Neutral endopeptidase depletion decreases colon cancer cell proliferation and TGF-ß1 synthesis in indirect co-cultures with normal colon fibroblasts.

PURPOSE: Although recent studies have suggested that neutral endopeptidase (NEP) is implicated in the regulation of colon cancer (CC) cell growth and metastasis, the influence of the tumor microenvironment on this role of NEP has not been investigated so far. Normal colon fibroblasts (NCFs) constitute a component of the stroma surrounding a tumor in an early stage of its development. NCFs can influence transformed cells via different paracrine factors, including TGF-ß1. This in vitro study was undertaken to evaluate the role of NEP in CC promotion in conditions of indirect co-culture of CC cells (LS180 and SW620) with NCFs (CCD-18Co) or their conditioned medium (CM-18Co). METHODS: We examined cell proliferation (with the BrdU assay) and invasiveness (using BME-coated inserts, 8 µm) of NEP-expressing, NEP-silenced (siRNA), and NEP-inhibited (with thiorphan, i.e. a NEP specific inhibitor) CC cells cultured alone or co-cultured with CCD-18Co or with their conditioned medium. The Western blot and ELISA methods were used to assess the level of TGF-ß1. RESULTS: The results showed that the co-culture of the NEP-depleted CC cells with NCFs or their conditioned medium resulted in a significant decrease in cell proliferation in comparison with the proliferative potential of NEP-silenced/inhibited CC cells cultivated alone. In contrast, the NEP depletion did not influence the invasiveness of CC cells in the co-cultures. The co-culture of CC cells with CCD-18Co or CM-C18Co resulted in increased synthesis of TGF-ß1, while the NEP downregulation decreased the synthesis of TGF-ß1 in CC cells and abolished the stimulatory effect of the co-cultures on TGF-ß1 production. CONCLUSIONS: The results suggest that the expression of NEP by colon cancer cells is essential for their proliferation and TGF-ß1 synthesis during paracrine interactions with NCFs.

A comparative study of the effects of meloxicam and flunixin meglumine (NSAIDs) as adjunctive therapy on interferon and tumor necrosis factor production in calves suffering from enzootic bronchopneumonia.

The study was performed on 18 Black-and-White Lowland Breed calves with clinical signs of enzootic bronchopneumonia divided into three groups and respectively treated with oxytetracycline and meloxicam--Group I (9 animals); oxytetracycline and flunixin meglumine--Group II (3 animals); and oxytetracycline only--Group III (6 animals--control). The following observations were recorded before treatment (1st day) and two days later (3rd day): body temperature, the serum level of interferon (IFN) and tumor necrosis factor (TNF) as well as cytokine production by bronchoalveolar lavage (BAL) cells. The treatment of calves with a combination of oxytetracycline and meloxicam (Group I) and especially with oxytetracycline and flunixin meglumine (Group II) caused a significantly faster, in comparison to the control group, normalization of body temperature. Both drugs, meloxicam and especially flunixin meglumine, inhibited excessive TNF production in the organism (measured as the serum level of cytokine). Moreover, BAL cells isolated from calves treated with both NSAIDs were still able, ex vivo, to release TNF, in contrast to the control group (treated only with tetracycline) which lost the ability to produce TNF. The treatment of the calves with meloxicam and flunixin meglumine did not significantly influence the levels of IFN in sera but normalized ex vivo IFN production in BAL cells. These results suggest that the combination of meloxicam with an antibiotic or flunixin meglumine with an antibiotic which does not exert an immunosuppressive influence on the organism of calves suffering from enzootic bronchopneumonia is equally effective in the treatment of calves and superior to the antibiotic alone.

Establishment and preliminary characterization of two cell lines derived from larynx carcinoma.

Two new cell lines, designated as RK-33 and RK-45, have been successfully established by an outgrowth technique from two different larynx tumours obtained from patients after laryngectomy. Both cell lineshave been maintained incultureforover 18 monthsandrecently have reached passage number 220 (RK-33) and 110 (RK-45). The cells display an epithelial morphology and multiply with a population doubling time of about 24 h (RK-33) and about 40 h (RK-45). The epithelial nature of the cells was also confirmed by expression of cytokeratins 8 and 18. Both lines were sensitive to antiproliferative effect of the tested cytostatic agents such as methotrexate. etoposide and thiotepa, with methotrexate being the most effective. We believe that both cell lines: RK-33 and RK-45 could be a suitable model for studying larynx cancer biology, however, further characterization of their properties is needed.

The influence of ketone bodies and glucose on interferon, tumor necrosis factor production and NO release in bovine aorta endothelial cells.

Bovine aorta endothelial cells (BAECs) were used to determine the effect of ketone bodies and glucose on in vitro interferon (IFN), tumor necrosis factor (TNF) and nitric oxide (NO) production. BAECs were incubated for 4 and 24h with the ketone bodies: 3.8mmol/l beta-hydroxybutyrate (BHB), 1mmol/l acetoacetate (AcAc) and 5. 2mmol/l acetone (Ac), used separately or in a mixture together with cytokine inducers: Newcastle disease virus (NDV) and lipopolysaccharide (LPS). BHB alone (but not AcAc or Ac) and a mixture of ketone bodies caused a significant decrease in IFN titers induced by NDV and LPS and in TNF titers induced by LPS. Glucose used at concentrations of 5.55, 3.33 and 1.66mmol/l did not influence cytokine production.NO measured by the nitrite content in culture medium was released spontaneously from BAECs. A slight enhancement of NO release was observed after infection of BAECs with NDV; however, treatment with LPS caused inhibition of the release. The mixture of ketone bodies used with NDV or LPS enhanced NO release. However, when cells were incubated in the medium with 1. 66mmol/l glucose (mimicking low plasma glucose level in ketotic cows) a significant decrease in NO release was observed. This enhancing effect of ketone bodies and inhibition by low glucose in the final effect balanced each other, and the amounts of NO released in the medium with 1.66mmol/l glucose and with the mixture of ketone bodies resembled those produced at 3.33mmol/l glucose without ketone bodies. The significance of these effects of ketone bodies and glucose concentrations on cytokine and NO production in the immunity of ketotic cows has been discussed.

The effect of steroidal and non-steroidal anti-inflammatory drugs on the cellular immunity of calves with experimentally-induced local lung inflammation.

We examined the effect of a single intravenous dose of flumetasone (SAID) and meloxicam (NSAID) treatment of calves with experimentally-induced localized lung inflammation on immunological and hematological variables such as total protein, gamma globulin, hemoglobin (Hb) concentrations, alkaline phosphatase activity, packed red cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts. The influence of drug treatment on the phagocytic activity of WBC and bronchoalveolar lavage (BAL) cells and their ex vivo ability to produce interferon (IFN) and tumor necrosis factor (TNF) after induction with Newcastle disease virus (NDV), as well as on the development of PHA-induced skin delayed hypersensitivity reaction was also determined. Two days after the treatment of calves with experimentally-induced local lung inflammation with flumetasone (5 mg per calf), we observed a significant increase in WBC count, especially neutrophils, and a decrease in gamma globulin concentration, in the percent of blood phagocytic cells and their random migration. Flumetasone treatment also inhibited the development of skin delayed hypersensitivity reaction. In contrast, the treatment of calves with meloxicam (50 mg per calf) did not influence any hematological parameters or skin reactivity. Both drugs, flumetasone and meloxicam, influenced TNF production in ex vivo cultures of blood and BAL cells, inhibiting excessive TNF production induced by local lung inflammation. Contrary to TNF, the treatment of calves with meloxicam and flumetasone enhanced ex vivo IFN production in blood and BAL cells. Histological examination of lung tissue revealed that in control calves (those not treated with anti-inflammatory drugs) and in calves treated with flumetasone, symptoms of stromo-purulent inflammation of pulmonary tissue developed. However, in calves treated with meloxicam, only interstitial inflammation with a slight thickening of interalveolar septa and infiltration of lymphoid cells was observed. These results suggest that in this model of pneumonia, it is more appropriate to use a single dose of meloxicam, rather than flumetasone, to modulate lung inflammation.

Alterations in peripheral blood leukocytes functions during enzootic bronchopneumonia of calves. Effect of treatment with antibiotics and immunomodulators.

Twelve calves from over veal calf farm were divided into two groups: group I-6 calves which developed typical signs of enzootic bronchopneumonia and group II-6 calves with no symptoms of the disease. Both groups of calves were compared with respect to changes in several hematological parameters. Some functions of peripheral blood leukocytes as random migration, phagocytic index, percent of phagocytic cells and percent of NBT positive cells were also scored. In addition, changes in serum levels of interferon (IFN) and tumor necrosis factor (TNF) and the ability of peripheral blood leukocytes (PBL) to produce IFN and TNF were quantitated by biological methods. On the day of diagnosis, in group I of calves a significant increase in the total serum protein concentration, hemoglobin content, red and white blood cells counts in comparison to control calves (group II) was observed. The increased number of NBT positive neutrophils and moderate levels of serum IFN and TNF correlated with elevated body temperature, breathing and heart rates. Calves with bronchopneumonia (group I) after diagnosis of the disease were treated with Tylbian (tylosine derivative), Flumetazon (glucocorticoid), Emulselvet (immunomodulator), bromhexinum and sulphonamides. Seven days after the beginning of treatment with medicaments a significant improvement in clinical symptoms was observed, however, the ability of PBL to cytokine production increased significantly 2 weeks after beginning of treatment and correlated with significant increase in random migration of neutrophils and their phagocytic activity, measured by the percent of phagocytic cells. Unexpectedly, in control calves (group II), not exhibiting any symptoms of bronchopneumonia at the beginning of experiment, high serum IFN titers were detected which decreased significantly during the first week of observation. In contrast to that the ability of PBL of control calves to produce IFN increased significantly within 3 weeks of observation. The correlations between the ability of PBL to produce cytokine and the development of clinical symptoms of bronchopneumonia are discussed.

[The role of nitric oxide in normal and pathologic immunologic reactions]. / Rola tlenku azotu w prawidlowych i patologicznych reakcjach odpornosciowych.

Nitric oxide is intercellular messenger that performs a number of important functions, including neurotransmission, vasodilation, inhibition of platelet aggregation and modulation of leukocyte adhesion. The paper presents a new aspects of NO synthesis regulation by bacterial products and cytokines. Problems concerning the role of NO in antimicrobial/antitumor resistance and pathomechanisms of autoimmune diseases are also discussed.

In vitro and in vivo interferon production by bovine colostral leukocytes.

Sixteen healthy pregnant cows were divided into two groups. Group I (8 cows) received immediately after calving two injections of Vaccina L containing the LaSota strain of Newcastle Disease Virus (total dose 10(9) TCID50/cow) into udder lymph nodes. The second group of 8 cows served as control. Newborn calves of both groups of cows were allowed to suckle the colostrum. Interferon activity was detected 24 h after injection in whey obtained from colostrum of Vaccina L-treated dams and in supernatants of cultures of colostral leukocytes. Interferon was also present in sera of Vaccina L treated dams 48 h after injection. No IFN activity was detected in sera of calves. Cultures of leukocytes obtained from colostrum of dams 48 h after Vaccina L-treatment exhibited hyporeactivity to the second induction in vitro and produced low IFN levels in response to NDV. In contrast to hyporeactivity observed in colostral leukocytes, peripheral blood leukocytes (PBL) of calves suckling colostrum from dams treated with Vaccina L produced higher IFN levels after induction in vitro with NDV than leukocytes of control calves.

Influence of the perinatal period on bovine interferon production.

Interferon (IFN) production in cultures of leukocytes of calves immediately after birth, 2--3-day-old calves, dams and nonpregnant lactating cows was compared. For IFN induction Radom strain of Newcastle disease virus (NDV-R), phytohemagglutinin (PHA) and concanavalin A (ConA) were used. Cultures of newborn-calf peripheral blood mononuclear cells (PBMC) separated from blood by Ficoll-Hypaque density centrifugation or leukocytes cultivated by whole blood technique responded to all inducers used with a significantly lower IFN level than those of 2--3-day-old calves, dams and lactating cows. Although the mechanism of this phenomenon is not clear, our data connot rule out a defect of IFN synthesis in blood leukocytes of newborn calves. Spontaneous antiviral activity was detected in fetal membranes and in colostral leukocytes. Colostral leukocytes were efficient producers of IFN after induction with NDV-R, but less efficient than blood leukocytes after induction with PHA and ConA. Low response to PHA and ConA correlated with low number of lymphocytes in colostrum.