Ethical hazards of the substituted judgement test in decision making concerning the end of life of dementia patients

Previously we reported that the South American gray short-tailed opossum, Monodelphis domestica, had an MHC class-I locus similar to that of eutherian species. In addition to the detection of lymphocyte antigens by cytotoxic antisera, we concluded that this marsupial rejected allogeneic skin grafts, as would be expected of animals with MHC class-I polymorphism. However, this conclusion was based on a limited number of skin transplants that were assayed for only a short period. Here we report the results of 22 reciprocal skin grafts made between individuals of known genetic relationships. On the basis of gross inspection of the grafts and histologic examination, we found that the average time of the onset of graft rejection was about 19 days and that the average time for complete graft rejection was about 31 days. In general, it took longer for the onset of graft rejection among pairs of genetically related animals than among less related animals. These results indicate unequivocally that this marsupial species has a high degree of class-I polymorphism and rejects allogeneic skin transplants in a manner similar to but more slowly than eutherian mammals.     

Relations of cigarette smoking and dietary antioxidants with placental calcification

The purpose of this study was to evaluate the diagnostic efficacy of the double inversion recovery fast spin echo (DIR-FSE) sequence for brain imaging compared to the fluid-attenuated inversion recovery (FLAIR) sequence. DIR-FSE and FLAIR-FSE sequences were obtained and compared side by side. Image assessment criteria included lesion conspicuity, contrast between different types of normal tissue, image quality, and artifacts. In addition, contrast ratios and contrast-to-noise ratios were determined. Scan time of DIR-FSE was 33% longer than scan time of FLAIR-FSE. Overall lesion conspicuity was equal on DIR-FSE and FLAIR-FSE; however, DIR-FSE showed particular advantages for infratentorial lesions and lesions with only poor contrast on T2-weighted images, whereas FLAIR was slightly superior for small superficial cerebral abnormalities. Gray-white differentiation was better with DIR-FSE. Cerebrospinal fluid suppression was equal on both sequences; cerebrospinal fluid pulsation artifacts were more pronounced on DIR-FSE but did not cause diagnostic difficulties on these images. We conclude that DIR-FSE might be obtained if infratentorial lesions and abnormalities with only slightly prolonged T2 relaxation times are suspected. Otherwise, FLAIR-FSE seems preferable.     

Structure-activity relationships for 1-phenylbenzimidazoles as selective ATP site inhibitors of the platelet-derived growth factor receptor

1-Phenylbenzimidazoles are shown to be a new class of ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR). Structure-activity relationships (SARs) are narrow, with closely related heterocycles being inactive. A systematic study of substituted 1-phenylbenzimidazoles showed clear SARs. Substituents at the 4'- and 3'-positions of the phenyl ring are tolerated but do not significantly improve activity, while substituents at the 2'-position abolish it. Substituents in the 2-, 4-, and 7-positions of the benzimidazole ring (with the exception of 4-OH) also abolish activity. Most substituents at the 5- and 6-positions maintain or increase activity, with the 5-OH, 5-OMe, 5-COMe, and 5-CO2Me analogues being >10-fold more potent than the parent 1-phenylbenzimidazole. The 5-OMe analogue was both the most potent inhibitor, and showed the highest selectivity (50-fold) between PDGFR and FGFR isolated enzymes, and also a moderately effective inhibitor (IC50 = 1.9 microM) of PDGF-stimulated PDGFR autophosphorylation in rat aorta smooth muscle cells.     

Evidence for sexual differentiation of glia in rat brain

It is well established that gonadal steroids mediate sexual differentiation of the brain via direct effects on neurons during a restricted critical period. In addition, estrogen can influence glial morphology in the adult brain, and in vitro studies suggest estrogen induces glial differentiation. However, there is a lack of in vivo evidence for steroid effects on glia during the critical period. We report here a hormone-mediated sexual differentiation of arcuate glia as early as Postnatal Day 1. Using glial fibrillary acidic protein immunoreactivity (GFAP-ir), we compared the responsiveness of astroglia in the rat arcuate nucleus among five hormonally different groups. The results indicate increased GFAP-ir cell surface area 24 hr after hormonal manipulation in castrate males compared to intact males, intact females (ANOVA; P < 0.01), and females injected with testosterone propionate (50 microg; ANOVA; P < 0.05). However, astroglia in intact males extended their processes significantly greater distances from the cell body compared to all other treatment groups (ANOVA; P < 0.01). The GFAP-ir cells were categorized into four distinct classes ranging from a simple bipolar to a fully stellate morphology. The frequency distribution of classes varied between groups with more stellate cells found in intact males. Finally, these sex differences in arcuate glia persisted into adulthood. We hypothesize that during the critical period, testosterone, or its metabolite estrogen, induce sexual differentiation of glia. We further hypothesize that in females glial cells remain partially undifferentiated and this may be important to glial plasticity seen in adult female arcuate.     

ANP and bradycardic reflexes in hypertensive rats: influence of cardiac hypertrophy

OBJECTIVES: Beta2-integrin (CD11b/CD18) expression, an indicator of neutrophil activation, has been associated with the development of acute respiratory distress syndrome. Leumedins act directly on leukocytes to inhibit the up-regulated expression of beta2-integrins involved in leukocyte adhesion. We examined the effect of such a new anti-inflammatory agent, NPC 15669 (N-[9H-(2,7-dimethylfluorenyl-9-methoxy)-carbonyl]-L-leucine), on neutrophil-mediated acute lung injury in an animal model. DESIGN: Prospective, randomized, blinded, controlled animal study. SETTING: An animal laboratory in a university setting. SUBJECTS: Adult New Zealand rabbits. INTERVENTIONS: After repeated lung lavages with normal saline to induce acute lung injury, anesthetized rabbits were randomly assigned to one of two groups (n = 6 per group): a) treatment group (pretreated with NPC 15669 [10 mg/kg i.v. bolus] 30 mins before lavage, followed by a continuous infusion [5 mg/kg/hr] for the duration [4 hrs] of the experiment); or b) control group (pretreatment and continuous infusion with placebo). All animals were mechanically ventilated with identical pressure settings over 4 hrs and were killed at the end of the experiment. MEASUREMENTS AND MAIN RESULTS: PaO2, PaCO2, and tidal volumes were repeatedly measured and airway pressure settings were noted every 30 mins. At the end of the experiment, lungs were taken out for measurements of the myeloperoxidase content, for conventional histology (hematoxylin and eosin staining), and for intracellular adhesion molecule-1 immunohistostaining. Pretreatment with NPC 15669 profoundly improved oxygenation from a PaO2 of 52 +/- 5 torr (6.9 +/- 0.7 kPa) to 250 +/- 161 torr (33.3 +/- 21.5 kPa) within 60 mins after lung lavage (p < .05). Oxygenation continued to improve throughout the study, reaching a maximal PaO2 value of 395 +/- 98 torr (52.7 +/- 13.1 kPa) at 4 hrs. In the control group, oxygenation remained poor throughout the observation period. PaO2 values differed significantly (51 +/- 20 torr [6.8 +/- 2.7 kPa] vs. 306 +/- 126 torr [40.8 +/- 16.8 kPa], p < .005) at 90 mins and at all subsequent measurements from those values in the NPC 15669 group. Dynamic lung compliance improved significantly 60 to 90 mins after repeated lung lavage. Histology demonstrated markedly less lung damage (hyaline membrane formation and leukocyte infiltration) in treated animals (p < .05) than in controls. CONCLUSIONS: NPC 15669 seems to block inflammatory reactions by inhibiting the sequestration of neutrophils in acute, ventilator-associated lung injury. As a result, gas exchange and total lung compliance improve. Application of this and similar compounds affecting neutrophil adhesion warrants further investigation as a treatment modality for acute lung injury.     

In vivo binding of mouse IgG via polyreactive surface IgM abrogates progressive lymphocytosis in prolymphocytic leukemia

Surface IgM expressed by malignant CD5+ B-cells from patients with B-chronic lymphocytic leukemia (B-CLL) has previously been shown to bind mouse Ig in what appears to be an example of polyreactive antigen-binding activity. This report demonstrates the in vitro and in vivo binding of mouse Ig to the surface of malignant B-cells from a patient with B-cell prolymphocytic leukemia (B-PLL). In vitro studies showed that K121, a mouse monoclonal antibody, bound to the B-PLL cells via the same low-affinity binding interaction demonstrated to occur between mouse Ig and surface IgM expressed by B-CLL cells rather than in the conventional sense against a specific antigen via its antigen-binding site. With the view to using this phenomenon to target malignant B-cells, it was important to determine whether the low-affinity interaction also occurred in vivo. Infusions of K121 totalling 286 mg were administered to a B-PLL patient over 7 days. Binding of K121 to circulating B-PLL cells was demonstrable after the administration of 36 mg of antibody and was preceded by the appearance of free antibody in the serum. Throughout the period of the infusion, the rapid rise in the peripheral blood white cell count normally observed after leukopheresis was abrogated. However, the count rose markedly after cessation of the antibody infusion in parallel with a decrease in both free and cell-bound K121. There were no observable side effects and no host immune response to either species specific or idiotypic determinants on the mouse Ig was detected. The in vivo binding of mouse Ig together with the previous in vitro data suggest the potential for a novel targeting mechanism using a region of the mouse Ig molecule to target polyreactive Ig expressed by malignant cells in B-CLL and B-PLL.     

Peptide growth factors in the prostate as mediators of stromal epithelial interaction

Peptide growth factors play a role in the maintenance of normal prostatic growth and differentiation (Fig. 2). It seems likely that the androgen sensitivity of human prostate is mediated by the production of peptide growth factors from stromal cells which act as the direct intermediate of androgen action on epithelial cells. TGF-beta 1 inhibition of epithelial cells is opposed by the stimulatory action of EGF, IGF and FGFs to maintain an equilibrium of epithelial cell numbers. The indirect mitogenic action of androgens appear to act by down-regulation of TGF-beta 1 and possibly EGF receptors. There is also interaction with the effects of IGF-II, produced by prostatic stromal cells and acting on epithelial cells to increase proliferation. The growth of normal prostatic fibroblasts is under the control of bFGF and TGF-beta 1. However, although our understanding of the actions of these growth factors in the normal prostate has improved over the last decade, their role in the development and maintenance of prostate cancer is less clearly defined. TGF-beta 1, classically considered to be inhibitory for epithelial cells, may be up-regulated in prostatic tumours, stimulating growth. Alternatively, autocrine production of such growth factors by tumour cells may lead to loss of inhibitory effects from exogenous TGF-beta 1, a mechanism also witnessed with TGF-alpha and bFGF. The role of EGF in the development of prostate cancer is confusing because results from the use of different cell types and experimental conditions is contradictory. It may be that a switch in the production of the predominant EGFr ligand from EGF to TGF-alpha is an important feature in the development and maintenance of the malignant phenotype. The presence of TGF-alpha autocrine loops has been shown clearly in some tumour cell lines. This switch in the production of a particular ligand may also be a feature of IGFs in prostate cancer. IGF-II may be replaced by IGF-I during malignant progression, both of which are able to act via the type 1 receptor. This change in IGF expression appears to be accompanied by altered expression of the IGF-BP2, with less detectable within prostatic tissues but elevated serum levels [58]. Basic FGF is normally produced by prostatic fibroblasts but is also produced by some prostatic cancer cell lines [64]. However, as with all growth factors, the expression of the bFGF protein and its receptor is dependent on the cell line examined. The autocrine and paracrine control of normal and abnormal prostatic growth by growth factors is important in determining their role in the development and maintenance of prostate cancer. Better understanding of such mechanisms is essential for the development of novel therapeutic strategies in the control and treatment of prostate cancer.     

Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis

Sphingolipid-related metabolites have been implicated as potential signaling molecules in many studies with mammalian cells as well as in some studies with yeast. Our previous work showed that sphingolipid-deficient strains of Saccharomyces cerevisiae are unable to resist a heat shock, indicating that sphingolipids are necessary for surviving heat stress. Recent evidence suggests that one role for the sphingolipid intermediate ceramide may be to act as a second messenger to signal accumulation of the thermoprotectant trehalose. We examine here the mechanism for generating the severalfold increase in ceramide observed during heat shock. As judged by compositional analysis and mass spectrometry, the major ceramides produced during heat shock are similar to those found in complex sphingolipids, a mixture of N-hydroxyhexacosanoyl C18 and C20 phytosphingosines. Since the most studied mechanism for ceramide generation in animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S. cerevisiae for an analogous enzyme. Using [3H]phytosphingosine and [3H]inositol-labeled yeast sphingolipids, a novel membrane-associated phospholipase C-type activity that generated ceramide from inositol-P-ceramide, mannosylinositol-P-ceramide, and mannose(inositol-P)2-ceramide was demonstrated. The sphingolipid head groups were concomitantly liberated with the expected stoichiometry. However, other data demonstrate that the ceramide generated during heat shock is not likely to be derived by breakdown of complex sphingolipids. For example, the water-soluble fraction of heat-shocked cells showed no increase in any of the sphingolipid head groups, which is inconsistent with complex sphingolipid hydrolysis. Rather, we find that de novo ceramide synthesis involving ceramide synthase appears to be responsible for heat-induced ceramide elevation. In support of this hypothesis, we find that the potent ceramide synthase inhibitor, australifungin, completely inhibits both the heat-induced increase in incorporation of [3H]sphinganine into ceramide as well as the heat-induced increase in ceramide as measured by mass. Thus, heat-induced ceramide most likely arises by temperature activation of the enzymes that generate ceramide precursors, activation of ceramide synthase itself, or both.