Influence of ureteric anastomosis technique on urological complications after kidney transplantation.

The best technique for anastomosis of the donor ureter remains unresolved and an individual surgeon's preferred technique is likely to be based on training background. This retrospective analysis compares three ureteroneocystostomy techniques in 204 consecutive transplants with an overall ureteric complication rate of 7.3%. Ureteric complications after kidney transplantation present early, are more common when using deceased donor kidneys, and, require complex reconstructive surgery to resolve. Three significant and related findings have been demonstrated with a common theme related to the length of the transplanted ureter, and not, the debate over extravesical versus intravesical techniques.

Inferior early posttransplant outcomes for recipients of right versus left deceased donor kidneys: an ANZDATA registry analysis.

Anatomical differences between right and left kidneys could influence transplant outcome. We compared graft function and survival for left and right kidney recipients transplanted from the same deceased organ donor. Adult recipients of 4900 single kidneys procured from 2450 heart beating deceased donors in Australia and New Zealand from 1995 to 2009 were included in a paired analysis. Right kidneys were associated with more delayed graft function (DGF) (25 vs. 21% for left kidneys, p < 0.001) and, if not affected by DGF, a slower fall in serum creatinine. One-year graft survival was lower for right kidneys (89.1 vs. 91.1% for left kidneys, p = 0.001), primarily attributed to surgical complications (66 versus 35 failures for left kidneys). Beyond the first posttransplant year, kidney side was not associated with eGFR, graft or patient survival. Receipt of a right kidney is a risk factor for inferior outcomes in the first year after transplantation. A higher incidence of surgical complications suggests the shorter right renal vein may be contributory. The higher susceptibility of right kidneys to injury should be considered in organ allocation.

Transplant glomerulopathy: ultrastructural abnormalities occur early in longitudinal analysis of protocol biopsies.

Transplant glomerulopathy (TXG) presents a distinctive pattern of glomerular abnormalities. The aim of this study was to describe its sequential ultrastructural pathology. A paired cohort study of 228 protocol biopsies, from our longitudinal database (n = 1345), compared TXG (7 patients, 95 biopsies) and controls (8 patients, 133 biopsies). Ultrastructural morphometry and C4d immunoperoxidase were evaluated from implantation to 5 years after transplantation against sequential histology and functional changes. TXG was predated by early glomerular endothelial cell activation; typified by vacuolation, hypertrophy, serration and expansion of lamina rara interna from 39 +/- 23 days after transplantation. Endothelial cells were transformed into an activated phenotype, containing numerous mitochondria, Golgi and ribosomes. Transition from fenestrated to continuous endothelium, mesangial matrix expansion and podocyte fusion occurred late. Endothelial cell activation also occurred in peritubular capillaries (PTC) followed by basement membrane multi-lamination (p < 0.05-0.001). Light microscopy changes of TXG occurred at 2.3 years. PTC C4d deposition was intermittently expressed over time, correlating with endothelial abnormalities, glomerular C4d and donor-specific antibodies (DSA) (p < 0.05-0.001). In summary, endothelial and subendothelial ultrastructural abnormalities in glomerular and peritubular capillaries are sensitive, early markers of TXG, likely due to stimulation of endothelial cells into an activated phenotype by antibody-mediated sub-lytic complement deposition.

Mycophenolate mofetil is associated with altered expression of chronic renal transplant histology.

Mycophenolate mofetil (MMF) reduces acute rejection in controlled trials of kidney transplantation and is associated with better registry graft survival. Recent experimental studies have demonstrated additional antifibrotic properties of MMF, however, human histological data are lacking. We evaluated sequential prospective protocol kidney biopsies from two historical cohorts treated with cyclosporine (CSA)-based triple therapy including prednisolone and either MMF (n = 25) or azathioprine (AZA, n = 25). Biopsies (n = 360) were taken from euglycemic kidney-pancreas transplant recipients. Histology was independently assessed by the Banff schema and electron microscopic morphometry. MMF reduced acute rejection and OKT3 use (p < 0.05) compared with AZA. MMF therapy was associated with limited chronic interstitial fibrosis, striped fibrosis and periglomerular fibrosis (p < 0.05-0.001), mesangial matrix accumulation (p < 0.01), chronic glomerulopathy scores (p < 0.05) and glomerulosclerosis (p < 0.05). MMF was associated with delayed expression of CSA nephrotoxicity, reduced arteriolar hyalinosis, striped fibrosis and tubular microcalcification (p < 0.05-0.001). The beneficial effects of MMF remained in recipients without acute rejection. Retrospective analysis shows that MMF therapy was associated with substantially reduced fibrosis in the glomerular, microvascular and interstitial compartments, and a delayed expression of CSA nephrotoxicity. These outcomes may be due to a limitation of immune-mediated injury and suggest a direct effect of reduced fibrogenesis.

Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene.

A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV 35S promoter was expressed in a hybrid poplar clone, Populus x euramericana ('Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and in vivo in transgenic 'Ogy'. The enzyme activity in the transformed 'Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic 'Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic 'Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from 'Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls.

Negative regulation of CD4 gene expression by a HES-1-c-Myb complex.

Expression of the CD4 gene is tightly controlled throughout thymopoiesis. The downregulation of CD4 gene expression in CD4(-) CD8(-) and CD4(-) CD8(+) T lymphocytes is controlled by a transcriptional silencer located in the first intron of the CD4 locus. Here, we determine that the c-Myb transcription factor binds to a functional site in the CD4 silencer. As c-Myb is also required for CD4 promoter function, these data indicate that depending on the context, c-Myb plays both positive and negative roles in the control of CD4 gene expression. Interestingly, a second CD4 silencer-binding factor, HES-1, binds to c-Myb in vivo and induces it to become a transcriptional repressor. We propose that the recruitment of HES-1 and c-Myb to the silencer leads to the formation of a multifactor complex that induces silencer function and repression of CD4 gene expression.

Effect of histological damage on long-term kidney transplant outcome.

BACKGROUND: Chronic renal allograft failure remains a major challenge to overcome. Factors such as donor quality, delayed graft function (DGF), acute rejection, and immunosuppression are known to affect long-term outcome, but their relationship to histological damage to graft outcome is unclear. METHODS: Protocol kidney biopsies (n=112) obtained at 3 months after transplantation yielded 102 with adequate tissue. Histology was scored by the Banff schema, and compared with implantation biopsies (n=91), repeat 12-month histology (n=39), decline in serum creatinine and serial isotopic glomerular filtration rate, onset of chronic allograft nephropathy (CAN), and actuarial graft survival censored for death with a functioning graft. RESULTS: At a median follow-up of 9.3 years, 20 patients had graft failure and 26 died with a functioning graft. Banff chronic nephropathy was present in 24% of 3-month biopsies, and was predicted by microvascular disease in the donor, cold ischemia, DGF, and acute vascular rejection (P<0.001). Acute glomerulitis at 3 months correlated with segmental glomerulosclerosis at 12 months, subsequent recurrent glomerulonephritis, and graft failure (P<0.01). Subclinical rejection at 3 months occurred in 29% of biopsies, correlated with prior acute rejection and HLA mismatch, and led to chronic histological damage by 12 months (r=0.25-0.67, P<0.05-0.001). Subclinical rejection, arteriolar hyalinosis, and tubulitis present at 3 months had resolved by 12 months. The 10-year survival rates for Banff chronic nephropathy were 90.4% for grade 0, 81.0% grade 1, and 57.9% for grades 2 or greater (P<0.01). Early tubulointerstitial damage at 3 months profoundly influenced graft survival beyond 10 years. CAN was predicted by kidney ischemia, 3-month chronic intimal vascular thickening, tubular injury, proteinuria, and late rejection. Chronic fibrointimal thickening of the small arteries and chronic interstitial fibrosis at 3 months independently predicted graft loss and decline in renal function (P<0.05-0.001). CONCLUSIONS: Early transplant damage occurs in the tubulointerstitial compartment from preexisting donor kidney injury and discrete events such as vascular rejection and DGF. Subsequent chronic damage and graft failure reflect accumulated previous injury and chronic interstitial fibrosis, vascular impairment, subclinical rejection, and injury from late rejection. CAN may be conceptualized as the sequelae of incremental and cumulative damage to the transplanted kidney. The duration of graft survival is dependent and predicted by the quality of the transplanted donor kidney combined with the intensity, frequency, and irreversibility of these damaging insults.

Cellular membranes that undergo cyclic changes in tension: Direct measurement of force generation by an in vitro contractile vacuole of Paramecium multimicronucleatum.

The contractile vacuole of the fresh water protozoan Paramecium is a membrane-bound vesicle that expels excess cytosolic water, acquired osmotically, through its periodic exocytotic activity. The in vitro contractile vacuole, isolated in a small amount of cytosol from the Paramecium cell and confined under mineral oil, showed periodic rounding and slackening at regular intervals for an extended time. The contractile vacuole rounded against the cytosol-mineral oil boundary tension. The tension at the surface of the contractile vacuole is, therefore, assumed to increase during the rounding phase. We first estimated the tension relative to the boundary tension from the degree of compression of the contractile vacuole by the boundary. We then determined the absolute value for the tension at the surface of the contractile vacuole from the degree of bending of an elastic carbon fiber microcantilever (8 microm thick; 2 mm long), whose free end was placed at the surface of an in vitro contractile vacuole. The tension was found to increase to its maximum value of approximately 5 mN m(-)(1) when the contractile vacuole rounded. This value was more than 35 times higher than that for the slackened contractile vacuole. Electron micrographs of conventional thin sections of chemically fixed in vitro contractile vacuoles as well as those of in vivo contractile vacuoles obtained from rapid frozen and cryosubstituted cells revealed the lack of any ultrastructural evidence for the presence of a fibrous network system surrounding the contractile vacuole. Thus we conclude that the mechanism(s) by which tension is developed at the surface of the contractile vacuole membrane resides in the contractile vacuole membrane itself. We propose a hypothesis that periodic changes in the spontaneous curvature of the contractile vacuole's lipid bilayer membrane is involved in the periodic development of higher contractile vacuole membrane tension. The isolated CV promises to be an excellent model system for understanding the molecular mechanisms of the dynamics of biological membrane.

How external osmolarity affects the activity of the contractile vacuole complex, the cytosolic osmolarity and the water permeability of the plasma membrane in Paramecium multimicronucleatum.

The rate of fluid expulsion, R(CVC), from the contractile vacuole complex (CVC) of Paramecium multimicronucleatum was estimated from the volume of the contractile vacuoles (CVs) immediately before the start of fluid discharge and from the time elapsing between discharges. The R(CVC) increased when the cell was exposed to a strongly hypotonic solution and decreased in a weakly hypotonic solution. When the cell was exposed to an isotonic or a hypertonic solution, R(CVC) fell to zero. The time constant, tau, used to describe the change in R(CVC) in response to a change in external osmolarity shortened after a short-term exposure to a strongly hypotonic solution and lengthened after a short-term exposure to a less hypotonic solution. A remarkable lengthening of tau occurred after a short-term exposure to isotonic or hypertonic solution. Under natural conditions, mechanisms for controlling R(CVC) are effective in maintaining the cytosolic osmolarity hypertonic within a narrow concentration range despite changes in the external osmolarity, which is normally hypotonic to the cytosol. Cells exposed to an isotonic or hypertonic solution resumed CV activity when left in the solution for 12 h. The cytosolic osmolarity was found to increase and to remain hypertonic to the external solution. This will permit cells to continue to acquire water. The increase in the cytosolic osmolarity occurred in a stepwise fashion, rather than linearly, as the external osmolarity increased. That is, the cytosolic osmolarity first remained more-or-less constant at an increased level until the external osmolarity exceeded this level. Thereupon, the cytosolic osmolarity increased to a new higher level in 12 h, so that the cytosol again became hypertonic to the external solution and the cells resumed CV activity. These results imply that the cell needs to maintain water segregation activity even after it has been exposed to an isotonic or hypertonic environment. This supports the idea that the CVC might be involved not only in the elimination of excess cytosolic water but also in the excretion of some metabolic waste substances.

Phagosome formation in Paramecium: roles of somatic and oral cilia and of solid particles as revealed by video microscopy.

The roles of somatic and oral cilia and solid particles during digestive vacuole (DV) formation in Paramecium multimicronucleatum were investigated using video-enhanced and immunofluorescence microscopy. Membrane incorporation into DVs was found to increase linearly with increasing particle concentration. The rate of discoidal vesicle transport to the cytopharynx was not affected by particles, showing that particles are not required for membrane trafficking to the cytopharynx. However, the presence of particles leads to an increased membrane fusion between the cytopharyngeal membrane and the discoidal vesicles. When live cells lost their somatic cilia on the left-ventral side anterior to the oral region due to deciliation, membrane incorporation into newly formed DVs was strongly inhibited. Using video-enhanced microscopy, latex beads were seen to be loaded along the quadrulus on the dorsal surface of the buccal cavity, but few beads were seen next to the dorsal and ventral peniculi. Particle sequestration into a pre-formed nascent digestive vacuole (NDV) was studied in Triton X-100-permeabilized cells whose ciliary beating was reactivated by the addition of Mg-ATP. Both beat frequency and the percentage of cells containing bead-labeled NDV were dependent on the Mg-ATP concentration: the higher the beat frequency, the higher the percentage of cells with a bead-labeled NDV. These results suggest that ciliary beating is probably the only mechanism required for particle accumulation in the NDV, while a coordinated beating of the somatic cilia on the left-ventral side anterior to the oral region as well as the quadrulus moves particles into the NDV. The beating of the peniculi may somehow prevent the backward flow of particles out of the NDV.

Genetic factors influencing the development of chronic graft-versus-host disease in a murine model.

Graft-versus-host disease (GVHD) is a major complication of bone marrow transplantation that can occur in either acute or chronic forms. Much of the long-term pathology seen in chronic GVHD is a result of autoantibody production. In the DBA/2-->B6D2F1 murine model of chronic GVHD, anti-ssDNA autoantibodies can be detected by 14 days post cell transfer. These autoantibodies are not observed in B6D2F1 recipients of cells from C57BL/6 or B10.D2 donors, which develop acute rather than chronic GVHD. Therefore, in this model, donor genetic factors predispose to the development of chronic GVHD in recipients. We performed a genetic analysis aimed at mapping donor loci that influence the magnitude of early autoantibody production in B6D2F1 recipients of cells from DBA/2 donor mice. Linkage analysis suggested an influence of two loci: a locus on chromosome 11 linked to D11Mit278 and a locus on chromosome 4 linked to D4Mit226. The locus on chromosome 11 also appeared to influence the development of renal pathology associated with chronic GVHD.

Stress tolerance in transgenic tobacco seedlings that overexpress glutathione S-transferase/glutathione peroxidase.

Overexpression of a tobacco glutathione S-transferase with glutathione peroxidase activity (GST/GPX) in transgenic tobacco (Nicotiana tabacum L.) enhanced seedling growth under a variety of stressful conditions. In addition to increased GST and GPX activity, transgenic GST/GPX-expressing (GST+) seedlings had elevated levels of monodehydroascorbate reductase activity. GST+ seedlings also contained higher levels of glutathione and ascorbate than wild-type seedlings and the glutathione pools were more oxidized. Thermal or salt-stress treatments that inhibited the growth of wild-type seedlings also caused increased levels of lipid peroxidation. These treatments had less effect on the growth of GST+ seedling growth and did not lead to increased lipid peroxidation. Stress-induced damage resulted in reduced metabolic activity in wild-type seedlings while GST+ seedlings maintained metabolic activity levels comparable to seedlings grown under control conditions. These results indicate that overexpression of GST/GPX in transgenic tobacco seedlings provides increased glutathione-dependent peroxide scavenging and alterations in glutathione and ascorbate metabolism that lead to reduced oxidative damage. We conclude that this protective effect is primarily responsible for the ability of GST+ seedlings to maintain growth under stressful conditions.

The contractile vacuole and its membrane dynamics.

The contractile vacuole (CV) is an osmoregulatory organelle whose mechanisms of function are poorly understood. Immunological studies in the last decade have demonstrated abundant proton-translocating V-type ATPases (V-ATPases) in its membrane that could provide the energy, from proton electrochemical gradients, for moving ions into the CV to be followed by water. This review emphasizes recent work on the contractile vacuole complex (CVC) of Paramecium including (1) CV expulsion, (2) a role for V-ATPases in sequestering fluid, (3) identifying ions in the cytosol and in the CV, (4) in situ electrophysiological parameters of the CVC membrane, and (5) a better understanding of the membrane dynamics of this organelle.

A locus closely linked to Mtv7 on mouse chromosome 1 influences development of acute versus chronic graft-versus-host disease in a murine model.

The relationship between acute and chronic graft-versus-host disease (GVHD) is not well understood. A murine model of acute and chronic GVHD is the B6D2F1 parent-->F1 model in which transfer of C57BL/6 parental strain lymphoid cells to B6D2F1 recipients results in development of Th1-mediated acute GVHD, whereas transfer of DBA/2 parental strain lymphoid cells to B6D2F1 recipients results in development of Th2-mediated chronic GVHD. Numerous studies have investigated the reason for the differential development of acute versus chronic GVHD in this model but have as yet failed to identify the factor that determines which type of T helper cell will predominate and thereby which type of GVHD will develop. In this report, we demonstrate, using congenic strains of mice, that a locus in the vicinity of the Mtv7 locus on Chromosome 1 of the mouse significantly influences development of acute versus chronic GVHD in the B6D2F1 model.

Membrane trafficking and processing in Paramecium.

Cellular membranes are made in a cell's biosynthetic pathway and are composed of similar biochemical constituents. Nevertheless, they become differentiated as membrane components are sorted into different membrane-limited compartments. We summarize the morphological and immunological similarities and differences seen in the membranes of the various interacting compartments in the single-celled organism, Paramecium. Besides the biosynthetic pathway, membranes of the regulated secretory pathway, endocytic pathway, and phagocytic pathway are highlighted. Paramecium is a multipolarized cell in the sense that several different pools of membrane-limited compartments are targeted for exocytosis at very specific sites at the cell surface. Thus, the method used by this cell to sort and package its membrane subunits into different compartments, the processes used to transport these compartments to specific locations at the plasma membrane and to other intracellular fusion sites, the processes of membrane retrieval, and the processes of membrane docking and fusion are reviewed. Paramecium has provided an excellent model for studying the complexities of membrane trafficking in one cell using both morphological and immunocytochemical techniques. This cell also promises to be a useful model for studying aspects of the molecular biology of membrane sorting, retrieval, transport, and fusion.

Ulceration of the urethral meatus after simultaneous pancreas-kidney transplantation.

A 39-year-old male developed painful ulceration of the glans penis following simultaneous pancreas and kidney transplantation for end-stage renal failure complicating insulin-dependent diabetes mellitus. Infection was excluded. Diversion of the pancreatic secretions away from the urinary bladder into the bowel resulted in healing.

Posttransplant cataract: lessons from kidney-pancreas transplantation.

INTRODUCTION: Cataract is a major cause of visual disturbance after transplantation. Although corticosteroid therapy has been linked with posterior subcapsular cataract, its natural history in the cyclosporine era is not well understood. METHODS: Baseline and regular postoperative slit-lamp biomicroscopy and ophthalmic examinations (n=432) were performed in 108 eyes of simultaneous kidney/pancreas (SPK) recipients (n=54) for up to 10 years after transplantation. Triple therapy immunosuppression of cyclosporine, azathioprine, and prednisolone was used. RESULTS: Cataract was present in 40% of eyes at simultaneous kidney/pancreas associated with duration of diabetes, lower insulin dose, and the use of pretransplant hemodialysis (P<0.05-0.01). Cataract became increasing more common 2 years after simultaneous kidney/pancreas, and lens abnormalities were virtually universal at 6-10 years by slit lamp biomicrosopy. The instantaneous hazard rate for new cataract formation was highest within the first 2 years and remained abnormal for the study duration. Nuclear and posterior subcapsular cataract increased significantly after transplantation (P<0.05) and were the predominant types of cataract presenting late. Risk factors for posttransplant cataract formation included older age and high-dose pulse methylprednisolone dose. Visual acuity was reduced by severity of cataract grade, presence of combined nuclear and subcapsular cataract, retinal hemorrhage and underlying diabetic retinopathy (P<0.05-0.001). Cataract formation imposed significant additional impairment of visual acuity above that of diabetic retinopathy. Cataract surgery was undertaken in 14% of eyes, improving visual acuity from mean decimalized score of 0.28 to 0.43, P<0.01 but did not normalize it to the noncataract level of 0.72. CONCLUSION: Transplantation substantially increases all types of cataract, and is highly prevalent by slit lamp examination. High-risk patients are older and diabetic, and received hemodialysis and pulse corticosteroid therapy. In contrast to older studies using high-dose corticosteroid and azathioprine, the pattern of cataract in the cyclosporine era is different with broader cataract types, a weaker association with corticosteroids and a progressive course. Regular screening of visual acuity and appropriate surgery for posterior subcapsular or severe cataract are recommended.