The train-of-four or double-burst ratios cannot reliably exclude residual neuromuscular block in cats.

This study investigated the relationship between train-of-four (TOF) or double burst (DBS) ratios (T4:T1 or B2:B1) and twitch (T1) or burst (B1) magnitudes during the recovery from rocuronium-induced neuromuscular block in dogs and cats. The main hypothesis was that TOF or DBS ratios recover after the recovery of T1 or B1, and hence high ratio levels are sensitive indicators of restoration of the neuromuscular function. Six anesthetized dogs and six anesthetized cats received 0.5 mg/kg of rocuronium intravenously. The amplitudes of T1 or B1 were measured with mechanomyography during neuromuscular block until the neuromuscular function recovered fully. The TOF or DBS ratio was recorded concurrently. In dogs, recovery of T1 and B1 preceded the recovery of the TOF and DBS ratios, and T1 and B1 were always ≥90% of recovery when the respective ratio reached 0.9. In contrast, T1 was still depressed in 5/6 cats when the TOF ratio reached 0.9. At that moment, T1 was 72.5 ± 19.8% of recovery. Similarly, the DBS ratio returned to 0.9 when B1 was still <90% in 3/6 cats of recovery. The TOF and DBS fade in dogs consistently disappeared after the magnitude of T1 or B1 were restored, and hence, ratios ≥0.9 are a sensitive indicator that the neuromuscular function recovered. Our observation in cats however show that the spontaneous recovery of neither the TOF nor the DBS ratio of 0.9 can reliably exclude residual block, as the magnitude of T1 or B1 was still depressed in several instances.

Functional cell phenotype induction with TGF-ß1 and collagen-polyurethane scaffold for annulus fibrosus rupture repair.

Appropriate cell sources, bioactive factors and biomaterials for generation of functional and integrated annulus fibrosus (AF) tissue analogues are still an unmet need. In the present study, the AF cell markers, collagen type I, cluster of differentiation 146 (CD146), mohawk (MKX) and smooth muscle protein 22α (SM22α) were found to be suitable indicators of functional AF cell induction. In vitro 2D culture of human AF cells showed that transforming growth factor ß1 (TGF-ß1) upregulated the expression of the functional AF markers and increased cell contractility, indicating that TGF-ß1-pre-treated AF cells were an appropriate cell source for AF tissue regeneration. Furthermore, a tissue engineered construct, composed of polyurethane (PU) scaffold with a TGF-ß1-supplemented collagen type I hydrogel and human AF cells, was evaluated with in vitro 3D culture and ex vivo preclinical bioreactor-loaded organ culture models. The collagen type I hydrogel helped maintaining the AF functional phenotype. TGF-ß1 supplement within the collagen I hydrogel further promoted cell proliferation and matrix production of AF cells within in vitro 3D culture. In the ex vivo IVD organ culture model with physiologically relevant mechanical loading, TGF-ß1 supplement in the transplanted constructs induced the functional AF cell phenotype and enhanced collagen matrix synthesis. In conclusion, TGF-ß1-containing collagen-PU constructs can induce the functional cell phenotype of human AF cells in vitro and in situ. This combined cellular, biomaterial and bioactive agent therapy has a great potential for AF tissue regeneration and rupture repair.

Context-sensitive recovery of neuromuscular function from vecuronium in dogs: Effects of dose and dosing protocol.

Recovery of neuromuscular function is a gradual phenomenon whereby function progresses from absent to normal. The speed of spontaneous recovery can be used to predict the time when neuromuscular function is expected to be restored. However, the speed of recovery might be affected by the dose of the neuromuscular blocker administered, and by the dosing regimen of that dose. The effects of both factors on the speed of spontaneous recovery from vecuronium were evaluated. Seven dogs were anesthetized three times and the train-of-four (TOF) ratio was measured with acceleromyography. Vecuronium was administered at 0.1 mg/kg, 0.2 mg/kg, or 0.1 mg/kg followed by two doses of 0.05 mg/kg was administered each time. In the divided-dose treatment group, aliquots were administered on return of the first twitch (T1) of the TOF from the previous dose. The duration of surgical block, from injection to return of T1, was longest for the divided-dose protocol, intermediate for 0.2 mg/kg single bolus, and shortest for 0.1 mg/kg (P < 0.0001). The recovery period, from return of T1 to a TOF ratio ≥0.9, was longer for 0.2 mg/kg administered as a single bolus than for the other two groups (P = 0.007). Doubling the dose of a single bolus of vecuronium extended the time of surgical block and prolonged the duration of the recovery period. However, dividing that dose into smaller aliquots extended the period of surgical block while shortening the recovery period. Hence, the spontaneous reappearance of T1 should not be used in isolation to predict the time to complete recovery of neuromuscular function.

Variability in the time to initiation of CPR in continuously monitored pediatric ICUs.

AIM: To study the influence of patient characteristics and unit ergonomics and human factors on the time to initiation of CPR. METHODS: A single center study of children, 0 to 21 years old, admitted to an ICU who experienced cardiopulmonary arrest (CPA) requiring >1 min of chest compressions. Time of CPA was determined by analysis of continuous ECG, plethysmography, arterial blood pressure, and end-tidal CO2 (EtCO2) waveforms. Initiation of CPR was identified by the onset of cyclic artifact in the ECG waveform. Patient characteristics and unit ergonomics and human factors were examined including CPA cause, identification on the High-Risk Checklist (HRC), existing monitoring, ICU type, time of day, nursing shift change, and outcome. RESULTS: The median time from CPA to initiation of CPR was 50.5 s (IQR 26.5 to 127.5) in 36 CPAs. Forty-seven percent of patients experienced time from CPA to initiation of CPR of >1 min. There was no difference in CPA cause, ICU type, time of day, or nursing shift change. CONCLUSION: Nearly half of pediatric patients who experienced CPA in an ICU setting did not meet AHA guidelines for early initiation of CPR. This is an opportunity to study the recognition phase of CPA using continuous monitoring data with the aim of improving the understanding of and factors contributing to delays in initiation of CPR.

Bias, limits of agreement, and percent errors between acceleromyography and mechanomyography in anesthetized dogs.

This study compared measurements of neuromuscular function with mechanomyography (MMG) and acceleromyography (AMG) in nine anesthetized dogs receiving 0.1mg/kg vecuronium intravenously. Train-of-four (TOF) stimulation was applied to each ulnar nerve every 15s. The resulting amplitude of the first twitch (T1) and the TOF ratio were measured with both monitors. The baseline TOF ratio (prior to vecuronium), onset time (time of injection to T1<5%), recovery index (time between T1 values of 25% and 75%) and duration of neuromuscular block (injection to TOF 0.9) were recorded. The MMG TOF ratios when the AMG first reached 0.7 (AMG 0.7) and 0.9 (AMG 0.9) during recovery were also recorded. Values were compared with paired tests and individual errors>25% between monitors were identified for each dog. Bias, limits of agreement (LOA) and percentage error (PE) between methods were calculated from Bland-Altman plots for T1 and TOF ratio for the complete data set, and for TOF≥0.7 during recovery. There were no statistical differences in baseline TOF ratio, onset, recovery index, duration, AMG 0.7 and AMG 0.9. Individual errors>25% were evident in onset, recovery index, AMG 0.7 and AMG 0.9. Overall, T1 and TOF ratio had a small bias, wide LOA and PE>100%. Percent error was reduced to 30% when TOF≥0.7 was analyzed. Although there were no statistical differences between MMG and AMG in any variable of interest, individual discrepancies, wide LOA and high PE suggest that these monitors should not be used interchangeably for serial measurements on the same animals.

Transplantation of activated nucleus pulposus cells after cryopreservation: efficacy study in a canine disc degeneration model.

Transplantation of activated nucleus pulposus (NP) cells obtained by coculturing NP cells and bone marrow mesenchymal stromal cells having cell-to-cell contact has been shown to be effective in animal models and, more recently, in human clinical trials. If the NP cells can be cryopreserved, then autologous cell transplantation could be offered to patients as and when required. In a previous study, we confirmed that activated NP cells can be obtained by coculturing with mesenchymal cells after cryopreservation. However, the in vivo effects of cell transplantation therapy using activated NP cells prepared from cryopreserved cells are not known. In this in vivo canine model, we compared indicators of disc degeneration in animals that received transplanted activated normal NP cells, transplanted cryopreserved NP cells, and no cell transplantation after induction of disc degeneration. The intervertebral disc height on radiographs and T2-weighted magnetic resonance imaging were significantly higher in both cell transplantation groups compared with the degenerated disc group. Macroscopic and histological findings demonstrated attenuated disc degeneration in the two transplanted groups. Intense staining of proteoglycan and collagen type II was seen in green fluorescent protein-labelled transplanted cells, which suggested that the cells had survived and were functioning after transplantation. No significant differences were observed between the two transplanted groups. Transplanted activated cryopreserved NP cells induced a similar attenuation of intervertebral disc degeneration as that of conventionally activated NP cells. These findings suggest that the use of cryopreserved cells specific to a patient's condition has potential in transplantation therapy.

Intervertebral disc repair with activated nucleus pulposus cell transplantation: a three-year, prospective clinical study of its safety.

Degeneration of the lumbar intervertebral discs is irreversible, with no treatment currently available. Building upon experimental studies that demonstrated the importance of the nucleus pulposus (NP) in preserving disc structure, we demonstrated that reinsertion of NP cells slowed further disc degeneration and that direct cell-to-cell contact co-culture with mesenchymal stromal cells (MSCs) significantly upregulated the viability of NP cells in basic and pre-clinical studies in vitro and in vivo using animal models and human cells. Here, we report a 3-year result of a prospective clinical study, aimed to assess the safety and efficacy of activated NP cell transplantation in the degenerate lumbar intervertebral disc. Candidates were 9 patients aged 20-29 years who had Pfirrmann's grade III disc degeneration at the level adjacent to the level scheduled for posterior lumbar intervertebral fusion. Viable NP cells from the fused disc were co-cultured in direct contact with autologous bone marrow-derived MSCs. One million activated NP cells were transplanted into the degenerated disc adjacent to the fused level at 7 d after the first fusion surgery. No adverse effects were observed during the 3-year follow-up period. Magnetic resonance imaging did not show any detrimental effects to the transplanted discs and revealed a mild improvement in 1 case. No cases reported any low back pain. Our clinical study confirmed the safety of activated NP cell transplantation, and the findings suggest the minimal efficacy of this treatment to slow the further degeneration of human intervertebral discs.

Fibrin-genipin adhesive hydrogel for annulus fibrosus repair: performance evaluation with large animal organ culture, in situ biomechanics, and in vivo degradation tests.

Annulus fibrosus (AF) defects from annular tears, herniation, and discectomy procedures are associated with painful conditions and accelerated intervertebral disc (IVD) degeneration. Currently, no effective treatments exist to repair AF damage, restore IVD biomechanics and promote tissue regeneration. An injectable fibrin-genipin adhesive hydrogel (Fib-Gen) was evaluated for its performance repairing large AF defects in a bovine caudal IVD model using ex vivo organ culture and biomechanical testing of motion segments, and for its in vivo longevity and biocompatibility in a rat model by subcutaneous implantation. Fib-Gen sealed AF defects, prevented IVD height loss, and remained well-integrated with native AF tissue following approximately 14,000 cycles of compression in 6-day organ culture experiments. Fib-Gen repair also retained high viability of native AF cells near the repair site, reduced nitric oxide released to the media, and showed evidence of AF cell migration into the gel. Biomechanically, Fib-Gen fully restored compressive stiffness to intact levels validating organ culture findings. However, only partial restoration of tensile and torsional stiffness was obtained, suggesting opportunities to enhance this formulation. Subcutaneous implantation results, when compared with the literature, suggested Fib-Gen exhibited similar biocompatibility behaviour to fibrin alone but degraded much more slowly. We conclude that injectable Fib-Gen successfully sealed large AF defects, promoted functional restoration with improved motion segment biomechanics, and served as a biocompatible adhesive biomaterial that had greatly enhanced in vivo longevity compared to fibrin. Fib-Gen offers promise for AF repairs that may prevent painful conditions and accelerated degeneration of the IVD, and warrants further material development and evaluation.

Cell therapy for intervertebral disc repair: advancing cell therapy from bench to clinics.

Intervertebral disc (IVD) degeneration is a major cause of pain and disability; yet therapeutic options are limited and treatment often remains unsatisfactory. In recent years, research activities have intensified in tissue engineering and regenerative medicine, and pre-clinical studies have demonstrated encouraging results. Nonetheless, the translation of new biological therapies into clinical practice faces substantial barriers. During the symposium "Where Science meets Clinics", sponsored by the AO Foundation and held in Davos, Switzerland, from September 5-7, 2013, hurdles for translation were outlined, and ways to overcome them were discussed. With respect to cell therapy for IVD repair, it is obvious that regenerative treatment is indicated at early stages of disc degeneration, before structural changes have occurred. It is envisaged that in the near future, screening techniques and non-invasive imaging methods will be available to detect early degenerative changes. The promises of cell therapy include a sustained effect on matrix synthesis, inflammation control, and prevention of angio- and neuro-genesis. Discogenic pain, originating from "black discs" or annular injury, prevention of adjacent segment disease, and prevention of post-discectomy syndrome were identified as prospective indications for cell therapy. Before such therapy can safely and effectively be introduced into clinics, the identification of the patient population and proper standardisation of diagnostic parameters and outcome measurements are indispensable. Furthermore, open questions regarding the optimal cell type and delivery method need to be resolved in order to overcome the safety concerns implied with certain procedures. Finally, appropriate large animal models and well-designed clinical studies will be required, particularly addressing safety aspects.

CCL5/RANTES is a key chemoattractant released by degenerative intervertebral discs in organ culture.

Release of chemotactic factors in response to tissue damage has been described for different musculoskeletal tissues, including the intervertebral disc (IVD). This study investigated the chemoattractants that are released by induced degenerative IVDs and may be involved in recruiting mesenchymal stem cells (MSCs). Bovine caudal discs were cultured within a bioreactor and loaded under conditions that mimicked physiological or degenerative settings. Between days 4-6, medium was replaced by PBS, which was subsequently used for proteomic, ELISA and immunoprecipitation analyses of secreted chemokines and cytokines. A Boyden chamber assay was used to observe human MSC migration towards native and chemokine depleted media. Gene expression levels of chemokine receptors in human MSCs were analysed, and CCL5 was localised in bovine and human IVD by immunohistochemistry. Proteomic analysis revealed the presence of CCL5 and CXCL6 within conditioned media. Higher concentrations of CCL5 were found in the degenerative media, and a relationship was found between interleukin-1ß and CCL5 concentration. Chemokine immunoprecipitation showed that MSCs had a significantly reduced chemotactic migration towards CCL5-immunoprecipitated and CCL5/CXCL6 co-immunoprecipitated media, whilst CXCL6 depletion did not change MSC chemotaxis. MSCs showed a significant increase in mRNA expression of the CCL5 receptors, CCR1 and CCR4, upon culture in degenerative media. Furthermore, CCL5 was identified in bovine and human disc tissue by immunohistochemistry. Hence, CCL5 may be a key chemoattractant that is produced and released by the intervertebral disc cells. Therefore, these factors could be used to enhance stem/progenitor cell mobilisation in regenerative therapies for early stages of disc degeneration.

Phase II study of trastuzumab in combination with S-1 plus cisplatin in HER2-positive gastric cancer (HERBIS-1).

BACKGROUND: S-1, an oral fluoropyrimidine, plus cisplatin (SP) is a standard regimen for advanced gastric cancer (AGC) in East Asia. To date, no studies have evaluated the efficacy and safety of trastuzumab combined with SP in patients with human epidermal growth factor receptor type 2 (HER2)-positive AGC. METHODS: Patients with HER2-positive AGC received S-1 (80-120 mg per day) orally on days 1-14, cisplatin (60 mg m(-2)) intravenously on day 1, and trastuzumab (course 1, 8 mg kg(-1); course 2 onward, 6 mg kg(-1)) intravenously on day 1 of a 21-day cycle. The primary end point was response rate (RR); secondary end points included overall survival (OS), progression-free survival (PFS), time to treatment failure (TTF), and adverse events. RESULTS: A total of 56 patients were enrolled. In the full analysis set of 53 patients, the confirmed RR was 68% (95% confidence interval (CI)=54-80%), and the disease control rate was 94% (95% CI=84-99%). Median OS, PFS, and TTF were estimated as 16.0, 7.8, and 5.7 months, respectively. Major grade 3 or 4 adverse events included neutropaenia (36%), anorexia (23%), and anaemia (15%). CONCLUSIONS: Trastuzumab in combination with SP showed promising antitumour activity and manageable toxic effects in patients with HER2-positive AGC.

Randomised phase II study comparing dose-escalated weekly paclitaxel vs standard-dose weekly paclitaxel for patients with previously treated advanced gastric cancer.

BACKGROUND: This randomised phase II trial compared dose-escalated weekly paclitaxel (wPTX) vs standard-dose wPTX for patients with previously treated advanced gastric cancer (AGC). METHODS: Ninety patients were randomised to a standard dose of wPTX (80 mg m(-2)) or an escalated dose of wPTX (80-120 mg m(-2)) to assess the superiority of overall survival (OS) with a one-sided alpha error of 0.3 and a power of 0.8. RESULTS: The median OS showed a trend towards longer survival in the dose-escalated arm (11.8 vs 9.6 months; hazard ratio (HR), 0.75; one-sided P=0.12), although it was statistically not significant. The median progression-free survival (PFS) was significantly longer in the dose-escalated arm (4.3 vs 2.5 months, HR, 0.55; P=0.017). Objective response rate was 30.3% with dose escalation and 17.1% with standard dose (P=0.2). The frequency of all grades of neutropenia was significantly higher with dose escalation (88.7% vs 60.0%, P=0.002); however, no significant difference was observed in the proportion of patients experiencing grade 3 or more (40.9% vs 31.1%, P=0.34). CONCLUSION: Dose-escalated wPTX in patients with pretreated AGC met our predefined threshold of primary end point, OS (P<0.3); however, it did not show a significantly longer OS. Progression-free survival was significantly better with dose escalation.

Challenges and strategies in the repair of ruptured annulus fibrosus.

Lumbar discectomy is the surgical procedure most frequently performed for patients suffering from low back pain and sciatica. Disc herniation as a consequence of degenerative or traumatic processes is commonly encountered as the underlying cause for the painful condition. While discectomy provides favourable outcome in a majority of cases, there are conditions where unmet requirements exist in terms of treatment, such as large disc protrusions with minimal disc degeneration; in these cases, the high rate of recurrent disc herniation after discectomy is a prevalent problem. An effective biological annular repair could improve the surgical outcome in patients with contained disc herniations but otherwise minor degenerative changes. An attractive approach is a tissue-engineered implant that will enable/stimulate the repair of the ruptured annulus. The strategy is to develop three-dimensional scaffolds and activate them by seeding cells or by incorporating molecular signals that enable new matrix synthesis at the defect site, while the biomaterial provides immediate closure of the defect and maintains the mechanical properties of the disc. This review is structured into (1) introduction, (2) clinical problems, current treatment options and needs, (3) biomechanical demands, (4) cellular and extracellular components, (5) biomaterials for delivery, scaffolding and support, (6) pre-clinical models for evaluation of newly developed cell- and material-based therapies, and (7) conclusions. This article highlights that an interdisciplinary approach is necessary for successful development of new clinical methods for annulus fibrosus repair. This will benefit from a close collaboration between research groups with expertise in all areas addressed in this review.

Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration.

OBJECTIVE: Regardless of recent progress in the elucidation of intervertebral disc (IVD) degeneration, the basic molecular characteristics that define a healthy human IVD are largely unknown. Although work in different animal species revealed distinct molecules that might be used as characteristic markers for IVD or specifically nucleus pulposus (NP) cells, the validity of these markers for characterization of human IVD cells remains unknown. DESIGN: Eleven potential marker molecules were characterized with respect to their occurrence in human IVD cells. Gene expression levels of NP were compared with annulus fibrosus (AF) and articular cartilage (AC) cells, and potential correlations with aging were assessed. RESULTS: Higher mRNA levels of cytokeratin-19 (KRT19) and of neural cell adhesion molecule-1 were noted in NP compared to AF and AC cells. Compared to NP cytokeratin-18 expression was lower in AC, and alpha-2-macroglobulin and desmocollin-2 lower in AF. Cartilage oligomeric matrix protein (COMP) and glypican-3 expression was higher in AF, while COMP, matrix gla protein (MGP) and pleiotrophin expression was higher in AC cells. Furthermore, an age-related decrease in KRT19 and increase in MGP expression were observed in NP cells. The age-dependent expression pattern of KRT19 was confirmed by immunohistochemistry, showing the most prominent KRT19 immunoreaction in the notochordal-like cells in juvenile NP, whereas MGP immunoreactivity was not restricted to NP cells and was found in all age groups. CONCLUSIONS: The gene expression of KRT19 has the potential to characterize human NP cells, whereas MGP cannot serve as a characteristic marker. KRT19 protein expression was only detected in NP cells of donors younger than 54 years.

Low-intensity pulsed ultrasound stimulates cell proliferation, proteoglycan synthesis and expression of growth factor-related genes in human nucleus pulposus cell line.

Low-intensity pulsed ultrasound (LIPUS) stimulation has been shown to effect differentiation and activation of human chondrocytes. A study involving stimulation of rabbit disc cells with LIPUS revealed upregulation of cell proliferation and proteoglycan (PG) synthesis. However, the effect of LIPUS on human nucleus pulposus cells has not been investigated. In the present study, therefore, we investigated whether LIPUS stimulation of a human nucleus pulposus cell line (HNPSV-1) exerted a positive effect on cellular activity. HNPSV-1 cells were encapsulated in 1.2% sodium alginate solution at 1x10(5) cells/ml and cultured at 10 beads/well in 6-well plates. The cells were stimulated for 20 min each day using a LIPUS generator, and the effects of LIPUS were evaluated by measuring DNA and PG synthesis. Furthermore, mRNA expression was analyzed by cDNA microarray using total RNA extracted from the cultured cells. Our study revealed no significant difference in cell proliferation between the control and the ultrasound treated groups. However, PG production was significantly upregulated in HNPSV cells stimulated at intensities of 15, 30, 60, and 120 mW/cm(2) compared with the control. The results of cDNA array showed that LIPUS significantly stimulated the gene expression of growth factors and their receptors (BMP2, FGF7, TGFbetaR1 EGFRF1, VEGF). These findings suggest that LIPUS stimulation upregulates PG production in human nucleus pulposus cells by the enhancement of several matrix-related genes including growth factor-related genes. Safe and non-invasive stimulation using LIPUS may be a useful treatment for delaying the progression of disc degeneration.

Stem cell applications in intervertebral disc repair.

There is increasing rise of interest in stem cell therapy, as it provides new options for treating a broad range of diseases. Several experimental methods are being explored for the use of stem cells in delaying or reversing the degenerative process of the intervertebral disc, a major cause of low back pain. In this article, we review the current strategies for stem cell applications in intervertebral disc repair and present three novel approaches. These are, first, the activation of nucleus pulposus cells by co-culture with mesenchymal stem cells for autologous disc cell reinsertion; second, the in vitro induction of nucleus pulposus-like or annulus fibrosus-like cells from mesenchymal stem cells; and third, the in vivo induction study by direct transplantation of mesenchymal stem cells to the intervertebral disc induced to degenerate experimentally. Although still untested, stem cell therapy may become a major option in the treatment of intervertebral disc degeneration.

A torque removal study on the primary stability of orthodontic titanium screw mini-implants in the cortical bone of dog femurs.

The aim of this study was to biomechanically evaluate the primary stability of pure titanium orthodontic mini-implants, inserted into pre-drilled cavities of differing diameters. Mini-implants (1.2 mm diameter) were placed into 1.0 mm and 1.2 mm diameter cavities prepared in the mid-region of the bilateral hind leg femurs of anesthetized beagles. Removal torque strengths were measured immediately, 1, 3, 6, 9 and 12 weeks post-insertion of the implant. For mini-implants placed into 1-mm cavities, removal torque values decrease over the first 6 weeks (p<0.01), after which values remained static. Average values obtained immediately, 1, 3 and 6 weeks post-insertion were 10.98, 8.83, 7.20 and 5.12 Ncm, respectively . Immediately post-insertion, removal torque values of mini-implants placed in a 1.2-mm cavity, were 11-fold lower than those placed in 1.0-mm cavities, which then demonstrated a significant increase in strength from 3 weeks (1.35 Ncm) to 6 weeks (5.17 Ncm) post-insertion (p<0.01). Measurements 6, 9 and 12 weeks post-insertion were similar to those in the 1.0-mm cavity. Initial stability of titanium mini-implants is considered necessary for immediate and early use in orthodontics, and an implant without this initial stability should be replaced or isolated until it develops the appropriate stability supported by osseointegration.

Cross talk between Smad transcription factors and TNF-alpha in intervertebral disc degeneration.

The transforming growth factor-beta (TGF-beta) and the tumor necrosis factor-alpha (TNF-alpha) families are known to play important roles in intervertebral disc degeneration (IVD). However, molecular interactions between the TGF-beta and TNF-alpha signaling pathways have yet to be elucidated. The purpose of this study was to analyze the expression patterns of Smad transcription factor signaling associated with IVDs with aging and to examine the modulation of Smad signaling by TNF-alpha in IVD cells using SD rats. According to these experimental results, BMP signals in the TGF-beta family were more likely to be a key factor in IVD degeneration by aging, and it was predicted that besides the involvement of catabolic factors like MMPs and ADAMS-TS, there may be a decrease in expression of anabolic factors through cross talk of signaling between TNF-alpha and TGF-beta pathway in pathogenesis of disc degeneration.

Real-time PCR for quantification of viable Renibacterium salmoninarum in chum salmon Oncorhynchus keta.

Quantification of msa gene mRNA of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was investigated using reverse transcription followed by real-time PCR assay on R. salmoninarum in culture, and in experimentally challenged chum salmon Oncorhynchus keta fry kidney tissues (total of 70 samples) after intraperitoneal (i.p.) injection and bath infection. Correlations of msa gene mRNA concentrations with culturable cell concentrations (as colony forming units [CFU]), determined by drop-plate culture method on selective kidney disease medium (SKDM) agar through a 12 wk incubation time, and msa gene DNA concentrations by real-time PCR assay were examined. Furthermore, ovarian fluid samples from wild chum salmon adults with no clinical signs of disease were collected from 8 rivers and from clinically infected kokanee 0. nerka and masu salmon O. masou that were reared in 1 and 2 hatcheries, respectively (total of 414 samples). All samples were examined by nested PCR assay. Then, positive samples were examined by real-time PCR assays for mRNA and DNA; mRNA was detectable at 8 log units (5.0 x 101 to 5.0 x 10(9) copies p11(-1)) with high correlation (R2 = 0.999). The mRNA concentration correlated with CFU in kidney tissue from fish infected by i.p. injection (R2 = 0.924), by bath infection (R2 = 0.502) and in culture (R2 = 0.888). R. salmoninarum was detected and quantified by real-time PCR assay for mRNA in ovarian fluid samples in both subclinically infected chum salmon adults and clinically infected kokanee and masu salmon adults; detection rates ranged from 0 to 44.4% and concentrations ranged from 9.7 x 10(2) to 5.6 x 10(5) copies pl(-1). These results indicate that real-time PCR assay for the mRNA is a rapid, sensitive and reliable method to detect and quantify the viability of R. salmoninarum in kidney and ovarian fluid samples of salmonid fishes with both clinical and subclinical infection of the pathogen.

Expression of CD90 on keratinocyte stem/progenitor cells.

BACKGROUND: The identification and purification of keratinocyte stem cells (KSCs) that are capable of self-renewal and maintenance of differentiating cell populations could contribute both to our understanding of the biology of these cells, and to significant clinical applications, such as the culturing of keratinocytes for transplantation to severe burn wounds. Here, we report the detection of CD90(+) cells in cultured normal human epidermal keratinocytes and adult skin. OBJECTIVES: To investigate the biological function of CD90(+) and CD90(-) keratinocytes. METHODS: CD90(+) and CD90(-) keratinocytes were purified from adult skin and cultured keratinocytes using fluorescent activated cell sorting, and their biological abilities were analysed using both in vitro and in vivo assays. RESULTS: Flow cytometry (FCM) analysis identified approximately 18% of post-primary neonatal keratinocytes as CD90(+). However, during expansion of the culture, the expression level of CD90 rapidly decreased to about 2.5% at passage 10, while most of the keratinocytes maintained expression of alpha6 integrin. Purified CD90(+) keratinocytes demonstrated a sixfold higher cell growth rate than CD90(-) cells and the ability to form large (over 3 mm in diameter) colonies. We then quantitatively evaluated both populations using a previously described in vivo human epidermal cyst formation assay. Enhanced green fluorescent protein (EGFP)-labelled CD90(+) or CD90(-) keratinocytes were subcutaneously injected into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Six weeks after transplantation, EGFP(+) cell clusters in human epidermal cysts were evaluated using image analysis software. EGFP(+) cell cluster areas in the basal layer, derived from EGFP(+) CD90(+) cells, were eightfold larger than clusters of EGFP(+) CD90(-) cells. Furthermore, immunohistochemical staining and FCM analysis indicated that CD90 was expressed in most of the basal layer of the normal human epidermis. CONCLUSIONS: These results indicated that CD90 is a useful marker for the detection of human KSC-enriched populations in cultured human keratinocytes.