Long-term follow up of childhood tuberculous meningitis.

The purpose of the present study was to determine the long-term outcome of 76 children (40 females and 36 males) diagnosed and treated with modern antituberculosis drugs. The median age of the children on admission was 29.5 months and on follow-up 9 years. Antituberculosis therapy consisted of daily isoniazid (20 mg/kg), rifampicin (20 mg/kg), ethionamide (20 mg/kg), and pyrazinamide (40 mg/kg) for 6 months. Twenty-three children received daily prednisone (2-4 mg/kg) for the first month of treatment. Raised intracranial pressure was actively monitored and treated. Patients with non-communicating hydrocephalus received ventriculo-peritoneal shunts shortly after admission while communicating hydrocephalus was treated with oral acetazolamide (100 mg/kg/day) and furosemide (1 mg/kg/day) in 3-4 divided doses. Communicating hydrocephalus that did not respond to this regimen within the first month of treatment also underwent ventriculo-peritoneal shunting. Only 20% of children were functionally completely normal at follow-up. Main areas of functional deficit were cognitive impairment (80%), poor scholastic progress (43%), and emotional disturbance (40%). Twenty-five per cent of children had evidence of motor impairment, but all could walk and only 5 of 76 children (6% of total) were unable to run. One child was blind but no child had sensori-neural deafness. It was concluded that these disabilities in children from mainly deprived socioeconomic backgrounds have serious implications for their future social, academic, and career prospects. A high index of suspicion of TBM in high tuberculosis incidence communities will help prevent the morbidity documented in this study.

Nucleosomes in serum as a marker for cell death.

The concentration of nucleosomes is elevated in blood of patients with diseases which are associated with enhanced cell death. In order to detect these circulating nucleosomes, we used the Cell Death Detection-ELISAplus (CDDE) from Roche Diagnostics (Mannheim, Germany) (details at http:\\biochem.roche.com). For its application in liquid materials we performed various modifications: we introduced a standard curve with nucleosome-rich material, which enabled direct quantification and improved comparability of the values within (CVintraassay:3.0-4.11%) and between several runs (CVinterassay:8.6-13.5%), and tested the analytical specificity of the ELISA. Because of the fast elimination of nucleosomes from circulation and their limited stability, we compared plasma and serum matrix and investigated in detail the pre-analytical handling of serum samples which can considerably influence the test results. Careless venipuncture producing hemolysis, delayed centrifugation and bacterial contamination of the blood samples led to false-positive results; delayed stabilization with EDTA and insufficient storage conditions resulted in false-negative values. At temperatures of -20 degrees C, serum samples which were treated with 10 mM EDTA were stable for at least 6 months. In order to avoid possible interfering factors, we recommend a schedule for the pre-analytical handling of the samples. As the first stage, the possible clinical application was investigated in the sera of 310 persons. Patients with solid tumors (n=220; mean=361 Arbitrary Units (AU)) had considerably higher values than healthy persons (n=50; mean=30 AU; p=0.0001) and patients with inflammatory diseases (n=40; mean= 296 AU; p=0.096). Within the group of patients with tumors, those in advanced stages (UICC 4) showed significantly higher values than those in early stages (UICC 1-3) (p=0.0004).

Nucleosomes in serum of patients with benign and malignant diseases.

High quantities of mono- and oligonucleosomes circulate in the blood of patients with malignant tumors. For their direct quantification in serum, we modified the Cell Death Detection(plus)-ELISA for its application in liquid materials. We examined sera samples from 590 persons, including 418 patients with malignant tumors, 109 patients with benign diseases and 63 healthy persons. We also observed the kinetics of the concentration of nucleosomes in serum samples from 20 patients undergoing chemotherapy and from 16 patients undergoing radiotherapy. Sera of patients with malignant tumors contained considerably higher concentrations of nucleosomes (mean = 350 arbitrary units [AU], median = 190 AU) compared with those of healthy persons (mean = 36 AU, median = 24 AU; p = 0.0001) and patients with benign diseases (mean = 264 AU, median = 146 AU; p = 0.072). Concerning the follow-up investigations, the concentration of nucleosomes in serum increased 24-72 hr after the first application of chemotherapy and 6-24 hr after the start of radiotherapy. A subsequent decrease was often correlated with regression of the tumor. In patients undergoing chemotherapy, an increase in the baseline values of circulating nucleosomes >50%, which were determined before each new therapeutic cycle, was correlated with progression of disease; all patients with disease regression showed a decrease >50% of the baseline values. In patients undergoing radiotherapy, an early decrease of the nucleosomal concentration (< or = 1 day after the initial peak during therapy) to low minimum levels (< or = 100 AU) correlated with good clinical outcome; a late decrease (>1 day) to higher minimum levels (>100 AU) was associated with a worse clinical outcome. Thus, the concentration of nucleosomes in serum might be a useful tool for monitoring the biochemical response during antitumor therapy, especially for the early estimation of therapeutic efficacy.

Plasma membrane phospholipid asymmetry precedes DNA fragmentation in different apoptotic cell models.

Biochemical alterations occurring in many cell types during apoptosis include the loss of plasma membrane phospholipid asymmetry and nuclear DNA fragmentation. Annexin V staining detects phosphatidylserine translocation into the outer plasma membrane layer occurring during cell death, while the in situ tailing (IST or TUNEL) reaction labels the DNA strand breaks typical of apoptosis. To compare the time course of these processes we investigated methylprednisolone-induced apoptosis of rat thymocytes, topoisomerase inhibitor-induced apoptosis in the human histiocytic lymphoma cell line U937, and serum deprivation-induced apoptosis in the rat pheochromocytoma cell line, PC12. At all time points, FACS analysis and quantitative fluorescence light microscopy showed a higher proportion of annexin V-positive than IST-positive cells, with significantly different time courses in the apoptotic cell models investigated (Anova test). Results were confirmed by confocal microscopy. Our data indicate that the exposure of phosphatidylserine, a potential phagocyte recognition signal on the cell surface of apoptotic cells in vivo, precedes DNA strand breaks during apoptosis in different cell types.

Biotechnological aspects of the production of human pro-kallikrein using the AcNPV-baculovirus-expression system.

We have investigated large scale production processes (up to 2 liters) of recombinant proteins using the baculovirus expression system in order to optimize the product yields. Experiments using cell lines of Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-Mb0503) were performed to show the different production capacities of the cell lines. The influence of the infection at different cell densities is described. Beyond that, TC100-, IPL41- and serum-free IPL41-medium were compared to demonstrate their different capabilities of supporting cell growth and protein expression. Additionally, the inhibitory effect of FCS on the protease activity of kallikrein, which is produced in its zymogenic form, is discussed Improved production parameters are described, which enabled us to produce up to 8000 units of activated pro-kallikrein within 14 days using perfusion cultivation.

Purification and characterization of human salivary-gland prokallikrein from recombinant baculovirus-infected insect cells.

A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion-exchange chromatography and gel-filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N-terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/l insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.

Multistage production of Autographa californica nuclear polyhedrosis virus in insect cell cultures.

The aim of our study was to establish an efficient system for the in vitro production of the insect pathogenic Autographa californica nuclear polyhedrosis virus in a Spodoptera frugiperda cell line. We optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. Cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. In order to provide sufficient nutrients during virus synthesis filtration and perfusion devices were connected to the bioreactor. A virus production procedure in a repeated batch mode by using a two stage bioreactor system is described. Stage I was optimized for cell production and stage II for virus production.

Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures.

Cell cycle kinetic of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G0) of mammalian cells is characterized by a 2c-DNA content whereas G0-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2'-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.

Flow-cytometric cell-cycle analysis of Chinese hamster cells following exposure to cytotoxicants.

Chinese hamster cell-cycle kinetics were studied following exposure to a cytotoxic agent. Different parameters like dependence on concentration and preparation interval were measured by flow cytometry. The results were compared with data from established methods. DNA histograms showed the cell-cycle effect of EMS (ethyl methanesulfonate) at different time intervals after treatment. The principle of quenching the fluorescence of Hoechst 33258 by staining 5-bromodeoxyuridine (BrdU) substituted DNA was applied and supports these results. Comparison to chromosome-aberration studies demonstrates the suitability of this method to screen quickly for adequate dosing of a cytotoxic substance and also gives information on the appropriate preparation interval.