Pilot study of a community pharmacist led program to treat hepatitis C virus among people who inject drugs.

Background: People who inject drugs (PWID) are a key population for treatment with direct-acting antiviral medications (DAAs) to eliminate hepatitis C virus (HCV). We developed a Pharmacist, Physician, and Patient Navigator Collaborative Care Model (PPP-CCM) for delivery of HCV treatment; this study describes clinical outcomes related to HCV treatment (initial evaluation, treatment initiation, completion, and cure), as well as patient satisfaction. Methods: We conducted a single-arm prospective pilot study of adult PWID living with HCV. Participants completed baseline and six-month follow-up surveys, and treatment and outcomes were abstracted from electronic health records. Primary outcome was linkage to pharmacist for HCV evaluation; secondary outcomes included DAA initiation, completion, and cure, as well as patient-reported satisfaction. Results: Of the 40 PWID enrolled, mean age was 43.6 years, 12 (30 %) were female, 20 (50 %) were non-white, and 15 (38 %) were unhoused. Thirty-eight (95 %) were successfully linked to the pharmacist for initial evaluation. Of those, 21/38 (55 %) initiated DAAs, and 16/21 (76 %) completed treatment. Among those completing treatment who had viral load data to document whether they achieved "sustained virologic response", i.e. cure, 10/11 (91 %) were found to be cured. There was high satisfaction with 100 % responding "agree or strongly agree" that they had a positive experience with the pharmacist. Conclusion: Nearly all participants in this pilot were successfully linked to the pharmacist for evaluation, and more than half were started on DAAs; results provide preliminary evidence of feasibility of pharmacist-led models of HCV treatment for PWID. Clinicaltrialsgov registration number: NCT04698629.

Ketone body ß-hydroxybutyrate (BHB) preserves mitochondrial bioenergetics.

The ketogenic diet is an emerging therapeutic approach for refractory epilepsy, as well as certain rare and neurodegenerative disorders. The main ketone body, ß-hydroxybutyrate (BHB), is the primary energy substrate endogenously produced in a ketogenic diet, however, mechanisms of its therapeutic actions remain unknown. Here, we studied the effects of BHB on mitochondrial energetics, both in non-stimulated conditions and during glutamate-mediated hyperexcitation. We found that glutamate-induced hyperexcitation stimulated mitochondrial respiration in cultured cortical neurons, and that this response was greater in cultures supplemented with BHB than with glucose. BHB enabled a stronger and more sustained maximal uncoupled respiration, indicating that BHB enables neurons to respond more efficiently to increased energy demands such as induced during hyperexcitation. We found that cytosolic Ca2+ was required for BHB-mediated enhancement of mitochondrial function, and that this enhancement was independent of the mitochondrial glutamate-aspartate carrier, Aralar/AGC1. Our results suggest that BHB exerts its protective effects against hyperexcitation by enhancing mitochondrial function through a Ca2+-dependent, but Aralar/AGC1-independent stimulation of mitochondrial respiration.

The TWEAK receptor Fn14 is a potential cell surface portal for targeted delivery of glioblastoma therapeutics.

UNLABELLED: Fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is the cell surface receptor for the tumor necrosis factor (TNF) family member TNF-like weak inducer of apoptosis (TWEAK). The Fn14 gene is normally expressed at low levels in healthy tissues but expression is significantly increased after tissue injury and in many solid tumor types, including glioblastoma (GB; formerly referred to as 'GB multiforme'). GB is the most common and aggressive primary malignant brain tumor and the current standard-of-care therapeutic regimen has a relatively small impact on patient survival, primarily because glioma cells have an inherent propensity to invade into normal brain parenchyma, which invariably leads to tumor recurrence and patient death. Despite major, concerted efforts to find new treatments, a new GB therapeutic that improves survival has not been introduced since 2005. In this review article, we summarize studies indicating that (i) Fn14 gene expression is low in normal brain tissue but is upregulated in advanced brain cancers and, in particular, in GB tumors exhibiting the mesenchymal molecular subtype; (ii) Fn14 expression can be detected in glioma cells residing in both the tumor core and invasive rim regions, with the maximal levels found in the invading glioma cells located within normal brain tissue; and (iii) TWEAK: Fn14 engagement as well as Fn14 overexpression can stimulate glioma cell migration, invasion and resistance to chemotherapeutic agents in vitro. We also discuss two new therapeutic platforms that are currently in development that leverage Fn14 overexpression in GB tumors as a way to deliver cytotoxic agents to the glioma cells remaining after surgical resection while sparing normal healthy brain cells.

Nanostructuring of Palladium with Low-Temperature Helium Plasma.

Impingement of high fluxes of helium ions upon metals at elevated temperatures has given rise to the growth of nanostructured layers on the surface of several metals, such as tungsten and molybdenum. These nanostructured layers grow from the bulk material and have greatly increased surface area over that of a not nanostructured surface. They are also superior to deposited nanostructures due to a lack of worries over adhesion and differences in material properties. Several palladium samples of varying thickness were biased and exposed to a helium helicon plasma. The nanostructures were characterized as a function of the thickness of the palladium layer and of temperature. Bubbles of ~100 nm in diameter appear to be integral to the nanostructuring process. Nanostructured palladium is also shown to have better catalytic activity than not nanostructured palladium.

Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate.

Cerebral ischemia and excitotoxic injury induce transient or permanent bioenergetic failure, and may result in neuronal apoptosis or necrosis. We have previously shown that ATP depletion and activation of AMP-activated protein kinase (AMPK) during excitotoxic injury induces neuronal apoptosis by transcription of the pro-apoptotic BH3-only protein, Bim. AMPK, however, also exerts pro-survival functions in neurons. The molecular switches that determine these differential outcomes are not well understood. Using an approach combining biochemistry, single-cell imaging and computational modeling, we here demonstrate that excitotoxic injury activated the bim promoter in a FOXO3-dependent manner. The activation of AMPK reduced AKT activation, and led to dephosphorylation and nuclear translocation of FOXO3. Subsequent mutation studies indicated that bim gene activation during excitotoxic injury required direct FOXO3 phosphorylation by AMPK in the nucleus as a second activation step. Inhibition of this phosphorylation prevented Bim expression and protected neurons against excitotoxic and oxygen/glucose deprivation-induced injury. Systems analysis and computational modeling revealed that these two activation steps defined a coherent feed-forward loop; a network motif capable of filtering any effects of short-term AMPK activation on bim gene induction. This may prevent unwanted AMPK-mediated Bim expression and apoptosis during transient or physiological bioenergetic stress.

A phase II study of a 5T4 oncofoetal antigen tumour-targeted superantigen (ABR-214936) therapy in patients with advanced renal cell carcinoma.

In a phase II study, 43 renal cell carcinoma patients were treated with individualised doses of ABR-214936; a fusion of a Fab recognising the antigen 5T4, and Staphylococcal enterotoxin A. Drug was given intravenously on 4 consecutive days, treatment was repeated 1 month later. Treatment was associated with moderate fever and nausea, but well tolerated. Of 40 evaluable patients, 28 had disease control at 2 months, and at 4 months, one patient showed partial response (PR) and 16 patients stable disease. Median survival, with minimum follow-up of 26 months was 19.7 months with 13 patients alive to date. Stratification by the Motzer's prognostic criteria highlights prolonged survival compared to published expectation. Patients receiving higher drug exposure had greater disease control and lived almost twice as long as expected, whereas the low-exposure patients survived as expected. Sustained interleukin-2 (IL-2) production after a repeated injection appears to be a biomarker for clinical effect, as the induced-IL-2 level on the day 2 of treatment correlated with survival. The high degree of disease control and the prolonged survival suggest that this treatment can be effective. These findings will be used in the trial design for the next generation of drug, with reduced antigenicity and toxicity.

In vitro and in vivo protein sampling by combined microdialysis and ultrafiltration.

Cytokines, chemokines and growth factors regulate inflammation, resistance to infection and tissue repair. Understanding their function within tissues is a priority in evolving therapy for a number of disease processes. Yet, the existence of complex networks of these factors in the tissue microenvironment has made understanding of their interactions difficult. We demonstrate the capability of microdialysis probes to recover small proteins efficiently in vitro. Further we show that microdialysis of human tissues allows for protein recovery from tissue interstitial fluid. This technology, combined with a multiplexed immunoassay, facilitates the simultaneous measurement of cytokines and chemokines in response to injury in the oral mucosa of human subjects in vivo.

Quantitation of monoclonal plasma cells in bone marrow biopsies in plasma cell dyscrasia.

Direct measurement of monoclonal plasma cell mass in bone marrow biopsies may be a useful parameter to establish in plasma cell dyscrasia. In this study monoclonal plasma cells/mm in light chain immunoglobulin immunostained archival bone marrow sections from 22 patients in whom a diagnosis of multiple myeloma (MM) had been excluded but who had monoclonal proteins were counted by two observers at light microscopic level. There was good correlation between the counts of the two observers. The levels of monoclonal plasma cells/mm in biopsies were not related to the % counts in the aspirates taken at the same time as the biopsies. Three of seven patients with biopsy levels in excess of the polyclonal levels in patients without plasma cell dyscrasia developed progressive MM within the observation time. Monoclonal plasma cell levels/mm of bone marrow biopsies can be measured and they provide a useful parameter for the assessment of patients with low volume plasma cell dyscrasia.

Magnitude of the inflammatory response to cardiopulmonary bypass and its relation to adverse clinical outcomes.

INTRODUCTION: Cardiopulmonary bypass (CPB) induces an inflammatory response believed to contribute to postoperative morbidity. We hypothesized that the magnitude of the inflammatory response following CPB would be associated with adverse clinical outcomes. METHODS: Twenty-nine patients had plasma TNF, IL-6, IL-8, elastase, histamine, complement C5a, and complement C3a measured by ELISA before, during, and after cardiac operations employing CPB. Inflammatory mediator levels were analyzed with respect to outcomes. RESULTS: Mediator levels peaked at 4 h post-CPB and either returned to baseline or substantially decreased by 24 h. Patients with peak mediator levels above the median for the group as a whole were classified as 'hyper-responders'; those with levels below the median were classified as 'normal responders'. While IL-8, C3a, and IL-6 levels were independently associated with adverse outcomes, TNF, histamine, and C5a levels were not. Elastase levels trended towards adverse outcomes. IL-8 'hyper-responders' experienced significantly greater postoperative weight gain and had higher IL-8 levels at 24 h (p<0.05), with trends towards renal impairment and protracted supplemental oxygen requirements. C3a 'hyper-responders' strongly trended towards increased bleeding, delayed extubation, greater postoperative weight gain, and decreased levels of independent functioning at discharge (p < or = 0.10). IL-6 'hyper-responders' experienced significantly more postoperative bleeding, delayed extubation, and higher IL-6 levels at 24 h compared to 'normal responders' (p < 0.05). They strongly trended towards greater postoperative weight gain and decreased levels of independent functioning at discharge (p < or = 0.10). CONCLUSIONS: Patients who have an exaggerated inflammatory response to CPB tend to bleed more, require more respiratory support, demonstrate greater capillary leak via weight gain, and display a decline in independent functioning relative to normal responders. Thus, it appears that the magnitude of the inflammatory response to CPB adversely influences clinical outcomes.

Phlebotomy overdraw in the neonatal intensive care nursery.

OBJECTIVE: Because blood loss attributable to laboratory testing is the primary cause of anemia among preterm infants during the first weeks of life, we quantified blood lost attributable to phlebotomy overdraw, ie, excess that might be avoided. We hypothesized that phlebotomy overdraw in excess of that requested by the hospital laboratory was a common occurrence, that clinical factors associated with excessive phlebotomy loss would be identified, and that some of these factors are potentially correctable. DESIGN, OUTCOME MEASURES, AND ANALYSIS: Blood samples drawn for clinical purposes from neonates cared for in our 2 neonatal special care units were weighed, and selected clinical data were recorded. The latter included the test performed; the blood collection container used; the infant's location (ie, neonatal intensive care unit [NICU] and intermediate intensive care unit); the infant's weight at sampling; and the phlebotomist's level of experience, work shift, and clinical role. Data were analyzed by univariate and multivariate procedures. Phlebotomists included laboratory technicians stationed in the neonatal satellite laboratory, phlebotomists assigned to the hospital's central laboratory, and neonatal staff nurses. Phlebotomists were considered experienced if they had worked in the nursery setting for >1 year. Blood was sampled from a venous or arterial catheter or by capillary stick from a finger or heel. Blood collection containers were classified as tubes with marked fill-lines imprinted on the outside wall, tubes without fill-lines, and syringes. Infants were classified by weight into 3 groups: <1 kg, 1 to 2 kg, and >2 kg. The volume of blood removed was calculated by subtracting the weight of the empty collection container from that of the container filled with blood and dividing by the specific gravity of blood, ie, 1.050 g/mL. The volume of blood withdrawn for individual laboratory tests was expressed as a percentage of the volume requested by the hospital laboratory. RESULTS: The mean (+/- standard error of the mean) volume of blood drawn for the 578 tests drawn exceeded that requested by the hospital laboratory by 19.0% +/- 1.8% per test. The clinical factors identified as being significantly associated with greater phlebotomy overdraw in the multiple regression model included: 1) collection in blood containers without fill-lines; 2) lighter weight infants; and 3) critically ill infants being cared for in the NICU. Because the overall R(2) of the multiple regression for these 3 clinical factors was only.24, the random factor of individual phlebotomist was added to the model. This model showed that there was a significant variation in blood overdraw among individual phlebotomists, and as a result, the overall R(2) increased to.52. An additional subset analysis involving 2 of the 3 groups of blood drawers (ie, hospital and neonatal laboratory phlebotomists) examining the effect of work shift, demonstrated that there was significantly greater overdraw for blood samples obtained during the evening shift, compared with the day shift when drawn using unmarked tubes for the group of heavier infants cared for in the NICU. CONCLUSION: Significant volumes of blood loss are attributable to overdraw for laboratory testing. This occurrence likely exacerbates the anemia of prematurity and may increase the need for transfusions in some infants. Attempts should be made to correct the factors involved. Common sense suggests that blood samples drawn in tubes with fill-lines marked on the outside would more closely approximate the volumes requested than those without. Conversely, the use of unmarked tubes could lead to phlebotomy overdraw because phlebotomists may overcompensate to avoid having to redraw the sample because of an insufficient volume for analysis. We were surprised to observe that the lightest and most critically ill infants experienced the greatest blood overdraw. (ABSTRACT TRUNCATED)

Neonatal laboratory blood sampling: comparison of results from arterial catheters with those from an automated capillary device.

PURPOSE: To compare neonatal laboratory results from capillary blood samples drawn using the Tenderfoot automated capillary sampling device with those drawn through arterial catheters. DESIGN: Prospective, paired comparisons of laboratory results from capillary and arterial blood. SAMPLE: Twenty-one infants being cared for in an NICU and having indwelling arterial catheters through which a variety of predominantly glucose-containing fluids were being administered. MAIN OUTCOME VARIABLES: Statistical comparisons of paired capillary and arterial results of pH, PO2, PCO2, lactate, sodium, potassium, ionized calcium, and hematocrit. RESULTS: No capillary-arterial differences were observed for pH, PCO2, lactate, or sodium. Although capillary results were slightly, but significantly (p < .01), higher for potassium (+0.4 mEq/liter), ionized calcium (+0.47 mg/dl), and hematocrit (+4 percent), these differences fell within acceptable Clinical Laboratories Improvement Act (CLIA) performance criteria. Markedly lower PO2 (-30.2 mmHg) and glucose (-61 mg/dl) values were observed with capillary sampling. With the exception of results for PaO2 and plasma glucose, capillary blood drawn using the Tenderfoot automated device yields laboratory results comparable to those from blood drawn from arterial catheters as assessed by CLIA performance criteria.

Audit of asthma care in Army general practice.

Asthma is a common condition. General Practitioners in the NHS now receive specific remuneration for running asthma disease management programmes. This paper presents the results of an audit of asthma management in an Army General Practice. The records of all patients were reviewed. In addition a questionnaire survey of patient knowledge and satisfaction was undertaken. The study identified several areas for improvement.

Properties of protein kinase C isoforms (beta II, epsilon, and zeta) in a macrophage cell line (J774) and their roles in LPS-induced nitric oxide production.

Northern analysis of poly(A)+ RNA extracted from J774 cells (a mouse macrophage cell line) showed that this cell line constitutively expresses mRNAs specific for protein kinase C (PKC)-beta I, -beta II, -epsilon and -zeta, but not those for PKC-alpha, -gamma or -delta. Western analysis of the total cell lysate showed that J774 cells express PKC-beta II, -epsilon and -zeta isoenzymes, but failed to show the expression of PKC-beta I. The exposure of J774 cells to > 10 nM PMA led to a loss of immunoreactive PKC-beta II in 4 h. The down-regulation of immunoreactive PKC-epsilon required more than 8 h of the exposure to > 100 nM PMA. Immunoreactive PKC-zeta was most resistant to PMA treatment and was not significantly reduced after the exposure to 300 to 600 nM PMA for 24 h. PMA-mediated, persistent down-regulation of PKC-beta II is probably a result of the inhibition of PKC-beta II biosynthesis at the posttranscriptional level, because PMA-exposed cells were found to gradually increase the expression of PKC-beta II specific mRNA. PMA-pretreated cells responded to a low dose (10 ng/ml), but not to a high dose (1 microgram/ml), of LPS by significantly lower expression of mRNA specific for the inducible nitric oxide synthase (i-NOS) gene and production of nitric oxide (NO) than the control cells did. Thus, PKC could be a part of the signal transduction apparatus involved in LPS-induced inducible nitric oxide synthase gene activation.

What practicing physicians in North Carolina rate as their most challenging geriatric medicine concerns.

We recently surveyed a random sample of 500 physicians in family practice, general practice, and internal medicine in North Carolina, to discover their most challenging geriatric concerns. Using a three-stage survey technique, respondents were asked to answer two open-ended questions about the most challenging geriatric problems they face and what specific geriatric content areas would attract their participation in an educational program. They were then asked to rank 34 topics on which they would like more information. A total of 242 responses were received for a 55% response rate (63 of the 500 were undeliverable). Responses indicated that physicians are more concerned with management than diagnosis and revealed considerable evidence of empathy and concern. The top three topics had to do with the management of dementia, multiple problems, and depression. Approximately 25% of physicians consider problems of financing health care as among the most challenging problems.

Developmental regulation of the human cathepsin G gene in myelomonocytic cells.

Cathepsin G is a neutral serine protease that is found in the azurophil granules of neutrophils and monocytes. Previous experiments had demonstrated that cathepsin G is actively produced by the promonocytic U937 cell line, and that 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of these cells toward macrophages resulted in a reduction of cathepsin G activity. In this study, we have analyzed the mechanism of this TPA-induced down-regulatory event. Using in situ hybridization techniques, we demonstrated that cathepsin G mRNA is detectable only at the promyelocytic stage of myeloid development. Using U937 promonocytic cells as a model, we demonstrated; 1) cathepsin G protein levels decline in TPA-treated cells; 2) this decline was due to a nearly complete loss of cathepsin G mRNA in cells treated with TPA for 24 h; and 3) the rate of cathepsin G mRNA loss with TPA treatment was similar to that with actinomycin D. These results suggested that cathepsin G transcription was down-regulated within several hours of TPA addition. This was directly tested by performing nuclear run-off assays of TPA-treated U937 cells; cathepsin G transcription was shown to be strand-specific, and declined within 4 h of TPA addition. Cathepsin G transcription was essentially undetectable 8 or more hours after TPA treatment, suggesting that down-regulation is predominantly transcriptional. Cycloheximide treatment of U937 cells resulted in a partial block of TPA-mediated cathepsin G down-regulation, indicating that continuous protein synthesis is required for down-regulation to occur. A newly synthesized protein or proteins may therefore be required for the transcriptional down-regulation of cathepsin G during the normal development of promyelocytes or promonocytes.

Interferon-gamma down-regulates membrane adenylate cyclase activity of a murine macrophage-like cell line (P388D1).

Effects of recombinant murine interferon-gamma (rIFN-gamma) on the membrane adenylate cyclase of a murine macrophage cell line (P388D1) were investigated in order to explore the nature of a signal transmitted by IFN-gamma receptor. Following the incubation of P388D1 cells with 40 U/ml of rIFN-gamma, the intracellular level of cAMP gradually increased about twofold over the control level within 60 min, and then began to gradually decline to about half the control level by 24 h incubation. The initial rise in cAMP level appeared to be due to the modest activation of adenylate cyclase and not due to the inhibition of cAMP-phosphodiesterase. Later decrease of intracellular cAMP may be due to quantitative down-regulation of the adenylate cyclase system. The basal enzymatic activity of the membrane prepared from P388D1 cells exposed to IFN-gamma for 24 h was found to be reduced to about 20% of that of the control membrane. However, the quality of the adenylate cyclase system appeared unchanged, because the relative degree of the response of the down-regulated membrane adenylate cyclase to prostaglandin PGE2, NaF, guanylimidodiphosphate (GppNHp), choleara toxin (CT), or forskolin was found to remain unchanged. This quantitative down-regulation of adenylate cyclase must be due to the action of rIFN-gamma, since the prior treatment of rIFN-gamma with either acid (pH 2) or monoclonal anti-IFN-gamma antibody inhibited the ability of IFN-gamma to induce the down-regulation. The rIFN-gamma-induced down-regulation is a reversible process, since the adenylate cyclase activity of the membrane was found to be restored when the rIFN-gamma-exposed cells were cultured for 72 h in the absence of rIFN-gamma. In addition, the 48 h-incubation of P388D1 cells with rIFN-beta or IFN-alpha was found not to significantly affect the membrane adenylate cyclase system.

Elastin degradation by human alveolar macrophages. A prominent role of metalloproteinase activity.

Macrophages are thought to play an important role in the turnover of extracellular matrix, but the capacity of human macrophages to degrade elastin, and the elastolytic mechanisms of these cells, have been controversial. Particular difficulty has been encountered in efforts to establish whether human macrophages secrete a metalloelastase that is analogous to the enzyme secreted by rodent macrophages. We studied elastin degradation by human alveolar macrophages cultured directly in contact with radiolabeled elastin using media containing 10% fetal bovine serum, and for comparison performed parallel studies of P388D1 murine macrophagelike cells that are known to secrete metalloelastase. With both cell types, we observed elastin degradation and the following: (1) direct contact between the cells and elastin substrate was required for elastin degradation; (2) elastin degradation was inhibited by the tissue inhibitor of metalloproteinases, but minimally or not at all by inhibitors of cysteine proteinases (E-64, CBZ-phe-phe-CHN2, CBZ-phe-ala-CHN2, and cystatin C), or by the serine proteinase inhibitor eglin-c; (3) elastin degradation increased sharply after the cells were in contact with elastin for 24 h, and required new protein synthesis as indicated by sensitivity to cycloheximide; (4) inclusion of dexamethasone (10(-6) to 10(-8) M) in the cultures led to decreased elastin degradation. Also, with both cell types, elastin degradation occurred despite the finding that cell-conditioned media did not contain elastase activity and could inhibit P388D1-derived metalloproteinase elastase. These results indicate a prominent role for metalloproteinase activity in elastin degradation by both human and murine macrophages and support the concept that events at the cell-substrate interface are critically important to macrophage-mediated elastin degradation.