Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Incidence of Nerve Palsies During Pavlik Harness Treatment for Developmental Dysplasia of the Hip: A Retrospective Cohort Study.

BACKGROUND: The Pavlik harness has been used for approximately a century to treat developmental dysplasia of the hip (DDH). Femoral nerve palsy is a documented complication of Pavlik harness use, with an incidence ranging from 2.5% to 11.2%. Rare reports of brachial plexus palsy have also been documented. The primary purpose of the current study was to evaluate the incidence of various nerve palsies in patients undergoing Pavlik harness treatment for DDH. Secondary aims were to identify patient demographic or hip characteristics associated with nerve palsy. METHODS: We performed a retrospective review of patients diagnosed with DDH and treated with a Pavlik harness from February 1, 2016, to April 1, 2023, at a single tertiary care orthopaedic hospital. Hip laterality, use of a subsequent rigid abduction orthosis, birth order, breech positioning, weight, and family history were collected. The median (and interquartile range [IQR]) or mean (and standard deviation [SD]) were reported for all continuous variables. Independent 2-sample t tests and Mann-Whitney U tests were conducted to identify associations between the variables collected at the initiation of Pavlik harness treatment and the occurrence of nerve palsy. RESULTS: Three hundred and fifty-one patients (547 hips) were included. Twenty-two cases of femoral nerve palsy (4% of all treated hips), 1 case of inferior gluteal nerve palsy (0.18%), and 2 cases of brachial plexus palsy (0.37%) were diagnosed. Patients with nerve palsy had more severe DDH as measured by the Graf classification (p < 0.001) and more severe DDH as measured on physical examination via the Barlow and Ortolani maneuvers (p = 0.003). CONCLUSIONS: Nerve palsies were associated with more severe DDH at the initiation of Pavlik harness use. Upper and lower-extremity neurological status should be scrutinized at initiation and throughout treatment to assess for nerve palsies. The potential for femoral, gluteal, and brachial plexus palsies should be included in the discussion of risks at the beginning of treatment. Families may be reassured that nerve palsies associated with Pavlik harness can be expected to resolve with a short break from treatment. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Application of Raman Spectroscopy to Dynamic Binding Capacity Analysis.

Protein A affinity chromatography is a key step in isolation of biotherapeutics (BTs) containing fragment crystallizable regions, including monoclonal and bispecific antibodies. Dynamic binding capacity (DBC) analysis assesses how much BT will bind to a protein A column. DBC reduces with column usage, effectively reducing the amount of recovered product over time. Drug regulatory bodies mandate chromatography resin lifetime for BT isolation, through measurement of parameters including DBC, so this feature is carefully monitored in industrial purification pipelines. High-performance affinity chromatography (HPAC) is typically used to assess the concentration of BT, which when loaded to the column results in significant breakthrough of BT in the flowthrough. HPAC gives an accurate assessment of DBC and how this changes over time but only reports on protein concentration, requires calibration for each new BT analyzed, and can only be used offline. Here we utilized Raman spectroscopy and revealed that this approach is at least as effective as both HPAC and ultraviolet chromatogram methods at monitoring DBC of protein A resins. In addition to reporting on protein concentration, the chemical information in the Raman spectra provides information on aggregation status and protein structure, providing extra quality controls to industrial bioprocessing pipelines. In combination with partial least square (PLS) analysis, Raman spectroscopy can be used to determine the DBC of a BT without prior calibration. Here we performed Raman analysis offline in a 96-well plate format, however, it is feasible to perform this inline. This study demonstrates the power of Raman spectroscopy as a significantly improved approach to DBC monitoring in industrial pipelines.

Thermal Conductivity of Carbon/Boron Nitride Heteronanotube and Boron Nitride Nanotube Buckypapers: Implications for Thermal Management Composites.

To date, there has been limited reporting on the fabrication and properties of macroscopic sheet assemblies (specifically buckypapers) composed of carbon/boron nitride core-shell heteronanotubes (MWCNT@BNNT) or boron nitride nanotubes (BNNTs). Herein we report the synthesis of MWCNT@BNNTs via a facile method involving Atmospheric Pressure Chemical Vapor Deposition (APCVD) and the safe h-BN precursor ammonia borane. These MWCNT@BNNTs were used as sacrificial templates for BNNT synthesis by thermal oxidation of the core carbon. Buckypaper fabrication was facilitated by facile sonication and filtration steps. To test the thermal conductivity properties of these new buckypapers, in the interest of thermal management applications, we have developed a novel technique of advanced scanning thermal microscopy (SThM) that we call piercing SThM (pSThM). Our measurements show a 14% increase in thermal conductivity of the MWCNT@BNNT buckypaper relative to a control multiwalled carbon nanotube (MWCNT) buckypaper. Meanwhile, our BNNT buckypaper exhibited approximately half the thermal conductivity of the MWCNT control, which we attribute to the turbostratic quality of our BNNTs. To the best of our knowledge, this work achieves the first thermal conductivity measurement of a MWCNT@BNNT buckypaper and of a BNNT buckypaper composed of BNNTs not synthesized by high energy techniques.

EyeCee One preloaded intraocular lens: are patients with glaucoma more at risk?

OBJECTIVE: The Medicine and Healthcare products Regulatory Agency reported links of raised intraocular pressure (IOP) with recently implanted EyeCee One intraocular lens (IOL). This work investigates if glaucomatous eyes were more susceptible to these postoperative IOP rises and if they required more intensive management. METHODS: Retrospective observational study of all phacoemulsification surgery with implanted EyeCee One IOL, performed between 1 October 2022 and 26 January 2023 inclusive. ANALYSIS: A significant IOP elevation was defined as an IOP rise of 10 mm Hg or more from preoperative to maximal postoperative IOP reading. The management of all patients who had a significant IOP elevation was reviewed. Glaucoma/ocular hypertension cases were identified and analysed against non-glaucomatous eyes and statistical analysis performed. RESULTS: 112 glaucoma and 671 non-glaucoma cases identified; 19.6% of the glaucoma cohort had a significant postoperative IOP rise compared with 8.9% of patients without glaucoma (OR 2.49 (95% CI 1.45 to 4.20) p=0.0014). In the glaucoma cohort, 12.5% had an increase in the number of topical IOP-lowering agents (mean increase 1.65±1.58), 6.3% required systemic treatment and 2.7% surgical intervention. In the non-glaucoma group, 3.3% required topical treatment (mean number of agents 0.88±1.34), 0.8% required systemic treatment and 0.2% surgical intervention. CONCLUSION: This study shows that during the time frame in question, patients with glaucoma or ocular hypertension who had an EyeCee One IOL were almost two and a half times more likely to have a postoperative rise of 10 mm Hg or more in IOP following routine cataract surgery, requiring more aggressive management.

Causes of Industrial Protein A Column Degradation, Explored Using Raman Spectroscopy.

Monoclonal antibodies (mAbs) are used extensively as biotherapeutics for chronic and acute conditions. Production of mAbs is lengthy and expensive, with protein A affinity capture the most costly step, due both to the nature of the resin and its marked reduction in binding capacity with repeated use. Our previous studies using in situ ATR-FTIR spectroscopy indicated that loss in protein A binding capacity is not the result of leaching or degradation of protein A ligand, suggesting fouling is the principal cause. Here we explore binding behavior and resin capacity loss using Raman spectroscopy. Our data reveal a distinct Raman spectral fingerprint for mAb bound to the protein A ligand of MabSelect SuRe. The results show that the drop in static binding capacity (SBC) previously observed for used protein A resin is discernible by Raman spectroscopy in combination with partial least-squares regression. The SBC is lowest (35.76 mg mL-1) for used inlet resin compared to used outlet (40.17 mg mL-1) and unused resin samples (70.35 mg mL-1). Depth profiling by Raman spectroscopy indicates that at below saturating concentrations (∼18 mg mL-1), binding of mAb is not homogeneous through used resin beads with protein binding preferentially to the outer regions of the bead, in contrast to fully homogeneous distribution through unused control MabSelect SuRe resin beads. Analysis of the Raman spectra indicates that one foulant is irreversibly bound mAb. The presence of irreversibly bound mAb and host cell proteins was confirmed by mass spectrometric analysis of used resin beads.

Modest changes in Spi1 dosage reveal the potential for altered microglial function as seen in Alzheimer's disease.

Genetic association studies have identified multiple variants at the SPI1 locus that modify risk and age of onset for Alzheimer's Disease (AD). Reports linking risk variants to gene expression suggest that variants denoting higher SPI1 expression are likely to have an earlier AD onset, and several other AD risk genes contain PU.1 binding sites in the promoter region. Overall, this suggests the level of SPI1 may alter microglial phenotype potentially impacting AD. This study determined how the microglial transcriptome was altered following modest changes to Spi1 expression in primary mouse microglia. RNA-sequencing was performed on microglia with reduced or increased Spi1/PU.1 expression to provide an unbiased approach to determine transcriptomic changes affected by Spi1. In summary, a reduction in microglial Spi1 resulted in the dysregulation of transcripts encoding proteins involved in DNA replication pathways while an increased Spi1 results in an upregulation of genes associated with immune response pathways. Additionally, a subset of 194 Spi1 dose-sensitive genes was identified and pathway analysis suggests that several innate immune and interferon response pathways are impacted by the concentration of Spi1. Together these results suggest Spi1 levels can alter the microglial transcriptome and suggests interferon pathways may be altered in individuals with AD related Spi1 risk SNPs.

Insight into purification of monoclonal antibodies in industrial columns via studies of Protein A binding capacity by in situ ATR-FTIR spectroscopy.

Therapeutic monoclonal antibodies (mAbs) are effective treatments for a range of cancers and other serious diseases, however mAb treatments cost on average ∼$100 000 per year per patient, limiting their use. Currently, industry favours Protein A affinity chromatography (PrAc) as the key step in downstream processing of mAbs. This step, although highly efficient, represents a significant mAb production cost. Fouling of the Protein A column and Protein A ligand leaching contribute to the cost of mAb production by shortening the life span of the resin. In this study, we assessed the performance of used PrAc resin recovered from the middle inlet, center and outlet as well as the side inlet of a pilot-scale industrial column. We used a combination of static binding capacity (SBC) analysis and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy to explore the used resin samples. SBC analysis demonstrated that resin from the inlet of the column had lower binding capacity than resin from the column outlet. ATR-FTIR spectroscopy with PLS (partial least square) analysis confirmed the results obtained from SBC analysis. Importantly, in situ ATR-FTIR spectroscopy also allowed both measurement of the concentration and assessment of the conformational state of the bound Protein A. Our results reveal that PrAc resin degradation after use is dependent on column location and that neither Protein A ligand leaching nor denaturation are responsible for binding capacity loss.

The role and uses of antibodies in COVID-19 infections: a living review.

Coronavirus disease 2019 has generated a rapidly evolving field of research, with the global scientific community striving for solutions to the current pandemic. Characterizing humoral responses towards SARS-CoV-2, as well as closely related strains, will help determine whether antibodies are central to infection control, and aid the design of therapeutics and vaccine candidates. This review outlines the major aspects of SARS-CoV-2-specific antibody research to date, with a focus on the various prophylactic and therapeutic uses of antibodies to alleviate disease in addition to the potential of cross-reactive therapies and the implications of long-term immunity.

Evaluation of a primary care-based collaborative care model (PARTNERS2) for people with diagnoses of schizophrenia, bipolar, or other psychoses: study protocol for a cluster randomised controlled trial.

BACKGROUND: Current NHS policy encourages an integrated approach to provision of mental and physical care for individuals with long term mental health problems. The 'PARTNERS2' complex intervention is designed to support individuals with psychosis in a primary care setting. AIM: The trial will evaluate the clinical and cost-effectiveness of the PARTNERS2 intervention. DESIGN & SETTING: This is a cluster randomised controlled superiority trial comparing collaborative care (PARTNERS2) with usual care, with an internal pilot to assess feasibility. The setting will be primary care within four trial recruitment areas: Birmingham & Solihull, Cornwall, Plymouth, and Somerset. GP practices are randomised 1:1 to either (a) the PARTNERS2 intervention plus modified standard care ('intervention'); or (b) standard care only ('control'). METHOD: PARTNERS2 is a flexible, general practice-based, person-centred, coaching-based intervention aimed at addressing mental health, physical health, and social care needs. Two hundred eligible individuals from 39 GP practices are taking part. They were recruited through identification from secondary and primary care databases. The primary hypothesis is quality of life (QOL). Secondary outcomes include: mental wellbeing, time use, recovery, and process of physical care. A process evaluation will assess fidelity of intervention delivery, test hypothesised mechanisms of action, and look for unintended consequences. An economic evaluation will estimate its cost-effectiveness. Intervention delivery and follow-up have been modified during the COVID-19 pandemic. CONCLUSION: The overarching aim is to establish the clinical and cost-effectiveness of the model for adults with a diagnosis of schizophrenia, bipolar, or other types of psychoses.

Wounds Inhibit Tumor Growth In Vivo.

OBJECTIVE: The aim of this study was to determine the interaction of full thickness excisional wounds and tumors in vivo. SUMMARY OF BACKGROUND DATA: Tumors have been described as wounds that do not heal due to similarities in stromal composition. On the basis of observations of slowed tumor growth after ulceration, we hypothesized that full thickness excisional wounds would inhibit tumor progression in vivo. METHODS: To determine the interaction of tumors and wounds, we developed a tumor xenograft/allograft (human head and neck squamous cell carcinoma SAS/mouse breast carcinoma 4T1) wound mouse model. We examined tumor growth with varying temporospatial placement of tumors and wounds or ischemic flap. In addition, we developed a tumor/wound parabiosis model to understand the ability of tumors and wounds to recruit circulating progenitor cells. RESULTS: Tumor growth inhibition by full thickness excisional wounds was dose-dependent, maintained by sequential wounding, and relative to distance. This effect was recapitulated by placement of an ischemic flap directly adjacent to a xenograft tumor. Using a parabiosis model, we demonstrated that a healing wound was able to recruit significantly more circulating progenitor cells than a growing tumor. Tumor inhibition by wound was unaffected by presence of an immune response in an immunocompetent model using a mammary carcinoma. Utilizing functional proteomics, we identified 100 proteins differentially expressed in tumors and wounds. CONCLUSION: Full thickness excisional wounds have the ability to inhibit tumor growth in vivo. Further research may provide an exact mechanism for this remarkable finding and new advances in wound healing and tumor biology.

Spectroscopy integration to miniature bioreactors and large scale production bioreactors-Increasing current capabilities and model transfer.

Spectroscopy techniques are being implemented within the biopharmaceutical industry due to their non-destructive ability to measure multiple analytes simultaneously, however, minimal work has been applied focussing on their application at small scale. Miniature bioreactor systems are being applied across the industry for cell line development as they offer a high-throughput solution for screening and process optimization. The application of small volume, high-throughput, automated analyses to miniature bioreactors has the potential to significantly augment the type and quality of data from these systems and enhance alignment with large-scale bioreactors. Here, we present an evaluation of 1. a prototype that fully integrates spectroscopy to a miniature bioreactor system (ambr®15, Sartorius Stedim Biotech) enabling automated Raman spectra acquisition, 2. In 50 L single-use bioreactor bag (SUB) prototype with an integrated spectral window. OPLS models were developed demonstrating good accuracy for multiple analytes at both scales. Furthermore, the 50 L SUB prototype enabled on-line monitoring without the need for sterilization of the probe prior to use and minimal light interference was observed. We also demonstrate the ability to build robust models due to induced changes that are hard and costly to perform at large scale and the potential of transferring these models across the scales. The implementation of this technology enables integration of spectroscopy at the small scale for better process understanding and generation of robust models over a large design space while facilitating model transfer throughout the scales enabling continuity throughout process development and utilization and transfer of ever-increasing data generation from development to manufacturing.

Craniofacial and Long Bone Development in the Context of Distraction Osteogenesis.

BACKGROUND: Bone retains regenerative potential into adulthood, and surgeons harness this plasticity during distraction osteogenesis. The underlying biology governing bone development, repair, and regeneration is divergent between the craniofacial and appendicular skeleton. Each type of bone formation is characterized by unique molecular signaling and cellular behavior. Recent discoveries have elucidated the cellular and genetic processes underlying skeletal development and regeneration, providing an opportunity to couple biological and clinical knowledge to improve patient care. METHODS: A comprehensive literature review of basic and clinical literature regarding craniofacial and long bone development, regeneration, and distraction osteogenesis was performed. RESULTS: The current understanding in craniofacial and long bone development and regeneration is discussed, and clinical considerations for the respective distraction osteogenesis procedures are presented. CONCLUSIONS: Distraction osteogenesis is a powerful tool to regenerate bone and thus address a number of craniofacial and appendicular skeletal deficiencies. The molecular mechanisms underlying bone regeneration, however, remain elusive. Recent work has determined that embryologic morphogen gradients constitute important signals during regeneration. In addition, striking discoveries have illuminated the cellular processes underlying mandibular regeneration during distraction osteogenesis, showing that skeletal stem cells reactivate embryologic neural crest transcriptomic processes to carry out bone formation during regeneration. Furthermore, innovative adjuvant therapies to complement distraction osteogenesis use biological processes active in embryogenesis and regeneration. Additional research is needed to further characterize the underlying cellular mechanisms responsible for improved bone formation through adjuvant therapies and the role skeletal stem cells play during regeneration.

The experiences of hospital staff who provide care for people living with dementia: A systematic review and synthesis of qualitative studies.

AIMS AND OBJECTIVES: To systematically review and synthesise qualitative data from studies exploring the experiences of hospital staff who care for people living with dementia (Plwd). BACKGROUND: In hospital, the number of Plwd continues to rise; however, their experiences of care remain problematic. Negative experiences of care are likely to contribute to poorer mental and physical health outcomes for Plwd while in hospital and after discharge. Experiences of the hospital staff who care for Plwd can also be poor or unrewarding. It is important to understand the experiences of staff in order to improve staff well-being and ultimately the experience of care for Plwd while in hospital. DESIGN: Systematic review and evidence synthesis of qualitative research. DATA SOURCES: We searched 16 electronic databases in March 2018 and completed forward and backward citation chasing. METHODS: Eligible studies explored the experiences of paid and unpaid staff providing care in hospital for Plwd. Study selection was undertaken independently by two reviewers, and quality appraisal was conducted. We prioritised included studies according to richness of text, methodological rigour and conceptual contribution. We adopted approaches of meta-ethnography to analyse study findings, creating a conceptual model to represent the line of argument. FINDINGS: Forty-five studies reported in 58 papers met the inclusion criteria, and of these, we prioritised 19 studies reported in 24 papers. The line of argument was that Institutions can improve staff experiences of care for Plwd by fostering person-centred care (PCC). PCC aligned with staff perceptions of 'good care'; however, staff often felt prevented from providing PCC because of care cultures that prioritised tasks, routines and physical health. Staff experienced conflict over the care they wanted to give versus the care they were able to give, and this caused moral distress. When staff were able to provide PCC, this increased experiences of job satisfaction and emotional well-being. CONCLUSIONS: Person-centred care not only has the potential to improve the experience of care for Plwd and their carers, but can also improve the experiences of hospital staff caring for Plwd. However, without institutional-level changes, hospital staff are often unable to provide PCC even when they have the experience and knowledge to do so. IMPLICATIONS FOR PRACTICE: Institutional-level areas for change include the following: training; performance indicators and ward cultures that prioritise psychological needs alongside physical needs; adequate staffing levels; inclusive approaches to carers; physical environments that promote familiarisation, social interaction and occupation; systems of documentation about individual needs of Plwd; and cultures of sharing knowledge across hierarchies.

Activity interventions to improve the experience of care in hospital for people living with dementia: a systematic review.

BACKGROUND: An increasingly high number of patients admitted to hospital have dementia. Hospital environments can be particularly confusing and challenging for people living with dementia (Plwd) impacting their wellbeing and the ability to optimize their care. Improving the experience of care in hospital has been recognized as a priority, and non-pharmacological interventions including activity interventions have been associated with improved wellbeing and behavioral outcomes for Plwd in other settings. This systematic review aimed at evaluating the effectiveness of activity interventions to improve experience of care for Plwd in hospital. METHODS: Systematic searches were conducted in 16 electronic databases up to October 2019. Reference lists of included studies and forward citation searching were also conducted. Quantitative studies reporting comparative data for activity interventions delivered to Plwd aiming to improve their experience of care in hospital were included. Screening for inclusion, data extraction and quality appraisal were performed independently by two reviewers with discrepancies resolved by discussion with a third where necessary. Standardized mean differences (SMDs) were calculated where possible to support narrative statements and aid interpretation. RESULTS: Six studies met the inclusion criteria (one randomized and five non-randomized uncontrolled studies) including 216 Plwd. Activity interventions evaluated music, art, social, psychotherapeutic, and combinations of tailored activities in relation to wellbeing outcomes. Although studies were generally underpowered, findings indicated beneficial effects of activity interventions with improved mood and engagement of Plwd while in hospital, and reduced levels of responsive behaviors. Calculated SMDs ranged from very small to large but were mostly statistically non-significant. CONCLUSIONS: The small number of identified studies indicate that activity-based interventions implemented in hospitals may be effective in improving aspects of the care experience for Plwd. Larger well-conducted studies are needed to fully evaluate the potential of this type of non-pharmacological intervention to improve experience of care in hospital settings, and whether any benefits extend to staff wellbeing and the wider ward environment.

Virtual Interviews in the Era of COVID-19: A Primer for Applicants.

PROBLEM: The COVID-19 pandemic is an evolving crisis with widespread impact upon our medical system, including senior trainee travel for fellowship interviews. Numerous institutions have conscientiously deferred in-person interviews or virtual formats. Given the competitive nature of fellowship interviews, candidates may express concern that they are at a disadvantage in engaging in online meetings versus live, on-site interviews, and similarly may feel ill prepared to perform optimally during online interviews. APPROACH: We draw upon our experience with online interview platforms in this guide for fellowship candidates who are rapidly adapting to new technology and styles associated with videoconference interviews so that they can best promote themselves for competitive positions.

Innate immunology in COVID-19-a living review. Part I: viral entry, sensing and evasion.

The coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a world health concern and can cause severe disease and high mortality in susceptible groups. While vaccines offer a chance to treat disease, prophylactic and anti-viral treatments are still of vital importance, especially in context of the mutative ability of this group of viruses. Therefore, it is essential to elucidate the molecular mechanisms of viral entry, innate sensing and immune evasion of SARS-CoV-2, which control the triggers of the subsequent excessive inflammatory response. Viral evasion strategies directly target anti-viral immunity, counteracting host restriction factors and hijacking signalling pathways to interfere with interferon production. In Part I of this review, we examine SARS-CoV-2 viral entry and the described immune evasion mechanisms to provide a perspective on how the failure in initial viral sensing by infected cells can lead to immune dysregulation causing fatal COVID-19, discussed in Part II.

Innate immunology in COVID-19-a living review. Part II: dysregulated inflammation drives immunopathology.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global health crisis and will likely continue to impact public health for years. As the effectiveness of the innate immune response is crucial to patient outcome, huge efforts have been made to understand how dysregulated immune responses may contribute to disease progression. Here we have reviewed current knowledge of cellular innate immune responses to SARS-CoV-2 infection, highlighting areas for further investigation and suggesting potential strategies for intervention. We conclude that in severe COVID-19 initial innate responses, primarily type I interferon, are suppressed or sabotaged which results in an early interleukin (IL)-6, IL-10 and IL-1ß-enhanced hyperinflammation. This inflammatory environment is driven by aberrant function of innate immune cells: monocytes, macrophages and natural killer cells dispersing viral pathogen-associated molecular patterns and damage-associated molecular patterns into tissues. This results in primarily neutrophil-driven pathology including fibrosis that causes acute respiratory distress syndrome. Activated leukocytes and neutrophil extracellular traps also promote immunothrombotic clots that embed into the lungs and kidneys of severe COVID-19 patients, are worsened by immobility in the intensive care unit and are perhaps responsible for the high mortality. Therefore, treatments that target inflammation and coagulation are promising strategies for reducing mortality in COVID-19.

Doxycycline Reduces Scar Thickness and Improves Collagen Architecture.

OBJECTIVE: To investigate the effects of local doxycycline administration on skin scarring. BACKGROUND: Skin scarring represents a major source of morbidity for surgical patients. Doxycycline, a tetracycline antibiotic with off-target effects on the extracellular matrix, has demonstrated antifibrotic effects in multiple organs. However, doxycycline's potential effects on skin scarring have not been explored in vivo. METHODS: Female C57BL/6J mice underwent dorsal wounding following an established splinted excisional skin wounding model. Doxycycline was administered by local injection into the wound base following injury. Wounds were harvested upon complete wound closure (postoperative day 15) for histological examination and biomechanical testing of scar tissue. RESULTS: A one-time dose of 3.90 mM doxycycline (2 mg/mL) within 12 hours of injury was found to significantly reduce scar thickness by 24.8% (P < 0.0001) without compromising tensile strength. The same effect could not be achieved by oral dosing. In doxycycline-treated scar matrices, collagen I content was significantly reduced (P = 0.0317) and fibers were favorably arranged with significantly increased fiber randomness (P = 0.0115). Common culprits of altered wound healing mechanics, including angiogenesis and inflammation, were not impacted by doxycycline treatment. However, engrailed1 profibrotic fibroblasts, responsible for scar extracellular matrix deposition, were significantly reduced with doxycycline treatment (P = 0.0005). CONCLUSIONS: Due to the substantial improvement in skin scarring and well-established clinical safety profile, locally administered doxycycline represents a promising vulnerary agent. As such, we favor rapid translation to human patients as an antiscarring therapy.

Developing methods for the overarching synthesis of quantitative and qualitative evidence: The interweave synthesis approach.

The incorporation of evidence derived from multiple research designs into one single synthesis can enhance the utility of systematic reviews making them more worthwhile, useful, and insightful. Methodological guidance for mixed-methods synthesis continues to emerge and evolve but broadly involves a sequential, parallel, or convergent approach according to the degree of independence between individual syntheses before they are combined. We present two case studies in which we used novel and innovative methods to draw together the findings from individual but related quantitative and qualitative syntheses to aid interpretation of the overall evidence base. Our approach moved beyond making a choice between parallel, sequential, or convergent methods to interweave the findings of individual reviews and offers three key innovations to mixed-methods synthesis methods: The use of intersubjective questions to understand the findings of the individual reviews through different lenses, Immersion of key reviewers in the entirety of the evidence base, and Commencing the process during the final stages of the synthesis of individual reviews, at a point where reviewers are developing an understanding of initial findings. Underlying our approach is the process of exploration and identification of links between and across review findings, an approach that is fundamental to all evidence syntheses but usually occurs at the level of the study. Adapting existing methods for exploring and identifying patterns and links between and across studies to interweave the findings between and across reviews may prove valuable.