In vivo stability of human chemokine and chemokine receptor expression.

Cross-sectional analyses of human PBMC, plasma, and tissue have reported altered chemokine and/or chemokine receptor expression in several inflammatory diseases. Interpretation of such studies is difficult without data on the in vivo stability of such parameters. Using four color flow cytometry, we longitudinally followed CXCR3, CCR5 (Th1-associated), and CCR3 (Th2-associated) expression within CD4+/CD45RO+ and CD8+/CD45RO+ T cell populations in peripheral blood of healthy individuals over a 21 day period. In parallel, we quantified plasma levels of IP-10, Mig, eotaxin and TARC. Chemokine and receptor expression differed markedly between subjects but was highly stable, varying by <5% within individuals. Differences in chemokine receptor expression between subjects were markedly altered when quantified as absolute cell numbers rather than frequencies. Finally, CCR3 expression by CD4+/CD45RO+ T cells was positively correlated with plasma levels of its ligand, eotaxin, whereas strong negative correlations were evident between CXCR3 expression and IP-10 or Mig. These data demonstrate longitudinal stability of chemokine receptor and ligand expression among healthy individuals; reveal that both frequency and absolute cell count analysis is essential for accurate assessment of chemokine receptor expression; and identify inverse relationships between type 1 and type 2 immunity-associated receptors and their ligands in vivo.

B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis.

The formation of liver metastases involves interactions between intravascular cancer cells and the hepatic microvasculature. Here we provide evidence that the arrest of intravascular B16F1 melanoma cells in the liver induces a rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases. B16F1 melanoma cells (5 x 10(5)) labeled with fluorescent microspheres were injected into the portal circulation of C57BL/6 mice. The production of NO in vivo was detected by electron paramagnetic resonance spectroscopy ex vivo using an exogenous NO-trapping agent. A burst of NO was observed in liver samples examined immediately after tumor cell injection. The relative electron paramagnetic resonance signal intensity was 667 +/- 143 units in mice injected with tumor cells versus 28 +/- 5 units after saline injection (P < 0.001). Two-thirds of cells arrested in the sinusoids compared with the terminal portal venules (TPVs). By double labeling of B16F1 cells with fluorescent microspheres and a TdT-mediated UTP end labeling assay, we determined that the melanoma cells underwent apoptosis from 4-24 h after arrest. The mean rate of apoptosis was 2-fold greater in the sinusoids than in the TPVs at 4, 8, and 24 h after injection (P < 0.05-0.01). Apoptotic cells accounted for 15.9 +/- 0.8% of tumor cells located in the sinusoids and 7.1 +/- 0.9% of tumor cells in the TPVs. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester completely blocked the NO burst (P < 0.001) and inhibited the apoptosis of B16F1 cells in the sinusoids by 77%. However, the rate of tumor cell apoptosis in the TPVs was not changed. There were 5-fold more metastatic nodules in the livers of N(G)-nitro-L-arginine methyl ester-treated mice (P < 0.05). The inactive enantiomer N(G)-nitro-D-arginine methyl ester had no effect on the initial NO burst or on apoptosis of tumor cells in vivo. Both annexin V phosphatidylserine plasma membrane labeling and DNA end labeling of apoptotic cells were demonstrated after a 5-min exposure (a time equivalent to the initial transient NO induction in vivo) of B16F1 cells to a NO donor in vitro. These results identify the existence of a natural defense mechanism against cancer metastasis whereby the arrest of tumor cells in the liver induces endogenous NO release, leading to sinusoidal tumor cell killing and reduced hepatic metastasis formation.

Cell cycle behavior and MyoD expression in response to T3 differ in normal and mdx dystrophic primary muscle cell cultures.

Since mdx limb muscle regeneration in vivo is accompanied by rapid myoblast proliferation and differentiation compared to normal, we tested the possibility that proliferation and differentiation were differentially regulated in normal and mdx dystrophic muscle cells. Cell cycle behavior, MyoD expression, and the effects of thyroid hormone (T3) treatment were examined in primary cultures. Using a 4-hour pulse time for bromodeoxyuridine (BrdU) incorporation during S-phase, the phases of the cell cycle (early S, late S, G(2)/M, and G(0)/G(1)) were separated by 2-colour fluorescence (BrdU/PI) analysis using flow cytometry. The G(0)/G(1)-early S and the late S-G(2)/M transitions were examined under the influence of T3 in cycling normal and mdx muscle cell cultures over a 20-hour time period. Myogenesis and differentiation were assessed morphologically and by immunostaining for MyoD protein. Mdx cultures had fewer cells in G(0)/G(1) at 20 hours and more cells in early and late S-phase compared to normal cultures. T3 significantly increased the proportion of normal cells in early S-phase by 20 hours, and reduced the proportions in G(2)/M phase. Over the same time interval in parallel cultures, the proportion of MyoD+ normal cells decreased significantly. In the absence of T3, mdx cell cultures showed greater proportions of cells in S-phase than normal cultures, and similar increases in S-phase and loss of MyoD expression over time. However, mdx cultures had no change in the proportion that were MyoD+ during T3 treatment. The results confirm that T3 in primary cultures increased proliferation and prevented the de-differentiation of mdx cells to a greater degree than was typical of normal cells. The different susceptibilities to T3-related shifts between proliferation and differentiation observed in vitro support the idea that committed mdx myoblasts may be more activated and proliferative than normal myoblasts during regeneration in vivo.

Depletion of natural killer cells from the graft reduces interferon-gamma levels and lipopolysaccharide-induced tumor necrosis factor-alpha release in F1 hybrid mice with acute graft-versus-host disease.

BACKGROUND: We wished to determine whether removal of NK1.1+ cells from the graft provides protection against acute graft-versus-host disease (GVHD) by obviating the Th1 immune response that underlies the development of this disease. METHODS: Graft-versus-host (GVH) reactions were induced in two groups of (C57BL/6 x DBA/2)F1 hybrid mice. The first received grafts harvested from polyinosinic:polycytidylic acid-stimulated, C57BL/6 donors and depleted in vitro of NK1.1+ cells. This treatment provides protection against GVHD-associated mortality and cachexia. The second received unmodified grafts. We compared interferon-gamma and interleukin-10 production as well as the levels of engraftment in these two groups. Lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha) release was also compared since TNF-alpha levels in GVH mice following injection of a sublethal dose of endotoxin provide an index of macrophage priming by Th1 cytokines. RESULTS: Interferon-gamma production was absent in recipients of NK1.1-depleted grafts at the time when high levels were seen in recipients of unmodified grafts. Following lipopolysaccharide injection, high levels of TNF-alpha were observed in recipients of unmodified grafts, whereas negligible amounts were present in recipients of NK1.1-depleted grafts. The use of NK1.1-depleted grafts did not result in a reduced level of engraftment of CD4+ or CD8+ cells. CONCLUSIONS: These results suggest that NK1.1 depletion of the graft confers protection against mortality by interfering with an immunoregulatory mechanism that results in the development of a Th1 response in GVH mice, and does not result in abortion of the graft. Because macrophage priming is prevented, recipients are also protected from the exaggerated sensitivity to endotoxin seen in mice with acute GVHD.

Heterogeneity of polyclonal IgE characterized by differential charge, affinity to protein A, and antigenicity.

Functional and physical heterogeneity of polyclonal IgE has been reported. Extremely low serum concentrations of IgE have limited the study of these important differences. We have purified polyclonal dog IgE and developed polyclonal and monoclonal (mAb C2) anti-dog IgE antibodies. In this study chromatofocusing of dog IgE revealed two biologically active IgE fractions: IgE1 eluted at pH 5.0, and IgE2 eluted at pH 4.7. The two IgE subforms (IgEs) exhibited typical IgE characteristics: positive in the 48-hour passive cutaneous anaphylaxis response, heat-labile, identical molecular weight, and reactive to polyclonal anti-dog IgE. However, the two IgEs were found to be significantly heterogeneous. IgE1 bound to protein A and did not react with mAb C2 in ELISA and isoelectric focusing-immunoblotting, whereas IgE2 did not bind to protein A and reacted with mAb C2. Further, in sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting, IgE2, but not IgE1, reacted with seven well-defined mAb anti-human IgE antibodies and an mAb anti-mouse IgE antibody, even though both IgE1 and IgE2 reacted with polyclonal anti-human and anti-mouse IgE. Neuraminidase or endoglycosidase treatment did not abolish the differential antigenicity and charge of IgE1 and IgE2, although the antigenicity of IgE2 was significantly reduced after incubation with endoglycosidase. These data suggest that carbohydrate moieties are not involved in the observed differences in antigenicity and charge and that the two IgE molecules represent distinct isotypes. In studies with seven purified IgE fractions obtained from different ragweed-allergic dogs, the distribution of ragweed IgE2 varied 200-fold, whereas ragweed total IgE levels varied only fourfold. This raises the possibility of a relationship between different IgEs and the allergic response.

Gamma delta T cells in the pathobiology of murine acute graft-versus-host disease. Evidence that gamma delta T cells mediate natural killer-like cytotoxicity in the host and that elimination of these cells from donors significantly reduces mortality.

NK-like cytotoxicity in F1-hybrid mice with acute GVH disease is mediated by donor-derived CD3+/CD4-/CD8- cells that can lyse both NK-sensitive YAC-1 target cells as well as NK-resistant targets such as BW1100 and P815. Our objective was to determine whether this activity is mediated by gamma delta TCR+ cells. We showed that NK-like cytotoxic activity in the spleen and lymph nodes of mice with acute GVH disease could be depleted by indirect complement-mediated lysis using an Ab against gamma delta TCR. When purified NK1.1+ spleen cells that had been positively selected on a magnetic cell separator were used as effector cells, we found that NK-like cytotoxicity was mediated only by gamma delta TCR+ cells, suggesting that cells with NK-like activity are gamma delta TCR+/NK1.1+. We showed by flow cytometry experiments that coexpression of NK1.1 and TCR-gamma delta occurred on a large proportion of large granular lymphocytes in the spleens of GVH mice, but was not detectable in normal control mice. In GVH mice, fewer than 10% of small agranular NK1.1+ lymphocytes coexpressed NK1.1+ and gamma delta TCR+. On the basis of this hypothesis, we postulate that graft-derived large granular lymphocytes develop the NK1.1+/gamma delta TCR+ phenotype during the reaction, and that these cells play a role in the pathogenesis of acute GVH disease. We performed experiments to determine whether depletion of gamma delta T cells from donor mice affected the outcome of lethal GVH disease and found that there was a significant reduction in mortality.

Phenotypic changes among hybrid rat mast cells.

Rat peritoneal mast cells and 6-thioguanine-resistant rat basophilic leukemia cells, representative of connective tissue-type (CTMC) and mucosal (MMC) mast cells, respectively, were fused using polyethylene glycol. Four out of 14 primary hybrid mast cell lines contained more than 50% of CTMC as demonstrated by histochemical staining. Two cell lines, one predominantly of the CTMC and the other of the MMC phenotype, were selected for further study. Among these, the phenotype was also confirmed by analysis for rat mast cell protease I and by mediator release triggered by compound 48/80 and ionophore A23187. The CTMC phenotype disappeared after culturing cells for 2 weeks. The change in phenotype did not significantly alter the mediator release due to calcium ionophore A23187. Repeated cloning of cells bearing the CTMC phenotype did not yield a cloned line of cells expressing the CTMC phenotype only, although it prolonged the persistence of this phenotype. During the period of CTMC phenotype loss, a drop in cellular DNA content occurred, suggesting that chromosome instability may, at least partially, have been responsible for the phenotypic changes.

Purification and identification of polyclonal IgE antibodies from ragweed-sensitized dog sera.

We have purified and characterized polyclonal dog IgE. Serum IgE was precipitated by (NH4)2SO4 and then purified by two different procedures. Ion exchange on DEAE-Sephacel, followed by HPLC using Tonen hydroxylapatite and then Protein G-Sepharose, produced a highly purified IgE fraction (No. 1) free of IgG, IgA and IgM as measured by ELISA, but recovery of IgE as measured by passive cutaneous anaphylaxis was low. Gel filtration on Sephacryl S-300, Con A-Sepharose and Protein G-Sepharose recovered 18% of initial IgE, 0.02% IgG, 0.4% IgM and 0.3% IgA. This IgE fraction (No. 2) was used to induce antibody production in rabbits. Western blot analysis was then performed for dog IgE fractions No. 1 and 2. Using the rabbit anti-dog IgE, a prominent IgE band with an apparent molecular mass of 226 kD was identified in fractions No. 1 and 2 subjected to nonreducing SDS-PAGE. This band also reacted with anti-human IgE, but not with anti-dog IgG or anti-dog IgA. Under reducing conditions the approximate molecular mass for the IgE & chain, estimated by Western blot using rabbit anti-dog IgE, was 73 kD, providing a molecular mass of 196 kD for dog IgE.

Characterization of suppressor T cell clones derived from a mouse tolerized with conjugates of ovalbumin and monomethoxypolyethylene glycol.

The induction of antigen-specific tolerance in mice by conjugates of ovalbumin (OVA) and monomethoxypolyethylene glycol (mPEG) previously had been shown to be associated with the generation of antigen-specific suppressor T (Ts) cells. For the elucidation of the nature of these Ts cells, five nonhybridized OVA-specific Ts cell clones were generated from the spleen cells of a BDF1 mouse which had been immunosuppressed by the tolerogenic conjugate, OVA(mPEG)12. The cloned Ts cells were maintained in vitro by periodic stimulation with OVA and feeder cells and were able to suppress the in vitro antibody production in an OVA-specific and MHC class I (H-2Kd or H-2Dd)-restricted manner. All these Ts cell clones were shown to be Thy1.2+, CD4-, CD5-, CD8+, and to express CD3 and the alpha beta heterodimer of the T cell receptor. The cell-free extracts of these cells contained soluble suppressor factors which could mimic in vitro the suppressive activity of the intact cells. In contrast to cytotoxic T lymphocytes (CTL), none of the cloned Ts cells were endowed with cytolytic activity as revealed in the perforin-mediated microhemolysis and in the 18-hr51Cr release assays. These results demonstrate that (i) OVA-specific Ts cell clones can be generated from mice pretreated with OVA(mPEG)12 by employing conventional T cell culture techniques, and (ii) these Ts cells are functionally different from conventional CD8+ CTL.

Suppression of antibody responses in rats to murine anti-CD4 monoclonal antibodies by conjugates with monomethoxypolyethylene glycol.

The effectiveness of therapeutically relevant xenogeneic monoclonal antibodies (MoAb) may be counteracted by their inherent immunogenicity. Since conjugates of diverse proteins with mono-methoxypolyethylene glycol (mPEG) were shown to induce Ag-specific tolerance in mice and rats, we used outbred rats in this study as an experimental model for establishing the tolerogenicity of mPEG conjugates of murine MoAb. The results demonstrate that: (i) murine anti-rat CD4 MoAb (W3/25) were more immunogenic than murine anti-human CD4 MoAb (MAX.16H5) in rats; (ii) W3/25 preferentially induced an anti-idiotypic (anti-id) antibody response; and (iii) antibodies to both common and idiotypic determinants could be suppressed in rats by treatment with W3/25(mPEG)28.

A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV.

A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

Development and characterization of ouabain-resistant human fusion partners.

The ouabain-resistant mutant cell lines, HOA-1 and HOA-20 were developed from WI-L2-729-HF2 by cloning with increasing concentration of ouabain. Both parent and mutant cell lines were resistant to base analogues, 6-thioguanine (6-TG) and 8-azaguanine (8-AG) to the level of 20 micrograms/ml in the culture medium. The parent cell line WI-L2-729-HF2 was highly sensitive to ouabain, whereas HOA-1 and HOA-20 were resistant to ouabain to the level of 1 microM and 20 microM, respectively. However, all the cell lines were sensitive to HAT-selective medium which is essential for hybrid selection after fusion. All three lymphoblastoid cell lines were positive for Epstein-Barr virus nuclear antigen (EBNA), secreted TNF-beta (lymphotoxin) without any external stimulation, secreted trace amounts of IgG(kappa), which was also present in their cytoplasm and had IgM(kappa) as surface bound immunoglobulin. They also expressed the CD20, CD71 (transferrin receptor) as surface antigens. In addition to these antigens, HOA-20 also expressed CD38 antigen. The karyotype analysis of these cell lines revealed modal chromosomal numbers ranging from 40 to 47. The HLA-A, -B and -C antigens expressed by WI-L2-729-HF2 and its mutants HOA-1 and HOA-20 were identical. Both the HOA-1 and HOA-20 mutants were found suitable for the generation of hybrids after fusion with EBV-transformed human B-lymphocytes.

Establishment and characterization of hybrid rat mast cells.

Rat peritoneal mast cells (RPMC) and rat basophilic leukemia (RBL) cells are representative of connective tissue-type (CTMC) and mucosal-type (MMC) mast cells, respectively. Using polyethylene glycol, we have fused RPMC with 6-thioguanine resistant, HAT (hypoxanthine, aminopterin, thymidine) sensitive RBL-CA10.7 or RBL-CK2 cells, yielding several hybrid rat mast cell lines (HRMC). The hybridomas exhibited different size and cytoplasmic granularity when compared with parental cell lines. Analysis of both high (Fc epsilon RI) and low affinity (Fc epsilon RL) receptors for IgE revealed that the hybrid lines had more variable receptor patterns than the parent lines. Three hybridoma lines were chosen for further study. Differential histochemical staining with alcian blue and safranin O dyes indicated the hybrids to be predominantly of the MMC type: however, a few cells of one of these uncloned hybridomas were found to be of the CTMC type. Attempts to isolate the CTMC hybridomas yielded one culture which was predominantly of the CTMC phenotype and in a number of other cultures, cells were found expressing simultaneously both the CTMC and the MMC phenotype. After 3 weeks in culture, however, all hybridomas, including those which were cloned further, expressed only the MMC histochemical phenotype. This was found to correlate with the presence of rat mast cell protease II (RMCPII) and the absence of RMCPI in all hybridomas, as detected by Western blot analysis. In addition, the histamine content of all cells was significantly lower than that of the parent RPMC. Most hybrid mast cells expressed both Fc epsilon RI and Fc epsilon RL which in some cases exhibited significant variations in the Mr. These results indicate that somatic cell hybrids expressing the MMC and CTMC phenotype can be produced by the fusion of RBL and RPMC. The CTMC phenotype, however, is unstable, and possible reasons for this are discussed.

Cloned suppressor T cells derived from mice tolerized with conjugates of antigen and monomethoxypolyethylene glycol. Relationship between monoclonal T suppressor factor and the T cell receptor.

Cloned Ts cells specific for the Ag, human monoclonal (myeloma) IgG, were derived from spleen cells of mice that had been immunosuppressed by treatment with a tolerogenic conjugate of HIgG and monomethoxypolyethylene glycol. The cloned Ts cells (clone 23.32) suppressed in vitro antibody responses in an Ag-specific and MHC-restricted manner. By FMF with appropriate antibody reagents, these cells were shown to be Thy-1+, CD4-, CD5-, and CD8+ and to express CD3 and the alpha beta-TCR. These results are consistent with the view that Ts cells use Ag recognition structures similar to those reported for Th cells and CTL. A soluble factor (TsF) extracted from the cloned Ts cells also suppressed in vitro antibody responses in an Ag-specific and H-2Kd-restricted manner, i.e., restricted to MHC class I molecules. The suppressive activity of this TsF could be abrogated by addition of mAb H28-710 that reacts with a determinant on the alpha-chain of TCR. Moreover, the TsF bound to and could be recovered from an immunosorbent consisting of the anti-alpha-TCR mAb H28-710 coupled to Sepharose 4B. In contrast, the TsF was not bound by immunosorbents consisting of mAb to the beta-chain of TCR (H57-597) or to V beta 8 (F23.1). It was, therefore, concluded that the TsF of clone 23.32 is serologically related to the alpha-chain of the TCR; however, it is not identical to TCR, because it lacks the determinants expressed on the TCR beta-chain that are recognized by the two anti-beta mAbs used in this study.

Low molecular weight B cell growth factor-responsive cloned human B cell lines. I. Phenotypic differences and lack of requirement for CD23 (Fc epsilon RII).

The EBV-transformed B cell line JR-2 proliferates in response to partially purified preparations of low m.w. B cell growth factor (LMW-BCGF). Two clones of JR-2 were generated that retained this LMW-BCGF responsiveness, exhibiting similar dose/response characteristics but differing phenotypically. The B10 clone grows as single, discrete, small round cells, whereas D3 grows in aggregates. The clones also differ in the expression of cell surface Ag, D3 being weakly DR+ and strongly CD23+, whereas B10 lacks these Ag. The CD23 on D3 cells binds IgE. Both clones are T9+, 4F2+, B1-, B2- and CALLA-. D3 expresses surface IgG and differentiates in the presence of LMW-BCGF, to secrete IgG. B10 lacks surface and cytoplasmic Ig and fails to differentiate in response to LMW-BCGF. CD23 cannot be induced on B10 by incubation with either LMW-BCGF or IL-4. B10 does not shed CD23 and shed CD23 is not a growth factor for either cloned line. Expression of CD23 on D3 cells is not affected by preincubation with LMW-BCGF. Neither B10 or D3 cells respond to rIL-1, rIL-2, rIL-4, rIL-6, rTNF-alpha/beta, rIFN-gamma, or to high m.w. BCGF (Namalwa), alone or in combination. Both clones absorb BCGF activity and crossover absorptions indicate that the clones remove growth factors required by each other. D3 and B10 both appear therefore to respond selectively to LMW-BCGF. These data suggest that the loss of CD23 from a cloned derivative of the cell line JR-2, although accompanied by considerable phenotypic change, is not associated with the disappearance of LMW-BCGF responsiveness, indicating that CD23 is not the essential receptor for LMW-BCGF.

Identification of two distinct allergenic sites on ryegrass-pollen allergen, Lol p IV.

Lol p IV is an important allergen of ryegrass pollen. For the immunochemical identification of antigenic and/or allergenic site(s), murine monoclonal antibodies (MAbs) were prepared against Lol p IV. The hybridoma cell-culture supernatants were screened for anti-Lol p IV antibodies by a combination of ELISA and Western immunoblot analyses. The MAbs were finally purified from ascites on a Mono Q ion-exchange column. In a competitive radioimmunoassay with Lol p IV as the solid phase and 125I-labeled MAbs, it was established that MAbs 90, 91, 92, 93, and 94, although they differed in their relative affinities, recognized in common with one another an epitope designated as antigenic site A, whereas MAb 12 recognized a different epitope referred to as site B. Sites A and B were also demonstrated to constitute allergenic determinants of Lol p IV. Differences in the repertoire of specificities of the human IgE antibodies directed to Lol p IV were also demonstrated. Interestingly, it was found that sera from both allergic as well as from nonatopic individuals had IgG antibodies to sites A and/or B.

Production and characterization of mouse monoclonal antibodies to allergenic epitopes on LolpI (Rye I).

This study describes the production and characterization of mouse monoclonal antibodies (Mab) to LolpI (Rye I), the major allergen of rye-grass pollen. Hybridoma cell lines were screened for the secretion of Mab, the binding of which to labelled LolpI could be inhibited by pooled human sera containing IgE and IgG anti-LolpI antibodies. Twenty monoclonal hybridoma cell lines were expanded, and their product was purified and characterized. Each Mab bound to LolpI but not to unrelated antigens. Cross-inhibition studies allowed the grouping of these 20 Mab into five families differing by their epitope specificity. One representative Mab of each family was tested for its ability to inhibit human IgE/IgG anti-Lolp-I from 15 allergic donors as well as for its inhibition by affinity-purified anti-LolpI antibodies isolated from 14 donors. The results indicated that each family of Mab was specific for an epitope that was identical or close to that recognized by some human anti-LolpI antibodies. It is further shown that human IgE/IgG anti-LolpI are heterogeneous with some patients reacting preferentially with one or two epitopes defined by the Mab LolpI, and others reacting with epitopes different from those identified by the Mab LolpI.

Production and characterization of a monoclonal antibody to biotin.

Monoclonal antibodies to biotin have been prepared by using biotin linked to keyhole limpet haemocyanin (KLH) as the antigen. Spleen cells obtained from mice immunized with biotin-KLH were fused with the myeloma cell line NS-1. The resulting hybridomas were screened for the production of antibodies to biotin using an enzyme-linked immunosorbent assay. Clones producing antibodies to biotin were isolated by limiting dilution methods. Four cell lines, each derived originally from a different fusion, were chosen for the production of monoclonal antibodies. The monoclonal antibodies obtained have been characterized with respect to their ability to interact with biotin, biotin-bovine serum albumin, biotin-KLH and biocytin as well as to inhibit biotin-dependent enzymes. They have been used to produce cellular biotin deficiency in vitro for studies of biotin function.

Immunochemical properties of some monoclonal IgE antibodies to 4-hydroxy-3-nitrophenylacetyl (NP).

Several hybridoma cell lines secreting NP-specific, murine IgE antibodies were generated by fusion of P3-X20 (gamma, kappa) tumour cells with spleen cells from (BALB/c X C57B1/6)F1 (CB6F1) mice previously immunized with NP-ovalbumin. Four subclones (designated NP-epsilon-3.57, NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31) were propagated in vivo and milligram quantities of the corresponding IgE antibodies were purified from ascitic fluid by gel filtration, ion exchange chromatography and affinity chromatography. Immunological analyses and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31 all possessed lambda 1 (or possibly lambda 3) light chains; and that NP-epsilon-3.57 possessed lambda 2 light chains; NP-epsilon-95.31 also expressed the P3-X20 derived, MOPC-21 kappa light chain. Radioallergosorbent test (RAST) titration curves, generated from the interaction of the four monoclonal IgE antibodies with NP-BSA attached to paper discs (NP-BSA-P) were found to be non-overlapping. Measurements of the relative amounts of NP-epsilon-aminocaproic acid (NP-CAP) and 4-hydro-3-iodo-5-nitrophenylacetyl-epsilon-aminocaproic acid (NIP-CAP) that were required to inhibit by 50% the binding of the 4 IgE antibodies to NP-BSA-P indicated that these antibodies were all heteroclitic, since their affinity for NIP appeared to be higher than their affinity for NP. These results, in conjunction with other findings reported in the literature, suggested that the V regions of NP-specific IgE antibodies are similar to the V regions of NP-specific IgM and IgG antibodies, produced by the same mouse strains. Finally, in vitro histamine release measurements demonstrated that two of these monoclonal IgE antibodies could mediate antigen induced histamine release from passively sensitized rat peritoneal mast cells.