Isolation and primary culture of rat Kupffer cells

The aim of this study was to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells. Kupffer cells were isolated following sequential pronase/collagenase digestion of the liver and enrichment of a non-parenchymal cell fraction by a single-density gradient centrifugation step using 30% metrizamide. Kupffer cells were isolated and further purified from this cell fraction by centrifugal elutriation. Kupffer cells were isolated at 1017 g at 48-110 mL/min. All Kupffer cell fractions exhibited phagocytosis of 3 microm latex beads. Kupffer cell fractions isolated at 48 and 60 mL/min were predominantly ED2 negative while later fractions (80-110 mL/min) were ED2 positive. Kupffer cells were adherent in culture after 2 h. This method for Kupffer cell isolation resulted in a yield of 80-120 x 10(6) Kupffer cells per liver.     

Synthesis and reactivity of coumarin 3,4-epoxide

Coumarin is used widely as a fragrance constituent and is administered clinically in the treatment of certain lymphedemas and malignancies. Although toxicity occurs only rarely in humans treated clinically with high-dose coumarin, it is well established that coumarin is hepatotoxic in the rat. This species difference in susceptibility to toxicity reflects the disparate metabolic processes occurring in humans and rodents. In humans, coumarin is converted extensively via cytochrome P450 2A6 to the nontoxic 7-hydroxycoumarin metabolite. In contrast, coumarin 3,4-epoxidation is thought to predominate in rodent species, resulting in the formation of several potentially toxic metabolites. Coumarin epoxide is thought to be highly unstable and has not been isolated synthetically or as a microsomal product. To address this issue, coumarin 3,4-epoxide was synthesized, and its stability and fate have been determined. Coumarin 3,4-epoxide was prepared by reacting coumarin with dimethyldioxirane. The epoxide was stable in organic solvents and survived conditions required for analysis by gas chromotography. Its structure was confirmed via 1H-NMR and gas chromatography-mass spectrometry-infrared spectroscopy (GC-MS-IR). In contrast, coumarin 3,4-epoxide was unstable in aqueous solution, converting within 20 sec to a ring-opened compound. Using GC-MS-IR analysis, the single coumarin 3,4-epoxide product was identified as o-hydroxyphenylacetaldehyde (o-HPA). Although other investigators have suggested that 3-hydroxycoumarin is an intermediate in o-HPA formation from coumarin 3,4-epoxide, we have demonstrated that 3-hydroxycoumarin, incubated in an aqueous system or with liver microsomal proteins, does not form o-HPA. Thus, the results of the present work establish that coumarin 3,4-epoxide can be synthesized and that o-HPA, which has previously been shown to be a prominent coumarin metabolite in rat liver microsomal incubations, is formed directly from coumarin 3,4-epoxide. These results suggest that both coumarin 3,4-epoxide and o-HPA may contribute to the hepatotoxicity of coumarin.     

Preoperative cortical localization with functional MRI for use in stereotactic radiosurgery

Accurate localization of the lesion with respect to functionally significant brain is essential to safe stereotactic radiosurgical dose planning. We report the use of functional MR imaging in 3 patients to identify critical areas of surrounding brain and to provide assistance with dose planning, especially with regard to shaping the peripheral isodose around the lesion. We used a functional MRI system employing a conventional 1.5-tesla MRI unit that can detect decreases in deoxyhemoglobin concentration occurring with performance of specific tasks. Two of the patients had supratentorial arteriovenous malformations and 1 patient had a recurrent parasagittal meningioma. Functional MRI provided information on the location of speech, motor, and sensory cortex in these patients. Radiosurgical dose plans were constructed that kept these cortical areas outside of the 30% isodose curves. We believe that the safety of supratentorial parenchymal radiosurgery will be enhanced by the localization of critical brain regions around the target.     

Extreme strength observed in limpet teeth

The teeth of limpets exploit distinctive composite nanostructures consisting of high volume fractions of reinforcing goethite nanofibres within a softer protein phase to provide mechanical integrity when rasping over rock surfaces during feeding. The tensile strength of discrete volumes of limpet tooth material measured using in situ atomic force microscopy was found to range from 3.0 to 6.5 GPa and was independent of sample size. These observations highlight an absolute material tensile strength that is the highest recorded for a biological material, outperforming the high strength of spider silk currently considered to be the strongest natural material, and approaching values comparable to those of the strongest man-made fibres. This considerable tensile strength of limpet teeth is attributed to a high mineral volume fraction of reinforcing goethite nanofibres with diameters below a defect-controlled critical size, suggesting that natural design in limpet teeth is optimized towards theoretical strength limits.     

A prospective, randomized study of the use of platelet concentrates irradiated with ultraviolet-B light in patients with hematologic malignancy

BACKGROUND: Irradiation of platelet concentrates (PCs) with ultraviolet-B (UVB) light inactivates the contaminating white cells and might be an alternative to filtration for the prevention of alloimmunization to HLA antigens and subsequent refractoriness to further platelet transfusions in multiply transfused patients with bone marrow failure. STUDY DESIGN AND METHODS: Patients with hematologic malignancy, mainly acute myeloid leukemia, were prospectively assigned in a random manner to receive either UVB-irradiated or control, nonirradiated PCs. All patients were given red cells that were white cell reduced by filtration. Transfusion efficacy and alloimmunization were assessed by means of corrected count increments, requirement for red cells and PCs, and measurement of lymphocyte-reactive antibodies. RESULTS: UVB-irradiated PCs had a clinical efficacy similar to controls as judged by corrected count increments at 1 to 6 and 12 to 24 hours and by the median requirement for red cell and platelet transfusions. Alloimmunization determined by measurements of lymphocyte-reactive antibodies using both conventional and antiglobulin-augmented lymphocytotoxicity techniques was not abolished in recipients of UVB-irradiated PCs (4/30, 13%) but was less than that in controls (5/20, 25%; p = NS). The mean number of platelet transfusion episodes prior to the occurrence of alloimmunization was greater in the control group (27 vs. 10; p = 0.017). CONCLUSION: In this trial, UVB irradiation did not diminish the clinical efficacy of platelet transfusions. There was a small but nonsignificant reduction alloimmunization, but no difference in refractoriness of the two groups was observed. Larger prospective randomized studies are required to confirm these findings and to compare UVB irradiation with white cell reduction.     

Effect of androstenedione administration on the maternal hypothalamo-pituitary-adreno-placental axis in the pregnant rhesus monkey

To assess the interaction among androgens, placenta, and the hypothalamo-pituitary-adrenal axis we studied effects of androstenedione administered intravascularly to the pregnant monkey on maternal plasma CRH, ACTH, dehydroepiandrosterone sulfate (DHEAS), cortisol, and estradiol concentrations. Ten monkeys (128 +/- 3 days gestation; mean +/- SEM) were instrumented under general halothane anesthesia with maternal femoral artery and venous catheters and uterine electromyogram electrodes. At 137-144 days gestation, baseline maternal femoral artery samples for CRH, ACTH, DHEAS, cortisol, and estradiol measurements were taken at 1.5-h intervals for 7 h starting 2 h before darkness. On the following day, a continuous iv androstenedione infusion (0.3 mg/kg.min at 0.25 ml/h) in 10% intralipid was started at 0930 h in four monkeys; the other six animals received vehicle alone at the same rate starting at the same time. Maternal blood sampling was repeated 1 and 3 days after androstenedione or vehicle administration. Maternal plasma CRH, ACTH, DHEAS, cortisol, and estradiol levels were unaffected by intralipid. In contrast, androstenedione infusion produced a sustained increase in maternal plasma estradiol and a sustained fall in maternal plasma ACTH, but did not affect maternal plasma CRH, DHEAS, or cortisol concentrations. These results provide evidence for negative feedback regulation by androgens at the hypothalamo-pituitary-adrenal axis in the pregnant monkey. Lack of inhibition of maternal plasma CRH after androstenedione administration supports differential regulation of hypothalamic and placental CRH by androgens.     

Recombinant subunit vaccines as an approach to study correlates of protection against primate lentivirus infection

Using pathogenic simian immunodeficiency virus (SIV) infection of macaques as a model, we explored the limits of the protective immunity elicited by recombinant subunit vaccines and examined factors that affect their efficacy. Envelope gp 160 vaccines, when used in a live recombinant virus-priming and subunit-protein-boosting regimen, protected macaques against a low-dose, intravenous infection by a cloned homologous virus SIVmne E11S. The same regimen was also effective against intrarectal challenge by the same virus and against intravenous challenge by E11S grown on primary macaque peripheral blood mononuclear cells (PBMC). However, only limited protection was observed against uncloned SIVmne. Priming with live recombinant virus was more effective than immunization with subunit gp 160 alone, indicating a potential advantage of native antigen presentation and the possible role of cell-mediated immunity in protection. Whole gp 160 was more effective than the surface antigen (gp 130), even though both antigens elicited similar levels of neutralizing antibodies. Animals immunized with the core (gag-pol) antigens failed to generate any neutralizing antibody and were all infected following challenge. However, their proviral load was 10-100-fold lower than that of the control animals, indicating that immune mechanisms such as cytotoxic T lymphocytes (CTL) may play a role. Finally, animals immunized with both the core and the envelope antigens generated significant protective immunity, even with relatively low neutralizing antibodies. Taken together, these results indicate that multiple mechanisms may contribute to protection. It may therefore be advantageous to incorporate multiple antigens in the design of recombinant subunit vaccines against acquired immunodeficiency syndrome (AIDS).