Phenotypic characterization of human chondrocyte cell line C-20/A4: a comparison between monolayer and alginate suspension culture

DNA microarray analysis was used to investigate the molecular phenotype of one of the first human chondrocyte cell lines, C-20/A4, derived from juvenile costal chondrocytes by immortalization with origin-defective simian virus 40 large T antigen. Clontech Human Cancer Arrays 1.2 and quantitative PCR were used to examine gene expression profiles of C-20/A4 cells cultured in the presence of serum in monolayer and alginate beads. In monolayer cultures, genes involved in cell proliferation were strongly upregulated compared to those expressed by human adult articular chondrocytes in primary culture. Of the cell cycle-regulated genes, only two, the CDK regulatory subunit and histone H4, were downregulated after culture in alginate beads, consistent with the ability of these cells to proliferate in suspension culture. In contrast, the expression of several genes that are involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin, biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to suspension culture in alginate. Also, nexin-1, vimentin, and IGFBP-3, which are known to be expressed by primary chondrocytes, were differentially expressed in our study. Consistent with the proliferative phenotype of this cell line, few genes involved in matrix synthesis and turnover were highly expressed in the presence of serum. These results indicate that immortalized chondrocyte cell lines, rather than substituting for primary chondrocytes, may serve as models for extending findings on chondrocyte function not achievable by the use of primary chondrocytes.     

Die Wirkung länger dauernder Hydrochlorothiazid-Gaben auf die Plasma-Renin-Aktivität und die Aldosteron-Exkretionsrate bei Normalpersonen

Zusammenfassung Die Wirkung von Hydrochlorothiazid auf die Plasma-Renin-Aktivität (PRA) und die Aldosteron-Exkretionsrate (AER) wird bei 10 Normalpersonen untersucht. Gleichzeitig werden Bestimmungen der Serum- und Urinelektrolyte, des Hämatokrits und der Flüssigkeitsbilanz durchgeführt. In derjenigen Gruppe, die während 7 Tagen täglich 50 mg Hydrochlorothiazid (Esidrex®) erhielt, steigen die PRA am 2. und 4. Tag und die AER am 3. Tag nach Beginn der Diuretikagabe signifikant an, während am 6. bzw. am 7. Tag ein Abfall beider Parameter zur Norm einsetzt. Wesentliche Änderungen der Serumelektrolyte und des Hämatokrits werden nicht registriert. Die Stimulation des Renin-Angiotensin-Aldosteron-Systems (RAAS) kann mit einer erhöhten Natriurese und Diurese korreliert werden. Die Kaliurese steigt ebenfalls deutlich an, so daß der Na/K-Quotient im Urin unter 1 abfällt.In derjenigen Gruppe, die während 6 Wochen täglich 50 mg Hydrochlorothiazid erhielt, werden nach 2, 4 und 6 Wochen der Diuretikaverabreichung keine signifikanten Veränderungen der PRA, AER, Serumelektrolyte und des Hämatokrits festgestellt. Es entwickelt sich demnach unter längerdauernden Saluretikagaben kein sekundärer Hyperaldosteronismus.Die Wirkung sowohl einer Natriumrestriktion als auch einer saluretikainduzierten Natriumexkretion auf das RAAS wird besprochen. Schließlich werden die Möglichkeiten diskutiert, die einen Rückgang der Stimulation des RAAS trotz andauernder Diuretikagaben bewirken können.

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Summary The effect of hydrochlorothiazide on plasma renin activity (PRA) and aldosterone excretion rate (AER) were examined in 10 normal persons. At the same time, determinations of serum and urine electrolytes, of hematocrit and of fluid balance were carried out. In that group which recieved 50 mg hydrochlorothiazide (Esidrex®) daily for 7 days, the PRA rose significantly on the 2nd and 4th day and the AER on the 3rd day after the beginning of diuretic treatment. A decline to normal in both parameters set in on the 6th and 7th day, respectively. Considerable changes in serum electrolytes and hematocrit were not registered. The stimulation of the renin-angiotensin-aldosterone system (RAAS) could be correlated with elevated natriuresis and diuresis. Kaliuresis rose considerably as well so that the Na/K quotient in urine fell under 1.In that group which recieved 50 mg hydrochlorothiazide daily for a period of 6 weeks, no significant changes were noticed in PRA, AER, serum electrolytes or hematocrit after 2, 4 and 6 weeks of diuretic treatment. There was no development of secondary hyperaldosteronism under extended saluretic treatment.The effect of sodium restriction as well as saluretica-induced sodium excretion on the RAAS is discussed. Finally, the possibilities are discussed which can cause a retreat in stimulation of the RAAS despite extended treatment with diuretics.

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Diese Arbeit wurde durch die Hilfe des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung und der Stiftung zur wissenschaftlichen Forschung an der Universität Zürich ermöglicht.     

No evidence for involvement of the calpain-10 gene 'high-risk' haplotype combination for non-insulin-dependent diabetes mellitus in early onset obesity

In light of evidence of linkage of obesity to chromosome 2q31-q37, we hypothesized that the calpain-10 gene 'high-risk' haplotype combination for non-insulin-dependent diabetes mellitus (NIDDM) is involved in early onset obesity. We screened the NIDDM 'high-risk'-haplotype combination formed by the alleles 112 and 121 of the polymorphisms UCSNP-43, -19, and -63 in 166 families consisting of an extremely obese child or adolescent (mean BMI percentile: 99.3+/-1.38), one or more obese sibs (mean BMI percentile: 97.42+/-2.88), and both of their parents. Genotyping for three calpain-10 gene polymorphisms was performed by polymerase chain reaction (PCR) with (a) length polymorphism detection (UCSNP-19) or (b) allele-specific PCR (UCSNP-43 and -63). To allow for correct haplotype assignment all individuals were additionally genotyped for two microsatellite markers (D2S125 and D2S2338). We followed a hierarchical test procedure. As the first step, model-free linkage analysis was performed using maximum likelihood binomial statistics. The second stage consisted of a one-sided asymptotic pedigree disequilibrium test for the UCSNP-43 and on an exploratory level for the other SNP-markers and all haplotypes formed by the three SNPs. The final stage investigated the reported haplotype combination. We failed to detect an initial linkage of obesity to this region (LOD score <0.4). All subsequent exploratory analyses were negative. Our analysis of the relationship between the NIDDM 'high-risk' haplotype combination and extreme early onset obesity revealed no evidence for linkage and association.     

Using probabilistic and decision-theoretic methods in treatment and prognosis modeling

Causal probabilistic networks, also called Bayesian networks, allow both qualitative knowledge about the structure of a problem and quantitative knowledge, derived from case databases, expert opinion and literature to be exploited in the construction of decision support systems for diagnosis, treatment and prognosis. This mixing of qualitative and quantitative knowledge will be illustrated, using the selection of antibiotics for a subset of patients with severe infections. The subset consists of patients where bacteria or fungi have been found in the blood. A simple pathophysiological model of infection is used to calculate a prognosis, dependent on the choice of antibiotics. A decision-theoretic approach is used to balance the therapeutic benefit of antibiotic treatment against the cost of antibiotics in the form of direct monetary cost, side effects and ecological cost. A retrospective trial on patients with bacteria or fungi in the blood stemming from the urinary tract indicates that with this approach, it may be possible to suggest balanced choices of antibiotics that not only achieve greater therapeutic benefit, but also reduce the cost of therapy.     

Efficacy of pyrimethamine/sulfadoxine in the prevention of toxoplasmic encephalitis relapses andPneumocystis carinii pneumonia in HIV-infected patients

The efficacy and safety of 25 mg pyrimethamine plus 500 mg sulfadoxine given twice a week in preventing relapses of AIDS-related toxoplasmic encephalitis was evaluated in an open study. The 56 HIV-infected patients evaluated had responded to intensive treatment with pyrimethamine/clindamycin prior to starting the present prophylactic regimen. Four patients (7 %) experienced relapse while on pyrimethamine/sulfadoxine. The probability of freedom from relapse was >90 % for 12 months and >80 % for 24 months. Side effects comprised mild or moderate allergic reactions which occurred in 23 patients (41 %), leading to discontinuation in four patients (7%). Forty-nine of the 56 patients did not have a history ofPneumocystis carinii pneumonia and did not receive antiparasitic prophylaxis other than pyrimethamine/sulfadoxine; two of them (4 %) developed pneumocystosis. The probability of freedom from pneumocystosis was about 90 % for 24 months. Pyrimethamine/sulfadoxine twice a week appears to be a promising regimen for prevention of toxoplasmic encephalitis, and also appears to provide protection againstPneumocystis carinii pneumonia. Although allergic reactions are usually mild and disappear on continuation, they may limit the value of this regimen.     

Connexin 26 mutations in cases of sensorineural deafness in eastern Austria

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with autosomal nonsyndromic sensorineural hearing loss. This study describes mutations in the Cx26 gene in cases of familial and sporadic hearing loss (HL) by gene sequencing and identifies the allelic frequency of the most common mutation leading to HL (35delG) in the population of eastern Austria. For this purpose we have developed and applied a molecular beacon based real-time mutation detection assay. Mutation frequencies in the Cx26 gene of individuals from affected families (14 out of 46) and sporadic cases (11 out of 40) were 30.4% and 27.5%, respectively. In addition to known disease related alterations, a novel mutation 262 G-->T (A88S) was also identified. 35delG accounted for almost 77% of all Cx26 mutations detected and displayed an allelic frequency in the normal hearing population of 1.7% (2 out of 120). The high prevalence of the 35delG mutation in eastern Austria would therefore allow screening of individuals and family members with Cx26 dependent deafness by a highly specific and semi-automated method.     

Two CD14 promoter polymorphisms and atopic phenotypes in Czech patients with IgE-mediated allergy

BACKGROUND: Immunoglobulin E (IgE)-mediated allergy belongs to common chronic disorders resulting from an interaction between both genetic and environmental factors. The gene encoding CD14 is a positional candidate gene for allergic diseases as it is localized on chromosome 5q31.1, a region that is linked to asthma and bronchial hyperresponsiveness. Recently, several polymorphisms in the promoter region of this gene have been associated with atopic phenotypes in various populations. METHODS: We investigated relationship among atopic phenotypes and two polymorphisms [C(-159)T and G(-1359)T] in the promoter of the CD14 gene in the Czech population. Polymerase chain reaction with restriction fragment length polymorphism analyses was used to determine the CD14 genotypes in subjects with IgE-mediated allergic diseases (n = 562) and random controls (n = 320). RESULTS: The CD14 allele or genotype distributions were similar in patients and control group. However, the frequency of the C allele of the C(-159)T polymorphism was higher in patients with positive skin prick tests for moulds than in patients without reactivity to this antigen (P < 0.002, Pcorr<0.01). In addition, we found that patients with homozygous genotype (GG) of the G(-1359)T polymorphism had marginally lower percentage of positive skin prick tests compared with the other genotypes (P < 0.029, Pcorr > 0.05). CONCLUSIONS: Our study supports the idea that CD14 gene variants may act as disease modifiers of IgE-mediated allergic diseases.