Effects of tail nerve electrical stimulation on the activation and plasticity of the lumbar locomotor circuits and the prevention of skeletal muscle atrophy after spinal cord transection in rats.

INTRODUCTION: Severe spinal cord injury results in the loss of neurons in the relatively intact spinal cord below the injury area and skeletal muscle atrophy in the paralyzed limbs. These pathological processes are significant obstacles for motor function reconstruction. OBJECTIVE: We performed tail nerve electrical stimulation (TNES) to activate the motor neural circuits below the injury site of the spinal cord to elucidate the regulatory mechanisms of the excitatory afferent neurons in promoting the reconstruction of locomotor function. METHODS: Eight days after T10 spinal cord transection in rats, TNES was performed for 7 weeks. Behavioral scores were assessed weekly. Electrophysiological tests and double retrograde tracings were performed at week 8. RESULTS: After 7 weeks of TNES treatment, there was restoration in innervation, the number of stem cells, and mitochondrial metabolism in the rats' hindlimb muscles. Double retrograde tracings of the tail nerve and sciatic nerve further confirmed the presence of synaptic connections between the tail nerve and central pattern generator (CPG) neurons in the lumbar spinal cord, as well as motor neurons innervating the hindlimb muscles. CONCLUSION: The mechanisms of TNES induced by the stimulation of primary afferent nerve fibers involves efficient activation of the motor neural circuits in the lumbosacral segment, alterations of synaptic plasticity, and the improvement of muscle and nerve regeneration, which provides the structural and functional foundation for the future use of cutting-edge biological treatment strategies to restore voluntary movement of paralyzed hindlimbs.

Nrf2 enhances the therapeutic efficiency of adipose-derived stem cells in the treatment of neurogenic erectile dysfunction in a rat model.

BACKGROUND: Erectile dysfunction (ED) caused by intraoperative nerve injury is a major complication of pelvic surgery. Adipose-derived stem cells (ADSCs) have presented therapeutic potential in a rat model of bilateral cavernous nerve injury (BCNI), while inadequate in vivo viability has largely limited their application. Nuclear factor-E2-related Factor (Nrf2) is a key transcription factor that regulates cellular anti-oxidative stress. In this work, we investigated the effect of Nrf2 expression regulation on the viability of ADSCs, and explore its repair potential in a BCNI rat model. RESULTS: The survival time of tert-Butylhydroquinone (tBHQ)-ADSCs in BCNI model increased obviously. In addition, the tBHQ-ADSCs group presented better restoration of major pelvic ganglion (MPG) nerve contents and fibers, better improvement of erectile function, and less penile fibrosis than the other groups. Moreover, the expression of Nrf2 and superoxide dismutase 1 (SOD1) were higher than those of other groups. CONCLUSION: Nrf2 could enhance the anti-oxidative stress ability of ADSCs, so as to improve the therapeutic effect of ADSCs on BCNI rat model.

RéSUMé: CONTEXTE: La dysfonction érectile (DE) causée par une lésion nerveuse peropératoire est une complication majeure de la chirurgie pelvienne. Les cellules souches dérivées du tissu adipeux (ADSC) ont constitué un potentiel thérapeutique dans un modèle de lésion bilatérale des nerfs caverneux (BCNI) chez le rat, mais une viabilité insuffisante in vivo a largement limité leur application. Nrf2 est un facteur de transcription clé qui régule le stress antioxydant cellulaire. Dans ce travail, nous avons étudié l'effet de la régulation de l'expression de Nrf2 sur la viabilité des ADSC, et exploré son potentiel de réparation dans un modèle de BCNI chez le rat. RéSULTATS: Le temps de survie des cellules tert-butylhydroquinone (tBHQ)-ADSC dans le modèle BCNI a significativement augmenté. De plus, le groupe tBHQ-ADSC a présenté une meilleure restauration du contenu et des fibres nerveuses des ganglions pelviens majeurs, une meilleure amélioration de la fonction érectile et une moindre fibrose pénienne que les autres groupes. Par ailleurs, l'expression de Nrf2 et de la superoxyde dismutase 1 était plus élevée dans ce groupe que dans les autres groupes. CONCLUSIONS: Nrf2 pourrait améliorer la capacité de stress anti-oxydatif des cellules ADSC, améliorant ainsi l'effet thérapeutique des ADSC sur le modèle de BCNI chez le rat.

The therapeutic mechanism of transcranial iTBS on nerve regeneration and functional recovery in rats with complete spinal cord transection.

Background: After spinal cord transection injury, the inflammatory microenvironment formed at the injury site, and the cascade of effects generated by secondary injury, results in limited regeneration of injured axons and the apoptosis of neurons in the sensorimotor cortex (SMC). It is crucial to reverse these adverse processes for the recovery of voluntary movement. The mechanism of transcranial intermittent theta-burst stimulation (iTBS) as a new non-invasive neural regulation paradigm in promoting axonal regeneration and motor function repair was explored by means of a severe spinal cord transection. Methods: Rats underwent spinal cord transection and 2 mm resection of spinal cord at T10 level. Four groups were studied: Normal (no lesion), Control (lesion with no treatment), sham iTBS (lesion and no functional treatment) and experimental, exposed to transcranial iTBS, 72 h after spinal lesion. Each rat received treatment once a day for 5 days a week; behavioral tests were administered one a week. Inflammation, neuronal apoptosis, neuroprotective effects, regeneration and synaptic plasticity after spinal cord injury (SCI) were determined by immunofluorescence staining, western blotting and mRNA sequencing. For each rat, anterograde tracings were acquired from the SMC or the long descending propriospinal neurons and tested for cortical motor evoked potentials (CMEPs). Regeneration of the corticospinal tract (CST) and 5-hydroxytryptamine (5-HT) nerve fibers were analyzed 10 weeks after SCI. Results: When compared to the Control group, the iTBS group showed a reduced inflammatory response and reduced levels of neuronal apoptosis in the SMC when tested 2 weeks after treatment. Four weeks after SCI, the neuroimmune microenvironment at the injury site had improved in the iTBS group, and neuroprotective effects were evident, including the promotion of axonal regeneration and synaptic plasticity. After 8 weeks of iTBS treatment, there was a significant increase in CST regeneration in the region rostral to the site of injury. Furthermore, there was a significant increase in the number of 5-HT nerve fibers at the center of the injury site and the long descending propriospinal tract (LDPT) fibers in the region caudal to the site of injury. Moreover, CMEPs and hindlimb motor function were significantly improved. Conclusion: Neuronal activation and neural tracing further verified that iTBS had the potential to provide neuroprotective effects during the early stages of SCI and induce regeneration effects related to the descending motor pathways (CST, 5-HT and LDPT). Furthermore, our results revealed key relationships between neural pathway activation, neuroimmune regulation, neuroprotection and axonal regeneration, as well as the interaction network of key genes.

Activating Endogenous Neurogenesis for Spinal Cord Injury Repair: Recent Advances and Future Prospects.

After spinal cord injury (SCI), endogenous neural stem cells are activated and migrate to the injury site where they differentiate into astrocytes, but they rarely differentiate into neurons. It is difficult for brain-derived information to be transmitted through the injury site after SCI because of the lack of neurons that can relay neural information through the injury site, and the functional recovery of adult mammals is difficult to achieve. The development of bioactive materials, tissue engineering, stem cell therapy, and physiotherapy has provided new strategies for the treatment of SCI and shown broad application prospects, such as promoting endogenous neurogenesis after SCI. In this review, we focus on novel approaches including tissue engineering, stem cell technology, and physiotherapy to promote endogenous neurogenesis and their therapeutic effects on SCI. Moreover, we explore the mechanisms and challenges of endogenous neurogenesis for the repair of SCI.

Identification of LSM family members as potential chemoresistance predictive and therapeutic biomarkers for gastric cancer.

Introduction: The Like-Smith (LSM) family plays a critical role in the progression of several cancers. However, the function of LSMs in chemoresistance of gastric cancer (GC) is still elusive. Methods: The Cancer Genome Atlas (TCGA) database, Gene Expression Omnibus (GEO) database and Tumor Immune Estimation Resource Analysis (TIMER) were utilized to analyze the expression, prognostic value and immune infiltration of LSMs in GC patients. Moreover, qPCR and immunohistochemistry (IHC) experiment were conducted with clinical samples. Results: The expression of LSMs was upregulated in GC tissues and most of LSMs were negatively correlated with overall survival of GC patients with 5-fluorouracil (5-FU) treatment. We further revealed that LSM5, 7 and 8 were hub genes of GEO (GSE14210). Besides, the qPCR results demonstrated that a higher level of LSM5 and LSM8 was associated with 5-FU chemoresistance in GC. Moreover, both TIMER and IHC revealed that a lower expression of LSM5 and LSM8 was correlated with high infiltration of T cells, regulatory T cells, B cells, macrophages, and neutrophils. Discussion: Our study systematically investigated the expression pattern and biological features of LSM family members in GC, and identified LSM5 and LSM8 as potential biomarkers in GC with 5-FU chemotherapy.

Tail nerve electrical stimulation promoted the efficiency of transplanted spinal cord-like tissue as a neuronal relay to repair the motor function of rats with transected spinal cord injury.

Following transected spinal cord injury (SCI), there is a critical need to restore nerve conduction at the injury site and activate the silent neural circuits caudal to the injury to promote the recovery of voluntary movement. In this study, we generated a rat model of SCI, constructed neural stem cell (NSC)-derived spinal cord-like tissue (SCLT), and evaluated its ability to replace injured spinal cord and repair nerve conduction in the spinal cord as a neuronal relay. The lumbosacral spinal cord was further activated with tail nerve electrical stimulation (TNES) as a synergistic electrical stimulation to better receive the neural information transmitted by the SCLT. Next, we investigated the neuromodulatory mechanism underlying the action of TNES and its synergism with SCLT in SCI repair. TNES promoted the regeneration and remyelination of axons and increased the proportion of glutamatergic neurons in SCLT to transmit brain-derived neural information more efficiently to the caudal spinal cord. TNES also increased the innervation of motor neurons to hindlimb muscle and improved the microenvironment of muscle tissue, resulting in effective prevention of hindlimb muscle atrophy and enhanced muscle mitochondrial energy metabolism. Tracing of the neural circuits of the sciatic nerve and tail nerve identified the mechanisms responsible for the synergistic effects of SCLT transplantation and TNES in activating central pattern generator (CPG) neural circuits and promoting voluntary motor function recovery in rats. The combination of SCLT and TNES is expected to provide a new breakthrough for patients with SCI to restore voluntary movement and control their muscles.

Complement-mediated thrombotic microangiopathy as a link between endothelial damage and steroid-refractory GVHD.

Transplant-associated thrombotic microangiopathy (TA-TMA), a complication of hematopoietic cell transplant (HCT), is associated with significant morbidity and mortality. The pathophysiology and overlap of TA-TMA with other posttransplant complications such as graft-versus-host disease (GVHD) is poorly understood. We retrospectively identified cases of TA-TMA among patients with grade 3/4 gastrointestinal (GI) GVHD, reviewed intestinal biopsy specimens, and performed correlative testing of biomarkers associated with TA-TMA. TA-TMA was more common in patients with steroid-refractory GVHD compared with steroid-responsive GVHD (79.3% vs 42.1%; P = .001). Among patients surviving 100 days post-HCT, 1-year survival from day 100 was significantly better for patients who had not developed TA-TMA in the first 100 days (69.5% vs 36.7%; P < .001). Only 1 of 7 proposed TA-TMA histology criteria (mucosal hemorrhage) differed significantly based on GVHD steroid response. In multivariable modeling, steroid-refractory GVHD was a risk factor for development of TA-TMA (hazard ratio, 3.09; 95% confidence interval, 1.68-5.67; P < .001). There were no differences in complement activation at GVHD onset; however, 2 to 6 weeks later, patients with TA-TMA had higher levels of BBPlus and C5b-9, markers of alternative and terminal pathway activation (BBPlus: median, 600 vs 209.3 ng/mL; P = .0045) (C5b-9: median, 425.9 vs 258.4 ng/mL; P = .029). TA-TMA is associated with poor overall survival (OS) following HCT and may be detected early by histologic findings and may be differentiated from GVHD by measurement of alternative and terminal complement pathway activation. It is unknown whether treatment of TA-TMA will improve survival in steroid-refractory GVHD.

Increased complement activation during platelet storage.

BACKGROUND: It has been shown that platelet transfusion carries a higher incidence of transfusion-related adverse events than any other blood components, and prolonged platelet storage is associated with more transfusion reactions, most of which are considered to be inflammatory responses. However, the role of complement, which has very important proinflammatory activities, in the pathogenesis of platelet-related adverse events has not been fully understood. STUDY DESIGN AND METHODS: Three units of platelets collected by apheresis were stored on a platelet rotator with the temperature controlled between 22 and 24°C. Plasma samples were obtained using a sterile technique on Days 2 through 7. Complement components were assayed to evaluate the activation of complement activation and included C4d (classical pathway), Factor Bb (alternative pathway), C3a (common pathway), C5a (terminal pathway), and C5b-9 (terminal pathway). RESULTS: Both C4d and C3a were elevated on the first day of testing (Day 2) compared with the established normal ranges and continued to increase over time in storage. In contrast, Factor Bb levels remained stable and were within the normal range over time. Over a span of 7 days in storage, the terminal complement factors C5a and C5b-9 were also significantly increased, although the magnitude of increases was not as striking as those in C4d and C3a levels. CONCLUSION: Our results demonstrate that substantial complement activation occurs in platelets under standard storage conditions, and this activation increases with the duration of storage. After transfusion, these activated complement components might result in accelerated complement activation in recipients, leading to transfusion-related adverse events.

Cyclosporine or steroids as an adjunct to plasma exchange in the treatment of immune-mediated thrombotic thrombocytopenic purpura.

Although steroids are routinely used as an adjunct to plasma exchange (PEX) therapy in the treatment of immune-mediated thrombotic thrombocytopenic purpura (iTTP), limited data regarding their efficacy or effect on ADAMTS13 biomarkers are available. We report the results of a prospective, randomized study that compared the effectiveness of prednisone or cyclosporine (CSA) as adjuncts to PEX in the treatment of iTTP. A total of 26 of the planned 72 subjects were enrolled and treated from November 2007 until February 2014 before the study was halted after a planned interim analysis. Fourteen patients were randomly assigned to the prednisone arm, and 12 to the CSA arm of the study. One patient died in each arm of the study, and 2 patients in the prednisone arm of the study failed to achieve a response and crossed over to the CSA arm, leaving 11 patients in each arm of the study evaluable for the primary end point of exacerbation. One of the 11 prednisone-treated subjects (9%) suffered an exacerbation, whereas 3 of the 11 (27%) patients in the CSA arm suffered an exacerbation. Although there was no significant difference in the exacerbation rate, suppression of the anti-ADAMTS13 antibodies and improvement in ADAMTS13 activity in the first month after stopping PEX were significantly better in the prednisone-treated subjects. Side effects were manageable and comparable in both arms of the study. These data demonstrate the superiority of prednisone over CSA as an adjunct to PEX in the suppression of the anti-ADAMTS13 antibodies and improvement in ADAMTS13 activity. This trial was registered at www.clinicaltrials.gov as #NCT00713193.

Effect of blood sampling, processing, and storage on the measurement of complement activation biomarkers.

OBJECTIVES: Recent studies have shown that complement hyperactivation contributes to development of thrombotic microangiopathy. The evaluation of complement biomarkers is known to be influenced by inappropriate specimen handling. However, there has been no study fully addressing this topic. METHODS: Blood from each donor was subjected to 62 different handling conditions prior to complement assays. RESULTS: Complement biomarkers (C4d/C3a/factor Bb/C5a/C5b-9) are stable at room temperature (RT) for up to 4 hours in whole blood containing citrate or EDTA. However, under similar conditions, levels of C4d and C3a were significantly higher in serum than those in plasma. Thawing of the samples on ice or at RT had no significant effect on complement levels. In contrast, thawing at 37°C resulted in striking increases in levels of the complement system in serum and citrated plasma but not in EDTA plasma. Up to four freeze/thaw cycles on ice or RT did not substantially increase the levels of C3a, factor Bb, C5a, and C5b-9 but had a significant effect on C4d. Long-term storage of citrated plasma at -80°C for up to 6 years had no significant effect on levels of complement factors. CONCLUSIONS: The results from this study thus provide crucial guidelines for future investigations using complement biomarkers to define the role of complement system in disease.

Diagnostic and prognostic values of ADAMTS13 activity measured during daily plasma exchange therapy in patients with acquired thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) requires immediate treatment with plasma exchange (PE) to prevent disease mortality and/or morbidity. Frequently, PE is initiated before blood sample is collected to confirm ADAMTS13 deficiency. However, the effect of PE treatments on the evaluation of ADAMTS13 is uncertain. Moreover, the pertinence of ADAMTS13 activity during PE therapy to prediction of treatment outcomes is unclear. Thus, clarification of the diagnostic and prognostic values of ADAMTS13 activity obtained during PE treatment is an unmet clinical need. STUDY DESIGN AND METHODS: A total of 212 sequential samples were obtained during the course of daily PE treatment from 19 patients with acquired TTP. ADAMTS13 activity levels were determined in these longitudinal samples for analysis. RESULTS: After the initial three daily PE procedures, the sensitivities of ADAMTS13 activity in diagnosis of TTP (<10%) were 89, 83, and 78%, respectively. To determine prognostic value, patients with (n = 7) and without (n = 12) a recovery of ADAMTS13 activity to more than 10% within seven sessions of daily PE treatment were compared. Recovery of ADAMTS13 activity to more than 10% within 7 days is significantly associated with a timely achievement of clinical response (p < 0.01). In contrast, the patients without more than 10% ADAMTS13 within 1 week appear at risk for worse treatment outcomes manifested as TTP exacerbation, treatment refractoriness, or death. CONCLUSION: The data suggest that ADAMTS13 activities measured during the initial period of PE therapy offer both diagnostic and prognostic values in acquired TTP.

P21-activated kinase 7 mediates cisplatin-resistance of esophageal squamous carcinoma cells with Aurora-A overexpression.

Aurora-A overexpression is common in various types of cancers and has been shown to be involved in tumorigenesis through different signaling pathways, yet how the deregulation affects cancer therapeutics remains elusive. Here we showed that overexpression of Aurora-A rendered esophageal cancer cells resistance to cisplatin (CDDP) by inhibiting apoptosis. By using an apoptosis array, we identified a downstream gene, p21-activated kinase 7 (PAK7). PAK7 was upregulated by Aurora-A overexpression at both mRNA and protein levels. Importantly, the expression levels of Aurora-A and PAK7 were correlated in ESCC primary samples. Chromatin immunoprecipitation (ChIP) assay revealed that binding of E2F1 to the promoter of PAK7 was significantly enhanced upon Aurora-A activation, and knockdown of transcription factor E2F1 decreased PAK7 expression, suggesting that Aurora-A regulated PAK7 through E2F1. Furthermore, we demonstrated that PAK7 knockdown led to increased apoptosis, and Aurora-A-induced resistance to CDDP was reversed by downregulation of PAK7, suggesting PAK7 was a downstream player of Aurora-A that mediated chemoresistance of ESCC cells to CDDP. Our data suggest that PAK7 may serve as an attractive candidate for therapeutics in ESCC patients with Aurora-A abnormality.

Biomarkers of terminal complement activation confirm the diagnosis of aHUS and differentiate aHUS from TTP.

Atypical hemolytic uremic syndrome (aHUS) is characterized by dysregulated complement activity, the development of a thrombotic microangiopathy (TMA), and widespread end organ injury. aHUS remains a clinical diagnosis without an objective laboratory test to confirm the diagnosis. We performed a retrospective analysis of 103 patients enrolled in the Ohio State University TTP/aHUS Registry presenting with an acute TMA. Nineteen patients were clinically categorized as aHUS based on the following criteria: (1) platelet count <100 × 10(9)/L, (2) serum creatinine >2.25 mg/dL, and (3) a disintegrin and metalloprotease with thrombospondin type 1 motif, 13 (ADAMTS13) activity >10%. Sixteen of 19 patients were treated with plasma exchange (PEX) therapy, with 6/16 (38%) responding to PEX. Nine patients were treated with eculizumab with 7/9 (78%) responding to therapy. In contrast to thrombotic thrombocytopenic purpura (TTP) patients, no aHUS patients demonstrated ultralarge von Willebrand factor multimers at presentation. Median markers of generalized complement activation (C3a), alternative pathway (Bb), classical/lectin pathway (C4d), and terminal complement activation (C5a and C5b-9) were increased in the plasma of these 19 patients. Compared with a cohort of ADAMTS13-deficient TTP patients (n = 38), C5a and C5-9 were significantly higher in the 19 patients clinically characterized as aHUS, suggesting that pretreatment measurements of complement biomarkers C5a and C5b-9 may confirm the diagnosis of aHUS and differentiate it from TTP.

Gender Differences in Pulmonary Function, Respiratory Symptoms, and Macrophage Proteomics among HIV-Infected Smokers.

Background. HIV-infected subjects have an increased incidence of pulmonary emphysema. There are known gender differences in COPD phenotypic expression and diagnosis, but this is not well characterized in lung disease related to HIV. We analyzed a group at risk for the development of COPD (HIV-infected smokers) to determine gender differences in pulmonary symptoms, pulmonary function tests, and HRCT appearances. Methods. This was a cross-sectional, baseline analysis of a prospective study performed between 2006 and 2010. We performed symptomatic, pulmonary function, and computed tomography assessments in 243 HIV-infected smokers. In a subset bronchoalveolar lavage was performed with proteomic analysis of their alveolar macrophages. Results. The majority of the participants were male 213 (87.6%). There was significantly higher percentage of cough and phlegm production in males. There was also a lower FEV1 and a higher RV in males than females. Proteomic analysis revealed 29 proteins with at least a 2-fold higher expression in males and 13 identified proteins that were higher in females. Conclusions. In this group of HIV-infected smokers, airway symptoms and pulmonary function test abnormalities were higher in men than women. These gender differences may be due to differential expression of certain proteins in this group.

c-Myc suppressed E-cadherin through miR-9 at the post-transcriptional level.

c-Myc oncoprotein is overexpressed in most human cancers and regulates different genes and pathways in different cell types. E-cadherin expression is repressed by MYC through a post-transcriptional mechanism, but the exact mechanism remains elusive. Since E-cadherin is a direct target of miR-9 and miR-9 can be activated by MYC and MYCN, this suggests that c-Myc negatively modulates E-cadherin through a microRNA pathway. We have established a c-Myc-inducible expression system in which the protein level and transcriptional activity of c-Myc is significantly upregulated upon doxycycline induction. Overexpressed c-Myc led to an EMT-like conversion in the T-REx-293 cells and resulted in a significant decrease in E-cadherin and an increase in Vimentin. Stem-loop RT-PCR showed elevated expression of miR-9 when c-Myc was induced to be overexpressed. Regarding the relationship of c-Myc, miR-9 and E-cadherin, the expression of miR-9 was curtailed by using antagomir-9 in induced overexpressing c-Myc. Restoration of E-cadherin expression became much stronger in the presence of c-Myc. Thus c-Myc represses E-cadherin at the post-transcriptional level through miR-9.

Residual plasmatic activity of ADAMTS13 is correlated with phenotype severity in congenital thrombotic thrombocytopenic purpura.

The quantification of residual plasmatic ADAMTS13 activity in congenital thrombotic thrombocytopenic purpura (TTP) patients is constrained by limitations in sensitivity and reproducibility of commonly used assays at low levels of ADAMTS13 activity, blunting efforts to establish genotype-phenotype correlations. In the present study, the residual plasmatic activity of ADAMTS13 was measured centrally by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (limit of detection = 0.5%) in 29 congenital TTP patients. The results were used to study correlations among ADAMTS13 genotype, residual plasmatic activity, and clinical phenotype severity. An ADAMTS13 activity above 0.5% was measured in 26 (90%) patients and lower levels of activity were associated with earlier age at first TTP episode requiring plasma infusion, more frequent recurrences, and prescription of fresh-frozen plasma prophylaxis. Receiver operating characteristic curve analysis showed that activity levels of less than 2.74% and 1.61% were discriminative of age at first TTP episode requiring plasma infusion < 18 years, annual rate of TTP episodes > 1, and use of prophylaxis. Mutations affecting the highly conserved N-terminal domains of the protein were associated with lower residual ADAMTS13 activity and a more severe phenotype in an allelic-dose dependent manner. The results of the present study show that residual ADAMTS13 activity is associated with the severity of clinical phenotype in congenital TTP and provide insights into genotype-phenotype correlations.