Effect of penicillin on the morphology of a Neisseria meningitidis strain liberating free endotoxin. An electron microscope study

An endotoxin-liberating strain of Neisseria meningitidis plasmolysed extensively after 2 h of exposure to 100 times MIC values of benzypenicillin. The peptidoglycan layer could be demonstrated after 2 h of treatment in places where the cytoplasm still was close to the cell wall. After 20 h, however, this layer was complete undetectable. In untreated cells the peptidoglycan layer could more easily be found in older cultures than in very young cultures. An increased adhesiveness and aggregation to other bacterial cells and to cell wall material could be observed after 2 h of penicillin treatment, and more pronouncedly after 20 h. A high yield of free cell wall material could be demonstrated after 2 h of penicillin treatment. This corresponded well to an increased content of free endotoxin in the filtrates from the cell cultures treated with penicillin, compared to untreated controls. After 20 h of treatment, free cell wall material had formed large aggregates or was adherent to the cell walls of ghost cells. The corresponding endotoxin analysis showed a reduced content of filtrable endotoxin. Possible implications of the structural changes in relation to penicillin treatment of patients are discussed.     

Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator.

Cell-binding experiments have indicated that murine cells on their surface have specific binding sites for mouse urokinase-type plasminogen activator (u-PA). In contrast to the human system, chemical cross-linking studies with an iodinated ligand did not yield any covalent adducts in the murine system, but in ligand-blotting analysis, two mouse u-PA-binding proteins could be visualized. To confirm that these proteins are the murine counterpart of the human u-PA receptor (u-PAR), a peptide was derived from the murine cDNA clone assigned to represent the murine u-PAR due to cross-hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Dan?, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand-blotting analysis. Binding of mouse u-PA to its receptor showed species specificity in ligand-blotting analysis, since mouse u-PA did not bind to human u-PAR and human u-PA did not bind to mouse u-PAR. The apparent M(r) of mouse u-PAR varied between different mouse cell lines and ranged over M(r) 45,000-60,000. In four of the cell lines, mouse u-PA bound to two mouse u-PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u-PA-binding protein. In the monocyte macrophage cell line P388D.1, trypsin-treatment of intact cells could remove only the large mouse u-PAR variant (M(r) 60,000) indicating that only this type was a cell-surface-exposed molecule. The smaller mouse u-PAR variant (M(r) 45,000), was deglycosylated by the enzyme endo-beta-N-acetylglucosaminidase H and is probably an intracellular precursor form carrying only high-mannose carbohydrate. Deglycosylation of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u-PAR variant proteins by diisopropylfluorophosphate-inactivated mouse u-PA-Sepharose affinity chromatography yielded two silver-stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oligotrophication as a result of planktivorous fish removal with rotenone in the small,eutrophic, Lake Mosvatn,Norway

In September 1987 the shallow, eutrophic, Lake Mosvatn was treated with rotenone to eliminate planktivorous fish (mainly whitefish,Coregonus lavaretus, L.), and the effects were studied. The first summer after treatment the zooplankton community changed markedly from rotifer dominance and few grazers, to a community with few rotifers and many grazers. Accordingly there was a fivefold increase in the biomass ofDaphnia galeata. Adult females ofD. galeata approximately doubled in weight. The decrease in rotifer biomass was probably mainly due to a loss of food by competition with the daphnids. The phytoplankton community was also markedly affected. Prior to treatment Secchi depth was 1.7 m and Chl-a 23μg l⁻¹ in the summer. After treatment there was an increase in the proportion of small and gelatinous algae and the mean chlorophyll concentration fell to 7μg Chl-a l⁻¹. Secchi depth increased to>2.3 m (bottom-sight most of the season). After the treatment there were also fewer cyanobacterial blooms. This seems to be related to oligotrophication caused indirectly by increased grazing by the zooplankton. Total nutrient concentrations were affected. Prior to treatment the mean summer concentration of total phosphate was 44μg P l⁻¹. This decreased to 29μg P l⁻¹ in the first summer and 23μg P l⁻¹ the second summer after the treatment. Total nitrogen decreased from 0.68 mg N l⁻¹ before treatment to 0.32 mg N l⁻¹ the first summer after the treatment. The phosphate loading was not reduced, therefor it can be concluded that the fish removal provided a biomanipulation which caused the more oligotrophic conditions.     

Finite element analysis of different loading conditions for implant-supported overdentures supported by conventional or mini implants

The effect of implants’ number on overdenture stability and stress distribution in edentulous mandible, implants and overdenture was numerically investigated for implant-supported overdentures. Three models were constructed. Overdentures were connected to implants by means of ball head abutments and rubber ring. In model 1, the overdenture was retained by two conventional implants; in model 2, by four conventional implants; and in model 3, by five mini implants. The overdenture was subjected to a symmetrical load at an angle of 20 degrees to the overdenture at the canine regions and vertically at the first molars. Four different loading conditions with two total forces (120, 300 N) were considered for the numerical analysis. The overdenture displacement was about 2.2 times higher when five mini implants were used rather than four conventional implants. The lowest stress in bone bed was observed with four conventional implants. Stresses in bone were reduced by 61% in model 2 and by 6% in model 3 in comparison to model 1. The highest stress was observed with five mini implants. Stresses in implants were reduced by 76% in model 2 and 89% increased in model 3 compared to model 1. The highest implant displacement was observed with five mini implants. Implant displacements were reduced by 29% in model 2, and increased by 273% in model 3 compared to model 1. Conventional implants proved better stability for overdenture than mini implants. Regardless the type and number of implants, the stress within the bone and implants are below the critical limits.     

Colonization of the rat liver by syngeneic tumor cells. An experimental approach by in vivo and in situ studies

Syngeneic colon carcinoma cells and glioma cells were injected into the portal vein of BD IX rats. After various time periods the animals were sacrificed and the livers and lungs were fixed and prepared for histology. Atypical cells were observed in the liver 4 and 7 days after the injection of tumor cells, whereas distinct colonies of both colon carcinoma and glioma cells were demonstrated after 14 days. Lung metastases of both tumor cell types were seen after 14 and 30 days. Furthermore, injection of glioma and carcinoma cells into the tail vein gave detectable lung metastases after 7 and 4 days respectively. Intraperitoneal injection of tumor cells resulted in the accumulation of large tumor masses, particularly in the mesentery. By in situ perfusions of the liver with tumor cells included in the perfusion medium it was possible to establish that all the tumor cells were arrested in the course of 4 min. In contrast, normal rat leukocytes were not trapped in the liver, whereas trypsin-treated leukocytes were, suggesting the importance of trypsin-sensitive structures for binding to hepatic tissue. The binding of both glioma and carcinoma cells to the liver and the ensuing growth of tumor nodules in this organ indicate a lack of specificity on part of the malignant cell types for metastasis to the liver in the rat. Both tumor cell types colonized the first organ encountered after injection.     

Strong genetic influences on measures of behavioral‐regulation among inbred rat strains

A fundamental challenge for any complex nervous system is to regulate behavior in response to environmental challenges. Three measures of behavioral‐regulation were tested in a panel of eight inbred rat strains. These measures were: (1) sensation seeking as assessed by locomotor response to novelty and the sensory reinforcing effects of light onset, (2) attention and impulsivity, as measured by a choice reaction time task and (3) impulsivity as measured by a delay discounting task. Deficient behavioral‐regulation has been linked to a number of psychopathologies, including ADHD, Schizophrenia, Autism, drug abuse and eating disorders. Eight inbred rat strains (August Copenhagen Irish, Brown Norway, Buffalo, Fischer 344, Wistar Kyoto, Spontaneous Hypertensive Rat, Lewis, Dahl Salt Sensitive) were tested. With n = 9 for each strain, we observed robust strain differences for all tasks; heritability was estimated between 0.43 and 0.66. Performance of the eight inbred rat strains on the choice reaction time task was compared to the performance of outbred Sprague Dawley (n = 28) and Heterogeneous strain rats (n = 48). The results indicate a strong genetic influence on complex tasks related to behavioral‐regulation and indicate that some of the measures tap common genetically driven processes. Furthermore, our results establish the potential for future studies aimed at identifying specific alleles that influence variability for these traits. Identification of such alleles could contribute to our understanding of the molecular genetic basis of behavioral‐regulation, which is of fundamental importance and likely contributes to multiple psychiatric disorders .     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Simultaneous determination of 6 beta-blockers, 3 calcium-channel antagonists, 4 angiotensin-II antagonists and 1 antiarrhythmic drug in post-mortem whole blood by automated solid phase extraction and liquid chromatography mass spectrometry. Method development and robustness testing by experimental design

A method for the simultaneous determination of the beta-blockers atenolol, sotalol, metoprolol, bisoprolol, propranolol and carvedilol, the calcium-channel antagonists diltiazem, amlodipine and verapamil, the angiotensin-II antagonists losartan, irbesartan, valsartan and telmisartan, and the antiarrhythmic drug flecainide, in whole blood samples from forensic autopsies was developed. Sample clean-up was achieved by precipitation and solid phase extraction (SPE) with a mixed-mode column. Quantification was performed by reversed phase high performance liquid chromatography with positive electrospray ionization mass spectrometric detection (HPLC-MS). The method has been developed and robustness tested by systematically searching for satisfactory conditions using experimental designs including factorial and response surface designs. With the exception of amlodipine, the concentration limit of quantification (cLOQ) covered low therapeutic concentration levels for all the compounds. Within assay precisions and accuracies (bias) were 3.4-21% RSD and from -24 to 21% for the concentration range 1.00-5.00 microM, respectively. Between assay precisions were 4.4-28% RSD for the concentration range from 0.1 to 5 microM and recoveries varied from 9 to 103%. The method is used for determination of cardiovascular drugs in post-mortem whole blood samples from forensic autopsy cases.