[Myocardial Crypt of the Left Ventricle Combined With Barlows syndrome].

Myocardial crypts were initially described in patients with hypertrophic cardiomyopathy. Modern diagnostic data show that this structural abnormality can be found in patients with other diseases, or might represent the variant of normal heart development in healthy individuals. The prognostic significance of this finding is uncertain. In this publication we present a clinical case of the combination of myocardial crypt and Barlows syndrome.

GABA transport and neuroinflammation are coupled in multiple sclerosis: regulation of the GABA transporter-2 by ganaxolone.

Interactions between neurotransmitters and the immune system represent new prospects for understanding neuroinflammation and associated neurological disease. GABA is the chief inhibitory neurotransmitter but its actions on immune pathways in the brain are unclear. In the present study, we investigated GABAergic transport in conjunction with neuroinflammation in models of multiple sclerosis (MS). Protein and mRNA levels of γ-amino butyric acid transporter 2 (GAT-2) were examined in cerebral white matter from MS and control (Non-MS) patients, in cultured human macrophages, microglia and astrocytes, and in spinal cords from mice with and without experimental autoimmune encephalomyelitis (EAE) using western blotting, immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). GABA levels were measured by HPLC. The GAT-2's expression was increased in MS patients' (n=6) white matter, particularly in macrophage lineage cells, compared to Non-MS patients (n=6) (p<0.05). Interferon-γ (IFN-γ) stimulation of human macrophage lineage cells induced GAT-2 expression and reduced extracellular GABA levels (p<0.05) but soluble GABA treatment suppressed HLA-DRα, GAT-2 and XBP-1/s expression in stimulated macrophage lineage cells (p<0.05). Similarly, the synthetic allopregnanolone analog, ganaxolone (GNX), repressed GAT-2, JAK-1 and STAT-1 expression in activated macrophage lineage cells (p<0.05). In vivo GNX treatment reduced Gat-2, Cd3ε, MhcII, and Xbp-1/s expression in spinal cords following EAE induction (p<0.05), which was correlated with improved neurobehavioral outcomes and reduced neuroinflammation, demyelination and axonal injury. These findings highlight altered GABAergic transport through GAT-2 induction during neuroinflammation. GABA transport and neuroinflammation are closely coupled but regulated by GNX, pointing to GABAergic pathways as therapeutic targets in neuroinflammatory diseases.

Mucosal injury and inflammatory cells in response to brief ischaemia and reperfusion in the equine large colon.

REASON FOR PERFORMING STUDY: Intestinal ischaemia and reperfusion (I/R) can activate inflammatory cells in the equine colon, although effects on different types of inflammatory cells have received little attention. OBJECTIVES: To assess early mucosal injury, the reaction of mucosal neutrophils, eosinophils, mast cells and macrophages, and cyclooxygenase (COX)-1 and -2 expression in response to I/R in the equine large colon. METHODS: Large colon ischaemia was induced for 1 h (1hI) followed by 4 h of reperfusion in 6 horses, and mucosal biopsies were sampled before and after ischaemia, and after 1, 2 and 4 h of reperfusion. Semithin sections (500 nm) of epon-embedded biopsies were stained with toluidine blue for histomorphometric evaluation. The number and distribution of mucosal macrophages (CD163), neutrophils (calprotectin), eosinophils (LUNA) and mast cells (toluidine blue) were determined, and mucosal COX-1 and -2 expression was identified. RESULTS: Ischaemia caused epithelial cell and nuclear swelling (mean ± s.e. nuclear width; control: 2.7 ± 0.2 µm vs. 1hI: 4.2 ± 0.2 µm; P<0.01), subepithelial oedema (control: 0.2 ± 0.1 µm vs. 1hI: 3.2 ± 0.2 µm; P<0.01) and increased epithelial apoptosis (control: 14.3 ± 4.1 apoptotic cells/mm mucosa vs. 1hI: 60.4 ± 14.0 apoptotic cells/mm mucosa; P<0.01). COX-2 expression (P<0.01) was evident after ischaemia. Reperfusion caused paracellular fluid accumulation (control: 0.9 ± 0.1 µm vs. 1hI: 0.6 ± 0.6 µm vs. 1hI + 4hR: 1.6 ± 0.2 µm; P<0.05). Epithelial repair started at 1 h of reperfusion (P<0.001), followed by migration of neutrophils into the mucosa after 2 h (control: 72.3 ± 18.4 cells/mm(2) mucosa vs. 1hI + 2hR: 1149.9 ± 220.6 cells/mm(2) mucosa; P<0.01). Mucosal eosinophils, mast cells and macrophages did not increase in numbers but were activated. CONCLUSIONS: Epithelial injury and COX-2 expression caused by short-term hypoxia were followed by intense inflammation associated with epithelial repair during reperfusion. POTENTIAL RELEVANCE: Equine colonic mucosa subjected to a brief period of ischaemia can repair during reperfusion, despite increased mucosal inflammation.

Ultrastructural changes in the equine colonic mucosa after ischaemia and reperfusion.

REASON FOR PERFORMING STUDY: Ultrastructural changes in the epithelium can provide information on early changes in barrier properties, repair and inflammation in equine colon after ischaemia and reperfusion (I/R). OBJECTIVES: To describe the morphology and ultrastructure of the epithelium in equine large colonic mucosa after I/R, and the response of inflammatory cells to injury. METHODS: Ischaemia was induced for 1 h followed by 4 h of reperfusion in a 40 cm segment of the pelvic flexure in 6 horses. Mucosal biopsies before and after ischaemia, and after 1, 2 and 4 h of reperfusion were fixed in glutaraldehyde/paraformaldehyde and osmium tetroxide, and embedded in epon. Morphological and ultrastructural changes were evaluated in toluidine blue-stained semithin sections by light microscopy and in thin sections stained with uranyl acetate/lead citrate by transmission electron microscopy. RESULTS: Ischaemia caused swelling of epithelial cells and their organelles, opening of tight junctions, detachment from the basement membrane, early apoptosis and single cell necrosis. Autophagy was a prominent feature in epithelial cells after ischaemia. Reperfusion was characterised by apoptosis, epithelial regeneration and restoration of apical cell junctions. Phagocytic-like vacuoles containing cellular debris and bacteria were evident in epithelial cells after reperfusion. Paracellular and subepithelial clefts formed, accompanied by infiltration of neutrophils, lymphocytes and eosinophils into the epithelium. Subepithelial macrophages and luminal neutrophils had increased phagocytic activity. CONCLUSIONS: Ischaemia caused ultrastructural damage to the colonic epithelium, but epithelial cells recovered during reperfusion. POTENTIAL RELEVANCE: Transmission electron microscopy can demonstrate subtle ultrastructural damage to epithelial cells and evidence of recovery after I/R in equine colon.

Effects of flunixin meglumine on the recovery of ischaemic equine colonic mucosa in vitro.

REASONS FOR PERFORMING STUDY: The effects of prostaglandins and nonsteroidal anti-inflammatory drugs (NSAIDs) on repair of equine intestinal mucosa are important since most horses with gastrointestinal diseases are routinely treated with NSAIDs, such as flunixin meglumine (FM), and these drugs can be toxic to equine gastrointestinal mucosa. HYPOTHESIS: Flunixin meglumine would not affect recovery of equine colonic mucosa in vitro, 18 h after a reversible ischaemic injury. METHODS: In 14 anaesthetised horses, a segment of pelvic flexure was subjected to 2 h of ischaemia and the horses were allowed to recover for 18 h. Seven horses received normal saline and 7 received FM, 1.1 mg/kg bwt i.v., at the end of ischaemia and 12 h later. Colonic mucosa was harvested during a second anaesthesia, 18 h after recovery from ischaemia and then horses were subjected to euthanasia. Transepithelial electrical resistance (TER) and transepithelial flux of tritiated mannitol were used to measure mucosal permeability during 4 h of incubation in Ussing chambers, with the following in vitro treatments: 1) no addition, 2) FM 14 µmol/l as powder, 3) FM 14 µmol/l in injectable form and 4) diluent for injectable FM. Histomorphological changes were assessed by light microscopy. RESULTS: There were no significant differences in any of the measurements between saline and FM treated horses. The mucosal height of the ischaemic FM tissues incubated in diluent was significantly decreased compared to the nonischaemic tissues. CONCLUSIONS: Flunixin meglumine did not adversely affect barrier integrity in ischaemic equine colonic mucosa.

Detection of calprotectin and its correlation to the accumulation of neutrophils within equine large colon during ischaemia and reperfusion.

REASON FOR PERFORMING STUDY: The cytosolic protein complex, calprotectin, is abundant in neutrophils and could be used to improve the ability to localise and assess neutrophil infiltration in the equine intestine during ischaemia and reperfusion (I/R), but further study is required. OBJECTIVES: To assess the number of calprotectin-containing cells by immunohistochemistry in correlation with direct counting and scoring of neutrophils in the equine colon during I/R. METHODS: One and 2 h ischaemia of the left dorsal colon were induced, followed by 30 min reperfusion under general anaesthesia or by 18 h reperfusion after anaesthetic recovery. Biopsies were processed for light microscopy and stained with H/E for detection of neutrophils. To identify calprotectin-containing cells, immunohistochemistry was performed on formalin-fixed tissues with the murine MAC 387 antibody and a biotin-free peroxidase staining procedure. The number of neutrophils within submucosal venules and the colonic mucosa were calculated and compared with the number of calprotectin-positive cells. RESULTS: The number of calprotectin-positive cells within submucosal venules and within the colonic mucosa correlated significantly with the accumulation of neutrophils within the corresponding tissue segments. Within the submucosal venules, both calprotectin-positive cells and H/E-stained neutrophils increased with duration of ischaemia and peaked after 30 min of reperfusion. After 18 h reperfusion the number of these cells declined within the vessels. After 2 h ischaemia, neutrophils started to migrate into the mucosa towards the epithelium, with a significant increase over time during reperfusion, and peak infiltration after 18 h reperfusion. CONCLUSIONS: Neutrophil infiltration into the colon after I/R is a time-dependent process, involving migration through the submucosa towards the epithelium.

Effect of a novel solution for organ preservation on equine large colon in an isolated pulsatile perfusion system.

REASONS FOR PERFORMING STUDY: Several therapeutic agents have been tested in models of ischaemia and reperfusion injury (IRI) in equine jejunum, with mixed results. This study was based on the use of an organ perfusion solution (OPS) designed to protect human allografts from IRI. HYPOTHESIS: A modified OPS can preserve the integrity of equine large colon during 12 h of isolated pulsatile perfusion, in the absence of oxygen and blood. METHODS: Segments of large colon were removed from anaesthetised horses, the contents removed and the mucosa rinsed with 0.9% saline. Experimental segments were perfused for 12 h with one litre modified OPS (n = 7) delivered by pulsatile flow through an extracorporeal circuit. Control segments (n = 4) were perfused on the same circuit with one litre of autologous blood. Vascular resistance, flow and pressure were measured serially, and aliquots of OPS and blood drawn hourly for routine biochemical analyses. Mucosal biopsies of the experimental and control segments were taken at 0, 6 and 12 h and in vivo mucosal tissue at 0 h for baseline comparison. All biopsies underwent histomorphometric analysis and immunohistochemical assessment of calprotectin activity. RESULTS: All colon segments were machine perfused without technical complications. Vascular and biochemical indices remained constant over 12 h in the OPS group, and were constant over 6 h in the control group, but deteriorated later. Mucosal integrity, expression of cyclooxygenases-1 and -2, and expression of mucosal calprotectin were unchanged in the OPS group compared with the baseline tissues, and mucosal integrity was superior to the control tissues. CONCLUSIONS: A modified OPS designed to target specific pathways of damage from IRI can preserve colonic mucosal integrity for 12 h in the absence of blood and oxygen.

Pushing the envelope in renal preservation; improved results with novel perfusate modifications for pulsatile machine perfusion of cadaver kidneys.

INTRODUCTION: Novel preservation techniques may diminish ischemia/reperfusion (I/R) injury. Our preservation laboratory has modified Belzer MPS for machine perfusion (MP) with prostaglandin E1 (PGE 1), nitroglycerin (NTG), and polyethylene glycol-superoxide dismutase (PEG-SOD) to attenuate I/R injury. We reviewed our recent experience using this novel formulation (NF) compared with standard perfusates. RESULTS: Between January 1998 and March 2000, 1060 consecutive kidneys were preserved in our laboratory. One hundred forty-eight kidneys (14%) were discarded. Fifty-eight percent of kidneys during this time period underwent MP (n = 532). En bloc kidney pairs were randomly assigned to pulsatile MP using Waters RM3 or MOX-100 perfusion systems using 1 of 3 perfusates; NF (NF; n = 119), Belzer MPS (MPS; n = 201), or Belzer II albumin gluconate (ALB; n = 212) Significant improvements in delayed graft function (DGF) rate were seen with NF versus other perfusates (8% vs 14% vs 19%, respectively; P =.03). At 6 months, graft survival was significantly improved with NF compared with MPS and ALB (96% vs 90% vs 87%, respectively; P =.03). NF also produced a significantly higher percentage of recipients with a serum creatinine level < or = 1.5 mg/dL. CONCLUSIONS: Novel modifications of standard MP perfusate improved outcomes after renal transplantation. Preservation-based interventions targeted to ameliorate I/R injury can improve outcomes and may allow expansion of the donor pool.

A cholesterol-dependent CD20 epitope detected by the FMC7 antibody.

Accurate diagnosis of lymphoid malignancies is essential for appropriate therapeutic intervention. In conjunction with other diagnostic determinants, immunophenotypic analysis of differentially expressed cell surface markers, such as CD5, CD20, CD23 and FMC7, is useful in the subclassification of lymphomas and leukemias arising from the B-cell lineage. Recent evidence suggesting that CD20 predicts FMC7 expression has prompted reappraisal of the utility of monitoring both markers. Here, we report that the FMC7 monoclonal antibody (mAb) specifically and strongly recognized CD20 ectopically expressed in hematopoietic and nonhematopoietic cell lines. The reactivity of FMC7 was abolished by mutations in the extracellular domain of CD20. These data confirm the CD20 specificity of FMC7. Like other CD20 mAbs, FMC7 binding was temperature dependent and induced detergent insolubility of CD20. Of significant interest, the CD20 epitope recognized by FMC7 was unusual in that it was exceptionally sensitive to membrane cholesterol. Cholesterol depletion profoundly reduced expression of the FMC7 epitope, whereas cholesterol enrichment enhanced its expression. FMC7 mAb binding thus appears to be a sensitive indicator of the level of plasma membrane cholesterol and reveals a conformational state of CD20 that is regulated by cholesterol.

Glutathione supplementation during cold ischemia does not confer early functional advantage in renal transplantation.

BACKGROUND: Reduced glutathione (GSH), a component of University of Wisconsin (UW) solution, is reported to oxidize during storage. Consequently the commercial manufacturer of UW recommends the supplemental addition of GSH to UW before utilization. We investigated the influence of supplemental GSH during cold ischemia on early renal allograft function. METHODS: One hundred kidneys were locally procured from heart-beating donors, preserved in our laboratory, and transplanted during an 18-month period. Selected donor, preservation, and outcome characteristics were collected and compared by presence of supplemental GSH and method of preservation. All kidneys were randomized to receive 3.0 mM supplemental GSH to perfusate or no supplementation (control) and were preserved by either cold storage (CS) in UW or machine perfused (MP) in UW-machine perfusate solution (MPS). During MP, perfusion characteristics (flow, resistance, perfusate electrolytes, and pH) were serially measured. RESULTS: There were no significant differences among the groups when the donor characteristics of age, serum creatinine, and intra-operative urine output were compared. Preservation characteristics were similar among the groups with the exception of cold ischemia time, which was longer in the MP group compared to CS (26.1 h vs. 21.9 h, P=0.03). When compared with CS, kidneys preserved by MP exhibited a 33.4% increase in immediate function (93% vs. 62%, P=0.01), a corresponding 29.4% decrease in the incidence of delayed graft function (10% vs. 34%, P=0.02), and a 10% improvement in short-term (6-month) graft survival (98% vs. 88%, P=0.02). The addition of GSH supplementation to perfusate resulted in no significant differences in graft outcomes. CONCLUSIONS: Despite recommendations by the manufacturer that UW solution be routinely supplemented with GSH, supplemental GSH does not influence early renal allograft function. Our data suggest that a far greater beneficial impact on early graft function is achieved by machine perfusion. We conclude that GSH supplementation of commercially available UW is not necessary.

The influence of pulsatile preservation on renal transplantation in the 1990s.

BACKGROUND: Unlike simple cold storage (CS), pulsatile machine preservation (MP) of kidneys for transplantation permits pharmacologic manipulation of the perfusate and aids in the pretransplant assessment of the kidney graft. These characteristics of MP may have importance in the era of increasing use of extended criteria donor kidneys. The overall aim of this article is to critically assess practices at our preservation unit with respect to graft function. Specific aims are to (1) compare the influence of MP versus CS on graft function, (2) determine which pretransplant variables have significance in pretransplant assessment, and (3) determine whether pharmacologic manipulation during MP is advantageous. METHODS: There were 650 consecutive kidneys preserved in our laboratory between January 1, 1993 and March 1, 999, by either MP or CS. All MP kidneys were preserved by continuous hypothermic pulsatile perfusion using Belzer-MPS or Belzer II solution. Perfusion parameters and electrolytes were measured serially during pulsatile perfusion. All CS kidneys were stored in University of Wisconsin solution. All kidneys obtained from donors exhibiting extended criteria features underwent pretransplant frozen section biopsies. Transmission electron microscopy (EM) was performed on a subset of kidneys undergoing pharmacologic manipulation. Four agents were assessed prospectively for their ability to influence MP characteristics when added to perfusate: PGE1, trifluoperazine, verapamil, and papaverine. RESULTS: MP was associated with improved immediate, 1-, and 2-year graft function and reduced length of initial hospital stay when compared with CS grafts. Changes in the machine perfusion variables flow and resistance, and the [Ca++] in perfusate, were significantly associated with delayed graft function (DGF) after the transplant. Biopsy information was not predictive of DGF. The addition of PGE1 to perfusate improved MP characteristics, reduced the release of [Ca++] into perfusate, and ameliorated mitochondrial ischemic injury in transmission EM images. Early graft function was improved in the presence of PGE1+MP, compared with function in the presence of other pharmacologic agents or CS alone. CONCLUSIONS: MP is associated with improved early and long term renal function. Moreover, PGE1 augments MP in improving graft function. The combination of MP+PGE1 may be important in optimizing the ability to use extended donor criteria kidneys and, thereby, improve the overall efficiency of cadaveric renal transplantation.

Donor treatment with phentolamine mesylate improves machine preservation dynamics and early renal allograft function.

BACKGROUND: It has been suggested that pharmacologic conditioning of the donor before organ procurement may protect the renal allograft from injuries associated with the cold ischemic period. We compared the administration of two vasoactive agents before organ procurement to: (1) determine their influence on machine perfusion characteristics and (2) determine their impact on delayed graft function (DGF) in transplanted renal allografts. METHODS: Between January 1997 and December 1998, 150 kidneys were procured from heart-beating donors and preserved in our laboratory by machine perfusion (MP) or cold storage (CS). The following vasoactive agents were randomly administered to the donor 5 min before aortic cross clamp: phentolamine mesylate (PM) or hydralazine (H). The control groups received no donor conditioning. Kidneys were grouped as follows: (1) MP+PM, (2) MP+H, (3) MP, (4) CS+PM, (5) CS+H, (6) CS. 10 mg PM/50 kg donor weight was administered to the PM groups and 20 mg H/50 kg donor weight was administered to the H groups. DGF was defined as the need for dialysis within the first 7 days after the transplant. RESULTS: MP+PM increased renal flow by 12% and decreased renal resistance by 18% compared with the MP+H group, and increased renal flow by 23% and decreased renal resistance by 30% compared with the MP group. Moreover, the MP+PM group was associated with improved early allograft function. CONCLUSIONS: Donor treatment with PM immediately before aortic cross-clamp is associated with improved machine perfusion dynamics (renal flow and renal resistance) and lower incidence of DGF compared with donor treatment with H or no treatment. Moreover, MP of renal allografts was associated with improved early function compared with CS grafts.

Calcium ion concentration of machine perfusate predicts early graft function in expanded criteria donor kidneys.

Pulsatile preservation offers the advantage of pretransplant assessment of donor kidneys. Selected electrolyte concentrations of machine perfusate were measured over time in order to: (1) describe electrolyte changes in perfusate during the pulsatile preservation of expanded-criteria donor (ECD) kidneys, and (2) to assess the prognostic significance of these characteristics to early graft function. One hundred and fifty ECD kidneys were preserved in our laboratory between 1 January 1995 and 11 January 1997. ECD kidneys were defined as those requiring pretransplant biopsy. Kidneys were grouped by the presence or absence of delayed graft function (DGF), and perfusion parameters were measured every hour during pulsatile perfusion. All kidneys were preserved by continuous hypothermic pulsatile perfusion using Belzer II solution. Renal flow is decreased and renal resistance is increased in the presence of DGF in machine-preserved ECD kidneys. In addition, ionized calcium concentration of the machine perfusate is significantly elevated in the DGF group compared with the No DGF group (0.091 vs 0.054, P = 0.0016). The incidence of DGF is significantly lower in the ECD kidney. Among the pretransplant variables of donor characteristics, perfusion parameters and histology, perfusion parameters are highly predictive of early graft function. In addition, we found that ionized calcium concentration in the perfusate is significantly elevated in kidneys exhibiting DGF, which may have implications for assessing the suitability of donor kidneys for transplantation.

Prostaglandin E1 influences pulsatile preservation characteristics and early graft function in expanded criteria donor kidneys.

INTRODUCTION: Unlike simple cold storage, machine preservation allows dynamic assessment and manipulation of the donor organ prior to transplantation. We prospectively compared the effects of five pharmacological agents added to the perfusate during machine preservation of expanded criteria donor (ECD) kidneys in order to (1) describe their influence on perfusion parameters and (2) determine their influence on early graft outcome. METHODS: Two hundred seventy-five consecutive ECD kidneys were preserved in our laboratory between 1/1/94 and 12/31/97 by either machine perfusion (MP) or cold storage (CS). ECD kidneys were defined as those requiring pretransplant biopsy. ECD kidneys were divided by method of preservation and MP kidneys were randomized to receive prostaglandin E1 (MP+PGE1), trifluoperazine (TFP), verapamil (VER), papaverine (PAP), mannitol (MAN), or no intervention during the period of machine perfusion. CS kidneys were randomized to receive PGE1 (CS+PGE1), TFP, VER, PAP, or no intervention. All MP kidneys were preserved by continuous hypothermic pulsatile perfusion using Belzer II solution and perfusion parameters were measured every hour during pulsatile perfusion. All CS kidneys were stored in 1.0 L of University of Wisconsin (UW) solution. RESULTS: The addition of PGE1 to machine perfusate increased renal flow and decreased renal resistance. Moreover, the MP+PGE1-treated group was associated with improved early graft function compared to all other groups. The addition of VER, TFP, PAP, or MAN influenced neither the perfusion characteristics nor the incidence of early graft function in MP kidneys. Similarly, the addition of VER, TFP, or PAP did not influence early graft function in the CS kidneys. The CS+PGE1 group exhibited a significantly lower incidence of early graft function than did the MP+PGE1 group. CONCLUSIONS: PGE1 treatment during machine preservation improves hydrostatic perfusion parameters and reduces the incidence of delayed graft function in ECD kidneys. Moreover, the addition of PGE1, TFP, VER, or PAP to UW does not influence early graft function in the CS kidney.

Prostaglandin E1 improves pulsatile preservation characteristics and early graft function in expanded criteria donor kidneys.

Unlike simple cold storage, machine preservation allows dynamic assessment and manipulation of the donor organ before transplantation. The effects of four pharmacologic agents added to the perfusate during machine preservation of expanded criteria donor (ECD) kidneys were prospectively compared to 1) describe their influence on perfusion parameters and 2) determine their influence on early graft outcome. Between 1 January 1995 and 1 October 1997, 125 consecutive ECD kidneys were preserved in the authors' laboratory. A definition of ECD was assigned to kidneys requiring pretransplant biopsy. The ECD kidneys were randomized to receive prostaglandin E1 (PGE1), trifluoperazine (TFP), verapamil (VER), mannitol (MAN), or no intervention (control) during machine preservation. All kidneys were preserved by continuous hypothermic pulsatile perfusion (CHPP) using Belzer II solution, and perfusion parameters were measured every 2 hours during pulsatile perfusion. The addition of PGE1 to the perfusate increased renal flow and decreased renal resistance. Moreover, the PGE1 treated group was associated with improved early graft function when compared with all other groups. The addition of VER, TFP, and MAN influenced neither the perfusion characteristics nor the incidence of early graft function. Treatment with PGE1 during machine preservation enhances hydrostatic perfusion parameters (renal flow and renal resistance) and reduces the incidence of delayed graft function in ECD kidneys.

Identification of a cytoplasmic region of CD20 required for its redistribution to a detergent-insoluble membrane compartment.

CD20 is a B lymphocyte integral membrane protein with signal-transducing properties. Abs directed toward extracellular CD20 epitopes activate nonreceptor tyrosine kinases and modulate cell cycle progression of B lymphocytes. Recently, we demonstrated that binding of CD20 Abs to B cells induces the rapid redistribution of up to 95% of CD20 molecules to low density, detergent-insoluble membrane microdomains and induces the appearance of an approximately 50-kDa tyrosine-phosphorylated protein in the same compartment. Active relocalization of CD20 may thus be critical to the initiation of signaling events by CD20. The CD20 cDNA sequence predicts a nonglycosylated protein with four transmembrane-spanning regions and intracellular amino and carboxyl termini. Here we provide verification of the location of both the intracellular and extracellular regions of the CD20 molecule and identify a membrane-proximal sequence in the cytoplasmic carboxyl tail that is required for CD20 to redistribute to detergent-insoluble membrane microdomains.

Rapid redistribution of CD20 to a low density detergent-insoluble membrane compartment.

CD20 is a B cell integral membrane protein capable of initiating growth-modulating signals in human B lymphocytes upon its engagement with monoclonal anti-CD20 antibodies. In this report, we demonstrate that treatment of B cells with CD20 antibodies induces rapid redistribution of CD20 into a detergent-insoluble membrane compartment. Redistribution is detected as early as 15 s, following antibody addition, and involves up to 95% of CD20 molecules, depending on the antibody used. All of the detergent-insoluble CD20 was found in the low density fractions of sucrose density gradients, indicating that CD20 redistributes to glycolipid-rich membrane domains, analogous to caveolae in some cell types. As CD20 has previously been shown to associate with Src family tyrosine kinases, their co-existence in these compartments suggests a link to the role of CD20 in signal transduction. This study provides insight into the mechanism by which CD20 communicates signals to the cell interior and indicates that the search for membrane-proximal intracellular signaling partners should be directed to the Triton-insoluble fraction.