LMWF5A suppresses cytokine release by modulating select inflammatory transcription factor activity in stimulated PBMC.

BACKGROUND: Dysregulation of transcription and cytokine expression has been implicated in the pathogenesis of a variety inflammatory diseases. The resulting imbalance between inflammatory and resolving transcriptional programs can cause an overabundance of pro-inflammatory, classically activated macrophage type 1 (M1) and/or helper T cell type 1 (Th1) products, such as IFNγ, TNFα, IL1-ß, and IL12, that prevent immune switching to resolution and healing. The low molecular weight fraction of human serum albumin (LMWF5A) is a novel biologic drug that is currently under clinical investigation for the treatment of osteoarthritis and the hyper-inflammatory response associated with COVID-19. This study aims to elucidate transcriptional mechanisms of action involved with the ability of LMWF5A to reduce pro-inflammatory cytokine release. METHODS: ELISA arrays were used to identify cytokines and chemokines influenced by LMWF5A treatment of LPS-stimulated peripheral blood mononuclear cells (PBMC). The resulting profiles were analyzed by gene enrichment to gain mechanistic insight into the biologic processes and transcription factors (TFs) underlying the identified differentially expressed cytokines. DNA-binding ELISAs, luciferase reporter assays, and TNFα or IL-1ß relative potency were then employed to confirm the involvement of enriched pathways and TFs. RESULTS: LMWF5A was found to significantly inhibit a distinct set of pro-inflammatory cytokines (TNFα, IL-1ß, IL-12, CXCL9, CXCL10, and CXCL11) associated with pro-inflammatory M1/Th1 immune profiles. Gene enrichment analysis also suggests these cytokines are, in part, regulated by NF-κB and STAT transcription factors. Data from DNA-binding and reporter assays support this with LMWF5A inhibition of STAT1α DNA-binding activity as well as a reduction in overall NF-κB-driven luciferase expression. Experiments using antagonists specific for the immunomodulatory and NF-κB/STAT-repressing transcription factors, peroxisome proliferator-activated receptor (PPAR)γ and aryl hydrocarbon receptor (AhR), indicate these pathways are involved in the LMWF5A mechanisms of action by reducing LMWF5A drug potency as measured by TNFα and IL-1ß release. CONCLUSION: In this report, we provide evidence that LMWF5A reduces pro-inflammatory cytokine release by activating the immunoregulatory transcription factors PPARγ and AhR. In addition, our data indicate that LMWF5A suppresses NF-κB and STAT1α pro-inflammatory pathways. This suggests that LMWF5A acts through these mechanisms to decrease pro-inflammatory transcription factor activity and subsequent inflammatory cytokine production.

Anti-Inflammatory Activity in the Low Molecular Weight Fraction of Commercial Human Serum Albumin (LMWF5A).

The innate immune system is increasingly being recognized as a critical component in osteoarthritis (OA) pathophysiology. An ex vivo immunoassay utilizing human peripheral blood mononuclear cells (PBMC) was developed in order to assess the OA anti-inflammatory properties of the low molecular weight fraction (<5 kDa) of commercial human serum albumin (LMWF5A). PBMC from various donors were pre-incubated with LMWF5A before LPS stimulation. TNFα release was measured by ELISA in supernatants after an overnight incubation. A ≥ 30% decrease in TNFα release was observed. This anti-inflammatory effect is potentially useful in assessing potency of LMWF5A for the treatment of OA.

Oxidation­reduction potential parameters worsen following intraarterial therapy in patients with reduced collateral circulation and middle cerebral artery occlusions.

Collateral circulation is important for cerebral perfusion in acute ischemic strokes. Monitoring the oxidation-reduction potential (ORP) may be useful to assess collateral status or treatment efficacy. The objectives of the present study were to determine if the ORP was associated with collateral circulation status in middle cerebral artery (MCA) occlusions and to identify patterns in the ORP and the collateral circulation status among patients treated with intraarterial therapy (IAT) over time. The present pilot study was nested within a prospective cohort study measuring the ORP of the peripheral venous plasma of stroke patients. The population included in the present study were patients with MCA (M1/M2) occlusions. Two ORP parameters were examined: Static ORP (sORP; mV), indicating oxidative stress, and capacity ORP (cORP; µC), indicating antioxidant reserves. Collateral status was retrospectively graded using Miteff's system as good (grade 1) or reduced (grade 2/3). Comparisons were made between collateral status groups (reduced vs. good collaterals) in all patients, within a subset including only patients who received IAT, and between thrombolysis in cerebral infraction scale score (TICI) groups (0-2a vs. 2b/3). The Fisher's exact test, Student's t-test and Wilcoxon tests were used (α<0.20). The 19 patients were categorized based on their collaterals: Good collaterals (53%) and reduced collaterals (47%). The baseline characteristics were similar with the exception that the patients with good collaterals had a lower international normalized ratio (P=0.12) and were more likely to have a stroke on the left side (P=0.18) or to have a mismatch (P=0.05). The admission sORP values were comparable (169.5 vs. 164.2 mV; P=0.65), as was admission cORP (P=0.73). When considering only the patients who received IAT (n=12), admission sORP (P=0.69) and cORP (P=0.90) were also statistically similar. On day 2, after IAT, both groups experienced a worsening in ORP measures; however, the patients with good collaterals had a significantly lower sORP (169.4 vs. 203.5 mV; P=0.02) and a higher cORP (0.2 vs. 0.1 µC; P=0.002) compared with the patients with reduced collaterals. Neither sORP nor cORP were significantly different between TICI score groups on admission or on day 2. Upon discharge, patients with a TICI of 2b-3 had a significantly better sORP (P=0.03) and cORP (P=0.12) compared with those with a TICI of 0-2a. In conclusion, upon patient admission, the ORP parameters were not significantly different between the collateral circulation status groups for MCA occlusions. The ORP parameters worsened after IAT regardless of the collateral circulation status; however, after IAT, on day 2, patients with good collaterals experienced less oxidative stress (sORP) and had higher antioxidant reserves (cORP) than patients with reduced collaterals.

Biphasic effect of danazol on human vascular endothelial cell permeability and f-actin cytoskeleton dynamics.

Breakdown of endothelial barrier function is a hallmark event across a variety of pathologies such as inflammation, cancer, and diabetes. It has also been appreciated that steroid hormones impart direct biological activity on endothelial cells at many levels. The purpose of this investigation was to explore the effect of the androgen-like steroid, danazol, on endothelial cell barrier function in vitro. Primary human endothelial cells exposed to 0.01-50 µM danazol were evaluated for changes in permeability. We found that danazol altered endothelial permeability in a biphasic manner in which nanomolar concentrations enhance barrier function while micromolar concentrations are detrimental. Monitoring of trans-endothelial electrical resistance demonstrated that these barrier enhancing effects were rapid (within 5 min) and lasted for over 24h. Analysis of intracellular f-actin organization showed that barrier enhancement also correlated with the formation of a submembranous cortical actin ring. Conversely, at higher danazol concentrations, contractile cell phenotypes were observed, represented by stress fiber formation. Competitive binding studies performed using steroid hormone receptor antagonists proved that this activity is the result of androgen and estrogen receptor ligation. These findings suggest that low dose danazol may provide a therapeutic window for diseases involving vascular leakage.

AMP5A modulates Toll-like receptors 7 and 8 single-stranded RNA immune responses in PMA-differentiated THP-1 and PBMC.

Background: Dysregulation of antiviral immunity has been implicated in the progression of acute respiratory syndrome coronavirus 2 infection into severe cases of coronavirus disease of 2019 (COVID-19). Imbalances in the inflammatory response drive the overabundant production of pro-inflammatory cytokines and chemokines. The low molecular weight fraction of 5% human serum albumin commercial preparation (AMP5A) is a novel biologic drug currently under clinical investigation for the treatment of osteoarthritis and the hyperinflammatory response associated with COVID-19. This study aims to elucidate AMP5A effects following the activation of immune cells with agonists of Toll-like receptor (TLR) 7 and/or 8, which detect ssRNA viral sequences. Methods: CXCL10 ELISAs were used to evaluate the dynamics of myeloid cells activated with CL075 and CL307, agonists of TLR7/8 and TLR7, respectively. In addition, enrichment analysis of gene sets generated by ELISA arrays was utilized to gain insight into the biologic processes underlying the identified differentially expressed cytokine profiles. Finally, relative potency (REP) was employed to confirm the involvement of mechanisms of action paramount to AMP5A activity. Results: AMP5A inhibits the release of CXCL10 from both CL075- and CL307-activated PMA-differentiated THP-1 and peripheral blood mononuclear cells. Furthermore, AMP5A suppresses a distinct set of pro-inflammatory cytokines (including IL-1ß, IL-6, IL-12, and CXCL10) associated with COVID-19 and pro-inflammatory NF-κB activation. REP experiments using antagonists specific for the immunomodulatory transcription factors, peroxisome proliferator-activated receptor γ, and aryl hydrocarbon receptor, also indicate that these pathways are involved in the ability of AMP5A to inhibit CXCL10 release. Conclusion: Due to the biphasic course of COVID-19, therapeutic approaches that augment antiviral immunity may be more beneficial early in infection, whereas later interventions should focus on inflammation suppression. In this study, we show that AMP5A inhibits TLR 7/8 signaling in myeloid cells, resulting in a decrease in inflammatory mediators associated with hyperinflammation and autoimmunity. Furthermore, data demonstrating that AMP5A activates immunomodulatory transcription factors found to be protective in lung disease is provided. These findings suggest that the modes and mechanisms of action of AMP5A are well suited to treat conditions involving dysregulated TLR 7/8 activation.

Characterization of peritoneal reactive ascites collected from acute appendicitis and small bowel obstruction patients.

BACKGROUND: Pathological abdominal adhesions can cause bowel obstructions. A history of appendectomy (appy) increases patient rehospitalization risk directly related to adhesions. To potentially identify strategies for adhesion treatment, we characterized reactive ascites (rA) collected during appy or adhesiolysis for small bowel obstruction (SBO). METHODS: This is a non-randomized, prospective observational study recruiting patients with non-perforated appendicitis or SBO from three Level 1 trauma centers in the United States. rA were analyzed via liquid chromatography-mass spectrometry (LC-MS) (n = 31), bead-based quantification cytokines and chemokines (n = 32) and soluble receptors (n = 30), and LC-MS metabolomics (n = 18). RESULTS: LC-MS showed that samples contained albumin, apolipoprotein A1, and transthyretin and that metabolites increased in SBO vs appy rA were biomarkers of oxidative stress. Multi-plex analyses showed levels of 17 cytokines/chemokines and 6 soluble receptors were significantly different in appy vs SBO rA. Top increased proteins in appy compared to SBO rA by 20.14-, 11.53-, and 8.18-fold were granulocyte-colony stimulating factor, C-X-C motif chemokine ligand 10, and interleukin-10, respectively. CONCLUSIONS: These data further define pro- and anti-inflammatory mediators and metabolites that may drive formation or perpetuate chronic abdominal adhesions. Future research is to further explore whether attenuation of these factors may decrease pathologic adhesion formation.

Pharmacologic thromboprophylaxis is a risk factor for hemorrhage progression in a subset of patients with traumatic brain injury.

BACKGROUND: Pharmacologic thromboprophylaxis (PTP) may exacerbate intracranial hemorrhage (ICH) in patients with traumatic brain injury (TBI). We examined risk factors for hemorrhage progression in patients with blunt TBI and hypothesized that PTP would increase ICH progression in a subset of these patients. METHODS: We retrospectively studied patients with TBI admitted to our level I trauma center during 19 months. Progression of hemorrhage was examined in two populations: patients with a stable initial follow-up (F/U) computed tomography (CT) and patients with hemorrhage progression on initial F/U CT. Risk factors potentially associated with hemorrhage progression were analyzed using logistic regression. Timing of PTP was defined two ways: exposed to PTP versus not exposed; early (<72 hours), late (>or=72 hours), or no PTP. RESULTS: Three hundred forty patients with TBI were reviewed and hemorrhage progression occurred in 32.4% (n = 110) of patients of which 59.1% were considered clinically significant. In patients with ICH progression on initial F/U CT, predictors of subsequent hemorrhage progression include exposure to PTP (odds ratio [OR]: 13.07, p = 0.01), extradural/subdural hemorrhage (OR: 5.15, p = 0.03), Glasgow Coma Score 3-8 (OR: 4.64, p = 0.03), and body mass index >or=25 (OR = 4.32, p = 0.03). PTP was not significantly associated with hemorrhage progression in patients with a stable initial F/U CT. CONCLUSIONS: These findings suggest that PTP use is associated with a 13-fold increased odds of further hemorrhage progression in patients whose F/U CT within 1 day of admission showed ICH progression; 16% of this risk can be attributed to receiving PTP. Conversely, PTP may be safe in a subgroup of patients with TBI with no ICH progression on initial F/U CT.

Current utilization and radiation dose from computed tomography in patients with trauma.

OBJECTIVE: To quantify the cumulative effective dose of radiation received during hospitalization after traumatic injury and to compare the computed tomography (CT) utilization practices for two time periods in patients with trauma. DESIGN: A retrospective analysis of radiologic and medical data. SETTING: A level I trauma center. PATIENTS: Consecutively admitted adult patients with trauma with moderate to severe injuries (injury severity score >8), an intensive care unit (ICU) length of stay of one or more days, who were directly admitted and not transferred to another acute care center. MEASUREMENTS AND MAIN RESULTS: CT examination means and utilization were compared for April through August, 2003 and April to August, 2007. Cumulative effective doses were calculated for the 2007 period, and patients with a high radiation dose (>100 mSv) were identified. One hundred sixty-five adult patients with trauma were included. An increase in mean CT examinations per patient was observed in the 2007 period compared with the 2003 period, overall (4.41 vs. 3.44, p = 0.002) and among subsets of patients. The overall increase remained significant after adjustment for patient demographics (p = 0.05). The mean cumulative effective dose per patient was 11.13 mSv in 2007; 9% of patients received a dose >or=100 mSv. CONCLUSIONS: Patients with trauma are at an increased risk of adverse effects from CT studies, because they receive high doses of radiation, and the number of CT examinations that patients receive is increasing with time. We recommend that risk of radiation be prospectively monitored and estimated by hospitals through the use of CT examination count per patient.

Staff commitment to trauma care improves mortality and length of stay at a level I trauma center.

BACKGROUND: Optimizing human resources at trauma facilities may increase quality of care. The purpose of this study was to assess whether staffing changes within a Level I trauma center improved mortality and shortened length of stay (LOS) for trauma patients. METHODS: Mortality, hospital LOS, and intensive care unit LOS were evaluated during three time periods: trauma service coverage by in-house general surgery residents and attendings ("group 1"), the creation of a core trauma panel with in-house trauma surgeons ("group 2"), and the addition of physician assistants (PAs) to the core trauma panel ("group 3"). Logistic regression and chi tests were used to compare mortalities, and multiple linear regression, t-tests, and median tests were used to compare LOS. RESULTS: There were 15,297 adult patients with trauma included in the analysis. After adjustment for transfers-in, mechanism of injury, injury severity score, age, and head injury, the presence of in-house trauma surgeons (group 2) decreased the following compared with group 1: overall mortality (3.12% vs. 3.82%, p = 0.05), mortality in the severely injured (11.41% vs. 14.83%, p = 0.02), and median intensive care unit LOS (3.03 days vs. 3.40 days, p = 0.006). The introduction of PAs to the core trauma panel (group 3 vs. group 2) decreased overall mortality (2.80% vs. 3.76%, p = 0.05), and reduced mean and median hospital LOS (4.32 days vs. 4.69 days, p = 0.05; and 3.74 days vs. 3.88 days, p = 0.02, respectively). CONCLUSION: The presence of in-house core trauma surgeons and PAs improves management and outcome of critically injured trauma patients within a level I trauma center.

The effect of storage on the accumulation of oxidative biomarkers in donated packed red blood cells.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a life-threatening condition characterized by oxidative stress. Longer storage times of packed red blood cells (PRBC) and other blood products have been implicated with an increased risk in developing TRALI in transfused patients. METHODS: A total of 10 units of blood containing PRBC stored in citrate-phosphate-dextrose buffer at 4 degrees C were included in the study. At Bonfils Blood Center (Denver, CO), samples were collected on storage day 1 and day 42. Samples were immediately centrifuged, and the supernatants were collected and stored at -80 degrees C until further analysis. Oxidation-reduction potential and protein oxidation were measured in both the day 1 and day 42 samples. RESULTS: Oxidation-reduction potential significantly increased (p < 0.05) in the day 42 sample (98.1 mV +/- 21.9 SD) versus the day 1 sample (62.6 mV +/- 21.5 SD). The oxidation of human serum albumin increased by 63.6% during the storage time. Other serum proteins such as apolipoprotein A1 and transthyretin demonstrated similar increases in oxidation. Also, proteins with a cleaved C-terminal amino acid were observed indicating the presence of carboxypeptidase activity, a marker of inflammation. CONCLUSIONS: The presence of an oxidative environment in transfused PRBC increases with storage time. This could partially explain the increased risk of developing TRALI related to the transfusion of older blood products.

Mechanisms of delayed wound healing by commonly used antiseptics.

BACKGROUND: The cytotoxic effects of antiseptics on pivotal cell types of the healing process have been well documented. The purpose of our investigation was to explore the ability of subcytotoxic levels of antiseptics to interfere with fibroblast function. METHODS: Cell proliferation assays were performed by culturing fibroblasts in the presence of commonly used antiseptics. Migration was evaluated using scratch assays in which monolayers were "wounded" and cellular movement was monitored by digital photography. Matrix metalloproteinase (MMP) release was analyzed by zymography. RESULTS: H2O2 and povidone-iodine reduced both migration and proliferation of fibroblasts in a dose-dependent fashion. Treatment with silver-containing antiseptics and chlorhexidine exhibited reductions in proliferation at high concentrations, but enhanced growth at lower doses. Silver-containing compounds and chlorhexidine also proved to be the least detrimental to migration in these assays. metalloproteinase release from the cells was differently affected depending on the dosage and class of antiseptic applied. CONCLUSIONS: When debridement of the wound bed is not sufficient to reduce bacterial loads, the application of broad-spectrum antiseptics maybe indicated. Our data would suggest that H2O2 and iodine are poor choices, potentially retarding the contribution of fibroblasts to the healing process. Silver sulfadiazine and chlorhexidine, at levels still proven to be bactericidal, had fewer detrimental effects on fibroblast activity in these assays. The silver-containing antiseptics may even increase the proliferative potential of these cells in culture.

On the Mechanisms of Action of the Low Molecular Weight Fraction of Commercial Human Serum Albumin in Osteoarthritis.

The low molecular weight fraction of commercial human serum albumin (LMWF5A) has been shown to successfully relieve pain and inflammation in severe osteoarthritis of the knee (OAK). LMWF5A contains at least three active components that could account for these antiinflammatory and analgesic effects. We summarize in vitro experiments in bone marrow-derived mesenchymal stem cells, monocytic cell lines, chondrocytes, peripheral blood mononuclear cells, fibroblast-like synoviocytes, and endothelial cells on the biochemistry of anti-inflammatory changes induced by LMWF5A. We then look at four of the major pathways that cut across cell-type considerations to examine which biochemical reactions are affected by mTOR, COX-2, CD36, and AhR pathways. All three components show anti-inflammatory activities in at least some of the cell types. The in vitro experiments show that the effects of LMWF5A in chondrocytes and bone marrow- derived stem cells in particular, coupled with recent data from previous clinical trials of single and multiple injections of LMWF5A into OAK patients demonstrated improvements in pain, function, and Patient Global Assessment (PGA), as well as high responder rates that could be attributed to the multiple mechanism of action (MOA) pathways are summarized here. In vitro and in vivo data are highly suggestive of LMWF5A being a disease-modifying drug for OAK.

Plasma Oxidized Albumin in Acute Ischemic Stroke Is Associated With Better Outcomes.

Introduction: Plasma oxidized human serum albumin (OxHSA) is evidence of an active antioxidant mechanism as measured by oxidized species of HSA. CXCL-10 is a pro-inflammatory chemokine associated with ischemic conditions. Accordingly, we examined the relationship of admission OxHSA and CXCL-10 with discharge mRS in acute ischemic stroke (AIS). Methods: Plasma samples and clinical data were collected prospectively at a Comprehensive Stroke Center. Admission biomarkers of oxidative stress, CXCL-10 and %OxHSA, were measured. We examined if CXCL-10 or %OxHSA correlated with age, admission NIHSS score, and discharge mRS score using Spearman's Rank correlation. Logistic regression was performed to identify independent predictors of a favorable discharge mRS (≤2). Results: In 106 consecutive AIS patients, the median age was 73 (IQR 61-84), 47% were male, and the median admission NIHSS score was 11 (IQR 5-19). %OxHSA and CXCL-10 were significantly correlated (r = 0.23, p = 0.02). Both biomarkers were significantly correlated with age: %OxHSA (r = 0.44, p < 0.001) and CXCL-10 (r = 0.32, p = 0.001). Neither biomarker was correlated with admission NIHSS. There was a borderline significant correlation with discharge mRS and %OxHSA (r = -0.17, p = 0.08), where higher %OxHSA correlated with lower discharge mRS scores. For every 1% increase in %OxHSA, the odds of a favorable discharge mRS increased 11%. The odds of a favorable discharge mRS decreased 18% for every 1-point increase in the initial NIHSS. Conclusions: OxHSA, the result of an oxidative environment and evidence of the strong antioxidant buffering capacity of HSA, correlated with CXCL-10 and discharge mRS, implying that strong antioxidant activity of albumin may confer better outcomes.

The presence of S-sulfonated transthyretin in commercial human serum albumin solutions: Potential contribution to neuropathy.

BACKGROUND: Commercial solutions of human serum albumin (HSA) are administered to critically ill patients for the treatment of shock, restoration of blood volume, and the acute management of burns. Previously, conflicting results on the effects of HSA administration have been reported varying from a favorable increase in total plasma antioxidant capacity to a higher mortality rate in traumatic brain injury (TBI) patients. These results could be partially explained due to the known heterogeneity of HSA solutions. We report the discovery of S-sulfonated human transthyretin (hTTR) in HSA solutions. METHODS: Proteomics was performed on commercially available solutions of 5% HSA by LC-MS analysis. The MS charge envelope for hTTR was deconvolved to the uncharged native hTTR parent mass (13,762 Da). The parent mass was then integrated, and relative proportions of the 2 major species of hTTR, native and S-sulfonated hTTR (13,842 Da), were calculated. RESULTS: The majority of hTTR found in 5% commercial HSA solutions is in the S-sulfonated form regardless of the age of the HSA solution. S-sulfonation of hTTR at the free cysteine residue in position 10 appears to be the result of a mixed disulfide exchange possibly with S-cysteinylated hTTR or S-cysteinylated HSA. hTTR is a tetramer composed of four identical monomers each containing a reduced cysteine residue in position 10. S-sulfonation of hTTR at this cysteine residue can destabilize the hTTR tetramer, an important step in the formation of TTR-related amyloid fibrils. CONCLUSIONS: Administration of a commercial HSA solution that already contains S-sulfonated hTTR could potentially contribute to the development of amyloid-related/polyneuropathy in the critically ill.

The cobalt-albumin binding assay: insights into its mode of action.

BACKGROUND: We previously hypothesized that the N-terminus of human serum albumin (HSA) is altered during ischemic events, thus establishing the foundation for the cobalt-HSA binding assay phenomenon. In this investigation, we attempt to clarify the mode of action of the cobalt-HSA binding assay by direct observations of cobalt binding to HSA. METHODS: High pressure liquid chromatography coupled to positive electrospray ionization mass spectrometry (HPLC/MS) was used to study cobalt binding to HSA in the plasma of patients with and without evidence of myocardial ischemia. RESULTS: Strong binding of cobalt to the N-terminus of HSA occurs at pH>7.0. No differences in cobalt binding to the N-terminus of HSA are observed in ischemic versus non-ischemic patients' plasma despite differences in the cobalt-HSA binding assay. Plasma free cysteine/cystine ratio appears to play a role in the quantitative response of the cobalt-HSA binding assay. CONCLUSIONS: The main determinants of the cobalt-HSA binding assay mechanism of action include but are not limited to: the proportion of intact N-terminus of HSA, HSA concentration, plasma cysteine/cystine ratio, plasma pH, and the state of oxidation of cys34 of HSA. Assay improvements that consider and take these factors into account could lead to an improved cobalt-HSA binding assay with greater clinical utility.

An artificial neural network derived trauma outcome prediction score as an aid to triage for non-clinicians.

In mass casualty events Emergency Medical Service Providers (EMS) choose treatment at Scene or a "scoop and run" approach. The latter requires clinically trained personnel at the reception site to triage patients. Current methodology based on Revised Trauma Score (tRTS) requires use of Glasgow Coma Scale, a method reliant on experience and clinical knowledge. This makes the system subjective and often inadequate for non-clinicians. This project attempts to develop a simplified outcome prediction score using an artificial neural network for use by a non-clinically trained EMS to aid triage. The project uses National Trauma Data Bank, Version 6.1. Tiberius Data Mining Software created Neural Network models. Variables considered were values that could easily be obtained during an event. Binary values were used for low SBP and low Respiratory Rate, coded using the RTS scoring table as a basis, and age indicators. A modified motor component of Glasgow Coma Score was created to negate the need for clinical knowledge. Best performing models, identified by Gini coefficient and ability to predict mortality, were with 8 and 10 neurons. On mortality prediction all even numbers of hidden neurons have similar performances. Training sets were compared to test sets, and found to be identical in Gini coefficient and performance. Models performed well in predicting mortality compared to standard outcome predictors. Possible additional variables such as gender or ethnicity might improve the Neural Network predictive ability. Pulse appears an essential variable not recorded by the NTDB.

The anti-inflammatory effect of LMWF5A and N-acetyl kynurenine on macrophages: Involvement of aryl hydrocarbon receptor in mechanism of action.

After a traumatic insult, macrophages can become activated leading to general inflammation at the site of injury. Activated macrophages are partially regulated by the aryl hydrocarbon receptor (AhR) which when activated suppresses inflammation by limiting the secretion of pro-inflammatory cytokines and promoting the over expression of immuno-modulatory mediators. This study aims to determine whether the low molecular weight fraction of 5% human serum albumin (LMWF5A) and N-acetyl kynurenine (NAK), an N-acetyl tryptophan (NAT) breakdown product in LMWF5A, can regulate inflammation by inhibiting macrophage activation through the AhR since kynurenine is a known AhR agonist. Using LCMS, we demonstrate that NAT is non-enzymatically degraded during accelerated aging of LMWF5A with high heat accelerating degradation. More importantly, NAK is a major degradation product found in LMWF5A. THP-1 monocytes were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA) and pre-treated with 2-fold dilutions of LMWF5A or synthetic NAK with or without an AhR antagonist (CH223191) prior to overnight stimulation with lipopolysaccharide (LPS). Treatment with LMWF5A caused a 50-70% decrease in IL-6 release throughout the dilution series. A dose-response inhibition of IL-6 release was observed for NAK with maximal inhibition (50%) seen at the highest NAK concentration. Finally, an AhR antagonist partially blocked the anti-inflammatory effect of LMWF5A while completely blocking the effect of NAK. A similar inhibitory effect was observed for CXCL-10, but the AhR antagonist was not effective suggesting additional mechanisms for CXCL-10 release. These preliminary findings suggest that LMWF5A and NAK partially promote the suppression of activated macrophages via the AhR receptor. Therefore, LMWF5A, which contains NAK, is potentially a useful therapeutic in medical conditions where inflammation is prevalent such as trauma, sepsis, and wound healing.

Mass spectrometry analysis of urine and catheter of a patient with purple urinary bag syndrome.

INTRODUCTION: Purple urinary bag syndrome (PUBS) is considered to be a benign condition observed in the urinary catheter and bag in some catheterized patients with urinary tract infections. This syndrome is usually reported to occur in alkaline urine. CASE REPORT: We report of a catheterized patient with PUBS and slightly acidic urine (pH 6-6.5). A novel analysis method was developed using high pressure liquid chromatography and mass spectrometry (HPLC/MS) to detect compounds that are thought to be associated with PUBS. Urine, urinary sediment, and the plastic collection system were assayed and quantitated using these methods. The potential toxicity of one of these compounds, indoxyl sulfate, is discussed. CONCLUSIONS: The presence of PUBS in a catheterized patient with slightly acidic urine is reported. A novel method for the analysis of chemical components of PUBS and the first direct confirmation of the presence of indigo in the urine sediment and collecting system are described.

Interaction of arsine with hemoglobin in arsine-induced hemolysis.

The mechanism of arsine (AsH3) toxicity is not completely understood, but hemoglobin (Hb) has long been recognized as a necessary component of the overall mechanism of AsH3-induced hemolysis. In this study, the role of Hb in AsH3-induced hemolysis was investigated. The purpose was to determine whether exposure to AsH3 altered the structure of the heme or globin constituents of Hb. Arsine was incubated with isolated, human oxyhemoglobin (oxyHb) and carboxyhemoglobin (carboxyHb), and the release of heme and formation of AsH3-induced hemoglobin modifications were examined. Arsine increased the amount of heme released from oxyHb by 18%. When carboxyHb was incubated with AsH3, there was no change in heme release, suggesting that the sixth ligand position on the heme iron may be critical in the interaction with AsH3. Arsine-Hb interactions were studied by mass spectral analysis of heme, alpha-chain globin, and beta-chain globin. Arsine had no significant effect on the alpha- or beta-chain LCMS spectra in oxyHb and carboxyHb, but in oxyHb, arsine consistently increased the frequency of methyl acetate ion fragment (.CH2OOH, m/z = 59) loss from heme in the matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) spectra. The formation of Hb-protein crosslinks was investigated by Western blotting using an anti-Hb antibody in isolated membranes from AsH3-treated erythrocytes, but no Hb-membrane adducts were found. These results suggest that the interaction between AsH3 and hemoglobin result in an increase in heme release which may contribute to the hemolytic mechanism of AsH3.

The formation and rapid clearance of a truncated albumin species in a critically ill patient.

INTRODUCTION: Hypoalbuminemia is known to occur in critically ill patients and is associated with increased mortality. We observed a potentially novel, partial explanation for the hypoalbuminemia noticed in a severely traumatized patient. CASE REPORT: We report of a severely, multi-system traumatized patient in whom hypoalbuminemia was present (1-2 g/dl). The plasma albumin (HSA) was analyzed by liquid chromatography/positive electrospray ionization mass spectrometry. A high percentage of a truncated albumin that lost its carboxy terminal amino acid leucine (HSA-L) associated with a 10-fold increase in plasma carboxypeptidase A (CPA) activity (R(2)=0.994) were found. We estimated the half life of this truncated albumin species to be <80 h. CONCLUSIONS: The increased CPA activity encountered following a traumatic event and subsequent rapid clearance of the resulting HSA-L from plasma might be a contributing factor to the hypoalbuminemia observed in the critically ill patients.