Transgastric Jejunostomy (PEG-J) for Continuous Infusion of Levodopa-Carbidopa Intestinal Gel: An Approach for Parkinson's Disease Treatment.

BACKGROUND Parkinson's disease (PD) is a neurodegenerative disorder that often requires long-term management of motor symptoms. Continuous infusion of levodopa-carbidopa intestinal gel (LCIG) has shown promising results in alleviating motor fluctuations and improving quality of life. This study aimed to evaluate the efficacy and safety of transgastric jejunostomy (PEG-J) as a delivery method for LCIG in a cohort of 43 PD patients. MATERIAL AND METHODS Forty-three PD patients who were candidates for LCIG therapy underwent transgastric jejunostomy to facilitate continuous infusion of LCIG. The primary outcomes assessed were motor symptom improvement, reduction in motor fluctuations, and medication-related adverse events. Secondary outcomes included changes in quality of life, dyskinesia severity, and healthcare resource utilization. RESULTS The results of this study demonstrated significant improvements in motor symptoms, reduction in motor fluctuations, and enhanced quality of life following PEG-J for LCIG infusion. The treatment was generally well-tolerated, with a low incidence of procedure-related complications. Notably, the use of PEG-J allowed for precise and continuous delivery of LCIG, minimizing variations in drug absorption and ensuring consistent therapeutic levels. CONCLUSIONS Transgastric jejunostomy (PEG-J) offers an effective approach for the continuous infusion of LCIG in Parkinson's disease treatment. This method provides a stable and reliable delivery system, leading to improved symptom control and enhanced quality of life for PD patients.

Can a Solitary Juvenile Polyp Be Regarded as a Nonmalignant Polyp?

Juvenile polyps (JPs) are common, developing mostly as solitary, hamartomatous lesions in the colorectum, and principally affect pediatric patients. Solitary JPs are recognized as benign, with a negligible malignant transformation rate. Primary signet ring cell carcinoma is a rare type of colorectal cancer (0.1%-2.6%) that presents mostly at an advanced stage in younger patients and affects the right-sided colon, with extensive lymphatic invasion and peritoneal dissemination, resulting in a poorer prognosis compared with conventional colorectal cancer. We report a rare case of signet ring cell carcinoma in a solitary JP treated with endoscopic mucosal resection.

Does Endoscopic Retrograde Cholangiopancreatography Carry Higher Risk for Patients 90 Years and Older? A Single-Institution Retrospective Study.

BACKGROUND Endoscopic retrograde cholangiography (ERCP) for patients aged ≥90 years is often required. The safety of ERCP for super-elderly patients is a major concern for gastrointestinal endoscopists. We retrospectively examined the safety of ERCP for super-elderly patients by comparison with patients in their 70s. MATERIAL AND METHODS We reviewed 66 patients aged ≥90 years (Group A) and 43 patients in their 70s (Group B) who underwent ERCP in our institution from January 2012 to October 2019. Data were collected on patients' backgrounds, corresponding procedures, and clinical outcomes, including adverse events. RESULTS Patients in Group A (mean age: 92.3±2.1 years) had significantly poorer performance status (median: 3 vs. 0; P<0.001) and American Society of Anesthesiologists classification (median: III vs. II; P<0.001) when compared to Group B (mean age: 75.1±2.7 years). Underlying cardiovascular, cerebrovascular, renal, and orthopedic comorbidity occurrence was significantly higher in Group A than in Group B (87.88% vs. 67.44%; P=0.0094). Group A comprised more patients with benign disease than Group B (90.91% vs. 76.74%; P=0.040). Group B comprised more patients with malignant disease (31.82% vs. 53.54%; P=0.041). Emergency ERCP was higher in Group A than in Group B (71.70% vs. 29.73%; P<0.0001). No significant between-group differences in adverse events (15.15% vs. 11.63%; P=0.602) and mortality rate (1.52% vs. 2.33%; P=0.758) were noted. CONCLUSIONS Indications for ERCP should not be determined simply based on the super-elderly age of patients. ERCP may not necessarily carry higher risks if endoscopists practice maximal caution against gastrointestinal perforation.

Gadoxetic Acid-Enhanced MR Imaging Predicts Simeprevir-Induced Hyperbilirubinemia During Hepatitis C Virus Treatment: A Pilot Study.

Simeprevir is a substrate for organic anion-transporting polypeptides (OATPs) that transport bilirubin. Hyperbilirubinemia is an adverse event reported during treatment of chronic hepatitis C patients with simeprevir plus pegylated interferon and ribavirin. Because gadoxetic acid is also a substrate of OATPs, pretreatment gadoxetic acid-enhanced magnetic resonance imaging (MRI) may predict hyperbilirubinemia during treatment. This prospective study therefore evaluated 11 consecutive patients with chronic hepatitis C who underwent gadoxetic acid-enhanced MRI prior to treatment with simeprevir plus pegylated interferon and ribavirin for 12 weeks, followed by pegylated interferon and ribavirin for an additional 12 weeks. Their contrast enhancement index (CEI), an index of liver parenchymal enhancement during the hepatobiliary phase, was assessed before treatment. Plasma trough concentrations (Ctrough ) of simeprevir were determined 7 days after its administration, and serum bilirubin concentrations were measured throughout the course of treatment. Six patients (55%) developed hyperbilirubinemia (≥1.6 mg/dL) during treatment. Ctrough was significantly higher in patients with than without hyperbilirubinemia (P = .009), with a strong inverse relationship between CEI and Ctrough (r = -0.911, P < .001). CEI was significantly lower in patients with than without hyperbilirubinemia (P = .009), but there were no significant differences between the 2 groups in pretreatment serum albumin concentrations and FIB-4 index, an index of liver fibrosis. Hepatic enhancement with gadoxetic acid was related to Ctrough of simeprevir. Gadoxetic acid-enhanced magnetic resonance imaging may predict the development of hyperbilirubinemia during treatment of hepatitis C patients with simeprevir plus pegylated interferon and ribavirin.

No contribution of the ABCB11 p.444A polymorphism in Japanese patients with drug-induced cholestasis.

European studies have revealed that the ABCB11 c.1331T>C (V444A) polymorphism (rs2287622) C-allele frequency is higher among patients with drug-induced cholestasis. Given the low incidence of this disease, however, this association has not been sufficiently elucidated. We aimed to investigate the significance of this polymorphism in Japanese patients. We determined ABCB11 V444A polymorphism frequencies and HLA genotypes in two independent drug-induced cholestasis cohorts. Expression and taurocholate transport activity of proteins from 444A variants were analyzed using Madin-Darby canine kidney II cells. In cohort 1 (n = 40), the V444A polymorphism C-allele frequency (66%) was lower than that in controls (n = 190, 78%), but this difference was not significant (P = 0.09). In cohort 2 (n = 119), comprising patients with cholestatic (n = 19), hepatocellular (n = 74), and mixed (n = 26) liver injuries, the C-allele frequency was lower among patients with cholestatic liver injury (68%) than among those with hepatocellular (75%) or mixed liver injury (83%), although this difference was not significant. In cohort 1, HLA-A*0201 was observed more frequently in patients (22%) than in controls [11%; P = 0.003; odds ratio, 2.4 (95% confidence interval, 1.4-4.0)]. Taurocholate transport activity of 444A-encoded protein was significantly lower than that of 444V-encoded protein (81% of 444V, P < 0.05) because of the reduced protein stability. In conclusion, ABCB11 444A had slightly reduced transport activity, but it did not contribute to the occurrence of drug-induced cholestasis in Japanese patients. Therefore, genetic susceptibility to acquired cholestasis may differ considerably by ethnicity.

Recessive inheritance of population-specific intronic LINE-1 insertion causes a rotor syndrome phenotype.

Sequences of long-interspersed elements (LINE-1, L1) make up ∼17% of the human genome. De novo insertions of retrotransposition-active L1s can result in genetic diseases. It has been recently shown that the homozygous inactivation of two adjacent genes SLCO1B1 and SLCO1B3 encoding organic anion transporting polypeptides OATP1B1 and OATP1B3 causes a benign recessive disease presenting with conjugated hyperbilirubinemia, Rotor syndrome. Here, we examined SLCO1B1 and SLCO1B3 genes in six Japanese diagnosed with Rotor syndrome on the basis of laboratory data and laparoscopy. All six Japanese patients were homozygous for the c.1738C>T nonsense mutation in SLCO1B1 and homozygous for the insertion of a ∼6.1-kbp L1 retrotransposon in intron 5 of SLCO1B3, which altogether make up a Japanese-specific haplotype. RNA analysis revealed that the L1 insertion induced deleterious splicing resulting in SLCO1B3 transcripts lacking exon 5 or exons 5-7 and containing premature stop codons. The expression of OATP1B1 and OATP1B3 proteins was not detected in liver tissues. This is the first documented case of a population-specific polymorphic intronic L1 transposon insertion contributing to molecular etiology of recessive genetic disease. Since L1 activity in human genomes is currently seen as a major source of individual genetic variation, further investigations are warranted to determine whether this phenomenon results in other autosomal-recessive diseases.

Interplay of neuropilin-1 and semaphorin 3A after partial hepatectomy in rats.

AIM: To elucidate the role of neuropilin-1 (Nrp-1) and semaphorin 3A (Sema3A) in sinusoidal remodeling during liver regeneration in rats. METHODS: Male Wistar/ST rats at 7 wk of age, weighing about 200 g, were used for all animal experiments. In vivo, at 24, 48, 72, 96, 144 and 192 h after two-thirds partial hepatectomy (PHx), the remnant livers were removed. Liver tissues were immunohistochemically stained for Nrp-1, Sema3A and SE-1, a liver sinusoidal endothelial cell (SEC) marker. Total RNA of the liver tissue was extracted and reversely transcribed into cDNA. The mRNA expression of Sema3A was analyzed by quantitative real-time polymerase chain reaction and normalized to that of ribosomal protein S18. In vitro, SECs were isolated from rat liver and cultured in endothelial growth medium containing 20 ng/mL vascular endothelial cell growth factor. Migration of SECs in primary culture was assessed by cell transwell assay with or without recombinant Sema3A. Apoptotic cells were determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method. RESULTS: In vitro, immunohistochemistry study revealed that Sema3A and Nrp-1 were constitutively expressed in hepatocytes and SECs, respectively, in normal rat liver tissues. Nrp-1 expression in SECs was quantified by the percentage of immunostained area with anti-Nrp-1 antibody in relation to the area stained with SE-1. Between 24 h and 96 h following resection of liver, Nrp-1 expression in SECs was transiently increased. Compared with the baseline (5.2% ± 0.1%), Nrp-1 expression in SECs significantly increased at 24 h (17.3% ± 0.7%, P < 0.05), 48 h (39.1% ± 0.6%, P < 0.01), 72 h (46.9% ± 4.5%, P < 0.01) and 96 h (29.9% ± 3.8%, P < 0.01) after PHx, then returned to the basal level at termination of liver regeneration. Interestingly, the expression of Sema3A was inversely associated with that of Nrp-1 in liver after PHx. Sema3A mRNA expression was significantly reduced by about 75% over the period 24-144 h after PHx (P < 0.05), and returned to basal levels at 192 h after PHx. In vitro, SECs isolated from rats after PHx (PHx-SECs) were observed to migrate to the lower chamber of the cell transwell system after incubation for 24 h, but not cells from normal rats (CONT-SECs), indicating that mobility of PHx-SECs increases as compared with that of CONT-SECs. Moreover, recombinant Sema3A significantly attenuated migration in PHx-SECs in primary culture (vehicle-treated 100% ± 7.9% vs Sema3A-treated 42.6% ± 5.4%, P < 0.01), but not in CONT-SECs. Compared with CONT-SECs, the apoptotic rate of PHx-SECs decreased by 78.3% (P < 0.05). There was no difference in apoptosis between CONT-SECs that were treated with vehicle and Sema3A. However, in PHx-SECs, apoptosis was induced by the presence of 5 nmol Sema3A for 24 h (vehicle-treated 21.7% ± 7.6% vs Sema3A-treated 104.3% ± 8.9%, P < 0.05). In addition, immunohistochemistry confirmed the increased expression of Nrp-1 in PHx-SECs, while it was noted to a lesser extent in CONT-SECs. CONCLUSION: The interplay of Nrp-1 and Sema3A shown in our results may lead to a better understanding of interaction between sinusoidal remodeling and SECs during liver regeneration.

Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy.

BACKGROUND: Autophagy has been reported to play a pivotal role on the replication of various RNA viruses. In this study, we investigated the role of autophagy on hepatitis C virus (HCV) RNA replication and demonstrated anti-HCV effects of an autophagic proteolysis inhibitor, chloroquine. METHODS: Induction of autophagy was evaluated following the transfection of HCV replicon to Huh-7 cells. Next, we investigated the replication of HCV subgenomic replicon in response to treatment with lysosomal protease inhibitors or pharmacological autophagy inhibitor. The effect on HCV replication was analyzed after transfection with siRNA of ATG5, ATG7 and light-chain (LC)-3 to replicon cells. The antiviral effect of chloroquine and/or interferon-alpha (IFNalpha) was evaluated. RESULTS: The transfection of HCV replicon increased the number of autophagosomes to about twofold over untransfected cells. Pharmacological inhibition of autophagic proteolysis significantly suppressed expression level of HCV replicon. Silencing of autophagy-related genes by siRNA transfection significantly blunted the replication of HCV replicon. Treatment of replicon cells with chloroquine suppressed the replication of the HCV replicon in a dose-dependent manner. Furthermore, combination treatment of chloroquine to IFNalpha enhanced the antiviral effect of IFNalpha and prevented re-propagation of HCV replicon. Protein kinase R was activated in cells treated with IFNalpha but not with chloroquine. Incubation with chloroquine decreased degradation of long-lived protein leucine. CONCLUSION: The results of this study suggest that the replication of HCV replicon utilizes machinery involving cellular autophagic proteolysis. The therapy targeted to autophagic proteolysis by using chloroquine may provide a new therapeutic option against chronic hepatitis C.

Anti-inflammatory strategies in alcoholic steatohepatitis.

The hepatotoxic effects of alcohol have been described in detail, but mechanisms underlying the hepatotoxicity have been only partially characterized. Recently, increasing lines of evidence indicate that Kupffer cells play multiple roles in initiation and progression of alcoholic steatohepatitis. After ethanol exposure, Kupffer cells are activated via a mechanism dependent on gut-derived endotoxin, and release active mediators such as proinflammatory cytokines and eicosanoids. These mediators are responsible for the pathophysiology of alcoholic steatohepatitis. This review discusses the current concept of Kupffer cell-mediated steatohepatitis and how it relates to the hypothesis on the mechanism by which alcoholic steatohepetitis is caused, as well as several key issues that have to be addressed in this field: (i) How do Kupffer cells undergo priming and activation during alcoholic steatohepatitis?; (ii) What kind of mediators are involved?; and (iii) How does the concept translate into a strategy for therapeutics of alcoholic steatohepatitis?

Low molecular weight heparin prevents hepatic fibrogenesis caused by carbon tetrachloride in the rat.

BACKGROUND/AIMS: In this study, we investigated the effect of dalteparin sodium, a low molecular weight (LMW)-heparin, on hepatic fibrogenesis caused by chronic carbon tetrachloride (CCl4) administration in the rat. METHODS: Female Wistar rats were given a single, or repeated intraperitoneal injections of CCl4 (1ml/kg, twice per week) and dalteparin (50IU/kg, daily) for 7 weeks. RESULTS: Dalteparin did not prevent acute CCl4-induced hepatic necrosis and elevation in serum aminotransferases levels; however, proliferating cell nuclear antigen (PCNA)-positive hepatocytes were dramatically increased 24h after simultaneous administration of CCl4 and dalteparin. Interestingly, serum hepatocyte growth factor (HGF) levels 12h after injection of CCl4 were almost doubled when dalteparin was given simultaneously. Hepatic fibrosis following 7-week CCl4 treatment was markedly ameliorated by daily co-administration of dalteparin. Indeed, dalteparin largely inhibited CCl4-induction of smooth muscle alpha-actin expression, alpha1(I)procollagen and transforming growth factor (TGF)-beta1 mRNA levels in the liver. Further, dalteparin blunted platelet-derived growth factor (PDGF)-induced increases in 5-bromo-2'deoxyuridine (BrdU) uptake in 3-day cultured hepatic stellate cells (HSCs) in a dose-dependent manner. CONCLUSIONS: Dalteparin enhances hepatic regeneration and minimizes hepatic fibrogenesis caused by chronic CCl4 treatment. The mechanism underlying these effects most likely involves both up-regulation of HGF and inhibition of HSC proliferation.

Sphingosine 1-phosphate protects rat liver sinusoidal endothelial cells from ethanol-induced apoptosis: Role of intracellular calcium and nitric oxide.

In alcoholic liver disease, ethanol-induced damage to sinusoidal endothelial cells (SECs) appears to be important in the progression of liver damage. However, little is known about the mechanisms responsible for protection of SECs against ethanol-induced injury. To elucidate the role of sphingosine 1-phosphate (S1P), which is stored in platelets and may be released from them on their activation, we investigated the effect of S1P on rat liver SECs in primary culture. Pretreatment of cells with 1 mumol/L S1P attenuated ethanol-induced apoptosis. Electron microscopy confirmed this protective effect of S1P on damaged SECs in liver tissues after perfusion of ethanol. In the absence of ethanol, S1P increased DNA synthesis as determined via incorporation of bromodeoxyuridine. S1P also ameliorated the decreased DNA synthesis of cells induced by ethanol. Addition of S1P to cells induced an increase in intracellular calcium concentrations and NO production in cells. Western blotting revealed that S1P significantly induced the activation of endothelial NO synthase (eNOS), but not Akt, and that S1P-induced activation of eNOS was blocked by trifluoperazine, a calmodulin inhibitor. Furthermore, N(G)-nitro-L-arginine methyl ester, a NO synthase inhibitor, cancelled the effect of S1P on DNA synthesis, apoptosis, and NO production in vitro as well as the protective effect of S1P on cell damage in situ. In conclusion, the biological effect of S1P is at least partially mediated by Ca(2+)-sensitive eNOS activation and subsequent NO formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury.

Molecular tracing of Japan-indigenous hepatitis E viruses.

The ancestor(s) of apparently Japan-indigenous strains of Hepatitis E virus (HEV) was probably of foreign origin, but it remains unclear when and from where it made inroads. In this study, 24 genotype 3 and 24 genotype 4 HEV strains recovered in Japan each showed a significant cluster, clearly distinct from those of foreign strains, in the phylogenetic tree constructed from an 821 nt RNA polymerase gene fragment. The evolutionary rate, approximately 0.8 x 10(-3) nucleotide substitutions per site per year, enabled tracing of the demographic history of HEV and suggested that the ancestors of Japan-indigenous HEV had made inroads around 1900, when several kinds of Yorkshire pig were imported from the UK to Japan. Interestingly, the evolutionary growth of genotype 3 in Japan has been slow since the 1920s, whereas genotype 4 has spread rapidly since the 1980s. In conclusion, these data suggest that the indigenization and spread of HEV in Japan were associated with the popularization of eating pork.

Protective effect of pioglitazone against endotoxin-induced liver injury through prevention of Kupffer cell sensitization.

BACKGROUND: Activation of Kupffer cells by lipopolysaccharide (LPS) plays a pivotal role in the onset of pathophysiological events that occur during endotoxemia and intracellular calcium ([Ca]i) is involved in LPS-stimulated cytokine production. TNF-alpha is produced exclusively by the monocyte-macrophage lineage, including Kupffer cells, and pioglitazone has been shown to reduce TNF-alpha production from macrophages. On the other hand, there is increasing evidence that TNF-alpha plays a major role in the initiation and/or progression of multiple organ failure syndrome. Therefore, the purpose of this work was to determine whether pioglitazone could prevent LPS-induced liver injury METHODS: Rats were given a single oral dose of pioglitazone (500 microg/kg). To assess the sensitization of Kupffer cells, LPS (5 mg/kg) was administered IV and transaminases were evaluated 24 hr later. Kupffer cells were isolated 2 hr after pioglitazone treatment. After addition of LPS, [Ca]i was measured using a microspectrofluorometer with the fluorescent indicator, fura-2, and TNF-alpha was measured by ELISA RESULTS: LPS increased transaminases dramatically and elevation of serum transaminases were diminished markedly by pioglitazone. In isolated Kupffer cells, the LPS-induced increase in [Ca]i and TNF-alpha production were suppressed by treated with pioglitazone CONCLUSIONS: Therefore, it is concluded that pioglitazone prevents LPS induced liver injury via a mechanism dependent on suppression of TNF-alpha production from Kupffer cells.

Ethanol-induced sensitization to endotoxin in Kupffer cells is dependent upon oxidative stress.

BACKGROUND: Activation of Kupffer cells by gut-derived endotoxin plays a pivotal role in alcoholic liver injury. On the other hand, it was reported that acute ethanol administration reduced activation of Kupffer cells. We found that Kupffer cells isolated from rat treated only once with ethanol were sensitized to endotoxin 24 hrs later correlatively with CD14 expression. Moreover, it was shown that Kupffer cell activation by endotoxin via Toll-like receptor (TLR)-4 is involved in alcohol-induced liver injury and ethanol-induced oxidative stress is important in the regulation of transcription factor NFkappaB activation and cytokine production by Kupffer cells. Here, we show that IRAK, one of signaling molecules of TLR-4, regulates tolerance and sensitization to LPS and acute ethanol increases in IRAK expression through a mechanism dependent upon oxidant production. METHODS: Female C57BL/6 mice were given ethanol (5 g/kg) intragastrically, and LPS was injected 1 or 21 hrs later. Serum transaminase levels were measured. Moreover, some mice were treated with NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI, 1 mg/kg/day) or infected with adenovirus (1 x 10 plaque-forming units, intravenously) containing IkappaB superrepressor gene, which prevent NFkappaB activation of Kupffer cells, for three days. Kupffer cells were isolated from mice 1 hr and 21 hrs after ethanol treatment. After the addition of LPS, TNF-alpha in the media was measured using ELISA, Electrophoretic mobility shift assay (EMSA) was performed to analyze DNA binding activity of NFkappaB. Further, expression of Interleukin-1 receptor-associated kinase (IRAK) was evaluated by Western blotting. RESULTS: LPS-induced increases in transaminases were blunted in mice treated with ethanol before 1 hr. However, ethanol treatment 21 hrs earlier augmented LPS-increased transaminases three-fold over controls. Pretreatment with nonabsorbable antibiotics blocked these effects of ethanol. LPS-induced TNF-alpha production by Kupffer cells isolated from mice 1 hr after ethanol was reduced to about 60% of values from control Kupffer cells, while LPS-induced TNF-alpha production by Kupffer cells isolated from mice treated with ethanol 21 hrs earlier increased 1.5-fold over control Kupffer cells. In Kupffer cells from mice 1 hr after ethanol treatment, expression of IRAK was decreased, and LPS-induced activation of NFkappaB was decreased correlatively. In contrast, ethanol treatment to mice increased expression of IRAK in Kupffer cells 21hrs later and LPS-induced activation of NFkappaB was elevated significantly. On the other hands, DPI treatment for three days prior to ethanol did not prevent decreases in IRAK expression due to ethanol treatment for 1 hr. However, DPI treatment blunted ethanol-induced increases in IRAK expression. Additionally, inhibition of NKkappaB activation with dominant-negative IkappaBalpha blunted ethanol-induced increase in IRAK expression. Contrary, inhibition of NKkappaB did not affect decrease of IRAK expression due to ethanol treatment for 1 hr. CONCLUSIONS: Ethanol causes tolerance in the early phase after ethanol consumption, while sensitization was observed later. Both tolerance and sensitization were induced by gut-derived endotoxin. These findings indicate that ethanol-induced both tolerance and sensitization of Kupffer cells to endotoxin involve IRAK expression. Further, NADPH oxidase plays a pivotal role in the increase in IRAK expression due to ethanol via activation of NFkappaB signaling pathway. In conclusion, these data indicate that acute ethanol causes sensitization to endotoxin through mechanisms dependent upon oxidative stress.

Burn injury sensitizes rat Kupffer cells via mechanisms dependent on gut-derived endotoxin.

BACKGROUND: Bacterial translocation occurs after thermal injury in association with intestinal barrier loss. Recently, we found that sensitization of Kupffer cells involved gut-derived endotoxin; therefore, the purpose of this work was to study the mechanisms of sensitization of Kupffer cells in burn injury. METHODS: Rats received a 30% body surface area full-thickness steam burn 24 h before experiments. Serum alanine aminotransferase (ALT) was measured to assess liver damage, and plasma endotoxin in the portal vein were measured. Kupffer cells were isolated 24 h after the burn. Intracellular calcium ([Ca2+]i) in Kupffer cells was measured using a microspectrofluorometer with the fluorescent indicator, fura-2, and tumor necrosis factor (TNF)-alpha was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Lipopolysaccharide (LPS)-induced mortality was increased by burn treatment. This increase was blocked by gadolinium chloride, a Kupffer-cell toxicant. Accordingly, Kupffer cells were involved in this system. The LPS-induced increase of ALT was upregulated by the burn injury. This increase was blocked by pretreatment with antibiotics. Endotoxin levels were increased to almost 300 pg/ml (normal, <20 pg/ml) in the portal veins of rats that received a burn. This increase was blunted by antibiotics. In Kupffer cells isolated from untreated control rats, [Ca2+]i increased to 82+/-7 nM after the addition of LPS (100 ng/ml). Levels were elevated twofold over control levels in the cells from rats with burn (174+/-15 nM). In addition, TNF-alpha production by Kupffer cells isolated from rats with burn was increased fourfold over the based level. Sterilization of the gut with antibiotics completely blocked all effects of the burn on [Ca2+]i and TNF-alpha release. CONCLUSIONS: Kupffer cells isolated from rats with burn exhibited sensitization to LPS, involving gut-derived endotoxin. It is concluded that burns sensitize Kupffer cells to LPS via mechanisms that are dependent on gut-derived endotoxin.

Leptin facilitates proliferation of hepatic stellate cells through up-regulation of platelet-derived growth factor receptor.

In the present study, we investigated the effect of leptin on proliferation of hepatic stellate cells (HSCs) in vitro. Proliferation of 3-day cultured rat HSCs was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the nuclei. The percentages of BrdU-positive cells were increased in the presence of PDGF-BB (5 ng/ml) for 8h as expected. Co-incubation with leptin (10-100 nM) potentiates this PDGF-dependent increase in BrdU positive cells in a dose-dependent manner. Messenger RNA for PDGF receptor alpha and beta subunits was increased almost 2- to 3-fold by incubation with leptin for 6h. Further, pre-incubation with leptin for 6h enhanced PDGF-induced increases in phospho-p44/42 MAP kinase and phospho-Akt levels in a dose-dependent manner. In the same condition, however, leptin per se did not increase phospho-STAT 3 and phospho-p44/42 MAP kinase levels. Instead, leptin increased phospho-Akt levels in HSCs within 30 min, suggesting that the phosphatidylinositol 3 kinase (PI3K)/Akt pathway is involved in the mechanism by which leptin accelerates the proliferation of HSCs. In conclusion, the present study clearly indicated that leptin potentiates PDGF-dependent proliferative responses of HSCs in vitro.

Senescence marker protein-30 regulates Akt activity and contributes to cell survival in Hep G2 cells.

Senescence marker protein-30 (SMP30) is highly expressed in cytosol of hepatocytes, and its amount decreases with aging. Human hepatocellular carcinoma cell line was transfected with pcDNA3/SMP30 (SMP30 transfectants), or as a control with pcDNA3 (mock transfectants). When cells were exposed to 20 ng/ml tumor necrosis factor-alpha (TNF-alpha) plus 10 ng/ml actinomycin D (Act-D) for 15 h, the viability of cells was decreased in both SMP30 and mock transfectants. However, the viability of cells was threefold higher in SMP30 transfectants than mock transfectants. Cell death was confirmed as apoptosis by TUNEL assay. The presence of trifluoperazine, a calmodulin (CaM) inhibitor, attenuated anti-apoptotic effect of SMP30 in both transfectants, but the effect was more prominent in SMP30 transfectants. Western blot analyses revealed that Akt, which acts as a survival factor in cells, was activated in SMP30, but not mock, transfectants either in the presence or absence of TNF-alpha plus Act-D. Further, trifluoperazine inhibited Akt activation in SMP30 transfectants. We therefore propose that interplay between CaM and SMP30 regulates Akt activity, and thus SMP30 acts as a survival factor in hepatocytes.

[Gender difference in alcoholic liver injury].

Gender differences of alcoholic liver injury have been described previously, but mechanisms have only partially characterized. For example, it is known that females develop alcoholic liver injury more rapidly and to a greater extent than males. It now appears that estrogen participates in several aspects of this phenomenon. On the other hand, attention has been directed towards the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut-derived endotoxins via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cell and alcohol-induced liver injury.

The phosphodiesterase III inhibitor olprinone decreases sensitivity of rat Kupffer cells to endotoxin.

BACKGROUND: Sensitivity of Kupffer cells to endotoxin [lipopolysaccharide (LPS)] and overproduction of tumor necrosis factor-alpha (TNF-alpha) are critical for progression of alcoholic liver injury. Therefore, suppression of TNF-alpha should prove useful for treatment of alcoholic liver injury. However, a transient increase of intracellular calcium ([Ca]i) is required for LPS-induced TNF-alpha production by the macrophage cell line. The phosphodiesterase III inhibitor olprinone has been shown to suppress [Ca]i level in vascular smooth muscle cells. Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin. METHODS: Kupffer cells were isolated by collagenase digestion and differential centrifugation. LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 micromol/liter). After addition of LPS (10 microg/ml) to culture media, [Ca]i was measured using a fluorescent indicator, fura-2. RESULTS: LPS increased [Ca]i of Kupffer cells in control rats from basal levels (28 +/- 4 nmol/liter) to 280 +/- 14 nmol/liter. This increase was blunted by olprinone (91 +/- 8 nmol/liter). Similarly, olprinone diminished the LPS (1 microg/ml)-induced TNF-alpha production by Kupffer cells by 30% (2220 +/- 116 vs. 1386 +/- 199 pg/ml; p < 0.05). CONCLUSIONS: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin.