Physiologically Based Pharmacokinetic Modeling of Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid membrane bound-cell-derived structures that are a key player in intercellular communication and facilitate numerous cellular functions such as tumor growth, metastasis, immunosuppression, and angiogenesis. They can be used as a drug delivery platform because they can protect drugs from degradation and target specific cells or tissues. With the advancement in the technologies and methods in EV research, EV-therapeutics are one of the fast-growing domains in the human health sector. Therapeutic translation of EVs in clinics requires assessing the quality, safety, and efficacy of the EVs, in which pharmacokinetics is very crucial. We report here the application of physiologically based pharmacokinetic (PBPK) modeling as a principal tool for the prediction of absorption, distribution, metabolism, and excretion of EVs. To create a PBPK model of EVs, researchers would need to gather data on the size, shape, and composition of the EVs, as well as the physiological processes that affect their behavior in the body. The PBPK model would then be used to predict the pharmacokinetics of drugs delivered via EVs, such as the rate at which the drug is absorbed and distributed throughout the body, the rate at which it is metabolized and eliminated, and the maximum concentration of the drug in the body. This information can be used to optimize the design of EV-based drug delivery systems, including the size and composition of the EVs, the route of administration, and the dose of the drug. There has not been any dedicated review article that describes the PBPK modeling of EV. This review provides an overview of the absorption, distribution, metabolism, and excretion (ADME) phenomena of EVs. In addition, we will briefly describe the different computer-based modeling approaches that may help in the future of EV-based therapeutic research.

Secretory phospholipase (sPLA2-IIA) regulates breast cancer stem cells differentiation and metastatic potential.

Breast cancer is the second most cancer worldwide in females. The primary factor responsible for tumor recurrence is the presence of breast cancer stem cells (BCSCs), which escape the chemo-radiotherapy. In this study, we have investigated the role of Secretory phospholipase-A2 Group 2A (sPLA2-IIA) that is overexpressed in BCSCs of MCF7 and MDA-MB-231 breast cancer cell lines. Further, overexpression of sPLA2-IIA revealed an increased EGFR/JNK/c-JUN/c-FOS signaling in BCSCs, while sPLA2-IIA knockdown significantly reduced the percentage of BCSCs and decreased signaling in both the cell lines. Importantly, sPLA2-IIA knockdown showed differentiation of BCSCs. Strikingly, PET imaging showed a decreased metastatic potential of BCSCs. Our study revealed a novel role of sPLA2-IIA in regulating BCSCs, which play a crucial role in regulating the differentiation and metastatic potential of BCSCs.

Epithelial-to-mesenchymal transition status correlated with ultrastructural features, and TP53 mutation in patient-derived oral cancer cell lines.

BACKGROUND: Oral Squamous Cell Carcinoma (OSCC) is a highly prevalent cancer in the Indian subcontinent. The major cause of mortality in OSCC patients is metastasis. Epithelial-to-mesenchymal transition (EMT) marks an important step in the metastatic process. Additionally, TP53, an important tumor suppressor gene, is also a significant determinant of the treatment outcome, and also plays a role in EMT. Therefore, understanding the interconnections between ultrastructural features, EMT status and TP53 mutational status is of vital importance. METHODS AND RESULTS: The ultrastructure of five OSCC cell lines was visualized by transmission electron microscopy. Trans-well invasion and migration assays as well as scratch-wound assay, and the expression of various EMT-related genes were utilized to assess the EMT status of the cell lines. The TP53 exons were amplified for the ACOSC3, ACOSC4 and ACOSC16 cell lines and sequenced and the mutations in the gene were identified by sequence alignment. The TP53 mutation in the UPCI:SCC029B cell line has been previously reported, while UPCI:SCC040 has been reported to harbor a wild type TP53. The ACOSC4 cell line which showed the shortest intercellular gaps, also had the least invasive and migratory potential. Interestingly, ACOSC4 showed the highest expression of E-cadherin and the lowest expression of Vimentin, TWIST1, ZEB1, and MMPs. Additionally, TP53 gene of ACOSC4 was unmutated, whereas the ACOSC3 and ACOSC16 harbored TP53 mutations. The mutation in ACOSC3 (R196*) was also found in 7 TCGA samples. Similarly, the UPCI:SCC040 cell line that harbors a wild type TP53 showed shorter intracellular gaps. CONCLUSIONS: Cellular migratory properties are associated with cellular ultrastructure, epithelial-to-mesenchymal transition status and the status of TP53 mutation in the genome.

BMP-AKT-GSK3ß Signaling Restores Hair Follicle Stem Cells Decrease Associated with Loss of Sfrp1.

Wnt signaling plays a pivotal role in regulating activation, proliferation, stem cell renewal, and differentiation of hair follicle stem cells (HFSCs). Secreted frizzled-related protein 1 (Sfrp1), a Wnt antagonist is upregulated in the HFSCs; however, its role in the HFSCs regulation is still obscure. Here, we show that Sfrp1 loss showed a depletion of HFSCs, enhanced HFSC proliferation, and faster hair follicle cycle at PD21-PD28; HFSC markers, such as Lgr5 and Axin2, were decreased in both the Sfrp1+/- and Sfrp1-/- HFSCs. In addition, the second hair follicle cycle was also faster compared with WT. Importantly, Sfrp1-/- showed a restoration of HFSC by second telogen (PD49), whereas Sfrp1+/- did not show restoration with still having a decreased HFSC. In fact, restoration of HFSCs was due to a pronounced downregulation of ß-catenin activity mediated through a cross-talk of BMP-AKT-GSK3ß signaling in Sfrp1-/- compared with Sfrp1+/-, where downregulation was less pronounced. In cultured keratinocytes, Sfrp1 loss resulted in enhanced proliferation and clonogenicity, which were reversed by treating with either BMPR1A or GSK3ß inhibitor thereby confirming BMP-AKT-GSK3ß signaling involved in ß-catenin regulation in both the Sfrp1+/- and Sfrp1-/- mice. Our study reveals a novel function of Sfrp1 by unraveling an in vivo molecular mechanism that regulates the HFSCs pool mediated through a hitherto unknown cross-talk of BMP-AKT-GSK3ß signaling that maintains stem cell pool balance, which in turn maintains skin tissue homeostasis.

Single Gene Mutations in Pkd1 or Tsc2 Alter Extracellular Vesicle Production and Trafficking.

Patients with autosomal dominant polycystic kidney disease (ADPKD) and tuberous sclerosis complex (TSC) are born with normal or near-normal kidneys that later develop cysts and prematurely lose function. Both renal cystic diseases appear to be mediated, at least in part, by disease-promoting extracellular vesicles (EVs) that induce genetically intact cells to participate in the renal disease process. We used centrifugation and size exclusion chromatography to isolate the EVs for study. We characterized the EVs using tunable resistive pulse sensing, dynamic light scattering, transmission electron microscopy, and Western blot analysis. We performed EV trafficking studies using a dye approach in both tissue culture and in vivo studies. We have previously reported that loss of the Tsc2 gene significantly increased EV production and here demonstrate that the loss of the Pkd1 gene also significantly increases EV production. Using a cell culture system, we also show that loss of either the Tsc2 or Pkd1 gene results in EVs that exhibit an enhanced uptake by renal epithelial cells and a prolonged half-life. Loss of the primary cilia significantly reduces EV production in renal collecting duct cells. Cells that have a disrupted Pkd1 gene produce EVs that have altered kinetics and a prolonged half-life, possibly impacting the duration of the EV cargo effect on the recipient cell. These results demonstrate the interplay between primary cilia and EVs and support a role for EVs in polycystic kidney disease pathogenesis.

Secretory phospholipase sPLA2-IIAloss impairs tumorigenic and metastatic potential in breast cancer cells.

Breast cancer stem cells (BCSCs) are slow cycling cells that escape the traditional chemo-radio-therapy, thereby contributing in resistance and recurrence. Although several markers have been identified, it is still challenging to develop strategies targeting them. In this study, we have isolated BCSCs from MCF-7 cell line using markers CD44+/CD24-/low, which showed higher percentage of mammospheres in CSC population. Moreover, in vivo tumorigenic potential of BCSCs showed as low as 10,000 cells had the ability to develop tumors when transplanted into NOD-SCID mice. We observed an increased level of EMT markers in CSC population. Overexpression of secretory phospholipase sPLA2-IIA was found in CSCs. Further, we have uncovered the upregulation of sPLA2-IIA mediated through JNK signaling in breast cancer cells whereas knockdown of sPLA2-IIA reduces JNK signaling, cell proliferation, EMT and in vivo tumorigenic potential in breast cancer cells. Our study reveals overexpression of sPLA2-IIA in two different breast cancer cells such as MCF7 (ER+,PR+) and a triple negative, MDA-MB-231 (ER-PR-HER2-). Further, the novel role of sPLA2-IIA was discerned by unraveling the molecular mechanism, which regulates the cell proliferation and metastasis in breast cancer cells.

Toxicokinetic and Genotoxicity Study of NNK in Male Sprague Dawley Rats Following Nose-Only Inhalation Exposure, Intraperitoneal Injection, and Oral Gavage.

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10-5, 5 × 10-3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9-10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK.

Apoptosis inducing factor: Cellular protective function in Dictyostelium discoideum.

Apoptosis Inducing Factor (AIF), a nuclear encoded mitochondrial inter-membrane space flavoprotein with intrinsic NADH oxidase activity, plays an important role in inducing cell death mechanisms. In response to cell death signals, it undergoes mitochondrio-nuclear translocation leading to DNA fragmentation. In addition to its role in cell death, AIF has a pro-survival role, wherein it contributes to the maintenance of mitochondrial structure and function in a coordinated manner. However, its exact mechanism of controlling mitochondrial homeostasis is unclear. The current study aims to explore the protective functions of AIF by its downregulation and overexpression in Dictyostelium discoideum. Constitutive AIF downregulated (dR) cells exhibited compromised oxidative phosphorylation along with elevated levels of cellular ROS. Interestingly, constitutive AIF dR cells showed amelioration in the activity of the ETC complexes upon antioxidant treatment, strengthening AIF's role as an ROS regulator, by virtue of its oxidoreductase property. Also, constitutive AIF dR cells showed lower transcript levels of the various subunits of ETC. Moreover, loss of AIF affected mtDNA content and mitochondrial fusion-fission mechanism, which subsequently caused morphometric mitochondrial alterations. Constitutive AIF overexpressed (OE) cells also showed higher cellular ROS and mitochondrial fission genes transcript levels along with reduced mitochondrial fusion genes transcript levels and mtDNA content. Thus, the results of the current study provide a paradigm where AIF is implicated in cell survival by maintaining mitochondrial bioenergetics, morphology and fusion-fission mechanism in D. discoideum, an evolutionarily significant model organism for mitochondrial diseases.

Mindfulness in Surgery.

In the past 20 years, interest in mindfulness and its cultivation through various meditative practices has increased astronomically. This is reflected in the popularity of mindfulness training programs, its ever-widening exposure in popular culture, and in the number of scientific articles published on the topic. With the recent focus on burnout in the medical profession and reports of high levels of anxiety and depression among residents and staff physicians, the potential applications of mindfulness are becoming increasingly apparent in the hospital setting. Mindfulness meditation may be particularly useful for surgeons because they are required to maintain their presence of mind and mental focus in the setting of challenging physical and mental tasks. Furthermore, personality traits such as perfectionism and intensity, which may have facilitated success in the competitive environment of medical school and residency training, may later manifest as intolerance and impatience, contributing to frustration and anger. A mindfulness meditation practice may help reduce the tendency to react to these emotions, yet still allow surgeons to remain motivated to excel. This article provides a definition of mindfulness and describes its introduction to Western culture. The connection of a regular meditation practice to improvements in focus and performance are reviewed. The potential benefits of mindfulness training to a surgical career are discussed, and an approach to introducing mindfulness and meditation to individual surgeons and surgical departments is outlined. We hypothesize that the introduction of department-wide training programs in mindfulness and meditation could benefit surgeons with regard to technical performance, empathy toward patients, academic productivity, and general life and career satisfaction.

Cardiovascular Disease & Cancer Risk Among South Asians: Impact of Sociocultural Influences on Lifestyle and Behavior.

A comprehensive literature review revealed cultural beliefs, societal obligations, and gender roles within the South Asian community to be indirect contributors to the health of South Asian immigrants (SAIs). Health professionals need to increase their work with SAI communities to change less beneficial cultural elements such as misconceptions about health and exercise, and lack of communication when using alternative medicines. Community engaged efforts and continuing medical education are both needed to improve the health of the South Asian immigrant population in a culturally appropriate manner.

Effect of Hepatitis C Treatment with Ombitasvir/Paritaprevir/R + Dasabuvir on Renal, Cardiovascular and Metabolic Extrahepatic Manifestations: A Post-Hoc Analysis of Phase 3 Clinical Trials.

INTRODUCTION: We analyzed phase 3 trial data of ombitasvir/paritaprevir/ritonavir and dasabuvir (3D) ± ribavirin (RBV) in genotype 1 chronic hepatitis C patients to investigate the impact of 3D ± RBV on renal, cardiovascular and metabolic extrahepatic manifestations (EHMs), including persistency 52 weeks post treatment and differential impact by EHM disease severity. METHODS: Estimated glomerular filtration rate (eGFR), fasting triglyceride and fasting glucose values from clinical trials were used to assess renal, cardiovascular and metabolic EHMs, respectively. Two placebo-controlled trials were used to study the effect of treatment, while the pooled sample of treated patients was used to study the persistency and differential effect of treatment by baseline EHM disease severity, as defined by baseline values of respective EHM biomarkers. Changes in EHM outcomes from baseline were assessed with mixed models adjusting for patient baseline demographic and clinical characteristics. RESULTS: Treatment with 3D ± RBV resulted in statistically significant declines from baseline of triglycerides and glucose and no statistical change in eGFR. By 52 weeks post treatment patients with elevated triglycerides (-35.3 mg/dl), pre-diabetes (-4.4 mg/dl), diabetes (-34.2 mg/dl) and CKD stage 3 (+1.6 ml/min/1.73 m2) at baseline experienced a statistically significant improvement in their respective EHM values. Patients with CKD stages 2, 4 and 5 experienced no statistically significant change in eGFR from baseline. CONCLUSION: Treatment with 3D ± RBV resulted in improvement or no worsening of cardiovascular, metabolic and renal EHM markers, especially in patients with severe EHMs at baseline, which persisted until 52 weeks post treatment. FUNDING: Abbvie Inc.

A systematic review of psychosocial therapies for children with rheumatic diseases.

BACKGROUND: To assess the quality of evidence for the effects of psychosocial therapies on pain and function in children with rheumatic diseases. METHODS: We conducted a literature search of MEDLINE and PsycINFO for randomized clinical trials of psychosocial interventions for pain and disability in children with rheumatic diseases from January 1969 to September 2015. Studies with a sample size less than 10 subjects were excluded. Study quality was assessed using the Jadad score. RESULTS: Five articles met inclusion criteria, for a total of 229 patients, aged 5 to 18 years. Two studies included children with fibromyalgia. Three studies included children with juvenile arthritis. Neither study in fibromyalgia reported the statistical significance of immediate between-group pre-post changes in functioning or pain. One study examining the effects of an internet-based psychosocial intervention in children with juvenile arthritis reported significant differences in post-intervention pain scores (p = 0.03). However, 2 studies did not show improvements in pain scores among children with juvenile arthritis treated with psychosocial interventions vs. a wait-list control or vs. an active control (massage). No studies reported significant between-group differences for functional outcomes in children with juvenile arthritis. CONCLUSIONS: The available data were limited by the scarcity of randomized trials. Definite conclusions about the immediate effect of psychosocial interventions on pain and function in children with fibromyalgia could not be made because between-group comparisons of post-treatment change scores were not reported. For children with juvenile inflammatory arthritis, results of between-group comparisons for pain differed across studies, and analyses examining disability revealed no significant differences between groups.

Building Resiliency in a Palliative Care Team: A Pilot Study.

CONTEXT: Palliative care clinicians (PCCs) are vulnerable to burnout as a result of chronic stress related to working with seriously ill patients. Burnout can lead to absenteeism, ineffective communication, medical errors, and job turnover. Interventions that promote better coping with stress are needed in this population. OBJECTIVES: This pilot study tested the feasibility of the Relaxation Response Resiliency Program for Palliative Care Clinicians, a program targeted to decrease stress and increase resiliency, in a multidisciplinary cohort of PCCs (N = 16) at a major academic medical center. METHODS: A physician delivered the intervention over two months in five sessions (12 hours total). Data were collected the week before the program start and two months after completion. The main outcome was feasibility of the program. Changes in perceived stress, positive and negative affect, perspective taking, optimism, satisfaction with life, and self-efficacy were examined using nonparametric statistical tests. Effect size was quantified using Cohen's d. RESULTS: The intervention was feasible; all participants attended at least four of the five sessions, and there was no attrition. After the intervention, participants showed reductions in perceived stress and improvements in perspective taking. CONCLUSION: Our findings suggest that a novel team-based resiliency intervention based on elicitation of the relaxation response was feasible and may help promote resiliency and protect against the negative consequences of stress for PCCs.

Promoting resiliency among palliative care clinicians: stressors, coping strategies, and training needs.

BACKGROUND: Palliative care clinicians (PCCs) are susceptible to burnout, as they regularly witness immense patient and family suffering; however, little is known about their specific challenges and training needs to enhance their long-term sustainability. OBJECTIVE: The purpose of this qualitative study was to explore common stressors, coping strategies, and training needs among PCCs in efforts to inform the development of a targeted Resiliency Program. METHODS: Utilizing a semistructured interview guide, we conducted a series of in-depth interviews with 15 PCCs at the Massachusetts General Hospital. RESULTS: Content analysis highlighted three main areas of stressors: (1) systematic challenges related to managing large, emotionally demanding caseloads within time constraints; (2) patient factors, such as addressing patients' mutable needs, managing family dynamics, and meeting patient and family demands and expectations; and (3) personal challenges of delineating emotional and professional boundaries. Engaging in healthy behaviors and hobbies and seeking emotional support from colleagues and friends were among the most common methods of coping with stressors. In terms of programmatic topics, PCCs desired training in mind-body skills (e.g., breathing, yoga, meditation), health education about the effects of stress, and cognitive strategies to help reduce ruminative thoughts and negative self-talk. A majority of clinicians stressed the need for brief strategies that could be readily integrated in the workplace. CONCLUSIONS: These results suggest that an intervention aimed to enhance PCC sustainability should focus on utilizing a skill-building approach to stress reduction that imparts strategies that can be readily utilized during work hours.

Cost-effectiveness of adding bevacizumab to first line therapy for patients with advanced ovarian cancer.

OBJECTIVE: To evaluate, from a societal perspective, the cost-effectiveness of adding bevacizumab to first-line therapy based on outcomes from the GOG-218 and ICON-7 trials. METHODS: A three-state Markov model was used. The time horizon was until the death of 99% of the initial cohort of 1000 individuals. Costs and quality-adjusted life-years (QALYs) were discounted at an annual rate of 3%. All costs were adjusted to 2013 USD. The incremental cost-effectiveness ratio (ICER) was reported as incremental cost per QALY gained. The robustness of the result was checked with one-way sensitivity analyses and for relevant clinical situations (i.e. varying the drug of choice to treat cancer recurrence). Subgroup analysis was conducted to identify subgroup of population for whom the strategy could be cost-effective. The potential impact of biosimilar bevacizumab was considered, using a 30% price reduction. RESULTS: For the GOG-218 study protocol, widely followed in US, the addition of bevacizumab results in an ICER of $2,420,691/QALY. For the ICON-7 study protocol, the ICER is $225,515/QALY. The results of the model were sensitive to the quality of life (QoL) and the median progression free survival (PFS). Biosimilar bevacizumab didn't reduce cost sufficiently to change conclusions. First-line augmentation is cost-effective, with biosimilar bevacizumab, for stage IV patients ($126,169/QALY), ECOG PS1 patients ($116,575/QALY) and for patients with suboptimal residual disease ($122,822/QALY) as per the ICON-7 protocol. CONCLUSION: Addition of bevacizumab, by in large, is cost-ineffective. It can become cost-effective with the ICON-7 protocol, in patients at high risk of progression using biosimilar bevacizumab.