Penetration injury of the hindfoot following intramedullary nail fixation of a tibial fracture.

Technical errors during intramedullary nail insertion are not uncommon. We report a case of tibial guide wire penetration into the distal tibial articular surface, the talus and the calcaneus during insertion of the nail with the ankle dorsiflexed. This has not been reported in the past. Computerized tomogram was a useful tool in the diagnosis. This complication was associated with long-standing ankle pain, which however eventually settled. We advise frequent use of biplanar C-arm image during the insertion of the guide wire, the reamer and tibial nail into the medullary canal of the tibia or other long bones. None of these instruments should be forced through. Once the knobbed guide wire is exchanged to a straight guide wire, the wire should not be forced through or reamed over, and the nail should be introduced over the guide wire with caution. Early intraoperative identification and recording of this iatrogenic accident is necessary in order to explain the situation to the patient and modify treatment accordingly.

The distribution of MUC1, an apical membrane glycoprotein, in mammary epithelial cells at the resolution of the electron microscope: implications for the mechanism of milk secretion.

The distribution of the glycoprotein, mucin 1 (MUC1), was determined in lactating guinea-pig mammary tissue at the resolution of the electron microscope. MUC1 was detected on the apical plasma membrane of secretory epithelial cells, the surface of secreted milk-fat globules, the limiting membranes of secretory vesicles containing casein micelles and in small vesicles and tubules in the apical cytoplasm. Some of the small MUC1-containing vesicles were associated with the surfaces of secretory vesicles and fat droplets in the cytoplasm. MUC1 was detected in much lower amounts on basal and lateral plasma membranes. By quantitative immunocytochemistry, the ratio of MUC1 on apical membranes and milk-fat globules to that on secretory vesicle membranes was estimated to be 9.2:1 (density of colloidal gold particles/microm membrane length). The ratio of MUC1 on apical membranes compared with basal/lateral membranes was approximately 99:1. The data are consistent with a mechanism for milk-fat secretion in which lipid globules acquire an envelope of membrane from the apical surface and possibly from small vesicles containing MUC1 in the cytoplasm. During established lactation, secretory vesicle membrane does not appear to contribute substantially to the milk-fat globule membrane, or to give rise in toto to the apical plasma membrane.

Identification of a single amino acid substitution in the diphtheria toxin A chain of CRM 228 responsible for the loss of enzymatic activity.

CRM 228 (T. Uchida, A. M. Pappenheimer, and R. Greany, J. Biol. Chem. 248:3838-3844, 1973), a mutant form of diphtheria toxin which completely lacks ADP-ribosyltransferase activity, contains five amino acid substitutions. The two amino acid changes that fall within the A chain of the toxin (G79D and E162K) were separately analyzed by substituting a variety of other amino acids at these sites. The substitution at position 79 (G79D) singularly appears to account for the loss of enzymatic activity found in CRM 228.

Histidine 21 does not play a major role in diphtheria toxin catalysis.

It has been proposed that the histidine at position 21 (H21) of the diphtheria toxin A subunit (DTA) plays an important role in the ADP-ribosyltransferase (ADPRT) activity of the toxin. The region of DT encompassing H21 demonstrates sequence similarity with other toxins exhibiting ADPRT activity, is located along the catalytic cleft of DTA, and when H21 is chemically modified, ADPRT activity is abolished. H21 was mutagenized by a polymerase chain reaction-based system whereby all alternative amino acids were substituted in place of the histidine. The majority of the substitutions virtually abolished enzymatic activity, the exception being a mutant in which H21 was replaced with asparagine (DTA-H21N). This mutant demonstrated only a slight increase in Km and relatively small decreases in both reaction rate (kcat) and catalytic efficiency (kcat/Km). Asparagine is a sterically conserved substitution, but its side-chain is unable to replace the imidazole group of histidine in general acid-base mechanisms or to participate in electrostatic interactions. This suggests that H21 is important in maintaining a steric conformation required for catalysis rather than in participating in an electrostatic or acid-base type of exchange.

An improved method for generating single-chain antibodies from hybridomas.

Cloning the correct VL kappa gene from hybridomas derived from MOPC-21 can be problematic because such cell lines variably express a transcript which is aberrantly rearranged at the VJ recombination site. Cellular levels of the aberrant transcript can exceed that of productive light chain RNA, so a large proportion of the VL gene-derived products obtained on PCR amplification of hybridoma cDNA may not encode a functional protein. We have developed a method in which antibody variable region genes are recovered from hybridoma cDNA using a unique set of V gene family-specific primers; the V region genes are then spliced by PCR, in the form 5'-VL-LINKER-VH-3' (where the linker encodes [GlyGlyGlyGlySer]3), and cloned into an expression vector under control of T7 RNA polymerase. Plasmid DNA is isolated from colonies, and the insert is expressed in an in vitro rabbit reticulocyte lysate-based coupled transcription/translation system, in a microtiter plate format. Since aberrantly rearranged VL kappa genes contain a translation termination codon at amino acid position 105, only constructs containing the correctly rearranged gene produce a protein of the predicted size. We demonstrate the method by producing the single-chain form of OKT9, a murine IgG1 which binds to the human transferrin receptor, and extend the results to show that the protein generated by the in vitro expression system retains the antigen binding properties of the parent antibody. Our method will be generally useful for screening single-chain antibodies for function prior to large scale production in vivo.

Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate.

Chimeric proteins consisting of a fusion between binding-deficient mutants of diphtheria toxin (DT) or Pseudomonas exotoxin A (PE) and a single-chain antibody (E6 sFv) against the human transferrin receptor (TfnR) were expressed in a rabbit reticulocyte lysate system. Molecules utilizing PE40 (the carboxyl terminus 40 kDa of PE, lacking the binding domain) exhibited significant E6 sFv-mediated, cell type-specific cytotoxicity (IC50 1 x 10(-10) M) against a human erythroleukemia-derived cell line, K562. In contrast, a fusion protein between the same sFv and a DT mutant, DTM1 (containing two amino acid substitutions in the binding domain [S(508)F, S(525)F]) was not significantly cytotoxic, despite being enzymatically active. A tripartite protein in the form NH2-DTM1-E6 sFv-PE40-COOH exhibited cytotoxicity comparable to that of the PE40-sFv fusion (IC50 1 x 10(-10) M), suggesting that the deficit in activity of DTM1-sFv is not a function of misfolding of the sFv moiety or of a reduced ability to bind TfnR. In contrast to DTM1-E6 sFv, a fusion protein between a second DT mutant, CRM 107 [S(525)F], and the E6 sFv was specifically cytotoxic (IC50 1 x 10(-9) M), and toxicity could be blocked by addition of excess E6 antibody. The cell-free in vitro expression system we describe is rapid and may be used to express functional toxin-sFv fusion proteins. No protein refolding procedures are required, and the technique may be used to express proteins which, due to restrictions imposed on manipulation of toxin-encoding genes in Escherichia coli, could not be produced by more conventional methods.

The role of proline 345 in diphtheria toxin translocation.

Diphtheria toxin (DT) can translocate across endosomal membranes in response to low pH. Buried hydrophobic domains localized in the 37-kDa toxin B chain become exposed in response to acidic conditions and are thought to participate in the membrane translocation process. The crystal structure of DT has revealed a structurally distinct translocation domain composed of nine alpha-helices with their interconnecting loops (Choe, S., Bennett, M., Fujii, G., Curmi, P., Kantardjieff, K., Collier, R., and Eisenberg, D. (1992) Nature 357, 216-222). Two of these alpha-helices, TH8 and TH9, are unusually apolar and constitute the central core of the translocation domain. It has been proposed that these domains and the highly charged interconnecting loop undergo a conformation change under acidic conditions producing a dagger-like structure capable of inserting into the membrane thus initiating the translocation process. Proline 345 occupies a strategic location at the end of the TH8 alpha-helix. Proline residues have the ability to undergo a cis-trans isomerization reaction and because of this have been proposed to play a role in the conformational change that is a prerequisite for toxin translocation. The role of the proline at position 345 in membrane translocation was investigated. Pro was mutagenized to Glu and to Gly using a two-step recombinant polymerase chain reaction procedure, and the mutant proteins were expressed in vitro. Glu, an alpha-helix former, and Gly, an alpha-helix breaker, were selected for mutagenesis to distinguish between a structural role for Pro as an alpha-helix breaker and alternative roles, perhaps involving cis-trans isomerization-related conformational changes. Replacing Pro at position 345 with Glu or Gly resulted in a 99% reduction in toxicity to Vero cells. The enzymatic and binding activity of the toxin were not altered by the mutations. Instead, the reduction in toxicity is due to decreased translocation ability, suggesting that the Pro at position 345 plays a specific role in toxin membrane translocation.

Suppression of the immune response to immunotoxins with anti-CD4 monoclonal antibodies.

Treatment of normal mice with a mAb to CD4 (GK1.5) was explored as a means of inhibiting the antibody response to an immunotoxin. Three days of pretreatment with 200 micrograms of GK1.5 completely abrogated the primary antibody response to a 3-micrograms dose of a mutant diphtheria toxin conjugated to an anti-transferrin receptor antibody. The same dose and schedule of anti-CD4 antibody significantly reduced and delayed, but did not prevent, the anamnestic antitoxin response in animals that had been previously primed to the immunotoxin. Three daily injections of anti-CD4 antibodies followed by weekly doses of immunotoxin resulted in a 3-wk delay in the development of antitoxin antibodies, and the kinetics of the antitoxin response correlated with the kinetics of recovery of CD4+ T cells in the spleen and lymph nodes. The antitoxin response to repeated doses of immunotoxin was completely abrogated when anti-CD4 antibodies were given every 2 wk throughout the course of immunotoxin treatment. Thus, transient depletion of Th cells during treatment can block the immune response to an immunotoxin. There was no evidence of tolerance induction with this regimen.

A point mutation of proline 308 in diphtheria toxin B chain inhibits membrane translocation of toxin conjugates.

Diphtheria toxin (DT) is a soluble protein that translocates across hydrophobic lipid bilayers in response to low pH. The translocation activity of DT has been localized to the 40-kDa toxin B chain and can be expressed independently of the C-terminal receptor binding site. Buried hydrophobic domains in DT are thought to participate in the membrane translocation process. We have identified a mutant form of DT, CRM 102, that has a point mutation at position 308 (Pro----Ser) within one of these hydrophobic domains. CRM 102 conjugated to a monoclonal antibody against the T cell receptor, the transferrin receptor, or transferrin itself is approximately 10-fold less toxic than native DT or a control DT mutant, CRM 103, linked to the same binding moieties. Direct measurement of membrane translocation activity by exposure of cells to low extracellular pH demonstrates that CRM 102 conjugates express only 10% of the translocation activity of the control toxin conjugates. However, when CRM 102 or 102 conjugates bind and kill cells via the DT receptor, no reduction in membrane translocation activity is observed. The defect in CRM 102 is not evident in the presence of 20 mM NH4Cl. The defect in translocation also has no effect on the ratio of the lag time before protein synthesis inhibition begins to the rate of protein synthesis inhibition. Thus, the proline-serine substitution at position 308 disrupts the membrane translocation process and distinguishes between two routes of DT entry: DT receptor-mediated entry and entry mediated by alternate receptors.

Improved tumor-specific immunotoxins in the treatment of CNS and leptomeningeal neoplasia.

A novel antibody-toxin conjugate has been developed for use in cancer therapy. This report demonstrates that this new reagent selectively kills glioblastoma- and medulloblastoma-derived cell lines, medulloblastoma cells in primary culture, and cell lines derived from tumors commonly metastatic to the cerebrospinal fluid (CSF). Efficient killing of human tumor cells occurred at concentrations between 3.9 X 10(-13) M and 1.1 X 10(-10) M, whereas guinea pigs and rhesus monkeys tolerated intrathecal levels of 2 X 10(-9) M. Cerebrospinal fluid from normal humans and from brain-tumor patients does not inhibit the in vitro efficacy of this reagent. The wide therapeutic window, extreme potency, and general applicability of this antibody-toxin conjugate against CSF-borne primary or metastatic tumors warrants clinical trials.

Purification and characterization of a differentiation-specific sialoglycoprotein of lactating-guinea-pig mammary tissue.

A large acidic glycoprotein, PAS-I, was purified from the fat-globule membrane of guinea-pig milk. Threonine and serine accounted for over 30 mol% of the amino acids, and galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose and sialic acid were the principal sugars detected. On a molar basis, sialic acid accounted for over 60% of the total sugar. Removal of sialic acid by treatment with neuraminidase revealed the presence of binding sites for peanut (Arachis hypogaea) agglutinin, a lectin specific for the sugar sequence beta-D-Gal-(beta 1----3)-D-GalNac (the T antigen). The distribution of PAS-I-related epitopes, defined by five monoclonal antibodies, was determined in the mammary gland and in other guinea-pig tissues. PAS-I was maximally expressed on the apical surfaces of secretory cells in lactating mammary tissue and was either absent, or present in much lower amounts, in the glands of virgin or pregnant animals. PAS-I epitopes were not detected in liver, heart, spleen, pancreas, ovary, uterus, lung or intestine, either by immunofluorescence microscopy or by immunoblotting techniques. Several of the PAS-I-specific antibodies bound to mucins of high Mr in human fat-globule membrane, and similarities and differences between PAS-I and the human mucins are discussed. PAS-I and epitopes of this glycoprotein will be useful as indicators of differentiation in mammary cells and of markers of the apical surface of these cells during lactation.

The role of the diphtheria toxin receptor in cytosol translocation.

The role of the receptor in the transport of diphtheria toxin (DT) to the cytosol was examined. A point-mutant form of DT, CRM 107 (CRM represents cross-reacting material), that has an 8,000-fold lower affinity for the DT receptor than native toxin was conjugated to transferrin and monoclonal antibodies specific for the cell-surface receptors T3 and Thy1. Conjugating the binding site-inactivated CRM 107 to new binding moieties reconstituted full toxicity, indistinguishable from native DT linked to the same ligand, indicating that the entry activity of the DT B chain can be fully separated from the receptor binding function. Like DT, the toxin conjugates exhibited a dose-dependent lag period before first-order inactivation of protein synthesis. Inactivation of the binding site of the toxin portion of the conjugate was found to have no effect on the kinetics of protein synthesis inactivation. The receptor used by the toxin determined the length of the lag period relative to the killing rate. Comparing the potency of CRM 107 conjugates with native DT, standardized for receptor occupancy, shows that new receptors can be as or more efficient than the DT receptor in transporting DT to the cytosol. The transferrin-CRM 107 conjugate, unlike native DT, was highly toxic to murine cells. All the data presented are consistent with a model that the DT receptor, other than initiating rapid internalization of the toxin to low pH compartments, is unnecessary for transport of the toxin to the cytosol and that membrane translocation activity is expressed by the DT B subunit independent of the receptor-binding site.

Mutations in diphtheria toxin separate binding from entry and amplify immunotoxin selectivity.

Monoclonal antibodies linked to toxic proteins (immunotoxins) can selectively kill some tumor cells in vitro and in vivo. However, reagents that combine the full potency of the native toxins with the high degree of cell type selectivity of monoclonal antibodies have not previously been designed. Two heretofore inseparable activities on one polypeptide chain of diphtheria toxin and ricin account for the failure to construct optimal reagents. The B chains (i) facilitate entry of the A chain to the cytosol, which allows immunotoxins to efficiently kill target cells, and (ii) bind to receptors present on most cells, which imparts to immunotoxins a large degree of non-target cell toxicity. This report identifies point mutations in the B polypeptide chain of diphtheria toxin that block binding but allow cytosol entry. Three mutants of diphtheria toxin have 1/1,000 to 1/10,000 the toxicity and 1/100 to 1/8,000 the binding activity of diphtheria toxin. Linking of either of two of the inactivated mutant toxins (CRM103, Phe508; CRM107, Phe390, Phe525) to a monoclonal antibody specific for human T cells reconstitutes full target-cell toxicity--indistinguishable from that of the native toxin linked to the same antibody--without restoring non-target cell toxicity. This separation of the entry function from the binding function generates a uniquely potent and cell type-specific immunotoxin that retains full diphtheria toxin toxicity, yet is four to five orders of magnitude less toxic than the native toxin is to nontarget cells.

Potent and specific killing of human malignant brain tumor cells by an anti-transferrin receptor antibody-ricin immunotoxin.

Immunotoxins are hybrid molecules which combine the exquisite selectivity of monoclonal antibodies with the potent toxicity of protein toxins. An immunotoxin was constructed by linking a murine monoclonal antibody against the human transferrin receptor (TR) to the plant toxin, ricin. The cytotoxic activity of the anti-TR-ricin immunotoxin was tested in vitro and demonstrated highly potent and cell type-specific killing of cells derived from human glioblastoma, medulloblastoma, and leukemia. The anti-TR-ricin immunotoxin killed more than 50% of "target" cells at a concentration of 5.6 X 10(-13) M after an 18-hour incubation with the ionophore, monensin. This potency exceeds that of any other anti-TR immunotoxin reported in the literature. When the activity of the anti-TR-ricin immunotoxin against "target" tumor-derived cells was compared with the immunotoxin's activity against "non-target" cells, it could be predicted that a selective toxicity of anti-TR-ricin immunotoxin between tumor cells and normal brain was more than 150- to 1380-fold. Solid-phase indirect radioimmunoassay techniques were used to demonstrate significantly higher levels of TR in the glioblastoma- and medulloblastoma-derived cell lines, as well as in surgical tissue samples of medulloblastoma and glioblastoma, as compared to normal brain. Immunotoxins targeted to the TR may possess sufficient specificity to be of therapeutic importance, particularly to treat neoplastic disease of the central nervous system involving compartments (such as intrathecal, intraventricular, or cystic) where delivery of immunotoxins to tumor would not require transvascular transport.

Identification and characterization of a monoclonal antibody recognizing a galactose-binding domain of the toxin ricin.

A monoclonal antibody raised against purified ricin B chain, 75/3B12, blocked ricin toxicity 30- to 100-fold in vitro. The 75/3B12 IgG and F(ab')2 blocked ricin binding to cell surface galactose-containing receptors. The 75/3B12 Fab bound ricin D with a Ka of 10(7) M-1, and this binding was blocked by asialofetuin, lactose, and N-acetylgalactosamine--molecules which interact with the ricin galactose-binding site--but not by fetuin, sucrose, or glucose. The 75/3B12 Fab contained no detectable carbohydrate and, according to several lines of evidence, did not bind ricin via Ig carbohydrate determinants. The monoclonal antibody appears to recognize a galactose-binding site on ricin D via the variable region of the antibody. The 75/3B12 Fab bound ricin E only 1/50 as well as ricin D and bound the Ricinus agglutinin only 1/80 as well as ricin D. The antibody specificity indicates that structural differences exist in the galactose-binding sites of the Ricinus communis lectins. Abrin and other lectins which bind galactose or N-acetylgalactosamine were not significantly bound by the monoclonal antibody. In vitro, the antibody blocked the nontarget toxicity of immunotoxins similarly to lactose. However, in vivo, unlike lactose, the 75/3B12 antibody protected mice from ricin toxicity.

Analysis of a human tumor-associated glycoprotein (TAG-72) identified by monoclonal antibody B72.3.

Monoclonal antibody B72.3 binds a high-molecular-weight tumor-associated glycoprotein identified as TAG-72. This study reports the partial purification and characterization of TAG-72 from a xenograft of a human carcinoma cell line, LS-174T, which expresses high levels of this antigen. The tumor homogenate was initially fractionated by Sepharose CL-4B chromatography. The high-molecular-weight TAG-72, found in the exclusion volume, was then subjected to two sequential passages through B72.3 antibody affinity columns. At each step of the procedure, TAG-72 content was quantitated using a competition radioimmunoassay, and the degree of purification was expressed as the ratio of antigen in units to total protein. The three-step procedure produced a purification of TAG-72 with minimal contamination by other proteins as shown by polyacrylamide gel electrophoresis, followed by staining with Coomassie Blue or periodic acid/Schiff reagent. The density of affinity-purified TAG-72, as determined by cesium chloride gradient ultracentrifugation, was found to be 1.45 g/ml. This density determination, together with the high molecular weight of TAG-72, its resistance to Chondroitinase digestion, the presence of blood group-related oligosaccharides, and sensitivity to shearing into lower-molecular-weight forms suggest that TAG-72 is a mucin-like molecule.

Identification and characterization of the principal proteins of the fat-globule membrane from guinea-pig milk.

The milk-fat-globule membrane (MFGM) was isolated from guinea-pig milk and the membrane-associated proteins and glycoproteins characterized by electrophoretic techniques. Major components of the membrane included PAS-I, a sialoglycoprotein of Mr greater than or equal to 200000, the redox enzyme xanthine oxidase and the glycoprotein, butyrophilin. Membrane preparations also contained two other glycoproteins, GP-80 and GP-55, of Mr 80000 and 55000, respectively. Comparison of guinea-pig xanthine oxidase and butyrophilin with proteins from bovine MFGM by peptide mapping procedures, showed that the two proteins in both species were similar, but not identical. GP-55 may also be related to glycoproteins of Mr 45000 and 48000 in the bovine membrane. The integral and peripheral components of guinea-pig MFGM were identified by treating membrane preparations with sodium carbonate solutions at high pH and by partitioning the membrane proteins in solutions of Triton X-114. By these criteria xanthine oxidase and GP-55 appeared to be peripheral components and GP-80 an integral protein of the membrane. PAS-I and butyrophilin displayed hydrophilic properties in Triton X-114 solutions, but could not be removed from membrane preparations with sodium carbonate. Possible reasons for these ambiguous data are discussed. The observed similarity between several of the proteins of guinea-pig and bovine MFGM implies that these proteins may have specific functions related to milk secretion in mammary tissue, e.g. in the budding of milk-fat globules or the exocytosis of milk protein and lactose at the apical surface.

Monoclonal antibodies prepared against PAS-I butyrophilin and GP-55 from guinea-pig milk-fat-globule membrane bind specifically to the apical pole of secretory-epithelial cells in lactating mammary tissue.

Monoclonal antibodies to the three major glycoproteins of guinea-pig milk-fat-globule membrane were isolated. The specificity of these antibodies was determined by solid-phase immunoassays and by immunoblotting and autoradiographic techniques after one- and two-dimensional gel electrophoresis. The antibodies bound to PAS-I, a sialoglycoprotein of Mr greater than or equal to 200 000 and the glycoproteins butyrophilin and GP-55, of Mr 63 000 and 55 000, respectively. Immunolocalization studies showed that all three proteins were highly concentrated in the apical pole of secretory-epithelial cells in mammary tissue during lactation. PAS-I, butyrophilin or GP-55, were not detected in either the basal cytoplasm of mammary epithelial cells or in myoepithelial cells, capillary endothelial cells or other cells found in the mammary gland. These proteins were either present in small amounts or were absent from mammary tissue taken in late pregnancy. The monoclonal antibodies characterized in this study will therefore be useful as probes for studies of the biogenesis of apical membrane proteins in mammary epithelial cells during lactation.