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Surface-enhanced Raman scattering (SERS) spectroscopy is a surface- or cavity-enhanced variant of Raman scattering spectroscopy that allows the detection of analytes with a sensitivity down to single molecules. This method involves the use of SERS-active surfaces or cavities capable of concentrating incident radiation into small mode volumes containing the analyte. Here, we have engineered an ultranarrow metal-dielectric nano-cavity out of a film of the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) glycoprotein and a silver surface, held together by interaction between reduced protein sulfhydryl groups and silver. The concentration of light in this nano-cavity allows the label-free recording of the characteristic Raman spectra of protein samples smaller than 1 pg. This is sufficient for the ultrasensitive detection of viral protein antigens at physiologically relevant levels. Moreover, the protein SERS signal can be increased by several orders of magnitude by coating the RBD film with a nanometer-thick silver shell, thereby raising the cavity Q-factor. This ensures a sub-femtogram sensitivity of the viral antigen detection. A simple theoretical model explaining the observed additional enhancement of the SERS signal from the silver-coated protein is proposed. Our study is the first to obtain the characteristic Raman and SERS spectra of the RBD of S glycoprotein, the key SARS-CoV-2 viral antigen, directly, without the use of Raman-reporter molecules. Thus, our approach allows label-free recording of the characteristic spectra of viral antigens at concentrations orders of magnitude lower than those required for detecting the whole virus in biological media. This makes it possible to develop a high-performance optical detection method and conformational analysis of the pathogen and its variants.
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COVID-19 , Espectrometría Raman , Antígenos Virales , COVID-19/diagnóstico , Humanos , SARS-CoV-2 , Plata/química , Espectrometría Raman/métodos , Glicoproteína de la Espiga del CoronavirusRESUMEN
The Indium Tin Oxide (ITO) platform is one of the promising solutions for state-of-the-art integrated optical modulators towards low-loss silicon photonics applications. One of the key challenges on this way is to optimize ITO-based thin films stacks for electro-optic modulators with both high extinction ratio and low insertion loss. In this paper we demonstrate the e-beam evaporation technology of 20 nm-thick ITO films with low extinction coefficient of 0.14 (Nc = 3.7·1020 cm-3) at 1550 nm wavelength and wide range of carrier concentrations (from 1 to 10 × 1020 cm-3). We investigate ITO films with amorphous, heterogeneously crystalline, homogeneously crystalline with hidden coarse grains and pronounced coarsely crystalline structure to achieve the desired optical and electrical parameters. Here we report the mechanism of oxygen migration in ITO film crystallization based on observed morphological features under low-energy growth conditions. Finally, we experimentally compare the current-voltage and optical characteristics of three electro-optic active elements based on ITO film stacks and reach strong ITO dielectric permittivity variation induced by charge accumulation/depletion (Δn = 0.199, Δk = 0.240 at λ = 1550 nm under ± 16 V). Our simulations and experimental results demonstrate the unique potential to create integrated GHz-range electro-optical modulators with sub-dB losses.
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We have performed mutational profiling of 25 genes involved in epigenetic processes on 135 gastric cancer (GC) samples. In total, we identified 79 somatic mutations in 49/135 (36%) samples. The minority (n = 8) of mutations was identified in DNA methylation/demethylation genes, while the majority (n = 41), in histone modifier genes, among which mutations were most commonly found in KMT2D and KMT2C. Somatic mutations in KMT2D, KMT2C, ARID1A and CHD7 were mutually exclusive (p = 0.038). Mutations in ARID1A were associated with distant metastases (p = 0.03). The overall survival of patients in the group with metastases and in the group with tumors with signet ring cells was significantly reduced in the presence of mutations in epigenetic regulation genes (p = 0.036 and p = 0.041, respectively). Separately, somatic mutations in chromatin remodeling genes correlate with low survival rate of patients without distant metastasis (p = 0.045) and in the presence of signet ring cells (p = 0.0014). Our results suggest that mutations in epigenetic regulation genes may be valuable clinical markers and deserve further exploration in independent cohorts.
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Somatic mutation profiling in gastric cancer (GC) enables main driver mutations to be identified and their clinical and prognostic value to be evaluated. We investigated 77 tumour samples of GC by next-generation sequencing (NGS) with the Ion AmpliSeq Hotspot Panel v2 and a custom panel covering six hereditary gastric cancer predisposition genes (BMPR1A, SMAD4, CDH1, TP53, STK11 and PTEN). Overall, 47 somatic mutations in 14 genes were detected; 22 of these mutations were novel. Mutations were detected most frequently in the CDH1 (13/47) and TP53 (12/47) genes. As expected, somatic CDH1 mutations were positively correlated with distant metastases (p = 0.019) and tumours with signet ring cells (p = 0.043). These findings confirm the association of the CDH1 mutations with diffuse GC type. TP53 mutations were found to be significantly associated with a decrease in overall survival in patients with Lauren diffuse-type tumours (p = 0.0085), T3-T4 tumours (p = 0.037), and stage III-IV tumours (p = 0.013). Our results confirm that the detection of mutations in the main driver genes may have a significant prognostic value for GC patients and provide an independent GC-related set of clinical and molecular genetic data.
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Antígenos CD/genética , Cadherinas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Neoplasias Gástricas/patología , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Análisis de Secuencia de ADN , Neoplasias Gástricas/genética , Análisis de SupervivenciaRESUMEN
INTRODUCTION: We have investigated aberrant methylation of genes CDH1, RASSF1A, MLH1, N33, DAPK, expression of genes hTERT, MMP7, MMP9, BIRC5 (survivin), PTGS2, and activity of telomerase of 106 gastric tumor samples obtained intra-operatively and 53 gastric tumor samples from the same group of patients obtained endoscopically before surgery. Biopsy specimens obtained from 50 patients with chronic calculous cholecystitis were used as a control group. Together with tissue samples obtained from different sites remote to tumors, a total of 727 samples have been studied. The selected parameters comprise a system of molecular markers that can be used in both diagnostics of gastric cancer and in dynamic monitoring of patients after surgery. Special attention was paid to the use of molecular markers for the diagnostics of malignant process in the material obtained endoscopically since the efficacy of morphological diagnostics in biopsies is compromised by intratumoral heterogeneity, which may prevent reliable identification of tumor cells in the sampling. Our data indicated that certain molecular genetic events provided more sensitive yet specific markers of the tumor. CONCLUSION: We demonstrated that molecular profiles detected in preoperative biopsies were confirmed by the material obtained intra-operatively. The use of endoscopic material facilitates gastric tumors pre-operative diagnostics, improving early detection of gastric cancer and potential effective treatment strategies.
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Optical properties of two dimensional periodic system of the silicon micro-cones are investigated. The metasurface, composed of the silicon tips, shows enhancement of the local optical field. Finite element computer simulations as well as real experiment reveal anomalous optical response of the dielectric metasurface due to excitation of the dielectric resonances. Various electromagnetic resonances are considered in the dielectric cone. The metal-dielectric resonances, which are excited between metal nanoparticles and dielectric cones, are also considered. The resonance local electric field can be much larger than the field in the usual surface plasmon resonances. To investigate local electric field the signal molecules are deposited on the metal nanoparticles. We demonstrate enhancement of the electromagnetic field and Raman signal from the complex of DTNB acid molecules and gold nanoparticles, which are distributed over the metasurface. The metasurfaces composed from the dielectric resonators can have quasi-continuous spectrum and serve as an efficient SERS substrates.
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The nature of intrinsic and impurity point defects in lead zirconate titanate (PZT) ceramics has been explored. Using electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and X-ray photoelectron spectroscopy (XPS) methods, several impurity sites have been identified in the materials, including the Fe(3+)-oxygen vacancy (VO) complex and Pb ions. Both of these centers are incorporated into the PZT lattice. The Fe(3+) VÐ paramagnetic complex serves as a sensitive probe of the local crystal field in the ceramic; the symmetry of this defect roughly correlates with PZT phase diagram as the composition is varied from PbTiO3 to PbZrO3. NMR spectra (207)Pb in PbTiO3, PbZrO3, and PZT with iron content from 0 to 0.4 wt% showed that increasing the iron concentration leads to a distortion of the crystal structure and to improvement of the electrophysical parameters of the piezoceramics. This is due to the formation of a phase which has a higher symmetry, but at high concentrations of iron (>0.4 wt%), it leads to sharp degradation of electrophysical parameters.
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Periodontitis and Chlamydia pneumoniae infection are independent risk factors for cardiovascular diseases. The aim of this study was to investigate the effect of C. pneumoniae and Aggregatibacter actinomycetemcomitans infection on hepatic inflammation and lipid homeostasis of apolipoprotein E-deficient mice. Mice were infected with viable C. pneumoniae intranasally three times for chronic infection or once for acute infection. Viable A. actinomycetemcomitans was administered 10 times intravenously alone or in concert with C. pneumoniae. Hepatic alterations were assessed by histochemistry, lipid quantification, and fatty acid profile analysis. The RNA expression levels and the presence of pathogens in the livers and lungs were detected by quantitative real-time PCR. Both pathogens were detected in the livers of the infected animals. Chronic C. pneumoniae infection induced marked changes in hepatic lipid homeostasis. A. actinomycetemcomitans infection resulted in inflammatory cell infiltration into the liver, accompanied by elevated hepatic RNA expression levels of inflammation-related genes and higher serum amyloid A and lipopolysaccharide concentrations. Our results indicate that proatherogenic pathogens infect the liver, causing proinflammatory alterations and lipid disturbances. This infection may maintain chronic systemic inflammation attributable to atherogenesis.
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Apolipoproteínas/deficiencia , Infecciones por Chlamydophila/patología , Ácidos Grasos/metabolismo , Hepatitis/microbiología , Hepatitis/patología , Infecciones por Pasteurellaceae/patología , Animales , Chlamydophila pneumoniae/patogenicidad , Lipopolisacáridos/sangre , Hígado/microbiología , Hígado/patología , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Pasteurellaceae/patogenicidad , Proteína Amiloide A Sérica/análisisRESUMEN
The complement system can provoke but also participate in the repair of liver injury. Here we investigated by microarray analysis the effect of chronic ethanol consumption on hepatic mRNA expression of complement components and acute-phase proteins in complement C3-deficient (C3(-/-)) and wild-type (C3(+/+)) mice. Up-regulation by ethanol of factor B, C1qA-chain and clusterin but down-regulation of factor H, Masp-2, factor D and the terminal components C6, C8alpha and C9 was seen in both strains. Ethanol up-regulated C2 and down-regulated C4bp only in C3(+/+) mice, while in C3(-/-) mice up-regulation of C1qB-chain and vitronectin was observed. The expression of factor B, C6, C1qB and factor I was lower but that of factor D higher in C3(-/-) than in C3(+/+) mice. Ethanol induced mRNA synthesis of many acute-phase proteins including SPARC and lipocalin-2, but reduced the expression of SAP. The induction of early classical and alternative pathway components and suppression of terminal pathway components and soluble regulators may thus contribute to alcohol-induced liver injury. Lipocalin-2 and SPARC emerge as new candidate markers for early detection of liver damage.
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Proteínas de Fase Aguda/biosíntesis , Consumo de Bebidas Alcohólicas/inmunología , Proteínas del Sistema Complemento/biosíntesis , Hepatopatías Alcohólicas/inmunología , Hígado/inmunología , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/inmunología , Consumo de Bebidas Alcohólicas/genética , Consumo de Bebidas Alcohólicas/metabolismo , Animales , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/inmunología , Etanol/administración & dosificación , Etanol/toxicidad , Expresión Génica , Lipocalina 2 , Lipocalinas , Hepatopatías Alcohólicas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/inmunología , Osteonectina/biosíntesis , Osteonectina/inmunología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The aim of the present study was to investigate whether d-glycerate (glycerate) could accelerate ethanol and acetaldehyde (AcH) oxidation in vivo in rats by circumventing the rate-limiting step, that is, the reoxidation of the reduced form of nicotinamide adenine dinucleotide. Male rats belonging to the ANA (Alko, nonalcohol) and AA (Alko, alcohol) rat lines were challenged with 1.2 g ethanol per kilogram with or without glycerate administration (0.1-1.0 g/kg). Blood ethanol, blood AcH, and liver free glycerol concentrations were determined during ethanol intoxication. Glycerate treatment, regardless of the dose, accelerated ethanol elimination by approximately 25% (P < .001) in the ANA animals. Glycerate also accelerated the AcH oxidation, but perhaps not as much as the ethanol oxidation, as indicated by a trend toward elevated AcH levels. In the experiments with the AA rats, glycerate treatment elevated hepatic free glycerol levels by about 50% (P < .05) during alcohol intoxication. The acceleration of ethanol and AcH oxidation in conjunction with elevated glycerol levels by the treatment with glycerate supports the hypothesis that the aldehyde dehydrogenase-mediated AcH oxidation can be coupled with the reduction of glycerate to d-glyceraldehyde catalyzed by the same enzyme. Such a coupling should increase the availability of the oxidized form of nicotinamide adenine dinucleotide and thus accelerate both ethanol and AcH oxidation. Further studies are needed to investigate how the AcH could be even more efficiently oxidized to reduce the harmful effects of ethanol-derived AcH.
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Acetaldehído/metabolismo , Etanol/metabolismo , Ácidos Glicéricos/farmacología , Animales , Glicerol/metabolismo , Hígado/metabolismo , Masculino , NAD/metabolismo , Oxidación-Reducción , RatasRESUMEN
BACKGROUND/AIMS: Fatty infiltration initiates alcohol-induced liver changes and complement component C3 affects lipid metabolism. We recently observed that ethanol-induced steatosis seen in normal (C3(+/+)) mice was absent in livers of C3-deficient (C3(-/-)) mice. To understand the underlying molecular mechanisms we analyzed lipid parameters and liver gene expression profiles in these mice. METHODS: A Western-type high-fat diet with ethanol or carbohydrates (control) was fed for 6 weeks to C3(+/+) and C3(-/-) mice. Serum and liver lipid parameters were analyzed and liver mRNA expression patterns studied by micro-array analysis and RT-PCR. RESULTS: In both genotypes ethanol markedly reduced serum cholesterol, apolipoprotein A-I, phospholipid transfer protein activity and hepatic mRNA levels of fatty acid-binding proteins and fatty acid beta-oxidation enzymes. In contrast, exclusively in C3(-/-) mice, ethanol treatment increased serum and liver adiponectin levels but down-regulated transcripts of lipogenic enzymes, adiponectin receptor 2 and adipose differentiation-related protein and up-regulated phospholipase D1. CONCLUSIONS: We propose that these ethanol-induced alterations observed exclusively in C3(-/-) mice contribute to protection against fatty infiltration and subsequent inflammatory processes in the liver of these mice. The results suggest important cross-talk between complement factor C3 and lipid regulators in ethanol-induced steatosis.
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Complemento C3/deficiencia , Hígado Graso Alcohólico/genética , Hígado Graso Alcohólico/metabolismo , Expresión Génica , Hígado/metabolismo , Adiponectina/metabolismo , Animales , Vías Biosintéticas/efectos de los fármacos , Complemento C3/genética , Complemento C3/metabolismo , Grasas de la Dieta/administración & dosificación , Etanol , Hígado Graso Alcohólico/patología , Interleucina-10/sangre , Metabolismo de los Lípidos , Hígado/efectos de los fármacos , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The effect of PLTP deficiency on hepatic lipid status and apolipoprotein A-I (apoA-I) biosynthesis in PLTP knockout (PLTP-KO) mice was investigated. PLTP-KO mice exhibited a marked reduction in HDL levels, but also increased triglycerides (TG), phospholipids (PL), and cholesterol in very-low-density lipoproteins (VLDL). Both male and female PLTP-KO mice displayed increased hepatic PL and decreased TG, and in the females, increased hepatic cholesterol was also detected. Primary hepatocytes from PLTP-KO mice displayed a different PL molecular species composition to the wild type (WT) controls, with prominent changes being a reduction of long chain fatty acid-containing and an increase of medium chain mono- or di-unsaturated fatty acid containing PL species. Cultured PLTP-KO hepatocytes synthesized and secreted apoA-I in similar quantities as the WT cells. However, the apoA-I secreted by PLTP-KO hepatocytes contained less choline PL, differing also in phosphatidylcholine/sphingomyelin ratio and fatty acyl species composition when compared to apoA-I from WT hepatocytes. Furthermore, the PLTP-KO-derived PL-deficient apoA-I was less stable in the hepatocyte culture medium than that produced by WT cells. These results demonstrate a complex regulatory role of PLTP in serum and liver lipid homeostasis, as well as in the formation of nascent apoA-I-PL complexes from the liver.
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Apolipoproteína A-I/sangre , Hepatocitos/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Fosfolípidos/metabolismo , Animales , Apolipoproteína A-I/biosíntesis , Apolipoproteína A-I/metabolismo , Células Cultivadas , Femenino , Hepatocitos/citología , Lipoproteínas/sangre , Hígado/citología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Transferencia de Fosfolípidos/deficiencia , Proteínas de Transferencia de Fosfolípidos/metabolismo , Triglicéridos/sangreRESUMEN
BACKGROUND: It is becoming increasingly clear that liver steatosis, a typical early consequence of alcohol exposure, sensitizes the liver to more severe inflammatory and fibrotic changes. On the other hand, activation of the key complement component C3, a central player in causing inflammation and tissue damage, is also known to be involved in the regulation of lipid metabolism. This prompted us to study the development of alcoholic liver steatosis in mice lacking C3 (C3-/-). RESULTS: Both C3-/- and normal C3+/+ mice were fed a steatosis-promoting high-fat diet with or without ethanol for 6 weeks. The diet without ethanol caused moderate liver steatosis in C3-/- but not in C3+/+ mice. As expected, ethanol-containing diet caused marked macrovesicular steatosis and increased the liver triglyceride content in C3+/+ mice. In contrast, ethanol diet tended to reduce steatosis and had no further effect on liver triglycerides in C3-/- mice. Furthermore, while in normal mice ethanol significantly increased the liver/body weight ratio, liver malondialdehyde level and serum alanine aminotransferase (ALT) activity, these effects were absent or small in C3-/- mice. A separate experiment with mice on chow diet confirmed the aberrant steatotic effect of ethanol in C3-/-mice: 4 hours after acute dosing of ethanol the liver triglyceride level had increased by 138% in C3+/+ mice (P<0.001), but only by 64% in C3-/- mice (n.s.). CONCLUSION: In C3-/- mice alcohol-induced liver steatosis is absent or strongly reduced after chronic or acute alcohol exposure. This suggests that the complement system and its component C3 contribute to the development of alcohol-induced fatty liver and its consequences.
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Complemento C3/metabolismo , Etanol/administración & dosificación , Hígado Graso Alcohólico/metabolismo , Alanina Transaminasa/sangre , Animales , Complemento C3/genética , Grasas de la Dieta/administración & dosificación , Hígado Graso Alcohólico/sangre , Hígado Graso Alcohólico/patología , Peroxidación de Lípido , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Triglicéridos/metabolismoRESUMEN
Niemann-Pick type C (NPC) disease is a neuro-visceral cholesterol storage disorder caused by mutations in the NPC-1 or NPC-2 gene. In the present paper, we studied IR (insulin receptor) activation and the plasma-membrane lipid assembly in primary hepatocytes from control and NPC1-/- mice. We have previously reported that, in hepatocytes, IR activation is dependent on cholesterol-sphingolipid rafts [Vainio, Heino, Mansson, Fredman, Kuismanen, Vaarala and Ikonen (2002) EMBO Rep. 3, 95-100]. We found that, in NPC hepatocytes, IR levels were up-regulated and the receptor activation was compromised. Defective IR activation was reproduced in isolated NPC plasma-membrane preparations, which displayed an increased cholesterol content and saturation of major phospholipids. The NPC plasma membranes were less fluid than control membranes as indicated by increased DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence anisotropy values. Both in NPC hepatocytes and plasma-membrane fractions, the association of IR with low-density DRMs (detergent-resistant membranes) was increased. Moreover, the detergent resistance of both cholesterol and phosphatidylcholine were increased in NPC membranes. Finally, cholesterol removal inhibited IR activation in control membranes but restored IR activation in NPC membranes. Taken together, the results reveal a lipid imbalance in the NPC hepatocyte, which increases lipid ordering in the plasma membrane, alters the properties of lipid rafts and interferes with the function of a raft-associated plasma-membrane receptor. Such a mechanism may participate in the pathogenesis of NPC disease and contribute to insulin resistance in other disorders of lipid metabolism.
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Hepatocitos/metabolismo , Microdominios de Membrana/metabolismo , Enfermedades de Niemann-Pick/metabolismo , Enfermedades de Niemann-Pick/patología , Proteínas/genética , Receptor de Insulina/metabolismo , Animales , Células Cultivadas , Colesterol/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Proteína Niemann-Pick C1 , Enfermedades de Niemann-Pick/genética , Proteínas/metabolismo , Transducción de SeñalRESUMEN
The complement system can promote tissue damage or play a homeostatic role in the clearance and disposal of damaged tissue. We assessed the role of the terminal complement pathway in alcohol-induced liver damage in complement C6 (C6-/-) genetically deficient rats. C6-/- and corresponding C6+/+ rats were continuously exposed to ethanol by feeding ethanol-supplemented liquid diet for six weeks. Liver samples were analyzed for histopathology and complement component deposition by immunofluorescence microscopy. Prostaglandin E receptors and cytokine mRNA levels were analyzed by RT-PCR and plasma cytokines by ELISA. Deposition of complement components C1, C3, C8 and C9 was observed in C6+/+ rats, but not in C6-/- animals. The histopathological changes, the liver weight increase and the elevation of the plasma pro-/anti-inflammatory TNF-alpha/IL-10 ratio were, on the other hand, more marked in C6-/- rats. Furthermore, ethanol enhanced the hepatic mRNA expression of the prostaglandin E receptors EP2R and EP4R exclusively in the C6-/- rats. Our results indicate that a deficient terminal complement pathway predisposes to tissue injury and promotes a pro-inflammatory cytokine response. This suggests that an intact complement system has a protective function in the development of alcoholic liver damage.