Decreased expression of the INK4 family of cyclin-dependent kinase inhibitors in Wilms tumor.

Cyclin-dependent kinase (CDK) inhibitors represented by the INK4 family (including p16(INK4a, CDKN2A), p15(INK4b, CDKN2B), p18(INK4c, CDKN2C), and p19(INK4d, CDKN2D)) are regulators of the cell cycle shown to be aberrant in many types of human cancer. We tested the hypothesis that these CDK inhibitors are a target for altered gene expression in Wilms tumor. Using RT-PCR, gene expression of the INK4 family was found to be decreased in 9 of 38 Wilms tumor samples obtained from the National Wilms Tumor Study Group (NWTSG) tissue bank. All the affected tumor samples were of favorable histology. Methylation-specific PCR revealed that methylation in the p16 promoter region may be responsible for altered expression. The incidence of loss of p16 expression may increase with increasing tumor stage, i.e., 1/10 (10%) with stage I/II FH Wilms tumor, 2/10 (20%) with stage III FH Wilms tumor, and 4/10 (40%) with stage IV FH Wilms tumor. Thus, determining the expression status of the INK4 family may have potential prognostic value in the management of Wilms tumor.

Pseudomonas aeruginosa lasR transcription correlates with the transcription of lasA, lasB, and toxA in chronic lung infections associated with cystic fibrosis.

The role of Pseudomonas aeruginosa quorum-sensing systems in the lung infections associated with cystic fibrosis (CF) has not been examined. The purpose of this study was to determine if genes regulated by the LasR-LasI quorum-sensing system were coordinately regulated by the P. aeruginosa populations during the lung infections associated with CF. We also wanted to ascertain if there was a relationship between the expression of lasR, a transcriptional regulator, and some P. aeruginosa virulence factors during these infections. We extracted RNAs from the bacterial populations of 131 sputa taken from 23 CF patients. These RNAs were blotted and hybridized with probes to P. aeruginosa lasA, lasB, and toxA. The hybridization signals from each probe were ranked, and the rankings were analyzed by a Spearman rank correlation to determine if there was an association between the population transcript accumulations for the three genes. The correlations between the transcript accumulation patterns of pairs of the genes suggested that lasA, lasB, and toxA might be coordinately regulated during CF lung infections. To determine if this coordinate regulation might be due to regulation by LasR, we probed RNAs, extracted from 84 sputa, with the lasR, lasA, lasB, toxA, and algD probes. Statistical analysis indicated that lasR transcript accumulation correlated to lasA, lasB, toxA, and algD transcript accumulations. These results indicated that lasR may at least partially regulate or be coordinately regulated with lasA, lasB, toxA, and algD during the lung infections associated with CF. These results also suggested that the LasR-LasI quorum-sensing system may control the expression of at least some virulence factors in the lungs of patients with CF.

Mesna inactivates platinum agents in vitro.

BACKGROUND: Platinum agents are frequently combined with ifosfamide. Mesna, originally coadministered to protect from ifosfamide side effects, might also react with the platinum agents in these combinations. METHODS: Malignant glioma cells were incubated with cisplatin, carboplatin and mesna. Cell numbers were measured by counting and by MTT-tests. RESULTS: In cell free solution mesna turned MTT to its blue farmazan product. Mesna's effect on cells were cell-line specific: It penetrated U87 cells without effect on growth, reduced cell numbers in C6 and T98G cells and did not alter U251 cells. The concentration of cisplatin killing 50% of the cells were 7 x 10(-7) in C6, 9.7 x 10(-6) in T98G, 1.2 x 10(-5) in U251 and 2.4 x 10(-4) in U87 cells. For the same effect, carboplatin required 3-10 times higher concentrations. Mesna protected all cell lines from the cytotoxicity of the platinum agents. CONCLUSION: Clinical studies should specify in detail, infusion schedules of mesna and platinum agents.

Positive correlation of algD transcription to lasB and lasA transcription by populations of Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis.

Pseudomonas aeruginosa causes a chronic infection in the lungs of individuals with cystic fibrosis. The P. aeruginosa isolates from these infections, when grown under laboratory conditions, characteristically are mucoid and produce low levels of the more destructive virulence factors, such as exotoxin A and the proteases. We wanted to determine if during the chronic lung infections associated with CF, the expression of alginate was inversely correlated to the expression of exotoxin A, elastase, and the LasA protease. We measured the transcript accumulation of algD, a marker of alginate, toxA, the structural gene for exotoxin A, lasB, the structural gene for elastase, and lasA, the structural gene for LasA protease, from the sputum bacterial populations of 23 patients. In the 131 samples tested, we frequently detected transcripts from the four genes. When a Spearman rank correlation analysis was done on the samples, we found no correlation between algD transcript accumulation and toxA transcript accumulation. This result suggested that toxA was regulated independently of algD. Curiously, we found a positive correlation between algD transcript accumulation and both lasB and lasA transcript accumulation levels. This correlation may not indicate a direct association between algD and either lasA or lasB. More likely, it indicates a common regulatory element in a cascade of regulators or a common environmental cue that triggers transcription.

Association between transcript levels of the Pseudomonas aeruginosa regA, regB, and toxA genes in sputa of cystic fibrosis patients.

In this study, we examined the regulation of exotoxin A (ETA) production by Pseudomonas aeruginosa during chronic lung infections of cystic fibrosis (CF) patients. We used a recently developed technique termed population transcript accumulation in hybridization studies with RNA extracted from sputa. With this technique, we demonstrated that the structural gene for ETA, toxA, as well as two genes encoding positive regulators of ETA synthesis, regA and regB, were expressed in the lungs of CF patients infected with P. aeruginosa. These genes were always expressed together, never alone or in pairs, suggesting coincident expression and a possible regulatory role for regA and regB in this environment. Fluctuations in the levels of the three gene products were observed among samples, consistent with a regulatory phenomenon. The level of regB RNA detected never exceeded that of regA, although the ratio of regA RNA to regB RNA detected did change between samples. These observations are in agreement with in vitro observations which have shown that regB is located 3' to regA in an operon which is expressed from two independently regulated promoters located upstream of regA. The presence of high levels of toxA, regA, and regB RNAs in some sputum samples prompted us to look for hyperproducing-toxin strains in the sputa of CF patients. In vitro, one such strain, 4384, had a transcript accumulation pattern for toxA, regA, and regB similar to that of a laboratory hyperproducer of ETA, strain PA103. These observations suggest that regA and regB are involved in the regulation of ETA production in strains of P. aeruginosa infecting the lungs of CF patients and that some of these strains may regulate ETA production in a manner similar to that of the hyperproducing-ETA strain PA103.

Population transcript accumulation of Pseudomonas aeruginosa exotoxin A and elastase in sputa from patients with cystic fibrosis.

The in vivo regulation of Pseudomonas aeruginosa virulence factors during the chronic lung infections associated with cystic fibrosis is poorly understood. We have developed an approach for the analysis of transcript accumulation of individual virulence factors from the P. aeruginosa populations found in the sputa of patients with cystic fibrosis. This method has been named population transcript accumulation, since we examine the transcript accumulation patterns in RNA extracted from the total bacterial population found in the sputum samples. DNA probes specific for P. aeruginosa elastase (lasB) and exotoxin A (toxA) were used to examine the population transcript accumulation of 21 sputum samples taken from 10 patients. We detected three patterns of population transcript accumulation: lasB and toxA, lasB alone, and neither lasB nor toxA. We also measured the relative levels of elastase and exotoxin A transcript accumulation in 19 of these samples. In the six samples containing both toxA and lasB transcripts, we found that the levels of lasB transcripts were consistently higher than those of toxA. Differences in the stability of the two mRNA species could not completely account for the higher level of lasB population transcript accumulation, since we showed that the mRNA half-life of lasB (11 min) was similar to that of toxA (10 min). Finally, we showed that elastase transcripts could be detected in some samples which contained only mucoid isolates. This finding suggests that both mucoid and nonmucoid populations may be transcribing lasB in the lungs of patients with cystic fibrosis.

Antibodies to major outer membrane proteins of Escherichia coli in urinary infection in the elderly.

The serologic response to infection in elderly bacteriuric subjects and young women with acute pyelonephritis was measured with an enzyme-linked immunosorbent assay (ELISA) using the major outer membrane protein complex (MOMP) of one Escherichia coli strain as antigen. Elderly controls and subjects with asymptomatic bacteriuria had variable titers; control titers were significantly lower than those with asymptomatic bacteriuria. Titers were stable over 2-12 w in asymptomatic subjects. Elderly subjects with invasive infection and women with pyelonephritis demonstrated increases in titer between acute and convalescent serum for E. coli and other Enterobacteriaceae. With a convalescent specimen with an antibody titer greater than or equal to 3 standard deviations (SD) above the acute, the sensitivity of the MOMP ELISA for identifying invasive infection was 74%, the specificity 86%, the positive predictive value 82%, and the negative predictive value 79%. With the criteria of greater than or equal to 3 SD or an initial serum to control ratio of greater than or equal to 15 these parameters were 95%, 82%, 82%, and 95%, respectively. These initial investigations suggest the MOMP of E. coli may be an antigen with wide cross-reactivity, suitable for use as an objective test to identify invasive Enterobacteriaceae urinary infection.

Immunoblot analysis of serologic response to outer membrane proteins of Escherichia coli in elderly individuals with urinary tract infections.

We used immunoblotting to examine the serologic antibody responses to outer membrane proteins (OMP) of Escherichia coli in both symptomatic and asymptomatic elderly subjects with urinary tract infections. Controls with no present or past urinary tract infections showed variable weak immunoglobulin G (IgG) antibodies to OMP of infecting strains. Elderly individuals with asymptomatic infections demonstrated antibody to both lipopolysaccharide (LPS) and OMP of their infecting strain, with consistent cross-reactivity to OMP of other infecting strains. Young females with acute pyelonephritis showed an IgG response to LPS and OMP with cross-reactivity to OMP of other strains. Elderly individuals with symptomatic invasive infections had strong reactions to both LPS and OMP in specimens collected during the acute phase, generally with an increase in intensity in specimens from convalescent patients. They also demonstrated extensive cross-reactivity to LPS and OMP from all other infecting strains. IgM antibody was not observed in any patients. These data confirm other reports of low levels of antibodies to OMP of E. coli in normal populations. Asymptomatic bacteriuria in this population is associated with antibody responses to the LPS and OMP of the infecting strain. Elderly individuals with invasive infections had initial reactions to the infecting strain with an apparent increase in intensity during convalescence. Antibodies to the major OMP appear to be broadly cross-reactive.