Blood Brain Barrier (BBB) breakdown is a secondary form of brain injury which has yet to be fully elucidated mechanistically. Existing research suggests that breakdown of tight junction proteins ...between endothelial cells is a primary driver of increased BBB permeability following injury, and intercellular signaling between primary cells of the neurovascular unit: endothelial cells, astrocytes, and pericytes; contribute to tight junction restoration. To expound upon this body of research, we analyzed the effects of severely injured patient plasma on each of the cell types in monoculture and together in a triculture model for the transcriptional and translational expression of the tight junction proteins Claudins 3 and 5, (CLDN3, CLDN5) and Zona Occludens 1 (ZO-1). Conditioned media transfer studies were performed to illuminate the cell type responsible for differential tight junction expression. Our data show that incubation with 5% human ex vivo severely injured patient plasma is sufficient to produce a differential response in endothelial cell tight junction mRNA and protein expression. Endothelial cells in monoculture produced a significant increase of CLDN3 and CLDN5 mRNA expression, (3.98 and 3.51 fold increase vs. control respectively, p<0.01) and CLDN5 protein expression, (2.58 fold change vs. control, p<0.01), whereas in triculture, this increase was attenuated. Our triculture model and conditioned media experiments suggest that conditioned media from astrocytes and pericytes and a triculture of astrocytes, pericytes and endothelial cells are sufficient in attenuating the transcriptional increases of tight junction proteins CLDN3 and CLDN5 observed in endothelial monocultures following incubation with severely injured trauma plasma. This data suggests that inhibitory molecular signals from astrocytes and pericytes contributes to prolonged BBB breakdown following injury via tight junction transcriptional and translational downregulation of CLDN5.
Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and ...carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage Qβ VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to Qβ and the CSP-Qβ vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the Qβ-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by Qβ presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the Qβ-CSP vaccine. The data presented here pave the way for further improvement in the Qβ conjugation chemistry and evaluation of both the Qβ-CSP and soluble CSP vaccines in the non-human primate model.
The availability of a highly purified and well characterized circumsporozoite protein (CSP) is essential to improve upon the partial success of recombinant CSP-based malaria vaccine candidates. ...Soluble, near full-length, Plasmodium falciparum CSP vaccine antigen (CS/D) was produced in E. coli under bio-production conditions that comply with current Good Manufacturing Practices (cGMP). A mouse immunogenicity study was conducted using a stable oil-in-water emulsion (SE) of CS/D in combination with the Toll-Like Receptor 4 (TLR4) agonist Glucopyranosyl Lipid A (GLA/SE), or one of two TLR7/8 agonists: R848 (un-conjugated) or 3M-051 (covalently conjugated). Compared to Alum and SE, GLA/SE induced higher CS/D specific antibody response in Balb/c mice. Subclass analysis showed higher IgG2:IgG1 ratio of GLA/SE induced antibodies as compared to Alum and SE. TLR synergy was not observed when soluble R848 was mixed with GLA/SE. Antibody response of 3M051 formulations in Balb/c was similar to GLA/SE, except for the higher IgG2:IgG1 ratio and a trend towards higher T cell responses in 3M051 containing groups. However, no synergistic enhancement of antibody and T cell response was evident when 3M051 conjugate was mixed with GLA/SE. In C57Bl/6 mice, CS/D adjuvanted with 3M051/SE or GLA/SE induced higher CSP repeat specific titers compared to SE. While, 3M051 induced antibodies had high IgG2c:IgG1 ratio, GLA/SE promoted high levels of both IgG1 and IgG2c. GLA/SE also induced more potent T-cell responses compared to SE in two independent C57/BL6 vaccination studies, suggesting a balanced and productive T(H1)/T(H2) response. GLA and 3M-051 similarly enhanced the protective efficacy of CS/D against challenge with a transgenic P. berghei parasite and most importantly, high levels of cytophilic IgG2 antibodies were associated with protection in this model. Our data indicated that the cGMP-grade, soluble CS/D antigen combined with the TLR4-containing adjuvant GLA/SE warrants further evaluation for protective responses in humans.
Background: We hypothesized that emergent placement of 7 French (Fr) common femoral artery (CFA) sheaths during trauma resuscitation for potential resuscitative endovascular balloon occlusion of the ...aorta (REBOA) carries a low complication rate.Methods: Trauma patients at a Level I trauma center with emergent CFA access from January 2016 through to December 2020 were reviewed. CFA access was categorized as (1) 7 Fr sheath plus REBOA (REBOA) and (2) 7 Fr sheathwithout REBOA (Sheath). Outcomes included mortality and vascular complications. Results: 157 patients underwent emergent CFA access. Sixty-nine (43.9%) patients had a 7 Fr CFA sheath, and 88 (56.1%) progressed to REBOA. The mortality rate was similar (Sheath 30.4% vs. REBOA 34.1%, p = 0.63). The REBOA cohort had a significantly higher complication rate (22.7%) compared to the Sheath cohort (4.3%, p = 0.001).Conclusions: Emergent 7 Fr CFA sheath placement during trauma resuscitation is low risk, suggesting empiric sheath placement is warranted in potential REBOA candidates.
Background
Trauma‐induced hypocalcemia is an underappreciated complication of severe injury but is well known to result in the derangement of an array of physiological regulatory mechanisms. Existing ...literature provides a compelling link between hypocalcemia and worse trauma‐induced coagulopathy and increased mortality after injury.
Study Design and Methods
This narrative review evaluates available data related to the risk factors, mechanisms, and treatment of hypocalcemia after severe injury. The authors did not perform a systemic review or meta‐analysis.
Results and Discussion
The interplay of acidosis, hypothermia, and coagulopathy with hypocalcemia potentiates the bloody vicious cycle of hemorrhagic shock which has been the paradigm of trauma resuscitation for over half a century. However, current screening and treatment of postinjury hypocalcemia are relegated to a secondary consideration in trauma resuscitation. We conclude calcium supplementation should be a primary tier intervention for life‐threatening injury.
Background: A 2021 report of the Aortic Occlusion for Resuscitation in Trauma and Acute Care Surgery multicenter registry described the outcomes of patients treated with Zone 3 resuscitative ...endovascular balloon occlusion of the aorta (REBOA zone 3). Our study builds upon that report, testing the hypothesis that REBOA zone 3 is associated with better outcomes than REBOA Zone 1 in the immediate treatment of severe, blunt pelvic injuries. Methods: We included adults who underwent aortic occlusion (AO) via REBOA zone 1 or REBOA Zone 3 in the emergency department for severe, blunt pelvic injuries Abbreviated Injury Score ≥ 3 or pelvic packing/embolization/first 24 hours in institutions with >10 REBOAs. Adjustment for confounders was accomplished with a Cox proportional hazards model for survival, generalized estimating equations for intensive care unit (ICU)-free days (IFD) and ventilation-free days (VFD) > 0 days, and mixed linear models for continuous outcomes (Glasgow Coma Scale GCS, Glasgow Outcome Scale GOS), accounting for facility clustering. Results: Of 109 eligible patients, 66 (60.6%) underwent REBOA Zone 3 and 43 (39.4%) REBOA Zone 1. There were no differences in demographics, but compared with REBOA Zone 3, REBOA Zone 1 patients were more likely to be admitted to high volume centers and be more severely injured. These patients did not differ in systolic blood pressure (SBP), cardiopulmonary resuscitation in the prehospital/hospital settings, SBP at the start of AO, time to AO start, likelihood of achieving hemodynamic stability or requirement of a second AO. After controlling for confounders, compared with REBOA Zone 3, REBOA Zone 1 was associated with a significantly higher mortality (adjusted hazard ratio, 1.51; 95% confidence interval CI, 1.04-2.19), but there were no differences in VFD > 0 (adjusted relative risk, 0.66; 95% CI, 0.33-1.31), IFD > 0 (adjusted relative risk, 0.78; 95% CI, 0.39-1.57), discharge GCS (adjusted difference, -1.16; 95% CI, -4.2 to 1.90) or discharge GOS (adjusted difference, -0.67; 95% CI -1.9 to 0.63). Conclusions: This study suggests that compared with REBOA Zone 1, REBOA Zone 3 provides superior survival and is not inferior regarding other adverse outcomes in patients with severe blunt pelvic injuries.
Activated Protein C (aPC) plays dual roles after injury, driving both trauma-induced coagulopathy (TIC) by cleaving, and thus inactivating, factors Va and VIIIa and depressing fibrinolysis while also ...mediating an inflammomodulatory milieu via protease activated receptor-1 (PAR-1) cytoprotective signaling. Because of this dual role, it represents and ideal target for study and therapeutics after trauma. A known aPC variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity rendering it potentially ideal as a cytoprotective therapeutic after trauma. We hypothesized that 3K3A-aPC would mitigate the endotheliopathy of trauma by protecting against endothelial permeability.
We used electric cell-substrate impedance sensing to measure permeability changes in real time in primary endothelial cells. These were cultured, grown to confluence, and treated with a 2 μg/mL solution of 3K3A-aPC at 180 minutes, 120 minutes, 60 minutes, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (Injury Severity Score > 15 and BD < -6) (trauma plasma TP). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via electric cell-substrate impedance sensing for 30 minutes after treatment with TP. We quantified permeability changes in the control and treatment groups as area under the curve (AUC). Rac1/RhoA activity was also compared between these groups. Statistical significance was determined by one-way ANOVA followed by a post hoc analysis using Tukey's multiple comparison's test.
Treatment with aPC mitigated endothelial permeability induced by ex vivo trauma plasma at all pre-treatment time points. The AUC of the 30-minute 3K3A-aPC pretreatment group was higher than TP alone (mean diff. 22.12 95% CI 13.75, 30.49, p < 0.0001) (Figure). Moreover, the AUC of the 60-minute, 120-minute, and 180-minute pretreatment groups was also higher than TP alone (mean diff., 16.30; 95% confidence interval CI, 7.93-24.67; 19.43; 95% CI, 11.06-27.80, and 18.65; 95% CI, 10.28-27.02;, all p < 0.0001, respectively). Rac1/RhoA activity was higher in the aPC pretreatment group when compared with all other groups ( p < 0.01).
Pretreatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anticoagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thromboinflammation for injured patients by minimizing aPC's role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function.
Prognostic and Epidemiological; Level III.
Zone 1 resuscitative endovascular balloon occlusion of the aorta has been recommended for refractory shock after a dismounted complex blast injury for the austere combat scenario. While resuscitative ...endovascular balloon occlusion of the aorta should enhance coronary perfusion, there is a potential risk of secondary brain injury due to loss of cerebral autoregulation. We developed a combat casualty relevant dismounted complex blast injury swine model to evaluate the effects of resuscitative endovascular balloon occlusion of the aorta zone I on intracranial pressure and cerebral edema. We hypothesized that zone 1 aortic occlusion with resuscitative endovascular balloon occlusion of the aorta would increase mean arterial pressure transmitted in excessive intracranial pressure, thereby worsening brain injury.
50 kg male Yorkshire swine were subjected to a combination dismounted complex blast injury model consisting of blast traumatic brain injury (50 psi, ARA Mobile Shock Laboratory), tissue injury (bilateral femur fractures), and hemorrhagic shock (controlled bleeding to a base deficit goal of 10 mEq/L). During the shock phase, pigs were randomized to no aortic occlusion (n = 8) or to 30 minutes of zone 1 resuscitative endovascular balloon occlusion of the aorta (zone 1 aortic occlusion group, n = 6). After shock, pigs in both groups received a modified Tactical Combat Casualty Care–based resuscitation and were monitored for an additional 240 minutes until euthanasia/death for a total of 6 hours. Intracranial pressure was monitored throughout, and brains were harvested for water content. Linear mixed models for repeated measures were used to compare mean arterial pressure and intracranial pressure between zone 1 aortic occlusion and no aortic occlusion groups.
After dismounted complex blast injury, the zone 1 group had a significantly higher mean arterial pressure during hemorrhagic shock compared to the control group (41.2 mm Hg vs 16.7 mm Hg, P = .002). During balloon occlusion, intracranial pressure was not significantly elevated in the zone 1 aortic occlusion group vs control, but intracranial pressure was significantly lower in the zone 1 group at the end of the observation period. In addition, the zone 1 aortic occlusion group did not have increased brain water content (zone 1 aortic occlusion: 3.95 ± 0.1g vs no aortic occlusion: 3.95 ± 0.3 g, P = .87). Troponin levels significantly increased in the no aortic occlusion group but did not in the zone 1 aortic occlusion group.
Zone 1 aortic occlusion using resuscitative endovascular balloon occlusion of the aorta in a large animal dismounted complex blast injury model improved proximal mean arterial pressure while not significantly increasing intracranial pressure during balloon inflation. Observation up to 240 minutes postresuscitation did not show clinical signs of worsening brain injury or cardiac injury. These data suggest that in a dismounted complex blast injury swine model, resuscitative endovascular balloon occlusion of the aorta in zone 1 may provide neuro- and cardioprotection in the setting of blast traumatic brain injury. However, longer monitoring periods may be needed to confirm that the neuroprotection is lasting.
Blunt cerebrovascular injury (BCVI) can result in devastating stroke. Because of operative inaccessibility, the most common treatment for BCVI is aspirin or a low-dose systemic heparin infusion. ...While it is assumed that low dose heparin infusion imparts venous thromboembolism (VTE) prophylaxis, this has not been evaluated in the BCVI population. The purpose of this study was to evaluate VTE rates in patients receiving low-dose heparin infusion as treatment for BCVI.
Patients diagnosed with BCVI between 2014 and 2018 were reviewed for initiation of low-dose systemic heparin treatment. VTE was defined as a deep vein thrombosis or pulmonary embolism. BCVI patients without systemic heparin treatment were compared to BCVI patients with heparin treatment for overall VTE rates. Comparisons were also made to injured patients without a BCVI in our Trauma Activation Protocol (TAP) database.
During the 5-year study period, 265 patients were identified with a BCVI. The majority (61%) were men with a median injury severity score (ISS) 22 (interquartile range IQR:14-33). Of these patients, 146 (55.1%) received a heparin infusion to treat BCVI. VTE was identified in eight of these patients (5.5%). Compared to TAP patients (n = 1020) who received standard dosing of VTE chemoprophylaxis, there was no difference in VTE rates compared to BCVI patients who were started on a low dose heparin infusion (3% versus 5.5%, P = 0.16). Area under the receiver operating characteristics (AUROC) was used to evaluate the predictive power of time to initiation of heparin infusion (AUC = 0.64 95% CI 0.42-0.85, P = 0.2) and time to reaching PTT goal (AUC = 0.52 95% CI 0.27-0.77, P = 0.83) as a predictor VTE events.
Low dose heparin infusion is frequently used as an initial treatment of BCVI. In injured patients with BCVI, a low dose heparin infusion is associated with a low rate of VTE, comparable to injured patients without BCVI that received standard VTE chemoprophylaxis.
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