Tumor necrosis factor-α promoter polymorphisms and the risk of rejection after liver transplantation: A case control analysis of 210 donor-recipient pairs

After orthotopic liver transplantation (OLT), allograft rejection remains an important problem and is the major reason that immunosuppressive therapy must be administered. Tumor necrosis factor-α (TNF-α) is a proinflammatory mediator that is central to the immune response, and intragraft expression of this cytokine is increased during acute cellular rejection (ACR). Polymorphisms within the TNF promoter have been identified and correlated with alterations in production. The aims of this study were to determine if an individual patient's propensity to develop ACR is related to the presence of these genetic polymorphisms (either alone or in combination) within donor and recipient tissue and to determine if these polymorphisms affect patient survival after OLT. The study group consisted of 210 patients who underwent OLT between 1989 and 1999 with at least 6 months survival, including 42 cases who had evidence of acute cellular rejection (biopsy-proven, elevated enzymes, and response to increased immunosuppression) and were matched 4:1 to controls (n = 168) with similar age, gender, underlying liver disease, date of transplant, and baseline immunosuppression. The underlying liver diseases were hepatisis C virus (HCV)/alcohol (70), HCV alone (50), alcohol (30), primary biliary cirrhosis (15), primary sclerosing cholangitis (15), autoimmune hepatitis/cirrhosis (10), cryptogenic (15), and hepatitis B virus (HBV) (5). DNA was extracted from paraffin-embedded donor and recipient liver tissue (total 420 samples), amplified, and sequenced for TNF single-nucleotide polymorphisms (TNFA-308 A/G and TNFA-238 A/G). We found no differences between the TNF allelic distributions among donors without liver disease (presumably representative of a normal control population) and patients with end-stage liver disease undergoing OLT. Multivariate analysis revealed no association with TNF polymorphisms (within donor or recipient tissue) and rejection risk or patient survival after transplantation. In this large case control analysis of patients undergoing liver transplantation for diverse etiologies, TNF promoter polymorphisms were not independently associated with rejection or survival. (Liver Transpl 2003;9:377-382.)     

Tumor necrosis factor-α production after esophageal cancer surgery: Differences in the response to lipopolysaccharide stimulation among whole blood, pleural effusion cells, and bronchoalveolar lavage fluid cells

Escherichia coli (1 μg/ml), Staphylococcus aureus (10 μg/ml), and lipopolysaccharide (LPS) (1 μg/ml), and pleural effusion cells and BALF cells were stimulated with LPS; 24-H incubation and TNF-α concentration in supernate was measured by enzyme-linked immunosorbent assay (ELISA). Within 3 h after starting the operation, TNF-α production in whole blood was significantly (P < 0.05) decreased compared with preoperative value by each stimulation, and this suppression persisted up to day 3. These reductions in postoperative TNF-α production correlated with intraoperative hemorrhage. On the other hand, the LPS-induced release of TNF-α into pleural effusion cells and BALF cells were markedly increased during the study period. These results indicate that large quantities of cytokines are produced by a second attack, such as infection, in areas where immunocytes accumulate. We believe that the body reacts to surgical stress in a variety of ways. Circulating blood and immunocytes that accumulate in damaged organs are thought to react very differently to stress. (Received for publication on Dec. 16, 1997; accepted on May 15, 1998)     

17β-Estradiol increases the receptor number and modulates the action of 1,25-dihydroxyvitamin D3 in human osteosarcoma-derived osteoblast-like cells

It is well known that 17-estradiol (E₂) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) have important roles in bone metabolism. This study was undertaken to examine E₂ regulation of 1,25(OH)₂D₃ receptor (VDR) expression and the biological action of 1,25(OH)₂D₃ in human osteoblast-like cells. When human osteosarcoma-derived osteoblast-like cells were treated with varying concentrations of E₂, the VDR levels increased by up to 100% in a dose-dependent manner. VDR levels significantly increased at 10 nM E₂ and this increase plateaued at 100 nM E₂. E₂-dependent increase of VDR was time dependent, plateauing, at 24 hours and was maintained for at least 48 hours in human osteoblast-like cells. Scatchard analysis showed that E₂ increased the number of VDR (12.3±0.4 versus 26.5±0.3 fmol/mg protein; mean ±SE of three independent experiments) rather than the Kd (0.15±0.02 versus 0.16±0.01 nM; mean±SE of three independent experiments). Tamoxifen (50 nM), a specific competitor with E₂, completely abolished the E₂-induced increase of VDR. The levels of VDR mRNA (4.5 kb) from the cells increased in a dose-dependent manner after E₂ treatment. With regard to the biological effects, E₂ increased by 10–25% the inhibitory effect of 1,25(OH)₂D₃ on cell growth. However, E₂ did not increase the stimulation of alkaline phosphatase activity by 1,25(OH)₂D₃. The present study suggests that E₂ modulates the biological action of 1,25(OH)₂D₃ through VDR levels in bone cells.     

Regulation of synthesis of osteoprotegerin and soluble receptor activator of nuclear factor-κB ligand in normal human osteoblasts via the p38 mitogen-activated protein kinase pathway by the application of cyclic tensile strain

Mechanical stress is thought to play an important role in bone remodeling. However, the correlation between mechanical stress and bone remodeling is poorly understood. In this context, using a model of cyclic tensile strain (CTS) toward human osteoblasts, synthesis of osteoprotegerin (OPG) and soluble receptor activator of nuclear factor-κB ligand (sRANKL), and the activation of mitogen-activated protein kinases (MAPKs) were examined. The application of 7%, 0.25-Hz CTS once a day for 4 h for 3 successive days simultaneously caused an increase of OPG synthesis and a decrease of sRANKL release and RANKL mRNA expression in osteoblasts. As for MAPKs activation in osteoblasts with the application of CTS, p38 MAPK was activated 10–20 min after the application of CTS, but extracellular signal-regulated kinase (ERK1/2) and c-Jun NH₂-terminal kinase (JNK) were not activated by such application. Furthermore, when CTS was applied once a day for 4 h for 1, 2, or 3 successive days to osteoblasts, p38 MAPK activation was maintained during the 3-day period but ERK1/2 activation was downregulated from day to day, simultaneously. Then, when CTS was applied once a day for 4 h for 3 successive days to osteoblasts pretreated with the p38 MAPK inhibitor SB203580 for 1 h, OPG synthesis was dose-dependently suppressed and inhibition of sRANKL release and RANKL mRNA expression was abrogated. These results indicate that biological responses of OPG and sRANKL synthesis in osteoblasts to the application of CTS are regulated via the p38 MAPK pathway and suggest that CTS might modulate and regulate bone metabolism.     

Enhanced expression of the CD71 mesangial IgA1 receptor in Berger disease and Henoch-Schönlein nephritis: association between CD71 expression and IgA deposits

ABSTRACT. IgA nephropathy (IgA-N) that comprises Berger disease and Henoch-Sch?nlein Purpura (HSP) nephritis is defined by mesangial IgA deposits. Recently, this group has characterized a new receptor for IgA, the transferrin receptor (CD71), expressed on mesangial cells. To assess whether CD71 was involved in the pathogenesis of IgA-N, its expression was analyzed together with IgA deposits on 16 kidney biopsies from 16 patients with Berger disease (n = 4) or HSP (n = 12). These biopsies were compared with 17 kidney biopsies of a group of 15 patients (control group) with other glomerulonephritis, including systemic lupus erythematosus, poststreptococcal acute glomerulonephritis, membranoproliferative glomerulonephritis, steroid-sensitive minimal change nephrotic syndrome, steroid-resistant idiopathic nephrotic syndrome with focal and segmental glomerulosclerosis, and persistent and isolated proteinuria with minimal change on kidney biopsy. In this control group, IgA deposits could be observed in eight kidney biopsies of seven patients. These biopsies were also compared with normal kidney specimens (normal group). In normal kidney, it was found that CD71 was linearly expressed on tubular epithelium but was either not expressed or very dimly in glomeruli. In contrast, CD71 was strongly expressed in 105 of the 107 glomeruli of the kidney biopsies from the IgA-N group. For the control group, it was found that expression of CD71 in glomeruli was correlated to the presence of IgA deposits. Indeed, among the 87 glomeruli of nine kidney biopsies (eight patients) without IgA fixation, 78 exhibited no CD71 expression and nine exhibited a very dim one. On the other hand, all 49 glomeruli of the eight kidney biopsies (seven patients) in which IgA deposits were detected exhibited CD71 expression (P < 10(-4)). Performance of dual-labeling studies with confocal microscopy on kidney biopsies of IgA-N patients demonstrated that most of the IgA deposits co-localized with CD71. It was also demonstrated that the intensity of the expression of CD71 was not linked to the intensity of clinical or biologic findings but to the intensity of cellular proliferation in both IgA-N and control groups. These results show that mesangial CD71 expression is not specific to IgA-N. However, the association between IgA deposits and CD71 expression and their co-localization in the mesangium provide strong evidence that CD71 is a major IgA receptor on mesangial cells.     

Activation of β-catenin signaling in MLO-Y4 osteocytic cells versus 2T3 osteoblastic cells by fluid flow shear stress and PGE2: Implications for the study of mechanosensation in bone

The osteocyte is hypothesized to be the mechanosensory cell in bone. However, osteoblastic cell models have been most commonly used to investigate mechanisms of mechanosensation in bone. Therefore, we sought to determine if differences might exist between osteocytic and osteoblastic cell models relative to the activation of β-catenin signaling in MLO-Y4 osteocytic, 2T3 osteoblastic and primary neonatal calvarial cells (NCCs) in response to pulsatile fluid flow shear stress (PFFSS). β-catenin nuclear translocation was observed in the MLO-Y4 cells at 2 and 16 dynes/cm² PFFSS, but only at 16 dynes/cm² in the 2T3 or NCC cultures. The MLO-Y4 cells released high amounts of PGE₂ into the media at all levels of PFFSS (2–24 dynes/cm²) and we observed a biphasic pattern relative to the level of PFFSS. In contrast PGE₂ release by 2T3 cells was only detected during 16 and 24 dynes/cm² PFFSS starting at > 1 h and never reached the levels produced by the MLO-Y4 cells. Exogenously added PGE₂ was able to induce β-catenin nuclear translocation in all cells suggesting that the differences between the cell lines observed for β-catenin nuclear translocation were associated with the differences in PGE₂ production. To investigate a possible mechanism for the differences in PGE₂ release by the MLO-Y4 and 2T3 cells we examined the regulation of Ptgs2 (Cox-2) gene expression by PFFSS. 2T3 cell Ptgs2 mRNA levels at both 0 and 24 h after 2 h of PFFSS showed biphasic increases with peaks at 4 and 24 dynes/cm² and 24-hour levels were higher than zero-hour levels. MLO-Y4 cell Ptgs2 expression was similarly biphasic; however at 24-hour post-flow Ptgs2 mRNA levels were lower. Our data suggest significant differences in the sensitivity and kinetics of the response mechanisms of the 2T3 and neonatal calvarial osteoblastic versus MLO-Y4 osteocytic cells to PFFSS. Furthermore our data support a role for PGE₂ in mediating the activation of β-catenin signaling in response to the fluid flow shear stress.     

Changing serine-485 to alanine in the opossum parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of phospholipase C in a stably transfected human kidney cell line: a useful model for PTH-analog screening?

Using site-directed mutagenesis, we have introduced a serine-485-to-alanine mutation in the opossum parathyroid hormone (PTH) receptor. This amino acid is considered to be phosphorylated by protein kinase A upon ligand binding. Both wild-type (WT) and mutant receptor were stably expressed in 293-EBNA HEK cells. The mutant receptor showed comparable binding characteristics and only a slight increase in cAMP production compared with WT. However, the PTH dose-dependent increase in inositol phosphate production was 24-fold for the mutant receptor vs. 6-fold for the WT receptor. This mutant might prove useful in the sensitive detection of phospholipase C activation through various ligands, as the PTH receptor becomes a target of therapeutic intervention in osteoporosis.