Establishment and characterization of a continuous human chondrosarcoma cell line,ch-2879: comparative histologic and genetic studies with its tumor of origin

Chondrosarcomas are malignant cartilage-forming tumors that represent the second most common malignant solid tumor of bone. These biologically poorly understood neoplasms vary considerably in clinical presentation and biologic behavior. Chemotherapy and radiation therapy are generally ineffective. Here we describe the establishment and characterization of a new human chondrosarcoma cell line named ch-2879, and we compare the cell line with its tumor of origin. The cell line was established from a recurrent grade 3 chondrosarcoma of the chest wall and characterized by growth kinetics and morphologic studies. Immunocytochemistry and RT-PCR were performed to examine the expression of cartilage-specific phenotypes. Genetic characterization was performed using cytogenetics, fluorescence in situ hybridization, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control, amplification of MDM2, CDK4, and Cyclin D1, and mutations in the p53 gene. ch-2879 cells were subcultured for more than 80 passages. They expressed vimentin, HNK-1, HBA-71, Ki-67, cyclin D1, Fli-1, S-100, p21, p27, and p53 and were negative for cytokeratin, EMA, p14, p16, MDM2, Rb, and c-erb-b2 antigens. Cytogenetically the recurrent tumor showed a hyperhaploid karyotype with clonal numerical and structural abnormalities. The sole structural abnormality was a chromosome derivative of a t(1;21) translocation. The cell line at passage 3 showed two populations: the hyperhaploid and an exactly duplicated, hypotriploid population. After the 18th passage, only the hypotriploid population was present. The cells expressed collagen 2. Molecular comparison of the primary and recurrent tumor evidenced an in vivo molecular change consisting of a deletion of 9p21 genes in the recurrence, probably caused by a selection process. Because of its gene expression profile, including expression of genes implicated in chondrogenesis in uncoated plastic dishes, this cell line may prove useful for cellular and molecular studies as well as studies of chondrosarcoma characterization and treatment.     

Dipolar sources of the early scalp somatosensory evoked potentials to upper limb stimulation Effect of increasing stimulus rates

Brain electrical source analysis (BESA) of the scalp electroencephalographic activity is well adapted to distinguish neighbouring cerebral generators precisely. Therefore, we performed dipolar source modelling in scalp medium nerve somatosensory evoked potentials (SEPs) recorded at 1.5-Hz stimulation rate, where all the early components should be identifiable. We built a four-dipole model, which was issued from the grand average, and applied it also to recordings from single individuals. Our model included a dipole at the base of the skull and three other perirolandic dipoles. The first of the latter dipoles was tangentially oriented and was active at the same latencies as the N20/P20 potential and, with opposite polarity, the P24/N24 response. The second perirolandic dipole showed an initial peak of activity slightly earlier than that of the N20/P20 dipolar source and, later, it was active at the same latency as the central P22 potential. Lastly, the third perirolandic dipole exaplaining the fronto-central N30 potential scalp distribution was constantly more posterior than the first one. In order to evaluate the effect of an increasing repetition frequency on the activity of SEP dipolar sources, we applied the model built from 1.5-Hz SEPs to traces recorded at 3-Hz and 10-Hz repetition rates. We found that the 10-Hz stimulus frequency reduced selectively the later of the two activity phases of the first perirolandic dipole. The decrement in strength of this dipolar source can be explained if we assume that: (a) the later activity of the first perirolandic dipole can represent the inhibitory phase of a “primary response”; (b) two different clusters of cells generate the opposite activities of the tangential perirolandic dipole. An additional finding in our model was that two different perirolandic dipoles contribute to the centro-parietal N20 potential generation. Received: 5 August 1997 / Accepted: 26 November 1997     

Alterations of 9p in squamous cell carcinoma and adenocarcinoma of the lung: association with smoking, TP53, and survival

Tobacco smoke is well recognized as the major etiological contributor to lung cancer, yet the relationship between tobacco smoke exposure and a specific pattern of molecular abnormalities at somatic loci is less well characterized. We analyzed 100 primary tumors from patients undergoing surgical resection of squamous cell carcinoma and adenocarcinoma of the lung for loss of heterozygosity (LOH) and homozygous deletions at two microsatellite markers in a recombinogenic region of 9p13. We describe the relationship of alterations at these markers with tumor characteristics (both clinical and molecular), patient demographics, survival, and measures of tobacco-smoke exposure. Homozygous deletions in this region occurred in 25% (21/85) and LOH in 33% (28/85) of informative tumors examined. These alterations occurred more often in tumors with intense TP53 protein staining by immunohistochemistry, suggesting that inactivation of the TP53 pathway may contribute to these LOH events. Duration of smoking was greatest in patients with the homozygous deletion, intermediate in patients with LOH, and shortest in patients whose tumor did not demonstrate loss in these markers. Unexpectedly, LOH at 9p13 was a significant predictor of improved survival in patients, while the homozygous deletion was associated with the poorest patient survival. Together, these results suggest that TP53 alteration and long-term tobacco smoke exposure may contribute to genetic alterations at 9p13, and that the mechanism and biologic consequences of allele loss reflect individual biologic differences that determine the extent of loss (LOH or homozygous deletion), such that those patients with the deletion of this region face a more aggressive and deadly disease.     

The B-cell activation pathway in human systemic lupus erythematosus: Imbalancedin vitro production of lymphokines and association with serum analytical findings

Recent knowledge of B-lymphocyte physiology has clarified the role of T cell-derived lymphokines in clonal proliferation and differentiation of B-cell responses. Lymphokine production was analyzed in 19 systemic lupus erythematosus (SLE) patients and sex- and age-matched controls in relation to clinical activity and steroid treatment. Whenin vitro production of interleukin-2 (IL-2) and B-cell growth factor (BCGF) was tested, both activities were found to be diminished in the group of patients (P<0.01), while B-cell differentiation factor (BCDF) activity was higher in this group with respect to normal controls (P<0.01). Interestingly enough, thisin vitro BCDF synthesis was positively correlated with clinical activity regardless of low-dose steroid treatment. A correlation was also found between BCDF production and the levels of IgG (r=0.64,P<0.01), anti-DNA antibodies (r=0.52,P<0.05), and the IgG/IgM ratio (r=0.7,P<0.01) in serum. Implications of these abnormal T-lymphocyte functions in SLE with respect toin vivo B-cell function are discussed.     

Prevalence of transmitted HIV-1 drug resistance and the role of resistance algorithms: data from seroconverters in the CASCADE collaboration from 1987 to 2003

OBJECTIVES: To examine factors influencing the rate of transmitted drug resistance (TDR) among seroconverters, with particular emphasis on 3 widely used genotypic drug resistance algorithms. METHODS: The study used data from CASCADE (Concerted Action on Seroconversion to AIDS and Death in Europe), a collaboration of seroconverter cohorts in Europe and Canada. Genotypic resistance data were derived within 18 months of the last seronegative test or date of laboratory evidence of acute infection and before the initiation of antiretroviral therapy. The Stanford algorithm was used to analyze each individual's nucleotide sequence. A multivariate logistic model was used to assess independent relationships between the presence of TDR and exposure category, sex, age at seroconversion, and year of seroconversion. The paper also describes 3 alternative definitions of resistance: the Stanford algorithm, the key resistance mutations defined by the International AIDS Society, and the Agence Nationale de Recherches sur le Sida (ANRS) algorithm. RESULTS: Forty-five of 438 patients (10.3%) seroconverting between 1987 and 2003 were infected with a drug-resistant HIV-1 variant. Forty patients (9.1%) showed resistance mutations to only 1 class of antiretroviral drugs, 2 (0.5%) to 2 classes, and 3 (0.7%) to 3 classes of antiretroviral therapy. It was suggested that individuals seroconverting later in calendar time were more likely to have TDR (relative risk 3.89 and 95% CI: 0.84 to 18.02, and relative risk 4.69 and 95% CI: 1.03 to 21.31, for 1996-1999 and 2000-2003, respectively, compared with pre-1996; P trend = 0.08). This trend was apparent regardless of the definition of TDR used. The total estimated proportion of individuals with TDR varied between 10.3% and 15.5% according to which definition was used. CONCLUSIONS: Evidence was found for the rise of TDR over time. A specific definition of what constitutes TDR rather than a simple list of mutations is needed.     

Principle and practice of in vitro fertilization. Role in the treatment of female sterility

In vitro fertilization (IVF) and embryo transfer (ET) appear to constitute a revolution in the reproductive sciences rather than merely a new technique in the treatment of sterility. Principle of IVF: IVF accomplishes in vitro the process than normally occurs in the oviduct between the ovulation of oocyte II and embryo implantation in the endometrium. This 4 day period (under normal conditions in the woman) involves 4 steps: recovery, fertilization, segmentation and transport. Performance of IVF: Recovery of the oocytes: The oocytes are recovered under celioscopic or echographic observation when they have completed cytoplasmic maturation and their first meiosis. A precise monitoring of ovulation (spontaneous or induced) should be performed using estrogen and LH assays. IVF provides an opportunity for evaluating the methods of ovulation induction and monitoring, as a function of the maturation of the oocytes recovered. Fertilization: When the oocyte has achieved maturing after several hours of incubation, fertilization is obtained 15 h contact with washed and capacitated spermatozoa (100 000/ml). This step is highly dependent on gametocyte quality: oocyte maturity and fecundity of spermatozoa, which can be estimated from the percentage of survival in the insemination medium. Segmentation occurs in culture at pH 7.28 in the presence of 5 per cent CO2 at 37 degrees C (pronucleus 15th, 2 blastomeres 26 h, 4-8 blastomeres 52 h). Embryo transfer is carried out when an embryo is present at 52 h. Only 1/10 of the embryo transfers result in successful implantation, which depends on the quality of the embryo; the quality can only be indirect criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erratum: Neurofibromatosis type 1 (NF1): Identification of eight unreported mutations in NF1 gene in Italian patients

In the original version of this article, the title was incorrect. Please find the correct title given here. The publisher deeply regrets this error. The original article to which this Erratum refers was published in Human Mutation 22:179–180 Human Mutation(2003) 22(2) 179–180     

Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda Is IRAK-4 dependent and redundant for protective immunity to viruses

Five TLRs are thought to play an important role in antiviral immunity, sensing viral products and inducing IFN-alpha/beta and -lambda. Surprisingly, patients with a defect of IRAK-4, a critical kinase downstream from TLRs, are resistant to common viruses. We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. In contrast, IFN-alpha/beta and -lambda were induced normally by TLR-3 and TLR-4 agonists. Moreover, IFN-beta and -lambda were normally induced by TLR-3 agonists and viruses in IRAK-4-deficient fibroblasts. We further show that IFN-alpha/beta and -lambda production in response to 9 of 11 viruses tested was normal or weakly affected in IRAK-4-deficient blood cells. Thus, IRAK-4-deficient patients may control viral infections by TLR-3- and TLR-4-dependent and/or TLR-independent production of IFNs. The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-alpha/beta and -lambda is strictly IRAK-4 dependent and paradoxically redundant for protective immunity to most viruses in humans.     

Heat shock proteins (HSPs) 27, 72 and 73 in normal and pre-ulcerative mucosa of the gastric pars oesophagea in swine

Heat shock proteins (HSPs), known to play a key role in cellular homeostasis, may also play a role in the defensive mechanisms of gastric mucosa. By means of appropriate immunohistochemical and immunobiochemical techniques, the expression of HSP27, HSP72 and HSP73 within the epithelium of normal and pre-ulcerative (hyperkeratinized) mucosa of the pars oesophagea of abattoir pigs was assessed. In normal mucosa, HSP72 and HSP73 expression was mainly limited to the basal epithelial cell layer, whereas HSP27 expression was consistently detected within the superficial epithelial cell layers. In hyperkeratinized mucosa, HSP72 and HSP73 immunoreactivity appeared to be more widespread, becoming very intense within epithelial cells affected by hydropic degeneration. Hyperkeratinized mucosa also showed HSP27 immunoreactivity, which was particularly intense in epithelial areas affected by hydropic degeneration. Western blot analysis confirmed HSP27, HSP72 and HSP73 expression in normal and in pre-ulcerative mucosa of the pars oesophagea. Semi-quantitative analysis showed that for all three HSPs the immunoreactivity was more intense in pre-ulcerative mucosa than in normal mucosa. The different expression patterns observed may have functional significance; further studies are needed, however, to define the role of HSPs in swine oesophagogastric lesions, the aetiology and pathogenesis of which are largely unknown.