A pilot study in patients with established advanced liver fibrosis using pirfenidone.
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Aged↗
Biomedical subjects
J Armendáriz-Borunda
Publications and source records attributed to J Armendáriz-Borunda.
Nitric oxide synthases inhibition results in renal failure improvement in cirrhotic rats.
UNLABELLED: Nitric oxide (NO) has been implicated in cirrhosis and might be implicated in renal failure end-stage cirrhosis. AIM: Our aim was to evaluate NO role in renal failure induced during decompensated cirrhosis, using the following inhibitors: aminoguanidine (AG), a specific inducible nitric oxide synthase (iNOS) inhibitor and NG-nitro-l-arginine methyl ester (L-NAME), a nonselective blocker of NOS isoforms. METHODS: Endothelial (eNOS) and iNOS gene expression was analyzed by reverse transcriptase-polymerase chain reaction. Cirrhotic rats received a single intragastric dose of CCl(4) to induce acute liver damage (ALD). RESULTS: After ALD, aspartate aminotransferase highest levels were observed in rats treated with AG and ALT in rats treated with L-NAME. Inhibitors decreased creatinine serum levels to normal values and serum sodium levels re-established after the third day of ALD. L-NAME diminished (P<0.05) eNOS RNA renal expression. Renal iNOS with no inhibitor was overexpressed but was down-regulated by AG treatment. Liver eNOS RNA expression had a decreased expression before ALD in cirrhotic rats, but L-NAME treatment down-regulated eNOS after ALD. AG induced an important iNOS liver decrease. CONCLUSION: Both inhibitors improved renal function, although AG displayed a better effect and did not aggravate liver function. We concluded that NOS isoforms are implicated in the renal pathophysiologic events induced by ALD.
Animals↗
Molecular diagnosis of Chagas' disease and use of an animal model to study parasite tropism.
Chagas' disease, which is an important health problem in humans, is caused by the protozoan Trypanosoma cruzi. The cellular and molecular mechanisms, involved in the selective tropism of T. cruzi to different organs remain largely unknown. In this study we designed a PCR-based molecular diagnosis method in order to study the tropism and growth kinetics of T. cruzi in a murine model infected with parasites isolated from an endemic area of Mexico. The growth kinetics and parasite tropism of T. cruzi were also evaluated in the blood and other tissues. We observed that T. cruzi isolates from the Western Mexico showed a major tropism to mouse heart and skeletal muscles in this murine model.
Adolescent↗
T(H)1/T(H)2 cytokine profile, metalloprotease-9 activity and hormonal status in pregnant rheumatoid arthritis and systemic lupus erythematosus patients.
During the course of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), several immune and neuroendocrine changes associated with pregnancy may exert positive (amelioration) or negative (exacerbation) effects on the clinical outcome. In order to shed light on the mechanisms underlying these responses, we performed a prospective longitudinal study in RA and SLE pregnant women, including healthy pregnant women as a control group. Cytokine messenger RNA (mRNA) expression assessed by quantitative competitive polymerase chain reaction (PCR) in peripheral blood mononuclear cells (PBMC), cytokine levels and lymphocyte proliferation responses (LPR) following phytohaemagglutinin (PHA) stimulation of PBMC, plasma metalloprotease-9 activity (MMP-9) and hormonal status during pregnancy were determined. TNFa was the most abundant cytokine mRNA expressed in PBMC in all groups studied (healthy pregnant women, RA and SLE pregnant patients). However, a general TH2 response reflected by high IL-10 levels was found in RA, as well as SLE, patients. A significant change in IFN-gamma was observed in RA patients but only during the first trimester of pregnancy. This compared with a major TH1 response in healthy pregnant women. Interestingly, our study showed a homogeneous hormonal pattern in RA and SLE patients. Although decreased cortisol levels were observed in all patients studied, this is possibly related to the remission of disease activity status brought about by steroid treatment before and during pregnancy. In summary, we suggest that complex immune and hormonal networks are involved in pregnancy and that rheumatic diseases are very dynamic immune processes that cannot be described with a clear-cut cytokine profile. Furthermore, the observations in this study may reflect treatment-related immune effects more than those associated with disease.
Adult↗
Cholestasis-induced fibrosis is reduced by interferon alpha-2a and is associated with elevated liver metalloprotease activity.
BACKGROUND/AIM: Several drugs have been tested for the treatment of hepatic cirrhosis induced by various etiologic agents. Although interferon (IFN)alpha-2a has mostly been used to treat viral hepatitis, its anti-fibrogenic properties remain to be established. METHODS: An experimental model of cholestasis-induced cirrhosis was used to test the effect of IFNalpha-2a. Cirrhosis was induced in rats via ligation of the common bile duct. IFNalpha-2a (100,000 IU/rat, s.c.) was administered daily throughout the experiment. Collagens and TIMP-1 mRNA transcripts were determined by semi-quantitative reverse transcriptase-polymerase chain reaction in liver tissue samples. Activity of metalloproteases (MMPs) was measured using gelatin (denatured collagen) as substrate and the specific size of the enzymes was estimated by zymograms. Histology was performed using Sirius red as a specific stain for collagenous material, and computer-assisted morphometric analyses were carried out. A polyclonal mouse anti-plasminogen activator inhibitor (PAI-1) antibody was used to evaluate the distribution during treatment with IFNalpha-2a. RESULTS/CONCLUSIONS: MMP-activity was up-regulated in bile duct ligated rats treated with IFNalpha-2a. MMP-activity in homogenates of total liver was minimal as compared with activity in non-parenchymal cells isolated from the same parental perfused liver, indicating a cryptic MMP activity which was completely abolished by EDTA and 1,10 phenanthroline. Three bands of gelatin degradation were detected by zymography, corresponding to 95, 75 and 65 kDa. IFNalpha-2a decreased PAI-1 immunoreactivity in liver tissue slices as well as biochemical activity in non-parenchymal cell extracts (3.3+/-0.08 vs 7.4+/-1.1 U/mg protein). Procollagen alpha1 (III) and alpha1 (IV) genes expression were also down-regulated 1.5 and 4-fold, respectively. Interestingly, TIMP-1 gene expression did not change. Functional hepatic tests: alanine aminotransferase, aspartate aminotransferase, bilirubins and alkaline phosphatase were significantly lower in IFNalpha-2a treated animals. Analysis of histology demonstrated that IFNalpha-2a promoted resolution of fibrosis and decreased bile duct proliferation.
Alanine Transaminase↗
Truncated active matrix metalloproteinase-8 gene expression in HepG2 cells is active against native type I collagen.
BACKGROUND/AIMS: Excess type I collagen accumulation is a major feature of fibrotic diseases such as liver cirrhosis. Reversion of this disease has not been fully accomplished. Physiologically, collagen is degraded by interstitial collagenases, neutrophil collagenase (MMP-8) being the most active against type I collagen. Introduction of MMP-8 gene into liver cells could be an advantageous tool to potentiate fibrosis degradation. METHODS: We cloned latent and active MMP-8 genes in prokaryotic and eukaryotic expression vectors and an adenoviral vector. Transfection of MMP-8 in HepG2 was effectuated by CaPO4, polylysine-lactose (P-L) and adenoviral transduction, and cells and culture supernatant were harvested 72 h after transfection for analysis of MMP-8 expression by reverse transcription-polymerase chain reaction and collagenolytic activity. RESULTS AND CONCLUSIONS: We show that a truncated neutrophil collagenase (tMMP-8) lacking a portion of the carboxy terminus and with an intact aminoterminus (latent; l-tMMP-8) or a truncated amino terminus (active; a-tMMP-8) has enzymatic activity against native type I collagen, and the activity was inhibited by EDTA, 1,10-phenanthroline and TIMP-1. Both MMP-8 mRNA (latent and active) were detected by polymerase chain reaction in cells transfected with CaPO4, P-L and adenoviral transduction; however, relative expression of MMP-8 was enhanced when the plasmid was delivered as a P-L complex and increased by adenoviral infection. Finally, a-tMMP-8 cDNA was cloned in a vector under transcriptional control of a regulated promoter (PEPCK-a-tMMP-8). HepG2 cells transfected with the PEPCK-a-tMMP-8 plasmid DNA up-regulated expression of a-tMMP-8 after incubation of the cells with butyryl-cAMP and glucagon, while stimulation with insulin slightly down-regulated its expression. Recombinant MMP-8 expressed by HepG2-transduced cells can efficiently degrade soluble type I collagen, which is potentially useful for gene transfer therapies.
Collagen↗
Differential effect of CCl4 on renal function in cirrhotic and non-cirrhotic rats.
The pathogenesis of renal function alteration associated with liver disease remains to be elucidated. Although different experimental animal models have been utilized in order to explain such pathophysiological state, none of them have completely explained the mechanisms involved. In this study we performed differential hemodynamic, hepatic and renal function alteration studies after induction of acute liver damage via intragastric administration of a single dose of CCl4 to cirrhotic and non-cirrhotic rats. Cirrhotic rats with acute liver damage exhibited a significant decrease in mean arterial pressure followed by a decreased glomerular filtration rate, urinary sodium concentration and an induction of plasma renin concentration and activity. At the same time, a significant association between oliguria and mortality was observed. The renal histopathological studies revealed glomeruli with mesangial hypercellularity and thickening of capillary wall, but not tubular epithelial injury. All these alterations were not detected in the control group, i.e. by non-cirrhotic rats with acute liver damage. This study suggests that the effect of CCl4 on kidney structure and function depends on the functional state of the liver. Since this experimental model of acute liver damage in cirrhotic rats presents hemodynamics and renal function alterations similar to those observed in the hepatorenal syndrome in man, it could be utilized to study the pathogenesis of renal function alterations associated with liver damage.
Acute Disease↗
Treatment with anti-transforming growth factor beta antibodies influences an altered pattern of cytokines gene expression in injured rat liver.
The role of transforming growth factor beta1 (TGF-beta1) in mediating hepatic inflammation and regeneration after acute liver injury is beginning to be elucidated, yet its in vivo effect on the gene expression of the major pro-inflammatory and anti-inflammatory cytokines produced during that process is unknown. Our previous experiments demonstrated that anti-TGF-beta-treated animals presented profound histological changes as compared with control animals. Therefore, our hypothesis was that by blocking in vivo TGF-beta1 action, with polyclonal anti-TGF-beta antibodies, we could monitor by RT-PCR significative alterations on the gene expression of IL-1beta, IL-6, TGF-beta, TNF-alpha, IL-4 and IL-10 in liver-regenerated rats after administration of a single CCl4 dosing. Accordingly, we here report a completely different pattern of cytokines gene expression amidst those groups of rats. Pro-inflammatory cytokines gene expression in control animals showed a clear-cut pattern peaking at 1-2 days postinjury and declining thereafter. Interestingly, IL-6 was present in the control animals only between 12 and 24 h after CCl4 dosing. In the experimental animals, TGF-beta1 was mainly increased at 4 and 6 days, while IL-6 mRNA was completely absent. IL-1beta mRNA expression was also altered in the experimental rats, albeit TNF-alpha was nearly unaffected. IL-4 was fully absent in control rats, but remarkably expressed in experimental animals throughout the study. IL-10 was also more expressed in experimental animals.
Animals↗
Antisense S-oligodeoxynucleotides down-regulate TGFbeta-production by Kupffer cells from CCl4-injured rat livers.
TGFbeta is a pleiotropic cytokine involved in multiple physiological and pathophysiological regulatory mechanisms. Since TGFbeta is a disparate modulator of cell recruitment, proliferation and extracellular matrix phenotype for mesenchymal and nonmesenchymal cells, we have been investigating the role of this cytokine in the pathophysiology of liver. In the present paper we investigate which hepatic cell types from CCl4-injured rat livers express TGFbeta mRNA and produce TGFbeta in culture, with the aim of further obliterating its biological activity by means of antisense technology. We performed a series of comprehensive molecular studies of in situ hybridization, northern blots, and RT-PCR and we found that only non-parenchymal cells produce TGFbeta while its expression in hepatocytes was absent. Consistent with the in situ hybridization findings, we observed that Kupffer cells expressed high steady-state levels of TGFbeta mRNA, while circulating monocytes expressed a smaller amount of TGFbeta transcripts. We did not detect TGFbeta gene expression in endothelial cells. These findings were further confirmed by RT-PCR analyses. TGFbeta activity, as measured by inhibition of [3H]thymidine incorporation by Mv 1 Lu mink lung epithelial cells, was down-regulated in culture by antisense phosphorothioate oligonucleotides. These effects of antisense oligomers were dose-dependent and the sense oligonucleotides had no effect at the same concentration.
Animals↗
Kupffer cells from CC1(4)-treated rat livers induce skin fibroblast and liver fat-storing cell proliferation in culture.
Conditioned media of Kupffer cells from normal rat liver produce in culture two factors that inhibit fibroblast proliferation. The inhibitory factors have molecular masses of approximately 25 and 5 kDa. In contrasts to these results, the conditioned media of Kupffer and mononuclear macrophagic cells, obtained 48 hours after CC1(4) administration to rats, contains a 17 kDa factor that stimulates fibroblast proliferation (FSF). FSF also stimulates [3H]-thymidine incorporation into DNA of cultured rat liver fat-storing cells. Two peaks with FSF activity were demonstrated after isoelectrofocusing; one with a pI of 6.1 and a second with a pI of 7.5. The fraction containing FSF is devoid of interleukin-1 (IL-1) activity and no inhibitory activity is detected in this conditioned media. Production of FSF is inhibitable by colchicine but not by indomethacin, it is thermolabile and trypsin-sensitive. In animals treated chronically with CC1(4) to produce cirrhosis, FSF activity is demonstrable from the first to the 8th week of treatment. However, the activity is lower at 8 weeks post-CC1(4) as compared with 2 weeks. The results suggest that homeostasis of cells in the liver is controlled by factors produced locally, that act by autocrine and paracrine mechanisms. When homeostasis is altered, fibroblast proliferation occurs, and excess collagen deposition leads to fibrosis. We propose that the antifibrogenic activity of colchicine is associated, in part, with its capacity to inhibit the release of FSF by Kupffer cells and liver mononuclear macrophage cells.
Animals↗
A simple quantitative method for collagen typing in tissue samples: its application to human liver with schistosomiasis.
Collagen chains separated on 5% SDS-acrylamide gels were stained with a 0.1% Sirius F3BA solution in saturated aqueous picric acid. After destaining the gels with methanol: acetic acid: water (30: 7 : 63), they were scanned at 540 nm and the area under each peak was determined. After that, the bands were sliced and the slices incubated overnight with trypsin at 37 degrees C. The color of the slices was eluted completely when the denatured collagen was ingested with trypsin. The absorbance of the color eluted was determined at 540 nm. The results obtained demonstrated that both procedures are reproducible and linear from 10 to at least 120 micrograms of protein. The correlation coefficient between both procedures was greater than 95%. The color is stable and the same end-point is obtained after destaining. In order to test the usefulness of the procedure in the typing of collagens from parenchymatous tissues, liver biomatrix was prepared from normal and cirrhotic human specimens obtained at autopsy. Over 95% of the collagen originally present in each liver was recovered in the corresponding biomatrix . We also showed that over 80-85% of biomatrix collagen could be solubilized with pepsin. These extracts were neutralized to pH 7.0 to inactivate pepsin and were applied onto the acrylamide gels. After electrophoresis and staining with Sirius red the types of collagen were determined by densitometric analysis. Our findings confirmed the results obtained by others using more complex and time consuming methodologies, mainly that type I and type III collagens are present in the normal liver in equal concentrations and that the ratio of I/III is 1. In all the cirrhotic livers investigated, the ratio of type I/type III collagen was greater than 1 due to an increase in type I collagen.
Azo Compounds↗
Expression of interleukin-1 beta, tumor necrosis factor alpha, interleukins-6, -10 and -4, and metalloproteases by freshly isolated mononuclear cells from early never-treated and non-acute treated rheumatoid arthritis patients.
OBJECTIVE: To determine IL-1 beta, TNF alpha, IL-6, IL-4, IL-10, MMP-1, MMP-3 and MMP-13 expression by freshly isolated peripheral blood (PBMC) and synovial fluid mononuclear cells (SFMC) in early, never-treated (ENT-RA) and non-acute, treated rheumatoid arthritis (NAT-RA) patients. To elucidate whether excessive or inadequate interleukin (IL) and metalloprotease (MMP) expression is influenced by the disease duration. METHODS: Fourteen RA patients, 7 with early RA (< 1 year of evolution) never treated with corticosteroids or disease-modifying antirheumatic drugs, and 7 patients with non-acute RA (> 2 years of evolution) treated with disease-modifying antirheumatic drugs, were studied by ELISA and quantitative and semiquantitative RT-PCR. A group of 14 healthy subjects matched for sex and age was included. RESULTS: No statistically significant difference in the protein or transcript levels for the cytokines of interest was found between the ENT-RA and NAT-RA groups. The cytokine mRNA expression by freshly isolated PBMC and SFMC in both groups was as follows: IL-1 beta > TNF alpha > IL-10 > IL-6, with no mRNA IL-4 expression. In contrast, cytokine serum levels in ENT-RA and NAT-RA patients were detected in inverse order as follows: IL-6 > IL-10, while IL-1 beta, TNF alpha and IL-4 were undetectable. MMP-3 mRNA expression by the PBMC of NAT-RA patients was statistically different to that in ENT-RA patients. Similar levels of mRNA expression of MMP-1, MMP-3 and MMP-13 by the PBMC and SFMC in both RA groups were observed. CONCLUSIONS: A close equilibrium between MMP and pro/anti-inflammatory cytokine production is observed in ENT-RA and NAT-RA patients. This balance is apparently not influenced by the length of the disease. Highly sensitive methods such as quantitative RT-PCR and ELISA, and even studying freshly isolated MC, showed sustained cytokine secretion at the local level (synovial fluid/SFMC) and scarce translation at the peripheral level (serum/PBMC). Expression of MMP mRNA needs to be further evaluated in order to know whether their peripheral expression reflects their local activity in RA patients.
Acute Disease↗