Variants selected by treatment of human immunodeficiency virus-infected cells with an immunotoxin.

An immunotoxin has been made by coupling anti-human immunodeficiency virus (HIV) envelope antibody 907 to ricin A chain (907-RAC). 907 recognizes an epitope within the immunodominant PB-1 loop of gp120. Variant cells were selected by cloning persistently infected H9/human T lymphocyte virus IIIB cells in the presence of the immunotoxin. Clones resistant to 907-RAC arose at a frequency of 0.1-1.0%. Seven clones were selected for intensive analysis. When studied, these clones fell into two distinct groups, members of which appeared to be identical, suggesting that the variation arose before the selection process. In contrast to the parent cells, none of the cloned variants produced infectious HIV. The first set of clones, designated the "E" variants, expressed decreased levels of the HIV envelope on the cell surface. However, levels of intracellular HIV antigens and reverse transcriptase were equal to or greater than that of the parental cell line. Radioimmunoprecipitation demonstrated that the gp160 was truncated to 145 kD (gp120 was normal length), capable of binding to CD4, and, unlike normal gp160, was released in its unprocessed form into the cellular supernatant. Sequence analysis demonstrated that a deletion at codon 687 of the envelope gene resulted in the production of this truncated protein. Ultrastructural analysis of E variants demonstrated some budding forms of virus, but also large numbers of HIV within intracellular vesicles. The second set of variants, the "F" series, produced no HIV antigens, reverse transcriptase, nor was there ultrastructural evidence of virus. However, proviral DNA was present. Virus could not be induced with agents known to activate latent HIV. These cells also lacked cell surface CD4 and could not be infected with HIV. These studies demonstrate that variation in HIV can affect the phenotype of the cells carrying the altered virus, allowing for escape from immunologic destruction. The E variants may serve as prototypes for attenuated HIV, which could be used as a vaccine. We have reconstructed the mutation found in the E variants within the infectious HIV clone HXB-2 and demonstrated that the resulting virus retains its noninfectious phenotype.

Hydrogen peroxide utilization in myeloperoxidase-deficient leukocytes: a possible microbicidal control mechanism.

Phagocytosis-induced formate and glucose C-1 oxidation by the polymorphonuclear leukocytes of a patient with hereditary myeloperoxidase deficiency was considerably greater than normal. The addition of catalase to the leukocyte suspension was required for optimum formate oxidation. Azide and cyanide increased glucose C-1 oxidation by normal leukocytes but had little or no effect on myeloperoxidase-deficient leukocytes suggesting that these agents normally stimulate glucose C-1 oxidation, in part, by inhibition of myeloperoxidase. It is suggested that the inhibition or absence of myeloperoxidase results in an increased utilization of H(2)O(2) in nonmyeloperoxidase-mediated H(2)O(2)-dependent reactions such as formate oxidation and hexose monophosphate pathway activation. The possibility of a microbicidal control mechanism in which a decrease in the microbicidal activity of myeloperoxidase is offset, in part, by an increase in the nonenzymatic microbicidal activity of H(2)O(2) is considered.

Effect of nonprotective vaccination on antibody response to subsequent human immunodeficiency virus infection.

We have investigated the systemic anti-HIV antibody response in chimpanzees who were immunized with live vaccinia containing either the HIV envelope glycoprotein (gp160IIIB) or a control antigen (herpes simplex virus glycoprotein D) and then challenged with either a high dose (300,000 TCID50) or low dose (100 TCID50) of HIVIIIB. HIV was subsequently isolated from all animals, indicating failure of the vaccination to protect against HIV infection. Serum antibody responses were evaluated before immunization, at the time of challenge with HIV, and at multiple time points in the 9 mo after challenge. Immunization resulted in a more rapid rise of antibody to gp160 in both high and low dose animals. Antibodies to the V3 loop induced upon infection were unaffected by immunization. In low dose animals, neutralizing antibody rose more rapidly and to higher levels in the immunized animals as compared with the control. There was no difference in neutralizing antibodies between immunized and control chimpanzees in the high dose group. Epitope mapping of the anti-gp 160 response indicated that immunization with gp160 vaccinia induced a postinfection antibody response to a region of gp41 (amino acids 718-743) that was not immunogenic in control-vaccinated animals. These data indicate that failed vaccination with the HIV envelope can alter both the timing and epitope specificity of the subsequent anti-HIV antibody response. These studies also define the evolution and fine specificity of the antibody response during the critical period immediately postinfection.

Temporal analysis of the antibody response to HIV envelope protein in HIV-infected laboratory workers.

Three laboratory workers have been infected with the IIIB strain of HIV; their antibody response to HIV has been studied in serial serum specimens. Because the infecting virus is known, the fine specificity of the antibody response was studied on the homologous strain of HIV. Anti-p17, anti-p24, anti-gp160, CD4/gp120 blocking and neutralizing antibodies developed in parallel. Epitope mapping of the anti-gp160 response indicated several regions that consistently induced an antibody response. Serum contained antibody which reacted with V3-specific peptides corresponding to the very tip of the loop and crossreactivity was seen with V3 loop peptides from other sequence divergent strains of HIV. Antibody to the V1 loop was produced at levels comparable with that seen for the V3-loop. Anti-V1 neutralized HIV with a titration curve equivalent to an anti-V3 monoclonal antibody. Because the infecting virus is known and serial reisolates have been obtained, we explored the relationship between production of antibody to a given epitope and mutation in the virus. The data suggest that an association exists, but do not clearly indicate that antibody drives the selection for mutant viruses. The findings presented here provide a fine specificity analysis of the evolution of the antibody response to HIV in greater detail than has previously been performed.

Differences in the antibody response to human immunodeficiency virus-1 envelope glycoprotein (gp160) in infected laboratory workers and vaccinees.

Studies of the immune response to the human immunodeficiency virus (HIV) have been hampered by the antigenic diversity of the HIV envelope protein. In an effort to predict the efficacy of vaccination we have compared the systemic anti-envelope antibody response in seronegative volunteers immunized with recombinant gp160 (either in vaccinia or as soluble protein produced in baculovirus) derived from the HTLV-IIIB strain of HIV-1 and in two laboratory workers accidentally infected with the same strain. 11 of 14 vaccinees responded to immunization by producing anti-gp160 of similar titer and the same isotype as that seen in the laboratory workers. Four vaccinees also had antibody to the principal neutralizing domain (V3 loop) that was comparable in titer with that seen in the laboratory workers, but the fine specificity of anti-V3 antibody was qualitatively different in the two groups. Antibody that can block the interaction between CD4 and gp120 was present at comparable levels in three vaccines and the lab workers. Neutralizing antibody titers were markedly lower in the vaccinees than in the laboratory workers. In seven of the vaccinees, an immunodominant epitope was at amino acid 720-740. Analyses of monoclonal antibodies to this region indicate that they do not neutralize, bind to infected cells, nor function as immunotoxins. Although the anti-gp160 antibody response was of similar magnitude in both infected and vaccinated individuals, there were important qualitative differences.

Activated human eosinophils synthesize new proteins.

In order to study the biochemical consequences of prolonged in vitro activation of human blood eosinophils, aqueous whole cell lysates, cell-free supernatants from resting eosinophils, and cells activated with opsonized zymosan, calcium ionophore (A23187), N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), and phorbol 12-myristate 13-acetate (PMA) were analyzed by polyacrylamide gel electrophoresis (PAGE). In comparison to resting eosinophils, opsonized zymosan-activated eosinophil extracts demonstrated altered protein composition on both the native PAGE and sodium dodecyl sulfate (SDS) -PAGE. Three new polypeptides of apparent molecular mass 24 kDa, 43 kDa and 60 kDa appeared on SDS-PAGE gels when opsonized zymosan-activated eosinophil extracts were electrophoresed. In contrast, extracts from fMet-Leu-Phe, A23187, and PMA-activated eosinophils demonstrated neither altered polypeptide composition nor new polypeptides. Opsonized zymosan also induced the incorporation of L-[35S]methionine into eosinophil proteins and this was completely blocked by pretreating the cells with cycloheximide. This finding suggests that eosinophils activated by certain stimuli synthesize new proteins. These newly synthesized proteins, which are freely secreted into the medium during cell activation, may possess important immunological functions.

Molecular preservation in Late Cretaceous sauropod dinosaur eggshells.

Exceptionally preserved sauropod eggshells discovered in Upper Cretaceous (Campanian) deposits in Patagonia, Argentina, contain skeletal remains and soft tissues of embryonic Titanosaurid dinosaurs. To preserve these labile embryonic remains, the rate of mineral precipitation must have superseded post-mortem degradative processes, resulting in virtually instantaneous mineralization of soft tissues. If so, mineralization may also have been rapid enough to retain fragments of original biomolecules in these specimens. To investigate preservation of biomolecular compounds in these well-preserved sauropod dinosaur eggshells, we applied multiple analytical techniques. Results demonstrate organic compounds and antigenic structures similar to those found in extant eggshells.

Variable regions of antibodies to synthetic polypeptides--III. Antibodies arising in response to administration of anti-idiotope.

Antibodies elicited by the synthetic polypeptide antigen (T,G)-A-L are directed against two distinct epitopes. The majority of antibodies bind to a GT containing epitope and bear an idiotope defined by monoclonal antibodies I7 and I9. In this study, we have examined the effect of in vivo administration of the I7 and I9 antibodies to mice. Administration of anti-idiotope elicits anti-(T,G)-A-L antibodies in all strains of mice tested, including genetic non-responders to (T,G)-A-L. These antibodies bind to GT and express the idiotope. Additionally, idiotope expressing antibodies that fail to bind to antigen are also produced. Monoclonal anti-(anti-idiotope) antibodies were made. One antibody bound to (T,G)-A-L, the other did not. Sequence analysis was performed and the V-regions of (T,G)-A-L binding antibodies were compared to those of the antibody that failed to bind antigen. Both sets of antibodies are derived from the same germline V-genes as the anti-(T,G)-A-L antibodies. These results have implications for understanding the nature of network regulation of the immune system and for those attempting idiotypic vaccination.

A defect in the humoral immune response to protein antigens and haptens in immunoglobulin mu heavy-chain transgenic mice.

We have examined the antibody response in mice expressing a functionally rearranged mu Ig heavy chain derived from a hybridoma antibody with specificity for the hapten 4-hydroxy-3-nitrophenyl (NP). Transgenic mice and their normal littermates were immunized with the antigens NP-OVA, the synthetic polypeptide (Tyr,Glu)-Ala-Lys ((T,G)-A-L), or saline. The presence of serum antibodies to NP-BSA, OVA, (T,G)-A-L, and BSA was examined by ELISA. Sera were evaluated prior to immunization and at periods of up to 4 months following immunization. Prior to immunization, transgenic mice had high levels of IgM anti-NP antibody but no detectable antibody to the other antigens. Both the primary and secondary antibody responses of transgenic mice to NP, OVA, and (T,G)-A-L were depressed when compared with the response of non-transgenic mice. Because of reports that these transgenic mice have increased proportions of CD5 + B-cells, a subpopulation associated with the production of autoantibodies, we examined these mice for the production of both IgG and IgM rheumatoid factors and anti-DNA antibodies. Transgenic mice had a modest increase in the spontaneous production of IgM anti-DNA. These data demonstrate a functional defect in the humoral immune response of mu transgenic mice.

Characterization of monoclonal antibodies with specificity for DNA and the synthetic polypeptide antigen (T,G)-A-L.

Because of evidence for structural similarity of variable region genes of anti-DNA and anti-(T,G)-A-L antibodies, polyspecific interactions of monoclonal anti-DNA and anti-(T,G)-A-L antibodies were investigated. Of 20 monoclonal antibodies from C57BL/10 mice with (T,G)-A-L binding, two bound DNA as determined by ELISA. In contrast, two of five anti-DNA monoclonal antibodies from MRL-lpr/lpr mice bound (T,G)-A-L. For both sets of antibodies, antigen binding was shown to be the activity of the same antibody by cross-inhibition studies. To determine whether such polyspecific antibodies were expressed during autoimmune disease, sera of autoimmune MRL-lpr/lpr mice were tested for anti-(T,G)-A-L activity. This analysis demonstrated minimal elevations of anti-(T,G)-A-L in comparison to BALB/c controls. These studies thus confirm predictions about the binding activity of anti-(T,G)-A-L and anti-DNA antibodies based on structural analysis of variable region genes. They further indicate, that while anti-(T,G)-A-L and anti-DNA antibodies may have overlapping specificity, polyspecific antibodies of this kind are not preferentially expressed during autoimmunity.

Monoclonal anti-(T,G)-A--L antibodies: characterization of fine specificity and idiotype expression.

The technique of polyethylene glycol mediated cell fusion was used to establish 22 monoclonal cell lines secreting anti-(T,G)-A--L antibody. Cell lines were derived from C3H.SW and B10 mice and produced antibody with kappa light chains and predominantly gamma 1, heavy chains. Fine-specificity analysis demonstrated that 15 cell lines made antibodies that also recognize a determinant present on GAT, GT (9:1) and GT (1:1), whereas little, if any, serum antibody demonstrates this cross-reaction. Fourteen antibodies, derived from both B10 and C3H.SW mice, bear idiotypic determinants defined by Lewis anti-[B10 anti-(T,G)-A--L], but only two, both from C3H.SW mice, react with Lewis anti-[C3H.SW anti-(T,G)-A--L]. Adsorption studies indicate that no hybridoma tested bore the complete set of idiotypic determinants defined by either serum.

Variable regions of antibodies to synthetic polypeptides--I. Characterization of an idiotope expressed on antibodies and T-cell factors.

Spleen cells from a Lewis rat immunized with affinity-purified B10 anti-(T,G)-A-L antibody were fused with the non-secreting murine hybridoma SP2/0. Cell lines secreting monoclonal antibodies specific for mu- and kappa-chains, as well as an idiotope on anti-(T,G)-A-L antibodies, were isolated and characterized. The anti-mu and -kappa antibodies, are true anti-isotypes, reacting with sera from all strains of mice tested. The anti-idiotope antibodies recognize a determinant on antibodies binding a GT-containing epitope. The proportion of anti-GAT antibody bearing the idiotope varies markedly in different murine strains. A 1000-fold higher level of antibody from Igha mice than from Ighb and Ighe mice is required to give an equivalent inhibition of the idiotope-anti-idiotope reaction. Analysis of monoclonal antibodies expressing the idiotope indicates that the affinity of binding between idiotope and anti-idiotope can vary by as much as two orders of magnitude. Immunoadsorbants prepared with anti-idiotope antibody bind suppressor factor secreted by a GAT-specific T-cell hybridoma.

Hydrogen peroxide release from eosinophils: quantitative, comparative studies of human and guinea pig eosinophils.

Eosinophils play an important role in host defense against parasitic infection, employing both oxidative and nonoxidative systems to effect damage. The active oxygen product, hydrogen peroxide, either alone or in combination with the enzyme eosinophil peroxidase, may damage parasites. These studies use a sensitive fluorometric assay to document the release of hydrogen peroxide from guinea pig peritoneal exudate eosinophils and human peripheral blood eosinophils. Guinea pig eosinophils released 0.13 +/- 0.02 (n = 10) nmol H2O2/10(5) eos/5 min under resting conditions which were markedly increased when stimulated by phorbal myristate acetate (PMA, 1 microgram/ml) [4.80 +/- 0.50 (19) nmol H2O2/10(5) eos/5 min], preopsonized zymosan [2.40 +/- 0.17 (4) nmol H2O2/10(5) eos/5 min], or latex beads [1.14 +/- 0.26 (5) nmol H2O2/10(5) eos/5 min]. Normal human peripheral blood eosinophils had greater resting release of hydrogen peroxide [0.34 +/- 0.05 (8) nmol H2O2/10(5) eos/5 min], but were less effectively stimulated by PMA [3.12 +/- 0.57 (9) nmol H2O2/10(5) eos/5 min], preopsonized zymosan [0.78 +/- 0.16 (8) nmol H2O2/10(5) eos/5 min] or latex beads [0.69 +/- 0.32 (6) nmol H2O2/10(5) eos] (p less than or equal to 0.05). Hydrogen peroxide release was markedly enhanced by the presence of exogenous glucose and was linearly dependent upon cell number when the soluble stimulus PMA was used. Particle-stimulated hydrogen peroxide was not necessarily enhanced by increases in the particle:cell ratio. The demonstration of the release of large amounts of hydrogen peroxide from eosinophils is further support for the concept that eosinophils play an active role in host defense.

Elevated plasma interleukin-1 levels in humans following ultraviolet light therapy for psoriasis.

Interleukin-1 (IL-1) is a potent cytokine with a wide range of biologic activities including induction of several acute phase responses. Ultraviolet radiation (UVR) is widely used as a therapeutic modality to treat many chronic skin diseases, including psoriasis. In the present study, we investigated whether ultraviolet B (UVB) radiation induced circulating IL-1 in the plasma of patients undergoing chronic UVB therapy. In order to remove plasma proteins which inhibit IL-1-induced T-cell proliferation, each plasma sample was chromatographed and each fraction was assayed for IL-1 activity. There was no detectable IL-1 before and 1 h after UVB radiation; IL-1 appeared 4 h after treatment and was absent after 24 h. Plasma IL-1 was neutralized by antibodies to recombinant human IL-1 beta and alpha. The anti-IL-1 alpha, but not anti-IL-1 beta, antibodies partially neutralized the IL-1 activity present in a keratinocyte cell line supernate. These results demonstrate that UVB therapy induces circulating IL-1 and that this IL-1 may originate from both keratinocyte and non-keratinocyte sources.

In situ localization of interleukin-1 in normal and psoriatic skin.

Interleukin-1 (IL-1) is a family of polypeptides that mediates a wide range of inflammatory and immune responses. In human skin, unstimulated keratinocytes produce a large amount of such cytokines. Although the precise role of IL-1 in the skin is unknown, there is experimental evidence supporting involvement of IL-1 in the pathogenesis of inflammatory skin diseases. In this study, we investigated in situ localization of IL-1 alpha and IL-1 beta in normal and psoriatic skin. Using polyclonal antibodies and the avidin-biotin peroxidase complex, we demonstrated the presence of both IL-1 alpha and IL-1 beta in normal and psoriatic formalin-fixed paraffin-embedded tissues. In both cases, IL-1 alpha was more prominent. However, the distribution of IL-1 alpha differed between normal and psoriatic skin. In normal skin, IL-1 alpha distribution was predominantly intercellular, whereas IL-1 alpha distribution was predominantly within the cytoplasm in psoriatic skin. These studies confirm that IL-1 alpha is the predominant form of IL-1 in the skin and provide further support for the hypothesis that IL-1 participates in the pathogenesis of psoriasis.

Anaphylaxis in mast cell-deficient mice.

Evidence of a functional deficit in mast cell-deficient mice was sought by testing for the development of systemic anaphylaxis. W/Wv mast cell-deficient mice and wild type control animals were sensitized by injection of bovine serum albumin and subsequently challenged. Nine of ten W/Wv mast cell-deficient mice and 10/10 control mice demonstrated signs of anaphylaxis, including death. Although histamine levels were higher in control animals, no differences were noted before and after antigen challenge. The numbers of peripheral blood basophils were the same in W/Wv and wild type mice. These studies suggest that caution is necessary in the use of these animals to study disease.

Superoxide production by eosinophils: activation by histamine.

Both guinea pig peritoneal exudate and human peripheral blood eosinophils produce large amounts of superoxide anion when stimulated by preopsonized zymosan or phorbol myristate acetate (PMA). Superoxide production is also activated by histamine but not the histamine metabolite, imidazole acetic acid. Supernatants from degranulated rat mast cells stimulate superoxide production. In studies of both human and guinea pig eosinophils, the H1-antagonist, chlorpheniramine (10-3 M and 10-4 M), preopsonized zymosan histamine) production of superoxide anion but the H2-antagonist, cimetidine, only modestly inhibited superoxide anion production (zymosan, PMA), These studies provide direct evidence for the influence of histamine on the oxidative metabolism of eosinophils. These results are consistent with the hypothesis that histamine interacts with eosinophils predominantly via an H1 receptor site. Furthermore, they suggest that eosinophils may participate in immediate hypersensitivity reactions by the release of superoxide anion in response to stimulation by histamine.

Chronic neutropenia in childhood. Analysis of 16 cases and a review of the literature.

We analyzed the clinical histories and hematologic information concerning 16 persons in whom chronic neutropenia was discovered early in life. Only two of them, those with the lowest mean neutrophil counts, had frequent and severe pyogenic infections. Almost all the infections were caused by Staphylococcus aureus or by enteric microorganisms and involved the skin, respiratory tract or gastrointestinal system. The prognosis in our subjects or in patients described in the literature could not be predicted from the bone marrow morphology, presence or absence of blood monocytosis, pattern of genetic transmission or results of special tests of neutrophil function. We propose that the complex nomenclature associated with chronic neutropenic states be discarded until a better basis for classification becomes available.

Use of a focal infectivity assay for testing susceptibility of HIV to antiviral agents.

A highly sensitive and quantitative focal immunoassay has been developed for detecting the human immunodeficiency virus (HIV). The assay can be used to measure cell-free virus or the production of HIV by virus-infected cells. Both laboratory-adapted strains of HIV and patient isolates can be studied with this assay. In this communication, we demonstrate the utility of this assay for measuring the effects of anti-HIV agents on viral isolates. We show that the anti-viral effects of such diverse agents as azidothymidine, interferon-alpha, immunotoxins, soluble CD4 and antibody can be accurately quantified. This assay may be used in the discovery and evaluation of new anti-HIV therapies or may be adapted for use in testing the sensitivity of patient isolates to standard therapeutic agents.

Human immunoglobulin heavy-chain-variable (VH) region gene families defined by hybridization with cloned human and murine VH-genes.

The Southern DNA hybridization technique was used to determine the organization of human immunoglobulin heavy-chain-variable region (VH) gene families and the extent of polymorphism within these families. Human DNA was digested with a restriction enzyme and then hybridized to a cloned human or murine VH gene. Six human and six murine genes were used. Hybridization patterns seen in the human genes fell into three groups, which corresponded to the Kabat subgroup assignments based on sequence. When DNA from different individuals was compared, polymorphism was demonstrated in each of the three gene families. The VH II family demonstrated a higher degree of polymorphism than the others. Hybridization with the murine probes, each of which represented a different murine VH-gene family, revealed six distinct patterns of hybridization. Five of the probes detected all or a portion of one of the previously defined human families. The sixth probe, 36-60, yields a unique pattern of hybridization, suggesting a fourth VH-gene family. Dot hybridizations show that 36-60 does not hybridize with human VH-genes representing each of the known families, further supporting this possibility.