Lentiviral gene transfer to the nonhuman primate brain.

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene beta galatosidase (beta Gal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the beta Gal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were beta Gal positive in monkeys 1 (n = 2) and 3 (n = 1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 beta Gal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the beta Gal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinson's disease.

Superinfection with drug-resistant HIV is rare and does not contribute substantially to therapy failure in a large European cohort.

BACKGROUND: Superinfection with drug resistant HIV strains could potentially contribute to compromised therapy in patients initially infected with drug-sensitive virus and receiving antiretroviral therapy. To investigate the importance of this potential route to drug resistance, we developed a bioinformatics pipeline to detect superinfection from routinely collected genotyping data, and assessed whether superinfection contributed to increased drug resistance in a large European cohort of viremic, drug treated patients. METHODS: We used sequence data from routine genotypic tests spanning the protease and partial reverse transcriptase regions in the Virolab and EuResist databases that collated data from five European countries. Superinfection was indicated when sequences of a patient failed to cluster together in phylogenetic trees constructed with selected sets of control sequences. A subset of the indicated cases was validated by re-sequencing pol and env regions from the original samples. RESULTS: 4425 patients had at least two sequences in the database, with a total of 13816 distinct sequence entries (of which 86% belonged to subtype B). We identified 107 patients with phylogenetic evidence for superinfection. In 14 of these cases, we analyzed newly amplified sequences from the original samples for validation purposes: only 2 cases were verified as superinfections in the repeated analyses, the other 12 cases turned out to involve sample or sequence misidentification. Resistance to drugs used at the time of strain replacement did not change in these two patients. A third case could not be validated by re-sequencing, but was supported as superinfection by an intermediate sequence with high degenerate base pair count within the time frame of strain switching. Drug resistance increased in this single patient. CONCLUSIONS: Routine genotyping data are informative for the detection of HIV superinfection; however, most cases of non-monophyletic clustering in patient phylogenies arise from sample or sequence mix-up rather than from superinfection, which emphasizes the importance of validation. Non-transient superinfection was rare in our mainly treatment experienced cohort, and we found a single case of possible transmitted drug resistance by this route. We therefore conclude that in our large cohort, superinfection with drug resistant HIV did not compromise the efficiency of antiretroviral treatment.

Antiretroviral treatment of adult HIV infection: 2012 recommendations of the International Antiviral Society-USA panel.

CONTEXT: New trial data and drug regimens that have become available in the last 2 years warrant an update to guidelines for antiretroviral therapy (ART) in human immunodeficiency virus (HIV)-infected adults in resource-rich settings. OBJECTIVE: To provide current recommendations for the treatment of adult HIV infection with ART and use of laboratory-monitoring tools. Guidelines include when to start therapy and with what drugs, monitoring for response and toxic effects, special considerations in therapy, and managing antiretroviral failure. DATA SOURCES, STUDY SELECTION, AND DATA EXTRACTION: Data that had been published or presented in abstract form at scientific conferences in the past 2 years were systematically searched and reviewed by an International Antiviral Society-USA panel. The panel reviewed available evidence and formed recommendations by full panel consensus. DATA SYNTHESIS: Treatment is recommended for all adults with HIV infection; the strength of the recommendation and the quality of the evidence increase with decreasing CD4 cell count and the presence of certain concurrent conditions. Recommended initial regimens include 2 nucleoside reverse transcriptase inhibitors (tenofovir/emtricitabine or abacavir/lamivudine) plus a nonnucleoside reverse transcriptase inhibitor (efavirenz), a ritonavir-boosted protease inhibitor (atazanavir or darunavir), or an integrase strand transfer inhibitor (raltegravir). Alternatives in each class are recommended for patients with or at risk of certain concurrent conditions. CD4 cell count and HIV-1 RNA level should be monitored, as should engagement in care, ART adherence, HIV drug resistance, and quality-of-care indicators. Reasons for regimen switching include virologic, immunologic, or clinical failure and drug toxicity or intolerance. Confirmed treatment failure should be addressed promptly and multiple factors considered. CONCLUSION: New recommendations for HIV patient care include offering ART to all patients regardless of CD4 cell count, changes in therapeutic options, and modifications in the timing and choice of ART in the setting of opportunistic illnesses such as cryptococcal disease and tuberculosis.

Sustained improvement in left ventricular function after bone marrow derived cell therapy in patients with acute ST elevation myocardial infarction. A 5-year follow-up from the Stem Cell Transplantation in Ischaemic Myocardium Study.

BACKGROUND: Intracoronary injection of autologous bone marrow-derived mononucleated cells (BM-MNC) may improve LV function shortly after acute ST elevation myocardial infarction (STEMI), but little is known about the long-term durability of the treatment effect. METHODS: In a single-centre trial a total of 60 patients with acute anterior STEMI, successful reperfusion therapy and a left ventricular ejection fraction (LVEF) of <50% were screened for the study. 23 patients were actively treated with intracoronary infusion of BM-MNC within a median of 3 days. The open-label control group consisted of 19 patients who did not consent to undergo BM-MNC treatment but agreed to undergo regular clinical and echocardiographic follow-up for up to 5 years after AMI. RESULTS: Whereas at 4 months there was no significant difference between the increase in LVEF in the BM-MNC group and the control group (+7.0%, 95%CI 3.6; 10.4) vs. +3.9%, 95%CI -2.1; 10), the absolute increase at 5 years remained stable in the BM-MNC but not in the control group (+7.95%, 95%CI 3.5; 12.4 vs. -0.5%, 95%CI -5.4; 4.4; p for interaction between groups = 0.035). DISCUSSION: In this single-centre, open-labelled study, intracoronary administration of BM-MNC is feasible and safe in the short term. It is also associated with sustained improvement of left ventricular function in patients with acute myocardial infarction, encouraging phase III studies to examine the potential BM-MNC effect on clinical outcome.

Phase II study of weekly plitidepsin as second-line therapy for small cell lung cancer.

OBJECTIVE: To evaluate the antitumor activity and safety profile of plitidepsin administered as a 1h weekly intravenous (i.v.) infusion of 3.2mg/m(2) to patients with small cell lung cancer (SCLC) who relapsed or progressed after one line of chemotherapy. PATIENTS AND METHODS: This was a multicenter, open-label, single-arm, exploratory, phase II clinical trial. Treatment lasted until disease progression, unacceptable toxicity, patient refusal or treatment delay for >2 weeks. Objective response rate (primary efficacy endpoint) was evaluated according to response evaluation criteria in solid tumors (RECIST). The rate of stable disease (SD) lasting for at least 6 months and time-to-event variables were secondary endpoints of efficacy. Toxicity was assessed using National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 2.0. RESULTS: Twenty pretreated SCLC patients (median age, 60 years) with extensive (n=13) or limited-stage disease (n=7) received a total of 24 treatment cycles (median, one cycle per patient; range, 1-2). Objective tumor responses were not observed and only one of the 17 evaluable patients had SD. With a median follow-up of 11.8 months, the progression-free survival and the median overall survival were 1.3 months and 4.8 months, respectively. The most troubling or common toxicities were fatigue, muscle weakness, lymphopenia, anemia (no patients showed neutropenia), and asymptomatic, non-cumulative increase of transaminases levels and alkaline phosphatase. CONCLUSION: This clinical trial shows that a cycle of 1h weekly i.v. infusion of plitidepsin (3.2mg/m(2)) was generally well tolerated other than fatigue and muscle weakness in patients with pretreated SCLC. One patient died due to multi-organ failure. The absence of antitumor activity found here precludes further studies of this plitidepsin schedule as second-line single-agent treatment of SCLC.

No experimental evidence that sibling competition induces young to switch nests in the colonial Alpine swift, Apus melba

Adoption is frequent in colonial animals where opportunities for dependent young to receive care from nonbiological parents are high. The departure of dependent young from their original family to seek adoption in neighbouring families is thought to be induced by sibling competition for access to limited resources provided by poor-quality parents. We tested this hypothesis in the colonial Alpine swift by manipulating the number of young reared per brood, with the prediction that offspring from enlarged broods switch nests more frequently than those from reduced broods. Although nestling swifts hatch with little locomotor activity, from 20 days until their first flight at 50-70 days they frequently move out of their nests to seek adoption in neighbouring families. Although nestlings reared in experimentally enlarged broods were lighter and their body mass at day 20 after hatching was more variable than in nestlings reared in reduced broods, there was no difference between the two treatments in the frequency of nests switching and in the age when nestlings switched nests for the first time. However, consistent with other evidence that nest switching by nestling swifts evolved as a strategy to reduce ectoparasite load, young from broods with naturally high numbers of the ectoparasitic louse fly Crataerina melbae were more prone to switch nests. This shows that ectoparasitism rather than sibling competition is a key proximate factor promoting the evolution of nest switching in the colonial Alpine swift. (c) 2006 The Association for the Study of Animal Behaviour Published by Elsevier Ltd. All rights reserved.

Electro-cortical correlates of motor inhibition : a comparison between selective and non-selective stop tasks

In this study, we compared a selective stop task (transition from a bimanual in-phase to a unimanual index fingers' tapping), with a non-selective stop task (stopping a bimanual in-phase tapping at all), and with a switching task (transition from in-phase to anti-phase bimanual tapping). The aim was twofold: 1) to identify the electro-cortical correlates of selective and non-selective inhibition processes and 2) to investigate which type of inhibition - selective or not - is required when switching between two bimanual motor patterns. The results revealed that all tasks led to enhanced activation (alpha power) of the left sensorimotor and posterior regions which seems to reflect an overall effort to stop the preferred bimanual in-phase tendency. Each task implied specific functional connectivity reorganizations (beta coherence) between cerebral motor areas, probably reflecting engagement in a new unimanual or bimanual movement.

Immunological mechanism of action and clinical profile of disease-modifying treatments in multiple sclerosis.

Multiple sclerosis (MS) is a life-long, potentially debilitating disease of the central nervous system (CNS). MS is considered to be an immune-mediated disease, and the presence of autoreactive peripheral lymphocytes in CNS compartments is believed to be critical in the process of demyelination and tissue damage in MS. Although MS is not currently a curable disease, several disease-modifying therapies (DMTs) are now available, or are in development. These DMTs are all thought to primarily suppress autoimmune activity within the CNS. Each therapy has its own mechanism of action (MoA) and, as a consequence, each has a different efficacy and safety profile. Neurologists can now select therapies on a more individual, patient-tailored basis, with the aim of maximizing potential for long-term efficacy without interruptions in treatment. The MoA and clinical profile of MS therapies are important considerations when making that choice or when switching therapies due to suboptimal disease response. This article therefore reviews the known and putative immunological MoAs alongside a summary of the clinical profile of therapies approved for relapsing forms of MS, and those in late-stage development, based on published data from pivotal randomized, controlled trials.

Adult human adipose tissue contains several types of multipotent cells.

Multipotent mesenchymal stromal cells (MSCs) are a type of adult stem cells that can be easily isolated from various tissues and expanded in vitro. Many reports on their pluripotency and possible clinical applications have raised hopes and interest in MSCs. In an attempt to unify the terminology and the criteria to label a cell as MSC, in 2006 the International Society for Cellular Therapy (ISCT) proposed a standard set of rules to define the identity of these cells. However, MSCs are still extracted from different tissues, by diverse isolation protocols, are cultured and expanded in different media and conditions. All these variables may have profound effects on the selection of cell types and the composition of heterogeneous subpopulations, on the selective expansion of specific cell populations with totally different potentials and ergo, on the long-term fate of the cells upon in vitro culture. Therefore, specific molecular and cellular markers that identify MSCs subsets as well as standardization of expansion protocols for these cells are urgently needed. Here, we briefly discuss new useful markers and recent data supporting the rapidly emerging concept that many different types of progenitor cells are found in close association with blood vessels. This knowledge may promote the necessary technical improvements required to reduce variability and promote higher efficacy and safety when isolating and expanding these cells for therapeutic use. In the light of the discussed data, particularly the identification of new markers, and advances in the understanding of fundamental MSC biology, we also suggest a revision of the 2006 ISCT criteria.

Six years of denosumab treatment in postmenopausal women with osteoporosis: results from the first three years of the freedom extension

Background/Purpose: Denosumab (DMAb) is an approved therapy for the treatment of postmenopausal women with osteoporosis at increased risk for fracture. A favorable risk/benefit profile was demonstrated in the pivotal, 3-year FREEDOM trial (Cummings et al NEJM 2009). The open-label, active-treatment FREEDOM Extension study is investigating the efficacy and safety of DMAb for up to 10 years. The Extension trial enrolled women who had received DMAb or placebo in FREEDOM and provides an opportunity to evaluate the long-term efficacy and safety of continuous DMAb treatment (long-term group), and to replicate the DMAb findings observed in FREEDOM (cross-over group). Here, we report the results from the first 3 years of the Extension, representing up to 6 continuous years of DMAb exposure.Methods: During the Extension, each woman is scheduled to receive 60 mg DMAb every 6 months and supplemental calcium and vitamin D daily. For the analyses reported here, women from the FREEDOM DMAb group received 3 more years of DMAb for a total of 6 years of exposure (long-term group) and women from the FREEDOM placebo group received 3 years of DMAb exposure (cross-over group).Results: Of the 5928 women eligible for the Extension, 4550 (77%) enrolled (N_2343 long-term; N_2207 cross-over). In the long-term group, further significant mean increases in bone mineral density (BMD) occurred 4044 for cumulative 6-year gains of 15.2% at the lumbar spine and 7.5% at the total hip (Figure). During the first 3 years of DMAb treatment during the Extension, the cross-over group had significant mean gains in BMD at the lumbar spine (9.4%) and total hip (4.8%), similar to those observed in the long-term DMAb group during the first 3 years of FREEDOM (lumbar spine, 10.1%; total hip, 5.7%). Serum CTX was rapidly and similarly reduced after the 1st (cross-over) or 7th (long-term) DMAb dose with the characteristic attenuation observed at the end of the dosing period. In the cross-over group, yearly incidences of new vertebral and nonvertebral fractures were lower than in the FREEDOM placebo group. Fracture incidence remained low in the long-term group. Incidences of adverse events (AEs) and serious AEs did not increase over time with DMAb treatment. There were 2 subjects with AEs adjudicated to ONJ in the cross-over group and 2 in the long-term group. Both cases in the cross-over group healed completely and without further complications; 1 of these subjects continues to receive DMAb. Both women in the long-term group continue to be followed. No atypical femur fractures have been observed to date. Figure. Percent changes in bone mineral density during FREEDOM and the Extension Conclusion: DMAb treatment for 6 continuous years (long-term group) remained well tolerated, maintained reduced bone turnover, and continued to significantly increase BMD. Fracture incidence remained low. DMAb treatment for 3 years in the cross-over group reproduced the original observations in FREEDOM.

Clinical benefits of an adherence monitoring program in the management of secondary hyperparathyroidism with cinacalcet: results of a prospective randomized controlled study.

BACKGROUND/AIMS: One of the causes of uncontrolled secondary hyperparathyroidism (sHPT) is patient's poor drug adherence. We evaluated the clinical benefits of an integrated care approach on the control of sHPT by cinacalcet. METHODS: Prospective, randomized, controlled, multicenter, open-label study. Fifty hemodialysis patients on a stable dose of cinacalcet were randomized to an integrated care approach (IC) or usual care approach (UC). In the IC group, cinacalcet adherence was monitored using an electronic system. Results were discussed with the patients in motivational interviews, and drug prescription adapted accordingly. In the UC group, drug adherence was monitored, but results were not available. RESULTS: At six months, 84% of patients in the IC group achieved recommended iPTH targets versus 55% in the UC group (P = 0.04). The mean cinacalcet taking adherence improved by 10.8% in the IC group and declined by 5.3% in the UC group (P = 0.02). Concomitantly, the mean dose of cinacalcet was reduced by 7.2 mg/day in the IC group and increased by 6.4 mg/day in the UC group (P = 0.03). CONCLUSIONS: The use of a drug adherence monitoring program in the management of sHPT in hemodialysis patients receiving cinacalcet improves drug adherence and iPTH control and allows a reduction in the dose of cinacalcet.

Photoaffinity labeling of the T cell receptor on living cytotoxic T lymphocytes.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of an antigenic peptide with MHC class I molecules and the TCR on living cells. Two photoreactive derivatives of the H-2Kd (Kd) restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) were used. The first derivative contained an N-terminal photoreactive iodo, 4-azido salicyloyl (IASA) group and biotin on the TCR contact residue Lys259 [IASA-YIPSAEK(biotin)I]. As previously described, this derivative selectively bound to and labeled the Kd molecule. The second photoreactive compound, the isomeric biotin-YIPSAEK(IASA)I, also efficiently bound to the Kd molecule, but failed to label this protein. A CTL clone derived from a mouse immunized with this derivative recognized this conjugate but not the parental P. berghei circumsporozoite peptide or the [IASA-YIPSAEK-(biotin)I] derivative in an Kd-restricted manner. Incubation of the cloned CTL cells with biotin-YIPSAEK(IASA)I, but not its isomer, followed by UV irradiation resulted in photoaffinity labeling of the TCR-alpha chain that was dependent on the conjugate binding to the Kd molecule. The TCR labeling was partially inhibited by anti-LFA 1 and anti-ICAM1 mAb, but was increased by addition of beta 2m or soluble KdQ10. The exquisite labeling selectivity of the two photoprobes opens a new, direct approach to the molecular analysis of antigen presentation and recognition by living CTL.

Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide.

PURPOSE: Temozolomide is a novel oral alkylating agent with demonstrated efficacy as second-line therapy for patients with recurrent anaplastic astrocytoma and glioblastoma multiforme (GBM). This phase II study was performed to determine the safety, tolerability, and efficacy of concomitant radiation plus temozolomide therapy followed by adjuvant temozolomide therapy in patients with newly diagnosed GBM. PATIENTS AND METHODS: Sixty-four patients were enrolled onto this open-label, phase II trial. Temozolomide (75 mg/m(2)/d x 7 d/wk for 6 weeks) was administered orally concomitant with fractionated radiotherapy (60 Gy total dose: 2 Gy x 5 d/wk for 6 weeks) followed by temozolomide monotherapy (200 mg/m(2)/d x 5 days, every 28 days for six cycles). The primary end points were safety and tolerability, and the secondary end point was overall survival. RESULTS: Concomitant radiation plus temozolomide therapy was safe and well tolerated. Nonhematologic toxicities were rare and mild to moderate in severity. During the concomitant treatment phase, grade 3 or 4 neutropenia, thrombocytopenia, or both were observed in 6% of patients, including two severe infections with Pneumocystis carinii. During adjuvant temozolomide, 2% and 6% of cycles were associated with grade 3 and 4 neutropenia or thrombocytopenia, respectively. Median survival was 16 months, and the 1- and 2-year survival rates were 58% and 31%, respectively. Patients younger than 50 years old and patients who underwent debulking surgery had the best survival outcome. CONCLUSION: Continuous daily temozolomide and concomitant radiation is safe. This regimen of concomitant chemoradiotherapy followed by adjuvant chemotherapy may prolong the survival of patients with glioblastoma. Further investigation is warranted, and a randomized trial is ongoing.

Comparative angiotensin II receptor blockade in healthy volunteers: the importance of dosing.

OBJECTIVES: We have reported previously that 80 mg valsartan and 50 mg losartan provide less receptor blockade than 150 mg irbesartan in normotensive subjects. In this study we investigated the importance of drug dosing in mediating these differences by comparing the AT(1)-receptor blockade induced by 3 doses of valsartan with that obtained with 3 other antagonists at given doses. METHODS: Valsartan (80, 160, and 320 mg), 50 mg losartan, 150 mg irbesartan, and 8 mg candesartan were administered to 24 healthy subjects in a randomized, open-label, 3-period crossover study. All doses were given once daily for 8 days. The angiotensin II receptor blockade was assessed with two techniques, the reactive rise in plasma renin activity and an in vitro radioreceptor binding assay that quantified the displacement of angiotensin II by the blocking agents. Measurements were obtained before and 4 and 24 hours after drug intake on days 1 and 8. RESULTS: At 4 and 24 hours, valsartan induced a dose-dependent "blockade" of AT(1) receptors. Compared with other antagonists, 80 mg valsartan and 50 mg losartan had a comparable profile. The 160-mg and 320-mg doses of valsartan blocked AT(1) receptors at 4 hours by 80%, which was similar to the effect of 150 mg irbesartan. At trough, however, the valsartan-induced blockade was slightly less than that obtained with irbesartan. With use of plasma renin activity as a marker of receptor blockade, on day 8, 160 mg valsartan was equivalent to 150 mg irbesartan and 8 mg candesartan. CONCLUSIONS: These results show that the differences in angiotensin II receptor blockade observed with the various AT(1) antagonists are explained mainly by differences in dosing. When 160-mg or 320-mg doses were investigated, the effects of valsartan hardly differed from those obtained with recommended doses of irbesartan and candesartan.

Zeitpunkt der Medikamenten-einnahme bei Hypertonie und Angina pectoris. Eine kontrollierte Uberwachungsstudie. [Time of drug intake in hypertension and angina pectoris. A controlled monitoring study]

A prospective cross-over study was performed in a general practice environment to assess and compare compliance data obtained by electronic monitoring on a BID or QD regimen in 113 patients with hypertension or angina pectoris. All patients were on a BID regimen (nifedipine SR) during the first month and switched to QD regimen (amlodipine) for another month. Taking compliance (i.e. the proportion of days with correct dosing) improved in 30% of patients (95% confidence interval 19 to 41%, p < 0.001), when switching from a BID to a QD regimen, but at the same time there was a 15% increase (95% confidence interval 5 to 25%, p < 0.02) in the number of patients with one or more no-dosing days. About 8% of patients had a low compliance rate, irrespective of the dosage regimen. Actual dosage intervals were used to estimate extent and timing of periods with unsatisfactory drug activity for various hypothetical drug durations of action, and it appears that the apparent advantage of QD regimen in terms of compliance is clinically meaningful only, when the duration of activity extents beyond the dosage interval in all patients.