Immunological properties of conjugates of ragweed pollen antigen E with methoxypolyethylene glycol or a copolymer of D-glutamic acid and D-lysine.

The major allergen of ragweed pollen, antigen E, was modified by coupling its amino acid groups with either methanol, methoxypolyethylene glycol (MPEG) of 5,000 daltons, or a synthetic copolymer of D-glutamic acid and D-lysine (DGL) of 34,000 daltons, all appropriately activated. The conjugates were characterized chemically and immunologically. Compared to the native antigen, the methoxy conjugate showed little reduction in allergenic activity, but the other two conjugates showed strong reductions, as measured by heterologous passive cutaneous anaphylaxis in rats sensitized with murine anti-antigen E reaginic sera. The MPEG conjugate was apparently nonimmunogenic in mice known to be high responders to the native antigen. MPEG and DGL conjugates retained the immunosuppressive property of the native antigen as subcutaneous treatment of antigen E sensitized mice with these two conjugates led to significant long-lasting depression of their antigen E-specific IgE and IgG antibody levels. These immunological changes are believed to result from reduction of antigenic valency and specificity upon coupling the bulky molecules to the protein antigens.

Paclitaxel and concurrent radiation for locally advanced pancreatic and gastric cancer: a phase I study.

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicities, and potential antitumor activity of weekly paclitaxel with concurrent radiation (RT) for locally advanced pancreatic and gastric cancer. PATIENTS AND METHODS: Thirty-four patients with locally advanced adenocarcinoma of the pancreas or stomach were studied. The initial dose of paclitaxel was 30 mg/m2 by 3-hour intravenous (I.V.) infusion repeated every week for 6 weeks with 50 Gy RT. Doses were escalated at 10-mg/m2 increments in successive cohorts of three new patients until dose-limiting toxicity was observed. RESULTS: The dose-limiting toxicities at 60 mg/m2/wk were abdominal pain within the RT field, nausea, and anorexia. Of 23 patients with assessable disease, 11 (seven with gastric, four with pancreatic cancer) had objective responses for an overall response rate of 48%. CONCLUSION: Concurrent paclitaxel with upper abdominal RT is well tolerated at dosages that have substantial activity. A phase II trial of neoadjuvant paclitaxel and RT at the MTD of 50 mg/m2/wk is underway.

Immunochemical observations of antigen 5, a major venom allergen of hornets, yellowjackets and wasps.

Antigen 5 is a venom protein of hornets, yellowjackets and wasps, and it is an important allergen for insect-sensitive patients. Antigen 5s from these insects are found to have similar amino-terminal sequences for their first 20 residues, and to have similar cyanogen bromide cleavage patterns. A partial sequence homology of antigen 5s and scorpion neurotoxins is observed. Antigen 5s from these insects show varying extents of cross reactivity when tested with mouse polyclonal antibodies by enzyme immunoassay and they show limited or no cross reactivity with mouse monoclonal antibodies. Chemically modified antigen 5s shown greatly decreased binding of antigen 5-specific mouse polyclonal antibodies. The modified antigen 5s include its reduced and carboxymethylated

Immunochemical studies of yellowjacket venom proteins.

The major proteins of yellowjacket venoms have been isolated and characterized immuno-chemically. They consist of hyaluronidase, phospholipase, and antigen 5. Venoms from three species of yellowjacket were studied. Vespula germanica, V. maculifrons, and V. vulgaris. The phospholipases could be isolated in good yield only when affinity chromatography was used to minimize limited proteolysis. A kallikrein-like peptidase was found present in the yellowjacket venom. Phospholipases from these three species were immunochemically indistinguishable from each other, as were their antigen 5s. Sera from individuals sensitive to yellowjacket venom contained IgE and IgG specific for antigen 5 and phospholipase.

Sequence similarity of a hornet (D. maculata) venom allergen phospholipase A1 with mammalian lipases.

We have determined the sequence of a venom allergen phospholipase A1 from white-faced hornet (Dolichovespula maculata) by cDNA and protein sequencings. This protein of 300 amino acid residues (Dol m I) has no sequence similarity with other known phospholipases. But it has sequence similarity with mammalian lipases; about 40% identity in overlaps of 123 residues. Tests suggest that hornet phospholipase has weak lipase activity. Hornet venom has 3 major allergens, and another hornet allergen antigen 5 (Dol m V) was previously found to have sequence similarity with a mammalian testis protein and a plant leaf protein.

A comparison of different enzyme-antibody conjugates for enzyme-linked immunosorbent assay.

Calf intestinal alkaline phosphatase (CIAP)- and horseradish peroxidase (HRP)-linked goat antibody (Ab) conjugates have been prepared by two procedures. One procedure is by glutaraldehyde coupling of the proteins; the other is by intermolecular disulfide bond formation of the appropriately modified proteins. The conjugates, specific for rabbit IgG, were tested for their effectiveness as reagents in enzyme-linked immunosorbent assays for ragweed antigen E specific rabbit antibody. The CIAP-Ab conjugate prepared by glutaraldehyde coupling and the HRP-Ab conjugate made by disulfide bond formation were equally effective reagents for immunoassay, but the HRP-Ab conjugate obtained by glutaraldehyde coupling was definitely less useful than the other two conjugates. The assay procedure utilized an antigen E coupled paper disc as the immunosorbent and it was sensitive in the range of 0.5--100 ng per test. Inhibition of the enzyme-linked immunosorbent assay was used as a sensitive technique for measuring antigenic activity of antigen E or its derivatives.

Enzyme and radioimmunoassays for specific murine IgE and IgG with different solid-phase immunosorbents.

Enzyme and radioimmunoassays of specific murine IgE and IgG were done using antigen-coated flex vinyl plates, antigen-coupled paper discs or Sepharose 4B beads as solid-phase immunosorbents. These 3 immunosorbents differ in their antibody binding capacities. Only Sepharose beads have a high capacity so that they can be used to assay specific IgE in serum samples without interference from specific IgG which can be present in concentrations of several thousand times greater than those of specific IgE. All 3 types of immunosorbents are equally useful for assaying specific IgG, but the flex vinyl plate is the immunosorbent of choice since it requires less time and gives lower blanks than do the other two. Under the conditions tried, the lowest detectable concentration of specific IgE or IgG was about 1 ng/ml on enzyme or radioimmunoassay. The immunoassays described in this paper are useful for following the kinetics of specific IgE and IgG responses in mice. This was demonstrated with sera from mice immunized with ragweed antigen E.

The photodecomposition of aminopterin.

Aminopterin is used in cell fusion experiments to select for hybrid cells by killing unfused cells which are deficient in enzymes for nucleotide salvage pathways. Aqueous aminopterin solutions exposed to fluorescent room light (wavelength greater than 300 nm) were found to lose cytotoxicity. Inactivation was accompanied by changes in the ultraviolet absorption spectra of the solutions. The ultraviolet spectrum of an irradiated aminopterin solution is used to provide a quantitative measure of its cytotoxicity. Aminopterin appears to be photolytically cleaved at the C9-N10 methylene-anilino bond in a reaction analogous to that known for the photolysis of folic acid irradiated at 365 nm (Lowry et al., 1949).

Chitin in egg shells of Onchocerca gibsoni and Onchocerca volvulus.

Chemical analysis of adult females of Onchocerca gibsoni gave estimated chitin contents of 200-500 micrograms (g dry weight)-1. Egg shells from both O. gibsoni and Onchocerca volvulus stained with Calcofluor white and with fluorescent wheat germ agglutinin as shown by fluorescent light microscopy, and bound gold-labelled wheat germ agglutinin as shown by electron microscopy, under conditions specific for chitin. The egg shells appeared as single electron dense layers from 50 to 85 nm in thickness. Purified chitinase digested these egg shells, leaving coiled microfilariae unattacked. We conclude that chitin is a major component of the egg shells.

Immunochemical studies of stinging insect venom allergens.

Several major venom allergens from different insects of the Hymenoptera order have been cloned and sequenced by different laboratories. These insects include fire ants, honey bees, hornets, yellowjackets and wasps. These venom allergens have different biochemical functions, but have one feature in common, their varying extents of sequence identity with other proteins in our environment. Our studies in mice suggest that recombinant fragments containing regions of sequence identity of venom allergen(s) and host protein(s) show antigenic cross-reactivity. These studies lead to the hypothesis that cross-reactivity of venom allergens with host proteins promotes the immunogenicity of venom allergens in susceptible people.

Recent advances in the study of the nutritional toxicity of kidney bean (Phaseolus vulgaris) lectins in rats.

The main toxic component isolated from several varieties of kidney bean (Phaseolus vulgaris) is a haemagglutinating lectin. The inclusion in rat diets of raw kidney beans or purified bean lectins results in abnormal development of microvilli in the small intestine. Immunocytochemical investigations have provided evidence that this lesion is associated with the binding of lectins to the luminal surfaces of enterocytes. The lectin-induced disruption of intestinal microvilli may result in interference with the intermediate and final stages of nutrient hydrolysis in the gut. In nitrogen balance studies it was found that rats ingesting pure bean lectins were in negative nitrogen balance. These nitrogen losses may have been partly the result of systemic effects, possibly caused by a selective uptake of lectins by the gut.

Escherichia coli K88 receptor expression in intestine of disease-susceptible weaned pigs.

A challenge trial was carried out in which Escherichia coli O157 K88ac was administered to a litter of weaned pigs and the development of the disease monitored over a five-day experimental period. The eight animals in the trial were assigned to two groups depending on whether they exhibited disease symptoms. Six pigs developed diarrhoea and two appeared unaffected; these were designated as the test (or K88-susceptible) group and the control (or K88-resistant) group, respectively. The animals were euthanised and the intestine was removed and sections processed for brush border membrane vesicle preparation. Microscopic and biochemical assays were undertaken on tissue samples from each animal and a strong correlation was observed between the expression of a glycoprotein receptor complex associated with the brush border membrane and the development of disease symptoms. Further investigation revealed the presence of an analogous glycoprotein complex in the K88-resistant group which did not bind the K88-fimbriae antigen. These results suggest that genetic differences in the glycosyl moieties of the receptor complex provide the basis for disease susceptibility to K88-positive E. coli.

Characterization and autoradiographic localization of the epidermal growth factor receptor in the jejunum of neonatal and weaned pigs.

Receptors for epidermal growth factor (EGF) were characterized on the intestinal membranes of newborn, sucking and weaned pigs. 125I-labelled EGF (125I-EGF) binding to membrane homogenates was time-dependent, saturable, linearly correlated to membrane protein and reversible. Analysis of saturation curve data revealed a single class of 125I-EGF binding sites in both newborn and weaned pigs. Receptor levels tended to be higher in weaned than in newborn pigs; the converse was true for the receptor affinity. In contrast, virtually no binding sites were found on the intestinal membranes of sucking pigs. Autoradiography in vitro of jejunal sections of newborn and weaned pigs demonstrated 125I-EGF receptors on both microvillar and basolateral surfaces of enterocytes, suggesting that luminal EGF could influence developmental processes in the intestine either directly or indirectly following transcytosis of the ligand.

Sensitivity of immunoassays for detecting cross-reactivity of homologous venom proteins of yellow jackets.

The venoms of yellow jackets of Vespula flavopilosa, V. pennsylvanica, V. squamosa, and V. vulgaris have similar proteins. Their major components comprise antigen 5, hyaluronidase, and phospholipase A1. The homologous venom proteins share very similar biochemical properties. With the exception of antigen 5 and phospholipase of V. squamosa, they also have very similar antigenic properties. The venom proteins of V. squamosa and V. vulgaris were equally effective in inducing secondary antibody responses in mice that were primed with V. squamosa proteins. No cross-reaction of V. squamosa and V. vulgaris venom proteins was detected on immunodiffusion with specific mouse antisera. A very weak cross-reaction was detected by inhibition of ELISA, and a moderate cross-reaction was detected by direct ELISA. The varying cross-reactivity is a consequence of the different sensitivities of the assays. The sensitivity of the direct ELISA is apparently caused by a greater enhancement of the binding of low-affinity antibodies for the solid-phase antigen than that of high-affinity antibodies. Most untreated yellow jacket-sensitive patients tested had about tenfold higher levels of IgG specific for V. vulgaris proteins than those specific for V. squamosa proteins. This pattern of antibody specificity can be accounted for on the basis of cross-reactivity of these yellow jackets as suggested by the results with the mouse system.