Immune cell populations and induced immune responses at admission in patients hospitalized with vaccine breakthrough SARS-CoV-2 infections.

Background: Vaccine breakthrough SARS-CoV-2 infections are common and of clinical and public health concern. However, little is known about the immunological characteristics of patients hospitalized due to these infections. We aimed to investigate and compare immune cell subpopulations and induced immune responses in vaccinated and non-vaccinated patients hospitalized with severe COVID-19. Methods: A nested case-control study on adults (≥ 18 years) who received at least two doses of a mRNA-COVID-19 vaccine and were hospitalized with SARS-CoV-2 breakthrough infections and severe COVID-19 between January 7, 2021, and February 1, 2022, were eligible for inclusion. Age- and sex-matched non-vaccinated controls were identified. Immunophenotyping was performed using a custom-designed 10-color flow cytometry prefabricated freeze-dried antibody panel (DuraClone, Beckman Coulter (BC), Brea, Calif). TruCulture (Myriad RBM, Austin, USA) was used to assess induced immune response in whole blood, revealing different critical signaling pathways as a proxy for immune function. All samples were obtained within 48 hours of admission. Results: In total, 20 hospitalized patients with severe COVID-19 and a breakthrough SARS-CoV-2 infection were included, ten vaccinated and ten non-vaccinated patients. Vaccinated patients had lower concentrations of CD19 B cells (p = 0.035), naïve CD4 T cells (p = 0.015), a higher proportion of γδ1 T cells (p = 0.019), and higher unstimulated immune cell release of IL-10 (p = 0.015). Conclusion: We observed immunological differences between vaccinated and non-vaccinated patients hospitalized due to severe COVID-19 that indicate that vaccinated patients had lower B cell concentrations, lower concentrations of CD4 naïve T cells, a skewed gamma-delta V1/V2 ratio, and an exaggerated IL-10 response at admission. These results could indicate a suboptimal immune response involved in SARS-CoV-2 breakthrough infections that cause severe COVID-19 in vaccinated adults. However, the sample size was small, and further research is needed to confirm these results.

The direct anterior approach for conversion to total hip arthroplasty after acetabular fixation is safe and effective.

INTRODUCTION: Total Hip Arthroplasty (THA) after prior acetabular fracture repair is known to be demanding as studies have shown inferior implant survival rates and higher infection rates for these procedures. The direct anterior (DA) approach might help mitigate some of these risks by utilizing a new surgical tissue plane. However, potential criticisms of the DA approach for these surgeries include the inability to access previous acetabular implants or heterotopic ossification (HO) if they were to inhibit implant placement. The goals of this study are to analyze the efficacy of the DA approach for conversion to hip arthroplasty surgery after previous acetabular fixation. METHODS: After reviewing all records at our institution using current procedural terminology codes, we isolated patients with previous acetabular repair who underwent conversion to THA through the DA approach. Patient records were reviewed, and patients were contacted to obtain Harris Hip Scores. RESULTS: 23 patients (16 males and 7 females) were found with a mean follow-up time of 46 months (range 16-156 months). The mean age was 50 (range 28 - 83) and mean BMI was 28.5 (range 15.2 - 39.2). The average blood loss was 400 ml (range 200 - 900). The average operative time was 140 min (range 85-200 min). In 7 cases (32%) implants were encountered during acetabular reaming but the implants were either removed entirely or removed partially with a burr so that the acetabular cup could be positioned within acceptable parameters. In 2 cases pre-operative HO was encountered and was resected. The average Harris Hip Score at final follow-up was 92 (range 75 - 100). There were no deep infections and no neurovascular injuries encountered. 2 patients (9%) underwent revision surgery for aseptic femoral stem loosening. There was 1 anterior dislocation (4.5%) at 3 days post-operatively that was successfully treated with closed reduction and maintenance of hip precautions. Otherwise, the remaining 19 (86%) patients went on to uncomplicated recovery. CONCLUSION: This is the largest known cohort analyzing the DA approach for conversion to hip arthroplasty after previous acetabular fixation. Overall, we demonstrate that the DAA is safe for conversion THA after acetabular fixation.

Retrograde Nailing for Extremely Proximal Fractures of the Femoral Shaft.

OBJECTIVES: To report the results of retrograde intramedullary nailing (RIMN) for the treatment of extremely proximal femur fractures. DESIGN: Retrospective cohort study. SETTING: Level I trauma center. PATIENTS/PARTICIPANTS: 63 patients with femoral shaft fractures involving the anatomic region within 10 centimeters of the inferior border of the lesser trochanter, which were treated with retrograde intramedullary nailing. INTERVENTION: Retrograde intramedullary femoral nail. MAIN OUTCOME MEASUREMENTS: Time to union, nonunion, malunion, and unplanned reoperation. RESULTS: Between 2009 and 2020, 63 fractures were followed up to fracture union, reoperation, or a minimum of 1 year clinically. The mean follow-up was 32 months, and 48 (76%) of the patients were followed up beyond 1 year clinically. The mean patient age was 34 years (range 18-84 years), and the mean BMI was 27 (range 14-45) kg/m 2 . Forty (64%) patients were polytraumatized. Clinical and radiographic union was achieved in 59 (94%) fractures after index operation at a mean time to union of 22 weeks (range 9-51 weeks). Delayed union requiring nail dynamization occurred in 1 (2%) instance. Malreduction was noted in 1 (2%) patient with a 12-degree flexion deformity that resulted in nonunion. In total, there were 3 (5%) nonunions requiring revision surgery, 1 treated with retrograde exchange nailing and 2 revised to cephalomedullary nails; all were united after revision. CONCLUSIONS: Retrograde intramedullary nailing can be an effective treatment strategy for extremely proximal femur fractures when necessary. Our series demonstrated a high rate of union and a low rate of malalignment and complications. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Erratum: Author Correction: Early stimulated immune responses predict clinical disease severity in hospitalized COVID-19 patients.

[This corrects the article DOI: 10.1038/s43856-022-00178-5.].

A randomized placebo-controlled trial of convalescent plasma for adults hospitalized with COVID-19 pneumonia.

Passive immunotherapy with convalescent plasma may be the only available agent during the early phases of a pandemic. Here, we report safety and efficacy of high-titer convalescent plasma for COVID-19 pneumonia. Double-blinded randomized multicenter placebo-controlled trial of adult patients hospitalized with COVID-19 pneumonia. The intervention was COVID-19 convalescent plasma and placebo was saline allocated 2:1. The primary outcome was clinical status 14 days after the intervention evaluated on a clinical ordinal scale. The trial was registered at ClinicalTrials.Gov, NCT04345289, 14/04/2020. The CCAP-2 trial was terminated prematurely due to futility. Of 147 patients randomized, we included 144 patients in the modified intention-to-treat population. The ordinal clinical status 14 days post-intervention was comparable between treatment groups (odds ratio (OR) 1.41, 95% confidence interval (CI) 0.72-2.09). Results were consistent when evaluating clinical progression on an individual level 14 days after intervention (OR 1.09; 95% CI 0.46-1.73). No significant differences in length of hospital stay, admission to ICU, frequency of severe adverse events or all-cause mortality during follow-up were found between the intervention and the placebo group. Infusion of convalescent plasma did not influence clinical progression, survival or length of hospitalization in patients with COVID-19 pneumonia.

Early stimulated immune responses predict clinical disease severity in hospitalized COVID-19 patients.

Background: The immune pathogenesis underlying the diverse clinical course of COVID-19 is poorly understood. Currently, there is an unmet need in daily clinical practice for early biomarkers and improved risk stratification tools to help identify and monitor COVID-19 patients at risk of severe disease. Methods: We performed longitudinal assessment of stimulated immune responses in 30 patients hospitalized with COVID-19. We used the TruCulture whole-blood ligand-stimulation assay applying standardized stimuli to activate distinct immune pathways, allowing quantification of cytokine responses. We further characterized immune cell subsets by flow cytometry and used this deep immunophenotyping data to map the course of clinical disease within and between patients. Results: Here we demonstrate impairments in innate immune response pathways at time of COVID-19 hospitalization that are associated with the development of severe disease. We show that these impairments are transient in those discharged from hospital, as illustrated by functional and cellular immune reconstitution. Specifically, we identify lower levels of LPS-stimulated IL-1ß, and R848-stimulated IL-12 and IL-17A, at hospital admission to be significantly associated with increasing COVID-19 disease severity during hospitalization. Furthermore, we propose a stimulated immune response signature for predicting risk of developing severe or critical COVID-19 disease at time of hospitalization, to validate in larger cohorts. Conclusions: We identify early impairments in innate immune responses that are associated with subsequent COVID-19 disease severity. Our findings provide basis for early identification of patients at risk of severe disease which may have significant implications for the early management of patients hospitalized with COVID-19.

Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay.

Virus neutralization assays provide a means to quantitate functional antibody responses that block virus infection. These assays are instrumental in defining vaccine and therapeutic antibody potency, immune evasion by viral variants, and post-infection immunity. Here we describe the development, optimization and evaluation of a live virus microneutralization assay specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this assay, SARS-CoV-2 clinical isolates are pre-incubated with serial diluted antibody and added to Vero E6 cells. Replicating virus is quantitated by enzyme-linked immunosorbent assay (ELISA) targeting the SARS-CoV-2 nucleocapsid protein and the standardized 50% virus inhibition titer calculated. We evaluated critical test parameters that include virus titration, assay linearity, number of cells, viral dose, incubation period post-inoculation, and normalization methods. Virus titration at 96 hours was determined optimal to account for different growth kinetics of clinical isolates. Nucleocapsid protein levels directly correlated with virus inoculum, with the strongest correlation at 24 hours post-inoculation. Variance was minimized by infecting a cell monolayer, rather than a cell suspension. Neutralization titers modestly decreased with increasing numbers of Vero E6 cells and virus amount. Application of two different normalization models effectively reduced the intermediate precision coefficient of variance to <16.5%. The SARS-CoV-2 microneutralization assay described and evaluated here is based on the influenza virus microneutralization assay described by WHO, and are proposed as a standard assay for comparing neutralization investigations.

Cytokine autoantibodies are stable throughout the haematopoietic stem cell transplantation course and are associated with distinct biomarker and blood cell profiles.

Cytokine-specific autoantibodies (c-aAbs) represent an emerging field in endogenous immunodeficiencies, and the immunomodulatory potential of c-aAbs is now well documented. Here, we investigated the hypothesis that c-aAbs affects inflammatory, immunoregulatory and injury-related processes and hence the clinical outcome of haematopoietic stem cell transplantation (HSCT). C-aAbs against IL-1α, IL-6, IL-10, IFNα, IFNγ and GM-CSF were measured in 131 HSCT recipients before and after (days + 7, + 14, + 28) HSCT and tested for associations with 33 different plasma biomarkers, leukocyte subsets, platelets and clinical outcomes, including engraftment, GvHD and infections. We found that c-aAb levels were stable over the course of HSCT, including at high titres, with few individuals seeming to acquire high-titre levels of c-aAbs. Both patients with stable and those with acquired high-titre c-aAb levels displayed significant differences in biomarker concentrations and blood cell counts pre-HSCT and at day 28, and the trajectories of these variables varied over the course of HSCT. No clinical outcomes were associated with high-titre c-aAbs. In this first study of c-aAbs in HSCT patients, we demonstrated that high-titre levels of c-aAb may both persist and emerge in patients over the course of HSCT and may be associated with altered immune biomarkers and cell profiles.

Application of a deep learning-based image analysis and live-cell imaging system for quantifying adipogenic differentiation kinetics of adipose-derived stem/stromal cells.

Quantitative methods for assessing differentiative potency of adipose-derived stem/stromal cells may lead to improved clinical application of this multipotent stem cell, by advancing our understanding of specific processes such as adipogenic differentiation. Conventional cell staining methods are used to determine the formation of adipose areas during adipogenesis as a qualitative representation of adipogenic potency. Staining methods such as oil-red-O are quantifiable using absorbance measurements, but these assays are time and material consuming. Detection methods for cell characteristics using advanced image analysis by machine learning are emerging. Here, live-cell imaging was combined with a deep learning-based detection tool to quantify the presence of adipose areas and lipid droplet formation during adipogenic differentiation of adipose-derived stem/stromal cells. Different detection masks quantified adipose area and lipid droplet formation at different time points indicating kinetics of adipogenesis and showed differences between individual donors. Whereas CEBPA and PPARG expression seems to precede the increase in adipose area and lipid droplets, it might be able to predict expression of ADIPOQ. The applied method is a proof of concept, demonstrating that deep learning methods can be used to investigate adipogenic differentiation and kinetics in vitro using specific detection masks based on algorithm produced from annotation of image data.

Functional immune reconstitution early after allogeneic haematopoietic cell transplantation: A comparison of pre- and post-transplantation cytokine responses in stimulated whole blood.

We aimed to use a novel standardized whole-blood stimulation system to evaluate differences in the functional immune reconstitution in patients early after allogeneic haematopoietic cell transplantation (HCT). Between April and September 2018, 30 patients undergoing HCT had whole blood samples collected around day -21 (day 0 being the day of haematopoietic cell infusion) and day +28. Whole blood was transferred to TruCulture assays comprising prefilled incubation tubes with cell culture medium and a standardized stimulus. We used a panel of four stimuli (lipopolysaccharide, resiquimod, heat-killed Candida albicans and polyinosinic:polycytidylic acid) and a blank, designed to evaluate the function of critical extra- and intracellular immunological signalling pathways. For each stimulus, the cytokine response was assessed by the concentration of interferon-γ, interleukin (IL)-12p40, IL-10, IL-1ß, IL-6, IL-8, IL-10, IL-12p40, IL-17A and tumour necrosis factor-α using a multiplex Luminex assay. Pre-HCT cytokine responses were globally decreased across several different stimuli. Despite patients receiving immunosuppressive prophylaxis at the time, post-HCT cytokine responses were higher and less intercorrelated than pre-HCT responses, also after adjusting for differences in the leukocyte differential counts. For the resiquimod and heat-killed Candida albicans stimuli, we identified a cluster of patients in whom post-HCT responses were lower than average across several cytokines, indicating a possible functional immune deficiency. Our findings suggest that the standardized whole blood stimulation system can be used to reveal heterogeneity in the in vitro cytokine responses to various stimuli after HCT. Larger studies are needed to address if the functional immune reconstitution after HCT can predict the risk of infections.

Estimation of SARS-CoV-2 Infection Fatality Rate by Real-time Antibody Screening of Blood Donors.

BACKGROUND: The pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has tremendous consequences for our societies. Knowledge of the seroprevalence of SARS-CoV-2 is needed to accurately monitor the spread of the epidemic and to calculate the infection fatality rate (IFR). These measures may help the authorities make informed decisions and adjust the current societal interventions. The objective was to perform nationwide real-time seroprevalence surveying among blood donors as a tool to estimate previous SARS-CoV-2 infections and the population-based IFR. METHODS: Danish blood donors aged 17-69 years giving blood 6 April to 3 May were tested for SARS-CoV-2 immunoglobulin M and G antibodies using a commercial lateral flow test. Antibody status was compared between geographical areas, and an estimate of the IFR was calculated. Seroprevalence was adjusted for assay sensitivity and specificity taking the uncertainties of the test validation into account when reporting the 95% confidence intervals (CIs). RESULTS: The first 20 640 blood donors were tested, and a combined adjusted seroprevalence of 1.9% (95% CI, .8-2.3) was calculated. The seroprevalence differed across areas. Using available data on fatalities and population numbers, a combined IFR in patients <70 years is estimated at 89 per 100 000 (95% CI, 72-211) infections. CONCLUSIONS: The IFR was estimated to be slightly lower than previously reported from other countries not using seroprevalence data. The IFR is likely severalfold lower than the current estimate. We have initiated real-time nationwide anti-SARS-CoV-2 seroprevalence surveying of blood donations as a tool in monitoring the epidemic.

Part Three of Three-Part Series: Colorectal Surgery Review for Primary Care Providers.

Malignant colon and rectal disorders must be identified and treated. Timing and indication for diagnostic and screening colonoscopy are extremely important. A high index of suspicion to exclude malignancy is imperative. This paper will focus on the screening for and treatment of colorectal and anal cancers.

Cryopreservation of adipose-derived stromal/stem cells using 1-2% Me2SO (DMSO) in combination with pentaisomaltose: An effective and less toxic alternative to comparable freezing media.

Mesenchymal stromal/stem cells (MSCs) derived from bone marrow, umbilical cord and especially adipose tissue are increasingly being explored for their therapeutic potential to treat a wide variety of diseases. A prerequisite for most allogeneic off-the-shelf and some autologous MSC therapies is the ability to safely and efficiently cryopreserve cells during production or for storage prior to treatment. Dimethyl sulfoxide (Me2SO) is still the commonly used gold standard cryoprotectant (CPA). However, undesirable cellular impacts and side effects of Me2SO have led to an increasing demand for the development of safe and effective alternatives. This study investigated the effect of pentaisomaltose as a CPA for cryopreservation of adipose-derived stromal/stem cells (ASCs). We compared pentaisomaltose-based freezing media containing 1% Me2SO (PIM1) or 2% Me2SO (PIM2) to our in-house freezing media formulation containing 10% Me2SO (STD10) and to CryoStor freezing media containing 2% or 10% Me2SO (CS2 and CS10). We assessed the recovery of viable ASCs, their phenotype, differentiation potential, proliferation potential, and migratory potential. Further, their immunomodulatory potential was assessed by measuring their ability to suppress T cell proliferation and express immunomodulatory markers. The results showed that the post-thaw viability of ASCs cryopreserved with STD10, CS10 and PIM2 was improved compared to that of CS2. The recovery of ASCs with PIM1 and PIM2 was also improved compared to that of CS2. Proliferation and migration were comparable among the tested freezing media. The results showed no difference in the induction of PDL1, PDL2 or IDO1 expression. Nevertheless, the potential of cryopreserved ASCs to suppress T cell proliferation was reduced when the Me2SO concentration was reduced (CS10>STD10>CS2 and PIM2>PIM1). Altogether, the migratory and immunomodulatory potential combined with improved recovery indicate that the addition of pentaisomaltose in the freezing media may allow for the reduction of the Me2SO concentration to 2% while retaining a more potent cell product that what is recovered using comparable freezing media. With the desire to reduce the amount of Me2SO, these results suggest that 2% and potentially even 1% Me2SO in combination with 10% pentaisomaltose could be an effective and less toxic alternative to comparable freezing media.

Adipose-Derived Stromal/Stem Cell Culture: Effects of Different Concentrations of Human Platelet Lysate in Media.

Adipose-derived stromal/stem cells (ASCs) are being tested as a possible treatment for a wide range of diseases to exploit the immunomodulatory and regenerative potential demonstrated in vitro. Pooled human platelet lysate (pHPL) has replaced fetal bovine serum (FBS) as the preferred growth supplement because of its xeno-free origin and improved cell proliferation. Much has been done toward reducing the concentration of pHPL required when expanding ASCs. However, little is known on how increasing the concentration of pHPL affects ASC potency, which could lead to changes with possible beneficial applications. This study investigated the effect of 5, 10, or 20% pHPL in culture media on ASC proliferation and phenotypic marker expression, including chemokine receptors CXCR2, CXCR3, CXCR4, and VLA-4. Adipogenic and osteogenic properties, as well as immunosuppressive properties, including the ability to induce indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and suppress T cell proliferation, were also examined. We observed a significant increase in cell yield (approximately 2-fold) and a corresponding reduction in population doubling time and cell volume when doubling the concentration of pHPL in the growth media. ASCs maintained expression of phenotypic surface markers CD73, CD90, and CD105 and were negative for CD45 and CD31. The ability to induce IDO1 and suppress T cell proliferation was observed as well. Adipogenesis and osteogenesis, however, seem to be increased at higher concentrations of pHPL (20% > 10% > 5%), while expression of chemokine receptors CXCR2 and CXCR3 was lower. In conclusion, increasing the pHPL concentration to 20% could be used to optimize culture conditions when producing cells for clinical treatments and may even be used to enhance beneficial ASC properties depending on the desired therapeutic effect.

Ventilator Alarms in Intensive Care Units: Frequency, Duration, Priority, and Relationship to Ventilator Parameters.

Ventilator alarms have long been presumed to contribute substantially to the overall alarm burden in the intensive care unit. In a prospective observational study, we determined that each ventilator triggered an alarm cascade of up to 8 separate notifications once every 6 minutes. In 1 intensive care unit with different ventilator manufacturers, the distribution of high-priority alarms was manufacturer dependent with 8.6% of alarms from 1 type and 89.8% of alarms from another type of ventilator. Alarm limits were not a function of patient-specific ventilator settings.

The impact of sanitation interventions on latrine coverage and latrine use: A systematic review and meta-analysis.

BACKGROUND: An estimated 2.4 billion people still lack access to improved sanitation and 946 million still practice open defecation. The World Health Organization (WHO) commissioned this review to assess the impact of sanitation on coverage and use, as part of its effort to develop a set of guidelines on sanitation and health. METHODS AND FINDINGS: We systematically reviewed the literature and used meta-analysis to quantitatively characterize how different sanitation interventions impact latrine coverage and use. We also assessed both qualitative and quantitative studies to understand how different structural and design characteristics of sanitation are associated with individual latrine use. A total of 64 studies met our eligibility criteria. Of 27 intervention studies that reported on household latrine coverage and provided a point estimate with confidence interval, the average increase in coverage was 14% (95% CI: 10%, 19%). The intervention types with the largest absolute increases in coverage included the Indian government's "Total Sanitation Campaign" (27%; 95% CI: 14%, 39%), latrine subsidy/provision interventions (16%; 95% CI: 8%, 24%), latrine subsidy/provision interventions that also incorporated education components (17%; 95% CI: -5%, 38%), sewerage interventions (14%; 95% CI: 1%, 28%), sanitation education interventions (14%; 95% CI: 3%, 26%), and community-led total sanitation interventions (12%; 95% CI: -2%, 27%). Of 10 intervention studies that reported on household latrine use, the average increase was 13% (95% CI: 4%, 21%). The sanitation interventions and contexts in which they were implemented varied, leading to high heterogeneity across studies. We found 24 studies that examined the association between structural and design characteristics of sanitation facilities and facility use. These studies reported that better maintenance, accessibility, privacy, facility type, cleanliness, newer latrines, and better hygiene access were all frequently associated with higher use, whereas poorer sanitation conditions were associated with lower use. CONCLUSIONS: Our results indicate that most sanitation interventions only had a modest impact on increasing latrine coverage and use. A further understanding of how different sanitation characteristics and sanitation interventions impact coverage and use is essential in order to more effectively attain sanitation access for all, eliminate open defecation, and ultimately improve health.

Industrial noise control.

Only option E reduced the overall A-weighted level to less than 90 dBA, and in fact, it reduced it to less than 85 dBA, the level at which OSHA requires a hearing conservation program. Thus, a worker could be exposed to the resulting sound level using option E for up to eight hours. The results of all of the other measures required limited exposure, according to Table 1. Untreated (and without hearing protection), the worker could be exposed to the 112-dBA sound level for only about 15 minutes during the day. Option B would allow the employee to be exposed to the 105-dBA sound level for one hour; Option C of 98 dBA for about 2 hours; and Option D of 91 dBA for about 7 hours. These times assume the worker is not exposed to other high noise levels; otherwise, these have to be accounted for, as well. The best solution depends on how long the parts tumbler is in operation and whether barriers or enclosures are practical. Other solutions may exist, as well. It's always best to have a qualified acoustician evaluate the problem and make recommendations.