Reduction of ultraviolet light-induced DNA damage in human colon cancer cells treated with a lactoferrin-derived peptide.

Treatment of Caco-2 cells with the peptide lactoferricin(4-14), results in reduction of the growth rate by prolongation of the S phase of the cell cycle. Lactoferricin(1-25) is formed in the gut by cleavage from lactoferrin and the bioactive amino acids are found within lactoferricin(4-14). Our hypothesis is that the reduction of the rate of S phase progression may result in increased DNA repair. To test this hypothesis, Caco-2 cells were subjected to UV light that caused DNA lesions and then the cells were grown in the absence or presence of 2.0 µM lactoferricin(4-14). Evaluation of DNA strand breaks using the comet assay showed that lactoferricin(4-14) treatment indeed resulted in a reduction of comets showing damaged DNA. In the search for a mechanism, we have investigated the levels of several proteins involved in cell cycle regulation, DNA replication, and apoptosis using Western blot. Lactoferricin(4-14) treatment resulted in an increased expression of flap endonuclease-1 pointing to increased DNA synthesis activity. Lactoferricin(4-14) treatment decreased the expression of the proapoptotic protein B-cell lymphoma 2-associated X protein (or Bax), indicating decreased cell death. As we have found previously, lactoferricin(4-14) treatment reduced the expression of cyclin E involved in the G(1)/S transition. Immunofluorescence microscopy showed that a lower γ-H2AX expression in lactoferricin(4-14)-treated cells, pointing to more efficient DNA repair. Thus, altogether our data show that lactoferricin(4-14) treatment has beneficial effects.

Identification of ubiquitin in bovine milk and its growth inhibitory effects on human cancer cell lines.

Bovine milk is associated with improved health and reduced risk of several diseases, among them cancer. Milk is a complex mixture of known and unknown components. The components and the mechanisms that contribute to the cancer-preventive effects are largely unknown. We set out to find new peptides in milk and identified ubiquitin (Ub) using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Western blot. Using quantitative Western blot, we estimated the Ub concentration to be about 0.003 micromol/L in milk. We then decided to investigate the effect of treating human colon cancer CaCo-2 cells with Ub, using higher concentrations than in milk. CaCo-2 cells treated with 0.02 to 2.0 micromol/L Ub showed significantly decreased proliferation compared with untreated control cells. A higher growth inhibitory effect than in CaCo-2 cells was found in the neuroblastoma cell line SH-SY5Y treated with 0.02 to 0.2 micromol/L Ub. A bromodeoxyuridine DNA flow cytometric method was used to study cell cycle kinetics in Ub-treated CaCo-2 cells. The data point toward a prolongation of the G(1) phase. The levels of several cell cycle regulatory proteins were affected. Our data point to Ub possibly being one of the components in milk reducing the risk of cancer.

Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line.

Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 microM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G(1)/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time.

A novel approach to monoclonal antibody separation using high performance liquid affinity chromatography (HPLAC) with SelectiSpher-10 protein G.

Protein G, a bacterial cell wall protein extracted from strains of Streptococci, has been employed as a ligand in high performance liquid affinity chromatography (HPLAC) for separation of monoclonal antibodies. Examples are given of rapid high-resolution separations of rat and mouse monoclonal antibodies belonging to various subclasses. In comparison with protein A chromatography, we were able to show superior binding characteristics for SelectiSpher-10 protein G columns under conditions of 'low' ionic strength (about 0.1 M) and neutral pH (pH approximately 7). The monoclonal antibodies were isolated in high purity (greater than 90%) and with good recovery of specific activity (80-100%). We believe that the HPLAC technology based on SelectiSpher-10 protein G is of potential value in the analysis and purification of monoclonal antibodies from various species and subclasses.

Apheresis.

Treatment of hemophilia patients with inhibitors is most effective if circulating levels of factor VIII (F.VIII) or factor IX (F.IX) can be obtained. This is possible not only in patients with low inhibitor concentration, but also in those with high inhibitor levels after temporary removal of the inhibitors by means of apheresis methods. Extensive plasma exchange combined with huge doses of F.VIII has occasionally been tried, but the method is of limited capacity, and exposure to plasma products can initiate an anamnestic response of the inhibitor before treatment with factor concentrates can be started. A better alternative is extracorporeal immunoadsorption for removal of antibodies against F.VIII or F.IX. Two methods developed at the center in Malmö are presented. 1) Group specific elimination using protein A. This is done by computer controlled extracorporeal adsorption to protein A Sepharose columns of the antibodies in plasma transferred on-line to the plasma treatment apparatus (Citem 10, Excorim, Lund, Sweden). The plasma depleted of inhibitor is returned to the patient together with the separated blood cells. At the Malmö center, high titer inhibitors have so far been removed in 10 hemophilia patients, and 4 patients with acquired hemophilia, on a total of 25 immunoadsorption episodes. Usually between 2 and 4 plasma volumes were processed in the course of 6 to 12 hours. The inhibitor decreased to zero or very low levels, enabling conventional replacement therapy to be given for control of severe bleeding or as cover for surgery. The treatment is well tolerated and no signs of activation of the coagulation, fibrinolytic, and complement systems were seen. 2) Specific elimination using coagulation factor. A method has been developed for the removal of F.IX antibodies directly from whole blood in a continuous extracorporeal system. A F.IX preparation with a specific activity of 92 units per mg protein was covalently coupled to Macro Beaded Sepharose, which allows passage of whole blood. One ml of the absorbent was capable of binding about 2100 BU. The F.IX antibodies could be specifically adsorbed directly from citrated whole blood with a minimal extracorporeal volume. In this way, high-titer antibodies could be removed from a boy with hemophilia B, by passing four times his blood volume through the F.IX column. No side effects were noted. In the future it will be possible to use immobilized F.VIII in a similar system for the removal of F.VIII antibodies.

r-Process in Neutron Star Mergers.

The production site of the neutron-rich heavy elements that are formed by rapid neutron capture (the r-process) is still unknown despite intensive research. Here we show detailed studies of a scenario that has been proposed earlier by Lattimer & Schramm, Symbalisty & Schramm, Eichler et al., and Davies et al., namely the merger of two neutron stars. The results of hydrodynamic and full network calculations are combined in order to investigate the relevance of this scenario for r-process nucleosynthesis. Sufficient material is ejected to explain the amount of r-process nuclei in the Galaxy by decompression of neutron star material. Provided that the ejecta consist of matter with a proton-to-nucleon ratio of Ye approximately 0.1, the calculated abundances fit the observed solar r-pattern excellently for nuclei that include and are heavier than the A approximately 130 peak.

Suppression of secondary antibody response by intravenous immunoglobulin in a patient with haemophilia b and antibodies.

A 39-year-old patient, suffering from severe haemophilia B and antibodies against factor IX, has twice been treated with extracorporeal protein A-Sepharose adsorption followed by conventional substitution therapy in combination with immunosuppression (cyclophosphamide). On both occasions, separated by a 2-year interval, the same procedure was followed except that, on the second, administration of i.v. immunoglobulin (Gammonativ. KabiVitrum) was added. Within a week of the first treatment the patient developed a 15-fold increase in the antibody titre. Following the second treatment described here, no secondary antibody response could be detected, and after a further 12 weeks only traces of antibodies are demonstrable. It seems that antibody synthesis was suppressed by the i.v. immunoglobulin. No evidence was found to demonstrate that the effect was due either to a non-specific suppression of the immune and reticuloendothelial systems or to the action of interfering antibodies. It has not yet been established whether or not the protein A-Sepharose adsorption technique, or the immunosuppressive treatment, contributed in any way to the result. The observations suggest a new approach to the treatment of haemophiliacs with antibodies of the high-responding type.

Immunoadsorption for removal of inhibitors: update on treatments in Malmö-Lund between 1980 and 1995.

Treatment of severe bleeding and the performance of surgery in haemophilia patients with inhibitors creates severe problems. It is generally agreed that treatment is most effective if circulating levels of factor VIII/IX can be achieved long enough for control of haemostasis. Immunoadsorption with protein A for the removal of inhibitor has improved treatment for patients with initial inhibitor titres too high to neutralize by infusion alone. This is a summary of our experience in Malmö regarding immunoadsorption and haemostasis. A total of 19 applications with immunoadsorption in 10 patients were performed. On all occasions it was possible to eliminate totally the inhibitor or reduce it to low levels that could easily be neutralized with factor concentrate. Haemostatic levels of coagulation factors could be maintained for 5-9 days in all but one patient. This period was sufficient to stop ongoing haemorrhage or prevent excessive bleeding at surgical interventions.

Tolerance induction using the Malmö treatment model 1982-1995.

The ultimate goal in the treatment of haemophilia patients with inhibitors is to eradicate permanently the inhibitor and induce tolerance. Here we summarize our experience at the Malmö centre regarding tolerance induction according to the Malmö Treatment Model. The protocol includes immunoadsorption if needed, neutralization of inhibitor and replacement with factor concentrates, cyclophosphamide intravenously for 2 days (12-15 mg kg-1 bw) and then orally (2-3 mg kg-1 bw) for an additional 8-10 days and intravenous gammaglobulin daily at dosages of 0.4 g kg-1 bw for 5 days. This protocol has been applied in 23 haemophilia patients with inhibitors, 16 haemophilia A patients and seven haemophilia B patients. Altogether 36 attempts have been made to induce tolerance. Ten of the 16 haemophilia A (62.5%) and 6/7 patients with haemophilia B (86%) became tolerant after the treatment. The chances of success or failure are roughly equal, if the series is considered in a historical perspective. The data showed that the chances of success in tolerance induction with the Malmö protocol were best in those patients with low inhibitor titres, with relatively low historical inhibitory peak and with a long interval since the previous replacement therapy. This was especially true where no inflammatory state was present at the start or during tolerance induction. The advantage with this method compared to the high-dose regimen is that in the successful cases tolerance can be achieved within 3-4 weeks.

Feasibility of extracorporeal on-line large-scale plasma adsorptions on protein A-sepharose columns in cancer patients.

The feasibility of extracorporeal adsorption of 1.5-3 L plasma on protein A-Sepharose was investigated in six patients with advanced cancer. Anticoagulation with heparin was associated with respiratory distress syndrome in two patients, most likely caused by complement activation as indicated by a transient leukopenia during plasma reinfusion and appearance of C3 degradation products in the extracorporeal circulation. Addition of citrate abolished the respiratory symptoms, C3 degradation, and leukopenia, and no adverse reactions were observed. No objective tumor regression was observed in any of the patients. Three patients progressed during therapy. In one of these, multifocal central tumor necrosis was observed as a possible, although unproven, therapeutic effect. Increased natural killer and/or killer cell activities were recorded in three patients and increased complement-dependent serum cytotoxicity in one patient. The level of circulating immune complexes decreased significantly (18-28%) in three patients studied. It is concluded that extracorporeal plasma adsorption on protein A-Sepharose is feasible when citrate is added to the extracorporeal system, but its therapeutic efficacy is uncertain.