Social and structural barriers and facilitators to HIV healthcare and harm reduction services for people experiencing syndemics in Manitoba: study protocol.

INTRODUCTION: In Manitoba, Canada, there has been an increase in the number of people newly diagnosed with HIV and those not returning for regular HIV care. The COVID-19 pandemic resulted in increased sex and gender disparities in disease risk and mortalities, decreased harm reduction services and reduced access to healthcare. These health crises intersect with increased drug use and drug poisoning deaths, houselessness and other structural and social factors most acutely among historically underserved groups. We aim to explore the social and structural barriers and facilitators to HIV care and harm reduction services experienced by people living with HIV (PLHIV) in Manitoba. METHODS AND ANALYSIS: Our study draws on participatory action research design. Guiding the methodological design are the lived experiences of PLHIV. In-depth semi-structured face-to-face interviews and quantitative questionnaires will be conducted with two groups: (1) persons aged ≥18 years living or newly diagnosed with HIV and (2) service providers who work with PLHIV. Data collection will include sex, gender, sociodemographic information, income and housing, experiences with the criminal justice system, sexual practices, substance use practices and harm reduction access, experiences with violence and support, HIV care journey (since diagnosis until present), childhood trauma and a decision-making questionnaire. Data will be analysed intersectionally, employing grounded theory for thematic analysis, sex-based and gender-based analysis and social determinants of health and syndemic framework to understand the experiences of PLHIV in Manitoba. ETHICS AND DISSEMINATION: We received approval from the University of Manitoba Health Ethics Research Board (HS25572; H2022:218), First Nations Health and Social Secretariat of Manitoba, Nine Circles Community Health Centre, Shared Health Manitoba (SH2022:194) and 7th Street Health Access Centre. Findings will be disseminated using community-focused knowledge translation strategies identified by participants, peers, community members and organisations, and reported in conferences, peer-reviewed journals and a website (www.alltogether4ideas.org).

Inequities in organ and tissue donation and transplantation for sexual orientation and gender identity diverse people: A scoping review.

Sexual orientation and gender identity (SOGI)-diverse populations experience discrimination in organ and tissue donation and transplantation (OTDT) systems globally. We assembled a multidisciplinary group of clinical experts as well as SOGI-diverse patient and public partners and conducted a scoping review including citations on the experiences of SOGI-diverse persons in OTDT systems globally to identify and explore the inequities that exist with regards to living and deceased OTDT. Using scoping review methods, we conducted a systematic literature search of relevant electronic databases from 1970 to 2021 including a grey literature search. We identified and screened 2402 references and included 87 unique publications. Two researchers independently coded data in included publications in duplicate. We conducted a best-fit framework synthesis paired with an inductive thematic analysis to identify synthesized benefits, harms, inequities, justification of inequities, recommendations to mitigate inequities, laws and regulations, as well as knowledge and implementation gaps regarding SOGI-diverse identities in OTDT systems. We identified numerous harms and inequities for SOGI-diverse populations in OTDT systems. There were no published benefits of SOGI-diverse identities in OTDT systems. We summarized recommendations for the promotion of equity for SOGI-diverse populations and identified gaps that can serve as targets for action moving forward.

Hydroxychloroquine as Pre-exposure Prophylaxis for Coronavirus Disease 2019 (COVID-19) in Healthcare Workers: A Randomized Trial.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly emerging virus causing the ongoing coronavirus disease 2019 (COVID-19) pandemic with no known effective prophylaxis. We investigated whether hydroxychloroquine could prevent SARS-CoV-2 in healthcare workers at high risk of exposure. METHODS: We conducted a randomized, double-blind, placebo-controlled clinical trial of healthcare workers with ongoing exposure to persons with SARS-CoV-2, including those working in emergency departments, intensive care units, COVID-19 hospital wards, and first responders. Participants across the United States and in the Canadian province of Manitoba were randomized to hydroxychloroquine loading dose then 400 mg once or twice weekly for 12 weeks. The primary endpoint was confirmed or probable COVID-19-compatible illness. We measured hydroxychloroquine whole-blood concentrations. RESULTS: We enrolled 1483 healthcare workers, of whom 79% reported performing aerosol-generating procedures. The incidence of COVID-19 (laboratory-confirmed or symptomatic compatible illness) was 0.27 events/person-year with once-weekly and 0.28 events/person-year with twice-weekly hydroxychloroquine compared with 0.38 events/person-year with placebo. For once-weekly hydroxychloroquine prophylaxis, the hazard ratio was .72 (95% CI, .44-1.16; P = .18) and for twice-weekly was .74 (95% CI, .46-1.19; P = .22) compared with placebo. Median hydroxychloroquine concentrations in whole blood were 98 ng/mL (IQR, 82-120) with once-weekly and 200 ng/mL (IQR, 159-258) with twice-weekly dosing. Hydroxychloroquine concentrations did not differ between participants who developed COVID-19-compatible illness (154 ng/mL) versus participants without COVID-19 (133 ng/mL; P = .08). CONCLUSIONS: Pre-exposure prophylaxis with hydroxychloroquine once or twice weekly did not significantly reduce laboratory-confirmed COVID-19 or COVID-19-compatible illness among healthcare workers. CLINICAL TRIALS REGISTRATION: Clinicaltrials.gov NCT04328467.

Safety of Hydroxychloroquine Among Outpatient Clinical Trial Participants for COVID-19.

BACKGROUND: Use of hydroxychloroquine in hospitalized patients with coronavirus disease 2019 (COVID-19), especially in combination with azithromycin, has raised safety concerns. Here, we report safety data from 3 outpatient randomized clinical trials. METHODS: We conducted 3 randomized, double-blind, placebo-controlled trials investigating hydroxychloroquine as pre-exposure prophylaxis, postexposure prophylaxis, and early treatment for COVID-19 using an internet-based design. We excluded individuals with contraindications to hydroxychloroquine. We collected side effects and serious adverse events. We report descriptive analyses of our findings. RESULTS: We enrolled 2795 participants. The median age of research participants (interquartile range) was 40 (34-49) years, and 59% (1633/2767) reported no chronic medical conditions. Overall 2544 (91%) participants reported side effect data, and 748 (29%) reported at least 1 medication side effect. Side effects were reported in 40% with once-daily, 36% with twice-weekly, 31% with once-weekly hydroxychloroquine, compared with 19% with placebo. The most common side effects were upset stomach or nausea (25% with once-daily, 19% with twice-weekly, and 18% with once-weekly hydroxychloroquine, vs 11% for placebo), followed by diarrhea, vomiting, or abdominal pain (23% for once-daily, 17% twice-weekly, and 13% once-weekly hydroxychloroquine, vs 7% for placebo). Two individuals were hospitalized for atrial arrhythmias, 1 on placebo and 1 on twice-weekly hydroxychloroquine. No sudden deaths occurred. CONCLUSIONS: Data from 3 outpatient COVID-19 trials demonstrated that gastrointestinal side effects were common but mild with the use of hydroxychloroquine, while serious side effects were rare. No deaths occurred related to hydroxychloroquine. Randomized clinical trials, in cohorts of healthy outpatients, can safely investigate whether hydroxychloroquine is efficacious for COVID-19. CLINICALTRIALSGOV IDENTIFIER: NCT04308668 for postexposure prophylaxis and early treatment trials; NCT04328467 for pre-exposure prophylaxis trial.

Hydroxychloroquine as pre-exposure prophylaxis for COVID-19 in healthcare workers: a randomized trial.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly emerging virus causing the ongoing Covid-19 pandemic with no known effective prophylaxis. We investigated whether hydroxychloroquine could prevent SARS CoV-2 in healthcare workers at high-risk of exposure. METHODS: We conducted a randomized, double-blind, placebo-controlled clinical trial of healthcare workers with ongoing exposure to persons with Covid-19, including those working in emergency departments, intensive care units, Covid-19 hospital wards, and first responders. Participants across the United States and in the Canadian province of Manitoba were randomized to hydroxychloroquine 400mg once weekly or twice weekly for 12 weeks. The primary endpoint was confirmed or probable Covid-19-compatible illness. We measured hydroxychloroquine whole blood concentrations. RESULTS: We enrolled 1483 healthcare workers, of which 79% reported performing aerosol-generating procedures. The incidence of Covid-19 (laboratory-confirmed or symptomatic compatible illness) was 0.27 events per person-year with once-weekly and 0.28 events per person-year with twice-weekly hydroxychloroquine compared with 0.38 events per person-year with placebo. For once weekly hydroxychloroquine prophylaxis, the hazard ratio was 0.72 (95%CI 0.44 to 1.16; P=0.18) and for twice weekly was 0.74 (95%CI 0.46 to 1.19; P=0.22) as compared with placebo. Median hydroxychloroquine concentrations in whole blood were 98 ng/mL (IQR, 82-120) with once-weekly and 200 ng/mL (IQR, 159-258) with twice-weekly dosing. Hydroxychloroquine concentrations did not differ between participants who developed Covid-19 (154 ng/mL) versus participants without Covid-19 (133 ng/mL; P=0.08). CONCLUSIONS: Pre-exposure prophylaxis with hydroxychloroquine once or twice weekly did not significantly reduce laboratory-confirmed Covid-19 or Covid-19-compatible illness among healthcare workers.

Safety of Hydroxychloroquine among Outpatient Clinical Trial Participants for COVID-19.

INTRODUCTION: Use of hydroxychloroquine in hospitalized patients with COVID-19, especially in combination with azithromycin, has raised safety concerns. Here, we report safety data from three outpatient randomized clinical trials. METHODS: We conducted three randomized, double-blind, placebo-controlled trials investigating hydroxychloroquine as pre-exposure prophylaxis, post-exposure prophylaxis and early treatment for COVID-19. We excluded individuals with contraindications to hydroxychloroquine. We collected side effects and serious adverse events. We report descriptive analyses of our findings. RESULTS: We enrolled 2,795 participants. The median age of research participants was 40 (IQR 34-49) years, and 59% (1633/2767) reported no chronic medical conditions. Overall 2,324 (84%) participants reported side effect data, and 638 (27%) reported at least one medication side effect. Side effects were reported in 29% with daily, 36% with twice weekly, 31% with once weekly hydroxychloroquine compared to 19% with placebo. The most common side effects were upset stomach or nausea (25% with daily, 18% with twice weekly, 16% with weekly, vs. 10% for placebo), followed by diarrhea, vomiting, or abdominal pain (23% for daily, 16% twice weekly, 12% weekly, vs. 6% for placebo). Two individuals were hospitalized for atrial arrhythmias, one on placebo and one on twice weekly hydroxychloroquine. No sudden deaths occurred. CONCLUSION: Data from three outpatient COVID-19 trials demonstrated that gastrointestinal side effects were common but mild with the use of hydroxychloroquine, while serious side effects were rare. No deaths occurred related to hydroxychloroquine. Randomized clinical trials can safely investigate whether hydroxychloroquine is efficacious for COVID-19.

Hydroxychloroquine in Nonhospitalized Adults With Early COVID-19 : A Randomized Trial.

BACKGROUND: No effective oral therapy exists for early coronavirus disease 2019 (COVID-19). OBJECTIVE: To investigate whether hydroxychloroquine could reduce COVID-19 severity in adult outpatients. DESIGN: Randomized, double-blind, placebo-controlled trial conducted from 22 March through 20 May 2020. (ClinicalTrials.gov: NCT04308668). SETTING: Internet-based trial across the United States and Canada (40 states and 3 provinces). PARTICIPANTS: Symptomatic, nonhospitalized adults with laboratory-confirmed COVID-19 or probable COVID-19 and high-risk exposure within 4 days of symptom onset. INTERVENTION: Oral hydroxychloroquine (800 mg once, followed by 600 mg in 6 to 8 hours, then 600 mg daily for 4 more days) or masked placebo. MEASUREMENTS: Symptoms and severity at baseline and then at days 3, 5, 10, and 14 using a 10-point visual analogue scale. The primary end point was change in overall symptom severity over 14 days. RESULTS: Of 491 patients randomly assigned to a group, 423 contributed primary end point data. Of these, 341 (81%) had laboratory-confirmed infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or epidemiologically linked exposure to a person with laboratory-confirmed infection; 56% (236 of 423) were enrolled within 1 day of symptoms starting. Change in symptom severity over 14 days did not differ between the hydroxychloroquine and placebo groups (difference in symptom severity: relative, 12%; absolute, -0.27 point [95% CI, -0.61 to 0.07 point]; P = 0.117). At 14 days, 24% (49 of 201) of participants receiving hydroxychloroquine had ongoing symptoms compared with 30% (59 of 194) receiving placebo (P = 0.21). Medication adverse effects occurred in 43% (92 of 212) of participants receiving hydroxychloroquine versus 22% (46 of 211) receiving placebo (P < 0.001). With placebo, 10 hospitalizations occurred (2 non-COVID-19-related), including 1 hospitalized death. With hydroxychloroquine, 4 hospitalizations occurred plus 1 nonhospitalized death (P = 0.29). LIMITATION: Only 58% of participants received SARS-CoV-2 testing because of severe U.S. testing shortages. CONCLUSION: Hydroxychloroquine did not substantially reduce symptom severity in outpatients with early, mild COVID-19. PRIMARY FUNDING SOURCE: Private donors.

A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19.

BACKGROUND: Coronavirus disease 2019 (Covid-19) occurs after exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For persons who are exposed, the standard of care is observation and quarantine. Whether hydroxychloroquine can prevent symptomatic infection after SARS-CoV-2 exposure is unknown. METHODS: We conducted a randomized, double-blind, placebo-controlled trial across the United States and parts of Canada testing hydroxychloroquine as postexposure prophylaxis. We enrolled adults who had household or occupational exposure to someone with confirmed Covid-19 at a distance of less than 6 ft for more than 10 minutes while wearing neither a face mask nor an eye shield (high-risk exposure) or while wearing a face mask but no eye shield (moderate-risk exposure). Within 4 days after exposure, we randomly assigned participants to receive either placebo or hydroxychloroquine (800 mg once, followed by 600 mg in 6 to 8 hours, then 600 mg daily for 4 additional days). The primary outcome was the incidence of either laboratory-confirmed Covid-19 or illness compatible with Covid-19 within 14 days. RESULTS: We enrolled 821 asymptomatic participants. Overall, 87.6% of the participants (719 of 821) reported a high-risk exposure to a confirmed Covid-19 contact. The incidence of new illness compatible with Covid-19 did not differ significantly between participants receiving hydroxychloroquine (49 of 414 [11.8%]) and those receiving placebo (58 of 407 [14.3%]); the absolute difference was -2.4 percentage points (95% confidence interval, -7.0 to 2.2; P = 0.35). Side effects were more common with hydroxychloroquine than with placebo (40.1% vs. 16.8%), but no serious adverse reactions were reported. CONCLUSIONS: After high-risk or moderate-risk exposure to Covid-19, hydroxychloroquine did not prevent illness compatible with Covid-19 or confirmed infection when used as postexposure prophylaxis within 4 days after exposure. (Funded by David Baszucki and Jan Ellison Baszucki and others; ClinicalTrials.gov number, NCT04308668.).

Post-exposure prophylaxis or pre-emptive therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): study protocol for a pragmatic randomized-controlled trial.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 causing the coronavirus disease (COVID-19) pandemic. Currently, there is a lack of evidence-based therapies to prevent COVID-19 following exposure to the virus, or to prevent worsening of symptoms following confirmed infection. We describe the design of a clinical trial of hydroxychloroquine for post-exposure prophylaxis (PEP) and pre-emptive therapy (PET) for COVID-19. METHODS: We will conduct two nested multicentre international double-blind randomized placebo-controlled clinical trials of hydroxychloroquine for: 1) PEP of asymptomatic household contacts or healthcare workers exposed to COVID-19 within the past four days, and 2) PET for symptomatic outpatients with COVID-19 showing symptoms for less than four days. We will recruit 1,500 patients each for the PEP and PET trials. Participants will be randomized 1:1 to receive five days of hydroxychloroquine or placebo. The primary PEP trial outcome will be the incidence of symptomatic COVID-19. The primary PET trial outcome will be an ordinal scale of disease severity (not hospitalized, hospitalized without intensive care, hospitalization with intensive care, or death). Participant screening, informed consent, and follow-up will be exclusively internet-based with appropriate regulatory and research ethics board approvals in Canada and the United States. DISCUSSION: These complementary randomized-controlled trials are innovatively designed and adequately powered to rapidly answer urgent questions regarding the effectiveness of hydroxychloroquine to reduce virus transmission and disease severity of COVID-19 during a pandemic. In-person participant follow-up will not be conducted to facilitate social distancing strategies and reduce risks of exposure to study personnel. Innovative trial approaches are needed to urgently assess therapeutic options to mitigate the global impact of this pandemic. TRIALS REGISTRATION: clinicaltrials.gov (NCT04308668); registered 16 March, 2020.

RéSUMé: CONTEXTE: Le syndrome respiratoire aigu sévère du coronavirus 2 (SARS-CoV-2) est apparu en décembre 2019, provoquant la pandémie de la COVID-19. À l'heure actuelle, il n'existe aucun traitement fondé sur des données probantes permettant de prévenir la COVID-19 suite à une exposition au virus ou de prévenir l'aggravation des symptômes suite à une infection confirmée. Nous décrivons la conception d'une étude clinique examinant l'utilisation d'hydroxychloroquine en tant que prophylaxie post-exposition (PPE) et de traitement préventif (TP) pour la COVID-19. MéTHODE: Nous réaliserons deux études cliniques imbriquées contrôlées par placebo, randomisées, à double insu, internationales et multicentriques examinant l'utilisation d'hydroxychloroquine pour : 1) la prophylaxie post-exposition des contacts asymptomatiques dans un même foyer ou les travailleurs de la santé exposés à la COVID-19 au cours des quatre derniers jours, et 2) le traitement préventif des patients symptomatiques en ambulatoire atteints de COVID-19 et présentant des symptômes pour une durée totale de moins de quatre jours. Nous recruterons 1500 patients pour chaque bras de l'étude (PPE et TP). Les participants seront randomisés à un ratio de 1 : 1 pour recevoir cinq jours d'hydroxychloroquine ou de placebo. Le critère d'évaluation principal de l'étude PPE sera l'incidence de COVID-19 symptomatique. Le critère d'évaluation principal de l'étude TP consistera en une échelle ordinale de la gravité de la maladie (pas d'hospitalisation, hospitalisation sans soins intensifs, hospitalisation avec soins intensifs, ou décès). La sélection des participants, le consentement éclairé et le suivi se feront exclusivement en ligne après avoir obtenu les consentements réglementaires et des comités d'éthique de la recherche appropriés au Canada et aux États-Unis. DISCUSSION: Ces études randomisées contrôlées complémentaires sont conçues de façon innovatrice et disposent de la puissance nécessaire pour répondre rapidement aux questions urgentes quant à l'efficacité de l'hydroxychloroquine pour réduire la transmission et la gravité de la maladie de la COVID-19 pendant une pandémie. Le suivi des participants ne sera pas réalisé en personne afin de faciliter les stratégies de distanciation sociale et de réduire le risque d'exposition du personnel de l'étude. Des approches innovatrices d'études sont nécessaires afin d'évaluer rapidement les options thérapeutiques pour mitiger l'impact global de cette pandémie. ENREGISTREMENT DE L'éTUDE: clinicaltrials.gov (NCT04308668); enregistrées le 16 mars 2020.

Anaplasmosis: An emerging tick-borne disease of importance in Canada.

Human Granulocytic Anaplasmosis (HGA) is an infection caused by the intracellular bacterium Anaplasma phagocytophilum. As a tick-borne disease, the public health impact of HGA continues to increase with range expansion of the disease vector. The clinical presentation of HGA is often a non-specific febrile illness. The presence of leukopenia, thrombocytopenia, and mild hepatic injury are frequently noted on laboratory investigations, which can be important diagnostic clues in attaining an appropriate diagnosis. Herein we present three cases of HGA, highlighting the spectrum of disease by which HGA can manifest. Although each case has their unique features, we outline important shared clinical elements to facilitate an empiric diagnosis while definitive laboratory investigations are pending. Our case series further serves to highlight the critical importance of prompt antimicrobial treatment to reduce morbidity and potential mortality.

Is there a rural/urban gap in the quality of HIV care for treatment-naïve HIV-positive individuals initiating antiretroviral therapy in British Columbia?

Rurally located people living with HIV (PLWH) face unique challenges associated with remoteness that may negatively affect their HIV care outcomes. The Programmatic Compliance Score (PCS) has been used previously as a quality of care metric, and is predictive of mortality for treatment-naïve individuals initiating combination antiretroviral therapy (cART). This study looked at whether the rurality of PLWH impacted their PCS. PCS was calculated for PLWH (≥19 years old) initiating cART in British Columbia between 2000 and 2013. Rurality was determined at the time of cART initiation using two methodologies: (1) a categorical postal code method; and (2) the General Practice Rurality Index (GPRI), a score representing an individual's degree of rurality. Ordinal logistic regression modeling was used to assess the relationship between rurality and PCS. Among 4616 PLWH with an evaluable PCS, 176 were classified as rural and 3512 as urban (928 had an unknown postal code). After adjusting for age, sex, hepatitis C status, Indigenous ancestry, and year of cART initiation, categorical rurality was not associated with a worse PCS (adjusted odds ratio (AOR) 1.04; 95% CI: 0.77-1.39). However, an increasing degree of rurality was associated with a worse PCS (AOR (per 10 increase in GPRI) 1.13; 95% CI: 1.06-1.20). Given that a poor PCS has been shown to be predictive of all-cause mortality for individuals initiating cART, strategies to improve access to HIV care for rural individuals should be evaluated.

The Continuum of HIV Care in Rural Communities in the United States and Canada: What Is Known and Future Research Directions.

The nature of the HIV epidemic in the United States and Canada has changed with a shift toward rural areas. Socioeconomic factors, geography, cultural context, and evolving epidemics of injection drug use are coalescing to move the epidemic into locations where populations are dispersed and health care resources are limited. Rural-urban differences along the care continuum demonstrate the implications of this sociogeographic shift. Greater attention is needed to build a more comprehensive understanding of the rural HIV epidemic in the United States and Canada, including research efforts, innovative approaches to care delivery, and greater community engagement in prevention and care.

Quintessential Culture-Negative Endocarditis.

Bartonella spp are important causes of culture-negative endocarditis, generally causing a subacute insidious form of endocarditis, often leading to a delay in diagnosis. Most patients have fever and often present with signs and symptoms of heart failure. The diagnosis is frequently established only on meticulous examination of the resected heart valve with the polymerase chain reaction technique. We present a case of B quintana mitral and aortic valve endocarditis with associated severe valvular insufficiency and decompensated heart failure precipitated by Streptococcus pneumoniae bacteremia, necessitating urgent surgical valve replacement. Pathologic examination of the valve complemented by serologic and molecular testing established the surprising diagnosis of B quintana endocarditis.

Initiation of antiretroviral therapy at high CD4+ cell counts is associated with positive treatment outcomes.

OBJECTIVE: There is limited research investigating the possible mechanisms of how starting combination antiretroviral therapy (cART) at a higher CD4 cell count decreases mortality. This study investigated the association between initiating cART with short-term and long-term achievement of viral suppression; emergence of any drug resistance and of an AIDS-defining illness (ADI); long-term treatment adherence; and all-cause mortality. METHODS: This retrospective cohort study included 4120 naive patients who initiated cART between 2000 and 2012. Patients were followed until 2013, death or until the last contact date (varied by outcome). The main exposure was the interaction between period of cART initiation (2000-2006 and 2007-2012) and CD4 cell count at cART initiation (<500 versus ≥500 cells/µl). We considered both baseline and longitudinal covariates. We fitted different multivariable models using cross-sectional and longitudinal statistical methods, depending on the outcome. RESULTS: Patients who initiated cART with a CD4 cell count at least 500 cells/µl in 2007-2012 had an increased likelihood of achieving viral suppression at 9 months and of maintaining an adherence level of at least 95% over time, and the lowest probability of developing any resistance and an ADI during follow-up. These patients were not the ones with the highest likelihood of maintaining viral suppression over time, most likely due to viral load blips experienced during the follow-up. CONCLUSION: The outcomes in this study likely play an important role in explaining the positive impact of early cART initiation on mortality. These results should alleviate some of the concerns clinicians may have when initiating cART in patients with high CD4s as recommended by current treatment guidelines.

Proteome profile of cytosolic component of zebrafish liver generated by LC-ESI MS/MS combined with trypsin digestion and microwave-assisted acid hydrolysis.

The zebrafish genome has recently been sequenced and annotated allowing for high-throughput proteomic analysis. Here, we report for the first time a proteomic subset of zebrafish liver, an important organ for metabolizing toxins. Using a newly developed analytical procedure, we have identified 1204 proteins from the cytosolic component of a zebrafish liver tissue sample. Our methods involve cell-compartment fractionation of liver tissue samples, four levels of protein digestion, and off-line two-dimensional liquid chromatography (2-D LC) separations of resultant peptides. Proteins are identified using an electrospray ionization quadrupole time-of-flight tandem mass spectrometer (ESI-QTOF MS/MS), which provides high-resolution and high-accuracy mass measurement of peptide ions and their fragment ions. We demonstrate that greater proteome coverage can be achieved by combining the results obtained from four methods of protein digestion: three tryptic digests (one in buffer, one in methanol, and another in SDS), and a microwave-assisted acid hydrolysate of the protein extracts. Identified proteins--which included several groups of established protein biomarkers--were functionally classified. We discuss the functions and implications of these biomarkers within the context of zebrafish toxicology.

The spatial pattern of atrial cardiomyocyte calcium signalling modulates contraction.

We examined the regulation of calcium signalling in atrial cardiomyocytes during excitation-contraction coupling, and how changes in the distribution of calcium impacts on contractility. Under control conditions, calcium transients originated in subsarcolemmal locations and showed local regeneration through activation of calcium-induced calcium release from ryanodine receptors. Despite functional ryanodine receptors being expressed at regular (approximately 2 microm) intervals throughout atrial myocytes, the subsarcolemmal calcium signal did not spread in a fully regenerative manner through the interior of a cell. Rather, there was a diminishing centripetal propagation of calcium. The lack of regeneration was due to mitochondria and SERCA pumps preventing the inward movement of calcium. Inhibiting these calcium buffering mechanisms allowed the globalisation of action potential-evoked responses. In addition, physiological positive inotropic agents, such as endothelin-1 and beta-adrenergic agonists, as well as enhanced calcium current, calcium store loading and inositol 1,4,5-trisphosphate infusion also led to regenerative global responses. The consequence of globalising calcium signals was a significant increase in cellular contraction. These data indicate how calcium signals and their consequences are determined by the interplay of multiple subcellular calcium management systems.

Nifedipine and diltiazem suppress ventricular arrhythmogenesis and calcium release in mouse hearts.

Ventricular arrhythmogenesis leading to sudden cardiac death remains responsible for significant mortality in conditions such as cardiac failure and the long-QT syndrome (LQTS). Arrhythmias may be accentuated by beta-adrenergic stimulation and, accordingly, the present study explored the possible effects of beta-adrenergic stimulation and L-type Ca(2+) channel blockade on ventricular arrhythmogenesis and Ca(2+) handling using the mouse heart as an experimental system. Studies in whole, Langendorff-perfused hearts using programmed electrical stimulation protocols adapted from clinical practice demonstrated sustained ventricular tachycardia following addition of 0.1 microM isoprenaline (n=15), whilst no arrhythmias were observed in the absence of the drug (n=15). Arrhythmias were suppressed by nifedipine or diltiazem pre-treatment (both 1 microM) (n=8 and 4 respectively) and were also induced by elevating external [Ca(2+)] (n=3). At the cellular level, 0.1 microM isoprenaline significantly increased normalized fluorescence (F/F(0)) in field-stimulated fluo-3-loaded mouse ventricular myocytes imaged using confocal microscopy, reflecting increases in sarcoplasmic reticulum Ca(2+) release (n=8). Elevated external [Ca(2+)] also increased F/F(0) (n=4) whilst 0.1 microM nifedipine or 0.1 microM diltiazem significantly decreased F/F(0) (n=13 and 6 respectively). Pre-treatment with 0.1 microM nifedipine or 0.1 microM diltiazem suppressed the increases in F/F(0) induced by 0.1 microM isoprenaline alone (n=14 and 6 respectively). The findings thus paralleled suppression of isoprenaline-induced arrhythmias seen with nifedipine or diltiazem at the whole-heart level. Taken together, the findings may have implications for the use of L-type Ca(2+) channel blockade in conditions associated with beta-adrenergically driven ventricular arrhythmias such as cardiac failure and LQTS.