[Hip joint biomechanical analysis of the acetabular anatomical reconstruction and nonanatomical reconstruction in total hip arthroplasty for Crowe type â ¢ developmental dysplasia of the hip by finite element method].

OBJECTIVE: To analyze the hip joint biomechanics of the acetabular anatomical reconstruction and nonanatomical reconstruction in total hip arthroplasty (THA) for Crowe type Ⅲ developmental dysplasia of the hip (DDH) by finite element method, which provided theoretical foundation and experimental basis for the anatomical acetabular reconstruction during THA in clinical practice. METHODS: One patient with left end-stage hip arthritis secondary to Crowe type Ⅲ DDH was selected in this study, who underwent total hip arthroplasty in the orthopedic department of the First Affiliated Hospital of Bengbu Medical College in April 2020. This patient was female, 57 years old. The preoperative and postoperative three dimentional CT scan of the patient's pelvis were performed. Fourteen acetabular cup models with different anteversion, inclination and rotation center height were established in Mimics and 3-Matic software. The boundary and load conditions were set in Abaqus software. The Von Mises and stress distribution of the hip joint were calculated and observed. RESULTS: In the Crowe type Ⅲ DDH THA, if the hip rotation center was restored anatomically and the acetabular cup's inclination was set as 40°, the cup's anteversion varied from 5° to 25°, the lowest Von Mises value of acetabular cup and polyethylene liner occured in 20°anteversioin;if the hip rotation center was restored anatomically and the acetabular cup's anteversion was set as 15°, the cup's inclination varied from 35° to 55°, the lowest Von Mises value of acetabular cup and polyethylene liner occured in 35° inclination;if the acetabular cup's anteversion and inclination were set as 15°and 40°respectively, the up migration of hip rotaion center varied from 0 mm to 20 mm, the lowest Von Mises value of acetabular cup and polyethylene liner occured in 10 mm up migration. In all fourteen models, the Von Mises value of the acetabulum, acetabulum cup and polyethylene liner were lowest when the acetabular cup's anteversion and inlcination were 15°, 35° respectively, as well as the rotation center was restored anatomically. CONCLUSION: In total hip arthroplasty for Crowe type Ⅲ DDH, the anatomical restoration of hip rotation center with 15° anteversion and 35° inclination of the acetabular cup are suggested, bone graft above the acetabular cup and additional screws are recommended simultaneously to further reduce the Von Mises of hip joint.

Cysteine carboxyethylation generates neoantigens to induce HLA-restricted autoimmunity.

Autoimmune diseases such as ankylosing spondylitis (AS) can be driven by emerging neoantigens that disrupt immune tolerance. Here, we developed a workflow to profile posttranslational modifications involved in neoantigen formation. Using mass spectrometry, we identified a panel of cysteine residues differentially modified by carboxyethylation that required 3-hydroxypropionic acid to generate neoantigens in patients with AS. The lysosomal degradation of integrin αIIb [ITGA2B (CD41)] carboxyethylated at Cys96 (ITGA2B-ceC96) generated carboxyethylated peptides that were presented by HLA-DRB1*04 to stimulate CD4+ T cell responses and induce autoantibody production. Immunization of HLA-DR4 transgenic mice with the ITGA2B-ceC96 peptide promoted colitis and vertebral bone erosion. Thus, metabolite-induced cysteine carboxyethylation can give rise to pathogenic neoantigens that lead to autoreactive CD4+ T cell responses and autoantibody production in autoimmune diseases.

Restoring Rotation Center in Total Hip Arthroplasty for Developmental Dysplasia of the Hip with the Assistance of Three Dimensional Printing Technology: A Pilot Study.

OBJECTIVE: To develop a new method to restore hip rotation center exactly and rapidly in total hip arthroplasty (THA) with the assistance of three dimensional (3D) printing technology and evaluate its clinical and radiological outcomes. METHODS: From March 2014 to July 2018, a total of 17 patients (five hips of four men and 16 hips of 13 women) with end-stage osteoarthritis secondary to developmental dysplasia of the hip who underwent THA were analyzed and followed up retrospectively. The average age is 58.00 ± 8.12 years (range from 45 to 71 years). Simulated operations were performed on 3D printed hip models for preoperative planning. The morphology of Harris fossa and acetabular notches were recognized and restored to locate the acetabular center. The size of bone defect was measured by the bone wax method. The agreement on the size of acetabular cup and bone defect between simulated operations and actual operations were analyzed. Harris Hip Score (HHS) was used to evaluate the recovery of hip joint function. The vertical distance and horizontal distance of the rotation center on the pelvis plain radiograph were measured, which were used to assess the efficacy of restoring hip rotation center and acetabular cup migration. RESULTS: The mean sizes of bone defect in simulated operations and THA were 4.58 ± 2.47 cm2 and 4.55 ± 2.57 cm2 respectively. There was no significant difference statistically between the sizes of bone defect in simulated operations and the actual sizes of bone defect in THA (t = 0.03, P = 0.97). The sizes of the acetabular cup of simulated operations on 3D print models showed a high rate of coincidence with the actual sizes in the operations (ICC = 0.93). All 17 patients were available for clinical and radiological follow-up. The average follow-up time was 18.35 ± 6.86 months (range, 12-36 months. The average HHS of the patients was improved from (38.33 ± 6.07) preoperatively to the last follow-up (88.61 ± 3.44) postoperatively. The mean vertical and horizontal distances of hip rotation center on the pelvic radiographs were restored to 15.12 ± 1.25 mm and 32.49 ± 2.83 mm respectively. No case presented dislocation or radiological signs of loosening until last follow-up. CONCLUSIONS: The application of 3D printing technology facilitates orthopedists to recognize the morphology of Harris fossa and acetabular notches, locate the acetabular center and restore the hip rotation center rapidly and accurately.

Safety and efficacy of meplazumab in healthy volunteers and COVID-19 patients: a randomized phase 1 and an exploratory phase 2 trial.

Recent evidence suggests that CD147 serves as a novel receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Blocking CD147 via anti-CD147 antibody could suppress the in vitro SARS-CoV-2 replication. Meplazumab is a humanized anti-CD147 IgG2 monoclonal antibody, which may effectively prevent SARS-CoV-2 infection in coronavirus disease 2019 (COVID-19) patients. Here, we conducted a randomized, double-blinded, placebo-controlled phase 1 trial to evaluate the safety, tolerability, and pharmacokinetics of meplazumab in healthy subjects, and an open-labeled, concurrent controlled add-on exploratory phase 2 study to determine the efficacy in COVID-19 patients. In phase 1 study, 59 subjects were enrolled and assigned to eight cohorts, and no serious treatment-emergent adverse event (TEAE) or TEAE grade ≥3 was observed. The serum and peripheral blood Cmax and area under the curve showed non-linear pharmacokinetic characteristics. No obvious relation between the incidence or titer of positive anti-drug antibody and dosage was observed in each cohort. The biodistribution study indicated that meplazumab reached lung tissue and maintained >14 days stable with the lung tissue/cardiac blood-pool ratio ranging from 0.41 to 0.32. In the exploratory phase 2 study, 17 COVID-19 patients were enrolled, and 11 hospitalized patients were involved as concurrent control. The meplazumab treatment significantly improved the discharged (P = 0.005) and case severity (P = 0.021), and reduced the time to virus negative (P = 0.045) in comparison to the control group. These results show a sound safety and tolerance of meplazumab in healthy volunteers and suggest that meplazumab could accelerate the recovery of patients from COVID-19 pneumonia with a favorable safety profile.

[Short term effect of total hip arthroplasty through direct anterior approach for the treatment of ankylosing spondylitis with hip flexion deformity].

OBJECTIVE: To investigate the short term clinical efficacy of direct anterior approach(DAA) total hip arthroplasty for the treatment of ankylosing spondylitis with hip flexion deformity. METHODS: From September 2014 to June 2017, 15 cases of ankylosing spondylitis with flexion deformity of the hip were treated with total hip arthroplasty through DAA approach including 12 males(17 hips) and 3 females(4 hips) with an average age of 34.4 years old ranging from 21 to 57 years old. Harris score system was used before and after operation to evaluate hip function, total hip activity and visual analogue scale (VAS) were used to evaluate the clinical efficacy. RESULTS: All 15 patients were followed up for an average of 26.2 months. In the operation, 1 case of great trochanter avulsion was given wire binding, and 1 case of linear split of the femur were given by wire binding. There were no hematoma, nerve injury and deep vein thrombosis of lower extremity. No prosthesis loosening and sinking were observed in the follow-up of X-ray film after operation. There was no heterotopic ossification after operation. After operation, 18 hips pain were relieved completely, and 3 hips pain were found when walking, which all satisfied with the daily life self-care requirements. Harris hip score, total hip motion and VAS score at 1 week after operation were significantly different from those before operation(P<0.05). There was no significant difference in the scores of HHS, total hip motion and VAS at 1, 6 months after operation(P>0.05). At the final follow-up, the Harris score was 91.2±5.3, the total hip mobility was (217.1±29.7)°, and the postoperative VAS pain score was 1.2±0.5, which was significantly different from the preoperative score(P<0.05). CONCLUSIONS: DAA approach THA has good effect in treating AS hip nonfunctional ankylosis with less trauma, less pain and quick recovery. It has a good short term effect, which can effectively improve the quality of life of patients.

Tbx18-dependent differentiation of brown adipose tissue-derived stem cells toward cardiac pacemaker cells.

A cell-sourced biological pacemaker is a promising therapeutic approach for sick sinus syndrome (SSS) or severe atrial ventricular block (AVB). Adipose tissue-derived stem cells (ATSCs), which are optimal candidate cells for possible use in regenerative therapy for acute or chronic myocardial injury, have the potential to differentiate into spontaneous beating cardiomyocytes. However, the pacemaker characteristics of the beating cells need to be confirmed, and little is known about the underlying differential mechanism. In this study, we found that brown adipose tissue-derived stem cells (BATSCs) in mice could differentiate into spontaneous beating cells in 15% FBS Dulbecco's modified Eagle's medium (DMEM) without additional treatment. Subsequently, we provide additional evidence, including data regarding ultrastructure, protein expression, electrophysiology, and pharmacology, to support the differentiation of BATSCs into a cardiac pacemaker phenotype during the course of early cultivation. Furthermore, we found that silencing Tbx18, a key transcription factor in the development of pacemaker cells, terminated the differentiation of BATSCs into a pacemaker phenotype, suggesting that Tbx18 is required to direct BATSCs toward a cardiac pacemaker fate. The expression of Tbx3 and shox2, the other two important transcription factors in the development of pacemaker cells, was decreased by silencing Tbx18, which suggests that Tbx18 mediates the differentiation of BATSCs into a pacemaker phenotype via these two downstream transcription factors.

Transcriptome profile of rat genes in injured spinal cord at different stages by RNA-sequencing.

BACKGROUND: Spinal cord injury (SCI) results in fatal damage and currently has no effective treatment. The pathological mechanisms of SCI remain unclear. In this study, genome-wide transcriptional profiling of spinal cord samples from injured rats at different time points after SCI was performed by RNA-Sequencing (RNA-Seq). The transcriptomes were systematically characterized to identify the critical genes and pathways that are involved in SCI pathology. RESULTS: RNA-Seq results were obtained from total RNA harvested from the spinal cords of sham control rats and rats in the acute, subacute, and chronic phases of SCI (1 day, 6 days and 28 days after injury, respectively; n = 3 in every group). Compared with the sham-control group, the number of differentially expressed genes was 1797 in the acute phase (1223 upregulated and 574 downregulated), 6590 in the subacute phase (3460 upregulated and 3130 downregulated), and 3499 in the chronic phase (1866 upregulated and 1633 downregulated), with an adjusted P-value <0.05 by DESeq. Gene ontology (GO) enrichment analysis showed that differentially expressed genes were most enriched in immune response, MHC protein complex, antigen processing and presentation, translation-related genes, structural constituent of ribosome, ion gated channel activity, small GTPase mediated signal transduction and cytokine and/or chemokine activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the most enriched pathways included ribosome, antigen processing and presentation, retrograde endocannabinoid signaling, axon guidance, dopaminergic synapses, glutamatergic synapses, GABAergic synapses, TNF, HIF-1, Toll-like receptor, NF-kappa B, NOD-like receptor, cAMP, calcium, oxytocin, Rap1, B cell receptor and chemokine signaling pathway. CONCLUSIONS: This study has not only characterized changes in global gene expression through various stages of SCI progression in rats, but has also systematically identified the critical genes and signaling pathways in SCI pathology. These results will expand our understanding of the complex molecular mechanisms involved in SCI and provide a foundation for future studies of spinal cord tissue damage and repair. The sequence data from this study have been deposited into Sequence Read Archive ( http://www.ncbi.nlm.nih.gov/sra ; accession number PRJNA318311).

[Genotypes of adenoviruses in infants and young children with diarrhea].

OBJECTIVE: To investigate the prevalence of adenoviruses (AdV) and their genotypes in infants and young children with diarrhea. METHODS: A total of 380 children with diarrhea aged less than 3 years were enrolled. The genomic DNA was extracted from stool and PCR was used to detect AdV. Clone sequencing and genotyping were performed for DNA in AdV-positive specimens. RESULTS: AdV was detected in 24 out of 380 specimens, and the detection rate was 6.3% (24/380). A majority of children with positive AdV were aged 2-3 years. The viral sequence analysis of positive specimens showed that the detection rates of enteric AdV41 and non-enteric AdV were 4.2% (16/380) and 2.1% (8/380), respectively, and among the children with non-enteric AdV, there were 2 with AdV1, 2 with AdV2, 1 with AdV7, 2 with AdV12, and 1 with AdV31. CONCLUSIONS: Diarrhea caused by AdV is commonly seen in children aged 2-3 years, and AdV41 is the major predominant strain.

Over-expression of Sox4 and ß-catenin is associated with a less favorable prognosis of osteosarcoma.

The purpose of this study was to examine the association of the expression of Sox4 and ß-catenin with the prognosis of osteosarcoma. A total of 108 cases of conventional osteosarcoma were involved in this study and 28 cases of osteochondroma served as controls. The expression of Sox4 and ß-catenin was detected by using immunohistochemical staining and Western blotting. The results showed that Sox4 and ß-catenin were over-expressed in 67 (62.03%) and 62 (57.41%) of 108 osteosarcoma cases, while in only 3 (10.71%) and 5 (17.86%) of 28 controls, respectively (P<0.05 for all). The expression of Sox4 and ß-catenin was associated with the distant metastasis, pathological grade and Enneking stage of patients with osteosarcoma (P<0.05 for all). The mean overall survival time and the 5-year-survival rate in osteosarcoma patients with Sox4 and ß-catenin over-expressed were significantly reduced as compared with those in Sox4 and ß-catenin low-expression group (P<0.05 for all). Cox multifactor regression analysis revealed that the distant metastasis, Enneking stage, and the expression of Sox4 and ß-catenin were independent risk factors of patients with osteosarcoma (P<0.05 for all). The findings indicated that overexpression of Sox4 and ß-catenin is associated with a poor prognosis of osteosarcoma.

Temporal kinetics of macrophage polarization in the injured rat spinal cord.

Local activated macrophages derived from infiltrating monocytes play an important role in the damage and repair process of spinal cord injury (SCI). The present study investigates the dynamic change of classically activated proinflammatory (M1) and alternatively activated anti-inflammatory (M2) cells in a rat model with contusive SCI by flow cytometry (FCM) and immunohistochemistry. The macrophage subsets were immunophenotyped by using antibodies against cluster of differentiation (CD)-68, C-C chemokine receptor type 7 (CCR7), CD163, and arginase 1 (Arg1). The CD68(+) CD163(-) and CD68(+) CCR7(+) cells were determined to be M1 subsets, whereas the CD68(+) CD163(+) and CD68(+) Arg1(+) cell subpopulations represented M2 cells. The subsets of macrophages in the injured spinal cord at 1, 3, 5, 7, 14, and 28 days postinjury (dpi) were examined. In the sham-opened spinal cord, few M1 or M2 cells were found. After SCI, the phenotypes of both M1 and M2 cells were rapidly induced. However, M1 cells were detected and maintained at a high level for up to 28 dpi (the longest time evaluated in this study). In contrast, M2 cells were transiently detected at high levels before 7 dpi and returned to preinjury levels at 14 dpi. These results indicate that M1 cell response is rapidly induced and sustained, whereas M2 induction is transient after SCI in rat. Increasing the fraction of M2 cells and prolonging their residence time in the injured local microenvironment is a promising strategy for the repair of SCI.

Silencing profilin-1 inhibits gastric cancer progression via integrin ß1/focal adhesion kinase pathway modulation.

AIM: To investigate the role of profilin-1 (PFN1) in gastric cancer and the underlying mechanisms. METHODS: Immunohistochemical analysis, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to detect PFN1 expression in clinical gastric carcinoma and adjacent tissues, and the association of PFN1 expression with patient clinicopathological characteristics was analyzed. PFN1 was knocked down to investigate the role of this protein in cell proliferation and metastasis in the SGC-7901 cell line. To explore the underlying mechanisms, the expression of integrin ß1 and the activity of focal adhesion kinase (FAK) and the downstream proteins extracellular-regulated kinase (ERK)1/2, P38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) were measured through Western blot or qRT-PCR analysis. Fibronectin (FN), a ligand of integrin ß1, was used to verify the correlation between alterations in the integrin ß1/FAK pathway and changes in tumor cell aggressiveness upon PFN1 perturbation. RESULTS: Immunohistochemical, Western blot and qRT-PCR analyses revealed that PFN1 expression was higher at both the protein and mRNA levels in gastric carcinoma tissues compared with the adjacent tissues. In addition, high PFN1 expression (53/75, 70.4%) was correlated with tumor infiltration, lymph node metastasis and TNM stage in gastric cancer, but not with gender, age, location, tumor size, or histological differentiation. In vitro experiments showed that PFN1 knockdown inhibited the proliferation of SGC-7901 cells through the induction G0/G1 arrest. Silencing PFN1 inhibited cell migration and invasion and down-regulated the expression of matrix metalloproteinase (MMP)-2 and MMP9. Moreover, silencing PFN1 reduced the expression of integrin ß1 at the protein level and inhibited the activity of FAK, and the downstream effectors ERK1/2, P38MAPK, PI3K, AKT and mTOR. FN-promoted cell proliferation and metastasis via the integrin ß1/FAK pathway was ameliorated by PFN1 silencing. CONCLUSION: These findings suggest that PFN1 plays a critical role in gastric carcinoma progression, and these effects are likely mediated through the integrin ß1/FAK pathway.

Adoptive transfer of M2 macrophages promotes locomotor recovery in adult rats after spinal cord injury.

Classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) macrophages populate the local microenvironment after spinal cord injury (SCI). The former type is neurotoxic while the latter has positive effects on neuroregeneration and is less toxic. In addition, while the M1 macrophage response is rapidly induced and sustained, M2 induction is transient. A promising strategy for the repair of SCI is to increase the fraction of M2 cells and prolong their residence time. This study investigated the effect of M2 macrophages induced from bone marrow-derived macrophages on the local microenvironment and their possible role in neuroprotection after SCI. M2 macrophages produced anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor ß and infiltrated into the injured spinal cord, stimulated M2 and helper T (Th)2 cells, and produced high levels of IL-10 and -13 at the site of injury. M2 cell transfer decreased spinal cord lesion volume and resulted in increased myelination of axons and preservation of neurons. This was accompanied by significant locomotor improvement as revealed by Basso, Beattie and Bresnahan locomotor rating scale, grid walk and footprint analyses. These results indicate that M2 adoptive transfer has beneficial effects for the injured spinal cord, in which the increased number of M2 macrophages causes a shift in the immunological response from Th1- to Th2-dominated through the production of anti-inflammatory cytokines, which in turn induces the polarization of local microglia and/or macrophages to the M2 subtype, and creates a local microenvironment that is conducive to the rescue of residual myelin and neurons and preservation of neuronal function.

JNK is necessary for oligodendrocyte precursor cell proliferation induced by the conditioned medium from B104 neuroblastoma cells.

The conditioned medium from B104 neuroblastoma cells (B104CM) induces proliferation of oligodendrocyte progenitor cells (OPCs) in vitro. Our previous study showed that phosphorylation of extracellular signal-regulated protein kinase(s), not PI3K or p38, is key to B104CM-induced OPC proliferation. However, whether there are still other signaling pathways that are also involved in B104CM-induced proliferation remains unknown. In the present study, we evaluated the implication of c-Jun N-terminal kinase (JNK) signaling pathway in the B104CM-induced proliferation of OPCs using the specific inhibitor of JNK. We provided convincing evidence for the first time that the phosphorylation of JNK is necessary for OPC proliferation induced by B104CM in vitro. Moreover, the activation of JNK results in subsequent expressions of c-jun, c-fos, and c-myc, which initiates proliferation of OPCs. Collectively, these results suggest that JNK is also necessary for B104CM-induced OPC proliferation.

Effects of adenoviral vector expressing hIGF-1 on apoptosis in nucleus pulposus cells in vitro.

The excessive apoptosis of cells of the nucleus pulposus may plays an important role in intervertebral disc (IVD) degeneration. It has been shown that the pro-inflammatory cytokine tumour necrosis factor (TNF)-α can induce disc cell apoptosis. Insulin-like growth factor (IGF)-1 can promote nucleus pulposus cell proliferation; however, whether or not IGF-1 inhibits TNF-α-induced apoptosis in the nucleus pulposus has not yet been elucidated. In this study, our objective was to create a potentially therapeutic viral vector, which could be used to achieve the enforced expression of IGF-1 in rabbit nucleus pulposus cells. Furthermore, we investigated the ability of IGF-1 to reverse TNF-α-induced apoptosis in cells of the nucleus pulposus. Isolated nucleus pulposus cells were cultured to a confluent monolayer, digested with collagenase Ⅱ and purified using trypsin and differential adhesion methods. Nucleus pulposus cells were positively identified using type Ⅱ collagen immunohistochemistry. Following transfection with adenoviral vectors engineered to overexpress recombinant human IGF-1 (Ad-hIGF-1) or TNF-α, the cells were observed under a light microscope. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) and flow cytometry (FCM) were used to assess the rate of apoptosis. The Ad-hIGF-1 viral vector was effectively transduced into the nucleus pulposus cells and increased IGF-1 expression as confirmed by RT-PCR and western blot analysis. In the TNF-α-treated group, a large number of apoptotic cells was observed that exhibited morphological changes associated with this form of cell death. Minimal apoptosis was observed in the Ad-hIGF-1-treated group and the control group showed no obvious signs of apoptosis. TUNEL assay revealed that the rate of apoptosis in the Ad-hIGF-1 group was significantly reduced compared with the TNF-α-treated group (P<0.01). This result was confirmed using FCM. The rate of apoptosis was also significantly increased in the TNF-α-exposed cells compared with the control group (P<0.01). Our findings strongly suggest that the adenoviral vector expressing hIGF-1 can successfully infect nucleus pulposus cells in vitro and effectively enhance the expression of IGF-1. In addition, IGF-1 reversed the TNF-α -induced apoptosis of nucleus pulposus cells. Thus, Ad-hIGF-1 may be useful in the development of clinical interventions for disc degeneration.

The molecular events involved in oligodendrocyte precursor cell proliferation induced by the conditioned medium from b104 neuroblastoma cells.

The conditioned medium from B104 neuroblastoma cells (B104CM) induces proliferation of oligodendrocyte progenitor cells (OPCs) in vitro. However, the molecular events that occur during B104CM-induced proliferation of OPCs has not been well clarified. In the present study, using OPCs immunopanned from embryonic day 14 Sprague-Dawley rat spinal cords, we explored the activation of several signaling pathways and the expression of several important immediate early genes (IEGs) and cyclins in OPCs in response to B104CM. We found that B104CM can induce OPC proliferation through the activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2), but not PI3K or p38 MAPK signaling pathways in vitro. The IEGs involved in B104CM-induced OPC proliferation include c-fos, c-jun and Id2, but not c-myc, fyn, or p21. The cyclins D1, D2 and E are also involved in B104CM-stimulated proliferation of OPCs. The activation of Erk results in subsequent expression of IEGs (such as c-fos, c-jun and Id-2) and cyclins (including cyclin D1, D2 and E), which play key roles in cell cycle initiation and OPC proliferation. Collectively, these results suggest that the phosphorylation of Erk1/2 is an important molecular event during OPC proliferation induced by B104CM.

Effects of IGF-1 on IL-1ß-induced apoptosis in rabbit nucleus pulposus cells in vitro.

Excessive apoptosis in intervertebral disc (IVD) cells is important in IVD degeneration. Interleukin (IL)-1ß has been shown to induce apoptosis in these cells. However, whether insulin-like growth factor-1 (IGF-1) inhibits IL-1ß-induced apoptosis in the nucleus pulposus remains unclear. The purpose of this study was to investigate the effects of IGF-1 on IL-1ß-induced apoptosis in the nucleus pulposus. Cells isolated from the nucleus pulposus were grown in culture to a monolayer. These cells were identified using immuno-histochemistry for type II collagen and toluidine blue staining for glycosaminoglycans. Following exposure to IGF-1 or IL-1ß, the cells were observed using light microscopy. Giemsa staining, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) and flow cytometry (FCM) were used to detect the rate of early cell death, which served as an indicator of apoptosis. In the IL-1ß group, a large number of these cells underwent apoptosis and demonstrated morphological changes associated with apoptosis. A small proportion of cells exposed to IGF-1 alone underwent apoptosis. No obvious signs of apoptosis were observed in the control group. TUNEL results revealed that the rate of apoptosis in the IGF-1 group was significantly reduced compared with that in the IL-1ß group (P<0.01), confirmed using FCM. Compared with the control group, the apoptotic rate was also significantly increased in IL-1ß-exposed cells (P<0.01). These findings strongly suggested that IGF-1 inhibits IL-1ß-induced apoptosis in the nucleus pulposus.

PDGF-AA and bFGF mediate B104CM-induced proliferation of oligodendrocyte precursor cells.

The conditioned medium from B104 neuroblastoma cells (B104CM) induces proliferation of οligodendrocyte precursor cells (OPCs) in vitro, which indicates that certain factors contained within B104CM give instructional signals that direct the proliferation of OPCs. However, the OPC-proliferative factors present in B104CM have yet to be identified. Platelet-derived growth factor AA (PDGF-AA), basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1) have been reported to act as potent mitogens for OPC proliferation. This raises the possibility that B104CM induces proliferation of OPCs through secretion of PDGF­AA, bFGF and/or IGF-1. In the present study, we detected the expression and levels of PDGF-AA, bFGF and IGF-1 in B104 cells and B104CM, and observed the expression of their receptors in OPCs. The results indicated that these growth factors were expressed in B104 cells and B104CM. All 3 receptors, PDGFR, FGFR2 and IGF-1R, were also detected in OPCs. Furthermore, B104CM-stimulated OPC proliferation could be markedly decreased by both AG1295 (an inhibitor of PDGFR) and PD173074 (an inhibitor of FGFR). However, the inhibition of IGF-1R with AG1204 did not affect the proliferation of OPCs. Our study suggests that the PDGF-AA and bFGF in B104CM are 2 key factors that stimulate OPC proliferation.

Expression of BMP-2 and BMP-4 proteins by type-1 and type-2 astrocytes induced from neural stem cells under different differentiation conditions.

Bone morphogenetic proteins (BMPs), a subgroup of the TGF-ß superfamily, play critical roles in neural progenitor cell fate determination. Neural stem cells (NSCs) are multipotent progenitor cells that can differentiate into neurons, oligodendrocytes and astrocytes under certain conditions. In our recent report, using an antibody that can recognize both BMP-2 and BMP-4 (BMP-2/4), we showed that BMP-2/4 is only expressed in astrocytes differentiated from NSCs in a medium containing 1% fetal bovine serum (FBS). In this in vitro model, the astrocytic differentiation of NSCs was mainly toward type-2. When NSCs were cultured in a medium containing 10% FBS, most of the cells differentiated into type-1 astrocytes. However, little information is available for BMP-2 and BMP-4 expression in type-1 and type-2 astrocytes induced from NSCs under these different culture conditions. In this study, using two antibodies specific for BMP-2 and BMP-4, respectively, we discriminated the presence of BMP-2 and BMP-4 in NSCs and their derivatives under 1% and 10% FBS culture conditions by RT-PCR, western blot and immunofluorescence staining. We found that BMP-2 and BMP-4 are highly expressed in both type-1 and type-2 astrocytes, and no detectable expression in NSCs, neurons and oligodendrocytes. This suggests that the astrocytes might be one source of BMPs during the differentiation of NSCs. However, in our model, we cannot exclude the possibility that microglia or endothelial cells could also be a source of BMPs.

Passive immunization with myelin basic protein activated T cells suppresses axonal dieback but does not promote axonal regeneration following spinal cord hemisection in adult rats.

The previous studies suggested that some subpopulations of T lymphocytes against central nervous system (CNS) antigens, such as myelin basic protein (MBP), are neuroprotective. But there were few reports about the effect of these T cells on axon regeneration. In this study, the neonatally thymectomied (Tx) adult rats which contain few T lymphocytes were subjected to spinal cord hemisection and then passively immunized with MBP-activated T cells (MBP-T). The regeneration and dieback of transected axons of cortico-spinal tract (CST) were detected by biotin dextran amine (BDA) tracing. The behavioral assessments were performed using the Basso, Beattie, and Bresnahan locomotor rating scale. We found that passive transferring of MBP-T could attenuate axonal dieback. However, no significant axon regeneration and behavioral differences were observed among the normal, Tx and sham-Tx (sTx) rats with or without MBP-T passive immunization. These results indicate that passive transferring of MBP-T cells can attenuate axonal dieback and promote neuroprotection following spinal cord injury (SCI), but may not promote axon regeneration.

Effects of Olig2-overexpressing neural stem cells and myelin basic protein-activated T cells on recovery from spinal cord injury.

Neural stem cell (NSC) transplantation is a major focus of current research for treatment of spinal cord injury (SCI). However, it is very important to promote the survival and differentiation of NSCs into myelinating oligodendrocytes (OLs). In this study, myelin basic protein-activated T (MBP-T) cells were passively immunized to improve the SCI microenvironment. Olig2-overexpressing NSCs were infected with a lentivirus carrying the enhanced green fluorescent protein (GFP) reporter gene to generate Olig2-GFP-NSCs that were transplanted into the injured site to differentiate into OLs. Transferred MBP-T cells infiltrated the injured spinal cord, produced neurotrophic factors, and induced the differentiation of resident microglia and/or infiltrating blood monocytes into an "alternatively activated" anti-inflammatory macrophage phenotype by producing interleukin-13. As a result, the survival of transplanted NSCs increased fivefold in MBP-T cell-transferred rats compared with that of the vehicle-treated control. In addition, the differentiation of MBP-positive OLs increased 12-fold in Olig2-GFP-NSC-transplanted rats compared with that of GFP-NSC-transplanted controls. In the MBP-T cell and Olig2-GFP-NSC combined group, the number of OL-remyelinated axons significantly increased compared with those of all other groups. However, a significant decrease in spinal cord lesion volume and an increase in spared myelin and behavioral recovery were observed in Olig2-NSC- and NSC-transplanted MBP-T cell groups. Collectively, these results suggest that MBP-T cell adoptive immunotherapy combined with NSC transplantation has a synergistic effect on histological and behavioral improvement after traumatic SCI. Although Olig2 overexpression enhances OL differentiation and myelination, the effect on functional recovery may be surpassed by MBP-T cells.