Structural relationship between the two IgY of the duck, Anas platyrhynchos: molecular genetic evidence.

cDNA clones encoding the H chains of the 7.8S and 5.7S IgY of the White Pekin duck have been isolated and sequenced. The H chain of the 7.8S IgY possesses four C region domains and thus resembles the H chain of chicken IgY with which it shows, in the C region, 54% inferred amino acid sequence identity, and complete conservation of the C region cysteine and tryptophan residues. The H chain of the 5.7S IgY possesses only two C region domains, that are virtually identical to CH1 and CH2 of the 7.8S IgY H chain. Although Southern blot genomic analysis did not resolve whether the two transcripts encoding the H chains of the 7.8S and 5.7S IgY are derived from one or two H chain-encoding genes, the CH 1, 2, 3, and 4 exons are apparently colinear, and no evidence was found for a separate locus in which CH1 and 2 exons were present and CH3 and 4 exons were lacking. The VH domain-encoding sequences of the cDNA for the two IgY H chains showed high similarity in the inferred VH gene (93% nucleotide and 91% inferred amino acid identity) and in the inferred JH segment (89% nucleotide and 93% inferred amino acid identity) but low similarity in the D region (26% nucleotide and 7% inferred amino acid identity). Genomic Southern blot hybridization analysis showed multiple VH-hybridizing sequences represented on up to 20 restriction fragments.


DRB1*0301 molecules recognize a structural motif distinct from the one recognized by most DR beta 1 alleles.

The DR3-restricted peptide, MT 65 kDa 3-13, was used to develop a DR3-specific binding assay. The binding activity detected was strictly pH-dependent, in that it was optimal in the pH 4 to 5 range, and no activity was detected at neutral pH. By means of affinity chromatography purifications and the use of DR3-transfected fibroblasts, it was shown that no cross-reactivity exists at the level of DR52a molecules, thus allowing use of only partially purified DR3/DR52a mixtures in high throughput binding assays. The immunologic relevance of the assay established was also verified by examining the correlation between DR3 restriction and binding for a panel of DR-restricted peptides. When the structural requirements for peptide DR3 interactions were further examined by using panels of analogues of two different epitopes (Myo 132-151 and TT 830-843), it was found that DR3 molecules recognized a peptide motif distinct from the one recognized by the other major DR beta 1 alleles.


Identification and characterization of the murine homologue of CD22, a B lymphocyte-restricted adhesion molecule.

The human B lymphocyte-specific Ag, CD22, is a cell adhesion molecule expressed on the surface during a narrow window of B cell development, coincident with surface IgD. A ligand for CD22 has recently been identified on human T cells as the low molecular mass isoform of the leukocyte common Ag, CD45RO. CD22 has been reported to function in the regulation of both T and B cell activation in vitro. In this study, we report the isolation and expression of a molecular cDNA clone encoding the murine homologue of CD22, mCD22. Within their predicted protein sequences, murine and human sequences overall have 62% identity, which includes 18 of 20 extracellular cysteines and six of six cytoplasmic tyrosines. BHK cells transfected with mCD22 cDNA specifically adhere to resting and activated T lymphocytes and in addition bound activated, but not resting, B cells. Five Th clones were analyzed for their ability to adhere to mCD22; two Th0 clones and one Th1 clone bound CD22+ BHK transfectants, but not all T cell clones bound CD22+ cells: another Th1 clone and a Th2 clone did not. mCD22+ BHK transfectants were also specifically bound by the B cell-specific mAb, NIM-R6, demonstrating that this mAb is specific for murine CD22. Human cell lines expressing the counter-receptors for human CD22 were also examined for adhesion to the murine CD22 homologue; the epitope responsible for B cell adhesion to CD22 is conserved, whereas the T cell epitope binding to CD22 is not. The cDNA and mAb to murine CD22 will be useful for defining the in vivo function of CD22.


Isolation, characterization, and expression of mouse ICAM-2 complementary and genomic DNA.

Intercellular adhesion molecule-2 (ICAM-2), a cell surface glycoprotein, is a second counter-receptor for lymphocyte function-associated Ag-1 (LFA-1). We report here the isolation and characterization of the cDNA and the gene that encode murine ICAM-2 (Accession numbers X65493 and X65490, respectively). The deduced sequence of the cDNA has 60% amino acid identity with its human counterpart and has the same expression pattern in cells and tissues. Furthermore, COS cells transfected with mouse ICAM-2 complementary and genomic DNA bind to purified human LFA-1, demonstrating the conservation of the function of ICAM-2 as a ligand for LFA-1 and conservation across species of sequences that are critical for binding to human LFA-1. COS cells transfected with the ICAM-2 cDNA do not react with mAb PA3, previously suggested to define ICAM-2 in the mouse. The mouse ICAM-2 gene was isolated and its structural organization determined. The gene is present in a single copy in the mouse genome and contains four exons spanning about 5.0 kb of DNA. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members. The gene for ICAM-2, which is constitutively expressed in endothelial cells, has several conserved sequence motifs in its promoter region, including a direct repeat, and lacks transcription factor-binding sites present in the ICAM-1 gene, which is inducible in endothelial cells.


IFN-gamma/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor.

IFN gamma/LPS treatment increases macrophage tumoricidal and microbicidal activity and inhibits CSF-1-induced macrophage proliferation. The mechanism underlying the latter effect was investigated in the CSF-1-dependent mouse macrophage cell line, BAC-1.2F5. IFN-gamma and LPS together dramatically reduced the total number of CSF-1 receptors (CSF-1R) via selective degradation of the cell surface form. Processing and transport of intracellular CSF-1R to the cell surface were unaffected. IFN-gamma alone had no effect but significantly enhanced LPS-induced CSF-1R down-regulation. The reduction in CSF-1R number was protein kinase C-dependent and involved changes in serine phosphorylation of the receptor at different sites. CSF-1R down-modulation by this mechanism may be important in switching off the energy-consuming processes of CSF-1R-mediated proliferation and chemotaxis in activated macrophages.


Interference with cyclophosphamide-induced skin allograft tolerance by cyclosporin A.

In a murine strain combination identical in H-2 Ag but disparate in minor histocompatibility (H) Ag consisting of C3H/He (C3H; H-2k, Mls-1b) mice as recipients and AKR/J (AKR; H-2k, Mls-1a) mice as donors, a permanent skin allograft tolerance can be achieved by the cyclophosphamide (CP)-induced tolerance system that consists of i.v. injection of donor spleen cells (day -2) and i.p. injection of CP 2 days later (day 0). Such permanent take of allografts in CP-induced tolerant mice was interfered with by intramuscular injection of cyclosporin A (CsA) from day -5 to day -1 and their grafts were rejected by 21 days after grafting. Mls-1a-reactive CD4+V beta 6+ T cells in the periphery, as the indicator to follow the kinetics of donor-reactive T cells, increased on day 0 and day 3 in the C3H mice treated with AKR spleen cells alone, whereas they disappeared rapidly from day 0 to day 3 in CP-induced tolerant mice. When CsA capable of interfering with IL-2 production and T cell proliferation was administered before CP treatment in CP-induced tolerance system, the number of CD4+V beta 6+ T cells in periphery did not increase on day 0 and 3, but increased on day 7 in contrast to the decreased number of those in CP-induced tolerant mice. On day 7, MLR against donor cells was decreased in CP-induced tolerant mice, but maintained in CsA-interfered tolerant mice. These result may indicate that the destruction of donor-Ag-stimulated, proliferating T cells by CP is interfered with by CsA, probably because CsA inhibits the proliferation of donor-reactive T cells at the time of CP treatment. Furthermore, these results also implicate that the protocol for immunosuppression with CsA and antimetabolites has to be designed carefully in clinical transplantation.


Localization of IFN-gamma receptor in first trimester placenta to trophoblasts but lack of stimulation of HLA-DRA, -DRB, or invariant chain mRNA expression by IFN-gamma.

Trophoblasts do not express MHC class II Ag on their cell surface, but it is not known at which level the regulation of expression of these IFN-gamma-responsive genes occurs. We localized the IFN-gamma R Ag to the trophoblasts of normal human first trimester placenta. As previously shown, treatment with IFN-gamma of short term cultures of placental explants did not induce expression of HLA-DR Ag in trophoblasts. We demonstrated that in situ hybridization with probes for DRA, DRB, and MHC class II-associated invariant chain genes gave no detectable signals for the presence of the corresponding mRNA in cytotrophoblasts or syncytiotrophoblasts of first trimester placenta with or without IFN-gamma. Maternal leukocytes in these placental preparations as well as IFN-gamma-treated HeLa cell control cultures expressed these mRNA. In addition, Northern analysis of RNA isolated from the trophoblast cell line Jar after treatment with IFN-gamma showed that induction of transcription did not occur from the HLA-DRA, HLA-DRB, or invariant chain genes. Jar cells expressed the IFN-gamma R Ag. These data suggest that down-regulation of MHC class II Ag on trophoblasts in the first trimester placenta and in a cell line include a lack of productive intracellular signaling by the IFN-gamma R and maintenance, in a coordinate manner, of low steady state levels of the mRNA encoded by the DRA, DRB, and invariant chain genes.


Identification of IL-8 as a locomotor attractant for activated human lymphocytes in mononuclear cell cultures with anti-CD3 or purified protein derivative of Mycobacterium tuberculosis.

On culture of human blood mononuclear cells for 24 to 48 h with anti-CD3 (aCD3) or purified protein derivative of Mycobacterium tuberculosis, chemoattractants are released into the medium which induce polarization and locomotion of activated (G1) lymphocytes but not resting lymphocytes. Here we show that, during a period of up to 72 h of culture, IL-8 is released in nanomolar quantities into the supernatant and that the lymphocyte chemoattractant activity of these supernatants is inhibited by incubation with anti-IL-8. Examination of the cultured mononuclear cells by immunofluorescence suggests that many monocytes, but almost no lymphocytes in aCD3 cultures contain IL-8 in cytoplasmic organelles, yet few monocytes direct from blood stained for IL-8. IL-8 is an attractant for only a small proportion (ca 10%) of lymphocytes direct from blood. The proportion of responding cells is increased after culture for 24 to 48 h in aCD3 or purified protein derivative of Mycobacterium tuberculosis, and these are a phenotypically distinct subpopulation consisting of large lymphocytes enriched for CD45RO. These cells respond to their own culture supernatants and to IL-8 in polarization assays and by invasion of collagen gels into which the attractants are incorporated. They also show orientation to a source of IL-8 in a chemotactic gradient. These responses are consistent with in vivo observations that the lymphocytes which migrate selectively into inflammatory sites are activated. The fact that many lymphocytes do not respond to IL-8 may reflect the diversity of migratory pathways shown by lymphocytes in vivo, the locomotion of small, recirculating, lymphocytes being regulated by other, unknown, locomotor stimuli.


Recognition of different domains of the Plasmodium falciparum CS protein by the sera of naturally infected individuals compared with those of sporozoite-immunized volunteers.

The fine specificities of antibodies to the circumsporozoite (CS) protein of Plasmodium falciparum, present in the sera of volunteers immunized with irradiated P. falciparum sporozoites, were defined and compared to those of sera from persons living in a malaria-endemic area in West Africa. The specificity of these anti-CS antibodies was determined by ELISA, using recombinant proteins and synthetic peptides containing repeat and nonrepeat sequences of this CS protein. All 10 serum samples of the five sporozoite-immunized volunteers displayed very high antibody titers to the immunodominant repeat (NANP)n of the CS protein. However, only three of the serum samples of these vaccinees reacted with a single nonrepeat region and only at low titers. In contrast, a high percentage of sera from adults living in the malaria-endemic area who had been exposed to sporozoites, as well as liver and blood stages of P. falciparum, had high antibody levels, not only to the repeats but also to several nonrepeat regions of the CS protein. Furthermore, a number of sera from children living in this endemic area displayed appreciable levels of antibodies to the nonrepeat regions, in the absence of any antirepeat reactivity. Sera of Saimiri monkeys, which had undergone multiple blood-induced P. falciparum infections, consistently contained high titers of antibodies to several nonrepeat sequences of the CS protein, whereas only a few of these sera had low titers of antirepeat antibodies. Antibody binding sites, in nonrepeat regions, were mapped using synthetic polymers containing multiple copies of selected C-terminal sequences of the P. falciparum CS protein. The binding to sporozoites of antibodies to nonrepeat regions of the CS protein was determined. The basis for the differences in antibody binding sites of sera from persons immunized with irradiated sporozoites, compared to those from an endemic area, is discussed.


Macrophage arginine metabolism and the inhibition or stimulation of cancer.

The potential of the immune system to inhibit or stimulate tumor growth is a vivid example of the "two-edged sword" nature of immune responses. Our results provide evidence that this dual capacity can be attributed, in part, to the dual pathways of arginine metabolism exhibited by intratumor macrophages. Specifically, i.p. tumor rejection in P815-preimmunized mice is accompanied by an upshift in intratumor macrophage arginine metabolism to the nitric oxide (NO) synthase pathway that yields citrulline and NO. A rapid and marked local increase in IFN-gamma (both mRNA and protein) in preimmunized mice during tumor rejection suggests that this cytokine plays a role in up-regulating nitric oxide production in vivo. Unlike tumor rejection, progressive i.p. P815 tumor growth in naive mice is associated with a marked decline in the production of citruline/NO by intratumor macrophages. Examination of macrophage arginine metabolism via arginase revealed a pattern opposite that of NO synthase. The local production of ornithine/urea markedly increases during progressive tumor growth whereas arginase activity decreases during tumor rejection. Inasmuch as nitric oxide inhibits tumor cell replication whereas ornithine is the precursor of polyamines required for cell replication, these results are consistent with the conclusion that the pathway macrophages use to metabolize arginine can influence the type of host immune responses against cancer and other conditions.


Expression of the 50-kDa integrin-associated protein on myeloid cells and erythrocytes.

Integrin-associated protein (IAP) is a 50-kDa intrinsic membrane protein that is involved in signal transduction during neutrophil activation by a variety of Arg-Gly-Asp-containing ligands. However, IAP does not itself directly bind these ligands, which are instead recognized by the leukocyte response integrin (LRI). In fact, IAP is more widely expressed than the LRI and is even on erythrocytes, which express no known integrins. This suggests that IAP may have additional functions besides signal transduction in association with the LRI. In this work we have quantitated IAP expression on several myeloid cells and cell lines, as well as erythrocytes, using a mAb which recognizes this protein. These data show that there are about 10,000 IgG-binding sites on each erythrocyte and 20 times that number on the myeloid cell lines U937 and HL60. Normal PMN and monocytes express about 25,000 IgG-binding sites. There are twice as many Fab'-binding sites on each cell, suggesting that each IgG binds via both Ag-combining sites. Binding data using several mAb to IAP suggest that IAP undergoes a temperature-dependent conformational change. Unlike some integrin receptors, IAP is not stored in a regulated secretory compartment in polymorphonuclear leukocytes (PMN). In addition, there is no evidence for internalization or shedding of IAP upon PMN activation. These data show that IAP is expressed at significant levels on myeloid cells and erythrocytes and that its expression is unaffected by the state of PMN activation.


Spontaneous development of protective anti-T cell receptor autoimmunity targeted against a natural EAE-regulatory idiotope located within the 39-59 region of the TCR-V beta 8.2 chain.

The development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats is mediated by V beta 8.2+ T cells specific for myelin basic protein. One consequence of this biased expression of V beta 8.2 is the spontaneous development of regulatory T cells and antibodies against residues 39-59 of the V beta 8.2 sequence. Moreover, a synthetic V beta 8.2-39-59 peptide could induce protection against and speed recovery from EAE. T cells and antibodies specific for V beta 8.2-39-59 could transfer protection from EAE. Recently, we reported that the protective T cell epitope is subsumed within the V beta 8-44-54 sequence. We now report that protection induced by V beta 8-44-54 lasted at least 102 days and produced "split tolerance," enhancing anti-myelin basic protein antibody titers but reducing anti-myelin basic protein T cell frequency. The shorter V beta 8-44-54 peptide induced a distinct set of antibodies that did not cross-react with the longer V beta 8.2-39-59 peptide, although both specificities could stain V beta 8.2+ T cells and were equally protective against EAE. However, the V beta 8.2-39-59 peptide, but not the V beta 8-44-54 peptide, would appear to represent the natural idiotope: antibodies to V beta 8.2-39-59 that develop spontaneously during EAE could be boosted to higher titers only by the V beta 8.2-39-59, but not by other TCR peptides from the V beta 8.2 sequence, including V beta 8-44-54 that contains the functional T cell epitope. These results suggest that natural processing of the TCR V beta-chain favors the formation of a peptide that resembles the V beta 8.2-39-59 sequence. The B cell epitope present on the V beta 8-44-54 sequence was evident only in the absence of residues 39-43 and 55-59, suggesting that the two peptides possess distinct conformations. However, the V beta 8-44-54 B cell epitope is most likely expressed on the V beta 8.2+ T cells, either as a low affinity determinant on the intact TCR alpha/beta heterodimer or as a cryptic epitope bound in the cleft of surface MHC molecules.


Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.


Regulation of low affinity IgE receptor (CD23) expression on mononuclear phagocytes in normal and asthmatic subjects.

Mononuclear phagocytic cells contain low affinity receptors for IgE (Fc epsilon RII or CD23) which induce cellular activation in the presence of specific allergen. These studies were performed to quantify the expression by monocytes and alveolar macrophages of Fc epsilon RII in asthma and to determine biologic response modifiers that regulate Fc epsilon RII. Whereas 2.5 +/- 1.0% of the monocytes obtained from normal volunteers were Fc epsilon RII positive, this increased to 16.7 +/- 2.4% in asthma (p < 0.001). Stimulation of Fc epsilon RII expression on monocytes was shown to be an activity of IL-4 (24.5 +/- 5.9%), granulocyte-macrophage-CSF (28.1 +/- 5.2%), IFN-alpha (15.8 +/- 5.3%), IFN-gamma (10.4 +/- 3.7%), and macrophage-CSF (7.3 +/- 0.7%) but not of IL-2, IL-6, or TNF-alpha. Expression of Fc epsilon RII by these cytokines was associated with the induction of specific mRNA transcripts. Using Fc epsilon RII subtype specific primers in the polymerase chain reaction expansion of cDNA, cytokine-induced receptors were shown to be Fc epsilon RIIb. Alveolar macrophages from nonasthmatic subjects displayed minimal expression of Fc epsilon RII (3.2 +/- 1.2%); however, these receptors were present on 69.2 +/- 6.3% of asthmatic volunteers (p < 0.001). Induction of Fc epsilon RII appears specific for allergic asthma insofar as these receptors are also not expressed in subjects with interstitial lung disease (1.3 +/- 1.3%). As assessed by shift in mean fluorescence, instillation of allergen in the asthmatic's airway further up-regulated Fc epsilon RII on alveolar macrophages by 151 +/- 7%. Up-regulation of Fc epsilon RII in atopic individuals may therefore reflect allergen-induced exposure of mononuclear phagocytes to one or more of these cytokines. These studies suggest a mechanism by which an immunologic stimulus that leads to the production of these cytokines (e.g., allergen or viral infection) would contribute to the development or exacerbation of allergic disease.


Engraftment of human peripheral blood leukocytes into severe combined immunodeficient mice results in the long term and dynamic production of human xenoreactive antibodies.

Severe combined immunodeficient (SCID) mice engrafted with human peripheral blood leukocytes (hu-PBL-SCID) represent a potentially important small animal model for the study of human immune function. Attempts to generate human primary immune responses to exogenous Ag in the hu-PBL-SCID have had limited success which raises questions about the functional capacity of human lymphocytes in the SCID environment. Here, we demonstrate that the spontaneously secreted human Ig in hu-PBL-SCID includes antibodies with specificity for several different mouse RBC (mRBC) proteins. These antibodies apparently reflect the transfer of peripheral B cells which are responsible for the production of naturally occurring xenoreactive antibodies in the donor. Western blot analysis showed that engraftment of anti-mRBC specificities was random among mice receiving PBL from the same donor sample. In at least one mouse, this engraftment was polyclonal and included human IgM and IgG which recognized at least 12 different mRBC proteins ranging in size from 35 to > 200 kDa. Anti-mRBC specificities were found to vary with time demonstrating a dynamic expression of the human xenoreactive repertoire in hu-PBL-SCID. In contrast to mice engrafted with human PBL, mice engrafted with another source of human B cells, i.e., tumor-infiltrating leukocytes, produced very little or no human anti-mRBC antibody. Ag-driven proliferation of xenoreactive clones may result in a skewing of the engrafted human B cells in hu-PBL-SCID which could account in part for the limited ability of hu-PBL-SCID to respond to exogenous Ag. The long term production of anti-mRBC antibodies and the modulation of the expressed xenoreactive repertoire observed in hu-PBL-SCID represents an opportunity to study the molecular genetics and cell biology of the human humoral immune response to a defined complex Ag.


Human pre-B and B cell membrane mu-chains are noncovalently associated with a disulfide-linked complex containing a product of the B29 gene.

B cell activation after Ag binding to membrane Ig (mIg) is mediated by a complex series of events that involves proximal activation of a tyrosine kinase and phospholipase C. Until recently it was unclear how mIgM and mIgD, with their limited cytoplasmic domains (three amino acids on each H chain), were able to couple to these secondary signal transducers. Studies of murine B cells conducted in several laboratories, including our own, suggest that products of the mb-1 (IgM-alpha or IgD-alpha) and B29 (Ig-beta, Ig-gamma) genes occur as disulfide-linked alpha/beta and alpha/gamma heterodimers that are noncovalently associated with mIgM and mIgD. Although studies utilizing Daudi and Raji cell lines indicate that human mIgM is also associated with a dimer containing the mb-1 gene product, the other molecules associated with the human receptor have not been identified. In this report we characterize the phosphoproteins that are noncovalently associated with mIgM on human tonsillar B cells and human pre-B cell lines. mIgM is noncovalently associated with a disulfide-linked heterodimer composed of variably glycosylated forms of two core proteins with apparent molecular mass of 26.5 and 27 kDa. Western blotting analysis reveals that the lower m.w. component of each of the mIgM-associated heterodimers and its 27-kDa deglycosylated core protein are reactive with antibodies against the murine B29 gene product. Thus, a product of the B29 gene is a component of the AgR complex in human and murine B cells, occurring as a disulfide linked dimer with product(s) of the mb-1 gene. Interestingly, mb-1 and B29 gene products expressed on human cells are much more heterogenously N-glycosylated than their murine B cell counterparts.


Characterization of gamma delta T lymphocytes at the maternal-fetal interface.

A specific subset of gamma delta T lymphocytes bearing a V gamma 6V delta 1-encoded TCR is known to populate the normal nonpregnant murine uterine and vaginal epithelium. However, gamma delta T lymphocytes residing at the maternal-fetal interface during pregnancy have not yet been investigated. Using mAb and cytofluorographic analysis, we analyzed gamma delta TCR-bearing lymphocytes obtained from placental/decidual tissues of allogeneic (C57B1/10 X BALB/c and BALB/c X C57B1/10) pregnancies. Gestations were analyzed at several time points during the second half of pregnancy, with lymphocytes from both maternal spleen and nonpregnant C57B1/10 uteri analyzed as controls. We found that all gamma delta T lymphocytes at the maternal-fetal interface are maternally derived. Relative to the total T lymphocyte population, the percentage of gamma delta TCR-bearing T lymphocytes at the maternal-fetal interface is enriched three- to four-fold compared with maternal spleen, and twofold compared with nonpregnant uteri. Although one-third of gamma delta T lymphocytes from pregnant animals express a cell-surface marker associated with activation (IL-2R), gamma delta cells from uteri of nonpregnant mice fail to express IL-2R. In terms of absolute numbers, we estimate that reproductive-tract gamma delta T cells are increased nearly 100-fold in pregnant animals compared with nonpregnant animals. To characterize the TCR-gamma delta repertoire in the placenta/decidua, we generated 21 TCR-gamma delta-bearing hybridomas from lymphocytes in this tissue. Analysis of these hybridomas revealed at least six distinct gamma delta receptor types expressed at the maternal-fetal interface, with V gamma 6V delta 1 encoded TCR representing the predominant population. As specific resident constituents of the reproductive tract, gamma delta T lymphocytes may be involved in regulating a variety of physiologic and pathophysiologic events in reproductive biology.


C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63.

C33 Ag was originally identified by mAb inhibitory to syncytium formation induced by human T cell leukemia virus type 1. The Ag was shown to be a highly heterogeneous glycoprotein consisting of a 28-kDa protein and N-linked oligosaccharides ranging from 10 to 50 kDa. In the present study, cDNA clones were isolated from a human T cell cDNA expression library in Escherichia coli by using mAb C33. The identity of cDNA was verified by immunostaining and immunoprecipitation of transfected NIH3T3 cells with mAb. The cDNA contained an open reading frame of a 267-amino acid sequence which was a type III integral membrane protein of 29.6 kDa with four putative transmembrane domains and three putative N-glycosylation sites. The C33 gene was found to belong to a newly defined family of genes for membrane proteins, such as CD9, CD37, CD53, CD63, and TAPA-1, and was identical to R2, a cDNA recently isolated because of its strong up-regulation after T cell activation. Availability of mAb for C33 Ag enabled us to define its distribution in human leukocytes. C33 Ag was expressed in CD4+ T cells, CD19+ B cells, CD14+ monocytes, and CD16+ granulocytes. Its expression was low in CD8+ T cells and mostly negative in CD16+ NK cells. PHA stimulation enhanced the expression of C33 Ag in CD4+ T cells by about 5-fold and in CD8+ T cells by about 20-fold. PHA stimulation also induced the dramatic size changes in the N-linked sugars previously shown to accompany human T cell leukemia virus type 1-induced transformation of CD4+ T cells.


Long term expression of IL-4 in vivo using retroviral-mediated gene transfer.

Th cell subsets regulate immune responses by cell-cell interaction and secretion of cytokines. IL-4 is one of the cytokines secreted by Th cells important for cellular and humoral, particularly IgG1 and IgE responses. To study the role of IL-4 in T cell development and regulation of immune responses in vivo, low IgE-responder C57BL/6 mice were reconstituted with bone marrow cells that had been infected with recombinant retrovirus expressing a high level of IL-4. The reconstituted mice expressed retroviral IL-4 transcripts (9/10) even 8 mo postreconstitution. Physiologically significant levels of IL-4 were detected in the majority of the provirus-positive animals tested (5/8). Ectopic expression of exogenous IL-4 in hematopoietic cells had dramatic effects on T cell development resulting in changes in CD4:CD8 ratios. Moreover, the levels of serum IgG1 and, with antigenic stimulation, IgE were also increased. These results demonstrate that the retrovirus gene transfer system can be used to study the effects of ectopic cytokine gene expression in vivo on Ig isotype regulation and T helper cell subset differentiation.


Regulation of transcription of the germ-line Ig alpha constant region gene by an ATF element and by novel transforming growth factor-beta 1-responsive elements.

Inasmuch as transcription of unrearranged, or germ-line, Ig CH genes appears to direct switch recombination, understanding the regulation of this transcription is essential for understanding the regulation of class switching. Transforming growth factor-beta 1 (TGF-beta 1) induces germ-line alpha transcripts and increases class switching to IgA in the I.29 mu B lymphoma and in Peyer's patch and splenic B cells. It has been previously demonstrated that induction of germ-line alpha transcripts by TGF-beta occurs at the transcriptional level in I.29 mu cells. We now demonstrate that the DNA segment located 5' to the initiation sites of germ-line alpha RNA drives expression of a luciferase reporter gene construct in transient transfection experiments. Full constitutive expression requires no more than 106 bp of the 5' flanking segment. By creating a series of deletion and substitution mutations, we have demonstrated that an ATF/CRE site residing within this region is very important for constitutive expression of the germ-line alpha promoter, but mutation of this motif does not diminish TGF-beta induction. Inducibility by TGF-beta requires additional sequences residing between -128 to -106 relative to the first RNA initiation site. Two copies of a tandemly repeated sequence 5' CA-CAG(G)CCAGAC 3' (termed Ig alpha TGF-beta-RE) are located in the region from -127 to -105. An oligonucleotide containing multimers of these repeats confers TGF-beta inducibility to a heterologous promoter. An additional copy of the TGF-beta-RE was identified at -41/-30 and its deletion reduces the TGF-beta response. Thus, we conclude that tandem repeats of a novel TGF-beta-RE are the positive regulatory element for the TGF-beta response. Our study provides further evidence that TGF-beta directs class switching to IgA through induction of transcription of the germ-line C alpha gene and demonstrates that TGF-beta can activate the promoter for the germ-line alpha gene.


Independent and synergistic effects of disulfide bond formation, beta 2-microglobulin, and peptides on class I MHC folding and assembly in an in vitro translation system.

We have examined the post-translational processing, intrachain disulfide bond formation, folding, and assembly of MHC class I H chains with beta 2-microglobulin after coupled in vitro translation of homogeneous mRNA and transport of nascent chains into canine microsomal vesicles. The formation of native alpha 3 domain conformation was dependent on conditions that optimized intrachain disulfide bond formation, and efficient folding of the alpha 1 alpha 2 domain required exposure to antigenic peptide. beta 2-microglobulin and peptide acted synergistically in forming native alpha 1 alpha 2 domain structure, and a small proportion of molecules with native alpha 1 alpha 2, but non-native alpha 3 structure were detected, indicating that alpha 3 domain folding is not an absolute prerequisite for the formation of native alpha 1 alpha 2 domain structure.


The HLA-E gene encodes two differentially regulated transcripts and a cell surface protein.

An HLA-E-specific oligonucleotide probe was used to study the expression of HLA-E. This probe detects two HLA-E transcripts, 1.8 and 2.7 kb in size, which are present in varying ratios in all tissues and cell lines investigated. We demonstrate that alternative poly(A) site usage accounts for the differential regulation of the two HLA-E mRNA species. Sequence analysis of three cDNA clones, representing the two transcripts of HLA-E, and of an HLA-E gene encoded by cosmid cd3.14, revealed identity of gene and cDNA in the 3' untranslated region. S1 nuclease protection assays confirmed that the two HLA-E transcripts are not alternative splicing products. Introduction of cd3.14, together with human beta 2 m into the murine myeloma cell line P3X63-Ag8.653, resulted in a cell surface expression of an HLA-class I heavy chain detectable by indirect immunofluorescence whereas transfection into the human beta 2m expressing mouse L cell line, J27 was negative with regard to cell surface expression. Cell surface labeling of transfectants and immunoprecipitation with a monomorphic HLA class I-specific antibody or an antibody against human beta 2m confirmed the presence of an HLA-E H chain on the cell surface. These results indicate that the HLA-E gene codes for a class I H chain that can be expressed on the cell surface.


Leukoregulin, a T cell-derived cytokine, induces IL-8 gene expression and secretion in human skin fibroblasts. Demonstration and secretion in human skin fibroblasts. Demonstration of enhanced NF-kappa B binding and NF-kappa B-driven promoter activity.

It was shown previously that leukoregulin (LR), a T cell-derived cytokine with unique antitumor properties, modulates fibroblast functions in vitro, including prostaglandin production, matrix synthesis, and protease gene expression. Here, we have focused on the ability of LR to modulate IL-8 gene expression in human dermal fibroblasts. Using a specific ELISA, we demonstrated a dose-dependent enhancement of IL-8 production by LR, accompanied by a parallel elevation of the corresponding mRNA levels, as measured by Northern hybridizations. Maximum accumulation of IL-8 mRNA was observed after 6 h of incubation with LR, and the elevation persisted over 24 h. Inhibition of protein synthesis by cycloheximide resulted in superinduction of IL-8 mRNAs by LR. Dexamethasone, all-trans-retinoic acid, and TGF-beta 1 failed to counteract the effect of LR on IL-8 gene expression. Transient cell transfections with an IL-8 promoter/CAT reporter gene construct showed a dose-dependent enhancement of the promoter activity by LR, suggesting transcriptional regulation. Gel shift assays with oligonucleotides containing the consensus NF-kappa B binding sequences of the IL-8 and Ig kappa light chain genes showed enhanced binding activity in nuclear extracts from cells incubated with LR. Transient transfection experiments using a NF-kappa B/SV2 promoter-CAT reporter gene construct showed enhanced CAT activity by LR. Taken together, these data suggest that LR may up-regulate IL-8 gene expression by activation of the binding of NF-kappa B to the corresponding cis-acting element in the IL-8 promoter. Our results demonstrate that LR, together with IL-1 and TNF-alpha, could participate in the recruitment of neutrophils to the sites of inflammation by induction of IL-8 production in fibroblasts.


IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones.

The cytolytic potential of a total number of 118 CD4+ human T cell clones specific for purified protein derivative (PPD) from Mycobacterium tuberculosis, tetanus toxoid, Lolium perenne group I allergen (Lol p I), Poa pratensis group IX allergen (Poa p IX), or Toxocara canis excretory/secretory antigen(s) (TES) was assessed by both a lectin (PHA)-dependent and a MHC-restricted lytic assay and compared with their profile of cytokine secretion. The majority of clones with Th1 or Th0 cytokine profile exhibited cytolytic activity in both assays, whereas Th2 clones usually did not. There was an association between the cytolytic potential of T cell clones and their ability to produce IFN-gamma, even though IFN-gamma produced by T cell clones was not responsible for their cytolytic activity. IL-4 added in bulk culture before cloning inhibited not only the differentiation of PPD-specific T cells into Th1-like cell lines and clones, but also the development of their cytolytic potential. The depressive effect of IL-4 on the development of PPD-specific T cell lines with both Th1 cytokine profile and cytolytic potential was dependent on early addition of IL-4 in bulk cultures. In contrast, the addition in bulk culture of IFN-gamma enhanced both the cytolytic activity of PPD-specific T cell lines, as well as the proportion of PPD-specific T cell clones with cytolytic activity. The addition in bulk cultures before cloning of IFN-gamma or IFN-alpha favored the development of TES-specific and Poa p IX-specific T cells into T cell clones showing a Th0 or even a Th1, rather than a Th2, cytokine profile. Accordingly, most of TES- and Poa p IX-specific T cell clones derived from cultures containing IFN-gamma or IFN-alpha displayed strong cytolytic activity. These data indicate that the majority of human T cell clones that produce IFN-gamma, but not IL-4 (Th1-like), as well as of T cell clones that produce IFN-gamma in combination with IL-4 (Th0-like) are cytolytic. More importantly, they demonstrate that the addition of IFN (alpha and gamma) or IL-4 in bulk cultures before cloning may influence not only the cytokine profile of human CD4+ T cell clones but also their cytolytic potential.


Kinetic analysis of cytokine gene expression in the livers of naive and immune mice infected with Listeria monocytogenes. The immediate early phase in innate resistance and acquired immunity.

The anamnestic response to infection with Listeria monocytogenes is characterized by the rapid elimination of normally lethal doses of bacteria and accelerated granuloma formation. These phenomena are mediated by listeria-specific memory T cells within the first 24 h after reinfection. In order to elucidate the mechanisms operative during this decisive phase of infection, we conducted a comprehensive kinetic and quantitative analysis of cytokine gene expression in the livers of naive and immune mice. Organs were removed at 30 min, and 1, 2, 6, and 24 h after primary and secondary infections, and PCR3-assisted messenger RNA (mRNA) amplification was performed on matched samples using primers specific for IL-1 beta, IL-6, M-CSF, GM-CSF, TNF-alpha, IFN-gamma, IL-10, IL-4, IL-2, IL-3 and I1-2Rp55. The cytokine pattern characteristic of secondarily infected animals differed qualitatively by the expression of mRNA for IL-2, IL-2Rp55, IL-3, and IL-4, demonstrating the accumulation and activation of specific T cells in the livers as early as 1 to 2 h after reinfection. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection almost completely abrogated the differentiated cytokine profile typical of the anamnestic response. Using competitive PCR for semiquantitative determination of mRNA levels, the amount of IL-1 beta and IL-6 mRNA was found to be very similar during primary and secondary infection, whereas TNF-alpha mRNA was found to be increased by approximately 10-fold 2 h and IFN-gamma mRNA by approximately 50 to 100-fold 6 h after reinfection when compared with a primary challenge. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection resulted in a substantial (approximately 10-fold) decrease in IFN-gamma mRNA expression. To correlate these findings with cytokine secretion, spleen cells from naive and immune as well as normal and CD4+ and CD8+ cell depleted mice infected 6 h previously were cultured for 48 h, and supernatants were analyzed for the amount of the above mentioned cytokines. Semiquantitative PCR-assisted mRNA amplification is demonstrated to be a superior tool in dissociating the mediators of innate resistance from those operative in protective immunity and granuloma formation.


IFN-gamma and IL-2 are protective in the skin but pathologic in the corneas of HSV-1-infected mice.

HSV type 1 (HSV-1) infection of the mouse cornea results in a tissue-destructive inflammatory reaction in the cornea, but little or no disease in the skin surrounding the eye. Depleting T lymphocytes from mice before HSV-1 corneal infection prevents the corneal inflammation but severely exacerbates the periocular skin lesions. Studies described in this communication investigated the role of T cell cytokines in the corneal and periocular skin disease induced by HSV-1 corneal infection. Mice received weekly i.p. injections of rat mAb specific for IL-2, IL-4, or IFN-gamma beginning 1 day before (day -1) or 6 days after (day +6) corneal infection with the RE strain of HSV-1. The severity of corneal inflammation and the area of periocular skin involvement were measured. Treatment with anti-IFN-gamma or anti-IL-2 significantly reduced the incidence and severity of corneal inflammation. Treatment was equally effective when initiated on day -1 (before T cell activation) or day +6 (after T cell activation but before the initiation of corneal inflammation). Treatment with anti-IL-4 had no effect. The histologic features of corneal inflammation in mock-treated mice included neovascularization, corneal edema, and cellular infiltration. Corneas of anti-IL-2-treated mice that developed inflammation had similar but less severe histologic features. Corneas of anti-IFN-gamma-treated mice that developed inflammation had neovascularization and edema but minimal cellular infiltration. Treatment with anti-IFN-gamma or anti-IL-2 significantly exacerbated periocular skin lesions when initiated at day -1, but not when initiated at day +6. Anti-IL-4 treatment had no effect on skin lesions. Treatment with either anti-IFN-gamma or anti-IL-2, when initiated at day -1, significantly inhibited the delayed-type hypersensitivity response to HSV Ag, but when treatment was begun at day +6 only anti-IFN-gamma significantly inhibited the delayed-type hypersensitivity response. Our findings suggest that IFN-gamma and IL-2 are important elements in both an immunopathologic T-lymphocyte response to HSV-1 Ag in the cornea and a protective T lymphocyte response in the skin.


Biochemical and immunologic characterization of a major IgE-inducing filarial antigen of Brugia malayi and implications for the pathogenesis of tropical pulmonary eosinophilia.

A major allergen of the human filarial parasite Brugia malayi has been identified by two-dimensional immunoblot analysis using a serum pool from patients with tropical pulmonary eosinophilia. The allergen is composed of two Ag with M(r) 23 and M(r) 25 and acidic isoelectric point (Bm23-25). Immunoblots using affinity-purified IgE antibodies to BM23-25 indicated that Bm23-25 is expressed mainly in the microfilarial stage. Digestion of the allergen with endoglycosidases indicates that it has N-linked oligosaccharide chains. Analysis of the reactivity of T cells derived from patients with lymphatic filariasis revealed that the Bm23-25 allergen was capable of stimulating T cell proliferation; Bm23-25 was also shown to induce IgE production in vitro from PBMC derived from patients with either TPE or other filarial symptoms. Bronchoalveolar lavage fluid of patients with TPE contained IgE antibodies that recognized Bm23-25 strongly, an observation suggesting that the microfilarial allergen might be involved in the pathogenesis of the TPE syndrome.


Cytokine expression in vivo during murine listeriosis. Infection with live, virulent bacteria is required for monokine and lymphokine messenger RNA accumulation in the spleen.

To examine the regulation of cytokine synthesis during murine listeriosis, we have monitored IFN-gamma, TNF-alpha, and IL-1 beta mRNA levels in the spleens of C57B1/6 mice after the i.v. infusion of virulent and nonvirulent preparations of Listeria monocytogenes (LM). Messenger RNA coding for TNF, IL-1, or IFN did not become detectable until approximately 12 to 15 h after the infusion of virulent LM. Levels of each cytokine mRNA then increased synchronously reaching peak or near peak levels around 24 h after infection. Levels gradually decreased over the next 4 to 5 days. Unlike virulent LM, neither heat-killed LM, nor nonvirulent LM variants lacking listeriolysin O, stimulated monokine or IFN mRNA accumulation even when administered in very large doses. To gain perspective concerning the response to LM, we examined the early pattern of cytokine mRNA accumulation induced by Salmonella typhimurium (ST), an intracellular pathogen expressing LPS. We noted at least three significant differences between the cytokine responses to LM and ST: 1) monokine mRNA levels increased much more rapidly (within 1 h) after ST infection; 2) unlike LM, ST retained the capacity to stimulate cytokine mRNA production when injected as heat-killed bacteria; 3) in contrast to LM, ST could not trigger the early IFN production characteristic of LM infection. Our data suggest that monokine and IFN production early in listeriosis are critically linked with the process of bacterial invasion of host cells. The timing and pattern of cytokine mRNA accumulation in this setting is qualitatively different from that induced by LPS. The pathway described in these studies may also play a role in the host cytokine response to other intracellular pathogens as well.


Lipopolysaccharide induces Egr-1 mRNA and protein in murine peritoneal macrophages.

Bacterial LPS has diverse effects on the function of immune cells, in general, and macrophages, in particular. The intracellular molecular events that mediate the effects of LPS are unclear. We undertook a series of studies in thioglycollate-elicited murine peritoneal macrophages to evaluate the effect of LPS on expression of Egr-1, a member of the immediate early response gene family. Egr-1 may function as an intranuclear "third messenger" because it is rapidly induced in a variety of cell types and encodes a 75- to 80-kDa nuclear phosphoprotein that activates transcription of genes containing the DNA consensus sequence GCGGGGGCG. LPS from Salmonella minnesota Re595 induced a maximal increase in Egr-1 mRNA in macrophages after 30 to 60 min of incubation that returned to baseline level by 120 min. LPS increased Egr-1 mRNA at 0.01 to 0.1 ng/ml with a maximal effect at 10 to 100 ng/ml. LPS markedly increased the transcription rate of Egr-1 by 10 min of incubation using nuclear run on analysis. Using a polyclonal anti-Egr-1 antibody, nuclear staining for Egr-1 protein was prominent after 1 to 2 h of incubation with LPS and declined to baseline by 4 h. Inasmuch as protein kinase C (PKC) has been implicated in mediating the effects of LPS, we determined whether PKC was required for LPS to increase Egr-1 mRNA. Two pharmacologic approaches were used to deplete PKC, PMA pretreatment, and H-7. The induction of Egr-1 mRNA by LPS was markedly reduced in PKC-depleted macrophages. These data reveal that LPS induces transcriptional activation of Egr-1 and increases Egr-1 protein in peritoneal macrophages. In addition, these findings support further study of the potential role of Egr-1 in mediating the effects of LPS in peritoneal macrophages.


Macrophage and monocyte IL-1 beta regulation differs at multiple sites. Messenger RNA expression, translation, and post-translational processing.

Maturation of blood monocytes into macrophages is accompanied by a number of functional changes including decreased IL-1 beta release in response to LPS. This limitation has previously been ascribed to transcriptional regulation. However, in seeming conflict with the observed depression in IL-1 beta mRNA levels, recent work demonstrates increased intracellular IL-1 beta in macrophages. Therefore, the present study sought to explain these differences by comparing IL-1 beta production from autologous alveolar macrophage and blood monocyte pairs at multiple regulatory sites, including endotoxin responsiveness, mRNA expression, protein translation, and post-translational processing. Macrophages did not differ from monocytes in endotoxin sensitivity, but when analyzed by both ELISA and Western blot, were confirmed to have limitations in IL-1 beta release. Gene expression studies demonstrated that at 4 h, macrophage IL-1 beta steady state mRNA levels were 3-fold lower than the monocyte's. However, total IL-1 beta protein production, as measured by [35S]methionine labeling with immunoprecipitation, demonstrated three- to sixfold higher amounts in macrophages at comparable time points. The enhanced protein production in the face of relatively low mRNA levels suggests that macrophages translate IL-1 beta mRNA more efficiently. Furthermore, characterization of IL-1 beta release into supernatants revealed that whereas monocyte release occurred early, represented 5 to 20% of the intracellular amounts, and contained largely processed IL-1 beta, macrophage release was delayed, represented 1 to 5% of the intracellular amounts, and contained primarily unprocessed IL-1 beta. Taken together, these data demonstrate that the limitations in alveolar macrophage IL-1 beta release occur due to slower export and conversion of 35- to 17-kDa protein and are not due to differences in sensitivity to endotoxin or to transcriptional control mechanisms.


Detection of alveolar macrophage-derived IL-1 beta in asthma. Inhibition with corticosteroids.

This study investigated alveolar macrophage function in asthma as assessed by the presence and source of the cytokine IL-1 beta. Bronchoalveolar lavage (BAL) fluid and cell pellets were obtained at 4 a.m. from a cohort of asthmatic subjects. Normal subjects were used as controls. Asthmatic BAL fluid contained 57.0 +/- 5.9 pg/ml of IL-1 beta as determined by ELISA. No IL-1 beta could be identified in the BAL fluid of the control group. This IL-1 represented synthesis by cells of the airway as assessed by the presence of IL-1 beta-specific mRNA transcripts in the BAL cell pellets. Inasmuch as IL-1 beta transcripts are known to be present for only a short time period after activation (approximately 0.5 to 16 h), the presence of transcripts represents recent cellular activation. Using the technique of in situ hybridization, IL-1 beta transcripts were found to be located exclusively within alveolar macrophages, and a mean of 40 +/- 14% of alveolar macrophage were activated at the time of the lavage. Corticosteroids are known to be efficacious in the treatment of asthma. Administration of a single 50-mg dose of prednisone at 3 p.m. resulted in diminished IL-1 beta protein concentration in the BAL fluid (28.3 +/- 5.7 pg/ml; p < 0.01 compared to baseline) and completely abrogated IL-1 beta mRNA expression. In summary, these studies demonstrate that IL-1 beta is synthesized within the airways by alveolar macrophage and suggest that IL-1 beta may be a mediator of asthma. Inhibition of IL-1 beta may be an additional mechanism for the efficacy of corticosteroids in the treatment of asthma.


Structure and binding properties of a monoclonal anti-idiotypic autoantibody to anti-DNA with epibody activity.

We report the isolation and characterization of a mouse autoanti-idiotypic mAb (D7.4 IgG2a), which is directed against a major public Id (A52 IgG2b) in the murine and human autoimmune response to DNA. The natural anti-Id mAb has been produced in the course of the SLE-like disease in a female NZB/NZW F1 mouse and showed a dual specificity (epibody activity) for the public Id (A52) and for the autoantigen (DNA). The two binding activities were shown to reside in the Fab portion of the epibody and were highly specific for their respective Ag. A complete nucleotide sequence analysis of the D7.4 H and L chain V-region genes combined with computer comparisons to available Ig sequences may suggest a charge interaction between the H chain CDR3 segments of the Id and anti-Id antibodies. The D7.4 epibody may be a component of the self-binding, idiotypically connected network of natural antibodies. Alternatively, it could be elicited against the potentially pathogenic, DNA-containing immune complexes in order to facilitate their removal from the circulation of diseased individuals.


Novel deletion and a new missense mutation (Glu 217 Lys) at the catalytic site in two adenosine deaminase alleles of a patient with neonatal onset adenosine deaminase- severe combined immunodeficiency.

Mutations at the adenosine deaminase (ADA) locus result in a spectrum of disorders, encompassing a fulminant neonatal onset severe combined immunodeficiency (SCID) and childhood onset immunodeficiency, as well as apparently normal immune function. The extent of accumulation of the toxic metabolite, deoxyATP, correlates directly with severity of disease. We have now determined the mutations on both alleles of a child with fulminant, neonatal onset ADA- SCID and accumulation of extremely high concentrations of deoxyATP. The genotype was consistent with the severely affected phenotype. One allele carried a large deletion that arose by non-homologous recombination and included the first five exons and promoter region. The second allele carried a missense mutation (G649A) resulting in replacement of Glu217, an amino acid involved in the catalytic site, by Lys and predicting a major alteration in charge. Expression of the mutant cDNA in Cos cells confirmed that the mutation abolished enzyme activity. We have previously reported that a missense mutation at the preceding codon is similarly associated with neonatal onset ADA- SCID and accumulation of extremely high deoxyATP. These findings suggest that genotype-phenotype correlations may be apparent for ADA- SCID, despite the role that random variation in exposure to environmental pathogens may play in the initial phenotype. Such genotype-phenotype correlations may be important to consider in evaluating results of ongoing trials of "gene" and enzyme replacement therapy.


The use of nylon wool for the isolation of T lymphocyte subpopulations.

A simple method for the affinity isolation of lymphocyte subpopulations using nylon wool as a solid phase matrix is described. This matrix was coated with an acid-treated IgG fraction of polyclonal antibodies against mouse immunoglobulins. Using this approach, a simple isolation procedure for lymphocyte subpopulations, yielding purities higher than 95% was developed. The method can be used on a large scale for positive and negative cell selection, with very little non-specific binding. The isolated populations are fully functional.


Effective blocking of natural cytotoxicity of young rabbit serum on murine thymocytes by high concentration of glucose in complement-dependent cytotoxicity method.

By an accidental observation, the natural cytotoxicity of 21-day-old rabbit sera to murine thymocytes was found to be almost completely inhibitable by 4.5 mg/ml glucose. This enabled us to use unabsorbed young rabbit serum as a source of complement for antibody-mediated cytotoxic treatment of murine thymocytes. This glucose-sensitive natural cytotoxic activity was recovered in the immunoglobulin fraction purified on an affinity column, and was dependent on complement activity. The unabsorbed 21-day-old rabbit sera had much higher complement activity than those from 14-day-old rabbits and commercially available low background complement. Indeed, complete depletion of CD4+ cells from murine thymocytes and splenocytes was achieved using unabsorbed 21-day rabbit serum as a source of complement and a monoclonal antibody GK1.5 which has been claimed to be inefficient in complement-dependent killing. Owing to the increase in complement titer and circulating blood volume 14-21 days after birth, the use of 21-day-old rabbit sera assures a much more abundant supply of complement for the treatment of murine lymphocytes.


The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry.

Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.


A rapid and sensitive cellular enzyme-linked immunoabsorbent assay (CELISA) for the detection and quantitation of antibodies against cell surface determinants. I. A comparison of cell fixation and storage techniques.

A solid phase cellular ELISA was designed and evaluated for the detection of antibodies specific for cell surface determinants. It was hypothesized that certain fixation and freezing procedures would result in stabilization of cell structures for prevention of antigen diffusion and extraction during washing procedures. This would assure assay accuracy and convenient sample management. It was hypothesized that fixation with certain reagents prior to analysis would not alter antigenicity of antibody targeted epitopes. In order to improve the preservation of the cells following cell binding to the solid phase matrix while still retaining antigenicity and morphology, a series of fixatives and storage procedures were screened to determine which were best suited for CELISA. Methanol, washing buffer (WB), Hanks' balanced salt solution (HBSS), and 0.5% formalin in HBSS were examined by comparing their relative cell binding capacity and the subsequent cell morphology. In consideration of all variables, fixation in 0.5% formalin provided the best maintenance of cell antigenicity, morphology, binding, and was associated with consistent results. Cells used immediately after fixation and fixed cells used after storage at -80 degrees C for up to 12 months were compared to determine if long term storage affected antigenicity. Since frozen cells and fresh cells demonstrated statistically identical positive to negative ratios and consistency of antibody binding, it was determined that long term frozen storage of formalin-fixed cells did not adversely affect antibody binding capacity to cell surface determinants.


A transient transfection system for identifying biosynthesized proteins processed and presented to class I MHC restricted T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTL) constitute a major portion of immune responses to foreign and self antigens. CTL recognize class I major histocompatibility complex molecules complexed to peptides of 8-10 residues derived from cytosolic proteins. To understand CTL responses to these antigens and to manipulate CTL responses optimally, it is necessary to identify the specific peptides recognized by CTL. The methods currently used for this purpose have significant drawbacks. We describe a plasmid transfection method that results in significant lysis of histocompatible target cells. Influenza virus-specific CTLs specifically lysed target cells that were transfected with plasmids bearing cDNAs encoding full length gene products, fragments containing the region that encodes the CTL epitope, or even a ten residue peptide. This significantly lessens the time and effort required to define genes, and gene segments that contain CTL epitopes.


Selective generation of antigen-specific human hybridomas optimized for large scale growth in serum-free medium.

The direct propagation of newly formed human hybridomas in serum-free medium selects for hybrids with a metabolism best suited to growth in this environment. Under optimal culture conditions, this procedure results in the generation of antigen-specific human hybridomas comparable in frequency, stability, and antibody secretion rate to that obtained with murine hybridomas. After a transient phase of a few days in the appropriate selection medium supplemented with 1% serum, hybridomas grow in serum-free medium in stationary cultures with a cell doubling time of 15-25 h and an antibody production rate averaging 12 micrograms/10(6) cells/day. Clones propagated in bioreactors exhibited a cell doubling time of 29-35 h and an antibody secretion rate of 10-21 micrograms/10(6) cells/day.


Monoclonal antibody based ELISAs for cryptococcal polysaccharide.

Mouse monoclonal antibody (MAb)-based enzyme-linked immunosorbent assays (ELISAs) have been developed to detect Cryptococcus neoformans capsular polysaccharide from the four serotypes A, B, C and D. The ELISAs avoid the problem of unreliable polysaccharide binding to polystyrene plates by using MAbs to capture and immobilize the polysaccharide antigen. The presence of polysaccharide is detected using MAbs of a different isotype from that of the capture MAb. The capturing MAbs are themselves immobilized on the plates using commercial goat anti-mouse polyclonal sera. The MAbs bind to the glucuronoxylomannan component of cryptococcal polysaccharide. The ELISAs can be used to measure the concentration of polysaccharide in biological fluids and are potentially useful tools for basic research and clinical studies.


Analysis of high complement levels in Mus hortulanus and BUB mice.

BUB/BnJ mice were previously identified as having exceptionally potent complement activity, relative to common mouse strains, in the lysis of antibody-coated human tumor cells. We describe herein our investigation into the molecular and genetic basis for this difference between mouse strains, and also our results with wild mice and mouse strains recently derived from the wild, to determine whether low complement levels are characteristic of wild mice. BUB complement was compared with complement from BALB/c and C57BL/6 mice. BUB mice had higher levels of most individual classical pathway components, except for C1, than the other two strains, but the difference was generally only 2-3-fold, so insufficient to fully explain the difference observed with tumor target cells. CH50 titers on antibody-coated sheep erythrocytes also demonstrated only a 2-4-fold difference. However, CH50 titers on antibody-coated human erythrocyte target cells demonstrated a difference similar in magnitude to that seen with human tumor targets. These results suggest that the difference between mouse strains depends partly on the use of human, rather than sheep, target cells. In an assay for alternative complement pathway activity using neuraminidase-treated human erythrocytes as targets, complements of BALB/c and BUB mice were similar in activity, suggesting that the difference between mouse strains is manifested in the early steps of complement activation. Analysis of F1 and backcross mice suggested that the difference in complement level between BUB and BALB/c or C57BL/6 mice is controlled by semi-dominant genes, and cannot be attributed to a single gene. Wild mice and mice recently derived from the wild generally had low complement levels, similar to most laboratory mice. However, three strains of aboriginal mice, including Mus hortulanus (spicilegus) and Mus spretus, had complement levels higher than that of BUB mice, and as high as sera from the rabbit or rat, which are the most potent known complement sources for the lysis of human tumor cells. In comparison with BUB mouse sera, M. hortulanus sera had at least four-fold higher levels of C3, C6, C8 and C9, and some or all of these differences may explain its higher total complement activity. In the lysis of antibody-coated human erythrocytes, M. hortulanus serum was more potent than any other complement source tested, including sera of the guinea pig, rat, rabbit or human. These strains may be useful in investigating the role of complement in various pathological processes, and in investigating the genetic regulation of the complement system.


Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies directed against intracellular antigens.

For investigations involving monoclonal antibodies (mAbs) against cellular antigens cell binding assays are routinely used to determine the immunoreactive fraction after radiolabeling. In general, surface antigens are targets for radioimmunodetection, but recent studies indicate that intracellular determinants may also prove useful for this purpose. Thus, there is a need to adapt the regular cell binding assay for use with antibodies directed against cytoplasmic antigens. Here we describe a fixation method which permits such mAbs to bind to cell surfaces as well as to intracellular determinants. Moreover, the procedure may be used for antigens that are sensitive to the commonly used aldehyde fixatives. The method is illustrated with two human IgM mAbs 16.88 and 28A32, which recognize cytoplasmic antigens. Human colon cancer cells in suspension were fixed with either acetone or glutaraldehyde. Intracellular antigens appeared to be best exposed for antibody binding after fixation with acetone as determined by immunofluorescent staining and flow cytometry. An antibody directed against the cell surface antigen HLA class I showed similar binding with both live cells and acetone-fixed cells. Double-inverse plots of the binding of radiolabeled 16.88 or 28A32 antibody with acetone-fixed cells gave reliable immunoreactive fraction values. Acetone-fixed cells stored at 4 degrees C could be used for immunoreactivity assays for at least 6 months without loss of performance.


Increased specificity and sensitivity of insulin antibody measurements in autoimmune thyroid disease and type I diabetes.

Insulin autoantibodies (IAA), a marker for insulin-dependent diabetes mellitus (IDDM), have been reported in other diseases such as thyroid disease and after treatment with sulfhydryl containing medications. Reported prevalences of IAA in non-diabetics vary widely, probably due in part to methodological differences between laboratories. In addition, certain sera may have a high non-specific binding to insulin. We compared a radioimmunoassay (RIA) for IAA which included non-specific binding with an RIA that incorporated a competitive displacement with cold insulin to remove non-specific binding. Using the RIA which measured specific plus non-specific binding, IAA positivity was found in 22/92 (23.9%) of sera from thyroid disease patients, 16/124 (12.9%) of random masked sera from a hospital laboratory, 27/335 (8.1%) of first degree relatives of IDDM patients, 63/178 (35.4%) of subjects with newly diagnosed IDDM, and 0/92 (0%) of normal controls. Insulin antibodies (IA) were found in 80/99 (80.8%) of insulin-treated diabetic subjects. In contrast, using the displacement assay which allowed measurement of specific binding, the frequency of IAA positivity was lower for subjects with thyroid disease (7/92 (7.6%)), random hospital sera (12/124 (9.8%)), and for first degree relatives of IDDM patients (8/335 (2.4%)), while higher for subjects with newly diagnosed IDDM (71/178 (39.9%)). Subjects with insulin-treated diabetes (78/99 (78.8%)) and normal subjects (1/92 (1.1%)) showed little change. Strikingly, three of the eight (37.5%) relatives of IDDM patients that were positive in the RIA measuring specific binding were detected only because cold displacement was utilized. We conclude: (1) subjects with thyroid disease and first degree relatives of IDDM patients frequently have high non-specific binding for IAA in an RIA not employing a cold displacement step, (2) in some newly diagnosed IDDM patients and first degree relatives of IDDM patients, IAA may be missed by an assay not optimized to measure specific binding, and (3) displacement with cold insulin increases both the specificity and sensitivity of RIAs measuring insulin autoantibodies.


Use of defined oligosaccharide epitopes in an ELISA for group B streptococcus.

A single-site ELISA for group B streptococcal polysaccharide based on a monoclonal antibody against an immunodominant trirhamnoside epitope was inhibited at high capture antibody coating densities. The inhibition was eliminated when less antibody was coated or when high antigen concentrations were tested. This antigen is polyvalent with respect to the terminal trirhamnoside epitope and therefore it appears that closely spaced capture antibodies bound the epitope completely, leaving no sites for attachment of the enzyme-labeled second antibody with the same specificity. To make use of the trirhamnoside epitope feasible, a two-site ELISA was evaluated with this monoclonal antibody and a polyclonal antibody isolated by affinity chromatography. ELISA inhibition studies using oligosaccharides derived from the group B polysaccharide were used to evaluate the specificity of the polyclonal antibody. This showed that the antibody recognized both alpha-L-rhamnose (1----3)-D-galactose and alpha-L-rhamnose(1----3)-D-glucitol side chains, which together represent 30 potential binding sites per antigen molecule. A two-site ELISA with the anti-trirhamnoside monoclonal antibody to capture the antigen and the polyclonal antibody as enzyme conjugated second antibody reacted with only group B streptococci when tested against a panel of bacteria representative of the vaginal flora and was able to detect 3 x 10(4) cfu/test of group B streptococci. This two-site ELISA, based on well defined oligosaccharide epitopes had the sensitivity and specificity necessary to identify women at risk of infecting their newborns with group B streptococcus.


Lymphocyte immunophenotyping by flow cytometry in normal adults. Comparison of fresh whole blood lysis technique, Ficoll-Paque separation and cryopreservation.

In the present report we have assessed the extent to which Ficoll-Paque separation and cryopreservation of mononuclear cells alter the measurement of lymphocyte subsets by flow cytometry. Standard Ficoll-Paque separation increased the percentage of CD4+, CD19+ and CD4+CD45RA+ cells, as well as decreasing that of CD8+, and CD4+CD29+ cells, compared to the fresh whole blood lysis technique. Moreover, cryopreservation caused a depletion of CD4+ p80+ cells, but normal whole blood values were restored following a short incubation.


Microplate washing: process description and improvements.

Heterogeneous immunoassays require wash steps in order to separate bound from free constituents. In this paper we demonstrate that in microplate assays the washing process includes two separate physical processes: (1) a rapid and wash volume-dependent direct dilution of the droplet-shaped residual volume, and (2) a diffusion-limited and strongly time-dependent dilution of a residual layer of liquid, which necessitates the use of time-consuming soak times in the immunoassay. We have shown that optimizing the motion of the wash fluid effectively reduces the residual layer thickness that results in extended soak times. This results not only in improved washing efficiency and reduced background variance in the immunoassay, it also yields a significantly improved immunoassay sensitivity.


The development of enzyme-linked immunosorbent assays (ELISA) for the catecholamines adrenalin and noradrenalin.

Monoclonal antibodies have been raised against adrenalin and noradrenalin and used as the basis of enzyme-linked immunosorbent assays (ELISA) to detect and estimate the concentrations of these catecholamines. Inhibition assays are described, with sensitive quantification in the range from 1 mg/ml to 100 pg/ml. Cross-reactivity assays reveal that neither assay is subject to interference by catecholamine metabolites at concentrations less than 100 ng/ml and 1 micrograms/ml respectively. Isolation and quantification of both catecholamines from clinical samples is discussed and the potential application of these ELISAs in a clinical setting is proposed.


A simple fluorescence method for surface antigen phenotyping of lymphocytes undergoing DNA fragmentation.

Apoptosis, a metabolically active process of programmed cell death characterized by DNA fragmentation, is believed to play an important role in development of lymphocyte repertoires and in embryogenesis. Studies of this phenomenon would be greatly facilitated by the development of a simple assay capable of identifying and isolating intact apoptotic cells. A rapid fluorescence assay which identifies relatively small, intact cells containing fragmented DNA is described in this report. Thymocytes in which DNA fragmentation is induced by culture with or without dexamethasone are readily identified by their bright blue fluorescence after a 15 min treatment with Hoechst 33342, a DNA binding fluorochrome which diffuses through cell membranes. Since Hoechst 33342 staining does not require destruction of the cell membrane, it is possible to directly phenotype cell surface antigen expression on Hoechst 33342bright lymphocytes by conventional immunofluorescence techniques and to evaluate membrane integrity of Hoechst 33342bright cells by dye exclusion criteria. The advantages of this system are that it: (1) is rapid and simple, (2) quantitates the percentage of cells fragmenting their DNA and presumably undergoing apoptosis, (3) permits standard immunofluorescence staining of cell surface markers to identify even minor cell subsets of presumably apoptotic cells within heterogeneous populations, (4) provides the tools (fluorescence activated cell sorting) for purifying intact cells containing fragmented DNA for further biochemical studies, and (5) provides a means for identifying cells which exclude vital dyes and in which DNA fragmentation will eventually result in cell death.


A quantitative objective method for the evaluation of anti-sperm cell-mediated immunity in humans.

Anti-sperm cell-mediated immunity (CMI) is considered as a crucial facet of infertility in patients of both sexes. A precise and objective method is designed, based on a one-step agarose leukocyte migration inhibition factor (LMIF) assay. The migration areas are evaluated by a computer-assisted image analysis system. Optimal concentrations of leukocytes and sperm, as well as technical conditions are described. The radial migration indexes (RMI) and area migration indexes (AMI) are computed and expressed as a migration index (MI) percentage for each patient or control. Preliminary clinical results indicate a highly significant association between migration inhibition and cases of 'immunopathological infertility'. The method described is considered a promising tool for a rapid and quantitative evaluation of a suspected anti-sperm CMI in infertile and recurrently aborting patients.


Analysis of human IgA subclasses by in situ hybridization and combined in situ hybridization/immunohistochemistry.

In this report we describe a method for the analysis of the cellular expression of the two human IgA subclasses by in situ hybridization (ISH). The technique permits the detection of specific mRNA in individual cells using 35S-labeled oligonucleotide probes without any detectable cross-hybridization between the subclasses. This method was applicable both on cytospin preparation of peripheral blood mononuclear cells as well as in formalin-fixed, paraffin-embedded tissue sections. Furthermore, we describe a combined ISH/immunohistochemistry technique for the simultaneous detection of IgA subclass mRNA and protein at the single cell level. Examination of tissue sections from tonsils revealed a striking localization of labeled cells within the germinal center of some of the lymphoid follicles. The implications of this novel finding and the development of the method are discussed.


Antibody to recombinant murine tumor necrosis factor (TNF) neutralizes guinea pig TNF.

Cotton dust has been found to cause acute pulmonary inflammation and fever in humans and in a guinea pig model of byssinosis. Following 3 h inhalation of cotton dust particles, guinea pig macrophages were found to release ex vivo a factor(s) toxic to WEHI fibrosarcoma cells. The cytotoxic factor(s) was also present in the bronchoalveolar fluid. We sought to investigate the mechanism of the inflammatory response to determine whether the factor was TNF. Antibodies to murine TNF were produced by immunizing sets of rabbits using two protocols. All animals produced anti-TNF antibodies with titers of 1/1000-1/25,000. Sera from one set of animals completely neutralized the cytotoxicity of murine TNF toward the WEHI cell line. The antisera neutralized up to 93% of the cytotoxicity of guinea pig samples but only 54% of human recombinant TNF. These results identify TNF in pulmonary tissues of guinea pigs following exposure to cotton dust. Moreover, the studies indicate that rabbit antibodies to murine TNF can be used to detect the guinea pig cytokine.


Rapid DNA typing utilizing immobilized oligonucleotide probe and a nonradioactive detection system. Application to HLA-DR typing of the Japanese population.

We established a rapid and simple method of HLA-DR genotyping, and applied it for analysis of the Japanese population. Our method includes rapid preparation of DNA samples from buccal mucosa, incorporation of biotin-dATP into DRB genes during amplification by the polymerase chain reaction, hybridization with sequence-specific oligonucleotide (SSO) probes immobilized on nylon membranes via poly (dT) tails, and detection of the hybridization signal as chemiluminescence. We carried out DR typing of 30 Japanese donors using 20 different immobilized SSO probes, and obtained unambiguous typing signals showing perfect correlation with their serologic DR types. The genotyping also enabled us to identify several DR types unique to the Japanese population, such as DRw12b (DRB1*1202), DRw14c (DRB1*1405), and serology blank type, DR'JX6' (DRB1*1403). The method presented here would be suitable for routine DR typing in tissue-typing laboratories.


Isolation of mouse Lyt2- mutant cells using magnetic particles.

A procedure is described for the selection of mouse Lyt2- mutant cells. The procedure is based upon repeated cycles of selection with rat monoclonal antibodies to the Lyt2 antigen and magnetic particles coated with goat anti-mouse IgG. Stable Lyt2- mutant clones were derived from cells previously mutagenized with X rays and, at a lower frequency, also from non-mutagenized cells. The procedure can be used to rescue Lyt2- cells when the ratio of Lyt2+:Lyt2- cells is 10(4):1.


A quantitative method for measuring the adherence of group B streptococci to murine peritoneal exudate macrophages.

We have developed a solid phase, direct binding, enzyme-linked immunosorbent assay (ELISA) to detect and quantify the adherence of group B streptococci to murine macrophages. The assay correlated well with direct microscopic quantification of adherence. As few as 3.8 x 10(4) bacteria/assay well or less than one bacterium per macrophage could be detected. This assay is both quantitative and selective, and is readily adaptable for multiple sample analysis. It provides a valuable alternative to visual detection of bacterial adherence.


Factors influencing HIV-1 banding patterns in miniaturized western blot testing of dried blood spot specimens.

In the HIV Seroprevalence Survey among Childbearing Women (SCBW), antibodies to human immunodeficiency virus type 1 are detected using enzyme immunoassays (EIA) and Western blot (WB) methods modified to accommodate samples of blood dried on special collection paper. Dried blood spot (DBS) eluates positive by EIA are tested by one of two WB methods, the miniblot technique using equipment from Immunetics Corporation and the PBS Integra assay (pageblot) from Genetic Systems. In this report we compared the performance of the two WB methods. The identity and position of the viral proteins on the WB were identified using monoclonal antibodies and monospecific antisera. The blots differed substantially in their composition and concentration of viral glycoproteins. Performance of the WB assays with DBS elution buffers from different EIA kits was equivalent except for samples eluted in the Abbott buffer, which reduced detection of antibodies to the p31, p51, p55, and p66 viral proteins. Case classification of DBS, positive sera, dilution curve samples, and seroconversion panels was equivalent by both tests in the presence of all elution buffers. Proficiency evaluation panels sent to SCBW participating laboratories over a 3-year period were used to note the differences between the two WB methods in detection of antibodies to the viral glycoproteins.


In vitro proliferation and the cytotoxic specificity of a cryopreserved cytotoxic T cell clone reacting against human autologous tumor cells.

Proliferation and functional maintenance of CTL after cell cryopreservation often proves to be quite difficult. We developed an improved method for proliferating cryopreserved CTL, and for gaining their specific cytotoxic function. T cells were cryopreserved at -180 degrees C in RPMI 1640 containing 50% FCS and 10% DMSO. The cryopreserved T cells were well recovered by culturing in a medium containing the supernatant of primary cultures with TIL and autologous tumor cells, in addition to a high concentration (350 U/ml) of rIL-2. Furthermore, these cells were proliferated more efficiently when MMC-treated autologous tumor cells were used in vitro as a feeder and an antigenic stimulant. However, such a high dose IL-2 cultivation resulted in the loss of cytotoxic reactivity of CTL clone. In contrast, the withdrawal of rIL-2 from in vitro cultivation for 24 h prior to the cytotoxic assays conferred the specificity of cytotoxicity on CTL. By these methods, one can obtain a large number of CTL, and pursue the physiologic detail of the specific cytotoxic mechanism of CTL against autologous human tumor cells.


High gradient magnetic separation of cells on the basis of expression levels of cell surface antigens.

The possibility of separating cells on the basis of levels of antigen expression was explored in a model system using fixed erythrocytes and high gradient magnetic separation (HGMS). Fixed human erythrocytes were labelled to varying degrees with tetrameric monoclonal antibody complexes specific for both dextran and glycophorin A-M. The cells were then mixed and incubated with dextran iron particles prior to magnetic separation. The small size of the dextran iron particles (less than 0.2 microns) resulted in quantitative magnetic labelling of cells as shown using fluoresceinated anti-dextran antibodies and flow cytometry. The relationships between the initial percentage of labelled cells, cell recovery, non-specific entrapment of unlabelled cells, the purity of the removed fraction, the degree of antigen expression and separation conditions (flow rate and field strength) were determined and used to establish separation conditions that allowed recovery of cells that differ only in the degree of antibody labelling.


Dendritic cells from mouse bone marrow: in vitro differentiation using low doses of recombinant granulocyte-macrophage colony-stimulating factor.

Dendritic cells (DC) are potent stimulator cells that are crucially involved in primary T cell responses. Since DC comprise a minor population in lymphoid cell suspensions tedious and time consuming procedures are required for their preparation. This work outlines an in vitro culture system that promotes the differentiation of DC from unfractionated mouse bone marrow (BM) cells in the presence of low doses of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Unlike co-induced BM-macrophages the outgrowing BM-DC express high levels of MHC class II molecules; they are negative for specific and nonspecific esterase and are nonphagocytic. A rapid one step purification procedure employing immunomagnetic bead selection yielded up to 95% BM-DC enriched cell fractions. The bead-selected BM-DC were powerful inducers of the allogeneic mixed leukocyte reaction. Thus, our findings demonstrate that low dose rGM-CSF-driven in vitro culture of BM cells provides convenient access to substantial numbers of DC and will greatly facilitate their further exploration.


Restoring antibody activity of a monoclonal anti-RNP antibody by dissociative HPLC. Demonstration of blocking antibody binding sites with antigen released from effete hybridoma cells.

In biomedical research, monoclonal anti-nuclear antibodies have a number of advantages over polyclonal antibodies in terms of both specificity and reproducibility. However, there are some potential problems in the preparation of monoclonal antibodies. A well characterized mouse monoclonal anti-ribonucleoprotein antibody (anti-RNP antibody, 2.73) known to function in Western blotting was found to lose this activity when produced in vitro from long term hybridoma cell culture. Whilst it could no longer detect RNP antigen by Western blotting, it could still function effectively in affinity purification of RNP antigen. Further studies suggested that this was due to blocking of antibody binding sites by RNP antigen released from effete hybridoma cells in culture. The activity of the antibody in affinity purification was retained because the antigen was stripped away by repeated elutions with 6 M urea. HPLC gel filtration in the presence of 6 M guanidine was able to restore the antibody activity of the protein A purified monoclonal antibody. This finding has important general consequences for the preparation of monoclonal antibodies against antigens present in hybridoma cell culture media.


An improved method for the purification of IgG monoclonal antibodies from culture supernatants.

A method for the purification of mouse monoclonal antibodies from hybridoma culture supernatants is described. The protocol involves the use of a combination of three previously described methods for the concentration and purification of monoclonal antibodies. Firstly, hybridomas were grown in a Diacult dialysis system (Inter Med Laboratory, Denmark) to yield milligram quantities of monoclonal antibody in a culture supernatant. Monoclonal antibodies were then purified from the culture supernatant by precipitation with polyethylene glycol 6000 (PEG 6000) and finally reprecipitated using an ammonium sulphate procedure. The PEG 6000 treatment caused a density change in the ammonium sulphate immunoglobulin precipitate, and resulted in the formation of a pellicle which contained pure mouse monoclonal antibody. The protocol removed contaminating bovine serum immunoglobulin as well as other serum and cellular protein from the monoclonal antibody preparations.


Effector-target interactions: saturability, affinity and binding isotherms. A study of such interactions in the human NK cell-K562 tumour cell system.

Effector-target interactions at the cell-to-cell level have been studied. This has revealed that saturability, i.e., the existence of a finite number of specific receptor sites, applies to both the effector and target cell populations and plays a key role in the formation of conjugates. As a result, two maximum conjugate frequencies, alpha max and beta max, are recognised for the effector and target cell populations, respectively. The dissociation constant of the conjugates formed, KD, characterizes effector-target affinity. This constant, together with the maximum conjugate frequencies, are the three parameters which make it possible to describe the binding process quantitatively. The existence of binding isotherms for effector-target interactions has been demonstrated. These isotherms contain all the relevant information necessary to interpret quantitatively the formation of conjugates. Quantitative procedures to determine the three binding parameters are described together with the modifications necessary to use Scatchard plots in the analysis of conjugate frequencies in these kinds of cell-to-cell interactions. A quantitative study of these interactions in the NK-K562 tumour cell system has been performed. For this purpose, nine different cell source donors were used to test the model proposed. Relationships with related phenomena--CMC and the adhesion process--are also discussed.


Optimization of cytotoxic assay by target cell retention of the fluorescent dye carboxyfluorescein diacetate (CFDA) and comparison with conventional 51CR release assay.

This paper describes the comparison between a fluorimetric NK assay based on the target cell retention of fluorescent dye carboxyfluorescein diacetate (cFDA) and standard 51Cr release assay. The results provide several suggestions to improve the cytotoxic assay based on the use of the fluorogenic substrate showing that the measurements of cFDA retained by target cells represent a method of evaluating cytotoxicity completely comparable to the 51Cr release assay.


Development of a new technique for recovery of cytokines from inflammatory sites in situ.

We attempted to recover cytokines from nasal mucosal surface following allergen challenge. Repeated lavage of nasal mucosa of seven allergic patients was done, but we failed to detect IL-1 beta in the lavage samples even in ten-fold concentrated materials. Therefore, we developed a new technique to recover cytokines using filter strips. Small filter strips were placed on nasal turbinates for 10 min at different time points after allergen challenge. The strips were air-dried, and stored. For recovery of cytokines individual strips were washed with small volumes of Hepes buffer containing 0.3% human serum albumin. Eluates were assayed for the presence of IL-1 beta and GM-CSF using commercially available ELISA. We were able to detect IL-1 beta and GM-CSF in eluates. Both cytokines were consistently detectable in the late phase allergic reaction peaking at 5 h. Nasal challenge with saline failed to detect any cytokine during the 7 h observation period. In standardization experiments known quantities of IL-1 beta and GM-CSF were applied to filter strips and the recovery ranged from 67 to 89%. Thus, we developed a simple technique of recovery of cytokines from inflammatory mucosa in situ.


Immunological properties of ribosome-inactivating proteins and a saporin immunotoxin.

Antibodies have been raised in rabbits against plant ribosome-inactivating proteins (RIPs) and used to demonstrate cross-reactivity between RIPs from plants belonging to the same family, but little or no cross-reactivity between RIPs from taxonomically unrelated plants. When an immunotoxin consisting of saporin conjugated to bovine IgG was injected into rabbits, the animals formed antibodies against both saporin and bovine IgG.


Acoustic probe-based ELISA.

The effects of acoustic microstreaming during incubation steps of a prototypical three-step ELISA were studied. Acoustic microstreaming, an orderly mixing of microwell contents induced by linear oscillation of immersible acoustic probes, was shown to be particularly effective when coupled with locating the solid phase on the surface of the probes. Optical densities achieved for acoustic probe-based assays were equivalent to those for uninsonated microwell-based assays with only 20% of the microwell solid phase surface area. Low-level antibody detection was significantly improved and antibody incubation times significantly shortened without loss of signal. Acoustic probe-based assays can enhance assay and laboratory efficiency through testing for multiple analytes in a single sample or increasing available binding surface area (by using probe and well surfaces simultaneously), and by eliminating quenching. Acoustic probe ELISA methodology has significant implications for cost-effective automation.


Human rabies: epidemiological and laboratory studies in 80 cases.

Epidemiological and laboratory studies were conducted on 80 clinically diagnosed human rabies cases. Incidence of rabies was more in adult males (55%) and boys (37.5%) than adult females (5%) and girls (2.5%) and more in rural areas (76.2%) than urban areas (23.8%). Dogs constituted the main vector of transmission (92.5%). Incomplete course of antirabic vaccine had been received by 31% of cases and 2.5% of cases had complete course of antirabic vaccine. Among the laboratory tests conducted 3.8% of corneal smears and 2.5% of saliva smears were positive by fluorescent antibody technique. Rabies virus was isolated from 3.8% of saliva and 3% of cerebrospinal fluid samples. Rabies neutralising antibody titre ranged from 1:64 to 1:128 in the serum of patients who were fully vaccinated.


Surgical manifestations and management of ascariasis in Kashmir.

Amongst 876 cases suffering from ascariasis 662 cases were managed conservatively and 214 cases were treated by surgery. Surgical complications were found to be more common in males in the age group of 6-10 years. Principal clinical features included pain abdomen (99.54%), constipation (80.25%), vomiting (67.46%), abdominal distension (47.03%), palpable worm masses in abdomen (35.50%), visible peristalsis (27.63%), worms in vomitus (24.20%) and palpable worm clumps on rectal examination (20.09%). Principal clinical diagnosis were worm colics (48.74%), sub-acute intestinal obstruction (27.74%), acute intestinal obstruction (11.42%) and acute intestinal obstruction with strangulation (5.71%); rest of the cases included worm cholecystitis (2.63%), obstructive jaundice (1.71%), bile peritonitis (0.91%), intestinal perforation (0.68%) and acute appendicitis (0.46%). Surgical procedures performed were milking of worms (34.12%), resection anastomosis of small intestine (23.36%), enterotomy with removal of worms (16.36%), cholecystectomy with T-tube drainage (12.15%), cholecystectomy (8.41%), appendectomy (1.87%), resection anastomosis with excision of Meckel's diverticulum (1.40%), repair of intestinal perforation with peritoneal toilet (1.40%) and cholecystectomy with choledochoduodenostomy (0.93%). In surgically managed patients 35 cases died of septicaemia and in conservatively managed cases 3 died of encephalitis with an overall mortality of 4.34%.