Safety of Autologous Umbilical Cord Blood Therapy for Acquired Sensorineural Hearing Loss in Children.

BACKGROUND AND OBJECTIVES: Sensorineural hearing loss (SNHL) in children is associated with neurocognitive morbidity. The cause of SNHL is a loss of hair cells in the organ of Corti. There are currently no reparative treatments for SNHL. Numerous studies suggest that cord blood mononuclear cells (human umbilical cord blood, hUCB) allow at least partial restoration of SNHL by enabling repair of a damaged organ of Corti. Our objective is to determine if hUCB is a safe treatment for moderate to severe acquired SNHL in children. Subjects and. METHODS: Eleven children aged 6 months to 6 years with moderate to severe acquired SNHL were treated with intravenous autologous hUCB. The cell dose ranged from 8 to 30 million cells/kg body weight. Safety was assessed by measuring systemic hemodynamics during hUCB infusion. Infusion-related toxicity was evaluated by measuring neurologic, hepatic, renal and pulmonary function before and after infusion. Auditory function, auditory verbal language assessments and MRI with diffusion tensor imaging (DTI) were obtained before and after treatment. RESULTS: All patients survived, and there were no adverse events. No infusionrelated changes in hemodynamics occurred. No infusion-related toxicity was recorded. Five subjects experienced a reduction in auditory brainstem response (ABR) thresholds. Four of those 5 subjects also experienced an improvement in cochlear nerve latencies. Comparison of MRI with DTI sequences obtained before and after treatment revealed increased fractional anisotropy in the primary auditory cortex in three of five subjects with reduced ABR thresholds. Statistically significant (p<0.05) reductions in ABR thresholds were identified. CONCLUSIONS: TIntravenous hUCB is feasible and safe in children with SNHL.

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.

Biphasic regulation of myosin light chain phosphorylation by p21-activated kinase modulates intestinal smooth muscle contractility.

Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

Autologous bone marrow mononuclear cell therapy for severe traumatic brain injury in children.

BACKGROUND: Severe traumatic brain injury (TBI) in children is associated with substantial long-term morbidity and mortality. Currently, there are no successful neuroprotective/neuroreparative treatments for TBI. Numerous preclinical studies suggest that bone marrow-derived mononuclear cells (BMMNCs), their derivative cells (marrow stromal cells), or similar cells (umbilical cord blood cells) offer neuroprotection. OBJECTIVE: To determine whether autologous BMMNCs are a safe treatment for severe TBI in children. METHODS: Ten children aged 5 to 14 years with a postresuscitation Glasgow Coma Scale of 5 to 8 were treated with 6×10 autologous BMMNCs/kg body weight delivered intravenously within 48 hours after TBI. To determine the safety of the procedure, systemic and cerebral hemodynamics were monitored during bone marrow harvest; infusion-related toxicity was determined by pediatric logistic organ dysfunction (PELOD) scores, hepatic enzymes, Murray lung injury scores, and renal function. Conventional magnetic resonance imaging (cMRI) data were obtained at 1 and 6 months postinjury, as were neuropsychological and functional outcome measures. RESULTS: All patients survived. There were no episodes of harvest-related depression of systemic or cerebral hemodynamics. There was no detectable infusion-related toxicity as determined by PELOD score, hepatic enzymes, Murray lung injury scores, or renal function. cMRI imaging comparing gray matter, white matter, and CSF volumes showed no reduction from 1 to 6 months postinjury. Dichotomized Glasgow Outcome Score at 6 months showed 70% with good outcomes and 30% with moderate to severe disability. CONCLUSION: Bone marrow harvest and intravenous mononuclear cell infusion as treatment for severe TBI in children is logistically feasible and safe.

Progenitor cell therapy for the treatment of central nervous system injury: a review of the state of current clinical trials.

Recent preclinical work investigating the role of progenitor cell therapies for central nervous system (CNS) injuries has shown potential neuroprotection in the setting of traumatic brain injury (TBI), spinal cord injury (SCI), and ischemic stroke. Mechanisms currently under investigation include engraftment and transdifferentiation, modulation of the locoregional inflammatory milieu, and modulation of the systemic immunologic/inflammatory response. While the exact mechanism of action remains controversial, the growing amount of preclinical data demonstrating the potential benefit associated with progenitor cell therapy for neurological injury warrants the development of well-controlled clinical trials to investigate therapeutic safety and efficacy. In this paper, we review the currently active or recently completed clinical trials investigating the safety and potential efficacy of bone marrow-derived progenitor cell therapies for the treatment of TBI, SCI, and ischemic stroke. Our review of the literature shows that while the preliminary clinical trials reviewed in this paper offer novel data supporting the potential efficacy of stem/progenitor cell therapies for CNS injury, a great deal of additional work is needed to ensure the safety, efficacy, and mechanisms of progenitor cell therapy prior to widespread clinical trials.

Bone marrow-derived mononuclear cell populations in pediatric and adult patients.

BACKGROUND AIMS: Many clinical trials are currently evaluating the safety and efficacy of autologous bone marrow (BM) mononuclear cells (MNC) for various pathologies in younger and older non-cancer patients. Concern has been raised that autologous MNC derived from elderly patients may be less effective as a therapeutic option. METHODS: We compared the MNC yield, viability, phenotypic markers and in vitro functionality in pediatric patients compared with adult patients enrolled in clinical trials evaluating autologous BM MNC transplantation. Thirty-six patients (n=10 pediatric and n=26 adult) were included in this analysis. All patients underwent BM harvest in which 1-3 mL/kg was aspirated under local anesthesia. MNC were isolated by gradient densitometry. The average age of the older and younger patient groups was 59+/-7 years and 9+/-3 years, respectively. RESULTS: The average total MNC recovered from the BM in pediatric patients was 2.1 x 10(6)/mL and in older patients was 3.2 x 10(6)/mL. There were no differences in cell viability (>97%) or phenotypic markers identifying T cells, natural killer (NK) cells and neutrophils between the two groups. Of note, the Lin(-)CD34(+) cell population was not different between the groups. Average post-processing CFU-F, CFU-GEMM and BFU-E were not statistically different but there were significantly increased levels of CFU-GM in the older population. CONCLUSIONS: These results suggest that MNC from younger and older non-cancer patients are similar, but the data must be interpreted with caution given the small sample size and limited general understanding of MNC mechanisms of action on target cells. It is still possible that cells from older patients may produce fewer cytokines or be functionally impaired.

Cell therapies for traumatic brain injury.

Preliminary discoveries of the efficacy of cell therapy are currently being translated to clinical trials. Whereas a significant amount of work has been focused on cell therapy applications for a wide array of diseases, including cardiac disease, bone disease, hepatic disease, and cancer, there continues to be extraordinary anticipation that stem cells will advance the current therapeutic regimen for acute neurological disease. Traumatic brain injury is a devastating event for which current therapies are limited. In this report the authors discuss the current status of using adult stem cells to treat traumatic brain injury, including the basic cell types and potential mechanisms of action, preclinical data, and the initiation of clinical trials.

Autoantibody from women with preeclampsia induces soluble Fms-like tyrosine kinase-1 production via angiotensin type 1 receptor and calcineurin/nuclear factor of activated T-cells signaling.

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Recent evidence indicates that maternal endothelial dysfunction in preeclampsia results from increased soluble Fms-like tyrosine kinase-1 (sFlt-1), a circulating antiangiogenic protein. Factors responsible for excessive production of sFlt-1 in preeclampsia have not been identified. We tested the hypothesis that angiotensin II type 1 (AT(1)) receptor activating autoantibodies, which occur in women with preeclampsia, contribute to increased production of sFlt-1. IgG from women with preeclampsia stimulates the synthesis and secretion of sFlt-1 via AT(1) receptor activation in pregnant mice, human placental villous explants, and human trophoblast cells. Using FK506 or short-interfering RNA targeted to the calcineurin catalytic subunit mRNA, we determined that calcineurin/nuclear factor of activated T-cells signaling functions downstream of the AT(1) receptor to induce sFlt-1 synthesis and secretion by AT(1)-receptor activating autoantibodies. AT(1)-receptor activating autoantibody-induced sFlt-1 secretion resulted in inhibition of endothelial cell migration and capillary tube formation in vitro. Overall, our studies demonstrate that an autoantibody from women with preeclampsia induces sFlt-1 production via angiotensin receptor activation and downstream calcineurin/nuclear factor of activated T-cells signaling. These autoantibodies represent potentially important targets for diagnosis and therapeutic intervention.

Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human mesangial cells and induce interleukin-6 and plasminogen activator inhibitor-1 secretion.

BACKGROUND: Preeclampsia affects 3-5% of all pregnancies. It is a major cause of maternal and fetal morbidity and mortality. Recent studies demonstrate that autoantibodies against the angiotensin II type 1 (AT(1)) receptor are present in the serum of preeclamptic patients. In this study, we investigated the role of AT(1) receptor-agonistic autoantibody (AT1-AA) regarding interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (Pai-1) secretion in human mesangial cells. METHODS: The study included ten patients: five severely preeclamptic and five normotensive pregnant women. Immunoglobulin-G (IgG) was purified from each individual. The presence of AT1-AA was determined based on its ability to stimulate an increase in the contraction rate of rat neonatal cardiomyocytes. Primary human mesangial cells were chosen to study IgG-induced secretion of IL-6 and Pai-1. Losartan and epitope peptides were used to determine whether AT1-AA interaction with AT(1) receptor was associated with stimulation of IL-6 and Pai-1 secretion and was mediated through AT(1) receptor activation. RESULTS: The IgG from preeclamptic patients stimulated an increased contraction rate in rat neonatal cardiomyocytes. The IgG from preeclamptic patients induced the AT(1) receptor-specific secretion of IL-6 and Pai-1 from human mesangial cells at a significantly higher level than that achieved with IgG from normotensive patients. Competition with an epitope peptide suggested that the AT(1) receptor was stimulated by AT1-AA. CONCLUSIONS: Our findings suggest that a maternal autoantibody with the ability to activate AT(1) receptors may account for the development of renal damage seen in preeclamptic patients.

Effects of nuchal cord on birthweight and immediate neonatal outcomes.

Our objective was to ascertain if nuchal cord is associated with adverse neonatal outcomes. Using a retrospective database of term neonates, outcomes were compared among infants with 0, 1, and 2 or more loops of cord encircling the neck. Of 4426 neonates, 3651 served as controls, 691 had one loop, and 84 had two or more loops. There were no significant differences in the mean birthweight, the frequency of nonreassuring fetal heart rate patterns, operative vaginal deliveries, or 5-minute Apgar scores of < 7. The cesarean delivery rate was significantly different among the three groups and was the highest among the group of women whose fetus had no nuchal cord ( p < 0.01). A nuchal cord at term is not associated with untoward pregnancy outcomes.

Antibodies from preeclamptic patients stimulate increased intracellular Ca2+ mobilization through angiotensin receptor activation.

BACKGROUND: Preeclampsia is a serious disorder of pregnancy characterized by hypertension, proteinuria, edema, and coagulation and vascular abnormalities. At the cellular level, abnormalities include increased calcium concentration in platelets, lymphocytes, and erythrocytes. Recent studies have shown that antibodies directed against angiotensin II type I (AT1) receptors are also highly associated with preeclampsia. METHODS AND RESULTS: We tested the hypothesis that AT1 receptor-agonistic antibodies (AT1-AAs) could activate AT1 receptors, leading to an increased intracellular concentration of free calcium and to downstream activation of Ca2+ signaling pathways. Sera of 30 pregnant patients, 16 diagnosed with severe preeclampsia and 14 normotensive, were examined for the presence of IgG capable of stimulating intracellular Ca2+ mobilization. IgG from all preeclamptic patients activated AT1 receptors and increased intracellular free calcium. In contrast, none of the normotensive individuals had IgG capable of activating AT1 receptors. The specific mobilization of intracellular Ca2+ by AT1-AAs was blocked by losartan, an AT1 receptor antagonist, and by a 7-amino-acid peptide that corresponds to a portion of the second extracellular loop of the AT1 receptor. In addition, we have shown that AT1-AA-stimulated mobilization of intracellular Ca2+ results in the activation of the transcription factor, nuclear factor of activated T cells. CONCLUSIONS: These results suggest that maternal antibodies capable of activating AT1 receptors are likely to account for increased intracellular free Ca2+ concentrations and changes in gene expression associated with preeclampsia.

Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells.

OBJECTIVES: Recent evidence indicates that preeclampsia is associated with the presence of autoantibodies capable of activating the angiotensin II receptor, AT1. We sought to evaluate the role of AT1 agonistic autoantibodies (AT1-AA) in two major features of preeclampsia-increased plasminogen activator inhibitor-1 (PAI-1) production and shallow trophoblast invasion. METHODS: This study included 38 pregnant patients, 20 of whom had severe preeclampsia and 18 normotensive individuals. Immunoglobulin (Ig)G was purified from these individuals, and the presence of AT1-AA was determined based on its ability to stimulate an increase in the contraction rate of cultured rat neonatal cardiac myocytes. Immortalized human trophoblasts were chosen to study PAI-1 production and secretion after treatment with IgG from normotensive and preeclamptic women. An in vitro Matrigel invasion assay was used to test the effect of AT1-AA on the invasive properties of human trophoblasts. Losartan and cyclosporin A were used to determine whether the AT1-AA-induced stimulation of PAI-1 secretion is through the AT1 receptor and the calcineurin-nuclear factor of activated t-cells (NFAT)-dependent pathway. RESULTS: The results show that IgG from 18 of 20 severely preeclamptic women stimulated increased cardiomyocyte contraction rates of 20-40 beats per minute. A significant stimulation of PAI-1 secretion from human trophoblasts was observed with IgG from the same 18 of 20 patients with severe preeclampsia. Of IgG obtained from 18 normotensive pregnant patients, only two showed a relatively low level of biologic activity in the cardiomyocyte contraction and PAI-1 secretion assays. Activation of AT1 receptors by AT1-AA was blocked by losartan (an AT1 receptor antagonist) and by a seven amino acid peptide corresponding to a sequence present on the second extracellular loop of the AT1 receptor. Activation of AT1 receptors by AT1-AA resulted in decreased trophoblast invasiveness as determined by the in vitro Matrigel invasion assay. Additional data indicate that AT1 receptor activation by AT1-AA is followed by the downstream activation of the calcium-dependent calcineurin-NFAT signaling pathway leading to increased PAI-1 gene expression. CONCLUSION: Our findings suggest that maternal autoantibody with the ability to activate AT1 receptors may account for two features of preeclampsia, increased PAI-1 production and shallow trophoblast invasion.

Effect of parental education on decision-making about neonatal circumcision.

BACKGROUND: We investigated the impact of unbiased, published, easily accessible brochures on the parental decision about circumcision. METHODS: A total of 190 women who were delivered of healthy male infants at Memorial-Hermann Hospital and Woman's Hospital of Texas from December 1, 1999, to April 30, 2000, were asked to complete a brief demographic self-description and questionnaire regarding their attitudes and beliefs about circumcision. The most recent American Academy of Pediatrics (AAP) brochure about circumcision was then distributed, and subjects completed the questionnaire a second time after reading the brochure. RESULTS: Eighty-five percent of participants opted for circumcision. No woman altered her decision on the basis of information in the AAP brochure. Regardless of parental desire for circumcision, responses to all questions before and after distribution of the AAP brochure were highly correlated. Circumcision status of the father and the parents' education and age were significantly associated with the decision to circumcise. There was no significant association between this decision and marital status, race, or religion. CONCLUSIONS: Parental education about the medical indications and possible risks of circumcision has no impact on the decision-making process about neonatal circumcision.