Functional role of P-glycoprotein in the human blood-placental barrier

OBJECTIVE: In vitro and animal experiments suggest that P-glycoprotein forms a functional barrier between maternal and fetal blood circulation in the placenta, thus protecting the fetus from exposure to xenobiotics during pregnancy. In this study we aimed to characterize the role of P-glycoprotein in the blood-placental barrier by use of dually perfused human placenta. METHODS: Twenty-eight human placentas were obtained after delivery, and both the maternal side and the fetal side were perfused for 2 hours. Saquinavir was used as a probe drug for P-glycoprotein-dependent active transfer, and PSC833 (valspodar) or GG918 was used as an inhibitor of P-glycoprotein function in a maternal-to-fetal and fetal-to-maternal perfusion setting. Genotyping for ABCB1 (C3435T and G2677A/T) polymorphism and quantification of P-glycoprotein expression were done for each placenta. RESULTS: The fetal-to-maternal transfer of saquinavir was 108-fold higher (P = .003) compared with transfer from the maternal to the fetal direction. Preperfusion with PSC833 increased the placental transfer of saquinavir by 7.9-fold (P < .001), and preperfusion with GG918 increased it by 6.2-fold (P < .001). The end-perfusion transfer (percentage) of saquinavir at 120 minutes was 11-fold (P < .001) and 6-fold (P < .001) higher in placentas preperfused with PSC833 and GG918, respectively, compared with control. However, PSC833 had no effect on the transfer of saquinavir from the fetal to the maternal direction (P = .79). P-glycoprotein expression was correlated with the PSC833-induced change in the saquinavir transfer (r = 0.75, P = .086). ABCB1 polymorphism did not affect the PSC833- or GG918-induced change in the saquinavir transfer. CONCLUSIONS: P-glycoprotein has a major functional role in the human blood-placental barrier but a negligible role in the removal of substances from the fetal circulation to maternal blood. Pharmacologic blockade of P-glycoprotein function can lead to disruption of the blood-placental barrier and increase the transfer of P-glycoprotein substrates to the fetal side by several-fold, which may be a noteworthy mechanism for teratogenicity.     

Cholecystokinin tetrapeptide effects on HPA axis function and elevated plus maze behaviour in maternally separated and handled rats

Deficits in the function of the hypothalamic-pituitary-adrenal (HPA) axis have been suggested to predispose to the development of depression and anxiety disorders. This is mirrored in the animal model "Maternal Separation (MS)" where the stress of repeated separation of rat pups from the dam during early postnatal development results in long lasting alterations in HPA axis function. Cholecystokinin increases serum concentrations of stress axis hormones and might be involved in the dam-pup interaction in rats. Therefore, we hypothesized that adult animals, which had been separated daily (postnatal days (PND) 2-14) for 180 min (MS180) would differ in HPA axis responsiveness to an intravenous challenge dose of cholecystokinin tetrapeptide (CCK-4) compared to handled rats, separated for 15 min daily. The study explored the effects of intravenous CCK-4 on elevated plus maze behaviour and HPA axis hormones. MS180 animals displayed reduced general activity but unaltered levels of open arm activity in the elevated plus maze. CCK-4 administration elevated general activity in the handled rats, while leaving MS180 rats unaffected. MS180 rats had increased baseline CRF mRNA expression in the paraventricular nucleus of the hypothalamus. When CRF mRNA was assessed in chronically catheter implanted and single housed rats, lower levels were found in the paraventricular nucleus of MS180 animals compared to handled animals and this parameter was not affected by CCK-4 treatment. Adrenocorticotropin concentrations in serum were equal in MS180 and handled rats and unaffected by CCK-4. Corticosterone serum concentrations were lower in saline treated MS180 rats compared to saline treated handled rats. CCK-4 injection raised serum corticosterone in MS180 rats to levels equal to the handled rats, while leaving handled rats unaffected. We suggest that the lower levels of hypothalamic CRF mRNA and serum corticosterone concentrations in MS180 rats might be due to the experimental set-up with chronic venous catheter implants and single housing. In conclusion, this study supports the hypothesis of elevated CCK sensitivity in separated rats as measured by corticosterone changes thus adding to the existing literature reporting early life stress having long-term impact on HPA axis function.     

Increased adult hippocampal brain-derived neurotrophic factor and normal levels of neurogenesis in maternal separation rats

Repeated maternal separation of rat pups during the early postnatal period may affect brain-derived neurotrophic factor (BDNF) or neurons in brain areas that are compromised by chronic stress. In the present study, a highly significant increase in hippocampal BDNF protein concentration was found in adult rats that as neonates had been subjected to 180 min of daily separation compared with handled rats separated for 15 min daily. BDNF protein was unchanged in the frontal cortex and hypothalamus/paraventricular nucleus. Expression of BDNF mRNA in the CA1, CA3, or dentate gyrus of the hippocampus or in the paraventricular hypothalamic nucleus was not affected by maternal separation. All animals displayed similar behavioral patterns in a forced-swim paradigm, which did not affect BDNF protein concentration in the hippocampus or hypothalamus. Repeated administration of bromodeoxyuridine revealed equal numbers of surviving, newly generated granule cells in the dentate gyrus of adult rats from the 15 min or 180 min groups. The age-dependent decline in neurogenesis from 3 months to 7 months of age did not differ between the groups. Insofar as BDNF can stimulate neurogenesis and repair, we propose that the elevated hippocampal protein concentration found in maternally deprived rats might be a compensatory reaction to separation during the neonatal period, maintaining adult neurogenesis at levels equal to those of the handled rats.     

Reproducible loss of CA1 neurons following carotid artery occlusion combined with halothane-induced hypotension

The 2-vessel occlusion approach to produce global ischemia in rats requires concomitant reduction of systemic blood pressure. We have utilized the hypotensive effect of halothane administrated by artificial respiration to prevent respiratory arrest and to ensure stable physiological conditions. Systemic blood pressure was reduced to 40-45 mmHg by instant adjustments of the halothane concentration. Bilateral occlusion of the carotid arteries caused a profound and reproducible ischemia, as analyzed by laser-Doppler flowmetry. In the rats exposed to 11, 12, or 13 min of ischemia, 5% died and 5% developed seizures. The extent of neuronal death in CA1 was highly correlated to the duration of ischemia. Following 11 min of ischemia, CA1 neuronal cell death, as analyzed by Fluoro-Jade, was absent 1 day after injury, variable at day 4, and consistent at day 7. The numbers of cresyl violet- and NeuN-positive neurons at day 7 were 8% and 20% of control, respectively. OX42 immunoreactivity was low and variable at day 4, but pronounced at day 7. In conclusion, this rat global ischemia model is relatively simple to perform, has a low mortality, and produces a profound and highly reproducible delayed cell death of hippocampal CA1 neurons.     

Immunization with the glutamate receptor-derived peptide GluR3B induces neuronal death and reactive gliosis, but confers partial protection from pentylenetetrazole-induced seizures

Do autoantibodies (Ab's) against glutamate/AMPA receptor subtype 3 affect the severity of seizures? Rats immunized with the GluR3B-peptide (amino acids (aa) 372-395) or with the control GluR3A-peptide (aa 245-274) produced the respective anti-GluR3B and anti-GluR3A Ab's (both types of Ab's found in some epilepsy patients). The GluR3B-immunized rats exhibited neuronal death and reactive gliosis in the brain, but not overt spontaneous seizures. Surprisingly, in response to the chemoconvulsant pentylenetetrazole, the GluR3B-immunized rats displayed fewer jerks, a lower percentage of generalized seizures, and a lower overall seizure-severity score than GluR3A-immunized, scrambled GluR3B-immunized or non-immunized control rats. These findings, combined with the previously demonstrated ability of anti-GluR3B Ab's to bind, activate, and kill neurons and glia, suggest that if these Ab's are present in the brain they may cause neuronal death, which by itself may be pro-epileptic, but they may also decrease the excitability of seizure-related neural circuits, thereby conferring partial protection from seizures induced by other exogenously applied epileptogenic stimuli. The present results could have clinical implications for epilepsy.     

Botox and collagen for glabellar furrows: advantages of combination therapy

Plastic surgeons frequently administer botulinum toxin A (Botox) or collagen as monotherapy to treat glabellar furrows. This study evaluates the possible advantages of combination therapy. Sixty-five patients with moderate to severe glabellar rhytids were prospectively randomized to receive standard injections of Botox, Zyderm II collagen, or a combination. Improvement in rhytids was assessed over 3 months using patient satisfaction scores and an independent physician evaluation. Baseline wrinkle severity was similar in all 3 groups. By 1 month posttreatment, the combination arm showed significantly greater improvement in furrows (79% compared with only 56% and 50% in the Botox and Zyderm arms, respectively; P < 0.05). At 3 months postinjection, the dual-therapy arm maintained better improvement (57% versus 33% and 27% in the monotherapy arms; P < 0.05). Patient satisfaction further highlighted the superiority of the combination approach. By simultaneously addressing the static and dynamic aspects of glabellar furrows, dual therapy provides optimal treatment of this problem.     

Effect of prolonged use of injectable hormonal contraceptives on blood pressure and body weight

The study was undertaken to determine the effect of prolonged use of injectable hormonal contraceptive on blood pressure and body weight in young women. Two hundred volunteers were selected for the purpose. Of them, one hundred and forty were taking injectable hormonal contraceptive, DMPA for 3 to 5 years uninterruptedly. Rest forty subjects served as control using no contraceptive steroid. Blood pressure, systolic as well as diastolic, was measured by sphygmomanometer. Body weight was measured by weighing machine. It was observed that there were insignificant (P>0.05) elevations of systolic and diastolic blood pressure from DMPA use. But body weight of the experimental subjects was significantly (P <0.05) increased in comparison to that of the control.     

Body composition and bone measurements in intra-uterine growth retarded and early postnatally undernourished male and female rats at the age of 6 months: comparison with puberty

Undernutrition in early life may permanently change body structure, physiology and metabolism and leads to chronic diseases in later life. To test whether malnutrition during different critical time periods around birth in the rat has long-lasting effects on body composition and skeletal growth, we examined body weight and body composition in pubertal rats and adult rats of 6 months after pre- and postnatal malnutrition. Prenatal malnutrition or intra-uterine growth retardation (IUGR) was induced by ligation of the uterine arteries on day 17 of gestation and postnatal food restriction (FR) by litter enlargement to 20 pups per mother from day 2 after birth until weaning (day 24). Pubertal markers were balanopreputial separation (BPS) in the male and vaginal opening (VO) in the female. IUGR as well as FR resulted in a persistent growth retardation. From birth in IUGR rats and from day 4 after birth in FR rats until 6 months of age body weight in male and female rats was significantly lower compared with controls (P < 0.01 and P < 0.05). Although total body bone mineral content (TBBMC) did not differ between male IUGR rats and controls at BPS, at the age of 6 months TBBMC was significantly lower (P < 0.01) compared with controls. At BPS as well as at 6 months of age, TBBMC was significantly lower in male FR rats compared with controls (P < 0.05 and P < 0.01). In the female IUGR rats TBBMC was significantly lower compared with controls at VO (P < 0.01) and 6 months (P < 0.05). TBBMC in the female FR rats was significantly lower at VO (P < 0.01), but did not differ from controls at the age of 6 months. For both IUGR and FR male and female rats these differences disappeared after adjusting for body weight. Body composition in terms of total fat mass, percentage fat and percentage lean did not differ from controls in male and female IUGR rats at 6 months and the same results were observed in the female FR rats. However, in the male FR rats, total fat mass and percentage fat were significantly lower compared with controls (P < 0.01 and P < 0.05), while the percentage lean mass was significantly higher (P < 0.05). We conclude that different critical time periods of malnutrition around birth have different effects later in life on growth, which do not disappear at least after 6 months of life. With respect to body composition, only in the FR male rats, differences are found in total fat mass and the balance of percentage fat mass and lean mass. At time of puberty and at the age of 6 months bone mass adjusted for body weight does not seem to be affected by perinatal undernutrition.     

Impact of information leaflets on HIV test uptake amongst GUM clinic attendees: an update

In the Sexual Health Strategy, the targets of HIV testing uptake in genitourinary medicine clinics (GUM) are set at 40% by year 2004 and 60% by 2007. We assessed the uptake of HIV test amongst GUM clinic attendees, more than six months after introducing an information leaflet in place of verbal pre-test counselling (PTC). The uptake rate was significantly higher than in a previous audit conducted in the same year, two weeks after introduction of the leaflets (62% vs 50%, P =0.001).     

Complex Coacervation of Lysozyme and Heparin: Complex Characterization and Protein Stability

No HeadingPurpose. To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability.Methods. Insoluble lysozyme-heparin complexes were formed at pH 7.2 and the binding stoichiometry determined using a solution depletion method. Protein structure was determined by infrared spectroscopy and intrinsic fluorescence. Protein stability was evaluated using differential scanning calorimetry and followed in a 12-weeks storage stability study at 37C.Results. Binding stoichiometry between heparin and lysozyme was found to be dependent on ionic strength of the solution. At low ionic strength (I 0.01) about 11 lysozyme molecules could bind to a 17 kDa heparin chain, 3 to a 6 kDa chain, and less than 2 to a 3 kDa chain. At higher ionic strength (I 0.1), only 7 lysozyme molecules could bind to a 18 kDa heparin chain.. Above ionic strengths of approximately 0.32 M, no insoluble complexes were observed. Infrared spectroscopy and intrinsic fluorescence did not show any major changes in protein structure upon complexation to heparin. In contrast, differential scanning calorimetry showed a large decrease in the melting temperature of the protein, from 77C to 61C. Moreover, after 12-weeks storage at 37C, only 60% protein recovery was observed for the complexes, with no loss of protein for the uncomplexed protein.Conclusions. Heparin has multiple binding sites for lysozyme, amounting to at most one lysozyme molecule per 3 disaccharide units of heparin. Complexation decreased lysozyme stability, suggesting that heparin has a higher affinity for the unfolded state than the native state. Similar destabilization may occur for other proteins upon interaction with highly charged polymeric compounds or surfaces.