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PubMed1992

Efficacy of nafamostat mesilate as a regional anticoagulant in experimental direct hemoperfusion and in plasma exchange on humans.

As an anticoagulant, we compared Nafamostat mesilate (FUT) to heparin in experimental direct hemoperfusion (DHP) and studied the efficacy of FUT in clinical plasma exchange (PE). In in vitro study, FUT (5 micrograms/ml) inhibited the activation of C4 more strongly than heparin (100 U/h), and larger dose of FUT (50 micrograms/ml) inhibited the activation of C3. Experimental DHP with FUT on jaundiced dogs was safely performed, but not with heparin. Clinical PE with FUT was safely performed and the hemostatic condition was not aggravated either during or after PE in patients with bleeding.

Animals↗

PubMed1991

Metalloproteases of Serratia liquefaciens: degradation of purified human serum proteins.

Two representative strains of Serratia liquefaciens, SL 5 (serotype O5:H1) and SL 11 (serotype O1:H1), produced proteases characterized by molecular weights of 52.5 kilodaltons and isoelectric points of 6.2; both enzymes were inhibited by 50 mM EDTA. As demonstrated with SDS-PAGE electrophoresis, the two metalloproteases attacked the following purified human serum proteins: complement components C3, C4, C5, C6, C7, C8, and C9, transferrin, alpha 1-antitrypsin, alpha 2-macroglobulin, fibronectin, type III fibrinogen, immunoglobulin G (heavy chains), and IgM (heavy chains). However, C1q, IgA, haptoglobin, and C-reactive protein were refractory.

Blood Proteins↗

PubMed1986

Inactivation of C3a by a monocarboxypeptidase present in culture supernatants of stimulated guinea pig peritoneal macrophages.

Hog C3a, as well as its derivative C3a-desArg were not found to act cytotoxically on starch gel-induced guinea pig peritoneal macrophages. Likewise, neither peptide significantly modified the secretion of N-acetyl-beta-D-glucosaminidase from these cells. However, C3a rapidly lost its spasmogenic activity during incubation in serum-free macrophage cultures and less rapidly in cellfree supernatants collected from cultured macrophages. The following results indicate that C3a is converted into its spasmogenically inactive derivative C3a-desArg by a macrophage-derived monocarboxypeptidase. The inactivated C3a product does not differ from native C3a in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; it elutes from CM cellulose in the same position as purified C3a-desArg; and it is devoid of the carboxyl-terminal arginyl residue of C3a, but still contains the carboxyl-terminal sequence of C3a-desArg as determined by analysis after treatment with carboxypeptidases B or Y. Furthermore, inactivation of C3a in supernatants of macrophage cultures is completely blocked by the specific carboxypeptidase inhibitors guanidinopropylsuccinic acid and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid in final concentrations of 10 mM and 2.1 mM, respectively. The monocarboxypeptidase is apparently supplied by biosynthesis of new material but is not stored as a preformed enzyme because cycloheximide markedly inhibits its expression.

Acetylglucosaminidase↗

PubMed1999

Primary biliary cirrhosis associated with membranous glomerulonephritis.

A 33-year-old woman was admitted to our department for evaluation of liver dysfunction and proteinuria. A liver biopsy specimen showed ductular proliferation and moderate portal fibrosis indicating stage II primary biliary cirrhosis. A renal biopsy specimen showed mild to moderate mesangial cell proliferation without crescent formation or interstitial nephritis. Immunofluorescent staining revealed deposition of immunoglobulin G (IgG), third component of complement (C3), and Clq on glomerular basement membranes. The findings indicated stage I membranous glomerulonephritis. Administration of ursodesoxycholic acid together with prednisolone, azathioprine, and dipyridamole decreased proteinuria and improved cholestatic liver dysfunction.

Adult↗

PubMed2002

Decay-accelerating factor expression in the rat kidney is restricted to the apical surface of podocytes.

BACKGROUND: Decay-accelerating factor (DAF) has inhibitory activity toward complement C3 and C5 convertases. DAF is present in human glomeruli and on cultured human glomerular visceral epithelial cells (GEC). We studied the distribution and function of rat DAF. METHODS: Function-neutralizing antibodies (Abs) were raised against DAF. The distribution of DAF in vivo was determined by immunoelectron microscopy. Functional studies were performed in cultured GEC and following IV injection of anti-DAF Abs into rats. RESULTS: DAF was present exclusively on the apical surfaces of GEC, and was not present on the basal surfaces of GEC, nor other glomerular or kidney cells. DAF was functionally active on cultured GEC, and served to limit complement activation in concert with CD59, an inhibitor of C5b-9 formation. Upon injection into normal rats, anti-DAF F(ab')2 Abs bound to GEC in vivo, yet there was no evidence for complement activation and animals did not develop abnormal albuminuria. Anti-megalin complement-activating IgG Abs were "planted" on GEC, which activated complement as evidenced by the presence of C3d on GEC. Attempts to inhibit DAF function with anti-DAF Abs did not affect the quantity of complement activation by these anti-megalin Abs, nor did it lead to development of abnormal albuminuria. In contrast, in the puromycin aminonucleoside model of GEC injury and proteinuria, anti-DAF Abs slowed the recovery from renal failure that occurs in this model. CONCLUSION: In cultured rat GEC, DAF is an effective complement regulator. In vivo, DAF is present on GEC apical surfaces. Yet, it appears that DAF is not essential to prevent complement activation from occurring under normal circumstances and in those cases in which complement-activating Abs are present on the basal surfaces of GEC in vivo. However, in proteinuric conditions, DAF appears to be protective to GEC.

Animals↗

PubMed1995

Modulation of antigen processing and presentation by covalently linked complement C3b fragment.

Ligands such as complement fragments (C3, C4), IgG or alpha 2-macroglobulin, which bind antigen (Ag) before their uptake by antigen-presenting cells (APC), are likely to modulate the different steps of Ag processing and presentation. These ligands contribute to internalization and endosomal targeting of Ag; they also influence its processing and, consequently, the binding of resulting peptides to major histocompatibility complex (MHC) class II molecules before presentation to T cells. Complement protein C3 contains, like other members of the alpha 2-macroglobulin family, an intrachain thiolester bond. Conformational alteration or limited proteolysis of C3 into C3b leads to breaking of the thiolester with transient capacity of the revealed carbonyl group to esterify hydroxyl groups of Ag. Ester-linked complexes including tetanus toxin (TT) and C3b were prepared to analyse the influence of bound C3b on TT processing and presentation by APC. Covalent binding of C3b to TT resulted in increased and prolonged stimulation of specific T-cell proliferation. This effect was observed with non-specific B cells, as well as with a TT-specific B-cell clone, as APC. On the other hand, SDS-PAGE analysis of proteolysates of TT or C3b-TT, obtained with endosome/lysosome-enriched subcellular fractions prepared from human Epstein-Barr virus (EBV)-transformed B cells, indicated a delay of TT proteolysis when TT was associated to C3b. Treatment of APC with protease inhibitors, before and during exposure of the cells to Ag, resulted in differences in the inhibition of TT and C3b-TT proteolysis. Using purified cathepsins B and D, we demonstrated that covalent binding of C3b to TT totally abolished TT proteolysis by cathepsin D, while proteolysis by cathepsin B was preserved. This finding and the absence of cathepsin B in endosomes may explain a delay in TT processing when it is associated to C3b. Confirming these data, presentation by formaldehyde-fixed cells of C3b-TT proteolysates showed higher stimulation of specific T-cell clones than formaldehyde-fixed TT proteolysates.

Antigen Presentation↗

PubMed2003

Dose-dependent inhibition of complement in baboons by vaccinia virus complement control protein: implications in xenotransplantation.

Vaccinia virus complement control protein (VCP) is a potent inhibitor of both the alternative and the classical complement pathways through its binding to activated third and fourth components. In addition to its complement inhibiting abilities, VCP can bind heparan sulfate on cell surfaces, resulting in further functional activities. Altogether, the multiple functions of VCP have been shown to reduce the inflammatory response of the host, helping the vaccinia virus to evade immune destruction. Recently, we reported that VCP is able to block hyperacute xenograft rejection, significantly prolonging graft survival in two separate in vivo heterotopic cervical cardiac xenograft models. Histopathological examination of the transplanted hearts receiving VCP revealed marked VCP deposition on the endothelium, a significant reduction in cardiac tissue damage, and significantly less C3, IgG and IgM deposition in the tissue. It is concluded that VCP may inhibit hyperacute xenorejection by binding to the endothelial surface, blocking complement fixation activation, thereby preventing xenoantibody attachment. In the current study, the level of serum complement inhibition was evaluated following different bolus dosages of VCP in baboons. The results indicated that to achieve a satisfactory level of complement inhibition higher doses of VCP are needed in baboons, than previously observed in rats. The current observations are critical for future assessment of the role of VCP to suppress hyperacute rejection following pig-to-baboon xenotransplantation.

Animals↗

PubMed2000

Comparison of fresh frozen plasma with a standardized serum protein solution following therapeutic plasma exchange in patients with autoimmune disease: a prospective controlled clinical trial.

The aim of the study was the comparison of the influence of fresh frozen plasma (FFP) (Freiburg, Germany) and Biseko, Biotest Pharma GmbH (Dreieich, Germany), as a plasma substitute (a standardized, virus inactivated human serum protein solution) on the coagulation factors, inhibitors, proteins, and complement factors in the plasma of autoimmune disease patients following membrane plasma separation. Patients (n = 24) with autoimmune disease were randomized to receive either FFP or Biseko for membrane plasma separation therapy. During each plasma exchange, 100% of the plasma volume was replaced by the respective substitute. Plasma exchange volume was performed once daily for 3 days. Target test parameters of the coagulation system were fibrinogen, fibrinopeptide A, factor VIII (FVIIIC), von Willebrand factor antigen (vWFAg), partial thromboplastin time (PTT), thromboplastin time (Quick value), and antithrombin (AT III). The immunoglobulins were IgG, IgA, and IgM and C-reactive protein (CRP). The thrombocytes were platelet factor 4 (PF4), and complement factors were C3 and C4. Biseko was well tolerated with 1 mild adverse drug reaction (ADR) (n = 1) while FFP gave rise to ADR on 7 occasions (n = 4). Statistically significant differences in the 2 groups were observed for fibrinogen, PTT, Quick value, and AT III. From the clinical point of view, all fluctuations and differences in parameter levels remained clinically silent. The differences had no clinical consequences. Reflecting on a potential decrease in the risk of infections in comparison to FFP therapy and the lower rate of adverse drug reactions, it is possible to postulate an advantage of Biseko for plasma exchange therapy.

Adult↗

PubMed1999

Complement-dependent accumulation and degradation of platelets in the lung and liver induced by injection of lipopolysaccharides.

We found unique behaviors among platelets within a few minutes of the intravenous injection of lipopolysaccharide (LPS) into mice. Platelets accumulated primarily in the liver at lower doses of LPS, but at higher doses they accumulated largely in the lungs. When the platelets accumulated in these organs were degraded, there was a rapid anaphylactoid shock. The platelet response depended on the strain of mouse and on the source of LPS. Of various LPSs tested, the LPS from the smooth type of Klebsiella O3 (KO3-S LPS) was the most potent at inducing the platelet response and shock. K-76 monocarboxylic acid, an inhibitor of complement C5, effectively prevented the KO3-S LPS-induced degradation (but not accumulation) of platelets and the ensuing rapid shock in BALB/c mice. Moreover, in DBA/2 mice (which are deficient in complement C5), platelets accumulated in the lungs and liver in response to KO3-S LPS but soon returned to the circulation without degradation, and there was no rapid shock. The LPS from the rough type of KO3 induced an accumulation of platelets in the liver and lungs but not a degradation of platelets. On the basis of these results and those reported by other investigators, we propose that in the platelet response to LPS, the lectin pathway to form C3 convertase from C4 and C2 is involved in the rapid accumulation of platelets in the liver and lungs and that the pathway from C5 to C9 is involved in the destruction of platelets and the consequent anaphylactoid shock.

Anaphylaxis↗

PubMed1983

Partial myeloperoxidase deficiency in preleukemia.

In seven subjects with partial and apparently acquired form of myeloperoxidase (MPO) deficiency, some functional properties of neutrophils (PMNs) were studied. Five patients suffered from preleukemia, one from diabetes mellitus and one from carcinoma of the breast with bone marrow metastases. Intracellular bactericidal activity, oxygen consumption and superoxide radical production were within normal limits. In three patients with preleukemia, the serum opsonic activity was markedly reduced (less than m-3SD) in an autologous system, but normal in the presence of pooled normal serum. Decreased opsonic activity was also found when these patient's sera were assayed in the presence of normal PMNs. Since the levels of IgG and C3 were comparable in the patients' sera and the pooled serum, a deficiency of another unknown opsonin or the presence of an opsonization inhibitor has to be postulated. The partial MPO defect apparently doesn't decrease the intracellular killing of Staphylococcus aureus by PMNs. The known susceptibility to bacterial infections in preleukemia may be explained by the reduction of serum opsonization conducing to a secondary decrease of the ingestion and killing of bacteria by the PMNs.

Blood Bactericidal Activity↗

PubMed1981

Polyanion, immunoprotein and proteinase inhibitor in ischemic glomerular change.

Human nephrectomy specimens with ischemic glomerular changes were studied by colloidal iron staining and immunofluorescence with respect to glomerular polyanion, immunoprotein deposition and proteinase inhibitor deposition. In the sclerotic area of ischemic glomerular changes, polyanion loss and deposition of IgM, Clq, C4 and C3 were observed. These findings were common in cases with and without chronic pyelonephritis showing a considerable similarity to the glomerular changes of experimental focal sclerosis in rats or focal sclerosing glomerulonephropathy in human. alpha 1-Antitrypsin and alpha 2-macroglobulin (proteinase inhibitors) were also found in the sclerotic area. In particular, alpha 1-antitrypsin was always present in the sclerotic portions of every atrophic nephron and an important role of alpha 1-antitrypsin in atrophy of nephrons was suggested.

Complement System Proteins↗

PubMed1974

Hereditary deficiency of the sixth component of complement in man. I. Immunochemical, biologic, and family studies.

An 18-yr-old black woman in good general health was found to lack serum hemolytic complement activity. The sixth component of complement (C6) was undetectable by functional assay of serum or plasma and by immunoprecipitin analysis of serum. Functional titers of all other complement components were normal. The absence of C6 in the patient's serum could not be accounted for by a circulating C6 inhibitor, and addition of functionally pure C6 to the patient's serum restored hemolytic activity to normal. Both parents of the proband and five of six available siblings had approximately half the normal levels of functional C6. The other sibling had a normal C6 level. These data suggest that both parents and five siblings are heterozygous for C6 deficiency, while the proband is homozygous and one sibling is normal. Thus, C6 deficiency appears to follow classic mendelian inheritance, with all three possible genotypes recognizable within the family. Functional properties of the proband's C6-deficient serum included total absence of bactericidal activity against Salmonella typhi 0 901 and Hemophilus influenzae, type b, and inability to mediate lysis of red blood cells from patients with paroxysmal nocturnal hemoglobinuria in either the acidified serum or "sugar water" tests. The proband's serum did, however, exhibit a normal capacity (a) to generate chemotactic activity during incubation with bacterial endotoxin or aggregated IgG, (b) to mediate the immune adherence phenomenon, and (c) to coat human red blood cells, sensitized by cold agglutinins, with C4 and C3.

Adolescent↗

PubMed2001

Immunization against tumor cell surface complement-regulatory proteins.

Complement is an enzymatic cascade that results in the release of pro-inflammatory anaphylatoxins, C3b deposition and the assembly of the membrane attack complex (MAC), which results in cell lysis. Cells express complement regulatory proteins or inhibitors to protect themselves from bystander attack by complement. Expression of the complement-regulatory proteins CD55, CD46 and CD59 are deregulated in cancer with tumors showing loss of one or more inhibitors and strong overexpression of others. This results in tumors that are resistant to attack by complement and is a major limitation in the use of monoclonal antibodies as monotherapies. However, tumor sensitivity to complement can be restored by co-administration of antibodies that bind to the functional domains of complement-regulatory proteins. Overexpression of complement-regulatory proteins on tumors also makes them potential targets for cancer vaccines. However, these vaccines have to be carefully designed to induce immune responses that recognize inhibitors overexpressed on tumors and that do not detect the levels expressed by normal cells. A human anti-idiotypic antibody that mimics CD55 has been used successfully in over 200 colorectal cancer and osteosarcoma patients. 70% Of patients show CD55-specific immune responses with no associated toxicity. Similar vaccines targeting CD46 and CD59 would eliminate any cell overexpressing a complement inhibitor. Any remaining tumor cell or any tumor cell that loses complement-regulatory proteins in response to therapy would become highly susceptible to in situ complement deposition. In summary, targeting complement-regulatory proteins is a very attractive approach to tumor therapy, although great care must be taken in preventing normal tissue recognition as this could lead to uncontrolled complement deposition and massive cell lysis.

Animals↗

PubMed2005

Anti-heat shock protein 90beta antibodies decrease pre-oligodendrocyte population in perinatal and adult cell cultures. Implications for remyelination in multiple sclerosis.

Lesions in the CNS of patients with multiple sclerosis (MS) often fail to remyelinate, resulting in neurological dysfunction. A key factor seems to be the inefficiency of oligodendrocyte precursor cells (OPCs). We recently reported antibodies against heat shock protein 90beta (Hsp90beta) in MS patients that recognized the antigen on the OPC surface. This study investigates the mechanism and result of anti-Hsp90beta antibody attack. These antibodies induced OPC death in culture in a complement-dependent fashion. Anti-Hsp90beta antibody-induced, complement-mediated OPC death only operated in these cells and caused a significant reduction in the number of O4-positive pro-oligodendrocytes (pre-oligodendrocytes). Adult cultured OPCs also expressed Hsp90beta on their cell surface and were attacked by anti-Hsp90beta antibodies leading to a significant decrease in the pre-oligodendrocyte population. In the presence of low levels of anti-Hsp90beta antibody--i.e. in the range seen in the CSF of MS patients--the complement concentration was critical to reduce the pre-oligodendrocyte population (via attack to OPCs). Higher concentrations of anti-Hsp90beta antibodies and complement became extinct the pre-oligodendrocytes. Complement 1-esterase inhibitor prevented these effects in the pre-oligodendrocyte population. These findings demonstrate, for the first time in vitro, a feasible mechanism to decrease the production of new oligodendrocytes, thus limiting the possibility of remyelination.

Adult↗

PubMed2005

Dextran sulfate as a leukocyte-endothelium adhesion molecule inhibitor of lung injury in pediatric open-heart surgery.

BACKGROUND: In spite of the progress in operative techniques and pre- and postoperative management for congenital heart disease, lung injury induced by the extracorporeal circulation is still a serious insult in pediatric open-heart operations. To prevent this injury, we used a leukocyte-endothelial cell adhesion molecule blocking agent, dextran sulfate, in a clinical setting. METHODS: Sixty mg/kg of dextran sulfate (DS) was intravenously infused to the patients just before cardiopulmonary bypass was started and 600 mg was added to the bypass circuit prime. Thirty patients (DS group, 14 patients with atrial septal defect, and 16 patients with ventricular septal defect) were compared with age and body-weight matched control patients (control group, 14 patients with atrial septal defect, 11 patients with ventricular septal defect). Postoperative respiratory index, white blood cell counts, complement C3 and plasma granulocyte elastase levels during and after the operation were measured. RESULTS: Respiratory index just after the termination of cardiopulmonary bypass was better preserved in the DS group than in the control group (0.50 +/- 0.08 versus 0.81 +/- 0.12, p < 0.01). The sum total amount of measured granulocyte elastase across whole study period was significantly lower in the DS group (p < 0.05). CONCLUSIONS: The data suggeste the possible effects of dextran sulfate in ameliorating post-perfusion lung damage by interfering with leukocyte-endothelial cell adhesion in pediatric open-heart operations. Future application to patients with more complex anomalies is anticipated.

Anticoagulants↗

PubMed1984

Contraction of guinea pig lung by synthetic oligopeptides related to human C3a.

Complement-derived human C3a is a 77 residue protein whose biological activities include the contraction of guinea pig ileum and parenchymal lung strips. The C3a molecule is active at submicromolar concentrations and the spasmogenic activities are absolutely dependent on a carboxy-terminal arginyl residue. Studies with synthetic peptide analogues of C3a have localized the active site for all spasmogenic functions at the carboxy-terminal portion of the native molecule. Studies reported here demonstrate that the spasmogenic action of C3a on guinea pig parenchymal lung tissue is mimicked by synthetic peptides based on the carboxy-terminal sequence of C3a. Synthetic peptides with sequences corresponding to the 5, 8, 13 and 21 carboxy-terminal residues of C3a all possess spasmogenic activity on lung tissue. Molar activities of the synthetic peptides relative to that of C3a increase as the length of the peptide increases. The activity of the pentapeptide C3a 73-77 is only 0.5% that of C3a, while those of C3a 70-77 and C3a 65-77 are 3.8 and 7.8%, respectively. A 21 residue peptide, C3a 57-77, exhibits activity equivalent to native C3a. The synthetic peptides, unlike C3a, fail to produce tachyphylaxis. We compared C3a reactivity of guinea pig parenchymal lung strips with those of the synthetic C3a peptides in the presence of various inhibitor combinations. Responses of lung strips to C3a or the C3a peptides were not significantly inhibited by the antihistamine pyrilamine. However, lung responses to synthetic C3a peptides, like those to C3a, were inhibited by indomethacin. Complete inhibition of responses to C3a or the synthetic C3a peptides was produced in the presence of indomethacin, FPL55712 and pyrilamine.

Acetylcholine↗

PubMed1990

Inhibition of factor I by diisopropylfluorophosphate. Evidence of conformational changes in factor I induced by C3b and additional studies on the specificity of factor I.

The factor I-mediated cleavage of C3b, using factor H as a cofactor was completely inhibited by diisopropylfluorophosphate (DFP) when factor I and C3b were incubated with DFP before the addition of factor H. Inhibition, although to a lesser degree, was observed when factor H was present during DFP-exposure. No inhibition in factor I activity was seen when factor I and H were incubated with DFP either alone or together. It was also demonstrated that the 38-kDa subunit of factor I bound radiolabeled DFP when factor I and C3b together were exposed to DFP. These observations suggest that factor I interacts with C3b in a manner that exposes its catalytic site to DFP, an interaction that is independent of factor H. The inhibitory effect by DFP on factor I led us to further investigate the factor I cleavage products of iC3b, inasmuch as previous reports were ambiguous as to whether digestion occurs in the presence of DFP. Digestion of C3b bound to activated thiol Sepharose (ATS-C3b) in the presence of factor H at low pH and ionic strength and in serum by complement activation produced C3d,g-like fragments with apparent molecular mass of 41 and 43 kDa. These fragments were shown to have three different N-terminal and two different C-terminal ends. The major fragments had N-terminal sequences starting with Glu933, as shown by sequence determination. Traces of fragments extending beyond this point were also found, shown by Western blot analysis using a panel of mAb previously shown to bind to epitopes exposed within a region of C3 spanning residues 929 to 943, as well as a shorter fragment starting with Glu938. When digestion of C3b is carried out in the presence of DFP, the factor I level necessary for digestion is elevated and may explain how the first two cleavages producing iC3b but not the following giving C3d,g, can occur. The finding of several factor I cleavage sites in the C3d,g region of C3 demonstrates that factor I has a broad specificity, mainly for arginyl bonds. It has also been shown to digest a lysyl bond exposed in ATS-bound C3b.

Amino Acid Sequence↗

PubMed2007

Changes in hepatic gene expression related to innate immunity, growth and iron metabolism in GH-transgenic amago salmon (Oncorhynchus masou) by cDNA subtraction and microarray analysis, and serum lysozyme activity.

Growth hormone (GH) transgenic amago salmon (Oncorhynchus masou) were generated with a construct containing the sockeye salmon GH1 gene fused to the metallothionein-B (MT-B) promoter from the same species. This transgene directed significant growth enhancement with transgenic fish reaching approximately four to five times greater weight than control salmon in F(2) and F(3) generations. This drastic growth enhancement by GH transgene is well known in fish species compared with mammals, however, such fish can show morphological abnormalities and physiological disorders like other GH transgenic animals. GH is known to have many acute effects, but currently there are no data describing the chronic effects of over-expression of GH on various hepatic genes in GH transgenic fish. Hepatic gene expression is anticipated to play very important roles in many physiological functions and growth performance of transgenic and control salmon. To examine these effects, we performed subtractive hybridization (using cDNA generated from liver RNA) in both directions to identify genes both increased and decreased in transgenic salmon relative to controls (576 clones were isolated and sequenced in total). Heme oxygenase, vitelline envelope protein, Acyl-coA binding protein, NADH dehydrogenase, mannose binding lectin-associated serine protease, hemopexin-like protein, leucyte-derived chemotaxin2 (LECT2), and many other genes were obtained in higher clone frequencies suggesting enhanced expression. In contrast, complement C3-1, lectin, rabin, alcohol dehydrogenase, Tc1-like transposase, Delta6-desaturase, and pentraxin genes were obtained in lower frequencies. Microarray analysis was also performed to obtain quantitative expression data for these subtracted cDNA clones. Analysis of fish across seasons was also conducted using both F(2) and F(3) salmon. Results of the microarray data essentially corresponded with those of the subtraction data when both F(2) and F(3) fish were completely immature, but the expression pattern was changed when fish approached maturation. Genes showing enhanced expression in GH transgenic fish in F(2) and F(3) by array analysis were vitelline envelope protein, hemopexin-like protein, heme-oxygenase, inter alpha-trypsin inhibitor, LECT2, GTP cyclohydrolase I feedback regulatory protein (GFRP), and bikunin. Reduced expression genes were lectin, Delta6-desaturase, apolipoprotein, and pentraxin. In particular, lectin was found to be highly suppressed in all F(2) and immature F(3) salmon. Further, serum lysozyme activity, one of innate immunity, was significantly (p<0.05) decreased in both F(2) and F(3) GH transgenic fish. These results indicate that the GH transgene fish had altered hepatic gene expression relating to iron-metabolism, innate immunity, reproduction, and growth.

Animals↗