Comparative Preclinical Study of Three Bone Marrow Purging Methods Using PCR Evaluation of Residual t(14;18) Lymphoma Cells

The t(14;18) chromosomal translocation occuring in most follicular lymphomas can be exploited by a Bcl2/JH polymerase chain reaction (PCR) to detect residual disease and to monitor the effectiveness of ex-vivo tumor cell immunological purging. We first demonstrated the 10^-5 Bcl2/JH PCR sensitivity with serial dilutions of OCY-LY8 lymphoma cell lines in normal mononuclear cells; and then the specificity and reproductibility of this technique by analysing follicular and non follicular lymphoma samples. With the Bcl2/JH PCR, we tested the efficiency of three marrow purging protocols with an experimentally contaminated bone marrow either treated by three anti-B cell monoclonal antibodies (mAb) followed by three rounds of rabbit complement or two rounds of immunomagnetics beads. Samples obtained after each purging were amplified by Bcl2/JH PCR and hybridized with PFL3 probe. We were able to produce a 2 to 3 log tumor cell reduction after three rounds of complement and a 4 to 5 log reduction after two rounds of beads.

This study showed that it is feasible to use the Bcl2/JH PCR technique for residual cell lymphoma detection in patients undergoing intensive chemotherapy or BM transplantation. These results indicate that ex-vivo immunomagnetic BM purging is probably superior to complement mediated lysis for the eradication of B lymphoma cells from the marrow of patients undergoing autologous transplantation.     

Genetics: Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities

In the present study we investigated the results of cyto-geneticanalysis in male and female patients included in an intracytoplasmicsperm injection (ICSI) programme for severe male infertilityas well as in conceptuses resulting from these ICSI treatments.In the 261 couples treated, 11 male (4.2%) and three female(1.2%) abnormal karyotypes were found, all consisting of structuralchromosome anomalies. Chromosomal translocation exhibited thehighest frequency (eight males and two females), and there werealso three cases of chromosomal inversion (two males and onefemale) and one male with one additional marker chromosome.There was no difference in fertilization rates among coupleswith abnormal (n = 14) and normal (n = 147) cytogenetic results,and the rates of clinical pregnancy per ICSI attempt were 25.0%(5/20) and 20.6% (78/378) respectively. In pregnancies obtainedin couples with normal karyotypes, all of the 108 fetuses werefree of chromosomal abnormalities. Among the eight fetuses fromcouples with chromosome structural anomalies, three out of fiveand two out of three inherited the cytogenetic defects foundin their father or mother respectively. In this series of 83ICSI pregnancies there were no chromosomal abnormalities otherthan those inherited from the parents. These findings suggestthat normal pregnancy rates can be obtained by ICSI in casesof chromosomal translocation in couples with severe male infertility.However, until further evaluations of available data can beperformed, cytogenetic analysis must be conducted prior to ICSIin men with low sperm counts, and genetic counselling must includeprenatal diagnosis for all growing conceptuses.     

Tissue inhibitor of metalloproteinase 1 is an independent predictor of prognosis in patients with nonsmall cell lung carcinoma who undergo resection with curative intent

BACKGROUND: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a role in the processes of extracellular matrix degradation. Changes in their expression levels have been observed in various tumor types, including lung carcinoma. However, their clinical significance and their prognostic importance in the progression of nonsmall cell lung carcinoma (NSCLC) remain to be specified. In this study, mRNA expression levels of MMP-1, MMP-9, TIMP-1, and TIMP-2 were evaluated in patients with resected NSCLC, and their associations with disease progression and prognosis were determined. METHODS: Between June 1996 and December 1999, 116 patients underwent resection for NSCLC. Expression levels of MMPs and TIMPs were evaluated using Northern blot analysis in these NSCLC tissue samples and in 39 matched samples of normal lung tissue. RESULTS: MMP-1, MMP-9, and TIMP-1 expression levels were increased in tumor samples compared with matched, corresponding normal tissues. In contrast, TIMP-2 expression was decreased in tumor samples. MMP-1 tumor expression was correlated significantly with the evolution of lymph node status and tumor-lymph node-metastasis (TNM) stage. In contrast, MMP-9 tumor expression was correlated significantly with increased T stage. TIMP-1 overexpression was an independent predictor of worse survival in patients with NSCLC that was not associated with other prognosis factors, such as TNM stage. CONCLUSIONS: The overexpression of TIMP-1 was an independent prognostic marker in patients with NSCLC, and evaluating TIMP-1 may be important for identifying patients who are at greater risk of disease recurrence.     

Longitudinal membrane function in functionally anuric patients treated with APD: data from EAPOS on the effects of glucose and icodextrin prescription

BACKGROUND: Peritoneal dialysis is associated with changes in membrane function that can lead eventually to ultrafiltration (UF) failure. Factors driving these changes are thought to include hypertonic glucose exposure, but previously reported associations are confounded by the presence of residual renal function. METHODS: Longitudinal membrane function (solute transport and UF capacity) were measured annually in a prospective cohort of 177 functionally anuric patients as part of the European Automated Peritoneal Dialysis Outcomes Study (EAPOS). Subgroup analysis was performed according to glucose exposure and icodextrin use at baseline. RESULTS: The whole cohort experienced an increase in solute transport and reduction in UF capacity at 12 and 24 months that could not be explained by informative censoring. These changes were accelerated and more severe in patients using either 2.27% or 3.86% glucose, or those not using icodextrin at baseline. These differences could not be explained by age, comorbidity score, previous time spent on renal replacement, differential dropout from the study, peritonitis rates, or, by definition, residual renal function. Patients using icodextrin at baseline had worse membrane function and were more likely to be diabetic. There was an association between membrane function changes and achieved 24-hour ultrafiltration over the 2-year study period. CONCLUSION: Anuric automated peritoneal dialysis (APD) patients experience significant detrimental changes in membrane function over a relatively short time period. Glucose appears to enhance these changes independent of residual renal function. Icodextrin use in these circumstances is associated with less deterioration in membrane function.     

The effects of acetaldehyde in vitro on proteasome activities and its potential involvement after alcoholization of rats by inhalation of ethanol vapours

Background/ AIMS: Some models of chronic ethanol administration resulted in decreased proteasome activities. The mechanisms still remain speculative. In the present study, we tested another model of alcoholization with high blood alcohol levels (BALs) and high acetaldehyde fluxes as well as the in vitro effect of acetaldehyde on proteasome. Methods/ RESULTS: Ethanol vapour chronically inhaled by adult Wistar rats up to a specific protocol, can reach high BALs (200 mg/dl) with significant circulating acetaldehyde levels. After 4 weeks of ethanol intoxication, although cytochrome CYP2E1 was increased, liver lipid peroxidation remained unchanged when protein carbonyls augmented selectively for high molecular weight with a decrease of the proteasome activities in ethanol rats. Several aldehydes inhibit proteasome function; we specifically explored the effects of acetaldehyde, the first alcohol metabolite. Adduction of acetaldehyde in vitro to cytosolic proteins inhibits proteasome in a dose-dependent manner. Acetaldehyde adducted to purified proteasome also exhibits a decrease in its activities. Furthermore, an acetaldehyde-adducted protein, i.e. bovine serum albumin (BSA) is less degraded than a native BSA by purified proteasome. These findings suggest that acetaldehyde, if overproduced, can inhibit proteasome activities and reduce the proteolysis of acetaldehyde-adducted proteins. CONCLUSIONS: Our study, for the first time, provided the evidence that acetaldehyde by itself inhibits proteasome activities. As the chronic inhalation model used in this study is not associated with an overt lipid peroxidation, one can suggest that high BALs and their subsequent high acetaldehyde fluxes contribute to impairment of proteasome function and accumulation of carbonylated proteins. This early phenomenon may have relevance in experimental alcohol liver disease.     

Brussels sprouts, inulin and fermented milk alter the faecal microbiota of human microbiota-associated rats as shown by PCR-temporal temperature gradient gel electrophoresis using universal, Lactobacillus and Bifidobacterium 16S rRNA gene primers

We investigated the effect of Brussels sprouts, inulin and a fermented milk on the faecal microbiota diversity of human microbiota-associated (HMA) rats by PCR-temporal temperature gradient gel electrophoresis (PCR-TTGE) using universal and group-specific 16S rRNA gene primers. The HMA rats were submitted to a control diet for 10 d (initial time), then switched to the experimental diets for 4 weeks (final time). Using universal primers, the mean degree of similarity between all faecal samples at initial time was 80.8 %. In the group consuming the control diet throughout the experiment, the mean degree of similarity between the PCR-TTGE profiles at initial v. final time was 76.8 %, reflecting a spontaneous temporal variation. The mean degree of similarity between control and experimental groups at final time was lower, 72.4 %, 74.4 % and 75.6 % for inulin, Brussels sprouts and fermented milk, respectively, indicating a dietary effect on the predominant populations. Using specific primers, bifidobacteria could be detected only in those rats that had consumed inulin, showing a specific increasing effect of this dietary compound. The Lactobacillus population was very heterogeneous at initial time but tended to homogenize within each dietary group. At final time, caecal contents were collected for analysis of SCFA and beta-glucuronidase activity. Inulin and Brussels sprouts increased the butyrate and acetate proportion, respectively, while the fermented milk did not modify the caecal biochemistry. This experiment shows for the first time that cruciferous vegetables are able to alter the diversity and the metabolic activities of the digestive microbiota in HMA rats.     

New thiophene analogues of kenpaullone: synthesis and biological evaluation in breast cancer cells

Thieno analogues of kenpaullone have been synthesized using an established method. We investigated the effect of five structural analogues of kenpaullone on vincristine sensitive and resistant MCF7 (human mammary adenocarcinoma) cells. One analogue, 8-Bromo-6,11-dihydro-thieno-[3',2':2,3]azepino[4,5-b]indol-5(4H)-one (3a), showed an antiproliferative activity in the drug sensitive cell line that led to cell accumulation in G2/M phase. In addition, repression of cdk1, a G2/M transition key regulator, as well as induction of p21 were observed at the mRNA level. Programmed cell death (apoptosis) was induced in early time treatments and was accompanied by p53 mRNA induction. The antiproliferative and proapoptotic properties of 3a make this CDK inhibitor an interesting candidate for further investigations.