Mosquito bite immunization with radiation-attenuated Plasmodium falciparum sporozoites: safety, tolerability, protective efficacy and humoral immunogenicity.

BACKGROUND: In this phase 1 clinical trial, healthy adult, malaria-naïve subjects were immunized with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) by mosquito bite and then underwent controlled human malaria infection (CHMI). The PfRAS model for immunization against malaria had previously induced >90 % sterile protection against homologous CHMI. This study was to further explore the safety, tolerability and protective efficacy of the PfRAS model and to provide biological specimens to characterize protective immune responses and identify protective antigens in support of malaria vaccine development. METHODS: Fifty-seven subjects were screened, 41 enrolled and 30 received at least one immunization. The true-immunized subjects received PfRAS via mosquito bite and the mock-immunized subjects received mosquito bites from irradiated uninfected mosquitoes. Sera and peripheral blood mononuclear cells (PBMCs) were collected before and after PfRAS immunizations. RESULTS: Immunization with PfRAS was generally safe and well tolerated, and repeated immunization via mosquito bite did not appear to increase the risk or severity of AEs. Local adverse events (AEs) of true-immunized and mock-immunized groups consisted of erythaema, papules, swelling, and induration and were consistent with reactions from mosquito bites seen in nature. Two subjects, one true- and one mock-immunized, developed large local reactions that completely resolved, were likely a result of mosquito salivary antigens, and were withdrawn from further participation as a safety precaution. Systemic AEs were generally rare and mild, consisting of headache, myalgia, nausea, and low-grade fevers. Two true-immunized subjects experienced fever, malaise, myalgia, nausea, and rigours approximately 16 h after immunization. These symptoms likely resulted from pre-formed antibodies interacting with mosquito salivary antigens. Ten subjects immunized with PfRAS underwent CHMI and five subjects (50 %) were sterilely protected and there was a significant delay to parasitaemia in the other five subjects. All ten subjects developed humoral immune responses to whole sporozoites and to the circumsporozoite protein prior to CHMI, although the differences between protected and non-protected subjects were not statistically significant for this small sample size. CONCLUSIONS: The protective efficacy of this clinical trial (50 %) was notably less than previously reported (>90 %). This may be related to differences in host genetics or the inherent variability in mosquito biting behavior and numbers of sporozoites injected. Differences in trial procedures, such as the use of leukapheresis prior to CHMI and of a longer interval between the final immunization and CHMI in these subjects compared to earlier trials, may also have reduced protective efficacy. This trial has been retrospectively registered at ISRCTN ID 17372582, May 31, 2016.

Effectiveness of active self-care complementary and integrative medicine therapies: options for the management of chronic pain symptoms.

OBJECTIVES: Chronic pain management typically consists of prescription medications or provider-based, behavioral, or interventional procedures that are often ineffective, may be costly, and can be associated with undesirable side effects. Because chronic pain affects the whole person (body, mind, and spirit), patient-centered complementary and integrative medicine (CIM) therapies that acknowledge the patients' roles in their own healing processes have the potential to provide more efficient and comprehensive chronic pain management. Active self-care CIM (ACT-CIM) therapies allow for a more diverse, patient-centered treatment of complex symptoms, promote self-management, and are relatively safe and cost-effective. To date, there are no systematic reviews examining the full range of ACT-CIM used for chronic pain symptom management. METHODS: A systematic review was conducted, using Samueli Institute's Rapid Evidence Assessment of the Literature methodology, to rigorously assess both the quality of the research on ACT-CIM modalities and the evidence for their efficacy and effectiveness in treating chronic pain symptoms. A working group of subject matter experts was also convened to evaluate the overall literature pool and develop recommendations for the use and implementation of these modalities. RESULTS: Following key database searches, 146 randomized controlled trials were included in the review, 18 of which directly compared ACT-CIM approaches with one another. CONCLUSIONS: This article summarizes the current evidence, quality, effectiveness, and safety of these modalities. Recommendations and next steps to move this field of research forward are also discussed. The entire scope of the review is detailed throughout the current Pain Medicine supplement.

Sterile protective immunity to malaria is associated with a panel of novel P. falciparum antigens.

The development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized.

Losartan in hospitalized patients with COVID-19 in North America: An individual participant data meta-analysis.

BACKGROUND: Angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers (ARBs) have been hypothesized to benefit patients with COVID-19 via the inhibition of viral entry and other mechanisms. We conducted an individual participant data (IPD) meta-analysis assessing the effect of starting the ARB losartan in recently hospitalized COVID-19 patients. METHODS: We searched ClinicalTrials.gov in January 2021 for U.S./Canada-based trials where an angiotensin-converting enzyme inhibitors/ARB was a treatment arm, targeted outcomes could be extrapolated, and data sharing was allowed. Our primary outcome was a 7-point COVID-19 ordinal score measured 13 to 16 days post-enrollment. We analyzed data by fitting multilevel Bayesian ordinal regression models and standardizing the resulting predictions. RESULTS: 325 participants (156 losartan vs 169 control) from 4 studies contributed IPD. Three were randomized trials; one used non-randomized concurrent and historical controls. Baseline covariates were reasonably balanced for the randomized trials. All studies evaluated losartan. We found equivocal evidence of a difference in ordinal scores 13-16 days post-enrollment (model-standardized odds ratio [OR] 1.10, 95% credible interval [CrI] 0.76-1.71; adjusted OR 1.15, 95% CrI 0.15-3.59) and no compelling evidence of treatment effect heterogeneity among prespecified subgroups. Losartan had worse effects for those taking corticosteroids at baseline after adjusting for covariates (ratio of adjusted ORs 0.29, 95% CrI 0.08-0.99). Hypotension serious adverse event rates were numerically higher with losartan. CONCLUSIONS: In this IPD meta-analysis of hospitalized COVID-19 patients, we found no convincing evidence for the benefit of losartan versus control treatment, but a higher rate of hypotension adverse events with losartan.

Pre-hospital resuscitation with HBOC-201 and rFVIIa compared to HBOC-201 alone in uncontrolled hemorrhagic shock in swine.

In a previous dose escalation study our group found that combining 90µg/kg rFVIIa with HBOC-201 reduced blood loss and improved physiologic parameters compared to HBOC alone. In this follow-up study in a swine liver injury model, we found that while there were no adverse hematology effects and trends observed in the previous study were confirmed, statistical significance could not be reached. Additional pre-clinical studies are indicated to identify optimal components of a multifunctional blood substitute for clinical use in trauma.

COVIDMED - An early pandemic randomized clinical trial of losartan treatment for hospitalized COVID-19 patients.

Objectives: To assess the efficacy and safety of losartan for COVID-19 patients. Methods: COVIDMED was a double-blinded, placebo-controlled platform RCT. Enrollees were randomized to standard care plus hydroxychloroquine, lopinavir/ritonavir, losartan, or placebo. Hydroxychloroquine and lopinavir/ritonavir arms were discontinued early. We report losartan data vs. combined (lopinavir-ritonavir and placebo) and prespecified placebo-only controls. The primary endpoint was the mean COVID-19 Ordinal Severity Score (COSS) slope of change. Slow enrollment prompted early termination. Results: Fourteen patients were included in our final analysis (losartan [N = 9] vs. control [N = 5] [lopinavir/ritonavir [N = 2], placebo [N = 3]]). Most baseline parameters were balanced. Losartan treatment was not associated with a difference in mean COSS slope of change vs. combined (p = 0.4) or placebo-only control (p = 0.05) (trend favoring placebo). 60-day mortality and overall AE/SAE rates were insignificantly higher with losartan. Conclusion: In this small RCT in hospitalized COVID-19 patients, losartan did not improve outcome and was associated with adverse safety signals.

Tick magnets: The occupational risk of tick-borne disease exposure in forestry workers in New York.

BACKGROUND: Outdoor workers, such as forestry workers, are at an increased risk for contracting tick-borne diseases due to their prolonged time spent in tick habitats. Although well studied in Europe, no studies have been conducted with forestry workers in the Northeastern United States since 1990s. METHODS: Full-time forestry workers and two comparison groups (volunteer firefighter/first responders and indoor/healthcare workers) within New York State Department of Environmental Conservation Regions 3, 4, 5, 6, and 7 were recruited for this cross-sectional seroprevalence study. Blood draws were conducted to test for antibodies to Lyme, anaplasmosis, babesiosis, and ehrlichiosis. Surveys were administered to determine personal risk factors and protective behaviors. RESULTS: Between November 2020 and May 2021, 256 (105 forestry, 101 firefighter/first responder, and 50 indoor/healthcare) workers participated in this study. Forestry workers had a probability of testing positive nearly twice as high for any tick-borne disease (14%) compared to firefighter/first responders (8%) and to indoor workers (6%); however, this difference was not statistically significant (P = .140). Forestry workers were more likely to find embedded ticks on themselves (f = 33.26, P < .0001 vs both comparison groups) and to have been previously diagnosed with a tick-borne disease (P = .001 vs firefighter/first responders, P = .090 vs indoor/healthcare workers). CONCLUSIONS: This pilot study suggests a higher proportion of tick-borne disease risk among forestry workers compared to firefighters/first responders and indoor/healthcare workers with lesser exposure. A larger study to confirm or refute this pilot data could help optimize mitigation/prevention strategies.

Comparison of antibody response durability of mRNA-1273, BNT162b2, and Ad26.COV2.S SARS-CoV-2 vaccines in healthcare workers.

OBJECTIVES: There are limited comparative immunologic durability data post COVID-19 vaccinations. METHODS: Approximately 8.4 months after primary COVID-19 vaccination, 647 healthcare workers completed surveys about COVID-19 vaccinations/infections and blood draws. The groups included participants vaccinated with mRNA-1273 (n = 387), BNT162b2 (n = 212), or Ad26.COV2.S (n = 10) vaccines; unvaccinated participants (n = 10); and participants who received a booster dose (n = 28). The primary outcome was immunoglobin anti-spike titer. Secondary/tertiary outcomes included neutralizing antibodies (enzyme-linked immunosorbent assay-based pseudoneutralization) and vaccine effectiveness (VE). Antibody levels were compared using analysis of variance and linear regression. RESULTS: Mean age was 49.7 and 75.3% of the participants were female. Baseline variables were balanced except for immunosuppression, previous COVID-19 infection, and post-primary vaccination time. Unadjusted median (interquartile range [IQR]) anti-spike titers (AU/ml) were 1539.5 (876.7-2626.7) for mRNA-1273, 751.2 (422.0-1381.5) for BNT162b2, 451.6 (103.0-2396.7) for Ad26.COV2.S, 113.4 (3.7-194.0) for unvaccinated participants, and 31898.8 (21347.1-45820.1) for participants administered with booster dose (mRNA-1273 vs BNT162b2, P <.001; mRNA-1273, BNT162b2, or boosted vs unvaccinated, P <.006; mRNA-1273, BNT162b2, Ad26.COV2.S, or unvaccinated vs boosted, P <.001). Unadjusted median (IQR) pseudoneutralization was as follows: 90.9% (80.1-95.0) for mRNA-1273, 77.2% (59.1-89.9) for BNT162b2, 57.9% (36.6-95.8) for Ad26.COV2.S, 40.1% (21.7-60.6) for unvaccinated, and 96.4% (96.1-96.6) for participants administered with booster dose (mRNA-1273 vs BNT162b2, P <.001; mRNA-1273, BNT162b2, or boosted vs unvaccinated, P <.028; mRNA-1273, BNT162b2, Ad26.COV2.S, or unvaccinated vs boosted, P <.001). VE was 87-89% for participants administered mRNA-1273 vaccine, BNT162b2 vaccine, and booster dose, and 33% for Ad26.COV2.S (none significantly different). CONCLUSION: Antibody responses 8.4 months after primary vaccination were significantly higher with mRNA-1273 than those observed with BNT162b2.

Hydroxychloroquine/chloroquine for the treatment of hospitalized patients with COVID-19: An individual participant data meta-analysis.

Background: Results from observational studies and randomized clinical trials (RCTs) have led to the consensus that hydroxychloroquine (HCQ) and chloroquine (CQ) are not effective for COVID-19 prevention or treatment. Pooling individual participant data, including unanalyzed data from trials terminated early, enables more detailed investigation of the efficacy and safety of HCQ/CQ among subgroups of hospitalized patients. Methods: We searched ClinicalTrials.gov in May and June 2020 for US-based RCTs evaluating HCQ/CQ in hospitalized COVID-19 patients in which the outcomes defined in this study were recorded or could be extrapolated. The primary outcome was a 7-point ordinal scale measured between day 28 and 35 post enrollment; comparisons used proportional odds ratios. Harmonized de-identified data were collected via a common template spreadsheet sent to each principal investigator. The data were analyzed by fitting a prespecified Bayesian ordinal regression model and standardizing the resulting predictions. Results: Eight of 19 trials met eligibility criteria and agreed to participate. Patient-level data were available from 770 participants (412 HCQ/CQ vs 358 control). Baseline characteristics were similar between groups. We did not find evidence of a difference in COVID-19 ordinal scores between days 28 and 35 post-enrollment in the pooled patient population (odds ratio, 0.97; 95% credible interval, 0.76-1.24; higher favors HCQ/CQ), and found no convincing evidence of meaningful treatment effect heterogeneity among prespecified subgroups. Adverse event and serious adverse event rates were numerically higher with HCQ/CQ vs control (0.39 vs 0.29 and 0.13 vs 0.09 per patient, respectively). Conclusions: The findings of this individual participant data meta-analysis reinforce those of individual RCTs that HCQ/CQ is not efficacious for treatment of COVID-19 in hospitalized patients.

Renin-Angiotensin System Inhibitors in Patients With COVID-19: A Meta-Analysis of Randomized Controlled Trials Led by the International Society of Hypertension.

Background Published randomized controlled trials are underpowered for binary clinical end points to assess the safety and efficacy of renin-angiotensin system inhibitors (RASi) in adults with COVID-19. We therefore performed a meta-analysis to assess the safety and efficacy of RASi in adults with COVID-19. Methods and Results MEDLINE, EMBASE, ClinicalTrials.gov, and the Cochrane Controlled Trial Register were searched for randomized controlled trials that randomly assigned patients with COVID-19 to RASi continuation/commencement versus no RASi therapy. The primary outcome was all-cause mortality at ≤30 days. A total of 14 randomized controlled trials met the inclusion criteria and enrolled 1838 participants (aged 59 years, 58% men, mean follow-up 26 days). Of the trials, 11 contributed data. We found no effect of RASi versus control on all-cause mortality (7.2% versus 7.5%; relative risk [RR], 0.95; [95% CI, 0.69-1.30]) either overall or in subgroups defined by COVID-19 severity or trial type. Network meta-analysis identified no difference between angiotensin-converting enzyme inhibitors versus angiotensin II receptor blockers. RASi users had a nonsignificant reduction in acute myocardial infarction (2.1% versus 3.6%; RR, 0.59; [95% CI, 0.33-1.06]), but increased risk of acute kidney injury (7.0% versus 3.6%; RR, 1.82; [95% CI, 1.05-3.16]), in trials that initiated and continued RASi. There was no increase in need for dialysis or differences in congestive cardiac failure, cerebrovascular events, venous thromboembolism, hospitalization, intensive care admission, inotropes, or mechanical ventilation. Conclusions This meta-analysis of randomized controlled trials evaluating angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers versus control in patients with COVID-19 found no difference in all-cause mortality, a borderline decrease in myocardial infarction, and an increased risk of acute kidney injury with RASi. Our findings provide strong evidence that RASi can be used safely in patients with COVID-19.

Hydroxychloroquine/chloroquine for the treatment of hospitalized patients with COVID-19: An individual participant data meta-analysis.

BACKGROUND: Results from observational studies and randomized clinical trials (RCTs) have led to the consensus that hydroxychloroquine (HCQ) and chloroquine (CQ) are not effective for COVID-19 prevention or treatment. Pooling individual participant data, including unanalyzed data from trials terminated early, enables more detailed investigation of the efficacy and safety of HCQ/CQ among subgroups of hospitalized patients. METHODS: We searched ClinicalTrials.gov in May and June 2020 for US-based RCTs evaluating HCQ/CQ in hospitalized COVID-19 patients in which the outcomes defined in this study were recorded or could be extrapolated. The primary outcome was a 7-point ordinal scale measured between day 28 and 35 post enrollment; comparisons used proportional odds ratios. Harmonized de-identified data were collected via a common template spreadsheet sent to each principal investigator. The data were analyzed by fitting a prespecified Bayesian ordinal regression model and standardizing the resulting predictions. RESULTS: Eight of 19 trials met eligibility criteria and agreed to participate. Patient-level data were available from 770 participants (412 HCQ/CQ vs 358 control). Baseline characteristics were similar between groups. We did not find evidence of a difference in COVID-19 ordinal scores between days 28 and 35 post-enrollment in the pooled patient population (odds ratio, 0.97; 95% credible interval, 0.76-1.24; higher favors HCQ/CQ), and found no convincing evidence of meaningful treatment effect heterogeneity among prespecified subgroups. Adverse event and serious adverse event rates were numerically higher with HCQ/CQ vs control (0.39 vs 0.29 and 0.13 vs 0.09 per patient, respectively). CONCLUSIONS: The findings of this individual participant data meta-analysis reinforce those of individual RCTs that HCQ/CQ is not efficacious for treatment of COVID-19 in hospitalized patients.

The effect of rFVIIa on pro- and anti-inflammatory cytokines in serum and cerebrospinal fluid in a swine model of traumatic brain injury.

Traumatic brain injury (TBI) is associated with significant infectious and inflammatory complications. Though increasing evidence suggests that rFVIIa administration may be efficacious for the pre-hospital treatment of TBI, the FVIIa-tissue factor complex has been shown to be immunologically active. To date the cytokine response to rFVIIa administration for the treatment of TBI has not been evaluated. Twenty anesthetized immature Yorkshire swine underwent fluid percussion TBI. At 15 min following injury, animals were randomized to receive either 90 µg/kg rFVIIa (rFVIIa) or nothing. Animals were observed for 6 h and then euthanized. Plasma and cerebrospinal (CSF) samples were collected at 0 min and 360 min, and ELISA analysis of TNF-α, IL-1ß and IL-10 was performed. Survival in both groups was 100%. Baseline cytokine concentrations were not statistically different between rFVIIa and control animals in plasma or CSF. Animals in both groups did not have significant changes in plasma cytokine concentrations following TBI. Control animals did not demonstrate significant changes from baseline of CSF cytokine concentrations following TBI. The administration of rFVIIa however, resulted in significant increases in CSF TNF-α concentration (232.0 pg/ml ± 75.9 vs 36.4 pg/ml ± 10.4, p = 0.036) and IL-10 concentration (10.7 pg/ml ± 0.6 vs 8.8 pg/ml ± 0.1, p = 0.015). IL-1ß concentrations were not significantly changed over the experimental time course. These results suggest that rFVIIa administration for the treatment of TBI is not immunologically inert, and is associated with increased CSF concentrations of TNF-α and IL-10.

Amylase and lipase detection in hemorrhaged animals treated with HBOC-201.

HBOC-201 may alter lipase and amylase detection on chemistry analyzers using optical methods and affect pancreatic function after trauma. Amylase and lipase measurements were correlated against HBOC-201 to evaluate interference on samples spiked with 0-6g/dL HBOC-201. The detection threshold was 2.5g/dL or none when measured, respectively, on Vitros 250 or Advia 1650 instruments. Amylase and lipase from blood samples collected from 55% EBV hemorrhaged Yucatan min-pigs showed peaks around 24-48 hours. Amylase increase was not significant between treatments but lipase was higher in HBOC-201-treated animals. Animals particularly affected by the injury had elevated enzymes after hemorrhagic shock, without significant clinical consequences.

Dose response of sodium nitrite on vasoactivity associated with HBOC-201 in a swine model of controlled hemorrhage.

Sodium nitrite (NaNO(2)) was evaluated in a 55% EBV hemorrhage swine model to mitigate the increased blood pressure due to HBOC-201. Animals were resuscitated by three 10 ml/kg infusions of either HBOC-201 or Hextend with and without NaNO(2). All vital signs, coagulation and blood chemistry were measured for 2 hr. HBOC-201-vasoconstriction was attenuated only after the first 10.8 µmol/kg NaNO(2) infusion. Complete abolition was obtained with the highest 3 NaNO(2) dose, but side effects were observed. There was no reduction in platelet function due to NaNO(2). NaNO(2) ability to reduce HBOC-201 vasoactivity was transient and 10.8 µmol/kg NaNO(2) seems an acceptable dose for further investigation.

Hemoglobin-based oxygen carrier (HBOC-201) and escalating doses of recombinant factor VIIa (rFVIIa) as a novel pre-hospital resuscitation fluid in a swine model of severe uncontrolled hemorrhage.

Exsanguinating hemorrhage and unavailability of blood are major problems in pre-hospital trauma care. We investigated if combining rFVIIa with HBOC-201 reduces blood loss and improves physiologic parameters compared to HBOC alone. Swine underwent liver injury and were resuscitated with HBOC-201 alone or HBOC+90, 180 or 360 µg/kg rFVIIa before hospital arrival at 240 min; animals survived to 72 hours. Blood loss was reduced; MAP, CI, transcutaneous oxygen saturation, and 72-hour survival improved in the 90 and 180 µg/kg rFVIIa groups. Lactate was cleared faster in the HBOC+rFVIIa 90 µg/kg group. Verification in a large, well-powered study is indicated.

HBOC-201 vasoactivity in a phase III clinical trial in orthopedic surgery subjects--extrapolation of potential risk for acute trauma trials.

BACKGROUND: Vasoactivity has hampered progress of hemoglobin-based oxygen carriers (HBOCs) due to concern for adverse blood pressure responses and secondary complications. A recent formulation, highly polymerized HBOC-201 (Biopure, Cambridge, MA), has been found to be less vasoactive than prior less polymerized formulations, and to improve outcome in animal models of hemorrhagic shock (HS) compared with standard resuscitation fluids. HBOCs are envisioned to have life- saving potential for severe trauma patients for whom death due to HS is common despite transport to level I trauma centers. As part of a benefit:risk analysis for a proposed clinical trial of HBOC-201 in patients with traumatic HS, we analyzed data from a previous phase III clinical trial of this HBOC that involved orthopedic surgery patients, for vasoactivity and related effects, with focus on patients more representative of the trauma population. STUDY DESIGN: In a previous phase III study involving orthopedic surgery patients, HEM-0115, consented/stabilized patients were randomized to receive HBOC-201 (N = 350) (up to ten 30 g Hb units) or red blood cells (RBC) (N = 338) (up to 9 units) at the first transfusion decision. Systolic blood pressure (SBP) responses, key system and individual adverse events (AEs) and serious adverse events, and cardiac biomarker elevation incidences, were compared in the overall population and subpopulations with stable trauma, hypotension, and with age stratification (Student's t and Fisher's exact tests, significance p < 0.05). RESULTS: Mild to moderate peak SBP responses were common in HBOC-201 subjects and more common than with RBC in the overall population (mean, 60.8 years old), but less frequent in HBOC-201 subjects with stable trauma, younger age (<50 years old), and hypotension, in whom group differences were narrowed. SBP Delta responses were more common with HBOC-201 than RBC in the overall population, but not in subjects with stable trauma and <50 year olds, in whom response rates were lower. In the overall population, AEs were more common than with RBC in most systems (also, hypertension and stroke); only cardiac system serious adverse events were more common with HBOC-201. In contrast, there were few significant group differences in stable trauma, hypotensive, and <70 and especially <50-year-old subjects, in whom AE incidences were generally lower. A disproportionate number of key AEs occurred in elderly subjects. Troponin (but not CK-MB) elevation was more frequent with HBOC-201 than RBC in the overall population but not in <50 year olds, and was not associated with acute coronary syndrome (ACS) or death. CONCLUSIONS: Our limited HEM-0115 safety analysis shows that key potentially vasoactivity-related adverse safety signals were more frequent with HBOC-201 than RBC in older patients undergoing orthopedic surgery with rapid access to safe blood transfusions. That incidences of these safety signals were generally lower and group differences narrowed in subpopulations with stable trauma, hypotension, and younger age, suggests an acceptable safety profile in younger acute trauma populations, especially in settings where rapid access to safe blood transfusions is unavailable; confirmation in controlled clinical trials is urgently warranted.

Profiling humoral immune responses to P. falciparum infection with protein microarrays.

A complete description of the serological response following exposure of humans to complex pathogens is lacking and approaches suitable for accomplishing this are limited. Here we report, using malaria as a model, a method which elucidates the profile of antibodies that develop after natural or experimental infection or after vaccination with attenuated organisms, and which identifies immunoreactive antigens of interest for vaccine development or other applications. Expression vectors encoding 250 Plasmodium falciparum (Pf) proteins were generated by PCR/recombination cloning; the proteins were individually expressed with >90% efficiency in Escherichia coli cell-free in vitro transcription and translation reactions, and printed directly without purification onto microarray slides. The protein microarrays were probed with human sera from one of four groups which differed in immune status: sterile immunity or no immunity against experimental challenge following vaccination with radiation-attenuated Pf sporozoites, partial immunity acquired by natural exposure, and no previous exposure to Pf. Overall, 72 highly reactive Pf antigens were identified. Proteomic features associated with immunoreactivity were identified. Importantly, antibody profiles were distinct for each donor group. Information obtained from such analyses will facilitate identifying antigens for vaccine development, dissecting the molecular basis of immunity, monitoring the outcome of whole-organism vaccine trials, and identifying immune correlates of protection.

Hematologic effects of recombinant factor VIIa combined with hemoglobin-based oxygen carrier-201 for prehospital resuscitation of swine with severe uncontrolled hemorrhage due to liver injury.

The combination of traumatic injury, hemorrhage, and fluid resuscitation results in consumption and dilution of coagulation factors, adversely impacting hematology outcome in trauma patients. The hemostatic effects of escalating doses of recombinant factor VIIa added to hemoglobin-based oxygen carrier-201 were assessed as prehospital fluid resuscitation in swine with severe uncontrolled hemorrhage. Swine underwent liver injury causing severe uncontrolled hemorrhage and shock. During a 4-h prehospital phase, either hypotensive or tachycardic, or both, animals were resuscitated with hemoglobin-based oxygen carrier-201 without (0x) or with escalating doses of recombinant factor VIIa [90 microg/kg (1x), 180 microg/kg (2x), or 360 microg/kg (4x)]. The animals received one initial full dose of 10 ml/kg at 15 min and up to four doses of 5 ml/kg thereafter. From 4 to 72 h (hospital phase), animals received either transfusions or isotonic saline or both as needed. Hematology profile (complete blood count), thromboelastography, in-vitro bleeding (platelet function analyzer), and coagulation (prothrombin time) were measured and the results were compared using mixed statistical models. In all groups, dilutional coagulopathy was evidenced by reduced hematocrit, platelets, and thromboelastography-maximum amplitude, and increased platelet function analyzer closure time and thromboelastography-reaction time. During the prehospital phase, hemoglobin-based oxygen carrier-201 restored hemoglobin in all groups. Recombinant factor VIIa decreased prothrombin time in recombinant factor VIIa groups compared with the hemoglobin-based oxygen carrier-201 group (P < 0.01). Unexpectedly, increasing recombinant factor VIIa dosage tended to increase fluid requirement (P > 0.05). Compared with hemoglobin-based oxygen carrier, 1x recombinant factor VIIa tended to decrease blood loss, lactate and thromboelastography-reaction time at 24 h but the 4x group increased these parameters. Platelets and thromboelastography-maximum amplitude decreased (P < 0.01) with the 4x group. In swine with severe uncontrolled hemorrhage, prehospital resuscitation with escalating doses of recombinant factor VIIa in combination with hemoglobin-based oxygen carrier-201 did not change survival or hemostasis. However, there were trends toward possible benefits of low recombinant factor VIIa doses, whereas high recombinant factor VIIa doses adversely affected hemostasis.

Innate immune response after resuscitation with hemoglobin-based oxygen carrier and recombinant factor VIIA in uncontrolled hemorrhagic shock in a swine model.

BACKGROUND: Rapid resuscitation with oxygen-carrying fluids is critically important in hemorrhagic shock (HS) combat casualties in remote areas where blood is not available. Hemoglobin-based oxygen carrier-201 (HBOC-201) has been shown to increase survival and reduce immune activation following HS in animal models. Recombinant factor VIIa (rfVIIa), a systemic hemostatic agent, is Food and Drug Administration approved for use in acute hemorrhage in hemophilic patients. The combination of HBOC-201 and rfVIIa may form the basis of a prospective multifunctional blood substitute and provide benefits in the rapid restoration of hemostasis, decreased inflammation and improved survival of HS combat casualties. In the present study, we evaluated innate immune responses in a swine model of uncontrolled HS following resuscitation with HBOC-201 +/- rfVIIa. MATERIALS: Thirty-two pigs underwent uncontrolled hemorrhage/liver crush injury, followed by resuscitation with five doses of HBOC-201 or HBOC + rfVIIa (90 microg/kg, or 180 microg/kg, or 360 microg/kg) and simulated 4 hours hospital arrival. Immune parameters were evaluated by flow cytometry and enzyme-linked immunosorbent assay. RESULTS: Survival differences to 72 hours of animals resuscitated with HBOC, HBOC + rfVIIa (90), (180), and (360) were not statistically significant and resulted in survival of 25%, 63%, 63% and 50%, respectively. At the prehospital phase all groups exhibited minimal immunomodulation, characterized by stable CD4/CD8 ratio, marginal increase of apoptosis and insignificant fluctuations of adhesion markers; increase of plasma cytokines was comparable across all groups, except tumor necrosis factor-alpha, that was significantly elevated in the HBOC group. CONCLUSION: HBOC-201 + rfVIIa triggered minimum immune activation in an uncontrolled HS swine and there was a nonsignificant survival benefit.

The effects of decreasing low-molecular weight hemoglobin components of hemoglobin-based oxygen carriers in swine with hemorrhagic shock.

BACKGROUND: Some hemoglobin-based oxygen carriers (HBOCs) improve outcome in animal models of hemorrhagic shock (HS) in comparison with standard asanguinous resuscitation fluids. Nevertheless, concern about intrinsic vasoactivity, linked in part to low-molecular weight (MW) hemoglobin (Hb), has slowed HBOC development. We assessed the impact of decreasing the low-MW Hb component of bovine HBOC on vasoactivity in severe HS. METHODS: Anesthetized invasively monitored swine were hemorrhaged 55% blood volume and resuscitated with bovine HBOC containing 31% (31 TD [HBOC-301]), 2% (2 TD [HBOC-201]), or 0.4% (0.4 TD) low-MW Hb. Pigs received four 10 mL/kg infusions over 60 minutes, hospital arrival was simulated at 75 minutes, organ blood flow (BF) was evaluated by microsphere injection, and monitoring was continued for 4 hours followed by complete necrotic evaluation. RESULTS: There were few differences between 2 TD and 0.4 TD. Thirty-one TD pigs had higher systemic and pulmonary blood pressure (BP), systemic vascular resistance index, and pulmonary artery wedge pressure, compared with 2 TD or 0.4 TD (p < 0.01); however, pigs in all groups had at least mildly elevated BP. Transcutaneous tissue oxygenation, base excess, and mixed venous oxygen saturation were similar across groups; lactate and methemoglobin were highest with 0.4 TD (p < 0.03). There were no group differences in BF. Over time, myocardial BF increased and hepatic BF decreased in all groups (for 31 TD, p < 0.05); renal BF was unchanged in all groups. There were no group differences in heart, lung, or liver histopathology, and survival. CONCLUSIONS: Although purification from 31% to 2% low-MW Hb content significantly decreased vasoactive responses, further purification to 0.4% had no additional clinically measurable effects in severe HS. If further diminution in HBOC vasoactivity is desired for use in HS, additional technical approaches may be required.