Patrick Muller

Lyophilized Plasma for Resuscitation in a Swine Model of Severe Injury

HYPOTHESIS Lyophilized plasma (LP) is as safe and effective as fresh frozen plasma (FFP) for resuscitation after severe trauma. DESIGN Multicenter animal study. SETTING Animal laboratories, 2 level I trauma centers. PARTICIPANTS Thirty-two Yorkshire crossbred swine. INTERVENTIONS Lyophilized plasma was analyzed for factor levels and clotting activity before lyophilization and after reconstitution. Swine were subjected to complex multiple trauma including extremity fracture, hemorrhage, severe liver injury, acidosis, and hypothermia. They were then resuscitated with FFP, LP, FFP and packed red blood cells (PRBCs) in a ratio of 1:1, or 1:1 LP and PRBCs. MAIN OUTCOME MEASURES Residual clotting activity of LP after reconstitution, swine mortality, hemodynamic measures, total blood loss, coagulation profiles, and inflammatory measures. RESULTS Lyophilization decreased clotting factor activity by an average of 14%. Survival and heart rate were similar between all groups. Swine resuscitated with LP had equivalent or higher mean arterial pressures. Swine treated with LP had similar coagulation profiles, plasma lactate levels, and postinjury blood loss compared with those treated with FFP. Swine treated with 1:1 FFP-PRBCs were similar to those treated with 1:1 LP-PRBCs. Resuscitation with LP resulted in a reduction in postresuscitation interleukin 6 expression compared with resuscitation with FFP. CONCLUSIONS The process of lyophilization and reconstitution of plasma reduces coagulation factor activity by 14%, without acute differences in blood loss. Lyophilized plasma can be used for resuscitation in a severe multiple trauma and hemorrhagic shock swine model with efficacy equal to that of FFP and with decreased interleukin 6 production.

Reproducibility of an Animal Model Simulating Complex Combat-Related Injury in a Multiple-Institution Format

We developed a complex combat-relevant model of abdominal and extremity trauma, hemorrhagic shock, hypothermia, and acidosis. We then simulated injury, preoperative, and operative phases. We hypothesized that this model is reproducible and useful for randomized multicenter preclinical trials. Yorkshire swine were anesthetized, intubated, and instrumented. They then underwent femur fracture, 60% total blood volume hemorrhage, a 30-min shock period, induced hypothermia to 33°C, and hemorrhage volume replacement with 3:1 isotonic sodium chloride solution (NS) at each of three centers. Hemodynamic parameters were measured continuously. Thromboelastography, arterial blood gas, and laboratory values were collected at baseline, after the shock period, and after NS replacement. Thirty-seven animals were used for model development. Eight (21%) died before completion of the study period. Twenty-nine survivors were included in the analysis. MAP (±SEM) after the shock period was 32 ± 2 mmHg and was similar between centers (P = 0.4). Mean pH, base deficit, and lactate levels were 7.29 ± 0.02, 8.20 ± 0.65 mmol/L, and 5.29 ± 0.44 mmol/L, respectively, after NS replacement. These were similar between centers (P > 0.05). Prothrombin time values increased significantly over time at all centers, reflecting a progressive coagulopathy (P < 0.02). Thromboelastography maximum amplitude values were similar among centers (P > 0.05) and demonstrated progressively weakened platelet interaction over time (P < 0.03). Hematocrit was similar after controlled hemorrhage (P = 0.15) and dilution (P = 0.9). The pH, lactate, base deficit, and coagulation tests reflect a severely injured state. A complex porcine model of polytrauma and shock canbe used for multi-institutional study with excellent reproducibility. A consistent severe injury profile was achieved, afterwhich experimental interventions can be applied. This is the first report of a reproducible multicenter trauma and resuscitation-related animal model.

Mechanism of peroxide-induced cellular injury in cultured adult cardiac myocytes.

Reactive oxygen species contribute to the tissue injury seen after reperfusion of ischemic myocardium. We propose that toxicity originates from the effect that mitochondrial peroxide metabolism has on substrate entry into oxidative pathways. To support our contention, cultured adult rat cardiomyocytes were incubated with physiological concentrations of peroxide. The cellular extract and incubation medium were analyzed for adenine nucleotides and purines by reverse‐phase high‐pressure liquid chromatography. Cellular glutathione efflux was determined by enzymatic analysis of the incubation medium. Pyruvate dehydrogenase (PDH) activity was determined in the cultured myocytes as well as in freshly isolated cardiac mitochondria using [1‐C14]pyruvate. Extracellular glutathione rose 3.3‐fold in response to small doses of peroxide (≈ 108 nmol/mg protein). Likewise, small quantities of peroxide reduced total cellular adenine nucleotides to 50–60% of control values with only a modest (0.95–0.91) reduction in energy charge ((ATP+ ½ ADP)/(ATP+ADP+AMP)). Peroxide‐treated myocytes selectively release inosine and adenosine, as only these two purine degradation products were detected in the incubation medium. The most dramatic response was a peroxide dose‐dependent inhibition of PDH activity in cultured myocytes as well as freshly isolated mitochondria; just 65 and 30 nmol peroxide/mg protein induced a 50% reduction in cellular and mitochondrial PDH activity, respectively. In conclusion, physiological quantities of peroxide potently inhibit PDH in cultured cardiomyocytes and isolated cardiac mitochondria. PDH inhibition blocks the aerobic oxidation of glucose and inhibits the oxidative phosphorylation of ADP, which in turn leads to cellular adenine nucleotide degradation.—Vlessis, A. A.; Muller, P.; Bartos, D.; Trunkey, D. Mechanism of peroxide‐induced cellular injury in cultured adult cardiac myocytes. FASEB J. 5: 2600‐2605; 1991.

ADP-ribosyltransferase from beef liver which ADP-ribosylates elongation factor-2

Fragment A of diphtheria toxin and Pseudomonas toxin A intoxicate cells by ADP‐ribosylating the diphthamide residue of elongation factor‐2 (EF‐2) resulting in an inhibition of protein synthesis [1–3]. A cellular enzyme from polyoma virus transformed baby hamster kidney (pyBHK) cells ADP‐ribosylates EF‐2 in an identical manner [4]. Here we describe a similar cellular enzyme from beef liver which transfers [adenosine‐14C]ADP‐ribose from NAD to EF‐2. The 14C‐label can be removed from the EF‐2 by snake venom phosphodiesterase as a soluble product which comigrates with AMP on TLC plates, indicating the 14C‐label is present on EF‐2 as monomeric units of ADP‐ribose. Furthermore, the forward transferase reaction catalyzed by the beef liver ADP‐ribosyltransferase is reversible by excess diphtheria toxin fragment A, with the formation of 14C‐labeled NAD, indicating that both transferases ADP‐ribosylate the same site on the diphthamide residue of EF‐2. Thus, beef liver and pyBHK mono(ADP‐ribosyl) transferases both modify the diphthamide residue of EF‐2, in a manner identical to diphtheria toxin fragment A and Pseudomonas toxin A. These results suggest the cellular enzyme is probably ubiquitous among eukaryotic cells.

A novel highly porous silica and chitosan-based hemostatic dressing is superior to HemCon and gauze sponges.

BACKGROUND Hemostatic dressings have become increasingly popular as the optimal initial treatment for severe hemorrhage. The purpose of this study was to compare the hemostatic properties of a novel highly porous silica and chitosan-based dressing (TraumaStat) to HemCon, and gauze dressing in a severe groin injury model in swine. METHODS Thirty swine were blindly randomized to receive TraumaStat, HemCon, or standard gauze dressing for hemostatic control. A complex groin injury involving complete transaction of the femoral artery and vein was made. After 30 seconds of uncontrolled hemorrhage, the randomized dressing was applied and pressure was held for 5 minutes. Fluid resuscitation was initiated to achieve and maintain the baseline mean arterial pressure and the wound was inspected for bleeding. Failure of hemostasis was defined as pooling of blood outside of the wound. Animals were then monitored for 120 minutes and surviving animals were euthanized. RESULTS Blood loss before treatment was similar between groups (p > 0.1). TraumaStat had one failure, compared with five for gauze, and eight for HemCon (p = 0.005, TraumaStat vs. HemCon). TraumaStat significantly reduced median blood loss when compared with both HemCon and gauze (117 vs. 774 and 268 mL respectively, p < 0.05). At study conclusion, TraumaStat animals had a greater median hematocrit than both HemCon (24 vs. 19, p = 0.033), and gauze (24 vs. 19, p = 0.049) animals. Median volume of fluid resuscitation and mortality were not different between groups (p > 0.1). CONCLUSIONS TraumaStat was superior to HemCon and gauze dressings in controlling bleeding from a severe groin injury. TraumaStat may be a better hemostatic dressing for control of active hemorrhage than current standards of care.

Resuscitation with lactated ringer's does not increase inflammatory response in a Swine model of uncontrolled hemorrhagic shock.

Lactated Ringer’s (LR) and normal saline (NS) are widely and interchangeably used for resuscitation of trauma victims. Studies show LR to be superior to NS in the physiologic response to resuscitation. Recent in vitro studies demonstrate equivalent effects of LR and NS on leukocytes. We aimed to determine whether LR resuscitation would produce an equivalent inflammatory response compared with normal saline (NS) resuscitation in a clinically relevant swine model of uncontrolled hemorrhagic shock. Thirty-two swine were randomized. Control animals (n = 6) were sacrificed following induction of anesthesia for baseline data. Sham animals (n = 6) underwent laparotomy and 2 h of anesthesia. Uncontrolled hemorrhagic shock animals (n = 10/group) underwent laparotomy, grade V liver injury, and blinded resuscitation with LR or NS to maintain baseline blood pressure for 1.5 h before sacrifice. Lung was harvested, and tissue mRNA levels of interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor-alpha (TNF-α) were determined using quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR). Sections of lung were processed and examined for neutrophils sequestered within the alveolar walls. Cytokine analysis showed no difference in IL-6 gene transcription in any group (P = 0.99). Resuscitated swine had elevated G-CSF and TNF-α gene transcription, but LR and NS groups were not different from each other (P= 0.96 and 0.10, respectively). Both resuscitation groups had significantly more alveolar neutrophils present than controls (P < 0.01) and shams (P < 0.05) but were not different from one another (P= 0.83). LR and NS resuscitation have equivalent effects on indices of inflammation in the lungs in our model of uncontrolled hemorrhagic shock.

A new hormonal therapy for estrogen receptor-negative breast cancer.

BackgroundWe postulate that the androgen dehydroepiandrosterone sulfate (DHEAS) may represent an innovative hormonal treatment for estrogen (ER), progesterone (PR) receptor–negative, but androgen receptor (AR)–positive breast cancers by inhibiting breast cancer cell growth through AR stimulation.MethodsThree ER,PR–negative breast cancer cell lines (HCC 1137, 1954, and 38), were treated with DHEAS. DHEAS-induced growth was measured by a methylthiotetrazole (MTT) proliferation assay and apoptosis by TUNEL fluorescence. Androgen receptor gene expression levels were determined using quantitative real-time polymerase chain reaction (q-RT-PCR).ResultsHCC cell lines 1954 and 1937 were positive for AR expression; HCC 38 was weakly positive. MTT analysis showed DHEAS-induced decreases in cell proliferation of 47% in HCC 1937, 27% in HCC 1954, and 0.4% in HCC 38. Ten days of culturing HCC 1954 cells after the removal of DHEAS resulted in a 3.5-fold increase in growth. Continuous treatment for the same duration induced a 2.8-fold decrease in growth. Parallel experiments showed no significant changes in HCC 38 cultures. TUNEL assays showed DHEAS-induced apoptosis fold increases of 2.8 in HCC 1937, 1.9 in HCC 1954, and no significant difference in HCC 38 cultures. Q-RT-PCR of HCC 1954 cells showed a 6-fold DHEAS-induced decrease in AR gene expression at 4 h. Co-treatment with Casodex nullified this effect.ConclusionsDHEAS inhibited growth of ER,PR–negative, AR–positive breast cancer cells. DHEAS was cytotoxic to these breast cancer cells via the apoptosis pathway. DHEAS may be an effective treatment for a population previously excluded from hormone therapy.

Improved breast cancer survival among hormone replacement therapy users is durable after 5 years of additional follow-up

BACKGROUND We previously reported that breast cancer patients who used hormone replacement therapy (HRT) had significantly lower stage tumors and higher survival than never-users. We present an update with longer follow-up, HRT use data, and in vitro research. METHODS Our database of 292 postmenopausal breast cancer patients was updated to include HRT type, duration, and disease status. In vitro effects of estrogen (E) and/or medroxyprogesterone (MPA) on breast cancer cell growth were measured. RESULTS Tumor prognostic factors were better and survival rates higher for both E and combination HRT users of any duration. Use greater than 10 years correlated with node-negative disease, mammographically detected tumors, and 100% survival. E supported minimal proliferation; MPA induced cell death; E+MPA results were similar to E alone. CONCLUSIONS HRT users, regardless of type or duration of HRT use, continued to have higher survival rates. In vitro results supported the clinical finding that outcomes for users of E and E+MPA were similar.

Ketamine-based Total Intravenous Anesthesia Versus Isoflurane Anesthesia in a Swine Model of Hemorrhagic Shock

BACKGROUND Inhalational anesthetics can cause profound hemodynamic effects including decreases in systemic vascular resistance and cardiac inotropy. Although widely used in uncontrolled hemorrhagic shock (UHS), their consequences compared with other anesthetic regimens are not well-studied. Ketamine-based total intravenous anesthesia (TIVA) may produce less profound cardiovascular depression, and has been used during elective surgery but rarely during traumatic shock. The purpose of this study was to compare the effects of isoflurane (ISO) and TIVA regimens in a swine grade V liver injury model. We hypothesized that TIVA would result in less hypotension and dysfunctional inflammation than ISO. METHODS Twenty swine were randomized blindly to receive either 1% to 3% ISO, or intravenous ketamine, midazolam, and buprenorphine for maintenance anesthesia. Six animals acted as controls. After sedation and intubation, randomized anesthesia was initiated and monitored by an independent animal technician. Invasive lines were placed followed by celiotomy and splenectomy. Baseline mean arterial pressure (MAP) was documented and a grade V liver injury created. After 30 minutes of UHS, animals were resuscitated with 8 mL of Ringers lactate per milliliter blood loss at 165 mL/min. MAP and tissue oxygen saturation (StO2) were continuously recorded. The animals were sacrificed 120 minutes after injury and lung tissue was harvested. Serum cytokines (interleukin-6 [IL-6], IL-8, and tumor necrosis factor-alpha [TNF-alpha]) were quantified with enzyme-linked immunosorbent assay. Lung cytokine mRNA levels were quantified with real time reverse transcriptase polymerase chain reaction. RESULTS Animal weight, liver injury pattern, and blood loss were similar (p > 0.1). The ISO group had a lower MAP at baseline (p = 0.02), at injury (p = 0.004), and study completion (p = 0.001). After resuscitation, MAP decreased in the ISO group but remained stable in the TIVA group. StO2 was significantly higher in the TIVA group immediately after injury (p = 0.004), but similar between groups throughout the remainder of the study. Animals who received TIVA trended toward higher levels of lactate and lower pH throughout the study, reaching significance at 30 minutes postinjury (p = 0.037 and 0.043). Inflammatory cytokine (IL-6, IL-8, and TNF-alpha) production did not differ between groups, however TNF-alpha mRNA production was significantly lower in the TIVA group (p = 0.04). CONCLUSION Although a TIVA regimen produced less pronounced hypotension in a swine model of UHS than did ISO, end-organ perfusion with TIVA appeared to be equivalent or inferior to ISO. In circumstances of limited resources, such as those experienced by forward Army surgical teams, a ketamine-based TIVA regimen may be an option for use in UHS.

Correlation of Breast Cancer Axillary Lymph Node Metastases With Stem Cell Mutations

IMPORTANCE Mutations in oncogenes AKT1, HRAS, and PIK3CA in breast cancers result in abnormal PI3K/Akt signaling and tumor proliferation. They occur in ductal carcinoma in situ, in breast cancers, and in breast cancer stem and progenitor cells (BCSCs). OBJECTIVES To determine if variability in clinical presentation at diagnosis correlates with PI3K/Akt mutations in BCSCs and provides an early prognostic indicator of increased progression and metastatic potential. DESIGN, SETTING, AND PARTICIPANTS Malignant (BCSCs) and benign stem cells were collected from fresh surgical specimens via cell sorting and tested for oncogene mutations in a university hospital surgical oncology research laboratory from 30 invasive ductal breast cancers (stages IA through IIIB). MAIN OUTCOMES AND MEASURES Presence of AKT1, HRAS, and PIK3CA mutations in BCSCs and their correlation with tumor mutations, pathologic tumor stage, tumor histologic grade, tumor hormone receptor status, lymph node metastases, and patient age and condition at the last follow-up contact. RESULTS Ten tumors had mutations in their BCSCs. In total, 9 tumors with BCSC mutations and 4 tumors with BCSCs without mutations had associated tumor present in the lymph nodes (P = .001). CONCLUSIONS AND RELEVANCE Tumors in which BCSCs have defects in PI3K/Akt signaling are significantly more likely to manifest nodal metastases. These oncogenic defects may be missed by gross molecular testing of the tumor and are markers of more aggressive breast cancer. Molecular profiling of BCSCs may identify patients who would likely benefit from PI3K/Akt inhibitors, which are being tested in clinical trials.