Frank W. Falkenberg

Purification and immunological characterization of the human hexokinase isoenzymes I and III (ATP-d-hexose 6-phosphotransferase EC 2.7.1.1)☆

Abstract In extracts of human tissues three different hexokinases were demonstrated by starch gel electrophoresis. Hexokinase I was purified from heart tissue, and hexokinase III was isolated from spleen tissue. The enzymes have a similar molecular size, but differ in their electrophoretic and chromatographic properties and in their Michaelis constants for glucose, hexokinase III being a substrate-inhibited form. In electroimmunodiffusion experiments hexokinase I did not crossreact with antihexokinase III and vice versa.

Isolierung, enzymatische und immunologische charakterisierung einer plasmamembranfraktion vom proximalen tubulus der menschliche niere

A plasma membrane fraction (PM) from proximal tubules of human kidney cortex was separated by fractionational centrifugation and sucrose density-gradient centrifugation. Marker enzymes, especially from the brush-border were determined. Alkaline phosphatase and an aminopeptidase with alanine specificity were both localized on the membranes. Human anti-PM sera from rabbits revealed—after absorption with human serum proteins—a specific reaction with structures of brush-border. This could be demonstrated by indirect immunofluorescence technique.Abstract A plasma membrane fraction (PM) from proximal tubules of human kidney cortex was separated by fractionational centrifugation and sucrose density-gradient centrifugation. Marker enzymes, especially from the brush-border were determined. Alkaline phosphatase and an aminopeptidase with alanine specificity were both localized on the membranes. Human anti-PM sera from rabbits revealed—after absorption with human serum proteins—a specific reaction with structures of brush-border. This could be demonstrated by indirect immunofluorescence technique.

Urinary antigens as markers of papillary toxicity. I. Identification and characterization of rat kidney papillary antigens with monoclonal antibodies.

Abstract Monoclonal antibodies were prepared in an attempt to develop diagnostic tools for the identification of toxic damage to the rat renal papilla. One IgG and five IgM monoclonal antibodies, reacting with antigens localized in the papilla were obtained. Three of the IgM class and the IgG class monoclonal antibodies were found to be specific for antigens localized in collecting ducts, two of them staining papillary collecting ducts more intensely than cortical collecting ducts. The IgG class antibody, termed Pap X 5C10, recognizes an antigen located at high density on the luminal side of papillary collecting duct epithelial cells and at lower density in cortical collecting duct cells. One of the IgM class monoclonal antibodies reacts with an antigen localized in epithelial cells of ascending and descending loops of Henle and of connecting tubules. Another of the IgM class monoclonal antibodies reacts with an antigen localized in the interstices of the inner medulla. All these monoclonal antibodies react with their antigens in native frozen as well as in Bouin-fixed and paraffin-embedded tissue slices. Molecular properties of the Pap X 5C10 antigen have been investigated by gel permeation chromatography, SDS-PAGE, Western blotting, and isoelectric focusing. The results indicate that the antigen in both its tissue-derived and urinary form is of large (150–200 kDa) molecular size and can be separated into two molecular species with isoelectric points of pH 7.2 and 7.3 respectively. In the urine the antigens recognized by the monoclonal antibodies form large complexes with Tamm-Horsfall protein. The antigen-containing complexes can be extracted from urine by adsorption to diatomaceous earth and elution with SDS-containing buffer. Using sandwich ELISA-type assays it is possible to determine the concentration of the antigens. In preliminary experiments we were able to show that at least three of the antigens are detected in the urine following toxic insults to the kidney. The monoclonal antibodies prepared and the tests developed thus may provide direct diagnostic access to the renal papilla and allow, for the first time, early detection of papillary damage.

Die LDH-isoenzyme als ursache für unspezifische tetrazoliumsalz-anfärbungen in gelzymogrammen (“nothing dehydrogenase”)

Abstract It was demonstrated that after tetrazolium salt staining of gel enzymograms of extracts containing NAD-dependent dehydrogenases, a pattern of lactate dehydrogenase ( l -lactate: NAD-oxidoreductase, E.G. 1.1.1.27) isoenzymes also became visible, although the incubation mixture contained no lactate, but only the specific substrate for the dehydrogenase examined. From the experiments reported it appears that the supporting media of the gel electrophoresis (starch, polyacrylamide, cellulose acetate) contain groups similar to lactate insolubly bound to the polymerous gel system. This is probably the case also with agar gel. This fault unavoidably occurs with all methods used. By differential staining of horizontal layers of a gel with different substrates it was shown that extracts of human tissue contained only a single anodic band of glycerol-3-phosphate dehydrogenase ( l -glycerol-3-phosphate: NAD-oxidoreductase, E.G. 1.1.1.8) and a single anodic band of glutamate dehydrogenase ( l -glutamate: NAD-oxidoreductase, deaminating, E.G. 1.4.1.3). The remaining bands that could be visualized by usual staining methods have been identified as lactate dehydrogenase isoenzymes. Confirmation was obtained by comparison with results of ion exchange Chromatography. These results offer a simple explanation for the unspecific staining of lactate dehydrogenase isoenzymes, and make it possible to avoid erroneous conclusions in the examination of multiple forms of enzymes, as seen in numerous reports recently.

A simple and inexpensive high density dialysis tubing cell culture system for the in vitro production of monoclonal antibodies in high concentration

This paper describes the construction and application of a low-cost roller bottle-like culture appliance in which hybridoma cells can be cultivated in high density in dialysis tubing. The appliance facilitates the simultaneous culture of up to four cell lines yielding 50 ml culture volume of each. Samples for follow-up analysis of the cultures can easily be taken when needed through sample ports. In order to obtain high cell densities (at least 10(7) cells/ml), high cell viability (at least 50%) and high antibody yield (at least 1.0 mg/ml) the bottle is rolled at a speed of 4-6 rpm and is gassed continuously by a micropump driven by rechargeable NiCd batteries fixed to the culture flask. Depending on the individual properties of the hybridoma lines tested, the cells may be cultured for 1-2 weeks, and cell densities of up to 30 x 10(6) cells/ml with viabilities of approximately 50% and monoclonal antibodies in concentrations of up to 2.8 mg/ml may be obtained. In their properties the monoclonal antibodies produced by this in vitro procedure are indistinguishable from those prepared in the form of conventional stationary culture supernatant or of ascitic fluid. Specific antibody content is within the same range as in ascitic fluid. Consequently, the monoclonal antibodies can be purified in one step, e.g., by ion exchange chromatography from the culture supernatant. Therefore, the newly developed culture device and the culture method described is a useful alternative to ascites production in live mice.

Characterization of mouse macrophage differentiation antigens by monoclonal antibodies

Monoclonal rat antibodies to mouse macrophage antigens were prepared. For immunization phagocytic cells in the spleens of mice recovering from sublethal irradiation were used. Specificities of the monoclonal antibodies obtained were determined on cells of normal mouse cell populations as well as on cells of a panel of mouse cell lines. In an attempt to monitor expression of differentiation-related antigens two models of in vitro-induced macrophage differentiation were used: differentiation of cells of the myeloblast line Ml; CSF-1-induced differentiation of bone marrow cells. The results obtained clearly show that during maturation from undifferentiated to highly differentiated cells of the macrophage lineage expression of antigens recognized by the MIV 38, MIV 55, MV 87, and MV 114 monoclonal antibodies is enhanced. At the same time, expression of antigens recognized by the MIV 52, MIV 113, and MIV 116 monoclonal antibodies diminishes at a similar rate. The suitability of these monoclonal antibodies for the characterization of differentiation states of mouse macrophages is discussed.

A B-cell lymphoma vaccine using a depot formulation of interleukin-2 induces potent antitumor immunity despite increased numbers of intratumoral regulatory T cells

Therapeutic vaccination holds great potential as complementary treatment for non-Hodgkin’s lymphoma. Here, we report that a therapeutic whole cell vaccine formulated with IL-2 adsorbed onto aluminum hydroxide as cytokine-depot formulation elicits potent antitumor immunity and induces delayed tumor growth, control of tumor dissemination and longer survival in mice challenged with A20-lymphoma. Therapeutic vaccination induced higher numbers of tumor’s infiltrating lymphocytes (CD4+ and CD8+ T cells and NK cells), and the production of IFN-γ and IL-4 by intratumoral CD4+ T cells. Further, strong tumor antigen-specific cellular responses were detected at systemic level. Both the A20-derived antigenic material and the IL-2 depot formulation were required for induction of an effective immune response that impacted on cancer progression. All mice receiving any form of IL-2, either as part of the vaccine or alone as control, showed higher numbers of CD4+CD25+/highFoxp3+ regulatory T cells (Treg) in the tumor, which might have a role in tumor progression in these animals. Nevertheless, for those animals that received the cytokine as part of the vaccine formulation, the overall effect was improved immune response and less disseminated disease, suggesting that therapeutic vaccination overcomes the potential detrimental effect of intratumoral Treg cells. Overall, the results presented here show that a simple vaccine formulation, that can be easily prepared under GMP conditions, is a promising strategy to be used in B-cell lymphoma and may have enough merit to be tested in clinical trials.

Cellular analysis of the phenotypic correction of the genetically controlled low immune response to the polyproline determinant by macrophages.

Abstract SJL mice are high responders to the polyproline region of poly(Tyr,Glu) -polyPro-polyLys, (T,G)-Pro-L and of poly (Phe,Glu) -polyPro-polyLys, (Phe,G)-Pro-L, whereas DBA/1 mice are the low responders to this moiety. The low responsiveness of DBA/1 mice to polyproline could be enhanced by immunization with (T,G)-Pro-L 4 days after stimulation of peritoneal cells by thioglycolate. The same effect was observed when DBA/1 mice were immunized with 10 7 syngeneic peritoneal exudate cells (PEC) preincubated in vitro with the immunogen. Similar treatments of SJL mice did not enhance the high response to polyproline, nor did it enhance low responses to other synthetic polypeptides tested. The enhancing effect of PEC on immunocompetent cells was established by transferring graded numbers of spleen cells together with 10 7 PEC into irradiated syngeneic DBA/1 recipients. The effective cell type in the PEC was found to be the macrophage as the same results were observed with the adherent-cell population. Furthermore, the effect was not abolished after in vitro irradiation of PEC with 5000 R or by anti-θ treatment. In vivo irradiation of the PEC donors 2 days before the cells were harvested also did not influence the phenotypic correction of the low responsiveness. Transfer experiments in which graded inocula of either marrow cells or thymocytes from DBA/1 donors were transferred into syngeneic recipients in the presence of an excess of the complementary cell type together with PEC indicated that the enhancing effect was reflected in the bone-marrow-cell population only.

Kidney-derived urinary antigens assayed with monoclonal antibodies for the detection of renal damage

For the quantitation of kidney-derived Urinary Antigens (UA) monoclonal antibodies specific for antigens localized in cells of defined subunits of the nephron were applied in sandwich ELISA. Antigen excretion was measured in the urine of healthy individuals, patients suffering from various diseases, kidney transplant recipients, and healthy volunteers receiving therapeutic doses of antibiotic drugs. In healthy individuals, in patients with diseases primarily affecting the glomerulus, and in inactive phases of chronic diseases antigen excretion was low. Toxic drug effects enhanced antigenuria. Excretion of some or all of the antigens always indicated tubular alterations. The tests thus provide information on location and extent of acute primary tubular damage.

Preparation and characterization of a mouse monoclonal antibody against a rat kidney papillary antigen

Monoclonal antibodies were prepared in an attempt to develop diagnostic tools for the identification of toxic damage to the renal papilla. One of these antibodies, termed PAP X 5C10, recognizes an antigen that is located on the luminal side of collecting duct epithelial cells. As shown by Western blotting experiments, this antigen can be extracted from papillary tissue and visualized as a broad band of high molecular weight. Enzyme-linked immunosorbent assays have shown that increased amounts of this antigen can be detected in urine of rats treated with bromoethaneamine. This procedure thus enables this antigen to be detected also in supernatants of cultured ductal cells.