Keng Chen

Reduced immunomodulation potential of bone marrow-derived mesenchymal stem cells induced CCR4+CCR6+ Th/Treg cell subset imbalance in ankylosing spondylitis.

IntroductionAnkylosing spondylitis (AS) is a chronic autoimmune disease, and the precise pathogenesis is largely unknown at present. Bone marrow-derived mesenchymal stem cells (BMSCs) with immunosuppressive and anti-inflammatory potential and Th17/Treg cells with a reciprocal relationship regulated by BMSCs have been reported to be involved in some autoimmune disorders. Here we studied the biological and immunological characteristics of BMSCs, the frequency and phenotype of CCR4+CCR6+ Th/Treg cells and their interaction in vitro in AS.MethodsThe biological and immunomodulation characteristics of BMSCs were examined by induced multiple-differentiation and two-way mixed peripheral blood mononuclear cell (PBMC) reactions or after stimulation with phytohemagglutinin, respectively. The interactions of BMSCs and PBMCs were detected with a direct-contact co-culturing system. CCR4+CCR6+ Th/Treg cells and surface markers of BMSCs were assayed using flow cytometry.ResultsThe AS-BMSCs at active stage showed normal proliferation, cell viability, surface markers and multiple differentiation characteristics, but significantly reduced immunomodulation potential (decreased 68 ± 14%); the frequencies of Treg and Fox-P3+ cells in AS-PBMCs decreased, while CCR4+CCR6+ Th cells increased, compared with healthy donors. Moreover, the AS-BMSCs induced imbalance in the ratio of CCR4+CCR6+ Th/Treg cells by reducing Treg/PBMCs and increasing CCR4+CCR6+ Th/PBMCs, and also reduced Fox-P3+ cells when co-cultured with PBMCs. Correlation analysis showed that the immunomodulation potential of BMSCs has significant negative correlations with the ratio of CCR4+CCR6+ Th to Treg cells in peripheral blood.ConclusionsThe immunomodulation potential of BMSCs is reduced and the ratio of CCR4+CCR6+ Th/Treg cells is imbalanced in AS. The BMSCs with reduced immunomodulation potential may play a novel role in AS pathogenesis by inducing CCR4+CCR6+ Th/Treg cell imbalance.

Effects and Safety of Allogenic Mesenchymal Stem Cell Intravenous Infusion in Active Ankylosing Spondylitis Patients who Failed NSAIDs: A 20-Week Clinical Trial

Our objective was to evaluate the feasibility, safety, and efficacy of intravenous (IV) infusion of allogenic mesenchymal stem cells (MSCs) in ankylosing spondylitis (AS) patients who are refractory to or cannot tolerate the side effects of nonsteroidal anti-inflammatory drugs (NSAIDs). AS patients enrolled in this study received four IV infusions of MSCs on days 0, 7, 14, and 21. The percentage of ASAS20 responders (the primary endpoint) at the fourth week and the mean ASAS20 response duration (the secondary endpoint) were used to assess treatment response to MSC infusion and duration of the therapeutic effects. Ankylosing Spondylitis Disease Activity Score Containing C-reactive Protein (ASDAS-CRP) and other preestablished evaluation indices were also adopted to evaluate the clinical effects. Magnetic resonance imaging (MRI) was performed to detect changes of bone marrow edema in the spine. The safety of this treatment was also evaluated. Thirty-one patients were included, and the percentage of ASAS20 responders reached 77.4% at the fourth week, and the mean ASAS20 response duration was 7.1 weeks. The mean ASDAS-CRP score decreased from 3.6 ± 0.6 to 2.4 ± 0.5 at the fourth week and then increased to 3.2 ± 0.8 at the 20th week. The average total inflammation extent (TIE) detected by MRI decreased from 533,482.5 at baseline to 480,692.3 at the fourth week (p > 0.05) and 400,547.2 at the 20th week (p < 0.05). No adverse effects were noted. IV infusion of MSCs is a feasible, safe, and promising treatment for patients with AS.

Epidemiology of spinal cord injuries and risk factors for complete injuries in Guangdong, China: A retrospective study

Background Spinal cord injuries are highly disabling and deadly injuries. Currently, few studies focus on non-traumatic spinal cord injuries, and there is little information regarding the risk factors for complete injuries. This study aims to describe the demographics and the injury characteristics for both traumatic and non-traumatic spinal cord injuries and to explore the risk factors for complete spinal cord injuries. Methods A retrospective study was performed by reviewing the medical records of 3,832 patients with spinal cord injuries who were first admitted to the sampled hospitals in Guangdong, China. The demographics and injury characteristics of the patients were described and compared between the different groups using the chi-square test. Logistic regression was conducted to analyze the risk factors for complete spinal cord injuries. Results The proportion of patients increased from 7.0% to 14.0% from 2003 to 2011. The male-to-female ratio was 3.0∶1. The major cause of spinal cord injuries was traffic accidents (21.7%). Many of the injured were workers (36.2%), peasants (22.8%), and unemployed people (13.9%); these occupations accounted for 72.9% of the total sample. A multivariate logistic regression model revealed that the OR (95% CI) for male gender compared to female gender was 1.25 (1.07–1.89), the OR (95%CI) for having a spinal fracture was 1.56 (1.35–2.60), the OR (95%CI) for having a thoracic injury was 1.23 (1.10–2.00), and the OR (95%CI) for having complications was 2.47 (1.96–3.13). Conclusion The proportion of males was higher than the proportion of females. Workers, peasants and the unemployed comprised the high-risk occupational categories. Male gender, having a spinal fracture, having a thoracic injury, and having complications were the major risk factors for a complete injury. We recommend that preventive measures should focus on high-risk populations, such as young males.

Epidemiological Characteristics of Traumatic Spinal Cord Injury in Guangdong, China.

Study Design. A hospital-based retrospective epidemiological study. Objective. The aim of this study was to examine the demographic and epidemiological characteristics of patients with traumatic spinal cord injury (TSCI) in Guangdong to help health-related institutions develop measures to determine the best allocation of medical resources. Summary of Background Data. TSCI is a highly disabling and deadly injury. Currently, there is little information regarding the epidemiological characteristics for TSCI in Guangdong. Methods. We retrospectively reviewed the medical records of partial second-grade class-A hospitals (mainly capturing general city and county hospitals and some large-scale affiliated hospitals) in Guangdong province according to the International Classification of Disease Version 10 (ICD-10) and diagnostic code of TSCI. Results. The study included the medical records of 1340 patients with TSCI, and the annual number of TSCI admissions increased during the 2003 to 2011 period. The male-to-female ratio was approximately 3.5:1. The major causes of spinal cord injuries were high falls (41.0%) and traffic accidents (37.8%). The most common injury among patients with TSCI was cervical injury (818 cases). In addition, 62.9% of the patients had spinal fractures, 24.0% had other fractures, and 13.7% had brain injuries. Furthermore, 25.1% (337/1340) of the patients experienced clinical complications. The differences in the number of patients with and without complete injury who accepted surgery and hyperbaric oxygen therapy were statistically significant (P < 0.05), and the difference in total medical cost was significant (P < 0.05). Conclusion. There are specific epidemiological characteristics of TSCI patients in Guangdong, and preventive measures are suggested to focus on high-risk populations, such as adult men. Level of Evidence: 3

Whole Genome Expression Profiling and Signal Pathway Screening of MSCs in Ankylosing Spondylitis

The pathogenesis of dysfunctional immunoregulation of mesenchymal stem cells (MSCs) in ankylosing spondylitis (AS) is thought to be a complex process that involves multiple genetic alterations. In this study, MSCs derived from both healthy donors and AS patients were cultured in normal media or media mimicking an inflammatory environment. Whole genome expression profiling analysis of 33,351 genes was performed and differentially expressed genes related to AS were analyzed by GO term analysis and KEGG pathway analysis. Our results showed that in normal media 676 genes were differentially expressed in AS, 354 upregulated and 322 downregulated, while in an inflammatory environment 1767 genes were differentially expressed in AS, 1230 upregulated and 537 downregulated. GO analysis showed that these genes were mainly related to cellular processes, physiological processes, biological regulation, regulation of biological processes, and binding. In addition, by KEGG pathway analysis, 14 key genes from the MAPK signaling and 8 key genes from the TLR signaling pathway were identified as differentially regulated. The results of qRT-PCR verified the expression variation of the 9 genes mentioned above. Our study found that in an inflammatory environment ankylosing spondylitis pathogenesis may be related to activation of the MAPK and TLR signaling pathways.

Quantitative assessment of spinal cord perfusion by using contrast-enhanced ultrasound in a porcine model with acute spinal cord contusion

Objectives:To quantify spinal cord perfusion by using contrast-enhanced ultrasound (CEUS) in a porcine model with acute spinal cord injury.Methods:Microcirculatory changes of acute spinal cord injury were shown by CEUS in a porcine model with spinal cord contusion at three selected time points, coupling with conventional ultrasound (US) and Color Doppler US (CDFI). Time-intensity curves and perfusion parameters were also obtained by autotracking contrast quantification (ACQ) software in the epicenter of contusion site, adjacent region and distant region, respectively. Neurologic and histologic examinations were used to confirm the severity of injury.Results:Conventional US revealed the spinal cord was hypoechoic and homogeneous, whereas the dura mater, pia mater and cerebral aqueduct were hyperechoic. On CDFI intramedullary blood vessels were displayed as segmental and columnar. It was homogeneous on CEUS. After spinal cord contusion, the injured region on gray scale US was hyperechoic. CDFI demonstrated intramedullary blood vessels of adjacent region had increased and dilated during the observation period. On CEUS the epicenter of contusion site was hypoperfusion, whereas its adjacent region was hyperperfusion compared with the distant region. Quantitative analysis showed that peak intensity decreased in epicenters of contusion but increased in adjacent regions significantly at all time points (P<0.05). Evaluation of neurological function for post-contusion demonstrated significantly deterioration in comparison before injury (P<0.05).Conclusions:CEUS is a practical technique that provides overall views for evaluating microcirculatory pattern in spinal cord injury. Quantitative analysis shows the efficacy in assessment of perfusion changes after spinal cord injury.

The Role of Increased Frequency of Treg Cells in Patients with Chronic Osteomyelitis

The aim of this study is to determine whether regulatory T cells (Treg cells) are increased in patients with chronic osteomyelitis and whether they suppress cellular immune responses to the bacteria. The frequency of circulating CD4(+)CD25(+) and CD4(+)CD25(+)FoxP3(+) T cells in 30 chronic osteomyelitis patients were compared with 30 healthy donors. Treg-depleted PBMCs from the patients were cultured together with autologous antigen, unfractioned PBMCs used as the control. The cell proliferation and production of IL-10 and IFN-γ were compared with those of the control. The results demonstrated that frequencies of CD4(+)CD25(+) (10.85±2.82% vs 6.08±1.62%, P<.001) and CD4(+)CD25(+)FoxP3(+) T cells (2.06±0.83% vs 1.43%±0.51%, P<.001) in blood from chronic osteomyelitis patients were significantly higher than in healthy donors. The level of IL-10 (117±91 pg/ml vs 323±189 pg/ml, P<.001) in supernatants of Treg-depleted PBMCs was decreased. Cell proliferation (4489±11876 cpm vs 3547±1517 cpm, P<.05) and IFN-γ (875±203 pg/ml vs 405±129 pg/ml, P<.001) production by CD4(+)CD25(+) T cell in response to antigen was significantly inhibited by CD4(+)CD25(+) T cells. These results indicate that specific Tregs can depress the T cell mediated immune responses to bacteria in chronic osteomyelitis, and may play an important role in the persistence of bacteria.

Evaluation of the neural function of nonhuman primates with spinal cord injury using an evoked potential-based scoring system

Nonhuman primate models of spinal cord injury (SCI) have been widely used in evaluation of the efficacy and safety of experimental restorative interventions before clinical trials. However, no objective methods are currently available for the evaluation of neural function in nonhuman primates. In our long-term clinical practice, we have used evoked potential (EP) for neural function surveillance during operation and accumulated extensive experience. In the present study, a nonhuman primate model of SCI was established in 6 adult cynomologus monkeys through spinal cord contusion injury at T8–T9. The neural function before SCI and within 6 months after SCI was evaluated based on EP recording. A scoring system including somatosensory evoked potentials (SSEPs) and transcranial electrical stimulation-motor evoked potentials (TES-MEPs) was established for the evaluation of neural function of nonhuman primates with SCI. We compared the motor function scores of nonhuman primates before and after SCI. Our results showed that the EP below the injury level significantly changed during the 6 months after SCI. In addition, a positive correlation was identified between the EP scores and motor function. The EP-based scoring system is a reliable approach for evaluating the motor function changes in nonhuman primates with SCI.

Influences of different lower cervical bone graft heights on the size of the intervertebral foramen: multiple planar dynamic measurements with laser scanning

The aim of this study is to evaluate the influences of different bone graft heights on the size of the intervertebral foramen, which will help determine the optimal graft height in clinical practice. Six fresh adult cadavers were used, with the C5-C6 vertebral column segment defined as the functional spinal unit (FSU). After discectomy, the C5/6 intervertebral height was set as the baseline height (normal disc height). We initially used spiral computed tomography (CT) to scan and measure the middle area of the intervertebral foramen when at the baseline height. Data regarding the spatial relationship of C5-C6 were subsequently collected with a laser scanner. Grafting with four different sized grafts, namely, grafts of 100, 130, 160, and 190% of the baseline height, was implanted. Moreover, we scanned to display the FSU in the four different states using Geomagic8.0 studio software. Multiple planar dynamic measurements (MPDM) were adopted to measure the intervertebral foramen volume, middle area, and areas of internal and external opening. MPDM with a laser scanner precisely measured the middle area of the intervertebral foramen as spiral CT, and it is easy to simulate the different grafts implanted. With the increase of the bone graft height, the size of the intervertebral foramen began to decrease after it increased to a certain point, when grafts of 160% of the baseline height implanted. MPDM of the intervertebral foramens with laser scanning three-dimensional (3D) reconstitution are relatively objective and accurate. The recommended optimal graft height of cervical spondylosis is 160% of the mean height of adjacent normal intervertebral spaces.

Intraoperative contrast-enhanced ultrasonography for microcirculatory evaluation in rhesus monkey with spinal cord injury

This study tried to quantify spinal cord perfusion by using contrast-enhanced ultrasound (CEUS) in rhesus monkey models with acute spinal cord injury. Acute spinal cord perfusion after injury was detected by CEUS, coupling with conventional ultrasound (US) and Color Doppler US (CDFI). Time-intensity curves and perfusion parameters were obtained by autotracking contrast quantification (ACQ) software in the epicenter and adjacent regions of injury, respectively. Neurological and histological examinations were performed to confirm the severity of injury. US revealed spinal cords were hypoechoic and homogeneous, whereas dura maters, pia maters, and cerebral aqueducts were hyperechoic. After spinal cord contusion, the injured spinal cord was hyperechoic on US, and intramedullary vessels of adjacent region of injury were increased and dilated on CDFI. On CEUS hypoperfusion were found in the epicenter of injury, while hyperperfusion in its adjacent region. Quantitative analysis showed that peak intensity (PI) decreased in epicenters of injury but significantly increased in adjacent regions at all time points (p < 0.05). Functional evaluation demonstrated significant deterioration compared to pre-contusion (p < 0.05). Quantitative analysis with CEUS is a promising method for monitoring perfusion changes of spinal cord injury in overall views and real-time.