Characterization of P-glycoprotein and multidrug resistance proteins in rat kidney and intestinal cell lines.

The activity of P-glycoprotein (Pgp/MDR1/ABCB1) and multidrug resistance proteins (MRP/ABCC) influence the pharmacokinetics and bioavailability of many drugs. Few suitable cell lines for the study of drug transport exist. Additional non-human cell lines may help clarify species differences and contribute to the current knowledge of drug transport. The aim of the present study was to characterize three rat epithelial cell lines for transporter expression and activity. Transporter expression was assessed in intestinal IEC-6 and renal GERP and NRK-52E cells using RT-PCR and Western blot analysis. Pgp and Mrp transport activity were analyzed by measuring calcein accumulation and glutathione-S-bimane efflux, respectively. The three cell lines showed Pgp expression and Pgp-dependent transport, both decreasing with culture time after reaching confluency. Besides Pgp, cells expressed Mrp1, Mrp3, Mrp4, and Mrp5, while Mrp2 and Mrp6 were absent. In addition, they showed temperature- and Mrp-dependent efflux of glutathione-S-bimane. Exposure to a panel of different inhibitors showed that this efflux was probably mediated by Mrp4. In conclusion, the three rat epithelial cell lines investigated showed Pgp and Mrp expression and transport. Mrp dependent transport was most likely mediated by Mrp4. In future, these cell lines may be used as in vitro models to study drug transport.     

Impairment of intramacrophagic Brucella suis multiplication by human natural killer cells through a contact-dependent mechanism

Brucella spp. are facultative intracellular bacteria that can establish themselves and cause chronic disease in humans and animals. NK cells play a key role in host defense. They are implicated in an early immune response to a variety of pathogens. However, it was shown that they do not control Brucella infection in mice. On the other hand, NK cell activity is impaired in patients with acute brucellosis, and recently it was demonstrated that human NK cells mediate the killing of intramacrophagic Mycobacterium tuberculosis in in vitro infection. Therefore, we have analyzed the behavior of Brucella suis infecting isolated human macrophages in the presence of syngeneic NK cells. We show that (i) NK cells impair the intramacrophagic development of B. suis, a phenomenon enhanced by NK cell activators, such as interleukin-2; (ii) NK cells cultured in the presence of infected macrophages are highly activated and secrete gamma interferon and tumor necrosis factor alpha; (iii) impairment of bacterial multiplication inside infected cells is marginally associated with the cytokines produced during the early phase of macrophage-NK cell cocultures; (iv) direct cell-to-cell contact is required for NK cells to mediate the inhibition of B. suis development; and (v) inhibition of B. suis development results from an induction of NK cell cytotoxicity against infected macrophages. Altogether, these findings show that NK cells could participate early in controlling the intramacrophagic development of B. suis in humans. It seems thus reasonable to hypothesize a role for NK cells in the control of human brucellosis. However, by impairing the activity of these cells in the acute phase of the illness, the pathogen should avoid this control.     

Analysis of the behavior of eryC mutants of Brucella suis attenuated in macrophages

The facultatively intracellular pathogen Brucella, characterized by its capacity to replicate in professional and non professional phagocytes, also causes abortion in ruminants. This property has been linked to the presence of erythritol in the placenta, as brucellae preferentially utilize erythritol. The ery operon encodes enzymes involved in erythritol metabolism, and a link with virulence has since been discussed. Allelic exchange mutants in eryC of Brucella suis were erythritol sensitive in vitro with a MIC of 1 to 5 mM of erythritol. Their multiplication in macrophage-like cells was 50- to 90-fold reduced, but complementation of the mutant restored wild-type levels of intracellular multiplication and the capacity to use erythritol as a sole carbon source. In vivo, the eryC mutant colonized the spleens of infected BALB/c mice to a significantly lower extent than the wild type and the complemented strain. Interestingly, eryC mutants that were in addition spontaneously erythritol tolerant nevertheless exhibited wild-type-like intramacrophagic and intramurine replication. We concluded from our results that erythritol was not an essential carbon source for the pathogen in the macrophage host cell but that the inactivation of the eryC gene significantly reduced the intramacrophagic and intramurine fitness of B. suis.     

Advanced heterotopic pregnancy after in-vitro fertilization and embryo transfer, with survival of both the babies and the mother.

A combination of an extra-uterine and an intra-uterine pregnancy is defined as heterotopic pregnancy. An infertile patient, pregnant at her fourth in-vitro fertilization/embryo transfer attempt, was diagnosed at 21 weeks' gestation as having simultaneous abdominal and intra-uterine pregnancy. Expectant management under strict hospitalization was proposed and accepted by the couple, fetal assessment was by serial ultrasound evaluation of growth and amniotic fluid volume and by non-stress tests. Planned operative delivery was accomplished at 34 weeks' gestation. Both the mother and infants are alive and well.     

Patient education in Parkinson's disease: Formative evaluation of a standardized programme in seven European countries

OBJECTIVE: To evaluate a newly developed education programme for Parkinson's disease (PD) patients. METHODS: The programme consisted of eight sessions and aimed at improving knowledge and skills related to self-monitoring, health promotion, stress management, depression, anxiety, social competence, and social support, all with special reference to PD. The programme was formatively evaluated in seven European countries (Spain, Finland, Italy, The Netherlands, United Kingdom, Estonia, Germany) with 151 patients diagnosed with idiopathic PD. The evaluation included patients' ratings of the comprehensibility and feasibility of the programme as well as mood ratings before and after each session. Patients also completed questionnaires at the beginning and end of the programme to explore possible changes in disease-related psychosocial problems, quality of life, and depression. RESULTS: The programme was feasible to run, and patients were able to understand its elements. Patients reported mood elevations following individual sessions and reduced disease-related psychosocial problems after completing the programme. There were no substantial differences in results between cultures. CONCLUSION: Patient education appears to have potential as a useful and feasible intervention, complementing medical treatment in PD. PRACTICE IMPLICATIONS: The present programme will soon be available in seven European languages and can be tested in different health care systems.     

Neuromotor function and school performance in 7-year-old children born as high-risk preterm infants

Neuromotor behavior was studied in 63 children at a mean age of 7 years. They were born at a gestational age less than 32 weeks and/or birthweight under 1500 g and were categorized according to their medical history in conformance with the Neonatal Medical Index (from category I to V, from few to serious complications). We included only children considered at high risk as categorized in III to V. The neuromotor behavior study focuses on different subcategories, such as hand function, quality of walking, posture, passive muscle tone, coordination, and diadochokinesia. Hand preference and/or lateralization, the presence of associated movements, and/or asymmetry were noted, as was school performance. Then gender, gestational age, birthweight, and dysmaturity were investigated as confounding factors. The outcome at 7 years was correlated with the Neonatal Medical Index and the neonatal brain ultrasonography classification. None of the children scored 100% on the combined subcategories. Nineteen children (30%) had an overall score between 75 and 99%. Significant relationships between all different subcategories were found. Lack of hand preference, poor lateralization, and male gender were related to poor overall outcome. Poor motor control was correlated to special schooling and education below age level. The Neonatal Medical Index proved to have a significant influence on total outcome and the subcategories at the age of 7 years, with the worst outcome in children formerly classified in category V. Neuromotor behavior at 7 years of age was not related to birthweight, gestational age, dysmaturity, and neonatal brain ultrasonography classification only.     

Intravenously administered short interfering RNA accumulates in the kidney and selectively suppresses gene function in renal proximal tubules.

Different gene-silencing methods, like antisense and short interfering RNA (siRNA), are widely used as experimental tools to inhibit gene expression. In the present study, the in vivo behavior of siRNA in rats and siRNA-mediated silencing of genes in the renal proximal tubule were investigated. To study the biodistribution of siRNA, rats were injected i.v. with radiolabeled siRNA or radiolabel alone (control), and scintigraphic images were acquired at different time intervals postinjection. The siRNA preferentially accumulated in the kidneys and was excreted in the urine. One hour after injection, the amount of siRNA present in both kidneys (1.7 +/- 0.3% of injected dose/g tissue) was on average 40 times higher than in other tissues (liver, brain, intestine, muscle, lung, spleen, and blood). Besides the biodistribution, the effect of siRNA on multidrug resistance protein isoform 2 (Mrp2/Abcc2, siRNAMrp2) in renal proximal tubules was investigated. Mrp2 function was assessed by measuring the excretion of its fluorescent substrate calcein in the isolated perfused rat kidney. Four days after administration, siRNAMrp2 reduced the urinary calcein excretion rate significantly (35% inhibition over the period 80-150 min of perfusion). This down-regulation was specific because another siRNA sequence directed against a different transporter in the proximal tubule, Mrp4 (Abcc4, siRNAMrp4), did not alter the Mrp2-mediated excretion of calcein. In conclusion, siRNA accumulates spontaneously in the kidney after i.v. injection, where it selectively suppresses gene function in the proximal tubules. Therefore, i.v. administered siRNA provides a novel experimental and potential therapeutic tool for gene silencing in the kidney.     

Cytogenetics of uncleaved oocytes and arrested zygotes in IVF programs

Purpose: Cytogenetic studies of arrested oocytes and zygotes were used to understand in vitro fertilization (IVF) failures. Methods: We investigated the cytogenetics (Giemsa banding and FISH) of 710 uncleaved oocytes and 94 arrested zygotes from 208 patients undergoing IVF procedures. Results and Conclusions: Of uncleaved oocytes without a polar body, 39% were judged cytogenetically abnormal (17% unbalanced predivision and 21.5% diploid). Of 575 oocytes with a polar body, 124 (21.5%) showed numerical or structural chromosome aberrations. In arrested zygotes, approximately equal cases were found with separate condensed haploid complements (no syngamy), nuclear asynchrony and pulverized DNA, and apparently cytogenetically normal zygotes arrested at mitosis. These data on chromosome abnormalities were also analyzed with respect to two ovarian stimulation protocols and to maternal age. Both ovarian stimulation protocols showed the same levels of chromosome abnormalities. Overall chromosome abnormalities and premature chromosome condensation were also unchanged with maternal age. These data illustrate the significance of chromosome aberrations in IVF failures.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.