American society for metabolic and bariatric surgery: intra-operative care pathway for minimally invasive Roux-en-Y gastric bypass.

BACKGROUND: Clinical care pathways help guide and provide structure to clinicians and providers to improve healthcare delivery and quality. The Quality Improvement and Patient Safety Committee (QIPS) of the American Society for Metabolic and Bariatric Surgery (ASMBS) has previously published care pathways for the performance of laparoscopic sleeve gastrectomy (LSG) and pre-operative care of patients undergoing Roux-en-Y gastric bypass (RYGB). OBJECTIVE: This current RYGB care pathway was created to address intraoperative care, defined as care occurring on the day of surgery from the preoperative holding area, through the operating room, and into the postanesthesia care unit (PACU). METHODS: PubMed queries were performed from January 2001 to December 2019 and reviewed according to Level of Evidence regarding specific key questions developed by the committee. RESULTS: Evidence-based recommendations are made for care of patients undergoing RYGB including the pre-operative holding area, intra-operative management and performance of RYGB, and concurrent procedures. CONCLUSIONS: This document may provide guidance based on recent evidence to bariatric surgeons and providers for the intra-operative care for minimally invasive RYGB.

Microphysiological systems for solid tumor immunotherapy: opportunities and challenges.

Immunotherapy remains more effective for hematologic tumors than for solid tumors. One of the main challenges to immunotherapy of solid tumors is the immunosuppressive microenvironment these tumors generate, which limits the cytotoxic capabilities of immune effector cells (e.g., cytotoxic T and natural killer cells). This microenvironment is characterized by hypoxia, nutrient starvation, accumulated waste products, and acidic pH. Tumor-hijacked cells, such as fibroblasts, macrophages, and T regulatory cells, also contribute to this inhospitable microenvironment for immune cells by secreting immunosuppressive cytokines that suppress the antitumor immune response and lead to immune evasion. Thus, there is a strong interest in developing new drugs and cell formulations that modulate the tumor microenvironment and reduce tumor cell immune evasion. Microphysiological systems (MPSs) are versatile tools that may accelerate the development and evaluation of these therapies, although specific examples showcasing the potential of MPSs remain rare. Advances in microtechnologies have led to the development of sophisticated microfluidic devices used to recapitulate tumor complexity. The resulting models, also known as microphysiological systems (MPSs), are versatile tools with which to decipher the molecular mechanisms driving immune cell antitumor cytotoxicity, immune cell exhaustion, and immune cell exclusion and to evaluate new targeted immunotherapies. Here, we review existing microphysiological platforms to study immuno-oncological applications and discuss challenges and opportunities in the field.

Microphysiological model reveals the promise of memory-like natural killer cell immunotherapy for HIV± cancer.

Numerous studies are exploring the use of cell adoptive therapies to treat hematological malignancies as well as solid tumors. However, there are numerous factors that dampen the immune response, including viruses like human immunodeficiency virus. In this study, we leverage human-derived microphysiological models to reverse-engineer the HIV-immune system interaction and evaluate the potential of memory-like natural killer cells for HIV+ head and neck cancer, one of the most common tumors in patients living with human immunodeficiency virus. Here, we evaluate multiple aspects of the memory-like natural killer cell response in human-derived bioengineered environments, including immune cell extravasation, tumor penetration, tumor killing, T cell dependence, virus suppression, and compatibility with retroviral medication. Overall, these results suggest that memory-like natural killer cells are capable of operating without T cell assistance and could simultaneously destroy head and neck cancer cells as well as reduce viral latency.

Griddient: a microfluidic array to generate reconfigurable gradients on-demand for spatial biology applications.

Biological tissues are highly organized structures where spatial-temporal gradients (e.g., nutrients, hypoxia, cytokines) modulate multiple physiological and pathological processes including inflammation, tissue regeneration, embryogenesis, and cancer progression. Current in vitro technologies struggle to capture the complexity of these transient microenvironmental gradients, do not provide dynamic control over the gradient profile, are complex and poorly suited for high throughput applications. Therefore, we have designed Griddent, a user-friendly platform with the capability of generating controllable and reversible gradients in a 3D microenvironment. Our platform consists of an array of 32 microfluidic chambers connected to a 384 well-array through a diffusion port at the bottom of each reservoir well. The diffusion ports are optimized to ensure gradient stability and facilitate manual micropipette loading. This platform is compatible with molecular and functional spatial biology as well as optical and fluorescence microscopy. In this work, we have used this platform to study cancer progression.

Microfluidic device with reconfigurable spatial temporal gradients reveals plastic astrocyte response to stroke and reperfusion.

As a leading cause of mortality and morbidity, stroke constitutes a significant global health burden. Ischemic stroke accounts for 80% of cases and occurs due to an arterial thrombus, which impedes cerebral blood flow and rapidly leads to cell death. As the most abundant cell type within the central nervous system, astrocytes play a critical role within the injured brain. We developed a novel microphysiological platform that permits the induction of spatiotemporally controlled nutrient gradients, allowing us to study astrocytic response during and after transient nutrient deprivation. Within 24 h of inducing starvation in the platform, nutrient deprivation led to multiple changes in astrocyte response, from metabolic perturbations to gene expression changes, and cell viability. Furthermore, we observed that nutrient restoration did not reverse the functional changes in astrocyte metabolism, which mirrors reperfusion injury observed in vivo. We also identified alterations in numerous glucose metabolism-associated genes, many of which remained upregulated or downregulated even after restoration of the nutrient supply. Together, these findings suggest that astrocyte activation during and after nutrient starvation induces plastic changes that may underpin persistent stroke-induced functional impairment. Overall, our innovative device presents interesting potential to be used in the development of new therapies to improve tissue repair and even cognitive recovery after stroke.

Towards Novel Biomimetic In Vitro Models of the Blood-Brain Barrier for Drug Permeability Evaluation.

Current available animal and in vitro cell-based models for studying brain-related pathologies and drug evaluation face several limitations since they are unable to reproduce the unique architecture and physiology of the human blood-brain barrier. Because of that, promising preclinical drug candidates often fail in clinical trials due to their inability to penetrate the blood-brain barrier (BBB). Therefore, novel models that allow us to successfully predict drug permeability through the BBB would accelerate the implementation of much-needed therapies for glioblastoma, Alzheimer's disease, and further disorders. In line with this, organ-on-chip models of the BBB are an interesting alternative to traditional models. These microfluidic models provide the necessary support to recreate the architecture of the BBB and mimic the fluidic conditions of the cerebral microvasculature. Herein, the most recent advances in organ-on-chip models for the BBB are reviewed, focusing on their potential to provide robust and reliable data regarding drug candidate ability to reach the brain parenchyma. We point out recent achievements and challenges to overcome in order to advance in more biomimetic in vitro experimental models based on OOO technology. The minimum requirements that should be met to be considered biomimetic (cellular types, fluid flow, and tissular architecture), and consequently, a solid alternative to in vitro traditional models or animals.

Octogenarians exhibit quality of life improvement but increased morbidity after paraesophageal hernia repair.

BACKGROUND: The benefit of elective laparoscopic paraesophageal hernia repair (eLPEHR) in the elderly is unclear. This study compared quality of life and symptom resolution and morbidity after eLPEHR between octogenarians and younger patients. METHODS: A retrospective review was conducted comparing octogenarians (n = 23) to younger patients (n = 162) undergoing eLPEHR. Primary outcomes were pre and post-operative disease-specific quality of life (GERD-HRQL) and symptom scales (GERSS) and post-operative morbidity and length of stay (LOS). RESULTS: Octogenarians presented with higher ASA and lower preoperative BMI. Compared to controls, octogenarians exhibited higher overall morbidity (34.8% vs 16.1%, p = 0.03), including cardiac events (8.7% vs 0.6%) and mortality (8.7% vs 0%), and longer LOS (3 vs 2 days, p < 0.005). Post-operative reduction in GERD-HRQL/GERSS scores was comparable between groups. CONCLUSION: Octogenarians who undergo eLPEHR exhibit significant improvement in quality of life and symptom burden but may experience increased morbidity, suggesting a role for watchful waiting in this population. SUMMARY: Symptom burden, quality of life, and immediate post-operative morbidity was compared between octogenarians and younger patients undergoing elective laparoscopic paraesophageal hernia repair at a single institution. While exhibiting comparable post-operative improvement in symptom burden and quality of life, octogenarians experience higher post-operative morbidity. This may suggest a role for watchful waiting in the elderly population.

Benefits of VCE-003.2, a cannabigerol quinone derivative, against inflammation-driven neuronal deterioration in experimental Parkinson's disease: possible involvement of different binding sites at the PPARγ receptor.

BACKGROUND: Neuroprotection with cannabinoids in Parkinson's disease (PD) has been afforded predominantly with antioxidant or anti-inflammatory cannabinoids. In the present study, we investigated the anti-inflammatory and neuroprotective properties of VCE-003.2, a quinone derivative of the non-psychotrophic phytocannabinoid cannabigerol (CBG), which may derive its activity at the peroxisome proliferator-activated receptor-γ (PPARγ). The compound is also an antioxidant. METHODS: We evaluated VCE-003.2 in an in vivo [mice subjected to unilateral intrastriatal injections of lipopolysaccharide (LPS)] model of PD, as well as in in vitro (LPS-exposed BV2 cells and M-213 cells treated with conditioned media generated from LPS-exposed BV2 cells) cellular models. The type of interaction of VCE-003.2 at the PPARγ receptor was furtherly investigated in bone marrow-derived human mesenchymal stem cells (MSCs) and sustained with transcriptional assays and in silico docking studies. RESULTS: VCE-003.2 has no activity at the cannabinoid receptors, a fact that we confirmed in this study using competition studies. The administration of VCE-003.2 to LPS-lesioned mice attenuated the loss of tyrosine hydroxylase (TH)-containing nigrostriatal neurons and, in particular, the intense microgliosis provoked by LPS in the substantia nigra, measured by Iba-1/Cd68 immunostaining. The analysis by qPCR of proinflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase (iNOS) in the striatum showed they were markedly elevated by the LPS lesion and strongly reduced by the treatment with VCE-003.2. The effects of VCE-003.2 in LPS-lesioned mice implied the activation of PPARγ receptors, as they were attenuated when VCE-003.2 was co-administered with the PPARγ inhibitor T0070907. We then moved to some in vitro approaches, first to confirm the anti-inflammatory profile of VCE-003.2 in cultured BV2 cells exposed to LPS. VCE-003.2 was able to attenuate the synthesis and release of TNF-α and IL-1ß, as well as the induction of iNOS and cyclooxygenase-2 (COX-2) elicited by LPS in these cells. However, we found such effects were not reversed by GW9662, another classic PPARγ antagonist. Next, we investigated the neuroprotective effects of VCE-003.2 in cultured M-213 neuronal cells exposed to conditioned media generated from LPS-exposed cultured BV2 cells. VCE-003.2 reduced M-213 cell death, but again, such effects were not reversed by T0070907. Using docking analysis, we detected that VCE-003.2 binds both the canonical and the alternative binding sites in the PPARγ ligand-binding pocket (LBP). Functional assays further showed that T0070907 almost abolished PPARγ transcriptional activity induced by rosiglitazone (RGZ), but it did not affect the activity of VCE-003.2 in a Gal4-Luc system. However, T0070907 inhibited the effects of RGZ and VCE-003.2 on the expression of PPARγ-dependent genes upregulated in MSCs. CONCLUSIONS: We have demonstrated that VCE-003.2 is neuroprotective against inflammation-driven neuronal damage in an in vivo model of PD and in in vitro cellular models of neuroinflammation. Such effects might involve PPARγ receptors, although in silico and in vitro experiments strongly suggest that VCE-003.2 targets PPARγ by acting through two binding sites at the LBP, one that is sensitive to T0070907 (canonical binding site) and other that is not affected by this PPARγ antagonist (alternative binding site).

Endoscopic stent placement for treatment of sleeve gastrectomy leak: a single institution experience with fully covered stents.

BACKGROUND: Laparoscopic sleeve gastrectomy (SG) has risen in prevalence as a standalone surgical option for treating obesity over the last 15 years. One of the most worrisome complications is development of a leak at the gastrectomy staple line. OBJECTIVE: The objective of this report is to describe our single-institution experience in managing SG staple-line leaks with fully covered endoscopic stents. SETTING: Academic medical center, United States. METHODS: Data for all patients who underwent endoscopic stent placement for an SG leak between 2010 and 2016 at a single academic institution were retrospectively reviewed. Patient medical history, perioperative information, stent placement details, outcomes, and subsequent interventions were recorded. RESULTS: Twenty-four patients with SG staple-line leaks treated with fully covered endoscopic stents were identified. Leaks were identified at a median of 31.5 days postoperatively (range, 1-1615 d). The majority of patients underwent other treatment(s) for their leak before stent placement at our institution. Stents remained in place for an average of 28.8 ± 16.8 days. Migration occurred in 22% of all stent placements. Three patients were lost to follow-up, and 14 of the remaining 21 patients (66.7%) healed after stent placement. Five patients (23.8%) ultimately required operative revision with partial gastrectomy and Roux-en-Y esophagojejunostomy for management of persistent leaks. CONCLUSION: Endoscopic management using fully covered stents for staple-line leaks after SG is effective in the majority of patients. However, algorithms are needed for the management of chronic staple-line leaks, which are less likely to heal with stent placement.

Surgeon-performed endoscopic retrograde cholangiopancreatography. Outcomes of 2392 procedures at two tertiary care centers.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is a common procedure that, in the United States, is traditionally performed by gastroenterologists. We hypothesized that when performed by well-trained surgeons, ERCP can be performed safely and effectively. The objectives of the study were to assess the rate of successful cannulation of the duct of interest and to assess the 30-day complication and mortality rates. METHODS: We retrospectively reviewed the charts of 1858 patients who underwent 2392 ERCP procedures performed by five surgeons between August 2003 and June 2016 in two centers. Demographic and historical data, indications, procedure-related data and 30-day complication and mortality data were collected and analyzed. RESULTS: The mean age was 53.4 (range 7-102) years and 1046 (56.3%) were female. 1430 (59.8%) of ERCP procedures involved a surgical endoscopy fellow. The most common indication was suspected or established uncomplicated common bile duct stones (n = 1470, 61.5%), followed by management of an existing biliary or pancreatic stent (n = 370, 15.5%) and acute biliary pancreatitis (n = 173, 7.2%). A therapeutic intervention was performed in 1564 (65.4%), a standard sphincterotomy in 1244 (52.0%), stent placement in 705 (29.5%) and stone removal in 638 (26.7%). When cannulation was attempted, the rate of successful cannulation was 94.1%. When cannulation was attempted during the patient's first ERCP the cannulation rate was 92.4%. 94 complications occurred (5.4%); the most common complication was post-ERCP pancreatitis in 75 (4.2%), significant gastrointestinal bleeding in 7 (0.4%), ascending cholangitis in 11 (0.6%) and perforation in 1 (0.05%). 11 mortalities occurred (0.5%) but none of which were ERCP-related. CONCLUSION: When performed by well-trained surgical endoscopists, ERCP is associated with high success rate and acceptable complication rates consistent with previously published reports and in line with societal guidelines.

Laparoscopic Nissen fundoplication controls reflux symptoms and improves disease-specific quality of life in patients with class I and II obesity.

BACKGROUND: Patients with class I obesity and refractory gastroesophageal reflux disease may not qualify for bariatric surgery, and the effectiveness of laparoscopic Nissen fundoplication remains controversial. This study evaluates the early efficacy of laparoscopic Nissen fundoplication in patients with class I and II obesity. METHODS: Data for patients who underwent laparoscopic Nissen fundoplication between 2009 and 2014 were collected prospectively. Cohorts were stratified based on body mass index at the time of surgery: Nonobese (body mass index <30 kg/m2), class I obese (body mass index 30-34.9 kg/m2), and class II obese (body mass index 35-39.9 kg/m2). Primary outcome measures were symptoms assessed using the gastroesophageal reflux symptom scale and the gastroesophageal reflux disease health-related quality of life questionnaires. RESULTS: One hundred seventy-six patients underwent laparoscopic Nissen fundoplication during the study period: 76 nonobese (body mass index 26.2 ± 2.9 kg/m2), 53 class I obese (body mass index 32.4 ± 1.5 kg/m2), and 47 class II obese (body mass index 37.7 ± 2.5 kg/m2). Baseline gastroesophageal reflux symptom scale scores were 35.5 (6-72), 37.0 (5-72), and 45.0 (5-72) in nonobese, class I obese, and class II obese groups, respectively. Six weeks postoperatively, scores decreased to 6.5 (0-72), 4.0 (0-27), and 9.0 (0-64), respectively (P < .001). After 18-months, scores remained improved at 8.0 (0-72), 5 (0-48), and 4 (0-62), respectively (P < .001). A similar trend was seen in gastroesophageal reflux disease-health-related quality of life scores. Overall, 86%, 83%, and 79% remained free of proton-pump inhibitor medications, respectively. CONCLUSION: Laparoscopic Nissen fundoplication provides similar symptom control and quality of life 18-months postoperatively in nonobese and class I and II obese patients. Thus, laparoscopic Nissen fundoplication represents a viable surgical option for patients with class I and II obesity.

Laparoscopic redo fundoplication improves disease-specific and global quality of life following failed laparoscopic or open fundoplication.

INTRODUCTION: Laparoscopic fundoplication is associated with failure rates of up to 30% and redo operation rates of 5-8%. Redo fundoplication improves patient symptoms, but its impact on patient quality of life remains unclear. We hypothesized that laparoscopic redo fundoplication improves disease-specific and global quality of life in patients with recurrent symptoms following failed laparoscopic or open fundoplication. METHODS: Data for all patients undergoing a redo fundoplication between August 2009 and June 2014 were collected prospectively. Reflux symptoms and quality of life were assessed using the Gastroesophageal Reflux Symptom Scale (GERSS), the Gastroesophageal Reflux Disease Health-Related Quality of Life (GERD-HRQL), and the global quality of life Short Form-36 (SF-36) questionnaires obtained at 4 weeks and 16 months post-operatively. RESULTS: Forty-six patients underwent laparoscopic redo fundoplication during the study period for symptomatic hernia (n = 11), GERD (n = 18), or dysphagia (n = 17). GERSS improved from 41 at baseline to 9 at late follow-up (p < 0.001), and GERD-HRQL scores improved from 30 at baseline to 7 at late follow-up (p < 0.001). Median dysphagia scores decreased from 4.5 to 1 (p = 0.035). SF-36 scores demonstrated a significant improvement in general health (p = 0.016) and emotional well-being (p = 0.036) and a trend toward improved physical function (p = 0.068) in the post-operative period, but these improvements were not statistically significant at longer-term follow-up. Overall, 82% of patients reported satisfaction with their operation, and 96% reported that they would have the operation performed again given the benefit of hindsight. CONCLUSIONS: While associated with long operative times and significant complications, laparoscopic redo fundoplication produces a durable improvement in reflux symptoms and disease-specific quality of life. These procedures also improve global quality of life in the short term and are associated with high patient satisfaction.

Classification of Indeterminate Melanocytic Lesions by MicroRNA Profiling.

PURPOSE: Identification of indeterminate melanocytic skin lesions capable of neoplastic progression is suboptimal and may potentially result in unnecessary morbidity from surgery. MicroRNAs (miRs) may be useful in classifying indeterminate Spitz tumors as having high or low risk for malignant behavior. METHODS: RNA was extracted from paraffin-embedded tissues of benign nevi, benign Spitz tumors, indeterminate Spitz tumors, and Spitzoid melanomas in adults (n = 62) and children (n = 28). The expression profile of 12 miRs in adults (6 miRs in children) was analyzed by real-time polymerase chain reaction. RESULTS: Benign Spitz lesions were characterized by decreased expression of miR-125b and miR-211, and upregulation of miR-22, compared with benign nevi (p < 0.05). A comparison of Spitzoid melanomas to benign nevi revealed overexpression of miR-21, miR-150, and miR-155 in the malignant primaries (p < 0.05). In adults, Spitzoid melanomas exhibited upregulation of miR-21, miR-150, and miR-155 compared with indeterminate Spitz lesions. Indeterminate Spitz lesions with low-risk pathologic features had lower miR-21 and miR-155 expression compared with Spitzoid melanoma tumors in adults (p < 0.05), while pathologic high-risk indeterminate Spitz lesions had increased levels of miR-200c expression compared with low-risk indeterminate lesions (p < 0.05). Pediatric Spitzoid melanomas exhibited increased miR-21 expression compared with indeterminate Spitz lesions (p < 0.05). Moreover, miR-155 expression was increased in indeterminate lesions with mitotic counts >1 and depth of invasion >1 mm, suggesting miR-155 expression is associated with histological characteristics. CONCLUSIONS: miR expression profiles can be measured in indeterminate Spitz tumors and correlate with markers of malignant potential.

Increased visceral to subcutaneous fat ratio is associated with decreased overall survival in patients with metastatic melanoma receiving anti-angiogenic therapy.

BACKGROUND: Body fat distribution is an emerging prognostic indicator in patients treated with anti-angiogenic (AA) therapy. We sought to evaluate the association of visceral and subcutaneous fat with progression free survival (PFS) and overall survival (OS) in patients with metastatic melanoma treated with AA therapy. METHODS: Stage IV melanoma patients received bevacizumab ± interferon-alpha. Total abdominal fat, visceral fat area (VFA) and subcutaneous fat area (SFA) were measured at L3-L4 on CT images (cm(2)). PFS and OS were estimated by the Kaplan-Meier method. Cox proportional hazards model was used to assess the association of fat and clinical variables with PFS and OS. Prediction accuracy was evaluated using receiver operating characteristic curve with area under the curve (AUC). RESULTS: Forty-two patients were evaluated. Median VFA/SFA and body mass index (BMI) were used to group patients into high and low cohorts. PFS and OS were significantly decreased in patients with high VFA/SFA versus low (PFS, p=0.009; OS, p = 0.007), but not for BMI (PFS, p=0.774; OS, p=0.881). VFA/SFA, LDH and liver metastasis (LM) were predictors of PFS and OS on multivariate analysis. A prognostic score combining VFA/SFA, LDH, and presence or absence of LM had a higher accuracy for predicting PFS at 3 months (AUC 0.759) and OS at 24 months (AUC 0.846) than LDH and LM alone (PFS, AUC 0.705; OS, AUC 0.786). CONCLUSION: Increased VFA/SFA is associated with decreased PFS and OS in patients with metastatic melanoma treated with AA therapy, indicating body fat distribution is an important prognostic factor.

Predicting Overall Survival in Patients With Metastatic Melanoma on Antiangiogenic Therapy and RECIST Stable Disease on Initial Posttherapy Images Using CT Texture Analysis.

OBJECTIVE: The purpose of this study was to use CT texture analysis to predict overall survival (OS) in patients with metastatic melanoma and stable disease (SD) according to the Response Evaluation Criteria in Solid Tumors (RECIST) on initial posttherapy CT images. MATERIALS AND METHODS: This retrospective study included 42 patients with metastatic melanoma who received bevacizumab therapy in the context of a randomized prospective phase II clinical trial. Target lesions on the baseline and initial posttherapy contrast-enhanced CT examinations were evaluated by CT texture analysis using TexRAD software before and after image filtering in patients with RECIST SD on initial posttherapy images. Cox proportional hazards models were used to assess the associations of CT texture analysis measurements and of other patient factors with OS. The AUC was used to evaluate predictive accuracy. RESULTS: In multivariate analysis (in 23 patients with RECIST SD; median OS, 1.51 years), absolute change in mean positive pixels at spatial scaling filter of 4 mm, change in tumor size, and baseline serum lactate dehydrogenase (LDH) level were predictors of OS (hazard ratio [HR] = 5.05 for decrease in mean positive pixels at spatial scaling filter of 4 mm vs increase, p = 0.007; HR = 4.14 for > 5% increase in tumor size vs otherwise, p = 0.025; and HR = 1.29 for every 100 IU/L increase in baseline LDH level, p = 0.068). A prognostic index containing these three factors was highly accurate for predicting OS at 18 months (AUC = 0.917). CONCLUSION: In patients with metastatic melanoma and RECIST SD on initial post-therapy CT images, a model incorporating CT texture analysis of target lesions, tumor size changes, and baseline LDH levels was highly accurate in predicting OS.

The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells.

Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 µM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 µM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells.

MiR-21 enhances melanoma invasiveness via inhibition of tissue inhibitor of metalloproteinases 3 expression: in vivo effects of MiR-21 inhibitor.

Metastatic melanoma is the most aggressive form of this cancer. It is important to understand factors that increase or decrease metastatic activity in order to more effectively research and implement treatments for melanoma. Increased cell invasion through the extracellular matrix is required for metastasis and is enhanced by matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits MMP activity. It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma. It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby enhance the invasiveness of melanoma cells. miR-21 over-expression in the melanoma cell lines WM1552c, WM793b, A375 and MEL 39 was accomplished via transfection with pre-miR-21. Immunoblot analysis of miR-21-overexpressing cell lines revealed reduced expression of TIMP3 as compared to controls. This in turn led to a significant increase in the invasiveness of the radial growth phase cell line WM1552c and the vertical growth phase cell line WM793b (p < 0.05), but not in the metastatic cell lines A375 or MEL 39. The proliferation and migration of miR-21 over-expressing cell lines was not affected. Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cell lines, mimicking the effects of miR-21 over-expression. Treatment of tumor cells with a linked nucleic acid antagomir to miR-21 inhibited tumor growth and increased tumor expression of TIMP3 in vivo in 01B74 Athymic NCr-nu/nu mice. Intra-tumoral injections of anti-miR-21 produced similar effects. This data shows that increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.