Optimized protocol for detection of native, full-length HIV-1 envelope on the surface of transfected cells.

AIMS: Designing therapeutics against the HIV envelope glycoprotein (Env) is only as accurate as the structure of the Env they are targeting. Conserving the structure of the Env trimer is crucial for proper experimental assessment of antibody binding and neutralization. However, Env is notably difficult to express by transfection of a recombinant Env plasmid. To increase surface expression, researchers commonly utilize c-tail mutants of the gp41 transmembrane glycoprotein of HIV-1, but mutations and deletions in this region can impact the overall conformation and stability of the Env trimer. Multiple studies have shown that while tail mutants have higher Env surface expression, they are easier to neutralize and have altered trimer conformations compared with wild-type Env found in vivo on infected cells. To assess and characterize native cell surface Env structures, we sought a protocol that could reliably detect wild-type Env surface expression by flow cytometry. METHODS AND RESULTS: By avoiding fetal bovine serum-based buffers, significantly increasing the amounts of transfected plasmid and Env-specific antibody and by selecting a bright, biotin + streptavidin-PE detection system, we were able to increase the surface expression of transfected Env protein. CONCLUSION: This protocol will allow for more precise assessment of antibody binding, epitope exposure, and Env structure, all of which will contribute to designing more effective vaccines and immunotherapeutics.

Acute myocardial infarction with isolated congenitally corrected transposition of the great arteries.

Congenital cardiac abnormalities diagnosed at the time of acute coronary syndrome are rare. A 43-year-old man presented to the emergency department complaining of recurring, severe chest pain. Subsequent emergent coronary angiography demonstrated unusual coronary anatomy: 1) one small caliber bifurcating vessel originating from the right sinus of Valsalva; 2) one very large vessel arising from the posterior sinus; and 3) no coronary artery from the normal left sinus of Valsalva. The large vessel from the posterior sinus was totally occluded in its midportion and was treated with intravascular ultrasound-guided percutaneous coronary intervention. Further diagnostic workup, including two-dimensional transthoracic echocardiogram and computed tomographic coronary angiography, demonstrated isolated corrected transposition of the great arteries with a dilated systemic ventricle and systolic dysfunction with an ejection fraction of 30%. The patient's clinical course was complicated by recurrent nonsustained ventricular tachycardia, treated with medical therapy and a dual-chamber implantable cardioverter defibrillator. This case is an example of a common clinical presentation with a very uncommon congenital heart disorder. Similar cases may become more frequent as the number of adult congenital heart patients increases in the population.

Lactic acidosis: relationship between metformin levels, lactate concentration and mortality.

AIM: The role of metformin in lactic acidosis is regularly questioned. Arguments against a causal role for metformin in lactic acidosis occurrence are the lack of correlation between plasma metformin and lactate levels, as well as between metformin plasma levels and mortality. We aim to analyse these correlations in a large series of lactic acidosis cases recorded in the French nationwide pharmacovigilance database. METHODS: All cases of lactic acidosis spontaneously reported between 1985 and October 2013 associated with metformin exposure were extracted from the pharmacovigilance database. We assessed the statistical correlations between prescribed daily doses of metformin, plasma concentrations of metformin and lactate, pH and plasma creatinine, as well as the relationship between mortality and these variables. RESULTS: Seven hundred and twenty-seven cases of lactic acidosis were reported during the period. Metformin plasma concentration was documented for 260 patients, lactate plasma concentration for 556 patients, pH for 502 patients, creatinine for 397 patients and the vital outcome for 713 patients. Metformin plasma concentration, lactate concentration, pH and plasma creatinine were all correlated (P < 0.001). There were significant differences between surviving and deceased patients in terms of metformin plasma levels (25.2 vs. 37.4 mg/l, P = 0.002) and lactate concentrations (10.8 vs. 16.3 mmol/l, P < 0.001). Thirty per cent of patients died when metformin concentration was > 5 mg/l compared with 11% for patients with concentration < 5 mg/l (P = 0.003). CONCLUSIONS: Our data suggest that metformin accumulation contributes to the pathogenesis and prognosis of lactic acidosis.

AKT is a therapeutic target in myeloproliferative neoplasms.

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F- or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with ruxolitinib in suppressing the growth of JAK2V617F-mutant SET2 cells. Importantly, MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.

Discrimination of auditory motion patterns: the mismatch negativity study.

The aim of the present study is to test whether mismatch negativity (MMN) response can be elicited by changes in auditory motion dynamics. The discrimination of auditory motion patterns was investigated using psychophysical and electrophysiological methods in the same group of subjects. Auditory event-related potentials (ERP) were recorded for stationary midline noises and moving noises shifting to the left/right from the head midline. Two patterns of auditory motion were used with gradual (Motion) and stepwise (Step) movements which started and ended at the same loci. Auditory motion was produced by linear and abrupt changes of interaural time differences (ITD) in binaurally presented stimuli. In Experiment 1, ERPs were recorded for stationary midline standards and for Motion and Step deviants. It was found that Step deviants result in larger MMN amplitudes than Motion deviants with the same distance travelled, which implies that information contained in the stimulus midportion could be involved in the processing of the auditory motion. The threshold ITD values for the detection of Step and Motion stimuli displacement obtained during psychoacoustic tests were greater than the minimal ITD changes which elicited significant MMN. Experiment 2 demonstrated that Step deviants elicited significant MMNs in the context of Motion standards, although these stimuli could not be discriminated behaviourally. MMNs elicited by Step deviants in different acoustic contexts are discussed from the viewpoint of different brain processes underlying the discrimination of the abrupt ITD change. These results suggest that the early cortical mechanism of auditory motion processing reflected by MMN could not be considered as a spatial discriminator of the onset/offset stimulus positions, that is, a simple onset-offset detector. Combining psychoacoustic data with MMN results we may conclude that motion discrimination in auditory system might be better at the preattentive level.

Phase I/II multicenter study to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of AZD4877 in patients with refractory acute myeloid leukemia.

Eg5 (kinesin spindle protein) is a microtubule motor protein, essential for centrosome separation during mitosis. This Phase I/II, open-label, multicenter, two-part study investigated AZD4877, a potent Eg5 inhibitor, in patients with acute myeloid leukemia. Primary objectives were to determine the maximum tolerated dose (MTD) (part A), assess efficacy (part B) and determine the pharmacokinetic profile (parts A and B). Secondary objectives included assessment of safety and tolerability. AZD4877 was administered at a range of doses (2, 4, 7, 10, 13, 16 and 18 mg/day) as a 1-hour intravenous infusion on three consecutive days of a continuous 2-week schedule. The MTD in part A was defined as 16 mg/day based on dose-limiting stomatitis at 16 and 18 mg/day, hyperbilirubinemia at 16 mg/day and palmar-plantar erythrodysesthesia syndrome at 18 mg/day. Systemic exposure to AZD4877 generally increased with increasing dose whereas half-life was not dose dependent. No evaluable patients experienced a complete remission (CR) or CR with incomplete blood count recovery (CRi), demonstrating no evidence of AZD4877 efficacy in this population. Evidence of monoasters in all but the 4 mg/day dose group provided proof of mechanism for AZD4877. This study was terminated due to lack of efficacy. (ClinicalTrials.gov identifier NCT00486265).

How does mismatch negativity reflect auditory motion?

Recent studies have shown that the mismatch negativity (MMN), a change-specific component of the auditory event-related potential (ERP), is accurately tracking the spatial location of the stationary sound source. The aim of the present study was to estimate the parameters of MMNs evoked by auditory motion and to compare the motion discrimination measured by MMN in normally hearing subjects with the psychophysical data obtained in the same group of subjects. The auditory motion was simulated by introducing variable interaural time differences (ITDs) into the deviant stimuli. The ERPs were recorded for frequently occurring stationary midline standards and for infrequent deviant sounds moving horizontally at different velocities. It was established that all the deviant stimuli elicited significant MMNs. The MMN increased monotonically in amplitude with growing angular distances travelled by the deviant stimuli. The deviants that travelled over the same angular distances at different velocities caused MMNs that agreed in magnitude but differed in latency. These results indicated that the angular distance rather than sound image velocity was the most essential cue involved in the MMN generation. To test the psychophysical performance, a two-interval forced-choice task was employed, in which the ITD was the main dependent variable. The deviants that evoked significant MMNs at the minimal ITDs were not discriminated behaviorally, indicating that the motion discrimination of the hearing system may be better at a preattentive level.

Actual or ideal body weight to calculate CD34+ cell dose in patients undergoing autologous hematopoietic SCT for myeloma?

CD34+ cell dose calculations are usually based on actual body weight (ABW). We have shown that ideal body weight (IBW) may provide a better basis for this in a small population of patients with hematologic malignancies. This was studied further in 514 myeloma autografts. The CD34+ cell doses (10(6)/kg) by IBW and ABW were 1.37-39.36 (median 6.03) and 1.15-29.67 (median 4.84), respectively. IBW-based cell doses correlated slightly better with engraftment than ABW-based doses (higher r(2)): 0.5 x 10(9)/l neutrophils 0.83 versus 0.82, 1.0 x 10(9)/l neutrophils 0.78 versus 0.77, 20 x 10(9)/l platelets 0.54 versus 0.53 and 50 x 10(9)/l platelets 0.57 versus 0.55. When outliers (hematologic recovery in <8 or >16 days) were excluded, the findings were similar: 0.5 x 10(9)/l neutrophils 0.85 versus 0.84, 1.0 x 10(9)/l neutrophils 0.85 versus 0.84, 20 x 10(9)/l platelets 0.86 versus 0.85 and 50 x 10(9)/l platelets 0.85 versus 0.84. CD34+ cell doses based on IBW as well as ABW significantly affected engraftment when analyzed separately as continuous variables. However, when analyzed together, only the dose based on IBW retained significance. We conclude that calculation of CD34+ cell numbers for autotransplantation should be based on IBW.

Clinical trial: alvimopan for the management of post-operative ileus after abdominal surgery: results of an international randomized, double-blind, multicentre, placebo-controlled clinical study.

BACKGROUND: Post-operative ileus (POI) affects most patients undergoing abdominal surgery. AIM: To evaluate the effect of alvimopan, a peripherally acting mu-opioid receptor antagonist, on POI by negating the impact of opioids on gastrointestinal (GI) motility without affecting analgesia in patients outside North America. METHODS: Adult subjects undergoing open abdominal surgery (n = 911) randomly received oral alvimopan 6 or 12 mg, or placebo, 2 h before, and twice daily following surgery. Opioids were administered as intravenous patient-controlled analgesia (PCA) or bolus injection. Time to recovery of GI function was assessed principally using composite endpoints in subjects undergoing bowel resection (n = 738). RESULTS: A nonsignificant reduction in mean time to tolerate solid food and either first flatus or bowel movement (primary endpoint) was observed for both alvimopan 6 and 12 mg; 8.5 h (95% CI: 0.9, 16.0) and 4.8 h (95% CI: -3.2, 12.8), respectively. However, an exploratory post hoc analysis showed that alvimopan was more effective in the PCA (n = 317) group than in the non-PCA (n = 318) group. Alvimopan was well tolerated and did not reverse analgesia. CONCLUSION: Although the significant clinical effect of alvimopan on reducing POI observed in previous trials was not reproduced, this trial suggests potential benefit in bowel resection patients who received PCA.

Phase I/II trial of total lymphoid irradiation and high-dose chemotherapy with autologous stem-cell transplantation for relapsed and refractory Hodgkin's lymphoma.

BACKGROUND: The standard approach to treatment of relapsed/refractory Hodgkin's lymphoma (HL) is high-dose chemotherapy conditioning followed by autologous hematopoietic stem-cell transplantation (aHSCT). We report the results of a prospective phase I/II clinical trial of accelerated hyperfractionated total lymphoid irradiation (TLI) immediately followed by high-dose chemotherapy for relapsed/refractory HL. PATIENTS AND METHODS: Forty-eight patients underwent aHSCT with either sequential TLI/chemotherapy (n = 32) or chemotherapy-alone conditioning (n = 16), based on prior radiation exposure. The first 22 patients enrolled on trial received escalating doses of etoposide (1600-2100 mg/m(2)) with high-dose carboplatin and cyclophosphamide. RESULTS: No dose-limiting toxicity was seen and TLI/chemotherapy was well tolerated. The 5-year event-free survival (EFS) estimate for all patients was 44% with overall survival (OS) of 48%. Five-year EFS and OS for the TLI/chemotherapy group was 63% and 61%, respectively, compared with 6% and 27%, respectively, for the chemotherapy-alone group (P < 0.0001 and P = 0.04, respectively). Patients with primary induction failure HL who received TLI/chemotherapy had 5-year EFS and OS rate of 83%. The 100-day treatment-related mortality was 4.2% and two secondary cancers were seen. Significant factors predicting survival by multivariate analysis included TLI/chemotherapy conditioning and B symptoms at relapse. CONCLUSIONS: Sequential TLI/chemotherapy conditioning for relapsed/refractory HL is safe and associated with excellent long-term survival rates.

Difficulties of smoking cessation in diabetic inpatients benefiting from a systematic consultation to help them to give up smoking.

AIM: To assess the value of systematic smoking cessation consultations for diabetic smokers admitted to hospital. METHODS: All diabetic smokers admitted to the Diabetes Department of Georges Pompidou European Hospital between February 2003 and February 2004 were systematically offered a consultation with a physician specialised in tobacco cessation. Follow-up visits at three, six and nine months were planned. RESULTS: Of the 306 diabetic patients admitted, 38 (12.4%) were smokers. There were more men than women in the group of smokers and the diabetic smokers were younger than the non-smokers. The smokers had fewer micro-angiopathic complications than the non-smokers, but there was no difference in the frequency of macro-angiopathic complications. The level of nicotine physical dependence was moderate or high for 60% of the smokers. Although all the smokers agreed to the consultation, less than half agreed to drug-based treatments to help them to give up smoking and only 15% returned for the six-month visit. Only one patient had stopped smoking at the six-month visit. CONCLUSION: This study demonstrates the difficulties in systematic interventions to help diabetic patients to stop smoking. Diabetic smokers probably constitute a specific population for which the barriers to giving up smoking should be explored.

Efficacy of rosiglitazone in a genetically defined population with mild-to-moderate Alzheimer's disease.

Mild-to-moderate AD patients were randomized to placebo or rosiglitazone (RSG) 2, 4 or 8 mg. Primary end points at Week 24 were mean change from baseline in AD Assessment Scale-Cognitive (ADAS-Cog) and Clinician's Interview-Based Impression of Change Plus Caregiver Input global scores in the intention-to-treat population (N=511), and results were also stratified by apolipoprotein E (APOE) genotype (n=323). No statistically significant differences on primary end points were detected between placebo and any RSG dose. There was a significant interaction between APOE epsilon4 allele status and ADAS-Cog (P=0.014). Exploratory analyses demonstrated significant improvement in ADAS-Cog in APOE epsilon4-negative patients on 8 mg RSG (P=0.024; not corrected for multiplicity). APOE epsilon4-positive patients did not show improvement and showed a decline at the lowest RSG dose (P=0.012; not corrected for multiplicity). Exploratory analyses suggested that APOE epsilon4 non-carriers exhibited cognitive and functional improvement in response to RSG, whereas APOE epsilon4 allele carriers showed no improvement and some decline was noted. These preliminary findings require confirmation in appropriate clinical studies.

When should ultrasonography be used to detect asymptomatic carotid atheroma in diabetic patients?

INTRODUCTION: While guidelines for detection of silent myocardial and lower limb ischemia are established, data on screening asymptomatic carotid lesions remain scarce. However, such screening would be costly. Since the prevalence of diabetes increases constantly, it is necessary to keep screening costs low by setting up criteria for the selection of patients at risk of ischemic cerebral attack and those who will need medical or surgical attention. Diabetic patients, particularly type 2, often have many reasons to take anti-platelet agents and lipid-lowering therapy. Therefore, carotid ultrasonography screening would have little effect on treatment modification or on glycaemia and blood pressure objectives, but could improve the prognosis of operable lesions. IN THE GENERAL POPULATION: A one-time screening program was considered worthwhile if the prevalence of severe asymptomatic stenosis was over 20%. The presence of another arterial occlusive disease or other cardio-vascular risk factors could be a major argument for screening. IN DIABETIC PATIENTS: Carotid intima-media thickness (IMT) was recognized as a reliable prognostic indicator of heart attack and stroke. It worsens with duration of diabetes, renal failure, cardiac neuropathy and poor long term glycaemic control. CONCLUSION: Our review suggests that a one-time carotid ultrasonography screening could be recommended for diabetic patients with coronary disease or lower limb atherosclerosis (secondary prevention), all diabetic patients above 60 years of age, smokers, hypertensive and with hypercholesterolemia; type 1 diabetic patients with poor long term glycaemic control; all type 2 diabetic patients with renal failure, a long duration of ill-controlled diabetes or with a carotid bruit. This literature review should be analyzed with caution. It would be helpful to organize a prospective long term study on all types of diabetic patients, including a carotid ultrasonography screening program by experienced radiologists.

Premonitory symptoms in migraine: an electronic diary study.

BACKGROUND: Migraine is frequently associated with nonheadache symptoms before, during, and after the headache. Premonitory symptoms occurring before the attack have not been rigorously studied. Should these symptoms accurately predict headache, there are considerable implications for the pathophysiology and management of migraine. METHODS: Electronic diaries were used in a 3-month multicenter study to record nonheadache symptoms before, during, and after migraine. The authors recruited subjects who reported nonheadache symptoms in at least two of three attacks that they believed predicted headache. Symptoms were entered in the diaries by patient initiation and through prompted entries at random times daily. Entries could not be altered retrospectively. Data recorded included nonheadache symptoms occurring during all three phases of the migraine, prediction of the attack from premonitory symptoms, general state of health, and action taken to prevent the headache. RESULTS: One hundred twenty patients were recruited: 97 provided usable data. Patients correctly predicted migraine headaches from 72% of diary entries with premonitory symptoms. A range of cognitive and physical symptoms was reported at a similar rate through all three phases of the migraine. The most common premonitory symptoms were feeling tired and weary (72% of attacks with warning features), having difficulty concentrating (51%), and a stiff neck (50%). Subjects who functioned poorly in the premonitory phase were the most likely to correctly predict headache. CONCLUSIONS: Using an electronic diary system, the authors show that migraineurs who report premonitory symptoms can accurately predict the full-blown headache.

Prolonged improvement of total pancreatic allograft function by previous intrathymic bone marrow cell injection in rats.

Donor-specific induction of tolerance was previously achieved in the diabetic rat by intrathymic injection of pancreatic islets. It allowed a secondary islet graft in any site without immunosuppression. Since total pancreatic graft in man is metabolically more proficient than islet graft, we attempted tolerance induction for total vascularized pancreas transplantation in diabetic BN recipient rats by an intrathymic bone marrow cell (BMC) injection from Lewis donor rats, associated to an antilymphocyte antibody (ALS) administration. Control groups consisted of isogenic grafts, allogenic grafts without tolerance induction and allogenic grafts with ALS alone. In all grafted groups, mean blood glucose and plasma insulin were normalised within 24 h. Graft rejection (clinically suggested by diabetes recurrence and later confirmed by histology) appeared at 18 +/- 2 postoperative days in the absence of intrathymic BMC injection and at 36 +/- 8 days in the group with BMC injection (p < 0.05). Intrathymic bone marrow graft was successful in delaying rejection in our study.

Design and in vivo immunogenicity of a polyvalent vaccine based on SIVmac regulatory genes.

Most vaccine modalities for human immunodeficiency virus type 1 (HIV-1) tested for immunogenicity and efficacy in the SIVmac (simian immunodeficiency virus) macaque model do not include the viral regulatory proteins. Because viral regulatory proteins are expressed early during the virus life cycle and represent an additional source of antigens, their inclusion as a vaccine component may increase the overall virus-specific immune response in vaccinees. However, at least two of the early proteins, Tat and Nef, may be immunosuppressive, limiting their usefulness as components of an SIV vaccine. We have constructed a polyvalent chimeric protein in which the open reading frames for Tat and Nef have been reassorted and the nuclear localization sequence for Tat and Rev and the myristoylation site for Nef have been removed. The resulting DNA plasmid (pDNA-SIV-Retanef) (pDNA-SIV-RTN) encodes a protein of 55 kDa (Retanef) that localizes at the steady state in the cytoplasma of transfected cells. Both the DNA-SIV-RTN and the highly attenuated recombinant poxvirus vector NYVAC-SIV-RTN were demonstrated to be immunogenic in SIVmac251-infected macaques treated with ART as well as in naive macaques. An equivalent strategy may be used for the generation of polyvalent antigens encoding the regulatory proteins in a HIV-1 vaccine candidate.

Adjustable gastric banding in a public university hospital: prospective analysis of 400 patients.

BACKGROUND: Laparoscopic application of an adjustable gastric band (LAGB) is considered the least invasive surgical option for morbid obesity. It has the advantage of being potentially reversible and can improve quality of life. METHOD: Between April 1997 and January 2001, 400 patients underwent LAGB. There were 352 women and 48 men with mean age 40.2 years (16-66). Preoperative mean body weight was 119 kg (85-195) and mean body mass index (BMI) was 43.8 kg/m2 (35.1-65.8). RESULTS: Mean operative time was 116 minutes (30-380), and mean hospital stay was 4.55 days (3-42). There was no death. There were 12 conversions (3%). 40 complications required an abdominal reoperation (10%), for perforation (n = 2), gastric necrosis (n = 1), slippage (n = 31), incisional hernia (n = 2) and reconnection of the tube (n = 4). We noticed 7 pulmonary complications (2 ARDS, 5 atelectasis) and 30 minor problems related to the access port. At 2 years, mean BMI had fallen from 43.8 to 32.7 kg/m2 and mean excess weight loss (EWL) was 52.7% (12-94). CONCLUSION: LAGB is a very beneficial operation with an acceptable complication rate. EWL is 50% at 2 years if multidisciplinary follow-up remains assiduous. Surveillance for late anterior stomach slippage within the band is essential.