Improving the integrity and reproducibility of research that uses antibodies: a technical, data sharing, behavioral and policy challenge.

Antibodies are one of the most important reagents used in biomedical and fundamental research, used to identify, and quantify proteins, contribute to knowledge of disease mechanisms, and validate drug targets. Yet many antibodies used in research do not recognize their intended target, or recognize additional molecules, compromising the integrity of research findings and leading to waste of resources, lack of reproducibility, failure of research projects, and delays in drug development. Researchers frequently use antibodies without confirming that they perform as intended in their application of interest. Here we argue that the determinants of end-user antibody choice and use are critical, and under-addressed, behavioral drivers of this problem. This interacts with the batch-to-batch variability of these biological reagents, and the paucity of available characterization data for most antibodies, making it more difficult for researchers to choose high quality reagents and perform necessary validation experiments. The open-science company YCharOS works with major antibody manufacturers and knockout cell line producers to characterize antibodies, identifying high-performing renewable antibodies for many targets in neuroscience. This shows the progress that can be made by stakeholders working together. However, their work so far applies to only a tiny fraction of available antibodies. Where characterization data exists, end-users need help to find and use it appropriately. While progress has been made in the context of technical solutions and antibody characterization, we argue that initiatives to make best practice behaviors by researchers more feasible, easy, and rewarding are needed. Global cooperation and coordination between multiple partners and stakeholders will be crucial to address the technical, policy, behavioral, and open data sharing challenges. We offer potential solutions by describing our Only Good Antibodies initiative, a community of researchers and partner organizations working toward the necessary change. We conclude with an open invitation for stakeholders, including researchers, to join our cause.

Validation of antibodies for the specific detection of human TRPA1.

The transient receptor potential cation channel family member ankyrin 1 (TRPA1) is a potential target for several diseases, but detection of human TRPA1 (hTRPA1) protein in cells and tissues is problematic as rigorous antibody validation is lacking. We expressed hTRPA1 in a TRPA1-negative cell line to evaluate 5 commercially available antibodies by western blotting, immunofluorescence, immunocytochemistry and flow cytometry. The three most cited anti-TRPA1 antibodies lacked sensitivity and/or specificity, but two mouse monoclonal anti-TRPA1 antibodies detected hTRPA1 specifically in the above assays. This enabled the development of a flow cytometry assay, which demonstrated strong expression of TRPA1 in human lung myofibroblasts, human airway smooth muscle cells but not lung mast cells. The most cited anti-TRPA1 antibodies lack sensitivity and/or specificity for hTRPA1. We have identified two anti-TRPA1 antibodies which detect hTRPA1 specifically. Previously published data regarding human TRPA1 protein expression may need revisiting.

General practice referral of 'at risk' populations to community leisure services: applying the RE-AIM framework to evaluate the impact of a community-based physical activity programme for inactive adults with long-term conditions.

BACKGROUND: In the UK a high proportion of adults with long-term conditions do not engage in regular physical activity. General practice (GP) referral to community-based physical activity is one strategy that has gained traction in recent years. However, evidence for the real-world effectiveness and translation of such programmes is limited. This study aimed to evaluate the individual and organisational impacts of the 'CLICK into Activity' programme - GP referral of inactive adults living with (or at risk of) long-term conditions to community-based physical activity. METHODS: A mixed methods evaluation using the RE-AIM framework was conducted with data obtained from a range of sources: follow-up questionnaires, qualitative interviews, and programme-related documentation, including programme cost data. Triangulation methods were used to analyse data, with findings synthesised across each dimension of the RE-AIM framework. RESULTS: A total of 602 individuals were referred to CLICK into Activity physical activity sessions. Of those referred, 326 individuals participated in at least one session; the programme therefore reached 30.2% of the 1080 recruitment target. A range of individual-, social-, and environmental-level factors contributed to initial physical activity participation. Positive changes over time in physical activity and other outcomes assessed were observed among participants. Programme adoption at GP surgeries was successful, but the GP referral process was not consistently implemented across sites. Physical activity sessions were successfully implemented, with programme deliverers and group-based delivery identified as having an influential effect on programme outcomes. Changes to physical activity session content were made in response to participant feedback. CLICK into Activity cost £175,000 over 3 years, with an average cost per person attending at least one programme session of £535. CONCLUSIONS: Despite not reaching its recruitment target, CLICK into Activity was successfully adopted. Positive outcomes were associated with participation, although low 6- and 12-month follow-up response rates limit understanding of longer-term programme effects. Contextual and individual factors, which may facilitate successful implementation with the target population, were identified. Findings highlight strategies to be explored in future development and implementation of GP referral to community-based physical activity programmes targeting inactive adults living with (or at risk of) long-term conditions.

Increased ß2-adrenoceptor phosphorylation in airway smooth muscle in severe asthma: possible role of mast cell-derived growth factors.

The purpose of this study was to investigate whether growth factors produced by activated human lung mast cells (HLMCs) impair ß2 -adrenoceptor (ß2 -AR) function in human airway smooth muscle (ASM) cells. Protein array analysis confirmed the presence of various growth factors, including transforming growth factor (TGF)-ß1, in the supernatants of high-affinity IgE receptor (FcεRI)-activated HLMCs which, when applied to ASM cells, impaired albuterol-induced cyclic adenosine monophosphate (cAMP) production, an effect that was prevented following neutralization of TGF-ß1. This blunted ß2 -AR response was reproduced by treating ASM cells with TGF-ß1 or fibroblast growth factor (FGF)-2, which induced ß2 -AR phosphorylation at tyrosine residues Tyr141 and Tyr350 , and significantly reduced the maximal bronchorelaxant responses to isoproterenol in human precision cut lung slices (PCLS). Finally, ASM cells isolated from severe asthmatics displayed constitutive elevated ß2 -AR phosphorylation at both Tyr141 and Tyr350 and a reduced relaxant response to albuterol. This study shows for the first time that abnormal ß2 -AR phosphorylation/function in ASM cells that is induced rapidly by HLMC-derived growth factors, is present constitutively in cells from severe asthmatics.

Clinical, Virologic, Immunologic Outcomes and Emerging HIV Drug Resistance Patterns in Children and Adolescents in Public ART Care in Zimbabwe.

OBJECTIVE: To determine immunologic, virologic outcomes and drug resistance among children and adolescents receiving care during routine programmatic implementation in a low-income country. METHODS: A cross-sectional evaluation with collection of clinical and laboratory data for children (0-<10 years) and adolescents (10-19 years) attending a public ART program in Harare providing care for pediatric patients since 2004, was conducted. Longitudinal data for each participant was obtained from the clinic based medical record. RESULTS: Data from 599 children and adolescents was evaluated. The participants presented to care with low CD4 cell count and CD4%, median baseline CD4% was lower in adolescents compared with children (11.0% vs. 15.0%, p<0.0001). The median age at ART initiation was 8.0 years (IQR 3.0, 12.0); median time on ART was 2.9 years (IQR 1.7, 4.5). On ART, median CD4% improved for all age groups but remained below 25%. Older age (≥ 5 years) at ART initiation was associated with severe stunting (HAZ <-2: 53.3% vs. 28.4%, p<0.0001). Virologic failure rate was 30.6% and associated with age at ART initiation. In children, nevirapine based ART regimen was associated with a 3-fold increased risk of failure (AOR: 3.5; 95% CI: 1.3, 9.1, p = 0.0180). Children (<10 y) on ART for ≥4 years had higher failure rates than those on ART for <4 years (39.6% vs. 23.9%, p = 0.0239). In those initiating ART as adolescents, each additional year in age above 10 years at the time of ART initiation (AOR 0.4 95%CI: 0.1, 0.9, p = 0.0324), and each additional year on ART (AOR 0.4, 95%CI 0.2, 0.9, p = 0.0379) were associated with decreased risk of virologic failure. Drug resistance was evident in 67.6% of sequenced virus isolates. CONCLUSIONS: During routine programmatic implementation of HIV care for children and adolescents, delayed age at ART initiation has long-term implications on immunologic recovery, growth and virologic outcomes.

Nurse-led clinic: effective and efficient delivery of assessment and review of patients with hepatitis B and C.

BACKGROUND: Hepatology and gastroenterology services are increasingly utilising the skills and experience of nurse practitioners and nurse specialists to help meet the increasing demand for healthcare. A new nurse-led assessment clinic has been established in the liver clinic at Geelong Hospital to utilise the expertise of nurses to assess and triage new patients and streamline their pathway through the healthcare system. AIM: The aim of this study is to assess quantitatively the first 2 years of operation of the nurse assessment clinic at Geelong Hospital, and to assess advantages and disadvantages of the nurse-led clinic. METHODS: Data were extracted retrospectively from clinical records of new patients at the liver clinic. Quarterly 1-month periods were recorded over 2 years. Patients were categorised according to the path through which they saw a physician, including missed and rescheduled appointments. The number of appointments, the waiting time from referral to appointments and the number of 'did-not-attend' occasions were analysed before and after the institution of the nurse-led assessment clinic. The Mann-Whitney rank sum test of ordinal data was used to generate median wait times. RESULTS: There was shown to be a statistically significant longer waiting time for physician appointment if seen by the nurse first. The difference in waiting time was 10 days. However, there was also a reduction in the number of missed appointments at the subsequent physician clinic. Other advantages have also been identified, including effective triage of patients and organisation of appropriate investigations from the initial nurse assessment.

Effects of storage time and thawing methods on the recovery of Mycoplasma species in milk samples from cows with intramammary infections.

This study was executed to determine the effects of storage and thawing on the viability of Mycoplasma spp. in milk from cows with intramammary infections. The trial was designed using a control sample and seven handling regimens subjected to two methods of thawing. There was a significant treatment effect on the recovery of colony-forming units in milk samples when comparing the control sample with handling regimens 1 through 7. There was a continuous decline in log (10) mean number of cfu/mL recovered. Mean concentrations were 6.29, 4.64, 3.69, 3.01, 1.86, 4.41, 4.13, and 3.18 for control and handling regimens 1 to 7, respectively. To determine the best thawing method, handling regimen 1 through 7 samples were thawed using two methods. On average, more mycoplasma were recovered from milk samples thawed at ambient temperature (4.04 cfu/mL) than milk samples thawed in a 37 degrees C water bath (3.76 cfu/mL). A final comparison was made between individual treatments. With the exception of the handling regimen 5 to 6 pair-wise comparison, all pair-wise comparisons between handling regimens were significantly different. The results of this study indicate that storage and thawing of milk samples is harmful to mycoplasma organisms. Fresh samples should be used to improve detection of Mycoplasma spp. from milk of infected cattle. If frozen samples are used, then length of storage time should be minimized, and thawing milk at ambient temperature will improve recovery of mycoplasma as opposed to thawing in a 37 degrees C water bath.

Changes in dialysis units' quality improvement practices from 1994 to 1996.

In 1994, the Health Care Financing Administration initiated a nationwide effort to improve care to Medicare's end stage renal disease (ESRD) beneficiaries by reshaping the manner in which the ESRD Network Organizations measure and assess the quality of dialysis services. The new approach was named the ESRD Health Care Quality Improvement Program (HCQIP). It embodies themes such as the development of quality indicators and support for continuous improvement. Projects such as the ESRD Core Indicators Project and the National Anemia Cooperative Project are geared toward assisting dialysis providers to improve patient care. In an effort to document changes in dialysis quality practices associated with the ESRD HCQIP, surveys were sent by Network staff to the head nurses of all dialysis units in 1994, and a random sample of units in 1996. Analysis of the survey responses was performed identifying self-reported changes in dialysis units' quality improvement activities. Results indicate that practice changes are taking place, that they are generalizable to all dialysis units in the country, and that they are associated with improvement in patient outcomes. Trends in quality improvement activities are identified and conclusions are drawn about what impact these activities have on patient care.

Home care information systems: strategies for the future.

As market reform of the US health care system propels home care providers onto center stage, savvy home care leaders are quietly preparing a new information technology strategy to cope with the challenges that lie ahead.

Improved transport incubator temperature control with insulating thermal cover.

INTRODUCTION: Cold stress, secondary to heat loss, can compromise infants in transport incubators during interfacility transfer. With current incubator designs, considerable radiant heat loss occurs. The use of additional external thermal insulation to reduce heat loss has been recommended for infant transports in cold environments. METHOD: A laboratory experiment was done to compare the rate of heat loss from a transport incubator with and without a commercially available, thermal insulating cover in place. The environment was a commercial freezer simulating subzero environmental temperatures. Measurements included air temperatures. inside the incubator and freezer, patterns of heater action and duration of battery power output. The significance of the different rates of cooling was compared using Pearson's r. Suboptimal battery performance was excluded by repeating one arm of the study with an external battery in place of the internal unit. RESULTS: The rate of heat loss from the incubator was: 1) significantly slower when the covered and uncovered incubators were compared (r2 = 0.52), and 2) essentially identical for the uncovered incubator with either the internal or the external battery (r2 = 0.96). CONCLUSION: In the laboratory setting, external thermal insulation slows transport incubator radiant heat loss. Clinically, this effect likely would benefit infants at risk of cold stress during interfacility transports.

Team mix can smooth information system acquisitions.

Through effective planning and preparation, a healthcare organization can avoid bad experience in acquiring an information system. While outside consultants may be helpful in considering a proposed computer system, resources needed for successful system acquisition may be found among an organization's staff members. An acquisition team of employees with expertise in financial management, information systems management, clinical matters, operations, and legal issues can help a team leader acquire a system that balances the needs of each department.

Coccidioides immitis meningitis with arthroconidia in cerebrospinal fluid: report of the first case and review of the arthroconidia literature.

The authors report the first case in which the spherules and arthroconidia of Coccidioides immitis were identified from an indwelling cerebrospinal fluid reservoir. The organism was recovered in culture. Present as isolated meningitis, the patient's infection disseminated over a 12-year period despite intrathecal and intravenous administration of amphotericin B. The unusual findings in this patient are compared with findings in 95 cases of infections containing arthroconidia reported since 1940. Arthroconidia is most likely to be found in the tissues of white male patients who are in the third or fourth decade of life and have pulmonary lesions.

Radionuclide imaging of experimental myocarditis.

The ability of 67Ga citrate and 99mTc pyrophosphate cardiac imaging to detect myocarditis was assessed in an experimental rabbit model. Twenty-three rabbits were imaged approximately 72 hours after infusion of i.v. norepinephrine. Diffuse cardiac uptake was found in 13 of 15 rabbits scanned with 67Ga. Tissue distribution studies documented significant myocardial uptake of 67Ga in those with positive scans. Precordial imaging and tissue distribution studies revealed no cardiac uptake in the eight rabbits scanned with 99mTc. Histologic examination of all 23 hearts revealed qualitatively similar, typical lesions of myocarditis. This study suggests that cardiac imaging with 67Ga may be useful in the detection of myocarditis.

Helminthosporium corneal ulcers.

Two cases of fungal corneal ulcer caused by Helminthosporium are discussed. Dematiacious fungi are pigmented filamentary molds of which Helminthosporium is representative. Ulcers due to such dematiacious fungi seem to have a better prognosis than other types of fungal corneal ulcers. The importance of classifying fungal ulcers is emphasized. Therapy is considered for these keratomycoses.