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Studies of the induction of suppressor cells by soluble factors and anti-idiotypic antibody in mice have progressed to the point where it now seems likely that patients' allergic responses to grass pollen can be reduced by manipulation through the idiotype-anti-idiotype network.

Glatiramer acetate (GA, Copaxone, copolymer 1) for injection is an approved drug for relapsing-remitting multiple sclerosis. The clinical and immunological effects of GA were extensively studied in experimental autoimmune encephalomyelitis (EAE), the experimental animal model for MS. The effect of oral administration of GA was tested in both rodents and primates in acute as well as in chronic relapsing (CR) models of EAE. Oral GA was found to suppress acute EAE induced in rats, mice, and rhesus monkeys. The effect of GA was also tested in several models of CR-EAE: proteolipid protein and myelin oligodendrocyte glycoprotein induced CR-EAE in mice, CR-EAE in Biozzi mice, and CR-EAE in cynomolgus monkeys. In all the murine models, oral treatment with GA initiated at the peak of first relapse reduced the severity of disease and suppressed further relapses. Suppression of EAE with oral GA was associated with marked inhibition of spleen cell proliferation and Th1 cytokine (IL-2 and IFN-gamma) response to the respective autoantigens. GA-specific T cell lines of the Th2/3 type that inhibit EAE induction in vivo, similarly to those induced by injection of GA, could be isolated from spleens of GA-fed mice and rats. Furthermore, as demonstrated previously for GA-specific cells induced by the parenteral route, the orally induced GA-specific cells accumulate in the CNS and secrete in situ Th2 cytokines in response to both GA and MBP as well as brain-derived neurotrophic factor (BDNF). Although a clinical trial in MS with two doses of oral GA in enteric-coated tablets did not show a significant effect either at the clinical or immunological level, the results presented here suggest that oral GA may still be developed into a therapeutic modality in MS.

Antigen-specific-effected immunoregulation by T lymphocytes is mediated by extracellular proteins produced by T lymphocytes. These immunoproteins bind specifically to nonprocessed antigen and either induce antigen-specific immunoregulatory T cells (tsfi) or effect regulation (tsfe). T cell proteins that bind specifically to nonprocessed antigen have ben termed "T cell antigen-binding molecules" (TABM), and by definition, tsfe and tsfi are, in part, TABM. To characterize tsfi, tsfe, and TABM and understand the relationships and function of these immunoproteins, we have combined the efforts of two laboratories to compare tsfi, tsfe, and TABM isolated by each laboratory. Data obtained in one laboratory were reproduced by the other, and all reagents prepared by each laboratory were exchanged. TABM, tsfi, and tsfe were found to express TCRCalpha epitopes but not TCRCbeta epitopes. The amino acid sequence of a tryptic peptide of a T cell hybridoma TABM specific for nitrophenylhydroxy acetate (NP) is similar to a TCRalpha chain and TCR pre-alpha chain amino acid sequence. ELISA and immunoblotting demonstrated that Mr 77,000 T cell hybrid-derived tsfi, tsfe, and TABM are noncovalently associated with Mr 15,000-16,000 interleukin-10 (IL-10). ELISA also demonstrated that tsfi and tsfe are associated with I-J. The ability of tsfi and tsfe to suppress a mixed lymphocyte reaction was prevented by anti-IL-10 or anti-I-J antibodies, suggesting that antigen-specific immunoregulatory T cell proteins function by an antigen-specific focusing of immunoregulatory cytokines.

The existence of extracellular, antigen-specific, T-cell factors has been a subject of heated debate over the years. Here, Robert Cone and Arthur Malley consider the nature of the structural and functional properties of these T-cell-derived proteins, particularly their similarity to T-cell receptor α chains and their extremely high specific activity. A genetic origin and mode of action of these factors in immunoregulation is proposed based on existing experimental data.

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.

Epitope variability is one of the greatest obstacles to development of synthetic peptide vaccines. Based on a recently described hypervariable epitope (aa 414–434) on the envelope glycoprotein (gp130) to simian immunodeficiency virus (SIV mac 142), we have developed a novel approach to account for epitope variability. We have prepared, in a single synthesis, a cocktail of peptides, designated a hypervariable epitope construct (HEC), which collectively represent all the in vivo variability seen in an epitope. The HEC represents permutations of amino acid substitutions found in the epitope and has been able to induce antibodies with enhanced binding to native SIV and broad immunoreactivity to related epitope analogues.

These studies defined SRV-2 envelope peptides 96-102, 127-152, and 233-249 as T cell epitopes that induce significant T cell proliferation. Peripheral blood lymphocytes of Celebes macaques (Macaca nigra) exposed to SRV-2 and currently virus- antibody+, cultured with SRV-2 virus show strongly suppressed T cell responses and have two immunoregulatory T cell populations.

We analyzed SIV-specific monkey sera to localize B-cell epitopes of the envelope glycoprotein of SIV (gp130), using overlapping synthetic peptides representing the entire SIV gp130 protein and sera from experimentally infected monkeys and monkeys immunized with whole, inactivated SIV. A B-cell epitope which induces neutralizing antibody production and T-cell responses was characterized as well as a new B-cell epitope and a previously described neutralizing epitopes. Vaccinated monkey sera recognize the three epitopes differentially relative to unimmunized controls, and a correlation appears to exist between degree of cross-neutralization by infected monkey sera and degree of binding to these three regions.

We previously reported that an area encompassing amino acids 147–162 of the envelope region of the simian (type D) retrovirus serotype 1 (SRV-1) constitutes an antigenic site for the binding of murine and rhesus neutralizing antibodies. Neutralizing antibodies to SRV-2 are directed to a different area, encompassing residues 96–102 of SRV-2. This paper presents data on the activity of an SRV-1 recombinant envelope protein (rEP) and of monoclonal hybridoma cell line, C11B8, produced from murine spleen cells immunized with SRV-1 rEP. Purified monoclonal antibodies from C11B8 bind to the SRV-1 rEP and to both SRV-1 and SRV-2. However, the monoclonal antibody exhibits strain specificity in the capacity to neutralize SRV-1 infection in vitro . Thus, C11B8 neutralizes SRV-1 infection but fails to neutralize four other known serotypes of the virus. C11B8 also binds to an SRV-1 synthetic peptide representing residues 142–167, which encompasses the previously defined antigenic site of recognition for neutralizing antibodies to SRV-1. This paper also contains evidence that the SRV-1 rEP construct binds the site for SRV-1 attachment to the cell receptor. This is indicated by the ability of SRV-1 rEP to compete with SRV-1 (but not with SRV-2) and inhibit its infectivity in vitro . In addition, SRV-1 rEP inhibits the neutralizing activity of C11B8 against SRV-1 infection in vitro . SRV-1 rEP has no inhibitory effect on rhesus neutralizing antibodies to SRV-2. Taken together, the above findings indicate that immunity conferred at the level of neutralizing antibodies during SRV infection is strain-specific and involves the recognition of envelope sequences unique to each strain.

Synthetic envelope peptides of a simian retrovirus (SRV-2) were used to define both T- and B-cell epitopes of the envelope protein. The SRV-2 peptide 100-106 specifically blocks rhesus anti-SRV-2 neutralizing antibody activity, and a peptide 100-106 keyhole limpet hemocyanin conjugate induces a strong antipeptide antibody response. SRV-2 peptide 100-106 and 233-249 induces good T-cell proliferation of murine spleen cells immunized with the SRV-2 virus. Thus, SRV-2 envelope peptide 100-106 represents both a T- and B-cell epitope, and peptide 233-249 a T-cell epitope.

We previously demonstrated that the area encompassing residues 147-162 of the envelope protein of simian retrovirus serotype-1 (SRV-1) is the target for rhesus anti-SRV-1 neutralizing antibodies. A peptide representing amino acids 142-167 of envelope protein (gp70) is capable of inducing virus-neutralizing antibodies in mice. The virus receptor was immunoprecipitated from Raji cells using gp70 as the ligand. Antibodies to peptide 142-167 inhibit the immunoprecipitation, indicating that the receptor recognizes residues 142-167 of the viral envelope protein.

We have made visible the binding of a mouse monoclonal anti-human interleukin 2 (IL-2) receptor (anti-Tac) antibody on the surface of phytohemagglutinin (PHA)-stimulated Xenopusthymocytes using a colloidal gold-conjugated goat anti-mouse antibody and transmission electron microscopy. No binding was found when a different mouse monoclonal antibody (mAb) of the same isotype and subclass was tested, or when the anti-Tac antibody was omitted from the procedure. After metabolic radiolabeling of the IL-2 receptors with [35S]methionine using PHA-stimulated thymocytes of Xenopus laevis, the South African clawed toad, we show that a concentrated preparation of the mouse anti-human Tac antibody will immunoprecipitate a radiolabeled molecule just slightly larger than 55 kDa. Phorbol dibutyrate (PDB), an effective T cell mitogen, and cyclosporin A, an inhibitor of T cell mitogenesis in this species, are both capable of regulating the expression of this IL-2-binding molecule on Xenopus immunocytes [1]. Here, we use the calcium ionophore A23187 to show that the relationship between IL-2 receptor expression and mitogenesis, which was previously established in X. laevis [1], is associated with a calciumion flux. Flow cytometry is used for assaying alterations in epitope expression after binding the lectin-stimulated cells under test with a fluorescence (Fl*) conjugate of the anti-Tac antibody or a control mAb, which is either anti-DNP or anti-keyhole limpet hemocyanin (KLH) in specificity, but of the same mouse isotype and subclass as the anti-IL-2 receptor antibody.

H-2 specific T suppressor inducer (Tsfi) and T suppressor effector (Tsfe) factors show a dose-dependent inhibition of one-way mixed lymphocyte responses (MLR) between CBA J responder spleen cells and C57BL 6 mitomycin C-treated stimulator spleen cells. The hydrophobic proteins Tsfi and Tsfe purified by ammonium sulfate precipitation and affinity methods were stabilized by the addition of Tris-saline pH 8 buffered octylglucopyranoside solution. The stabilized Tsfi and Tsfe fractions stored at 4°C for 3–7 months retained a significant (> 72%) amount of their ability to inhibit MLR. Tsfi and Tsfe purified by salt precipitation and affinity methods were analyzed by SDS-PAGE. Enzyme-linked immunoassay (ELISA) and Western blots indicated that these molecules had T cell receptor (TcR) α chain, I-J, and IL-10 epitopes, but not TcR β chain epitopes.

The induction of H-2-specific Ts cells was accomplished by the i.v. injection of X-irradiated (2000R) C57BL/6 spleen cells into CBA/J mice. These Ts cells significantly (78%) suppressed the delayed type hypersensitivity (DTH) response in CBA/J mice injected s.c. with X-irradiated (2000R) C57BL/6 spleen cells and given a footpad challenge with the same cell population 5 days later. We have shown that both CD4 and CD8 T cells were involved in the observed suppression, and these cells secrete T suppressor inducer (Tsfi) and T suppressor effector (Tsfe) molecules. Both Tsfi and Tsfe molecules were shown to significantly inhibit (> 87%) one-way mixed lymphocyte responses between CBA/J and mitomycin C-treated C57BL/6 spleen cells. Using an adoptive transfer method, we showed that mice given both a primary and secondary immunization with X-irradiated C57BL/6 spleen cells to induce H-2-specific Ts cells contain a significantly greater number of Ts cells than mice given only a primary immunization, suggesting the presence of memory Ts cells.

SIVmac infection of macaques is an important animal model for HIV infection and AIDS; this model is being utilized for development of antiviral therapies and vaccines. In the present article, we sought to identify neutralization epitopes of SIVmac envelope surface glycoprotein (gp130). Algorithms were used to predict antigenicity of specific regions. Four regions from the primary amino acid sequence of the viral surface glycoprotein were selected. A synthetic peptide representing one of these regions (414-434) induced virus-neutralizing antibodies in mice; in addition, this peptide induced T cell-proliferative responses in macaques. To address the in vivo relevance of these observations, we demonstrated that experimentally infected macaques produce antibodies to the neutralization epitope. In addition, rhesus macaques protected against infection by an inactivated SIV vaccine develop antibodies that bind to peptide 414-434. These observations demonstrate that the region that includes the sequence 414-434 in the fourth variable domain (V4) of SIVmac gp130 contains both a linear neutralization epitope and a T cell epitope.

It has been recently demonstrated that two serotypes of type D simian retroviruses, namely SRV-1 and SRV-2, exhibit extensive immunological cross-reactivity but do not exhibit cross-reactivity at the level of neutralizing antibodies. We have also shown recently that an area which includes residues 147–162 of the envelope protein of SRV-1 constitutes an epitope to which neutralizing antibodies against SRV-1 but not against SRV-2 are directed. However, in spite of the capacity of various immunogenic preparations to induce antibodies which react with SRV-1 these antibodies were incapable of neutralizing in vitro viral infectivity. Work reported herein demonstrates that various immunogens consisting of a larger peptide, namely 142–167 of the envelope protein of SRV-1, induce antibodies capable of binding with the envelope protein of SRV-1 and with the whole virus. Moreover, these antibodies exhibit the capacity to neutralize in vitro the infectivity of SRV-1 but not of SRV-2.

An area encompassing residues 142-167 of the envelope protein of type D simian retrovirus (SRV-1) has been shown to contain the epitope to which neutralizing antibodies are directed. This area has been synthesized and shown to bind to monkey and mouse antiviral antibodies and to a virus neutralizing mouse monoclonal antibody. Protein conjugates of this peptide as well as the cross-linked or the free peptide induce antibodies capable of neutralizing, in vitro, viral infectivity. The cell receptor to the virus was isolated following extraction of Raji cells with non-ionic detergents. The receptor was isolated and characterized following radioimmuno-precipitation of 125I labeled cell extract bound to viral envelope protein. This immunoprecipitation could be inhibited by antiserum to peptide 142-167. Analysis in gels indicate that the receptor is of molecular weight of approximately 60 KDa. These results indicate that the neutralizing antibodies and the receptor recognize the same area on the viral envelope protein and that neutralization is the result of blocking the virus-receptor interaction by antibodies.

There are at least three distinct serotypes of simian type D retrovirus (SRV) which exhibit extensive serological cross-reactivity, but no cross-reactivity exists at the level of serum neutralizing antibodies. Amino acid sequence analysis and hydrophobicity plots of SRV-1 and SRV-2 envelope proteins were compared in order to identify unique potential antigenic determinants to which respective neutralizing antibodies may be directed. Peptides representing residues 147-162 of SRV-1 and 96-102 of SRV-2 were synthesized and assessed for their immunoreactivity. Free peptide inhibition of strain-specific serum (rhesus) neutralizing antibodies to SRV-1 and SRV-2 was demonstrated using the SRV-1 147-162 peptide and the SRV-2 peptide, 96-102, respectively. Inhibition of serum neutralizing activity by these peptides was also strain-specific, showing no cross-inhibition. SRV-1 147-162 conjugated to a protein carrier and cross-linked to Sepharose beads specifically adsorbed neutralizing antibodies from SRV-1 immune rhesus sera. The antibodies eluted from the immunoadsorbent possessed SRV-1 neutralizing activity, but showed no effect on the infectivity of SRV-2. Peptide SRV-1 147-162 also exhibited the capacity to bind specifically with a mouse monoclonal antibody which neutralizes the infectivity of SRV-1. Mice immunized with a recombinant SRV-1 envelope protein or with whole, inactivated SRV-1 produced antibodies which bound the SRV-1 147-162 conjugate, but not the protein carrier itself. Mouse antibodies to the SRV-1 147-162 conjugate exhibited specific binding with both native SRV-1 and with recombinant SRV-1 envelope protein. These findings provide strong evidence that SRV-1 147-162 and SRV-2 96-102 constitute at least two unique antigenic determinants, or parts thereof, which participate in the strain-specific neutralizing antibody response. Moreover, the findings indicate that the SRV-1 neutralizing antibodies produced by monkeys and at least a certain population of neutralizing antibodies produced by mice recognize the same epitope of SRV-1.

Experiments were conducted using an excess of biosynthetically labeled timothy grass pollen antigen-B-specific (AgB-specific) T helper factor (THF) with enriched T cells, enriched B cells, or normal spleen cells to determine the site of the THF action. These studies indicated that the labeled THF bound preferentially to T cells, and analysis of the cell surface characteristics suggested that these T cells were Lyt 123+ cells. A panning technique using partially purified AgB-specific THF-coated Petri dishes depleted a population of idiotype-binding T cells that appears to be required for the formation of AgB-specific IgE antibody.

The general use of enzymatic cleaners for soft contact lenses has led to reports of incidents of allergic reactions in previously sensitized patients. In light of this, experiments were performed to assess the relative allergenic potential of four such enzymes: papain, subtilisin A, subtilisin B, and pancreatin. Mice immunized intraperitoneally (i.p.) with either subtilisin A or papain exhibited little serum immunoglobulin E (IgE), even after tertiary doses of protein. Primates immunized intradermally (i.d.) with the four enzymes showed various levels of response to challenges: none to papain, mild to moderate to subtilisin A and pancreatin, and severe to subtilisin B. Finally, guinea pigs instilled ocularly with enzymes weekly for 15 weeks exhibited significant hyperemia and chemosis only to the positive control protein, ovalbumin, and not to any of the enzymes tested. We conclude that three of the four enzymes tested have low potential to sensitize human beings, and, further, that animal models may find increased use in screening products of biotechnology.

The Sixteenth Midwinter Conference of Immunologists was held January 22–25, 1977, at Asilomar Conference Grounds, Pacific Grove, California, with Joel W. Goodman and Leon Wofsy serving as Cochairmen. The subject of the conference, “Structure and Specificity: Antibodies and Receptors”, was discussed during four half-day sessions by invited speakers, and in six concurrent working sessions. Topics of the individual workshops were (1) identification and significance of surface immunoglobulin on B and T lymphocytes, (2) isolation and molecular characterization of surface antigens, (3) use of surface markers in lymphocyte differentiation, (4) separation of lymphocyte subpopulations and functional analysis, (5) molecular techniques for detecting antibody genes, and (6) how membranes transmit signals. The Third Annual Dan H. Campbell Memorial Lecture was given by Dr. Elvin A. Kabat (Columbia University, New York).

Enzyme-linked immunoassays were used to examine the binding specificity of affinity-purified anti-idiotypic antibody [against the idiotypic determinant on timothy grass pollen antigen B (AgB)-specific IgE] with murine AgB-specific IgG and IgE antibodies, and the serum from timothy-sensitive patients. In addition, the proliferative response of peripheral blood lymphocytes from timothy-sensitive patients with either antigen or anti-idiotypic antibody was examined. These studies suggest that the idiotypic determinant expressed on murine AgB-specific IgE is highly conserved on human AgB-specific IgE and T cells of timothy-sensitive patients.

Previous studies demonstrated that antigen B (AgB), a major antigen of timothy grass pollen, modified by photooxidation (Ox-AgB), does not react with rabbit, human, or mouse antibodies directed against AgB and does not induce antibodies reactive with either native or modified AgB. In this paper we show that Ox-AgB retains an estimated 70% of the lymphocyte-activating properties and a significant amount of the T-cell-priming capabilities of AgB.

Selective modification of the antigenic determinant of the major allergen of timothy grass pollen, antigen B,was achieved by a photooxidation reaction. The modified antigen B, OX-AgB, does not react with either rabbit or human (reaginic) antibodies directed against the native antigen B (AgB). LAP 1 mice immunized with either 10 or 100 μg protein of OX-AgB adsorbed to 1 mg alum and given two additional boosts with the same dose of OX-AgB over a 63-day period did not produce IgE or IgG 1 antibodies that react with either native or OX-AgB. In contrast, LAF 1 mice similarly immunized with 10 μg AgB produced significant levels of IgE and IgG 1 antibodies. Antigen-induced lymphocyte transformation was used to determine the effect of the chemical modification upon the carrier determinants of AgB. These studies indicate that OX-AgB retains a significant portion of the T-cell activating properties of AgB.

Treatment with rabbit anti-moneky thymus cell sera whether limited (3 days) or extensive (15 days), did not alter the development of experimental allergic encephalomyelitis (EAE) in rhesus monkeys challenged with myelin basic protein or central nervous system tissue (CNS) when compared to similarly challenged control monkeys treated with normal rabbit serum. No consistent difference in disease incidence or intensity as measured by incubation period, neurologic signs or CNS pathology was observed between experimental and control monkeys. This finding is in contrast to previous reports on the efficacy of ATS treatment in prevention of EAE in rodents.

The IgG1 and IgE homocytotropic antibody responses of LAF1 and C3H mice to timothy pollen antigens are defined. Both mouse strains responded to low doses of crude timothy pollen extract (WST) or a major antigen of timothy pollen coupled to a purified fraction of Ascaris suum (Antigen B-Ascaris). Titers in LAF1 mice were greater than those in C3H mice. Regardless of the immunogen, antigen B was the major determinant recognized by the homocytotropic antibodies; PCA titers with WST or antigen B for challenge were equivalent and PCA activity could be inhibited by antigen D, a dialyzable fraction of timothy pollen possessing the antigen B determinant in monovalent form. The possible usefulness of antigen D for in vivo and in vitro studies of specific immune suppression of cellular activity is discussed.

Purified populations of splenic B and T lymphocytes from LAF1 mice immunized with a crude extract of timothy pollen (WST) responded specifically to pollen antigens in an in vitro lymphocyte transformation system. The peak lymphocyte transformation response occurred 5 days after a secondary immunization and was the result of T-B cell cooperation in vitro. With two purified pollen antigens as in vitro stimulants we were able to define at least two antigen-specific populations of B cells and one population of T cells. These results were confirmed by inhibition studies with a monovalent hapten from WST, Antigen D.

The immune system of offspring mice from mothers immunized with photo-oxidized timothy grass pollen antigen B (OX-AgB) or Trinitrophenyl-bovine gamma globulin (TNP-BGG) during their pregnancy was examined. Offspring mice immunized 6 or 8 weeks after delivery with the same antigen administered to their mothers have completely suppressed primary responses and greater than 80% suppressed secondary responses. The observed immunosuppression in offspring mice appears to persist until about 16 weeks after delivery, and is antigen-specific. Adoptive transfer studies show that spleen cells from adult OX-AgB primed mice injected i.v. into lethally X-irradiated (800 rads) syngeneic recipients and challenged with antigen produce significant levels of AgB-specific IgG antibody. Spleen cells (5 x 10(6] from offspring mice of mothers immunized with OX-AgB during their pregnancy were added to spleen cells from adult OX-AgB primed mice and injected i.v. into lethally X-irradiated syngeneic recipients and challenged with antigen. These recipients showed a significantly (greater than 85%) immunosuppressed secondary response. The observed immunosuppression appears to be mediated by CD4+ and CD8+ T cells suggesting a requirement for both T suppressor inducer and effector cell populations. The reported findings are discussed in relation to possible mechanisms to explain the immunosuppression obtained in the offspring of mothers immunized during pregnancy.

The Sixteenth Midwinter Conference of Immunologists was held January 22–25, 1977, at Asilomar Conference Grounds, Pacific Grove, California, with Joel W. Goodman and Leon Wofsy serving as Cochairmen. The subject of the conference, “Structure and Specificity: Antibodies and Receptors”, was discussed during four half-day sessions by invited speakers, and in six concurrent working sessions. Topics of the individual workshops were (1) identification and significance of surface immunoglobulin on B and T lymphocytes, (2) isolation and molecular characterization of surface antigens, (3) use of surface markers in lymphocyte differentiation, (4) separation of lymphocyte subpopulations and functional analysis, (5) molecular techniques for detecting antibody genes, and (6) how membranes transmit signals. The Third Annual Dan H. Campbell Memorial Lecture was given by Dr. Elvin A. Kabat (Columbia University, New York).

Spleen cells from LAF1 mice were stimulated in a microculture system with T and B cell mitogens or antigens of timothy pollen. Only cells from mice immunized with crude timothy pollen extract (WST) or a major antigen of timothy pollen conjugated to Ascaris (antigen B-Ascaris) responded to timothy antigens in vitro. Optimum responses were obtained at 120 to 144 hr of culture with 5 to 10 μg WST per culture and ranged from three to 10 times greater than cell background. No correlations could be found between the optimum antigen concentration or the maximum response and the immune status of the spleen cell donor. Response could be inhibited by a dialyzáble fraction of timothy pollen, antigen D, which is a monovalent form of a major antigen of timothy pollen.

Summary The isolation of a fraction (WST-Fr2) from the dialysate of timothy grass pollen extract is described. This fraction (WST-Fr2) appears homogenous by chromatography on Sephadex-G25, Amberlite IRC-50 and electrophoresis, but immunologically heterogenous as demonstrated by inhibition of several lines of precipitation in agar diffusion. WST-Fr2 possesses haptenic properties as demonstrated by its inhibition of specific precipitation and inhibition of the formation of lines of precipitation in agar diffusions. The results indicate the importance of considering the possible presence and significance of haptenic material in pollen extracts for laboratory studies as well as for clinical use.

Summary A spectrofluorometric method for the determination of histamine liberated from monkey lung tissue passively sensitized with human atopic sera is described. The close correlation of histamine liberated from sensitized tissue with P-K titer, the loss of activity by heating sera at 56°C for 1 hr, and the failure of both rabbit and rhesus monkey precipitating antibody or human blocking antibody to release histamine indicate that this method provides an in vitro estimation of the reagin content of the sera from allergic patients. The sensitivity of the method described may also permit the detection of reagin in the sera of allergic patients not detectable by the P-K test. It is suggested that the use of adult monkey lung tissue may provide a means to study the mechanism of antibody fixation.

Summary The close antigenic relationship of antigen D and allergen B was demonstrated by inhibition of both passive cutaneous anaphylaxis (PCA) and precipitation in agar gel. The antigenic relationship of allergen A and B was demonstrated by a line of partial identity in microdouble diffusion analysis. Antigen D possesses haptenic properties as demonstrated by its inhibition of PCA, anaphylaxis and precipitin reactions of either rabbit or guinea pig antisera to timothy pollen antigens. The antigenic character of antigen D is demonstrated by its direct positive skin reactions in timothy-sensitive patients and by its induction in rabbits with varying amounts of antibody demonstrable with allergen B but not with antigen D.

A radioimmunoassay was developed for measuring testosterone (T) in the plasma of the rhesus monkey (Macaca mulatto). The method is sensitive and specific and can distinguish 20 pg of T when added to plasma from the adrenalectomized- ovariectomized monkey. When plasma obtained from cord blood at various times during gestation was analyzed by this method, the levels of T in plasma from the umbilical artery of males were significantly higher than those in females. Plasma from females did, however, contain small amounts of this hormone with little variation between animals. Large fluctuations in the concentrations of T in plasma from the fetal male were observed. Significantly higher amounts of T were found in the umbilical artery than in the vein in male but not in female fetuses. Castration of the fetus on day 100 of gestation abolished the sex difference in the amounts of T found on day ISO to 156 of gestation. These data indicate that the fetal testis of the monkey is the source of the elevated levels of ...

Rhesus monkeys injected intravenously with TNP-KLH produced homocytotropic antibody separable into two components on ion exchange chromatography, a heat-labile γ1- and a heat-stable γ2-immunoglobulin. Homocytotropic antibody was directed against the TNP group only, though both groups of determinants gave rise to large amounts of precipitating antibody. Arthus reactions induced by TNP on heterologous carrier were more severe than those caused by KLH. Prolonged TNP-induced reactions resembled delayed hypersensitivity reactions grossly and histologically. Anaphylaxis occurred in a few of the animals in response to TNP-KLH, but not to KLH alone, although no relationship to levels of homocytotropic antibody was discernible.

Summary A method of obtaining a highly purified preparation of horseshoe crab hemocyanin which was found to be highly antigenic in rabbits is described. The horseshoe crab hemocyanin migrated as a single band by cellulose acetate electrophoresis at pH 7.0 and 7.5 and showed a single line of precipitation with rabbit anti-HCH serum in agar-gel diffusion studies. Cross-reactivity between the hemocyanins of the horseshoe crab and the giant keyhole limpet by quantitative precipitation, and by immunogenicity tests both in vitro and in vivo, were found to be negative.

Transfer factor from guinea pigs sensitive to dinitrochlorobenzene was not bound to an immunoadsorbent column that is specific for the dinitrophenyl determinant. The absence of the dinitrophenyl determinant on transfer factor suggested that the factor does not function as superantigen. The duration of the adoptive sensitivity, the small molecular weight, and the polypeptide or polynucleotide (or a combination) composition of the transfer factor are consistent with a derepressor function of the molecule.

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.

TREATMENT of murine T lymphocytes with concanavalin A (con A) at concentrations as low as 10−17 g ml−1 produces significant increases in their electrophoretic mobility. The effect is blocked by α-methyl-D-mannopyranoside (α-MM). Phytohaemagglutinin (PHA) produces similar changes in T-cell mobility while treatment with lipopolysaccharide (LPS), a B-lymphocyte mitogen, does not. We present here evidence indicating that the altered mobility of con A-treated T cells is due to the rapid release of a factor from the few cells reacting directly with the lectin.

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.

Human r-DNA IL-2 and fluorescent (F1∗) mouse anti-human IL-2 receptor antibody have been tested separately and in competition with each other for their capacities to bind to the splenocytes of Xenopus laevis , the South African clawed toad. Binding by F1∗-mouse anti-DNP antibody of the same subclass (IgG 1 , κ) was used as a control. The results of visual tests using rIL-2 coated fluorescent Covaspheres demonstrate that the human mediator will bind cells of the toad spleen. Moreover, the mediator inhibits binding of the antibody against the human IL-2 receptor, as detected by cytofluorimetry. Some of the IL-2 receptors on the toad cells appear to be constitutive, since they are expressed on freshly biopsied lymphocytes. Activation of these cells in vitro will increase the percentage of those cells able to bind both the anti-receptor antibody and rIL-2. Since the human mediator is only able to modulate in vivo immune activity in antigen-activated toads, it appears that in spite of having some constitutive IL-2 receptors, a quantitative increase in receptor expression is required before immunological behavior can be effected. More stringent controls of receptor expression may have provided an additional regulatory level as mammalian mechanisms evolved.

Neutralizing antibodies that block the ability of simian acquired immunodeficiency syndrome (SAIDS) retrovirus type 2 (SRV-2) to induce syncytium formation in cultures of Raji cells have been found in the serum of nonviremic Celebes black macaques (Macaca nigra). Serum from Celebes macaques that are viremic have little or no neutralizing activity. The neutralizing antibodies were shown to block viral infectivity. The group of monkeys with neutralizing antibodies in their serum exhibited a dramatic improvement in their health from 1982 to 1984. The correlation of neutralizing antibodies with clinical improvement suggests that neutralizing antibodies may play a critical role in limiting the pathogenic effects of SAIDS retrovirus infection and in helping eliminate the infection.

Currently, 5 distinct Simian type D retrovirus serotypes have been defined: SRV-1 (Calif., New Eng.), SRV-2 (2R, 2C), SRV-3 (MPMV), SRV-4 (Calif. Cyno), and SRV-5 (Chinese). Neutralizing antibodies made against the indicated Simian viruses do not cross react. Thus, we may be able to produce synthetic envelope peptides specific for each Simian virus serotype and make antibodies against the neutralizing epitopes. The experiments presented here with SRV-1 and SRV-2 viruses represent our attempts to develop a model approach for the preparation of synthetic envelope peptide vaccines.

Publisher Summary This chapter provides details of a workshop held to analyze results of experiments conducted to study purification and characterization of allergens. New immunological control methods, useful during separation and standardization of extracts were presented, such as the laiirell immunoelectrophoretic technique with its newer modification and the radioallergosorbent test (RAST test). Studies on nondialyzable constituents of allergenic materials were also presented. Several studies were reported on dialyzable constituents of pollens and of peas. It was observed that delayed-type skin reactions in streptococcal hypersensitivity are provoked by the preparation of streptococcal enzymes called streptokinase/streptodornase (SK/SD). The search for strain-specific antigens of the Mycobacterium tuberculosis group is proceeding. This project is different from the objectives of others currently directed toward further purifications of skin-test materials for the diagnosis of clinical tuberculosis. A proposal was submitted in advance regarding adoption of nomenclature which would assign a codifying symbol to isolated allergens as these are reported.