Sustained nontumorigenic phenotype correlates with a largely stable chromosome content during long-term culture of the human keratinocyte line HaCaT

Altered growth and differentiation and a highly abnormal karyotype are generally believed to be indicators for tumorigenic conversion of human cells. Inactivation of TP53 is supposedly one possible mechanism for accelerated genetic aberrations via reduced control of the genetic integrity. To examine the significance of this functional relationship, we investigated the long-term development of the spontaneously immortalized human skin keratinocyte line HaCaT, carrying UV-specific mutations in both alleles of the TP53 tumor suppressor gene. During >300 passages, proliferation, clonogenicity, and serum-independent growth potential increased. In addition, HaCaT cells gained anchorage independence and at late passages showed reduced differentiation. Karyotypic analysis up to passage 225 revealed a high frequency of translocations and deletions, with a particular increase during passages 30 and 50. Nevertheless, the HaCaT cells remained nontumorigenic when injected subcutaneously, and noninvasive in surface transplants in nude mice. By comparative genomic hybridization, we confirmed the karyotypically identified phase of increased chromosomal aberrations between passages 30 and 50. However, before and thereafter, the CGH profiles of the individual chromosomes were largely unchanged, demonstrating that those translocations—also maintained in later passages—did not cause a gross chromosomal imbalance. Thus, our data suggest that multiple changes often correlated with a “transformed phenotype,” including extensive karyotypic alterations and mutational inactivation of TP53, are well compatible with a nontumorigenic phenotype of the HaCaT cells, and that preserved chromosomal balance may be crucial for this stability during long-term propagation. Genes Chromosom. Cancer 19:201–214, 1997. © 1997 Wiley-Liss, Inc.     

移植胚鼠神经干细胞对大鼠脊髓损伤后红核神经元损伤的影响

目的研究神经干细胞(NSCs)移植对大鼠脊髓损伤(SCI)后红核神经元的作用。方法5-溴脱氧尿嘧啶核苷(BrdU)法标记处于对数生长期的NSCs,采用电控脊髓损伤打击装置制作大鼠脊髓损伤模型。实验分为3组:NSCs组、SCI组和假手术组(Sham组)。SCI后3 d进行NSCs移植,用免疫组化法观察移植细胞的存活及迁移情况,用辣根过氧化物酶(HRP)逆行示踪技术标记红核神经元,并用四甲基联苯胺(TMB)呈色反应显示红核脊髓束神经元的存活情况,用行为学(BBB)评分法观察大鼠瘫痪肢体的恢复情况。结果在损伤脊髓区域可检测到BrdU标记的阳性NSCs,中脑HRP标记红核神经元数目明显多于SCI组(P<0.01),BBB评分亦明显高于SCI组(P<0.01)。结论体外培养的胚胎大鼠NSCs在移植到脊髓损伤区域后可存活和迁移,对SCI后中脑红核神经元具有保护作用,从而促进了大鼠肢体功能的恢复。     

The role of size, sequence and haplotype in the stability of FRAXA and FRAXE alleles during transmission

Factors involved in the stability of trinucleotide repeats during transmission were studied in 139 families in which a full mutation, premutation or intermediate allele at either FRAXA or FRAXE was segregating. The transmission of alleles at FRAXA, FRAXE and four microsatellite loci were recorded for all individuals. Instability within the minimal and common ranges (0-40 repeats for FRAXA, 0-30 repeats for FRAXE) was extremely rare; only one example was observed, an increased in size at FRAXA from 29 to 39 repeats. Four FRAXA and three FRAXE alleles in the intermediate range (41-60) repeats for FRAXA, 31-60 for FRAXE) were unstably transmitted. Instability was more frequent for FRAXA intermediate alleles that had a tract of pure CGG greater than 37 although instability only occurred in two of 13 such transmissions: the changes observed were limited to only one or two repeats. Premutation FRAXA alleles over 100 repeats expanded to a full mutation during female transmission in 100% of cases, in agreement with other published series. There was no clear correlation between haplotype and probability of expansion of FRAXA premutations. Instability at FRAXA or FRAXE was more often observed in conjunction with a second instability at an independent locus suggesting genomic instability as a possible mechanism by which at least some FRAXA and FRAXE mutations arise.      

Different modulation by histamine of IL-4 and interferon-gamma (IFN-γ) release according to the phenotype of human Th0, Th1 and Th2 clones

Histamine, an important inflammatory mediator in allergic diseases and asthma, has been reported to have modulator effects on T cells, suggesting that the bronchial microenvironment may regulate the function of resident T cells. We examined the effect of histamine on the release of the Th2-associated cytokines IL-4 and IL-5 and the Th1-associated cytokine IFN-γ by 30 CD4⁺ T cell clones from peripheral blood or bronchial biopsy of one atopic subject. Based on the IL-4/IFN-γ ratio, the clones were ascribed to the Th2 (ratio >1), Th0 (ratio 0.1 and 1) or Th1 (ratio <0.1) phenotype. Histamine inhibited IFN-γ production by Th1-like cells (P<0.02, Kruskall–Wallis), especially from bronchial biopsy, but had no effect on IL-4 release. Regarding Th0 clones, histamine inhibited IL-4 production (P<0.02) in a dose-dependent manner and slightly inhibited IFN-γ production, but had no effect on Th2-like cells. Histamine had a heterogeneous and insignificant effect on IL-5 production. The H₂-receptor antagonist ranitidine completely reversed the inhibition of IL-4 and IFN-γ production, whereas the agonist dimaprit mimicked this effect. In contrast, H₁- and H₃-receptor agonists and antagonists had no significant effect. These data demonstrate that histamine has different effects on IL-4 and IFN-γ release by T helper cells according to their phenotype via H₂-receptors. This study extends the immunomodulatory effects of histamine which may contribute to the perpetuation of airway inflammation in asthma.     

Nosocomial respiratory syncytial virus infection in Canadian pediatric hospitals: a Pediatric Investigators Collaborative Network on Infections in Canada Study

OBJECTIVE: To determine nosocomial transmission of respiratory syncytial virus (RSV) in Canadian pediatric hospitals, outcomes associated with nosocomial disease, and infection control practices. DESIGN: A prospective cohort study in the 1992 to 1994 winter respiratory seasons. SETTING: Nine Canadian pediatric university-affiliated hospitals. PARTICIPANTS: Hospitalized children with symptoms of lower respiratory tract infection (at least one of cough, wheezing, dyspnea, tachypnea, and apnea) and RSV antigen identified in a nasopharyngeal aspirate. RESULTS: Of 1516 children, 91 (6%) had nosocomial RSV (NRSV), defined as symptoms of lower respiratory tract infection and RSV antigen beginning >72 hours after admission. The nosocomial ratio (NRSV/[com-munity-acquired RSV {CARSV})] + NRSV) varied by site from 2.8% to 13%. The median length of stay attributable to RSV for community-acquired illness was 5 days, but 10 days for nosocomial illness. Four children with NRSV (4. 4%) died within 2 weeks of infection, compared with 6 (0.42%) with CARSV (relative risk = 10.4, 95% confidence interval: 3.0, 36.4). All sites isolated RSV-positive patients in single rooms or cohorted them. In a multivariate model, no particular isolation policy was associated with decreased nosocomial ratio, but gowning to enter the room was associated with increased risk of RSV transmission (incidence rate ratio 2.81; confidence interval: 1.65, 4.77). CONCLUSIONS: RSV transmission risk in Canadian pediatric hospitals is generally low. Although use of barrier methods varies, all sites cohort or isolate RSV-positive patients in single rooms. Children with risk factors for severe disease who acquire infection nosocomially have prolonged stays and excess mortality.     

Cell tracking technologies for acute ischemic brain injury

Stem cell therapy has showed considerable potential in the treatment of stroke over the last decade. In order that these therapies may be optimized, the relative benefits of growth factor release, immunomodulation, and direct tissue replacement by therapeutic stem cells are widely under investigation. Fundamental to the progress of this research are effective imaging techniques that enable cell tracking in vivo. Direct analysis of the benefit of cell therapy includes the study of cell migration, localization, division and/or differentiation, and survival. This review explores the various imaging tools currently used in clinics and laboratories, addressing image resolution, long-term cell monitoring, imaging agents/isotopes, as well as safety and costs associated with each technique. Finally, burgeoning tracking techniques are discussed, with emphasis on multimodal imaging.     

VJ REARRANGEMENTS OF THE TCRγ LOCUS IN PERIPHERAL T-CELL LYMPHOMAS: ANALYSIS BY POLYMERASE CHAIN REACTION AND DENATURING GRADIENT GEL ELECTROPHORESIS

Using Southern blotting for the diagnosis of clonality in peripheral T-cell lymphomas (PTCLs), analysis of the T-cell receptor (TCR) γ gene rearrangement was shown to be more informative than that of the TCR β gene rearrangement. In order to amplify every VJγ rearrangement, a polymerase chain reaction (PCR) procedure using newly designed GC-clamp primers has been developed. All primers can be mixed in a single multiplex PCR. PCR products are analysed by denaturing gradient gel electrophoresis (DGGE), providing tumour-specific imprints inasmuch as the procedure characterizes N sequence polymorphism at the VJ junctions. In a series of 30 PTCL cases, the PCR procedure demonstrated 27 cases to be clonally rearranged and failed in three cases. PCR was more accurate than Southern blotting, showing 47 rearranged γ alleles, four of which were undetectable on the Southern blot. When lymphomas were studied at different sites and at relapse, the DGGE pattern remained unchanged. In PTCL, the proposed PCR is helpful for the diagnosis and staging of the disease and should improve the follow-up monitoring. The undetectability of clonal rearrangements in a few cases is discussed in the light of concepts of lymphomagenesis and T-cell differentiation.