Jennifer Gurney

Direct measurement of peptide-specific CD8+ T cells using HLA-A2:Ig dimer for monitoring the in vivo immune response to a HER2/neu vaccine in breast and prostate cancer patients.

HER2/neu is a proto-oncogene and a member of the epidermal growth factor receptor family of proteins that is overexpressed in numerous types of human cancer. We are currently conducting clinical trials with the HER2/neu E75 peptide vaccine in breast and prostate cancer patients. We have evaluated the use of HLA-A2 dimer molecule for the immunological monitoring of cancer patients receiving the E75 peptide vaccine. Peripheral blood samples from patients receiving the vaccine were stained with HLA-A2 dimers containing the vaccine peptide E75 or control peptides and analyzed by flow cytometry. We compared the HLA-A2 dimer assay to standard methods of immunologic monitoring (IFN-γ release, lymphocyte proliferation, and cytotoxicity). The HLA-A2 dimer assay was also compared with the HLA-A2 tetramer assay. E75 peptide-specific CD8 T cells were detected directly in the peripheral blood of patients by staining with E75-HLA-A2 dimers and CD8 antibodies. T cell cultures generated by repeated stimulations using E75 peptide-pulsed dendritic cells showed increased staining with E75-peptide loaded HLA-A2 dimers. Simultaneously analysis by the dimer assay and standard immunologic assays demonstrated that the dimer-staining assay correlated well with these methods of immunologic monitoring. A direct comparison using E75-specific HLA-A2 tetramers and HLA-A2 dimers for the detection of E75-specific CD8 T cells in peripheral blood showed comparable results with the two assays. Our findings indicate that the HLA-A2 dimer is a powerful new tool for directly quantifying and monitoring immune responses of antigen-specific T cells in peptide vaccine clinical trials.

Bovine polymerized hemoglobin (hemoglobin-based oxygen carrier-201) resuscitation in three swine models of hemorrhagic shock with militarily relevant delayed evacuation--effects on histopathology and organ function.

Objective:To test our hypothesis that hemoglobin-based oxygen carrier (HBOC)-201 resuscitation in hemorrhagic shock (HS) will not lead to increased organ injury and dysfunction. Design:Three swine HS models simulating military-relevant delayed evacuation: a) moderate controlled HS, b) severe controlled HS, and c) severe uncontrolled HS. Setting:Military research laboratory. Subjects:Swine. Interventions:Swine were anesthetized/intubated and instrumented. To induce HS, in two controlled hemorrhage experiments, 40% (moderate controlled HS) or 55% (severe controlled HS) of blood volume was withdrawn; in an uncontrolled HS experiment, the liver was crushed/lacerated. During a 4-hr “prehospital phase,” pigs were resuscitated with HBOC-201 (HBOC) or Hextend (HEX) or were nonresuscitated (NON). Upon “hospital arrival,” liver injury was repaired (severe uncontrolled HS), blood or saline was infused, hemodynamics were monitored, and blood was collected. Upon animal death and/or 72 hrs, necropsy was followed by histopathologic evaluation of organ injury (hematoxylin and eosin, electron microscopy) and immunohistochemistry of oxidative potential (3-nitrotyrosine). Significance (p < .05) was assessed by Kruskal-Wallis, analysis of variance/Bonferroni, and mixed procedure tests. Measurements and Main Results:Survival was significantly higher with HBOC than HEX only with severe uncontrolled HS (p = .002). Myocardial necrosis/fibroplasia, fluid requirements, cardiac output, and cardiac enzymes were generally similar or lower in HBOC than HEX pigs, but creatine kinase-MB (but not creatine kinase-MB/creatine kinase ratio) was higher with HBOC in moderate controlled HS. Alveolar/interstitial pulmonary edema was similar with HBOC and HEX, but Po2 was higher with HBOC in severe uncontrolled HS. Jejunal villar epithelial and hepatocellular necrosis were similarly minimal to moderate in all groups. Minimal biliary changes occurred exclusively with HBOC. Aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase were generally higher with HBOC than HEX. Mild renal papillary injury occurred more frequently with HBOC, but consistent patterns for urine output, blood urea nitrogen, and creatinine, were not seen. The 3-nitrotyrosine staining intensity was not different. Conclusions:In comparison with hetastarch, HBOC-201 resuscitation of swine with HS increased survival (with severe HS), did not increase evidence of oxidative potential, and had histopathologic and/or functional effects on organs that were clinically equivocal (myocardium, lungs, hepatic parenchyma, jejunum, and renal cortex/medulla) and potentially adverse (hepatobiliary and renal papilla). The effects of HBOC-201-resuscitation in HS should be corroborated in controlled clinical trials.

Analysis of naïve and memory CD4 and CD8 T cell populations in breast cancer patients receiving a HER2/neu peptide (E75) and GM-CSF vaccine

We are conducting clinical trials of the E75 peptide as a vaccine in breast cancer (BrCa) patients. We assessed T cell subpopulations in BrCa patients before and after E75 vaccination and compared them to healthy controls. We obtained 17 samples of blood from ten healthy individuals and samples from 22 BrCa patients prior to vaccination. We also obtained pre- and post-vaccination samples of blood from seven BrCa patients who received the E75/GM-CSF vaccine. CD4, CD8, CD45RA, CD45RO, and CCR7 antibodies were used to analyze the CD4+ and CD8+ T cells by four-color flow cytometry. Compared to healthy individuals, BrCa patients have significantly more memory and less naïve T cells and more effector-memory CD8+ and less effector CD4+ T cells. Phenotypic differences in defined circulating CD4+ and CD8+ T cell subpopulations suggest remnants of an active immune response to tumor distinguished by a predominant memory T cell response and by untapped recruitment of naïve helper and cytotoxic T cells. E75 vaccination induced recruitment of both CD4+ and CD8+ naïve T cells while memory response remained stable. Additionally, vaccination induced global activation of all T cells, with specific enhancement of effector CD4+ T cells. E75 vaccination causes activation of both memory and naïve CD4+ and CD8+ T cells, while recruiting additional naïve CD4+ and CD8+ T cells to the overall immune response.

Immune effects of resuscitation with HBOC-201, a hemoglobin-based oxygen carrier, in swine with moderately severe hemorrhagic shock from controlled hemorrhage.

HBOC-201, a hemoglobin-based oxygen carrier, improved physiologic parameters and survival in hemorrhagic shock (HS) animal models. However, resuscitation from HS and the properties of different fluids influence immune responses. The aim of this study was to determine if HBOC-201 significantly alters immune function in traumatic HS. Anesthetized pigs underwent soft tissue injury, controlled hemorrhage of 40% of blood volume, and resuscitation with HBOC-201 or Hextend, or no resuscitation. Sequential whole-blood samples were collected for analyses of leukocyte differential (hematology analyzer), T-lymphocyte subsets (CD3+, CD4+, and CD8+) (FACS), lymphocyte adhesion marker CD49d (α4-integrin) expression (FACS), plasma cytokines-tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10-(ELISA), and lymphocyte apoptosis (annexin-V/propidium iodide staining) (FACS). Statistical analyses were performed by the mixed procedure. Total WBC counts decreased posthemorrhage in both resuscitation groups. Lymphocyte percentages decreased and PMN percentages increased around 4 h posthemorrhage in all groups. CD3 cells decreased in all groups, but CD4 and CD8 cells decreased only in the resuscitation groups. TNF-α levels were not detectable in any groups. IL-6 levels were similar across treatment groups (P > 0.05); however, IL-10 levels were higher in the HBOC group, as early as 1 h posthemorrhage (P = 0.04). Increases in lymphocytic CD49d expression levels and apoptosis occurred only in nonresuscitation and Hextend groups, respectively (P ≤ 0.01). In comparison with Hextend, HBOC-201 had no significant adverse or beneficial effects on immune function in this model of moderately severe HS in swine, suggesting that it may be safe as a resuscitation fluid in HS patients.

Association of Prehospital Blood Product Transfusion During Medical Evacuation of Combat Casualties in Afghanistan With Acute and 30-Day Survival

Importance Prehospital blood product transfusion in trauma care remains controversial due to poor-quality evidence and cost. Sequential expansion of blood transfusion capability after 2012 to deployed military medical evacuation (MEDEVAC) units enabled a concurrent cohort study to focus on the timing as well as the location of the initial transfusion. Objective To examine the association of prehospital transfusion and time to initial transfusion with injury survival. Design, Setting, and Participants Retrospective cohort study of US military combat casualties in Afghanistan between April 1, 2012, and August 7, 2015. Eligible patients were rescued alive by MEDEVAC from point of injury with either (1) a traumatic limb amputation at or above the knee or elbow or (2) shock defined as a systolic blood pressure of less than 90 mm Hg or a heart rate greater than 120 beats per minute. Exposures Initiation of prehospital transfusion and time from MEDEVAC rescue to first transfusion, regardless of location (ie, prior to or during hospitalization). Transfusion recipients were compared with nonrecipients (unexposed) for whom transfusion was delayed or not given. Main Outcomes and Measures Mortality at 24 hours and 30 days after MEDEVAC rescue were coprimary outcomes. To balance injury severity, nonrecipients of prehospital transfusion were frequency matched to recipients by mechanism of injury, prehospital shock, severity of limb amputation, head injury, and torso hemorrhage. Cox regression was stratified by matched groups and also adjusted for age, injury year, transport team, tourniquet use, and time to MEDEVAC rescue. Results Of 502 patients (median age, 25 years [interquartile range, 22 to 29 years]; 98% male), 3 of 55 prehospital transfusion recipients (5%) and 85 of 447 nonrecipients (19%) died within 24 hours of MEDEVAC rescue (between-group difference, −14% [95% CI, −21% to −6%]; P = .01). By day 30, 6 recipients (11%) and 102 nonrecipients (23%) died (between-group difference, −12% [95% CI, −21% to −2%]; P = .04). For the 386 patients without missing covariate data among the 400 patients within the matched groups, the adjusted hazard ratio for mortality associated with prehospital transfusion was 0.26 (95% CI, 0.08 to 0.84, P = .02) over 24 hours (3 deaths among 54 recipients vs 67 deaths among 332 matched nonrecipients) and 0.39 (95% CI, 0.16 to 0.92, P = .03) over 30 days (6 vs 76 deaths, respectively). Time to initial transfusion, regardless of location (prehospital or during hospitalization), was associated with reduced 24-hour mortality only up to 15 minutes after MEDEVAC rescue (median, 36 minutes after injury; adjusted hazard ratio, 0.17 [95% CI, 0.04 to 0.73], P = .02; there were 2 deaths among 62 recipients vs 68 deaths among 324 delayed transfusion recipients or nonrecipients). Conclusions and Relevance Among medically evacuated US military combat causalities in Afghanistan, blood product transfusion prehospital or within minutes of injury was associated with greater 24-hour and 30-day survival than delayed transfusion or no transfusion. The findings support prehospital transfusion in this setting.

Comparison of Military and Civilian Methods for Determining Potentially Preventable Deaths: A Systematic Review

Importance Military and civilian trauma experts initiated a collaborative effort to develop an integrated learning trauma system to reduce preventable morbidity and mortality. Because the Department of Defense does not currently have recommended guidelines and standard operating procedures to perform military preventable death reviews in a consistent manner, these performance improvement processes must be developed. Objectives To compare military and civilian preventable death determination methods to understand the existing best practices for evaluating preventable death. Evidence Review This systematic review followed the PRISMA reporting guidelines. English-language articles were searched from inception to February 15, 2017, using the following databases: MEDLINE (Ovid), Evidence-Based Medicine Reviews (Ovid), PubMed, CINAHL, and Google Scholar. Articles were initially screened for eligibility and excluded based on predetermined criteria. Articles reviewing only prehospital deaths, only inhospital deaths, or both were eligible for inclusion. Information on study characteristics was independently abstracted by 2 investigators. Reported are methodological factors affecting the reliability of preventable death studies and the preventable death rate, defined as the number of potentially preventable deaths divided by the total number of deaths within a specific patient population. Findings Fifty studies (8 military and 42 civilian) met the inclusion criteria. In total, 1598 of 6500 military deaths reviewed and 3346 of 19 108 civilian deaths reviewed were classified as potentially preventable. Among military studies, the preventable death rate ranged from 3.1% to 51.4%. Among civilian studies, the preventable death rate ranged from 2.5% to 85.3%. The high level of methodological heterogeneity regarding factors, such as preventable death definitions, review process, and determination criteria, hinders a meaningful quantitative comparison of preventable death rates. Conclusions and Relevance The reliability of military and civilian preventable death studies is hindered by inconsistent definitions, incompatible criteria, and the overall heterogeneity in study methods. The complexity, inconsistency, and unpredictability of combat require unique considerations to perform a methodologically sound combat-related preventable death review. As the Department of Defense begins the process of developing recommended guidelines and standard operating procedures for performing military preventable death reviews, consideration must be given to the factors known to increase the risk of bias and poor reliability.

Pelvic Fracture Care

While combat-related pelvis fractures are more commonly open, higher energy, and complex in pattern than those seen in the civilian setting, the principles of management are similar. The primary differences are related to the austere setting in which the initial management takes place, and the lack of resources typically available. Initial management consists of cessation of hemorrhage, along with the multi-disciplinary prioritized management of associated injuries, and skeletal stabilization. This is most commonly achieved with a compressive sheet or pelvic binder, with pelvic external fixation when resources allow, and debridement of open wounds as necessary. Definitive, internal fixation is delayed until the patient arrives at a higher echelon of care.

Military Preventable Death Conceptual Framework: A Systematic Approach for Reducing Battlefield Mortality

The National Academies of Sciences, Engineering, and Medicine Report on Zero Preventable Deaths highlighted the need for a military and civilian partnership to take lessons learned from the wars in Iraq and Afghanistan and incorporate them into a national learning trauma care system. One of the specific objectives in the report was to establish military preventable death metrics. Upon further inspection, it became apparent that the Department of Defense did not have an established methodology to reliably estimate and report preventable death metrics and opportunities to improve the military trauma care system. Over the last year, clinical and non-clinical subject matter experts from the Joint Trauma System and Armed Forces Medical Examiners System began the process of establishing a standard military preventable death review process to meet the objective outlined in the report. Based on an assessment to understand methodological best practices for preventable death reviews, this manuscript presents the conceptual framework that is guiding our effort to establish the first ever battle-related mortality surveillance system with preventable death metrics and opportunities to improve the trauma care system.

Frostbite and Immersion Foot Care

Historically, cold injury, hypothermia, and frostbite have been severe problems for military units on the battlefield. Kenneth D. Orr and David C. Fainer captured these difficulties in their book, Cold Injuries in Korea During Winter of 1950-51, still cited in military medical readiness training. While not common in modern conflicts, the potential exists for large numbers of these casualties in war and during training.

En Route Critical Care Transfer From a Role 2 to a Role 3 Medical Treatment Facility in Afghanistan

&NA; Background En route care is the transfer of patients requiring combat casualty care within the US military evacuation system. No reports have been published about en route care of patients during transfer from a forward surgical facility (role 2) to a combat support hospital (role 3) for comprehensive care. Objective To describe patients transferred from a role 2 to a role 3 US military treatment facility in Afghanistan. Methods A retrospective review of data from the Joint Trauma System Role 2 Database was conducted. Patient characteristics were described by en route care medical attendants. Results More than one‐fourth of patients were intubated at transfer (26.9%), although at transfer fewer than 10% of patients had a base deficit of more than 5 (3.5%), a pH of less than 7.3 (5.2%), an international normalized ratio of more than 2 (0.8%), or temporary abdominal or chest closure (7.4%). The en route care medical attendant was most often a nurse (35.5%), followed by technicians (14.1%) and physicians (10.0%). Most patients (75.3%) were transported by medical evacuation (on rotary‐wing aircraft). Conclusion This is the first comprehensive review of patients transported from a forward surgical facility to a more robust combat support hospital in Afghanistan. Understanding the epidemiology of these patients will inform provider training and the appropriate skill mix for the transfer of postsurgical patients within a combat setting.