Accumulation In Kupffer Cells Research Articles

Zaluzanin C Alleviates Inflammation and Lipid Accumulation in Kupffer Cells and Hepatocytes by Regulating Mitochondrial ROS.

Zaluzanin C (ZC), a sesquiterpene lactone isolated from Laurus nobilis L., has been reported to have anti-inflammatory and antioxidant effects. However, the mechanistic role of ZC in its protective effects in Kupffer cells and hepatocytes has not been elucidated. The purpose of this study was to elucidate the efficacy and mechanism of action of ZC in Kupffer cells and hepatocytes. ZC inhibited LPS-induced mitochondrial ROS (mtROS) production and subsequent mtROS-mediated NF-κB activity in Kupffer cells (KCs). ZC reduced mRNA levels of pro-inflammatory cytokines (Il1b and Tnfa) and chemokines (Ccl2, Ccl3, Ccl4, Cxcl2 and Cxcl9). Tumor necrosis factor (TNF)-α-induced hepatocyte mtROS production was inhibited by ZC. ZC was effective in alleviating mtROS-mediated mitochondrial dysfunction. ZC enhanced mitophagy and increased mRNA levels of fatty acid oxidation genes (Pparα, Cpt1, Acadm and Hadha) and mitochondrial biosynthetic factors (Pgc1α, Tfam, Nrf1 and Nrf2) in hepatocytes. ZC has proven its anti-lipid effect by improving lipid accumulation in hepatocytes by enhancing mitochondrial function to facilitate lipid metabolism. Therefore, our study suggests that ZC may be an effective compound for hepatoprotection by suppressing inflammation and lipid accumulation through regulating mtROS.

GPAT3 regulates the synthesis of lipid intermediate LPA and exacerbates Kupffer cell inflammation mediated by the ERK signaling pathway

In the process of inflammatory activation, macrophages exhibit lipid metabolism disorders and accumulate lipid droplets. Kupffer cells (KCs) are the resident hepatic macrophage with critical defense functions in the pathogenesis of several types of liver disease. How dysregulated lipid metabolism contributes to perturbed KCs functions remains elusive. Here we report that glycerol-3-phosphate acyltransferase 3 (GPAT3) plays a key role in KCs inflammation response. Our findings indicate that lipopolysaccharide (LPS)-mediated inflammatory activation markedly increased lipid droplets (LDs) accumulation in KCs. This increase could be attributed to significantly up-regulated GPAT3. The loss of GPAT3 function obviously reduced KCs inflammation reaction both in vivo and in vitro, and was accompanied by improved mitochondrial function and decreased production of lysophosphatidic acid (LPA), in turn inhibiting extracellular regulated protein kinases (ERK) signaling pathway. Overall, this study highlights the role of GPAT3 in inflammatory activation of KCs and could thus be a potential therapeutic target for the treatment of inflammation-related liver disease.

Noninvasive Visualization of Sub-5 mm Orthotopic Hepatic Tumors by a Nanoprobe-Mediated Positive and Reverse Contrast-Balanced Imaging Strategy.

Delineation of small malignant lesions and their vasculature enables early and accurate diagnosis of hepatocellular carcinoma (HCC). However, it remains challenging to identify these features simultaneously by noninvasive imaging technology. Reverse contrast imaging emerges as a powerful means to detect early-stage HCC by taking inspiration from the intrinsic liver phagocytosis toward exogenous agents to generate negative tumor-to-normal tissue signals. However, this mechanism conflicts with the signal-enhancing requirements for vasculature visualization. Here, we solve this conundrum by designing a positive and reverse contrast-balanced imaging strategy based on a multifunctional PEG-Ta2O5@CuS nanoprobe that combines advanced gemstone spectral computer tomography (GSCT) with photoacoustic (PA) imaging. The nanoprobe exhibits preferential accumulation in Kupffer cells and hepatocytes over tumor cells, and its spectral properties are well matched with GSCT, leading to the enhancement of reverse contrast signals that enable clear delineation of 2-4 mm orthotopic HCC lesions. Meanwhile, its strong PA imaging capability at the second near-infrared (NIR-II) window makes vascular evaluation accessible by monitoring the positive signal enhancement derived from the limited tumor accumulation of the nanoprobe. In addition, the nanoprobe enables NIR-II photohyperthermia for timely tumor ablation. Overall, this proposed strategy shows potential in early detection and theranostics of HCC for improved clinical outcomes.

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease

Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and exvivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. Invitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.

Macrophage LAMTOR1 Deficiency Prevents Dietary Obesity and Insulin Resistance Through Inflammation-Induced Energy Expenditure.

Here, we studied the metabolic function of LAMTOR1 from macrophages using LAMTOR1 macrophage-specific knockout (MKO) mice. LAMTOR1 MKO mice showed resistance to high-fat diet (HFD)-induced obesity, lipid steatosis, and glucose metabolic disorders, with elevated levels of pro-inflammatory cytokines. The energy expenditure, oxygen consumption, and CO2 production increased significantly in HFD-fed MKO vs. wild-type (WT) mice. HE and immunohistochemistry staining showed a remarkable CD68+ Kupffer cell accumulation in the liver. Additionally, flow cytometry revealed that the proportion of macrophages and monocytes increased significantly in the liver of MKO mice. Of note, these macrophages were probably derived from the bone marrow since the proportion of CD11b+ cells as well as the proliferative activity was also increased in the context of femoral bone marrow cells. In addition, the Kupffer cells of both WT and KO mice were double-positive for the M1 (CD86) and M2 (CD206) markers. However, the expression of both M1 and M2 macrophage-related genes was increased in the liver of HFD-fed KO mice. Murine primary hepatocytes and Kupffer cells were further isolated and incubated with oleic acid for 24 h. The glucose output of primary hepatocytes from MKO mice was not affected. However, decreased lipid tolerance was observed in LAMTOR1-deficient Kupffer cells. Overall, our results suggest that LAMTOR1 deficiency in macrophages prevents obesity and metabolic disorders via the accumulation of Kupffer cells in the liver and the consequent hyper-inflammation and increased energy expenditure. Therefore, our results provide a new perspective for macrophage-derived LAMTOR1 in the context of systemic metabolism.

Pathology of Hepatic Iron Overload

Pathology of Hepatic Iron Overload

Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice

Tissue macrophage-derived apoptosis inhibitor of macrophage (AIM, encoded by cd5l gene) is a circulating protein that has suppressive functions in a broad range of diseases including obesity, liver steatosis, hepatocellular carcinoma (HCC), and acute kidney injury (AKI). In healthy states, high levels of AIM circulate in the inactivated state by associating with the immunoglobulin M (IgM) pentamer in the blood, whereas during AKI, AIM dissociates from IgM and gains disease repair activity. Here, we assessed whether AIM activation via its release from IgM is required to ameliorate other diseases. To this end, we employed a mouse line in which mouse AIM was replaced with feline AIM (AIM-felinized mice). Because feline AIM rarely dissociates from IgM due to its extremely high binding affinity for IgM, these mice exhibited deficient AKI repair as in cats. When fed a high-fat diet (HFD), similar to AIM-deficient (AIM−/−) mice, AIM-felinized mice exhibited enhanced triacylglycerol deposition in visceral adipocytes and hepatocytes, resulting in more prominent obesity and fatty liver than in wild-type mice. In contrast, the incidence of HCC after a 1-year HFD was remarkably lower in AIM-felinized mice than in AIM−/− mice, suggesting that AIM produced by liver Kupffer macrophages might directly facilitate the elimination of HCC cells. Accordingly, the marked deposition of AIM accompanied by accumulation of Kupffer cells was obvious during HCC tumour development in AIM-felinized mice. Δsµ mice, which harbour almost no circulating AIM due to the lack of secreted IgM, showed a phenotype comparable with that of AIM-felinized mice in prevention of those diseases. Thus, blood AIM released from IgM contributes to suppression of obesity and fatty liver as in AKI, whereas macrophage-derived noncirculating AIM mainly prevents HCC development. Our study depicted two different modes of disease prevention/repair facilitated by AIM, which could be the basis for HCC therapy that works by increasing AIM expression in macrophages.

Blood-Stage Plasmodium Berghei ANKA Infection Promotes Hepatic Fibrosis by Enhancing Hedgehog Signaling in Mice

Background/Aims: Malaria is the most deadly parasitic infection in the world, resulting in damage to various organs, including the liver, of the infected organism; however, the mechanism causing this damage in the liver remains unclear. Liver fibrosis, a major characteristic of liver diseases, occurs in response to liver injury and is regulated by a complex network of signaling pathways. Hedgehog (Hh) signaling orchestrates a number of hepatic responses including hepatic fibrogenesis. Therefore, we investigated whether Hh signaling influenced the liver’s response to malarial infection. Methods: Eight-week-old male C57BL/6 mice inoculated with blood containing Plasmodium berghei ANKA (PbA)-infected erythrocytes were sacrificed when the level of parasitemia in the blood reached 10% or 30%, and the livers were collected for biochemical analysis. Liver responses to PbA infection were examined by hematoxylin and eosin staining, real-time polymerase chain reaction, immunohistochemistry and western blot. Results: Severe hepatic injury, such as ballooned hepatocytes, sinusoidal dilatation, and infiltrated leukocytes, was evident in the livers of the malaria-infected mice. Hypoxia was also induced in 30% parasitemia group. With the accumulation of Kupffer cells, inflammation markers, TNF-α, interleukin-1β, and chemokine (C-X-C motif) ligand 1, were significantly upregulated in the infected group compared with the control group. Expression of fibrotic markers, including transforming growth factor-β, α-smooth muscle actin (α-SMA), collagen 1a1, thymosin β4, and vimentin, were significantly higher in the infected groups than in the control group. With increased collagen deposition, hepatic stellate cells expressing α-SMA accumulated in the liver of the PbA-infected mice, whereas those cells were rarely detected in the livers of the control mice. The levels of Hh signaling and Yes-associated protein (YAP), two key regulators for hepatic fibrogenesis, were significantly elevated in the infected groups compared with the control group. Treatment of mice with Hh inhibitor, GDC-0449, reduced hepatic inflammation and fibrogenesis with Hh suppression in PbA-infected mice. Conclusion: Our results demonstrate that HSCs are activated in and Hh and YAP signaling are associated with this process, contributing to increased hepatic fibrosis in malaria-infected livers.

Successful Exchange Transfusions in a Patient With Sickle-Beta Thalassemia Having Recurrent Episodes of Liver Failure From Intrahepatic Cholestasis

Sickle cell intrahepatic cholestasis (SCIC) is a rare and often fatal manifestation of sickle cell disease. Microvascular and sinusoidal occlusion from sickling RBCs results in bile canalicular cholestasis with ballooning hepatocytes and hyperbilirubinemia often progressing to acute liver failure and death. Previous case reports have shown mixed success with early exchange transfusion with a goal of hemoglobin S (HgbS) < 30%. We present the first case report of a patient with sickle-beta thalassemia who was successfully treated on three seperate occasions with exchange transfusion for intrahepatic cholestasis liver failure. A 44 year-old African American female with sickle cell-β° thalassemia and pulmonary hypertension presented with acute lower extremity pain, fatigue, scleral icterus and fever. A previous liver biopsy showed moderate portal chronic inflammation (Figure 1A), mild sinusoidal and portal fibrosis (Figure 1B), marked sinusoidal dilatation with RBCs (Figure 1C), and mild iron accumulation in Kupffer cells. She denied recent use of alcohol or acetaminophen. She was altered with jaundice, asterixis and clonus on exam. Abdomen was soft and non-tender without hepatosplenomegaly or ascites. Abdominal ultrasound showed hepatopetal flow without biliary dilatation. Labs notable for total bilirubin 68.0 (conjugated 38.0 mg/dL), AST/ALT 191/51 U/L, INR 2.8 and platelets 186 x103/μL. Anemia stable at Hgb of 7.8 g/dL. HgbS of 44.1%. She had leukocytosis, elevated creatinine and evidence of hemolysis. Blood and urine culture and viral studies were negative. Her liver failure was felt secondary to SCIC and she was emergently treated with exchange transfusion. Her post-exchange HgbS dropped to 17.1%, bilirubin downtrended, coagulopathy reversed, and her mental status cleared. She was discharged 3 days later in her regular state of health. Over the next 15 months she presented with two additional episodes of liver failure from SCIC that each resolved with a single exchange transfusion (Figure 2). To the best of our knowledge, this is the only case of a sickle-beta thalassemia patient presenting with multiple, distinct episodes of liver failure SCIC successfully treated with exchange transfusion. Although her extensive comorbidities precluded liver transplant, the success of exchange transfusion in SCIC liver failure may provide a bridge to definitive therapies such as stem cell and/or liver transplant for sickle cell patients in the future.Figure: Prior liver biopsy demonstrating moderate portal chronic inflammation (A), sinusoidal and portal fibrosis (B) and marked diffuse sinusoidal dilatation (C) with red blood cells.Figure: Labs during the three separate acute liver failure episodes. Bold line between Day 0 and Day 1 indicates exchange transfusion performed. AST, aspartate aminotransferase; ALT, alanine aminotransferase; INR, international normalized ratio; mg, milligrams; g, grams; dL, deciliter; L, liter.

Macrophage Specific Caspase-1/11 Deficiency Protects against Cholesterol Crystallization and Hepatic Inflammation in Hyperlipidemic Mice

Background & AimsWhile non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.Methods Ldlr -/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11-/- mice were incubated with oxLDL for 24h and autophagy was assessed. ResultsIn line with our hypothesis, caspase-1/11-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11-/- mice had normal autophagy activity.ConclusionAltogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.

New malignant grading system for hepatocellular carcinoma using the Sonazoid contrast agent for ultrasonography

The ultrasonography contrast agent Sonazoid provides parenchyma-specific contrast imaging (Kupffer imaging) based on its accumulation in Kupffer cells. This agent also facilitates imaging of the fine vascular architecture in tumors through maximum intensity projection (MIP). We examined the clinical utility of the malignancy grading system for hepatocellular carcinoma (HCC) using a combination of 2 different contrast-enhanced ultrasonography images. We studied 121 histologically confirmed cases of HCC (well-differentiated, 45; moderately differentiated, 70; poorly differentiated, 6). The results of Kupffer imaging were classified as (1) iso-echoic pattern or (2) hypo-echoic pattern. The MIP patterns produced were classified into one of the following categories: fine, tumor vessels were not clearly visualized and only fine vessels were visualized; vascular, tumor vessels were visualized clearly; irregular, tumor vessels were thick and irregular. Based on the combined assessment of Kupffer imaging and the MIP pattern, the samples were classified into 4 grades: Grade 1 (iso-fine/vascular), Grade 2 (hypo-fine), Grade 3 (hypo-vascular), and Grade 4 (hypo-irregular). The distribution of moderately and poorly differentiated HCCs was as follows: Grade 1, 4% (1/24); Grade 2, 52% (15/29); Grade 3, 85% (44/52); and Grade 4, 100% (16/16). The grading system also predicted portal vein invasion in 72 resected HCCs: Grade 1, 0% (0/4); Grade 2, 13% (1/8); Grade 3, 23% (11/48); and Grade 4, 67% (8/12). This new malignant grading system is useful for estimation of histological differentiation and portal vein invasion of HCC.

Ad5:Ad48 Hexon Hypervariable Region Substitutions Lead to Toxicity and Increased Inflammatory Responses Following Intravenous Delivery

The development of adenoviral vectors for intravascular (i.v.) delivery will require improvements to their in vivo safety and efficacy. The hypervariable regions (HVRs) of the Ad5 hexon are a target for neutralizing antibodies, but also interact with factor X (FX), facilitating hepatocyte transduction. Ad48, a species D adenovirus, does not bind FX and has low seroprevalence. Therefore, it has been suggested that Ad5HVR48(1-7), a hexon-chimeric vector featuring the seven HVRs from Ad48, should display advantageous properties for gene therapy, by evading pre-existing Ad5 immunity and blocking FX interactions. We investigated the in vivo biodistribution of Ad5, Ad5HVR48(1-7), and Ad48 following i.v. delivery. Ad5HVR48(1-7) displayed reduced hepatocyte transduction and accumulation in Kupffer cells (KCs), but triggered a robust proinflammatory response, even at relatively low doses of vector. We detected elevated serum transaminases (48 hours) and increased numbers of periportal CD11b(+)/Gr-1(+) cells in the livers of Ad5HVR48(1-7)-treated animals following i.v., but not intramuscular (i.m.), delivery. In contrast, Ad48 did not elevate transaminases or result in the accumulation of CD11b(+)/Gr-1(+) cells. Collectively, these findings suggest that substantial hexon modifications can lead to unexpected properties which cannot be predicted from parental viruses. Therefore, refined mutations may be preferential for the successful development of targeted vector systems which require i.v. administration.

Combined Fiber Modifications Both to Target αvβ6and Detarget the Coxsackievirus–Adenovirus Receptor Improve Virus Toxicity ProfilesIn Vivobut Fail to Improve Antitumoral Efficacy Relative to Adenovirus Serotype 5

Achieving high-efficiency tumor targeting after systemic delivery is a considerable challenge facing oncolytic gene therapists. Efficient retargeting should be combined with efforts to improve in vivo safety, reduce hepatotoxicity, minimize off-target interactions, and improve antitumoral potency and efficacy. We previously described the successful retargeting of adenovirus serotype 5 (Ad5) to α(v)β(6), an integrin that is highly overexpressed in numerous human carcinomas. In this study, we have further modified this construct by introducing mutations that ablate coxsackievirus-adenovirus receptor (CAR) binding and putative interactions with factor IX (FIX)/C4b-binding protein (C4BP). We have found that the resulting vector, Ad5-477dlTAYT(A20), displays a desirable in vivo safety profile. This vector does not agglutinate human erythrocytes, fails to cause thrombocytopenia after intravenous delivery, has limited induction of proinflammatory cytokines, and results in low-level toxicity (aspartate aminotransferase/alanine aminotransferase) when compared with Ad5-EGFP(WT). Furthermore, it has reduced accumulation in Kupffer cells (1 hr) and limited hepatocyte transduction at later time points (24 and 96 hr). The parental vector, Ad5-EGFP(A20), also displayed many of these desirable properties. As a result of the improved safety profile of both A20-modified vectors, we escalated the dose from 2×10(10) to 4×10(10) viral particles in an antitumoral efficacy study. We observed improvements in reducing percent tumor growth at early time points (96 hr) when compared with Ad5-EGFP(WT), although increasing the dose did not affect the therapeutic outcome beneficially. On completion of the experiment, we detected increased E1A staining in the tumors of all A20-treated groups and we determined that E1A expression was localized largely within α(v)β(6)(+) tumor cells. However, in spite of apparently efficient tumor transduction, this did not result in enhanced antitumoral efficacy as the virus failed to disseminate effectively throughout the tumor mass, presumably due to physical intratumoral restrictions. This highlights a remaining challenge that needs to be overcome before such vectors can be developed for future cancer gene therapy applications.

Non-HFEHepatic Iron Overload

Numerous clinical entities have now been identified to cause pathologic iron accumulation in the liver. Some are well described and have a verified hereditary basis; in others the genetic basis is still speculative, while in several cases nongenetic iron-loading factors are apparent. The non- HFE hemochromatosis syndromes identifies a subgroup of hereditary iron loading disorders that share with classic HFE-hemochromatosis, the autosomal recessive trait, the pathogenic basis (i.e., lack of hepcidin synthesis or activity), and key clinical features. Yet, they are caused by pathogenic mutations in other genes, such as transferrin receptor 2 ( TFR2), hepcidin ( HAMP), hemojuvelin ( HJV) , and ferroportin ( FPN), and, unlike HFE-hemochromatosis, are not restricted to Caucasians. Ferroportin disease, the most common non- HFE hereditary iron-loading disorder, is caused by a loss of iron export function of FPN resulting in early and preferential iron accumulation in Kupffer cells and macrophages with high ferritin levels and low-to-normal transferrin saturation. This autosomal dominant disorder has milder expressivity than hemochromatosis. Other much rarer genetic disorders are associated with hepatic iron load, but the clinical picture is usually dominated by symptoms and signs due to failure of other organs (e.g., anemia in atransferrinemia or neurologic defects in aceruloplasminemia). Finally, in the context of various necro-inflammatory or disease processes (i.e., chronic viral or metabolic liver diseases), regional or local iron accumulation may occur that aggravates the clinical course of the underlying disease or limits efficacy of therapy.

Characterization of rat and human Kupffer cells after cryopreservation

Kupffer cells (KC) are the resident macrophages of the liver and represent about 80% of the total fixed macrophage population. They are involved in disease states such as endotoxin shock, alcoholic liver diseases and other toxic-induced liver injury. They release physiologically active substances such as eicosanoids and inflammatory cytokines (IL-1, IL-6, TNFα), and produce free radical species. Thus, KC are attractive targets for anti-inflammatory therapies and potential candidates responsible for differences in inflammation in liver disease seen between different individuals. However, to perform parallel in vitro experiments with KC from different donors a suitable method for conservation of KC would be necessary. Therefore, the present study evaluated, whether rat and human KC can be frozen, stored and recovered without losing their functional integrity. Rat and human KC were isolated and either cultured under standard conditions (fresh KC) or cryopreserved in special freezing medium (cryopreserved KC). At least 24 h later, cryopreserved KC were thawed, brought into suspension and seeded in the same density as fresh cells for subsequent experiments. Viability of cultured KC was analyzed by trypan blue exclusion. LPS (or PBS as control) stimulation was performed at different time points and cytokine release was analyzed with IL-6 and TNFα ELISAs, respectively. Phagocytic capacity was investigated by using a specific phagocytosis assay and FACS analysis. The recovery rate after thawing was around 57% for rat and around 65% for human cryopreserved KC. The results indicate, that KC can successfully be cryopreserved with an adequate recovery rate of viable cells. The properties of fresh and frozen KC can also be compared after thawing. Freshly isolated and cryopreserved cultured KC showed near-normal morphology and did not differ in the cultivation profiles over a period of 72 h. One to three days after seeding, frozen rat or human KC also retained inducible functions such as the production of TNFα or IL-6 after LPS challenge. Finally, regardless if they were cryopreserved or not, no differences in the phagocytic activities of the cells were obtained. Taken together, it is concluded that cryopreservation of KC does not change the physiological characteristics of the cells in vitro. Therefore, the method used here for cryopreservation of especially human KC allows the accumulation of KC from several donors for parallel in vitro experiments.

Cystinosis as a Cause of Noncirrhotic Portal Hypertension

Cystinosis is a rare autosomal recessive storage disorder, characterized by the abnormal accumulation of cystine in cellular lysosomes. This accumulation, which can occur in any organ system, leads to crystallization of trapped cystine and ultimately cellular death. Hepatic manifestations of Cystinosis although rare, have been described in the literature. However, to our knowledge, only one other case of non-cirrhotic portal hypertension secondary to cystine accumulation in Kupffer cells has been reported. In this case and ours, portal hypertension was found in the absence of bridging fibrosis. Furthermore, in our case, for the majority of the patient's course, hepatic synthetic function remained normal. Cysteamine is therapeutic in this disorder, and can lead to significant removal of cystine, and thus to reversibility of disease, however, it requires high doses and must be taken regularly. Porto-systemic shunting in combination with aggressive medical therapy could potentially benefit patients who develop non-cirrhotic portal hypertension in this clinical setting.

Tolerogenic role of Kupffer cells in immune-mediated adverse drug reactions

Immune-mediated adverse drug reactions (IADR) account for approximately 6–10% of all adverse drug reactions. Although IADR are often referred to as rare (afflicting 1/100 to 1/100,000 patients), their unpredictable and serious nature makes them a significant economic burden and safety concern to the health care community and the pharmaceutical industry. Current studies suggest that IADR are caused by immunogenic drug–protein adducts; however, it remains unclear why only a small percentage of patients are susceptible to developing these reactions. We hypothesized that most individuals may be resistant to IADR because they develop immunological tolerance to drug–protein adducts in the liver, an organ with tolerogenic properties. We tested this hypothesis using a murine model of T-cell-mediated reaction against a hapten, 2,4-dinitrochlorobenzene (DNCB). We showed that pre-treatment of mice with a protein adduct of DNCB led to its accumulation in Kupffer cells (KC) of the liver and induced tolerance to subsequent DNCB sensitization. KC depletion and adoptive transfer experiments further supported that KC may act as a primary inducer of immunological tolerance against protein adducts of haptens or drugs. Functional activities of KC, which are regulated by genetic and/or environmental factors, may play an important role in determining individual susceptibility to IADR.

Experimentally induced selenosis of adult mallard ducks: clinical signs, lesions, and toxicology.

Selenosis is thought to be a significant problem among waterfowl populations in selenium-contaminated wetlands in the western United States. Chemical analysis of avian tissues is currently the principal basis for diagnosis. The purpose of these two 150-day studies was to establish whether morphological criteria for selenosis could be developed to supplement chemical analysis. Forty-eight flightling male mallard ducks were fed either a proprietary waterfowl ration (< 1 ppm selenium) or the same ration amended to contain 10, 25, and 60 ppm selenium supplied as seleno-L-methionine (n = 12/group). In a separate study, 12 birds fed twice daily were offered either a proprietary ration or a selenium-supplemented ration (120 microg/g) for one of two daily feedings. Selenium in whole blood increased from baseline concentrations (< 0.4 microg/ml) to means of 4.5, 8.9, and 16.0 microg/ml in the 10-, 25-, and 60-ppm groups, respectively. All birds in the 60-ppm-dose group rapidly lost weight and were killed (11/12) or died (1/12) between 22 and 50 days of dietary exposure. In addition to emaciation, six of 12 birds (50%) fed the 60-microg/g diet developed mild to moderate generalized hepatopathy with single-cell necrosis, karyomegaly of hepatocytes, hyperplastic bile duct epithelium, and/or iron accumulation in Kupffer cells. The principal lesions in birds exposed to other dietary concentrations of selenium involved integumentary structures containing hard keratin. Gross lesions developed after 76 days of dietary exposure and consisted of bilaterally symmetrical alopecia of the scalp and dorsal cervical midline, broken or lost digital nails, and necrosis of the tip of the beak (maxillary nail). One or more of these three lesions were present in 0/12 birds (0%) fed 10 ppm selenium, 5/12 birds (42%) fed 25 ppm selenium, and 4/9 (44%) birds fed a split-feed diet containing 120 ppm selenium. Controls were unaffected. Histologic lesions in digital and maxillary nails consisted of single-cell to full-thickness necrosis of keratinocytes and multifocal parakeratosis in stratum corneum. Histologic lesions in alopecic skin (necrosis of the epidermal collar, inflammation of the feather pulp, and follicular keratosis) were mild. Some birds with alopecia had no detectable lesions in feather follicles from affected areas of skin. The highest tissue concentrations of selenium were in liver, kidney, and feathers, respectively. Mean hepatic tissue concentrations were 14.5 microg/g (10 ppm group), 29.6 microg/g (25 ppm group), 60.6 microg/g (60 ppm group), 13.0 microg/g (120 ppm split-feed group), and 2.0 microg/g (controls). Integumentary and hepatic lesions may be of value in corroborating a diagnosis of selenosis based on chemical analysis of tissues from naturally intoxicated waterfowl. Some birds with fatal selenosis may have no morphologic lesions other than emaciation.

Dynamic liver imaging with iron oxide agents: effects of size and biodistribution on contrast.

In vivo effective relaxation rates in normal rat liver were evaluated for four dextran coated iron oxide agents: monocrystalline iron oxide nanocolloid (MION) with a mean particle diameter of 3.9 nm, a polycrystalline agent (PION) with a larger mean diameter of 12 nm, and these two agents labeled with the asialofetuin (ASF) protein for high hepatocytic receptor binding affinity (MION-ASF and PION-ASF). Using echo planar imaging at 2 Tesla, dose response was measured with measured with high temporal resolution for 3 h after injection of agent, and by comparing with relaxivities in vitro and in brain, dominant in vivo contrast phenomena were elucidated. While transverse relaxivity for PION-ASF exceeded that for MION-ASF by almost a factor of 2 in solution, relaxation rates in vivo became equivalent. Liver relaxation using non-ASF agents was consistent with rapid water exchange between vascular and extravascular compartments, which dominated relaxation as a result of agent accumulation in Kupffer cells.

Lipid deposition in Kupffer cells after parenteral fat nutrition in rats: a biochemical and ultrastructural study.

To study fat metabolism and evaluate lipid deposition in hepatocytes and Kupffer cells during parenteral nutrition (PN) with or without fat. 20 male Sprague-Dawley rats, divided into four groups, were investigated. Rats fed orally were used as a reference group and compared to three groups of rats receiving PN either without fat or with 33% of non-protein energy as fat or with 66% of non-protein energy as fat. The PN regimens were equicaloric and administered continuously via a jugular catheter for 7 days. After suffocation, blood was collected for determination of serum lipids. Epididymal fat and heart were collected for analysis of lipoprotein lipase activities, and pieces of liver were saved for analyses of liver triglyceride concentration and hepatic lipase activity. Light and electron microscopy were used for examination of lipid deposition in Kupffer cells. Directly after termination of parenteral feeding, the serum levels of triglycerides were similar in all PN groups, while the levels of non-high-density lipoprotein (HDL) cholesterol and non-HDL phospholipids were significantly increased in parallel with increased doses of fat. Lipid-free PN resulted in significantly less liver steatosis than high-fat PN. Lipid PN also resulted in downregulated hepatic lipase activity, signs of lipid accumulation in Kupffer cells and hepatocytes and an increased number of phagosomes in Kupffer cells. Fat vacuoles were found in Kupffer cells after lipid PN, although serum levels of triglycerides were not elevated and lipoprotein lipase activity were not depressed. The cells were distended by fat vacuoles after administration of PN solutions with a high fat concentration. Morphological signs of increased Kupffer cell activity were also found, suggesting that intravenous fat emulsions may activate macrophages.