Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies.

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a 'Serological Assay based on a Tri-part split-NanoLuc® (SATiN)' to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Here, we expand on our previous work and describe a reconfigured version of the SATiN assay, called Neutralization SATiN (Neu-SATiN), which measures neutralization activity of antibodies directly from convalescent or vaccinated sera. The results obtained with our assay and other neutralization assays are comparable but with significantly shorter preparation and run time for Neu-SATiN. As the assay is modular, we further demonstrate that Neu-SATiN enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

Black and Hispanic Patients Do Not Stay Longer After Primary Total Knee Arthroplasty: Results From an Urban Center Serving a Predominantly Minority Cohort.

INTRODUCTION: Previous reports identified minority race/ethnicity to be an independent risk factor for prolonged length of stay (LOS); however, these cohorts consisted of predominantly White patients. This study sought to evaluate minority status as an independent risk factor for prolonged LOS after primary total knee arthroplasty (TKA) in a predominantly Hispanic and Black cohort. METHODS: This was a retrospective study using an institutional database of patients who underwent primary TKA between the years 2016 and 2019. Demographic and socioeconomic data, smoking, body mass index (BMI), medical comorbidities, discharge disposition, and 30-day readmission rates were collected. Patients were first categorized into racial/ethnic groups (Hispanic, Black, or White). An univariate analysis was performed comparing patient characteristics between racial/ethnic groups using the Wilcoxon rank sum, chi-squared, and Fisher exact tests. We then categorized patients into two groups-normal LOS (discharged on postoperative day 1 to 2) and prolonged LOS (discharged after postoperative day 2). An univariate analysis was again performed comparing patient characteristics between LOS groups using Wilcoxon rank sum, chi-squared, and Fisher exact tests. After identifying risk factors markedly associated with LOS, a multivariate logistic regression analysis was performed to identify independent risk factors for prolonged LOS. RESULTS: A total of 3,093 patients were included-47.9% Hispanic and 38.3% Black. Mean LOS was 2.9 ± 1.6 days. An univariate analysis found race/ethnicity, age, low socioeconomic status (SES), discharge disposition, insurance type, weekday of surgery, BMI >40, smoking, increased American Society of Anesthesiologists (ASA)/Charlson Comorbidity Index (CCI) and several medical comorbidities to be associated with prolonged LOS (P < 0.05). A multivariate logistic regression analysis found Black and Hispanic patients were less likely to have prolonged LOS after adjusting for associated risk factors. White race/ethnicity, nonhome discharge, low SES, weekday of surgery, smoking, BMI >40, and increased ASA and CCI were identified as independent risk factors for prolonged LOS (P < 0.05). The overall 30-day readmission rate was 3.6%, with no notable difference between racial/ethnic and LOS groups (P = 0.98 and P = 0.78). CONCLUSION: In contrast to previous reports, our study found that after adjusting for associated risk factors, minority patients do not have prolonged LOS after primary TKA in an urban, socioeconomically disadvantaged, predominantly minority patient cohort. White race/ethnicity, nonhome discharge, low SES, weekday of surgery, smoking, BMI >40, increased CCI, and ASA were all found to be independent risk factors for prolonged LOS. These findings highlight the need to further investigate the role of race/ethnicity on LOS after primary TKA using large-scale, randomized controlled trials with equally represented patient cohorts.

Therapeutic effiacy of T cells expressing chimeric antigen receptor derived from a mesothelin-specific scFv in orthotopic human pancreatic cancer animal models.

Novel CAR T cells targeting mesothelin (MSLN) expressed on pancreatic cancer cells were developed to overcome the limit of the clinical efficacy of CAR T cell therapy for pancreatic cancer patients. Optimal single-chain variable fragments (scFv) binding to MSLN were selected based on the binding activity and the functional effectiveness of various scFv containing CAR-expressing T cells. Engineered MSLN CAR T cells showed successful anti-tumor activity specific to MSLN expression level. Furthermore, MSLN CAR T cells were evaluated for the anti-cancer efficacy in orthotopic mouse models bearing pancreatic cancer cells, MIA Paca-2, MSLN-overexpressed MIA Paca-2 or endogenously MSLN-expressing AsPC-1. Mice were randomized into control, mock treated, MS501 BBz treated, MS501 28z treated or MS501 28BBz treated group. Mice were monitored by weekly IVIS imaging and tumors were harvested and analyzed by immunohistochemical analyses. MSLN CAR T cells produced the therapeutic effect in orthotopic animal models with complete remission in significant number of mice. Histopathological analysis indicated that CD4+ and CD8+ MSLN CAR T cells infiltrated pancreatic tumor tissue and led to cancer cell eradication. Our results demonstrated the anti-tumor efficacy of MSLN CAR T cell therapy against pancreatic cancer, suggesting its therapeutic potential.

Split-enzyme immunoassay to monitor EGFR-HER2 heterodimerization on cell surfaces.

Over 30,000 protein-protein interactions with pathological implications have been identified; yet, discovering and investigating drugs that target these specific interactions is greatly limited by the inability to monitor native protein-protein interactions (PPIs) efficiently. The two most frequently used tools to monitor PPIs, resonance-energy transfer (RET) assays and protein complementation assays (PCA), face significant limitations. RET assays have a narrow working range of 10 to 50 Å, while PCA require permanent attachment of a reporter probe to a protein of interest by chemical conjugation or genetic engineering. We developed a non-invasive assay platform to measure PPIs without modifications to the proteins of interest and is functional at a greater working range than RET assays. We demonstrate our approach by monitoring the EGFR-HER2 heterodimerization on relevant cell surfaces, utilizing various EGFR- and HER2-specific binders (e.g., Fab, DARPin, and VHH) fused with small fragments of a tri-part split-luciferase derived from NanoLuc®. Following independent binding of the binder fusions to their respective targets, the dimerization of EGFR and HER2 induces complementation of the luciferase fragments into a functional native structure, producing glow-type luminescence. We have confirmed the functionality of the platform to monitor EGFR-HER2 dimerization induction and inhibition. STATEMENT OF SIGNIFICANCE: We describe a platform technology for rapid monitoring of protein-protein interactions (PPIs). Our approach is uses a luciferase split into three parts - two short peptide "tags" and a large third fragment. Each of the short peptides can be fused to antibodies which bind to domains of a target antigens which orients the two tags and facilitates refolding of an active enzyme. To our knowledge this is the first example of a split-enzyme used to monitor PPIs without requiring any modification of the target proteins. We demonstrate our approach on the important PPI of HER2 and EGFR. Significantly, we quantify stimulation and inhibition of these partners, opening the possibility of using our approach to assess potential drugs without engineering cells.

Homogeneous Immunoassay Using a Tri-Part Split-Luciferase for Rapid Quantification of Anti-TNF Therapeutic Antibodies.

Anti-TNF therapeutics bind and sequester tumor necrosis factor (TNF) to prevent downstream signaling and are clinically important in the treatment of several autoimmune diseases. Effective treatment with these drugs requires frequent therapeutic drug monitoring (TDM). Current analytical methods, including reporter gene assay (RGA), enzyme-linked immunosorbent assay (ELISA), and mobility shift assay (MSA), can be technically rigorous, slow, and expensive. These qualities prevent the implementation of point-of-care testing and ultimately limit the frequency and utility of monitoring. An assay simple enough to be performed in the clinic would enable increased TDM frequency, more accurate dosing, and improved patient outcomes. Toward this end, we developed a homogeneous immunoassay based on a tri-part split-luciferase system for "add-and-read" detection of anti-TNF therapeutics. In our platform, two small fragments of the split-luciferase, called ß9 and ß10, are each fused to a different interacting protein. The binding of each of these proteins to anti-TNF antibodies forces the split-luciferase components into proximity where they reform the active luciferase. We identified the fusion proteins, ß9-protein A (ß9-A) and ß10-TNF, as promising binding pairs. We systematically adjusted assay conditions to optimize the signal/background (S/B) ratio, limit of detection (LOD), and percent recovery. The assay has a large dynamic range (0.5-32 µg/mL) and is sensitive enough to monitor both subtherapeutic and supratherapeutic serum concentrations of anti-TNF antibodies, as demonstrated in clinical samples.

Hyaluronic acid binding to CD44S is indiscriminate of molecular weight.

The ubiquitous presence of hyaluronic acid (HA) in the extracellular matrix (ECM) of both healthy and diseased tissues underscores its importance in human physiology. Previous studies suggest that HA can be used as a probe to qualitatively monitor cell surface levels of CD44 and other important HA receptors; however, these studies use mixtures of HA at various molecular weights. Using fluorescently labeled HA, we evaluated the apparent differences of low (25 kilodalton) and high (700 kilodalton) molecular weight HA interacting with breast cancer cell lines of varying levels of CD44. Our results confirm that CD44 expression and the apparent level of HA interaction correlates with molecular weight. Importantly, we show that HA only binds a small fraction of the major CD44 isoform, CD44S, on cell surfaces and that CD44S interactions account for <50% of the total HA bound to cell surfaces. Although increased fluorescence level correlates with higher molecular weight of HA, this appears to be an artifact of chain length and not a result of multivalent binding between HA and CD44S. Accordingly, we verify that HA binding characteristics of cell surfaces is similar to previous artificial membrane models which proposed that HA anchors to CD44S and forms a non-binding corona of HA that extends beyond the surface.

Early Experience Managing a High-volume Academic Orthopaedic Department During the Coronavirus Pandemic in New York City.

Our orthopaedic surgery department at Montefiore Medical Center and Albert Einstein College of Medicine is located within the Bronx, a borough of New York City, and serves a densely populated urban community. Since the beginning of the novel coronavirus outbreak in New York City, the medical center was forced to rapidly adapt to the projected influx of critically ill patients. The aim of this report is to outline how our large academic orthopaedic surgery department adopted changes and alternative practices in response to the most daunting challenge to public health in our region in over a century. We hope that this report provides insight for others facing similar challenges.

Correction to: Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse posttraumatic osteoarthritis model.

Following publication of the original article [1], the authors reported an error in Figs. 2C and 5C.

Relationship between Health Behaviors and Marital Adjustment and Marital Intimacy in Multicultural Family Female Immigrants.

BACKGROUND: This study analyzed the relationship between health behaviors and marital adjustment in multicultural couples to evaluate their health status. METHODS: Married couples (70 Korean men and their immigrant wives) completed a structured interview on health behaviors and sociodemographic factors, the Revised Dyadic Adjustment Scale (RDAS), and the Marital Intimacy Scale. Based on the cutoff value of the RDAS, respondents were classified into two groups: high or low dyadic adaptation groups. The collected data were compared with health behavior regarding smoking, alcohol consumption, exercise, and weight. RESULTS: The odds ratio (OR) (95% confidence interval [CI]) by logistic regression with adjustment for age, educational level, career, occupation, length of residence in Korea, nationality, religion, age difference between couple, number of children, monthly income, and proficiency in Korean was 1.279 (1.113-1.492) for unhealthy exercise and 1.732 (1.604-1.887) for unhealthy body weight in female immigrants with low marital adjustment. In Korean husbands with low marital adjustment, the OR (95% CI) was 1.625 (1.232-2.142) for smoking and 1.327 (1.174- 1.585) for unhealthy exercise. No significant relationship was found between marital intimacy and health behaviors in female immigrants or Korean husbands. CONCLUSION: More desirable health behaviors were observed in highly adapted couples. Therefore, family physicians should be concerned with marital adjustment and other associative factors to evaluate and improve multicultural couples' health status.

A-to-I miR-378a-3p editing can prevent melanoma progression via regulation of PARVA expression.

Previously we have reported that metastatic melanoma cell lines and tumor specimens have reduced expression of ADAR1 and consequently are impaired in their ability to perform A-to-I microRNA (miRNA) editing. The effects of A-to-I miRNAs editing on melanoma growth and metastasis are yet to be determined. Here we report that miR-378a-3p is undergoing A-to-I editing only in the non-metastatic but not in metastatic melanoma cells. The function of the edited form is different from its wild-type counterpart. The edited form of miR-378a-3p preferentially binds to the 3'-UTR of the PARVA oncogene and inhibits its expression, thus preventing the progression of melanoma towards the malignant phenotype. Indeed, edited miR-378a-3p but not its WT form inhibits melanoma metastasis in vivo. These results further emphasize the role of RNA editing in melanoma progression.

A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays.

Protein-fragment complementation is a valuable tool for monitoring protein interactions. In complementation assays, the reporter fragments are directly fused to the interacting proteins, eliminating the possibility of monitoring native interactions. In principle, complementation could be achieved by placing the reporter fragments on antibodies which bind to the proteins of interest, enabling the monitoring of endogenous protein interactions or detection of a single protein in a homogeneous immunoassay. Previous reports have demonstrated proof-of-concept of this approach; however, current complementation systems have not met the practical requirements as suitable fusion partners for antibodies while providing the sensitivity needed for immunoassays. To surmount these challenges, we created a first-in-class, tri-part split luciferase consisting of two 11-residue peptides that are used as the antibody appendages. As an initial proof-of-concept, we used antibody-peptide fusions and found them to be capable of quantifying pg/mL concentrations of soluble or cell-bound HER2, proving this unique complementation system overcomes previous limitations and transforms this approach from merely possible to practical and useful. As shown herein, this dual-peptide system provides a rapid, simple, and sensitive "add-and-read" homogeneous immunoassay platform that can be broadly adapted as an alternative to traditional immunoassays, and in the future should enable complementation to be expanded to monitoring endogenous protein interactions.

Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells.

Worldwide, breast cancer (BCa) is the most common cancer in women. Among its subtypes, triple-negative breast cancer (TNBC) is an aggressive form associated with diminished survival. TNBCs are characterized by their absence, or minimal expression, of the estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (i.e. ER-/-, PR-/-, Her2-/Low). Consequently, treatment for this subtype of BCa remains problematic. Silibinin, a derivative of the flavonoid silymarin, is reported to have anticancer activities against hepatic and non-small cell lung cancers. We hypothesized that silibinin might inhibit cell-extracellular matrix interactions via the regulation, expression, and activation of STAT3 in TNBCs, which could directly inhibit metastasis in silibinin-treated BCa cells. Using proliferation assays, we found that exposure to silibinin at a concentration of 200 µM inhibited the proliferation of breast cancer (BCa) cells; this concentration also inhibited phosphorylation of STAT3 and its principal upstream kinase, Jak2. Furthermore, we found that silibinin inhibited the nuclear translocation of STAT3, as well as its binding to the MMP2 gene promoter. The ability of silibinin to inhibit metastasis was further studied using an in vitro invasion assay. The results confirm the role of STAT3 as a critical mediator in the invasive potential of BCa cells, and STAT3 knock-down resulted in inhibition of invasion. The invasion ability of silibinin-treated BCa cells was studied in detail with the expression of MMP2. Prevention of STAT3 activation also resulted in the inhibition of MMP2 expression. Use of a small interfering RNA to knock down STAT3 (siSTAT3) allowed us to confirm the role of STAT3 in regulating MMP2 expression, as well as the mechanism of action of silibinin in inhibiting MMP2. Taken together, we found that silibinin inhibits the Jak2/STAT3/MMP2 signaling pathway, and inhibits the proliferation, migration, and invasion of triple-negative BCa cells.

Transcriptomic features of primary prostate cancer and their prognostic relevance to castration-resistant prostate cancer.

Although various mechanisms of castration-resistant prostate cancer (CRPC) have been discovered, reliable biomarkers for monitoring CRPC progression are lacking. We sought to identify molecules that predict the progression of advanced prostate cancer (AdvPC) into CRPC. The study used primary-site samples (N=45 for next-generation sequencing (NGS); N=243 for real-time polymerase chain reaction) from patients with prostate cancer (PC). Five public databases containing microarray data of AdvPC and CRPC samples were analyzed. The NGS data showed that each progression step in PC associated with distinct gene expression profiles. Androgen receptor (AR) associated with tumorigenesis, advanced progression, and progression into CRPC. Analysis of the paired and unpaired AdvPC and CRPC samples in the NGS cohort showed that 15 genes associated with progression into CRPC. This was validated by cohort-1 and public database analyses. Analysis of the third cohort with AdvPC showed that higher serine peptidase inhibitor, Kazal type 1 (SPINK1) and lower Sp8 transcription factor (SP8) expression associated with progression into CRPC (log-rank test, both P<0.05). Multivariate regression analysis showed that higher SPINK1 (Hazard Ratio (HR)=4.506, 95% confidence intervals (CI)=1.175-17.29, P=0.028) and lower SP8 (HR=0.199, 95% CI=0.063-0.632, P=0.006) expression independently predicted progression into CRPC. Gene network analysis showed that CRPC progression may be mediated through the AR-SPINK1 pathway by a HNF1A-based gene network. Taken together, our results suggest thatSPINK1 and SP8 may be useful for classifying patients with AdvPC who have a higher risk of progressing to CRPC.

Procyanidins Mitigate Osteoarthritis Pathogenesis by, at Least in Part, Suppressing Vascular Endothelial Growth Factor Signaling.

Procyanidins are a family of plant metabolites that have been suggested to mitigate osteoarthritis pathogenesis in mice. However, the underlying mechanism is largely unknown. This study aimed to determine whether procyanidins mitigate traumatic injury-induced osteoarthritis (OA) disease progression, and whether procyanidins exert a chondroprotective effect by, at least in part, suppressing vascular endothelial growth factor signaling. Procyanidins (extracts from pine bark), orally administered to mice subjected to surgery for destabilization of the medial meniscus, significantly slowed OA disease progression. Real-time polymerase chain reaction revealed that procyanidin treatment reduced expression of vascular endothelial growth factor and effectors in OA pathogenesis that are regulated by vascular endothelial growth factor. Procyanidin-suppressed vascular endothelial growth factor expression was correlated with reduced phosphorylation of vascular endothelial growth factor receptor 2 in human OA primary chondrocytes. Moreover, components of procyanidins, procyanidin B2 and procyanidin B3 exerted effects similar to those of total procyanidins in mitigating the OA-related gene expression profile in the primary culture of human OA chondrocytes in the presence of vascular endothelial growth factor. Together, these findings suggest procyanidins mitigate OA pathogenesis, which is mediated, at least in part, by suppressing vascular endothelial growth factor signaling.

DUSP1 induces paclitaxel resistance through the regulation of p-glycoprotein expression in human ovarian cancer cells.

The heterogeneity and genetic instability of ovarian cancer cells often lead to the development of drug resistance, closely related with the increased cancer-related mortality. In this study, we investigated the role of dual-specificity phosphatase 1 (DUSP1) in the development of the resistance in human ovarian cancer cells against paclitaxel. Overexpression of DUSP1 in HeyA8 human ovarian cancer cells (HeyA8-DUSP1) up-regulated the expression of the drug efflux pump, p-glycoprotein. Consequently, HeyA8-DUSP1 cells are highly resistant to paclitaxel, with the resistance comparable to that of a multi-drug resistance cell line (HeyA8-MDR). Moreover, over expression of DUSP1 significantly increased the activation of p38 MAPK, leaving the activation of ERK1/2 and JNK1/2 unaffected. Pharmacological suppression of p38 MAPK activity prevents the up-regulation of p-glycoprotein expression and the consequent resistance against paclitaxel in HeyA8-DUSP1 cells. By contrast, HeyA8-MDR cells expressed a significantly higher level of DUSP1, but treatment with small interference RNA against DUSP1 significantly suppressed the expression of p-glycoprotein and the resistance against paclitaxel in HeyA8-MDR cells. Ectopic expression of MKK3, an upstream activator of p38 MAPK, significantly up-regulated the expression of p-glycoprotein and increased the consequent resistance against paclitaxel in HeyA8 cells. Collectively, these data indicated that DUSP1 may induce the resistance against paclitaxel through the p38 MAPK-mediated overexpression of p-glycoprotein in human ovarian cancer cells.

Cognitive Reserve and Postoperative Delirium in Older Adults.

OBJECTIVES: To examine the role of cognitive reserve in reducing delirium incidence and severity in older adults undergoing surgery. DESIGN: Prospective cohort study. SETTING: Hospital. PARTICIPANTS: Older adults (mean age 71.2, 65% women) undergoing elective orthopedic surgery (N = 142). MEASUREMENTS: Incidence (Confusion Assessment Method) and severity (Memorial Delirium Assessment Scale) of postoperative delirium were the primary outcomes. Predictors included early- (literacy) and late-life (cognitive activities) proxies for cognitive reserve. RESULTS: Forty-five participants (32%) developed delirium. Greater participation in cognitive activity was associated with lower incidence (odds ratio = 0.92 corresponding to increase of 1 activity per week, 95% confidence interval (CI) = 0.86-0.98, P = .006) and severity (B = -0.06, 95% CI = -0.11 to -0.01, P = .02) of delirium after adjustment for age, sex, medical illnesses, and baseline cognition. Greater literacy was not associated with lower delirium incidence or severity. Of individual leisure activities, reading books, using electronic mail, singing, and computer games were associated with lower dementia incidence and severity. CONCLUSION: Greater late-life cognitive reserve was associated with lower delirium incidence and severity in older adults undergoing surgery. Interventions to enhance cognitive reserve by initiating or increasing participation in cognitive activities may be explored as a delirium prophylaxis strategy.

Treatment of experimental human breast cancer and lung cancer brain metastases in mice by macitentan, a dual antagonist of endothelin receptors, combined with paclitaxel.

BACKGROUND: We recently demonstrated that brain endothelial cells and astrocytes protect cancer cells from chemotherapy through an endothelin-dependent signaling mechanism. Here, we evaluated the efficacy of macitentan, a dual endothelin receptor (ETAR and ETBR) antagonist, in the treatment of experimental breast and lung cancer brain metastases. METHODS: The effect of macitentan on astrocyte- and brain endothelial cell-mediated chemoprotective properties was measured in cytotoxic assays. We compared survival of mice bearing established MDA-MB-231 breast cancer or PC-14 non-small cell lung cancer (NSCLC) brain metastases that were treated with vehicle, macitentan, paclitaxel, or macitentan plus paclitaxel. Cell division, apoptosis, tumor vasculature, and expression of survival-related proteins were assessed by immunofluorescent microscopy. RESULTS: Cancer cells and tumor-associated endothelial cells expressed activated forms of AKT and MAPK in vehicle- and paclitaxel-treated groups in both metastasis models, but these proteins were downregulated in metastases of mice that received macitentan. The survival-related proteins Bcl2L1, Gsta5, and Twist1 that localized to cancer cells and tumor-associated endothelial cells in vehicle- and paclitaxel-treated tumors were suppressed by macitentan. Macitentan or paclitaxel alone had no effect on survival. However, when macitentan was combined with paclitaxel, we noted a significant reduction in cancer cell division and marked apoptosis of both cancer cells and tumor-associated endothelial cells. Moreover, macitentan plus paclitaxel therapy significantly increased overall survival by producing complete responses in 35 of 35 mice harboring brain metastases. CONCLUSIONS: Dual antagonism of ETAR and ETBR signaling sensitizes experimental brain metastases to paclitaxel and may represent a new therapeutic option for patients with brain metastases.

Macitentan, a Dual Endothelin Receptor Antagonist, in Combination with Temozolomide Leads to Glioblastoma Regression and Long-term Survival in Mice.

PURPOSE: The objective of the study was to determine whether astrocytes and brain endothelial cells protect glioma cells from temozolomide through an endothelin-dependent signaling mechanism and to examine the therapeutic efficacy of the dual endothelin receptor antagonist, macitentan, in orthotopic models of human glioblastoma. EXPERIMENTAL DESIGN: We evaluated several endothelin receptor antagonists for their ability to inhibit astrocyte- and brain endothelial cell-induced protection of glioma cells from temozolomide in chemoprotection assays. We compared survival in nude mice bearing orthotopically implanted LN-229 glioblastomas or temozolomide-resistant (LN-229(Res) and D54(Res)) glioblastomas that were treated with macitentan, temozolomide, or both. Tumor burden was monitored weekly with bioluminescence imaging. The effect of therapy on cell division, apoptosis, tumor-associated vasculature, and pathways associated with cell survival was assessed by immunofluorescent microscopy. RESULTS: Only dual endothelin receptor antagonism abolished astrocyte- and brain endothelial cell-mediated protection of glioma cells from temozolomide. In five independent survival studies, including temozolomide-resistant glioblastomas, 46 of 48 (96%) mice treated with macitentan plus temozolomide had no evidence of disease (P < 0.0001), whereas all mice in other groups died. In another analysis, macitentan plus temozolomide therapy was stopped in 16 mice after other groups had died. Only 3 of 16 mice eventually developed recurrent disease, 2 of which responded to additional cycles of macitentan plus temozolomide. Macitentan downregulated proteins associated with cell division and survival in glioma cells and associated endothelial cells, which enhanced their sensitivity to temozolomide. CONCLUSIONS: Macitentan plus temozolomide are well tolerated, produce durable responses, and warrant clinical evaluation in glioblastoma patients.

Multifunctional Delivery Systems for Advanced oral Uptake of Peptide/Protein Drugs.

In recent years, advances in biotechnology and protein engineering have enabled the production of large quantities of proteins and peptides as important therapeutic agents. Various researchers have used biocompatible functional polymers to prepare oral dosage forms of proteins and peptides for chronic use and for easier administration to enhance patient compliance. However, there is a need to enhance their safety and effectiveness further. Most macromolecules undergo severe denaturation at low pH and enzymatic degradation in the gastrointestinal tract. The macromolecules' large molecular size and low lipophilicity cause low permeation through the intestinal membrane. The major strategies that have been used to overcome these challenges (in oral drug carrier systems) can be classified as follows: enteric coating or encapsulation with pH-sensitive polymers or mucoadhesive polymers, co-administration of protease inhibitors, incorporation of absorption enhancers, modification of the physicochemical properties of the macromolecules, and site-specific delivery to the colon. This review attempts to summarize the various advanced oral delivery carriers, including nanoparticles, lipid carriers, such as liposomes, nano-aggregates using amphiphilic polymers, complex coacervation of oppositely charged polyelectrolytes, and inorganic porous particles. The particles were formulated and/or surface modified with functional polysaccharides or synthetic polymers to improve oral bioavailability of proteins and peptides. We also discuss formulation strategies to overcome barriers, therapeutic efficacies in vivo, and potential benefits and issues for successful oral dosage forms of the proteins and peptides.

Functional interaction between CTGF and FPRL1 regulates VEGF-A-induced angiogenesis.

Vascular endothelial growth factor-A (VEGF-A) is a master regulator of angiogenesis that controls several angiogenic processes in endothelial cells. However, the detailed mechanisms of VEGF-A responsible for pleiotropic functions and crosstalk with other signaling pathways have not been fully understood. Here, we found that VEGF-A utilizes the connective tissue growth factor (CTGF)/formyl peptide receptor-like 1 (FPRL1) axis as one of its mediators in angiogenesis. Using a proteomic approach, we found increased secretion of a matricellular protein, CTGF, from VEGF-A-treated human umbilical vein endothelial cells (HUVECs). Then, we studied the effect of CTGF binding to FPRL1 in VEGF-A-induced angiogenesis. CTGF directly binds to FPRL1 through a linker region and activates the downstream signals of FPRL1, such as increase in extracellular signal-regulated kinase (ERK) phosphorylation and intracellular Ca(2+) concentration. We found that linker region-induced FPRL1 activation promotes the migration and network formation of HUVECs, while disruption of FPRL1 inhibits VEGF-A-induced HUVEC migration and network formation. In addition, similar results were observed by the chorioallantoic membrane (CAM) assay based evaluation of angiogenesis in vivo. To summarize, our data reveal a novel working model for VEGF-A-induced angiogenesis via the VEGF-A/CTGF/FPRL1 axis that might prolong and enhance the signals initiated from VEGF-A.