Human Renal Allograft Rejection Despite the Absence of Allogeneic Passenger Leukocytes

Passenger leukocytes have been suggested to be both pro-tolerant and immunogenic. The opportunity to evaluate the role of allogeneic passenger leukocytes in humans was presented by a 47-year-old man who donated bone marrow to his HLA-identical leukemic sister. Eleven years later he developed renal failure. The sister's marrow was noted to be 100% XY karyotype and free of malignancy. She donated a kidney to her brother. Immunosuppression was tapered following transplantation. After 6 months, the recipient was on monotherapy sirolimus, 1 mg every third day. A surveillance biopsy was normal and sirolimus was stopped. Eight weeks later, he presented with severe rejection that reversed with Thymoglobulin. Renal function returned to baseline and has been stable on conventional immunosuppression.     

麦胚提取物对辐射损伤修复的实验研究

目的 研究麦胚提取物对小鼠辐射损伤修复的调节和保护作用。方法 采用辐射前或辐射同时饲喂一定量的麦胚提取物，观察小鼠经X射线一次性全身照射后的临床症状、30d存活率、骨髓微核率、外周血白细胞总数在不同时间的变化。结果 与对照组(单纯照射)比较，饲喂麦胚提取物可使小鼠的头面部皮肤、小肠黏膜、肾脏损伤症状得到明显改善；30d存活率为86．17％(P＜0．01)，提高存活率41．79％，保护系数为1．72；骨髓微核率4．62‰；比对照组(12．14‰)降低(P＜0．01)；外周血白细胞总数在7，13，20，30d均显回升(P＜0.05，P＜0．01，P＜0．01，P＜0．01)。结论 麦胚提取物对小鼠辐射损伤修复有一定程度的调节和保护作用，对于辅助肿瘤放射治疗具有重要意义。     

Secretion and function of the third component of complement (C3) by murine leukocytes.

Our new finding of de novo synthesis and secretion of C3 by both murine peritoneal macrophages and polymorphonuclear leukocytes (PMN) was confirmed by the incorporation of [35S]methionine into C3 molecules and their complete inhibition by cycloheximide. The methods of secretion of C3 from these two types of cells were compared by examining the C3 contents in their culture supernatants. Completely different modes of secretion were observed, i.e. although macrophages synthesize and secrete C3 constitutively, PMN has a mechanism to store the already synthesized C3 in the cell and secrete it in response to stimuli. Protein kinase C (PKC) activators, e.g. 12-O-tetradecanoyl-phorbol 13-acetate, dioctanoyl glycerol, and mezerein, as well as calcium ionophore A23187 stimulate the secretion of C3 from PMN. These results suggest the involvement of PKC and the calmodulin pathway. A very sensitive method for measuring C3 activity was developed which enabled us to show for the first time that C3 secreted by PMN had opsonizing activity and that particles cultured with PMN were phagocytosed effectively.     

Chemoattractant-induced firm adhesion of leukocytes to vascular endothelium in vivo is critically dependent on initial leukocyte rolling

Leukocyte rolling and firm adhesion at the venular endothelium are two discrete events in the cellular inflammatory response mediated via selectin and integrin adhesion molecules, respectively. The dependency of chemoattractant-induced firm leukocyte adhesion on the preceding rolling interaction was investigated in rat mesenteric microvessels through use of intravital microscopy. Leukocyte rolling was dose-dependently inhibited by systemic treatment with the sulphated polysaccharide fucoidin. The firm leukocyte adhesion following stimulation with the chemotactic peptide fMLP was similarly inhibited when fMLP challenge was performed subsequent to inhibition of leukocyte rolling by fucoidin. Thus, based on paired observations in single venules before and after fucoidin treatment, reduced rolling leukocyte flux prior to fMLP challenge was paralleled over a wide range by a proportional decrease in fMLP-induced leukocyte adhesion. The results demonstrate quantitatively a close relationship between the extent of leukocyte rolling and the magnitude of the subsequent firm adhesion response, and, that an initial rolling interaction is a precondition for firm adhesion to occur at physiological blood flow rates in vivo.     

On the Use of Paramagnetic Beads and Ferrofluids to Assess and Eliminate the Leukocytic Contribution to Oxygen Radical Generation by Human Sperm Suspensions

PROBLEM: To develop a methodology to determine a) the leukocytic contribution to reactive oxygen species generation by human sperm suspensions and b) the therapeutic value of removing these cellular contaminants. METHODS: Leukocytes were removed with paramagnetic beads or colloidal ferrofluids coated with anti-CD45 antibody. The sperm suspensions were monitored for oxidant generation by chemiluminescence, leukocyte contamination by immunocytochemistry, and fertilizing potential using zona-free hamster oocytes. RESULTS: Percoll®-prepared human sperm suspensions exhibited a competence for PMA-induced reactive oxygen species generation which was significantly correlated with leukocyte contamination. However, the purified spermatozoa remaining after paramagnetic bead treatment, also demonstrated an intrinsic capacity for PMA-responsive reactive oxygen species generation and, freed from the oxidative stress created by the leukocytes, exhibited a significantly enhanced capacity for sperm-oocyte fusion. CONCLUSIONS: Although human spermatozoa can generate reactive oxygen species, sperm function is inhibited by the additional oxidative stress created by contaminating leukocytes. Removal of these cells with paramagnetic beads enhances fertilizing potential.     

Cell-associated adhesion molecules as early markers of bioincompatibility

BACKGROUND.: Transient nature of adhesive interactions occurring during cellmargination is mainly dependent on expression of selectins whichare shed by activated cells. This shedding in the circulationmay play an important role as anti-inflammatory mediator. Haemodialysisis also associated with P-selectin (CD62P)/sialyl-Lewisx (CD15s)interactions which mediate platelet-leukocyte coaggregation.We further investigated the mechanisms underlying leukocytemargination during haemodialysis. METHODS.: CD15s, CD11b and CD61 expression on circulating leukocytes frompatients dialysed on synthetic membranes (modified polyacrylonitrile(SPAN), polysulphone (PS), and polyacrylonitrile (AN69) wasassessed by cytofluorometry in a prospective crossover trial.We measured plasma levels C3a/C3a desArg, soluble CD62P, andCD62E molecules obtained from patients and healthy individuals. RESULTS.: Expression of CD11b and CD15s was upregulated on neutrophilsfrom patients dialysed with SPAN and PS membranes during thedialysis session. A significant negative correlation was foundbetween the expression of CD11b or CD15s molecules and neutrophilcounts as well as between CD15s expression and monocyte countsduring haemodialysis. As assessed by CD61 expression on leukocytes,we observed that platelets bound significantly onto both neutrophilsand monocytes during dialysis with both membranes. A significantpositive correlation was found between the expression of CD11bmolecules and the percentage of CD61 ⁺ monocytes counts duringSPAN and PS dialysis. We found a significant increase of solubleCD62P in plasma samples obtained from haemodialysed patientsbefore the dialysis session as compared to the levels detectedin plasma from healthy individuals. CONCLUSIONS.: This study documents a major role of CD15s, CD11b, CD61, CD62Pmolecules in the transient leukocytes activation and marginationduring haemodialysis on synthetic membranes despite their lowcomplement-activating properties.     

Andrology: Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma

The addition of luminol to unprocessed semen samples resultedin the generation of chemiluminescent signals, the intensityof which was highly correlated with the level of leukocyte contamination.Despite the spontaneous oxidant-generating capacity of seminalleukocytes, no correlations were observed between leukocytecontamination and the fertility status of the subjects or anyaspect of the semen profile, including the motility of the spermatozoaor their performance in a hyaluronate penetration assay. Luminol-dependentchemiluminescence and leukocyte contamination were also correlatedin washed sperm suspensions prepared either by repeated centrifugationor on discontinuous Percoll gradients. However, in such spermsuspensions, the spontaneous generation of oxidants by contaminatingleukocytes (>2x10⁴ leukocytes/ml) was invariably associatedwith a decreased capacity for movement. Moreover, causativeassociations between leukocyte contamination, reactive oxygenspecies generation, lipid peroxidation and impaired sperm motilitywere revealed by experiments involving the selective additionor removal of activated leukocytes. From these observationswe can conclude that low concentrations of leukocytes are acommon feature of the human ejaculate and can impair sperm function,particularly in the absence of seminal plasma. These findingshave implications for our understanding of the importance ofleukocytospermia in defining the fertility of human spermatozoain vivo and in vitro.