Relative Telomere Length in Blood Leukocytes of Patients with Anterior Cruciate Ligament Injury: A Pilot Study.

Introduction: Anterior cruciate ligament (ACL) tear is the most common knee ligament injury, especially in athletes. The objective of this study was to investigate relative telomere length (RTL) in blood leukocytes of patients with ACL injury compared with that of controls. Materials and Methods: A total of 187 subjects were invited to participate in this study. Ninety-two patients with clinically diagnosed ACL rupture were enrolled. Ninety-five age and gender-matched healthy controls were also recruited. Blood leukocyte RTL were analysed using quantitative real-time polymerase chain reaction. Results: Patients with ACL rupture had significantly longer relative telomere length than healthy controls (P=0.002). The patients with ACL rupture were classified into two groups according to the sport history of patients which are contact sports and non-contact sports. RTL in patients with non-contact sports was significantly greater than those with contact sports (P=0.006). Moreover, RTL was inversely correlated with body mass index of patients with ACL injury (r=-0.34, P=0.001). Logistic regression analysis indicated that long RTL was associated with a higher risk of ACL rupture. Conclusion: The present study showed that subjects with ACL rupture had significantly greater telomere length compared with their age and gender-matched controls. This finding may result from the increases in physical activity and overexpression of telomerase which acts as a protective mechanism against ACL injury. RTL in blood leukocytes is associated with a risk of ACL rupture.

Relative Telomere Length in Blood Leukocytes of Patients with Anterior Cruciate Ligament Injury: A Pilot Study

@#Introduction: Anterior cruciate ligament (ACL) tear is the most common knee ligament injury, especially in athletes. The objective of this study was to investigate relative telomere length (RTL) in blood leukocytes of patients with ACL injury compared with that of controls. Materials and Methods: A total of 187 subjects were invited to participate in this study. Ninety-two patients with clinically diagnosed ACL rupture were enrolled. Ninety-five age and gender-matched healthy controls were also recruited. Blood leukocyte RTL were analysed using quantitative real-time polymerase chain reaction. Results:Patients with ACL rupture had significantly longer relative telomere length than healthy controls (P=0.002). The patients with ACL rupture were classified into two groups according to the sport history of patients which are contact sports and non-contact sports. RTL in patients with non-contact sports was significantly greater than those with contact sports (P=0.006). Moreover, RTL was inversely correlated with body mass index of patients with ACL injury (r=-0.34, P=0.001). Logistic regression analysis indicated that long RTL was associated with a higher risk of ACL rupture. Conclusion: The present study showed that subjects with ACL rupture had significantly greater telomere length compared with their age and gender-matched controls. This finding may result from the increases in physical activity and overexpression of telomerase which acts as a protective mechanism against ACL injury. RTL in blood leukocytes is associated with a risk of ACL rupture.

Assessment of chondrogenic differentiation potential of autologous activated peripheral blood stem cells on human early osteoarthritic cancellous tibial bone scaffold.

INTRODUCTION: Current therapeutic regimens in osteoarthritis (OA) address mainly pain but not the slow progressive degradation of the extracellular matrix (ECM) and the loss of a chondrogenic phenotype in articular cartilage. In the present study, using an early OA cancellous bone scaffold, we aimed to uncover evidence of the successful hyaline cartilage regenerative capacity of autologous human granulocyte colony-stimulating factor (hG-CSF)-activated peripheral blood stem cells (AAPBSC) with growth factor addition. MATERIALS AND METHODS: AAPBSC were harvested in ten patients (median age 58 years, 8 females), and flow cytometry was performed for cell surface markers. Arthroscopically obtained cancellous bone scaffold specimens were seeded with AAPBSC. In Group 1, the scaffold was seeded with AAPBSC only, in Group 2, AAPBSC plus hyaluronic acid (HA), and in Group 3, AAPBSC plus HA, hG-CSF, and double-centrifuged platelet-rich plasma (PRP). The specimens were analyzed for cell attachment and proliferation by the fluorometric quantification of cellular DNA assay and scanning electron microscopy. Chondrogenic gene expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) of Sox9, collagen type II (COL-2), and aggrecan. Histological sections of scaffold constructs for cartilaginous matrix formation were stained with toluidine blue (proteoglycan) and safranin O (sGAG) after 3 weeks. RESULTS: AAPBSC displayed especially high levels of CD29 and CD44 surface markers, as well as CD90, and CD105, while only a small proportion expressed CD34. Almost half of the seeded cells attached on the bone scaffolds in all three groups (not statistically significant), whereas the means of cell proliferation on day 7 compared to day 1 were statistically significant difference with the order of increase as group 3 > group 2 > group 1. RT-PCR showed statistically significant sequential increases in Sox9, COL-2, and Aggrecan all being highest in group 3. Histological analysis demonstrated cells in the cancellous bone scaffold with a round morphology, and ECM was positively stained by toluidine blue and safranin O indicating increased proteoglycan and glycosaminoglycan content, respectively, in the newly formed cartilage matrix. CONCLUSIONS: AAPBSC initiated chondrocyte differentiation on an autologous cancellous bone scaffold, and the addition of PRP and hG-CSF further stimulated cell proliferation toward a chondrocyte phenotype with potentiated Sox9 transcription resulting in sequential COL-2 and aggrecan mRNA increases that ultimately resulted in histologically confirmed increased proteoglycan and glucosaminoglycan content in newly formed hyaline cartilage.

ADAMTS14 gene polymorphism associated with knee osteoarthritis in Thai women.

Osteoarthritis (OA) is the most prevalent form of arthritis in the elderly. This disease is characterized by breakdown and loss of articular cartilage due to genetic, mechanical and environmental factors. Although the pathophysiology of OA is not completely known, several candidate genes have been reported to be associated with OA susceptibility. We assessed the association between genetic variation in the ADAMTS14 region and knee osteoarthritis susceptibility in the Thai population. The rs4747096 SNP was genotyped by PCR-RFLP on genomic DNA extracted from peripheral blood of 108 OA patients and 119 controls. The PCR product (196 bp) was digested with BspEI. A sample with the GG genotype showed two band sizes of 158 and 38 bp, while a sample with the AA genotype showed a single band size of 196 bp. Heterozygotes with the AG genotype showed all three corresponding bands. Genotype distributions, allele frequencies and model of inheritance in patients and controls were compared. In females, the frequency of the AA genotype and the A allele were significantly higher in knee OA patients than in controls [odds ratio (OR) = 2.79, 95% confidence interval (CI) = 1.05-7.59 and OR = 1.58, 95%CI = 1.00-2.45, respectively]. Moreover, genotypic AA and AG were associated with significantly increased risk for knee OA when compared to GG (OR = 2.72, 95%CI = 1.10-6.87). No significant associations were observed in males. In conclusion, the nsSNP rs4747096 in ADAMTS14 was associated with knee OA in female Thai patients; therefore, the role of ADAMTS14 in OA seems to be gender-dependent.

Elevated serum IL-18 and interferon-gamma in medium-term survivors of biliary atresia.

INTRODUCTION: Biliary atresia (BA) is a fatal disease in children. Its main pathological feature is progressive immune-mediated cholangiopathy. Interleukin (IL)-12, IL-18, and interferon-gamma (IFN-gamma) play important roles in various immunological diseases. THE OBJECTIVE: was to investigate whether these serum markers were associated with clinical outcome in BA. METHODS: Serum levels of IL-12, IL-18, and IFN-gamma were determined using enzyme-linked immunosorbent assay from 46 BA patients (median age of 9 years) and 19 normal controls. The BA patients were then categorized into three groups according to their outcome: jaundice-free (29 cases), mild to moderate jaundice (10 cases), and marked jaundice (7 cases). The comparisons of serum IL-12, IL-18, and IFN-gamma levels among groups of the patients were performed using one-way analysis of variance with post-hoc tests. Data are expressed as mean + standard deviation. RESULTS: Serum IL-18 and IFN-gamma in BA patients were higher than the normal controls (IL-18: 113.3 + 82.6 vs. 80.5 + 9.9 pg/mL, p = 0.011 and IFN-gamma: 41.7 + 5.1 vs. 38.0 + 1.9 pg/mL, p < 0.001). There was no difference in serum IL-12 between BA and controls. Further analysis demonstrated that, in BA patients, only serum IL-18 levels significantly increased with the degree of jaundice (test for trend, p = 0.004). CONCLUSIONS: Serum IL-18 and IFN-gamma levels were increased in medium-term survivors of BA. The elevated serum IL-18 in BA patients was associated with worse clinical outcome. These results suggest that IL-18 and IFN-gamma play roles in the pathophysiology of BA. Additionally, IL-18 is likely to be involved in the disease progression.

Association between matrix metalloproteinase-3 polymorphism and anterior cruciate ligament ruptures.

Anterior cruciate ligament (ACL) ruptures are considered to be the most severe joint injury in sports. However, the precise etiologies of ACL injuries are not fully understood. Recently, the gene encoding the matrix metalloproteinase-3 (MMP-3, stromelysin-1) was shown to be associated with anterior cruciate ligament ruptures. The 5A/6A polymorphism in the promoter of the MMP-3 gene affects the regulation of MMP-3 gene expression. We examined the association between polymorphism within -1612 of the MMP-3 gene and ACL rupture in an independent population. Eighty-six participants between 20 and 40 years of age with surgically diagnosed ACL ruptures and 100 healthy controls between 18 and 28 years of age without history of ligament or tendon injuries were recruited for the study. All participants were genotyped for the MMP-3 polymorphism (-1612 5A/6A). Statistical analyses of genotype frequencies between patients and healthy controls were performed by the chi-square test. A significant difference was found between ACL rupture subgroups in terms of genotype association (5A+ (5A/5A, 5A/6A): 37.5% in contact sports vs 20% in non-contact sports; P = 0.02). In allelic association, there were significant differences (6A: 81.2% in contact sports vs 89.1% in non-contact sports, 5A: 18.8% in contact sports vs 10.9% in non-contact sports, P = 0.01). The 5A+ genotype of MMP-3 was represented in ACL ruptures in contact sport participants. We propose that this sequence variant is a specific genetic element that should be included in a multifactorial model to understand the etiologies and risk factors for ACL rupture.

Gene expression analysis of demineralized bone matrix-induced osteogenesis in human periosteal cells using cDNA array technology.

Demineralized bone matrix (DBM) has been widely investigated as a biomaterial to promote new bone formation and is utilized clinically for bone repair and regeneration. We investigated gene expression patterns of osteogenic differentiation in human periosteal (HPO) cells cultured with demineralized bone matrix, using cDNA array technology. Osteogenic differentiation of HPO cells was determined using alkaline phosphatase assay. In order to examine differential gene expression during osteogenic differentiation, total RNA was isolated from HPO cells in the absence or presence of DBM on day seven and analyzed using osteogenesis cDNA gene array. The selected genes were verified using reverse transcriptase (RT)-PCR analysis. Human periosteal cells differentiated along an osteogenic lineage after treatment of DBM. The alkaline phosphatase activity assay showed that HPO cells differentiated into an osteogenic lineage. Gene expression of HPO cells treated with DBM for seven days was analyzed with cDNA array and RT-PCR analyses. Expression of biglycan, TGF-ß1, and TGF-ßR1 was upregulated, whereas collagen14A1 expression was downregulated, as confirmed by RT-PCR. Human periosteal cells expressed osteogenesis genes when treated with DBM. These findings provide new insight into the capability of demineralized bone matrix to modulate the osteogenic differentiation of human periosteal cells.

Association of the IL-6 -174G/C gene polymorphism with knee osteoarthritis in a Thai population.

Osteoarthritis is a chronic progressive degenerative joint disease characterized by age-related regressive change in articular cartilage. A single nucleotide polymorphism has been described at position -174 of the interleukin-6 (IL-6) promoter region, leading to three possible genotypes, GG, GC, and CC. We investigated a possible association of the IL-6 -174G/C gene polymorphism with knee osteoarthritis in a Thai population. Genotype distributions and allelic frequencies of the IL-6 -174G/C polymorphism were investigated in 115 knee osteoarthritis patients and 100 healthy controls. Genotyping was performed using PCR-RFLP. The genotype distribution of IL-6 was 79 GG, 36 GC, 0 CC in knee osteoarthritis patients and 88 GG, 12 GC, 0 CC in controls. The frequency of the GC genotype in subjects with knee osteoarthritis was higher than in controls (P< 0.001). Logistic regression analysis showed that the GC genotype was independently associated with increased risk of knee osteoarthritis (odds ratio = 3.3, 95% confidence interval = 1.6-6.9, P = 0.001). These findings suggest that the -174G/C polymorphism of the IL-6 gene promoter plays a role in the pathogenesis of knee osteoarthritis.

Inflammation related to synovectomy during total knee replacement in patients with primary osteoarthritis: a prospective, randomised study.

We compared inflammation in the knee after total knee replacement (TKR) for primary osteoarthritis between two groups of patients undergoing joint replacement with and without synovectomy. A total of 67 patients who underwent unilateral TKR were randomly divided into group I, TKR without synovectomy, and group II, TKR with synovectomy. Clinical outcomes, serial serum inflammatory markers (including interleukin-6 (IL-6), CRP and ESR) and the difference in temperature of the skin of the knee, compared with the contralateral side, were sequentially evaluated until 26 weeks after surgery. Pre-operatively, there were no statistically different clinical parameters between groups I and II. At the 26-week follow-up, both groups had a similarly significantly improved American Knee Society clinical score (p < 0.001) and functional score (p < 0.001) with no differences between the groups. Similar changes in serial inflammatory markers were found in both groups, including mean peak levels of IL-6 (189 pg/ml (SD 53.4) versus 201 pg/ml (SD 49.4) for groups I and II, respectively) and CRP (91 mg/L (SD 24.1) versus 88 mg/L (SD 23.4), respectively) on the first post-operative day, returning to pre-operative values at two and six weeks, respectively. The mean peak level of ESR for the respective two groups was 46 mm/hr versus 48 mm/hr at two weeks, which had still not returned to its pre-operative mean value at 26 weeks. The elevation in the skin temperature appeared to mirror the peak elevation of the ESR, with a range of 2.5° C to 4.5° C with some reduction at 26 weeks but still exceeding the pre-operative value. We concluded that synovectomy at the time of TKR does not provide any benefit to the clinical outcome or shorten the duration of the inflammatory response after surgery.

Elevation of serum galectin-3 and liver stiffness measured by transient elastography in biliary atresia.

BACKGROUND AND AIM: Biliary atresia (BA) is an intractable neonatal liver disease characterized by progressive fibrosclerotic obliteration of the extrahepatic biliary tree. The aim of this study was to evaluate serum galectin-3 in postoperative BA patients and the association between galectin-3, clinical outcome and liver stiffness score. METHODS: 58 BA patients post Kasai operation and 20 controls were enrolled. None of the patients had undergone liver transplantation. BA patients were classified into 2 groups according to their serum total bilirubin (TB) levels (TB<2 mg/dL, no jaundice vs. TB≥2 mg/dL, persistent jaundice) and alanine aminotransferase (ALT) levels (ALT<45 IU/L, normal ALT vs. ALT≥45 IU/L, elevated ALT). Serum galectin-3 levels were determined by enzyme-linked immunosorbent assay. Liver stiffness scores were measured by transient elastography (FibroScan). RESULTS: BA patients had higher serum galectin-3 levels (5.1±0.3 vs. 3.8±0.4 ng/mL, p=0.01) and greater liver stiffness values than healthy controls (29.7±3.0 vs. 5.1±0.5 kPa, p<0.001). Serum galectin-3 levels were markedly elevated in BA patients with jaundice compared to those without jaundice (6.4±0.5 vs. 4.4±0.3 ng/mL, p=0.001). Furthermore, BA patients with elevated ALT displayed significantly higher levels of serum galectin-3 than those with normal ALT (5.9±0.4 vs. 3.8±0.3 ng/mL, p=0.001). Additionally, BA patients with portal hypertension had considerably higher serum galectin-3 levels than those without portal hypertension (6.1±0.4 vs. 3.7±0.3 ng/mL, p<0.001). CONCLUSIONS: Increased serum galectin-3 is associated with a poor outcome in postoperative BA patients. Serum galectin-3 could be used as a biochemical parameter reflecting the deterioration of liver function and the severity of liver fibrosis in postoperative BA.

Correlation of circulating endoglin with clinical outcome in biliary atresia.

BACKGROUND AND AIM: Biliary atresia (BA) is a chronic progressive inflammatory disorder of the extrahepatic and intrahepatic biliary system in children. The aim of the present study was to investigate circulating endoglin levels in BA patients compared with healthy controls and to determine the relationship between plasma endoglin levels and outcome parameters of BA patients after Kasai operation. METHODS: Fifty-five postoperative BA patients and 14 healthy controls were recruited. The patients were divided into two groups based on their serum total bilirubin levels (TB<34.2, no jaundice vs. TB>or=34.2 micromol/L, persistent jaundice) and serum alanine aminotransferase (ALT<45, normal ALT vs. ALT>or=45 IU/L, high ALT). Circulating endoglin levels were analyzed by enzyme-linked immunosorbent assay. RESULTS: Average levels of plasma endoglin were significantly higher in BA patients compared to healthy controls (7.8+/-0.4 vs. 6.5+/-0.4 ng/mL; P=0.02). BA patients with persistent jaundice had higher plasma endoglin levels than those without jaundice (9.2+/-0.8 vs. 6.9+/-0.3 ng/mL; P=0.006). Furthermore, the concentrations of plasma endoglin in BA patients with high ALT were significantly higher compared to those with normal ALT (8.5+/-0.5 vs. 6.3+/-0.5 ng/mL, P=0.003). In addition, BA patients with portal hypertension had more elevated plasma endoglin levels than those without portal hypertension (8.8+/-0.6 vs. 6.1+/-0.3 ng/mL, P=0.001). Plasma endoglin was positively correlated with serum ALT (r=0.36, P=0.007) and serum GGT (r=0.44, P=0.001). CONCLUSION: High circulating endoglin correlated with a poor outcome for BA. Plasma endoglin can be utilized as a potential biomarker reflecting the severity of ongoing liver injury and biliary obstruction in BA patients after Kasai procedure.

Cyclooxygenase-2 overexpression is associated with clinical outcome in biliary atresia.

OBJECTIVE: The purpose of this study was to investigate the association between cyclooxygenase-2 (COX-2) expression and clinical outcome in biliary atresia (BA) patients. METHODS: Six months after surgery, twenty-eight BA patients were divided into three groups according to their liver function tests: group A with satisfactory liver function (n=11), group B with moderate liver dysfunction (n=8), and group C with severe liver dysfunction (n=9). COX-2 expression was determined by immunohistochemistry. Choledochal cysts (n=5) and normal liver samples (n=4) served as controls. RESULTS: Our data have shown that the intrahepatic biliary epithelium in BA specimens expressed COX-2. The mean immunoreactive score of COX-2 in BA patients was significantly higher than that in choledochal cyst and normal liver (4.0+/-0.6, 0.9+/-0.3, and 0.7+/-0.3, respectively, p<0.002). Strong expression of COX-2 was observed in BA patients with severe liver dysfunction. Subgroup analysis showed that the mean COX-2 immunoreactive scores of patients in group A, B, and C were 2.1+/-0.6, 3.6+/-1.1, and 5.9+/-0.9, respectively. The COX-2 immunoreactive score in BA patients with severe liver dysfunction was higher than in patients with satisfactory liver function (p<0.005). CONCLUSION: Increased COX-2 expression of biliary epithelial cells at the time of Kasai operation was associated with an adverse therapeutic outcome in BA, suggesting that COX-2 could play a plausible role in the liver pathology of BA.

Elevated serum bone morphogenetic protein 7 levels and clinical outcome in children with biliary atresia.

BACKGROUND AND AIM: Biliary atresia (BA) is one of the most serious liver disorders in children. The purposes of the present study were to investigate serum levels of bone morphogenetic protein 7 (BMP7) in BA children compared with healthy controls and to evaluate the association between serum BMP7 and the clinical outcome of BA patients post Kasai operation. METHODS: Sixty-two BA patients post Kasai operation and 14 healthy controls were enrolled. The patients were divided into two groups according to their serum total bilirubin levels (TB<2, no jaundice vs. TB> or =2 mg/dL, persistent jaundice) and alanine aminotransferase levels (ALT<45, normal ALT vs. ALT> or =45 IU/L, elevated ALT). Serum BMP7 levels were determined by commercial enzyme-linked immunoabsorbent assay. RESULTS: The mean serum BMP7 was higher in BA patients compared with that of healthy controls (35.4+/-3.6 vs. 20.6+/-2.7 pg/mL, p=0.002). The BA patients with persistent jaundice had more elevated serum BMP7 levels than those without jaundice (59.5+/-6.5 vs. 20.3+/-1.6 pg/mL, p=0.001). There was also a correlation between serum total bilirubin and serum BMP7 levels (r=0.57, p<0.001). Moreover, the levels of serum BMP7 in BA patients with elevated ALT were significantly higher than those with normal ALT (41.6+/-4.7 vs. 22.4+/-4.2 pg/mL, p=0.003). Additionally, BA patients with portal hypertension had higher increased serum BMP7 levels compared to those without portal hypertension (45.3+/-4.9 vs. 18.7+/-2.8 pg/mL, p<0.001). CONCLUSION: The significant increment of serum BMP7 was associated with a deterioration of hepatic function and the progression of liver fibrosis. Serum BMP7 could be used as a prognostic marker to reflect disease severity and monitor disease progression in BA patients post Kasai operation.

Indolinone tyrosine kinase inhibitors block Kit activation and growth of small cell lung cancer cells.

Six indolinone tyrosine kinase inhibitors were characterized for their ability to inhibit Kit kinase and for their effects on the growth of small cell lung cancer (SCLC) cell lines. All of the six compounds were potent inhibitors of Kit kinase in a biochemical assay. A homology model of compound binding to the ATP binding site could account for the increased potency observed with the addition of a propionate moiety to the indolinone core but not the increase observed with addition of a chloride moiety. Although all of the compounds tested were potent in the biochemical assay, several exhibited significantly less potency in cellular kinase assays. Their effects on stem cell factor (SCF)-dependent Kit autophosphorylation and SCLC cell growth were also examined. Inhibition of SCF-stimulated Kit activation and cell growth in the H526 cell line was dose-dependent. At concentrations that inhibited SCF-stimulated H526 cell growth, there was little effect on insulin-like growth factor-1-stimulated growth, suggesting that these compounds exhibit reasonable selectivity for inhibition of Kit-mediated proliferation. Higher doses of the compounds were needed to inhibit serum-stimulated growth. Of the six compounds examined, SU5416 and SU6597 demonstrated the best cellular potency and, therefore, their effect on the growth of multiple SCLC cell lines in serum-containing media was examined. In addition to inhibiting proliferation, these compounds also induced significant cell death of several SCLC cell lines, but not of normal human diploid fibroblasts, in complete media. These observations suggest that Kit kinase inhibitors such as these may offer a new approach for inhibiting Kit-mediated proliferation of tumors such as SCLC, gastrointestinal stromal tumors, seminomas, and leukemias.

The selective tyrosine kinase inhibitor STI571 inhibits small cell lung cancer growth.

At least 70% of small cell lung cancers express the Kit receptor tyrosine kinase and its ligand, stem cell factor (SCF). Numerous lines of evidence have demonstrated that this coexpression constitutes a functional autocrine loop, suggesting that inhibitors of Kit tyrosine kinase activity could have therapeutic efficacy in this disease. STI571, formerly known as CGP 57148B, is a p.o. bioavailable 2-phenylaminopyrimide derivative that was designed as an Abl tyrosine kinase inhibitor, but also has efficacy against the platelet-derived growth factor receptor and Kit in vitro. Pretreatment of the H526 small cell lung cancer (SCLC) cell line with STI571 inhibited SCF-mediated Kit activation with an IC50 of 0.1 microM as measured by inhibition of receptor tyrosine phosphorylation and 0.2 microM as measured by immune complex kinase assay. This paralleled the inhibition of SCF-mediated growth by STI571, which had an IC50 of approximately 0.3 microM. Growth inhibition in SCF-containing medium was accompanied by induction of apoptosis. STI571 efficiently blocked SCF-mediated activation of mitogen-activated protein kinase and Akt, but did not affect insulin-like growth factor-1 or serum-mediated mitogen-activated protein kinase or Akt activation. Growth of five of six SCLC cell lines in medium containing 10% FCS was inhibited by STI571 with an IC50 of approximately 5 microM. Growth inhibition in serum-containing medium appeared to be cytostatic in nature because no increase in apoptosis was observed. Despite this growth inhibition, STI571 failed to enhance the cytotoxicity of either carboplatinum or etoposide when coadministered. However, taken together with the minimal toxicity that this compound has shown in preclinical studies, these data suggest that STI571 could have a role in the treatment of SCLC, possibly to block or slow recurrence after chemotherapy-induced remissions.