Molecular characterization, expression and functional analysis of two Kazal-type serine protease inhibitors from Venerupis philippinarum.

Kazal-type serine protease inhibitors (KSPIs) act as negative regulators in immune signaling pathway by controlling the extent of serine protease (SP) activities. In this study, the full-length cDNA of two KSPIs (designed as VpKSPI-1 and VpKSPI-2) were identified from Venerupis philippinarum by rapid amplification of cDNA ends (RACE) approaches. The open reading frame (ORF) of VpKSPI-1 and VpKSPI-2 was of 552 bp and 402 bp, encoding a polypeptide of 183 and 133 amino acids, respectively. The transcripts of VpKSPI-1 and VpKSPI-2 were ubiquitously expressed in all tissues tested with the highest expression level in hepatopancreas. After Vibrio anguillarum challenge, the relative mRNA expression of VpKSPI-1 and VpKSPI-2 in hepatopancreas was both up-regulated within 96 h. The recombinant VpKSPI-1 (rVpKSPI-1) displayed weak activities towards chymotrypsin, moderate inhibitory activity to trypsin, while rVpKSPI-2 showed significant inhibitory activities against chymotrypsin and trypsin. When the molar ratio of rVpKSPI-2 to chymotrypsin and trypsin reached 1:4 and 1:2, the protease activities could be almost entirely inhibited. All these results suggested that both VpKSPI-1 and VpKSPI-2 perhaps play a vital role in the innate immunity of V. philippinarum.

Mechanisms of IL-8 suppression by Treponema denticola in gingival epithelial cells.

The purpose of this study was to investigate the mechanism(s) of interleukin (IL)-8 suppression by Treponema denticola, one of the major periodontal pathogens, in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with wild-type (WT), dentilisin-deficient (K1) or flagellin-deficient (flgE) T. denticola in the presence or absence of 2% human serum for 24 h. The levels of IL-8 expression were measured with real-time reverse transcription PCR and ELISA. In the absence of human serum, the WT and flgE, but not K1, substantially reduced not only the levels of IL-8 protein but also of IL-8 mRNA. Such downregulation of IL-8 mRNA was independent of bacterial invasion. Degradation of cytokine mixture by the WT, K1 and flgE revealed dentilisin-dependent preferential degradation of tumor necrosis factor (TNF)-α, an IL-8-inducing cytokine. WT and flgE significantly decreased the levels of TNFα secreted by HOK-16B cells, suggesting modulation of IL-8 through dentilisin-mediated degradation of TNFα. The addition of human serum to the culture potentiated the suppressive effect of T. denticola, resulting in substantial reductions of IL-8 and TNFα levels, even by K1. The serum-dependent effects of T. denticola were attributed to its ability to suppress the accumulation of intracellular reactive-oxygen species (ROS), a group of ubiquitous signaling molecules. Pretreatment with an antioxidant suppressed TNFα-induced IL-8 expression, confirming the role of ROS in TNFα signaling. Collectively, T. denticola targeted a key inflammatory cytokine and its signaling molecule to modulate the host innate immune response, which provides a new insight into modulation of host immunity by a periodontal pathogen.

Pattern of MHC class I and immune proteasome expression in Walker 256 tumor during growth and regression in Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis.

Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.

Comparison of the peptidase activity in the oncosphere excretory/secretory products of Taenia solium and Taenia saginata.

We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluorometrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.

A chymotryptic-type serine protease is required for IL-2 production by Jurkat T cells.

Interleukin-2 (IL-2) production by activated Jurkat T cells was markedly delayed when these cells were treated with low concentrations of the chymotryptic-type protease inhibitor N-alpha-p-tosyl-L-phenylalanine chloromethylketone (TPCK). This increased lag time observed in the presence of TPCK directly correlates with the interaction of the inhibitor with a unique 42,000 molecular weight (MW) serine protease, which can be labelled with [3H]DFP, and was not due to an intracellular accumulation of a non-mature form of IL-2 nor to a non-specific inhibition of overall protein synthesis. The results presented in this report indicate that a 42,000 MW chymotryptic-like serine protease is required for IL-2 production by activated Jurkat T cells.

Monoclonal antibody to macrophages (EMB/11) labels macrophages and microglial cells in human brain.

Normal and diseased human central nervous system (CNS) tissues were studied immunohistochemically by a monoclonal antibody to human macrophages (EBM/11), antisera to glial fibrillary acidic protein (anti-GFAP), and alpha-1-antichymotrypsin (alpha 1-ACT). EBM/11 reacted with brain macrophages located mainly around blood vessels in normal brain; it also reacted with resting microglia in normal brain and with numerous reactive microglia and macrophages in brain tumours and inflammatory lesions. Microglia did not react with anti-GFAP or alpha 1-ACT. An EBM/11 positive phenotype, therefore, is shared by microglia and macrophages and suggests that microglial cells form a specialised part of the mononuclear phagocyte system.

Expression of an active proteinase inhibitor, alpha 1-antichymotrypsin, by human breast epithelial cells.

The synthesis of an active proteinase inhibitor, gp 66, by human breast epithelial cells is reported. This glycoprotein is identical to serum alpha 1-antichymotrypsin, which inhibits proteinases that cleave at hydrophobic residues. Immunohistological studies show the in vivo expression on normal secretory and ductal epithelial cells and on primary and metastatic adenocarcinomas. Immunoaffinity-purified gp 66 from MCF-7 culture supernatants is an active inhibitor of chymotrypsin as determined in a fluorogenic enzyme assay and can form stable 88 kDa enzyme-inhibitor complexes. The synthesis of a functional inhibitor may represent the epithelial cell's attempt to stabilize its extracellular milieu.

Immunohistochemistry of primary gastrointestinal lymphomas: a study of 76 cases.

A retrospective study of 76 primary gastrointestinal lymphomas utilizing an avidin: biotinylated horseradish peroxidase complex (ABC) technique demonstrated 22 B-cell lymphomas, including two associated with alpha-heavy chain disease. Seven cases were classified as true histiocytic lymphomas based on a positive reaction for one or more of three histiocytic enzyme markers utilized, predominantly alpha-1-antitrypsin and alpha-1-antichymotrypsin. However, in 20 cases, an intense admixture of reactive histiocytes was noted and these cells stained preferentially for the enzyme, lysozyme. Twenty cases, which stained for both kappa and lambda light chains and positively or negatively for albumin, could not be classified and 27 cases failed to stain with any of the antisera utilized.

Combined immunoreaction and Papanicolaou's stain on cytological smears.

The method described below combines an immunoreaction with Papanicolaou's stain on cytological smears. For the immunoreaction, the avidin-biotin-complex (ABC) method was used. The method was tested on various cytological material with the monoclonal antibody lu-5 and two polyclonal antibodies (anti-keratin and anti-chymotrypsin). Wet fixation of the smears with a modified Delaunay's solution is recommended. Drying of the material impairs immunoreactivity. The main advantages of the technique are the clear-cut permanent immunostaining and the preservation of the nuclear structure, permitting a combined immuncytological characterization of cellular products and conventional cyto-diagnosis.

A chymotrypsin-type serine protease in rat basophilic leukemia cells: evidence for its immunologic identity with atypical mast cell protease.

Activity of a chymotrypsin-type serine protease was found in a subline of rat basophilic leukemia (RBL-2H3) cells. The protease was immunologically cross-reactive with anti-atypical mast cell protease immunoglobulin (Ig) G, and its activity was inhibited in a dose-dependent manner by the antibody. The apparent m.w. of the protease that reacted with the antibody was 25,000, which was identical with that of atypical mast cell protease in rat mucosal mast cells. These results show that the chymotrypsin type serine protease in RBL-2H3 cells is immunologically identical with atypical mast cell protease, which was first purified from rat small intestine. Immunohistochemical studies showed that the protease was located not only in intracytoplasmic granules but also in organelles synthesizing protein, such as cisternae of the rough endoplasmic reticulum, perinuclear spaces, and the Golgi apparatus. However, no immunoreactivity was demonstrated in rat basophils. The activity of the protease increased in the exponential phase of growth of RBL-2H3 cells in which some activity was also detected in the medium, and it decreased in the late stationary phase.

The role of immunoperoxidase staining in diagnostic cytology.

A survey was made of the immune staining characteristics of 60 malignant neoplasms. Cytologically positive smears from each case were tested against a panel of six antibodies (alpha-antichymotrypsin, carcinoembryonic antigen, cytokeratin, desmin, vimentin and S-100 protein). The smears were decolorized and stained with polyclonal sera using the standard avidin-biotin immunoperoxidase procedure. In selected cases, the application of Diatex compound for partition of smears was necessary to obtain optimal results. Most staining reactions reflected the histogenesis of the neoplasms. However, more than one of five reactions was nonconclusive due to background staining, scanty cellularity or poor cytoplasmic preservation; furthermore, the 238 reactions scored as positive or negative included 33 unexpected positives and 15 unexpected negatives. In a series of 20 additional cases, selective immunoperoxidase staining was used in an attempt to solve specific diagnostic problems; the results in 13 of 15 cases with conclusive staining agreed with the cytologic impression. It is concluded that standard immunoperoxidase techniques can contribute to the solution of certain diagnostic problems in cytology; however, the results should be interpreted with caution and with full knowledge of the limitations of the technique.

Primary malignant hepatic tumours in childhood.

Twenty-four cases of hepatoblastoma, 14 cases of hepatocellular carcinoma and three cases of malignant mesenchymoma out of a total of 54 primary liver tumours were studied by light microscopy and immunohistochemistry. A remarkable finding in one case of hepatoblastoma and one case of hepatocellular carcinoma was a sarcoid-like reaction in the tumour tissue. Three cases of hepatoblastoma presented a macrotrabecular pattern. Among hepatocellular carcinomas, three cases corresponded to the fibrolamellar variant. By immunohistochemistry, the proportion of cases with positive staining for alpha 1-fetoprotein was higher in hepatoblastoma than in hepatocellular carcinoma. HBs-antigen could be demonstrated in non-neoplastic liver cells in two cases of hepatocellular carcinoma, but not in the tumour cells. No strong correlation between histological pattern and prognosis could be established in hepatoblastoma. However, there was a tendency to more aggressive biological behavior in cases with pronounced mitotic activity. The number of mitoses in hepatoblastoma varied widely. As in previous studies, patients with the fibrolamellar variant of hepatocellular carcinoma fared better than those with the classical type of this tumour. Prognosis in malignant mesenchymoma was not as poor as suggested from previous studies.

Isolation and characterization of two proteins copurifying with carcinoembryonic antigen.

Using common purification procedures CEA was eluted as a symmetrical peak after gel-chromatography with a molecular weight (mw) of 180,000. The purity was assessed by the Ouchterlony test and immunoelectrophoresis, and by SDS-PAGE, where only one precipitation line and one band were obtained. However, two weak bands with a mw of 45,000 and 58,000 appeared, when iodinated CEA preparations were analyzed on SDS-PAGE. It was impossible to separate these proteins from CEA by a great variety of purification procedures. Out of several different immune sera, only two, anti-alpha-antitrypsin and anti-alpha-1-antichymotrypsin, reacted with the proteins. Furthermore, antisera against these protease inhibitors also immunoprecipitated the typical 180,000 mw band of CEA. This was also true for CEA prepared by other laboratories. We have purified and partially characterized both proteins. Although reacting with antisera against alpha-1-antitrypsin and alpha-1-antichymotrypsin they were not identical to the protease inhibitors, because they possess a different N-terminal amino acid sequence than published for them. However, the comparison of their sequences to 1900 total protein sequences made by computer search revealed a strong homology of the N-terminal sequence of the 45,000 mw protein with an internal sequence of alpha-1-antitrypsin. For the 58,000 mw protein no significant homology was found.

Production of human alpha 1-antichymotrypsin-like protein by a human malignant melanoma transplanted into nude mice.

A tissue culture line of a human malignant melanoma, SEKI, induced cachexia in nude mice (BALB/c-nu/nu) (Kondo et al., Cancer Res., 41: 2912-2916, 1981). During the investigation of the cause of the cachexia, the melanoma was found to produce a protein immunologically identical to human alpha 1-antichymotrypsin (alpha 1-Ach). Tissues of the SEKI melanoma contained the protein immunologically equivalent to 0.29 +/- 0.11 (S.D.) mg of human alpha 1-Ach per g of wet tissue, while the other six human malignant tumors transplanted into nude mice did not contain a detectable amount of it. In the serum of nude mice bearing the melanoma, this protein appeared soon after the tumor growth occurred and gradually increased up to the level equivalent to 5 mg of human alpha 1-Ach per dl. Removal of the tumor resulted in a rapid decrease of the protein in the serum to an undetectable level within 1 day. This problem was never detected in the serum of nude mice bearing the other 27 human malignant tumors or controls. Purification of this protein was carried out by the column chromatography using DE-52, Blue-Sepharose, and SP-Sephadex. The elution patterns were the same as those of alpha 1-Ach in human serum, and the molecular weight of the protein was estimated as 69,000 by Sephadex G-100 column chromatography and 65,000 by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. This purified protein, however, did not exhibit inhibitory activity against chymotrypsin. These results show that this melanoma produced a protein immunologically identical and physicochemically very similar to human alpha 1-Ach. This melanoma-nude mouse system may provide a useful model for investigating the synthesis of human alpha 1-Ach and analysis of its physiological roles.

Migration inhibition factor-like activity in inflammatory synovial fluids might be due to proteases.

Using an assay of macrophage migration, where the cells emigrate from an agarose droplet, it was found that the neutral proteases trypsin, chymotrypsin, Pronase and elastase have MIF-like activity. Appropriate enzyme inhibitors counteract this effect. To twelve synovial fluids from patients with inflammatory arthritis, which have MIF-like activity (migration index between 0-3 and 0-7) protease indhibitors (Trasylol, ovomucoid and soybean inhibitor) were added. Ten of the fluids lost some of their MIF-like activity with at least one inhibitor. Phenylmethylsulphonylfluoride counteracted totally the MIF-like activity of the two fluids tested. It is concluded that MIF-like activity of inflammatory synovial fluids is due, at least partially, to proteases.