Monocyte-derived macrophages (MoMF) play a pivotal role in the resolution of acetaminophen-induced liver injury (AILI). Timely termination of neutrophil activity and their clearance are essential for ...liver regeneration following injury. Here, we show that infiltrating Ly6Chi monocytes, their macrophage descendants, and neutrophils spatially and temporally overlap in the centrilobular necrotic areas during the necroinflammatory and resolution phases of AILI. At the necroinflammatory phase, inducible ablation of circulating Ly6Chi monocytes resulted in reduced numbers and fractions of reactive oxygen species (ROS)-producing neutrophils. In alignment with this, neutrophils sorted from monocyte-deficient livers exhibited reduced expression of NADPH oxidase 2. Moreover, human CD14+ monocytes stimulated with lipopolysaccharide or hepatocyte apoptotic bodies directly induced ROS production by cocultured neutrophils. RNA-seq-based transcriptome profiling of neutrophils from Ly6Chi monocyte-deficient versus normal livers revealed 449 genes that were differentially expressed with at least twofold change (p ≤ 0.05). In the absence of Ly6Chi monocytes, neutrophils displayed gene expression alterations associated with decreased innate immune activity and increased cell survival. At the early resolution phase, Ly6Chi monocytes differentiated into ephemeral Ly6Clo MoMF and their absence resulted in significant accumulation of late apoptotic neutrophils. Further gene expression analysis revealed the induced expression of a specific repertoire of bridging molecules and receptors involved with apoptotic cell clearance during the transition from Ly6Chi monocytes to MoMF. Collectively, our findings establish a phase-dependent task division between liver-infiltrating Ly6Chi monocytes and their MoMF descendants with the former regulating innate immune functions and cell survival of neutrophils and the later neutrophil clearance.
Background/Aims The aims of the present study were to elucidate whether oxidative stress has a role in Con A-induced hepatitis and to examine if antioxidants may protect against liver damage in this ...model. Methods Hepatitis was induced in Balb/c mice by administration of Con A (18 mg/kg) to the tail vein. Liver enzymes and histology were determined 24 h after Con A injection. Tumor necrosis factor alpha (TNFα) and interleukin-10 (IL-10) levels were assayed 2 h after Con A injection. Hepatic malondialdehyde levels were measured at 1, 3, 8, 12, 18, and 24 h after Con A injection in order to examine the timing of free-radicals formation. Nuclear factor kappa B (NF-κβ) activation was determined by electrophoresis mobility shift assay (EMSA) 1 and 2 h after Con A injection. In separate experiments, mice were pretreated with either dimethylsulfoxide or dimethylthiourea before Con A inoculation. The antioxidant and NF-κβ inhibitor pyrrolidine dithiocarbamate (PDTC) was used as positive control. Results Hepatic malondialdehyde levels increased 12, 18, and 24 h after Con A inoculation but not earlier. Serum levels of liver enzymes and TNFα, hepatic malondialdehyde, and protein carbonyls and the histologic necroinflammatory score were significantly reduced in the antioxidants-treated mice, while IL-10 levels were increased. Dimethylsulfoxide, dimethylthiourea, and PDTC inhibited oxidative stress, but only PDTC inhibited Con A-induced NF-κB activation. Conclusions Reactive oxygen species play a role in immune-mediated Con A-induced hepatitis probably secondary to immune-mediated liver damage. Scavenging of reactive oxygen species by antioxidants prevents hepatitis independently of NF-κB inhibition and may be a new therapeutic target in this experimental model.
Background
Ras proteins are crucial for cell differentiation and proliferation. Targeting Ras with farnesylthiosalicylic acid (FTS), a Ras antagonist, has been suggested as a therapeutic strategy in ...proliferative and inflammatory diseases.
Aims
To examine the role of Ras and the therapeutic potential of FTS in experimental colitis.
Methods
Colitis was induced in 26 mice by adding 2.5% dextran sodium sulfate to their drinking water for 7 days during which 12 study mice were treated with FTS and 14 control mice were given normal saline. Two additional controls included 10 naïve mice treated with FTS and 7 naïve non-treated mice. The animals were followed clinically and sacrificed after 7 days. Their colons were isolated for histological assessment and for measurement of myeloperoxidase activity (MPO), tumor necrosis factor-α(TNF-α), and interleukin-1β(Il-1β) levels. Ras and activated Ras expression was determined by immunoblotting assays. T cell populations in the colon and spleen were analyzed by flow-cytometry.
Results
FTS induced a 2.1-fold reduction in activated Ras levels (
P
< 0.004). FTS-treated mice had lower disease activity scores (3.9 ± 1.7 vs. 7.5 ± 2.3,
P
< 0.001), and lower levels of MPO activity (1.65 ± 0.6 vs. 2.6 ± 0.8 units/g,
P
< 0.007), Il-1β (2.4 ± 3.6 vs. 24.3 ± 17.5 pg/mg,
P
< 0.01) and TNF-α (0.63 ± 0.5 vs. 1.9 ± 1 pg/mg,
P
< 0.04). FTS increased regulatory T cell population in the spleen (1.9 ± 0.4-fold,
P
< 0.04), and decreased effector T cell populations in the colon and spleen by 24 ± 3% (
P
< 0.03) and 27 ± 1% (
P
< 0.02), respectively. FTS had no remarkable side effects.
Conclusions
Ras is involved in the inflammatory processes of induced colitis in mice and its inhibition by FTS ameliorates the severity of the inflammation.
Current strategies for follow up of murine models of liver disease are flawed by inability to continuously monitor disease progression in the tissue level, and necessitate sacrifice of animals for ...tissue sampling.
In this study we aimed at developing a safe repetitive tool for sampling livers in vivo, by utilization of a miniaturized endoscopy system for laparoscopic liver biopsies and for injection of tumor cells into livers.
We report the development of a protocol for murine laparoscopy that allows repeated visualization of murine intra-abdominal organs. The system enables safe and repeated liver biopsies in mice and rats, yielding adequate tissue for histological staining and RNA extraction. In addition, injection of tumor cells into livers facilitates under-vision implantation of hepatic tumors in liver, followed by visualization of tumor growth.
Murine laparoscopy may be employed as a novel imaging modality for continuous assessment and manipulation of chronic liver disease models.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hepatic stellate cells (HSCs) are key participants in liver fibrosis development. 1,25(OH)(2)D(3), the active form of vitamin D, has antiproliferative properties and antifibrotic potential, as well ...as a role in extracellular matrix and matrix metalloproteinase (MMP) regulation in renal and lung fibrosis. Little is known about the role of 1,25(OH)(2)D(3) in liver and its involvement in liver fibrosis. Therefore, we investigated the antiproliferative and antifibrotic effects of 1,25(OH)(2)D(3) in primary cultured HSCs and in a rat model of liver fibrosis induced by thioacetamide (TAA).
Primary HSCs were isolated from rats' livers and treated with 1,25(OH)(2)D(3). Proliferation was examined by bromodeoxyuridine. Vitamin D receptor (VDR) expression and several fibrotic markers were detected by western blot analysis and real-time PCR. Collagen Iα1 and MMP-9 promoter activity were measured by luciferase assay. MMP-9 enzymatic activity was investigated by zymography. VDR silencing was performed by sh-RNA. An in vivo study was performed on TAA-induced liver fibrosis model in rats treated with or without 1,25(OH)(2)D(3). The fibrotic score and collagen deposition were determined by Masson and by Sirius red staining.
While VDR was highly expressed in quiescent HSCs, its expression decreased up to 40% during activation. Addition of 1,25(OH)(2)D(3) to activated HSCs stimulated VDR expression. 1,25(OH)(2)D(3) suppressed HSC proliferation and cyclin D1 expression by ~50% and tissue inhibitor of metalloproteinase 1 (TIMP-1) by 60% and led to a 40% downregulation of collagen Iα1 expression. Moreover, 1,25(OH)(2)D(3) increased MMP-9 activity by 30%. Silencing VDR by sh-RNA demonstrated that suppression of cyclin D1 and collagen Iα1 protein expression was VDR dependent. Treatment with 1,25(OH)(2)D(3) significantly reduced extracellular matrix deposition and lowered the fibrotic score in TAA-induced liver fibrosis.
1,25(OH)(2)D(3) has antiproliferative and antifibrotic effects on liver fibrosis in in vitro and in vivo models and may be considered as having potential therapeutic value.
Background: Studies on cancer patients undergoing chemotherapy have shown inhibitory effects of anticancer drugs on certain
immune activities. Materials and Methods: The in vitro effect of 5-(FU) ...fluorouracil was studied on both lymphocyte proliferation
and natural killer (NK) cell antitumor cytotoxicity following incubation of peripheral mononuclear cells (PBMCs) from healthy
donors with interleukin (IL)-2, phytohemagglutinin A (PHA) and pokeweed mitogen (PWM). Results: Activation of PBMCs with IL-2,
PHA and PWM in the presence of 250 and 2500 μM of 5-FU caused a marked decrease in both the induction of activated natural
killer (ANK) cell cytotoxic activity and DNA synthesis, while 2.5 μM increased DNA synthesis by 195%, 58% and 222% for IL-2,
PHA and PWM, respectively, more than cells cultured without the drug. No effect of 5-FU was noted on mature ANK cells. Conclusion:
5-FU exhibits diverse effects on lymphocyte proliferation and on the generation of ANK antitumor cytotoxic activity.
Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and may be involved in intestinal immune responses. Fungi are important components of the intestinal microflora. The ...potential role of fungi, and in particular their cell wall component β‐glucan, in modulating human intestinal epithelial responses is still unclear. Here we examined whether human IECs are capable of recognizing and responding to β‐glucans, and the potential mechanisms of their activation. We show that human IECs freshly isolated from surgical specimens, and the human IEC lines HT‐29 and SW480, express the β‐glucan receptor Dectin‐1. The β‐glucan‐consisting glycans curdlan and zymosan stimulated IL‐8 and CCL2 secretion by IEC lines. This was significantly inhibited by a Dectin‐1 blockade using its soluble antagonist laminarin. Spleen tyrosine kinase (Syk), a signaling mediator of Dectin‐1 activation, is expressed in human IECs. β‐glucans and Candida albicans induced Syk phosphorylation, and Syk inhibition significantly decreased β‐glucan‐induced chemokine secretion from IECs. Thus, IECs may respond to β‐glucans by the secretion of pro‐inflammatory chemokines in a Dectin‐1‐ and Syk‐dependent pathway, via receptors and a signaling pathway described to date only for myeloid cells. These findings highlight the importance of fungi–IEC interactions in intestinal inflammation.
Quantification of liver steatosis is clinically relevant in various liver diseases but cannot be done by conventional sonography, which only provides a qualitative assessment with significant ...observer variability. The aim of this study was to assess sonography as an objective tool for the quantification of liver steatosis.
Files of 111 patients with chronic liver disease who were referred for sonographically guided liver biopsy were collected. A hepatorenal sonographic index was calculated on the basis of the ratio between the echogenicity of the liver and that of the right kidney cortex using histogram echo intensity. Liver steatosis was graded by histology.
A significant correlation was found between histologic steatosis and the hepatorenal sonographic index (r = 0.82, p < 0.001). The validity of the hepatorenal sonographic index for the diagnosis of fatty liver was compared with liver biopsies with a steatosis level > 5%. The area under the receiver operating characteristic curve was 99.2% (95% CI, 98-100%). The optimal hepatorenal sonographic index cutoff point for the prediction of steatosis > 5% was 1.49, with sensitivity of 100% and specificity of 91%. The optimal hepatorenal sonographic index cutoff point for the prediction of steatosis >/= 25% was 1.86, with sensitivity of 90% and specificity of 90%. The optimal hepatorenal sonographic index cutoff point for the prediction of steatosis >/= 60% was 2.23, with sensitivity of 90% and specificity of 93%.
The hepatorenal sonographic index is a sensitive noninvasive method for steatosis quantification. It can diagnose small amounts of liver fat that would be missed by conventional sonography. It is reproducible and operator independent and can serve as an efficient tool to follow patients with steatosis and evaluate the efficacy of new treatment techniques.
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