Overall, 47 (36%) of patients had either vascular invasion or satellite tumors and did not meet the pathological criteria for very early HCC defined as T1 by the Japanese Society of Hepatology or as BCLC stage 0. The overall recurrence rate of 68% at 5 years and the 1-year recurrence rate of 17% seemed, at first, surprisingly high to us for such small cancers. A recurrence rate of 61% at 5 years for small tumors without vascular invasion or satellites was
particularly unexpected. However, a Japanese study of 70 patients with HCC ≤2 cm undergoing resection found an overall recurrence rate of 88% for the entire buy Midostaurin cohort.24 The same study demonstrated 1- and 5-year recurrence rates of 8% and 53%, respectively, for patients found to have T1 tumors.24 These numbers are very similar to what we have reported (12% at 1 year and 61% at 5 years) for our patients with pathologically proven very early tumors. Again, the recurrence rates for the entire cohort from our study (17% at
1 year and 68% at 5 years) compare favorably with the 1- and 5-year recurrence rates of 34% and 80%, respectively, reported for RFA of similarly sized HCC.10 The vast majority of the recurrences occurred within the first 3 years after surgery after which there were very few events. The pattern selleck kinase inhibitor of the instantaneous risk of recurrence for these small tumors was also very different from that published for more advanced tumors.25, 26 Instead of the two peaks generally seen for larger tumors—one at approximately 12 months representing early metastatic recurrence and another at approximately 36 months representing late de novo recurrence—we see only a single and delayed
peak at 30 months. This pattern may reflect a reduction in early metastatic recurrences given the early stage of the tumors but deserves further investigation. The presence of satellites, underlying cirrhosis, and nonanatomic resection were associated with time to recurrence. The presence of satellites has been found to be a significant predictor of outcome 上海皓元 after resection of HCC in many other studies.27 Likewise, the nature of the nontumoral liver around the HCC has also been shown to be a strong predictor of recurrence of HCC after resection.28 Generally, neither variable is known preoperatively to help guide patient selection or the selection of the most appropriate therapy. The success of sorafenib in the treatment of advanced HCC has opened the door for the testing of targeted molecules in the adjuvant setting.29 The degree of fibrosis and the presence of satellites can help select or stratify patients who are most at risk for recurrence and who may benefit most from sorafenib after resection if the drug is eventually found to be an effective agent in the adjuvant setting. Alternatively, patients with satellites who are at risk for early metastatic recurrence can be referred for salvage liver transplantation, as has been proposed by the Barcelona group.
Because preoperative staging was deemed highly unreliable, a majority of panelists voted for combined pre- and postoperative chemotherapy (even in localized cancers without lymph node involvement, T2N+ or T2N0), combined with a modified D2 resection (i.e., without resection of the pancreatic tail and without routine splenectomy). Adjuvant chemotherapy or radiochemotherapy was considered for patients MLN8237 molecular weight who had not received perioperative treatment. There were comments that radiochemotherapy should be
preferred in patients with lymph node involvement or inadequate lymphadenectomy. For pre- or perioperative treatment, a doublet of fluoropyrimidine/platinum was the most widely recommended regimen (e.g., cisplatinum/5-FU, cisplatinum/capecitabine and oxaliplatin/capecitabine). Some considered the addition of taxane as appropriate for the preoperative induction regimen. The addition of trastuzumab to a platinum/fluoropyrimidine combination is a new standard first-line therapeutic regimen for patients with advanced
HER2-positive GC. Trastuzumab is a monoclonal antibody that interferes with the HER2/neu receptor. In the ToGA trial, trastuzumab in addition selleck compound to chemotherapy prolonged the median overall survival from 11.8 months to 16.0 months in a selected group of patients with advanced GC overexpressing the HER-2 protein . However, the addition of trastuzumab to chemotherapy in patients with HER2-positive GC led to an additional absolute increase in response rate of only 12%, indicating the existence of a high primary trastuzumab resistance in this subpopulation. Furthermore, the majority of patients who had initially responded to the treatment developed acquired or secondary resistance. The molecular mechanisms underlying trastuzumab resistance are not yet well characterized. New targeted therapies 上海皓元 to overcome trastuzumab resistance
as well as to improve the outcome of patients with HER2-negative GC are currently being evaluated in clinical trials. HER2 is the preferred heterodimerization partner of the other HER family members, and the HER2-HER3 heterodimer is the most active HER signaling dimer, playing a critical role in oncogenic transformation in HER2-driven tumors . Dimerization is followed by transactivation of the receptor, which subsequently activates downstream proteins, including members of the Ras/Raf/mitogen-activated protein kinase (Ras/Raf/MAPK) and phosphatidylinositol-3 kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathways, as well as gene transcriptional programs . Trastuzumab binds to domain IV of the HER2 extracellular domain and does not inhibit the dimerization of HER2 with ligand-activated HER3 . In contrast, pertuzumab, a humanized monoclonal antibody directed against the extracellular heterodimerization domain II of HER2, effectively blocks HER2/HER3 heterodimerization.
The study was approved by the Institutional Review Board of Mayo Foundation and the Ethics Committee of the Korean National Cancer Center. The HCC lesions were characterized by cross-sectional radiographic characteristics, which included (1) the number of tumor nodules, (2) the diameter of the largest nodule, (3) vascular invasion (enhancing vascular tumor thrombi), and (4) extrahepatic metastasis. Based on these radiographic information and laboratory data at entry into the study, individual patients were staged according to the BCLC, the CLIP score, and the JIS score. The original MELD score (before
modification for the purpose of organ allocation) was calculated as published.18 For survival analysis, patients were followed from the first visit date for HCC assessment B-Raf cancer forward until July 22, 2010 and September 1, 2004 in the derivation and validation cohorts, respectively. To ascertain complete capture of all decedents, a proprietary information source (Accurint) was used to supplement information in the medical records in the derivation cohort and the National Cancer Registry data in the validation
cohort. Death from any causes was considered an event in this analysis. In the base-case analysis, liver transplantation was not considered an event, whereas a subsequent sensitivity analysis was conducted censoring liver transplantation. Patient survival probability was estimated using the Kaplan-Meier buy Depsipeptide method. The main tool for survival analysis was the proportional hazards model. Based on variables with univariate significance (P < 0.10) and clinical relevance, multivariate models were created. The output of the model was expressed as coefficients, which were used to compute hazard ratios. In addition, the coefficients were used to calculate
a risk score, which, in turn, was used to predict survival. In the derivation cohort, cross-validation was used to examine the reproducibility of the survival model. The data were 上海皓元医药股份有限公司 randomly divided into four equal subsets and the coefficients were recalculated after removing one subset of the data at a time. The concordance (c)-statistic was computed using the new coefficients in the remainder of the data. The c-statistic from each of the four subsets was compared to one another. In testing the accuracy of the model prediction in the validation cohort, patients were divided into three groups at the 25th and 75th percentiles of the risk score. The observed survival in the validation cohort was compared with survival estimated by the survival model. The goodness-of-fit of the models was assessed using the c-statistics.
Conversely, apparent mortality rates to age 1+ (range: 0.34–0.52) and 2+ (range: 0.15–0.59) were higher than values reported elsewhere. Target Selective Inhibitor high throughput screening The high apparent calf mortality in conjunction with a decline in local abundance, highlight the vulnerability of bottlenose dolphins in the Bay of Islands. Long-term studies are required to understand the causes of high calf mortality and the decline in local abundance. Meanwhile, management should focus on minimizing sources of anthropogenic disturbance and enforcing compliance with current legislation. “
“Increased terrestrial pup mortality
in small colonies due to harassment by subadult males has been proposed as a mechanism to explain the stagnation of South American sea lion populations after sealing ended. To test this hypothesis, pup survival rate was assessed in five northern Patagonia colonies with different sizes. Female diet quality as well as pup growth rate and immune status from the largest and smallest of these colonies BAY 57-1293 purchase were also assessed. Results indicated that the pup survival rate increased with colony size and pup-to-subadult male ratio. Furthermore, pups grew faster in the smallest colony, although female diet composition and pup immune status did not differ between the two colonies. Inverse relationship between pup growth rate and survival rate indicated that mortality
was independent of food supply. In absence of terrestrial predators,
infanticide by subadult males is the only mortality source other than starvation and illness and the relationship between pup survival rate and pup-to-subadult male ratio approached a type II functional response curve. Thus, infanticide stands as the most likely reason for the observed positive relationship between colony size and pup survival rate, supporting the hypothesis that post-sealing population stasis was caused by inverse density dependence. “
“Molecular phylogenetic analyses conducted over the past 15 yr have consistently had difficulties resolving relationships among the cetacean species in the subfamily Delphininae. In addition, paraphyly of the genera Tursiops and Stenella in these molecular phylogenies has been a recurrent problem since the first appearance of such a phylogeny in 1999, suggesting that these genera do not accurately reflect MCE公司 the evolutionary relationships of the species they contain. Morphological analyses have not resolved the issues. The genera in Delphininae originated in the 19th Century on questionable morphological grounds. The species were nearly all originally described in the genus Delphinus of Linnaeus. Recent molecular phylogenies based on various mitochondrial and nuclear DNA markers have suggested a wide range of possible relationships among these taxa, and several authors have suggested synonymizing all the taxa (Lagenodelphis, Stenella, Sousa, and Tursiops) under Delphinus.
OraQuick Rapid HCV antibody test was CP-868596 utilized to provide point of care HCV screening with results
available in 20-40 minutes. All patients completed a questionnaire prior to endoscopy regarding risk factors and exposures. Patients who tested positive for HCV were contacted for further testing and possible therapy. Results: HCV screening was offered to 254 patients and 220 agreed to be tested. Thirty-four patients declined testing (14%). The majority (62%) who declined were women. Two patients were found to be hepatitis C positive (1%); however both were already known to have hepatitis C. No new diagnoses of chronic hepatitis were made. Traditional risk factors for HCV transmission were prevalent, including illicit drug use (21.4%), acupuncture (19.5%), STD’s (12.3%), tattoos (8.2%), and IVDU (2.3%). There was a Caucasian predominance to the cohort of 62%. Point of care testing Small molecule library and counseling did
not affect the work-flow or efficiency of the endoscopy center. Prior to initiation of this study, average patient time after the nurse’s interview to patient discharge was 1 hour 52 minutes. During the enrollment and HCV testing period the patient time to proceed through endoscopy was largely unchanged at 1 hour 47 minutes. Conclusions This prospective study demonstrates that it is possible to test patients for viral hepatitis during routine colonoscopy and endoscopy in an efficient and time effective manner. Earlier studies documented a 1% new diagnosis rate, however despite prevalent traditional HCV risk factors in our study cohort we had no new diagnoses at this interval analysis. Point of care testing allows for high rates of patient acceptance. Also, testing did not delay workflow at the endoscopy center. Given recent CDC recommendations for birth cohort screening for HCV, gastroenterologists MCE should strongly consider PoC testing in non-traditional
patient interactions such as endoscopy. Disclosures: The following people have nothing to disclose: Raja Taunk, Daniel I. Zapata, Jennifer Stone, Victoria Menashy, llan Weisberg Introduction: Progressive liver damage due to hepatitis C virus infection is a major cause of co-morbidity in haemophilia patients. The need for accurate diagnostic tools for assessing the extent of liver fibrosis is high in these patients. The aim of this study was to investigate the additional value of the enhanced liver fibrosis (ELF) test combining hyaluronic acid, procollagen-III-amino terminal peptide and tissue inhibitor of metalloproteinase-1 compared to transient elastography (TE). Methods: We evaluated the ELF score in 58 HCV-infected haemophilia patients on the ADVIA Centaur XP (Siemens). TE was performed by using the FibroScan (Echosens, Paris, France). In 15 patients (25.
120% ± 0.005%. However, this number varied according to the considered amino acid position. Therefore, only substitution frequencies higher than the mean maximal error rate for the corresponding amino acid position plus 2 SDs were taken into account in the analysis. Plasmids were tested in triplicate at different dilutions ranging from Sotrastaurin mouse 5 × 103 to 108 copies/mL. No effect of the
DNA amount on UDPS results was observed (data not shown). UDPS was then applied to serum samples taken at baseline and frequently during therapy, representing a total of 119 serial samples from 7 patients who developed resistance to adefovir monotherapy (15-24
samples per patient). Approximately 480,000 sequences (111 Mbp) were generated, with 4,010 ± 843 sequences per sample and a mean length of 382 ± 31 nucleotides (nt) after eliminating excessively short sequences (≤50 nt). Overall, 10.2% of the generated sequences were eliminated by the software because of inadequate quality. Table 1 shows the prevalence of amino acid substitutions known to confer HBV resistance to nucleoside/nucleotide analogs detected by UDPS at baseline in the 7 patients. Five samples were found to harbor rtA181V/T substitutions, click here one harbored the rtN236T substitution, and two harbored rtM204V/I substitutions. No substitutions known to improve the fitness of rtM204V/I variants in
the presence of lamivudine, telbivudine, or entecavir (rtV173L and rtL180M) were found in baseline samples. One sample harbored the rtT184S/A/I/L substitution and none 上海皓元 harbored the rtR202G substitution; both these substitutions are known to confer full resistance to entecavir when associated with rtM204V/I and rtL180M. Table 1 also shows the prevalence of amino acid substitutions known to confer HBV resistance to nucleoside/nucleotide analogs detected by UDPS in the two comparator groups described in the Patients and Methods section. Among the 5 HBeAg-positive patients who seroconverted and maintained undetectable HBV DNA levels after adefovir therapy, 2 were found to harbor rtA181V/T substitutions at baseline, whereas none harbored the rtN236T substitution; 2 harbored rtM204V/I substitutions, including 1 in whom it represented nearly 10% of the viral quasispecies and was associated with an rtL180M substitution, suggesting earlier exposure to lamivudine; rtT184S/A/I/L and rtR202G substitutions were found in 1 and 1 patient, respectively.
Model performance was quantified [area under the curve (AUC), calibration plot] and internal validation (bootstrapping) was performed. A nomogram for clinical application was developed. Of the 825 patients, 225 (28%) developed inhibitors. The predictors family history of inhibitors, F8 gene mutation learn more and an interaction variable of dose and number of EDs of intensive treatment were independently associated with inhibitor development. Age and reason for first treatment were not associated with inhibitor development. The AUC was 0.69 (95% CI 0.65–0.72) and calibration was good. An improved prediction
model for inhibitor development and a nomogram for clinical use were developed in a cohort of 825 PUPs with severe haemophilia A. Clinical applicability was improved by combining dose and duration of intensive treatment, allowing the assessment
of the effects of treatment decisions on inhibitor risk and potentially modify treatment. “
“Diagnosis of haemophilia A is usually made by the measurement of factor VIII (FVIII) H 89 activity that allows categorization of the disease severity. However, tests that assess global haemostasis may better reflect clinical features and give additional clinically relevant information. The aim of this study was to develop a new quantitative activated partial thromboplastin time (aPTT) waveform analysis and compare it with FVIII activities to find out whether waveform parameters are superior determinants 上海皓元 of clinical phenotype. A total of 81 haemophilia A patients divided into two groups (37 severe, 44 non-severe) were included in the study. The control group comprised 101 healthy male volunteers. Quantitative aPTT waveform analysis was performed with Actin FS on BCS (Siemens Healthcare Diagnostics, Marburg, Germany) using three parameters (DELTA, RATIO-1, RATIO-2) obtained from a single aPTT measurement with two evaluation modes. FVIII activities were measured by
one-stage clotting and two-stage chromogenic assay. Statistically significant difference (P < 0.001) between control group and all haemophilia A patients, as well as between severe and non-severe haemophilia A patients was obtained for all quantitative waveform parameters. Our study revealed parameter DELTA as the best waveform parameter, showing significant correlation with FVIII activities and clinical parameters, and excellent performance for distinguishing between severe and non-severe haemophilia A patients (ROC analysis: sensitivity 97.3%, specificity 93.2%). The results obtained by new quantitative aPTT waveform analysis were superior to those obtained by standard laboratory methods. The simplicity and cost-benefit of the method make this approach a reasonable and promising tool for assessing coagulation in haemophilia A patients. "
“Summary. Many persons with severe haemophilia reach seniority thanks to effective treatment.
SF-treated HSC had significantly increased apoptosis rate compared with the untreated; after addition of GGPP, the
apoptosis rate decreased. SF reduced basal intrahepatic resistance and responsiveness of methoxamine on hepatic vascular smooth muscle. Conclusion: SF reduces fibrogenesis and decreases portal pressure of cirrhotic rats by down-regulating RhoA/Rho-kinase signaling pathway. Key Word(s): 1. sodium ferulate; 2. portal hypertension; 3. RhoA; 4. HSC; Presenting Author: SHAHRAM AGAH Corresponding Author: SHAHRAM AGAH Affiliations: colorectal Objective: About check details 10% of cirrhotic patients are unresponsive to sodium restriction and diuretics and develop refractory ascites. Such patients usually require recurrent large volume paracentesis and lots of hospital
admissions. Hereby, we introduce a method applying a central vein (CV) catheter for large volume paracentesis in patients with refractory ascites. Methods: Non-tunneled triple lumen central vein catheter was used to drainage the ascites fluid of 30 cirrhotic patients. After precise percussion the point of highest fluid accumulation was marked for puncture. Then, the skin and subcutaneous tissue were anesthetized. CV catheter set guide-wire was entered into the peritoneal cavity and the dilator of the CV catheter set was passed through the guide wire and extracted after some rotations around its insertion site on the skin. The catheter was passed over the guide wire; the guide wire was extracted gradually from one of the lumens and fixed to the skin. Results: Nineteen males and 11 females with mean age of 59.4 ± 11.7 year old underwent the procedure. A minimum of 9 and maximum of 29 liters (12 ± 6.6 L) click here ascites fluid drained during a minimum of 2 and maximum of 5 days of hospital stay. None of the patients develop hemodynamic instability. Number of rehospitalizations for paracentesis was 1.9 times during the following year. No complication occurred. medchemexpress Conclusion: This technique seems to be a simple noninvasive method that can be performed in the
endoscopy unit or even at patient’s bedside and may reduce the need for repeated admissions. Key Word(s): 1. catheter; 2. Refractory ascites; 3. Paracentesis; 4. Cirrhosi; Presenting Author: WEI-FEN XIE Additional Authors: HUI QIAN, XING DENG, ZHAO-WEI HUANG, XIN ZENG, CHUAN YIN, YUE-XIANG CHEN, XIN ZHANG, JUN-PING ZHANG Corresponding Author: WEI-FEN XIE Affiliations: the second military medical university; Department of Biochemical Pharmacology, School of Pharmacy, Second Military Medical University; Department of Gastroenterology, Changzheng Hospital, Second Military Medical University Objective: Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor which play a key role in hepatocyte differentiation. Our previous study revealed a significant inhibitory effect of HNF1α on hepatocellular carcinoma. Herein, we sought to investigate the role of HNF1α in hepatic fibrogenesis.
Male TIMP-1−/− knockout (KO) mice in the C57BL/6 background (B6.129S4-Timp1tm1Pd/J) and respective TIMP-1+/+ wildtype (WT) C57BL-6 controls were obtained from the Jackson Laboratory. Hepatic IRI was performed as described.4 Briefly, arterial and portal venous blood supplies were interrupted to the cephalad lobes of the liver for 90 minutes using an atraumatic clip and mice Palbociclib in vitro were sacrificed after reperfusion. The animal studies were performed according to approved guidelines by the American Association of Laboratory Animal Care. Serum
alanine transaminase (ALT) and serum aspartate transaminase (AST) levels were measured with an autoanalyzer by ANTECH Diagnostics (Los Angeles, CA), as described.4 Liver specimens were fixed with a 10% buffered formalin solution, embedded in paraffin, and processed for hematoxylin and eosin (H&E) staining; to determine the percentage of necrotic area, 10 random sections per slide were evaluated in duplicate Ceritinib using National Institutes of Health (NIH) Image-J. Immunostaining was performed in cryostat sections as described.4, 11 Mac-1 (M1/70) and Ly-6G (1A8), from BD Biosciences, TIMP-1 (Ab86482; Abcam), MMP-9 (AF909; R&D Systems), and cleaved-caspase-3 (ASP175; Cell Signaling) antibodies were used at optimal dilutions. Sections were
blindly evaluated by counting 10 high-powered fields (HPFs)/section in triplicate. Dual/triple staining was detected by immunofluorescence with Alexa Fluor 594-red antigoat immunoglobulin G (IgG) (H+L) (Molecular Probes), and Texas Red antirat IgG (H+L) antibodies (Vector Laboratories). Alexa Fluor 488 phalloidin (Molecular Probes) and Vectashield mounting media with DAPI (Vector Laboratories) were used for F-actin and nuclear staining, respectively. Slides were analyzed using a Leica Confocal Microscope (UCLA Brain Research Institute). Mice were injected intraperitoneally with 50 mg/kg of 5-bromodeoxyuridine
(BrdU) (Sigma) 2 hours prior to liver harvest as described.12 BrdU incorporation, proliferating cell nuclear antigen (PCNA), and phosphorylated histone H3 were detected by immunohistochemistry MCE公司 in paraffin sections using anti-BrdU (Bu20a; Neomarkers), anti-PCNA (PC-10; Neomarkers), and anti-pH3 (Ser10; Cell Signaling) antibodies. Proliferation indexes were determined in triplicate and quantified under light microscopy by counting 10, randomly chosen, HPFs/section. Data are expressed as the percentage of BrdU, PCNA, or pH3 stained hepatocytes per total number of hepatocytes. MPO activity was evaluated in frozen tissue homogenized in an iced solution of 0.5% hexadecyltrimethyl-ammonium and 50 mmol/L of potassium phosphate buffer solution.4 After centrifugation the supernatants were mixed in a solution of hydrogen peroxide-sodium acetate and tetramethyl benzidine (Sigma).
As shown in Fig. 4C, E2F1 and Wip1 proteins were clearly down-regulated in miR-17-5p-overexpressing cells. To investigate whether Wip1 is responsible for the activation of the p38 MAPK-HSP27 signaling pathway, we overexpressed Wip1 in clone 1 cells. Figure 4D shows that the phosphorylation of HSP27and p38 MAPK is reduced when Wip1 is up-regulated. Together, these results
suggest that E2F1-dependent down-regulation of Wip1 is necessary for p38 MAPK-HSP27 pathway activation by miR-17-5p. To verify whether the p38 MAPK-HSP27 pathway contributes to miR-17-5p-facilitated cell growth, we performed CCK-8 assays to detect cell proliferation in vitro. In accordance with the results of the xenograft models in nude mice, clone 1 cells proliferated much faster than control cells, and
SB203580 was able to reduce this effect TGF-beta inhibitor in clone 1 cells. However, siRNAs against HSP27 had no effect (Fig. 5A). These results indicate that miR-17-5p facilitates cell growth, but this process does not involve the p38 MAPK-HSP27 pathway. To verify whether the p38 MAPK-HSP27 pathway contributes to the miR-17-5p-induced increase in HCC cell motility, we performed a series of assays to detect cell migration in vitro. Migration of Huh-7 cells, as assayed by a scrape assay, was significantly increased in clone 1 and 2 cells compared with pEZX-MR01-Huh-7 cells. Treatment Crizotinib ic50 with SB203580 or the siRNA against HSP27 abolished this effect (Fig. 5B). Transwell experiments were then performed with clone 1, clone 2, and pEZX-MR01-Huh-7 cells with or without SB203580 or siRNA against HSP27. As shown in Fig. 6A,B, miR-17-5p induced Huh-7 cell migration in the transwell experiments, and this migration was inhibited by SB203580 or siRNA against HSP27. Together, these results indicate that p38 MAPK and HSP27 play a crucial role in the control of Huh-7 cell migration. Similar phenomena were also observed in HepG2 cells (Supporting Fig. 1B). We investigated
the effect of miR-17-5p on the organization of the actin cytoskeleton in Huh-7 cells. The actin cytoskeleton was stained with phalloidin (Fig. 6C). miR-17-5p increased the cortical localization of actin, which was inhibited by the siRNA against HSP27 or treatment with SB203580, demonstrating the involvement of miR-17-5p-p38 MAPK-HSP27 in actin cytoskeleton remodeling. To address in greater detail the function of miR-17-5p in HCC cells medchemexpress and to avoid potential overexpression artifacts, we transfected clone 1 cells with 2′-Ome-modified antisense oligoribonucleotides against miR-17-5p. As shown in Fig. 7A, when introduced into clone 1 cells, phosphorylated HSP27, total HSP27, and phosphorylated p38 MAPK levels were reduced by 50%-60% compared with negative controls, indicating efficient up-regulation by miR-17-5p. Transfection with 2′-Ome-modified antisense oligoribonucleotides, but not the scrambled oligoribonucleotides, significantly reduced the migration of clone 1 cells (Fig.