Evidence for the in vivo safety of insulated foamy viral vectors.

Retroviral vector-mediated stem cell gene therapy is a promising approach for the treatment of hematopoietic disorders. However, genotoxic side effects from integrated vector proviruses are a significant concern for the use of retroviral vectors in the clinic. Insulated foamy viral (FV) vectors are potentially safer retroviral vectors for hematopoietic stem cell gene therapy. We evaluated two newly identified human insulators, A1 and A2, for use in FV vectors. These insulators had moderate insulating capacity and higher titers than previously developed insulated FV vectors. The A1-insulated FV vector was chosen for comparison with the previously described 650cHS4-insulated FV vector in human cord blood CD34+ repopulating cells in an immunodeficient mouse model. To maximize the effects of the insulators on the safety of FV vectors, FV vectors containing a highly genotoxic spleen focus forming virus promoter were used to elicit differences in genotoxicity. In vivo, the A1-insulated FV vector showed an approximate 50% reduction in clonal dominance compared with either the 650cHS4-insulated or control FV vectors, although the transduction efficiency of the A1-insulated vector was higher. This data suggests that the A1-insulated FV vector is promising for future preclinical and clinical studies.

Systematic review and critical appraisal of the impact of acellular dermal matrix use on the outcomes of implant-based breast reconstruction.

BACKGROUND: Acellular dermal matrix (ADM) may improve outcomes in implant-based breast reconstruction (IBBR). The aim of this study was critically to appraise and evaluate the current evidence for ADM-assisted IBBR. METHODS: Comprehensive electronic searches identified complete papers published in English between January 2000 and August 2013, reporting any outcome of ADM-assisted IBBR. All systematic reviews, randomized clinical trials (RCTs) and non-randomized studies (NRSs) with more than 20 ADM recipients were included. Studies were critically appraised using AMSTAR for systematic reviews, the Cochrane risk-of-bias tool for RCTs and its adaptation for NRSs. Characteristics and results of identified studies were summarized. RESULTS: A total of 69 papers (8 systematic reviews, 1 RCT, 40 comparative studies and 20 case series) were identified, all of which were considered at high risk of bias, mostly due to patient selection and selective outcome reporting. The median ADM group sample size was 51.0 (i.q.r. 33.0-127.0). Most studies were single-centre (54), and they were often single-surgeon (16). ADM was most commonly used for immediate (40) two-stage IBBR (36) using human ADM (47), with few studies evaluating ADM-assisted single-stage procedures (10). All reported clinical outcomes (for example implant loss) and more than half of the papers (33) assessed process outcomes, but few evaluated cosmesis (16) or patient-reported outcomes (10). Heterogeneity between study design and, especially, outcome measurement precluded meaningful data synthesis. CONCLUSION: Current evidence for the value of ADMs in IBBR is limited. Use in practice should therefore be considered experimental, and evaluation within registries or well designed and conducted studies, ideally RCTs, is recommended to prevent widespread adoption of a potentially inferior intervention.

Invasive Propionibacterium acnes infections in a non-selective patient cohort: clinical manifestations, management and outcome.

An increasing number of reports suggest that Propionibacterium acnes can cause serious invasive infections. Currently, only limited data exist regarding the spectrum of invasive P. acnes infections. We conducted a non-selective cohort study at a tertiary hospital in the UK over a 9-year-period (2003-2012) investigating clinical manifestations, risk factors, management, and outcome of invasive P. acnes infections. Forty-nine cases were identified; the majority were neurosurgical infections and orthopaedic infections (n = 28 and n = 15 respectively). Only 2 cases had no predisposing factors; all neurosurgical and 93.3 % of orthopaedic cases had a history of previous surgery and/or trauma. Foreign material was in situ at the infection site in 59.3 % and 80.0 % of neurosurgical and orthopaedic cases respectively. All neurosurgical and orthopaedic cases required one or more surgical interventions to treat P. acnes infection, with or without concomitant antibiotic therapy; the duration of antibiotic therapy was significantly longer in the group of orthopaedic cases (median 53 vs 19 days; p = 0.0025). All tested P. acnes isolates were susceptible to penicillin, ampicillin and chloramphenicol; only 1 was clindamycin-resistant. Neurosurgical and orthopaedic infections account for the majority of invasive P. acnes infections. Most cases have predisposing factors, including previous surgery and/or trauma; spontaneous infections are rare. Foreign material is commonly present at the site of infection, indicating that the pathogenesis of invasive P. acnes infections likely involves biofilm formation. Since invasive P. acnes infections are associated with considerable morbidity, further studies are needed to establish effective prevention and optimal treatment strategies.

Demonstration of ignition radiation temperatures in indirect-drive inertial confinement fusion hohlraums.

We demonstrate the hohlraum radiation temperature and symmetry required for ignition-scale inertial confinement fusion capsule implosions. Cryogenic gas-filled hohlraums with 2.2 mm-diameter capsules are heated with unprecedented laser energies of 1.2 MJ delivered by 192 ultraviolet laser beams on the National Ignition Facility. Laser backscatter measurements show that these hohlraums absorb 87% to 91% of the incident laser power resulting in peak radiation temperatures of T(RAD)=300 eV and a symmetric implosion to a 100 µm diameter hot core.

Attenuation of low-frequency sound in freshwater.

Lake Superior was chosen as an experimental site to compare sound absorption of freshwater with the results of absorption measurements in seawater. The relaxation-like absorption at 1 kilohertz occurring in seawater is also present in freshwater. A relaxation related to the structural characteristics rather than to the salt content of water may be responsible for the anomalous absorption.

Ouabain effects on intracellular potassium activity and contractile force in cat papillary muscle.

The relationship between the positive inotropic and toxic effects of cardiac glycosides and their effects on intracellular ionic composition is incompletely defined. We measured intracellular potassium activity (alpha ik), extracellular potassium activity (alpha ek), resting potential, action potential duration, and contractile force at 32 degrees C in paired papillary muscles from feline right ventricles exposed to ouabain. Muscles used for electrophysiological measurements were quiescent except for isolated stimulation to confirm impalement and record action potential duration. Muscles used for contractile force measurements were quiescent except for 4-min periods when force was measured at a cycle length of 1,400 ms. Muscle length was adjusted to achieve 50% of maximal tension at this cycle length before each experiment. In four experiments, alpha ik and contractile force were measured in the same muscle. Alpha iK was measured with single and double-barrel K-sensitive electrodes. At 10 nM ouabain, action potential duration is prolonged. Among the concentrations tested, the threshold for a clear positive inotropic effect is 0.1 microM ouabain. The threshold for decrease in alpha iK, increase in alpha eK, and decrease in membrane potential is 1 microM, at which concentration toxic signs develop, including arrhythmias, aftercontractions, and alteration in the staircase response of contractile force to repetitive stimulation. Ouabain need not change alpha iK to effect positive inotropy in ventricular muscle, a relationship different from that reported between [K]i (intracellular potassium concentration) and positive inotropy. Higher ouabain concentrations, which others have shown to clearly inhibit active Na and K transport, are shown to upset intracellular potassium activity homeostasis and to consistently produce toxicity.

Bradykinin stimulates NF-kappaB activation and interleukin 1beta gene expression in cultured human fibroblasts.

Bradykinin (BK), a pluripotent nonameric peptide, is known for its proinflammatory functions in both tissue injury and allergic inflammation of the airway mucosa and submucosa. To understand the mechanisms by which BK serves as an inflammatory mediator, the human lung fibroblast cell line WI-38 was stimulated with BK and the expression of IL-1beta gene was examined. BK at nanomolar concentrations induced a marked increase in immunoreactive IL-1beta, detectable within 2 h in both secreted and cell-associated forms. BK-induced IL-1beta synthesis was inhibited by a B2-type BK receptor antagonist and by treatment of the cells with pertussis toxin, indicating the involvement of a BK receptor that couples to the G(i)/G(o) class of heterotrimeric G proteins. Whereas cycloheximide and actinomycin D both inhibited BK-induced IL-1beta synthesis, results from Northern blot and nuclear run-on assays suggested that BK acted primarily at the transcription level which led to the accumulation of IL-1beta message in stimulated cells. Gel mobility shift assays were used with nuclear extracts from stimulated WI-38 cells to examine the transcription mechanism for BK-induced IL-1beta expression. A DNA binding activity specific for the decameric kappaB enhancer was detected and was found to contain the p50 and p65 subunits of the NF-kappaB/rel protein family. BK-induced NF-kappaB activation correlated with IL-1beta message upregulation with respect to agonist concentration, time course, sensitivity to bacterial toxins, and blockade by the B2 receptor antagonist. After BK stimulation, a significant increase in the activity of chloramphenicol acetyltransferase was observed in WI-38 cells transfected with a reporter plasmid bearing the kappaB enhancers from the IL-1beta gene. Deletion of the kappaB enhancer sequence significantly reduced BK-induced chloramphenicol acetyltransferase activity. These findings suggests a novel function of BK in the activation of NF-kappaB and the induction of cytokine gene expression.

Randomized trial of peribulbar triamcinolone acetonide with and without focal photocoagulation for mild diabetic macular edema: a pilot study.

OBJECTIVE: To provide pilot data on the safety and efficacy of anterior and posterior sub-Tenon injections of triamcinolone either alone or in combination with focal photocoagulation in the treatment of mild diabetic macular edema (DME). DESIGN: Prospective, phase II, multicenter, randomized clinical trial. PARTICIPANTS: One hundred nine patients (129 eyes) with mild DME and visual acuity 20/40 or better. METHODS: The participants were assigned randomly to receive either focal photocoagulation (n = 38), a 20-mg anterior sub-Tenon injection of triamcinolone (n = 23), a 20-mg anterior sub-Tenon injection followed by focal photocoagulation after 4 weeks (n = 25), a 40-mg posterior sub-Tenon injection of triamcinolone (n = 21), or a 40-mg posterior sub-Tenon injection followed by focal photocoagulation after 4 weeks (n = 22). Follow-up visits were performed at 4, 8, 17, and 34 weeks. MAIN OUTCOME MEASURES: Change in visual acuity and retinal thickness measured with optical coherence tomography (OCT). RESULTS: At baseline, mean visual acuity in the study eyes was 20/25 and mean OCT central subfield thickness was 328 mum. Changes in retinal thickening and in visual acuity were not significantly different among the 5 groups at 34 weeks (P = 0.46 and P = 0.94, respectively). There was a suggestion of a greater proportion of eyes having a central subfield thickness less than 250 mum at 17 weeks when the peribulbar triamcinolone was combined with focal photocoagulation. Elevated intraocular pressure and ptosis were adverse effects attributable to the injections. CONCLUSIONS: In cases of DME with good visual acuity, peribulbar triamcinolone, with or without focal photocoagulation, is unlikely to be of substantial benefit. Based on these results, a phase III trial to evaluate the benefit of these treatments for mild DME is not warranted.

Differential gene expression between normal and tumor-derived ovarian epithelial cells.

The majority of ovarian tumors arise from the transformation of the ovarian surface epithelial cells, a single layer of cells surrounding the ovary. To identify genes that may contribute to the malignant phenotype of ovarian cancers, cDNA representational difference analysis was used to compare expressed genes in primary cultures of normal human ovarian surface epithelium (HOSE) and ovarian tumor-derived epithelial cells from the Cedars-Sinai Ovarian Cancer (CSOC) repository. A total of 255 differentially expressed genes were identified, of which 160 and 95 were specifically expressed in HOSE and CSOC cells, respectively. Using cDNA array hybridization, the expression profiles of the genes identified by cDNA-representational difference analysis were examined in an additional 5 HOSE and 10 CSOC lines. The comparison of average signal of each gene revealed 44 HOSE-specific and 16 CSOC-specific genes that exhibited at least a 2.5-fold difference in expression. A large number of genes identified in this study encode membrane-associated or secreted proteins and, hence, may be useful as targets in the development of serum-based diagnostic markers for ovarian cancer. Very few genes associated with protein synthesis or metabolism were identified in this study, reflecting the lack of observable differences in phenotypic or growth characteristics between HOSE and CSOC cells. Northern blot analysis on a subset of these genes demonstrated comparable levels of gene expression as observed in the cDNA array hybridization.

An essential role of Ffar2 (Gpr43) in dietary fibre-mediated promotion of healthy composition of gut microbiota and suppression of intestinal carcinogenesis.

Composition of the gut microbiota has profound effects on intestinal carcinogenesis. Diet and host genetics play critical roles in shaping the composition of gut microbiota. Whether diet and host genes interact with each other to bring specific changes in gut microbiota that affect intestinal carcinogenesis is unknown. Ability of dietary fibre to specifically increase beneficial gut microbiota at the expense of pathogenic bacteria in vivo via unknown mechanism is an important process that suppresses intestinal inflammation and carcinogenesis. Free fatty acid receptor 2 (FFAR2 or GPR43) is a receptor for short-chain fatty acids (acetate, propionate and butyrate), metabolites of dietary fibre fermentation by gut microbiota. Here, we show FFAR2 is down modulated in human colon cancers than matched adjacent healthy tissue. Consistent with this, Ffar2(-/-) mice are hypersusceptible to development of intestinal carcinogenesis. Dietary fibre suppressed colon carcinogenesis in an Ffar2-dependent manner. Ffar2 played an essential role in dietary fibre-mediated promotion of beneficial gut microbiota, Bifidobacterium species (spp) and suppression of Helicobacter hepaticus and Prevotellaceae. Moreover, numbers of Bifidobacterium is reduced, whereas those of Prevotellaceae are increased in human colon cancers than matched adjacent normal tissue. Administration of Bifidobacterium mitigated intestinal inflammation and carcinogenesis in Ffar2(-/-) mice. Taken together, these findings suggest that interplay between dietary fibre and Ffar2 play a key role in promoting healthy composition of gut microbiota that stimulates intestinal health.

Signal transduction during cannibalistic sexual phagocytosis: calcium is not the trigger but GTP-binding protein function is essential.

After fertilization, the zygote giant cell of Dictyostelium discoideum chemoattracts and subsequently engulfs hundreds of amoebae of the same species and strains from which it was derived. A pharmacological approach indicates that, while it may have some role, calcium is not the trigger for this cannibalistic phagocytic process. Of several agents that perturb intracellular calcium levels [A23187, LaCl, 8-diethylamino-octyl-3,4,5-trimethoxylbenzoate (TMB-8), and chlorotetracycline], only A23187 had an effect in reducing amoebal ingestion. In keeping with this, agents which interfered with downstream effectors of calcium function did not alter sexual phagocytosis. Calmidazolium and trifluoperazine, which inhibit calmodulin function, were ineffective, as were a protein kinase C inhibitor (staurosporine) and activator (phorbol 12-myristate 13-acetate). On the other hand, the nucleotide analogues GTP gamma S and GDP beta S both inhibited sexual phagocytosis indicating a role for GTP-binding protein activity at some stage in the process. Sub-fractionation of cells from non-phagocytic and phagocytic stage cell cultures followed by immunolocalization after SDS-PAGE and western blotting revealed a number of GTP-binding proteins in both the cell membrane and intracellular membrane fractions that might function during the events of sexual phagocytosis.

Comparative analysis of chemotaxis in Dictyostelium using a radial bioassay method: protein tyrosine kinase activity is required for chemotaxis to folate but not to cAMP.

The role of signal transduction during chemotaxis of Dictyostelium discoideum cells to cAMP and folic acid was investigated using a radial bioassay technique. The effects of signalling agonists were assessed by measuring the diameters of visible rings formed by the outward migration of amoebae up radial gradients of chemoattractant. This rapid and simple bioassay method yields chemotactic rates equivalent to more complex assay systems. In support of previous studies, chemotaxis toward both cAMP and folic acid was inhibited in a dose-dependent manner by LaCl3, EDTA, chlorotetracycline and A1F3, supporting the importance of calcium ions and G protein-mediated signalling in both chemotactic events. The work was extended by examining the effects of the protein tyrosine kinase inhibitor genistein. This agent inhibited chemotaxis to folate in a dose-dependent manner but had no observable effect on chemotaxis toward cAMP. The notion that phosphorylation of proteins on tyrosine residues is critical for chemotaxis to folic acid was supported by Western blotting experiments with monoclonal anti-phosphotyrosine antibodies which detected two candidate proteins of M(r) 52,000 and 38,000 in the membranes of folate-responsive amoebae. These two bands disappeared with starvation which leads to the loss of responsiveness of folic acid and the acquisition of responsiveness to cAMP. Time-lapse videomicrography also revealed some unique differences in chemotactic response. Starved cells responded to cAMP as individuals but feeding cells chemoattracted to folic acid on a populational basis. The ability to compare two different types of chemotaxis using a simple, rapid and accurate bioassay system should enhance future studies of chemotaxis in wild-type and mutant strains of D. discoideum.

Decreased release of norepinephrine in the isolated kidney of the adult spontaneously hypertensive rat.

Renal resistance vessels of the mature spontaneously hypertensive rat (SHR) exhibit an increased reactivity to exogenous norepinephrine, but a normal response to renal nerve stimulation. This difference could be due either to depression of the exocytotic process or to accelerated disposition of the released transmitter. We compared the overflow of norepinephrine in isolated perfused kidneys from adult SHR and normotensive rats. After previous incubation with 3H-norepinephrine, renal nerve stimulation caused smaller increases in the overflow of intact tritiated transmitter and its metabolites in kidneys form SHR than in those from normotensive controls. A similar difference was found when the amounts of endogenous norepinephrine were measured radioenzymatically. The tissue content of norepinephrine was comparable in kidneys from both hypertensive and normotensive animals. The uptake of 3H-norepinephrine was comparable in kidneys from SHR and normotensive controls; cocaine caused a comparable depression of the 3H-uptake in which then explains the normal vasoconstrictor response to renal nerve stimulation despite the increased responsiveness of the vascular smooth muscle cells to norepinephrine.

Incest: children at risk.

The typical family constellation in this study of 14 cases of incest was that of a chronically depressed mother, an alcoholic and violent father or stepfather, and an eldest daughter who was forced to assume many of her mother's responsibilities, with ensuing role confusion. The authors stress the need for physicians to be alert to the possibility of incest in such high-risk families.

Radiation therapy technology: manpower survey 1987.

A chronic shortage of radiation therapy technologists exists in the United States. This report presents the data obtained in a 1987 survey of all radiation oncology facilities identified by the Patterns of Care master list. Of the 1,142 questionnaires mailed, 52% were returned. The survey identified 2,328 credentialed (ARRT) and 554 non-credentialed technologists employed in radiation therapy. Of this total of 2,882, 2,141 were identified as staff technologists. A total of 1,186 megavoltage units were identified, giving a ratio of 1.8 technologist/megavoltage unit. Two hundred sixty open funded positions and 230 unfunded positions for radiation therapy technologists were identified. The report compares the results of this 1987 Radiation Therapy Technology Manpower Survey with the results of the three previous surveys conducted in 1975, 1977, and 1981. Whereas this comparison demonstrates that progress has been made, current trends in allied health education are expected to have a negative impact on recruitment into radiation therapy technology. Program directors report that the number and quality of applicants to all allied health education programs is declining, and this trend in radiation therapy technology is substantiated by a drop in the number of students enrolled in programs, from 970 in 1986-87 to 777 in 1987-88. Our discussion addresses the areas of new program development, recruitment, retention, innovative programs, and overseas recruitment.

Radiation therapy technology manpower needs 1982.

A shortage of radiation therapy technologists has existed in the United States for many years. This report analyzes the data from the third manpower survey of ACR/ASTR carried out in 1981 to 1982, using the Patterns of Care master facility list. Of 1106 questionnaires mailed, 77% were returned. The survey identified 3757 technologists performing radiation therapy technology duties. Of these, 2537 of these were Registered Technologists (Therapy) American Registry of Radiologic Technologists RTT (ARRT), 1220 were not. There has been a good growth in the total number of RTT (ARRT) members as indicated by the 1982 ARRT Annual Report (1148 in May, 1977, 2878 in May, 1982). Using the "Blue Book" Criteria of 1981 of 2 RTT/megavoltage unit or 2 RTT/300 new patients, the technology need (2900) would appear filled. However, 860 of the RTT were performing supervisory or dosimetry duties and 42% of the 2897 staff technologists were non RTT personnel. At the time of the survey, 597 funded vacancies existed (241 in 1977). A trend toward a changing standard of 3 RTT/megavoltage unit, reflecting the increased complexity of modern radiation therapy techniques, especially in Patterns of Care Strata A1, A2, C1 institutions was identified. While great progress has been made, there is a continuing need for recruitment into the 113 existing educational programs to try to stabilize the supply of technologists. Attention should also be given to measures for upgrading the skills and knowledge of the non RTT personnel in the field and retention of the RTT personnel.

Reliability of brightness comparison testing in predicting afferent pupillary defects.

We tested the reliability of brightness comparison testing in predicting relative afferent pupillary defects (RAPDs) in 206 patients. For unselected patients, brightness comparison testing correctly predicted the RAPD in 62% of patients. For subgroups of patients with glaucoma and optic nerve lesions, correct predictions were made in 92% and 83% of patients, respectively. Sensitivity and specificity of brightness comparison testing with a 6-V test light were 85% and 49%, respectively. In subgroups of patients who are strongly suspected of having an RAPD but in whom pupillary testing is not possible, brightness comparison testing can reliably predict presence or absence of an RAPD.

Pyoderma gangrenosum involving the eyelid.

A 62-year-old man was seen with an ulcer of the left upper eyelid of two weeks' duration. Over the previous 40 years, similar ulcers had intermittently developed elsewhere on his skin and spontaneously resolved over several months leaving atrophic scars. A biopsy specimen of the eyelid lesion showed epidermal ulceration with acute inflammation and liquefactive necrosis of the underlying dermis. Microorganisms and vasculitis were not present. On the basis of the clinical history and a compatible biopsy specimen, the diagnosis of pyoderma gangrenosum was made. This is an uncommon, idiopathic, ulcerating skin disease that may also have ocular manifestations. Eyelid ulcers have not previously been reported, to our knowledge. The diagnosis is established by clinical history and exclusion of other causes of ulceration.

A comparative survey of the results of analyses of blood serum in clinical chemistry laboratories in the United Kingdom.

Since July 1969, portions of the same blood serum have been dispatched to clinical chemistry laboratories in the United Kingdom at 14-day intervals. The results of each serum survey were reported to each of the 390 participants within 11 days of their originally receiving the specimen. During the first 18 months of the survey no overall improvement in the results was seen. Therefore a summary of each laboratory's ability consistently to produce results close to the mean of the method used was calculated and reported as a single figure, the variance index, and sent to all participants at regular intervals together with a histogram distribution of the variance indices of other participants. The subsequent improvement in the overall results is described.