Hepatic Sinusoidal Disorders.

Hepatic sinusoids are highly specialized microcirculatory conduits within the hepatic lobules that facilitate liver functions. The sinusoids can be affected by various disorders, including sinusoidal dilatation, sinusoidal obstruction syndrome (SOS), sinusoidal cellular infiltration, perisinusoidal infiltration, and endothelial neoplasms, such as hemangioendothelioma and angiosarcoma. While these disorders, particularly SOS and neoplasms, can be life threatening, their clinical manifestation is often nonspecific. Patients may present with right upper quadrant pain, jaundice, hepatomegaly, ascites, splenomegaly, and unexplained weight gain, although the exact manifestation depends on the cause, severity, and duration of the disease. Ultimately, invasive tests may be necessary to establish the diagnosis. A comprehensive understanding of imaging manifestations of various sinusoidal disorders contributes to early diagnosis and can help radiologists detect subclinical disease. Additionally, specific imaging features may assist in identifying the cause of the disorder, leading to a more focused and quicker workup. For example, a mosaic pattern of enhancement of the liver parenchyma is suggestive of sinusoidal dilatation; peripheral and patchy reticular hypointensity of the liver parenchyma on hepatobiliary MR images is characteristic of SOS; and associated diffuse multiple hyperintensities on diffusion-weighted images may be specific for malignant sinusoidal cellular infiltration. The authors provide an overview of the pathogenesis, clinical features, and imaging appearances of various hepatic sinusoidal disorders, with a special emphasis on SOS. ©RSNA, 2024 Supplemental material is available for this article.

Percutaneous Image Guided Cryoablation of Adrenal Metastases From Renal Cell Carcinoma: A Single Institution Review.

OBJECTIVES: To assess the safety, technical success, disease progression, and survival associated with percutaneous image-guided cryoablation of renal cell carcinoma metastasis (mRCC) in the adrenal gland. METHODS: Retrospective, single-institution review of adult patients undergoing percutaneous cryoablation for adrenal mRCC between the years of 2007-2021. Technical parameters, technical success, safety, and survival were analyzed according to standard criteria. RESULTS: Forty-six patients (39 male; mean age 66 ± 8.8 years) with 57 tumors ablated over 51 sessions with a median hospital length of stay of 1 day (range 0-3 days). Forty-four (96%) had primary of clear cell histology. Aim of ablation was curative intent in 39 of 57 tumors (72%) with local tumor control in the remainder. There were 2 (4%) technical failures and technique efficacy was achieved in 52 out of the remaining 55 (95%). There were no Common Terminology Criteria for Adverse Events' immediate complications and 4 of 51 (8%) delayed complications. Twenty-five of 57 (44%) had disease progression anywhere, away from ablation site. One-, 3-, and 5-year recurrence free survival rates were 100%, 89%, and 89% and overall survival was 98%, 85%, and 71%. Fifty-one of 57 (89%) underwent preprocedural alpha blockade with hypertensive crisis in 27 of 56 (54%) available records, of which there were no adverse outcomes. CONCLUSION: Percutaneous cryoablation of mRCC to the adrenal glands is safe with robust local control, leading to advocacy for its ongoing use in this patient population. Multi-disciplinary management is recommended for successful treatment.

The development of a rapid patient-derived xenograft model to predict chemotherapeutic drug sensitivity/resistance in malignant glial tumors.

BACKGROUND: High-grade gliomas (HGG) are aggressive brain tumors associated with short median patient survival and limited response to therapies, driving the need to develop tools to improve patient outcomes. Patient-derived xenograft (PDX) models, such as mouse PDX, have emerged as potential Avatar platforms for personalized oncology approaches, but the difficulty for some human grafts to grow successfully and the long time required for mice to develop tumors preclude their use for HGG. METHODS: We used a rapid and efficient ex-ovo chicken embryo chorioallantoic membrane (CAM) culture system to evaluate the efficacy of oncologic drug options for HGG patients. RESULTS: Implantation of fresh glioma tissue fragments from 59 of 60 patients, that include difficult-to-grow IDH-mutated samples, successfully established CAM tumor xenografts within 7 days, with a tumor take rate of 98.3%. These xenografts faithfully recapitulate the histological and molecular characteristics of the primary tumor, and the ability of individual fragments to form tumors was predictive of poor patient prognosis. Treatment of drug-sensitive or drug-resistant xenografts indicates that the CAM-glioma assay enables testing tumor sensitivity to temozolomide and carboplatin at doses consistent with those administered to patients. In a proof-of-concept study involving 14 HGG patients, we observed a correlation of 100% between the CAM xenograft response to temozolomide or carboplatin and the clinical response of patients. CONCLUSION: The CAM-glioma model is a fast and reliable assay that has the potential to serve as a complementary model to drug discovery and a real-time Avatar platform to predict the best treatment for HGG patients.

Establishment of a ccRCC patient-derived chick chorioallantoic membrane model for drug testing.

Clear cell renal cell carcinoma (ccRCC) is an aggressive subtype of renal cell carcinoma accounting for the majority of deaths in kidney cancer patients. Advanced ccRCC has a high mortality rate as most patients progress and develop resistance to currently approved targeted therapies, highlighting the ongoing need for adequate drug testing models to develop novel therapies. Current animal models are expensive and time-consuming. In this study, we investigated the use of the chick chorioallantoic membrane (CAM), a rapid and cost-effective model, as a complementary drug testing model for ccRCC. Our results indicated that tumor samples from ccRCC patients can be successfully cultivated on the chick chorioallantoic membrane (CAM) within 7 days while retaining their histopathological characteristics. Furthermore, treatment of ccRCC xenografts with sunitinib, a tyrosine kinase inhibitor used for the treatment of metastatic RCC, allowed us to evaluate differential responses of individual patients. Our results indicate that the CAM model is a complementary in vivo model that allows for rapid and cost-effective evaluation of ccRCC patient response to drug therapy. Therefore, this model has the potential to become a useful platform for preclinical evaluation of new targeted therapies for the treatment of ccRCC.

Hypoxia Selectively Increases a SMAD3 Signaling Axis to Promote Cancer Cell Invasion.

Transforming growth factor ß (TGFß) plays a paradoxical role in cancer, first inhibiting then promoting its progression, a duality that poses a real challenge for the development of effective TGFß-targeted therapies. The major TGFß downstream effectors, SMAD2 and SMAD3, display both distinct and overlapping functions and accumulating evidence suggests that their activation ratio may contribute to the dual effect of TGFß. However, the mechanisms responsible for their selective activation remain poorly understood. Here, we provide experimental evidence that hypoxia induces the pro-invasive arm of TGFß signaling through a selective increase in SMAD3 interaction with SMAD-Anchor for Receptor Activation (SARA). This event relies on HDAC6-dependent SMAD3 bioavailability, as well as increased SARA recruitment to EEA1+ endosomes. A motility gene expression study indicated that SMAD3 selectively increased the expression of ITGB2 and VIM, two genes that were found to be implicated in hypoxia-induced cell invasion and associated with tumor progression and metastasis in cohorts of cancer patients. Furthermore, CAM xenograft assays show the significant benefit of selective inhibition of the SMAD3 signaling pathway as opposed to global TGFß inhibition in preventing tumor progression. Overall, these results suggest that fine-tuning of the pro-invasive HDAC6-SARA-SMAD3 axis could be a better strategy towards effective cancer treatments.

Human Lung Tissue Implanted on the Chick Chorioallantoic Membrane as a Novel In Vivo Model of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with no curative pharmacological treatment. Current preclinical models fail to accurately reproduce human pathophysiology and are therefore poor predictors of clinical outcomes. Here, we investigated whether the chick embryo chorioallantoic membrane (CAM) assay supports the implantation of xenografts derived from IPF lung tissue and primary IPF lung fibroblasts and can be used to evaluate the efficacy of antifibrotic drugs. We demonstrate that IPF xenografts maintain their integrity and are perfused with chick embryo blood. Size measurements indicate that the xenografts amplify on the CAM, and Ki67 and pro-collagen type I immunohistochemical staining highlight the presence of proliferative and functional cells in the xenografts. Moreover, the IPF phenotype and immune microenvironment of lung tissues are retained when cultivated on the CAM and the fibroblast xenografts mimic invasive IPF fibroblastic foci. Daily treatments of the xenografts with nintedanib and PBI-4050 significantly reduce their size, fibrosis-associated gene expression, and collagen deposition. Similar effects are found with GLPG1205 and fenofibric acid, two drugs that target the immune microenvironment. Our CAM-IPF model represents the first in vivo model of IPF that uses human lung tissue. This rapid and cost-effective assay could become a valuable tool for predicting the efficacy of antifibrotic drug candidates for IPF.

The Chicken Chorioallantoic Membrane Tumor Assay as a Relevant In Vivo Model to Study the Impact of Hypoxia on Tumor Progression and Metastasis.

Hypoxia in the tumor microenvironment is a negative prognostic factor associated with tumor progression and metastasis, and therefore represents an attractive therapeutic target for anti-tumor therapy. To test the effectiveness of novel hypoxia-targeting drugs, appropriate preclinical models that recreate tumor hypoxia are essential. The chicken ChorioAllantoic Membrane (CAM) assay is increasingly used as a rapid cost-effective in vivo drug-testing platform that recapitulates many aspects of human cancers. However, it remains to be determined whether this model recreates the hypoxic microenvironment of solid tumors. To detect hypoxia in the CAM model, the hypoxic marker pimonidazole was injected into the vasculature of tumor-bearing CAM, and hypoxia-dependent gene expression was analyzed. We observed that the CAM model effectively supports the development of hypoxic zones in a variety of human tumor cell line-derived and patient's tumor fragment-derived xenografts. The treatment of both patient and cell line-derived CAM xenografts with modulators of angiogenesis significantly altered the formation of hypoxic zones within the xenografts. Furthermore, the changes in hypoxia translated into modulated levels of chick liver metastasis as measured by Alu-based assay. These findings demonstrate that the CAM xenograft model is a valuable in vivo platform for studying hypoxia that could facilitate the identification and testing of drugs targeting this tumor microenvironment.

14-3-3η Promotes Invadosome Formation via the FOXO3-Snail Axis in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

Erosive destruction of joint structures is a critical event in the progression of rheumatoid arthritis (RA), in which fibroblast-like synoviocytes (FLS) are the primary effectors. We previously reported that the ability of RA FLS to degrade extracellular matrix (ECM) components depends on the formation of actin-rich membrane protrusions, called invadosomes, through processes that remain elusive. 14-3-3η belongs to a family of scaffolding proteins involved in a wide range of cellular functions, and its expression is closely related to joint damage and disease activity in RA patients. In this study, we sought to assess the role of 14-3-3η in joint damage by examining its contribution to the invadosome formation phenotype of FLS. Using human primary FLS, we show that 14-3-3η expression is closely associated with their ability to form invadosomes. Furthermore, knockdown of 14-3-3η using shRNAs decreases the level of invadosome formation in RA FLS, whereas addition of the recombinant protein to FLS from healthy individuals promotes their formation. Mechanistic studies suggest that 14-3-3η regulates invadosome formation by increasing Snail expression, a mechanism that involves nuclear exclusion of the transcription repressor FOXO3. Our results implicate the 14-3-3η-FOXO3-Snail axis in promoting the aggressive ECM-degrading phenotype of RA FLS, and suggest a role for this scaffolding protein in cartilage degradation.

A comparative evaluation of telehealth and direct assessment when screening for spasticity in residents of two long-term care facilities.

OBJECTIVE: To evaluate the performance of telehealth as a screening tool for spasticity compared to direct patient assessment in the long-term care setting. DESIGN: Cross-sectional, observational study. SETTING: Two long-term care facilities: a 140-bed veterans' home and a 44-bed state home for individuals with intellectual and developmental disabilities. SUBJECTS: Sixty-one adult residents of two long-term care facilities (aged 70.1 ± 16.2 years) were included in this analysis. Spasticity was identified in 43% of subjects (Modified Ashworth Scale rating mode = 2). Contributing diagnoses included traumatic brain injury, spinal cord injury, birth trauma, stroke, cerebral palsy, and multiple sclerosis. MAIN MEASURES: Movement disorders neurologists conducted in-person examinations to determine whether spasticity was present (reference standard) and also evaluated subjects with spasticity using the Modified Ashworth Scale. Telehealth screening examinations, facilitated by a bedside nurse, were conducted remotely by two teleneurologists using a three-question screening tool. Telehealth screening determinations of spasticity were compared to the reference standard determination to calculate sensitivity, specificity, and the area under the curve (AUC) in receiver operating characteristics. Teleneurologist agreement was evaluated using Cohen's kappa. RESULTS: Teleneurologist 1 had a specificity of 89% and sensitivity of 65% to identify the likely presence of spasticity (n = 61; AUC = 0.770). Teleneurologist 2 showed 100% specificity and 82% sensitivity (n = 16; AUC = 0.909). There was almost perfect agreement between the two examiners at 94% (kappa = 0.875, 95% CI: 0.640-1.000). CONCLUSION: Telehealth may provide a useful, efficient method of identifying residents of long-term care facilities that likely need referral for spasticity evaluation.

A review of imaging of surface sarcomas of bone.

Surface lesions of bone are uncommon. Although their imaging features generally mirror those of their intramedullary counterparts, surface lesions may demonstrate distinct characteristics which along with their unusual location present a diagnostic challenge. Surface sarcomas are usually of a lower grade compared with intramedullary variants, leading to differences in management. Osteosarcoma arising from the cortical surface of the bone is termed juxtacortical or surface osteosarcoma and includes three distinct entities: parosteal, periosteal, and high-grade surface osteosarcoma. We also review the features intracortical osteosarcoma, which some authors include under the umbrella term surface osteosarcoma. These lesions exhibit biologic features distinct from those of conventional intramedullary osteosarcoma, which underlines the importance of accurate imaging diagnosis. Periosteal chondrosarcoma and periosteal Ewing sarcoma also have distinctive imaging appearances. The purpose of this article is to review surface sarcomas of bone with regard to their clinical and radiological features and to discuss the differential diagnosis for each condition.

Mutations disrupting neuritogenesis genes confer risk for cerebral palsy.

In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent-offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1A and CTNNB1) met genome-wide significance. We identified two novel monogenic etiologies, FBXO31 and RHOB, and showed that the RHOB mutation enhances active-state Rho effector binding while the FBXO31 mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in a Drosophila reverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.

A Simple Bedside Screening Tool for Spasticity Referral.

BACKGROUND AND OBJECTIVES: Spasticity is common in long-term care facilities; however, this often-disabling condition is largely underdiagnosed in this setting and therefore left untreated. This study aimed to test the ability of a three-question flowchart used at the bedside by primary care providers in the long-term care setting to identify residents in need of referral to a specialist for spasticity consultation. METHODS: All residents of a single long-term care facility were approached for participation in this cross-sectional, observational study. Spasticity diagnostic evaluations by a movement disorders specialist neurologist (reference standard) were compared with referral determinations made by two primary care providers [a primary care physician (PCP) and a nurse practitioner (NP)] using the simple flowchart. RESULTS: The analysis included 49 residents (80% male, age 78.2±9.0 years) who were evaluated by the reference standard neurologist and at least one primary care provider. The bedside referral tool demonstrated high sensitivity and moderate specificity when used by the PCP (92% and 78%, respectively; AUC=0.84) and NP (80% and 53%, respectively; AUC=0.67). CONCLUSION: This simple tool may be useful for primary care providers to identify residents to be referred to a specialist for evaluation and treatment of spasticity. These results warrant further investigation of the potential utility of this screening tool across multiple long-term care facilities and various types of care providers.

Hypoxia Downregulates LPP3 and Promotes the Spatial Segregation of ATX and LPP1 During Cancer Cell Invasion.

Hypoxia is a common characteristic of advanced solid tumors and a potent driver of tumor invasion and metastasis. Recent evidence suggests the involvement of autotaxin (ATX) and lysophosphatidic acid receptors (LPARs) in cancer cell invasion promoted by the hypoxic tumor microenvironment; however, the transcriptional and/or spatiotemporal control of this process remain unexplored. Herein, we investigated whether hypoxia promotes cell invasion by affecting the main enzymes involved in its production (ATX) and degradation (lipid phosphate phosphatases, LPP1 and LPP3). We report that hypoxia not only modulates the expression levels of lysophosphatidic acid (LPA) regulatory enzymes but also induces their significant spatial segregation in a variety of cancers. While LPP3 expression was downregulated by hypoxia, ATX and LPP1 were asymmetrically redistributed to the leading edge and to the trailing edge, respectively. This was associated with the opposing roles of ATX and LPPs in cell invasion. The regulated expression and compartmentalization of these enzymes of opposing function can provide an effective way to control the generation of an LPA gradient that drives cellular invasion and migration in the hypoxic zones of tumors.

Development and initial validation of the Support for Healthy Lifestyle (SHeL) questionnaire for adolescents.

This study developed and provided initial validation for the Support for Healthy Lifestyle (SHeL), a set of scales designed to measure adolescent-perceived social support of healthy eating and physical activity. Item pool development utilized a prior focus group study of adolescents' perceptions of socially supportive behavior and a review of the literature on social support for health behavior change in adolescents. Exploratory factor analysis of the item pool completed by 220 adolescents, internal consistency estimates, and expert review of items and consensus resulted in 9 scales for the SHeL: Family Healthy Eating Support, Family Physical Activity Support, Family Hypocritical Control, Peer Health Eating Support, Peer Physical Activity Support, Peer Undermining, Professional Healthy Eating Support, Professional Physical Activity Support, and Professional General Support. Scale internal reliability estimates were α = 0.73-0.96. Supporting construct validity, the SHeL showed a pattern of stronger correlations between measures of the same source (parent/peer) and target behavior (healthy eating/physical activity) and stronger correlations with corresponding Sallis scales vis-à-vis other Sallis scales, with exceptions related to peer support for healthy eating. Divergent validity was somewhat limited, including in two instances, the SHeL scale was more strongly correlated with another SHeL scale. Supporting criterion validity, often the SHeL scales were correlated with related health behaviors. This study provided important psychometric information for a new measurement of social support for health behavior for adolescents. Further research with larger, more diverse, and treatment-seeking populations is needed to provide further validation of the SHeL and to begin to establish normative scores.

Outcomes of hip and knee total joint arthroplasty in a Canadian inmate population over a 10-year period

Background: Little information exists regarding the risk of complications in inmates who receive total hip or knee arthroplasties in Canada. Inmates tend to be less healthy owing to socioeconomic factors and an increased number of medical and psychiatric comorbidities. We compared revision and complication rates of total joint replacements in a cohort of incarcerated Canadians versus a cohort of non-inmates. Methods: We obtained a list of all Canadian inmate inpatient hospital visits with diagnostic/procedure codes of primary hip or knee arthroplasty within the last 10 years from our institution's discharge abstract database. Demographic data and information related to the perioperative course, along with any data related to postoperative complications/readmissions, were obtained through manual chart review. Results: The inmate group consisted of 20 men (mean age 58 yr) with a total of 24 primary total joint arthroplasties; the comparison group included 171 men (mean age 62 yr). Postoperatively, the inmates had a 4-fold increased risk of major complication compared with non-inmates (33.3% v. 7.6%; odds ratio 4.21, p = 0.01). The inmates' revision rate was 20.8% compared with 5.8% in the comparison group (p = 0.03). The most common cause for revision in the inmate group was infection, with a rate of 16.7% compared with 3.5% in the comparison group (p = 0.03). Conclusion: Patients requiring total joint arthroplasty who are inmates in the Canadian penitentiary system are at increased risk of complication and revision surgeries following total joint arthroplasty.

Contexte: Il y a peu de données concernant le risque de complications chez les détenus opérés pour une prothèse totale de la hanche ou du genou au Canada. Les détenus tendent à être en moins bonne santé en raison de divers facteurs socioéconomiques et d'un nombre accru de comorbidités médicales et psychiatriques. Nous avons comparé les taux de révision et de complications suite à des arthroplasties articulaires totales dans une cohorte de Canadiens incarcérés comparée à une cohorte d'individus non incarcérés. Méthodes: Nous avons extrait de la base de données sur les congés des patients de notre établissement la liste de toutes les consultations de détenus hospitalisés assorties aux codes de diagnostics et d'interventions concernant les arthroplasties de la hanche ou du genou au cours des 10 dernières années. Les données démographiques et les renseignements relatifs à la période périopératoire, de même que les données concernant les complications postopératoires ou réadmissions ont été obtenus au moyen d'un examen manuel des dossiers. Résultats: Le groupe de détenus se composait de 20 hommes (âgés en moyenne de 58 ans) totalisant 24 arthroplasties articulaires totales primaires; le groupe de comparaison incluait 171 hommes (âgés en moyenne de 62 ans). En période postopératoire, les détenus étaient exposés à un risque 4 fois plus élevé de complications majeures, comparativement aux individus non incarcérés (33,3 % c. 7,6 %; risque relatif 4,21, p = 0,01). Le taux de révision chez les détenus a été de 20,8 %, contre 8 % dans le groupe de comparaison (p = 0,03). La plus fréquente cause de révision chez les détenus était l'infection, avec un taux de 16,7 %, contre 3,5 % dans le groupe de comparaison (p = 0,03).

The Hypoxic Tumor Microenvironment Promotes Invadopodia Formation and Metastasis through LPA1 Receptor and EGFR Cooperation.

Hypoxia, a common feature of solid tumors, has been critically involved in cell invasion and metastasis, but the underlying mechanisms remain poorly understood. Previously, it has been observed that the lysophosphatidic acid receptor 4 (LPA4) signaling axis mediates production of the degradative subcellular structures invadopodia, which are known to be required for metastasis. Here, it is demonstrated that LPA1 (LPAR1) is a common and major receptor used for hypoxia-induced invadopodia production in various cancer cell lines. The widespread use of LPA1 was not due to increased LPA1 expression but rather relied on Src-mediated cross-talk with EGFR. LPA1-mediated phosphorylation of Y845-EGFR under hypoxia led to PI3K/Akt activation, an event that increases the ability of cells to produce invadopodia. Moreover, phospho-Y845-EGFR was upregulated in hypoxic zones of tumors and a combination of EGFR and LPA1 inhibition synergistically suppressed metastasis in vivo Implications: This study uncovers an LPA1-EGFR signaling axis that is used for cell invasion in hypoxia and suggests a potential target to impede cancer metastasis. Mol Cancer Res; 16(10); 1601-13. ©2018 AACR.

Hypoxia-induced mobilization of NHE6 to the plasma membrane triggers endosome hyperacidification and chemoresistance.

The pH-dependent partitioning of chemotherapeutic drugs is a fundamental yet understudied drug distribution mechanism that may underlie the low success rates of current approaches to counter multidrug resistance (MDR). This mechanism is influenced by the hypoxic tumour microenvironment and results in selective trapping of weakly basic drugs into acidified compartments such as the extracellular environment. Here we report that hypoxia not only leads to acidification of the tumour microenvironment but also induces endosome hyperacidification. The acidity of the vesicular lumen, together with the alkaline pH of the cytoplasm, gives rise to a strong intracellular pH gradient that drives intravesicular drug trapping and chemoresistance. Endosome hyperacidification is due to the relocalization of the Na+/H+ exchanger isoform 6 (NHE6) from endosomes to the plasma membrane, an event that involves binding of NHE6 to the activated protein kinase C-receptor for activated C kinase 1 complex. These findings reveal a novel mechanism of hypoxia-induced MDR that involves the aberrant intracellular distribution of NHE6.

Platelet-Derived Growth Factor Receptor Activation Promotes the Prodestructive Invadosome-Forming Phenotype of Synoviocytes from Patients with Rheumatoid Arthritis.

Fibroblast-like synoviocytes (FLS) play a major role in invasive joint destruction in rheumatoid arthritis (RA). This prodestructive phenotype has been shown to involve autocrine TGF-ß that triggers formation of matrix-degrading invadosomes through molecular mechanisms that are not fully elucidated. The platelet-derived growth factor (PDGF) receptor (PDGFR) family of receptor tyrosine kinases (RTK) has been shown to cooperate with TGF-ß in various pathological conditions. We therefore sought to determine whether RTK activity played a role in invadosome biogenesis. We demonstrated that, among the common RTKs, PDGFR-αß was specifically phosphorylated in FLS from RA patients. Phosphorylation of PDGFR-αß was also elevated in RA synovial tissues. Interference with PDGFR activation or PDGF neutralization inhibited invadosome formation in RA synoviocytes, indicating the presence of an autocrine PDGFR activation loop that involved endogenous PDGF. Among the PDGF-A-D isoforms, only PDGF-B was found both significantly elevated in FLS lines from RA patients, and related to high-invadosome forming cells. Addition of TGF-ß upregulated invadosome formation, PDGF-B mRNA expression, and phosphorylation of PDGFR. All of these functions were efficiently suppressed by TGF-ß neutralization or interference with the Smad/TßR1or PI3K/Akt pathway. Among the class 1 PI3K family proteins known to be expressed in RA synoviocytes, PI3Kα was selectively involved in PDGF-B expression, whereas both PI3Kα and PI3Kδ participated in invadosome formation. Our findings demonstrate that PDGFR is a critical RTK required for the prodestructive phenotype of RA synovial cells. They also provide evidence for an association between autocrine TGF-ß and PDGFR-mediated invadosome formation in RA synoviocytes that involves the production of PDGF-B induced by TGF-ß.

Adolescents' preferences for social support for healthful eating and physical activity.

OBJECTIVE: This study explored adolescents' views on the sources and types of social support they would prefer when trying to eat more healthfully and be more active, as well as their opinions regarding means of enhancing social support in interventions. METHODS: A total of 28 adolescents (14 males, 14 females) ages 13 to 18 years participated in 4 focus groups that were stratified by gender and age to enhance participation. RESULTS: As expected, participants most readily named parents and friends as important sources of support and described wanting instrumental and emotional support from parents, companionship and emotional support from friends, and informational support from professionals. The focus groups revealed rich information regarding parents' and peers' behaviors that are and are not received as emotionally supportive, the helpfulness of parents' concurrent changes in lifestyle, and the importance of parents not ignoring or colluding with unhealthful behavior. Most participants expressed a need for frequent contact and a trusting relationship with professionals. Opinions were mixed regarding inclusion of others in appointments, use of electronic communications and social media, and group treatment formats. CONCLUSION: Results have implications for enhancing social support in behavioral weight management interventions that are developmentally relevant for adolescents.

Simultaneous pH measurement in endocytic and cytosolic compartments in living cells using confocal microscopy.

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported(1). Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases(2). Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells(3). This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis(3,4). Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.