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Background: Non-alcoholic steatohepatitis (NASH) is a serious end-stage spectrum of non-alcoholic fatty liver disease (NAFLD) with associated high risk of hepatic and extrahepatic complications. Several studies showed the significant beneficial effect of dapagliflozin on body composition, hepatic and metabolic parameters on NAFLD/NASH patients. The study aimed to investigate the efficacy and safety of dapagliflozin in both diabetic and non-diabetic biopsy-proven NASH patients; compared to pioglitazone., Methods: This was a four-group, prospective, randomized, parallel, open label study in which 100 biopsy-proven NASH patients were selected, stratified to diabetics and non-diabetics and randomized with 1:1 allocation to either 30 mg pioglitazone or 10 mg dapagliflozin, once daily for 24 weeks. Histological evaluation, anthropometric measures, hepatic, metabolic biochemical markers, fibrosis non-invasive markers, quality of life (QOL) and medications adverse events were examined., Results: Dapagliflozin showed a comparable histological effect to pioglitazone in both diabetic and non-diabetic patients (P>0.05). As assessed by transient elastography, it also showed a comparable effect on liver fibrosis grade improvement from baseline in diabetics (P=0.287) versus a significant superiority in non-diabetics (P=0.018). Dapagliflozin showed a significant superiority in all anthropometric measures (P<0.001) and QOL (P<0.05) among both diabetics and non-diabetics. There was a significant interaction between interventions and diabetes status on change from baseline of hepatic and metabolic panel collectively (P=0.023) in favor to dapagliflozin among diabetics., Conclusion: Compared to pioglitazone, dapagliflozin had a comparable effect histologically, superior effect biochemically among diabetics and superior effect on liver fibrosis, steatosis and insulin resistance among non-diabetics., Trial Registration: The study was registered on clinicaltrials.gov, identifier number NCT05254626., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest in this article., (Copyright © 2025 Elsevier Masson SAS. All rights reserved.)

Objective: Academic resilience, a critical determinant of academic achievement, is affected by various factors. There is a paucity of large-scale international assessments of academic resilience among pharmacy students. Therefore, this study aimed to assess academic resilience among pharmacy students in 12 countries and to evaluate factors associated with their academic resilience levels., Methods: A cross-sectional online survey-based study was conducted among randomly selected pharmacy students in 12 countries: Egypt, Türkiye, Indonesia, Pakistan, Bangladesh, Iraq, Jordan, Nigeria, Malaysia, Saudi Arabia, Sudan, and the United Arab Emirates. After pilot testing, the validated 30-item academic resilience scale (ARS) was used for the assessment. The data were collected between November 1, 2022 and April 15, 2023. Descriptive and inferential statistics were performed, as appropriate., Results: A total of 3950 were received from the 12 participating countries. The mean age was 21.68 ± 2.62 years. About two-thirds of the responses were from female participants and those studying for Bachelor of Pharmacy degrees. Overall, the findings show moderate academic resilience, which varied across countries. The median (IQR) of the total ARS-30 was 114 (103-124). Females exhibited lower negative affective and emotional response subscale levels than males. There were significant cross-country variations in the ARS-30 and all subscales. The highest overall levels were reported for Sudan, Pakistan, and Nigeria and the lowest were reported for Indonesia and Türkiye. Students in private universities tended to have higher overall ARS levels than public university students. Higher academic performance was significantly associated with ARS levels, whereas those with excellent performance exhibited the highest ARS levels. Students with exercise routines had higher ARS levels than those without exercise routines. Finally, students who were engaged in extracurricular activities had higher ARS levels than those who did not participate in these activities., Conclusion: The study offers insights into the factors affecting academic resilience in pharmacy students across several countries. The findings could guide interventions and support activities to improve resilience and academic outcomes., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Artificial intelligence models, like ChatGPT, have the potential to revolutionize higher education when implemented properly. This study aimed to investigate the factors influencing university students' attitudes and usage of ChatGPT in Arab countries. The survey instrument "TAME-ChatGPT" was administered to 2240 participants from Iraq, Kuwait, Egypt, Lebanon, and Jordan. Of those, 46.8% heard of ChatGPT, and 52.6% used it before the study. The results indicated that a positive attitude and usage of ChatGPT were determined by factors like ease of use, positive attitude towards technology, social influence, perceived usefulness, behavioral/cognitive influences, low perceived risks, and low anxiety. Confirmatory factor analysis indicated the adequacy of the "TAME-ChatGPT" constructs. Multivariate analysis demonstrated that the attitude towards ChatGPT usage was significantly influenced by country of residence, age, university type, and recent academic performance. This study validated "TAME-ChatGPT" as a useful tool for assessing ChatGPT adoption among university students. The successful integration of ChatGPT in higher education relies on the perceived ease of use, perceived usefulness, positive attitude towards technology, social influence, behavioral/cognitive elements, low anxiety, and minimal perceived risks. Policies for ChatGPT adoption in higher education should be tailored to individual contexts, considering the variations in student attitudes observed in this study., (© 2024. The Author(s).)

Background: Corticosteroids (CS) are essential drugs in the treatment of several medical conditions. Assuming different roles, physicians and pharmacists play a primary role in prescribing and dispensing these medications to optimize patients' clinical management. The data on assessing knowledge and experience of healthcare professionals toward CS is scarce. Therefore, this study aimed to assess and compare knowledge, experience, and fears towards CS among Egyptian physicians and pharmacists., Methods: A cross-sectional, self-administrated, validated online questionnaire was used to collect the data from Egyptian healthcare professionals. The questionnaire consisted of four sections with multiple choice questions: sociodemographic (7 questions), knowledge about CS (13 questions), experience with CS prescription/dispensing (5 questions), and fears and preferences toward CS prescription/dispensing (13 questions). Descriptive and inferential statistics were used to analyze the data., Results: A total of 600 responses were analyzed in this study. The study sample was almost two-half of healthcare providers: 303 (50.5%) pharmacists and 297 (49.5%) physicians. Pharmacists had marginally higher knowledge scores as compared to those recorded for physicians (11.29 versus 10.16, respectively; P = 0.047). Physicians had more experience choosing corticosteroids in treatment plans based on their experience (51.8% vs 38.5%) and guideline recommendations (72.8% vs 50.9%) than pharmacists. However, pharmacists had more experience dealing with corticosteroid use based on patients' preferences (19.5% vs 4.9%) and showed a broader scope of experiencing side effects of corticosteroids with their patients. The two professions demonstrated high levels of fear, with pharmacists acknowledging significantly lower concerns about CS than physicians (3.72 versus 4.0, respectively; P = 0.003)., Conclusion: Discrepancies exist among healthcare professionals in knowledge and experience, favoring better scientific knowledge of pharmacists related to corticosteroids. Based on these findings, the interprofessional collaborative efforts would provide comprehensive, patient-centered care that maximizes the benefits of CS while minimizing their risks., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Objectives: Traumatic brain injury (TBI) is one of the top causes of morbidity and mortality worldwide. The review aimed to discuss and summarize the current evidence on the effectiveness of adjuvant neuroprotective treatments in terms of their effect on brain injury biomarkers in TBI patients. Methods: To identify relevant studies, four scholarly databases, including PubMed, Cochrane, Scopus, and Google Scholar, were systematically searched using predefined search terms. English-language randomized controlled clinical trials reporting changes in brain injury biomarkers, namely, neuron-specific enolase (NSE), glial fibrillary acid protein (GFAP), ubiquitin carboxyl-terminal esterase L1 (UCHL 1 ) and/or S100 beta (S100 ß), were included. The methodological quality of the included studies was assessed using the Cochrane risk-of-bias tool. Results: A total of eleven studies with eight different therapeutic options were investigated; of them, tetracyclines, metformin, and memantine were discovered to be promising choices that could improve neurological outcomes in TBI patients. The most utilized serum biomarkers were NSE and S100 ß followed by GFAP, while none of the included studies quantified UCHL 1 . The heterogeneity in injury severity categories and measurement timing may affect the overall evaluation of the clinical efficacy of potential therapies. Therefore, unified measurement protocols are highly warranted to inform clinical decisions. Conclusion: Few therapeutic options showed promising results as an adjuvant to standard care in patients with TBI. Several considerations for future work must be directed towards standardizing monitoring biomarkers. Investigating the pharmacotherapy effectiveness using a multimodal biomarker panel is needed. Finally, employing stratified randomization in future clinical trials concerning potential confounders, including age, trauma severity levels, and type, is crucial to inform clinical decisions. Clinical Trial Registration: [https://www.crd.york.ac.uk/prospero/dis], identifier [CRD42022316327]., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mansour, Elnaem, Abdelaziz, Barakat, Dehele, Elrggal and Abdallah.)

Introduction: Grit is proposed as an essential trait for academic achievement. Thus, evaluating its current status and the associated factors could aid academic support planning., Objective: The present study aimed to assess grit level and its related factors among undergraduate pharmacy students from 14 countries amid the COVID-19 pandemic., Methods: A cross-sectional survey-based study was conducted among pharmacy students from 14 countries in Asia and the Middle East. A 31-item questionnaire was developed, validated, and pilot-tested, including the validated short scale for grit assessment. The data was collected between 1 February and 15 April 2022. Descriptive and inferential statistics were employed as appropriate., Results: A total of 2665 responses were received, mainly from females (68.7 %), living in urban areas (69.2 %) and studying at private universities (59.1 %). The average grit score on a scale of 5 was 3.15 ± 0.54. The responses revealed higher favourable responses to items on the perseverance of efforts (34.9 % to 54 %) compared to items on the consistency of interests (26.5 % to 31.1 %). Students who did not exercise (AOR: 0.47, 95 %CI: 0.33-0.67) or exercised irregularly (AOR: 0.64, 95 %CI: 0.45-0.90) were less likely to have higher grit scores than those who exercised regularly. Additionally, students who did not receive COVID-19 vaccination (AOR: 0.50, 95 %CI: 0.36-0.71) or received only one dose (AOR: 0.67, 95 %CI: 0.46-0.99) were less likely to have higher grit scores than those who received their booster vaccination. Interestingly, students who chose the pharmacy program as their only available or reasonable choice (AOR: 0.33, 95 %CI: 0.17-0.62) and students from public universities (AOR: 0.82, 95 %CI: 0.68-0.98) were less likely to have higher grit scores. On the other hand, students who did not face educational challenges with online learning (AOR: 1.19, 95 %CI: 1.003-1.416) and students with excellent (AOR: 2.28, 95 %CI: 1.57-3.31) and very good (AOR: 2.16, 95 %CI: 1.53-3.04) academic performance were more likely to have higher grit scores., Conclusion: The findings revealed moderate grit levels. Higher grit levels were thought to be associated with several personal, lifestyle and academic factors. Further interventions to support students' grit attributes are required, particularly concerning the consistency of interests., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Background: Corticosteroids play a significant role in managing the vast majority of inflammatory and immunologic conditions. To date, population-based studies on knowledge and attitudes concerning corticosteroids are scarce. This study aims to comprehensively assess knowledge, perception, experience and phobia toward corticosteroid use among the general population in the era of COVID-19., Methods: A cross-sectional self-administrated questionnaire was used to collect the data from 6 countries. Knowledge and corticophobia scores, descriptive statistics and logistic regression were computed., Results: A total of 2354 participants were enrolled in this study; the majority were females (61.6%) with an average age of 30. Around 61.9% had been infected previously with COVID-19, and about one-third of the participants had experience with corticosteroid use. The mean knowledge score was relatively satisfactory (8.7 ± 4.5 out of 14), and Corticophobia ranked a high score in all countries. Age, female gender, and history of COVID-19 were positively correlated with developing corticophobia., Conclusion: Our study highlights that the general knowledge about steroids was satisfactory. However, the phobia toward its use upon indication is high. Therefore, enhancing awareness and providing essential counseling regarding the rational use of corticosteroids may reduce corticophobia.

Controlling hypertension (HTN) remains a challenge, as it is affected by various factors in different settings. This study aimed to describe the disparities in the prevalence and barriers to hypertension control across countries of various income categories. Three scholarly databases-ScienceDirect, PubMed, and Google Scholar-were systematically examined using predefined search terms to identify potentially relevant studies. Original research articles published in English between 2011 and 2022 that reported the prevalence and barriers to HTN control were included. A total of 33 studies were included in this systematic review. Twenty-three studies were conducted in low and middle-income countries (LMIC), and ten studies were from high-income countries (HIC). The prevalence of hypertension control in the LMIC and HIC studies ranged from (3.8% to 50.4%) to (36.3% to 69.6%), respectively. Concerning barriers to hypertension control, patient-related barriers were the most frequently reported ( n = 20), followed by medication adherence barriers ( n = 10), lifestyle-related barriers ( n = 8), barriers related to the affordability and accessibility of care ( n = 8), awareness-related barriers ( n = 7), and, finally, barriers related to prescribed pharmacotherapy ( n = 6). A combination of more than one category of barriers was frequently encountered, with 59 barriers reported overall across the 33 studies. This work reported disparities in hypertension control and barriers across studies conducted in LMIC and HIC. Recognizing the multifactorial nature of the barriers to hypertension control, particularly in LMIC, is crucial in designing and implementing customized interventions.

Background: Pharmacy students will assume future roles as frontline healthcare providers. Therefore, evaluating their current state of mental wellbeing and its associated factors is essential for better planning students' support initiatives. This study aimed to assess mental wellbeing and its associated factors among undergraduate pharmacy students from 14 countries during the pandemic., Methods: A cross-sectional study was conducted among undergraduate pharmacy students in 14 countries in Asia and the Middle East. The validated Warwick-Edinburgh Mental Wellbeing Scale (the 14-item WEMWBS) was adopted to assess mental wellbeing. Data collection was performed online between February and April 2022. Descriptive and inferential statistics were used as appropriate., Results: A total of 2,665 responses were received, mainly from females (68.7%) with a higher presence of private universities (59.1%). About 34.9% had low mental wellbeing levels, while 57 and 8.1% had medium, and high levels, respectively. Binary logistic regression showed that males (AOR: 1.34; CI 95%: 1.11-1.61; p < 0.01) and students with no chronic illnesses (AOR: 2.01; CI 95%: 1.45-2.80; p < 0.001) were more likely to have higher mental wellbeing. Also, participants who did not engage in any exercise (AOR: 0.71; CI 95%: 0.52-0.98; p = 0.04) and those in public universities (AOR: 0.82; CI 95%: 0.69-0.97; p = 0.02) were less likely to have higher mental wellbeing. Additionally, students who had interest/passion for pharmacy (AOR: 1.69; CI 95%: 1.07-2.68; p = 0.02), and those who known pharmacists inspired (AOR: 1.81; CI 95%: 1.06-3.12; p = 0.03), were more likely to have higher mental wellbeing compared with those who had no specific reason for their choice to study pharmacy. The participants with excellent (AOR: 1.87; CI 95%: 1.29-2.70; p = 0.001) or very good self-reported academic performance (AOR: 1.57; CI 95%: 1.12-2.22; p = 0.01) were more likely to have higher mental wellbeing compared to those with fair academic performance., Conclusion: More than a third of the participants had low mental wellbeing. Various demographic, lifestyle, medical and academic factors appeared to affect students' mental wellbeing. Careful consideration of these factors and their integration into the pharmacy schools' plans for student support services and academic advising would be essential to improve students' mental wellbeing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elnaem, Mubarak, K. T., Barakat, Abdelaziz, Mansour, Thabit, Ramatillah, Al-Jumaili, Syed, Adam, Hossain, Baraka, Jose, Elkalmi, Chandran, Singh Dehele, Elrggal and Fathelrahman.)

Background: The pain pattern after laparoscopic cholecystectomy (LC) is complex and distinct from postoperative pain after other laparoscopic procedures, suggesting that procedure-specific optimal analgesic management plans should be proposed. Duloxetine, a non-opioid neuromodulator, has been widely used to manage pain with dual central and peripheral analgesic properties. Aims: To assess the effect of preoperative administration of duloxetine compared to placebo on postoperative pain control in patients undergoing LC. Patients and Methods: This study was a randomized, parallel-group, placebo-controlled, double-blinded study performed on patients undergoing LC. Patients were randomly divided into two groups of 30 each on the day of surgery in the preoperative holding area, using a computer-generated random number to receive 60 mg duloxetine as a single oral dose 2 h before the procedure or placebo. The primary outcome was the difference in the mean of visual analogue scale (VAS) scores between the two studied groups, as measured by the area under the curve (AUC) of the VAS scores. Results: The derived AUC of VAS scores in the duloxetine group (757.89 ± 326.01 mm × h) was significantly lower than that calculated for the control group (1005.1 ± 432.5 mm × h). The mean postoperative VAS scores recorded at 4 and 24 h were statistically different between the study groups ( p = 0.041 and 0.003, respectively). As observed in the survival curve analysis, there was no significant difference ( p = 0.665) for the time until the patient's first request for rescue medications in the two groups. The frequency of postoperative nausea and vomiting (PONV) was lower in patients of the duloxetine group than that recorded in those allocated to the control group at 8 and 24-h time intervals ( p = 0.734 and 0.572, respectively). Conclusion: Preoperative use of duloxetine reduces postoperative pain significantly compared with placebo. In addition, its use is associated with a reduction in PONV. These preliminary findings suggest that duloxetine could play a role in the acute preoperative period for patients undergoing LC. Clinical Trial Registration: [https://clinicaltrials.gov/ct2/show/NCT05115123, identifier NCT05115123]., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mansour, Boraii, Elnaem, Elrggal, Omar, Abdelraouf and Abdelaziz.)

Background and Aim: Rifaximin is an oral antibiotic with promising efficacy in the reduction of hepatic encephalopathy (HE) recurrence. Development of microbial resistance to rifaximin is not studied yet in HE. The study aim was to assess the microbial resistance, safety and efficacy of rifaximin as secondary prophylaxis of HE., Method: In this open-label parallel, prospective interventional study, 100 patients were randomly allocated either to receive 400 mg rifaximin 3 times/d plus 30-45 mL lactulose 3 times/d (intervention group) or to receive the standard of care only which is lactulose alone (control group) for 6 months. The primary outcome of the study was the difference between minimum inhibitory concentration (MIC) of rifaximin among the two studied groups at the end of treatment. The secondary outcomes included the time to first episode of HE, time to first hospitalisation, and patient's survival., Results: The MIC did not differ significantly after treatment exposure compared with baseline either between groups or within the same group. The time to new episode of HE was 18.84 ± 6.49 weeks (mean ± SD) in the intervention group and was significantly longer (P = .002) than that in the control group 14 ± 7.52 weeks. Moreover, only 23 (46%) patients developed overt HE in the intervention group compared with 35 patients (70%) in the control group (P = .005). Also, there was an observed 32% reduction in the risk of hospitalisation in intervention group compared with control group., Conclusion: Rifaximin succeeded to maintain remission from new episodes of HE in hepatitis C virus cirrhotic patients with limited potential for development of microbial resistance over the study period. ClinicalTrials.gov Identifier: NCT04736836., (© 2021 John Wiley & Sons Ltd.)

Background: Pain after laparoscopic cholecystectomy remains a major challenge. Ondansetron blocks sodium channels and may have local anesthetic properties., Aims: To investigate the effect of intraperitoneal administration of ondansetron for postoperative pain management as an adjuvant to intravenous acetaminophen in patients undergoing laparoscopic cholecystectomy., Methods: Patients scheduled for elective laparoscopic cholecystectomy were randomized into two groups (n = 25 each) to receive either intraperitoneal ondansetron or saline injected in the gall bladder bed at the end of the procedure. The primary outcome was the difference in pain from baseline to 24-h post-operative assessed by comparing the area under the curve of visual analog score between the two groups., Results: The derived area under response curve of visual analog scores in the ondansetron group (735.8 ± 418.3) was 33.97% lower than (p = 0.005) that calculated for the control group (1114.4 ± 423.9). The need for rescue analgesia was significantly lower in the ondansetron (16%) versus in the control group (54.17%) (p = 0.005), indicating better pain control. The correlation between the time for unassisted mobilization and the area under response curve of visual analog scores signified the positive analgesic influence of ondansetron (r s =0.315, p = 0.028). The frequency of nausea and vomiting was significantly lower in patients who received ondansetron than that reported in the control group (p = 0.023 (8 h), and 0.016 (24 h) respectively)., Conclusions: The added positive impact of ondansetron on postoperative pain control alongside its anti-emetic effect made it a unique novel option for patients undergoing laparoscopic cholecystectomy., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Terlipressin is the first-line pharmacological treatment for hepatorenal syndrome. When terlipressin is unavailable, midodrine/octreotide or norepinephrine, with albumin, represent the alternative treatments. The comparative efficacy of these alternative regimens remains unclear. Objective: To compare the efficacy of midodrine/octreotide to that of norepinephrine for the treatment of patients with hepatorenal syndrome. Methods: In the intensive care setting, sixty patients with hepatorenal syndrome were randomized to initially receive either 0.5 mg/h of norepinephrine (maximum 3 mg/h) or 5 mg of oral midodrine three times/day (maximum 12.5 mg three times/day) plus octreotide (100 μg/6 h) as subcutaneous injection (maximum 200 μg/6 h), together with albumin (20-40 g/day). Treatment was allowed for a maximum of 10 days. Survival was analyzed for up to 30 days. The primary efficacy outcome was the proportion of patients who achieved full response, defined as the return of serum creatinine to a value within 0.3 mg/dl of the baseline at the end of treatment. Results: There was a significantly higher rate of full response in the norepinephrine group (15/26, 57.60%) than the midodrine/octreotide group (5/25, 20%) ( p = 0.006). Eleven (42.30%) patients in the norepinephrine group and 6 (24%) in the midodrine/octreotide group survived ( p = 0.166). Conclusion: Norepinephrine plus albumin is significantly more effective than midodrine and octreotide plus albumin in improving renal function in patients with hepatorenal syndrome. (ClinicalTrials.gov, identifier: NCT03455322). https://clinicaltrials.gov/ct2/show/NCT03455322?cond = Hepatorenal+Syndrome&cntry = EG&draw = 2&rank = 1., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 El-Desoki Mahmoud, Abdelaziz, Abd-Elsalam and Mansour.)

Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrP C ) into the pathological isoform PrP Sc Elucidating the molecular and cellular mechanisms underlying prion propagation may help to develop disease interventions. Cell culture systems for prion propagation have greatly advanced molecular insights into prion biology, but translation of in vitro to in vivo findings is often disappointing. A wider range of cell culture systems might help overcome these shortcomings. Here, we describe an immortalized mouse neuronal astrocyte cell line (C8D1A) that can be infected with murine prions. Both PrP C protein and mRNA levels in astrocytes were comparable with those in neuronal and non-neuronal cell lines permitting persistent prion infection. We challenged astrocytes with three mouse-adapted prion strains (22L, RML, and ME7) and cultured them for six passages. Immunoblotting results revealed that the astrocytes propagated 22L prions well over all six passages, whereas ME7 prions did not replicate, and RML prions replicated only very weakly after five passages. Immunofluorescence analysis indicated similar results for PrP Sc Interestingly, when we used prion conversion activity as a readout in real-time quaking-induced conversion assays with RML-infected cell lysates, we observed a strong signal over all six passages, comparable with that for 22L-infected cells. These data indicate that the C8D1A cell line is permissive to prion infection. Moreover, the propagated prions differed in conversion and proteinase K-resistance levels in these astrocytes. We propose that the C8D1A cell line could be used to decipher prion strain biology., Competing Interests: Conflict of interest—Conflict of interest statement: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Tahir et al.)

Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrP C ) into the infectious isoform PrP Sc . These diseases have the potential to transmit within or between species, and no cure is available to date. Targeting the unfolded protein response (UPR) as an anti-prion therapeutic approach has been widely reported for prion diseases. Here, we describe the anti-prion effect of the chemical compound Sephin1 which has been shown to protect in mouse models of protein misfolding diseases including amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) by selectively inhibiting the stress-induced regulatory subunit of protein phosphatase 1, thus prolonging eIF2α phosphorylation. We show here that Sephin1 dose and time dependently reduced PrP Sc in different neuronal cell lines which were persistently infected with various prion strains. In addition, prion seeding activity was reduced in Sephin1-treated cells. Importantly, we found that Sephin1 significantly overcame the endoplasmic reticulum (ER) stress induced in treated cells, as measured by lower expression of stress-induced aberrant prion protein. In a mouse model of prion infection, intraperitoneal treatment with Sephin1 significantly prolonged survival of prion-infected mice. When combining Sephin1 with the neuroprotective drug metformin, the survival of prion-infected mice was also prolonged. These results suggest that Sephin1 could be a potential anti-prion drug selectively targeting one component of the UPR pathway.

Prion diseases are fatal infectious neurodegenerative disorders in human and animals that are caused by misfolding of the cellular prion protein (PrP C ) into the infectious isoform PrP Sc . No effective treatment is available for prion diseases. Metformin is a first-line medication for treatment of type 2 diabetes which is known to activate AMPK and induce autophagy through the inhibition of mammalian target of rapamycin (mTOR1) signaling. Metformin was reported to be beneficial in various protein misfolding and neurodegenerative diseases like Alzheimer's and Huntington's diseases. In this study we investigated the anti-prion effect of metformin in persistently prion-infected neuronal cells. Our data showed that metformin significantly decreased the PrP Sc load in the treated cells, as shown by less PK resistant PrP in Western blots and reduced prion conversion activity in Real-Time Quaking-Induced Conversion (RT-QuIC) assay in both 22L-ScN2a and RML-ScCAD5 cells. Additionally, metformin induced autophagy as shown by higher levels of LC3-II in treated cells compared with control cells. On the other hand, our mouse bioassay showed that oral metformin at a dose of 2 mg/ml in drinking water had no effect on the survival of prion-infected mice. In conclusion, our findings describe the anti-prion effect of metformin in two persistently prion-infected neuronal cell lines. This effect can be explained at least partially by the autophagy inducing activity of metformin. This study sheds light on metformin as an anti-prion candidate for the combination therapy of prion diseases., Competing Interests: Declaration of competing interest There are no financial interests to be disclosed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Prions use cellular machineries for autocatalytic propagation by conformational conversion of the cellular prion protein into the pathological isoform PrP Sc . Autophagy is a basic cellular degradation and recycling machinery that delivers cargo to lysosomes. Increase of autophagic flux in cells results in enhanced delivery of PrP Sc in late endosomes to lysosomal degradation, providing a therapeutic target for prion diseases. Application of chemical enhancers of autophagy to cell or mouse models of prion infection provided a solid experimental proof-of-concept for this anti-prion strategy. In addition, increasing autophagy also reduces exosomal release of prions and transfer of prion infectivity between cells. Taken together, pharmacological induction of autophagy is a promising target for containing prion diseases, and ideal candidate for future combination therapies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Chronic wasting disease (CWD) is a fatal neurodegenerative disease that affects cervids in North America and now Europe. No effective measures are available to control CWD. We hypothesized that active vaccination with homologous and aggregation-prone recombinant prion protein (PrP) could overcome self-tolerance and induce autoantibody production against the cellular isoform of PrP (PrP C ), which would be protective against CWD infection from peripheral routes. Five groups of transgenic mice expressing elk PrP (TgElk) were vaccinated with either the adjuvant CpG alone or one of four recombinant PrP immunogens: deer dimer (Ddi); deer monomer (Dmo); mouse dimer (Mdi); and mouse monomer (Mmo). Mice were then challenged intraperitoneally with elk CWD prions. All vaccinated mice developed ELISA-detectable antibody titers against PrP. Importantly, all four vaccinated groups survived longer than the control group, with the Mmo-immunized group exhibiting 60% prolongation of mean survival time compared with the control group (183 versus 114 days post-inoculation). We tested for prion infection in brain and spleen of all clinically sick mice. Notably, the attack rate was 100% as revealed by positive CWD signals in all tested tissues when assessed with Western blotting, real-time quaking-induced conversion, and immunohistochemistry. Our pilot study in reindeer indicated appreciable humoral immune responses to Mdi and Ddi immunogens, and the post-immune sera from the Ddi-vaccinated reindeer mitigated CWD propagation in a cell culture model (CWD-RK13). Taken together, our study provides very promising vaccine candidates against CWD, but further studies in cervids are required to investigate vaccine efficacy in the natural CWD hosts., (© 2018 Abdelaziz et al.)

Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrP C ) into the pathological isoform PrP Sc These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrP Sc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type- or prion strain-specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrP Sc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy., (© 2018 Thapa et al.)

Prions are protein-based infectious agents that autocatalytically convert the cellular prion protein PrP C to its pathological isoform PrP Sc Subsequent aggregation and accumulation of PrP Sc in nervous tissues causes several invariably fatal neurodegenerative diseases in humans and animals. Prions can infect recipient cells when packaged into endosome-derived nanoparticles called exosomes, which are present in biological fluids such as blood, urine, and saliva. Autophagy is a basic cellular degradation and recycling machinery that also affects exosomal processing, but whether autophagy controls release of prions in exosomes is unclear. Our work investigated the effect of autophagy modulation on exosomal release of prions and how this interplay affects cellular prion infection. Exosomes isolated from cultured murine central neuronal cells (CAD5) and peripheral neuronal cells (N2a) contained prions as shown by immunoblotting for PrP Sc , prion-conversion activity, and cell culture infection. We observed that autophagy stimulation with the mTOR inhibitor rapamycin strongly inhibited exosomal prion release. In contrast, inhibition of autophagy by wortmannin or CRISPR/Cas9-mediated knockout of the autophagy protein Atg5 (autophagy-related 5) greatly increased the release of exosomes and exosome-associated prions. We also show that a difference in exosomal prion release between CAD5 and N2a cells is related to differences at the level of basal autophagy. Taken together, our results indicate that autophagy modulation can control lateral transfer of prions by interfering with their exosomal release. We describe a novel role of autophagy in the prion life cycle, an understanding that may provide useful targets for containing prion diseases., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Chronic wasting disease (CWD) is the most contagious prion disease. It is expanding rapidly in North America, was found recently in Europe, and the potential for transmission to humans cannot be excluded yet. We hypothesized that it is possible to prevent peripheral CWD infection and CWD prion shedding by inducing auto-antibodies against the cellular prion protein (PrP C ) by active vaccination. Our objective is to overcome self-tolerance against PrP by using a multimeric recombinant PrP (recPrP) as an immunogen. We expressed in E. coli, purified and refolded four immunogens: cervid and murine recPrP in monomeric and dimeric form. Testing immunogenicity in sera of the vaccinated transgenic mice expressing cervid PrP revealed that all four immunogens effectively overcame self-tolerance against the prion protein as shown by high antibody titers. Confocal microscopy analysis revealed effective binding of post-immune sera to surface-located PrP C in both murine and cervid PrP expressing cultured cells. Remarkably, the post-immune auto-antibodies effectively inhibited CWD-induced prion conversion in RT-QuIC assay when incubated with either PrP substrate or CWD seed. Furthermore, they mitigated prion propagation in CWD-infected cervid-PrP expressing RK13 cells. Together, multimeric recombinant cervid PrP effectively overcomes self-tolerance to PrP and induces auto-antibodies that interfere with CWD conversion in vitro.

Individualized therapy is a recent approach aiming to specify dosage regimen for each patient according to its genetic state. Cancer chemotherapy requires continuous monitoring of the plasma concentration levels of active forms of cytotoxic drugs and subsequent dose adjustment. In order to attain optimum therapeutic efficacy, correlation to pharmacogenetics data is crucial. In this study, a specific, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed for determination of methotrexate (MTX), 6-mercaptopurine (MP) and its metabolite 6-thioguanine nucleotide (TG) in human plasma. Based on the basic character of the studied compounds, solid phase extraction using a strong cation exchanger was found the optimum approach to achieve good extraction recovery. Chromatographic separation was carried out using RP-HPLC and isocratic elution by acetonitrile: 0.1% aqueous formic acid (85:15v/v) with a flow rate of 0.8mL/min at 40°C. The detection was performed by tandem mass spectrometry in MRM mode via electrospray ionization source in positive ionization mode. Analysis was carried out within 1.0min over a concentration range of 6.25-200.00ng/mL for the studied analytes. Validation was carried out according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the monitoring of the MTX, MP and TG and subsequent application to personalized therapy was demonstrated in a clinical study on children with acute lymphoblastic leukemia (ALL). Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The aim of the present study is to determine the correlation of hepatitis C virus (HCV) infection and polymorphisms in different genes with toxicity of either methotrexate (MTX) or 6-mercaptopurine (6-MP) administered to children with acute lymphoblastic leukemia (ALL)., Procedure: One hundred children with low-risk ALL, who were treated according to the St. Jude Total therapy XV, were recruited. The recruited children were receiving MTX and 6-MP during maintenance phase. Patients were excluded from the study if they had other types of leukemia. Genotyping analyses for the thiopurine methyltransferase (TPMT), methylenetetrahydrofolate reductase (MTHFR), and glutathione S-transferase (GST) genes were performed using a combination of polymerase chain reaction (PCR) and PCR-RFLP (where RFLP is restriction fragment length polymorphism) protocols. Relevant clinical data on adverse drug reactions were collected objectively (blinded to genotypes) from the patient medical records., Results: There was a significant correlation between the combined presence of HCV and TPMT*3B G460A gene polymorphisms and grades 2-4 hepatotoxicity as aspartate aminotransferase (AST) elevation (P < 0.04). The same observation was seen when comparing either the presence of HCV alone or the presence of the gene polymorphism alone. A significant association between the combined presence of HCV and MTHFR C677T polymorphism and grades 2-4 hepatotoxicity as alanine aminotransferase (ALT), AST, and alkaline phosphatase (ALP) elevation was observed (P values <0.001, 0.02, and 0.001, respectively). The presence of HCV infection had a significant negative effect on hepatic transaminases., Conclusions: The present data support a role for combining analysis of genetic variation in drug-metabolizing enzymes and the presence of HCV in the assessment of specific drugs toxicities in multiagent chemotherapeutic treatment regimens., (© 2016 Wiley Periodicals, Inc.)

Background: Insulin resistance (IR) and type 2 diabetes mellitus (T2DM) are serious extrahepatic manifestations of chronic hepatitis C virus (HCV) infection. However, the mechanism underlying the IR in chronic HCV is obscure. Hepatokines are group of liver-derived protein, which affect the glucose and lipid metabolism in several tissues. Fetuin A (also known as human α2-HS-glycoprotein) is one of the hepatokines, which was recognized as a natural inhibitor of the insulin receptor tyrosine kinase in liver and skeletal muscle. Additionally, selenoprotein P has emerged as an important hepatokine, which primarily acts as selenium transporter and has been reported to be implicated in glucose homeostasis in human., Objective: The aim of the current case-control study was to investigate the serum levels of both fetuin A and selenoprotein P in chronic hepatitis C patients with or without T2DM and to correlate their levels with other biochemical parameters of insulin resistance., Main Findings: Our results showed that, serum fetuin A levels increased significantly in HCV patients compared with controls (P<0.01) and surplus increase was found in HCV with concomitant T2DM (P>0.001). However, selenoprotein P levels significantly elevated only in patients with both HCV and T2DM (P<0.05) compared with the healthy subjects. Both fetuin A and selenoprotein P were positively correlated with fasting blood glucose. Yet, only fetuin A was significantly correlated to the HOMA-IR (r=0.28; P=0.03)., Conclusions: These results indicate crucial roles played by fetuin A and selenoprotein P in the IR caused by HCV and that both hepatokines may be targets for the development of therapies to treat or inhibit insulin resistance associated to HCV. However, further studies on large scale should be conducted to confirm our findings., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Context: In Arabic folk medicine, the seeds of Phoenix dactylifera L. (Arecaceae) have been used to manage diabetes for many years. Few studies have reported the antidiabetic effect of P. dactylifera seeds; however, their effect on diabetic complications is still unexplored., Objective: The present study investigates the protective effect of P. dactylifera seeds against diabetic complications in rats., Material and Methods: The aqueous suspension of P. dactylifera seeds (aqPDS) (1 g/kg/d) was orally administered to streptozotocin-induced diabetic rats for 4 weeks. The serum biochemical parameters were assessed spectrophotometrically. Furthermore, oxidative stress was examined in both liver and kidney tissues by assessment of thiobarbituric acid reactive substances (TBARS), nitric oxide (NO), reduced glutathione, superoxide dismutase (SOD), glutathione S-transferase, and catalase., Results: Oral administration of aqPDS significantly ameliorated the elevated levels of glucose (248 ± 42 versus 508 ± 60 mg/dl), urea (32 ± 3.3 versus 48.3 ± 5.6 mg/dl), creatinine (2.2 ± 0.35 versus 3.8 ± 0.37 mg/dl), ALT (29.6 ± 3.9 versus 46.4 ± 5.9 IU/l), and AST (73.3 ± 13 versus 127.8 ± 18.7 IU/l) compared with the untreated diabetic rats. In addition to significant augmentation in the activities of antioxidant enzymes, there was reduction in TBARS and NO levels and improvement of histopathological architecture of the liver and kidney of diabetic rats., Discussion and Conclusion: The aqPDS showed potential protective effects against early diabetic complications of both liver and kidney. This effect may be explained by the antioxidant and free radical scavenging capabilities of P. dactylifera seeds.

Autophagy is originally described as the main catabolic pathway responsible for maintaining intracellular nutritional homeostasis that involves the formation of a unique vacuole, the autophagosome, and the interaction with the endosome-lysosome pathways. This conserved machinery plays a key role in immune-protection against different invaders, including pathogenic bacteria, intracellular parasites, and some viruses like herpes simplex and hepatitis C virus. Importantly, autophagy is linked to a number of human diseases and disorders including neurodegenerative disease, Crohn's disease, type II diabetes, tumorigenesis, cardiomyopathy, and fatty liver disease. On the other hand, inflammasomes are multiprotein platforms stimulated upon several environmental conditions and microbial infection. Once assembled, the inflammasomes mediate the maturation of pro-inflammatory cytokines and promote phagosome-lysosome fusion to sustain an innate immune response. The intersections between autophagy and inflammasome have been observed in various diseases and microbial infections. This review highlights the molecular aspects involved in autophagy and inflammasome interactions during different medical conditions and microbial infections., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Ethnopharmacological Relevance: In traditional Egyptian medicine, Phoenix dactylifera L. (date palm) seeds are listed in folk remedies for the management of diabetes, liver diseases and gastrointestinal disorders. The present study was conducted to investigate the protective effect of Phoenix dactylifera L. seeds aqueous suspension against the chemically-induced hepatic injury in rats., Methods: Liver injury was achieved by exposing Wistar rats to CCl4 (10% in olive oil; 0.5 mL/rat; IP) twice a week for 4 weeks. Along with CCl4, aqueous suspensions of raw or roasted Phoenix dactylifera seeds (1.0 g/kg) were administered orally in a daily manner., Results: Our results demonstrated that Phoenix dactylifera seeds significantly improved the CCl4-induced alterations in liver function parameters (AST, ALT, ALP and albumin). Moreover, the CCl4-induced oxidative stress, represented by elevated thiobarbituric acid reactive substance (TBARS), nitric oxide and oxidative DNA damage, was ameliorated by Phoenix dactylifera seeds treatment. In addition, Phoenix dactylifera seeds restored the activities of hepatic antioxidant enzymes (superoxide dismutase and glutathione S-transferase) that were declined after CCl4 treatment. Examination of liver histopathology revealed that Phoenix dactylifera seeds attenuate the incidence of liver lesions (including vacuolization and fibroblast proliferation) triggered by CCl4 intoxication., Conclusion: The Phoenix dactylifera seeds could be a promising candidate for protection against the CCl4-induced liver intoxication, and this hepatoprotective effect might be attributed to the antioxidant and free radical scavenging activities., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

A series of pyrrole and pyrrolopyrimidine derivatives were examined for their in vivo antihyperglycemic activity. Compounds Ia-c,e, and IVg showed promising antihyperglycemic activity equivalent to a well-known standard antihyperglycemic drug, Glimepiride (Amaryl, 4 mg/kg). In this paper, we examine and discuss the structure-activity relationships and antihyperglycemic activity of these compounds.

Objectives: Omentin-1 is a novel adipokine that has a pivotal role in modulating the insulin sensitivity, immunity and inflammation. The current study was conducted to evaluate the serum omentin-1 level in hepatitis C virus (HCV) infected patients, with or without type 2 diabetes, and to investigate its correlation with liver function parameters and insulin resistance., Methods: Eighty subjects were enrolled in this study divided into four groups: chronic HCV infected patients (n=20), chronic hepatitis C patients with concomitant type 2 diabetes (n=18), type 2 diabetic patients (n=22) and 20 healthy controls. Serum omentin-1 levels were assessed using enzyme-linked immunosorbent assay (ELISA). Fasting blood glucose, insulin levels, and liver parameters including aminotransferases (ALT and AST) were determined., Results: Serum omentin-1 levels were significantly elevated in HCV infected patients compared to all other groups. Omentin-1 levels were positively correlated with AST and ALT levels (r =0.43, p< 0.001; r =0.423, p<0.001, respectively). Additionally, a significant negative correlation was found between omentin-1 and both fasting insulin levels and homeostasis model assessment of insulin resistance (HOMA-IR) (r = -0.238, p<0.05; r = -0.277, p<0.05, respectively). Furthermore, fasting blood glucose, HbA1c and HOMA-β were negatively correlated to serum omentin-1 levels however these correlations were not statistically significant., Conclusion: Serum omentin-1 level is elevated in HCV infected patients and is positively associated with liver enzymes AST and ALT. This suggested that omentin-1 may be implicated in the pathogenesis of hepatitis C and its metabolic complications. However its role needs to be elucidated by further studies.

Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.

Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type macrophages but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 (ΔF508) macrophages than in WT macrophages. An autophagosome is a compartment that engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, Rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.

The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase(-1-/-) and Nlrc4(-/-) allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc(-/-) macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.

The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-κB activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-κB pathway simultaneously.

Nlrc4 is a member of the Nod-like receptors (NLRs), a family of cytosolic receptors involved in sensing bacterial molecules. NLRs are a group of proteins containing spans of leucine-rich repeats that senses bacterial factors within the eukaryotic cytosol. The recognition of bacterial factors provokes the formation of the inflammasome complex which includes specific NLRs. The inflammasome is responsible for caspase-1 activation which leads to the cleavage and maturation of inflammatory cytokines such as IL-1beta and IL-18. Nlrc4 was considered to be a devoted flagellin sensor in eukaryotic cells. However, studies using a variety of pathogens such as Salmonella, Legionella, Shigella and Pseudomonas at high bacterial burdens revealed that Nlrc4 can mediate caspase-1 activation independent of bacterial flagellin. On the other hand, new reports showed that Nlrc4 can restrict bacterial infection independently of caspase-1. Therefore, Nlrc4 maybe involved in sensing more than one bacterial molecule and may participate in several immune complexes., (Published by Elsevier Ltd.)