Patients taking benralizumab, dupilumab, or mepolizumab have lower postvaccination SARS-CoV-2 immunity.

BACKGROUND: Biologic therapies inhibiting the IL-4 or IL-5 pathways are very effective in the treatment of asthma and other related conditions. However, the cytokines IL-4 and IL-5 also play a role in the generation of adaptive immune responses. Although these biologics do not cause overt immunosuppression, their effect in primary severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunization has not been studied completely. OBJECTIVE: Our aim was to evaluate the antibody and cellular immunity after SARS-CoV-2 mRNA vaccination in patients on biologics (PoBs). METHODS: Patients with severe asthma or atopic dermatitis who were taking benralizumab, dupilumab, or mepolizumab and had received the initial dose of the 2-dose adult SARS-CoV-2 mRNA vaccine were enrolled in a prospective, observational study. As our control group, we used a cohort of immunologically healthy subjects (with no significant immunosuppression) who were not taking biologics (NBs). We used a multiplexed immunoassay to measure antibody levels, neutralization assays to assess antibody function, and flow cytometry to quantitate Spike-specific lymphocytes. RESULTS: We analyzed blood from 57 patients in the PoB group and 46 control subjects from the NB group. The patients in the PoB group had lower levels of SARS-CoV-2 antibodies, pseudovirus neutralization, live virus neutralization, and frequencies of Spike-specific B and CD8 T cells at 6 months after vaccination. In subgroup analyses, patients with asthma who were taking biologics had significantly lower pseudovirus neutralization than did subjects with asthma who were not taking biologics. CONCLUSION: The patients in the PoB group had reduced SARS-CoV-2-specific antibody titers, neutralizing activity, and virus-specific B- and CD8 T-cell counts. These results have implications when considering development of a more individualized immunization strategy in patients who receive biologic medications blocking IL-4 or IL-5 pathways.

Implementation of an EMS protocol to improve prehospital sepsis recognition.

PURPOSE: Optimal sepsis outcomes are achieved when sepsis is recognized early. Recognizing sepsis in the prehospital, EMS setting can be challenging and unreliable. The purpose of this study is to evaluate whether implementation of an EMS sepsis screening and prehospital alert protocol called PRESS (PREhospital SepsiS) is associated with improved sepsis recognition by EMS providers. DESIGN: We conducted a 12-month, before-after implementation study of the PRESS protocol in a large, public EMS system. The study intervention was a PRESS training program delivered to EMS providers. EMS patient inclusion criteria included: age ≥ 18 years, EMS systolic blood pressure < 110 mmHg, EMS heart rate > 90 bpm, and EMS respiratory rate > 20 bpm. Study exclusion criteria included the presence of any of following EMS conditions: trauma, cardiac arrest, pregnancy, toxic ingestion, or psychiatric emergency. Retrospective chart review was performed on all eligible EMS encounters during the study period. The primary outcome variable was the proportion of patients with sepsis who were identified by EMS providers. RESULTS: Approximately 300 EMS providers were trained to use PRESS. A total of 498 patient encounters met criteria for study inclusion; 222 were excluded, primarily due to trauma. A total of 276 patient encounters were analyzed. Sepsis recognition by EMS providers increased from 12% pre-PRESS protocol to 59% post-PRESS protocol (p < 0.001). In a post-hoc analysis of the post-PRESS cohort, septic patients who were identified by EMS received antibiotics 24 min faster than septic patients who were not identified by EMS [28 min (IQR 18-48) vs 52 (IQR 27-98), respectively, p = 0.021]. CONCLUSION: Implementation of an EMS sepsis screening and prehospital alert protocol was associated with an increase in sepsis recognition rates by EMS providers and a decrease in time to first antibiotic administration in the emergency department. Further studies are needed to evaluate the impact of this protocol in other populations.

Effect of Vitamin C, Thiamine, and Hydrocortisone on Ventilator- and Vasopressor-Free Days in Patients With Sepsis: The VICTAS Randomized Clinical Trial.

Importance: Sepsis is a common syndrome with substantial morbidity and mortality. A combination of vitamin C, thiamine, and corticosteroids has been proposed as a potential treatment for patients with sepsis. Objective: To determine whether a combination of vitamin C, thiamine, and hydrocortisone every 6 hours increases ventilator- and vasopressor-free days compared with placebo in patients with sepsis. Design, Setting, and Participants: Multicenter, randomized, double-blind, adaptive-sample-size, placebo-controlled trial conducted in adult patients with sepsis-induced respiratory and/or cardiovascular dysfunction. Participants were enrolled in the emergency departments or intensive care units at 43 hospitals in the United States between August 2018 and July 2019. After enrollment of 501 participants, funding was withheld, leading to an administrative termination of the trial. All study-related follow-up was completed by January 2020. Interventions: Participants were randomized to receive intravenous vitamin C (1.5 g), thiamine (100 mg), and hydrocortisone (50 mg) every 6 hours (n = 252) or matching placebo (n = 249) for 96 hours or until discharge from the intensive care unit or death. Participants could be treated with open-label corticosteroids by the clinical team, with study hydrocortisone or matching placebo withheld if the total daily dose was greater or equal to the equivalent of 200 mg of hydrocortisone. Main Outcomes and Measures: The primary outcome was the number of consecutive ventilator- and vasopressor-free days in the first 30 days following the day of randomization. The key secondary outcome was 30-day mortality. Results: Among 501 participants randomized (median age, 62 [interquartile range {IQR}, 50-70] years; 46% female; 30% Black; median Acute Physiology and Chronic Health Evaluation II score, 27 [IQR, 20.8-33.0]; median Sequential Organ Failure Assessment score, 9 [IQR, 7-12]), all completed the trial. Open-label corticosteroids were prescribed to 33% and 32% of the intervention and control groups, respectively. Ventilator- and vasopressor-free days were a median of 25 days (IQR, 0-29 days) in the intervention group and 26 days (IQR, 0-28 days) in the placebo group, with a median difference of -1 day (95% CI, -4 to 2 days; P = .85). Thirty-day mortality was 22% in the intervention group and 24% in the placebo group. Conclusions and Relevance: Among critically ill patients with sepsis, treatment with vitamin C, thiamine, and hydrocortisone, compared with placebo, did not significantly increase ventilator- and vasopressor-free days within 30 days. However, the trial was terminated early for administrative reasons and may have been underpowered to detect a clinically important difference. Trial Registration: ClinicalTrials.gov Identifier: NCT03509350.

Prehospital recognition of severe sepsis: development and validation of a novel EMS screening tool.

OBJECTIVE: To derive and validate a predictive model and novel emergency medical services (EMS) screening tool for severe sepsis (SS). DESIGN: Retrospective cohort study. SETTING: A single EMS system and an urban, public hospital. PATIENTS: Sequential adult, nontrauma, nonarrest, at-risk, EMS-transported patients between January 1, 2011, and December 31, 2012 were included in the study. At-risk patients were defined as having all 3 of the following criteria present in the EMS setting: (1) heart rate greater than 90 beats/min, (2) respiratory rate greater than 20 beats/min, and (3) systolic blood pressure less than 110 mm Hg. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among 66,439 EMS encounters, 555 met the criteria for analysis. Fourteen percent (n = 75) of patients had SS, of which 19% (n = 14) were identified by EMS clinical judgment. In-hospital mortality for patients with SS was 31% (n = 23). Six EMS characteristics were found to be predictors of SS: older age, transport from nursing home, Emergency Medical Dispatch (EMD) 9-1-1 chief concern category of "sick person," hot tactile temperature assessment, low systolic blood pressure, and low oxygen saturation. The final predictive model showed good discrimination in derivation and validation subgroups (areas under curves, 0.843 and 0.820, respectively). Sensitivity of the final model was 91% in the derivation group and 78% in the validation group. At a predefined threshold of 2 or more points, prehospital severe sepsis (PRESS) score sensitivity was 86%. CONCLUSIONS: The PRESS score is a novel EMS screening tool for SS that demonstrates a sensitivity of 86% and a specificity of 47%. Additional validation is needed before this tool can be recommended for widespread clinical use.

Navigating the institutional review board approval process in a multicenter observational critical care study.

OBJECTIVE: To characterize variation in the institutional review board application process of a multicenter, observational critical care study. DESIGN, SETTING, AND SUBJECTS: Survey analysis of 36 investigators who applied for participation in the United States Critical Illness and Injury Trials Group: Critical Illness and Outcomes Study, an observational study of 69 adult ICUs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Analysis of investigator-specific characteristics, institutional review board process, application and approval dates, and level of difficulty in obtaining approval. Surveys were analyzed from 36 sites (95%) that applied for institutional review board approval. Level of review ranged from full board, expedited, to exempt. Seventy-five percent of applications were submitted by an experienced investigator while 25% were submitted by a less experienced investigator. Median time to institutional review board approval was 30 days (interquartile range, 14-54) and ranged from 5 days to 5.5 months. Time to approval was 29 days (interquartile range, 17-48) for applications submitted by an experienced investigator compared with 97 days (interquartile range, 25-159) for those submitted by a less experienced investigator (p = 0.08). Subjective level of difficulty was significantly higher for less experienced investigators (4 of 10; interquartile range, 2-8) vs experienced investigators (2 of 10; interquartile range, 1-3) (p = 0.04). Four sites cited institutional review board concern regarding waiver of consent as a major barrier to approval and were required to perform revisions or participate in board meetings regarding this concern. CONCLUSIONS: In a multicenter, observational critical care study, significant variation was observed between sites in all aspects of the institutional review board evaluation and approval process. The level of difficulty was significantly higher for less experienced investigators with a trend toward longer time to institutional review board approval. Variation in institutional review board interpretation of waiver of informed consent regulations was cited as a major barrier to approval.

Hepatocellular ultrastructure after ischemia/reperfusion injury in human orthotopic liver transplantation.

The number of patients requiring organ transplants still outpaces the number of available transplantable organs. During the process of orthotopic liver transplantation (OLTx), donor organs undergo significant stress resulting from ischemia and reperfusion. Healthy organs respond to this stressful environment with compensatory mechanisms that ideally allow for complete recovery. However, "marginal" organs do not compensate as well. Hepatic steatosis typically renders an organ nontransplantable; a liver with 30% or more fat has a 25% chance of primary nonfunction (PNF) or graft failure after a technically sound operation. In this study, we report on the significant markers of cellular ultrastructural change in steatotic livers. These include glycogen content, mitochondrial swelling, and hepatocellular blebbing. The data disclosed here argue that further investigation of these factors in marginal organs subjected to I/R may better facilitate our understanding of PNF.

Mitochondrial uncoupling protein 2 induces cell cycle arrest and necrotic cell death.

Uncoupling protein 2 (UCP2) is a mitochondrial membrane protein that regulates energy metabolism and reactive oxygen species (ROS) production. We generated mouse carboxy- and amino-terminal green fluorescent protein (GFP)-tagged UCP2 constructs to investigate the effect of UCP2 expression on cell proliferation and viability. UCP2-transfected Hepa 1-6 cells did not show reduced cellular adenosine triphosphate (ATP) but showed increased levels of glutathione. Flow cytometry analysis indicated that transfected cells were less proliferative than nontransfected controls, with most cells blocked at the G1 phase. The effect of UCP2 on cell cycle arrest could not be reversed by providing exogenous ATP or oxidant supply, and was not affected by the chemical uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). However, this effect of UCP2 was augmented by treatment with genistein, a tyrosine kinase inhibitor, which by itself did not affect cell proliferation on control hepatocytes. Western blotting analysis revealed decreased expression levels of CDK6 but not CDK2 and D-type cyclins. Examination of cell viability in UCP2-transfected cells with Trypan Blue and Annexin-V staining revealed that UCP2 transfection led to significantly increased cell death. However, characteristics of apoptosis were absent in UCP2-transfected Hepa 1-6 cells, including lack of oligonucleosomal fragmentation (laddering) of chromosomal DNA, release of cytochrome c from mitochondria, and cleavage of caspase-3. In conclusion, our results indicate that UCP2 induces cell cycle arrest at G1 phase and causes nonapoptotic cell death, suggesting that UCP2 may act as a powerful influence on hepatic regeneration and cell death in the steatotic liver.

Rare Elizabethkingia meningosepticum meningitis case in an immunocompetent adult.

Though Elizabethkingia meningosepticum typically causes meningitis in neonates, its occurrence in adult is rare, with sixteen cases described worldwide. We report a case of E. meningosepticum meningitis in an immunocompetent adult. Bacterial identification was made a day earlier than conventional method by using matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) Vitek mass spectrometry RUO (VMS), which resulted in successful treatment with rifampin, trimethoprim-sulfamethoxazole, levofloxacin and minocycline.

Decrease of intracellular ATP content downregulated UCP2 expression in mouse hepatocytes.

Mitochondrial uncoupling protein 2 (UCP2) plays an important role in regulating energy metabolism. We previously reported that UCP2 expression in steatotic livers is increased which leads to diminished hepatic ATP stores and renders steatotic hepatocytes vulnerable to ischemic damage. In this study, reagents that inhibit the production of ATP were used to mimic an ischemic state in the liver in order to investigate the effects of decreased intracellular ATP levels on UCP2 expression in a murine hepatocyte cell line (HEP6-16). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an oxidative phosphorylation uncoupler, was found to decrease intracellular ATP levels in a dose- and time-dependent manner. Relatively high concentrations of FCCP from 8 to 80 microM were required to reduce the intracellular concentration of ATP. The inhibitory effect of FCCP on intracellular ATP was significantly potentiated by 2-deoxy-D-glucose, an inhibitor of glycolysis that when administered alone had no negative effect on cellular ATP levels in mouse hepatocytes. Decreased intracellular ATP levels were accompanied by lower UCP2 mRNA expression. Upon removal of FCCP and/or 2-deoxy-D-glucose and reculture with normal medium, ATP and UCP2 mRNA levels returned to normal within a few hours. Mitochondrial membrane potential in HEP6-16 cells was dissipated by 80 microM FCCP but not 8 microM FCCP, suggesting that the downregulation of UCP2 expression by FCCP was not related to mitochondrial potential changes. Consequently, the in vitro manipulation of ATP stores is consistent with the in vivo observations associated with ischemia/reperfusion injury.

Development of an unbiased method for the estimation of liver steatosis.

BACKGROUND: Steatosis significantly contributes to an organ's transplantability. Livers with >30% fat content have a 25% chance of developing primary non-function (PNF). The current practice of evaluating a hematoxylin and eosin (H&E) stained donor biopsy by visual interpretation is subjective. We hypothesized that H&E staining of frozen sections fails to accurately estimate the degree of steatosis present within a given liver biopsy. To address this problem of evaluating steatosis in prospective donor organs, we developed a fast, user friendly computer methodology to objectively assess fat content based on the differential quantification of color pixels in Oil Red O (ORO) stained liver biopsies. METHODS: The accuracy of human visual estimation of fat content by H&E and ORO stains was compared with computer-based measurements of the same slides from 25 frozen sections of donor biopsies. RESULTS: Samples with a fat content >20% showed marked variation between human interpretation and computer analysis. There was also a significant difference in the human interpretation of fat based on the method of staining. This difference ranged from 3 to 37% with H&E. DISCUSSION: Use of ORO resulted in a more consistent estimation of liver steatosis compared with H&E, but human interpretations failed to correlate with computer measurements. Such differences in fat content estimations might result in the rejection of a potentially transplantable organ or the acceptance of a marginal one. Ideally, our protocol can rapidly be applied to clinical practice for accurate and consistent measurement of fat in liver sections for the ultimate purpose of increasing the number of successful transplantable organs.

Steatotic liver transplantation in the mouse: a model of primary nonfunction.

BACKGROUND: The number of potential donor organs deemed suboptimal for transplantation because of hepatic steatosis is rising as the obesity rate increases. However, no mouse transplant model has been described within the framework of hepatic steatosis. We describe the development of and our initial experience with a steatotic mouse orthotopic liver transplant model using the ob/ob mouse. This model is technically achievable and functionally mimics primary nonfunction. MATERIALS AND METHODS: Adapting techniques of a nonarterialized murine transplant model, C57BL6 ob/ob mice aged 5-7 weeks (26-35 g) and lean controls served as liver donors and recipients. Orthotopic liver transplantation (OLT) was performed using a two-cuff technique at the infrahepatic cava and portal vein. The suprahepatic cava was anastomosed end to end, and the bile duct was stented. The hepatic artery was not reconstructed. RESULTS: Lean-to-lean OLT was performed with 70% (n = 10) long-term survival. ob/ob-to-age-matched lean recipients had 0% (n = 10) survival because of size discrepancy. ob/ob livers were transplanted to size-matched lean recipients (>3 months old) with short-term survival of 30% (n = 10). These mice survived the operation, awakened, but expired within 24 h. Serum transaminases revealed a significantly higher injury profile in the recipients of the steatotic livers, and histology showed massive centrilobular coagulative necrosis with hemorrhage, the overall picture being that of primary nonfunction. CONCLUSIONS: This novel use of the ob/ob mouse for OLT provides us with a model for steatotic transplantation with primary nonfunction as the end point and may help to better understand the response of the steatotic liver to the insult of transplantation.

Fatty acid synthase blockade protects steatotic livers from warm ischemia reperfusion injury and transplantation.

Cerulenin has been shown to reduce body weight and hepatic steatosis in murine models of obesity by inhibiting fatty acid synthase (FAS). We have shown that attenuating intrahepatocyte lipid content diminished the sensitivity of ob/ob mice to ischemia/reperfusion injury and improved survival after liver transplantation. The mechanism of action is by inhibition of fatty acid metabolism by downregulating PPARalpha, as well as mitochondrial uncoupling protein 2 (UCP2), with a concomitant increase in ATP. A short treatment course of cerulenin prior to I/R injury is ideal for protection of steatotic livers. Cerulenin opens the potential for expanding the use of steatotic livers in transplantation.

Anti-endotoxin monoclonal antibodies are protective against hepatic ischemia/reperfusion injury in steatotic mice.

Steatotic mice are particularly susceptible to hepatic ischemia/reperfusion injury compared with their lean littermates. We have previously demonstrated that livers of mice having a spontaneous mutation in the leptin gene (ob/ob), resulting in global obesity and liver steatosis, are ATP depleted, are endotoxin sensitive, and do not survive (I/R) injury. We hypothesize that administration of an anti-LPS monoclonal antibody (mAb) prior to initiation of I/R would be protective from that insult. Steatotic mice (ob/ob) were subjected to 15 min of ischemia via complete porta-hepatis occlusion and varying lengths of reperfusion with or without pre-treatment with an anti-LPS mAb. There was 14-31% survival of isotype matched control mAb treated ob/ob mice after 15 min of ischemia and 24 h of reperfusion. In contrast, 75-83% of ob/ob mice pre-treated with an anti-LPS mAb prior to initiation of I/R survived both ischemia and 24 h of reperfusion. Furthermore, there was a decrease in ALT and circulating endotoxin levels when treated with an anti-LPS mAb compared with control antibodies. Attenuation of the endotoxin load with anti-LPS mAb, prior to initiation of I/R, was cytoprotective and improved survival. Consequently, these studies might offer a solution to the problems associated with using steatotic livers in clinical transplantation.