Production of Recombinant Human Transferrin in Eukaryotic Pichia pastoris Expression System.

The development and manufacturing of serum-free culture media allowing reducing the costs of preparations and standardizing the biotechnological process are important trends in biotechnology. Substitution of protein compounds in the serum-free media with recombinant analogues reduces the risk of contamination with various infectious agents. Human transferrin is a protein component of serum-free media responsible for the transport of Fe3+ ions into cells. We generated a producing strain P. pastoris secreting human transferrin to the culture medium. The use of constitutive GAP promoter and maintenance of medium pH at 6.5 allows attaining maximum level of transferrin expression (20 mg/liter).

Expression of human transferrin can be regulated effectively by rabbit transferrin regulatory elements in transgenic mice.

Human transferrin (hTF) belongs to the iron-binding glycoprotein family. It plays an important role in iron transport throughout the body. Transgenic mice are a good model to study how to produce functional hTF on a large-scale. We have improved the expression of hTF and investigated its regulatory mechanism in transgenic mice. Three expression constructs were prepared in which hTF expression was controlled by different regulatory cassettes of rabbit transferrin (rTF). hTF was secreted into serum of transgenic mice when its expression was controlled by the rTF promoter and enhancer, whereas the rTF enhancer in tandem with the rTF promoter repressed hTF secretion into milk. A significant inverse relationship between methylation of the rTF promoter and hTF expression was observed in liver, heart, mammary gland, and muscle of transgenic mice. The highest concentration of hTF was 700 µg/ml in milk.

Evaluation of liver fat in the presence of iron with MRI using T2* correction: a clinical approach.

OBJECTIVES: To assess magnetic resonance imaging (MRI) with conventional chemical shift-based sequences with and without T2* correction for the evaluation of steatosis hepatitis (SH) in the presence of iron. METHODS: Thirty-one patients who underwent MRI and liver biopsy because of clinically suspected diffuse liver disease were retrospectively analysed. The signal intensity (SI) was calculated in co-localised regions of interest (ROIs) using conventional spoiled gradient-echo T1 FLASH in-phase and opposed-phase (IP/OP). T2* relaxation time was recorded in a fat-saturated multi-echo-gradient-echo sequence. The fat fraction (FF) was calculated with non-corrected and T2*-corrected SIs. Results were correlated with liver biopsy. RESULTS: There was significant difference (P < 0.001) between uncorrected and T2* corrected FF in patients with SH and concomitant hepatic iron overload (HIO). Using 5 % as a threshold resulted in eight false negative results with uncorrected FF whereas T2* corrected FF lead to true positive results in 5/8 patients. ROC analysis calculated three threshold values (8.97 %, 5.3 % and 3.92 %) for T2* corrected FF with accuracy 84 %, sensitivity 83-91 % and specificity 63-88 %. CONCLUSIONS: FF with T2* correction is accurate for the diagnosis of hepatic fat in the presence of HIO. Findings of our study suggest the use of IP/OP imaging in combination with T2* correction. KEY POINTS: • Magnetic resonance helps quantify both iron and fat content within the liver • T2* correction helps to predict the correct diagnosis of steatosis hepatitis • "Fat fraction" from T2*-corrected chemical shift-based sequences accurately quantifies hepatic fat • "Fat fraction" without T2* correction underestimates hepatic fat with iron overload.

[The use of EPR spectroscopy to control the changes of organism radiosensitivity/radioresistance. Experimental and clinical results].

The responses of deoxyribonucleotide (dNTP), DNA and protein synthesis systems in blood-forming organs of animals (dogs, mice) as well as changes in Fe(3+)-transferrin (Fe(3+)-TF) and Cu(2+)-ceruloplasmin (Cu(2+)-CP) pools in blood to gamma-irradiation and the administration of radioprotectors have been studied. It has been shown that changes in Fe(3+)-TF and Cu(2+)-CP pools in blood are indices of changes in the body radioresistance and are reliably controlled by the EPR technique. An increase in the Fe(3+)-TF pool promotes the activated synthesis of dNTP, DNA and Fe(3+)-containing proteins which are essential for the repair efficiency during the early post-irradiation time as well as for the development of compensatory and restorative reactions of cellular systems; i.e., they are responsible for the body resistance to DNA-damaging factors. It is important that the intensity of responses depends on the initial state of the organism. It has been shown, that changes in Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools, which are trust-worthy controlled by the EPR technique in whole blood, blood plasma, and serum, as well as the changes in the extracellular DNA content in blood plasma are the markers of the changes in the organism radioresistance. This has been proved during the medical examination of the Chernobyl accident recovery workers and civil population, including children, exposed to low-intensity radiation.

Characterizing antiviral mechanism of interleukin-32 and a circulating soluble isoform in viral infection.

Interleukin-32 (IL-32) is an inflammatory cytokine, and its activity is associated with various auto-inflammatory disorders as well as infectious pathogens such as Mycobacterium tuberculosis, and viral infections. However, the precise antiviral mechanism of IL-32 remains unclear. We assessed the IL-32 level in the sera of H1N1 influenza A patients and IL-32 level was significantly elevated. Next we examined the antiviral activity of recombinant IL-32γ (rIL-32γ) with WISH cells infected by vesicular stomatitis virus (VSV) but no antiviral activity was observed. Therefore we investigated the supernatant of rIL-32-treated THP-1 cells since this cell line effectively responded to rIL-32γ. The supernatant of rIL-32-treated THP-1 cell possessed an antiviral effect and in addition, an agonistic monoclonal antibody further enhanced a specific antiviral activity of rIL-32γ. The fractionation and mass spectrometer analysis of the THP-1 cell supernatant revealed that the antiviral activity of rIL-32γ is via a THP-1 cell-produced factor, transferrin, rather than the direct effects of rIL-32γ on epithelial cells. We also characterized a secreted soluble IL-32γ protein in serum of IL-32γ transgenic mouse (TG), but not in that of IL-32α TG. The present results suggest that IL-32γ expression and its genetic variation in individual could be an important aspect of viral infections.

Bovine prolactin elevates hTF expression directed by a tissue-specific goat ß-casein promoter through prolactin receptor-mediated STAT5a activation.

Prolactin promotes the expression of exogenous human transferrin gene in the milk of transgenic mice. To elucidate this, a recombinant plasmid of bovine prolactin plus human transferrin vector was co-transfected into cultured murine mammary gland epithelial cells. Prolactin-receptor antagonist and shRNA corresponding to prolactin-receptor mRNA were added into the cell culture mixture to investigate the relations between prolactin-receptor and human transferrin expression after bovine prolactin inducement. Levels of human transferrin in the supernatants were increased under the presentation of bovine prolactin (from 1,076 ± 115 to 1,886 ± 114 pg/ml). With the treatment of prolactin-receptor antagonist or shRNA, human transferrin in cells was declined (1,886 ± 113 vs. 1,233 ± 85 pg/ml or 1,114 ± 75 pg/ml, respectively). An inverse correlation was found between the dosage of prolactin-receptor antagonist and expression level of human transferrin. Real-time qRT-PCR analysis showed that the relative level of signal transducer and activator of transcription 5a (STAT5a) transcript in transfected cells correlated with expression levels of human transferrin in the supernatant of the same cells. Bovine prolactin thus improved the expression of human transferrin through such a possible mechanism that bovine prolactin activated STAT5a transcription expression via combined with prolactin-receptor and suggest a potential utility of the bovine prolactin for efficient expression of valuable pharmaceutical proteins in mammary glands of transgenic animals.

N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans.

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. ß1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human ß1,4-mannosyl-ß1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-ß1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-ß1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.

Immune-related genes expression profile in orange-spotted grouper during exposure to Cryptocaryon irritans.

Cryptocaryon irritans is one of the most important ectoparasites of marine fish. To identify the potential role of immune-related genes in antiparasitic immune responses in fish, we monitored the expression change of IL-8, COX-2, C-type lectin and transferrin in local and systemic immune organs of orange-spotted grouper post-C. irritans infection. IL-8 expression was up-regulated during the course of infection in the skin, while COX-2 and transferrin expression was up-regulated in the gill. COX-2 expression was significantly down-regulated in the spleen (0·7-5% of its control) and head kidney (0·5-4% of its control) post-primary infection. Transferrin expression was also down-regulated in the spleen and head kidney from 6 h to 5 days post-primary infection. However, C-type lectin expression was up-regulated in all tested organs post-infection, with the exception of day 7 in the spleen post-primary infection where the expression level was slightly down-regulated (44% of its control). These results suggest that these four immune-related genes play an important role in grouper anti-C. irritans infection and that local immune organs as the active organs contribute more than systemic immune organs to this course.

Activity of secretory sphingomyelinase is increased in plasma of alcohol-dependent patients.

BACKGROUND: Acid sphingomyelinase (ASM, EC 3.1.4.12) hydrolyzes sphingomyelin to ceramide and represents a major regulator of sphingolipid metabolism. Increased activity of ASM has been observed in a variety of human diseases, and a critical contribution of ASM to medical conditions was demonstrated in several mouse models. In agreement with increased ASM activity in cell lines treated with ethanol, we have recently found higher levels of ASM activity in peripheral blood cells of active drinkers. However, the influence of ethanol on secretory ASM (S-ASM) has not been investigated so far. METHODS: ASM activity and routine blood parameters were determined in plasma samples of 27 patients with alcohol dependence during physical withdrawal and compared to a group of 36 healthy volunteers. RESULTS: Compared to the control group, patients with alcohol dependence had S-ASM activity increased by about 3-fold (141 ± 69 vs. 428 ± 220 pmol/ml/h; p < 0.001) at the beginning of physical withdrawal. During withdrawal, S-ASM activity decreased by about 50% (p < 0.001; day 0 vs. day 7 to 10) and finally approximated nearly normal values. On the day of admission, S-ASM activity correlated positively with levels of carbohydrate-deficient transferrin (r = 0.410, p = 0.034) and high-density lipoprotein cholesterol (r = 0.440, p = 0.022) and inversely with body mass index (r = -0.509; p = 0.007), glucose (r = -0.480; p = 0.011), triglycerides (r = -0.592; p = 0.001), and large unstained cells (r = -0.526; p = 0.017). CONCLUSIONS: Activity of S-ASM is increased in alcohol-dependent patients and correlates with established biomarkers of excessive drinking. The increased S-ASM activity is implicated in alcohol-induced lipid alterations and might be relevant for the occurrence of alcohol-related disorders.

Ontogeny and tissue-specific expression of innate immune related genes in rohu, Labeo rohita (Hamilton).

The innate immune response in fish represents an early and rapid defense against pathogens. The present study aims at looking into ontogeny of innate immune system in the teleost, Labeo rohita using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs, and hatchlings (hatched at 28 ± 2 °C) at 0, 1, 3, 6, 12, 24 h, and 3, 7, 16, 21, 31 days post-fertilization were subjected to RT-PCR using self-designed or earlier published primers to amplify some innate immune relevant genes (lysozyme C, lysozyme G, beta-2 microglobulin, toll-like receptor 22-like and transferrin). The constitutive expression of ß-actin was detected in unfertilized eggs and further developmental stages. Transferrin and TLR22-like mRNA transcripts were detected by RT-PCR from 6 h post-fertilization to 31 day post-fertilization, whereas ß-2 microglobulin transcripts were detected only from 7 day post-fertilization onwards. Lysozyme C mRNA transcripts were detected from 24 h post-fertilization to 31 day post-fertilization. Lysozyme G mRNA transcripts were detected early from unfertilized egg stage onwards. Similarly, tissues viz. intestine, heart, ovary, gill, spleen, muscle, liver, brain, skin, anterior kidney, posterior kidney, and blood collected from juveniles of rohu were subjected to detection of all above mentioned gene transcripts by RT-PCR. ß2-microglobulin mRNA transcript was expressed in all tissues. Lysozyme C mRNA expression is confined to blood and posterior kidney only whereas lysozyme G mRNA is expressed in all tissues. TLR22-like mRNA is expressed in all tissues except ovary and liver whereas transferrin mRNA transcript is detected only in liver. Finally, all these information taken are likely to shed light on the ontogeny of innate immunity in L. rohita, which offers new insights to developmental biology when compared to higher vertebrates and also helpful in the development of preventive measures against problems concerning infectious diseases.

Effective treatment of anemia in pediatric kidney transplant recipients with methoxy polyethylene glycol-epoetin beta.

MPG-EPO is a continuous erythropoietin receptor activator with a longer half-life than darbepoetin, hence requires less frequent injections. It has been successfully used in adults, but currently, there are no published data available for its use in children. This pilot study was performed to verify the effect of MPG-EPO on Hb levels in children. Twelve patients (age 6.4-17.2 yr) were treated with MPG-EPO as an individual "Heilversuch" according to German law after RTx. Five patients were switched from DA, and seven were naïve to erythropoietin. Over a period of six months, Hb levels were measured monthly. A median MPG-EPO dose of 2.5 µg/kg was administered intravenously in a single dose every four wk. The median Hb value increased in naïve patients from 9.9 to 11.2 g/dL (median, p = 0.004) and from 10.3 to 11.6 g/dL (median, p = 0.39) in patients switched from DA to MPG-EPO. No adverse events secondary to MPG-EPO therapy were detected. Our results indicate that a once-monthly injection of MPG-EPO is an effective treatment of anemia in children after renal transplantation. Larger randomized trials will have to confirm our findings.

Toluene diisocyanate (TDI) regulates haem oxygenase-1/ferritin expression: implications for toluene diisocyanate-induced asthma.

Diisocyanate is a leading cause of occupational asthma (OA). Diisocyanate-induced OA is an inflammatory disease of the airways that is associated with airway remodelling. Although the pathogenic mechanisms are unclear, oxidative stress may be related to the pathogenesis of diisocyanate-induced OA. In our previous report, we observed that the expression of ferritin light chain (FTL) was decreased in both of bronchoalveolar lavage fluid and serum of patients with diphenyl-methane diisocyanate (MDI)-induced OA compared to those of asymptomatic exposed controls and unexposed healthy controls. In this study of toluene diisocyanate (TDI)-OA, we found identical findings with increased transferrin and decreased ferritin levels in the serum of patients with TDI-OA. To elucidate whether diisocyanate suppresses FTL synthesis directly, we tested the effect of TDI on the FTL synthesis in A549 cells, a human airway epithelial cell line. We found that haem oxygenase-1 as well as FTL was suppressed by treatment with TDI in dose- and time-dependent manners. We also found that the synthesis of other anti-oxidant proteins such as thioredoxin-1, glutathione peroxidase, peroxiredoxin 1 and catalase were suppressed by TDI. Furthermore, TDI suppressed nuclear translocation of Nrf2 through suppressing the phosphorylation of mitogen-activated protein kinases (MAPKs); extracellular-regulated kinase 1/2 (ERK1/2); p38; and c-Jun N-terminal kinase (JNK). Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists, 15-deoxy-Delta(12,14)-PGJ2 and rosiglitazone rescued the effect of TDI on HO-1/FTL expression. Collectively, our findings suggest that TDI suppressed HO-1/FTL expression through the MAPK-Nrf2 signalling pathway, which may be involved in the pathogenesis of TDI-induced OA. Therefore, elucidating these observations further should help to develop the therapeutic strategies of diisocyanate-induced OA.

High-level production of animal-free recombinant transferrin from Saccharomyces cerevisiae.

BACKGROUND: Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin. RESULTS: The first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 µm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin. CONCLUSIONS: A Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics.

Structure and expression of transferrin gene of channel catfish, Ictalurus punctatus.

Transferrin is important in iron metabolism and has been reported to be involved in disease defence responses after bacterial infection. In this study, we identified, sequenced, and characterized the transferrin gene from channel catfish, Ictalurus punctatus. The catfish transferrin gene was similar to those of other vertebrate species with 17 exons and 16 introns. Sequence analysis indicated the presence of the two duplicated lobes, each containing two sub-domains separated by a cleft harboring the iron-binding site, suggesting their structural conservation. The channel catfish transferrin cDNA encodes 679 amino acids with 42-56% similarity to known transferrin genes from various species. Southern blot analysis suggested the presence of two copies of the transferrin gene in the catfish genome, perhaps arranged in a tandem fashion. The catfish transferrin gene was mapped to a catfish BAC-based physical map. The catfish transferrin gene was highly expressed in the liver, but expression was low in most other tested tissues. Transferrin expression was significantly up-regulated after infection with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish. Such induction was also found with co-injection of iron-dextran and E. ictaluri, while transferrin expression was not significantly induced with the injection of iron-dextran alone.

Congenital disorder of glycosylation Ia: new differentially expressed proteins identified by 2-DE.

Congenital disorders of glycosylation (CDG) comprise a family of inherited multisystemic disorders resulting from the deficiency of glycosylation pathways. N-glycosylation defects are classified as two biochemical and genetic established types, of which CDG-Ia is the most frequent. We performed 2-DE proteomic analysis on serum from two functional hemizygous CDG-Ia patients bearing T237M and D65Y missense changes. Comparative analysis of control/patient serum proteome allowed us to identify differential expression of 14 proteins. The most remarkable groups included proteins involved in immune response, coagulation mechanism and tissue protection against oxidative stress. The patient bearing D65Y mutation had less favourable clinical outcome and showed more abnormalities in the spot patterns, suggesting that the proteomic results might also be correlated with the phenotype of CDG patients. This study describes for the first time the differential expression of alpha(2)-macroglobulin, afamin, fibrin and fibrinogen in CDG disorder and shows how the proteomic approach might be useful for understanding its physiopathology.

A loop in the N-lobe of human serum transferrin is critical for binding to the transferrin receptor as revealed by mutagenesis, isothermal titration calorimetry, and epitope mapping.

Transferrin (TF) is a bilobal transport protein that acquires ferric iron from the diet and holds it tightly within the cleft of each lobe (thereby preventing its hydrolysis). The iron is delivered to actively dividing cells by receptor mediated endocytosis in which diferric TF preferentially binds to TF receptors (TFRs) on the cell surface and the entire complex is taken into an acidic endosome. A combination of lower pH, a chelator, inorganic anions, and the TFR leads to the efficient release of iron from each lobe. Identification of residues/regions within both TF and TFR required for high affinity binding has been an ongoing goal in the field. In the current study, we created human TF (hTF) mutants to identify a region critical to the interaction with the TFR which also constitutes part of an overlapping epitope for two monoclonal antibodies (mAbs) to the N-lobe, one of which was previously shown to block binding of hTF to the TFR. Four single point mutants, P142A, R143A, K144A, and P145A in the N-lobe, were placed into diferric hTF. Isothermal titration calorimetry (ITC) revealed that three of the four residues (Pro142, Lys144, and Pro145) in this loop are essential to TFR binding. Additionally, Lys144 is common to the recognition of both mAbs which show different sensitivities to the three other residues. Taken together these studies prove that this loop is required for binding of the N-lobe of hTF to the TFR, provide a more precise description of the role of each residue in the loop in the interaction with the TFR, and confirm that the N-lobe is essential to high affinity binding of diferric hTF to TFR.

Intracellular interactions of transferrin and its receptor during biosynthesis.

The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during biosynthesis were studied in the human hepatoma cell line HepG2, which synthesizes both. Early during biosynthesis the Tfr monomer is converted to a disulfide-linked Tfr dimer. The Tfr monomer is not able to bind Tf, but Tf binding is observed as soon as the covalent Tfr dimer is formed and can take place in the ER. The Tf-Tfr complex is transported through the Golgi reticulum and trans-Golgi reticulum (TGR) and is ultimately delivered to an acidic compartment, where Tf releases its Fe3+. We did not observe conversion of Tf to apoTf in the TGR, showing that the part of the TGR passed by secreted Tf has a pH higher than 5.5. We conclude that when a ligand-receptor combination is synthesized by one and the same cell, ligand and receptor can interact during biosynthesis and be transported to the cell surface.

Study of liver differentiation in vitro.

A clonal rat fetal liver cell line that expresses the functions of differentiated liver cells under controllable conditions has been established. Normal fetal liver cells were transformed by a temperature-sensitive A (tsA) mutant (tsA209) of simian virus 40. At the permissive temperature (33 degrees C), the tsA209-transformed liver cell line (RLA209-15) can be cultured indefinitely and cloned readily. The RLA209-15 cells were temperature sensitive for maintenance of the transformed phenotype. These transformed liver cells selectively lost four characteristics of the transformed phenotype at the restrictive temperature (40 degrees C): generation time of the cells increased, the saturation density decreased, the efficiency of growth on nontransformed cell layers decreased, and the ability to clone in soft agar was lost. The transformation can be reversed simply by a shift in temperature. RLA209-15 fetal liver cells synthesized alpha-fetoprotein albumin, and transferrin. At 33 degrees C, the levels of these liver proteins were relatively low. At 40 degrees C the transformed phenotype was lost and the levels of alpha-fetoprotein, albumin, and transferrin were greatly increased. At the restrictive temperature, maximal induction of the synthesis of alpha-fetoprotein, albumin, and transferrin was achieved 3-4 d after the upward shift in temperature. The synthesis of alpha-fetoprotein then decreased; the synthesis of albumin and transferrin, however, was maintained. A second phase of albumin and transferrin synthesis was observed in all cultures after 6 d or more at 40 degrees C. Alpha-Fetoprotein, albumin, and transferrin secreted by RLA209-15 cells were immunologically indistinguishable from authentic alpha-fetoprotein, albumin, and transferrin, respectively. RLA209-15 cells, like primary cultures of hepatocytes and a simian virus 40 tsA255-transformed fetal liver cell line (RLA255-4) reported earlier from this laboratory, responded to glucagon with markedly elevated levels of cyclic AMP. Thus, it appears that glucagon receptors characteristic of hepatocytes are retained in the simian virus 40 tsA-transformed fetal liver cells.

Inhibition of synthesis of alpha-fetoprotein by glucocorticoids in cultured hepatoma cells.

alpha-Fetoprotein (AFP) synthesis was studied in the presence and absence of glucocorticoids in rat hepatoma Mc-A-RH-7777 cells. Radioimmunoassay of media from cell cultures grown in the presence of glucocorticoid (dexamethasone or cortisol) showed a reduction in AFP, an increase in albumin, and no significant change in transferrin accumulation, as compared to controls. Labeling experiments with L-[35S]methionine indicated that in both cells and media of dexamethasone-treated cultures there was a 50--80% reduction in polypeptide precipitated by anti-AFP serum, as compared with controls; no change was seen in polypeptide precipitated by anti-transferrin serum. Pulse and pulse-chase experiments demonstrated that dexamethasone inhibited the synthesis of AFP but not its secretion. The half-time for secretion of AFP in the presence and absence of dexamethasone was 43 min.

Synthesis and regulation of acute phase plasma proteins in primary cultures of mouse hepatocytes.

Adult mouse hepatocytes respond in vivo to experimentally induced acute inflammation by an increased synthesis and secretion of alpha 1-acid glycoprotein, haptoglobin, hemopexin, and serum amyloid A. Concurrently, the production of albumin and apolipoprotein A-1 is reduced. To define possible mediators of this response and to study their action in tissue culture, we established primary cultures of hepatocytes. Various hormones and factors that have been proposed to regulate the hepatic acute phase reaction were tested for their ability to modulate the expression of plasma proteins in these cells. Acute phase plasma and conditioned medium from activated monocytes influenced the production of most acute phase plasma proteins, and the regulation appears to occur at the level of functional mRNA. Purified hormones produced a significant anabolic response in only a few cases: dexamethasone was found to be effective in maintaining differentiated expression of the cells; and glucagon produced a specific inhibition of haptoglobin synthesis. When cells were treated with a combination of conditioned monocyte medium and dexamethasone, secretion of proteins was markedly reduced. The carbohydrate moieties of all plasma glycoproteins were incompletely modified, apparently as a result of decreased intracellular transport of newly synthesized plasma proteins. Although primary hepatocytes were not phenotypically stable in tissue culture, the cells nevertheless retained a broad response spectrum to exogenous signals. We propose this as a useful system to study the production of plasma proteins and thereby pinpoint the nature and activity of effectors mediating the hepatic acute phase reaction.