Delayed viral suppression during antiviral therapy is associated with increased hepatocellular carcinoma rates in HBeAg-positive high viral load chronic hepatitis B.

The treatment option in chronic hepatitis B (CHB) patients with persistent low-level viremia despite entecavir or tenofovir monotherapy is unclear. This study investigated the development of hepatocellular carcinoma (HCC) or cirrhosis in hepatitis B e antigen (HBeAg)-positive high viral load CHB patients, according to the time needed to achieve complete viral suppression. A total of 325 HBeAg-positive CHB patients with high viral load who were recently started on antiviral therapy with entecavir or tenofovir were included. The enrolled patients were divided into 2 groups with 4 separate criteria based on the time needed to achieve complete viral suppression: within 1, 2, 3 or 4 years of therapy initiation. The outcomes were development of HCC and cirrhosis. The cumulative incidence of HCC was significantly higher in patients failing complete viral suppression within 1 year (hazard ratio (HR), 4.54; 95% confidence interval (CI), 1.03-19.93; P = .045) or 2 years (HR, 3.38; 95% CI, 1.24-9.23; P = .018), than patients who achieved complete viral suppression within 1 or 2 years, respectively. Cumulative incidence of cirrhosis was also significantly higher in patients failing suppression within 1 year (HR, 1.95; 95% CI, 1.04-3.66; P = .037) or 2 years (HR, 2.44; 95% CI, 1.41-4.22; P = .001). When the time for achieving viral suppression exceeded 2 years, the cumulative incidence of HCC or cirrhosis was not different regardless of viral suppression. Complete hepatitis B virus suppression within 2 years of antiviral therapy initiation is associated with risk reduction in HCC or cirrhosis development.

Entecavir and tenofovir reduce hepatitis B virus-related hepatocellular carcinoma recurrence more effectively than other antivirals.

Nucleos(t)ide analogues (NAs) have been shown to decrease the risk of hepatocellular carcinoma (HCC) recurrence. This study evaluated whether high-potency NAs (entecavir and tenofovir disoproxil fumarate [TDF]) reduce the risk of tumour recurrence more potently than low-potency NAs after curative treatment of hepatitis B virus (HBV)-related HCC. This study included 607 consecutive HBV-related HCC patients treated with surgical resection or radiofrequency ablation. The patients were categorized into three groups according to antiviral treatment: group A (no antiviral; n = 261), group B (low-potency NA; n = 90) and group C (high-potency NA; n = 256). The primary end-point was recurrence-free survival (RFS). During the duration of follow-up, the median RFS was 29.4, 25.1, and 88.2 months in groups A, B and C, respectively (P < .001, log-rank test). The multivariate Cox analysis indicated that group C had a significantly longer RFS than both group A (adjusted hazard ratio [HR] = 0.39, P < .001) and group B (adjusted HR = 0.47, P < .001). When baseline characteristics were balanced using inverse probability weighting, group C still had a significantly longer RFS than group A (adjusted HR = 0.46, P < .001) and group B (adjusted HR = 0.59, P = .007). Group C had significantly lower risk of viral breakthrough than group B (HR = 0.19, P < .001). Viral breakthrough was an independent risk factor for shorter RFS among groups B and C (adjusted HR = 2.03, P = .007, time-dependent Cox analysis). Antiviral agents with high genetic barrier to resistance (entecavir and TDF) reduced the risk of HCC recurrence compared with other antivirals and no antiviral treatment, especially in patients with high baseline viral load.

Comparison of overall survival between antiviral-induced viral suppression and inactive phase chronic hepatitis B patients.

Nucleot(s)ide analogues (NAs) reduce the risk of hepatocellular carcinoma (HCC) in chronic hepatitis B (CHB) patients. However, the risk of HCC is reportedly higher for NA-treated patients than for patients in the inactive CHB phase. This study aimed to compare the long-term outcomes of CHB patients with NA-induced viral suppression and those of patients with inactive CHB. This retrospective study involved 1118 consecutive CHB patients whose HBV DNA level was continuously <2000 IU/mL during follow-up with/without antiviral agents. The patients were classified into inactive CHB (n = 373) or NA groups (n = 745). The primary endpoint was overall survival. Secondary endpoints included development of HCC and other liver-related events. The median duration of follow-up was 41.0 (interquartile range = 26.5-55.0) months. The difference in overall survival between the NA group vs. the inactive CHB group was not significant (hazard ratio [HR] = 0.78; 95% confidence interval [CI] = 0.33-1.85; P = .57). The NA group showed a significantly higher risk of HCC (HR = 3.44; 95% CI = 1.82-6.52; P < .01), but comparable risk for non-HCC liver-related events (HR = 1.02; 95% CI = 0.66-1.59; P = .93), compared with the inactive CHB group. Among patients with cirrhosis, the NA group showed a significantly lower risk of death (HR = 0.31; 95% CI = 0.097-0.998; P = .05) and non-HCC liver-related events (HR = 0.51; 95% CI = 0.31-0.83; P < .01), but a slightly higher risk of HCC (HR = 2.39; 95% CI = 0.85-6.75; P = .09), compared to the inactive CHB group. The overall survival of untreated patients with inactive CHB and of CHB patients achieving viral suppression with NA was comparable. However, NA treatment of cirrhotic patients was significantly associated with longer overall survival and lower risk of liver-related events.

Comparison of the characteristics of Mycobacterium tuberculosis isolates from sputum and lung lesions in chronic tuberculosis patients.

Mycobacterium tuberculosis (Mtb) in sputum originates from lung cavities in tuberculosis (TB) patients. But drug susceptibility testing (DST) of sputum Mtb can not be conducted the same as in the lung because mutagenesis of bacilli may be happening in the lung during treatment and result in the possibility of the presence of heterogeneous drug-resistant subpopulations in the different lung lesions. This could be one of the reasons for low cure rates for multi-drug resistant (MDR)-TB. We studied the resected lungs of nine surgery patients with chronic TB. The isolates isolated from the sputum and different lung lesions of each patient were tested for phenotypic DST and genotyped using restriction fragment length polymorphism (RFLP) typing method. Genetic analysis to resistance to first and second line drugs was also performed. Five of nine patients were MDR-TB and three XDR-TB. DST results for ten anti-TB drugs were in accordance among different lung lesions in eight patients. However, only three of these eight patients showed the concordance of DST with sputum. Even though the isolates were heteroresistant, genotyping them by RFLP showed the clonal population in each individual patient. Six of eight followed-up patients achieved successful cure. In conclusion, the heteroresistance between sputum and lung lesions and a clonal population without mixed infection might provide useful information in establishing treatment regimen and surgery decision for MDR- and XDR-TB.

HDL-C levels modify the association between C-reactive protein and coronary artery calcium score.

BACKGROUNDS AND AIMS: C-reactive protein (CRP) levels predict incident and recurrent cardiovascular disease (CVD) events; however, associations between CRP and pre-clinical atherosclerosis is less certain. Since high concentrations of high-density lipoprotein cholesterol (HDL-C) are inversely associated with CVD risk, we investigated whether HDL-C modified the association between CRP concentration and measures of preclinical atherosclerosis. METHODS AND RESULTS: Data were analyzed from a Korean occupational cohort of 12,030 male subjects who underwent a cardiac computed tomography (CT) estimation of coronary artery calcification (CAC) score and an assessment of CVD risk factors. Logistic regression was used to describe associations between CRP and measures of pre-clinical atherosclerosis, such as CAC scores >0. As many as 1351 (11.2%) participants had a CAC score>0. CRP was stratified into 3 groups based on clinical category: <1 mg/L, 1 to <2 mg/L, and ≥ 2 mg/dL. In the bottom CRP group, 907/8697 (10.4%) of subjects had a CAC score >0, compared with 242/1943 (12.5%) in the middle group and 202/1396 (14.5%) in the top CRP group (p < 0.0001). After adjustment for multiple CVD risk factors, there was a positive association between CRP and CAC score>0 (OR between top and bottom CRP groups, 1.41 [1.04, 1.90], p = 0.027) in the lowest HDL-C quartile but not in the highest HDL-C (OR between top and bottom CRP group, 0.80 [0.46, 1.39], p = 0.425). CONCLUSION: The association between CRP concentration and CAC score differed according to HDL-C levels.

Identification and biochemical characterization of small molecule inhibitors of ERK2 that target the D-recruitment site.

Extracellular signal-regulated kinase (ERK) is the culmination of a mitogen-activated protein kinase cascade that regulates cellular processes like proliferation, migration, and survival. Consequently, abnormal ERK signaling often plays a role in the tumorigenesis and metastasis of numerous cancers. ERK inhibition is a sought-after treatment for cancers, especially since clinically approved drugs that target signaling upstream of ERK often induce acquired resistance. Furthermore, the ERK2 isoform may have a differential role in various cancers from the other canonical isoform, ERK1. We demonstrate that small molecules can inhibit ERK2 catalytic and noncatalytic functions by binding to the D-recruitment site (DRS), a protein-protein interaction site distal to the enzyme active site. Using a fluorescence anisotropy-based high-throughput screening, we identify compounds that bind to the DRS and exhibit dose-dependent inhibition of ERK2 activity and ERK2 phosphorylation. We characterize the dose-dependent potency of ERK2 inhibitors using fluorescence anisotropy-based binding assays, fluorescence-based ERK2 substrate phosphorylation assays, and in vitro ERK2 activation assays. In our example, the binding of a DRS inhibitor can be prevented by mutating the DRS residue Cys-159 to serine, indicating that this residue is essential for the interaction. Resulting inhibitors from this process can be assessed in cellular and in vivo experiments for inhibition of ERK signaling and can be evaluated as potential cancer drugs.

Evaluation of the MolecuTech® REBA MTB-XMDR kit for detection of pre-extensively drug-resistant TB.

BACKGROUND: Rapid diagnosis of drug-resistant TB is critical for early initiation of effective therapy. YD Diagnostics in South Korea recently developed the MolecuTech® REBA MTB-XMDR test to rapidly detect multidrug-resistant TB (MDR-TB), pre-extensively drug-resistant TB (pre-XDR-TB) and resistance to second-line injectable drugs (SLIDs) simultaneously using a fully automated test platform. This study aimed to evaluate the MolecuTech® test for the detection of MDR- and pre-XDR-TB, as well as SLID resistance.METHODS: A total of 151 clinical Mycobacterium tuberculosis isolates from South Korea were tested using the MolecuTech test, and the results were analysed by comparing these with phenotypic drug susceptibility testing (pDST) and sequencing.RESULTS: Compared to pDST, the MolecuTech test showed a sensitivity and specificity of respectively 97.7% and 100.0% for rifampicin (RIF), 82.4% and 100.0% for isoniazid (INH), 97.5% and 97.2% for fluoroquinolones (FQs), and 94.0% and 98.8% for SLIDs. Concordances with the sequencing results of each resistance determinant were 99.3% for RIF, 96.7% for INH, 98.7% for FQs and 99.3% for SLIDs.CONCLUSION: The MolecuTech test is an efficient and reliable rapid molecular diagnostic tool for the simultaneous screening of MDR- and pre-XDR-TB.

Prognosis of surgical hernia repair in cirrhotic patients with refractory ascites.

BACKGROUND: Abdominal wall hernias are common in patients with ascites. Elective surgical repair is recommended for the treatment of abdominal wall hernias. However, surgical hernia repair in cirrhotic patients with refractory ascites is controversial. In this study, we aimed to evaluate the outcomes of elective surgical hernia repair in patients with liver cirrhosis with and without refractory ascites. METHOD: From January 2005 to June 2018, we retrospectively reviewed the records of consecutive patients with liver cirrhosis who underwent a surgical hernia repair. RESULTS: This study included 107 patients; 31 patients (29.0%) had refractory ascites. Preoperatively, cirrhotic patients with refractory ascites had a higher median model for end-stage liver disease (MELD) score (13.0 vs 11.0, P = 0.001) than those without refractory ascites. The 30-day mortality rate (3.2% vs 0%, P = 0.64) and the risk of recurrence (hazard ratio 0.410; 95% CI 0.050-3.220; P = 0.39) did not differ significantly between cirrhotic patients with refractory ascites and cirrhotic patients without refractory ascites. Among cirrhotic patients with refractory ascites, albumin (P = 0.23), bilirubin (P = 0.37), creatinine (P = 0.97), and sodium levels (P = 0.35) did not change significantly after surgery. CONCLUSION: In advanced liver cirrhosis patients with refractory ascites, hernias can be safely treated with elective surgical repair. Mortality rate within 30 days did not differ by the presence or absence of refractory ascites. Elective hernia repair might be beneficial for treatment of abdominal wall hernia in cirrhotic patients with refractory ascites.

Intensity of surveillance for hepatocellular carcinoma determines survival in patients at risk in a hepatitis B-endemic area.

BACKGROUND: Data are insufficient regarding the survival benefit of surveillance for hepatocellular carcinoma (HCC). AIM: To investigate the effectiveness of HCC surveillance in a hepatitis B-endemic population. METHODS: This retrospective cohort study included 1402 consecutive patients who were newly diagnosed with HCC between 2005 and 2012 at a single tertiary hospital in Korea. The primary endpoint was overall survival. Lead-time and length-time biases were adjusted (sojourn time = 140 days) and sensitivity analyses were performed. RESULTS: The most common aetiology was hepatitis B (80.4%). Cirrhosis was present in 78.2%. HCC was diagnosed during regular surveillance (defined as mean interval of ultrasonography <8 months, n = 834), irregular surveillance (n = 104) or nonsurveillance (n = 464). Patients in the regular surveillance group were diagnosed at earlier stages ([very] early stage, 64.4%) than the irregular surveillance (40.4%) or nonsurveillance (26.9%) groups and had more chance for curative treatments (52.4%) than the irregular surveillance (39.4%) or nonsurveillance (23.3%) groups (all P < 0.001). Mortality risk was significantly lower in the regular surveillance group (adjusted hazard ratio [aHR], 0.69; 95% [CI], 0.57-0.83) but not in the irregular surveillance group (aHR, 0.94; 95% CI, 0.69-1.28) compared with the nonsurveillance group after adjusting for confounding factors and lead-time. When the subjects were restricted to cirrhotic patients or Child-Pugh class A/B patients, similar results were obtained for mortality risk reduction between groups. CONCLUSIONS: HCC surveillance was associated with longer survival owing to earlier diagnosis and curative treatment. Survival advantage was significant with regular surveillance but not with irregular surveillance.

The impact of post-thaw colony-forming units-granulocyte/macrophage on engraftment following unrelated cord blood transplantation in pediatric recipients.

We retrospectively reviewed the engraftment kinetics following unrelated cord blood transplantation (CBT) in association with the post-thaw colony-forming units-granulocyte/macrophage (CFU-GM) number along with the numbers of total nucleated cells (TNC), CD34(+) cells and CD3(+) cells. A total of 71 cord blood units prepared for 53 patients (double-unit CBT in 18 patients) were evaluated. Either the number of infused CFU-GM or CD34(+) cells was significantly lower in patients who failed to achieve engraftment (P=0.028 and 0.005, respectively). Post-thaw CFU-GM, TNC and CD34(+) cells correlated with the speed of neutrophil engraftment (P=0.004, 0.037 and 0.004, respectively), whereas only CFU-GM showed a significant correlation with platelet engraftment (r=-0.385, P=0.024). In double-unit transplants, the number of CFU-GM was the only significant factor predicting engraftment of the predominating unit (P=0.006). We conclude that the post-thaw CFU-GM number is a reliable predictor of rapid engraftment after CBT as well as of the predominating unit in double-unit transplants. Thus, it would be important to perform post-thaw CFU-GM assay on cryopreserved aliquots from several candidate cord blood units in advance before CBT to avoid selecting the unit that might possess a low clonogenic potential.

Tandem high-dose chemotherapy and autologous stem cell rescue in patients over 1 year of age with stage 4 neuroblastoma.

From June 1997 to August 2005, 52 consecutive newly diagnosed stage 4 neuroblastoma patients over 1 year of age were assigned to receive tandem high-dose chemotherapy and autologous stem cell rescue (HDCT/ASCR) as consolidation therapy. Fifty of the 52 patients underwent a first HDCT/ASCR and 44 patients underwent a second HDCT/ASCR. Eight patients (15.4%) died from treatment-related toxicity (seven during the second HDCT/ASCR). Total body irradiation (TBI) in the first HDCT/ASCR and a shorter interval (< 12 weeks) between the first and second HDCT/ASCR were associated with a higher rate of treatment-related death in the second HDCT/ASCR (P = 0.032 and 0.095, respectively). The tumor relapsed or progressed in 11 patients, and 33 patients remained event free with a median follow-up of 53 months (range 19-117) from diagnosis. The 5-year event-free survival (EFS) (+/- 95% confidence interval) for all 52 patients was 62.1+/-13.7%. The application of TBI and local radiotherapy, and a longer interval between the first and second HDCT/ASCR were independently associated with a better EFS (P = 0.026, 0.007 and 0.020, respectively). However, further studies will be needed to decrease the toxic death rate in the second HDCT/ASCR while reducing the relapse rate.

Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II.

The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

Diagnosis of bovine freemartinism by fluorescence in situ hybridization on interphase nuclei using a bovine Y chromosome-specific DNA probe.

A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.

A study on setting of the fatigue limit of temporary dental implants.

A temporary dental implant is a medical device which is temporarily used to support a prosthesis such as an artificial tooth used for restoring patient's masticatory function during implant treatment. It is implanted in the oral cavity to substitute for the role of tooth. Due to the aging and westernization of current Korean society, the number of tooth extraction and implantation procedures is increasing, leading to an increase in the use and development of temporary dental implants. Because an implant performs a masticatory function in place of a tooth, a dynamic load is repeatedly put on the implant. Thus, the fatigue of implants is reported to be the most common causes of the fracture thereof. According to the investigation and analysis of the current domestic and international standards, the standard for fatigue of implant fixtures is not separately specified. Although a test method for measuring the fatigue is suggested in an ISO standard, it is a standard for permanent dental implants. Most of the test standards for Korean manufacturers and importers apply 250 N or more based on the guidance for the safety and performance evaluation of dental implants. Therefore, this study is intended to figure out the fatigue standard which can be applied to temporary dental implants when measuring the fatigue according to the test method suggested in the permanent dental implant standard. The results determined that suitable fatigue standards of temporary dental implants should be provided by each manufacturer rather than applying 250 N. This study will be useful for the establishment of the fatigue standards and fatigue test methods of the manufacturers and importers of temporary dental implants.

Rifaximin treatment is associated with reduced risk of cirrhotic complications and prolonged overall survival in patients experiencing hepatic encephalopathy.

BACKGROUND: Rifaximin might decrease the risk of portal hypertension-related complications by controlling small intestinal bacterial overgrowth. AIM: To evaluate whether rifaximin was associated with the risk of death and cirrhotic complications. METHODS: We conducted a retrospective study that included 1042 patients experiencing hepatic encephalopathy (HE): 421 patients without hepatocellular carcinoma (HCC; the non-HCC cohort) and 621 patients with HCC (the HCC cohort). The primary endpoint was overall survival and secondary endpoints were recurrence of HE and the development of spontaneous bacterial peritonitis (SBP), hepatorenal syndrome (HRS) and variceal bleeding. RESULTS: In the non-HCC cohort, 145 patients received rifaximin plus lactulose (the rifaximin group) and 276 patients received lactulose alone (the control group). The multivariate analysis revealed that rifaximin was significantly associated with lower risk of death (adjusted hazard ratio [aHR], 0.697; P = .024) and reduced the risk of recurrent HE (aHR, 0.452; P < .001), SBP (aHR, 0.210; P < .001) and variceal bleeding (aHR, 0.425; P = .011) but not HRS (aHR, 0.598; P = .08). In the HCC cohort, 173 patients received rifaximin plus lactulose and 448 patients received lactulose. Rifaximin was not associated with the risk of death (aHR, 1.177; P = .121). Rifaximin was associated with lower risk of SBP (aHR, 0.323; P < .001) but not with variceal bleeding (aHR, 0.660; P = .104) or recurrent HE (aHR, 0.689; P = .057). The risk of Clostridium difficile-associated diarrhoea was not different between the groups (aHR, 0.028; P = .338). CONCLUSIONS: In patients without HCC, rifaximin treatment was significantly associated with prolonged overall survival and reduced risks of spontaneous bacterial peritonitis, variceal bleeding and recurrent hepatic encephalopathy.

Transcriptional repression of cancer stem cell marker CD133 by tumor suppressor p53.

Novel therapeutic strategies are needed to overcome cancer recurrence, metastasis, and resistance to chemo- and radiotherapy. Cancer stem cells (CSCs) are major contributors to the malignant transformation of cells due to their capacity for self-renewal. Although various CSC markers have been identified in several types of tumors, they are primarily used as cancer-prediction markers and for the isolation of CSC populations. CD133, one of the best-characterized CSC markers in distinct solid tumor types, was shown to be correlated with CSC tumor-initiating capacity; however, the regulation of CD133 expression and its function in cancer are poorly understood. Here, we show that CD133 expression is negatively regulated by direct binding of the p53 tumor suppressor protein to a noncanonical p53-binding sequence in the CD133 promoter. Binding of p53 recruits Histone Deacetylase 1 (HDAC1) to the CD133 promoter and subsequently suppresses CD133 expression by reducing histone H3 acetylation. Furthermore, CD133 depletion suppresses tumor cell proliferation, colony formation, and the expression of core stemness transcription factors including NANOG, octamer-binding transcription factor 4 (OCT4), SOX2, and c-MYC. Critically, the anti-proliferative effects of p53 are antagonized by rescue of CD133 expression in a p53 overexpressing cell line, indicating that the tumor suppressive activity of p53 might be mediated by CD133 suppression. Taken together, our results suggest that p53-mediated transcriptional regulation of CD133 is a key underlying mechanism for controlling the growth and tumor-initiating capacity of CSCs and provide a novel perspective on targeting CSCs for cancer therapy.

Successive double high-dose chemotherapy with peripheral blood stem cell rescue collected during a single leukapheresis round in patients with high-risk pediatric solid tumors: a pilot study in a single center.

In total, 18 of 26 double high-dose chemotherapies (HDCT) in pediatric solid tumors were rescued with peripheral blood stem cells collected during a single leukapheresis round (single-harvest group, SHG). In the remaining eight HDCT, additional leukapheresis were necessary after the first HDCT (HDCT1) to rescue the second HDCT (HDCT2) (double-harvest group, DHG). Stem cell collection after HDCT1 was inefficient and delayed in patients who had received prior chemotherapy before HDCT1. The interval between HDCT1 and HDCT2 was shorter in SHG than in DHG (median 62.5 days vs 178.5 days, P-value=0.002). Hematologic recovery in HDCT2 was delayed compared to HDCT1. However, there was no difference in hematologic recovery between SHG and DHG. A high rate of treatment-related mortality (TRM) was recorded during HDCT2, but there was no evidence that the shorter interval caused a higher rate of TRM (P-value=0.454). The probability of disease-free survival at 2 years after HDCT2 in the SHG and DHG were 66.7 and 25.0%, respectively (P-value=0.031). Therefore, to administer the second HDCT earlier in double HDCT, and thus to improve the survival of patients with high-risk solid tumors, the single-harvest approach is recommended rather than the double-harvest approach.

Antioxidative activity of green tea treated with radical initiator 2, 2'-azobis(2-amidinopropane) dihydrochloride.

This study investigated the antioxidative activity of green tea extract, and a green tea tannin mixture and its components, under conditions of radical generation using the hydrophilic azo compound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate peroxyl radicals at a constant and measurable rate in the cultured renal epithelial cell line, LLC-PK(1), which is susceptible to oxidative damage. Treatment with AAPH decreased cell viability and increased the formation of thiobarbituric acid-reactive substances. However, green tea extract, and the tannin mixture and its components, comprising (-)-epigallocatechin 3-O-gallate (EGCg), (-)-gallocatechin 3-O-gallate (GCg), (-)-epicatechin 3-O-gallate (ECg), (-)-epigallocatechin (EGC), (+)-gallocatechin (GC), (-)-epicatechin (EC), and (+)-catechin (C), showed protective activity against AAPH-induced cellular damage. The tannin mixture and its components exhibited higher antioxidative activity than the green tea extract. Furthermore, EGCg and GCg had higher activity than EGC and GC, respectively. In particular, EGCg exerted the most significant cellular protective activity against AAPH. These results indicate that green tea tannin may inhibit cellular loss and lipid peroxidation resulting from the peroxyl radical generated by AAPH, and that the chemical structure of tannin is also involved in the activity, suggesting that the O-dihydroxy structure in the B ring and the galloyl groups are important determinants for radical scavenging and antioxidative potential.

Effects of green tea tannin on cisplatin-induced nephropathy in LLC-PK1 cells and rats.

A study was conducted to clarify whether green tea tannin ameliorated cisplatin-induced renal injury in terms of lactate dehydrogenase and malondialdehyde leakage from a renal epithelial cell line, swine-derived LLC-PK1 cells in culture. Green tea tannin was shown to suppress the cytotoxicity of cisplatin, the suppressive effect increasing with the dose of green tea tannin. The effect of cisplatin was then investigated in rats given green tea tannin for 40 days before cisplatin administration and in control rats given no green tea tannin. In control rats, blood, urinary and renal parameters and the activities of antioxidative enzymes in renal tissue deviated from the normal range, indicating dysfunction of the kidneys. In contrast, rats given green tea tannin showed decreased blood levels of urea nitrogen and creatinine, and decreased urinary levels of protein and glucose, reflecting less damage to the kidney. In this group, the activity of catalase in the renal tissue was increased, while the level of malondialdehyde was decreased, suggesting the involvement of radicals in the normalizing of kidney function. Based on the evidence available it appeared that green tea tannin eliminated oxidative stress and was beneficial to renal function.

CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).

Checkpoint kinase 2 (CHK2) kinase is a key mediator in many cellular responses to genotoxic stresses, including ionizing radiation (IR) and topoisomerase inhibitors. Upon IR, CHK2 is activated by ataxia telangiectasia mutated kinase and regulates the S-phase and G1-S checkpoints, apoptosis and DNA repair by phosphorylating downstream target proteins, such as p53 and Brca1. In addition, CHK2 is thought to be a multi-organ cancer susceptibility gene. In this study, we used a tandem affinity purification strategy to identify proteins that interact with CHK2 kinase. Cyclin-dependent kinase 11 (CDK11)(p110) kinase, implicated in pre-mRNA splicing and transcription, was identified as a CHK2-interacting protein. CHK2 kinase phosphorylated CDK11(p110) on serine 737 in vitro. Unexpectedly, CHK2 kinase constitutively phosphorylated CDK11(p110) in a DNA damage-independent manner. At a molecular level, CDK11(p110) phosphorylation was required for homodimerization without affecting its kinase activity. Overexpression of CHK2 promoted pre-mRNA splicing. Conversely, CHK2 depletion decreased endogenous splicing activity. Mutation of the phosphorylation site in CDK11(p110) to alanine abrogated its splicing-activating activity. These results provide the first evidence that CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).