Determination of penetration profiles of topically applied substances by means of tape stripping and optical spectroscopy: UV filter substance in sunscreens

Penetration profiles of topically applied drugs and cosmetic products provide important information on their efficacy. The application of tape stripping in combination with UV/VIS spectroscopy is checked to determine the local position of topically applied substances inside the stratum corneum, the penetration profile. The amount of corneocytes removed with each tape strip is quantified via the particle-dependent absorption, the pseudoabsorption, in the visible spectral range. The concentration of a typical UV filter substance, 4-methylbenzylidene camphor, is determined by optical spectroscopy using the tape strips removed originally. In this case, a time-dependent increase in the absorbance must be taken into account. Laser scanning microscopic investigations confirm that the nonhomogeneous distribution of the filter substance, on the strips, can explain this spectroscopic behavior. When reaching a homogeneous distribution, the UV spectroscopic signal reflects the correct concentration. These spectroscopic values are compared with high performance liquid chromatography (HPLC) data. The values obtained with both methods for the concentrations of 4-methylbenzylidene camphor are in good agreement. The data obtained are used to illustrate the determination of a penetration profile of a UV filter substance. The results demonstrate that the described protocol is well suited to characterize, in a simple manner, topically applied substances that have a characteristic UV/VIS absorption band.     

Quality assessment of heparin coatings by their binding capacities of coagulation and complement enzymes

In vitro testing of blood contacting materials before clinical application is generally advisable. Four heparin coatings from different manufacturers were tested for adsorbed proteins and soluble activation markers. The surface with the highest antithrombin, thrombin, high-molecular-weight-kininogen (HMWK) and the lowest fibrinogen binding capacity (Carmeda, Medtronic) showed significantly lower levels of granulocytes and platelet activation (beta-TG, PMN-elastase release). No statistically significant differences in soluble markers of the coagulation system could be detected (F1 + 2, TAT). Interestingly, complement activation (TCC) was significantly reduced within the group of the lowest adsorption of the complement factor C3. Our data demonstrate that there is a relation between the binding affinity of proteins (C1-inhibitor, C3-complement) and the consecutive changes in complement activation (TCC). Therefore, measuring adsorbed proteins on artificial surfaces is a suitable, sensitive and very reproducible method for assessing the thrombogenicity of biomaterials.     

Formation of viscoelastic protein layers on polymeric surfaces relevant to platelet adhesion

The hemocompatibility of biomaterials is highly dependent on the adhesion and activation of platelets. Surface-adsorbed fibrinogen has a dominant role in promoting platelet adhesion to artificial surfaces by binding glycoprotein IIb-IIIa (GPIIb-IIIa), the major platelet membrane receptor. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we have investigated the material-dependent binding kinetics of purified GPIIb-IIIa to polymer-adsorbed fibrinogen. The following ranking of polymer-adsorbed mass (fibrinogen and GPIIb-IIIa) to test polymers could be established: poly[desaminotyrosyl-tyrosine ethyl (DTE) carbonate]/poly(lactide-co-glycolide)>poly[DTE co-5% poly(ethylene glycol) carbonate]. The QCM-D fibrinogen adsorption data were confirmed using an immunofluorescence assay. A synthetic RGD-containing peptide, but not a control peptide, inhibited GPIIb-IIIa binding to polymer-adsorbed fibrinogen, demonstrating the specificity of binding. Importantly, the binding efficiency of purified GPIIb-IIIa to polymer-adsorbed fibrinogen correlated with increased platelet adhesion in an in vitro model. Theoretical simulations using a Voight-based model provided quantitative data on the thickness and viscoelastic properties of the polymer-adsorbed protein layers. The precision of the modeling technique was limited with respect to the shear moduli values, leading to large variations. However, the other modeling parameters showed reproducible results. The thickness of both protein layers was polymer-dependent and ranged from 5 to 35 nm and the viscosity from 0.001 to 0.005 kg/ms, whereas the protein layer densities showed little differences between the test polymers. These results suggest that material-dependent changes in the thickness and viscoelastic properties of adsorbed fibrinogen-GPIIb-IIIa layers are crucial factors in the binding behavior of platelets to biomaterials.     

Hemocompatibility of PMEA coated oxygenators used for extracorporeal circulation procedures

An inflammatory response to cardiopulmonary bypass (CPB) caused by bioincompatibility of extracorporeal circuits is one of the major clinical issues in cardiac surgery. Recently a new coating material, poly-2-methoxyethylacrylate (PMEA), was developed to improve the biocompatibility of blood contacting surfaces. In a simulated cardiopulmonary bypass model, using fresh human whole blood, 15 membrane oxygenators (Capiox SX18, Terumo Corp., Tokyo, Japan) were compared. Five of them had the PMEA coating, five had a heparin-coated surface, and five had no surface treatment. Blood samples were taken at several time-points during a 90 minute circulation period. Changes in coagulation, complement, and blood cell alteration factors were measured by ELISA methods, plasma bradykinin levels were measured by radioimmunoassay, and expression of genes encoding cytokines TNF-alpha, interleukin-1beta, interleukin-6, and interleukin-8 was determined by semiquantitative real time RT-PCR. Platelet adhesion was significantly reduced in both the PMEA and the heparin coated circuits. Release of platelet activation marker beta-thromboglobulin was significantly higher in the uncoated control group (p < 0.01). After 5 minutes of blood circulation bradykinin levels significantly increased in all three groups (p < 0.01); however, the group with the PMEA coated oxygenators showed the lowest values. Expression of genes encoding proinflammatory cytokines in monocytes was increased in all groups, with the lowest being in the PMEA coated group. PMEA coated CPB surfaces in an in vitro experimental model showed an improved thrombogenicity, reduced bradykinin release, less platelet activation and less proinflammatory cytokines gene expression in comparison with a noncoated group. The authors assume that PMEA coating may ameliorate some of intra- and postperfusion syndromes, particularly hypotension, unspecific inflammation, hyperfibrinolysis, and blood loss.     

Enhanced proliferation of endogenous virus in Chinese hamster cells associated with microtubules and the mitotic apparatus of the host cell.

Chinese hamster ovary cells harbour intracytoplasmic virus-like particles of type A which are closely associated with sites of microtubule formation. We report here the enhanced proliferation of these particles and their release at the cell membrane by using either 5-bromodeoxyuridine or dibutyryl cyclic AMP. The extracellular mature particles are similar in morphology to retroviruses of type B. Close association of the type A virus precursors with microtubule organizing centres, i.e. kinetochores, centrioles and basal bodies, and with microtubules per se, is confirmed by studying the effects of the microtubule inhibitors Colcemid and vincristine sulphate. The role of microtubules in the activation and transport of the intracytoplasmic type A particles is discussed.     

Regioselectivity and Enantioselectivity of Metoprolol Oxidation by Two Variants of cDNA-Expressed P4502D6

Purpose. The oxidative metabolism of metoprolol was investigated in two human lymphoblastoma cell-lines transfected with variants of cDNA for cytochrome P4502D6. Methods. The regioselective and enantioselective features of the oxidations of deuterium-labeled pseudoracemic metoprolol were characterized by GC/MS analysis of the substrate and products. Results. There were significant differences between the two P4502D6 variants in the formation kinetics of O-demethylmetoprolol and -hydroxymetoprolol. The h2D6-Val microsomes highly favored the formation of the O-demethylmetoprolol regioisomer 6.3:1 and 2.8:1, respectively from (R)-metoprolol-d₀ and (S)-metoprolol-d₂, while the corresponding ratios for h2D6v2 microsomes were much lower. For both variants, O-demethylmetoprolol formation favored the (R)-substrate 1.5 to 2-fold, while -hydroxymetoprolol formation was non-enantioselective. Similar Km values of metoprolol oxidation, 10-20 µM, were observed for the two microsomal preparations. Conclusions. The regioselectivity, enantioselectivity, and Km values for the h2D6-Val microsomes resemble those observed for the native P4502D6 in human liver microsomes, whereas the h2D6v2 microsomes deviated remarkably in regioselectivity.     

Acute obstructive hydrocephalus due to cysticercosis during pregnancy

Background: Cysticercosis, due to the parasite Taenia solium, can involve any organ. When central nervous system infection occurs, signs and symptoms depend on the location of the cerebral lesions. Most patients develop seizures, focal symptoms, or headaches with nausea and vomiting.Case: A case of extraparenchymal (intraventricular) cysticercosis was diagnosed in a patient at term who presented with acute alteration in mental status. Ventriculostomy was performed because of acute obstructive hydrocephalus. Labor ensued and was augmented with oxytocin. Intrapartum management included magnesium sulfate seizure prophylaxis and corticosteroids. Intracranial pressures ranged between 4 and 12 cm H(2)O peripartum with approximately 300 mL of cerebrospinal fluid drained over the first 24 hours. Postpartum management included craniotomy with resection of a larval cyst and oral praziquantel therapy.Conclusion: This case describes an uncommon presentation of neurocysticercosis that should be considered in gravidas with acute mental status changes.     

Hantavirus Dobrava infection with pulmonary manifestation

Dobrava virus infection was diagnosed serologically by enzyme-linked immunosorbent and immunofluorescence assays. To determine which hantavirus serotype was involved, sera were analyzed by a focus reduction neutralization test. The clinical data indicated that only pulmonary manifestation was present. Our data support the presence of Dobrava virus infection outside the Balkan region. In conclusion, a previously healthy adult with unexplained pulmonary perfusion failure should be investigated for hantavirus infection. Received: 15 March 1999     

Surgical repair of cytomegalovirus-related retinal detachment without silicone oil in patients with AIDS.

PURPOSE: To analyze visual and anatomic results following surgical repair of cytomegalovirus (CMV)-related retinal detachment (RD) without silicone oil permanent tamponade. METHODS: We analyzed five consecutive patients (six eyes) with acquired immunodeficiency syndrome and CMV-related RD that were repaired with pars plana vitrectomy with peeling of the posterior hyaloid, laser photocoagulation, encircling scleral buckle, and intraocular gas tamponade. RESULTS: Preoperative vision ranged from 20/40 to hand motion. Total retinal reattachment was achieved in five of six eyes (83%). Macular reattachment was achieved in all eyes. Mean postoperative visual acuity was 20/40 (range 20/30-20/60). Mean postoperative follow-up was 12 months (range 7-19 months). All patients in this series presented with low preoperative CD4+ T-lymphocyte counts (mean, 24 cells per microL) and received highly active antiretroviral therapy. One retina (Patient 1) redetached 7 months after initial repair and was successfully reattached without using silicone oil. Postoperatively, visual acuity remains 20/30, and total retinal reattachment has been maintained for 16 months. CONCLUSION: Good anatomic and visual success can be achieved and maintained in CMV-related RD without the use of silicone oil.