Molecular Determinant Underlying Selective Coupling of Primary G-Protein by Class A GPCRs.

G-protein-coupled receptors (GPCRs) transmit downstream signals predominantly via G-protein pathways. However, the conformational basis of selective coupling of primary G-protein remains elusive. Histamine receptors H2R and H3R couple with Gs- or Gi-proteins respectively. Here, three cryo-EM structures of H2R-Gs and H3R-Gi complexes are presented at a global resolution of 2.6-2.7 Å. These structures reveal the unique binding pose for endogenous histamine in H3R, wherein the amino group interacts with E2065.46 of H3R instead of the conserved D1143.32 of other aminergic receptors. Furthermore, comparative analysis of the H2R-Gs and H3R-Gi complexes reveals that the structural geometry of TM5/TM6 determines the primary G-protein selectivity in histamine receptors. Machine learning (ML)-based structuromic profiling and functional analysis of class A GPCR-G-protein complexes illustrate that TM5 length, TM5 tilt, and TM6 outward movement are key determinants of the Gs and Gi/o selectivity among the whole Class A family. Collectively, the findings uncover the common structural geometry within class A GPCRs that determines the primary Gs- and Gi/o-coupling selectivity.

Physical activity trajectory during pregnancy and associations with maternal fatigue using a growth mixture modeling approach.

The purpose of this study was to investigate the associations of physical activity trajectories with maternal fatigue. Pregnant women provided objectively assessed physical activity data by Pregnancy Physical Activity Questionnaire four times. Fatigue scale-14 was used to assess fatigue during pregnancy. Growth mixture modelling characterized physical activity trajectories across pregnancy. The generalized estimating equations was used to analyze the relationship between different physical activity profiles and fatigue in pregnant women. A total of 626 pregnant women were included in analysis in a teaching hospital in Nantong city. Fatigue (total, mental and physical) was not different between two groups based on total energy expenditure of PA (constantly high vs. constantly low). The pregnant women in "constantly high household PA" group had the higher fatigue compared to "constantly low household PA" (P < 0.05) and "constantly medium household PA" (P < 0.05). The pregnant women in "constantly high sport PA" group had lower fatigue compared to "constantly low sport PA" (P < 0.05). Household PA and sport PA were still an independent influencing factor for fatigue after controlling for confounding variables. Specifically, we observed that higher household PA and lower sport PA were associated with higher fatigue during pregnancy.

Platelet-to-Lymphocyte Ratio as an Independent Factor Associated With Atrial Tachyarrhythmia.

Objective To investigate the relationship between the presence of atrial tachyarrhythmia (AT) and the platelet-to-lymphocyte ratio (PLR), which is a recently described inflammatory marker. Methods A total of 149 patients with AT and 187 healthy volunteers were included in this study. Complete blood count, serum lipids, and serum creatinine were tested, and dynamic electrocardiograms were performed routinely in all subjects. Student's t-test, Mann-Whitney U test, logistic regression analysis, and receiver operating characteristic curve analysis were used for statistical analysis. Results In the AT group, the proportions of patients with diabetes, hypertension, and coronary heart disease were higher than those in the control group. Higher blood platelet, low-density lipoprotein, neutrophil-to-lymphocyte ratio, and PLR were detected in the AT group. In addition, haemoglobin, lymphocytes, and the fastest ventricular rate were significantly lower in the AT group. Higher PLR was identified as independently associated with the presence of AT. When a cut-off value of 119.47 was used, the sensitivity and specificity of PLR for predicting AT were 79.2% and 81.3%, respectively. Conclusion Elevated PLR was associated with AT, suggesting that it might be useful in the future as an adjunct biomarker for the detection of the disease.

Safety, pharmacokinetics and target engagement of novel RIPK1 inhibitor SAR443060 (DNL747) for neurodegenerative disorders: Randomized, placebo-controlled, double-blind phase I/Ib studies in healthy subjects and patients.

RIPK1 is a master regulator of inflammatory signaling and cell death and increased RIPK1 activity is observed in human diseases, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). RIPK1 inhibition has been shown to protect against cell death in a range of preclinical cellular and animal models of diseases. SAR443060 (previously DNL747) is a selective, orally bioavailable, central nervous system (CNS)-penetrant, small-molecule, reversible inhibitor of RIPK1. In three early-stage clinical trials in healthy subjects and patients with AD or ALS (NCT03757325 and NCT03757351), SAR443060 distributed into the cerebrospinal fluid (CSF) after oral administration and demonstrated robust peripheral target engagement as measured by a reduction in phosphorylation of RIPK1 at serine 166 (pRIPK1) in human peripheral blood mononuclear cells compared to baseline. RIPK1 inhibition was generally safe and well-tolerated in healthy volunteers and patients with AD or ALS. Taken together, the distribution into the CSF after oral administration, the peripheral proof-of-mechanism, and the safety profile of RIPK1 inhibition to date, suggest that therapeutic modulation of RIPK1 in the CNS is possible, conferring potential therapeutic promise for AD and ALS, as well as other neurodegenerative conditions. However, SAR443060 development was discontinued due to long-term nonclinical toxicology findings, although these nonclinical toxicology signals were not observed in the short duration dosing in any of the three early-stage clinical trials. The dose-limiting toxicities observed for SAR443060 preclinically have not been reported for other RIPK1-inhibitors, suggesting that these toxicities are compound-specific (related to SAR443060) rather than RIPK1 pathway-specific.

Pathological effect of different avian infectious bronchitis virus strains on the bursa of Fabricius of chickens.

Infectious bronchitis is an acute and highly contagious disease caused by avian infectious bronchitis virus (IBV). As well as the typical clinical respiratory signs, such as dyspnoea and tracheal rales, QX genotype strains can also cause damage to the urinary system and reproductive system. Our previous studies found that chickens infected with QX-type IBV also displayed damage to the bursa of Fabricius. To investigate the effects of different genotypes of IBV on the bursa of Fabricius, we challenged one-week-old SPF chickens with Mass, QX and TW genotype IBV strains and compared the clinical signs, gross lesions, histopathological damage, viral loads, and expression levels of inflammatory cytokines (IL-6, IL-8, IL-1ß, IFN-α,ß, γ and TNF-α). The results showed that all three strains caused tissue damage, while significant temporal variations in the viral loads of the different infected groups were detected. IBV infection seriously interfered with the natural immune response mediated by inflammatory cytokines (IFN-α, IFN-ß, IL-6 and IFN-γ) in chickens. Our results suggested that IBV has potential immunological implications for chickens that may lead to poor production efficiency. RESEARCH HIGHLIGHTSAvian coronavirus IBV is an important pathogen of chickens.IBV has potential immunological implications in chickens.The bursal viral load of different IBV strains varies significantly.

CD47-blocking Antibody ZL-1201 Promotes Tumor-associated Macrophage Phagocytic Activity and Enhances the Efficacy of the Therapeutic Antibodies and Chemotherapy.

Tumor-associated macrophages (TAM) are the most abundant immune cells in the tumor microenvironment. They consist of various subsets but primarily resemble the M2 macrophage phenotype. TAMs are known to promote tumor progression and are associated with poor clinical outcomes. CD47 on tumor cells and SIRPα on TAMs facilitate a "don't-eat-me" signal which prevents cancer cells from immune clearance. Therefore, blockade of the CD47-SIRPα interaction represents a promising strategy for tumor immunotherapy. Here, we present the results on ZL-1201, a differentiated and potent anti-CD47 antibody with improved hematologic safety profile compared with 5F9 benchmark. ZL-1201 enhanced phagocytosis in combination with standards of care (SoC) therapeutic antibodies in in vitro coculture systems using a panel of tumor models and differentiated macrophages, and these combinational effects are Fc dependent while potently enhancing M2 phagocytosis. In vivo xenograft studies showed that enhanced antitumor activities were seen in a variety of tumor models treated with ZL-1201 in combination with other therapeutic mAbs, and maximal antitumor activities were achieved in the presence of chemotherapy in addition to the combination of ZL-1201 with other mAbs. Moreover, tumor-infiltrating immune cells and cytokine analysis showed that ZL-1201 and chemotherapies remodel the tumor microenvironment, which increases antitumor immunity, leading to augmented antitumor efficacy when combined with mAbs. Significance: ZL-1201 is a novel anti-CD47 antibody that has improved hematologic safety profiles and combines with SoC, including mAbs and chemotherapies, to potently facilitate phagocytosis and antitumor efficacy.

Two newly isolated GVI lineage infectious bronchitis viruses in China show unique molecular and pathogenicity characteristics.

During 2016 to 2020, GVI-1 type infectious bronchitis virus (IBV) strains were sporadically reported across China, indicating a new epidemic trend of the virus. Here we investigated the molecular characteristics and pathogenicity of two newly isolated GVI-1 type IBV virus strains (CK/CH/TJ1904 and CK/CH/NP2011) from infected chicken farms in China. Genetic evolution analysis of the S1 gene showed the highest homology with the GVI-1 representative strain, TC07-2. Phylogenetic analysis and recombination analysis of the virus genomes indicated that newly isolated strains in China may be independently derived from recombination events that occurred between GI-19 and GI-22 strains and early GVI-1 viruses. Interestingly, unlike the deduced parental GI-19 or GI-22 strains, CK/CH/TJ1904 and CK/CH/NP2011 showed affinity for the trachea rather than the kidney and were less pathogenic. This difference may be because of recombination events that occurred during the long co-existence of the GVI-1 viruses with prevalent GI-19 and GI-22 strains. Considering the new trend, it is very important to permanently monitor circulating strains and to develop new vaccines to counteract emerging new-type IBVs.

Longitudinal biomarkers in amyotrophic lateral sclerosis.

OBJECTIVE: To investigate neurodegenerative and inflammatory biomarkers in people with amyotrophic lateral sclerosis (PALS), evaluate their predictive value for ALS progression rates, and assess their utility as pharmacodynamic biomarkers for monitoring treatment effects. METHODS: De-identified, longitudinal plasma, and cerebrospinal fluid (CSF) samples from PALS (n = 108; 85 with samples from ≥2 visits) and controls without neurological disease (n = 41) were obtained from the Northeast ALS Consortium (NEALS) Biofluid Repository. Seventeen of 108 PALS had familial ALS, of whom 10 had C9orf72 mutations. Additional healthy control CSF samples (n = 35) were obtained from multiple sources. We stratified PALS into fast- and slow-progression subgroups using the ALS Functional Rating Scale-Revised change rate. We compared cytokines/chemokines and neurofilament (NF) levels between PALS and controls, among progression subgroups, and in those with C9orf72 mutations. RESULTS: We found significant elevations of cytokines, including MCP-1, IL-18, and neurofilaments (NFs), indicators of neurodegeneration, in PALS versus controls. Among PALS, these cytokines and NFs were significantly higher in fast-progression and C9orf72 mutation subgroups versus slow progressors. Analyte levels were generally stable over time, a key feature for monitoring treatment effects. We demonstrated that CSF/plasma neurofilament light chain (NFL) levels may predict disease progression, and stratification by NFL levels can enrich for more homogeneous patient groups. INTERPRETATION: Longitudinal stability of cytokines and NFs in PALS support their use for monitoring responses to immunomodulatory and neuroprotective treatments. NFs also have prognostic value for fast-progression patients and may be used to select similar patient subsets in clinical trials.

Pulsed Electromagnetic Fields Reduce Interleukin-6 Expression in Intervertebral Disc Cells Via Nuclear Factor-κß and Mitogen-Activated Protein Kinase p38 Pathways.

STUDY DESIGN: This is an in vitro study of bovine disc cells exposed to pulsed electromagnetic fields. OBJECTIVE: The purpose of the present study was to investigate whether pulsed electromagnetic fields (PEMF) effects on the expression of interleukin-6 (IL-6) expression is mediated by two known inflammation regulators, nuclear factor-κB (NF-κß) and phosphorylated mitogen-activated protein kinase p38 (p38-MAPK) signaling pathways SUMMARY OF BACKGROUND DATA.: Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Increasing evidence showed that PEMF, a noninvasive biophysical stimulation, can have physiologically beneficial effects on inflammation and tissue repair. Our previous research shows that PEMF treatment can reduce IL-6 expression by intervertebral disc cells. However, the underlying mechanisms of PEMF action are yet to be uncovered. METHODS: Intervertebral disc nuclear pulposus cells were challenged with interleukin-1α (IL-1α) (for mimicking inflammatory microenvironment) and treated with PEMF simultaneously up to 4 hours. Cells were then collected for NF-κß and phosphorylated p38-MAPK protein detection with Western blot. Additionally, the RelA (p65) subunit of NF-κß was examined with immunostaining for assessment of NF-κß activation. RESULTS: As expected, Western blot results showed that both NF-κß and phosphorylated p38 expression were significantly increased by IL-1α treatment. This induction was significantly inhibited to control condition levels by PEMF treatment. Immunostaining demonstrated similar trends, that PEMF treatment reduced the NF-κß activation induced by IL-1α exposure. CONCLUSION: Our data indicate that the previously-reported inhibitory effect of PEMF treatment on disc inflammation is mediated by NF-κß and phosphorylated p38-MAPK signaling pathways. These results further establish PEMFs anti-inflammatory activity, and may inform potential future clinical uses for management of inflammation associated with disc degeneration. LEVEL OF EVIDENCE: N/A.

Pulsed electromagnetic fields reduce acute inflammation in the injured rat-tail intervertebral disc.

Pro-inflammatory cytokines are recognized contributors to intervertebral disc (IVD) degeneration and discogenic pain. We have recently reported the anti-inflammatory effect of pulsed electromagnetic fields (PEMF) on IVD cells in vitro. Whether these potentially therapeutic effects are sufficiently potent to influence disc health in vivo has not been demonstrated. We report here the effect of PEMF on acute inflammation arising from a rat-tail IVD injury model. Disc degeneration was induced by percutaneously stabbing the Co6-7, Co7-8, and Co8-9 levels using a 20-gauge needle. Seventy-two (72) rats were divided into three groups: sham control, needle stab, needle stab+PEMF. Treated rats were exposed to PEMF immediately following surgery and for either 4 or 7 days (4 hr/d). Stab and PEMF effects were evaluated by measuring inflammatory cytokine gene expression (RT-PCR) and protein levels (ELISA assay), anabolic and catabolic gene expression (RT-PCR), and histologic changes. We observed in untreated animals that at day 7 after injury, inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor α, and IL-1ß) were significantly increased at both gene and protein levels (P < .05). Similarly, catabolic factors (MMP [metalloproteinases]-2, MMP-13 and the transcriptional factor NF-kß gene expression) were significantly increased (P < .05). At day 7, PEMF treatment significantly inhibited inflammatory cytokine gene and protein expression induced by needle stab injury (P < .05). At day 4, PEMF downregulated FGF-1 and upregulated MMP-2 compared to the stab-only group. These data demonstrate that previously reported anti-inflammatory effects of PEMF on disc cells carry over to the in vivo situation, suggesting potential therapeutic benefits. Though we observed an inhibitory effect of PEMF on acute inflammatory cytokine expression, a consistent effect was not observed for acute changes in disc histology and anabolic and catabolic factor expression. Therefore, these findings should be further investigated in studies of longer duration following needle-stab injury.

Dynamic imaging demonstrates that pulsed electromagnetic fields (PEMF) suppress IL-6 transcription in bovine nucleus pulposus cells.

Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Pulsed electromagnetic fields (PEMF) are noninvasive biophysical stimulus that has been used extensively in the orthopaedic field for many years. However, the specific cellular responses and mechanisms involved are still unclear. The objective of this study was to assess the time-dependent PEMF effects on pro-inflammatory factor IL-6 expression in disc nucleus pulposus cells using a novel green fluorescence protein (GFP) reporter system. An MS2-tagged GFP reporter system driven by IL-6 promoter was constructed to visualize PEMF treatment effect on IL-6 transcription in single living cells. IL-6-MS2 reporter-labeled cells were treated with IL-1α to mimic the in situ inflammatory environment of degenerative disc while simultaneously exposed to PEMF continuously for 4 h. Time-lapse imaging was recorded using a confocal microscope to track dynamic IL-6 transcription activity that was demonstrated by GFP. Finally, real-time RT-PCR was performed to confirm the imaging data. Live cell imaging demonstrated that pro-inflammatory factor IL-1α significantly promoted IL-6 transcription over time as compared with DMEM basal medium condition. Imaging and PCR data demonstrated that the inductive effect of IL-1α on IL-6 expression could be significantly inhibited by PEMF treatment in a time-dependent manner (early as 2 h of stimulus initiation). Our data suggest that PEMF may have a role in the clinical management of patients with chronic low back pain. Furthermore, this study shows that the MS2-tagged GFP reporter system is a useful tool for visualizing the dynamic events of mechanobiology in musculoskeletal research. © 2017 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:778-787, 2018.

Quantification of Propionic Acid in the Bovine Spinal Disk After Infection of the Tissue With Propionibacteria acnes Bacteria.

STUDY DESIGN: Research. OBJECTIVE: The goal of this study was to investigate whether Propionibacteria acnes infection of the intervertebral disc can be detected noninvasively by nuclear magnetic resonance (NMR) spectroscopy. SUMMARY OF BACKGROUND DATA: Microbiological studies of surgical samples suggest that a significant subpopulation of back pain patients may have occult disc infection with P. acnes bacteria. This hypothesis is further supported by a double-blind clinical trial showing that back pain patients with Modic type 1 changes may respond to antibiotic treatment. Because significant side effects are associated with antibiotic treatment, there is a need for a noninvasive method to detect whether specific discs in back pain patients are infected with P acnes bacteria. METHODS: P. acnes bacteria were obtained from human patients. NMR detection of a propionic acid (PA) in the bacteria extracts was conducted on 500 MHz high-resolution spectrometer, whereas in vivo NMR spectroscopy of an isolated bovine disk tissue infected with P. acnes was conducted on 7 T magnetic resonance imaging scanner. RESULTS: NMR spectra of P. acnes metabolites revealed a distinct NMR signal with identical chemical shits (1.05 and 2.18 ppm) as PA (a primary P. acne metabolite). The 1.05 ppm signal does not overlap with other bacteria metabolites, and its intensity increases linearly with P. acnes concentration. Bovine disks injected with P. acnes bacteria revealed a very distinct NMR signal at 1.05 ppm, which linearly increased with P. acnes concentration. CONCLUSION: The 1.05 ppm NMR signal from PA can be used as a marker of P. acnes infection of discs. This signal does not overlap with other disc metabolites and linearly depends on P. acnes concentration. Consequently, NMR spectroscopy may provide a noninvasive method to detect disc infection in the clinical setting. LEVEL OF EVIDENCE: N/A.

Low intensity pulsed ultrasound (LIPUS) for the treatment of intervertebral disc degeneration.

Discogenic back pain presents a major public health issue, with current therapeutic interventions limited to short-term symptom relief without providing regenerative remedies for diseased intervertebral discs (IVD). Many of these interventions are invasive and can diminish the biomechanical integrity of the IVDs. Low intensity pulsed ultrasound (LIPUS) is a potential treatment option that is both non-invasive and regenerative. LIPUS has been shown to be a clinically effective method for the enhancement of wound and fracture healing. Recent in vitro studies have shown that LIPUS stimulation induces an upregulation functional matrix proteins and downregulation of inflammatory factors in cultured IVD cells. However, we do not know the effects of LIPUS on an in vivo model for intervertebral disc degeneration. The objective of this study was to show technical feasibility of building a LIPUS system that can target the rat tail IVD and apply this setup to a model for acute IVD degeneration. A LIPUS exposimetry system was built using a 1.0 MHz planar transducer and custom housing. Ex vivo intensity measurements demonstrated LIPUS delivery to the center of the rat tail IVD. Using an established stab-incision model for disc degeneration, LIPUS was applied for 20 minutes daily for five days. For rats that displayed a significant injury response, LIPUS treatment caused significant upregulation of Collagen II and downregulation of Tumor Necrosis Factor - α gene expression. Our preliminary studies indicate technical feasibility of targeted delivery of ultrasound to a rat tail IVD for studies of LIPUS biological effects.

Effects of cell type and configuration on anabolic and catabolic activity in 3D co-culture of mesenchymal stem cells and nucleus pulposus cells.

Tissue engineering constructs to treat intervertebral disc degeneration must adapt to the hypoxic and inflammatory degenerative disc microenvironment. The objective of this study was to determine the effects of two key design factors, cell type and cell configuration, on the regenerative potential of nucleus pulposus cell (NPC) and mesenchymal stem cell (MSC) constructs. Anabolic and catabolic activity was quantified in constructs of varying cell type (NPCs, MSCs, and a 50:50 co-culture) and varying configuration (individual cells and micropellets). Anabolic and catabolic outcomes were both dependent on cell type. Gene expression of Agg and Col2A1, glycosaminoglycan (GAG) content, and aggrecan immunohistochemistry (IHC), were significantly higher in NPC-only and co-culture groups than in MSC-only groups, with NPC-only groups exhibiting the highest anabolic gene expression levels. However, NPC-only constructs also responded to inflammation and hypoxia with significant upregulation of catabolic genes (MMP-1, MMP-9, MMP-13, and ADAMTS-5). MSC-only groups were unaffected by degenerative media conditions, and co-culture with MSCs modulated catabolic induction of the NPCs. Culturing cells in a micropellet configuration dramatically reduced catabolic induction in co-culture and NPC-only groups. Co-culture micropellets, which take advantage of both cell type and configuration effects, had the most immunomodulatory response, with a significant decrease in MMP-13 and ADAMTS-5 expression in hypoxic and inflammatory media conditions. Co-culture micropellets were also found to self-organize into bilaminar formations with an MSC core and NPC outer layer. Further understanding of these cell type and configuration effects can improve tissue engineering designs. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:61-73, 2017.

Identifying molecular phenotype of nucleus pulposus cells in human intervertebral disc with aging and degeneration.

Previous study claimed that disc degeneration may be preceded by structure and matrix changes in the intervertebral disc (IVD) which coincide with the loss of distinct notochordally derived nucleus pulposus (NP) cells. However, the fate of notochordal cells and their molecular phenotype change during aging and degeneration in human are still unknown. In this study, a set of novel molecular phenotype markers of notochordal NP cells during aging and degeneration in human IVD tissue were revealed with immunostaining and flow cytometry. Furthermore, the potential of phenotype juvenilization and matrix regeneration of IVD cells in a laminin-rich pseudo-3D culture system were evaluated at day 28 by immunostaining, Safranin O, and type II collagen staining. Immunostaining and flow cytometry demonstrated that transcriptional factor Brachyury T, neuronal-related proteins (brain abundant membrane attached signal protein 1, Basp1; Neurochondrin, Ncdn; Neuropilin, Nrp-1), CD24, and CD221 were expressed only in juvenile human NP tissue, which suggested that these proteins may be served as the notochordal NP cell markers. However, the increased expression of CD54 and CD166 with aging indicated that they might be referenced as the potential biomarker for disc degeneration. In addition, 3D culture maintained most of markers in juvenile NP, and rescued the expression of Basp1, Ncdn, and Nrp 1 that disappeared in adult NP native tissue. These findings provided new insight into molecular profile that may be used to characterize the existence of a unique notochordal NP cells during aging and degeneration in human IVD cells, which will facilitate cell-based therapy for IVD regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1316-1326, 2016.

A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells.

Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, "tissue incubation", for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7-10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3-4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.

Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging.

Intervertebral disc (IVD) disorder and age-related degeneration are believed to contribute to low back pain. Cell-based therapies represent a promising strategy to treat disc degeneration; however, the cellular and molecular characteristics of disc cells during IVD maturation and aging still remain poorly defined. This study investigated novel molecular markers and their age-related changes in the rat IVD. Affymetrix cDNA microarray analysis was conducted to identify a new set of genes characterizing immature nucleus pulposus (NP) cells. Among these markers, select neuronal-related proteins (Basp1, Ncdn and Nrp-1), transcriptional factor (Brachyury T), and cell surface receptors (CD24, CD90, CD155 and CD221) were confirmed by real-time PCR and immunohistochemical (IHC) staining for differential expression between IVD tissue regions and among various ages (1, 12 and 21 months). NP cells generally possessed higher levels of mRNA or protein expression for all aforementioned markers, with the exception of CD90 in anulus fibrosus (AF) cells. In addition, CD protein (CD24 and CD90) and Brachyury (T) expression in immature disc cells were also confirmed via flow cytometry. Similar to IHC staining, results revealed a higher percentage of immature NP cells expressing CD24 and Brachyury, while higher percentage of immature AF cells was stained positively for CD90. Altogether, this study identifies that tissue-specific gene expression and age-related differential expression of the above markers do exist in immature and aged disc cells. These age-related phenotype changes provide a new insight for a molecular profile that may be used to characterize NP cells for developing cell-based regenerative therapy for IVD regeneration.

The profile of MMP and TIMP in injured rat ACL.

Human anterior cruciate ligament (ACL) has poor healing ability after injury. The devastating effects of matrix metalloproteinases (MMPs) excess expression are regarded as the main reason. Tissue inhibitor metalloproteinases (TIMPs) may be independent of MMPs inhibition. In this paper, a rat ACL rotating injury apparatus was designed to produce ACL injury. After inducing injury, joint fluids and ACL tissue total proteins were immediately extracted. In addition, ACL tissue was isolated in a culture plate with 1%FBS medium for the ex vivo study. We found MMP-2 in joint fluids increased significantly by 4 folds after ACL injury as a function of time. Ex vivo study showed MMP-2 in the medium and ACL cultured tissue increased significantly respectively to 3 folds and to 6 folds. The joint fluids global MMP increased to 3.5 folds with non-treatment and APMA-treatment in day three. On the gene expression level, the changes in MMP-1 and CD147 have the similar tendency. The ratio of MMP-1/TIMP-1 increased with time after ACL injury. We conclude that MMP-2 increased significantly in the early phase in the joint cavity after ACL injury. The ex vivo study demonstrated the same tendency. Generic MMP Activity Assay (global MMP assay) an dzymography also showed significant increase in MMP activity in joint fluids. These results showed ACL having poor healing ability after injury may not be only due to ACL release of large quantities of MMPs. The important factor may be the alterations in the whole joint cavity's internal environment.

Differential MMP-2 activity induced by mechanical compression and inflammatory factors in human synoviocytes.

The anterior cruciate ligament, posterior cruciate ligament, cartilage and meniscus in human knee joint have poor healing ability. Accumulation of MMPs in the joint fluids due to knee injury has been considered as the main reason. Our previous experiments showed that synovium may be the major regulator of MMPs in joint cavity after injury. In this paper, we used human synoviocytes harvested from synovium to determine whether mechanical injury and inflammatory factors will induce MMP-2 production in synoviocytes. With zymography, we found that mechanical compression increased the MMP-2 production by 23% under 6% compressions, 61% under 12% compression and 109% under 14% compressions. In addition, TNF-alpha can also elevate the activity of MMP-2 in a dose dependent manner, while IL-1alpha does not. However, mixture of these two factors dramatically increased MMP-2 production by 201%. In addition, mechanical injury had a strong synergistic effect on MMP-2 production with TNF-alpha, IL-1alpha and their mixture, increasing by 207%, 354% and 468% individually. The generic MMP activity assayrevealed that mechanical compression increased the generic activity. APMA treatment increased the generic activity of MMPs induced by compres-

[Advance of researches in nitric oxide biological function on wound repair].

Nitric oxide (NO) is a short-life free radical that acts as the small biological molecule, and exists in body extensively. Since its discovery over 20 years ago, NO has been found to play an important regulation role in angiogenesis, nerve and immune system. The subsequent studies also showed that NO exerted an important biological action in wound repairing and healing, which involved in the following phases of wound repair, inflammation, cell proliferation, matrix deposition and remodeling. This paper reviews recent findings from in vitro & in vivo studies of NO in wound repair, and the biological function and mechanisms of NO in wound repair.