Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.     

肝再生中α2-巨球蛋白的表达动态分析

目的确定更多的与肝再生相关的基因.方法借助4～8正向抑制性消减文库(0-4-8-12短间隔连续部分肝切除)产生的α2-巨球蛋白基因片段,运用cDNA芯片分析了其mRNA在再生肝中的表达动态.结果肝再生中α2-巨球蛋白呈上调表达,而且变化幅度很大,至高点超过21倍.大部分肝切除中α2-巨球蛋白的表达高峰出现在8 h和16 h.短间隔连续部分肝切除中α2-巨球蛋白的表达呈现不同的变化趋势,4 h短间隔的连续肝切除诱导α2-巨球蛋白表达不断升高,而第二次的36 h短间隔的连续肝切除才能诱导α2-巨球蛋白表达显著升高.结论肝再生中α2-巨球蛋白可能在早期的正相急性反应中起重要作用,减少切除伤害带来的炎症,促进肝再生的顺利进行.     

Polyclonal B cell activator associated with alpha-2-macroglobulin in the serum of patients with rheumatoid arthritis

We investigated whether patients with rheumatoid arthritis have a polyclonal B cell activator (PBA) in their serum by using three methods: (1) the ability of any PBA to maintain the surface Ig of rabbit or human B cells in vitro; (2) the induction of blast transformation in human B lymphocyte cultures, and (3) stimulation of nude mouse spleen cells in vitro. All three methods indicated that a PBA is present in the serum of patients with rheumatoid arthritis but not in normal individuals or in patients with arthritis in which autoimmune phenomena have not been demonstrated. The entire PBA activity in rheumatoid arthritis patient serum was found associated with the macroglobulin fraction obtained by Sephadex G-200 chromatography and was precipitated by rabbit anti-human alpha 2-macroglobulin, but not by rabbit anti-Ig antibody. When alpha 2-macroglobulin was purified from patient serum the entire PBA activity was recovered in this fraction. Normal alpha 2-macroglobulin prepared by the same procedure had no PBA activity. Thus, the existence of a PBA associated with alpha 2-macroglobulin was demonstrated in serum of patients with rheumatoid arthritis.     

Immunological studies of human placentae: identification and distribution of proteins in immature chorionic villi.

Cryostat sections of normal human first-trimester placentae have been studied by indirect immunofluorescence to localize numerous protein antigens to well-defined anatomical regions. Transferrin was identified in the apical aspect of all trophoblasts. Fibrinogen/fibrin was also clearly demonstrated in speckled pattern in this location and similar reactions were also observed for alpha-2-macroglobulin, IgG and C3. Plasminogen was identifiable in trophoblasts, and beta-2-microglobulin was uniformly absent from these cells. Collagen and fibrinogen/fibrin were demonstrated on trophoblastic basement membrane, whereas IgG and C3 were only very rarely identified at this site. Few antisera gave strong staining of first-trimester villous stroma, although collagen was shown to contribute much of the stromal matrix. Fibrinogen/fibrin, plasminogen, alpha-2-macroglobulin, C4, C3, C1q, IgG and caeruloplasmin gave positive reactions in fibrin and fibrinoid areas, and phytohaemagglutinin was demonstrated to bind strongly to these areas as well as to trophoblastic basement membrane.     

Quantitative study of alpha 1 antitrypsin and alpha 2 macroglobulin in the sputum]

A quantitative study of alpha-1-antirypsin and alpha-2-macroglobulin in the sputum showed an increase in both during attacks of asthma and in patients with chronic obstructive bronchitis. The levels were much lower in asthma patients during remissions, in emphysema and in patients with chronic simple bronchitis, without associated bronchospasm. The parallel variations in levels of alpha-1-antitrypsin, albumin and transferrin in the sputum, contrasts with the course of secretory IgA and are in favour of a transudation process of alpha-1-antitrypsin across the respiratory mucosa.     

Monoclonal antibodies to rabbit alpha-2-macroglobulin and their use in a sensitive ELISA assay

alpha-2-macroglobulin (alpha-2-M), a plasma proteinase inhibitor, plays an important role in the pathogenesis of lung inflammation. The purpose of this study was to develop a sensitive ELISA assay for rabbit alpha-2-M to allow us to define the role of this protein in a rabbit model of lung inflammation/injury. Therefore, we developed hybridomas which secrete monoclonal antibodies (mAbs) against rabbit alpha-2-M. From the antibodies produced, two (5B6 and 3C5) were selected. Both of them were of the IgG1 subclass. 5B6 reacted with native alpha-2-M as well as with the "fast" form of alpha-2-M (native alpha-2-M or the "slow" form is converted to the "fast" form by reaction with a proteinase). On the other hand, 3C5 reacted only with "fast" form of alpha-2-M. With these antibodies, we developed two ELISA assays which were used to determine the concentration of alpha-2-M in the lung fluids from rabbits with lung injury.     

Monoclonal antibodies to rabbit α-2-macroglobulin and their use in a sensitive ELISA assay

alpha-2-macroglobulin (alpha-2-M), a plasma proteinase inhibitor, plays an important role in the pathogenesis of lung inflammation. The purpose of this study was to develop a sensitive ELISA assay for rabbit alpha-2-M to allow us to define the role of this protein in a rabbit model of lung inflammation/injury. Therefore, we developed hybridomas which secrete monoclonal antibodies (mAbs) against rabbit alpha-2-M. From the antibodies produced, two (5B6 and 3C5) were selected. Both of them were of the IgG1 subclass. 5B6 reacted with native alpha-2-M as well as with the "fast" form of alpha-2-M (native alpha-2-M or the "slow" form is converted to the "fast" form by reaction with a proteinase). On the other hand, 3C5 reacted only with "fast" form of alpha-2-M. With these antibodies, we developed two ELISA assays which were used to determine the concentration of alpha-2-M in the lung fluids from rabbits with lung injury.     

Vascular protein deposits in temporal arteritis with special reference to failure of histological findings

The object of an immunohistochemical search for vascular protein deposits in temporal arteritis is to assess the diagnostic possibilities in cases which are clinically typical but unconfirmed by biopsy results. In a group of older patients with arteritis, however, vascular aging may give rise to intimal thickening and a broad-spectrum deposition of protein. In an inter- and intra-individual comparison of vascular segments with and without arteritis we, however, found a few protein markers in arteritis which are essentially different from those in vascular aging. The intimal thickening and immune reaction in 9 selected marker proteins were graded 0-2, using the tunica media as reference for both properties. Of the nine proteins studied, alpha-2-macroglobulin was significantly increased, not only in segments affected with arteritis, but also in unaffected segments from the same biopsy as compared with biopsies from patients not suffering from this disease. 79% of patients with biopsy-confirmed arteritis also showed a significantly elevated serum alpha-2-macroglobulin as compared to 27% of those having only changes attributed to aging. In conclusion, immunohistochemical demonstration of deposits in the arterial wall and elevated serum levels of alpha-2-macroglobulin substantiate the clinical suspicion of arteritis in the absence of histological and inflammatory changes.     

Study of various serum proteins in lung cancer. Immunoglobulins A, G, M, haptoglobin, alpha-1-antitrypsin, alpha-2-macroglobulin]

A statistical study of the plasma concentration of immunoglobulin, alpha-1-antitrypsin, haptoglobin, and alpha-2-macroglobulin in 153 patients with primary carcinoma of the lung showed a strong increase in alpha-1-antitrypsin, haptoglobin and A and C immunoglobulins, whilst alpha-2-macroglobulin increases very moderately, and IgM does not vary. The relationships between variables are also modified, thus there appears a correlation between IgA and IgG to the detriment of the IgG/IgM relationship. Furthermore the coefficient of the correlation IgG/alpha-2-macroblobulin is lower in cancer patients.     

Hemodialysis and the acute-phase response in chronic uremic patients

In comparison with healthy persons, chronic uremic patients on regular hemodialysis treatment had significantly higher blood serum concentrations of alpha 1-acid glycoprotein, ceruloplasmin and C4 complement component, while levels of haptoglobin, C3 and transferrin were lower. Serum alpha 2-macroglobulin and alpha 1-antitrypsin levels were similar in both groups. Hemodialysis with cuprophan membrane induced only slight changes in some of these glycoproteins during a 48-hour follow-up period. Seven hours after termination of hemodialysis slight, but significant, decreases in blood serum transferrin and alpha 1-antitrypsin concentrations were observed. Hemodialysis thus does not seem to induce a conspicuous acute-phase reaction.     

Modifications of serum glycoproteins the days following a prolonged physical exercise and the influence of physical training

Summary Eight male subjects (mean age 24.1±2.6 years) performed at intervals of 2 weeks successively a 3 h and two 2 h runs of different running speed. The days following the running there were moderate elevations of C-reactive protein, haptoglobin, alpha-1-acid glycoprotein, coeruloplasmin, transferrin, alpha-1-antitrypsin and plasminogen. There were small or no changes of albumin, alpha-2-macroglobulin and hemopexin. The elevations of the acute phase reactants were examined in three male subjects following a 2 h run before and after an endurance training period of 9 weeks. This demonstrated a decreased acute phase response after training as illustrated by the changes of C-reactive protein, haptoglobin and alpha-1-acid glycoprotein in spite of higher posttraining running speeds. Well-trained athletes have elevated levels of the serum protease inhibitors alpha-1-antitrypsin, alpha-2-macroglobulin and C₁-inhibitor. These antiproteolytic glycoproteins might limit exercise-induced inflammatory reactions.This research was supported by the Bundesinstitut für Sportwissenschaften (Köln-Lövenich)     

Serum folic acid activity (L. casei) in Sephadex gel chromatography

The folic acid activity (FAA) of the protein zone in DEAE Sephadex A-50 chromatography of normal serum showed the following distribution: most (about 40%) was eluted with protein zone IV (alpha-2-macroglobulin), about 33% with protein zones II-III (transferrin), and the balance with zone V (albumin). In healthy human subjects the variations were relatively small. The repeatability of the chromatography in an individual was good.Towards the end of pregnancy the binding of FAA to protein zones II-III increased, while that to zone IV decreased. The return to normal started a few days after delivery.The binding to zones II-III was greatly increased in patients with pernicious anaemia but no neuropathy. It became normal during B(12) therapy. If the patient had neuropathy, his FAA chromatogram was close to normal. During therapy the amount of unbound FAA decreased rapidly.The use of diphenylhydantoin increased the binding of FAA to protein zone I and to zones II-III. In these patients the binding to protein zone IV was slighter than normal.     

Immunological studies of human placentae: identification and distribution of proteins in mature chorionic villi.

Cryostat sections of fifteen full-term, normal, human placentae have been studied by indirect immunofluorescence, using characterized antisera to a large number of proteins, in order to localize particular protein antigens to well-defined anatomical regions. Actin, plasminogen, and transferrin were identified in trophoblasts. Beta-2-microglobulin was uniformly absent from these cells. The complement component C3, the immunoglobulin IgG, and fibrinogen and collagen were identified in trophoblastic basement membranes. Of these IgG was the most sparingly represented, and C3 was not found on all trophoblastic basement membranes. Many proteins were identified within the stroma of chorionic billi, collagen being the most abundant. Many of these persisted following prolonged washing of the tissues.All four IgG subclasses were present in stroma, IgG1 and IgG3 being most prominent. Stromal cells were positive for beta-2-microglobulin, suggesting a unique sequestration of this protein on stromal cells but not on trophoblasts. Placental stem vessels were surrounded by collagen, and the walls of these vessels stained brightly for myosin: the vascular endothelium contained beta-2-microglobulin and actin. Peri- and inter-villous fibrin were faintly positive for several proteins, but these areas stained intensely for fibrinogen, plasminogen, alpha-2-macroglobulin and C4. This approach has proven to be useful in establishing precise definitions of the molecular morphology of normal human placentae.     

The use of conventional antisera in the production of specific monoclonal antibodies

Monoclonal antibodies specific for human alpha-2-macroglobulin (alpha 2M), a plasma glycoprotein, have been produced using a novel immunization method. Commercially available antisera to human alpha 2M was used to precipitate the antigen from whole serum. Immunization of animals with this immunoprecipitate resulted in the production of hybridomas with a specificity for human alpha-2-macroglobulin as confirmed by immunoprecipitation.     

Comparison of protein concentrations in saliva and serum

Saliva and serum protein profiles composed of 12 proteins: IgA, IgG, IgM, albumin, transferrin, alpha 1-glycoprotein acid, alpha 2-macroglobulin, alpha 1-proteinase inhibitor, haptoglobine, C3C, C4 and CRP were obtained by laser nephelometry of aliquots of samples of non-stimulated whole saliva and blood taken at the same time. The population of 138 controls of both sexes was divided into 4 age groups: 1 (20 to 29 years), 2 (30 to 39 years), 3 (40 to 49 years), 4 (50 to 60 years). Using the techniques developed for saliva assay, 10 proteins were measurable: IgA, IgG, IgM, albumin, transferrin, alpha 1-glycoprotein acid, alpha 2-macroglobulin, alpha 1-proteinase inhibitor, haptoglobulin and C3C. The C4 fraction and CRP were present at concentrations lower than the detection limit i.e. at 1.8 and 2.05 mg/l. In the serum 11 proteins were analysed, CRP occurring at less than 0.01 g/l. The statistical two-factor variance analysis test (sex and age) demonstrated the statistically significant influence of age in the saliva for alpha 1-PI and alpha 2-M in the serum for Tf, alpha 2-M and alpha 1-PI, and sex in the serum for IgG, IgM, alpha 1-PI, alpha 2-M and also the interaction of both factors in the serum for alpha 2-M.     

Immunohistochemical and ultrastructural studies on histiocytosis in children

Thirty-four cases of eosinophilic granulomas, 18 cases of diffuse histiocytosis-X, 2 cases of Letterer-Siwe-like syndrome with immunodeficiency, 4 cases of malignant histiocytosis and virus associated hemophagocytic syndrome were studied. On paraffin section, S100 protein, lysozyme, alpha-1-anti-trypsin, alpha-1-antichymotrypsin, alpha-2-macroglobulin, Transferrin, Ferritin, peanuts agglutinin, Concanavalin-A, and dolichos biflorus associated antigen were stained by the immunoperoxidase method. In a few fresh materials, T-cell subpopulation by use of monoclonal antibodies (OKT-3, 4, 6, and OK-M1) was examined by the immunoperoxidase method. Two types of Langerhans' cells were found, one is positive for Ferritin and alpha-2-macroglobulin in diffuse histiocytosis-X cells, and another is negative for them in both eosinophilic granulomas. Diffuse histiocytosis-X cell resembled the transformed type of Langerhans cell more than eosinophilic granuloma cells in cellular differentiation. It seemed that the term prolangerhans' cell proliferation disorder might be responsible for it.     

Serum protein levels in primary biliary cirrhosis

Serum levels of albumin, transferrin, alpha(2)-macroglobulin, beta(1)C/beta(1)A, IgA, IgG, and IgM have been determined in 73 patients with primary biliary cirrhosis and in age- and sex-matched controls. A highly significant fall in albumin was demonstrated, and there were highly significant increases in alpha(2)-macroglobulin and all three immunoglobulin levels. Transferrin and beta(1)/Cbeta(1)A levels were unchanged. No significant correlations were found between the titre of antimitochondrial antibody, the duration of symptoms, and any of the serum proteins estimated. A highly significant positive correlation was present between serum albumin and transferrin levels in both patient and control groups.     

Serum alpha2-macroglobulin, transferrin, albumin, and IgG levels in preeclampsia

A radial immunodiffusion technique has been used to measure levels of four serum proteins in preeclampsia with or without proteinuria and in normal pregnant and non-pregnant controls. In preeclampsia unaccompanied by proteinuria, albumin and transferrin levels are similar to those found in the normal pregnant controls, but there are significant falls in alpha(2)-macroglobulin and IgG. When preeclampsia is accompanied by proteinuria there is a marked fall in albumin and an increase in alpha(2)-macroglobulin. Since alpha(2)-macroglobulin has antiplasmin activity it is possible that increased levels of this protein in preeclampsia accompanied by proteinuria contribute to the intravascular coagulation which has been described in this disorder.     

The humoral immune response in recent pulmonary tuberculosis (author's transl)]

The average serum concentration of immunoglobulins, alpha-1-antitrypsin, haptoglobin and alpha-2-macroglobulin, calculated in 45 adults with pulmonary tuberculosis increased in a variable way for each of the six parameters studied. Tuberculosis does not induce new correlations between the parameters but modifies the intensity of existing links.