The Role of Social Media Advertisement and Physical Activity on Eating Behaviors among the General Population in Saudi Arabia.

Over the past few decades, people in Saudi Arabia have become less inclined to adopt active lifestyles and healthy eating habits due to the increasing use of digital technologies such as social media. The objective of this online-based cross-sectional study was to assess the role of social media food advertisements and physical activity on eating behaviors among the general population in Saudi Arabia (n = 471). Data were collected through a structured questionnaire, which consisted of four parts: (i) sociodemographic information, (ii) attitude towards social media, (iii) eating behaviors-related information, and (iv) exposure to and engagement with social media advertisements. The study's outcome variable, eating behaviors (healthy vs. unhealthy), was assessed using the following question: "Are you on a healthy diet (such as a balanced diet, keto, or low carb)?" A multiple binary logistic regression analysis was performed to investigate the factors that influence unhealthy eating behaviors. Approximately 79.6% of the participants had unhealthy eating behaviors. Participants who were not involved in daily physical activity were more likely to have unhealthy eating behaviors compared to their counterparts (adjusted odds ratio, AOR = 4.86). Participants who watched food ads on social media channels 1-3 times a week (AOR = 2.58) or daily (AOR = 3.49) were more likely to have unhealthy eating behaviors than their counterparts. Participants whose appetite to try foods increases always (AOR = 1.42) or usually (AOR = 2.88) after viewing ads on social media were more likely to have unhealthy eating behaviors. These findings suggest that policymakers should take immediate action to regulate food advertising policy to promote a healthy food environment across the country. Saudis should be encouraged to engage in more physical activity, which could support the maintenance of healthy eating patterns and lifestyles.

Treatment strategies and treatment-related adverse events in MG according to the age of onset.

Introduction: Early-onset (EOMG) and late-onset (LOMG) are distinct groups of MG patients. It is unclear if treatment strategies and treatment-related adverse events may differ according to the age of MG onset. Methods: This single-center retrospective study includes all MG patients followed at a tertiary center since 2007. We reviewed the electronic clinical records. Results: In total, 212 patients were identified, 142 (67.0%) females, with a median disease duration of 10 years. The median age of symptom onset was 42.0 (26.0-64.5) years, with 130 (61.3%) EOMG cases and 82 (38.7%) LOMG. EOMG were more frequently female, had longer disease duration and often more generalized MG (p < 0.001). Comorbidities were significantly more frequent in LOMG (67.1%) compared to EOMG (53.1%) (p = 0.002). Steroid-related adverse effects motivating the switch to steroid-sparing agents (82.0%) were different between groups, with hypertension, hypercholesterolemia, diabetes mellitus and malignancies being more common in LOMG. At the same time, osteoporosis and dyspepsia were more frequent in EOMG (p < 0.001). The most common first-line choice was azathioprine (45.8%), and rituximab was used in 4 patients (1.9%). Conclusion: Our study shows that treatment modalities are similar between EOMG and LOMG, while steroid-related adverse events appear to be distinct.

Consumer Knowledge about Dietary Relevance of Fruits and Vegetables: A Study Involving Participants from Portugal and France.

Fruits and vegetables are recommended as low-calorie foods that contribute to the proper intake of necessary micronutrients, macronutrients, and bioactive compounds with health benefits. However, the recommendations for the dietary intake of these foods fail to be attained in most European countries. For this reason, promoting more knowledge about the health effects of fruits and vegetables is essential to decrease the incidence of chronic diseases. This study was conducted to investigate the knowledge of the health benefits of fruits and vegetables among the population of Portugal and France. The present work involved a questionnaire survey of 639 participants (257 from Portugal and 382 from France). The results revealed that most participants were young females (68.9%) with good education (76%) and an average weight range. They consumed a varied diet (57%) but had body dissatisfaction (63.2%). The respondents had good knowledge about the health effects of fruits and vegetables. However, the French population knew more about the theme than the Portuguese. Portuguese individuals were more likely to have incomplete information. Gender and education significantly influenced knowledge levels, with females and highly educated individuals demonstrating greater understanding. Dissatisfaction with body weight drives individuals to seek nutrition information. This investigation enhances our comprehension of the factors that affect knowledge of vegetable and fruit consumption among young adults in Portugal and France. Moreover, it highlights the importance of implementing focused educational programs to enhance nutrition literacy, particularly for less-aware demographic groups. Going forward, a more in-depth analysis of these factors could assist in creating more efficient strategies to encourage healthier dietary habits and improve nutrition literacy among these communities.

The platelet-to-lymphocyte ratio as an indirect outcome predictor in primary hypertension: a retrospective study.

Background: Nondipper hypertensive patients have higher levels of platelet-to-lymphocyte ratio, a new studied inflammatory biomarker in primary hypertension. Furthermore, these patients have a higher risk of cardiovascular morbidity and mortality. This study aimed to assess the relationship between platelet-to-lymphocyte ratio and hypertensive pattern (dipper vs nondipper) and the association between the hypertensive pattern and major adverse cardiovascular events. Methods: A retrospective analysis was performed. One hundred fifty-three patients were included and classified as dipper or nondipper according to 24-hour ambulatory blood pressure measurements. Platelet-to-lymphocyte ratio was calculated based on complete blood count data. Results: The dipper group included 109 patients, and the nondipper group included 44 patients. Nondipper patients have 2.11 more risk of presenting a higher platelet-to-lymphocyte ratio than dipper individuals (odds ratio [OR] = 2.11; 95% CI, 1.220-3.664; P = .007). Nondipper patients also registered earlier cardiovascular events, such as acute myocardial infarction and stroke (P < .001). Conclusions: Nondipper hypertensive individuals registered higher levels of platelet-to-lymphocyte ratio and earlier cardiovascular events than dipper patients. Therefore, platelet-to-lymphocyte ratio could be used as an indirect predictor of cardiovascular risk in primary hypertension and contribute to optimize preventive strategies.

Study protocol for a pilot randomised controlled trial evaluating the feasibility and effectiveness of non-pharmacological interventions to recover functionality after a transient ischaemic attack or a minor stroke: the 'Back to Normal' trial.

INTRODUCTION: Transient ischaemic attack (TIA) and minor stroke are frequently assumed as temporary or non-disabling events. However, evidence suggests that these patients can experience relevant impairment and functional disability. Therefore, the present study aims to evaluate the feasibility and effectiveness of a 3-month multidomain intervention programme, composed of five non-pharmacological strategies, aimed at accelerating return to pre-event level of functionality in patients with TIA or minor stroke. METHODS AND ANALYSIS: Patients diagnosed with a TIA or a minor stroke are being recruited at the emergency or neurology departments of the Hospital Pedro Hispano, located in Matosinhos, Portugal (n=70). Those who accept to participate will be randomly allocated to two groups (1:1): (a) Intervention-receives a 3 months combined approach, initiating early post-event, composed of cognitive training, physical exercise, nutrition, psychoeducation and assessment/correction of hearing loss; (b) Control-participants will not be subject to any intervention. Both groups will receive the usual standard of care provided to these diseases. Recruitment began in May 2022 and is expected to continue until March 2023. Socio-demographic characteristics, lifestyles, health status, cognitive function, symptoms of anxiety and depression and quality of life will be assessed; as well as anthropometry, blood pressure and physical condition. Time to complete or partial recovery of instrumental activities of daily living will be assessed using an adapted version of the Frenchay Activities Index. All participants will be evaluated before the intervention and after 3 months. ETHICS AND DISSEMINATION: This study was approved by the Ethics Committee of the Local Health Unit of Matosinhos (Ref. 75/CES/JAS). Written informed consent will be required from all the participants; data protection and confidentiality will be also ensured. The findings of this project are expected to be submitted for publication in scientific articles, and the main results will be presented at relevant scientific meetings. TRIAL REGISTRATION NUMBER: NCT05369637.

Characterisation of institutionalised Portuguese older adult fallers: is there a place for pharmacist intervention? A preliminary study

Background: Falls are a major public health issue, given their prevalence and social impact. Older adults living in long-term care facilities (LTCF) are at greater risk of injury resulting from a fall due to multiple factors, such as nutritional, functional/cognitive impairment, postural instability, polypharmacy, and the presence of potentially inappropriate medications (PIMs). Medication management in LTCF is complex and often sub-optimal and might be crucial for falls. Pharmacist intervention is important, since they have a unique knowledge of medication. However, studies mapping the impact of pharmaceutical activities in Portuguese LTC settings are scarce. Objective: This study aims to assess the characteristics of older adult fallers living in LTFCs and examine the relationship between falling and several factors in this population. We also intend to explore the prevalence of PIMs and their relationship with the occurrence of falls. Methods: The study was conducted in two long-term care facilities for elderly people, in the central region of Portugal. We included patients aged 65 and older with no reduced mobility or physical weakness and with the ability to understand spoken and written Portuguese. The following information was assessed: sociodemographic characteristics, comorbidities, polypharmacy, fear of falling, functional, nutritional and cognitive status. PIMs were evaluated according to the Beers criteria (2019). Results: A total of 69 institutionalised older adults, 45 women and 24 men, with a mean age of 83.14 ± 8.87 years were included. The prevalence of falls was 21.74% Out of these, 46.67% (n=7) fell once, 13.33% (n=2) fell twice, and 40% (n=6) fell 3 or more times. Fallers were mainly women, had lower levels of education, were well nourished, had moderate to severe levels of dependence, and displayed moderate cognitive impairment. (AU)

Short- and long-term outcome and predictors in an international cohort of patients with neuro-COVID-19.

BACKGROUND AND PURPOSE: Despite the increasing number of reports on the spectrum of neurological manifestations of COVID-19 (neuro-COVID), few studies have assessed short- and long-term outcome of the disease. METHODS: This is a cohort study enrolling adult patients with neuro-COVID seen in neurological consultation. Data were collected prospectively or retrospectively in the European Academy of Neurology NEuro-covid ReGistrY ((ENERGY). The outcome at discharge was measured using the modified Rankin Scale and defined as 'stable/improved' if the modified Rankin Scale score was equal to or lower than the pre-morbid score, 'worse' if the score was higher than the pre-morbid score. Status at 6 months was also recorded. Demographic and clinical variables were assessed as predictors of outcome at discharge and 6 months. RESULTS: From July 2020 to March 2021, 971 patients from 19 countries were included. 810 (83.4%) were hospitalized. 432 (53.3%) were discharged with worse functional status. Older age, stupor/coma, stroke and intensive care unit (ICU) admission were predictors of worse outcome at discharge. 132 (16.3%) died in hospital. Older age, cancer, cardiovascular complications, refractory shock, stupor/coma and ICU admission were associated with death. 262 were followed for 6 months. Acute stroke or ataxia, ICU admission and degree of functional impairment at discharge were predictors of worse outcome. 65/221 hospitalized patients (29.4%) and 10/32 non-hospitalized patients (24.4%) experienced persisting neurological symptoms/signs. 10/262 patients (3.8%) developed new neurological complaints during the 6 months of follow-up. CONCLUSIONS: Neuro-COVID is a severe disease associated with worse functional status at discharge, particularly in older subjects and those with comorbidities and acute complications of infection.

Characterisation of institutionalised Portuguese older adult fallers: is there a place for pharmacist intervention? A preliminary study.

Background: Falls are a major public health issue, given their prevalence and social impact. Older adults living in long-term care facilities (LTCF) are at greater risk of injury resulting from a fall due to multiple factors, such as nutritional, functional/cognitive impairment, postural instability, polypharmacy, and the presence of potentially inappropriate medications (PIMs). Medication management in LTCF is complex and often sub-optimal and might be crucial for falls. Pharmacist intervention is important, since they have a unique knowledge of medication. However, studies mapping the impact of pharmaceutical activities in Portuguese LTC settings are scarce. Objective: This study aims to assess the characteristics of older adult fallers living in LTFCs and examine the relationship between falling and several factors in this population. We also intend to explore the prevalence of PIMs and their relationship with the occurrence of falls. Methods: The study was conducted in two long-term care facilities for elderly people, in the central region of Portugal. We included patients aged 65 and older with no reduced mobility or physical weakness and with the ability to understand spoken and written Portuguese. The following information was assessed: sociodemographic characteristics, comorbidities, polypharmacy, fear of falling, functional, nutritional and cognitive status. PIMs were evaluated according to the Beers criteria (2019). Results: A total of 69 institutionalised older adults, 45 women and 24 men, with a mean age of 83.14 ± 8.87 years were included. The prevalence of falls was 21.74% Out of these, 46.67% (n=7) fell once, 13.33% (n=2) fell twice, and 40% (n=6) fell 3 or more times. Fallers were mainly women, had lower levels of education, were well nourished, had moderate to severe levels of dependence, and displayed moderate cognitive impairment. All adult fallers had a fear of falling. The main comorbidities of this population were related to the cardiovascular system. Polypharmacy was present in every patient, and at least one PIM was identified in 88.41% of the subjects. Fear of falling (FOF) and cognitive impairment (in subjects with 1 to 11 years of education) showed statistically significant associations with the occurrence of falls (p=0.005 and p=0.05, respectively). No significant differences were found between fallers and non-fallers for any other factors. Conclusions: This present study is a preliminary contribution to characterise a group of older adult fallers living in Portuguese LTCFs and demonstrated that fear of falling and cognitive impairment are associated with the occurrence of falls in this population. The high prevalence of polypharmacy and PIMs emphasises the need for tailored interventions featuring the collaboration of a pharmacist to optimise medication management in this population.

Edible Mushrooms: A Comprehensive Review on Bioactive Compounds with Health Benefits and Processing Aspects.

Mushrooms are well-known functional foods due to the presence of a huge quantity of nutraceutical components. These are well recognized for their nutritional importance such as high protein, low fat, and low energy contents. These are rich in minerals such as iron, phosphorus, as well as in vitamins like riboflavin, thiamine, ergosterol, niacin, and ascorbic acid. They also contain bioactive constituents like secondary metabolites (terpenoids, acids, alkaloids, sesquiterpenes, polyphenolic compounds, lactones, sterols, nucleotide analogues, vitamins, and metal chelating agents) and polysaccharides chiefly ß-glucans and glycoproteins. Due to the occurrence of biologically active substances, mushrooms can serve as hepatoprotective, immune-potentiating, anti-cancer, anti-viral, and hypocholesterolemic agents. They have great potential to prevent cardiovascular diseases due to their low fat and high fiber contents, as well as being foremost sources of natural antioxidants useful in reducing oxidative damages. However, mushrooms remained underutilized, despite their wide nutritional and bioactive potential. Novel green techniques are being explored for the extraction of bioactive components from edible mushrooms. The current review is intended to deliberate the nutraceutical potential of mushrooms, therapeutic properties, bioactive compounds, health benefits, and processing aspects of edible mushrooms for maintenance, and promotion of a healthy lifestyle.

Neuro-COVID frequency and short-term outcome in the Northern Portuguese population.

BACKGROUND AND PURPOSE: COVID-19-related acute neurological phenotypes are being increasingly recognised, with neurological complications reported in more than 30% of hospitalised patients. However, multicentric studies providing a population-based perspective are lacking. METHODS: We conducted a retrospective multicentric study at five hospitals in Northern Portugal, representing 45.1% of all hospitalised patients in this region, between 1 March and 30 June 2020. RESULTS: Among 1261 hospitalised COVID-19 patients, 457 (36.2%) presented neurological manifestations, corresponding to a rate of 357 per 1000 in the North Region. Patients with neurologic manifestations were younger (68.0 vs. 71.2 years, p = 0.002), and the most frequent neurological symptoms were headache (13.4%), delirium (10.1%), and impairment of consciousness (9.7%). Acute well-defined central nervous system (CNS) involvement was found in 19.1% of patients, corresponding to a rate of 217 per 1000 hospitalised patients in the whole region. Assuming that all patients with severe neurological events were hospitalised, we extrapolated our results to all COVID-19 patients in the region, estimating that 116 will have a severe neurological event, corresponding to a rate of nine per 1000 (95% CI = 7-11). Overall case fatality in patients presenting neurological manifestations was 19.8%, increasing to 32.6% among those with acute well-defined CNS involvement. CONCLUSIONS: We characterised the population of hospitalised COVID-19 patients in Northern Portugal and found that neurological symptoms are common and associated with a high degree of disability at discharge. CNS involvement with criteria for in-hospital admission was observed in a significant proportion of patients. This knowledge provides the tools for adequate health planning and for improving COVID-19 multidisciplinary patient care.

Prevalence of cardiovascular risk factors and other comorbidities in patients with hypertension in Portuguese primary health care populations: The PRECISE study. / Prevalência de fatores de risco cardiovascular e outras comorbilidades em doentes com hipertensão arterial assistidos nos Cuidados de Saúde Primários: estudo Precise.

INTRODUCTION: Cardiovascular (CV) disease is the leading cause of death in Portugal. The prevalence of hypertension, the second most important risk factor accounting for overall disability-adjusted life years (DALYs), is significant. Hypertension rarely occurs in isolation, but is usually associated with other determining risk factors that contribute to greater overall CV risk. The main objective of the PRECISE study, a cross-sectional epidemiological study, was to determine the prevalence of other concomitant modulating CV risk factors in hypertensive patients. METHODS: The prevalence of other CV risk factors and target organ damage was assessed in 2848 hypertensive patients of both sexes followed in primary health care centers. Demographic, anthropometric and clinical data and antihypertensive and lipid-lowering therapies prescribed were collected. RESULTS: Of the study population (mean age 65.8±11.0 years, 60.8% women), 98.0% were treated for hypertension, but only 56.7% had controlled blood pressure. Hypercholesterolemia was the most frequent concomitant CV risk factor (82.1%), followed by sedentary behavior (71.4%). Prevalences of concomitant modulating risk factors were significantly different between the sexes and age groups. Overall, 81.7% of hypertensive patients had three or more concomitant CV risk factors. CONCLUSIONS: The study showed that, in Portugal, hypertensive patients have a high prevalence of other CV risk factors, confirming the need to identify these factors, calculate overall CV risk and continuously monitor the care provided and the results obtained.

Physiological and operative severity score for the enumeration of mortality and morbidity, frailty, and perioperative quality of life in the elderly.

BACKGROUND: Physiological and Operative Severity Score for the Enumeration of Mortality and Morbidity (POSSUM) is a validated instrument used to predict morbidity. The aim of our study was to evaluate the performance of the POSSUM score system on predicting perioperative frailty and quality of life (QOL) in elderly surgical patients. PATIENTS AND METHODS: An observational prospective study was conducted during 3 months. POSSUM was used to determine operative morbidity risk. Patients with a POSSUM score ≥26 were considered as having a high POSSUM (PHP). WHODAS 2.0, EuroQOL-5 dimensions (EQ-5D), Charlson score, and the Clinical Frailty Scale were used to assess the QOL and frailty. Chi-square, Fisher's exact, or Mann-Whitney tests were used for comparisons. RESULTS: Two hundred and thirty-five patients were included. Median age was 69 years; 58% were ASA I/II and 42% ASA III/IV. Frailty was present in 53 patients (23%). Median POSSUM score was 26. Patients PHP were older (median age 71 vs. 68, P = 0.008), more frequently ASA III/IV (P = 0.001), had higher median Charlson scores (7 vs. 5, P = 0.006) and were more frail (49% vs. 26%, P < 0.001). PHP presented more problems in EQ-5D dimensions preoperatively (mobility: 59% vs 41%, P = 0.008; care: 41% vs. 25%, P = 0.013; activity: 52% vs. 32%, P = 0.002; pain: 59% vs. 45%, P = 0.041) but not anxiety (P = 0.137). Three months after surgery, PHP patients presented more problems in mobility: 63% vs. 38%, P < 0.001; care: 48% vs. 31%, P = 0.009; activity: 58% vs. 44%, P = 0.036; pain 59% vs. 37%, P = 0.001 and anxiety: 54% vs. 50%, P = 0.025. CONCLUSIONS: Patients PHP were frailer and had worse perioperative QOL.

Autonomously hyperfunctioning cystic nodule harbouring thyroid carcinoma - Case report and literature review.

INTRODUCTION: Hyperthyroidism is rarely associated with malignancy, but it cannot rule out thyroid cancer. Although there is published data describing this coexistence, thyroid carcinomas inside autonomously functioning nodules are uncommon. PRESENTATION OF CASE: A 49-year-old woman presented with a cervical mass, unexplained weight loss and anxiousness, sweating and insomnia. On physical examination, she had a palpable left thyroid nodule. Thyroid function tests showed suppressed TSH (<0,1 uUI/mL), thyroxine 1,44 ng/dL (normal range 0,70-1,48) and triiodothyronine 4,33 pg/mL (normal range 1,71-3,71). Ultrasound imaging revealed a left lobe, 4 cm partial cystic nodule. 99mTC thyroid scintigraphy showed a hyperfunctioning nodule with suppression of the remainder parenchyma. Fine-needle aspiration cytology was nondiagnostic (cystic fluid). The patient was started on thiamazole 5 mg daily with subsequent normalization of thyroid function, but she developed cervical foreign body sensation and a left hemithyroidectomy was performed. Histology showed a 4 cm cystic nodule with a follicular variant papillary carcinoma and the patient underwent completion thyroidectomy, followed by radio-iodine ablation. DISCUSSION: Published literature showed an increased prevalence of autonomously functioning nodules, harbouring thyroid carcinomas in adults. Papillary carcinoma is the most frequently described but the follicular variant is rare. CONCLUSION: Although rare, thyroid cancer is not definitively excluded in hyperthyroid patients and it should always be considered as differential diagnosis.

Cochrane Corner: Antihypertensive efficacy of beta-1 selective beta blockers for primary hypertension. / Cochrane Corner: eficácia anti­hipertensora dos betabloqueadores seletivos beta­1 na hipertensão essencial.

Beta blockers are commonly used to treat hypertension. This Cochrane systematic review assessed the effect of beta-1 selective beta blockers on blood pressure (BP), pulse pressure (PP), heart rate (HR) and withdrawal due to adverse effects in patients with primary hypertension. Fifty-six randomized placebo-controlled trials were included, with a total of 7812 patients. These drugs reduced systolic/diastolic BP by 10/8 mmHg, PP by 2 mmHg and HR by 11 bpm; no difference was found between treatment and placebo regarding withdrawal due to adverse effects. Differences in efficacy were observed between the various beta-1 selective beta blockers, which may be due to methodological differences in the trials. The choice of an antihypertensive drug should take into account not only its efficacy in reducing BP but also its tolerability, its efficacy in preventing cardiovascular events, and other factors such as undesirable metabolic effects.

Translation and cultural adaptation of the Hill-Bone Compliance to High Blood Pressure Therapy Scale to Portuguese.

INTRODUCTION: Hypertension is an extremely prevalent disease worldwide and hypertension control rates remain low. Lack of adherence contributes to poor control and to cardiovascular events. No questionnaire in Portuguese is readily available for the assessment of adherence to antihypertensive drugs. We aimed to perform a translation and cultural adaptation to Portuguese of the Hill-Bone Compliance to High Blood Pressure Therapy Scale, a validated instrument to measure adherence in hypertensive patients. METHODS: A formal process was employed, consisting of a forward translation by two independent translators and a back translation by a third translator. Discrepancies were resolved after each step. Hypertensive patients were involved to identify and resolve phrasing and wording difficulties and misunderstandings. RESULTS: The forward and back translation did not produce significant discrepancies. However, important issues were identified when the questionnaire was presented to patients, which led to changes in the wording of the questions and in the format of the questionnaire. CONCLUSION: Questionnaires are important instruments to assess adherence to therapy, particularly in hypertension. A formal translation and cultural adaptation process ensures that the new version maintains the same concepts as the original. After translation, several changes were necessary to ensure that the questionnaire was understandable by elderly, low literacy patients, such as the majority of hypertensive patients. We propose a Portuguese version of the Hill-Bone Compliance Scale, which will require validation in further studies.

Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture.

A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic ß-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Krüppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells. Islet-enriched pancreatic cells, allowed to dedifferentiate and expand in adherent cell culture, were transduced with an adenovirus containing KLF4 (Ad-Klf4). Cells were subsequently analysed for changes in cell morphology by light microscopy, and for the presence of epithelial and pancreatic markers by immunocytochemistry and quantitative RT/PCR. Infection with Ad-Klf4 resulted in morphological changes, down-regulation of mesenchymal markers, and re-expression of both epithelial and pancreatic cell markers including insulin and transcription factors specific to ß-cells. This effect was further enhanced by culturing cells in suspension. However, the effects of Ad-KLf4 were transient and this was shown to be due to increased apoptosis in Klf4-expressing cells. Klf4 has been recently identified as a pioneer factor with the ability to modulate the structure of chromatin and enhance reprogramming/transdifferentiation. Our results show that Klf4 may have a role in the redifferentiation of expanded pancreatic cells in culture, but before this can be achieved the off-target effects that result in increased apoptosis would need to be overcome.

Suppression of epithelial-to-mesenchymal transitioning enhances ex vivo reprogramming of human exocrine pancreatic tissue toward functional insulin-producing ß-like cells.

Because of the lack of tissue available for islet transplantation, new sources of ß-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting ß-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-ß1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.

Pancreatic transcription factors containing protein transduction domains drive mouse embryonic stem cells towards endocrine pancreas.

Protein transduction domains (PTDs), such as the HIV1-TAT peptide, have been previously used to promote the uptake of proteins into a range of cell types, including stem cells. Here we generated pancreatic transcription factors containing PTD sequences and administered these to endoderm enriched mouse embryonic stem (ES) cells under conditions that were designed to mimic the pattern of expression of these factors in the developing pancreas. The ES cells were first cultured as embryoid bodies and treated with Activin A and Bone morphogenetic protein 4 (BMP4) to promote formation of definitive endoderm. Cells were subsequently plated as a monolayer and treated with different combinations of the modified recombinant transcription factors Pdx1 and MafA. The results demonstrate that each transcription factor was efficiently taken up by the cells, where they were localized in the nuclei. RT-qPCR was used to measure the expression levels of pancreatic markers. After the addition of Pdx1 alone for a period of five days, followed by the combination of Pdx1 and TAT-MafA in a second phase, up-regulation of insulin 1, insulin 2, Pdx1, Glut2, Pax4 and Nkx6.1 was observed. As assessed by immunocytochemistry, double positive insulin and Pdx1 cells were detected in the differentiated cultures. Although the pattern of pancreatic markers expression in these cultures was comparable to that of a mouse transformed ß-cell line (MIN-6) and human islets, the expression levels of insulin observed in the differentiated ES cell cultures were several orders of magnitude lower. This suggests that, although PTD-TFs may prove useful in studying the role of exogenous TFs in the differentiation of ES cells towards islets and other pancreatic lineages, the amount of insulin generated is well below that required for therapeutically useful cells.

Efficient differentiation of AR42J cells towards insulin-producing cells using pancreatic transcription factors in combination with growth factors.

The AR42J-B13 rat pancreatic acinar cell line was used to identify pancreatic transcription factors and exogenous growth factors (GFs) that might facilitate the reprogramming of exocrine cells into islets. Adenoviruses were used to induce exogenous expression of the pancreatic transcription factors (TFs) Pdx1, MafA, Ngn3 and Pax4. Individually Pdx1, MafA and Pax4 had no effect on the expression of endocrine markers, whilst adeno-Ngn3 on its own increased the expression of Pax4, Ngn3 and NeuroD. In combination the four TFs had a significant effect on the expression of insulin 1 and 2 that was associated with a change in cell morphology from a rounded to a spindle-like shape. Amongst a range of growth factors, Betacellulin and Nicotinamide were shown to enhance the effects of the four TFs. The presence of adeno-Pax4 in the differentiation cocktail was important in limiting the expression of glucagon and in generating glucose sensitive insulin secretion. Further experiments asked whether the adenoviral TFs could be replaced by protein transduction domain (PTD)-containing TFs. The results showed that the PTD-TFs could mimic in part the effects of the adeno-TFs, but the resultant cells did not undergo the important morphological change associated with differentiation to endocrine lineages and levels of endogenous markers were very much lower. In summary, the results describe a cocktail of four TFs and two GFs that can be used to induce formation of glucose sensitive insulin secreting cells from ARJ42 cells, and demonstrate that it would be difficult to replace adenoviral transduction with PTD-TFS.