Arthur Malley

The isolation of allergens from the green pea

The aqueous extract of green peas was separated into 3 fractions (albumin, legumin, and vicilin) by dialysis against distilled water and isoelectric precipitation. The major antigenic and all of the allergenic activity of the pea extract was associated with the albumin fraction. The albumin fraction retains its allergenicity upon heating at 60 degrees C for 30 min or boiling at 100 degrees C for 5 min, but becomes partially inactivated by autoclaving at 120 degrees C for 15 min. The allergenic determinant expressed by the albumin fraction appears to be common to several other members of the legume family. In addition, the pea dialysate fraction was shown to specifically inhibit precipitin and passive cutaneous anaphylaxis (PCA) reactions involving rabbit antipea serum and the pea albumin fraction, and histamine release from passively sensitized monkey lung tissue using the serum of pea-sensitive patients.

Timothy pollen fractions in treatment of hay fever:I. Clinical and immunological response to small and higher molecular weight fractions

Abstract Forty-two timothy-sensitive patients have been treated with timothy pollen fractions in alginate during the 1967 and 1968 pollen seasons. The results of these studies indicate that patients treated with a low molecular weight fraction (antigen D) showed better clinical protection than patients treated with the crude pollen extract (WST). The observed clinical protection in antigen D-treated patients was correlated with a significant drop in reagin titer rather than an increase in blocking antibody. Eighty per cent (1215) of patients treated with antigen D showed a tenfold or greater decrease in reagin titer during the first year of treatment. The observed decrease in reagin titer suggested that the mechanism of hyposensitization may involve either in vivo neutralization of reagin or the induction of partial immune tolerance.

Induction of both reaginic and blocking antibodies with a low molecular weight fraction of timothy pollen extract

Abstract In preliminary studies to evaluate the potential of the nonprecipitating antigen D for treatment of timothy-sensitive patients, a group of 8 nonallergic individuals was given a series of intracutaneous injections of antigen D at weekly intervals for 4 weeks. Each of the persons had no clinical history of allergy and gave negative skin reactions to antigen D, crude pollen extract (WST), and allergens A and B of timothy pollen. Four weeks after the last injection of antigen D, 6 of the 8 volunteers gave positive skin reactions when skin tested with various timothy allergens. Passive transfer studies with sera obtained from these volunteers not only showed significant blocking antibody titers but also varying amounts of reaginic antibody titers up to 1/25. The successful induction of homocytotropic antibodies in rhesus monkeys by repeated intracutaneous injections with antigen D was demonstrated by both direct skin reactions and histamine release from passively sensitized monkey lung fragments. The homocytotropic antibodies produced in both man and rhesus monkeys are heat labile, persist at sensitized sites for at least 48 hours, and can be passively transferred by sera.

Isolation of a Reaginic Antibody Fraction with Properties of γG-Globulin.∗

Summary 1. Serum from untreated timothy sensitive patient (E.F.) was fractionated by Sephadex G-200 gel-filtration and DEAE-cellulose ion-exchange chromatography, and resulted in the isolation of a single fraction with reagin activity. 2. The isolated reagin possesses chromatographic and immunochemi-cal characteristics of γG-globulin.

Virus-associated deficiencies in the mitogen reactivity in celebes black macaques (Macaca nigra)

Celebes black macaques (Macaca nigra) with a history of diabetes mellitus, recurrent bacterial and protozoal infections, diarrhea, anemia, weight loss, anorexia, and a high mortality were studied to determine their immune status. Two groups of monkeys, healthy and unhealthy, were formed on the basis of a clinical assessment. The proliferative response and the pokeweed-mitogen-induced polyclonal IgG response of peripheral blood mononuclear cells of unhealthy monkeys were significantly less than the responses of healthy monkeys. The percentage of HLA-DR+ cells varied greatly in unhealthy monkeys. The OKT4/OKT8 ratios of unhealthy monkeys were generally greater than the ratios of healthy monkeys. Unhealthy monkeys usually had smaller percentages of OKT8+ cells than did healthy monkeys. The two groups of monkeys were examined for the presence of a syncytial forming retrovirus by a coculture assay involving Raji cells, a human B lymphoblastoid cell line. A type D retrovirus was detected in the unhealthy group but not in the healthy group. Retroperitoneal fibromatosis was detected in several monkeys in the unhealthy group.

The site of action of antigen D immunotherapy

Abstract Attempts to determine the mechanism and site of action of antigen D immunotherapy involve the culture of gelatin-separated human peripheral lymphocytes with varying concentrations of timothy allergens. Lymphocytes obtained from patients at different stages of treatment were cultured for 6 days, and the degree of reactivity was quantitated by 3 H-thymidine uptake of the antigen-reactive cells. These studies suggest that in vitro lymphocyte transformation may serve as a good index for the effectiveness of antigen D immunotherapy. The site of action and the mechanism of antigen D immunotherapy are discussed.

The separation of substances in timothy pollen extract producing allergic skin reactions from those producing hemagglutination reactions

Abstract The hemagglutination of rabbit red blood cells coupled to timothy pollen extract by diazo bonds was explored as in vitro assay for timothy allergen during fractionation and purification procedures. We found that rabbit red blood cells coupled to the fraction of timothy pollen precipitated by 20 per cent saturated ammonium sulfate were agglutinated by sera of patients allergic to timothy pollen, but that this fraction gave very weak skin test reactions. The fraction soluble in 49 per cent but precipitated by 55 per cent saturated ammonium sulfate gave low hemagglutination titers, but strongly positive skin tests. Passive transfer tests indicated that the hemagglutinating factor is distinct from reagin and blocking antibody. The separation of the fraction in timothy pollen producing hemagglutination from the fraction producing the skin reaction indicated that reagin and the antigen in timothy pollen responsible for the skin reaction are not involved in the hemagglutination reaction.

Polymerase chain reaction detection of type D simian retrovirus proviral DNA from infected macaques.

A simple polymerase chain reaction (PCR) approach was developed for detection of Type D simian retrovirus (SRV) serogroup 2 proviral DNA using peripheral blood lymphocytes (PBLs) obtained from infected macaques. PCR primer pairs were developed against serogroup 2 envelope (env) gene sequence, and fidelity of PCR fragment amplification was determined using molecularly cloned SRV serogroup 2 (D2/RHE/OR) DNA, and genomic DNA from Raji cells independently infected with different SRV serogroups. One primer pair exhibiting high fidelity was then utilized for PCR detection of serogroup 2 proviral DNA from PBLs, and from cells sorted into immune cell subpopulations by fluorescent-activated cell sorting (FACS). Env PCR fragments were readily detected from as few as 10(4) PBLs or immune cell subpopulations. In addition, highly specific PCR primers against serogroups 1 and 3 were utilized to detect proviral DNA from Raji cells infected with SRV serogroups. In all cases, primers designed to amplify serogroups 1, 2, and 3 proviral DNA were specific for their intended serogroup. This primer information and development of a PCR approach for detection of specific SRV proviral DNA will be of potential utility as a rapid surveillance tool in monitoring type D simian retrovirus infection within Asian macaque colonies.

Transmission of simian acquired immunodeficiency syndrome with a type D retrovirus: immunological aspects

Simian acquired immunodeficiency syndrome (SAIDS) was transmitted to four of four rhesus macaques with blood from rhesus macaques naturally infected with a type D retrovirus, simian retrovirus-2 (SRV-2). Three of the four blood recipients died with SAIDS at 13, 15, and 26 weeks postinoculation. The fourth animal is alive with SAIDS. All four test monkeys became viremic and produced antiviral antibody. None of the inoculated monkeys produced measureable neutralizing antibody to SRV-2. The survivor produced higher levels of antiviral antibody than the monkeys that died. Phytohemagglutinin and concanavalin A reactivity of peripheral blood lymphocytes was depressed from weeks 6 to 12 after inoculation. Clinical findings included development of splenomegaly in all four monkeys, and diarrhea in two monkeys. Blood counts remained within the normal range except for a depression in the number of polymorphonuclear lymphocytes in two monkeys. Hematocrits were decreased in two monkeys just prior to their death. All four test monkeys developed lymph node atrophy and bone marrow hypoplasia. Total proteins and immunoglobulin production were normal. This report provides evidence that SRV-2, as well as other type D retroviruses, causes SAIDS in macaque species.

Characterization of the IgG Antibody Response to Timothy Grass Pollen Antigens

The IgG and IgE antibody responses against timothy grass pollen antigen B (AgB) in several mouse strains were determined. Considerable variability in both responses was demonstrated, and (CBA/J X C57BL/6)F1 mice were selected for use in subsequent experiments. Anti-idiotypic-antibody-induced T suppressor cells almost completely suppressed AgB-specific IgE, but the IgG response was not altered. Further studies with photooxidized AgB (Ox-AgB) indicated that the IgG response was directed against an antigenic determinant expressed on both native antigen and Ox-AgB. Our data indicates that AgB possesses two distinct antigenic determinants, one that induces an IgE response, and one that induces an IgG response.