Alternative microbial testing: a novel DNA-based detection system for specified microorganisms in pharmaceutical preparations.

Fluorescence-coupled PCR technology was employed to quantify DNA segments specific for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae. The PCR procedure is put forward as an alternative method for detecting microbial contaminations in pharmaceutical preparations and is compared to the tests for specified microorganisms described in European Pharmacopoeia (EP) 2, 2.6.13 and the USP, chapter 61. Data presented here describe the validation of this analytical method when used for proof of absence of specified microorganisms. The detection systems were specific for the microorganisms analyzed, and led to linear results over a wide range (more than 6-7 log intervals). The correlation coefficients lay above 0.99. The precision of replicate determinations within a single test was observed to be high, the relative standard deviation being between 0.39% and 1.53%. The precision between different tests was also high, with a relative standard deviation between 0.76% and 1.91%. The sensitivity without pre-enrichment amounted to 1-10 CFU. Since determination of the specified bacteria was performed following pre-enrichment, the limit of detection amounted to 1 CFU. Equivalent results were obtained in a study on nine batches of a milky hydrophilic cream (SH-No. M 440 A) with the conventional test for microbial contamination and the PCR procedure. The data presented here strongly indicate that the use of fluorescence-coupled PCR techniques can prove the absence of specified bacteria faster and more efficiently than conventional methods.

Molecular and biological analysis of eight genetic islands that distinguish Neisseria meningitidis from the closely related pathogen Neisseria gonorrhoeae.

The pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels. N. meningitidis can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike N. gonorrhoeae. We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to N. meningitidis and absent from N. gonorrhoeae. In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in N. meningitidis.

Two-dimensional structure of the Opc invasin from Neisseria meningitidis.

A two-dimensional structural model was devised for the Opc outer membrane protein invasin which contains 10 transmembrane strands and five surface-exposed loops. One continuous epitope recognized by three monoclonal antibodies was localized to the tip of loop 2 by synthetic peptides and site-directed mutagenesis while a second, discontinuous epitope recognized by a fourth antibody was localized to loops 4 and 5 by insertion mutagenesis. These monoclonal antibodies are bactericidal and inhibit adhesion and invasion. Most of the T-cell epitopes defined by Wiertz et al. (1996) were localized to the transmembrane strands. Oligonucleotides encoding a foreign epitope (nabla) from Semliki Forest virus were inserted into BglII restriction sites created by site-directed mutagenesis. The nabla epitopes inserted in all five predicted loops were recognized on the cell surface of live Escherichia coli bacteria by a monoclonal antibody and are exposed while nabla epitopes in the N-terminus or three predicted turns were not. The results thus confirm important predictions of the model and define five permissive sites within surface-exposed loops which can be used to insert foreign epitopes.

Correlation of relaxometry and histopathology: the transplantable human glioblastoma SF295 grown in athymic nude mice.

Human glioblastomas of the brain are characterized by a wide range of proton relaxation rates in vitro (1/T1 and 1/T2) and heterogeneous appearance in magnetic resonance imaging. It was previously found that their 1/T1 values vary widely at magnetic field strengths much below imaging fields, even at the same water content. In the present study, we measure 1/T1 at different magnetic field strengths (NMRD profile) for a specific transplantable, human glioblastoma (SF295), grown subcutaneously in athymic nude mice, to search for histologic characteristics that might correlate with the variability of 1/T1 at low fields (1/T1L). Using a field-cycling relaxometer, NMRD profiles were obtained for 32 fresh, histologically characterized, tumor specimens, 7 to 24 days post implantation of cryopreserved SF295 fragments. Tumor volume, dry weight, and pH of specimens were determined, the extent of hemorrhage and necrosis rated, and specimen location within the tumor recorded. A statistically significant increase in the average 1/T1 was found with increasing level of necrosis at 0.0024 T and below, possibly reflecting progressive protein aggregation in samples with up to 40% necrosis. This correlation was not significant at imaging fields. Although pH was increased in central necrosis, neither pH, dry weight, sample location, nor fresh hemorrhage could explain the changes in 1/T1L. The variability of 1/T1L among SF295 samples is much reduced compared to that of fresh surgical specimens of human glioblastomas of the brain. The heterogeneous appearance of glioblastomas in MRI may have a histologic correlate which reflects molecular changes involved with induction of cell death and necrosis. Further investigations may identify the factors responsible for affecting 1/T1L (hypoxia, radiation, chemotherapy).

Pharmacokinetics of resorcinol in the rat.

Pharmacokinetic data on resorcinol were obtained from studies in the rat. The drug, administered subcutaneously in an aqueous solution, rapidly cleared plasma vir urine and did not accumulate in tissues. In the urine, the major metabolite of resorcinol was the glucuronide. Repeated dosing for 30 days with maximum tolerated daily doses of 100 mg/kg did not alter pharmacokinetic parameters, nor did it cause overt toxic signs or adverse reactions. The animals' body weight, blood values, levels of serum T3 and T4' and the gross and microscopic appearance of the thyroid gland and spinal cord remained within normal limits throughout the study.

Combination chemotherapy against B16 melanoma: bleomycin/vinblastine, bleomycin/cis-diamminedichloroplatinum, 5-fluorouracil/BCNU and 5-fluorouracil/methyl-CCNU.

Four antitumor drug combinations which are currently in clinical use were evaluated experimentally using the murine B16 melanoma model. Bleomycin plus vinblastine produced an increase in life span over either of the two agents alone against both intraperitoneal and subcutaneous B16. This combination also resulted in a large number of long-term survivors. Bleomycin plus cis-platinum produced slight enhancement against subcutaneous B16, but showed no advantage against intraperitoneal B16. The combination of 5-fluorouracil plus methyl-CCNU significantly increased survival time against the intraperitoneal tumor, and produced long-term survivors as well. The combination of 5-fluorouracil plus BCNU was not more effective than BCNU or 5-fluorouracil alone. These data were compared with the degree of success reported from the clinics against a variety of solid human neoplasms.

Neuropharmacological effects of methotrexate perfused through the cerebrospinal fluid system of the rhesus monkey.

Thirteen adult Rhesus monkeys were repeatedly perfused through the ventriculocisternal or ventriculolumbar spaces with Elliott's B solution containing various concentrations of methotrexate (MTX) and trace amounts of [3H]MTX and [carboxy-14C]inulin. The concentrations of MTX ranged from 4.8 to 0.15 mg/ml representing perfusion dosages of 551 mg/sq m to 16 mg/sq m. The average steady-state concentration out-concentration in (Co/Ci) value for MTX was 0.78 +/- 0.04 for the ventriculocisternal and 0.66 +/- 0.01 for the ventriculolumbar routes. MTX treatments did not significantly affect mean inulin steady-state Co/Ci values or CSF formation rate. With the exception of a monkey perfused with MTX at an inflow concentration of 4.8 mg/ml, body weight, food intake, and urine output, analyzed at weekly intervals, generally were not remarkably affected by MTX perfusions. In five monkeys perfused with MTX in concentrations of 4.8 to 0.6 mg/ml, gross neurological toxicity was observed, principally in the form of seizures and hypokinesia during perfusion series with occasional residual motor deficit. Significant cerebral damage was associated with the brains of two monkeys perfused with MTX at concentrations of 2.4 and 0.6 mg/ml and two monkeys perfused at concentrations of 1.2 and 0.3 mg/ml; there of the four animals displayed signs of gross neurotoxicity, and two animals developed permanent motor deficits. However, the extent to which neurotoxic signs could be attributed solely to MTX was difficult to judge because some changes in central nervous system morphology were associated with the mechanical aspects of the procedure. Overall behavioral performance as measured by a visual pattern discrimination reinforced by avoidance or escape from an electric shock was not significantly affected by repeated perfusions of MTX (0.6 mg/ml) in two monkeys not otherwise studied in detail.

The effect of nitrogen mustard intoxication on glucose absorption from the small intestine of the rat.

Methyl bis (beta-chlorethyl) amine (HN(2)) was administered as a single intraperitoneal dose to male Sprague-Dawley rats. Animals were given either a median lethal dose and perfused one day after treatment or were given a larger dose and perfused two days after treatment. Intestinal segments were perfused in vivo and samples of effluent were collected and measured for glucose using the glucose oxidase method. Animals treated with the larger dose of HN(2) displayed significant reduction in intestinal dry weight. The average water content of intestinal segments derived from treated animals did not differ significantly from that in controls.Steady-state glucose absorption, obtained from data collected during the last 40 minutes of the perfusion period, was found to be significantly reduced in animals treated with drugs when intestinal absorption for control and treated animals was adjusted for water movement but not for dry intestinal weight and length. However, when glucose absorption was adjusted for dry tissue mass and length as well as for water movement, no significant differences in intestinal glucose absorption between control and treated rats were observed. Alterations can therefore be attributed to loss of intestinal tissue rather than to loss in the ability of the intestinal mass to absorb glucose.