Enrolling High-Acuity Emergency General Surgery Patients in a Prospective Longitudinal Cohort Study.

Over three million patients are admitted to hospitals annually with high-acuity conditions mandating emergency abdominal or skin/soft-tissue operation. Patients with these high-acuity emergency general surgery (HA-EGS) diseases experience significant morbidity/mortality, yet the quality of life (QOL) impact on survivors is not well studied. Acuity, transfer patterns, and adverse social determinants of health (SDOH) documented in epidemiologic studies are cited reasons for inability to measure patient-reported outcomes (PROs) among HA-EGS survivors. We conducted a feasibility study to understand facilitators and barriers of conducting a prospective study of changes in QOL after surviving HA-EGS. From September 2019 to April 2021, we collected baseline (pre-admission) and 30/60-days post-surgery data on activities of daily living, depression, self-efficacy, resilience, pain, work limitations, social support, and substance use from patients who enrolled during index hospitalization. 100 patients were consented to participate in the study (71.9% enrollment rate). The retention rate was 65.9% for 30-day calls and 53.8% for 60-day calls. Median time to complete each time point remained under 25 minutes. Patients with a longer length of stay and nicotine users didn't complete their 30-day interview while those with systemic complications didn't complete their 60-day interview. These results set the foundation for future PRO studies.

Microvesicle-Mediated Transfer of DNA Methyltransferase Proteins Results in Recipient Cell Immunosuppression.

INTRODUCTION: Patients with sepsis exhibit significant, persistent immunologic dysfunction. Evidence supports the hypothesis that epigenetic regulation of key cytokines plays an important role in this dysfunction. In sepsis, circulating microvesicles (MVs) containing elevated levels of DNA methyltransferase (DNMT) mRNA cause gene methylation and silencing in recipient cells. We sought to examine the functional role of MV DNMT proteins in this immunologic dysfunction. METHODS: In total, 33 patients were enrolled within 24 h of sepsis diagnosis (23 sepsis, 10 critically ill controls). Blood and MVs were collected on days 1, 3, and 5 of sepsis, and protein was isolated from the MVs. Levels of DNMT protein and activity were quantified. MVs were produced in vitro by stimulating naïve monocytes with lipopolysaccharide. Methylation was assessed using bisulfate site-specific qualitative real-time polymerase chain reaction. RESULTS: The size of MVs in the patients with sepsis decreased from days 1 to 5 compared to the control group. Circulating MVs contained significantly higher levels of DNMT 1 and 3A, protein. We recapitulated the production of these DNMT-containing MVs in vitro by treating monocytes with lipopolysaccharide. We found that exposing naïve monocytes to these MVs resulted in increased promoter methylation of tumor necrosis factor alpha. CONCLUSIONS: An analysis of the isolated MVs revealed higher levels of DNMT proteins in septic patients than those in nonseptic patients. Exposing naïve monocytes to DNMT-containing MVs produced in vitro resulted in hypermethylation of tumor necrosis factor alpha, a key cytokine implicated in postsepsis immunosuppression. These results suggest that DNMT-containing MVs cause epigenetic changes in recipient cells. This study highlights a novel role for MVs in the immune dysfunction of patients with sepsis.

Analyzing Outcomes Among Older Adults With Necrotizing Soft-Tissue Infections in the United States.

BACKGROUND: Necrotizing soft-tissue infections (NSTIs) encompass a group of severe, life-threatening diseases with high morbidity and mortality. Evidence suggests advanced age is associated with worse outcomes. To date, no large data sets exist describing outcomes in older individuals, and risk factor identification is lacking. METHODS: Retrospective data were obtained from the 2015 Medicare 100% sample. Included in the analysis were those aged ≥65 y with a primary diagnosis of an NSTI (gas gangrene, necrotizing fasciitis, cutaneous gangrene, or Fournier's gangrene). Risk factors for in-hospital mortality and discharge disposition were examined. Continuous variables were assessed using central tendency, t-tests, and Wilcoxon rank-sum tests. Categorical variables were assessed using the chi-squared and Fisher's exact tests. Statistical significance was defined as P < 0.05. RESULTS: 1427 patient records were reviewed. 59% of patients were male, and the overall mean age was 75.4±8.6 y. 1385 (97.0%) patients required emergency surgery for their NSTI diagnosis. The overall mortality was 5.3%. Several underlying comorbidities were associated with higher rates of mortality including cancer (OR: 3.50, P = 0.0009), liver disease (OR: 2.97, P = 0.03), and kidney disease (OR: 2.15, P = 0.01). While associated with high in-hospital mortality, these diagnoses were not associated with a difference in the rate of discharge to home compared with skilled nursing or rehab. Overall, patients discharged to skilled nursing facilities or rehab had higher rates of underlying comorbidities than patients who were discharged home (3 or more comorbid illness 84.3% versus 68.6%, P < 0.0001); however, no individual comorbid illness was associated with discharge location. CONCLUSIONS: In our Medicare data set, we identified several medical comorbidities that are associated with increased rates of in-hospital mortality. Patients with underlying cancers had the highest odds of increased mortality. The effect on outcomes of the potentially immunosuppressive cancer treatments in these patients is unknown. These data suggest that patients with underlying illnesses, especially cancer, kidney disease, or liver disease have higher mortalities and are more likely to be discharged to skilled nursing facilities or rehab. It is unclear why these illnesses were associated with these worse outcomes while others including diabetes and heart disease were not. These data suggest that these particular comorbid illnesses may have special prognostic implications, although further analysis is necessary to identify the causative factors.

Outcomes of Patients with Necrotizing Soft Tissue Infections: A Propensity-Matched Analysis Using the National Inpatient Sample.

Background: Necrotizing soft tissue infections (NSTIs) are severe, rapidly spreading infections with high morbidity and mortality. Attempts to identify risk factors for mortality and morbidity have produced variable results. We hope to determine which factors across the NSTI population impact mortality, morbidities, and discharge disposition. Patients and Methods: Retrospective data from the National Inpatient Sample from 2012-2018 of patients with primary diagnosis of NSTI (gas gangrene, necrotizing faciitis, cutaneous gangrene, or Fournier gangrene) were identified for analysis. A 1:4 greedy match was performed and risk factors for in-hospital mortality and discharge disposition were examined. Continuous variables were assessed using t-tests and Wilcoxon rank sum tests. Categorical variables were assessed using χ2 and Fisher exact tests. Statistical significance was defined as p < 0.05. Results: A total of 6,608 patients were identified. Weighted, this represents 33,040 patients; 32,390 are in the no-mortality cohort and 650 in the mortality cohort. Advanced age group was a risk factor for both in-hospital mortality and morbidity, but not for discharge to a skilled nursing or rehabilitation facility. Having two or more comorbidities was a risk factor for mortality, morbidity, and discharge to skilled nursing or rehabilitation facility. Cancer, liver disease, and kidney disease were predictors of in-hospital mortality. Diabetes mellitus and kidney disease were predictors of experiencing an in-hospital complication. Diabetes mellitus, heart disease, and kidney disease were predictors for discharge to skilled nursing or rehabilitation facility. Conclusions: Necrotizing soft tissue infections are associated with substantial morbidity and mortality. Identifying patients at higher risk for mortality, morbidity, and higher level of care at discharge can help providers properly allocate resources to improve patient outcomes and reduce the financial burden on patients and healthcare facilities. Special attention should be paid to those with existing or acute kidney dysfunction because this was the only comorbidity associated with increased risk mortality, morbidity, and discharge to higher level of care.

Exosomal Transfer of DNA Methyl-Transferase mRNA Induces an Immunosuppressive Phenotype in Human Monocytes.

INTRODUCTION: Survivors of sepsis exhibit persistent immunosuppression. Epigenetic events may be responsible for some of these immunosuppressive changes. During sepsis circulating exosomes contain large quantities of DNA methyltransferase (DNMT) mRNAs. We hypothesized that exosomes directly transfer DNMT mRNAs to recipient monocytes with resultant methylation events and immunosuppression. METHODS: Exosomes containing DNMT mRNA were generated by stimulating monocytes with LPS. Confocal microscopy was used to determine uptake kinetics in the presence of pharmacologic inhibition. Expression and packaging of specific DNMT mRNA was controlled using DNMT siRNAs. Whole genome and gene specific methylation was assessed using bisulfite sequencing. Ingenuity pathway analysis was performed to determine the biological function of significance of differentially methylated regions. RESULTS: Exosomes effectively transferred DNMT mRNA to recipient monocytes. Pharmacologic inhibition of exosome uptake prevented this increase in DNMT mRNA expression. Recipient monocytes exhibited hypermethylation changes and gene suppression. siRNAs decreased the packaging of DNMT mRNAs and prevented TNFα gene suppression, restoring immunocompetence. CONCLUSION: These data support a role for exosome-mediated transfer of DNMT mRNA with resultant methylation and gene silencing. Pharmacologic uptake inhibition or targeted siRNA mediated DNMT gene silencing prevented DNMT mRNA transfer and maintained the cell's ability to express TNFα in response to LPS. This highlights the potential therapeutic value of targeting these exosome-mediated epigenetic events to maintain the host immune response during sepsis.

Proteomic Pathway Analysis of Monocyte-Derived Exosomes during Surgical Sepsis Identifies Immunoregulatory Functions.

Background: Patients with sepsis exhibit significant long-term immunosuppressive sequelae. Monocyte dysfunction is a hallmark of this damage. Circulating exosomes are an important mediator of the systemic signaling events that occur during the septic response; thus, we sought to characterize the contribution of circulating exosomes to the inflammatory process induced during sepsis Methods: Monocyte-derived exosomes were isolated from cultured monocytes from healthy adult donors via stimulation with lipopolysaccharide (LPS) or phosphate-buffered saline (PBS). The proteome was determined by capillary-liquid chromatography-nanospray tandem mass spectrometry (capillary-LC/NT/MS). Using pathway analysis, proteomic networks of exosomes derived from LPS-stimulated monocytes were compared with those isolated from patients with surgical sepsis. Naïve monocytes were then treated with these exosomes and stimulated with LPS to determine the effects on recipient-cell immune function. Results: Proteomic analysis demonstrated 18 differentially expressed proteins (17 down-regulated, one up-regulated) in sepsis-derived exosomes, with 15 differentially expressed proteins (14 down-regulated, one up-regulated) in the LPS-stimulated exosomes. Functional enrichment analysis demonstrated several down-regulated processes, including localization, biogenesis, and metabolic and cellular processes in addition to immune system processes. In LPS-stimulated macrophages, similar down-regulated processes were seen, including metabolic and cellular processes, as well as the response to stimulus. Cells treated with sepsis-derived exosomes or exosomes from LPS-stimulated monocytes demonstrated significant reductions in tumor necrosis factor (TNF)-α generation in response to LPS stimulation. Conclusions: Proteomic analysis of sepsis-derived exosomes and LPS-stimulated, macrophage-derived exosomes exhibited down-regulation of several important protein networks, including the immune response. In addition, human monocytes treated with exosomes from patients with sepsis or LPS-stimulated monocytes demonstrated significant reductions in TNF-α generation in response to LPS stimulation. These data suggest the contribution of circulating exosomes to systemic signaling and immunomodulation during sepsis.