Distribution of drugs over whole blood: II. The transport function of whole blood for phenytoin

Phenytoin (DPH) partition between the three main blood compartments, i.e., plasma proteins, erythrocytes, and plasma water, was studied at various concentrations in vitro and in vivo. In vitro, the partition ratio of DPH in a system of erythrocytes in plasma water was 4.5 at concentrations between 0.8 and 100.8 micrograms DPH/ml. In vitro in whole blood (hence, in the presence of plasma proteins), this ratio was approximately 3.9. At 38 degrees C, blank erythrocytes were already in equilibrium with DPH-spiked plasma 3 min after contact, whereas at 20 degrees C, equilibration took 10 minutes or more. By adding blank ultrafiltrate to blood containing DPH, DPH concentrations of blood compartments shifted. It appeared that with the added blank ultrafiltrate, DPH was delivered overproportionally from erythrocytes and less from the protein fraction. In vivo, the elimination half-life of DPH in erythrocytes was 21.4 h and in plasma proteins 67.9 h. These results are similar to those obtained with valproate. It is concluded that erythrocytes have a low affinity for DPH. Their high-capacity transport system, having a "last-come-first-go" mechanism, plays a quantitatively important role in the transport of DPH.     

Assessment of virulence of pigeon isolates of Salmonella enterica subsp. enterica serovar typhimurium variant copenhagen for humans

Salmonella enterica serovar Typhimurium variant Copenhagen was isolated from 5 of 152 (3.3%) feral pigeons from the city of Ghent (Belgium) and from 26 pooled fecal samples from 114 pigeon lofts (22.8%). These isolates belonged to phage type (PT) 99. Seven of the pigeon isolates were further compared in vitro to five human variant Copenhagen isolates, 2 isolates of PT 208, 1 isolate each of PT 120 and U302, and a nontypeable isolate. No differences in invasiveness in human intestinal epithelial Caco-2 cells were found. The human strains, however, were able to multiply significantly more inside human THP-1 macrophages than the pigeon strains. After inoculation of mice with a pigeon PT 99 strain, high numbers of Salmonella bacteria were shed with the feces, the internal organs were heavily colonized, and the animals showed severe clinical symptoms resulting in death. In conclusion, the less-pronounced ability of the pigeon variant Copenhagen strains to multiply inside human macrophages than human strains as well as the lack of human PT 99 isolates during 2002, despite the relatively high frequency of this PT in the pigeon population, suggest these strains to be of low virulence to humans. However, the high virulence for mice of the tested strain implies that rodents may act as reservoirs.     

Distinct effects of amantadine and memantine on dopaminergic transmission in the rat striatum

Striatal glutamatergic inputs are known to participate in the modulation of dopaminergic transmission. Accordingly, the non-competitive N-methyl-D-aspartate receptor antagonists memantine and amantadine increase striatal dopamine levels, the latter being widely used in Parkinson's disease therapy. Based on our previous work revealing increased function of dopamine receptors and dopamine transporter after amantadine treatment, we studied the effects of repeated memantine administration on dopaminergic neurotransmission. On rat striatal membranes, dopamine-stimulated [(35)S]GTPgammaS binding was significantly reduced (20%) after 2 days injection with memantine (20 mg/kg per day, i.p.) but not after longer treatments (4 or 7 days). Evaluation of [(3)H]SCH 23390 and [(3)H]spiperone specific bindings only revealed a significant increase in D1 receptor density after 4 or 7 days treatment. Finally, none of these treatments were found to change the activity of the neuronal dopamine transporter in striatal synaptosomes. This shows that amantadine and memantine differentially affect striatal dopaminergic transmission, which could indicate that these two related aminoadamantanes display distinct pharmacodynamic properties.     

Intelligence indices in people with a high/low risk for developing Huntington''s disease.

Intelligence in 20 presymptomatic subjects with an increased risk (> 95%) for carrying the gene for Huntington's disease (HD) was studied in a prospective, case-control, single blind study. No significant differences between the groups were detected for intelligence indices and subtest scores (Wechsler Adult Intelligence Scale). The high level of the performance IQ and the significant discrepancy between performance IQ and verbal IQ found in both the high risk and the low risk groups contrasted with our expectations based on anamnestic information, general clinical opinion, and the results of previously conducted studies. We propose that psychosocial circumstances could explain the test results and discuss the consequences of our findings for clinical genetics practice.     

Genetic relatedness within serotypes of penicillin-susceptible Streptococcus pneumoniae isolates

The molecular epidemiological characteristics of all Streptococcus pneumoniae strains isolated in a nationwide manner from patients with meningitis in The Netherlands in 1994 were investigated. Restriction fragment end labeling analysis demonstrated 52% genetic clustering among these penicillin-susceptible strains, a value substantially lower than the percentage of clustering among Dutch penicillin-nonsusceptible strains. Different serotypes were found within 8 of the 28 genetic clusters, suggesting that horizontal transfer of capsular genes is common among penicillin-susceptible strains. The degree of genetic clustering was much higher among serotype 3, 7F, 9V, and 14 isolates than among isolates of other serotypes, i.e., 6A, 6B, 18C, 19F, and 23F. We further studied the molecular epidemiological characteristics of pneumococci of serotype 3, which is considered the most virulent serotype and which is commonly associated with invasive disease in adults. Fifty epidemiologically unrelated penicillin-susceptible serotype 3 invasive isolates originating from the United States (n = 27), Thailand (n = 9), The Netherlands (n = 8), and Denmark (n = 6) were analyzed. The vast majority of the serotype 3 isolates (74%) belonged to two genetically distinct clades that were observed in the United States, Denmark, and The Netherlands. These data indicate that two serotype 3 clones have been independently disseminated in an international manner. Seven serotype 3 isolates were less than 85% genetically related to the other serotype 3 isolates. Our observations suggest that the latter isolates originated from horizontal transfer of the capsular type 3 gene locus to other pneumococcal genotypes. In conclusion, epidemiologically unrelated serotype 3 isolates were genetically more related than those of other serotypes. This observation suggests that serotype 3 has evolved only recently or has remained unchanged over long periods.     

Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA.

The detection of amplified products resulting from polymerase chain reactions (PCRs) remains a complicated process. To simplify the detection procedures, we developed a colorimetric microtiter plate hybridization assay for the specific detection of 5'-biotinylated PCR fragments of Mycobacterium leprae DNA. For this assay, an M. leprae DNA capture probe was made and immobilized on the wells of a microtiter plate. Hybridization of the biotin-labeled PCR fragments was detected through enzymatic color development. The resulting optical densities showed a logarithm-linear relationship with the amount of template DNA and corresponded to the intensity of the bands obtained through gel analysis and Southern blotting of the PCR products. The sensitivity of the assay was found to be 125 fg of genomic M. leprae DNA, or 20 lysed bacilli, revealing a detection limit similar to that of agarose gel analysis. The efficient coamplification of human DNA was used as a positive control for the presence of inhibitory substances in clinical material. For detection of human PCR products, a human DNA capture probe was also constructed for the colorimetric assay. This dual setup for hybridization, which thus detected both M. leprae and human DNA PCR products, was useful for ascertaining the presence of inhibiting substances in clinical specimens. All biopsy specimens (n = 10) from untreated patients with leprosy were positive. Apparently, this assay is more sensitive than microscopy, because biopsy specimens from half of the patients were negative upon histopathological examination. Biopsy specimens from three treated patients were negative, as were those from the three patients who did not have leprosy. We conclude that this colorimetric assay can replace agarose gel analysis and Southern hybridization, because it is as sensitive as those methods. Its advantages over conventional gel analysis and Southern hybridization are that it is less cumbersome and more rapid.     

Pneumococcal carriage in children in The Netherlands: a molecular epidemiological study

In 1999, Engelen and coworkers investigated colonization in Amsterdam among 259 children attending 16 day-care centers (DCCs) and among 276 children who did not attend day-care centers (NDCCs). A 1.6- to 3.4-fold increased risk for nasopharyngeal colonization was observed in children attending DCCs compared with NDCC children, while no difference in antibiotic resistance was found between groups. The serotype and genotype distributions of 305 nasopharyngeal Streptococcus pneumoniae isolates of the latter study were investigated. The predominant serotypes in both the DCC and the NDCC groups included 19F (19 and 18%, respectively), 6B (14 and 16%, respectively), 6A (13 and 7%, respectively), 23F (9 and 7%, respectively), and 9V (7 and 7%, respectively). The theoretical vaccine coverage of the 7-valent conjugate vaccine was 59% for the DCC children and 56% for the NDCC group. Genetic analysis of the pneumococcal isolates revealed 75% clustering among pneumococci isolated from DCC attendees versus 50% among the NDCC children. The average pneumococcal cluster size in the DCC group was 3.8 and 4.6 isolates for two respective sample dates (range, 2 to 13 isolates per cluster), while the average cluster size for the NDCC group was 3.0 (range, 2 to 6 isolates per cluster). Similar to observations made in other countries, these results indicate a higher risk for horizontal spread of pneumococci in Dutch DCCs than in the general population. This study emphasizes the importance of molecular epidemiological monitoring before, during, and after implementation of pneumococcal conjugate vaccination in national vaccination programs for children.     

Molecular epidemiology of penicillin-susceptible non-beta-lactam-resistant Streptococcus pneumoniae isolates from Greek children

A total of 128 Streptococcus pneumoniae isolates that were susceptible to penicillin but resistant to non-beta-lactam agents were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and antibiotic resistance genotyping. The serotypes 6A/B (49%), 14 (14%), 19A/F (11%), 11A (9%), 23A/F (4%), 15B/C (2%), and 21 (2%) were most prevalent in this collection. Of the isolates, 65% were erythromycin resistant, while the remaining isolates were tetracycline and/or trimethoprim-sulfamethoxazole resistant. Fifty-nine distinct RFEL types were identified. Twenty different RFEL clusters, harboring 2 to 19 strains each, accounted for 76% of all strains. Confirmatory multilocus sequence typing analysis of the genetic clusters showed the presence of three international clones (Tennessee(23F)-4, England(14)-9, and Greece(6B)-22) representing 30% of the isolates. The erm(B) gene was present in 70% of the erythromycin-resistant isolates, whereas 18 and 8% contained the mef(A) and mef(E) genes, respectively. The pneumococci representing erm(B), erm(A), and mef genes belonged to distinct genetic clusters. In total, 45% of all isolates were tetracycline resistant. Ninety-six percent of these isolates contained the tet(M) gene. In conclusion, penicillin-susceptible pneumococci resistant to non-beta-lactams are a genetically heterogeneous group displaying a variety of genotypes, resistance markers, and serotypes. This suggests that multiple genetic events lead to non-beta-lactam-resistant pneumococci in Greece. Importantly, most of these genotypes are capable of disseminating within the community.     

Relative importance of C4 binding protein in the modulation of the classical pathway C3 convertase in patients with systemic lupus erythematosus

Serum concentrations of C1q, C4, C4 binding protein (C4bp), C3 and C2 haemolytic activity have been measured in 110 samples from 20 patients with systemic lupus erythematosus (SLE). Significant reductions in comparison to normal levels were found in the mean serum concentrations of C4, C3 and C4bp as well as C2 haemolytic activities. For patients serum concentrations of C4 correlated with C2 haemolytic activities (r = 0.91) and C4bp (r = 0.79); the C2 haemolytic levels correlated with the concentration of C4b (r = 0.72). It is concluded that serum concentrations of the complement components C4 and C2, which are the constituents of the classical pathway C3 convertase, are regulated by C4bp in vivo. Further metabolic studies are required to determine the causes of decreased serum concentrations of C4bp in patients with SLE.