Peritoneal dissemination is inhibited by treatment with antibodies against CD44H, beta(1) integrin, and carcinostatic agents in NUGC-4 human gastric cancer cells

Peritoneal dissemination frequently occurs after surgery in patients with gastric cancer. The presence of peritoneal metastasis after surgery affects the prognosis, therefore, a way must be found to effectively prevent the development of peritoneal dissemination. Very little is known about the biochemical processes involved in the initial attachment of gastric cancer cells to peritoneal mesothelial cells. We conducted in vitro and in vivo studies to assess the role of adhesion molecules and TGF-beta 1 in this process, using 4 gastric cancer cell lines. NUGC-4 cells, which disseminate early after inoculation into the abdominal cavity of nude mice, predominantly expressed CD44H and beta(1) integrin. We found that NUGC-4 cells adhered to monolayers of mesothelial cells more firmly than other cell lines. Adhesion of NUGC-4 cells to mesothelial cells was partially inhibited by antibodies against CD44H or the beta(1) subunit of integrin, and was completely blocked by a combination of these 2 antibodies. Treatment with ligands for CD44H and beta(1) integrin also inhibited this adhesion. In the NUGC-4 cell culture medium, larger amounts of transforming growth factor beta 1(TGF-beta 1) was detected in proportion to the increase in cancer cells than in the other cell lines. TGF-beta 1 increased the expression of CD44H in NUGC-4 cells and in mesothelial cells, and augmented the adhesion and implantation of NUGC-4 cells to mesothelial cells accompanied by accumulation of extracellular matrix (ECM) components. Carcinostatic agents decreased the expression of CD44H but increased the expression of E-cadherin in NUGC-4 cells. Treatment with carcinostatic agents and antibodies against CD44H and beta(1) integrin inhibited the dissemination of NUGC-4 cells in the peritoneal cavity of nude mice, and prolonged their survival time. These findings suggest that CD44H and integrins mediate in the initial attachment of gastric cancer cells to mesothelial cells, and TGF-beta 1 participates in the promotion of the disease. It is possible that a treatment strategy that interferes with CD44H or integrins function and increases the functions of E-cadherin immediately after surgery may result in the decreased intra-abdominal spread of gastric cancer.     

Production of monoclonal antibody to human esophageal cancer cell line

The immunization of Balb/C mice with esophageal cell line KYSE-50 established from poorly-differentiated esophageal squamous cell carcinoma, resulted in obtaining the monoclonal antibody KYMN-28-5. This monoclonal antibody is of the IgM class and recognizes a carbohydrate antigen contained in glycoproteins with molecular weights of 53 and 56K, and in neutral glycolipids extracted from teratomas. Tissue staining revealed that this monoclonal antibody reacts strongly with malignant tumors but only weakly, or not at all, with normal tissues, apart from squamous epithelial tissue. KYMN-28-5 is thus a useful tumor marker which will improved the accuracy of serological diagnosing squamous cell carcinoma when combined with the measurement of SCC antigen.     

Calpain activates two transglutaminases from porcine skin

Summary Two transglutaminases (TGase) with estimated molecular weight of 55,000 (55-K TGase) and 120,000 (120-K TGase) were partially purified from the cytosolic fraction of porcine skin (epidermis-rich preparation) using DEAE-cellulose and gel-filtration chromatographies. The enzyme activities of both trans-glutaminases were enhanced more than 20-fold by treatment with calpain (Ca²⁺-dependent cysteine proteinase) in the presence of Ca²⁺, and this enhancement was inhibited by adding EDTA, leupeptin, or an endogenous calpain-specific inhibitor protein (calpastatin). 55-K TGase was effectively activated by a smaller amount of calpain than was 120-K TGase, while known activating reagents such as thrombin and dimethyl sulfoxide or heat treatment preferentially activated 120-K TGase. One of the physiological functions of calpain in the epidermis may be the activation of epidermal transglutaminases.     

Purification and characterization of calpains from pig epidermis and their action on epidermal keratin

Two forms of Ca++-dependent cysteine proteinases, calpain I, requiring low Ca++ (microM concentration), and calpain II, requiring high Ca++ (mM concentration), were purified from the cytosolic fraction of pig epidermis. Calpains I and II were separated on DEAE-cellulose chromatography, and thereafter they were purified by separate but almost identical procedures, which included chromatographies on Sephacryl S-300, Blue Sepharose CL-6B, and DEAE Bio-Gel A. Purified calpains I and II required 10 and 450 microM Ca++ for half-maximal activation, respectively, and had an optimal pH of 7.0 to 8.0. Both enzymes were heterodimers and composed of one heavy subunit (83 kDa for calpain I and 80 kDa for calpain II) and one light subunit (29 kDa for both enzymes). The action of calpains I and II on keratin extracted from the same tissue was studied. Both enzymes rapidly cleaved keratin into small fragments. The cleavage depends on Ca++ and could be blocked by leupeptin and calpastation, an endogenous calpain-specific inhibitor, which was also found in the cytosolic fraction of pig epidermis and partially purified.     

Carbohydrate auto-antigens in auto-immune hemolytic anemia

Anti-erythrocyte antibodies which appear in the sera of patients with auto-immune hemolytic anemia frequently recognize carbohydrate auto-antigens. Most of cold agglutinins are known to recognize the Ii-antigens, and Donath-Landsteiner antibodies which appear in patients with paroxismal cold hemoglobinuria are almost exclusively directed to the P-antigen. These carbohydrate auto-antigens are strongly expressed in various tissues and organs other than erythrocytes, and behave as differentiation- or developmental-antigens, in both humans and mice. The study of nucleotide sequences of human and murine anti-Ii antibodies shows that these antibodies share a highly homologous antigen-binding site in their VH regions. These results indicate that the carbohydrate auto-antigens in autoimmune hemolytic anemia are evolutionally conserved developmental antigens, and suggest that the immunoglobulin genes which encode variable regions of the auto-antibodies directed to these antigens are also conserved evolutionally.     

Potential enhancement of osteoclastogenesis by severe acute respiratory syndrome coronavirus 3a/X1 protein

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a lung disease with high mortality. In addition, osteonecrosis and bone abnormalities with reduced bone density have been observed in patients following recovery from SARS, which were partly but not entirely explained by the short-term use of steroids. Here, we demonstrate that human monocytes, potential precursors of osteoclasts, partly express angiotensin converting enzyme 2 (ACE2), a cellular receptor of SARS-CoV, and that expression of an accessory protein of SARS-CoV, 3a/X1, in murine macrophage cell line RAW264.7 cells, enhanced NF-κB activity and differentiation into osteoclast-like cells in the presence of receptor activator of NF-κB ligand (RANKL). Furthermore, human epithelial A549 cells expressed ACE2, and expression of 3a/X1 in these cells up-regulated TNF-α, which is known to accelerate osteoclastogenesis. 3a/X1 also enhanced RANKL expression in mouse stromal ST2 cells. These findings indicate that SARS-CoV 3a/X1 might promote osteoclastogenesis by direct and indirect mechanisms.     

Immunological and virological status of a hemophiliac infected with human T cell lymphotropic virus type 1 and human immunodeficiency virus type 1, and results of therapy

The immunological and virological status of three hemophiliacs infected with human immunodeficiency virus type 1 (HIV-1) was monitored for 11 months. One of these patients was also infected with human T cell lymphotropic virus type 1 (HTLV-1), and HIV-1 could be isolated only from this patient among the three subjects. The doubly infected subject had the fewest T4 (helper) lymphocytes and the highest proportion of T8 lymphocytes with DR human leukocyte antigens (DR-Ag). The serum level of HIV-1 antigen increased in this patient during the observation period, and this increase was accompanied by a decrease in the proportion of DR-Ag-positive cells among the T8 lymphocytes. This patient was treated with 800 mg of zidovudine daily for 50 days. With treatment, the nonspecific clinical symptoms improved and the proportions of DR-Ag positive cells among the T8 lymphocytes decreased. Serum levels of HIV-1 antigen decreased immediately when therapy was started but later increased during therapy. In persons infected with both HTLV-1 and HIV-1, HIV-1 seems to proliferate readily.     

Carnitine: a neuromodulator in aged rats

A wide range of morphological and biochemical changes occur in the central nervous system with increasing age. L-carnitine, a naturally occurring compound, plays a vital role in fatty acid transport across the mitochondrial membrane. L-carnitine (300 mg/kg body wt/day) was administered intraperitoneally to young and old male Wistar rats for 7, 14, and 21 days. Carnitine, dopamine, epinephrine, and serotonin levels were assayed in discrete regions of the brain. Carnitine supplementation increased the levels of dopamine, epinephrine, and serotonin in the experimental animals in our study. Response to carnitine supplementation varied among the brain regions that have been studied. The regions rich in cholinergic neurons such as the cortex, hippocampus, and striatum showed more response after 21 days of carnitine treatment. The results of the present study suggest the role of L-carnitine as a neuromodulator and antiaging medication.     

Monoclonal antibodies directed to a disulfated glycosphingolipid, SB1a (GgOse4Cer-II3IV3-bis-sulfate), associated with human hepatocellular carcinoma

Two murine monoclonal antibodies, 2H6G5 (IgM) and 4A9E10 (IgG3), were obtained by using either cultured human hepatocellular carcinoma cells (PLC/PRF/5) or the acidic glycolipid mixture prepared from the same cells as immunogens. The antigen in PLC/PRF/5 cell membranes recognized by both antibodies was identified as a disulfated acidic glycolipid, GgOse4Cer-II3IV3-bis-sulfate (SB1a). Both antibodies reacted specifically with SB1a, and no significant reactivity was noted with other sulfated glycolipids or gangliosides except that the antibody 2H6G5 showed a weak cross-reactivity with LacCer-II3-sulfate (SM3), another sulfated glycolipid which partly shares the same carbohydrate structure as SB1a. The SB1a antigen is a relatively minor glycolipid in PLC/PRF/5 cells, but it was strongly expressed at the surface of PLC/PRF/5 cells as ascertained by cytofluorometry using both antibodies. A significant amount of SB1a antigen was present in 3 of the acidic glycolipid fractions isolated from 15 human hepatocellular carcinoma tissues as well as in the acidic glycolipid fraction prepared from PLC/PRF/5 cells, while all the acidic glycolipid fractions prepared from cirrhotic livers and a normal liver were essentially negative for SB1a, as ascertained by both solid phase enzyme immunoassay and the thin-layer chromatography-immunostaining method. These results strongly suggest that the SB1a antigen as defined by these new monoclonal antibodies is associated with human hepatocellular carcinoma.