Transcriptomic responses from improved murine sepsis models can better mimic human surgical sepsis.

Historically, murine models of inflammation in biomedical research have been shown to minimally correlate with genomic expression patterns from blood leukocytes in humans. In 2019, our laboratory reported an improved surgical sepsis model of cecal ligation and puncture (CLP) that provides additional daily chronic stress (DCS), as well as adhering to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. This model phenotypically recapitulates the persistent inflammation, immunosuppression, and catabolism syndrome observed in adult human surgical sepsis survivors. Whether these phenotypic similarities between septic humans and mice are replicated at the circulating blood leukocyte transcriptome has not been demonstrated. Our analysis, in contrast with previous findings, demonstrated that genome-wide expression in our new murine model more closely approximated human surgical sepsis patients, particularly in the more chronic phases of sepsis. Importantly, our new model of murine surgical sepsis with chronic stress did not reflect well gene expression patterns from humans with community-acquired sepsis. Our work indicates that improved preclinical murine sepsis modeling can better replicate both the phenotypic and transcriptomic responses to surgical sepsis, but cannot be extrapolated to other sepsis etiologies. Importantly, these improved models can be a useful adjunct to human-focused and artificial intelligence-based forms of research in order to improve septic patients' morbidity and mortality.

Old Mice Demonstrate Organ Dysfunction as well as Prolonged Inflammation, Immunosuppression, and Weight Loss in a Modified Surgical Sepsis Model.

OBJECTIVES: Our goal was to "reverse translate" the human response to surgical sepsis into the mouse by modifying a widely adopted murine intra-abdominal sepsis model to engender a phenotype that conforms to current sepsis definitions and follows the most recent expert recommendations for animal preclinical sepsis research. Furthermore, we aimed to create a model that allows the study of aging on the long-term host response to sepsis. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Young (3-5 mo) and old (18-22 mo) C57BL/6j mice. INTERVENTIONS: Mice received no intervention or were subjected to polymicrobial sepsis with cecal ligation and puncture followed by fluid resuscitation, analgesia, and antibiotics. Subsets of mice received daily chronic stress after cecal ligation and puncture for 14 days. Additionally, modifications were made to ensure that "Minimum Quality Threshold in Pre-Clinical Sepsis Studies" recommendations were followed. MEASUREMENTS AND MAIN RESULTS: Old mice exhibited increased mortality following both cecal ligation and puncture and cecal ligation and puncture + daily chronic stress when compared with young mice. Old mice developed marked hepatic and/or renal dysfunction, supported by elevations in plasma aspartate aminotransferase, blood urea nitrogen, and creatinine, 8 and 24 hours following cecal ligation and puncture. Similar to human sepsis, old mice demonstrated low-grade systemic inflammation 14 days after cecal ligation and puncture + daily chronic stress and evidence of immunosuppression, as determined by increased serum concentrations of multiple pro- and anti-inflammatory cytokines and chemokines when compared with young septic mice. In addition, old mice demonstrated expansion of myeloid-derived suppressor cell populations and sustained weight loss following cecal ligation and puncture + daily chronic stress, again similar to the human condition. CONCLUSIONS: The results indicate that this murine cecal ligation and puncture + daily chronic stress model of surgical sepsis in old mice adhered to current Minimum Quality Threshold in Pre-Clinical Sepsis Studies guidelines and met Sepsis-3 criteria. In addition, it effectively created a state of persistent inflammation, immunosuppression, and weight loss, thought to be a key aspect of chronic sepsis pathobiology and increasingly more prevalent after human sepsis.

Myeloid-derived suppressor cell function and epigenetic expression evolves over time after surgical sepsis.

BACKGROUND: Sepsis is an increasingly significant challenge throughout the world as one of the major causes of patient morbidity and mortality. Central to the host immunologic response to sepsis is the increase in circulating myeloid-derived suppressor cells (MDSCs), which have been demonstrated to be present and independently associated with poor long-term clinical outcomes. MDSCs are plastic cells and potentially modifiable, particularly through epigenetic interventions. The objective of this study was to determine how the suppressive phenotype of MDSCs evolves after sepsis in surgical ICU patients, as well as to identify epigenetic differences in MDSCs that may explain these changes. METHODS: Circulating MDSCs from 267 survivors of surgical sepsis were phenotyped at various intervals over 6 weeks, and highly enriched MDSCs from 23 of these samples were co-cultured with CD3/CD28-stimulated autologous T cells. microRNA expression from enriched MDSCs was also identified. RESULTS: We observed that MDSC numbers remain significantly elevated in hospitalized sepsis survivors for at least 6 weeks after their infection. However, only MDSCs obtained at and beyond 14 days post-sepsis significantly suppressed T lymphocyte proliferation and IL-2 production. These same MDSCs displayed unique epigenetic (miRNA) expression patterns compared to earlier time points. CONCLUSIONS: We conclude that in sepsis survivors, immature myeloid cell numbers are increased but the immune suppressive function specific to MDSCs develops over time, and this is associated with a specific epigenome. These findings may explain the chronic and persistent immune suppression seen in these subjects.

Intraoperative hemidiaphragm electrical stimulation reduces oxidative stress and upregulates autophagy in surgery patients undergoing mechanical ventilation: exploratory study.

BACKGROUND: Mechanical ventilation (MV) during a cardio-thoracic surgery contributes to diaphragm muscle dysfunction that impairs weaning and can lead to the ventilator- induced diaphragm dysfunction. Especially, it is critical in older adults who have lower muscle reparative capacity following MV. Reports have shown that the intraoperative intermittent hemidiaphragm electrical stimulation can maintain and/or improve post-surgery diaphragm function. In particular, from a molecular point of view, intermittent ES may reduce oxidative stress and increase regulatory autophagy levels, and therefore improve diaphragm function in animal studies. We have recently shown in humans that intraoperative ES attenuates mitochondrial dysfunction and force decline in single diaphragm muscle fibers. The aim of this study was to investigate an effect of ES on oxidative stress, antioxidant status and autophagy biomarker levels in the human diaphragm during surgery. METHODS: One phrenic nerve was simulated with an external cardiac pacer in operated older subjects (62.4 ± 12.9 years) (n = 8) during the surgery. The patients received 30 pulses per min every 30 min. The muscle biopsy was collected from both hemidiaphragms and frozen for further analyses. 4-hydroxynonenal (4-HNE), an oxidative stress marker, and autophagy marker levels (Beclin-1 and the ratio of microtubule-associated protein light chain 3, I and II-LC3 II/I) protein concentrations were detected by the western blot technique. Antioxidant enzymatic activity copper-zinc (CuZnSOD) and manganese (MnSOD) superoxide dismutase were analyzed. RESULTS: Levels of lipid peroxidation (4-HNE) were significantly lower in the stimulated side (p < 0.05). The antioxidant enzyme activities (CuZnSOD and MnSOD) in the stimulated side of the diaphragm were not different than in the unstimulated side (p > 0.05). Additionally, the protein concentrations of Beclin-1 and the LC3 II/I ratio were higher in the stimulated side (p < 0.05). CONCLUSION: These results suggest that the intraoperative electrical stimulation decreases oxidative stress levels and upregulates autophagy levels in the stimulated hemidiaphragm. These results may contribute future studies and clinical applications on reducing post-operative diaphragm dysfunction.

The post-septic peripheral myeloid compartment reveals unexpected diversity in myeloid-derived suppressor cells.

Introduction: Sepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness. Methods: Cellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression, RNA velocities, and biologic process clustering. Results: We identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally, we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome. Discussion: The origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however, these cells exhibit a dynamic phenotype with considerable plasticity.

Adverse outcomes and an immunosuppressed endotype in septic patients with reduced IFN-γ ELISpot.

BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.

Adverse Long-Term Outcomes and an Immune Suppressed Endotype in Sepsis Patients with Reduced Interferon-γELISpot: A Multicenter, Prospective Observational Study.

BACKGROUND: Sepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients has generally relied on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision. METHODS: An ex vivo whole blood enzyme-linked immunosorbent (ELISpot) assay for cellular production of interferon-γ (IFN-γ) was evaluated in 107 septic and 68 non-septic patients from five academic health centers using blood samples collected on days 1, 4 and 7 following ICU admission. RESULTS: Compared with 46 healthy subjects, unstimulated and stimulated whole blood IFNγ expression were either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole blood IFNγ expression was significantly reduced on ICU days 1, 4 and 7 (all p<0.05), due to both significant reductions in total number of IFNγ producing cells and amount of IFNγ produced per cell (all p<0.05). Importantly, IFNγ total expression on day 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6 and procalcitonin. Septic patients with low IFNγ expression were older and had lower ALC and higher sPD-L1 and IL-10 concentrations, consistent with an immune suppressed endotype. CONCLUSIONS: A whole blood IFNγ ELISpot assay can both identify septic patients at increased risk of late mortality, and identify immune-suppressed, sepsis patients.

Evaluation of a Multivalent Transcriptomic Metric for Diagnosing Surgical Sepsis and Estimating Mortality Among Critically Ill Patients.

Importance: Rapid and accurate discrimination of sepsis and its potential severity currently require multiple assays with slow processing times that are often inconclusive in discerning sepsis from sterile inflammation. Objective: To analyze a whole-blood, multivalent, host-messenger RNA expression metric for estimating the likelihood of bacterial infection and 30-day mortality and compare performance of the metric with that of other diagnostic and prognostic biomarkers and clinical parameters. Design, Setting, and Participants: This prospective diagnostic and prognostic study was performed in the surgical intensive care unit (ICU) of a single, academic health science center. The analysis included 200 critically ill adult patients admitted with suspected sepsis (cohort A) or those at high risk for developing sepsis (cohort B) between July 1, 2020, and July 30, 2021. Exposures: Whole-blood sample measurements of a custom 29-messenger RNA transcriptomic metric classifier for likelihood of bacterial infection (IMX-BVN-3) or 30-day mortality (severity) (IMX-SEV-3) in a clinical-diagnostic laboratory setting using an analysis platform (510[k]-cleared nCounter FLEX; NanoString, Inc), compared with measurement of procalcitonin and interleukin 6 (IL-6) plasma levels, and maximum 24-hour sequential organ failure assessment (SOFA) scores. Main Outcomes and Measures: Estimated sepsis and 30-day mortality performance. Results: Among the 200 patients included (124 men [62.0%] and 76 women [38.0%]; median age, 62.5 [IQR, 47.0-72.0] years), the IMX-BVN-3 bacterial infection classifier had an area under the receiver operating characteristics curve (AUROC) of 0.84 (95% CI, 0.77-0.90) for discriminating bacterial infection at ICU admission, similar to procalcitonin (0.85 [95% CI, 0.79-0.90]; P = .79) and significantly better than IL-6 (0.67 [95% CI, 0.58-0.75]; P < .001). For estimating 30-day mortality, the IMX-SEV-3 metric had an AUROC of 0.81 (95% CI, 0.66-0.95), which was significantly better than IL-6 levels (0.57 [95% CI, 0.37-0.77]; P = .006), marginally better than procalcitonin levels (0.65 [95% CI, 0.50-0.79]; P = .06), and similar to the SOFA score (0.76 [95% CI, 0.62-0.91]; P = .48). Combining IMX-BVN-3 and IMX-SEV-3 with procalcitonin or IL-6 levels or SOFA scores did not significantly improve performance. Among patients with sepsis, IMX-BVN-3 scores decreased over time, reflecting the resolution of sepsis. In 11 individuals at high risk (cohort B) who subsequently developed sepsis during their hospital course, IMX-BVN-3 bacterial infection scores did not decline over time and peaked on the day of documented infection. Conclusions and Relevance: In this diagnostic and prognostic study, a novel, multivalent, transcriptomic metric accurately estimated the presence of bacterial infection and risk for 30-day mortality in patients admitted to a surgical ICU. The performance of this single transcriptomic metric was equivalent to or better than multiple alternative diagnostic and prognostic metrics when measured at admission and provided additional information when measured over time.

Sex differences associate with late microbiome alterations after murine surgical sepsis.

BACKGROUND: Sepsis-induced gut microbiome alterations contribute to sepsis-related morbidity and mortality. Given evidence for improved postsepsis outcomes in females compared with males, we hypothesized that female mice maintain microbiota resilience versus males. METHODS: Mixed-sex C57BL/6 mice underwent cecal ligation and puncture (CLP) with antibiotics, saline resuscitation, and daily chronic stress and were compared with naive (nonsepsis/no antibiotics) controls. For this work, the results of young (3-5 months) and old (18-22 months) adult mice were analyzed by sex, independent and dependent of age. Mice were sacrificed at days 7 and 14, and 16S rRNA gene sequencing was performed on fecal bacterial DNA. α and ß diversity were determined by Shannon index and Bray-Curtis with principal coordinate analysis, respectively. False discovery rate (FDR) correction was implemented to account for potential housing effect. RESULTS: In control mice, there was no difference in α or ß diversity between male and female mice (FDR, 0.76 and 0.99, respectively). However, male mice that underwent CLP with daily chronic stress had a decrease in microbiota α diversity at 7 days post-CLP (Shannon FDR, 0.005), which was sustained at 14 days post-CLP (Shannon FDR, 0.001), compared with baseline. In addition, male mice maintained differences in ß diversity even at day 14 compared with controls (FDR, <0.0001). In contrast, female mice had a decreased microbiota α diversity (Shannon FDR, 0.03) and ß diversity (FDR, 0.02) 7 days post-CLP but recovered their α and ß diversity by post-CLP day 14 (Shannon FDR, 0.5, and FDR, 0.02, respectively). Further analysis of females revealed that only young female mice were not different (ß diversity) post-CLP day 14 to controls. CONCLUSION: Although sepsis-induced perturbations of the intestinal microbiota occur initially in both male and female C57BL/6 mice, females demonstrate different microbiota by day 14. This may be seen primarily in younger females. This difference in recovery may play a role in outcome differences between sexes after sepsis.

Overlapping but Disparate Inflammatory and Immunosuppressive Responses to SARS-CoV-2 and Bacterial Sepsis: An Immunological Time Course Analysis.

Both severe SARS-CoV-2 infections and bacterial sepsis exhibit an immunological dyscrasia and propensity for secondary infections. The nature of the immunological dyscrasias for these differing etiologies and their time course remain unclear. In this study, thirty hospitalized patients with SARS-CoV-2 infection were compared with ten critically ill patients with bacterial sepsis over 21 days, as well as ten healthy control subjects. Blood was sampled between days 1 and 21 after admission for targeted plasma biomarker analysis, cellular phenotyping, and leukocyte functional analysis via enzyme-linked immunospot assay. We found that circulating inflammatory markers were significantly higher early after bacterial sepsis compared with SARS-CoV-2. Both cohorts exhibited profound immune suppression through 21 days (suppressed HLA-DR expression, reduced mononuclear cell IFN-gamma production), and expanded numbers of myeloid-derived suppressor cells (MDSCs). In addition, MDSC expansion and ex vivo production of IFN-gamma and TNF-alpha were resolving over time in bacterial sepsis, whereas in SARS-CoV-2, immunosuppression and inflammation were accelerating. Despite less severe initial physiologic derangement, SARS-CoV-2 patients had similar incidence of secondary infections (23% vs 30%) as bacterial sepsis patients. Finally, COVID patients who developed secondary bacterial infections exhibited profound immunosuppression evident by elevated sPD-L1 and depressed HLA-DR. Although both bacterial sepsis and SARS-CoV-2 are associated with inflammation and immune suppression, their immune dyscrasia temporal patterns and clinical outcomes are different. SARS-CoV-2 patients had less severe early inflammation and organ dysfunction but had persistent inflammation and immunosuppression and suffered worse clinical outcomes, especially when SARS-CoV-2 infection was followed by secondary bacterial infection.

Septic Stability? Gut Microbiota in Young Adult Mice Maintains Overall Stability After Sepsis Compared to Old Adult Mice.

BACKGROUND: Older adults have worse outcomes after sepsis than young adults. Additionally, alterations of the gut microbiota have been demonstrated to contribute to sepsis-related mortality. We sought to determine if there were alterations in the gut microbiota with a novel sepsis model in old adult mice, which enter a state of persistent inflammation, immunosuppression, and catabolism (PICS), as compared with young adult mice, which recover with the sepsis model. METHODS: Mixed sex old (∼20 mo) and young (∼4 mo) C57Bl/6J mice underwent cecal ligation and puncture with daily chronic stress (CLP+DCS) and were compared with naive age-matched controls. Mice were sacrificed at CLP+DCS day 7 and feces collected for bacterial DNA isolation. The V3-V4 hypervariable region was amplified, 16S rRNA gene sequencing performed, and cohorts compared. α-Diversity was assessed using Chao1 and Shannon indices using rarefied counts, and ß-diversity was assessed using Bray-Curtis dissimilarity. RESULTS: Naive old adult mice had significantly different α and ß-diversity compared with naive adult young adult mice. After CLP+DCS, there was a significant shift in the α and ß-diversity (FDR = 0.03 for both) of old adult mice (naive vs. CLP+DCS). However, no significant shift was displayed in the microbiota of young mice that underwent CLP+DCS in regards to α-diversity (FDR = 0.052) and ß-diversity (FDR = 0.12), demonstrating a greater overall stability of their microbiota at 7 days despite the septic insult. The taxonomic changes in old mice undergoing CLP+DCS were dominated by decreased abundance of the order Clostridiales and genera Oscillospira. CONCLUSION: Young adult mice maintain an overall microbiome stability 7 days after CLP+DCS after compared with old adult mice. The lack of microbiome stability could contribute to PICS and worse long-term outcomes in older adult sepsis survivors. Further studies are warranted to elucidate mechanistic pathways and potential therapeutics.

A Novel Single Cell RNA-seq Analysis of Non-Myeloid Circulating Cells in Late Sepsis.

Background: With the successful implementation of the Surviving Sepsis Campaign guidelines, post-sepsis in-hospital mortality to sepsis continues to decrease. Those who acutely survive surgical sepsis will either rapidly recover or develop a chronic critical illness (CCI). CCI is associated with adverse long-term outcomes and 1-year mortality. Although the pathobiology of CCI remains undefined, emerging evidence suggests a post-sepsis state of pathologic myeloid activation, inducing suboptimal lymphopoiesis and erythropoiesis, as well as downstream leukocyte dysfunction. Our goal was to use single-cell RNA sequencing (scRNA-seq) to perform a detailed transcriptomic analysis of lymphoid-derived leukocytes to better understand the pathology of late sepsis. Methods: A mixture of whole blood myeloid-enriched and Ficoll-enriched peripheral blood mononuclear cells from four late septic patients (post-sepsis day 14-21) and five healthy subjects underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). Results: We identified unique transcriptomic patterns for multiple circulating immune cell subtypes, including B- and CD4+, CD8+, activated CD4+ and activated CD8+ T-lymphocytes, as well as natural killer (NK), NKT, and plasmacytoid dendritic cells in late sepsis patients. Analysis demonstrated that the circulating lymphoid cells maintained a transcriptome reflecting immunosuppression and low-grade inflammation. We also identified transcriptomic differences between patients with bacterial versus fungal sepsis, such as greater expression of cytotoxic genes among CD8+ T-lymphocytes in late bacterial sepsis. Conclusion: Circulating non-myeloid cells display a unique transcriptomic pattern late after sepsis. Non-myeloid leukocytes in particular reveal a host endotype of inflammation, immunosuppression, and dysfunction, suggesting a role for precision medicine-guided immunomodulatory therapy.

Single-Cell RNA-seq of Human Myeloid-Derived Suppressor Cells in Late Sepsis Reveals Multiple Subsets With Unique Transcriptional Responses: A Pilot Study.

BACKGROUND: Increased circulating myeloid-derived suppressor cells (MDSCs) are independently associated with poor long-term clinical outcomes in sepsis. Studies implicate subsets of MDSCs having unique roles in lymphocyte suppression; however, characterization of these cells after sepsis remains incomplete. We performed a pilot study to determine the transcriptomic landscape in MDSC subsets in sepsis using single-cell RNAseq (scRNA-seq). METHODS: A mixture of whole blood myeloid-enriched and Ficoll-enriched PBMCs from two late septic patients on post-sepsis day 21 and two control subjects underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). RESULTS: We successfully identified the three MDSC subset clusters-granulocytic (G-), monocytic (M-), and early (E-) MDSCs. Sepsis was associated with a greater relative expansion of G-MDSCs versus M-MDSCs at 21 days as compared to control subjects. Genomic analysis between septic patients and control subjects revealed cell-specific and common differential expression of genes in both G-MDSC and M-MDSC subsets. Many of the common genes have previously been associated with MDSC proliferation and immunosuppressive function. Interestingly, there was no differential expression of several genes demonstrated in the literature to be vital to immunosuppression in cancer-induced MDSC. CONCLUSION: This pilot study successfully demonstrated that MDSCs maintain a transcriptomic profile that is immunosuppressive in late sepsis. Interestingly, the landscape in chronic critical illness is partially dependent on the original septic insult. Preliminary data would also indicate immunosuppressive MDSCs from late sepsis patients appear to have a somewhat unique transcriptome from cancer and/or other inflammatory diseases.

Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist.

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.

The effect of backbone cyclization on PK/PD properties of bioactive peptide-peptoid hybrids: the melanocortin agonist paradigm.

A peptide-peptoid hybrid (peptomer) library was designed and synthesized, based on the sequence Phe-d-Phe-Arg-Trp-Gly. This sequence was previously found to specifically activate the melanocortin-4-receptor (MC4R) which participates in regulation of energy homeostasis and appetite. The library of peptomers included a peptoid bond in the Phe and/or d-Phe position and consisted of linear and backbone cyclic analogs, differed in their ring size. While the linear peptides rapidly degraded in serum and in brush border membrane vesicles (BBMV's), the linear peptomers were more stable. Backbone cyclic peptomers were also stable under the same conditions. Backbone cyclization significantly increased the intestinal permeability of two peptomers compared to their linear counterparts, in the Caco-2 model. Pharmacological evaluation revealed that two linear and one backbone cyclic peptomer, were found active towards MC4R at the nanomolar range. Thus, the conformational constrains imposed by these local and global modifications affect both the pharmacokinetic and pharmacodynamic properties of the parent peptide. This study demonstrates the potential of imposing backbone cyclization on bioactive peptomers as a promising approach in developing an orally available peptidomimetic drug leads.

Semi-rigid tripeptide agonists of melanocortin receptors.

A series of 30 RCO-HfR-NH(2) derivatives show preference for the mouse MC1R vs MC3-5Rs. trans-4-HOC(6)H(4)CH=CHCO-HfR-NH(2) (13) [EC(50) (nM): MC1R 83, MC3R 20500, MC4R 18130 and MC5R 935; ratio 1:246:217:11] is 11 times more potent than the lead compound LK-394 Ph(CH(2))(3)CO-HfR-NH(2) (2) and only 11 times less potent than the native tridecapeptide alpha-MSH at mMC1R. Differences in conformations of 2 and 13 are discussed.

Evidence for Persistent Immune Suppression in Patients Who Develop Chronic Critical Illness After Sepsis.

BACKGROUND: Many sepsis survivors develop chronic critical illness (CCI) and are assumed to be immunosuppressed, but there is limited clinical evidence to support this. We sought to determine whether the incidence of secondary infections and immunosuppressive biomarker profiles of patients with CCI differ from those with rapid recovery (RAP) after sepsis. METHODS: This prospective observational study evaluated 88 critically ill patients with sepsis and 20 healthy controls. Cohorts were defined based on clinical trajectory (early death, RAP, or CCI), whereas immunosuppression was clinically determined by the presence of a postsepsis secondary infection. Serial blood samples were collected for absolute lymphocyte counts (ALCs), monocytic human leukocyte antigen-DR (mHLA-DR) expression, and plasma-soluble programmed death-ligand 1 (sPD-L1) concentrations. RESULTS: Of the 88 patients with sepsis, 3 (3%) died within 14 days of sepsis onset, 50 (57%) experienced RAP, and 35 (40%) developed CCI. Compared with RAP patients, CCI patients exhibited a higher incidence and overall number of infections adjusted for hospital length of stay. ALC and mHLA-DR levels were dramatically reduced at the time of sepsis diagnosis when compared with healthy controls, whereas sPD-L1 concentrations were significantly elevated. There were no differences between RAP and CCI patients in ALC, sPD-L1, or mHLA-DR at the time of diagnosis or within 24 h after sepsis diagnosis. However, in contrast to the RAP group, CCI patients failed to exhibit any trend toward restoration of normal values of ALC, HLA-DR, and sPD-L1. CONCLUSIONS: Septic patients demonstrate clinical and biological evidence to suggest they are immunosuppressed at the time of sepsis diagnosis. Those who develop CCI have a greater incidence of secondary infections and persistently aberrant markers of impaired host immunity, although measurements at the time of sepsis onset did not distinguish between subjects with RAP and CCI.

Sepsis is associated with reduced spontaneous neutrophil migration velocity in human adults.

Sepsis is a common and deadly complication among trauma and surgical patients. Neutrophils must mobilize to the site of infection to initiate an immediate immune response. To quantify the velocity of spontaneous migrating blood neutrophils, we utilized novel microfluidic approaches on whole blood samples from septic and healthy individuals. A prospective study at a level 1 trauma and tertiary care center was performed with peripheral blood samples collected at <12 hours, 4 days, and/or 14 days relative to study initiation. Blood samples were also collected from healthy subjects. Ex vivo spontaneous neutrophil migration was measured on 2 µl of whole blood using microfluidic devices and time-lapse imaging. For each sample, individual neutrophils were tracked to calculate mean instantaneous velocity. Forty blood samples were collected from 33 patients with sepsis, and 15 blood samples were collected from age- and gender-matched healthy, control subjects. Average age was 61 years for septic patients with a male predominance (67%). Overall, average spontaneous neutrophil migration velocity in septic samples was 16.9 µm/min, significantly lower than controls samples at 21.1 µm/min (p = 0.0135). Neutrophil velocity was reduced the greatest at <12 hours after sepsis (14.5 µm/min). Regression analysis demonstrated a significant, positive correlation between neutrophil velocity and days after sepsis (p = 0.0059). There was no significant association between neutrophil velocity and age, gender, APACHE II score, SOFA score, sepsis severity, total white blood cell count, or percentage of neutrophils. Circulating levels of the cytokines IL-6, IL-8, IL-10, MCP-1, IP-10, and TNF were additionally measured using bead-based multiplex assay and found to peak at <12 hours and be significantly increased in patients with sepsis at all three time points (<12 hours, 4 days, and 14 days after sepsis) compared to healthy subjects. In conclusion, these findings may demonstrate an impaired ability of neutrophils to respond to sites of infection during the proinflammatory phase of sepsis.

Human ß-Defensin 1 and ß-Defensin 3 (Mouse Ortholog mBD14) Function as Full Endogenous Agonists at Select Melanocortin Receptors.

ß-Defensin 3 (BD3) was identified as a ligand for the melanocortin receptors (MCRs) in 2007, although the pharmacology activity of BD3 has not been clearly elucidated. Herein, it is demonstrated that human BD3 and mouse BD3 are full micromolar agonists at the MCRs. Furthermore, mouse ß-defensin 1 (BD1) and human BD1 are also MCR micromolar agonists. This work identifies BD1 as an endogenous MCR ligand and clarifies the controversial role of BD3 as a micromolar agonist.

Effects of blood-flow restriction on biomarkers of myogenesis in response to resistance exercise.

We investigated the acute myogenic response to resistance exercise with and without blood-flow restriction (BFR). Six men and women (age, 22 ± 1 years) performed unilateral knee extensions at 40% of 1-repetition maximum with or without (CNTRL) BFR applied via pressure cuff inflated to 220 mm Hg. Muscle biopsies were collected at 4 h and 24 h postexercise. Addition of BFR increased myoD and c-Met messenger RNA expression relative to CNTRL. Expression of hepatocyte growth factor protein was significantly higher following CNTRL.