Colchicine protects against cartilage degeneration by inhibiting MMP13 expression via PLC-γ1 phosphorylation.

OBJECTIVE: Low molecular weight compounds that reduce the expression of MMP13 at the mRNA level might serve as disease-modifying osteoarthritis (OA) drugs (DMOADs). The objective of this study was to identify a candidate DMOAD that targets MMP13 expression. DESIGN: High-throughput screening was performed to identify compounds that suppress inflammatory cytokine-induced MMP13 expression. Ingenuity pathway analysis (IPA) using isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analysis was conducted to identify signaling pathways related to cytokines. MMP13 expression in chondrocytes was evaluated through RT-qPCR and western blotting analyses. Additionally, 10-week-old mice were subjected to destabilization of the medial meniscus (DMM) surgery to induce OA and were sacrificed 12 weeks post-surgery for pathological examination. OA was evaluated using the OARSI scoring system. RESULTS: Colchicine was identified as a DMOAD candidate as it inhibited inflammatory cytokine-induced MMP13 expression in vitro, and the colchicine-administered mice with DMM presented significantly lower OARSI scores (adjusted P: 0.0242, mean difference: 1.6, 95% confidence interval (CI) of difference: 0.1651-3.035) and significantly lower synovial membrane inflammation scores (adjusted P: 0.0243, mean difference: 0.6, 95% CI of difference: 0.06158-1.138) than mice with DMM. IPA further revealed that components of the Rho signaling pathways are regulated by cytokines and colchicine. IL-1ß and TNF-α activate RAC1 and SRC signals, respectively, leading to the phosphorylation of PLC-γ1 and synergistic induction of MMP13 expression. Most notably, colchicine abrogates inflammatory cytokine-induced phosphorylation of PLC-γ1, leading to the induction of MMP13 expression. CONCLUSIONS: Colchicine is a potential DMOAD candidate that inhibits MMP13 expression and consequent cartilage degradation by disrupting the SRC/RAC1-phospho-PLCγ1-Ca2+ signaling pathway.

Heparan sulfate deficiency leads to hypertrophic chondrocytes by increasing bone morphogenetic protein signaling.

OBJECTIVE: Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext-/- mouse model. METHOD: We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice. RESULTS: Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007). CONCLUSIONS: HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.

An analysis of osteoporosis in patients with hereditary multiple exostoses.

We analyzed osteoporosis in 20 HME patients. According to the T-score of BMD, 30% and 67.5% of the patients fell in the range of osteopenia in the lumbar spine and femoral neck. Our results indicate HME patients have low bone mass. They do not have abnormal bone metabolism. INTRODUCTION: There are few reports of osteoporosis in hereditary multiple exostoses (HME) patients. Therefore, the purpose of this study was to analyze osteoporosis in HME patients. METHODS: This retrospective cohort study included 20 patients diagnosed with HME. Patients underwent bone mineral density (BMD) measurement of the lumbar spine (n = 20) and femoral neck (n = 40). Bone metabolic parameters, including serum osteocalcin and urinary cross-linked N-telopeptide of type 1 collagen (NTx), were analyzed in all subjects. EXT1 and EXT2 genes were sequenced using genomic DNA. We also examined the correlation between genotype and BMD Z-score and T-score. RESULTS: The mean BMD values of the lumbar spine were 1.085 ± 0.116 g/cm2 (n = 11) in male and 1.108 ± 0.088 g/cm2 (n = 9) in female. The mean BMD values of the femoral neck area were 0.759 ± 0.125 g/cm2 (n = 22) in male and 0.749 ± 0.115 g/cm2 (n = 18) in female. Z-score of most HME patients show < 0, indicating that these patients tend to have low bone mass compared with the age-matched population. According to the T-score of BMD, 30% (6 of 20) and 67.5% (27 of 40) of the patients fell in the range of osteopenia in the lumbar spine and femoral neck areas, respectively. Serum osteocalcin and urinary NTx were in the normal range in most patients. There was no significant correlation between genotypes and Z-score. CONCLUSION: HME patients have low bone mass, especially in the femoral neck area. They do not have abnormal bone metabolism, and there was no correlation between genotypes and Z-score.

Classification of osteonecrosis of the femoral head: Who should have surgery?

OBJECTIVES: Using a simple classification method, we aimed to estimate the collapse rate due to osteonecrosis of the femoral head (ONFH) in order to develop treatment guidelines for joint-preserving surgeries. METHODS: We retrospectively analyzed 505 hips from 310 patients (141 men, 169 women; mean age 45.5 years (sd 14.9; 15 to 86)) diagnosed with ONFH and classified them using the Japanese Investigation Committee (JIC) classification. The JIC system includes four visualized types based on the location and size of osteonecrotic lesions on weightbearing surfaces (types A, B, C1, and C2) and the stage of ONFH. The collapse rate due to ONFH was calculated using Kaplan-Meier survival analysis, with radiological collapse/arthroplasty as endpoints. RESULTS: Bilateral cases accounted for 390 hips, while unilateral cases accounted for 115. According to the JIC types, 21 hips were type A, 34 were type B, 173 were type C1, and 277 were type C2. At initial diagnosis, 238/505 hips (47.0%) had already collapsed. Further, the cumulative survival rate was analyzed in 212 precollapsed hips, and the two-year and five-year collapse rates were found to be 0% and 0%, 7.9% and 7.9%, 23.2% and 36.6%, and 57.8% and 84.8% for types A, B, C1, and C2, respectively. CONCLUSION: Type A ONFH needs no further treatment, but precollapse type C2 ONFH warrants immediate treatment with joint-preserving surgery. Considering the high collapse rate, our study results justify the importance of early diagnosis and intervention in asymptomatic patients with type C2 ONFH.Cite this article: Y. Kuroda, T. Tanaka, T. Miyagawa, T. Kawai, K. Goto, S. Tanaka, S. Matsuda, H. Akiyama. Classification of osteonecrosis of the femoral head: Who should have surgery?. Bone Joint Res 2019;8:451-458. DOI: 10.1302/2046-3758.810.BJR-2019-0022.R1.

Ir(iii) complex-based oxygen imaging of living cells and ocular fundus with a gated ICCD camera.

Phosphorescence lifetime imaging methods using oxygen-sensitive probes are very useful for visualizing the oxygen status of living cells and tissues with high spatial resolution. We aim to develop a useful oxygen detection technique combining a phosphorescent oxygen probe and an optimal detection method. Herein we present a biological oxygen imaging method using a microscope equipped with a gated intensified charge-coupled device (ICCD) camera as a detector and an Ir(iii) complex as a phosphorescent oxygen probe. Microscopic luminescence images of monolayer HT-29 cells (human colorectal adenocarcinoma cells) obtained using the cell-penetrating Ir(iii) complex BTPDM1 and an inverted microscope demonstrated that this method allowed visualization of the oxygen gradient produced in a monolayer of cultured cells when the monolayer is covered with a thin coverslip. Furthermore, combining the IR-emitting Ir(iii) complex DTTPH-PEG24 with a macrozoom microscope equipped with a gated ICCD camera enabled both the visualization of retinal vessels near the optic disc and the monitoring of oxygen level changes in a rabbit retina upon changing the inhaled oxygen content.

Sagittal alignment and mobility of the thoracolumbar spine are associated with radiographic progression of secondary hip osteoarthritis.

OBJECTIVE: To identify predictors of radiographic progression of hip osteoarthritis (OA) over 12 months among functional hip impairments and spinal alignment and mobility. DESIGN: Fifty female patients with secondary hip OA, excluding those with end-stage hip OA, participated in this prospective cohort study. Joint space width (JSW) of the hip was measured at baseline and 12 months later. With radiographic progression of hip OA over 12 months (>0.5 mm in JSW) as dependent variable, logistic regression analyses were performed to identify predictors for hip OA progression among functional impairments of the hip and spine with and without adjustment for age, body mass index (BMI), and minimum JSW at baseline. The independent variables were hip pain, Harris hip score (HHS), hip morphological parameters, hip passive range of motion (ROM) and muscle strength, and alignment and mobility of the thoracolumbar spine at baseline. RESULTS: Twenty-one (42.0%) patients demonstrated radiographic progression of hip OA. Multivariable logistic regression analysis showed that larger anterior inclination of the spine in standing position (adjusted OR [95% CI], 1.37 [1.04-1.80]; P = 0.028) and less thoracolumbar spine mobility (adjusted OR [95% CI], 0.96 [0.92-0.99]; P = 0.037) at baseline were statistically significantly associated with radiographic progression of hip OA, even after adjustment for age, BMI, and minimum JSW. CONCLUSIONS: The findings suggest that spinal alignment and mobility should be considered when assessing risk and designing preventive intervention for radiographic progression of secondary hip OA.

The prevention of a lateral hinge fracture as a complication of a medial opening wedge high tibial osteotomy: a case control study.

AIMS: We aimed to investigate factors related to the technique of medial opening wedge high tibial osteotomy which might predispose to the development of a lateral hinge fracture. PATIENTS AND METHODS: A total of 71 patients with 82 osteotomies were included in the study. Their mean age was 62.9 years (37 to 80). The classification of the type of osteotomy was based on whether it extended beyond the fibular head. The level of the osteotomy was classified according to the height of its endpoint. RESULTS: At a mean follow-up of 20 months (6 to 52), a total of 15 lateral hinge fractures (18.3%) were identified. A sufficient osteotomy, in which both anterior and posterior tibial cortices were involved with extension into the lateral aspect of the plateau in relation to an anteroposterior line tangential to the medial edge of the fibular head in the CT axial plane, was seen in 48 knees (71.6%) in those without a lateral hinge fracture and in seven (46.7%) in those with a lateral hinge fracture. An osteotomy which ended above the level of the fibular head was seen in nine (13.4%) of the knees without a lateral hinge fracture and seven (46.7%) of the those with a lateral hinge fracture. There was a significant relationship between the absence of a lateral hinge fracture and both a sufficient osteotomy and one whose endpoint was at the level of the fibular head (p = 0.0451 and p = 0.0214, respectively). CONCLUSION: A sufficient osteotomy involving both the anterior and posterior cortices, whose endpoint is at the level of the fibular head, should be performed when undertaking a medial opening wedge high tibial osteotomy if a lateral hinge fracture is to be avoided as a complication. Cite this article: Bone Joint J 2017;99-B:887-93.

Daily cumulative hip moment is associated with radiographic progression of secondary hip osteoarthritis.

OBJECTIVE: To investigate whether higher daily cumulative hip moment at baseline is associated with subsequent radiographic progression of hip osteoarthritis (OA) over 12 months. DESIGN: Fifty patients with secondary hip OA, excluding patients with end-stage hip OA, participated in this prospective cohort study. Joint space width (JSW) of the hip was measured at baseline and 12 months later. With radiographic progression of hip OA (>0.5 mm/year in JSW) as dependent variable (yes/no), univariable and multivariable logistic regression analyses were performed to assess the association between load-related parameters during gait (i.e., peak hip moment, hip moment impulse, and daily cumulative hip moment [product of hip moment impulse and mean steps/day]) and hip OA progression with and without adjustment for age, body weight, and minimum JSW. RESULTS: Of the 50 patients (47.4 ± 10.7 years old), 21 (42.0%) were classified into the progression group. The higher daily cumulative hip moment in the frontal plane at baseline was statistically significantly associated with radiographic progression of hip OA (adjusted odds ratio (OR) [95% confidence interval (CI)], 1.34 [1.06-1.70]; P = 0.013). The higher daily cumulative hip moment in the sagittal plane was also approaching significance in its association with hip OA progression (adjusted OR, 1.80 [0.99-3.26]; P = 0.052). CONCLUSIONS: In the female patients with secondary hip OA, higher daily cumulative hip moment, particularly in the frontal plane, was a predictor of radiographic progression of hip OA over 12 months. Reduction in daily cumulative hip moment by modification in gait and physical activity may potentially slow hip OA progression.

Evaluation of the safety and efficacy of Glycyrrhiza uralensis root extracts produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems.

Glycyrrhiza uralensis roots used in this study were produced using novel cultivation systems, including artificial hydroponics and artificial hydroponic-field hybrid cultivation. The equivalency between G. uralensis root extracts produced by hydroponics and/or hybrid cultivation and a commercial Glycyrrhiza crude drug were evaluated for both safety and efficacy, and there were no significant differences in terms of mutagenicity on the Ames tests. The levels of cadmium and mercury in both hydroponic roots and crude drugs were less than the limit of quantitation. Arsenic levels were lower in all hydroponic roots than in the crude drug, whereas mean lead levels in the crude drug were not significantly different from those in the hydroponically cultivated G. uralensis roots. Both hydroponic and hybrid-cultivated root extracts showed antiallergic activities against contact hypersensitivity that were similar to those of the crude drug extracts. These study results suggest that hydroponic and hybrid-cultivated roots are equivalent in safety and efficacy to those of commercial crude drugs. Further studies are necessary before the roots are applicable as replacements for the currently available commercial crude drugs produced from wild plant resources.

Systemic effects of surgical intervention on disease activity, daily function, and medication in patients with rheumatoid arthritis.

OBJECTIVES: Although tight control of rheumatoid arthritis (RA) has been achieved through the development of effective medication, surgical intervention is still required for a certain subpopulation of patients. To examine the systemic effects of orthopaedic surgery, we evaluated improvements in disease activity, daily function, and medication after surgery. METHOD: A prospective cohort study was conducted in 196 cases of elective orthopaedic surgery in 150 patients with RA from January 2011 to March 2014 in our institution. The 28-joint count Disease Activity Score based on erythrocyte sedimentation rate (DAS28-ESR) and modified Health Assessment Questionnaire (mHAQ) scores just before surgery and at 6 and 12 months after surgery were examined prospectively. Concomitant medications were also investigated. RESULTS: Significant improvement was seen in the DAS28-ESR and mHAQ scores for replacement surgery in both the upper and lower extremities, and for arthroplasty/arthrodesis in the upper extremities at the 12-month follow-up. Partial mHAQ scores for the lower extremities were significantly reduced in lower replacement surgery, and partial mHAQ scores for the upper extremities were significantly reduced in upper arthroplasty/arthrodesis surgery. Although the use of conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) did not decrease after surgery, the dose of prednisolone (PSL) decreased significantly at 12 months after surgery, especially in the well-controlled group and in surgical procedures in the lower extremities. CONCLUSIONS: Elective orthopaedic surgery improves both systemic disease activity and general functional impairment. Orthopaedic surgery is effective in reducing the amount of medication required postoperatively.

Fish collagen is an important panallergen in the Japanese population.

Collagen was identified as a fish allergen in early 2000s. Although its allergenic potential has been suggested to be low, risks associated with collagen as a fish allergen have not been evaluated to a greater extent. In this study, we aimed to clarify the importance of collagen as a fish allergen. Our results showed that 50% of Japanese patients with fish allergy had immunoglobulin E (IgE) against mackerel collagen, whereas 44% had IgE against mackerel parvalbumin. IgE inhibition assay revealed high cross-reactivity of mackerel collagen to 22 fish species (inhibition rates: 87-98%). Furthermore, a recently developed allergy test demonstrated that collagen triggered IgE cross-linking on mast cells. These data indicate that fish collagen is an important and very common panallergen in fish consumed in Japan. The high rate of individuals' collagen allergy may be attributable to the traditional Japanese custom of raw fish consumption.

Subcycle Optical Response Caused by a Terahertz Dressed State with Phase-Locked Wave Functions.

The coherent interaction of light with matter imprints the phase information of the light field on the wave function of the photon-dressed electronic state. A driving electric field, together with a stable phase that is associated with the optical probe pulses, enables the role of the dressed state in the optical response to be investigated. We observed optical absorption strengths modulated on a subcycle time scale in a GaAs quantum well in the presence of a multicycle terahertz driving pulse using a near-infrared probe pulse. The measurements were in good agreement with the analytical formula that accounts for the optical susceptibilities caused by the dressed state of the excitons, which indicates that the output probe intensity was coherently reshaped by the excitonic sideband emissions.

Periarticular osteoporosis of the forearm correlated with joint destruction and functional impairment in patients with rheumatoid arthritis.

UNLABELLED: The relationship between periarticular osteoporosis in the distal forearm and joint destruction or functional impairment in patients with rheumatoid arthritis (RA) is not sufficiently elucidated. From a single institutional cohort study, we found a strong correlation between periarticular forearm bone mineral density (BMD) and joint destruction or functional impairment. INTRODUCTION: This study was conducted to investigate (1) the difference between various periarticular regions of interest (ROIs) of BMD of the forearm, (2) the correlation between periarticular forearm BMD and joint destruction and physical function, (3) the independent variables for predicting BMD of the forearm, and (4) the forearm BMD of different ROIs in the early stage of RA. METHODS: We conducted a cross-sectional study in an RA cohort. Measurements included BMD of the distal forearm, joint destruction of the hands assessed by modified total Sharp score (mTSS), functional impairment assessed by a health assessment questionnaire (HAQ), and other clinical data. Variables affecting the forearm BMD values were analyzed by correlation and stepwise regression analyses. RESULTS: Of the 405 patients enrolled in the present study, 370 (average age; 62.9 years) were identified as having definite RA with a complete set of data. BMD in the distal end of the forearm (BMDud) was significantly reduced compared with that in the distal third of the forearm (BMD1/3). In a stepwise regression analysis, the mTSS in BMD1/3 was an independent predicting variable, while age and partial HAQ scores associated with the upper extremity were common independent variables in BMDud and BMD1/3. BMDud was significantly less than BMD1/3, even in patients with a short duration of the disease. BMD1/3 was significantly less in non-remission group compared with that in remission group in patients with a short duration of the disease. CONCLUSION: Periarticular BMD in the distal forearm is closely correlated with joint destruction and functional impairment in RA. Periarticular BMD in the distal forearm may be already reduced at the clinical manifestation of the disease.

Evaluation of the Glasgow Prognostic Score in patients receiving chemoradiotherapy for stage III and IV esophageal cancer.

High Glasgow Prognostic scores (GPSs) have been associated with poor outcomes in various tumors, but the values of GPS and modified GPS (mGPS) in patients with advanced esophageal cancer receiving chemoradiotherapy (CRT) has not yet been reported. We have evaluated these with respect to predicting responsiveness to CRT and long-term survival. Between January 2002 and December 2011, tumor responses in 142 esophageal cancer patients (131 men and 11 women) with stage III (A, B and C) and IV receiving CRT were assessed. We assessed the value of the GPS as a predictor of a response to definitive CRT and also as a prognostic indicator in patients with esophageal cancer receiving CRT. We found that independent predictors of CRT responsiveness were Eastern Cooperative Oncology Group (ECOG) performance status, GPS and cTNM stage. Independent prognostic factors were ECOG performance status and GPS for progression-free survival and ECOG performance status, GPS and cTNM stage IV for disease-specific survival. GPS may be a novel predictor of CRT responsiveness and a prognostic indicator for progression-free and disease-specific survival in patients with advanced esophageal cancer. However, a multicenter study as same regime with large number of patients will be needed to confirm these outcomes.

On the importance of cavity-length and heat dissipation in GaN-based vertical-cavity surface-emitting lasers.

Cavity-length dependence of the property of optically pumped GaN-based vertical-cavity surface-emitting lasers (VCSELs) with two dielectric distributed Bragg reflectors was investigated. The cavity lengths were well controlled by employing etching with inductively coupled plasma and chemical mechanical polishing. It was found that the lasing characteristics including threshold, slope efficiency and spontaneous emission coupling factor were substantially improved with reducing the cavity length. In comparison with the device pumped by a 400 nm pulsed laser, the lasing spectrum was featured by a red shift and simultaneous broadening with increasing the pumping energy of a 355 nm pulsed laser. Moreover, the lasing threshold was much higher when pumped by a 355 nm pulsed laser. These were explained by taking into account of the significant heating effect under 355 nm pumping. Our results demonstrate that a short cavity length and good heat-dissipation are essential to GaN-based VCSELs.

Functional performance of female patients more than 6 months after total hip arthroplasty shows greater improvement with weight-bearing exercise than with non-weight-bearing exercise. Randomized controlled trial.

BACKGROUND: Impaired functional performance and decreased muscle strength and muscle atrophy generally persist for a long period after total hip arthroplasty (THA). AIM: To investigate the effects of weight-bearing (WB) and non-weight-bearing (NWB) exercises on functional performance, Harris hip score (HHS), muscle strength and muscle thickness in women after THA. DESIGN: A randomised, controlled trial. SETTING: Community. POPULATION: Sixty-five women who had undergone unilateral or bilateral THA at least 6 months before enrolment in the study. METHODS: Participants were randomly allocated to the following groups: WB (n = 22), NWB (n = 21) and control (n = 22) groups. Participants in the WB and NWB groups performed daily home exercise programs for 8 weeks. Functional performance (timed up and go, sit-to-stand, stair climbing, walking speed and 3-min walk test), HHS, isometric muscle strength of the hip and knee muscle and gluteus and quadriceps muscle thickness were measured at baseline and after 8 weeks. RESULTS: An intention-to-treat analysis revealed that both the intervention groups exhibited significant improvements in almost all functional performance measures compared with the control group. Furthermore, the WB group showed significantly greater pre-post changes in the sit-to-stand and 3-min walk test compared with the NWB group. In terms of HHS, only the WB group showed significantly greater improvement compared with the control group. Relative to the control group, improvements in all isometric strength measures were observed in both the intervention groups. Significant improvement in quadriceps muscle thickness was observed in the WB group compared with the control group. However, no significant difference was observed among the three groups in pre-post changes in gluteus muscle thickness. CONCLUSION: WB and NWB home exercise programs were both effective for improving functional performance and muscle strength in women after THA. However, the WB exercise was more effective than the NWB exercise for improving sit-to-stand ability and walking endurance. CLINICAL REHABILITATION IMPACT: In this study, we demonstrated that the WB exercise was more effective than the NWB exercise for improving functional performance in patients after THA.

Elevated receptor for activated C kinase 1 expression is involved in intracellular Ca2+ influx and potentially associated with compromised regulatory T cell function in patients with asthma.

BACKGROUND: Regulatory T cells (T(regs)) are activated during anergy in response to T cell receptor (TCR) activation and functional immune suppression. Anergy of paediatric T(regs) is partially dependent on intracellular calcium mobility; following TCR activation, T(regs) do not exhibit increased intracellular Ca(2+) concentration ([Ca(2+) ](i)). OBJECTIVE: We determined whether [Ca(2+) ](i) in adult T(regs) defined their anergy, if intracellular Ca(2+) movement was linked to regulatory functions, whether [Ca(2+)](i) was indicative of asthma pathology, and the potential molecular mechanism responsible for Ca(2+) movement in T(regs). METHODS: T(regs) were purified by the magnetic bead method, and their regulatory functions were assessed by monitoring carboxyfluorescein succinimidyl ester-labelled responder T cell proliferation. The Ca(2+) response of Fura-2-labelled cells was measured using a video image analysis system. To analyse the functions of T(regs) at the molecular level, we generated Jurkat Tet-On(®) clones with doxycycline (Dox)-induced forkhead box P3 (FOXP3) protein expression. RESULTS: CD4(+) CD25(+) CD127(-/low) T(regs) from participants without asthma did not elicit Ca(2+) influx in response to TCR activation, exhibited little proliferation and suppressed proliferation of CD4(+) CD25(-) T cells. In contrast, under similar conditions, T(regs) from patients with asthma exhibited increased [Ca(2+)](i) and robust proliferation with partial loss of regulatory functions. FOXP3 protein levels in Tet-On(®) clones were high after both 2- and 5-day Dox treatment; however, 5-day cells were comparable with T(regs) from patients with asthma, whereas 2-day cells were similar to T(regs) from participants without asthma. Increasing [Ca(2+)](i) induced a high level of receptor for activated C kinase 1 (RACK1) expression in 5-day cells. CONCLUSIONS AND CLINICAL RELEVANCE: We confirmed that T(regs) in patients with asthma are functionally impaired and that the abnormal regulatory functions of these cells can be analysed by [Ca(2+)](i) following TCR engagement. Furthermore, the impaired functioning of T(regs) evident in patients with asthma may be due to a high level of RACK1.

Prognostic significance of CD44 variant 2 upregulation in colorectal cancer.

BACKGROUND: CD133 and CD44 are putative cancer stem cell (CSC) markers in colorectal cancer (CRC). However, their clinical significance is currently unclear. Here, we evaluated primary CRC cell isolates to determine the significance of several CSC markers, including CD133 and CD44, as predictors of tumourigenesis and prognosis. METHODS: CD133- and CD44-positive cells from fresh clinical samples of 77 CRCs were selected by flow cytometric sorting and evaluated for tumourigenicity following subcutaneous transplantation into NOD/SCID mice. Cancer stem cell marker expression was examined in both xenografts and a complementary DNA library compiled from 167 CRC patient samples. RESULTS: CD44(+), CD133(+) and CD133(+)CD44(+) sub-populations were significantly more tumourigenic than the total cell population. The clinical samples expressed several transcript variants of CD44. Variant 2 was specifically overexpressed in both primary tumours and xenografts in comparison with the normal mucosa. A prognostic assay using qRT-PCR showed that the CD44v2(high) group (n=84, 5-year survival rate (5-OS): 0.74) had a significantly worse prognosis (P=0.041) than the CD44v2(low) group (n=83, 5-OS: 0.88). CONCLUSIONS: CD44 is an important CSC marker in CRC patients. Furthermore, CRC patients with high expression of CD44v2 have a poorer prognosis than patients with other CD44 variants.