Antibiotic resistance-the need for global solutions.

The causes of antibiotic resistance are complex and include human behaviour at many levels of society; the consequences affect everybody in the world. Similarities with climate change are evident. Many efforts have been made to describe the many different facets of antibiotic resistance and the interventions needed to meet the challenge. However, coordinated action is largely absent, especially at the political level, both nationally and internationally. Antibiotics paved the way for unprecedented medical and societal developments, and are today indispensible in all health systems. Achievements in modern medicine, such as major surgery, organ transplantation, treatment of preterm babies, and cancer chemotherapy, which we today take for granted, would not be possible without access to effective treatment for bacterial infections. Within just a few years, we might be faced with dire setbacks, medically, socially, and economically, unless real and unprecedented global coordinated actions are immediately taken. Here, we describe the global situation of antibiotic resistance, its major causes and consequences, and identify key areas in which action is urgently needed.

New business models for antibiotic innovation.

The increase in antibiotic resistance and the dearth of novel antibiotics have become a growing concern among policy-makers. A combination of financial, scientific, and regulatory challenges poses barriers to antibiotic innovation. However, each of these three challenges provides an opportunity to develop pathways for new business models to bring novel antibiotics to market. Pull-incentives that pay for the outputs of research and development (R&D) and push-incentives that pay for the inputs of R&D can be used to increase innovation for antibiotics. Financial incentives might be structured to promote delinkage of a company's return on investment from revenues of antibiotics. This delinkage strategy might not only increase innovation, but also reinforce rational use of antibiotics. Regulatory approval, however, should not and need not compromise safety and efficacy standards to bring antibiotics with novel mechanisms of action to market. Instead regulatory agencies could encourage development of companion diagnostics, test antibiotic combinations in parallel, and pool and make transparent clinical trial data to lower R&D costs. A tax on non-human use of antibiotics might also create a disincentive for non-therapeutic use of these drugs. Finally, the new business model for antibiotic innovation should apply the 3Rs strategy for encouraging collaborative approaches to R&D in innovating novel antibiotics: sharing resources, risks, and rewards.

Durability of antiretroviral therapy and predictors of virologic failure among perinatally HIV-infected children in Tanzania: a four-year follow-up.

BACKGROUND: In Tanzania, HIV-1 RNA testing is rarely available and not standard of care. Determining virologic failure is challenging and resistance mutations accumulate, thereby compromising second-line therapy. We evaluated durability of antiretroviral therapy (ART) and predictors of virologic failure among a pediatric cohort at four-year follow-up. METHODS: This was a prospective cross-sectional study with retrospective chart review evaluating a perinatally HIV-infected Tanzanian cohort enrolled in 2008-09 with repeat HIV-1 RNA in 2012-13. Demographic, clinical, and laboratory data were extracted from charts, resistance mutations from 2008-9 were analyzed, and prospective HIV RNA was obtained. RESULTS: 161 (78%) participants of the original cohort consented to repeat HIV RNA. The average age was 12.2 years (55% adolescents ≥12 years). Average time on ART was 6.4 years with 41% receiving second-line (protease inhibitor based) therapy. Among those originally suppressed on a first-line (non-nucleoside reverse transcriptase based regimen) 76% remained suppressed. Of those originally failing first-line, 88% were switched to second-line and 72% have suppressed virus. Increased level of viremia and duration of ART trended with an increased number of thymidine analogue mutations (TAMs). Increased TAMs increased the odds of virologic failure (p = 0.18), as did adolescent age (p < 0.01). CONCLUSIONS: After viral load testing in 2008-09 many participants switched to second-line therapy. The majority achieved virologic suppression despite multiple resistance mutations. Though virologic testing would likely hasten the switch to second-line among those failing, methods to improve adherence is critical to maximize durability of ART and improve virologic outcomes among youth in resource-limited settings.

CD4 enumeration technologies: a systematic review of test performance for determining eligibility for antiretroviral therapy.

BACKGROUND: Measurement of CD4+ T-lymphocytes (CD4) is a crucial parameter in the management of HIV patients, particularly in determining eligibility to initiate antiretroviral treatment (ART). A number of technologies exist for CD4 enumeration, with considerable variation in cost, complexity, and operational requirements. We conducted a systematic review of the performance of technologies for CD4 enumeration. METHODS AND FINDINGS: Studies were identified by searching electronic databases MEDLINE and EMBASE using a pre-defined search strategy. Data on test accuracy and precision included bias and limits of agreement with a reference standard, and misclassification probabilities around CD4 thresholds of 200 and 350 cells/μl over a clinically relevant range. The secondary outcome measure was test imprecision, expressed as % coefficient of variation. Thirty-two studies evaluating 15 CD4 technologies were included, of which less than half presented data on bias and misclassification compared to the same reference technology. At CD4 counts <350 cells/μl, bias ranged from -35.2 to +13.1 cells/μl while at counts >350 cells/μl, bias ranged from -70.7 to +47 cells/μl, compared to the BD FACSCount as a reference technology. Misclassification around the threshold of 350 cells/μl ranged from 1-29% for upward classification, resulting in under-treatment, and 7-68% for downward classification resulting in overtreatment. Less than half of these studies reported within laboratory precision or reproducibility of the CD4 values obtained. CONCLUSIONS: A wide range of bias and percent misclassification around treatment thresholds were reported on the CD4 enumeration technologies included in this review, with few studies reporting assay precision. The lack of standardised methodology on test evaluation, including the use of different reference standards, is a barrier to assessing relative assay performance and could hinder the introduction of new point-of-care assays in countries where they are most needed.

Mechanobiology of the meniscus.

The meniscus plays a critical biomechanical role in the knee, providing load support, joint stability, and congruity. Importantly, growing evidence indicates that the mechanobiologic response of meniscal cells plays a critical role in the physiologic, pathologic, and repair responses of the meniscus. Here we review experimental and theoretical studies that have begun to directly measure the biomechanical effects of joint loading on the meniscus under physiologic and pathologic conditions, showing that the menisci are exposed to high contact stresses, resulting in a complex and nonuniform stress-strain environment within the tissue. By combining microscale measurements of the mechanical properties of meniscal cells and their pericellular and extracellular matrix regions, theoretical and experimental models indicate that the cells in the meniscus are exposed to a complex and inhomogeneous environment of stress, strain, fluid pressure, fluid flow, and a variety of physicochemical factors. Studies across a range of culture systems from isolated cells to tissues have revealed that the biological response of meniscal cells is directly influenced by physical factors, such as tension, compression, and hydrostatic pressure. In addition, these studies have provided new insights into the mechanotransduction mechanisms by which physical signals are converted into metabolic or pro/anti-inflammatory responses. Taken together, these in vivo and in vitro studies show that mechanical factors play an important role in the health, degeneration, and regeneration of the meniscus. A more thorough understanding of the mechanobiologic responses of the meniscus will hopefully lead to therapeutic approaches to prevent degeneration and enhance repair of the meniscus.

Improving Indwelling Glucose Sensor Performance: Porous, Dexamethasone-Releasing Coatings that Modulate the Foreign Body Response

Inflammation and the formation of an avascular fibrous capsule have been identified as the key factors controlling the wound healing associated failure of implantable glucose sensors. Our aim is to guide advantageous tissue remodeling around implanted sensor leads by the temporal release of dexamethasone (Dex), a potent anti-inflammatory agent, in combination with the presentation of a stable textured surface.

First, Dex-releasing polyurethane porous coatings of controlled pore size and thickness were fabricated using salt-leaching/gas-foaming technique. Porosity, pore size, thickness, drug release kinetics, drug loading amount, and drug bioactivity were evaluated. In vitro sensor functionality test were performed to determine if Dex-releasing porous coatings interfered with sensor performance (increased signal attenuation and/or response times) compared to bare sensors. Drug release from coatings monitored over two weeks presented an initial fast release followed by a slower release. Total release from coatings was highly dependent on initial drug loading amount. Functional in vitro testing of glucose sensors deployed with porous coatings against glucose standards demonstrated that highly porous coatings minimally affected signal strength and response rate. Bioactivity of the released drug was determined by monitoring Dex-mediated, dose-dependent apoptosis of human peripheral blood derived monocytes in culture.

The tissue modifying effects of Dex-releasing porous coatings were accessed by fully implanting Tygon® tubing in the subcutaneous space of healthy and diabetic rats. Based on encouraging results from these studies, we deployed Dex-releasing porous coatings from the tips of functional sensors in both diabetic and healthy rats. We evaluated if the tissue modifying effects translated into accurate, maintainable and reliable sensor signals in the long-term. Sensor functionality was accessed by continuously monitoring glucose levels and performing acute glucose challenges at specified time points.

Sensors treated with porous Dex-releasing coatings showed diminished inflammation and enhanced vascularization of the tissue surrounding the implants in healthy rats. Functional sensors with Dex-releasing porous coatings showed enhanced sensor sensitivity over a 21-day period when compared to controls. Enhanced sensor sensitivity was accompanied with an increase in sensor signal lag and MARD score. These results indicated that Dex-loaded porous coatings were able to elicit a favorable tissue response, and that such tissue microenvironment could be conducive towards extending the performance window of glucose sensors in vivo.

The diabetic pilot animal study showed differences in wound healing patters between healthy and diabetic subjects. Diabetic rats showed lower levels of inflammation and vascularization of the tissue surrounding implants when compared to their healthy counterparts. Also, functional sensors treated with Dex-releasing porous coatings did not show enhanced sensor sensitivity over a 21-day period. Moreover, increased in sensor signal lag and MARD scores were present in porous coated sensors regardless of Dex-loading when compared to bare implants. These results suggest that the altered wound healing patterns presented in diabetic tissues may lead to premature sensor failure when compared to sensors implanted in healthy rats.

Interception of host angiogenic signalling limits mycobacterial growth.

Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimens are largely ineffective.

Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response.

Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.

The inoculum effect and band-pass bacterial response to periodic antibiotic treatment.

The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

Mental Health and Substance Use Among Patients in a North Carolina HIV Clinic.

BACKGROUND: The HIV/AIDS epidemic is a significant public health concern in North Carolina, and previous research has pointed to elevated mental health distress and substance use among HIV-infected populations, which may impact patients' adherence to medications. The aims of this study were to describe the prevalence of mental health and substance use issues among patients of a North Carolina HIV clinic, to examine differences by demographic characteristics, and to examine factors associated with suboptimal adherence to HIV medications. METHODS: This study was a secondary analysis of clinical data routinely collected through a health behavior questionnaire at a large HIV clinic in North Carolina. We analyzed data collected from February 2011 to August 2012. RESULTS: The sample included 1,398 patients. Overall, 12.2% of patients endorsed current symptomology indicative of moderate or severe levels of depression, and 38.6% reported receiving a psychiatric diagnosis at some point in their life. Additionally, 19.1% had indications of current problematic drinking, and 8.2% reported problematic drug use. Nearly one-quarter (22.1%) reported suboptimal adherence to HIV medications. Factors associated with poor adherence included racial/ethnic minority, age less than 35 years, and indications of moderate or severe depression. LIMITATIONS: The questionnaire was not completed systematically in the clinic, which may limit generalizability, and self-reported measures may have introduced social desirability bias. CONCLUSION: Patients were willing to disclose mental health distress, substance use, and suboptimal medication adherence to providers, which highlights the importance of routinely assessing these behaviors during clinic visits. Our findings suggest that treating depression may be an effective strategy to improve adherence to HIV medications.

Ribonucleic acid metabolism in unfertilized and fertilized sea-urchin eggs.

Journal Article

Comparative antioxidant, prooxidant and cytotoxic activity of sigmoidin A and eriodictyol

Sigmoidin A (SGN) is a prenylated flavanone derivative of eriodictyol (ERD) with reported moderate antioxidant, antimicrobial and anti-inflammatory activity. Since ERD and other structurally similar antioxidant phenolic compounds have been shown to induce prooxidative macromolecular damage and cytotoxicity in cancer cells, the comparative in vitro effects of these structural analogues on cancer cell viability and Cu(II)-dependent DNA damage were studied. In the presence of Cu(II) ions, both SGN and ERD (7.4-236 µM) caused comparable concentration-dependent pBR322 plasmid DNA strand scission. The DNA damage induced by SGN and ERD could be abolished by ROS scavengers, glutathione (GSH) and catalase as well as EDTA and a specific Cu(I) chelator neocuproine. Both ERD and SGN readily reduce Cu(II) to Cu(I) suggesting a prooxidative mechanism of DNA damage. In a cell free system, ERD and SGN did also show comparable radical scavenging activity. SGN was, however, by an order of magnitude more cytotoxic to cancer cells than ERD and this effect was significantly attenuated by GSH suggesting a prooxidative mechanism of cell death. A depletion of intracellular GSH level by SGN in cancer cells is also demonstrated.

Polyunsaturated fatty acids in the pathogenesis and treatment of multiple sclerosis

Epidemiological, biochemical, animal model and clinical trial data described in this overview strongly suggest that polyunsaturated fatty acids, particularly n-6 fatty acids, have a role in the pathogenesis and treatment of multiple sclerosis (MS). Data presented provides further evidence for a disturbance in n-6 fatty acid metabolism in MS. Disturbance of n-6 fatty acid metabolism and dysregulation of cytokines are shown to be linked and a "proof of concept clinical trial" further supports such a hypothesis. In a randomised double-blind, placebo controlled trial of a high dose and low dose selected GLA (18:3n-6)-rich oil and placebo control, the high dose had a marked clinical effect in relapsing-remitting MS, significantly decreasing the relapse rate and the progression of disease. Laboratory findings paralleled clinical changes in the placebo group in that production of mononuclear cell pro-inflammatory cytokines (TNF-alpha, IL-1 beta) was increased and anti-inflammatory TGF-beta markedly decreased with loss of membrane n-6 fatty acids linoleic (18:2n-6) and arachidonic acids (20:4n-6). In contrast there were no such changes in the high dose group. The improvement in disability (Expanded Disability Status Scale) in the high dose suggests there maybe a beneficial effect on neuronal lipids and neural function in MS. Thus disturbed n-6 fatty acid metabolism in MS gives rise to loss of membrane long chain n-6 fatty acids and loss of the anti-inflammatory regulatory cytokine TGF-beta, particularly during the relapse phase, as well as loss of these important neural fatty acids for CNS structure and function and consequent long term neurological deficit in MS.

Epilobium parviflorum Schreb. - in vitro study of biological action

Epilobium parviflorum Schreb. (Onagraceae) is used for the treatment of benign prostatic hyperplasia (BPH), which is regarded as an endocrine disorder caused by age-related hormone imbalance and increased oxidative damage [1,2,3]. Epilobium can moderate the obstructive and the irritative symptoms of BPH [1] but its biological action is not entirely identified. E. parviflorum is rich in phytosterols, flavonoids (myricetin, quercetin, kaempferol and their glycosides), phenolic acids, catechins, ellagi- and gallotannins [4]. The potential biological effects of Epilobium parviflorum Schreb. have been investigated, in respect to its antioxidant, anti-inflammatory, enzyme-inhibitory and anti-androgenic effect. The whole-plant water extract showed higher antioxidant effect (IC50=1.65±0.05µg/mL) in DPPH assay than Trolox or ascorbic acid and inhibited the lipid peroxidation examined in TBA assay (IC50=2.31±0.18mg/mL). In concentrations 0.20-15.00µg/mL the extract possessed a protective effect comparable to catalase enzyme (2500 IU/mL), against oxidative damage generated on fibroblast cells. The examination of the COX-inhibitory effect showed that E. parviflorum had an anti-inflammatory effect (IC50=1.38±0.08µg/mL). Investigation of steroid receptor binding ability and the aromatase enzyme-inhibition showed negative results in the concentration range examined.