A proposed methodology for uncertainty extraction and verification in priority setting partnerships with the James Lind Alliance: an example from the Common Conditions Affecting the Hand and Wrist Priority Setting Partnership.

BACKGROUND: To report our recommended methodology for extracting and then confirming research uncertainties - areas where research has failed to answer a research question - derived from previously published literature during a broad scope Priority Setting Partnership (PSP) with the James Lind Alliance (JLA). METHODS: This process was completed in the UK as part of the PSP for "Common Conditions Affecting the Hand and Wrist", comprising of health professionals, patients and carers and reports the data (uncertainty) extraction phase of this. The PSP followed the robust methodology dictated by the JLA and sought to identify knowledge gaps, termed "uncertainties" by the JLA. Published Cochrane Systematic Reviews, Guidelines and Protocols, NICE (National Institute for Health and Care Excellence) Guidelines, and SIGN (Scottish Intercollegiate Guidelines Network) Guidelines were screened for documented "uncertainties". A robust method of screening, internally verifying and then checking uncertainties was adopted. This included independent screening and data extraction by multiple researchers and use of a PRISMA flowchart, alongside steering group consensus processes. Selection of research uncertainties was guided by the scope of the Common Conditions Affecting the Hand and Wrist PSP which focused on "common" hand conditions routinely treated by hand specialists, including hand surgeons and hand therapists limited to identifying questions concerning the results of intervention, and not the basic science or epidemiology behind disease. RESULTS: Of the 2358 records identified (after removal of duplicates) which entered the screening process, 186 records were presented to the PSP steering group for eligibility assessment; 79 were deemed within scope and included for the purpose of research uncertainty extraction (45 full Cochrane Reviews, 18 Cochrane Review protocols, 16 Guidelines). These yielded 89 research uncertainties, which were compared to the stakeholder survey, and added to the longlist where necessary; before derived uncertainties were checked against non-Cochrane published systematic reviews. CONCLUSIONS: In carrying out this work, beyond reporting on output of the Common Conditions Affecting the Hand and Wrist PSP, we detail the methodology and processes we hope can inform and facilitate the work of future PSPs and other evidence reviews, especially those with a broader scope beyond a single disease or condition.

Partnering with survivors & families to determine research priorities for adult out-of-hospital cardiac arrest: A James Lind Alliance Priority Setting Partnership.

BACKGROUND: Research priority setting in health care has historically been done by expert health care providers and researchers and has not involved patients, family or the public. Survivors & family members have been particularly absent from this process in the field of resuscitation research and specifically adult out of hospital cardiac arrest (OHCA). As such, we sought to conduct a priority setting exercise in partnership with survivors, lay responders and their families in order to ensure that their priorities were visible. We partnered with the James Lind Alliance (UK) and used their commonly used consensus methodology for Public Priority Setting Partnerships (PSPs) to identify research priorities that reflected the perspectives of all stakeholders. METHODS: We used two rounds of public and health care professional surveys to create the initial priority lists. The initial survey collected open-ended questions while the second round consolidated the list of initial questions into a refined list for prioritization. This was done by reviewing existing evidence and thematic categorization by the multi-disciplinary steering committee. An in-person consensus workshop was conducted to come to consensus on the top ten priorities from all perspectives. The McMaster PPEET tool was used to measure engagement. RESULTS: The initial survey yielded more than 425 responses and 1450 "questions" from survivors and family members (18%), lay responders, health care providers and others. The second survey asked participants to rank a short list of 125 questions. The final top 25 questions were brought to the in-person meeting, and a top ten were selected through the JLA consensus process. The final list of top ten questions included how to improve the rate of lay responder CPR, what interventions used at the scene of an arrest can improve resuscitation and survival, how survival can be improved in rural areas of Canada, what resuscitation medications are most effective, what care patient's family members need, what post-discharge support is needed for survivors, how communication should work for everyone involved with a cardiac arrest, what factors best predict neurologically intact survival, whether biomarkers/genetic tests are effective in predicting OHCA and more research on the short and long-term psycho-social impacts of OHCA on survivors. The PPEET showed overwhelmingly positive results for the patient and family engagement experience during the final workshop. CONCLUSIONS: This inclusive research priority setting provides essential information for those doing resuscitation research internationally. The results provide a guide for priority areas of research and should drive our community to focus on questions that matter to survivors and their families in our work. In particular the Canadian Resuscitation Outcomes Consortium will be incorporating the top ten list into its strategic plan for the future.

Priorities for research in multiple conditions in later life (multi-morbidity): findings from a James Lind Alliance Priority Setting Partnership.

INTRODUCTION: multiple conditions in later life (multi-morbidity) is a major challenge for health and care systems worldwide, is of particular relevance for older people, but has not (until recently) received high priority as a topic for research. We have identified the top 10 research priorities from the perspective of older people, their carers, and health and social care professionals using the methods of a James Lind Alliance Priority Setting Partnership. METHODS: in total, 354 participants (162 older people and carers, 192 health professionals) completed a survey and 15 older people and carers were interviewed to produce 96 'unanswered questions'. These were further refined by survey and interviews to a shortlist of 21 topics, and a mix of people aged 80+ living with three or more conditions, carers and health and social care providers to prioritised the top 10. RESULTS: the key priorities were about the prevention of social isolation, the promotion of independence and physical and emotional well-being. In addition to these broad topics, the process also identified detailed priorities including the role of exercise therapy, the importance of falls (particularly fear of falling), the recognition and management of frailty and Comprehensive Geriatric Assessment. CONCLUSION: these topics provide a unique perspective on research priorities on multiple conditions in later life and complement existing UK and International recommendations about the optimisation of health and social care systems to deliver essential holistic models of care and the prevention and treatment of multiple co-existing conditions.

Setting the top 10 research priorities to improve the health of people with Type 2 diabetes: a Diabetes UK-James Lind Alliance Priority Setting Partnership.

AIMS: To describe processes and outcomes of a priority setting partnership to identify the 'top 10 research priorities' in Type 2 diabetes, involving people living with the condition, their carers, and healthcare professionals. METHODS: We followed the four-step James Lind Alliance Priority Setting Partnership process which involved: gathering uncertainties using a questionnaire survey distributed to 70 000 people living with Type 2 diabetes and their carers, and healthcare professionals; organizing the uncertainties; interim priority setting by resampling of participants with a second survey; and final priority setting in an independent group of participants, using the nominal group technique. At each step the steering group closely monitored and guided the process. RESULTS: In the first survey, 8227 uncertainties were proposed by 2587 participants, of whom 18% were from black, Asian and minority ethnic groups. Uncertainties were formatted and collated into 114 indicative questions. A total of 1506 people contributed to a second survey, generating a shortlist of 24 questions equally weighted to the contributions of people living with diabetes and their carers and those of healthcare professionals. In the final step the 'top 10 research priorities' were selected, including questions on cure and reversal, risk identification and prevention, and self-management approaches in Type 2 diabetes. CONCLUSION: Systematic and transparent methodology was used to identify research priorities in a large and genuine partnership of people with lived and professional experience of Type 2 diabetes. The top 10 questions represent consensus areas of research priority to guide future research, deliver responsive and strategic allocation of research resources, and improve the future health and well-being of people living with, and at risk of, Type 2 diabetes.

Top ten research priorities for spinal cord injury: the methodology and results of a British priority setting partnership.

STUDY DESIGN: This is a mixed-method consensus development project. OBJECTIVES: The objective of this study was to identify a top ten list of priorities for future research into spinal cord injury (SCI). SETTING: The British Spinal Cord Injury Priority Setting Partnership was established in 2013 and completed in 2014. Stakeholders included consumer organisations, healthcare professional societies and caregivers. METHODS: This partnership involved the following four key stages: (i) gathering of research questions, (ii) checking of existing research evidence, (iii) interim prioritisation and (iv) a final consensus meeting to reach agreement on the top ten research priorities. Adult individuals with spinal cord dysfunction because of trauma or non-traumatic causes, including transverse myelitis, and individuals with a cauda equina syndrome (henceforth grouped and referred to as SCI) were invited to participate in this priority setting partnership. RESULTS: We collected 784 questions from 403 survey respondents (290 individuals with SCI), which, after merging duplicate questions and checking systematic reviews for evidence, were reduced to 109 unique unanswered research questions. A total of 293 people (211 individuals with SCI) participated in the interim prioritisation process, leading to the identification of 25 priorities. At a final consensus meeting, a representative group of individuals with SCI, caregivers and health professionals agreed on their top ten research priorities. CONCLUSION: Following a comprehensive, rigorous and inclusive process, with participation from individuals with SCI, caregivers and health professionals, the SCI research agenda has been defined by people to whom it matters most and should inform the scope and future activities of funders and researchers for the years to come. SPONSORSHIP: The NIHR Oxford Biomedical Research Centre provided core funding for this project.

Research priorities in mesothelioma: A James Lind Alliance Priority Setting Partnership.

BACKGROUND: In the UK, despite the import and use of all forms of asbestos being banned more than 15 years ago, the incidence of mesothelioma continues to rise. Mesothelioma is almost invariably fatal, and more research is required, not only to find more effective treatments, but also to achieve an earlier diagnosis and improve palliative care. Following a debate in the House of Lords in July 2013, a package of measures was agreed, which included a James Lind Alliance Priority Setting Partnership, funded by the National Institute for Health Research. The partnership brought together patients, carers, health professionals and support organisations to agree the top 10 research priorities relating to the diagnosis, treatment and care of patients with mesothelioma. METHODS: Following the established James Lind Alliance priority setting process, mesothelioma patients, current and bereaved carers, and health professionals were surveyed to elicit their concerns regarding diagnosis, treatment and care. Research questions were generated from the survey responses, and following checks that the questions were currently unanswered, an interim prioritisation survey was conducted to identify a shortlist of questions to take to a final consensus meeting. FINDINGS: Four hundred and fifty-three initial surveys were returned, which were refined into 52 unique unanswered research questions. The interim prioritisation survey was completed by 202 responders, and the top 30 questions were taken to a final meeting where mesothelioma patients, carers, and health professionals prioritised all the questions, and reached a consensus on the top 10. INTERPRETATION: The top 10 questions cover a wide portfolio of research (including assessing the value of immunotherapy, individualised chemotherapy, second-line treatment and immediate chemotherapy, monitoring patients with pleural thickening, defining the management of ascites in peritoneal mesothelioma, and optimising follow-up strategy). This list is an invaluable resource, which should be used to inform the prioritisation and funding of future mesothelioma research.

IGF2BP1 controls cell death and drug resistance in rhabdomyosarcomas by regulating translation of cIAP1.

Rhabdomyosarcoma (RMS), a neoplasm characterised by undifferentiated myoblasts, is the most common soft tissue tumour of childhood. Although aggressive treatment of RMS could provide long-term benefit, resistance to current therapies is an ongoing problem. We report here that insulin-like growth factor 2-binding protein 1 (IGF2BP1), an oncofetal protein, is expressed in RMS patient-derived cell lines and in primary tumours where it drives translation of the cellular inhibitor of apoptosis 1 (cIAP1), a key regulator of the nuclear factor-κB signalling pathway and of caspase-8-mediated cell death. We demonstrate that reducing the levels of cIAP1 in RMS, either by IGF2BP1 knockdown or by IAP antagonists, sensitises these cells to tumour necrosis factor-α-mediated cell death. Finally, we show that targeting cIAP1 by IAP antagonists delays RMS tumour growth and improve survival in mice. Our results identify IGF2BP1 as a critical translational regulator of cIAP1-mediated apoptotic resistance in RMS and advocate for the combined use of IAP antagonists and tumour necrosis factor-α as a therapeutic approach for this type of cancer.

A novel function of HER2/Neu in the activation of G2/M checkpoint in response to γ-irradiation.

In response to γ-irradiation (IR)-induced DNA damage, activation of cell cycle checkpoints results in cell cycle arrest, allowing time for DNA repair before cell cycle re-entry. Human cells contain G1 and G2 cell cycle checkpoints. While G1 checkpoint is defective in most cancer cells, commonly due to mutations and/or alterations in the key regulators of G1 checkpoint (for example, p53, cyclin D), G2 checkpoint is rarely impaired in cancer cells, which is important for cancer cell survival. G2 checkpoint activation involves activation of ataxia telangiectasia-mutated (ATM)/ATM- and rad3-related (ATR) signalings, which leads to the inhibition of Cdc2 kinase and subsequent G2/M cell cycle arrest. Previous studies from our laboratory show that G2 checkpoint activation following IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. As HER receptor tyrosine kinases (RTKs), which have important roles in cell proliferation and survival, have been shown to activate ERK1/2 signaling in response to various stimuli, we investigated the role of HER RTKs in IR-induced G2/M checkpoint response in breast cancer cells. Results of the present studies indicate that IR exposure resulted in a striking increase in the phosphorylation of HER1, HER2, HER3 and HER4 in MCF-7 cells, indicative of activation of these proteins. Furthermore, specific inhibition of HER2 using an inhibitor, short hairpin RNA and dominant-negative mutant HER2 abolished IR-induced activation of ATM/ATR signaling, phosphorylation of Cdc2-Y15 and subsequent induction of G2/M arrest. Moreover, the inhibition of HER2 also abrogated IR-induced ERK1/2 phosphorylation. In contrast, inhibition of HER1 using specific inhibitors or decreasing expression of HER3 or HER4 using short hairpin RNAs did not block the induction of G2/M arrest following IR. These results suggest an important role of HER2 in the activation of G2/M checkpoint response following IR.

Protein phosphatase 2A has an essential role in the activation of gamma-irradiation-induced G2/M checkpoint response.

G2/M checkpoint activation after DNA damage results in G2/M cell cycle arrest that allows time for DNA repair before the entry of cells into mitosis. Activation of G2/M checkpoint involves a series of signaling events, which include activation of ataxia telangiectecia-mutated and Rad3-related (ATR) and Chk1 kinases and inhibition of Cdc2/Cyclin B activity. Studies presented in this report show that serine (Ser)/threonine (Thr) protein phosphatase 2A (PP2A) has an important role in G2/M checkpoint activation in response to gamma-irradiation (IR) exposure. Using PP2A inhibitors, as well as siRNA targeting various forms of Ser/Thr protein phosphatases, results presented in this report show that specific PP2A inhibition abrogates IR-induced activation of ATR and Chk1 kinases, as well as phosphorylation of Cdc2-Tyr15, and attenuates IR-induced G2/M arrest. These results suggest an important regulation of PP2A on IR-induced G2/M checkpoint signaling response.

Irradiation-induced G2/M checkpoint response requires ERK1/2 activation.

Following DNA damage, cells undergo G2/M cell cycle arrest, allowing time for DNA repair. G2/M checkpoint activation involves activation of Wee1 and Chk1 kinases and inhibition of Cdc25A and Cdc25C phosphatases, which results in inhibition of Cdc2 kinase. Results presented in this report indicate that gamma-irradiation (IR) exposure of MCF-7 cells resulted in extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation and induction of G2/M arrest. Furthermore, inhibition of ERK1/2 signaling resulted in >or=85% attenuation in IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ataxia telangiectasia mutated- and rad3-related (ATR), Chk1 and Wee1 kinases as well as phosphorylation of Cdc25A-Thr506, Cdc25C-Ser216 and Cdc2-Tyr15. Moreover, incubation of cells with caffeine, which inhibits ataxia telangiectasia mutated (ATM)/ATR, or transfection of cells with short interfering RNA targeting ATR abrogated IR-induced Chk1 phosphorylation and G2/M arrest but had no effect on IR-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling resulted in marked attenuation in IR-induced ATR activity with little, if any, effect on IR-induced ATM activation. These results implicate IR-induced ERK1/2 activation as an important regulator of G2/M checkpoint response to IR in MCF-7 cells.

Thromboembolism following removal of femoral venous apheresis catheters in patients with breast cancer.

BACKGROUND: Apheresis catheters have simplified collection of peripheral blood stem cells (PBSC), but may be associated with thrombosis of the instrumented vessels. We performed a retrospective analysis to study the prevalence of thromboembolism associated with the use of femoral apheresis catheters in patients with breast cancer. PATIENTS AND METHODS: Patients were participants in clinical trials of high-dose chemotherapy with autologous PBSC rescue. They underwent mobilization with either high-dose cyclophosphamide (n = 21) or cyclophosphamide/paclitaxel (n = 64), followed by filgrastim. Double lumen catheters (12 or 13 Fr) were placed in the femoral vein and removed within 12 h of the last apheresis procedure. Apheresis was performed using a continuous flow cell separator and ACD-A anticoagulant. Thromboembolism was diagnosed by either venous ultrasonography or ventilation-perfusion scan. RESULTS: Nine of 85 patients (10.6%) undergoing large volume apheresis with use of a femoral catheter developed thromboembolic complications. Pulmonary embolus (PE) was diagnosed in five and femoral vein thrombosis in four patients. Four of the five patients who developed PE were symptomatic; one asymptomatic patient had a pleural-based, wedge-shaped lesion detected on a staging computed tomography scan. The mean number of apheresis procedures was 2.4 (range one to four) and the mean interval between removal of the apheresis catheter and diagnosis of thrombosis was 17.6 days. In contrast, none of 18 patients undergoing apheresis using jugular venous access and none of 54 healthy allogeneic donors undergoing concurrent filgrastim-mobilized PBSC donation (mean 1.7 procedures/donor) using femoral access experienced thromboembolic complications. CONCLUSIONS: Thromboembolism following femoral venous catheter placement for PBSC collection in patients with breast cancer may be more common than previously recognized. Healthy PBSC donors are not at the same risk. Onset of symptoms related to thrombosis tended to occur several weeks after catheter removal. This suggests that the physicians not only need to be vigilant during the period of apheresis, but also need to observe patients for thromboembolic complications after the catheter is removed. The long interval between the removal of apheresis catheter and the development of thromboembolism may have a potential impact on prophylactic strategies developed in future, such as the duration of prophylactic anticoagulation. Avoidance of the femoral site in breast cancer patients, and close prospective monitoring after catheter removal, are indicated.

Polyglutamine protein aggregation and toxicity are linked to the cellular stress response.

Chronic exposure of cells to expanded polyglutamine proteins results in eventual cell demise. We constructed mouse cell lines expressing either the full-length androgen receptor (AR), or truncated forms of AR containing 25 or 65 glutamines to study the cellular consequences of chronic low-level exposure to these proteins. Expression of the polyglutamine-expanded truncated AR protein, but not the full-length expanded protein, resulted in the formation of cytoplasmic and nuclear aggregates and eventual cell death. Nuclear aggregates preferentially stained positive for heat shock protein (hsp)72, a sensitive indicator of a cellular stress response. Biochemical studies revealed that the presence of nuclear aggregates correlated with activation of the c-jun NH2-terminal kinase (JNK). Different metabolic insults, including heat shock treatment, and exposure to sodium arsenite or menadione, proved more toxic to those cells expressing the polyglutamine-expanded truncated protein than to cells expressing the non-expanded form. Cells containing cytoplasmic polyglutamine-protein aggregates exhibited a delayed expression of hsp72 after heat shock. Once expressed, hsp72 failed to localize normally and instead was sequestered within the protein aggregates. This was accompanied by an inability of the aggregate-containing cells to cease their stress response as evidenced by the continued presence of activated JNK. Finally, activation of the cellular stress response increased the overall extent of polyglutamine protein aggregation, especially within the nucleus. Inclusion of a JNK inhibitor reduced this stress-dependent increase in nuclear aggregates. Abnormal stress responses may contribute to enhanced cell vulnerability in cells expressing polyglutamine-expanded proteins and may increase the propensity of such cells to form cytoplasmic and nuclear inclusions.

Nonmyeloablative bone marrow transplantation from partially HLA-mismatched related donors using posttransplantation cyclophosphamide.

Cyclophosphamide (Cy) is a potent immunosuppressive agent that is selectively toxic to lymphocytes proliferating in response to recent antigen stimulation. In animal models, both graft rejection and GVHD after histoincompatible BMT can be inhibited by the posttransplantation administration of high-dose Cy. Therefore, a phase I clinical trial was undertaken to determine the minimal conditioning, including posttransplantation Cy, that permits the stable engraftment of partially HLA-mismatched marrow (up to 3 HLA antigens) from first-degree relatives. Thirteen patients (median age, 53 years) with high-risk hematologic malignancies received conditioning with fludarabine, 30 mg/m2 per day from days -6 to -2, and TBI, 2 Gy on day -1. All patients received Cy, 50 mg/kg on day 3, mycophenolate mofetil from day 4 to day 35, and tacrolimus from day 4 to day > or = 50. Three patients in cohort 1 received no additional conditioning, and 2 experienced graft rejection. Ten patients in cohort 2 received identical conditioning with the addition of Cy 14.5 mg/kg on days -6 and -5. Sustained donor cell engraftment occurred in 8 of these patients, with a median time to absolute neutrophil count > 500/microL of 15 days (range, 13-16 days) and to unsupported platelet count > 20,000/microL of 14 days (range, 0-26 days). All patients with engraftment achieved > or = 95% donor chimerism within 60 days of transplantation. Two patients with myelodysplastic syndrome rejected their grafts but experienced autologous neutrophil recovery at 24 and 44 days. Histologic acute GVHD developed in 6 patients (grade II in 3 patients, grade III in 3 patients) at a median of 99 days (range, 38-143 days) after transplantation and was fatal in 1 patient. At a median follow-up of 191 days (range, 124-423 days), 6 of 10 patients in cohort 2 were alive, and 5 were in complete remission of their disease, including both patients with graft rejection. These data demonstrate that partially HLA-mismatched bone marrow can engraft rapidly and stably after nonmyeloablative conditioning that includes posttransplantation Cy. Clinically significant antitumor responses occur, even among patients who reject their donor grafts.

Protein kinase and phosphatase responses to anoxia in crayfish, Orconectes virilis: purification and characterization of cAMP-dependent protein kinase.

The freshwater crayfish, Orconectes virilis, shows good anoxia tolerance, enduring 20 h in N(2)-bubbled water at 15 degrees C. Metabolic responses to anoxia by tolerant species often include reversible phosphorylation control over selected enzymes. To analyze the role of serine/threonine kinases and phosphatases in signal transduction during anoxia in O. virilis, changes in the activities of cAMP-dependent protein kinase (PKA) and protein phosphatases 1, 2A, and 2C were measured in tail muscle and hepatopancreas over a time course of exposure to N(2)-bubbled water. A strong increase in the percentage of PKA present as the free catalytic subunit (% PKAc) occurred between 1 and 2 h of anoxia exposure whereas phosphatase activities were strongly reduced. This suggests that PKA-mediated events are important in the initial response by tissues to declining oxygen availability. As oxygen deprivation became severe and prolonged (5-20 h) these changes reversed; the % PKAc fell to below control values and activities of phosphatases returned to or rose above control values. Subcellular fractionation also showed a decrease in PKA associated with the plasma membrane after 20 h anoxia whereas cytosolic PKA content increased. PKAc purified from tail muscle showed a molecular weight of 43.8+/-0.4 kDa, a pH optimum of 6.8, a high affinity for Mg ATP (K(m)=131.0+/-14.4 microM) and Kemptide (K(m)=31.6+/-5.2 microM). Crayfish PKAc was sensitive to temperature change; a break in the Arrhenius plot occurred at approximately 15 degrees C with a 2.5-fold rise in activation energy at temperatures <15 degrees C. These studies demonstrate a role for serine/threonine protein kinases and phosphatases in the metabolic adjustments to oxygen depletion by crayfish organs.

New subscales for an anchored version of the Brief Psychiatric Rating Scale: construction, reliability, and validity in acute psychiatric admissions.

Attending psychiatrists completed an anchored version of the 18-item Brief Psychiatric Rating Scale (BPRS-A) based on admission and evaluation information on a total of 2,921 adult patients treated at 1 public sector acute psychiatric teaching hospital. Exploratory factor analysis was applied to a 6-month sample to construct 4 nonoverlapping subscales: Resistance, Positive Symptoms, Negative Symptoms, and Psychological Discomfort. Confirmatory factor analysis compared these new subscales to 3 other published subscale models using a second 6-month sample. Internal consistency, rater influence, and interrater agreement were estimated in separate studies. Discriminant validity was explored by comparison of diagnosis-based samples. Application of the BPRS-A as a debriefing instrument in the study of symptomatic change and the multiple challenges inherent in psychometric study of such a rating scale in realistic hospital practice are discussed.

Tyrosine kinases and phosphatases in the estivating spadefoot toad.

To endure seasonally arid conditions, spadefoot toads (Scaphiopus couchii) spend 9-10 months underground each year in a hypometabolic state, termed estivation. Protein tyrosine kinases (PTKs) and phosphatases (PTPs) were evaluated in organs of control and estivating toads to assess their possible role in signal transduction during estivation. Total PTK activity decreased by 27-52 % in liver, lung and skeletal muscle during estivation but rose by 66% in heart. Total PTP activity changed only in liver (55 % decrease) and heart (74 %increase). Analysis of the distribution of PTKs between cytoplasmic and membrane-associated forms showed that estivation-linked changes in both fractions occurred in heart (increase) and liver (decrease) whereas in lung and skeletal muscle only the soluble fraction was affected. PTPs were assessed using both a general substrate (ENDpYINASL) and a substrate (DADEpYLIPQQG) specific for PTPs containing the SH2 binding site; both revealed estivation-associated changes in activities and subcellular distribution of PTPs in all tissues. DEAE-Sephadex chromatography showed multiple forms of skeletal muscle PTKs and PTPs in both soluble and insoluble fractions. Each fraction showed three major peaks of PTK activity, two of which shifted in elution position during estivation. The data show that PTKs and PTPs are modified in an organ-specific fashion during estivation by three mechanisms: changes in total activity, changes in subcellular distribution and possible protein covalent modification.

Phase I clinical trial of alitretinoin and tamoxifen in breast cancer patients: toxicity, pharmacokinetic, and biomarker evaluations.

PURPOSE: To determine the overall and dose-limiting toxicities (DLTs) of alitretinoin (9-cis-retinoic acid) in combination with tamoxifen and the pharmacokinetics of alitretinoin alone and when combined with tamoxifen in patients with metastatic breast cancer. The effect of tamoxifen and alitretinoin on MIB-1, a marker of proliferation, in unaffected breast tissue was explored. PATIENTS AND METHODS: Eligible patients had metastatic breast cancer. Previous tamoxifen therapy was allowed. Planned dose levels for alitretinoin ranged from 50 to 140 mg/m2/d with 20 mg/d tamoxifen in all patients after 4 weeks of alitretinoin as a single agent. Plasma concentrations of alitretinoin and retinol were measured at baseline and after 1, 2, and 3 months. Breast core biopsies were obtained at baseline and after 2 months of therapy. RESULTS: Twelve patients with metastatic breast cancer received a total of 86 cycles of therapy. At 90 mg/m2/d, three of five patients experienced a DLT: grade 3 headache, grade 3 hypercalcemia, and grade 3 noncardiogenic pulmonary edema. At 70 mg/m2/d, one of six patients experienced a DLT (headache), and this level was considered the maximal tolerated dose in this study. Three toxicities occurred that had not been reported previously with alitretinoin: an asymptomatic delay in dark adaptation, a marked decrease in high-density lipoprotein cholesterol, and the occurrence of enthesopathy. Two of the nine assessable patients had a durable clinical response: one partial response and stable disease for 18 months and one complete response in continuous remission for 48+ months. Both responding patients were estrogen receptor-positive and had had previous tamoxifen therapy. There was a high degree of interpatient variability of plasma alitretinoin concentrations, although a significant decline in alitretinoin plasma levels over time was observed. MIB-1 scores declined in four of the eight paired breast specimens obtained. CONCLUSION: The combination of tamoxifen and alitretinoin is well tolerated and has antitumor activity in metastatic breast cancer. The recommended phase II dose is 70 mg/m2/d with 20 mg/d tamoxifen.

Freeze-thaw effects on metabolic enzymes in wood frog organs.

To determine whether episodes of natural freezing and thawing altered the metabolic makeup of wood frog (Rana sylvatica) organs, the maximal activities of 28 enzymes of intermediary metabolism were assessed in six organs (brain, heart, kidney, liver, skeletal muscle, gut) of control (5 degrees C acclimated), frozen (24 h at -3 degrees C), and thawed (24 h back at 5 degrees C) frogs. The enzymes assessed represented pathways including glycolysis, gluconeo-genesis, amino acid metabolism, fatty acid metabolism, the TCA cycle, and adenylate metabolism. Organ-specific responses seen included (a) the number of enzymes affected by freeze-thaw (1 in gut ranging to 17 in heart), (b) the magnitude and direction of response (most often enzyme activities decreased during freezing and rebounded with thawing but, liver showed freeze-specific increases in several enzymes), and (c) the response to freezing versus thawing (enzyme activities in gut and kidney changed during freezing, whereas most enzymes in skeletal muscle responded to thawing). Overall, the data show that freeze-thaw implements selected changes to the maximal activities of various enzymes of intermediary metabolism and that these may aid organ-specific responses that alter fuel use during freeze-thaw, support cryoprotectant metabolism, and aid organ endurance of freeze-induced ischemia.

Inhibition of glioma cells in vitro and in vivo using a recombinant adenoviral vector containing an astrocyte-specific promoter.

Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene in combination with the drug ganciclovir (GCV) is a promising approach for the treatment of cancer-inducing gliomas, a tumor with a poor prognosis. In an attempt to limit the toxic effects on normal tissues, we constructed a recombinant adenoviral vector, Adgfa2TK, in which the HSV-TK gene is driven by the promoter for the gene encoding glial fibrillary acidic protein, an intermediate filament protein expressed primarily in astrocytes. Infection by Adgfa2TK of a glial cell line (C6) and a non-glial cell line (MDA-MB-231) revealed markedly increased expression of HSV-TK in glial cells as determined by Western blot. In comparison, high HSV-TK protein levels were produced in both cell lines after infection with a control virus, AdCMVTK, in which the constitutive cytomegalovirus viral promoter was used to direct HSV-TK expression. Infection of two glial cell lines (C6, U251) and two non-glial cell lines (HepG2, MDA-MB-231) with Adgfa2TK followed by GCV treatment revealed high toxicity in glial cell lines (50% growth inhibitory concentration: <2 microg/mL of GCV) with little or no toxicity (50% growth inhibitory concentration: >75 microg/mL) in the non-glial cell lines. In vivo, injection of Adgfa2TK into C6 tumors grown in nude mice followed by intraperitoneal GCV treatment significantly repressed tumor growth compared with the controls. Adgfa2TK may be useful for directing expression of the HSV-TK gene to gliomas.

Identification of shc as the primary protein binding to the tyrosine-phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin platelet signaling.

Outside-in signaling mediated by the integrin alpha(IIb)beta(3) (GPIIbIIIa) is critical to platelet function and has been shown to involve the phosphorylation of tyrosine residues on the cytoplasmic tail of beta(3). To identify proteins that bind directly to phosphorylated beta(3), we utilized an affinity column consisting of a peptide modeled on the tyrosine-phosphorylated cytoplasmic domain of beta(3). Tandem mass spectrometric sequencing and immunoblotting demonstrated that Shc was the primary protein binding to phosphorylated beta(3). To determine the involvement of Shc in outside-in alpha(IIb)beta(3) signaling, the phosphorylation of Shc during platelet aggregation was examined; transient Shc phosphorylation was observed when thrombin-stimulated platelets were allowed to aggregate or when aggregation was induced by an LIBS (ligand-induced binding site) antibody, D3. Moreover, Shc was co-immunoprecipitated with tyrosine-phosphorylated beta(3) in detergent lysates of aggregated platelets. Using purified, recombinant protein, it was found that the binding of Shc to monophosphorylated (C-terminal tyrosine) and diphosphorylated beta(3) peptides was direct, demonstrating Shc recognition motifs on phospho-beta(3). Aggregation-induced Shc phosphorylation was also observed to be robust in platelets from wild-type mice, but not in those from mice expressing (Y747F,Y759F) beta(3), which are defective in outside-in alpha(IIb)beta(3) signaling. Thus, Shc is the primary downstream signaling partner of beta(3) in its tyrosine phosphorylation outside-in signaling pathway.