Vacuum-assisted early transanal closure of leaking low colorectal anastomoses: the CLEAN study.

INTRODUCTION: Non-healing of anastomotic leakage can be observed in up to 50% after total mesorectal excision for rectal cancer. This study investigates the efficacy of early transanal closure of anastomotic leakage after pre-treatment with the Endosponge® therapy. METHODS: In this prospective, multicentre, feasibility study, transanal suturing of the anastomotic defect was performed after vacuum-assisted cleaning of the presacral cavity. Primary outcome was the proportion of patients with a healed anastomosis at 6 months after transanal closure. Secondary, healing at last follow-up, continuity, direct medical costs, functionality and quality of life were analysed. RESULTS: Between July 2013 and July 2015, 30 rectal cancer patients with a leaking low colorectal anastomosis were included, of whom 22 underwent neoadjuvant radiotherapy. Median follow-up was 14 (7-29) months. At 6 months, the anastomosis had healed in 16 (53%) patients. At last follow-up, anastomotic integrity was found in 21 (70%) and continuity was restored in 20 (67%) patients. Non-healing at 12 months was observed in 10/29 (34%) patients overall, and in 3/14 (21%) when therapy started within three weeks following the index operation. Major LARS was reported in 12/15 (80%) patients. The direct medical costs were €8933 (95% CI 7268-10,707) per patient. CONCLUSION: Vacuum-assisted early transanal closure of a leaking anastomosis after total mesorectal excision with 73% preoperative radiotherapy showed that acceptable anastomotic healing rates and stoma reversal rates can be achieved. Early diagnosis and start of treatment seems crucial.

Do all patients with advanced HER2 positive breast cancer need upfront-chemo when receiving trastuzumab? Randomized phase III trial SAKK 22/99.

Background: HER2-targeted therapy plus chemotherapy is standard treatment in advanced HER2+ breast cancer. Trastuzumab alone followed by addition of chemotherapy at disease progression versus upfront combination therapy has not been elucidated. Patients and methods: One-hundred seventy-five patients with measurable/evaluable HER2+ advanced disease without previous HER2-directed therapy were randomized to trastuzumab alone followed, at disease progression, by the combination with chemotherapy (Arm A) or upfront trastuzumab plus chemotherapy (Arm B). Chemotherapy could be stopped after ≥6 cycles in responding patients, trastuzumab was continued until progression. The primary endpoint of this superiority trial was time to progression (TTP) on combined trastuzumab-chemotherapy (Combination-TTP) in both arms. Secondary endpoints included response rate, TTP, overall survival, quality of life and toxicity. Results: Combination-TTP was longer than expected in both arms, 12.2 months in Arm A and 10.3 months in Arm B and not significantly different (hazard ratio [HR] 0.7; 95% CI 0.5-1.1; P =0.1). Overall survival was also not significantly different (HR 0.9; 95% CI 0.6-1.5; P = 0.55). In Arm A, the median TTP before introduction of chemotherapy was 3.7 months (95% CI 2.3-5.4), yet at 2 years 6% of patients were still on trastuzumab alone. Patients without visceral disease had a Combination-TTP of 21.8 months in arm A, compared with 10.1 months in arm B (unplanned analysis HR 2.1, 95% CI 1.1-4.2, P = 0.03). Patients with visceral disease showed no difference. Toxicity was chemotherapy-related. Conclusion: The outcome of patients receiving sequential trastuzumab-chemotherapy or upfront combination was similar. We failed to demonstrate superiority of the sequential approach. These results nevertheless suggest chemotherapy and its toxicity can be deferred, especially in patients with indolent, non-visceral disease. Despite a larger non-inferiority confirmatory study would be needed, these findings represent an additional proof of concept that de-escalation strategies can be discussed in individual patients.

Phase I trial of the oral smoothened inhibitor sonidegib in combination with paclitaxel in patients with advanced solid tumors.

Purpose To establish a recommended phase II dose (RP2D) for the oral smoothened inhibitor sonidegib in combination with paclitaxel; secondary objectives include evaluation of safety, tolerability, markers of Hedgehog (Hh) signaling and preliminary antitumor activity. Methods Patients with advanced solid tumors were enrolled in cohorts of escalating sonidegib dose levels (400mg, 600mg and 800mg orally, once daily on days 1-28) in combination with paclitaxel 80 mg/m2 on days 1, 8 and 15 in 4-weekly cycles. Dose-limiting toxicities (DLTs) were assessed using CTCAE v4. Once the RP2D was defined, patients with advanced ovarian carcinoma were treated at this dose level in an expansion phase. Biomarkers of Hh signaling were assessed by immunohistochemistry in archival tissue and antitumor activity evaluated using RECIST 1.1. Results 18 patients were treated: 3 at 400 mg, 3 at 600 mg and 12 at 800 mg sonidegib. Only one patient treated at 800 mg presented a DLT (prolonged neutropenia resulting in failure to receive 75% of the planned sonidegib dose). However, 4 of 12 patients treated at 800 mg had their sonidegib dose reduced for toxicity after cycle 1. Hh biomarker (SHH, Patched, SMO and GLI1) staining did not correlate with clinical activity. Best response was partial response in 3 patients (2 ovarian, 1 breast cancer) and stable disease >4 cycles in 3 patients (2 ovarian, 1 anal cancer). Conclusions The combination of sonidegib and paclitaxel is tolerable and evidence of antitumor activity was identified. The RP2D of sonidegib was 800 mg in combination with paclitaxel 80mg/m2.

Phase-I dose finding and pharmacokinetic study of the novel hydrophilic camptothecin ST-1968 (namitecan) in patients with solid tumors.

PURPOSE: This is a first-in-human, phase I, dose-escalation study to determine the maximum tolerated dose (MTD) of intravenous, flat-dosed ST-1968 (namitecan), a new hydrophilic camptothecan derivative. METHODS: Namitecan was administered intravenously over 2 h on day 1 and day 8 every 21 days (D1-D8-Q21D), starting at a flat dose of 2.5 mg, and increased according to a 3 + 3 cohort design. Due to frequent skipping of day 8 dosing for cytopenias, the study was expanded to test namitecan dosing on day 1 every 21 days (D1-Q21) at a starting dose of 17.5 mg. Major dose-limiting toxicity (DLT) was defined as grade (G) 4 neutropenia persisting >5 days, febrile neutropenia, G3 thrombocytopenia or G2 non-hematological toxicity. RESULTS: Thirty-four patients were included into the D1-D8-Q21D group (2.5, 5, 10, 15, 17.5, 20 mg dosing cohorts), 29 patients into the D1-21D group (17.5, 20, 23, 27, 30 mg dosing cohorts). Neutropenia was the DLT in both groups, with 15 mg being defined as the recommended dose (RD) for the D1-D8-Q21D group, and 23 mg for the D1-Q21D group. Non-hematological toxicity was negligible. One patient with endometrial cancer in the D1-D8-Q21D group and one patient with cholangiocellular carcinoma in the D1-Q21D group experienced a partial remission. Namitecan exhibited fully dose-proportional pharmacokinetics. CONCLUSIONS: This study demonstrates clinical safety, favourable pharmacokinetics and preliminary antitumor activity of the novel hydrophilic camptothecin analogue namitecan in patients with heavily pretreated solid malignancies, when given either on a 2 out of 3 weeks or 3-weekly regimen.

Integrative population pharmacokinetic and pharmacodynamic dose finding approach of the new camptothecin compound namitecan (ST1968).

AIMS: Namitecan is a new camptothecan compound undergoing early clinical development. This study was initiated to build an integrated pharmacokinetic (PK) and pharmacodynamic (PD) population model of namitecan to guide future clinical development. METHODS: Plasma concentration-time data, neutrophils and thrombocytes were pooled from two phase 1 studies in 90 patients with advanced solid tumours, receiving namitecan as a 2 h infusion on days 1 and 8 every 3 weeks (D1,8) (n = 34), once every 3 weeks (D1) (n = 29) and on 3 consecutive days (D1-3) (n = 27). A linear three compartment PK model was coupled to a semiphysiological PD-model for neutrophils and thrombocytes. Data simulations were used to interrogate various dosing regimens and give dosing recommendations. RESULTS: Clearance was estimated to be 0.15 l h(-1), with a long terminal half-life of 48 h. Body surface area was not associated with clearance, supporting flat-dosing of namitecan. A significant and clinically relevant association was found between namitecan area under the concentration-time curve (AUC) and the percentage drop of neutrophils (r(2) = 0.51, P < 10(-4)) or thrombocytes (r(2) = 0.49, P < 10(-4)). With a target for haematological dose-limiting toxicity of <20%, the recommended dose was defined as 12.5 mg for the D1,8 regimen, 23 mg for the once every 3 week regimen and 7 mg for the D1-3 regimen. CONCLUSION: This is the first integrated population PK-PD analysis of the new hydrophilic topoisomerase I inhibitor namitecan, that is currently undergoing early clinical development. A distinct relationship was found between drug exposure and haematological toxicity, supporting flat-dosing once every 3 weeks as the most adequate dosing regimen.

Learning and implementation of TransREctus sheath PrePeritoneal procedure for inguinal hernia repair.

PURPOSE: The TransREctus sheath PrePeritoneal procedure (TREPP) was introduced as an alternative open and preperitoneal technique for inguinal hernia mesh repair, demonstrating safety and efficacy in retro- and prospective studies. However, little is known about the technique's inherent learning curve. In this study, we aimed to determine TREPP learning curve effects after its implementation in high-volume surgical practice. METHODS: All primary, unilateral TREPP procedures performed in the first three years after implementation (between January 2016 and December 2018) were included out of a large preconstructed regional inguinal hernia database. Data were analyzed on outcome (i.e., surgical complications, hernia recurrences, postoperative pain). Learning curve effects were analyzed by assessing outcome in relation to surgeon experience. RESULTS: In total, 422 primary, unilateral TREPP procedures were performed in 419 patients. In three patients a unilateral TREPP procedure was performed on both sides separated in time. A total of 99 surgical complications were registered in 83 procedures (19.6% of all procedures), most commonly inguinal postoperative pain (8%) and bleeding complications (7%). Hernia recurrences were observed in 17 patients (4%). No statistically significant differences on outcome were found between different surgeon experience (< 40 procedures, 40-80 procedures, > 80 procedures). CONCLUSION: Implementation of TREPP seems not to be associated with a notable increase of adverse events. We were not able to detect a clear learning curve limit, potentially suggesting a relatively short learning curve among already experienced hernia surgeons compared to other guideline techniques.

Heterozygosity for an inactivating mutation in low-density lipoprotein-related receptor 6 (Lrp6) increases osteoarthritis severity in mice after ligament and meniscus injury.

OBJECTIVE: Wnt/ß-catenin signaling plays an integral and complex role in cartilage development and maintenance. ß-catenin signaling has been linked to osteoarthritis (OA), but the role of Lrp6-mediated Wnt/ß-catenin signaling during OA remains unexplored. Mutations in the Wnt/ß-catenin co-receptors LRP5 and LRP6 (low-density lipoprotein-related receptors 5 and 6) result in skeletal abnormalities, which tend to be more severe in Lrp6 mutant mice. We examined OA development, chondrocyte and osteoblast behavior, and ß-catenin signaling after ligament and meniscus damage in mice with global heterozygous deletion of Lrp6. DESIGN: Ligament and meniscus damage was surgically induced in Lrp6(+/-) and wild-type (WT) mice, and evidence of joint disease was assessed by Microcomputed tomography (micro-CT) and histology. Wnt/ß-catenin signaling, proliferation, apoptosis, chondrogenesis, osteogenesis, and catabolic enzyme activity were measured. RESULTS: Relative to WT mice, Lrp6(+/-) mice had lower nuclear ß-catenin signaling within articular cartilage. After surgery, osteophytes and reduced articular cartilage were apparent in WT mice, but more severe in Lrp6(+/-) animals. Impairments to trabecular bone geometry occurred for WT and Lrp6(+/-) mice after surgery. Relative to WT mice, Lrp6(+/-) mice had reduced trabecular BMD and thickness, and Cyclin D1 and Lrp6 gene expression after surgery. There was an increase in apoptotic cells and serum matrix metalloproteinase-9 (MMP9) for Lrp6(+/-) mice after surgery, but no differences in cell proliferation occurred. CONCLUSIONS: Heterozygous loss-of-function mutation in Lrp6 leads to less ß-catenin signaling within articular cartilage and to increased degenerative joint disease after ligament and meniscus injury. Modulation of Lrp6 function could attenuate joint disease after damage to ligaments and the meniscus.

Intrapancreatic delivery of human umbilical cord blood aldehyde dehydrogenase-producing cells promotes islet regeneration.

AIMS/HYPOTHESIS: We sought to investigate the stimulation of islet regeneration by transplanted human umbilical cord blood (UCB) cells purified according to high aldehyde dehydrogenase (ALDH) activity (ALDH(hi)), a conserved characteristic of multiple progenitor lineages. We hypothesised that direct intrapancreatic (iPan) delivery of ALDH(hi) progenitors would augment islet regeneration via timely and localised exposure to islet-regenerative stimuli. METHODS: Cells were purified from UCB based on flow cytometry for low ALDH activity (ALDH(lo)) vs ALDH(hi). UCB ALDH(lo) or ALDH(hi) cells were compared for surface marker expression, as well as haematopoietic, endothelial and multipotent stromal progenitor content in vitro. UCB ALDH(lo) or ALDH(hi) cells were i.v. or iPan injected into streptozotocin-treated non-obese diabetic/severe combined immune-deficient mice temporally monitored for blood glucose, serum insulin and glucose tolerance. Human cell recruitment and survival in the pancreas, insulin content, islet-associated cell proliferation and islet vascularisation were documented in situ. RESULTS: UCB-derived ALDH(hi) cells were highly enriched for haematopoietic and endothelial progenitor frequency, and showed increased expression of progenitor and myeloid cell surface markers. Although i.v. transplantation of ALDH(hi) cells demonstrated low pancreas engraftment and only transient blood glucose lowering capacity, iPan injected ALDH(hi) cells reversed established hyperglycaemia, increased serum insulin and improved the response to a glucose challenge. iPan injected ALDH(hi) cells surrounded damaged islets at early time points and increased islet-associated cell proliferation, resulting in the recovery of beta cell mass. CONCLUSIONS/INTERPRETATION: iPan delivery of UCB ALDH(hi) cells potentiated islet-associated cell proliferation, insulin production and islet revascularisation, resulting in the recovery of host islet function. Elucidation of the progenitor-specific pathways stimulated during islet regeneration may provide new approaches to promote islet expansion during diabetes.

Healthcare-associated infection impact with bioaerosol treatment and COVID-19 mitigation measures.

BACKGROUND: The real-world impact of breathing zone air purification and coronavirus disease 2019 (COVID-19) mitigation measures on healthcare-associated infections is not well documented. Engineering solutions to treat airborne transmission of disease may yield results in controlled test chambers or single rooms, but have not been reported on hospital-wide applications, and the impact of COVID-19 mitigation measures on healthcare-associated infection rates is unknown. AIM: To determine the impact of hospital-wide bioaerosol treatment and COVID-19 mitigation measures on clinical outcomes. METHODS: The impact of the step-wise addition of air disinfection technology and COVID-19 mitigation measures to standard multi-modal infection control on particle counts, viral and bacterial bioburden, and healthcare-associated infection rates was investigated in a 124-bed hospital (>100,000 patient-days over 30 months). FINDINGS AND CONCLUSION: The addition of air disinfection technology and COVID-19 mitigation measures reduced airborne ultrafine particles, altered hospital bioburden, and reduced healthcare-associated infections from 11.9 to 6.6 (per 1000 patient-days) and from 6.6 to 1.0 (per 1000 patient-days), respectively (P<0.0001, R2=0.86). No single technology, tool or procedure will eliminate healthcare-associated infections, but the addition of a ubiquitous facility-wide engineering solution at limited expense and with no alteration to patient, visitor or staff traffic or workflow patterns reduced infections by 45%. A similar impact was documented with the addition of comprehensive, restrictive, and labour- and material-intensive COVID-19 mitigation measures. To the authors' knowledge, this is the first direct comparison between traditional infection control, an engineering solution and COVID-19 mitigation measures.

Laparoscopic versus open ventral hernia repair in the elderly: a propensity score-matched analysis.

BACKGROUND: Ventral hernia repair is common in the expanding aging population, but remains challenging due to their frequent comorbidities. The purpose of this study is to compare the surgical outcomes of open vs. laparoscopic ventral hernia repair in elderly patients. METHODS: Patients ≥ 65 years of age that underwent elective open or laparoscopic ventral hernia repair were identified from the American College of Surgeons National Surgical Quality Improvement Project (NSQIP) database. To reduce potential selection bias, propensity scores were created for the likelihood of undergoing laparoscopic surgery based on patients' demographics and comorbidities. Patients were matched based on the logit of the propensity scores. Thirty-day surgical outcomes were compared after matching using Chi-square test for categorical variables and the Wilcoxon Rank-Sum test for continuous variables. RESULTS: 35,079 (71.1%) and 14,270 (28.9%) patients underwent open and laparoscopic ventral hernia repairs, respectively. Laparoscopic surgery was associated with a lower overall morbidity (5.9% vs. 9.1%; p < 0.001) compared to open repair. The incidence of surgical site infections (1.1% vs. 3.5%; p < 0.001), post-operative infections (2.7% vs. 3.6%; p < 0.001), and reoperation (1.7% vs. 2.1%; p = 0.009) were all lower after laparoscopic repair. All other major surgical outcomes were either better with laparoscopy or comparable between both treatment groups except for operative time. CONCLUSION: Although open surgery remains the most prevalent in the elderly population, the results of this study suggest that laparoscopic surgery is safe and associated with a lower risk of overall morbidity, surgical site infections, and reoperation.

Transrectus sheath pre-peritoneal (TREPP) procedure versus totally extraperitoneal (TEP) procedure and Lichtenstein technique: a propensity-score-matched analysis in Dutch high-volume regional hospitals.

PURPOSE: Results of the most commonly used inguinal hernia repair techniques often originate from expert centers or from randomized controlled studies. In this study, we portray daily-practice results of a high-volume, regional surgical group in the Netherlands, comparing TREPP (open (posterior) transrectus sheath pre-peritoneal) with Lichtenstein (open anterior) and TEP (endoscopic (posterior) totally extraperitoneal). We hypothesize that the TREPP shows more favorable outcome compared to the current gold standard procedures: TEP and Lichtenstein. METHODS: Between January 2016 and December 2018, 3285 consecutive patients underwent surgical treatment and were included for analysis. The outcome measures were postoperative pain, recurrence rate and other surgical complications. Propensity-score matching was used to address potential selection bias. RESULTS: After propensity-score matching, there was no statistically significant difference in postoperative pain in the TREPP group compared to the Lichtenstein group (TREPP 7.3% versus Lichtenstein 6.3%; p = 0.67) nor in TREPP compared to TEP (TREPP 7.4% versus TEP 4.1%; p = 0.064). There was no statistically significant difference in recurrences in the TREPP group compared to Lichtenstein (3.8% vs 2.5%; p = 0.42), nor in the TREPP versus TEP comparison (3.9% vs 2.8%; p = 0.55) CONCLUSION: This study compares TREPP with Lichtenstein and TEP in the presence of postoperative pain, recurrences and other adverse outcomes. After propensity-score matching, no statistically significant difference in postoperative pain or recurrences remained between either TREPP compared to Lichtenstein, or TREPP compared to TEP. Based on these results, TREPP, Lichtenstein and TEP showed comparable results in postoperative pain, recurrences and other surgical site complications.

Phase Ib study of weekly mammalian target of rapamycin inhibitor ridaforolimus (AP23573; MK-8669) with weekly paclitaxel.

BACKGROUND: The additive cytotoxicity in vitro prompted a clinical study evaluating the non-prodrug rapamycin analogue ridaforolimus (AP23573; MK-8669; formerly deforolimus) administered i.v. combined with paclitaxel (PTX; Taxol). MATERIALS AND METHODS: Patients with taxane-sensitive solid tumors were eligible. The main dose escalation foresaw 50% ridaforolimus increments from 25 mg with a fixed PTX dose of 80 mg/m(2), both given weekly 3 weeks in a 4-week cycle. Collateral levels with a lower dose of either drug were planned upon achievement of the maximum tolerated dose in the main escalation. Pharmacodynamic studies in plasma, peripheral blood mononuclear cells (PBMCs) and skin biopsies and pharmacokinetic (PK) interaction studies at cycles 1 and 2 were carried out. RESULTS: Two recommended doses were determined: 37.5 mg ridaforolimus/60 mg/m(2) PTX and 12.5 mg/80 mg/m(2). Most frequent toxic effects were mouth sores (79%), anemia (79%), fatigue (59%), neutropenia (55%) and dermatitis (48%). Two partial responses were observed in pharyngeal squamous cell and pancreatic carcinoma. Eight patients achieved stable disease > or =4 months. No drug interaction emerged from PK studies. Decrease of eukaryotic initiation factor 4E-binding protein1 (4E-BP1) phosphorylation was shown in PBMCs. Similar inhibition of phosphorylation of 4E-BP1 and mitogen-activated protein kinase was present in reparative epidermis and vascular tissues, respectively. CONCLUSION: Potential antiangiogenic effects and encouraging antitumor activity justify further development of the combination.

Antiangiogenic drugs in oncology: a focus on drug safety and the elderly - a mini-review.

Angiogenesis is essential for normal tissue and even more so for solid malignancies. At present, inhibition of tumor angiogenesis is a major focus of anticancer drug development. Bevacizumab, a humanized antibody against VEGF, was the first antiangiogenic agent to be approved for advanced non-small cell lung cancer, breast cancer and colorectal cancer. The most commonly observed adverse events are hypertension, proteinuria, bleeding and thrombosis. Sunitinib, a small molecule blocking intracellular VEGF, KIT, Flt3 and PDGF receptors, which regulate angiogenesis and cell growth, is approved for the treatment of advanced renal cell cancer (RCC) and malignant gastrointestinal stromal tumor. The most frequent adverse events include hand-foot syndrome, stomatitis, diarrhea, fatigue, hypothyroidism and hypertension. Sorafenib, an oral multikinase inhibitor, is approved for the second-line treatment of advanced RCC and upfront treatment of advanced hepatocellular carcinoma. Most common adverse events with sorafenib are dermatologic (hand-foot skin reaction, rash, desquamation), fatigue, diarrhea, nausea, hypothyroidism and hypertension. More recently, cardiovascular toxicity has increasingly been recognized as a potential adverse event associated with sunitinib and sorafenib treatment. Elderly patients are at increased risk of thromboembolic events when receiving bevacizumab, and potentially for cardiac dysfunction when receiving sunitinib or sorafenib. The safety of antiangiogenic drugs is of special concern when taking these agents for longer-term adjuvant or maintenance treatment. Furthermore, newer investigational antiangiogenic drugs are briefly reviewed.

Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity.

Activity and stability of the proto-oncogene c-Myb are regulated by post-translational modifications, though the molecular mechanisms underlying such control are only partially understood. Here we describe the functional interaction of c-Myb with Pin1, an isomerase that binds to phosphorylated Ser/Thr-Pro motifs. We found that co-expression of c-Myb and Pin1 led to a net increase of c-Myb transactivation activity, both on reporter constructs as well as on an endogenous target gene. DNA-binding studies revealed that Pin1 did not increase the association of c-Myb with its response element in DNA. The increase of c-Myb transactivation activity was strictly dependent on the presence of an active catalytic center in Pin1. We provide evidence that c-Myb and Pin1 physically interacted, both upon ectopic expression of the proteins in HEK-293 cells as well as in the more physiological setting of HL60 cells, where c-Myb and Pin1 are resident proteins. By point mutating each individual Ser/Thr-Pro motif in c-Myb as well as by using deletion mutants we show that S528 in the EVES-motif was the docking site for Pin1. Mass spectrometry confirmed that S528 is phosphorylated in vivo. Finally, functional studies showed that mutation of S528 to alanine almost abolished the increase of transactivation activity by Pin1. This study reveals a new paradigm by which phosphorylation controls c-Myb function.

Heavy-electron metals: new highly correlated States of matter.

Heavy-electron metals exhibit highly correlated electronic behavior at liquid helium temperatures, with conduction-electron masses some hundred times the free-electron mass. Whether "normal," antiferromagnetic, or superconducting, their electronic behavior differs drastically from their ordinary metallic counterparts. The physical origin of the large mass and unusual superconducting and magnetic properties is the strong coupling between the conduction electrons and the local f-electron moment fluctuations characteristic of these materials.

Fas-induced caspase denitrosylation.

Only a few intracellular S-nitrosylated proteins have been identified, and it is unknown if protein S-nitrosylation/denitrosylation is a component of signal transduction cascades. Caspase-3 zymogens were found to be S-nitrosylated on their catalytic-site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol. Protein S-nitrosylation/denitrosylation can thus serve as a regulatory process in signal transduction pathways.

Fabrication of fully enclosed paper microfluidic devices using plasma deposition and etching.

A fully enclosed paper microfluidic device has been fabricated using pentafluoroethane (PFE) plasma deposition followed by O2 plasma etching. Structures with one and two non-interacting, fully enclosed hydrophilic channels were generated in a single paper sheet using metal masks. Furthermore, by performing an additional O2 plasma step with a secondary mask, pinholes were created at the reaction zones for reagent loading. Finally, to demonstrate the functionality of the device, a glucose assay was performed. Quantitative analysis of glucose assays showed that the device can be used for the clinically relevant range of glucose. To our knowledge, this is the first time that such structures have been fabricated without paper stacking. Multi-layer devices have enhanced functionality relative to a single channel device, because the design space for creating networks of channels within the paper substrate is greatly expanded. The fluid-filled channels in the fabricated device are isolated, thereby preventing contamination due to handling and environmental exposure. Fluid evaporation can be inhibited by sealing the device with adhesive tape without contaminating the enclosed channels. The method described is a dry process and compatible with roll-to-roll technology, thus facilitating large scale production. The novel method to fabricate enclosed µ-PADs overcomes many of the limitations experienced with current approaches and thus offers an alternative means to develop low-cost point-of-care diagnostics for resource-limited settings.

Nelfinavir and lenalidomide/dexamethasone in patients with lenalidomide-refractory multiple myeloma. A phase I/II Trial (SAKK 39/10).

The antiretroviral agent nelfinavir has antimyeloma activity and can overcome resistance to bortezomib. Our phase I/II trial investigated whether adding nelfinavir to lenalidomide-dexamethasone can overcome lenalidomide resistance in lenalidomide-refractory multiple myeloma (MM). Twenty-nine patients were included (high-risk cytogenetic aberrations 31%; ≥2 prior therapy lines 93%; lenalidomide-bortezomib double-refractory 34%). Twenty-four patients (83%) had prior bortezomib and 10 (34%) were lenalidomide-bortezomib double-refractory. They received four cycles of nelfinavir 2500 mg/day with standard-dose lenalidomide (25 mg days 1-21) and dexamethasone (40/20 mg days 1, 8, 15, 22). Minor response or better was achieved in 16 patients (55%; 95% CI 36-74%), including 40% of those who were lenalidomide-bortezomib double-refractory, and partial response or better in nine patients (31%; 95% CI 15-51%). Median progression-free survival was 3.4 (95% CI 2.0-4.9) months and median overall survival 21.6 (13.0-50.1) months. Lenalidomide-related pneumonitis, pneumonia, and neutropenic fever occurred, but there were no unexpected adverse events. Peripheral blood mononuclear cells showed a 45% (95% CI 40-51%) reduction in total proteasome activity from baseline and significant induction of unfolded protein response and autophagy. Thus, nelfinavir-lenalidomide-dexamethasone is an active oral combination in lenalidomide-refractory MM.

Effect of exercise on perfusion of collateral-dependent myocardium in dogs with chronic coronary artery occlusion.

Since the ability of mature intercoronary collateral channels to increase myocardial blood flow in response to drug-induced coronary vasodilation has been questioned, the present study was undertaken to evaluate the response of coronary collateral circulation to the stress of exercise. Studies were performed at rest and during two levels of treadmill exercise in six dogs a minimum of 6 mo after placement of an Ameroid constrictor on the left circumflex coronary artery. Regional myocardial blood flow was estimated in normally perfused anterior and predominantly collateral-dependent posterior left ventricular wall with left atrial injections of radio-nuclide-labeled microscheres 7-10 mum in diameter. At rest, heart rate was 87 +/- 7 beats/min and mean myocardial blood flow was comparable in control and collateral-dependent regions (0.96 +/- 0.13 and 0.97 +/- 0.14 ml/min-g, respectively). During exercise, heart rates increased to 180 +/- 13 and 228 +/- 14 beats/min and myocardial blood flow (MBF) in the anterior control region increased linearly with heart rate (HR), (MBF = 0.133 HR - 0.202, r = 0.88). MBF to the posterior collateral-dependent region was similarly augmented during exercise (MBF = 0.140 HR - 0.252, r = 0.89), so that the linear correlation between HR and MBF was similar for the control and collateral-dependent regions. In addition, the transmural distribution of MBF was uniform at rest and during exercise in both the anterior control and posterior collateral-dependent regions. Thus, not only could the mature intercoronary collateral vasculature supply adequate flow at rest, but when subjected to the natural stress of exercise, the increase in flow to the predominantly collateral-dependent area was similar to that in the normally perfused area.

Activity-dependent release of transforming growth factor-beta in a neuronal network in vitro.

For neurotrophins and also for members of the transforming growth factor beta (TGF-beta) family an activity-dependent regulation of synthesis and release has been proposed. Together with the observation that the secretion of neurotransmitters is initiated by neurotrophic factors, it is reasonable to assume that they might act as retrograde modulators enhancing the efficacy and stabilization of synapses. In the present study, we have tested this hypothesis and studied the release and regulation of TGF-beta in vitro using mouse primary hippocampal neurons at embryonic day E16.5 as model. We show that neuronal activity regulates TGF-beta release and TGF-beta expression in vitro. Treatment of the cultures with KCl, 3-veratroylveracevine (veratridine), glutamate or carbamylcholine chloride (carbachol) increased the levels of secreted TGF-beta, as assessed by the MLEC/plasminogen activator inhibitor (PAI)-luciferase-assay, whereas TGF-beta release stimulated by KCl or veratridine was reduced in the presence of tetrodotoxin or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). In addition, application of glutamate significantly upregulated expression of TGF-beta2 and TGF-beta3 in the culture. Notably, KCl stimulation caused Smad (composite term from SMA (C. elegans) and MAD=mothers against dpp (Drosophila)) translocation into the nucleus and upregulated TGF-beta inducible early gene (Tieg1) expression, demonstrating that activity-dependent released TGF-beta may exert autocrine actions and thereby activate the TGF-beta-dependent signaling pathway. Together, these results suggest an activity-dependent release and gene transcription of TGF-beta from mouse hippocampal neurons in vitro as well as subsequent autocrine functions of the released TGF-beta within the hippocampal network.