Interferon-{alpha}2b treatment of chronic hepatitis C in haemodialysis patients

Nineteen haemodialysis (HD) patients with chronic hepatitisC were treated with interferonalpha2b (IFN-) at a dose of 3or 1 MU thrice weekly for 6 months and were followed-up foranother 14 months without treatment. Six patients discontinuedtreatment because they either presented severe side-effectsto IFN- or had complications of their primary disease. Levelsof AST and ALT were within normal limits on the 2nd month oftreatment and remained so throughout the treatment and the follow-upperiod in all patients except one who showed an elevation oftransaminase levels 2 months after the end of treatment. SerumHCVRNA became negative in 10/13 patients at the end of treatmentand was negative in all patients on the 6th month and in 12/13patients on the 14th month during the follow-up period. Levelsof 2'5' oligosynthetase were increased significantly on the2nd and 4th month of treatment and returned to pretreatmentvalues the 2nd month after treatment. These findings demonstratethat haemodialysis patients with chronic hepatitis C respondwell to interferon treatment and that a long-term response isachieved in a high proportion of patients.     

Alphav integrin regulates TNF-alpha-induced nitric oxide synthesis in rat mesangial cells--possible role of osteopontin.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) induces nitric oxide (NO) synthesis in rat mesangial cells (MCs). We previously demonstrated that osteopontin (OP), a matrix protein that mainly interacts with the alphav integrin family, increased time-dependently by TNF-alpha stimulation at gene and protein levels. The regulation of NO synthesis by integrins or matrix proteins is unclear. METHODS: We examined whether integrin, especially alphav integrin, regulates NO synthesis in rat MCs and whether OP, an alphav integrin ligand, has an effect on TNF-alpha-induced NO synthesis. Furthermore, OP and inducible NO synthase (iNOS) gene expression was examined by Northern blotting. RESULTS: TNF-alpha increased NO synthesis in MCs in a time-dependent manner. Synthetic GRGDSP peptide, which is known to inhibit various integrins that interact with RGD-containing extracellular matrices, increased TNF-alpha-induced NO levels in a dose-dependent manner. Cyclical RGD peptide, the specific inhibitor of alphav integrin, also exhibited a dose-dependent effect of increasing NO levels, while GRGESP peptide, which has very low affinity to integrins, had no effect. In addition, NO synthesis was found to be significantly reduced when MCs were plated on OP-coated dishes compared to type I or IV collagen-coated dishes. Furthermore, anti-OP antibody increased NO synthesis in MCs. iNOS mRNA levels were increased by TNF-alpha, and were abruptly diminished after OP mRNA was significantly induced. CONCLUSIONS: The present study demonstrated the involvement of alphav integrin in TNF-alpha-induced NO synthesis in rat MCs, and the possible role of OP was suggested in the mechanism. TNF-alpha and extracellular matrices can co-operate to regulate the behaviour of MCs at least partly through NO synthesis, which may participate in the course of glomerular diseases.     

Hypoxia reduces the expression and anti-inflammatory effects of peroxisome proliferator-activated receptor-gamma in human proximal renal tubular cells.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-gamma may counteract tissue fibrosis via its anti-inflammatory actions, while hypoxia, a new pro-fibrotic factor, reportedly modifies PPAR-gamma expression. However, the effects of hypoxia on the expression and anti-inflammatory actions of PPAR-gamma have yet remained to be clarified in renal tubular cells. METHODS: Confluent human proximal renal tubular epithelial cells (HPTECs) were exposed to hypoxia (1% O2) and/or TNF-alpha at 10 ng/ml for up to 48 h. The cells were incubated with PPAR-gamma agonists, 15d-PGJ2 or pioglitazone, for 30 min before stimulation. Precise amounts of PPAR-gamma and MCP-1 mRNA and protein were measured by TaqMan quantitative PCR and immunoblot or ELISA, respectively. RESULTS: A cDNA array analysis identified PPAR-gamma as one of the hypoxia-affected genes in HPTECs. Hypoxia reduced mRNA levels of PPAR-gamma at 24 and 48 h and protein levels at 6 and 48 h. Knockout of hypoxia-inducible factor-1alpha (HIF-1alpha) with its dominant negative form did not block the hypoxia-induced reduction in PPAR-gamma expression. PPAR-gamma's activation with 15d-PGJ2 or pioglitazone reduced basal and TNF-alpha-stimulated MCP-1 expression at mRNA and protein levels at 24 h under normoxia. MCP-1 reduction rates at basal mRNA and protein levels were slightly but significantly lower during hypoxia than normoxia (9 vs 69% and 36 vs 42%, respectively, for 15d-PGJ2, and 0 vs 34% and 12 vs 21%, respectively, for pioglitazone). Finally, a specific inhibitor for PPAR-gamma, GW9662, weakened the MCP-1-decreasing effect of 15d-PGJ2 by about 30%, under basal conditions, while it abolished the effect of pioglitazone almost completely. CONCLUSIONS: Hypoxia-induced loss of function of PPAR-gamma reduces anti-inflammatory effects of PPAR-gamma activation, possibly modulating inflammatory responses in the diseased kidney.     

Morphology and distribution of chandelier cell axon terminals in the mouse cerebral cortex and claustroamygdaloid complex

Chandelier cells represent a unique type of cortical --aminobutityricacidergic interneuron whose axon terminals (Ch-terminals) onlyform synapses with the axon initial segments of some pyramidalcells. Here, we have used immunocytochemistry for the high-affinityplasma membrane transporter GAT-1 and the calcium-binding proteinparvalbumin to analyze the morphology and distribution of Ch-terminalsin the mouse cerebral cortex and claustroamygdaloid complex.In general, 2 types of Ch-terminals were distinguished on thebasis of their size and the density of the axonal boutons thatmade up the terminal. Simple Ch-terminals were made up of 1or 2 rows of labeled boutons, each row consisting of only 3–5boutons. In contrast, complex Ch-terminals were tight cylinder-likestructures made up of multiple rows of boutons. Simple Ch-terminalswere detected throughout the cerebral cortex and claustroamygdaloidcomplex, the complex type was only occasionally found in certainregions, whereas in others they were very abundant. These resultsindicate that there are substantial differences in the morphologyand distribution of Ch-terminals between different areas andlayers of the mouse cerebral cortex. Furthermore, we suggestthat the distribution of complex Ch-terminals may be relatedto the developmental origin of the different brain regions analyzed.     

Plasma concentrations of alpha-melanocyte-stimulating hormone are elevated in patients on chronic haemodialysis.

BACKGROUND: Clinical and/or laboratory signs of systemic inflammation occur frequently in patients undergoing long-term haemodialysis. It is likely, therefore, that a compensatory release of endogenous anti-inflammatory molecules occurs to limit host reactions. The aim of the present research was to determine if the potent anti-inflammatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH), a pro-opiomelanocortin derivative, is increased in plasma of haemodialysis patients. Because endotoxin and cytokines induce alpha-MSH in vivo and in vitro, we also measured plasma concentrations of endotoxin, interleukin-6 (IL-6), and tumour necrosis factor alpha (TNF-alpha), and the two circulating products of activated monocytes, nitric oxide (NO) and neopterin. METHODS: Thirty-five chronic haemodialysis patients, 20 patients with chronic renal failure not yet on dialysis, and 35 normal controls were included in the study. In the haemodialysis group, blood samples were obtained before and at the end of a dialysis session. Plasma alpha-MSH was measured using a double antibody radioimmunoassay, and IL-6, TNF-alpha, and neopterin using specific enzyme-linked immunosorbent assays. Plasma nitrites were determined by a colorimetric method, and endotoxin with the quantitative chromogenic LAL (limulus amoebocyte lysate) method. RESULTS: Mean plasma alpha-MSH was higher in haemodialysis patients than in control subjects, with the peptide concentrations being particularly elevated in dialysed patients with detectable endotoxin. High alpha-MSH concentrations were observed in the pre-dialysis samples, with no substantial change at the end of the dialysis session. Plasma concentrations of IL-6, TNF-alpha, neopterin, and NO were generally elevated in chronic haemodialysis patients and there was a negative correlation between circulating alpha-MSH and IL-6. In patients with renal failure not yet on dialysis, mean plasma alpha-MSH was similar to that of normal subjects. CONCLUSIONS: alpha-MSH is increased in the circulation of chronic haemodialysis patients and particularly so in case of detectable endotoxaemia. Reduction of renal clearance is unlikely to contribute to the observed rise of the peptide because alpha-MSH concentration is not increased in patients with chronic renal failure who are not yet on dialysis. It is likely that dialysis-associated endotoxaemia, directly and/or through cytokine release, enhances the production of the anti-inflammatory mediator alpha-MSH that limits host reactions.     

THIP, a hypnotic and antinociceptive drug, enhances an extrasynaptic GABAA receptor-mediated conductance in mouse neocortex

THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a selective GABA(A) receptor agonist with a preference for delta-subunit containing GABA(A) receptors. THIP is currently being tested in human trials for its hypnotic effects, displaying advantageous tolerance and addiction properties. Since its cellular actions in the neocortex are uncertain, we studied the effects of THIP on neurons in slices of frontoparietal neocortex of 13- to 19-day-old (P13-19) mice. Using whole-cell patch-clamp recordings, we found that the clinically relevant THIP concentration of 1 muM induced a robust tonic GABA(A)-mediated current in layer 2/3 neurons. In comparison, only a minute tonic current was induced by mimicking in vivo endogenous GABA levels. Miniature IPSCs were not affected by 1 muM THIP suggesting an extrasynaptic site of action. The EC(50) for THIP was 44 muM. In accordance with the stronger expression of delta-containing receptors in superficial neocortical layers, THIP induced a 44% larger tonic current in layer 2/3 than in layer 5 neurons. Finally, monitoring spontaneously active neocortical neurons, THIP caused an overall depression of inhibitory activity, while enhancing excitatory activity prominently. Our studies suggest that THIP activates an extrasynaptic GABA(A) receptor-mediated conductance in the neocortex, which may alter the cortical network activity.     

p38 MAPK phosphorylation and NF-kappa B activation in human crescentic glomerulonephritis.

BACKGROUND: p38 mitogen-activated protein kinase (p38 MAPK) followed by the activation of NF-kappa B participates in the intracellular signal transduction and production of cytokines and chemokines. The pathophysiological roles of p38 MAPK and NF-kappa B in human glomerulonephritis, however, remain to be investigated. METHODS: We investigated the phosphorylated p38 MAPK (p-p38 MAPK) and activated NF-kappa B immunohistochemically in the kidneys of 34 patients with crescentic glomerulonephritis and 26 control patients with thin basement membrane disease and minimal change nephrotic syndrome. We also explored the co-localization of p-p38 MAPK with CCR5, the signal of which leads to p38 MAPK activation. Furthermore, urinary levels of MIP-1 alpha, the cognate ligand for CCR5, were determined by enzyme-linked immunosorbent assay. RESULTS: p-p38 MAPK-positive cells and activated NF-kappa B-positive cells were mainly detected in crescentic lesions, tubular epithelial cells, and interstitial mononuclear infiltrates. The number of p-p38 MAPK-positive cells in patients with crescentic glomerulonephritis was higher than that in control patients. The number of p-p38 MAPK-positive cells in glomeruli was well correlated with the percentage of cellular crescents, the number of CD68-positive cells, and urinary MIP-1 alpha levels. In addition, the number of activated NF-kappa B-positive cells was well correlated with the number of p-p38 MAPK-positive cells in glomeruli. Dual staining revealed that most of CCR5-positive cells were positive for p-p38 MAPK. Finally, p-p38 MAPK-positive cells and activated NF-kappa B-positive cells decreased during glucocorticoid therapy-induced convalescence. CONCLUSIONS: We conclude that the phosphorylation of p38 MAPK associated with the activation of NF-kappa B may be involved in the upregulation of intrarenal MIP-1 alpha and the utilization of CCR5 signalling, which may result in human crescentic glomerulonephritis.     

Role of atrophic changes in proximal tubular cells in the peritubular deposition of type IV collagen in a rat renal ablation model.

BACKGROUND: Tubular atrophy, dilation and interstitial fibrosis are common in tubulointerstitial lesions, but the precise roles and inter-relationships of these components in the development of interstitial lesions have not been determined. This study focused on the origin and roles of atrophic tubules in the peritubular deposition of type IV collagen in a rat renal ablation model. METHODS: Male Wistar rats underwent 5/6 nephrectomy or sham operation, and then were sacrificed at 4, 8 or 12 weeks, their remaining kidneys removed for histological and immuno-histochemical studies as well as in situ hybridization for type IV collagen mRNA. RESULTS: Immuno-histochemistry demonstrated the positive staining of atrophic tubules to vimentin, platelet-derived growth factor-B chain (PDGF) and heat shock protein 47 (HSP47). Cells positive to one or more of PDGF receptor beta, alpha-smooth muscle actin (alpha-SMA), and HSP47 accumulated around atrophic tubules. Type IV collagen was also increased in the proximity of the atrophic tubules. These intimate relationships were more clearly demonstrated in 'mosaic tubules', which are composed of both intact and atrophic proximal tubular epithelial cells, and which had a mixed pattern of staining with vimentin, PDGF and HSP47. The interstitial cells positive to alpha-SMA or HSP47, or both, were in close contact with atrophic but not with intact epithelial cells. Type IV collagen was exclusively deposited between atrophic tubules and HSP47-positive interstitial cells. In situ hybridization of type IV collagen mRNA demonstrated predominant expression in atrophic tubular epithelial cells, but not in surrounding interstitial cells. CONCLUSIONS: These findings suggest that atrophic proximal tubular cells are active in the development of collagen deposition in the peritubular space, i.e. in this model type IV collagen in the interstitial fibrotic area may be produced mainly by atrophic proximal tubules.     

Interleukin 6 production by human proximal tubular epithelial cells in vitro: analysis of the effects of interleukin-1{alpha} (IL-1{alpha}) and other cytokines

Proximal tubular epithelial cells (PTEC) from human renal tissueobtained from biopsy or nephrectomy were grown in monocultureand evaluated in vitro at passage 2–4 for interleukin6 (IL-6) production in response to medium alone or to interleukin1 alpha (IL-1), tumour necrosis factor alpha (TNF), interleukin2 (IL-2), interferon gamma (INF) or lipopolysaccharide (LPS).IL-6 bioactivity was quantitated using the IL-6-dependent murinehybridoma cell line (B9) and expressed as IL-6 units/ml/10⁵PTEC. PTEC cell lines exposed to medium alone produced intermediateamounts of IL-6 with substantial variability between cell lines.Introduction of IL-1 resulted in a dose- and time-dependentincrease in IL-6 production by PTEC that was maximal at 1 ng/mlIL-1 at 24 h. All PTEC cell lines showed an increased IL-6 productionon exposure to IL-1 varying from 1.3- to 24-fold increase overbaseline production. This response was completely blocked byanti-rIL-1. No significant IL-6 production by PTEC could beinduced by TNF, IL-2, IFN, or LPS over a broad dosage range.Cycloheximide inhibited IL-6 production without irreversiblecell toxicity, indicating de-novo synthesis. IL-6 produced byPTEC had a molecular weight of 26-29 kDa as demonstrated byWestern blot analysis. Using PCR analysis we could demonstrateupregulation by IL-1 of IL-6 mRNA in a dose-response fashion,indicating that IL-1 regulates IL-6 production at a pretranslationalvalue of protein synthesis. These results show that human culturedPTEC produce IL-6 under both normal and IL-1-stimulated conditions,and suggest that they may have a regulatory function in responseto cytokines in the setting of inflammation in the renal cortex.     

T gamma/delta lymphocytes in renal transplant recipients.

T gamma/delta lymphocytes are able to perform allospecific cytotoxicity and natural killer cytotoxicity in vitro. However, very little is known about their function in vivo. To investigate the possible involvement of T gamma/delta lymphocytes in the immune response to renal allografts, fine-needle aspiration biopsies and peripheral blood of 15 renal transplant recipients were studied during the first 4 weeks after transplantation. In addition peripheral blood of patients before transplantation, half a year and one year after transplantation was studied. No increase in the percentage of T gamma/delta lymphocytes in the fine-needle aspiration biopsies, including those taken during acute rejection episodes, was found. A significant decrease in the percentage of T gamma/delta lymphocytes was observed in peripheral blood after transplantation. We conclude that T gamma/delta lymphocytes seem to play no major role in the immune response to renal allografts.     

Rosiglitazone ameliorates cisplatin-induced renal injury in mice.

BACKGROUND: Inflammatory mechanisms may play an important role in the pathogenesis of cisplatin nephrotoxicity. Agonists of the peroxisome proliferator-activated receptor-gamma (PPARgamma), such as rosiglitazone, have been recently demonstrated to regulate inflammation by modulating the production of inflammatory mediators and adhesion molecules. The purpose of this study was to examine the protective effects of rosiglitazone on cisplatin nephrotoxicity and to explore the mechanism of its renoprotection. METHODS: Mice were treated with cisplatin with or without pre-treatment with rosiglitazone. Renal functions, histological findings, aquaporin 2 (AQP2) and adhesion molecule expression, macrophage infiltration and tumour necrosis factor-alpha (TNF-alpha) levels were investigated. The effect of rosiglitazone on nuclear factor (NF)-kappaB activity and on viability was examined using cultured human kidney (HK-2) cells. RESULTS: Rosiglitazone significantly decreased both the damage to renal function and histological pathology after cisplatin injection. Pre-treatment with rosiglitazone reduced the systemic levels of TNF-alpha and down-regulated adhesion molecule expression in addition to the infiltration of inflammatory cells after cisplatin administration. Rosiglitazone restored the decreased AQP2 expression after cisplatin treatment. Pre-treatment with rosiglitazone blocked the phosphorylation of the p65 subunit of NF-kappaB in cultured HK-2 cells. Rosiglitazone had a protective effect via a PPARgamma-dependent pathway in cisplatin-treated HK-2 cells. CONCLUSION: These results showed that pre-treatment with rosiglitazone attenuates cisplatin-induced renal damage through the suppression of TNF-alpha overproduction and NF-kappaB activation.