Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification.

Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed.Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay. The developed assays were applied for the quantification of bacteria in soil samples.

Longitudinal follow-up circulating immune complexes of the serum in patients with breast cancer.

The values of circulating immune complexes (CIC) in patients with breast cancer were followed for one year at 4-month intervals by means of the "PEGIKEM" test. From the total of 67 patients, 37 were treated surgically and a significant decrease in mean values of CIC occurred 8 months after the operation. 30 patients with a more advanced stage of the disease treated with radiotherapy and chemotherapy showed a decrease in mean CIC values already after 4 months but later on the CIC values increased again. No significant differences in the mean CIC values prior to treatment between individual clinical stages were found, only a certain tendency towards a more frequent occurrence of higher CIC values in more advanced stages was noted. Nor were the findings in lymph nodes reflected in the mean CIC values prior to treatment. Comparison of CIC values prior to treatment and the results of 2-hour blood sedimentation, revealed conformity in only 40% of the patients. In the other cases higher values of blood sedimentation were more frequent. The study of prognostic value of CIC after 3 and 5 years follow-up is in progress.

Haptoglobin type in connection with mean age at first breast cancer signs?

In breast cancer patients divided into groups according to haptoglobin (Hp) type (519 patients), blood group (ABO) (433 patients) or Rh factor (358 patients) the mean age at first disease manifestation was calculated. The results indicate that in Hp 1 bearers breast cancer appears at significantly lower age (49 years) as compared to Hp type 2-1 (53.3 years) or Hp 2 (53 years). In blood groups the mean age at first cancer signs raises from 48 years in the AB group to 53.1 years in the A group, only the difference between A and AB being significant. However, these preliminary results require confirmation in larger patient series.

Alterations of plasma levels in malignant lymphoma.

Fibrinogen levels were investigated in the plasma of a group of patients with malignant lymphoma consisting of M. Hodgkin's and non-Hodgkin's lymphoma, and were found to be significantly higher in an acute onset of the disease and in relapses, than in a control group of healthy blood donors. In the terminal stages, fibrinogen levels in the plasma were observed to decline towards the upper normal limits. Plasma fibrinogen levels are considered to be a good indicator of the activity of the disease.