of 100μl iron-dextran solution . Three days after the last injection mice were sacrificed, kidneys were collected, frozen in liquid nitrogen and stored at -80°C.Kidneys and spleens were collected and fixed in 10% neutral buffered formalin or 4% PFA, respectively. The kidneys were trimmed mid-longitudinally, to include the cortex, medulla and papilla, and then embedded in paraffin, sectioned to a thickness of approximately 5 microns and stained with Hematoxylin & Eosin (H&E) for histology and Perl’s Prussian blue (PPB) for ferric iron detection. Histopathological changes were analyzed and scored using a semi-quantitative grading of five grades (0–4), taking into consideration the severity of the changes . Spleens were prepared and analyzed similarly.2O. For IHC, quenching of endogenous peroxidase was performed by incubating the slides in 3% H2O2 in a humid chamber following by ddH2O washes. Next, sections were blocked with 10% normal goat/donkey serum (Jackson) in PBS containing 0.1% bovine serum albumin  that was chosen according to the animal in which the secondary antibody was raised. Then, sections were incubated overnight (ON) at room temperature (RT) in a humidified chamber with the following primary antibodies; polyclonal goat-anti-mouse cubilin , monoclonal mouse-anti-human TfR1 , polyclonal affinity purified rabbit-anti-mouse H- or L-ferritin  and polyclonal goat-anti-mouse CD68 . For IHC staining, sections were washed with PBS and incubated for 1 hour at RT with biotinylated goat-anti-rabbit or rabbit-anti-goat antibodies . Then, sections were incubated with Vectastain ABC kit following manufacturer's instructions. The immunohistochemical reaction was visualized using DAB (Sigma). Sections were dehydrated and mounted with Eukitt resin (Sigma). For IF staining, donkey-anti-goat 488, donkey-anti-rabbit 568 and goat-anti-mouse 488 were used as secondary antibodies and incubated for 1 hour at RT . Next, slides were washed with PBS and mounted with VECTASHIELD mounting medium containing DAPI (Vector laboratories). Negative controls were incubated with secondary antibodies only.Paraffin-embedded kidney sections from iron overloaded and control mice were heated in the microwave for 10 minutes for antigen retrieval with 6M urea in PBS  or with 0.01M citrate buffer (for H-ferritin) and rinsed with ddH2O2 in PBS), stained with PPB solution (2% K2Fe(CN)6, 2% HCL mixed freshly in a 1:1 ratio) for 1 h and washed several times with ddH2O. Positive staining appeared as a blue color under light microscope.Paraffin-embedded kidney sections from iron overloaded and control mice were blocked with peroxidase blocking solution . Samples were treated with DNase I recombinant, RNase free kit (Roche) according to the manufacturer's instructions. cDNA was synthesized using total RNA (1 μg) by qScript cDNA synthesis kit (Quanta biosciences), and was amplified using SYBER Green (Quanta biosciences) in AB 7300 (Thermo Fisher Scientific).Primers:TfR1 sense 5' TGG GTC TAA GTC TAC AGT GGC 3' and anti-sense 5' AGA TAC ATA GGG CGA CAG GAA 3';Cubilin sense 5' AGG CTG TGG AGG CAA TCT CA 3' and anti-sense 5' GGT AGT AGG GCA TCG GGT AGT 3';Megalin sense 5' AGG CCA CCA GTT CAC TTG CT 3' and anti-sense 5' AGG ACA CGC CCA TTC TCT TG 3';β-actin sense 5' AGC CTT CCT TCT TGG GTA TGG 3' and anti-sense 5' TCA ACG TCA CAC TTC ATG ATG G 3'.airSEM (B-nano LTD) analysis. First, slides were imaged with an optical microscope for sample orientation and selection of region of interest, images were acquired using 20X, 40X and 100X objectives, then the sample was moved automatically to the SEM, where matching fields were imaged using backscattered electron imaging as described.[Paraffin-embedded kidney sections were fixed on Superfrost microscope slides. High iron kidney slides were H&E stained and left uncovered for escribed., 36 ElemProteins separated on SDS-PAGE were transferred to a nitrocellulose membrane. For blocking, membranes were incubated in 5% BSA for 1 h at RT , in 3% BSA, ON, at 4°C for ferritin, and in 2% BSA and 3% milk ON, at 4°C for FPN. Next, the membranes were incubated ON, at 4°C, with the following first antibodies: Cubilin , actin , TfR1 , while the incubation with anti-ferritin antibodies was for 1 h at RT  and with anti-FPN antibodies was for 3 h at RT . HRP-conjugated anti-goat  or anti-rabbit  antibodies were used as secondary antibodies for analysis of cubilin/actin and TfR1/ferritin/FPN, following 1 hr incubation at RT.To study how iron transport is regulated during PIO, we caused PIO in mice by injecting iron dextran into the peritoneum. Histological analysis of kidney and spleen sections of these mice showed completely normal kidney morphology with well-defined glomeruli and tubules. In the kidney, no iron accumulation can be detected in H&E staining, without specific visualization of iron . In contTo understand how Tf-iron uptake systems are regulated in the kidney during iron-overload, we studied the expression of these systems, which are known to be expressed at the apical epithelial membrane. Both cubilin and TfR1 play a role in Tf and iron uptake to the epithelium and thus we localized both candidates . At the As expected, TfR1 protein and mRNA levels were decreased in the kidneys of iron overloaded mice , supportTo further investigate the elevated cubilin levels in kidneys of iron overloaded mice we wondered if megalin, that is part of the megalin-cubilin complex, is affected by iron as well. Indeed, megalin mRNA expression was highly elevated in the kidneys of iron overloaded mice , suggestIn renal epithelial cells of control mice, ferritin has a polarized appearance near the apical brush-border of the PT , 34. We To further study the route of iron through kidney epithelium, we asked whether the ferrous iron exporter FPN was regulated also in the kidney by the iron status of the mouse. We found that FPN was not reduced by PIO in the kidney, but was significantly reduced in the spleen of the same mice . In miceairSEM and metal content was analyzed by EDX (3+), which is mainly found in ferritin and hemosiderin. Indeed, ferritin immune-staining presented a very similar pattern to PPB stain and localized predominantly to the interstitium in the kidneys of PIO mice. Most of the ferritin co-localized with the CD68-macrophage-marker  and NCOA4 –45. In cTogether with albumin and many other plasma proteins, also a fraction of plasma–transferrin is filtered through the glomerulus, and re-absorbed from the primary urine into kidney epithelial cells. TfR1 is found apically on kidney epithelium  and Fig and takeOnce in the epithelial cells, iron needs to be transported across the cells. We have previously shown a possible role for ferritin in both intra- and intercellular iron trafficking in the Sertoli cells of the testis . There wFerrous iron can be exported through FPN, which is located basolaterally in kidney epithelium . In our 2) support an iron homeostasis with slightly higher concentrations of ferrous iron in solution than in the well-oxygenated cortex (6.6% O2) [Iron chemistry is dominated by the inter-conversion of ferrous and ferric iron , which a6.6% O2) , 59. ThuWe can think of two biological functions for efficient iron re-absorption of the kidney also when systemic iron is high. 1) Erythropoiesis is the most iron consuming process in the body and is regulated by erythropoietin (epo), made in the kidney interstitium . Epo is Taken together, we have evidence that during PIO, iron is transported efficiently across the epithelial barrier. TfR1 levels are low, but cubilin levels are high and the cubilin-megalin heterodimer likely plays a major role in iron transport from the primary urine back to the body. Ferritin is distributed throughout the epithelial cells and does not accumulate at the apical brush-border, suggesting that it may contribute to intra- and inter-cellular iron trafficking. FPN is not down-regulated by the high iron conditions and thus may export iron efficiently from the basolateral epithelium into the renal interstitium and also from interstitial macrophages. We suggest that the highly expressed cubilin-megalin complex mediates Tf-iron re-absorption during PIO both in the cortex and the medulla, where excess iron is stored predominantly in interstitial medullar macrophages. The strategy of shifting a significant part of iron re-absorption to the medulla during PIO may accelerate renal iron flux. In conclusion, we suggest that iron transport through the kidney epithelium is unique in its regulation, re-absorbing iron even when systemic iron is high. This may protect the host from uropathogenic bacteria and provide erythropoietin producing cells with important information on body iron stores.


To examine the epidemiology and outcomes of in-hospital cardiopulmonary resuscitation (CPR) among patients with cirrhosis.We used the Texas Inpatient Public Use Data File to identify hospitalizations aged ≥ 18 years with and without cirrhosis during 2009–2014 and those in each group who have undergone in-hospital CPR. Short-term survival  following in-hospital CPR was examined. Multivariate logistic regression modeling was used to assess the prognostic impact of cirrhosis following in-hospital CPR and predictors of short-term survival among cirrhosis hospitalizations.In-hospital CPR was reported in 2,511 and 51,969 hospitalizations with and without cirrhosis, respectively. The rate of in-hospital CPR  was 7.6 and 4.0 among hospitalizations with and without cirrhosis, respectively. The corresponding rate of in-hospital CPR among decedents was 10.7% and 13.4%, respectively. Short-term survival following in-hospital CPR among hospitalizations with and without cirrhosis was 14.9% and 27.3%, respectively, and remained unchanged over time on adjusted analyses among the former (p = 0.1753), while increasing among the latter (p = 0.0404). Cirrhosis was associated with lower odds of short-term survival following in-hospital CPR (adjusted odds ratio [aOR] 0.55 [95% CI: 0.49–0.62]). Lack of health insurance (vs. Medicare) (aOR] 0.47 [95% CI: 0.34–0.67]) and sepsis ([aOR] 0.67 [95% CI: 0.53–85]) were associated with lower odds of short-term survival following in-hospital CPR among cirrhosis hospitalizations.The rate of in-hospital CPR was nearly 2-fold higher among hospitalizations with cirrhosis than among those without it, though it was used more selectively among the former. Short-term survival following in-hospital CPR remained markedly lower among cirrhosis hospitalizations, while progressively improving among those without cirrhosis. Strategies to increase access to health insurance and improve early identification and control of infection should be explored in future preventive and interventional efforts. Cirrhosis is increasingly prevalent in the United States (US)  and affeDespite the abovementioned burdens, hospital survival of patients with cirrhosis has been improving , includiData are scarce on the contemporary epidemiology and outcomes of in-hospital CPR among patients with cirrhosis, with reported studies focusing on select older age groups , describContemporary data addressing these latter questions can inform future preventive and interventional efforts by clinicians and healthcare systems and may provide benchmark data for performance improvement initiatives.We conducted a population-level study of hospitalized patients with and without cirrhosis to: a) examine the comparative patterns of utilization of in-hospital CPR and its associated outcomes, overall and over time b) characterize the patients undergoing in-hospital CPR and c) determine the prognostic impact of cirrhosis following in-hospital CPR and the factors associated with short-term survival among those with cirrhosis.This was a retrospective, population-based cohort study. Because we used a publicly available, de-identified data set, the study was determined to be exempt from formal review by the Texas Tech Health Sciences Center’s Institutional Review Board. A detailed description of the study methodology is included in the Supplementary Materials (Appendix A in We used the Texas Inpatient Public Use Data File (TIPUDF), an administrative data set maintained by the Texas Department of State Health Services . The TIPWe initially extracted data on all patients aged ≥18 years, who were hospitalized between the years 2009 and 2014. We then identified among these hospitalizations those with a diagnosis of cirrhosis and further identified, among the latter, hospitalizations with in-hospital CPR. This latter group formed the primary analytic cohort. A diagnosis of cirrhosis was based on the presence of International Classification of Diseases, Ninth Revision, Clinical Modification codes (ICD-9) 571.2, 571.5, and 571.6 in the principal or secondary diagnosis fields. These codes were used in prior studies of cirrhosis in administrative data  and wereIn order to identify secular trends not captured through analysis of the primary cohort, we anchored the data to similar-aged hospitalizations during 2009–2014 without a diagnosis of cirrhosis who had in-hospital CPR. In addition, we have examined all hospitalizations aged ≥18 years during the years 2009–2014 with and without a diagnosis of cirrhosis and those who died during hospitalization within each group to estimate the annual rates of in-hospital CPR among all hospitalizations and among hospital decedents.The co-primary outcomes were rates of in-hospital CPR among all hospitalizations with and without cirrhosis and rates of short-term survival among hospitalizations with in-hospital CPR. We defined short-term survival as that of hospitalizations without in-hospital death or discharge to hospice.The secondary outcomes included a) overall and over time rates of in-hospital CPR among all hospitalizations with and without cirrhosis who died in the hospital and b) overall and over time short-term survival among those with and without shockable rhythm, and hospital disposition of those without in-hospital death or discharge to hospice, both among hospitalizations with and without cirrhosis who have undergone in-hospital CPR.Study variables were selected based on clinical plausibility and prior reports , 6, 10. We summarized categorical variables as numbers and percentages, while continuous variables were reported as means (standard deviation [SD]). Group comparisons were performed using chi-square test for categorical variables and t-test or Mann-Whitney test for continuous variables. We used hospitalizations as the unit of analysis because the TIPUDF does not identify individual patients and thus does not capture repeated hospitalizations.In order to provide further anchoring context to the trends of outcome trajectories between hospitalizations with and without cirrhosis who had in-hospital CPR, we examined their respective temporal trends of the burden of chronic illness and illness severity using the Deyo comorbidity index and the number of failing organs, respectively, as proxy measures.Temporal trends of the primary and secondary outcomes were examined using weighted least square regression, with calendar year as predictor. Modeled findings are reported as average annual percent change (AAPC) and 95% confidence intervals (95% CI). All temporal analyses were carried out separately for hospitalizations with and without cirrhosis. Comparison of regression slopes was performed using the methods described by Armitage et al .We estimated the risk-adjusted of short-term survival following in-hospital CPR among hospitalizations with and without cirrhosis using empirical Bayesian posterior estimates from multivariate logistic regression models for each (see modeling approach below) and then derived the corresponding annual rates of short-term survival.A multivariate logistic regression model was fitted to estimate the association between cirrhosis as independent predictor and short-term survival following in-hospital cardiopulmonary resuscitation as dependent variable. Predictors with p < 0.1 on univariate logistic regression were considered for multivariate analysis, following examination for multicollinearity. Candidate predictor covariates were then entered into the model using backward stepwise selection. Results of the multivariate models were expressed as adjusted odds ratios (aOR) and their 95% CI.The potential predictors of short-term survival following in-hospital cardiopulmonary resuscitation among hospitalizations with cirrhosis were examined using multivariate logistic regression modeling, employing the approach described above for the whole cohort.In order to examine the robustness of our findings and to facilitate comparison to prior studies we re-examined both temporal trends and logistic regression models by substituting the outcome of short-term survival, as defined earlier, with hospital survival, defined as hospitalization without in-hospital death, among hospitalizations with in-hospital CPR, with and without cirrhosis, as well as among the corresponding strata with and without a shockable rhythm and those aged ≥ 65 years. Because the number of independent predictors of short-term survival among hospitalizations with cirrhosis was unexpectedly substantially more limited than among those without cirrhosis, we have repeated the multivariate logistic regression model without the cirrhosis-related complications to examine whether the findings of the original model were driven by the latter complications.p value < 0.05 was considered statistically significant.Data analyses were carried out on MedCalc version 17.5.5 . A 2-sided The total number of hospitalizations with and without cirrhosis during the study period was 330,069 and 12,962,435, respectively. There were 19,320 in-hospital deaths among hospitalizations with cirrhosis and 269,978 deaths among those without cirrhosis, corresponding hospital mortality rates of 5.9% and 2.1%, respectively. The details of the annual hospitalization volumes and the corresponding hospital mortality of all hospitalizations with and without cirrhosis are provided in Table A in There were 54,280 hospitalizations with in-hospital CPR during the study period, of which 2,511 were with a diagnosis of cirrhosis and 51,969 without cirrhosis. The characteristics of these hospitalizations are detailed in The short-term survival following in-hospital CPR was markedly lower among hospitalizations with cirrhosis than among those without cirrhosis . When stratified by the presence or absence of a shockable rhythm, short-term survival was similar between the two groups among hospitalizations with cirrhosis , while being markedly higher among those with shockable rhythm among hospitalizations without cirrhosis .The temporal patterns of the rates of in-hospital CPR, presence of a shockable rhythm, and related short-term survival among hospitalizations with and without cirrhosis are described in The annual rates of in-hospital CPR remained unchanged among hospitalizations with cirrhosis, while rising on average by 2.6%/year among those without cirrhosis. Similarly, the annual rates of in-hospital CPR among hospital decedents remained unchanged among hospitalizations with cirrhosis, while rising slightly, but statistically significantly, among those without cirrhosis.There was no significant change over time in the crude short-term survival following in-hospital CPR among hospitalizations with or without cirrhosis. However, while the risk-adjusted short-term survival following in-hospital CPR remained unchanged among hospitalizations with cirrhosis, it increased over time among those without cirrhosis . The DeyThe annual rates of discharge to hospice following in-hospital cardiopulmonary resuscitation remained unchanged among hospitalizations with cirrhosis , while rising 6.7%/year among those without cirrhosis . Transfers to another hospital following in-hospital cardiopulmonary resuscitation did not change over time for hospitalizations with (p = 0.9901) or without cirrhosis (p = 0.9910). Discharges to a nursing facility remained unchanged overtime among hospitalizations with cirrhosis (p = 0.2004), while rising 8.2%/year among those without cirrhosis .The predictors of short-term survival following in-hospital CPR for the whole cohort and among hospitalizations with cirrhosis are described in Tables Cirrhosis was associated with 45% lower odds of short-term survival following in-hospital CPR on adjusted analysis. Among hospitalizations with cirrhosis who had in-hospital CPR, hepatocellular cancer (aOR = 0.35 [95% CI 0.16–0.76]) and lack of health insurance (aOR = 0.47 [95% CI 0.33–0.67]) were associated with the lowest odds of short-term survival. The predictors of short-term survival following in-hospital CPR among hospitalizations without cirrhosis are detailed in Table C in When hospital survival following in-hospital CPR was used as the outcome instead of short-term survival, the former remained significantly lower among hospitalizations with cirrhosis than among those without cirrhosis . The hospital survival following in-hospital CPR among those aged ≥65 years with and without cirrhosis was 20.1% vs. 28.0%, respectively (p < 0.0001). On trend analysis  Click here for additional data file.


A multilayer approach was used to achieve robust coatings. The first coating was a polymeric layer  and the second one was a composite made of BG particles in a polymer matrix. The coatings were characterized in terms of morphology by scanning electron microscopy (SEM), in vitro bioactivity, and antibacterial properties. Chitosan/Ag-MBG coatings showed the ability to form hydroxyl-carbonate-apatite on their surfaces after immersion in SBF. An antibacterial effect against Gram (+) and Gram (-) bacteria was confirmed, highlighting the potential application of the coated sutures for antibiotic-free approaches.Resorbable (Vicryl Sutures like Vicryl® are often used because of their good mechanical properties, high reproducibility, and minimal tissue reaction. To further strengthen the bond between suture and tissue, several researchers have investigated the coating of sutures with bioactive substances such as bioactive glass [®  sutures with Ag-doped BG by a conventional slurry-dipping technique. Ag-doped BG was used to make the suture bioactive and antibacterial. The coated sutures showed antibacterial properties against Staphylococcus epidermidis, limiting the bacteria adhesion to the surface of the samples. The use of antibacterial bioactive glasses is being considered to replace the controversially discussed triclosan [® Plus  sutures.Due to their widespread application in medicine, e.g., routine skin laceration or organ transplantation ,2, suturve glass ,6,7,8. Bve glass . Stambouriclosan ,13,14 usThe introduction of a foreign device in the human body can induce inflammatory reactions or introduce pathogens on site, which in turn could cause infections. Studies have shown that in 2%–5% of all surgeries, surgical site infections (SSIs) can occur  with con® Plus) with silver containing ordered mesoporous bioactive glass (Ag-MBG) and with melt derived BG-doped with zinc (Zn-BG) particles applying, a dip coating technique. To improve the adhesion of the BG particles on the surface, two different polymers were used: chitosan and poly(ε-caprolactone) (PCL) and layered coatings were produced. Sutures were characterized in terms of acellular in vitro bioactivity by immersion in simulated body fluid (SBF) and antibacterial properties against Gram-positive Staphylococcus carnosus and Gram-negative Escherichia coli. The knot test was also performed to verify the stability and attachment of the coatings to the substrate. The BG/polymer-coated sutures developed in this study represent an innovative advance from the first BG-coated sutures reported earlier [In the present work, composite sutures were produced combining commercially available sutures  sutures were coated with chitosan/silver-doped ordered mesoporous bioactive glass (Ag-MBG), PCL/Ag-MBG, chitosan/Zn-BG, and PCL/Zn-BG, using a slurry dipping method previously applied in other studies [® and Vicryl® sutures with silver-doped BG. The authors observed the formation of crystalline HA after three days in SBF. In another work, Stamboulis et al. [The aim of this work was the development of composite coatings based on Zn-BG or Ag-MBG particles embedded in a polymer matrix of either chitosan or PCL for surgical sutures. Commercially available Vicryl studies ,7. The d studies , who coa® Plus sutures showed an inhibition zone. This can be explained by the fact that this type of resorbable suture is subject to a surface treatment with triclosan, a widespread antiseptic. Nowadays, the use of triclosan is being critically discussed because some studies have indicated that this compound could promote liver tumors and provoke muscle weakness [− or H2O2) can provoke the oxidative stress in the bacteria, leading to perturbation of the cell membrane and damage of cell proteins and DNA [+ can cause the detachment of the cytoplasm membrane of bacteria from the cell wall, compromising the bacteria’s ability to replicate [The antibacterial properties of the coated sutures were tested by means of agar diffusion tests. BG-coated sutures showed clear inhibition zones, which demonstrate their antibacterial effect. However, it should be noted that also pristine Vicrylweakness ,13,14. Fweakness ,24,25. Nweakness . The aut and DNA . The ant and DNA ,24,27 haeplicate . Moreoveeplicate . Studieseplicate ,31 have However, further tests should be carried out to better understand the antibacterial mechanism of the present antibacterial sutures, in particular the possible combined effects of BGs and triclosan, which may lead to a reduction or even elimination of triclosan to achieve the desired antibacterial effect.® Plus sutures  were used. The material is braided from fine filaments of Polyglactin 910, a copolymer of glycolide and lactide at a ratio of 90:10. The braided sutures are additionally coated with Irgacare® MP (Triclosan). The sutures were cut into pieces of 1 cm length  and they were coated by either zinc-doped melt derived BG  or silver-doped mesoporous ordered BG , developed in previous works [n 8000) were purchased from Sigma-Aldrich  and used without further purification. A two-step coating was used to improve the adhesion between suture and coating; the first layer consists of a polymeric layer (chitosan or PCL) and the second one was a mixture of chitosan or PCL and Ag-MBG or Zn-BG particles. A slurry dipping method was performed manually in a glass beaker placed on a magnetic stirrer. An optimization process based on trial-and-error was used to determine the optimal composition of the slurry required to obtain uniform and homogeneous coatings.Undyed braided 4-0 Vicrylus works ,27. Chitv/v acetic acid and 2% w/v chitosan) or PCL solution  and letting them dry for 24 h at room temperature. The second layer was applied by dipping the suture for 2 min into a suspension made of BG powder and chitosan/PCL. The second layer of chitosan/BG was prepared as follows: the chitosan solution mentioned beforehand was mixed with an aqueous slurry containing 40 wt.% BG (mixed for 2 h) and then stirred for 4 days. On the other hand, the second layer of PCL/BG was prepared by adding to the afore-mentioned PCL solution 30% w/v (with respect to PCL) of BG particles and the resultant suspension was stirred for 2 h. All slurries were produced by using benign solvents, which led to an increase in preparation time but enabled a safer work environment and may lead to a better biological compatibility of the coatings.The first layer was obtained by dipping the suture for 1 min in an aqueous chitosan (with 4% ® sutures to form hydroxyl-carbonate-apatite (HCA) once in contact with biological fluids was assessed by immersion in simulated body fluid (SBF) for different time periods. The standard procedure described by Kokubo et al. [Escherichia coli (Gram-negative) and Staphylococcus carnosus (Gram-positive). These bacteria were chosen because they are common bacteria responsible for infections [The microstructure and uniformity of the coatings were investigated using a light microscope  and scanning electron microscopy (SEM) . The ability of coated and non-coated Vicrylo et al.  was usedfections  and theySurgical sutures were successfully coated by a two-step coating process to improve the adhesion between suture and coating: The first layer consisted of a polymeric layer (chitosan or PCL), and the second one formed by a mixture of chitosan or PCL and Ag-MBG or Zn-BG particles. Ag-MBG coated sutures showed a high reactivity once in contact with simulating body fluid, developing a layer of HCA after three days of immersion, while Zn-BG did not lead to HCA formation. Moreover, the chitosan coated samples showed promising results in terms of antibacterial properties against both Gram-positive and Gram-negative strains. Coatings with PCL did not show any antibacterial properties, which might be due to the low glass concentration present in the outer layer of the coating. Future investigations to determine the mechanical properties of coated sutures should be performed, e.g., by combining both polymers, in different layer structures and by optimizing the BG content.


Objective: To evaluate real-world persistence and adherence in patients with benign prostate hyperplasia (BPH) receiving a fixed-dose combination of dutasteride plus tamsulosin (DUT-TAM FDC) versus α-blocker plus 5-α reductase inhibitor (AB/5ARI) free-combination therapy. Materials and methods: This retrospective, observational cohort study utilized the German IMS LRx (IQVIA) database. Patients ≥ 45 years old with BPH receiving DUT-TAM FDC or AB/5ARI free-combination therapy from July 1, 2011 to November 30, 2017 were included. Data were analyzed for 48 months from index date (date of first prescription). Persistence, measured as time to discontinuation (defined as a 90-day gap in therapy), was evaluated using Kaplan-Meier curves (log-rank tests). Adherence, measured as medication possession ratio (MPR), was based on a comparison of mean prescribing duration and expected treatment duration. Results: A total of 141,667 patients were included . Small differences in persistence were observed between treatment arms. At month 12, 41.8% of DUT-TAM FDC-treated and 41.0% of AB/5ARI free-combination therapy-treated patients were persistent; at month 24, 28.2% and 27.1% were persistent, respectively. A higher proportion of DUT-TAM FDC-treated patients had MPR ≥ 0.80, ≥ 0.75 and ≥ 0.70 compared with AB/5ARI free-combination therapy (p < 0.0001). Conclusion: Small differences observed in persistence between treatment arms may not translate to meaningful clinical relevance. Adherence was significantly better in the FDC arm, which may be clinically relevant as improved adherence is associated with better outcomes. Persistence and adherence to BPH therapy in Germany is low; further studies exploring the reasons behind this are required.  What is known about this subject α-blocker (AB) plus 5-α reductase inhibitor (5ARI) combination therapy is superior to AB monotherapy in improving quality of life, reducing lower urinary tract symptoms, and reducing the risk of disease progression in patients with benign prostate hyperplasia (BPH). Poor persistence and adherence to medication is common in patients with BPH, which is associated with an increased risk of complications, such as acute urinary retention and BPH-related hospitalization and surgery. Patients with chronic diseases are more likely to adhere to a once-daily medication regimen compared with more frequent doses; therefore, medication in fixed-dose combinations, which reduce dosing frequency, may be beneficial. What this study adds This is the first study to compare real-world persistence and adherence with dutasteride-tamsulosin fixed-dose combination (DUT-TAM FDC) therapy compared with AB/5ARI in free-combination therapy in patients with BPH. Although persistence and adherence in both treatment groups was low overall, adherence was significantly higher in patients receiving DUT-TAM FDC compared with those receiving AB/5ARI free-combination therapy. Small differences in persistence were observed between groups; however, the magnitude of differences in persistence may not translate to meaningful clinical relevance. DUT-TAM FDC was associated with increased adherence in patients with BPH; this may be of clinical relevance to prescribers as increased adherence is associated with improved clinical outcomes, but further studies are required to investigate reasons behind low persistence and adherence overall. Benign prostatic hyperplasia (BPH) is a condition characterized by a non-malignant overgrowth of the prostate gland. It is the most common cause of lower urinary tract symptoms (LUTS) in ageing men , 2, 3 wimax)) . Finasteride and tamsulosin, the most commonly prescribed free AB/5ARI combination , are avaIn general, persistence was low in both groups. Despite a conservative definition of treatment discontinuation , over 30% of patients in both groups discontinued AB plus 5ARI combination therapy, possibly after the first prescription. Less than 42% of patients were persistent for at least 12 months, the minimal treatment period required for a beneficial clinical effect of 5ARI therapy to be observed , 9, and This low rate of persistence is consistent with findings in previous studies , 26. In A significantly higher percentage of patients receiving DUT-TAM FDC therapy were adherent during the current study compared with those receiving AB/5ARI in free combination; however, this may have a lesser impact on clinical outcomes due to low persistence overall. This reflects findings in previous studies, which have shown that adherence is better in medication regimens of 1 tablet per day, compared with 2 per day , 20, 21.Poor persistence and adherence is associated with less favorable clinical outcomes; therefore, it is important for patients to commit to taking pharmacological therapy for LUTS/BPH as prescribed . In chroThere are several limitations of this study. Firstly, due to the large sample size, even small differences with potentially limited clinical relevance may have led to significant p-values and therefore should be interpreted with caution. No data was available to show whether patients took the medications according to the prescribed treatment regimen. Rather, it was assumed that if the medication was dispensed, it was correctly administered by the patient. However, regardless of whether all doses are correctly taken, differences between a once-daily regimen (DUT-TAM FDC) and a multiple-dose daily regimen (AB/5ARI free-combination therapy) cannot be discounted. Another limitation, which is common to retrospective observational studies using routinely collected electronic data platforms (not designed for specific research investigations), was the lack of availability of important variables in the IMS LRx (IQVIA) database such as diagnosis, severity of disease, and laboratory data . FurthermAs the IMS LRx (IQVIA) database collects anonymized patient histories and patient-level prescription data from pharmacy coding centers, patients were analyzed who actually had the medication in their hands  which eliminates the uncertainty of prescription data from medical records, where it is unknown if patients actually pick up their prescriptions in the pharmacy. Another strength of this study is the large sample size which may reduce the impact of the above-mentioned variables. Further, due to the retrospective design of the study the observed results may better reflect the “real-world” situation than more interventional study designs like controlled clinical trials or prospective observational trials. These approaches may use more sophisticated tools like medication event monitoring systems (medication bottles with an electronic cap counting every opening of the bottle) to reduce the uncertainty if patients actually take their medication, and maybe questionnaires for physicians and patients to further explore behavior and reasons for non-compliance. However, it is well known that such tools and study designs may influence the behavior of patients and thereby artificially increase persistence and adherence. The findings presented in this study indicate that persistence and adherence to AB/5ARI therapy in patients with LUTS/BPH in Germany is lower than DUT-TAM FDC overall. Differences in adherence were statistically significant in favor of the FDC once-daily regimen compared with the multiple-daily dosing combination therapy regimens. There were small differences in persistence between DUT-TAM FDC and AB/5ARI free-combination therapy over the 48-month period of evaluation; however, the magnitude of difference observed between treatment arms may not translate to meaningful clinical relevance. These findings may be useful clinically as improved adherence is associated with improved outcomes in patients with LUTS/BPH. Therefore, educating patients on the importance of persistence with medical therapy in long-term prevention of disease progression and its associated complications may be beneficial. Further studies are required to explore the key drivers for early treatment discontinuation and poor adherence to DUT-TAM FDC therapy in patients with LUTS/BPH. Medical writing support was provided by Gillian Wallace, MSc, and Lisa Auker, PhD, of Fishawack Indicia Ltd, UK, and funded by GSK. This study was funded by GlaxoSmithKline . Christoph Eisen, Michael Hennig, Burkay Adalig, Juan Manuel Palacios-Moreno, Vanessa Cortes and Zrinka Lulic are all employees of GSK and hold shareholder status in the company. Florian Gilg was an employee of GSK at the time of the study being performed. Karel Kostev is an employee of IQVIA.