Cysteamine-mediated blockade of the glycine cleavage system modulates epithelial cell inflammatory and innate immune responses to viral infection.

Transient blockade of glycine decarboxylase (GLDC) can restrict de novo pyrimidine synthesis, which is a well-described strategy for enhancing the host interferon response to viral infection and a target pathway for some licenced anti-inflammatory therapies. The aminothiol, cysteamine, is produced endogenously during the metabolism of coenzyme A, and is currently being investigated in a clinical trial as an intervention in community acquired pneumonia resulting from viral (influenza and SARS-CoV-2) and bacterial respiratory infection. Cysteamine is known to inhibit both bacterial and the eukaryotic host glycine cleavage systems via competitive inhibition of GLDC at concentrations, lower than those required for direct antimicrobial or antiviral activity. Here, we demonstrate for the first time that therapeutically achievable concentrations of cysteamine can inhibit glycine utilisation by epithelial cells and improve cell-mediated responses to infection with respiratory viruses, including human coronavirus 229E and Influenza A. Cysteamine reduces interleukin-6 (IL-6) and increases the interferon-λ (IFN-λ) response to viral challenge and in response to liposomal polyinosinic:polycytidylic acid (poly I:C) simulant of RNA viral infection.

Validation of automated fluorescent-based technology for measuring total nucleated cell viability of hematopoietic progenitor cell products.

BACKGROUND: A reliable rapid method for measuring total nucleated cell (TNC) viability is essential for cell-based products manufacturing. The trypan blue (TB) exclusion method, commonly used to measure TNC viability of hematopoietic progenitor cell (HPC) products, is a subjective assay, typically uses a microscope, and includes a limited number of cells. The NucleoCounter NC-200 is an automated fluorescent-based cell counter that uses pre-calibrated cartridges with acridine orange and DAPI dyes to measure cell count and viability. This study describes the validation of the NC-200 for testing HPC's viability. METHODS: Samples from 189 fresh and 60 cryopreserved HPC products were included. Fresh products were tested for viability after collection by both TB and NC-200. 7-aminoactinomycin D (7AAD) CD45+ cell viability results were obtained from a flow cytometry test. Cryopreserved products thawed specimens were tested for viability by both TB and NC-200. The NC-200 viability results were compared with the other methods. Acceptability criteria were defined as ≤10% difference between the NC-200 method and the other methods for at least 95% of the samples. RESULTS: Fresh products' mean viability difference between NC-200 and TB or 7AAD CD45+ method was 4.9% (95%CI 4.6-5.4) and 2.8% (95%CI 2.2-3.4), respectively. Thawed products' mean viability difference between NC-200 and TB was 3.0% (95%CI 0.4-5.6). CONCLUSION: The NC-200 automated fluorescent-based method can be used effectively to determine HPC's viability for both fresh and cryopreserved products. It can help eliminate human bias and provide consistent data and operational ease.

Evaluating the effect of short-course rifapentine-based regimens with or without enhanced behaviour-targeted treatment support on adherence and completion of treatment for latent tuberculosis infection among adults in the UK (RID-TB: Treat): protocol for an open-label, multicentre, randomised controlled trial.

INTRODUCTION: The successful scale-up of a latent tuberculosis (TB) infection testing and treatment programme is essential to achieve TB elimination. However, poor adherence compromises its therapeutic effectiveness. Novel rifapentine-based regimens and treatment support based on behavioural science theory may improve treatment adherence and completion. METHODS AND ANALYSIS: A pragmatic multicentre, open-label, randomised controlled trial assessing the effect of novel short-course rifapentine-based regimens for TB prevention and additional theory-based treatment support on treatment adherence against standard-of-care. Participants aged between 16 and 65 who are eligible to start TB preventive therapy will be recruited in England. 920 participants will be randomised to one of six arms with allocation ratio of 5:5:6:6:6:6: daily isoniazid +rifampicin for 3 months (3HR), routine treatment support (control); 3HR, additional treatment support; weekly isoniazid +rifapentine for 3 months (3HP), routine treatment support; weekly 3HP, additional treatment support ; daily isoniazid +rifapentine for 1 month (1HP), routine treatment support; daily 1HP, additional treatment support. Additional treatment support comprises reminders using an electronic pillbox, a short animation, and leaflets based on the perceptions and practicalities approach. The primary outcome is adequate treatment adherence, defined as taking ≥90% of allocated doses within the pre-specified treatment period, measured by electronic pillboxes. Secondary outcomes include safety and TB incidence within 12 months. We will conduct process evaluation of the trial interventions and assess intervention acceptability and fidelity and mechanisms for effect and estimate the cost-effectiveness of novel regimens. The protocol was developed with patient and public involvement, which will continue throughout the trial. ETHICS AND DISSEMINATION: Ethics approval has been obtained from The National Health Service Health Research Authority (20/LO/1097). All participants will be required to provide written informed consent. We will share the results in peer-reviewed journals. TRIAL REGISTRATION NUMBER: EudraCT 2020-004444-29.

Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF.

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.

Antimicrobial immunotherapeutics: past, present and future.

In this age of antimicrobial resistance (AMR) there is an urgent need for novel antimicrobials. One area of recent interest is in developing antimicrobial effector molecules, and even cell-based therapies, based on those of the immune system. In this review, some of the more interesting approaches will be discussed, including immune checkpoint inhibitors, Interferons (IFNs), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Chimeric Antigen Receptor (CAR) T cells, Antibodies, Vaccines and the potential role of trained immunity in protection from and/or treatment of infection.

Practising critical resilience as an advanced peer support worker in London: A qualitative evaluation of a peer-led hepatitis C intervention amongst people experiencing homelessness who inject drugs.

BACKGROUND: Peer support has been used as a mechanism to facilitate active engagement with healthcare amongst underserved populations. The HepCare project upskilled experienced peer support workers (PSWs) to become equal members of a service provider team, taking on advanced clinical roles normally carried out by medical or nursing specialists. METHOD: A participatory case study approach was taken to the study following the methodological guidance of Merriam (1998). The subject of the case in our study is the advanced peer support workers (APSWs) functioning in the HepCare project as service providers. The object of the case is an exploration of their transition to service provider in the HCV screening and treatment support service. Five peer led in-depth interviews with APSWs were supplemented by a survey of health professionals, interviews with service users, documentary evidence in the form of job descriptions, observational notes and a blog from the field. Thematic analysis of the data was conducted, refined and finalised in a workshop with the research team and APSW participants. RESULTS: Three themes were generated from the data that explore the peer support worker's transition to APSW in the programme: Transition to Integration, Retaining 'Peerness', and Practising Critical Resilience. The advocacy and support enacted by the APSWs through the HepCare project, offer purpose and meaning alongside integration into a new social group. This is buffered by the supportive context of the programme and facilitates a motivating sense of worth. CONCLUSION: The programme offers policy guidance for the structured career development of APSWs and a platform for enactment of critical resilience as they transition to their advanced role, in the healthcare provider team.

From peer-based to peer-led: redefining the role of peers across the hepatitis C care pathway: HepCare Europe.

BACKGROUND: HCV infection disproportionately affects underserved populations such as homeless individuals, people who inject drugs and prison populations. Peer advocacy can enable active engagement with healthcare services and increase the likelihood of favourable treatment outcomes. OBJECTIVES: This observational study aims to assess the burden of disease in these underserved populations and describe the role of peer support in linking these individuals to specialist treatment services. METHODS: Services were identified if they had a high proportion of individuals with risk factors for HCV, such as injecting drug use or homelessness. Individuals were screened for HCV using point-of-care tests and a portable FibroScan. All positive cases received peer support for linkage to specialist care. Information was gathered on risk factors, demographics and follow-up information regarding linkage to care and treatment outcomes. RESULTS: A total of 461 individuals were screened, of which 197 (42.7%) were chronically infected with HCV. Referral was made to secondary care for 176 (89.3%) and all received peer support, with 104 (52.8%) individuals engaged with treatment centres. Of these, 89 (85.6%) started treatment and 76 (85.4%) had a favourable outcome. Factors associated with not being approved for treatment were recent homelessness, younger age and current crack cocaine injecting. CONCLUSIONS: Highly trained peer support workers working as part of a specialist outreach clinical team help to identify a high proportion of individuals exposed to HCV, achieve high rates of engagement with treatment services and maintain high rates of treatment success amongst a population with complex needs.

Hepatitis B vaccination uptake in hard-to-reach populations in London: a cross-sectional study.

BACKGROUND: In the UK, hepatitis B virus (HBV) incidence is associated with migrants from particular high-burden countries and population groups deemed 'hard-to-reach' by standard healthcare services: the homeless, people who inject drugs and ex-prisoners. Currently, there is a national targeted HBV vaccine policy for such at-risk groups, but there is limited recent evidence about 1) the levels of vaccine uptake, 2) the factors associated with incomplete vaccination, and 3) reasons for incomplete vaccination. METHODS: A questionnaire capturing social and medical history, demographic factors and information about HBV vaccination status was completed by individuals deemed hard-to-reach due to socio-structural factors that criminalise, isolate and stigmatise who consented to participate in a randomised controlled trial of a peer intervention to promote engagement with hepatitis C services. The questionnaire also captured the reasons for incomplete vaccination. Descriptive, univariable and multivariable regression analyses were undertaken. RESULTS: Three hundred fourty six participants completed the questionnaire. 1) 52.3% (n = 181) reported full HBV vaccination. 2) Within a multivariable model adjusting for sociodemographic variables, the presence of one or two or more socio-structural factors that are included in the national targeted vaccination policy was associated with protection against incomplete HBV vaccination (51.7% vaccine coverage in those with one factor, odds ratio 0.43 [95% confidence interval 0.20-0.92]); 70.1% coverage with two or more factors, 0.19 [0.09-0.39]; overall p-value < 0.001). Being female was also associated with lower vaccine uptake (2.37 [1.24-4.57], 0.01). Examining the socio-structural factors individually, intravenous drug use was associated with protection against incomplete HBV vaccination. 3) The most common reasons declared for incomplete vaccination were never being offered the vaccine or not returning for further doses. CONCLUSION: Within this study of HBV vaccination uptake among hard-to-reach population groups in London, UK, we document 52.3% coverage of the full vaccine course. Critically, although participants recommended for immunisation within national guidelines had an increased likelihood of receiving a complete vaccine course, we note surprisingly low coverage in the presence of the risk factors that are national indicators for vaccination. Public health bodies should make additional efforts to improve coverage in the hard-to-reach through improved vaccine delivery systems. TRIAL REGISTRATION: ISRCTN24707359 , Registered 19th October 2012.

Improving engagement with healthcare in hepatitis C: a randomised controlled trial of a peer support intervention.

BACKGROUND: Peer support can enable patient engagement with healthcare services, particularly for marginalised populations. In this randomised controlled trial, the efficacy of a peer support intervention at promoting successful engagement with clinical services for chronic hepatitis C was assessed. METHODS: In London, UK, potential participants were approached through outreach services for problematic drug use and homelessness. Individuals positive for hepatitis C virus (HCV) after confirmatory testing were randomised using an online service to the intervention (peer support) or standard of care. The primary outcome of interest was successful engagement with clinical hepatitis services. The study was non-blinded. Absolute differences were calculated using a generalised linear model and the results compared to logistic regression. RESULTS: Three hundred sixty-four individuals consented to participate. One hundred one had chronic hepatitis C and were randomised, 63 to receive the intervention (peer support). A successful outcome was achieved by 23 individuals in this arm (36.5%) and seven (18.4%) receiving the standard of care, giving an absolute increase of 18.1% (95% confidence interval 1.0-35.2%, p value = 0.04). This was mirrored in the logistic regression (odds ratio 2.55 (0.97-6.70), p = 0.06). No serious adverse events were reported. CONCLUSIONS: Peer support can improve the engagement of patients with chronic HCV with healthcare services. TRIAL REGISTRATION: ISRCTN24707359 . Registered 19th October 2012.

Directional Porin Binding of Intrinsically Disordered Protein Sequences Promotes Colicin Epitope Display in the Bacterial Periplasm.

Protein bacteriocins are potent narrow spectrum antibiotics that exploit outer membrane porins to kill bacteria by poorly understood mechanisms. Here, we determine how colicins, bacteriocins specific for Escherichia coli, engage the trimeric porin OmpF to initiate toxin entry. The N-terminal ∼80 residues of the nuclease colicin ColE9 are intrinsically unstructured and house two OmpF binding sites (OBS1 and OBS2) that reside within the pores of OmpF and which flank an epitope that binds periplasmic TolB. Using a combination of molecular dynamics simulations, chemical trimerization, isothermal titration calorimetry, fluorescence microscopy, and single channel recording planar lipid bilayer measurements, we show that this arrangement is achieved by OBS2 binding from the extracellular face of OmpF, while the interaction of OBS1 occurs from the periplasmic face of OmpF. Our study shows how the narrow pores of oligomeric porins are exploited by colicin disordered regions for direction-specific binding, which ensures the constrained presentation of an activating signal within the bacterial periplasm.

Impact of Hepatitis C Treatment as Prevention for People Who Inject Drugs is sensitive to contact network structure.

Treatment as Prevention (TasP) using directly-acting antivirals has been advocated for Hepatitis C Virus (HCV) in people who inject drugs (PWID), but treatment is expensive and TasP's effectiveness is uncertain. Previous modelling has assumed a homogeneously-mixed population or a static network lacking turnover in the population and injecting partnerships. We developed a transmission-dynamic model on a dynamic network of injecting partnerships using data from survey of injecting behaviour carried out in London, UK. We studied transmission on a novel exponential-clustered network, as well as on two simpler networks for comparison, an exponential unclustered and a random network, and found that TasP's effectiveness differs markedly. With respect to an exponential-clustered network, the random network (and homogeneously-mixed population) overestimate TasP's effectiveness, whereas the exponential-unclustered network underestimates it. For all network types TasP's effectiveness depends on whether treated patients change risk behaviour, and on treatment coverage: higher coverage requires fewer total treatments for the same health gain. Whilst TasP can greatly reduce HCV prevalence, incidence of infection, and incidence of reinfection in PWID, assessment of TasP's effectiveness needs to take account of the injecting-partnership network structure and post-treatment behaviour change, and further empirical study is required.

The anti-sigma factor RsrA responds to oxidative stress by reburying its hydrophobic core.

Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm for sensing oxidative stress centred on the hydrophobic core of a sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor that responds to disulfide stress in the cytoplasm of Actinobacteria. We show that RsrA utilizes its hydrophobic core to bind the sigma factor σ(R) preventing its association with RNA polymerase, and that zinc plays a central role in maintaining this high-affinity complex. Oxidation of RsrA is limited by the rate of zinc release, which weakens the RsrA-σ(R) complex by accelerating its dissociation. The subsequent trigger disulfide, formed between specific combinations of RsrA's three zinc-binding cysteines, precipitates structural collapse to a compact state where all σ(R)-binding residues are sequestered back into its hydrophobic core, releasing σ(R) to activate transcription of anti-oxidant genes.

Aerococcus urinae endocarditis: A report of two cases and review of literature

BACKGROUND: Aerococcus urinae is a rare pathogen of endocarditis with high rates of embolic events, valvular damage and mortality.CASE 1:A 24 y/o male, with mitral valve prolapse, presented with recurrent fever and body malaise for four months. ?-hemolytic streptococci was isolated in his blood 3 months prior, antibiotics for 10 days temporarily relieved his symptoms. He denied illicit drug use, recent dental, genitourinary manipulations. On admission, he was febrile with a 4/6 holosystolic murmur at the apex. He had leukocytosis and elevated acute phase reactants. Blood cultures: Aerococcus urinae. A TEE revealed: ruptured chordae and vegetation at the posterior mitral valve leaflet. Gentamicin for 14 days and Ceftriaxone for 28 days was completed. Mitral valve replacement was done and LV dimension returned to normal.CASE 2:A 51 y/o male presented with 9 days of fever, chills, and malaise. He was treated with norfloxacin with no relief of symptoms. On admission, he was febrile, with a grade 2/6 holosystolic murmur at the apex and left parasternal area. He had leukocytosis and blood culture grew Aerococcus urinae. Echocardiogram showed mitral stenosis, aortic stenosis and vegetations at the mitral valve and non coronary cusp. Pen-G plus Gentamicin for 14 days and upon discharge, amoxicillin for 2 weeks was completed.DISCUSSION: Risk factors associated with A. urinae endocarditis are >65 year s of age, male, urologic abnormalities, malignancy and diabetes. Diagnosis isusually made by culture as our cases and both were found to have vegetations by echocardiography. B-lactam and amino glycoside treatment is effective and although mortality is high, both patients improved and were discharged.CONCLUSION: A. urinae endocarditis does occur in a young population and to those without urologic abnormality.

Perceived efficacy of herbal remedies by users accessing primary healthcare in Trinidad.

BACKGROUND: The increasing global popularity of herbal remedies requires further investigation to determine the probable factors driving this burgeoning phenomenon. We propose that the users' perception of efficacy is an important factor and assessed the perceived efficacy of herbal remedies by users accessing primary health facilities throughout Trinidad. Additionally, we determined how these users rated herbal remedies compared to conventional allopathic medicines as being less, equally or more efficacious. METHODS: A descriptive cross-sectional study was undertaken at 16 randomly selected primary healthcare facilities throughout Trinidad during June-August 2005. A de novo, pilot-tested questionnaire was interviewer-administered to confirmed herbal users (previous or current). Stepwise multiple regression analysis was done to determine the influence of predictor variables on perceived efficacy and comparative efficacy with conventional medicines. RESULTS: 265 herbal users entered the study and cited over 100 herbs for the promotion of health/wellness and the management of specific health concerns. Garlic was the most popular herb (in 48.3% of the sample) and was used for the common cold, cough, fever, as 'blood cleansers' and carminatives. It was also used in 20% of hypertension patients. 230 users (86.8%) indicated that herbs were efficacious and perceived that they had equal or greater efficacy than conventional allopathic medicines. Gender, ethnicity, income and years of formal education did not influence patients' perception of herb efficacy; however, age did (p = 0.036). Concomitant use of herbs and allopathic medicines was relatively high at 30%; and most users did not inform their attending physician. CONCLUSION: Most users perceived that herbs were efficacious, and in some instances, more efficacious than conventional medicines. We suggest that this perception may be a major contributing factor influencing the sustained and increasing popularity of herbs. Evidence-based research in the form of randomized controlled clinical trials should direct the proper use of herbs to validate (or otherwise) efficacy and determine safety. In the Caribbean, most indigenous herbs are not well investigated and this points to the urgent need for biomedical investigations to assess the safety profile and efficacy of our popular medicinal herbs.

Perceived efficacy of herbal remedies by users accessing primary healthcare in Trinidad

BACKGROUND: The increasing global popularity of herbal remedies requires further investigation to determine the probable factors driving this burgeoning phenomenon. We propose that the users' perception of efficacy is an important factor and assessed the perceived efficacy of herbal remedies by users accessing primary health facilities throughout Trinidad. Additionally, we determined how these users rated herbal remedies compared to conventional allopathic medicines as being less, equally or more efficacious. METHODS: A descriptive cross-sectional study was undertaken at 16 randomly selected primary healthcare facilities throughout Trinidad during June-August 2005. A de novo, pilot-tested questionnaire was interviewer-administered to confirmed herbal users (previous or current). Stepwise multiple regression analysis was done to determine the influence of predictor variables on perceived efficacy and comparative efficacy with conventional medicines. RESULTS: 265 herbal users entered the study and cited over 100 herbs for the promotion of health/wellness and the management of specific health concerns. Garlic was the most popular herb (in 48.3% of the sample) and was used for the common cold, cough, fever, as 'blood cleansers' and carminatives. It was also used in 20% of hypertension patients. 230 users (86.8%) indicated that herbs were efficacious and perceived that they had equal or greater efficacy than conventional allopathic medicines. Gender, ethnicity, income and years of formal education did not influence patients' perception of herb efficacy; however, age did (p = 0.036). Concomitant use of herbs and allopathic medicines was relatively high at 30%; and most users did not inform their attending physician. CONCLUSION: Most users perceived that herbs were efficacious, and in some instances, more efficacious than conventional medicines. We suggest that this perception may be a major contributing factor influencing the sustained and increasing popularity of herbs. Evidence-based research in the form of randomized controlled clinical trials should direct the proper use of herbs to validate (or otherwise) efficacy and determine safety. In the Caribbean, most indigenous herbs are not well investigated and this points to the urgent need for biomedical investigations to assess the safety profile and efficacy of our popular medicinal herbs.