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Background Clinical and biologic phenotypes of sepsis are proposed in human studies, yet it is unknown whether prognostic or drug response phenotypes are present in animal models of sepsis. Using a biotelemetry-enhanced, murine cecal ligation and puncture (CLP) model, we determined phenotypes of polymicrobial sepsis prior to physiologic deterioration, and the association between phenotypes and outcome in a randomized trial of prompt or delayed antibiotics and fluids. Methods We performed a secondary analysis of male C57BL/6J mice in two observational cohorts and two randomized, laboratory animal experimental trials. In cohort 1, mice (n = 118) underwent biotelemetry-enhanced CLP, and we applied latent class mixed models to determine optimal number of phenotypes using clinical data collected between injury and physiologic deterioration. In cohort 2 (N = 73 mice), inflammatory cytokines measured at 24 h after deterioration were explored by phenotype. In a subset of 46 mice enrolled in two trials from cohort 1, we tested the association of phenotypes with the response to immediate (0 h) vs. delayed (2 to 4 h) antibiotics or fluids initiated after physiologic deterioration. Results Latent class mixture modeling derived a two-class model in cohort 1. Class 2 (N = 97) demonstrated a shorter time to deterioration (mean SD 7.3 (0.9) vs. 9.7 (3.2) h, p p p = 0.75). In cohort 2 used for biomarker measurement, class 2 mice had greater plasma concentrations of IL6 and IL10 at 24 h after CLP (p = 0.05). In pilot randomized trials, the effects of sepsis treatment (immediate vs. delayed antibiotics) differed by phenotype (p = 0.03), with immediate treatment associated with greater survival in class 2 mice only. Similar differential treatment effect by class was observed in the trial of immediate vs. delayed fluids (p = 0.02). Conclusions We identified two sepsis phenotypes in a murine cecal ligation and puncture model, one of which is characterized by faster deterioration and more severe inflammation. Response to treatment in a randomized trial of immediate versus delayed antibiotics and fluids differed on the basis of phenotype.

Background: There is wide heterogeneity in sepsis in causative pathogens, host response, organ dysfunction, and outcomes. Clinical and biologic phenotypes of sepsis are proposed, but the role of pathogen data on sepsis classification is unknown.Methods: We conducted a secondary analysis of the Recombinant Human Activated Protein C (rhAPC) Worldwide Evaluation in Severe Sepsis (PROWESS) Study. We used latent class analysis (LCA) to identify sepsis phenotypes using, (i) only clinical variables (“host model”) and, (ii) combining clinical with microbiology variables (e.g., site of infection, culture-derived pathogen type, and anti-microbial resistance characteristics, “host-pathogen model”). We describe clinical characteristics, serum biomarkers, and outcomes of host and host-pathogen models. We tested the treatment effects of rhAPC by phenotype using Kaplan-Meier curves.Results: Among 1,690 subjects with severe sepsis, latent class modeling derived a 4-class host model and a 4-class host-pathogen model. In the host model, alpha type (N = 327, 19%) was younger and had less shock; beta type (N=518, 31%) was older with more comorbidities; gamma type (N = 532, 32%) had more pulmonary dysfunction; delta type (N = 313, 19%) had more liver, renal and hematologic dysfunction and shock. After the addition of microbiologic variables, 772 (46%) patients changed phenotype membership, and the median probability of phenotype membership increased from 0.95 to 0.97 (P < 0.01). When microbiology data were added, the contribution of individual variables to phenotypes showed greater change for beta and gamma types. In beta type, the proportion of abdominal infections (from 20 to 40%) increased, while gamma type patients had an increased rate of lung infections (from 50 to 78%) with worsening pulmonary function. Markers of coagulation such as d-dimer and plasminogen activator inhibitor (PAI)-1 were greater in the beta type and lower in the gamma type. The 28 day mortality was significantly different for individual phenotypes in host and host-pathogen models (both P < 0.01). The treatment effect of rhAPC obviously changed in gamma type when microbiology data were added (P-values of log rank test changed from 0.047 to 0.780).Conclusions: Sepsis host phenotype assignment was significantly modified when microbiology data were added to clinical variables, increasing cluster cohesiveness and homogeneity.

Sepsis is defined as a dysregulated host response to infection that leads to life-threatening acute organ dysfunction. It afflicts approximately 50 million people worldwide annually and is often deadly, even when evidence-based guidelines are applied promptly. Many randomized trials tested therapies for sepsis over the past 2 decades, but most have not proven beneficial. This may be because sepsis is a heterogeneous syndrome, characterized by a vast set of clinical and biologic features. Combinations of these features, however, may identify previously unrecognized groups, or "subclasses" with different risks of outcome and response to a given treatment. As efforts to identify sepsis subclasses become more common, many unanswered questions and challenges arise. These include: 1) the semantic underpinning of sepsis subclasses, 2) the conceptual goal of subclasses, 3) considerations about study design, data sources, and statistical methods, 4) the role of emerging data types, and 5) how to determine whether subclasses represent "truth." We discuss these challenges and present a framework for the broader study of sepsis subclasses. This framework is intended to aid in the understanding and interpretation of sepsis subclasses, provide a mechanism for explaining subclasses generated by different methodologic approaches, and guide clinicians in how to consider subclasses in bedside care.

Purpose/Objective(s) Tumor Treating Fields (TTFields) therapy is a clinically applied loco-regional, non-invasive, anti-mitotic treatment modality delivering to the tumor low intensity (1-3 V/cm) alternating electric fields (100-300 kHz). While TTFields therapy has demonstrated proven efficacy in management of glioblastoma and malignant pleural mesothelioma, tumor recurrence still occurs in some patients. In order to anticipate potential molecular mechanisms of resistance to TTFields, we developed TTFields-resistant cancer cell lines, and analyzed the changes in their signaling pathways. We also examined the in vitro efficacy of TTFields when combined with agents targeting the molecular candidates that possibly confer resistance to TTFields. Materials/Methods To evaluate the mechanisms of cellular resistance to TTFields, we developed resistant ovarian (A2780), glioma (U-87-MG), and non-small cell lung carcinoma (H1299) cancer cell lines through continued long-term application of TTFields (one or two weeks, depending on the cell line), without treatment breaks. We then assessed the concomitant changes in cellular signaling pathways using an immunoassay followed by Western blot analysis. Finally, we characterized the mechanism of resistance by examining the in vitro efficacy of continued long-term application of TTFields in combination with pharmacological inhibitors including a PI3K/mTOR dual inhibitor (BGT226), and Pan- and isoform-specific inhibitors of PI3K (alpelisib, pictilisib, and BKM120), using cell count, apoptosis, and colony formation assays. Results Continued long-term application of TTFields resulted in reduced sensitivity to TTFields in all tested cell lines. Immunoassay and Western blot analysis showed significant activation of the PI3K/AKT/mTOR signaling pathway in response to TTFields treatment. The combination therapy of TTFields with either alpelisib, pictilisib, BKM120 or BGT226, inhibited AKT S473 phosphorylation and sensitized cancer cells to “long-term” TTFields application. Conclusion Our study demonstrates that the PI3K/ AKT/ mTOR signaling pathway confers resistance to TTFields. This provides a rationale for the combined targeting of PI3K in patients treated with TTFields.

Background: Patients with acute illness who receive intravenous (IV) fluids prior to hospital arrival may have a lower in-hospital mortality. To better understand whether this is a direct treatment effect or epiphenomenon of downstream care, we tested the association between a prehospital fluid bolus and the change in inflammatory cytokines measured at prehospital and emergency department timepoints in a sample of non-trauma, non-cardiac arrest patients at risk for critical illness. Methods: In a prospective cohort study, we screened 4,013 non-trauma, non-cardiac arrest encounters transported by City of Pittsburgh Emergency Medical Services (EMS) to 2 hospitals from August 2013 to February 2014. In 345 patients, we measured prehospital biomarkers (IL-6, IL-10, and TNF) at 2 time points: the time of prehospital IV access placement by EMS and at ED arrival. We determined the relative change for marker X as: ([XED – XEMS]/XEMS). We determined the risk-adjusted association between prehospital IV fluid bolus and relative change for each marker using multivariable linear regression. Results: Among 345 patients, 88 (26%) received a prehospital IV fluid bolus and 257 (74%) did not. Compared to patients who did not receive prehospital fluids, median prehospital IL-6 was greater initially in subjects receiving a prehospital IV fluid bolus (22.3 [IQR 6.4–113] vs. 11.5 [IQR 5.5–47.6]). Prehospital IL-10 and TNF were similar in both groups (IL-10: 3.5 [IQR 2.2–25.6] vs. 3.0 [IQR 1.9–9.0]; TNF: 7.5 [IQR 6.4–10.4] vs. 6.9 [IQR 6.0–8.3]). After adjustment for demographics, illness severity, and prehospital transport time, we observed a relative decrease in IL-6 at hospital arrival in those receiving a prehospital fluid bolus (adjusted β = −10.0, 95% CI: −19.4, −0.6, p = 0.04), but we did not detect a significant change in IL-10 (p = 0.34) or TNF (p = 0.53). Conclusions: Among non-trauma, non-cardiac arrest patients at risk for critical illness, a prehospital IV fluid bolus was associated with a relative decrease in IL-6, but not IL-10 or TNF.

Background: Numerous techniques exist for repairing large congenital diaphragmatic hernias (CDHs) including prosthetic patches, tissue-engineered grafts, and various muscle flaps. A split abdominal wall muscle flap is a simple, durable way to repair a large diaphragmatic hernia. This technique has not gained widespread use, and some have suggested that it would be inappropriate in the setting of extracorporeal membrane oxygenation (ECMO) because of bleeding risk. We present our series of diaphragmatic hernias with a focus on those repaired with the split abdominal wall technique while on ECMO. Methods: A retrospective, single-institution chart review was performed on all patients who underwent surgical repair for CDH over 6 years beginning in August 2000. Results: Seventy-five patients underwent repair. Sixteen were performed with patients on ECMO. Of these, 4 were closed primarily, 7 used a prosthetic patch, and 5 used a split abdominal wall muscle flap. Two patients in the prosthetic group developed a recurrent hernia, and 2 required reoperation for bleeding while on ECMO. No reoperations for bleeding were required in the abdominal muscle flap group. Conclusions: The split abdominal wall muscle flap can be safely performed on anticoagulated patients. We believe it is a practical option for repairing large CDHs.

The amyloid precursor protein (APP) is cleaved by two enzymes, beta-secretase and gamma-secretase, to generate the pathological amyloid beta (Abeta) peptide. Expression of familial Alzheimer's disease (FAD) mutants of APP in primary neurons causes both intracellular accumulation of the C-terminal beta-secretase cleavage product of APP and increased secretion of Abeta, and eventually results in apoptotic death of the cells. To determine whether either of these two processing products of APP is involved in this apoptotic pathway, we first modeled experimentally the accumulation of the beta-secretase cleavage product in neurons. The C-terminal 100 amino acids (C100) of APP, with and without a signal peptide, was expressed in cells via recombinant herpes simplex virus (HSV) vectors. Both transgene products were targeted to the membrane, and both caused apoptosis in the neurons, implicating the beta-secretase cleavage product of APP in apoptosis caused by FAD APPs. Expression in neurons of a mutant of FAD APP that inhibited beta-secretase cleavage inhibited its ability to cause apoptosis. However, expression in neurons of a mutant of FAD APP that inhibited gamma-secretase cleavage did not inhibit the ability of this mutant to cause apoptosis. These data suggested that the C-terminal beta-secretase cleavage product of APP, but not Abeta, mediates the apoptosis caused by FAD mutants of APP. Consistent with this hypothesis, C31, which is generated from the beta-secretase cleavage product, itself caused neuronal apoptosis. Inhibitors of caspases 3, 6 and 8, but not of caspase 9, inhibited the apoptosis caused by FAD mutants of APP. It may be inferred from these data that beta-secretase cleavage of FAD mutants of APP allows the appropriate caspase access to its site of action to produce C31, which directly causes neuronal apoptosis.

Isolated mycotic common iliac artery aneurysms are rare, and as such, there is no consensus opinion on management. Traditional surgical options include resection with extra-anatomic bypass, placement of allograft or antibiotic treated synthetic graft, or autogenous vein reconstruction. We report the case of a 46-year-old, human immunodeficiency virus-positive male who presented with a recurrent pneumonia and new onset of right lower quadrant abdominal pain associated with right lower extremity swelling. computed tomographic scan revealed an isolated 9.5 cm right common iliac artery aneurysm with no evidence of rupture. Preoperative blood cultures grew out Streptococcus pneumoniae. Operative repair included aneurysm resection and reconstruction using an autogenous femoropopliteal vein interposition graft from the ipsilateral thigh. The patient had an uneventful recovery with resolution of his lower extremity swelling and a normal duplex exam at follow-up. Large mycotic common iliac artery aneurysms can be successfully treated with aneurysm resection and reconstruction using an autogenous femoropopliteal vein conduit. This technique obviates the need for extra-anatomic bypass or other forms of reconstruction using prosthetic material.