Autologous bone marrow mononuclear cells to treat severe traumatic brain injury in children.

Autologous bone marrow mononuclear cells (BMMNCs) infused after severe traumatic brain injury have shown promise for treating the injury. We evaluated their impact in children, particularly their hypothesized ability to preserve the blood-brain barrier and diminish neuroinflammation, leading to structural CNS preservation with improved outcomes. We performed a randomized, double-blind, placebo-sham-controlled Bayesian dose-escalation clinical trial at two children's hospitals in Houston, TX and Phoenix, AZ, USA (NCT01851083). Patients 5-17 years of age with severe traumatic brain injury (Glasgow Coma Scale score ≤ 8) were randomized to BMMNC or placebo (3:2). Bone marrow harvest, cell isolation and infusion were completed by 48 h post-injury. A Bayesian continuous reassessment method was used with cohorts of size 3 in the BMMNC group to choose the safest between two doses. Primary end points were quantitative brain volumes using MRI and microstructural integrity of the corpus callosum (diffusivity and oedema measurements) at 6 months and 12 months. Long-term functional outcomes and ventilator days, intracranial pressure monitoring days, intensive care unit days and therapeutic intensity measures were compared between groups. Forty-seven patients were randomized, with 37 completing 1-year follow-up (23 BMMNC, 14 placebo). BMMNC treatment was associated with an almost 3-day (23%) reduction in ventilator days, 1-day (16%) reduction in intracranial pressure monitoring days and 3-day (14%) reduction in intensive care unit (ICU) days. White matter volume at 1 year in the BMMNC group was significantly preserved compared to placebo [decrease of 19 891 versus 40 491, respectively; mean difference of -20 600, 95% confidence interval (CI): -35 868 to -5332; P = 0.01], and the number of corpus callosum streamlines was reduced more in placebo than BMMNC, supporting evidence of preserved corpus callosum connectivity in the treated groups (-431 streamlines placebo versus -37 streamlines BMMNC; mean difference of -394, 95% CI: -803 to 15; P = 0.055), but this did not reach statistical significance due to high variability. We conclude that autologous BMMNC infusion in children within 48 h after severe traumatic brain injury is safe and feasible. Our data show that BMMNC infusion led to: (i) shorter intensive care duration and decreased ICU intensity; (ii) white matter structural preservation; and (iii) enhanced corpus callosum connectivity and improved microstructural metrics.

Removing Implicit Bias From Cardiothoracic Surgery Resident Recruitment: Changing the Paradigm.

INTRODUCTION: The cardiothoracic (CT) surgery workforce continues to suffer from underrepresentation of women and minority physicians. The presence of implicit bias in the recruitment process may impair efforts to enhance the diversity of our training programs. Using a systematic approach, we aimed to investigate and optimize our candidate selection processes to minimize implicit bias. METHODS: Internal review of a single center's CT fellowship program selection process was conducted. Areas of potential bias were evaluated. Specifically, we investigated how interview questions were selected, how candidates were assessed during interviews, and how they were compared after interviews. Proactive measures were implemented to remove identified sources of bias. RESULTS: Several areas of potential bias were identified, including variability in types of questions asked and disparities in how candidates were scored. We noted the presence of potentially gendered language, cultural bias, and stereotyping within traits being scored. With the goals of intentionally promoting diversity and inclusion, we selected five traits as likely predictors of success which served as the framework from which standardized interview questions were created. The interview scoresheet was modified to include all attributes felt to be important, while eliminating irrelevant confounders and language that could carry potential advantage to specific groups. CONCLUSIONS: By implementing strategies to identify and remove sources of implicit bias in the interview and recruitment process, our training program improved its process for the recruitment of a diverse cadre of matriculants. We must aim not only to diversify the composition of our trainee classes, but also to ensure equitable support, mentorship, and sponsorship throughout training and career advancement.

Clinical parameters affecting multipotent adult progenitor cells in vitro.

BACKGROUND: Human multipotent adult progenitor cells (MAPC®) are an emerging therapy for traumatic brain injury (TBI); however, clinically translating a therapy involves overcoming many factors in vivo which are not present in pre-clinical testing. In this study we examined clinical parameters in vitro that may impact cell therapy efficacy. METHODS: MAPC were infused through varying gauged needles and catheters with and without chlorhexidine, and their viability tested with trypan blue exclusion. MAPC were co-cultured with phenytoin and celecoxib at relevant clinical concentrations for 1 h and 24 h. Anti-inflammatory potency was tested using a stimulated rat splenocyte co-culture and ELISA for TNF-α production. MAPC were cultured under different osmolar concentrations and stained with propidium iodide for viability. Anti-inflammatory potency was tested by co-culture of MAPC with naïve lymphocytes activated by CD3/CD28 beads, and Click-iT® Plus EdU was used to quantify proliferation by flow cytometry. RESULTS: The mean viability of the MAPC infused via needles was 95 ± 1%; no difference was seen with varying flow rate, but viability was notably reduced by chlorhexidine. MAPC function was not impaired by co-culture with phenytoin, celecoxib, or combination with both. Co-culture with phenytoin showed a decrease in TNF-α production as compared to the MAPC control. MAPC cultured at varying osmolar concentrations all had viabilities greater than 90% with no statistical difference between them. Co-culture of MAPC with CD3/CD28 activated PBMCs showed a significant reduction in proliferation as measured by EdU uptake. DISCUSSION: Needle diameter, phenytoin, celecoxib, and a relevant range of osmolarities do not impair MAPC viability or anti-inflammatory potency in vitro.

Treatment of Severe Adult Traumatic Brain Injury Using Bone Marrow Mononuclear Cells.

Preclinical studies using bone marrow derived cells to treat traumatic brain injury have demonstrated efficacy in terms of blood-brain barrier preservation, neurogenesis, and functional outcomes. Phase 1 clinical trials using bone marrow mononuclear cells infused intravenously in children with severe traumatic brain injury demonstrated safety and potentially a central nervous system structural preservation treatment effect. This study sought to confirm the safety, logistic feasibility, and potential treatment effect size of structural preservation/inflammatory biomarker mitigation in adults to guide Phase 2 clinical trial design. Adults with severe traumatic brain injury (Glasgow Coma Scale 5-8) and without signs of irreversible brain injury were evaluated for entry into the trial. A dose escalation format was performed in 25 patients: 5 controls, followed 5 patients in each dosing cohort (6, 9, 12 ×106 cells/kg body weight), then 5 more controls. Bone marrow harvest, cell processing to isolate the mononuclear fraction, and re-infusion occurred within 48 hours after injury. Patients were monitored for harvest-related hemodynamic changes, infusional toxicity, and adverse events. Outcome measures included magnetic resonance imaging-based measurements of supratentorial and corpus callosal volumes as well as diffusion tensor imaging-based measurements of fractional anisotropy and mean diffusivity of the corpus callosum and the corticospinal tract at the level of the brainstem at 1 month and 6 months postinjury. Functional and neurocognitive outcomes were measured and correlated with imaging data. Inflammatory cytokine arrays were measured in the plasma pretreatment, posttreatment, and at 1 and 6 month follow-up. There were no serious adverse events. There was a mild pulmonary toxicity of the highest dose that was not clinically significant. Despite the treatment group having greater injury severity, there was structural preservation of critical regions of interest that correlated with functional outcomes. Key inflammatory cytokines were downregulated. Treatment of severe, adult traumatic brain injury using an intravenously delivered autologous bone marrow mononuclear cell infusion is safe and logistically feasible. There appears to be a treatment signal as evidenced by central nervous system structural preservation, consistent with previous pediatric trial data. Inflammatory biomarkers are downregulated after cell infusion. Stem Cells 2016 Video Highlight: https://youtu.be/UiCCPIe-IaQ Stem Cells 2017;35:1065-1079.

Human Neural Stem Cell Transplantation-Mediated Alteration of Microglial/Macrophage Phenotypes after Traumatic Brain Injury.

Neural stem cells (NSCs) promote recovery from brain trauma, but neuronal replacement is unlikely the sole underlying mechanism. We hypothesize that grafted NSCs enhance neural repair at least partially through modulating the host immune response after traumatic brain injury (TBI). C57BL/6 mice were intracerebrally injected with primed human NSCs (hNSCs) or vehicle 24 h after a severe controlled cortical impact injury. Six days after transplantation, brain tissues were collected for Western blot and immunohistochemical analyses. Observations included indicators of microglia/macrophage activation, M1 and M2 phenotypes, axonal injury detected by amyloid precursor protein (APP), lesion size, and the fate of grafted hNSCs. Animals receiving hNSC transplantation did not show significant decreases of brain lesion volumes compared to transplantation procedures with vehicle alone, but did show significantly reduced injury-dependent accumulation of APP. Furthermore, intracerebral transplantation of hNSCs reduced microglial activation as shown by a diminished intensity of Iba1 immunostaining and a transition of microglia/macrophages toward the M2 anti-inflammatory phenotype. The latter was represented by an increase in the brain M2/M1 ratio and increases of M2 microglial proteins. These phenotypic switches were accompanied by the increased expression of anti-inflammatory interleukin-4 receptor α and decreased proinflammatory interferon-γ receptor ß. Finally, grafted hNSCs mainly differentiated into neurons and were phagocytized by either M1 or M2 microglia/macrophages. Thus, intracerebral transplantation of primed hNSCs efficiently leads host microglia/macrophages toward an anti-inflammatory phenotype that presumably contributes to stem cell-mediated neuroprotective effects after severe TBI in mice.

Autologous bone marrow mononuclear cells reduce therapeutic intensity for severe traumatic brain injury in children.

OBJECTIVES: The devastating effect of traumatic brain injury is exacerbated by an acute secondary neuroinflammatory response, clinically manifest as elevated intracranial pressure due to cerebral edema. The treatment effect of cell-based therapies in the acute post-traumatic brain injury period has not been clinically studied although preclinical data demonstrate that bone marrow-derived mononuclear cell infusion down-regulates the inflammatory response. Our study evaluates whether pediatric traumatic brain injury patients receiving IV autologous bone marrow-derived mononuclear cells within 48 hours of injury experienced a reduction in therapeutic intensity directed toward managing elevated intracranial pressure relative to matched controls. DESIGN: The study was a retrospective cohort design comparing pediatric patients in a phase I clinical trial treated with IV autologous bone marrow-derived mononuclear cells (n = 10) to a control group of age- and severity-matched children (n = 19). SETTING: The study setting was at Children's Memorial Hermann Hospital, an American College of Surgeons Level 1 Pediatric Trauma Center and teaching hospital for the University of Texas Health Science Center at Houston from 2000 to 2008. PATIENTS: Study patients were 5-14 years with postresuscitation Glasgow Coma Scale scores of 5-8. INTERVENTIONS: The treatment group received 6 million autologous bone marrow-derived mononuclear cells/kg body weight IV within 48 hours of injury. The control group was treated in an identical fashion, per standard of care, guided by our traumatic brain injury management protocol, derived from American Association of Neurological Surgeons guidelines. MEASUREMENTS AND MAIN RESULTS: The primary measure was the Pediatric Intensity Level of Therapy scale used to quantify treatment of elevated intracranial pressure. Secondary measures included the Pediatric Logistic Organ Dysfunction score and days of intracranial pressure monitoring as a surrogate for length of neurointensive care. A repeated-measure mixed model with marginal linear predictions identified a significant reduction in the Pediatric Intensity Level of Therapy score beginning at 24 hours posttreatment through week 1 (p < 0.05). This divergence was also reflected in the Pediatric Logistic Organ Dysfunction score following the first week. The duration of intracranial pressure monitoring was 8.2 ± 1.3 days in the treated group and 15.6 ± 3.5 days (p = 0.03) in the time-matched control group. CONCLUSIONS: IV autologous bone marrow-derived mononuclear cell therapy is associated with lower treatment intensity required to manage intracranial pressure, associated severity of organ injury, and duration of neurointensive care following severe traumatic brain injury. This may corroborate preclinical data that autologous bone marrow-derived mononuclear cell therapy attenuates the effects of inflammation in the early post-traumatic brain injury period.

Far-red tracer analysis of traumatic cerebrovascular permeability.

BACKGROUND: Blood brain barrier (BBB) compromise is a key pathophysiological component of secondary traumatic brain injury characterized by edema and neuroinflammation in a previously immune-privileged environment. Current assays for BBB permeability are limited by working size, harsh extraction processes, suboptimal detection via absorbance, and wide excitation fluorescence spectra. In this study, we evaluate the feasibility of Alexa Fluor 680, a far-red dye bioconjugated to dextran, as an alternative assay to improve resolution and sensitivity. METHODS: Alexa Fluor was introduced intravenously on the day of sacrifice to three groups: sham, controlled cortical impact (CCI), and CCI treated with a cell based therapy known to reduce BBB permeability. The brains were sectioned coronally and imaged using an infrared laser scanner to generate intensity plot profiles as well as signal threshold images to distinguish regions with varying degrees of permeability. RESULTS: Linear plot profile analysis demonstrated greater signal intensity from CCI than treated rats at corresponding injury depths. Threshold analysis identified rims of signal at low + narrow threshold ranges. The integrated signals from a treatment group known to preserve the BBB were significantly less than the groups with CCI injury alone. There was no significant difference at high + wide signal intensity threshold ranges. CONCLUSIONS: Alexa Fluor 680 infrared photodetection and image analysis can aid in detecting differential degrees of BBB permeability after traumatic brain injury and maybe particularly useful in demonstrating BBB preservation of at-risk regions in response to therapeutic agents.

Differential MSC activation leads to distinct mononuclear leukocyte binding mechanisms.

Advances in the field of Multipotent Mesenchymal Stromal cell (MSC) biology have demonstrated that MSCs can improve disease outcome when 'activated' to exert immunomodulatory effects. However, the precise mechanisms modulating MSC-immune cells interactions remain largely elusive. In here, we activated MSC based on a recent polarization paradigm, in which MSCs can be polarized towards a pro- or anti-inflammatory phenotype depending on the Toll-like receptor stimulated, to dissect the mechanisms through which MSCs physically interact with and modulate leukocytes in this context. Our data show that MSCs activated through the Toll-like receptor (TLR) 4 pathway increased VCAM-1 and ICAM-1 dependent binding of leukocytes. On the other hand, TLR3 stimulation strongly increases leukocytes affinity to MSC comparatively, through the formation of cable-like hyaluronic acid structures. In addition, TLR4 activation elicited secretion of pro-inflammatory mediators by MSCs, whereas TLR3-activated MSCs displayed a milder pro-inflammatory phenotype, similar to inactivated MSCs. However, the differently activated MSCs maintained their ability to suppress leukocyte activation at similar levels in our in vitro model, and this immunomodulatory property was shown here to be partially mediated by prostaglandin. These results reinforce the concept that alternate activation profiles control MSC responses and may impact the therapeutic use of MSCs.

Immunomagnetic enrichment and flow cytometric characterization of mouse microglia.

BACKGROUND: The inflammatory response after a CNS injury is regulated by microglia/macrophages. Changes in the ratio of M1 classically activated pro-inflammatory cells versus M2 alternatively activated anti-inflammatory cells reveal the direction of the immune response. These cells are routinely identified and enumerated by morphology and cell-surface markers using immunohistochemistry. NEW METHOD: We used a controlled cortical impact (CCI) mouse model for traumatic brain injury (TBI), then isolated microglia/macrophages from neural cell suspensions using magnetic beads conjugated to CD11b monoclonal antibody to obtain the entire myeloid population. Polarization states of CD11b(+)CD45(lo) microglia were evaluated by expression of M1 surface marker FcγRII/III and M2 surface marker CD206. RESULTS: After TBI, we observed an increase in M1:M2 ratio in the ipsilateral hemisphere when compared to the contralateral side, indicating that 24h after CCI, a shift in microglia polarization occurs localized to the hemisphere of injury. Comparison with existing method(s): The major impetus for developing and refining the methods was the need to accurately quantify microglial activation states without reliance on manual morphometric counting of serial immunohistochemistry slides. Flow cytometric analysis of enriched cell suspensions provides quantitative measurement of microglial polarization states complementary to existing methods, but for entire populations of cells. CONCLUSIONS: In summary, we used immunomagnetic beads to isolate myeloid cells from injured brain, then stained surface antigens to flow cytometrically identify and categorize microglia as either classically activated M1 or alternatively activated M2, generating a ratio of M1:M2 cells which is useful in studying attempts to reduce or redirect neuroinflammation.

Autologous bone marrow mononuclear cells therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury.

BACKGROUND: Autologous bone marrow-derived mononuclear cells (AMNCs) have shown therapeutic promise for central nervous system insults such as stroke and traumatic brain injury (TBI). We hypothesized that intravenous injection of AMNC provides neuroprotection, which leads to cognitive improvement after TBI. METHODS: A controlled cortical impact (CCI) rodent TBI model was used to examine blood-brain barrier (BBB) permeability, neuronal and glial apoptosis, as well as cognitive behavior. Two groups of rats underwent CCI with AMNC treatment (CCI-autologous) or without AMNC treatment (CCI-alone), consisting of 2 million AMNC per kilogram body weight harvested from the tibia and intravenously injected 72 hours after injury. CCI-alone animals underwent sham harvests and received vehicle injections. RESULTS: Ninety-six hours after injury, AMNC significantly reduced the BBB permeability in injured animals, and there was an increase in apoptosis of proinflammatory activated microglia in the ipsilateral hippocampus. At 4 weeks after injury, we observed significant improvement in probe testing of CCI-Autologous group in comparison to CCI-Alone in the Morris Water Maze paradigm. CONCLUSION: Our data demonstrate that the intravenous injection of AMNC after TBI leads to neuroprotection by preserving early BBB integrity, increasing activated microglial apoptosis and improving cognitive function.

Progenitor cells: therapeutic targets after traumatic brain injury.

Traumatic brain injuries and their associated treatments carry high cost in both financial impact and morbidity to human life. Recent studies and trials present promising results in reducing secondary injury in the days and weeks following the primary insult. A number of studies, both pre-clinical and clinical, have found that different populations of stem/progenitor cells result in a reduction of inflammation, maintenance of the blood brain barrier, and an overall improved prognosis. The mechanism of action of these cellular therapies appears to rely upon the ability of the cells to influence microglia/macrophage phenotype and alter the state of the inflammatory response. The spleen has become an area of intense interest as an arena where therapeutic cells interact with reactive macrophages to cause system-level changes in immune activity. Additionally, the spleen enacts anti-inflammatory responses originating in the CNS, delivered through vagal activity with a recently described mechanism culminating in acetylcholine release. This review provides a summary of recent findings as to the mechanisms of action observed in current cellular therapies.