Potential availability of cadaver organs for transplantation.

OBJECTIVE--To determine the potential number of cadaver kidney donors by applying defined donor criteria to people dying in hospital. DESIGN--Prospective study of all deaths occurring in 21 hospitals from 1 September 1988 to 31 August 1989. Questionnaires were administered to medical and nursing staff and families of potential donors aged 1-69. SETTING--Acute care hospitals in Gwent, South Glamorgan, Mid Glamorgan, West Glamorgan, Pembrokeshire, and East Dyfed health authorities, serving a population of 2.2 million. MAIN OUTCOME MEASURES--Cause of death, age, ventilation at time of death, diagnosis of brain death, and consideration of consent. RESULTS--Adequate data were available for 9840 of 10,095 hospital deaths (97.5% coverage). 188 patients aged 0-69 were identified as potential organ donors (widest definition), and of these 108 died without being ventilated at the time of death. Tests of brain stem death were formally completed in 57 cases, and organ donation was considered by the families of 47 of these potential donors. 26 patients became organ donors. Patients aged 50-69 with stroke were less likely to be ventilated than those aged less than or equal to 49 (21/96 v 24/34). Families of potential donors aged 20-39 were least likely to give permission. CONCLUSIONS--The supply of donor organs (specifically kidneys) could be increased by altering the management of patients aged 50-69 dying of severe cerebrovascular disease in general medical wards, in particular by increasing the proportion ventilated. The ethics of elective ventilation for the purposes of organ donation require discussion.     

DNA methylation status of bovine blastocysts obtained from peripubertal oocyte donors

In the dairy industry, the high selection pressure combined with the increased efficiency of assisted reproduction technologies (ART) are leading toward the use of younger females for reproduction purposes, with the aim to reduce the interval between generations. This situation could impair embryo quality, decreasing the success rate of the ART procedures and the values of resulting offspring. Young Holstein heifers (n = 10) were subjected to ovarian stimulation and oocyte collection at 8, 11, and 14 months of age. All the oocytes were fertilized in vitro with semen from one adult bull, generating three pools of embryos per animal. Each animal was its own control for the evaluation of the effects of age. The EmbryoGENE platform was used to compare the DNA methylation status of blastocysts obtained from oocytes collected at 8 versus 14 and 11 versus 14 months of age. Age‐related contrast analysis identified 5,787 and 3,658 differentially methylated regions (DMRs) in blastocysts from heifers at 8 versus 14 and 11 versus 14 months of age, respectively. For both contrasts, the DMRs were distributed nonrandomly in the different DNA regions. The DNA from embryos from 8‐month‐old donors was more hypermethylated, while the DNA from embryos from 11‐month‐old donors displayed an intermediate phenotype. According to Ingenuity Pathway Analysis, the upstream regulator genes cellular tumor antigen p53, transforming growth factor β1, tumor necrosis factor, and hepatocyte nuclear factor 4α are particularly associated with methylation sensitive targets, which were more hypermethylated in embryos from younger donors.     

Anti-leishmanial activity of alkaloidal extracts obtained from different organs of Aspidosperma ramiflorum

The present study was designated to evaluate semi-quantitative antileishmanial activity of alkaloidal extracts that were obtained from 1g of different parts of Aspidosperma ramiflorum (leaves, roots, seeds, and stem barks). Alkaloidal extracts of barks and leaves presented a good activity against the extracellular form (promastigotes) of Leishmania (L.) amazonensis. It is known that compounds responsible for the antileishmanial activity in the alkaloidal extracts from A. ramiflorum are the monoterpenoid indole alkaloids ramiflorine A and ramiflorine B, therefore extracts obtained from different plant parts were analyzed by electrospray ionization mass spectrometry (ESI-MS) in order to evidence the presence of these bioactive alkaloids. Based on these findings, alkaloidal extract from leaves was fractionated on preparative thin-layer chromatography in a bioassay-guided fractionation affording individual purified ramiflorines A and B. Both ramiflorines A and B showed significant activity against Leishmania (L.) amazonensis (LD(50) values of 18.5±6.5μg/ml and 12.63±5.52μg/ml, respectively). Our results are showing that alkaloidal extract from leaves is a promising alternative to the use of stem barks from A. ramiflorum.     

Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a Ficoll-Hypaque gradient

Polymorphonuclear neutrophils (PMN) have an important role in the host defence response to infection. These cells produce large amounts of reactive oxygen species (O(2).(-), H(2)O(2) and ONOO(-)) with microbicidal activity. PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density (Ficoll-Hypaque gradient and dextran), yielding a highly homogeneous cellular population. However, some cellular activation due to membrane perturbation is also expected. We studied how the production of reactive oxygen species and release of myeloperoxidase (MPO) from blood PMN are affected by the use of the Ficoll-Hypaque density gradient. PMN isolated by spontaneous sedimentation and total blood were used for comparisons. Lucigenin- and luminol-enhanced chemiluminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli. The release of MPO, estimated by the chemiluminescence of the luminol/H(2)O(2) reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B, was also higher for PMN isolated by a density gradient. In conclusion, it was shown that the PMN isolation procedure affects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results.     

The functional relevance of passenger leukocytes and microchimerism for heart allograft acceptance in the rat.

With an organ transplant, hematopoietic donor cells are transferred to the recipient. To study the relevance of the resulting microchimerism for allograft acceptance, we analyzed a rat model of cyclosporine-induced tolerance for strongly incompatible heart allografts. Using a monoclonal antibody that detects a donor-specific CD45 allotype (RT7a), we selectively depleted donor leukocytes at different times after transplantation (days 0 or 18). Depletion was similarly effective at both times. However, only depletion on day 0 prevented tolerance induction and was associated with severe acute or chronic graft rejection. This indicates that passenger leukocytes have an essential immunomodulatory effect on the induction phase of allograft acceptance.     

Influence of the recipient's age on the effect of transplanting lymphoid organs from newborn donors

The development of immunological capacity of newborn thymus and spleen grafted under the kidney capsule of different-age recipients was investigated. The grafts functions appear to depend strongly upon the macroenvironment of the organism where their development occurs. Therefore+ the favourable influence of young environmental factors, gradually decreasing with the recipient age to become immunosuppressive in old animals is obtained. These data indicate a predominant significance of the macroenvironmental factors both in maturation of the immune system and its alteration during aging.     

Quantitative measurement of DNA strand breaks and repair in gamma-irradiated human leukocytes from normal and ataxia telangiectasia donors

Fluorimetric analysis of DNA unwinding, which allows measurement of DNA strand breaks in human leukocytes, has been optimized by reducing the amount of cells required for the test and by modifying the DNA alkali unwinding conditions. This permitted measurement of DNA strand-break induction in cells irradiated with low (0.5-7 Gy) or high doses (5-20 Gy) of gamma rays. Linear dose-response curves were obtained for both dose ranges. Presence of cysteamine during irradiation caused a decrease in the extent of DNA strand breaks. The kinetics of the DNA strand-break rejoining process appeared to be biphasic over the dose range of 2-20 Gy when plotted on a linear vs linear axis (percentage of damage as a function of time). Since the rate of disappearance of damaged DNA was similar for any given dose and for all postirradiation incubation times tested, we have expressed the extent of repair after a given postirradiation incubation as the ratio of the slopes of the regression lines obtained from incubated and nonincubated cells. Leukocytes from 25 healthy donors were analyzed to determine an average value for controls. No difference in the level of DNA strand breaks and the rate of repair of these breaks was observed between leukocytes from three ataxia telangiectasia patients and those from normal donors.     

Significant increase of interleukin 6 production in blood mononuclear leukocytes obtained from patients with active inflammatory bowel disease

In the present study, we compared the potency of interleukin 6 production in peripheral blood mononuclear leukocytes between paired patients with active stage and inactive stage of inflammatory bowel disease. Subjects included nine patients with ulcerative colitis, ten patients with Crohn's disease and sex-matched nine healthy volunteers. Mononuclear leukocytes were stimulated with concanavalin A for 24 h to induce interleukin 6 production. Interleukin 6 content in the culture medium was assayed by using specific ELISA and interleukin 6 dependent cell line MH-60. Interleukin 6 production was found to be significantly increased in mononuclear leukocytes from both active ulcerative colitis and Crohn's disease as compared to that from control subjects. There was no significant difference in interleukin 6 production between ulcerative colitis and Crohn's disease. The potency of interleukin 6 production was returned to the control level when the diseases became inactive. The present results, therefore, may indicate some important role of interleukin 6 in the pathogenesis of inflammatory bowel disease and also the potency of interleukin 6 production in mononuclear leukocytes can be an indicator of the activity of inflammatory bowel disease.     

Spontaneous death of mononuclear cells, obtained from health donors and patients with systemic lupus erythematosus]

Unstimulated human peripheral blood mononuclear cells (MNC) underwent death during incubation in vitro. According to morphological criteria the type of death was identified as apoptosis. There was a good correlation between a fraction of apoptotic cells qualified morphologically, and fraction of "apoptotic" comets. The use of DNA-comets for studying the spontaneous death in vitro of MNC from patients with systemic lupus erythematosus (SLE) showed that 24 h after isolation cells from SLE patients demonstrated a higher level of apoptosis than MNC from normal donors. It is likely that the increased apoptosis of SLE-MNC in vitro may reflect changes occurring in those cells in vivo which are bound with pathogenesis of disease. In this context the comet assay may be promising in diagnostics and monitoring of therapeutic treatments.