V. Větvička

Antibody-directed affinity therapy applied to the immune system: In vivo effectiveness and limited toxicity of daunomycin conjugated to HPMA copolymers and targeting antibody

The applicability of targeting therapy intervention in lymphatic tissue was studied. The effect was measured as the inhibition of anti-sheep red blood cell antibody response expressed in plaque-forming cells. Daunomycin was used as the effective drug and polyclonal and monoclonal anti-Thy 1.2 or anti-Iak antibody served for targeting. Both components were coupled to a soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer with oligopeptidic side sequences which permitted a controlled release of the drug in the target tissue. HPMA copolymer conjugates with side sequences Gly-Phe-Leu-Gly cleavable by lysosomal enzymes decreased in vivo the antibody reaction by 60-85%. A comparable amount of free targeting antibody was without a significant effect. Injection of targeted daunomycin decreased the toxicity of the drug against hematopoietic precursors in bone marrow colony-forming unit-spleen 80 times compared to the same amount of free drug. The in vivo effectiveness of targeted daunomycin was confirmed morphologically. Application of free daunomycin lead to a significant irritation of Kupffer cells in liver while none of the daunomycin-antibody-copolymer conjugate had such an effect.

Phagocytosis of human blood leukocytes: A simple micromethod

A simple micromethod for testing human blood leukocyte phagocytosis employing synthetic hydrophilic particles based on 2-hydroxyethylmethacrylate is described. The normal level of phagocytosing leukocytes in healthy children was 20.1 +/- 2.7%, and in healthy adult donors 34.3 +/- 6.1%. The method was found suitable for routine testing in both clinical and laboratory practice.

Action of polymeric prodrugs based on N-(2-hydroxypropyl)-methacrylamide copolymers. II. Body distribution and T-cell accumulation of free and polymer-bound [125i]daunomycin

Abstract N -(2-hydroxypropyl)methacrylamide copolymers containing oligopeptide side-chains terminated in [ 125 I]daunomycin (DNM), and targeting moieties [anti-Thy 1.2 antibodies or nonspecific rabbit gammaglobulin (RGG)], were synthesized. The body distribution of these copolymers after i.v. administration was studied in vivo in an inbred strain of mice (C57L/J). Covalent binding of [ 125 I]DNM to an HPMA copolymer containing anti-Thy 1.2 antibodies increased its level in blood 5–20 times and decreased its rate of elimination compared to the free drug. The maximal organ accumulation of [ 125 I]DNM bound to the targetable conjugate in the spleen, thymus and liver was detected after 2 hours. Liver accumulation was observed only when the specific anti-Thy 1.2 antibody was replaced with nonspecific RGG in the conjugates. Similar results were obtained when using i.p. administration of the copolymers studied. The accumulation of HPMA copolymer conjugates in thymocytes was studied in vitro . Intracellular radioactivity of thymocytes cultivated in the presence of [ 125 I]DNM-HPMA copolymer conjugates with anti-Thy 1.2 antibodies was maximal after 2 hours while the maximum accumulation of free drug was observed after 1 hour. Comparing HPMA copolymers without targeting moieties, the copolymer with biodegradable oligopeptide side-chains (Gly-Phe-Leu-Gly) demonstrated a higher binding and accumulation of radioactivity in the T cells than the copolymer containing nonbiodegradable sidechains (Gly-Gly). The higher hydrophobicity of the former may contribute to the observed phenomenon.

Alpha-fetoprotein and phagocytosis in athymic nude mice

Alpha-fetoprotein (AFP) was found to suppress the phagocytic activity of the blood monocytes and neutrophils in vitro. The amounts of AFP detectable by immunofluorescence in the livers of nu/nu, nu/+ and +/+ mice were quite comparable, and thus could not have been responsible for the alterations in phagocytosis found in leukocytes of athymic nude mice during their ontogenetic development.

The effect of bilirubin on the Fc receptor expression and phagocytic activity of mouse peritoneal macrophages.

The effect of bilirubin on the phagocytic activity of mouse peritoneal macrophages and on the expression of Fc receptors and receptors for SRBC was studied. Intraperitoneally administered bilirubin influenced the expression of Fc receptors for IgM, IgG2B, IgA and IgE, whereas the expression of other receptors as well as the phagocytic activity of peritoneal macrophages remained unchanged. The possible mechanism of the effect of bilirubin on Fc receptors is discussed.

An advantageous method for detection of Fc-receptors and for studying Fc-receptor-mediated phagocytosis

A method employing fluorescent 2-hydroxyethyl methacrylate copolymer particles with dinitrophenyl haptenic group and anti-haptenic murine monoclonal antibodies of different isotypes is described for tracing Fc-receptors and for studies of phagocytosis via these cell surface structures. The possibility of quenching the fluorescence of non-phagocytosed particles enables us to distinguish clearly and reliably between internalized and cell surface-bound particles.

The immunosuppressive effects of bilirubin

The strong effects of bilirubin on various levels of the immune system are multifactorial. Concerning the mechanisms of these effects, we hypothesize that the primary causes of the described actions of bilirubin are the direct interaction of bilirubin molecules with cell membranes.

Action of polymeric prodrugs based on N-(2-hydroxypropyl)meth- acrylamide copolymers. I. Suppression of the antibody response and proliferation of mouse splenocytes in vitro

Abstract Splenocytes were removed from A/J mice 14 days after i.p. injection of 1 × 10 8 sheep red blood cells and were incubated for 10 min, 3 h and 5 days with free daunomycin, free anti Thy 1.2 antibodies or daunomycin conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates with biodegradable (Gly-Leu-Gly; Gly-Phe-Leu-Gly) or non-biodegradable (Gly-Gly) side-chains containing targeting anti Thy 1.2 antibodies. The effect on the antibody response, represented by decrease in the numbers of IgM and IgG plaque-forming, i.e., antibody-releasing mouse splenocytes (plaque-forming cells), was detected. It was shown that a 10 min contact of immunocompetent cells with daunomycin-HPMA copolymer conjugates is sufficient for suppression of antibody formation. However, 3 hours incubation were necessary to obtain significant decrease in a number of PFC. Lymphocytes (mouse splenocytes) were very sensitive to free daunomycin. Concentration of 50 μg/ml eliminated all lymphocytes from tissue culture. At 1–10 ug/ml, a high proportion of cells remained viable, but the antibody response was completely suppressed. A comparable amount of daunomycin (35–70 μg/ml) bound to copolymer with targeting antibodies did not kill lymphocytes in tissue culture, but the antibody response was substantially suppressed (50–90%). In all experiments IgG antibody formation was more sensitive to suppression than IgM response. Biodegradability of the bond between the HPMA copolymer carrier and daunomycin substantially increased the pharmacological effect although a certain degree of immunosuppression was detected with the drug conjugated to targeted non-biodegradable HPMA copolymer. Free daunomycin inhibited [ 3 H]thymidine incorporation by mouse T lymphocytes (cultivation in presence of Con A) at the concentration of 0.1 μg/ml. To achieve the same effect, 5 times more daunomycin bound to biodegradable HPMA copolymer with targeting anti Thy 1.2 antibodies or 500 times more daunomycin bound to non-targeted biodegradable HPMA copolymer was necessary.

The effect of Bilirubin on the phagocytic activity of mouse peripheral granulocytes and monocytesin vivo

Using anin vitro phagocytic assay with synthetic 2-hydroxyethylmethaerylate copolymer particles, the phagocytic activity of leukocytes of bilirubin-treated mice (85 and 170 μmol/L in 0.5 mL intraperitoneally) was studied. Bilirubin treatment significantly stimulated the phagocytosis of both peripheral blood granulocytes and monocytes; the increase of phagocytosis persisted for 6 h after bilirubin injection. The potential immunostimulating and/or immunotoxic effect of bilirubin is discussed.

Macrophage function in high- and low-responder strains of mice

Unstimulated peritoneal macrophages of the high-responder A/J mice compared to the low-responder B10 strain have a lower number of cells with the different Fc receptor (FcR) subtypes and correspondingly the FcR mediated phagocytosis is also lower. The intraperitoneal immunization with SRBC leads to a prompt increase of FcR expression and of phagocytic and pinocytic activity in the A/J mice, while in the B10 strain the activity of macrophages remains unchanged.