Neutrophil extracellular traps are present in the airways of ENaC-overexpressing mice with cystic fibrosis-like lung disease.

BACKGROUND: Neutrophils are key components of the exacerbated inflammation and tissue damage in cystic fibrosis (CF) airways. Neutrophil extracellular traps (NETs) trap and kill extracellular pathogens. While NETs are abundant in the airways of CF patients and have been hypothesized to contribute to lung damage in CF, the in vivo role of NETs remains controversial, partially due to lack of appropriate animal models. The goal of this study was to detect NETs and to further characterize neutrophil-mediated inflammation in the airways of mice overexpressing the epithelial sodium channel (ßENaC-Tg mice on C57BL/6 background) in their lung with CF-like airway disease, in the absence of any apparent bacterial infections. METHODS: Histology scoring of lung tissues, flow cytometry, multiplex ELISA, immunohistochemistry and immunofluorescence were used to characterize NETs and the airway environment in uninfected, ßENaC-Tg mice at 6 and 8 weeks of age, the most chronic time points so far studied in this model. RESULTS: Excessive neutrophilic infiltration characterized the lungs of uninfected, ßENaC-Tg mice at 6 and 8 weeks of age. The bronchoalveolar lavage fluid (BALF) of ßENaC-Tg mice contains increased levels of CF-associated cytokines and chemokines: KC, MIP-1α/ß, MCP-1, G-CSF, IL-5, and IL-6. The BALF of ßENaC-Tg mice contain MPO-DNA complexes, indicative of the presence of NETs. Immunofluorescence and flow cytometry of BALF neutrophils and lung tissues demonstrated increased histone citrullination, a NET-specific marker, in ßENaC-Tg mice. CONCLUSIONS: NETs are detected in the airways of ßENaC-Tg mice, in the absence of bacterial infections. These data demonstrate the usefulness of the ßENaC-Tg mouse to serve as a model for studying the role of NETs in chronic CF airway inflammation.

A Role for Mycobacterium tuberculosis Sigma Factor C in Copper Nutritional Immunity.

Sigma factor C (SigC) contributes to Mycobacterium tuberculosis virulence in various animal models, but the stress response coordinated by this transcription factor was undefined. The results presented here indicate that SigC prevents copper starvation. Whole genome expression studies demonstrate short-term (4-h) induction of sigC, controlled from a tetracycline-inducible promoter, upregulates ctpB and genes in the nonribosomal peptide synthase (nrp) operon. These genes are expressed at higher levels after 48-h sigC induction, but also elevated are genes encoding copper-responsive regulator RicR and RicR-regulated copper toxicity response operon genes rv0846-rv0850, suggesting prolonged sigC induction results in excessive copper uptake. No growth and global transcriptional differences are observed between a sigC null mutant relative to its parent strain in 7H9 medium. In a copper-deficient medium, however, growth of the sigC deletion strain lags the parent, and 40 genes (including those in the nrp operon) are differentially expressed. Copper supplementation reverses the growth defect and silences most transcriptional differences. Together, these data support SigC as a transcriptional regulator of copper acquisition when the metal is scarce. Attenuation of sigC mutants in severe combined immunodeficient mice is consistent with an inability to overcome innate host defenses that sequester copper ions to deprive invading microbes of this essential micronutrient.

A mixed-method investigation of patient monitoring and enhanced feedback in routine practice: Barriers and facilitators.

OBJECTIVE: To investigate the barriers and facilitators of an effective implementation of an outcome monitoring and feedback system in a UK National Health Service psychological therapy service. METHOD: An outcome monitoring system was introduced in two services. Enhanced feedback was given to therapists after session 4. Qualitative and quantitative methods were used, including questionnaires for therapists and patients. Thematic analysis was carried out on written and verbal feedback from therapists. Analysis of patient outcomes for 202 episodes of therapy was compared with benchmark data of 136 episodes of therapy for which feedback was not given to therapists. RESULTS: Themes influencing the feasibility and acceptability of the feedback system were the extent to which therapists integrated the measures and feedback into the therapy, availability of administrative support, information technology, and complexity of the service. There were low levels of therapist actions resulting from the feedback, including discussing the feedback in supervision and with patients. CONCLUSIONS: The findings support the feasibility and acceptability of setting up a routine system in a complex service, but a number of challenges and barriers have to be overcome and therapist differences are apparent. More research on implementation and effectiveness is needed in diverse clinical settings.

One problem, multiple potential targets: Where are we now in the development of small molecule inhibitors against Shiga toxin?

Shiga toxin-producing Escherichia coli (STEC) are a group of enteric pathogens which carry phage-encoded Shiga toxins (Stx). STEC infections begin with severe abdominal pain and non-bloody diarrhoea, which can progress to bloody diarrhoea after approximately 4-days post-infection. In high-risk groups such as children and the elderly, patients may develop haemolytic uremic syndrome (HUS). HUS is characterised by microangiopathic haemolytic anaemia, thrombocytopenia, and in severe disease acute renal failure. Traditional antibiotics have been linked with increased toxin production due to the activation of recA-mediated bacterial stress response, resulting in poorer patient outcomes. Therefore, treatment relies on supportive therapies. Antivirulence strategies have been explored as an alternative treatment for bacterial infections and blockers of virulence factors such as the Type III Secretion System. Recent improvements in the mechanistic understanding of the Stx pathway have led to the design of inhibitors to disrupt the pathway, leading to toxin-mediated ribosome damage. However, compounds have yet to progress beyond Phase III clinical trials successfully. This review explores the progress in developing small molecule inhibitors by collating lead compounds derived from in-silico and experimental approaches.

Evaluation Design for The Two Georgias Initiative: Assessing Progress Toward Health Equity in the Rural South.

As persistent inequities in health gained increased attention nationally due to COVID-19 and racial justice protests in 2020, it has become increasingly important to evaluate both the process and outcomes associated with coalition-based efforts to address health inequities. The Two Georgias Initiative supports coalitions in 11 rural counties to (1) achieve greater health equity, (2) improve health and health care, (3) build healthier rural communities and improve social conditions that impact the health of rural populations, and (4) build community, organizational, and individual leadership capacity for health equity. Rural communities suffer significant health disparities relative to urban areas, and also experience internal inequities by race and poverty level. The evaluation framework for The Two Georgias Initiative provides a comprehensive mixed methods approach to evaluating both processes and outcomes. Early results related to community readiness and capacity to address health inequities, measured through a coalition member survey (n = 236) conducted at the end of the planning phase, suggest coalitions were in the preparation stage, with higher levels of readiness among coalition members and organizations/groups similar to the coalition members' own, lower levels among public officials and other leaders, and the lowest levels among county residents. In addition, coalition members reported more experience with downstream drivers (e.g., access to care) of health than upstream drivers (e.g., affordable housing, environmental or racial justice). By providing a logic model, evaluation questions and associated indicators, as well as a range of data collection methods, this evaluation approach may prove practical to others aiming to evaluate their efforts to address health equity.

Short chain fatty acids reduce the respiratory burst of human neutrophils in response to cystic fibrosis isolates of Staphylococcus aureus.

Short chain fatty acids (SCFA) are produced by anaerobic bacteria. The most common SCFAs are acetate, propionate and butyrate. SCFAs have been implicated in several inflammatory diseases including cystic fibrosis (CF) where they are present in the airways at millimolar concentrations. Staphylococcus aureus is one of the main respiratory pathogens in CF. Polymorphonuclear neutrophil granulocytes (PMN) represent the most important immune defense the host uses against S. aureus. However, the reason why PMNs are unable to clear S. aureus in CF remains largely unclear. We hypothesized that SCFAs impair effector functions of PMNs in response to S. aureus. To test this, human PMNs were exposed to CF clinical isolates of S. aureus in vitro in the presence or absence of SCFAs and effector functions of PMNs were assessed. Our data show that SCFAs do not affect the viability of PMNs and do not stimulate the release of neutrophil extracellular traps (NET) from human PMNs. Production of reactive oxygen species (ROS), another important antimicrobial function of PMNs, on the other hand, was significantly inhibited by SCFAs in response to the bacterium. SCFAs did not compromise the ability of PMNs to kill CF isolates of S. aureus in vitro. Overall, our results provide new knowledge into the interactions between SCFAs and the immune system, and indicate that SCFAs produced by anaerobic bacteria in the CF lung could interfere with reactive oxidant production of PMNs in response to S. aureus, one of the prominent respiratory pathogens in this disease.

Differences in Health Care Access and Use by Gender, Race, Income, Age and Employment among Residents of the Rural South.

Understanding how disparities are experienced by subpopulations within rural areas may inform efforts to mitigate persistent inequities in access to health care. Among 2,545 randomly sampled adults who completed a mailed survey in ten rural counties in Georgia as part of a health equity initiative, 50.8% of respondents were aged 35-64, 65.9% were women, 16.6% identified as Black, 36.0% worked full-time, and 39% had a high school degree or less. Significant disparities were observed in health care access, use and financial burden by age, employment status, race, and annual household income. In an examination of intersectionality of race and income, all sub-groups except for higher income Black respondents were more likely to report no health insurance and not seeing a doctor in the past 12 months due to cost relative to higher income White respondents. The findings shed insight into inequities in health care access within rural communities.

Granulocytic Myeloid-Derived Suppressor Cells in Cystic Fibrosis.

Cystic Fibrosis (CF) is a genetic disease that causes chronic and severe lung inflammation and infection associated with high rates of mortality. In CF, disrupted ion exchange in the epithelium results in excessive mucus production and reduced mucociliary clearance, leading to immune system exacerbation and chronic infections with pathogens such as P. aeruginosa and S. aureus. Constant immune stimulation leads to altered immune responses including T cell impairment and neutrophil dysfunction. Specifically, CF is considered a Th17-mediated disease, and it has been proposed that both P. aeruginosa and a subset of neutrophils known as granulocytic myeloid suppressor cells (gMDSCs) play a role in T cell suppression. The exact mechanisms behind these interactions are yet to be determined, but recent works demonstrate a role for arginase-1. It is also believed that P. aeruginosa drives gMDSC function as a means of immune evasion, leading to chronic infection. Herein, we review the current literature regarding immune suppression in CF by gMDSCs with an emphasis on T cell impairment and the role of P. aeruginosa in this dynamic interaction.

PI3K/ NF-κB-dependent TNF-α and HDAC activities facilitate LPS-induced RGS10 suppression in pulmonary macrophages.

Regulator of G-protein signaling 10 (RGS10) is a member of the superfamily of RGS proteins that canonically act as GTPase activating proteins (GAPs). RGS proteins accelerate GTP hydrolysis on the G-protein α subunits and result in termination of signaling pathways downstream of G protein-coupled receptors. Beyond its GAP function, RGS10 has emerged as an anti-inflammatory protein by inhibiting LPS-mediated NF-κB activation and expression of inflammatory cytokines, in particular TNF-α. Although RGS10 is abundantly expressed in resting macrophages, previous studies have shown that RGS10 expression is suppressed in macrophages following Toll-like receptor 4 (TLR4) activation by LPS. However, the molecular mechanism by which LPS induces Rgs10 silencing has not been clearly defined. The goal of the current study was to determine whether LPS silences Rgs10 expression through an NF-κB-mediated proinflammatory mechanism in pulmonary macrophages, a unique type of innate immune cells. We demonstrate that Rgs10 transcript and RGS10 protein levels are suppressed upon LPS treatment in the murine MH-S alveolar macrophage cell line. We show that pharmacological inhibition of PI3K/ NF-κB/p300 (NF-κB co-activator)/TNF-α signaling cascade and the activities of HDAC (1-3) enzymes block LPS-induced silencing of Rgs10 in MH-S cells as well as microglial BV2 cells and BMDMs. Further, loss of RGS10 generated by using CRISPR/Cas9 amplifies NF-κB phosphorylation and inflammatory gene expression following LPS treatment in MH-S cells. Together, our findings strongly provide critical insight into the molecular mechanism underlying RGS10 suppression by LPS in pulmonary macrophages.

RGS10 Reduces Lethal Influenza Infection and Associated Lung Inflammation in Mice.

Seasonal influenza epidemics represent a significant global health threat. The exacerbated immune response triggered by respiratory influenza virus infection causes severe pulmonary damage and contributes to substantial morbidity and mortality. Regulator of G-protein signaling 10 (RGS10) belongs to the RGS protein family that act as GTPase activating proteins for heterotrimeric G proteins to terminate signaling pathways downstream of G protein-coupled receptors. While RGS10 is highly expressed in immune cells, in particular monocytes and macrophages, where it has strong anti-inflammatory effects, its physiological role in the respiratory immune system has not been explored yet. Here, we show that Rgs10 negatively modulates lung immune and inflammatory responses associated with severe influenza H1N1 virus respiratory infection in a mouse model. In response to influenza A virus challenge, mice lacking RGS10 experience enhanced weight loss and lung viral titers, higher mortality and significantly faster disease onset. Deficiency of Rgs10 upregulates the levels of several proinflammatory cytokines and chemokines and increases myeloid leukocyte accumulation in the infected lung, markedly neutrophils, monocytes, and inflammatory monocytes, which is associated with more pronounced lung damage. Consistent with this, influenza-infected Rgs10-deficent lungs contain more neutrophil extracellular traps and exhibit higher neutrophil elastase activities than wild-type lungs. Overall, these findings propose a novel, in vivo role for RGS10 in the respiratory immune system controlling myeloid leukocyte infiltration, viral clearance and associated clinical symptoms following lethal influenza challenge. RGS10 also holds promise as a new, potential therapeutic target for respiratory infections.

Cystic Fibrosis Sputum Impairs the Ability of Neutrophils to Kill Staphylococcus aureus.

Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of inflammatory polymorphonuclear (PMN) granulocytes. Staphylococcus aureus (S. aureus) is a major lung pathogen in CF that persists despite the presence of PMNs and has been associated with CF lung function decline. While PMNs represent the main mechanism of the immune system to kill S. aureus, it remains largely unknown why PMNs fail to eliminate S. aureus in CF. The goal of this study was to observe how the CF airway environment affects S. aureus killing by PMNs. PMNs were isolated from the blood of healthy volunteers and CF patients. Clinical isolates of S. aureus were obtained from the airways of CF patients. The results show that PMNs from healthy volunteers were able to kill all CF isolates and laboratory strains of S. aureus tested in vitro. The extent of killing varied among strains. When PMNs were pretreated with supernatants of CF sputum, S. aureus killing was significantly inhibited suggesting that the CF airway environment compromises PMN antibacterial functions. CF blood PMNs were capable of killing S. aureus. Although bacterial killing was inhibited with CF sputum, PMN binding and phagocytosis of S. aureus was not diminished. The S. aureus-induced respiratory burst and neutrophil extracellular trap release from PMNs also remained uninhibited by CF sputum. In summary, our data demonstrate that the CF airway environment limits killing of S. aureus by PMNs and provides a new in vitro experimental model to study this phenomenon and its mechanism.

The clinical communication and information challenges associated with the psychosexual aspects of prostate cancer treatment.

RATIONALE: Prostate cancer and its treatment have significant sexual side effects that necessitate timely patient information and open communication with healthcare professionals. However, very little is known about men's experiences of talking to clinicians about the psychosexual difficulties associated with the disease. OBJECTIVE: This study aims to advance understanding of men's perceptions of the communication and information challenges associated with the psychosexual aspects of prostate cancer and its treatment. METHOD: Between October 2013 and April 2014, semi-structured interviews were conducted with 21 men from the UK who had been treated for prostate cancer. Interview transcripts were analysed using thematic analysis. RESULTS: Three themes describe the communication challenges men face: (1) It can be too soon to talk about sex; (2) the psychology of sex is missing; (3) communication is not individually tailored. CONCLUSIONS: Clinicians might usefully (1) consider and discuss with patients how their psychosexual communication needs and information processing abilities may fluctuate across the cancer timeline; (2) initiate discussions about the consequences of treatment that extend beyond biological and mechanical aspects to include emotional and relational factors; (3) tailor communication to the dynamic mix of attributes that shape men's individual psychosexual needs, including their relationship status, sexual orientation, sexual motivations and values. Skills-based training in communication and psychosexual awareness may facilitate the proactive and permissive stance clinicians need to discuss sexual side effects with a heterogeneous group of patients.

Development of an explanatory model of sexual intimacy following treatment for localised prostate cancer: A systematic review and meta-synthesis of qualitative evidence.

RATIONALE: Prostate cancer is a leading cause of cancer in men, affecting one in eight. An ageing population coupled with increased testing indicates that the incidence of early-stage prostate cancer is rising rapidly. Treatments are effective, but all can result in chronic sexual side effects and impact on the psychological, emotional and relational components of sexual functioning. Whilst the physical consequences of treatment are well documented, we lack a comprehensive picture of the effects of localised prostate cancer treatment on men's experience of sexual intimacy and how this may affect survivorship and recovery. OBJECTIVE: This study synthesises the qualitative literature related to men's experience of sexual intimacy in the context of localised prostate cancer. METHODS: A systematic search strategy identified 12 studies, which were assessed using a modified version of the Critical Appraisal Skills Programme. Using Noblit and Hare's (1988) approach, a meta-synthesis was conducted. RESULTS: Findings are organised within four inter-related themes that form the basis of a new conceptual explanatory model: (i) Loss and grief: Destroyed intimacy; (ii) Going through the motions: Artificial intimacy; (iii) Fear of failure: Avoiding intimacy and (iv) Breaking barriers: Constructing an alternative intimacy. CONCLUSION: The LMAC (Loss, Motions, Avoidance and Construction) model provides a new way of conceptualising sexual recovery following prostate cancer treatment and opportunities for health care professionals to support men and their partners.