[Daratumumab for the treatment of primary systemic amyloidosis: a multicenter retrospective analysis].

Objective: To analyze the efficacy and safety of Daratumumab for the treatment of primary AL light chain systemic amyloidosis. Methods: Twenty one patients who were diagnosed as primary AL light chain systemic amyloidosis and treated with Daratumumab from 7 centers were retrospectively analyzed. Daratumumab was administrated as first line therapy in seven patients and 14 patients with relapsed settings. Hematological response, safety and survival were analyzed. Results: All 7 patients achieved very good partial response (VGPR) or better with first-line application of daratumumab. Three patients died, and the other four achieved organ remission. Among 14 relapsed patients, 2 patients had a difference of free light chain (dFLC) less than 20 mg/L before treatment, and 9 with a dFLC of more than 50 mg/L. All patients reached partial response (PR) or better, including 4 patients with complete response (CR), 3 with VGPR and 2 with PR. The response rate was 100% in 3 patients with dFLC 20-50 mg/L at baseline. The organ remission rate was 50% in patients with heart involvement and 58.3% in patients with kidney impairment. The overall median follow-up period was 5.3 months, and 11 months in surviving patients. One patient died of severe infection and disseminated intravascular coagulation (DIC) with stable amyloidosis. One patient switched to other regimens because dFLC elevated but did not fulfill progressive disease after 2 year application. As to safety, no grade 3/4 infusion reaction developed, and grade 1 infusion reaction occurred in 3 cases during the first infusion. Lymphocytopenia was seen in 75% patients including grade 3 or more in 30% patients. Conclusion: Daratumumab is effective to eliminate serum free light chain in both newly diagnosed and relapsed patients with systemic amyloidosis.

Characteristics and prognostic significance of circRNA-100876 in patients with colorectal cancer.

OBJECTIVE: The purpose of this study was to explore the correlation between circRNA-100876 and the clinicopathological parameters of patients with colorectal cancer (Cc). PATIENTS AND METHODS: Quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect the circRNA-100876 expression in Cc tissues and cell lines. Overall survival analysis was carried out to explore the correlation between circRNA-100876 and the prognosis of Cc patients by Kaplan-Meier method and Log-rank method. Subsequently, Chi-square test was used to investigate the clinical significance of circRNA-100876 in the clinicopathological parameters of Cc patients. Moreover, the expression of circRNA-100876 was inhibited by small interfering RNAs (siRNAs) in loss-of-function assay. Finally, the invasion ability of Cc cells was determined by transwell assay. RESULTS: The results of this study manifested that circRNA-100876 was abnormally overexpressed in Cc tissues and cell lines, and the high expression of circRNA-100876 was clearly associated with the Clinical stage, T classification and Lymph node metastasis of Cc patients. Besides, Cc patients with high expression worsened overall survival. In addition, it was demonstrated that the inhibition of circRNA-100876 reduced the invasion ability of Cc cells. CONCLUSIONS: Acting as a tumor promoter, circRNA-100876 might be regarded as a new potential biomarker for the diagnosis and therapy of Cc.

Long non-coding RNA BCAR4 accelerates cell proliferation and suppresses cell apoptosis in gastric cancer via regulating MAPK/ERK signaling.

OBJECTIVE: As the fourth most common malignant tumor with high mortality rate, gastric cancer (GC) seriously threatens people's health and life quality worldwide. The aim of this study was to explore the functional role of long non-coding RNA (lncRNA) BCAR4 in GC. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay was used to detect the expression level of lncRNA BCAR4 in GC cell lines and tissues. Subsequently, cell counting kit-8 (CCK-8) assay, colony formation assay, and flow cytometry were recruited to investigate the role of lncRNA BCAR4 in the proliferation and apoptosis of GC cells, respectively. Western blotting was used to detect the protein expression level of mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) in GC. Besides, tumor formation assay was applied to examine the ability of lncRNA BCAR4 in vivo. RESULTS: LncRNA BCAR4 was highly expressed in both GC tissues and cell lines. CCK-8 assay, colony formation assay, and flow cytometry results indicated that up-regulated lncRNA BCAR4 significantly promoted cell proliferation and suppressed cell apoptosis in GC. Besides, over-expression of lncRNA BCAR4 could activate the MAPK/ERK signaling pathways. Tumor xenograft formation assay demonstrated that over-expression of lncRNA BCAR4 promoted tumor formation in vivo. CONCLUSIONS: LncRNA BCAR4 was proved significantly up-regulated in GC. Over-expression of lncRNA BCAR4 promoted cell proliferation and suppressed cell apoptosis in vitro and promoted tumor formation in vivo. Besides, Western blotting revealed that lncRNA BCAR4 played an oncogenic role in GC via regulating MAPK/ERK signaling.

Application of Virtual Anatomy Technology in Postmortem Examination of Medical Dispute Cases.

ABSTRACT: Objective To discuss the application value of CT scanning technology in cause of death determination of medical dispute cases. Methods From July 2017 to December 2018, postmortem CT imaging data of 12 medical dispute cases were collected. CT imaging diagnosis results and anatomy findings as well as differences between antemortem and postmortem CT diagnosis were compared. The advantages and disadvantages of CT routine tests of the cadavers in terms of the diagnosis of disease and damage were analyzed. Results The comparison between CT imaging diagnosis and anatomical findings showed that CT scans had advantages in the diagnosis of disease and damage with large differences in density changes, such as atelectasis, pneumonia, calcification, fracture and hemorrhage, etc. The comparison of CT diagnosis in antemortem and postmortem examination showed that the cadavers of medical dispute cases were well preserved and that postmortem CT scan was meaningful for the diagnosis of antemortem diseases. Conclusion Virtual anatomy technology has a relatively high application value in postmortem examination of medical dispute cases. It can provide effective information for the appraisers before the autopsy and can also provide a reference for cause of death analysis when the anatomy cannot be performed.

[Value of dual-energy CT-based volumetric iodine-uptake in the evaluation of chemotherapy efficacy in advanced gastric cancer].

Objective: To explore the value of dual-energy CT-based volumetric iodine-uptake (VIU) in the evaluation of chemotherapy efficacy in advanced gastric cancer. Methods: Inclusion criteria of subjects: (1) without previous systematic therapy; (2) with complete clinical information before and after chemotherapy; (3) without contraindications of chemotherapy. Exclusion criteria of subjects: (1) unfinished duration and times of chemotherapy; (2) unmeasurable primary lesions; (3) poor imaging quality or poor gastric filling. Clinical and image data of 52 patients with advanced gastric cancer who were diagnosed by pathology from gastroscopic biopsy, and needed chemotherapy evaluated by imaging and clinical information in the First Affiliated Hospital of Wenzhou Medical University from February 2017 to February 2018 were collected and analyzed. Of 52 patients, 38 were male and 14 were female with the median age of 65 (31-88) years old. All the patients underwent a dual-energy, dual phase-enhanced CT scanning before chemotherapy and after the third chemotherapy session. The parameters of the lesions measured before and after chemotherapy in portal vein phase were as follows: the maximum diameter (the largest diameter among those measured in the cross-sectional, coronal, and sagittal planes), average CT value (the regions of interest were manually pinpointed under cross-sectional planes with largest diameter of the tumor, which did not include regions less than 2 mm to the edge of the tumor) and VIU (lesion volume × iodine concentration). The change rates of maximum lesion diameter, average CT value and VIU before and after chemotherapy were calculated [(post-chemotherapy parameters-pre-chemotherapy parameters)/ pre-chemotherapy parameters]. The efficacy of chemotherapy was evaluated by RECIST 1.1 (the change of maximum tumor diameter after chemotherapy), Choi (the change of average CT value after chemotherapy) and VIU (the change of VIU after chemotherapy), respectively, which was categorized by complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). Patients with CR, PR, and SD were assigned to the effective group, while those with PD were classified as the ineffective group. Paired t - test or Wilcoxon signed ranks test was used to compare the changes of parameters before and after chemotherapy, whereas Spearman correlation analysis and Kappa test were used for the correlation analysis and the consistency test between the three evaluation criteria (Kappa≥0.75 indicated good consistency). Results: After chemotherapy, the average CT value [(74.01±16.75) HU vs. (81.06±15.87) HU, t=2.202, P=0.030] and median VIU (668.53×10(2) µg vs. 272.52×10(2) µg, Z=4.761, P<0.001) decreased significantly, while the difference of the maximum diameter was not statistically significant [(66.71±34.49) mm vs. (78.45±35.62) mm, t=1.708, P=0.091]. The median change rate of VIU (-53.33%) was greater than that of CT values (-5.75%) with significant difference (Z=-5.408, P<0.001). According to the RECIST 1.1 criteria, 47 patients (90.4%, including 19 with PR and 28 with SD) were effective and 5 patients (9.6%) were ineffective. According to the Choi criteria, 45 patients (86.5%, including 37 with PR and 8 with SD) were effective and 7 patients (13.5%) were ineffective. According to the VIU criteria, 46 patients (88.5%, including 41 with PR and 5 with SD) were effective and 6 patients (11.5%) were ineffective. Efficacy comparison among these three criteria showed no significant difference (χ(2)=0.377, P=0.828). As compared to RECIST 1.1 evaluation, the proportion of PR evaluated by Choi and VIU was significantly higher (χ(2)=16.861, P<0.001), whereas the proportion of SD was significantly lower (χ(2)=24.089, P<0.001). There was no significant difference in the proportions of PR and SD between VIU and Choi criteria (χ(2)=0.887, P=0.346). Consistency and correlation analysis showed that the VIU and Choi evaluation criteria presented the highest consistency and correlation (Kappa=0.912, P<0.001; r=0.916, P<0.001). Conclusion: VIU is a feasible parameter for the evaluation of chemotherapy efficacy in advanced gastric cancer, and may be more sensitive than the evaluation criteria based on maximum diameter or change of CT value in the tumor.

[Associations of parental thalassemia with preterm birth and low birth weight].

Objective: To investigate the association between the preterm birth and low birth weight and parental thalassemia. Methods: Pregnant women and their husbands receiving prenatal examination in local hospitals or maternal and child health centers in Jingxi and Debao in Guangxi from January to December 2017 were selected as study subjects. A total of 758 pregnant women with pregnancy outcomes and their husbands, who were both or alone diagnosed with thalassemia through thalassemia gene detection, were selected as case group and 758 pregnant women with pregnancy outcomes and their husbands, who were negative in thalassemia gene detection and hemoglobin electrophoresis test were selected as control groups. The case group were further divided into mother group, father group and both mother and farther group. Clinical and pregnancy outcome data of the study subjects were collected for the analysis on the association between parental thalassaemia and preterm birth or low birth weight by the independent sample t test, χ(2) test and Cox regression analysis. Results: The incidence of preterm birth in case group and control group was about 6.5% and 1.6% and the incidence of low birth weight in case group and control group was about 7.3% and 0.8%. After adjusting for possible confounding factors, Cox regression analysis results showed that mother suffering from thalassemia (aRR=3.45, 95%CI: 1.35-8.81, P=0.010), fathers suffering from thalassemia (aRR=4.93, 95%CI: 2.16-11.21, P<0.001) and both mother and farther suffering from thalassemia (aRR=5.13, 95%CI: 2.62-10.04, P<0.001) were associated with preterm birth. Mother suffering from thalassemia (aRR=12.98, 95%CI: 4.91-34.30, P<0.001), fathers suffering from thalassemia (aRR=9.40, 95%CI: 3.40-25.95, P<0.001) and both mother and farther suffering from thalassemia (aRR=10.74, 95%CI: 4.44-26.00, P<0.001) were associated with low birth weight. The newborn whose parent all suffered from thalassemia had higher risks for preterm birth (χ(2)=22.72, P<0.001)and low birth weight (χ(2)=34.03, P<0.001) compared with those only with mother or father suffering from thalassemia. Conclusion: Parental thalassaemia, including both sides and single side, might increase the risks of preterm birth and low birth weight for newborn, and the risks might be higher in newborn with both mother and father suffering from thalassaemia.

[Dose-response relationship between maternal hemoglobin concentration and preterm birth, in pregnant women].

Objective: To investigate the dose-response relationship between hemoglobin concentration and preterm birth, during pregnancy. Methods: With Zhuang ethnicity, a total of 12 780 pregnant women and their infants that admitted to Wuming、Pingguo、Jingxi、Debao、Longan and Tiandong hospitals, were recruited, in Guangxi Zhuang Autonomous Region, from January 2015 to December 2017. Non-conditional logistic regression method was used to analyze the effect of anemia on preterm birth during pregnancy. Dose-response relationship between hemoglobin concentration and preterm birth was explored, using the restrictive cubic spline model. Results: After excluding 2 053 pregnant women with hypertension or aged 35 years and over, results from the non-conditional logistic regression analysis showed that the risk of preterm birth in the anemia group was 1.29 times (OR=1.29, 95%CI: 1.04-1.59, P=0.019) of the non-anemia group in the first trimester. Data from the restricted cubic sample showed that there appeared nonlinear "L" dose-response relationship between hemoglobin concentration and preterm birth in the first trimester and "U" shape in the third trimester (non-linearity test P<0.001). Conclusion: There appeared nonlinear dose-response relationship between the hemoglobin concentration and preterm birth, both in the first and third trimesters.

[Clinical analysis of rituximab-induced interstitial lung disease].

Objective: To investigate the clinical characteristics, diagnosis-treatment points and prognosis of rituximab-induced interstitial lung disease (R-ILD), and to improve the recognition of this disease. Methods: The clinical data on 4 cases of R-ILD were analyzed retrospectively, and the related literatures were reviewed. The literature review was carried out respectively in Wanfang Data, CNKI and PubMed by October 2016 with"rituximab"and"interstitial lung disease"or"interstitial pneumonitis"as the search terms. Results: The all 4 patients received chemotherapy including rituximab, had respiratory symptoms after 2 to 5 cycles chemotherapy respectively. The chest computerized tomography findings of all 4 cases showed diffuse ground glass opacities. In all of the patients, the diagnosis of R-ILD was made and glucocorticoids therapy was initiated. After treatment, the clinical symptoms improved promptly and follow-up chest computerized tomography showed pulmonary lesions significantly resolved. Literature review found 48 articles (2 reviews, 6 original articles, 39 case reports and 1 other article) . 50 cases of R-ILD were collected and the chief complaint were dyspnea, cough and fever. The ground-glass pattern on the CT scan of the chest was the important feature of this disease. Therapy included glucocorticoids, discontinuation of rituximab, and any other clinically necessary measures. Conclusions: Rituximab can cause interstitial lung disease. The diagnosis relies on clinical manifestation and radiological findings. The good prognosis depends on prompt discontinuation of rituximab and treatment with glucocorticoids.

[Efficacy and safety of IA regimen containing different doses of idarubicin in de-novo acute myeloid leukemia for adult patients].

Objective: To investigate the efficacy and safety of IA regimen which contains idarubicin (IDA) 8 mg/m(2), 10 mg/m(2) or 12 mg/m(2) as induction chemotherapy for adult patients with de-novo acute myeloid leukemia (AML) . Methods: A total of 1 215 newly diagnosed adult AML patients, ranging from May 2011 to March 2015 in the First Affiliated Hospital of Soochow University and other 36 clinical blood centers in China were enrolled in the multicenter, single-blind, non-randomized, clinical controlled study. To compare the response rate of complete remission (CR) , adverse events between different dose idarubicin combined with cytarabine (100 mg/m(2)) as induction chemotherapy in newly diagnosed patients of adult AML. Results: Of 1 207 evaluable AML patients were assigned to this analysis of CR rate. The CR rates of IDA 8 mg/m(2) group, IDA 10 mg/m(2) group and IDA 12 mg/m(2) group were 73.6% (215/292) , 84.1% (662/787) and 86.7% (111/128) , respectively (P<0.001) . After adjusted for age, blast ratio of bone marrow, FAB classification and risk stratification, the odds ratios (95% CI) of IDA 10 mg/m(2) group and IDA 12 mg/m(2) group were 0.49 (0.34-0.70) and 0.36 (0.18-0.71) , as compared with the IDA 8 mg/m(2) group (P<0.001, P=0.003) . In the intermediate and favorable groups, CR rates was 76.5% (163/213) , 86.9% (506/582) and 86.1% (68/79) in different doses of IDA (P=0.007) . Interestingly, IA regimen with IDA 10 mg/m(2) was the only beneficial factor affecting CR in this group after adjusted for age, blast ratio of bone marrow and FAB classification[OR=0.47 (95% CI 0.31-0.71) , P<0.001]. CR rates in adverse group was 50.0% (18/36) , 60.6% (43/71) and 81.8% (18/22) respectively (P=0.089) . However, the odds ratios (95% CI) of IDA 12 mg/m(2) when compared with the IDA 8 mg/m(2) was 0.22 (0.06-0.80) , after adjusted for age, blast ratio of bone marrow and FAB classification. The median time (days) of neutrophil count less than 0.5×10(9)/L in IDA 8 mg/m(2) group, IDA 10 mg/m(2) group and IDA 12 mg/m(2) group were 14 (11-18) , 15 (11-20) and 18 (14-22) , respectively (P=0.012) and of platelet count lower than 20×10(9)/L were 14 (7-17) , 15 (11-20) and 17 (15-21) , respectively (P=0.001) . The incidences of lung infection in the three groups were 9.8%, 13.5% and 25.2%, respectively (P<0.001) . Conclusions: For young adult patients (aged 18-60 years) with AML in China, intensifying induction therapy with idarubicin 10 mg/m(2) is clinically superior to IDA 8 mg/m(2) and IDA 12 mg/m(2) in favorable intermediate AML subgroup. However, idarubicin 12 mg/m(2) is more suitable to adverse AML subgroup.

Influence of interleukin-1ß and interleukin-6 gene polymorphisms on the development of acute pancreatitis.

We investigated the association between 3 main proinflammatory cytokines [interleukin (IL)-1ß and IL-6] and the risk of acute pancreatitis. Polymerase chain reaction-restriction fragment length polymorphism was used to genotype IL-1ß+3954 C/T (rs1143634) and IL-1ß-511 C/T (rs16944) and IL-6 -174 G/C (rs1800795) and IL-6 -634 C/G (rs1800796). The genotype distributions of IL-1ß+3954 C/T (rs1143634) and IL-1ß-511 C/T (rs16944) and IL-6 -174 G/C (rs1800795) and IL-6 -634 C/G (rs1800796) were in Hardy-Weinberg equilibrium for the control group. Multivariate regression analyses showed that subjects carrying the rs1143634 TT genotype had a significantly increased risk of acute pancreatitis, with an adjusted odds ratio (95% confidence interval) of 2.11 (1.03-4.51). Subjects carrying the IL-1ß rs1143634 TT genotype had a significantly increased risk of acute pancreatitis in our Chinese population.

Identification of RASSF1A modulated genes in nasopharyngeal carcinoma.

RASSF1A is a tumor suppressor gene on 3p21.3 frequently inactivated by promoter hypermethylation in nasopharyngeal carcinoma (NPC). To identify RASSF1A target genes in NPC, we have investigated the expression profile of the stable RASSF1A transfectants and controls by high-density oligonucleotide array. A total of 57 genes showed differential expression in the RASSF1A-expressing cells. These RASSF1A target genes were involved in multiple cellular regulatory processes such as transcription, signal transduction, cell adhesion and RNA processing. The RASSF1A-modulated expression of eight selected genes with the highest fold changes (ATF5, TCRB, RGS1, activin betaE, HNRPH1, HNRPD, Id2 and CKS2) by RASSF1A was confirmed in both stable and transient transfectants. Compared with the RASSF1A transfectants, an inverse expression pattern of activin betaE, Id2 and ATF5 was shown in the immortalized nasopharyngeal epithelial cells treated with siRNA against RASSF1A. The findings imply that the expression of activin betaE, Id2 and ATF5 was tightly regulated by RASSF1A and may associate with its tumor suppressor function. Strikingly, overexpression of Id2 is common in NPC and RASSF1A-induced repression of Id2 was mediated by the overexpression of activin betaE. The results suggest a novel RASSF1A pathway in which both activin betaE and Id2 are involved.

Epstein-Barr virus latent membrane protein 1 (LMP1) upregulates Id1 expression in nasopharyngeal epithelial cells.

Nasopharyngeal carcinoma is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded LMP1 has cell transformation property. It suppresses cellular senescence and enhances cell survival in various cell types. Many of the downstream events of LMP1 expression are mediated through its ability to activate NF-kappaB. In this study, we report a novel function of LMP1 to induce Id1 expression in nasopharyngeal epithelial cells (NP69) and human embryonal kidney cells (HEK293). The Id1 is a basic helix-loop-helix (bHLH) protein and a negative transcriptional regulator of p16(INK4a). Expression of Id1 facilitates cellular immortalization and stimulates cell proliferation. With the combination of both specific chemical inhibitors and genetic inhibitors of cell signaling, we showed that induction of Id1 by LMP1 was dependent on its NF-kappaB activation domain at the carboxy-terminal region, CTAR1 and CTAR2. Induction of Id1 by LMP1 may facilitate clonal expansion of premalignant nasopharyngeal epithelial cells infected with EBV and may promote their malignant transformation.

Combined in vivo bioluminescence and fluorescence imaging for cancer gene therapy.

Nasopharyngeal carcinoma is intimately associated with the Epstein-Barr virus (EBV), which we have exploited therapeutically by constructing an EBV-specific synthetic enhancer sequence, within an adenoviral vector, denoted as adv.oriP. The achievement of tumor targeting provides therapeutic potential when delivered systemically, which could impact on distant metastases. We demonstrate here the feasibility and potential utility of combined, minimally invasive in vivo bioluminescence and fluorescence imaging to monitor adenoviral infection of subcutaneous C666-1 nasopharyngeal xenograft tumors stably expressing the DsRed2 gene. Fluorescence imaging was used to monitor the location and size of the C6661.DsRed2 tumors, whereas bioluminescence imaging demonstrated the distribution and specificity of a transcriptionally targeted adenoviral vector, adv.oriP.fluc, expressing the firefly luciferase gene. Fluorescence, bioluminescence, and photographic images were aligned using grids to examine colocalization of adenovirus and tumors. Bioluminescence and fluorescence co-localized in 92% (11/12) of tumors at 24 hr and 100% (12/12) at 96 hr after adv.oriP.fluc (10(9) ifu) was administered intravenously. Nonspecific luciferase signal was detected in the liver area. The combined imaging was therefore successful in monitoring the uptake of systemically administered adenovirus in implanted tumors. This may ultimately lead to an effective noninvasive method to monitor the response of metastases to adenoviral gene therapy.

Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes.

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially in southern China. One of the most striking features of this disease is its close relationship with Epstein-Barr Virus (EBV). However, to date there is no direct study on the mechanisms involved in the role of EBV in the tumorigenesis of NPC, largely due to lack of an experimental model. Available hypotheses on the association between EBV and NPC are generated from non-nasopharyngeal epithelial cell systems such as human keratinocytes or mouse epithelial cells, which may not truly represent the biological properties of nasopharyngeal epithelial (NP) cells. In this study, we report the establishment of two immortalized NP cell lines, NP69SV40T and NP39E6/E7, using SV40T and HPV16E6/E7 oncogenes. We found that NP60SV40T and NP39E6/E7 cell lines not only maintained many characteristics of normal NP cells (i.e. keratin profile and responsive to TGFbeta inhibition) but also highly responsive to one of the EBV encoded genes, LMP1. Comparative genome hybridization (CGH) analysis showed that these two cell lines contained multiple genetic alterations, some of which have been described in NPC. The immortalized NP cell lines are non-tumorigenic and exhibit anchorage-dependent growth. These cell lines may provide a possible cell model system for studying the mechanisms involved in the tumorigenesis of NPC.

Identification of downstream target genes of latent membrane protein 1 in nasopharyngeal carcinoma cells by suppression subtractive hybridization.

Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China and is closely associated with infection of Epstein-Barr virus (EBV). The EBV encoded latent membrane protein 1 (LMP1) is frequently detected in NPC and may play a role in its pathogenesis. Previous studies have shown that LMP1 transformed rodent fibroblasts and altered growth properties in B cells and epithelial cells. However, the pathological role of LMP1 in NPC cells is still poorly understood. In order to investigate the downstream target genes of LMP1 in NPC cells, suppression subtractive hybridization was used to clone and identify the genes differentially expressed in a LMP1 expressing NPC cell line, CNE-2. Two subtractive cDNA libraries were constructed: one enriched for the genes upregulated by LMP1 and one was for the genes downregulated by LMP1. A total of 192 clones were screened by reverse Northern blotting. Fourteen of them were confirmed to be overexpressed while eight of them were suppressed. The upregulation of integrin alpha6, laminin 5gamma2, TAP1 and downregulation of p54nrb, RACK1 and p66Shc were further confirmed in three sets of LMP1 expressing NPC cell lines. The expression profiles of differentially expressed genes identified in this study suggest a role of LMP1 in promotion of cell survival and facilitation of tumor invasion.

High frequency of promoter hypermethylation of RASSF1A in nasopharyngeal carcinoma.

We have investigated the genetic and epigenetic changes of a newly isolated tumor suppressor gene on 3p21.3, RASSF1A, in nasopharyngeal carcinoma (NPC). Four xenografts, four cell lines and 21 primary tumors were examined. Promoter hypermethylation of the 5'CpG island of RASSF1A was detected in 4 of 4 (100%) xenografts, in 3 of 4 (75%) cell lines, and in 14 of 21 (66.7%) primary tumors but not in the normal nasopharyngeal epithelia. Mutations were found in 2 of 21 (9.5%) primary tumors. In the cell lines and xenografts with extensive methylation, no RASSF1A gene expression was found. After treatment with 5'-aza-2'deoxycytidine, reexpression and demethylation of the RASSF1A gene were detected in a NPC cell line. These findings suggest that promoter hypermethylation may participate in the transcriptional inactivation of the RASSF1A gene in NPC. The high incidence of RASSF1A alterations suggest that it is the critical target gene on chromosome 3p21.3 involved in the development of NPC.

Dextran sedimentation in a semi-closed system for the clinical banking of umbilical cord blood.

BACKGROUND: The results of current processing procedures for reducing volume and recovering HPCs from umbilical cord blood (UCB) before cryopreservation vary. STUDY DESIGN AND METHODS: Dextran was added to bags containing UCB, followed by sedimentation for 30 minutes. The processed UCB was then frozen. RBCs, nucleated cells, MNCs, CD34+ cells, CFUs and long-term culture-initiating cells (LTC-ICs), viability, and sterility were evaluated. Fractionations in ficoll-hypaque and hydroxyethyl starch (HES) were also run in parallel for comparison. RESULTS: The nucleated cell (NC) recovery and RBC depletion were 86.1 percent and 94.3 percent, respectively (n = 50). Sedimentation with dextran also enabled the recovery of 80.7 percent MNCs and 82.6 percent CD34+ cells (n = 30). Postsedimentation samples displayed no impairment of CFU growth (n = 42, 108.7% CFU-C, 104.6% CFU-GEMM, 107% CFU-GM, and 95.7% BFU-E). Long-term cultures on five paired samples before and after sedimentation generated similar numbers of CFU-C each week (p = 0.88). Limiting dilution analysis of 12 paired pre/postsedimentation samples showed comparable median proportions of LTC-ICs (1/6494 vs. 1/5236; p = 0.18). The cell viability of 24 samples of thawed UCB after sedimentation was 90.3 percent (77.5-96%) and the recovery of CFU-C, CFU-GEMM, CFU-GM, and BFU-E of 11 postsedimentation samples was 93.4 percent, 84.9 percent, 92.3 percent, and 83.4 percent, respectively. NC recovery was significantly higher after treatment with dextran than with ficoll-hypaque (n = 30; 88.5% vs. 29.1%; p<0.005) and HES treatment (n = 21; 88.5% vs. 76.4%; p = 0.004). However, MNCs, CD34+ cells, CFUs, LTC-ICs, and RBCs were comparable. Two cycles of dextran sedimentation recovered 93.9 percent of NCs with cell viability of 98.6 percent (96.5-100%), whereas 11.7 percent of RBCs were retained (n = 20). The final yield volume was 33.5 (28-41) mL. CONCLUSION: In a semi-closed system, dextran sedimentation enabled volume reduction of UCB without significant quantitative and qualitative losses of HPCs.