Organophosphorus Poisoning: Acute Respiratory Distress Syndrome (ARDS) and Cardiac Failure as Cause of Death in Hospitalized Patients.

Industrial production of food for animals and humans needs increasing amounts of pesticides, especially of organophosphates, which are now easily available worldwide. More than 3 million cases of acute severe poisoning are estimated to occur worldwide every year, and even more cases remain unreported, while 200,000-350,000 incidentally or intentionally poisoned people die every year. Diagnostic and therapeutic procedures in organophosphate poisoning have, however, remained unchanged. In addition to several neurologic symptoms (miosis, fasciculations), hypersecretion of salivary, bronchial, and sweat glands, vomiting, diarrhea, and loss of urine rapidly induce dehydration, hypovolemia, loss of conscience and respiratory distress. Within hours, signs of acidosis due to systemic hypoxia can be observed at first laboratory investigation after hospitalization. While determination of serum-cholinesterase does not have any diagnostic value, it has been established that hypoalbuminemia alone or accompanied by an increase in creatinine, lactate, or C-reactive protein serum levels has negative prognostic value. Increased serum levels of C-reactive protein are a sign of systemic ischemia. Protective mechanical ventilation should be avoided, if possible. In fact, acute respiratory distress syndrome characterized by congestion and increased weight of the lung, accompanied by heart failure, may become the cause of death. As the excess of acetylcholine at the neuronal level can persist for weeks until enough newly, locally synthesized acetylcholinesterase becomes available (the value of oximes in reducing this time is still under debate), after atropine administration, intravenous albumin and fluid infusion should be the first therapeutic interventions to reestablish normal blood volume and normal tissue oxygenation, avoiding death by cardiac arrest.

SARS-CoV-2-Infection (COVID-19): Clinical Course, Viral Acute Respiratory Distress Syndrome (ARDS) and Cause(s) of Death.

SARS-CoV-2-infected symptomatic patients often suffer from high fever and loss of appetite which are responsible for the deficit of fluids and of protein intake. Many patients admitted to the emergency room are, therefore, hypovolemic and hypoproteinemic and often suffer from respiratory distress accompanied by ground glass opacities in the CT scan of the lungs. Ischemic damage in the lung capillaries is responsible for the microscopic hallmark, diffuse alveolar damage (DAD) characterized by hyaline membrane formation, fluid invasion of the alveoli, and progressive arrest of blood flow in the pulmonary vessels. The consequences are progressive congestion, increase in lung weight, and progressive hypoxia (progressive severity of ARDS). Sequestration of blood in the lungs worsens hypovolemia and ischemia in different organs. This is most probably responsible for the recruitment of inflammatory cells into the ischemic peripheral tissues, the release of acute-phase mediators, and for the persistence of elevated serum levels of positive acute-phase markers and of hypoalbuminemia. Autopsy studies have been performed mostly in patients who died in the ICU after SARS-CoV-2 infection because of progressive acute respiratory distress syndrome (ARDS). In the death certification charts, after respiratory insufficiency, hypovolemic heart failure should be mentioned as the main cause of death.

Interleukin-6-Production Is Responsible for Induction of Hepatic Synthesis of Several Chemokines as Acute-Phase Mediators in Two Animal Models: Possible Significance for Interpretation of Laboratory Changes in Severely Ill Patients.

A mild to moderate increase in acute-phase proteins (APPs) and a decrease in serum albumin levels are detected in hospitalized COVID-19 patients. A similar trend is also observed for acute-phase cytokines (APC), mainly IL6, besides chemokines (e.g., CXCL8 and CCL2). However, the source of the chemokines in these patients at different stages of disease remains to be elucidated. We investigated hepatic gene expression of CXC- and CC-chemokines in a model of a localized extrahepatic aseptic abscess and in a model of septicemia produced by the intramuscular injection of turpentine oil (TO) into each hindlimb or lipopolysaccharide (LPS) intraperitoneally (i.p.) in rats and mice (wild-type (WT) and IL6-KO). Together with a striking increase in the serum IL6 level, strong serum CXCL2 and CXCL8 concentrations were detected. Correspondingly, rapid (2 h) upregulation of CXCL1, CXCL2, CXCL5, and CXCL8 was observed in rat liver after intramuscular TO injection. The induction of the gene expression of CXCL1 and CXCL8 was the fastest and strongest. The hepatic CXC-chemokines behaved like positive APPs that depend on IL6 production by activated macrophages recruited to extrahepatic damaged tissue. Chemokine upregulation was greatly reduced in IL6-KO mice. However, IL6 was dispensable in the LPS-APR model, as massive induction of hepatic chemokines studied was measured in IL6-KO mice.

Albumin Infusion in Critically Ill COVID-19 Patients: Hemodilution and Anticoagulation.

Hypercoagulation is one of the major risk factors for ICU treatment, mechanical ventilation, and death in critically ill patients infected with SARS-CoV-2. At the same time, hypoalbuminemia is one risk factor in such patients, independent of age and comorbidities. Especially in patients with severe SARS-CoV-2-infection, albumin infusion may be essential to improve hemodynamics and to reduce the plasma level of the main marker of thromboembolism, namely, the D-dimer plasma level, as suggested by a recent report. Albumin is responsible for 80% of the oncotic pressure in the vessels. This is necessary to keep enough water within the systemic circulatory system and for the maintenance of sufficient blood pressure, as well as for sufficient blood supply for vital organs like the brain, lungs, heart, and kidney. The liver reacts to a decrease in oncotic pressure with an increase in albumin synthesis. This is normally possible through the use of amino acids from the proteins introduced with the nutrients reaching the portal blood. If these are not sufficiently provided with the diet, amino acids are delivered to the liver from muscular proteins by systemic circulation. The liver is also the source of coagulation proteins, such as fibrinogen, fibronectin, and most of the v WF VIII, which are physiological components of the extracellular matrix of the vessel wall. While albumin is the main negative acute-phase protein, fibrinogen, fibronectin, and v WF VIII are positive acute-phase proteins. Acute illnesses cause the activation of defense mechanisms (acute-phase reaction) that may lead to an increase of fibrinolysis and an increase of plasma level of fibrinogen breakdown products, mainly fibrin and D-dimer. The measurement of the plasma level of the D-dimer has been used as a marker for venous thromboembolism, where a fourfold increase of the D-dimer plasma level was used as a negative prognostic marker in critically ill SARS-CoV-2 hospitalized patients. Increased fibrinolysis can take place in ischemic peripheral sites, where the mentioned coagulation proteins can become part of the provisional clot (e.g., in the lungs). Although critically ill SARS-CoV-2-infected patients are considered septic shock patients, albumin infusions have not been considered for hemodynamic resuscitation and as anticoagulants. The role of coagulation factors as provisional components of the extracellular matrix in case of generalized peripheral ischemia due to hypoalbuminemia and hypovolemia is discussed in this review.

PECAM-1 modulates liver damage induced by synergistic effects of TNF-α and irradiation.

The mechanisms of radiation-induced liver damage are poorly understood. We investigated if tumour necrosis factor (TNF)-α acts synergistically with irradiation, and how its activity is influenced by platelet endothelial cell adhesion molecule-1 (PECAM-1). We studied murine models of selective single-dose (25 Gy) liver irradiation with and without TNF-α application (2 µg/mouse; i.p.). In serum of wild-type (wt)-mice, irradiation induced a mild increase in hepatic damage marker aspartate aminotransferase (AST) in comparison to sham-irradiated controls. AST levels further increased in mice treated with both irradiation and TNF-α. Accordingly, elevated numbers of leucocytes and increased expression of the macrophage marker CD68 were observed in the liver of these mice. In parallel to hepatic damage, a consecutive decrease in expression of hepatic PECAM-1 was found in mice that received radiation or TNF-α treatment alone. The combination of radiation and TNF-α induced an additional significant decline of PECAM-1. Furthermore, increased expression of hepatic lipocalin-2 (LCN-2), a hepatoprotective protein, was detected at mRNA and protein levels after irradiation or TNF-α treatment alone and the combination of both. Signal transducer and activator of transcription-3 (STAT-3) seems to be involved in the signalling cascade. To study the involvement of PECAM-1 in hepatic damage more deeply, the liver of both wt- and PECAM-1-knock-out-mice were selectively irradiated (25 Gy). Thereby, ko-mice showed higher liver damage as revealed by elevated AST levels, but also increased hepatoprotective LCN-2 expression. Our studies show that TNF-α has a pivotal role in radiation-induced hepatic damage. It acts in concert with irradiation and its activity is modulated by PECAM-1, which mediates pro- and anti-inflammatory signalling.

Modulation of Chemokine- and Adhesion-Molecule Gene Expression and Recruitment of Neutrophil Granulocytes in Rat and Mouse Liver after a Single Gadolinium Chloride or Zymosan Treatment.

Kupffer cells are professional phagocytes of the liver clearing bacteria from portal blood. Their clearance capacity, however, can be overwhelmed, transforming them into critical mediators of hepatic-injury. We investigated the consequences of selective Kupffer cell-overload by intraperitoneally administering pyrogen-free gadolinium chloride (GdCl3) or Zymosan into rats and into endotoxin-resistant mice (C3H/HeJ). The number of myeloperoxidase-positive (MPO⁺) cells increased at 3 h mainly around the portal vessel after both GdCl3 and Zymosan treatment. Simultaneously, GdCl3 administration reduced detectability of ED-1⁺ (but not ED-2) cells near the portal vessel. Serum chemokine (C-X-C motif) ligand 1 (CXCL-1), CXCL-2 and chemokine (C-C motif) ligand 2 (CCL-2) showed a peak at 3 h after both treatment regimens although at a higher extent after Zymosan administration. Accordingly, CXCL-1, CXCL-5 and CCL-2 gene expression in the liver was up-regulated after GdCl3 treatment at 3 h. After Zymosan administration a significant up-regulation of CXCL-1, CXCL-2, CXCL-10, CCL-2, CCL-3 and CCL-20 gene expression in liver at 3 h was observed. After Zymosan administration intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) gene expression was up-regulated in rat liver tissue. In C3H/HeJ mice both treatment regimens up-regulated CCL-2 and ICAM-1 gene expression after 3 h and down-regulated platelet endothelial cell adhesion molecule 1 (PECAM-1) gene expression. In conclusion, phagocytosis overload of Kupffer cells causes induction of several CXC, CC-chemokines, upregulation of "positive" adhesion molecule gene expression, down-regulation of the "negative" adhesion molecule PECAM-1 and a recruitment of neutrophil granulocytes in the portal area of the liver of treated rats and mice mainly in close contact to the liver macrophages.

Case report: 8 years after liver transplantation: de novo hepatocellular carcinoma 8 months after HCV clearance through IFN-free antiviral therapy.

BACKGROUND: After orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC), recurrent HCC mostly develops within 2 years. All cases of de novo HCC described so far occurred later than 2 years after OLT. Prevention of post-transplantation HCC has usually been tried to achieve by curing or controlling recurrent liver disease. This has been rationale for treatment with interferon (IFN)/ribavirin of HCV-recurrence in patients after OLT, transplanted for advanced HCV-induced liver disease and/or HCC. The availability of new and more efficient drugs has improved chances also for previously difficult-to-treat HCV-positive patients. CASE PRESENTATION: A 75 year-old male patient who had undergone OLT for decompensated HCV-cirrhosis in 2009, and bilio-digestive surgery in 2011 under tracrolimus (0.5 mg/day) and prednisone (5 mg/day) immunosuppressive therapy, started to receive antiviral treatment for recurrent HCV-infection of graft with 200 mg/day ribavirin in combination with ledipasvir and sofosbuvir by the end of October 2015. Because of multiple side effects (anemia, asthenia, infections, and reduction of kidney functions - palliated by treatment with erythropoietin), treatment was stopped after 16 weeks. At the third control, a minimal increase in alpha-fetoprotein (AFP) serum level to 10 µg/L was measured 8 months after therapy, whereas both liver sonography and serum transaminases were normal. The patient's general condition; however, remained poor, and a magnetic resonance imaging (MRI) of abdomen was performed 2 months later. A nodule of 3 cm in diameter with a pseudocapsule was found centrally in the liver. The patient had to be hospitalized for recurrent infections of the lung, overt ascites and peritonitis. Rapid tumor growth (10 cm) was detected during last stay in hospital (April 2017), concomitant with a rise of AFP-serum levels to 91 µg/L. The family decided to take the patient home, and best supportive care was provided by a general practitioner, local nurses and the patient's dedicated wife until his death. CONCLUSION: Before treating OLT patients with HCV graft reinfection one should not only consider possible advantages of newly effective antiviral-therapies, but also life expectancy and possible side effects (difficult to manage at an outpatient service basis), including severe disadvantages such as the development of HCC.

Reabsorption of iron into acutely damaged rat liver: A role for ferritins.

AIM: To studied iron metabolism in liver, spleen, and serum after acute liver-damage, in relation to surrogate markers for liver-damage and repair. METHODS: Rats received intraperitoneal injection of the hepatotoxin thioacetamide (TAA), and were sacrificed regularly between 1 and 96 h thereafter. Serum levels of transaminases and iron were measured using conventional laboratory assays. Liver tissue was used for conventional histology, immunohistology, and iron staining. The expression of acute-phase cytokines, ferritin light chain (FTL), and ferritin heavy chain (FTH) was investigated in the liver by qRT-PCR. Western blotting was used to investigate FTL and FTH in liver tissue and serum. Liver and spleen tissue was also used to determine iron concentrations. RESULTS: After a short initial decrease, iron serum concentrations increased in parallel with serum transaminase (aspartate aminotransferase and alanine aminotransferase) levels, which reached a maximum at 48 h, and decreased thereafter. Similarly, after 48 h a significant increase in FTL, and after 72h in FTH was detected in serum. While earliest morphological signs of inflammation in liver were visible after 6 h, increased expression of the two acute-phase cytokines IFN-γ (1h) and IL-1ß (3h) was detectable earlier, with maximum values after 12-24 h. Iron concentrations in liver tissue increased steadily between 1 h and 48 h, and remained high at 96 h. In contrast, spleen iron concentrations remained unchanged until 48 h, and increased mildly thereafter (96 h). Although tissue iron staining was negative, hepatic FTL and FTH protein levels were strongly elevated. Our results reveal effects on hepatic iron concentrations after direct liver injury by TAA. The increase of liver iron concentrations may be due to the uptake of a significant proportion of the metal by healthy hepatocytes, and only to a minor extent by macrophages, as spleen iron concentrations do not increase in parallel. The temporary increase of iron, FTH and transaminases in serum is obviously due to their release by damaged hepatocytes. CONCLUSION: Increased liver iron levels may be the consequence of hepatocyte damage. Iron released into serum by damaged hepatocytes is obviously transported back and stored via ferritins.

Insulin Production and Resistance in Different Models of Diet-Induced Obesity and Metabolic Syndrome.

The role of the liver and the endocrine pancreas in development of hyperinsulinemia in different types of obesity remains unclear. Sedentary rats (160 g) were fed a low-fat-diet (LFD, chow 13% kcal fat), high-fat-diet (HFD, 35% fat), or HFD+ 30% ethanol+ 30% fructose (HF-EFr, 22% fat). Overnight-fasted rats were culled after one, four or eight weeks. Pancreatic and hepatic mRNAs were isolated for subsequent RT-PCR analysis. After eight weeks, body weights increased three-fold in the LFD group, 2.8-fold in the HFD group, and 2.4-fold in the HF-EFr (p < 0.01). HF-EFr-fed rats had the greatest liver weights and consumed less food during Weeks 4-8 (p < 0.05). Hepatic-triglyceride content increased progressively in all groups. At Week 8, HOMA-IR values, fasting serum glucose, C-peptide, and triglycerides levels were significantly increased in LFD-fed rats compared to that at earlier time points. The greatest plasma levels of glucose, triglycerides and leptin were observed in the HF-EFr at Week 8. Gene expression of pancreatic-insulin was significantly greater in the HFD and HF-EFr groups versus the LFD. Nevertheless, insulin: C-peptide ratios and HOMA-IR values were substantially higher in HF-EFr. Hepatic gene-expression of insulin-receptor-substrate-1/2 was downregulated in the HF-EFr. The expression of phospho-ERK-1/2 and inflammatory-mediators were greatest in the HF-EFr-fed rats. Chronic intake of both LFD and HFD induced obesity, MetS, and intrahepatic-fat accumulation. The hyperinsulinemia is the strongest in rats with the lowest body weights, but having the highest liver weights. This accompanies the strongest increase of pancreatic insulin production and the maximal decrease of hepatic insulin signaling, which is possibly secondary to hepatic fat deposition, inflammation and other factors.

Response of Hepatic Stellate Cells to TGFB1 Differs from the Response of Myofibroblasts. Decorin Protects against the Action of Growth Factor.

Regardless to the exact nature of damage, hepatic stellate cells (HSCs) and other non-parenchymal liver cells transform to activated myofibroblasts, synthesizing the accumulating extracellular matrix (ECM) proteins, and transforming growth factor-ß1 (TGF-ß1) plays a crucial role in this process. Later it was discovered that decorin, member of the small leucin rich proteoglycan family is able to inhibit this action of TGF-ß1. The aim of our present study was to clarify whether HSCs and activated myofibroblasts of portal region exert identical or different response to TGF-ß1 exposure, and the inhibitory action of decorin against the growth factor is a generalized phenomenon on myofibroblast of different origin? To this end we measured mRNA expression and production of major collagen components (collagen type I, III and IV) of the liver after stimulation and co-stimulation with TGF-ß1 and decorin in primary cell cultures of HSCs and myofibroblasts (MFs). Production of matrix proteins, decorin and members of the TGF-ß1 signaling pathways were assessed on Western blots. Messenger RNA expression of collagens and TIEG was quantified by real-time RT-PCR. HSCs and MFs responded differently to TGF-ß1 exposure. In contrast to HSCs in which TGF-ß1 stimulated the synthesis of collagen type I, type III, and type IV, only the increase of collagen type IV was detected in portal MFs. However, in a combined treatment, decorin seemed to interfere with TGF-ß1 and its stimulatory effect was abolished. The different mode of TGF-ß1 action is mirrored by the different activation of signaling pathways in activated HSCs and portal fibroblasts. In HSCs the activation of pSMAD2 whereas in myofibroblasts the activation of MAPK pathway was detected. The inhibitory effect of decorin was neither related to the Smad-dependent nor to the Smad-independent signaling pathways.

Adjuvant Imatinib for High-Risk GI Stromal Tumor: Analysis of a Randomized Trial.

PURPOSE: Three years of adjuvant imatinib therapy are recommended for patients with GI stromal tumor (GIST) with high-risk features, according to survival findings in the Scandinavian Sarcoma Group XVIII/AIO (Arbeitsgemeinschaft Internistische Onkologie) trial. To investigate whether the survival benefits have persisted, we performed the second planned analysis of the trial. PATIENTS AND METHODS: Eligible patients had macroscopically completely excised, KIT-positive GIST with a high risk of recurrence, as determined by using the modified National Institutes of Health criteria. After surgery, the patients were randomly assigned to receive imatinib for either 1 or 3 years. The primary objective was recurrence-free survival (RFS), and the secondary objectives included survival. RESULTS: A total of 400 patients were entered onto this open-label study between February 4, 2004, and September 29, 2008. During a median follow-up of 90 months, 171 recurrences and 69 deaths were detected. Patients assigned to the 3-year group had longer RFS than those assigned to the 1- year group; 5-year RFS was 71.1% versus 52.3%, respectively (hazard ratio [HR], 0.60; 95% CI 0.44 to 0.81; P < .001), and survival was 91.9% versus 85.3% (HR, 0.60; 95% CI, 0.37 to 0.97; P = .036). Patients in the 3-year group survived longer in the subset with centrally confirmed GIST and without macroscopic metastases at study entry (93.4% v 86.8%; HR, 0.53; 95% CI, 0.30 to 0.93; P = .024). Similar numbers of cardiac events and second cancers were recorded in the groups. CONCLUSION: Three years of adjuvant imatinib therapy results in longer survival than 1 year of imatinib. High 5-year survival rates are achievable in patient populations with high-risk GIST.

Upregulation of hepatic melanocortin 4 receptor during rat liver regeneration.

BACKGROUND: Melanocortin 4 receptor (MC4R) is predominantly recognized to mediate energy metabolism and anti-inflammation through the central nervous system. However, the expression of MC4R has recently been identified in rat liver and was shown to be upregulated during acute phase response. This study aims to investigate potential roles of MC4R in liver regeneration. MATERIALS AND METHODS: Rat partial hepatectomy (PH) was performed, and MC4R expression was analyzed at different time points after resection. Sham-operated animals (SH) served as controls. In vitro primary hepatocytes (HCs) were isolated from normal rat liver and stimulated with α-melanocyte-stimulating hormone (MC4R agonist). Real-time polymerase chain reaction, Western blot, and immunofluorescence staining were applied to detect gene expression. RESULTS: Up to 8 h after PH, hepatic messenger RNA of proinflammatory cytokines interleukin 6 and tumor necrosis factor α reached peak values. Between 8 and 72 h after PH, rat liver regeneration was extremely active as assessed by the regeneration indices labeled by Ki-67. Immunofluorescence staining indicated that MC4R was mostly expressed in hepatocyte nuclear factor 4(+) cells (HCs) and upregulated during rat liver regeneration. Concurrently, the expression of hepatic MC4R protein was significantly higher in PH than in SH animals, and phosphorylated extracellular signal-regulated kinase 1/2 was remarkably increased in PH compared with SH animals (P < 0.05, respectively). In vitro experiments showed that the expression of proliferating cell nuclear antigen was significantly higher in HCs treated with α-melanocyte-stimulating hormone than in control HCs, which was correlated to the increase of phosphorylated extracellular signal-regulated kinase 1/2 and reduction of phosphorylated signal transducer and activator of transcription 3 (P < 0.05, respectively). CONCLUSIONS: MC4R is predominantly expressed in HCs and upregulated during rat liver regeneration. In vitro stimulation of HC MC4R is associated with a modulation of extracellular signal-regulated kinase and signal transducer and activator of transcription 3 pathways regulating liver regeneration.

Hepatic fat accumulation and regulation of FAT/CD36: an effect of hepatic irradiation.

Irradiation is known to induce inflammation and affect fat metabolic pathways. The current study investigates hepatic fat accumulation and fatty acid transportation in a rat model of single dose liver irradiation (25-Gy). Rat livers were selectively irradiated in-vivo (25-Gy), sham-irradiated rats served as controls. Hepatic lipids were studied by colorimetric assays in liver and serum. Intracellular lipids, protein and mRNA were studied by Nile red staining, immunohistology, Western Blot analysis and RT-PCR in liver, respectively. Changes in FAT/CD36 expression were studied in-vitro in a human monocyte cell line U937 after irradiation in presence or absence of infliximab (IFX). Nile Red staining of liver cryosections showed a quick (12-48 h) increase in fat droplets. Accordingly, hepatic triglycerides (TG) and free fatty acids (FFA) were elevated. An early increase (3-6 h) in the serum level of HDL-C, TG and cholesterol was measured after single dose irradiation followed by a decrease thereafter. Furthermore, expression of the fat transporter protein FAT/CD36 was increased, immunohistochemistry revealed basolateral and cytoplasmic expression in hepatocytes. Moreover, apolipoprotein-B100, -C3 and enzymes (acetyl-CoA carboxylase, lipoprotein-lipase, carnitine-palmitoyltransferase, malonyl-CoA-decarboxylase) involved in fat metabolism were induced at 12-24 h. Early activation of the NFkß pathway (IκBα) by TNF-α was seen, followed by a significant elevation of serum markers for liver damage (AST and GLDH). TNF-α blockage by anti-TNF-α in cell culture (U937) prevented the increase of FAT/CD36 caused by irradiation. Selective liver irradiation is a model for rapid induction of steatosis hepatis and fat accumulation could be triggered by irradiation-induced inflammatory mediators (e.g. TNF-α).

Combination of alcohol and fructose exacerbates metabolic imbalance in terms of hepatic damage, dyslipidemia, and insulin resistance in rats.

Although both alcohol and fructose are particularly steatogenic, their long-term effect in the development of a metabolic syndrome has not been studied in vivo. Consumption of fructose generally leads to obesity, whereas ethanol can induce liver damage in the absence of overweight. Here, Sprague-Dawley rats were fed ad libitum for 28 days on five diets: chow (control), liquid Lieber-DeCarli (LDC) diet, LDC +30%J of ethanol (L-Et) or fructose (L-Fr), and LDC combined with 30%J ethanol and 30%J fructose (L-EF). Body weight (BW) and liver weight (LW) were measured. Blood and liver samples were harvested and subjected to biochemical tests, histopathological examinations, and RT-PCR. Alcohol-containing diets substantially reduced the food intake and BW (≤3rd week), whereas fructose-fed animals had higher LW than controls (P<0.05). Additionally, leukocytes, plasma AST and leptin levels were the highest in the fructose-administered rats. Compared to the chow and LDC diets, the L-EF diet significantly elevated blood glucose, insulin, and total-cholesterol levels (also vs. the L-Et group). The albumin and Quick-test levels were the lowest, whereas ALT activity was the highest in the L-EF group. Moreover, the L-EF diet aggravated plasma triglyceride and reduced HDL-cholesterol levels more than 2.7-fold compared to the sum of the effects of the L-Et and L-Fr diets. The decreased hepatic insulin clearance in the L-EF group vs. control and LDC groups was reflected by a significantly decreased C-peptide:insulin ratio. All diets except the control caused hepatosteatosis, as evidenced by Nile red and H&E staining. Hepatic transcription of insulin receptor substrate-1/2 was mainly suppressed by the L-Fr and L-EF diets. The L-EF diet did not enhance the mitochondrial ß-oxidation of fatty acids (Cpt1α and Ppar-α expressions) compared to the L-Et or L-Fr diet. Together, our data provide evidence for the coaction of ethanol and fructose with a high-fat-diet on dyslipidemia and insulin resistance-accompanied liver damage.

Immune cells in primary and metastatic gastrointestinal stromal tumors (GIST).

We have previously described immune cells in untreated primary gastrointestinal stromal tumors (GIST). Here we compare immune cells in metastatic and primary GIST, and describe their chemoattractants. For this purpose, tissue microarrays from 196 patients, 188 primary and 51 metastasized GIST were constructed for paraffin staining. Quantitative analysis was performed for cells of macrophage lineage (Ki-M1P, CD68), T-cells (CD3, CD56) and B-cells (CD20). Chemokine gene-expression was evaluated by real-time RT-PCR. Immuno-localisation was verified by immunofluorescence. Ki-M1P+ cells were the predominant immune cells in both primary and metastatic GIST (2 8.8% ± 7.1, vs. 26.7% ± 6.3). CD68+ macrophages were significantly fewer, with no significant difference between primary GIST (3.6% ± 2.1) and metastases (4.6% ± 1.5). CD3+ T-cells were the most dominant lymphocytes with a significant increase in metastases (7.3% ± 2.3 vs. 2.2% ± 1.8 in primary GIST, P < 0.01). The percentage of CD56+ NK-cells was 1.1% ± 0.9 in the primary, and 2.4 ± 0.7 (P < 0.05) in the metastases. The number of CD20+ B-cells was generally low with 0.6% ± 0.7 in the primary and 1.8% ± 0.3 (P < 0.05) in the metastases. Analysis of the metastases showed significantly more Ki-M1P+ cells in peritoneal metastases (31.8% ± 7.4 vs. 18.2% ± 3.7, P < 0.01), whilst CD3+ T-cells were more common in liver metastases (11.7% ± 1.8 vs. 4.4% ± 2.6, P < 0.01). The highest transcript expression was seen for monocyte chemotactic protein 1 (MCP1/CCL2), macrophage inflammatory protein 1α (MIP-1α/CCL3) and the pro-angiogenic growth-related oncoprotein 1 (Gro-α/CXCL-1). Whilst the ligands were predominantly expressed in tumor cells, their receptors were mostly present in immune cells. This locally specific microenvironment might influence neoplastic progression of GIST at the different metastatic sites.

Risk factors for gastrointestinal stromal tumor recurrence in patients treated with adjuvant imatinib.

BACKGROUND: Little is known about the factors that predict for gastrointestinal stromal tumor (GIST) recurrence in patients treated with adjuvant imatinib. METHODS: Risk factors for GIST recurrence were identified, and 2 risk stratification scores were developed using the database of the Scandinavian Sarcoma Group (SSG) XVIII trial, where 358 patients with high-risk GIST with no overt metastases were randomly assigned to adjuvant imatinib 400 mg/day either for 12 or 36 months after surgery. The findings were validated in the imatinib arm of the American College of Surgeons Oncology Group Z9001 trial, where 359 patients with GIST were randomized to receive imatinib and 354 were to receive placebo for 12 months. RESULTS: Five factors (high tumor mitotic count, nongastric location, large size, rupture, and adjuvant imatinib for 12 months) were independently associated with unfavorable recurrence-free survival (RFS) in a multivariable analysis in the SSGXVIII cohort. A risk score based on these 5 factors had a concordance index with GIST recurrence of 78.9%. When a simpler score consisting of the 2 strongest predictive factors (mitotic count and tumor site) was devised, the groups with the lowest, intermediate high, and the highest risk had 5-year RFS of 76.7%, 47.5%, and 8.4%, respectively. Both scores were strongly associated with RFS in the validation cohort (P < .001 for each comparison). CONCLUSIONS: The scores generated were effective in stratifying the risk of GIST recurrence in patient populations treated with adjuvant imatinib. Patients with nongastric GIST with a high mitotic count are at a particularly high risk for recurrence.

Diet high in fructose leads to an overexpression of lipocalin-2 in rat fatty liver.

AIM: To explore lipocalin-2 (LCN-2) expression and its possible role and mechanism(s) of production in rat models of diet-inducible fatty liver. METHODS: Fatty liver was triggered in male Sprague-Dawley rats fed either with liquid Lieber-DeCarli (LDC) or LDC + 70% cal fructose (L-HFr) diet for 4 or 8 wk. Chow-nourished animals served as controls. Hepatic expression of LCN-2 and other metabolic and inflammatory mediators was assessed by quantitative reverse transcription polymerase chain reaction and Western blotting. Serum LCN-2, fasting leptin, and lipid profile were evaluated via Enzyme-Linked Immunosorbent Assay, Radioimmunoassay, and colorimetric assays, respectively. The localization of LCN-2 in the liver was detected by using immunofluorescence staining. Furthermore, HE stain was used to evaluate hepatic fat degeneration and inflammation. RESULTS: Both LDC-fed and L-HFr-fed rat histologically featured fatty liver. In the liver, mRNA transcriptions of Mcp-1, a2-m, Il-8 and Glut5 were increased in the L-HFr group at both time points (P < 0.001), while the transcription of Tlr4, Inos, and Tnf-α was significantly up-regulated at week 4. Interestingly, hepatic Lcn-2 expression was 90-fold at week 4 and 507-fold at week 8 higher in L-HFr-subjected rats vs control (P < 0.001). In contrast to HDL-cholesterol, systemic levels of LCN-2, fasting leptin and triglycerides were elevated in the L-HFr regimen (P < 0.001). Moreover, protein expression of hepatic LCN-2, CD14, phospho-MAPK, caspase-9, cytochrome c and 4-hydroxynonenal was increased in the L-HFr group. Conversely, the hepatic expression of PGC-1α (a mitochondrial-biogenic protein) was reduced in the L-HFr category at week 8. The localization of LCN-2 in the liver was predominantly restricted to MPO⁺ granulocytes. CONCLUSION: Fructose diet up-regulates hepatic LCN-2 expression, which correlates with the increased indicators of oxidative stress and mitochondrial dysfunction. The LCN-2 may be involved in liver protection.

Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines: role of cytokines.

AIM: To study KRAS/BRAF mutations in colorectal-cancer (CRC) that influences the efficacy of treatment. To develop strategies for overcoming combination of treatment. METHODS: Five colonic cell-lines were investigated: DLD-1 with KRAS (G13D) mutation, HT 29 and Colo 205 with BRAF (V600E) mutation as well as the wild type (Wt) cell-lines Caco2 and Colo-320. DLD-1 (KRAS), HT-29 (BRAF) and Caco2 (Wt) cell lines were treated with cytokines (TNFα 50 ng, IL-1ß 1 ng and IFNγ 50 ng) and harvested at different time points (1-24 h). KRAS inhibition was performed by the siRNA-approach. Two colorectal cancer cells DLD-1 and Caco2 were used for KRAS inhibition. About 70% confluency were confirmed before transfection with small interferring RNA (siRNA) oligonucleotides. All the synthetic siRNA sequences were designed in our laboratory. Total RNA and protein was isolated from the cells for RT-PCR and Western blotting. Densitometry of the Western blotting was analyzed with the Image J software (NIH). Results are shown as mean ± SD. RESULTS: RT-PCR analysis in non-stimulated cells showed a low basal expression of TNFα and IL-1ß in the DLD-1 KRAS-mutated cell-line, compared to Caco2 wild type. No detection was found for IL-6 and IFNγ in any of the studied cell lines. In contrast, pro-angiogenic chemokines (CXCL1, CXCL8) showed a high constitutive expression in the mutated cell-lines DLD-1 (KRAS), HT-29 and Colo205 (BRAF), compared to wild type (Caco2). The anti-angiogenic chemokine (CXCL10) showed a high basal expression in wild-type, compared to mutated cell-lines. KRAS down-regulation by siRNA showed a significant decrease in CXCL1 and CXCL10 gene expression in the DLD-1 (KRAS) cell-line in comparison to wild type (Caco2) at 72 h after KRAS silencing. In contrast, the specific KRAS inhibition resulted in an up-regulation of CXCL1 and CXCL10. The results of our study show a higher expression of pro-angiogenic chemokines at basal level in mutated cell-lines, which was further increased by cytokine treatment. CONCLUSION: To summarize, basal chemokine gene expression for pro-angiogenic chemokines was high in mutated as compared to wild type cell-lines. This reflects the likely existence of a different microenvironment in tumours consistent of wild type or mutated cells. This may help to rationalize the choice of molecular targets for suitable therapeutic investigation in clinical studies.

Plasma triglyceride levels may modulate hepatitis C viral replication.

BACKGROUND: Plasma and hepatic lipid abnormalities are frequent in hepatitis C infected individuals. METHODS: Plasma lipid and medical records profiles were prospectively obtained in 130 consecutive individuals seen by a single hepatologist in a university liver disease clinic. The relationships between viral load, genotype, plasma lipid fractions, HDL, LDL particle number and particle size were examined. RESULTS: Of 130 individuals studied, 74 had hepatitis C while 15 had NAFLD/NASH and 30 had alcohol related liver disease. The LDL particle number and LDL-C levels did not differ between those with and without hepatitis C although the number of small LDL particles was greater in those with hepatitis C infection. The HDL-C and total cholesterol levels were greater in those without hepatitis C than those with hepatitis C (P = 0.009). In contrast, the serum triglyceride level was greater in the hepatitis C viral group (P = 0.013). Importantly, the hepatitis C viral load regardless of the genotype correlated directly with the triglyceride and VLDL levels with r values of 0.73 and 0.84, respectively. CONCLUSIONS: There are: (1) important differences in lipid classes, number and the size of lipid particles exist between hepatitis C virus infected and noninfected liver disease groups, (2) the serum total triglyceride and the LDL levels correlate significantly with the hepatitis C viral load and, (3) Serum triglyceride level may play an important role in viral replication. These data further suggest that therapies directed at lowering plasma triglyceride levels may enhance the efficacy of current antiviral treatment regimens.