Interleukin 6 Influences Germinal Center Development and Antibody Production via a Contribution of C3 Complement Component

Mice rendered deficient for interleukin (IL) 6 by gene targeting were evaluated for their response to T cell–dependent antigens. Antigen-specific immunoglobulin (Ig)M levels were unaffected whereas all IgG isotypes showed varying degrees of alteration. Germinal center reactions occurred but remained physically smaller in comparison to those in the wild-type mice. This concurred with the observations that molecules involved in initial signaling events leading to germinal center formation were not altered (e.g., B7.2, CD40 and tumor necrosis factor R1). T cell priming was not impaired nor was a gross imbalance of T helper cell (Th) 1 versus Th2 cytokines observed. However, B7.1 molecules, absent from wild-type counterparts, were detected on germinal center B cells isolated from the deficient mice suggesting a modification of costimulatory signaling. A second alteration involved impaired de novo synthesis of C3 both in serum and germinal center cells from IL-6–deficient mice. Indeed, C3 provided an essential stimulatory signal for wild-type germinal center cells as both monoclonal antibodies that interrupted C3-CD21 interactions and sheep anti–mouse C3 antibodies caused a significant decrease in antigen-specific antibody production. In addition, germinal center cells isolated from C3–deficient mice produced a similar defect in isotype production. Low density cells with dendritic morphology were the local source of IL-6 and not the germinal center lymphocytes. Adding IL-6 in vitro to IL-6–deficient germinal center cells stimulated cell cycle progression and increased levels of antibody production. These findings reveal that the germinal center produces and uses molecules of the innate immune system, evolutionarily pirating them in order to optimally generate high affinity antibody responses.     

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15–25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80–150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20°C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.     

Immunology: Diversity of the blocking effects of antisperm antibodies on fertilization in human and mouse

The blocking effects of complement-dependent sperm immobilizingantibodies in the sera of infertile women and monoclonal antispermantibodies against humans and mice on fertilization were investigated.The hemizona assay (HZA) and sperm penetration assay (SPA) wereused to study the inhibitory effects of sera from 22 infertilepatients positive for sperm immobilizing antibodies. Use ofthese tests allowed us to differentiate whether the antibodyblocked sperm—zona pellucida tight binding and/or spermpenetration into the ooplasm. The zona pellucida penetrationassay (ZPA) was also used to study the effects of four monoclonalantibodies (mAbs) on human sperm penetration into the zona pellucida.Seven mAbs against murine spermatozoa were tested for theirinhibitory effects on in-vitro fertilization (IVF) and HZA inmice. Of 22 patient sera with sperm immobilizing antibodies,21 (95.5%) inhibited HZA attachment and penetration, whereasthis did not occur in any of 13 patient sera without these antibodies.However, 19 of 22 (86.4%) patient sera with sperm immobilizingantibodies and eight of 13 (61.5%) patient sera without theseantibodies inhibited the SPA. Two (2C6, 1G12) of four mAbs againsthuman spermatozoa showed strong inhibitory effects in all theassays (HZA, ZPA and SPA). One mAb (3B10) did not inhibit HZAbut blocked ZPA and SPA. Another mAb (H6-3C4) seemed to haveno inhibitory effects on fertilization. Two (Vx 5 and Vx 8)of seven mAbs against murine spermatozoa inhibited IVF in micebut did not block mouse HZA. These findings suggest that antispermantibodies block fertilization at specific stages. Some of themmay inhibit sperm capacitation and thus prevent all processesof fertilization that follow. Some other antibodies may notaffect capacitation and sperm binding to zona pellucida butinhibit the acrosome reaction, followed by the blocking of spermpenetration through zona pellucida and ooplasm.     

Binding of cardiolipin to polystyrene beads: evidence for a lamellar phase orientation

Summary. The association of cardiolipin with polystyrene beads was studied using ³¹P-NMR and electron microscopy. In the presence and absence of fetal calf serum, cardiolipin appeared to bind to the polystyrene beads in lamellar phase as assessed by ³¹P-NMR imaging. Electron microscopic analysis revealed an even coating of phospholipid about the beads with extensive micelle binding. Cardiolipin-coated beads challenged with ACA-positive sera followed by immunogold indicated antibody bound to micelles associated with the bead. Studies conducted with ACA IgG purified from patient sera indicated that some ACA bound to CL beads in the absence of a source of ACA cofactor (i.e. gelatin-blocked beads), some ACA required β₂-GPI for binding (i.e. no binding in the presence of β₂-GPI-depleted plasma), whereas other ACA which showed negliglible binding with gelatin-blocked beads, showed enhanced binding in the presence of /?₂-GPI-depleted plasma. The data indicate that: (1) cardiolipin binds to polystyrene beads in lamellar phase, (2) ACA bind to phospholipid micelles bound directly to the polystyrene beads, and (3) ACA differ between individuals displaying varying phospholipid and phospholipid/cofactor substrate specificities.     

Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis and cell division in normal glia. At least half of malignant human gliomas (MHG) express high levels of the EGF receptor (EGFR), which are above those detected in normal brain. The demonstration that antibodies against the EGFR inhibit the growth of squamous cell carcinoma line A-431, with large numbers of EGFR, in vitro and in vivo raises the possibility that these agents could be used therapeutically against malignant human gliomas either alone or conjugated to other agents. We have measured the growth effects of EGF and an anti-EGFR monoclonal antibody, 528 (Ab-528), on four well-characterized human malignant glioma cell lines, D-263 MG, D-247 MG, U-343 MGa Cl 26, and D-37 MG, with 2.9×10⁴, 1.5×10⁵, 8.6×10⁵ and 1.59×10⁶ EGFRs per cell, respectively. EGF significantly increased cell number in D-263 MG and D-37 MG by 65% and 74%, respectively, had no effect on D-247 MG, and significantly decreased cell number in U-343 MGa Cl 26 by 39%. U-343 MGa Cl 26 growth was inhibited 19% by Ab-528, but Ab-528 had no effect on growth of the other MHG lines. Ab-528 significantly inhibited all EGF-mediated growth effects. These studies demonstrate that, although Ab-528 alone has little antiproliferative activity on MGH, it successfully competes with EGF to reduce the biological effects of EGF-EGFR binding. Therefore, this antibody could potentially be used to target radioisotopes to MHG via the EGFR for diagnosis and therapy.Supported by Grants CA-11898, NS-20023, CA-43722, and the Association for Brain Tumor Research (MHW, PAH)     

猪卵透明带单克隆抗体的研制

用热溶猪卵透明带抗原免疫BALB/C 小鼠,取其脾细胞和SP2/O 细胞融合产生的杂交瘤。用间接免疫荧光和ELISA 方法筛选,经四次克隆化后,获得三株分泌猪卵透明带单克隆抗体杂交瘤细胞株(LPD_8,LPC_4,LPD_2)。通过间接免疫荧光试验,其中二株单克隆抗体(LPD_8,LPC_4)与人卵透明带有交叉反应。但它们在猪与人卵透明带上呈现的荧光部位有明显的不同,LPD_8位于整个透明带上,而LPC_4只见于透明带外层。LPD_2荧光位于透明带内层且与人卵无交叉反应。我们认为这种差别是由于抗原决定簇的不同所致。     

Histochemical study of pituitary adenomas with Ki-67 and anti-DNA polymerase α monoclonal antibodies,bromodeoxyuridine labeling,and nucleolar organizer region counts

Summary The growth potential of 65 pituitary adenomas was determined by histochemical analysis with Ki-67 and anti-DNA polymerase monoclonal antibodies, bromodeoxyuridine (BrdUdR) labeling, and counts of argyrophilic nucleolar organizer regions (Ag-NORs). The mean proliferating cell indices (PCIs) determined by Ki-67 and anti-DNA polymerase and the BrdUdR labeling index (LI) were generally very low [1.0±0.2%, 1.1±0.2%, and 0.5±0.1% (±SE), respectively]. Apart from adrenocorticotropic hormone-positive adenomas, which had significantly higher indices, there were no statistically significant differences in the indices among the other subtypes of pituitary adenomas. Recurrent tumors had higher Ki-67 and DNA polymerase PCIs and BrdUdR LIs (3.6%, 4.2%, 1.4%) than primary tumors (0.8%, 0.8%, 0.3%; P<0.005). The number of Ag-NORs did not correlated significantly with any of the three indices. The mean number of Ag-NORs was higher in nonfunctioning adenomas than in functioning adenomas (2.04 vs 1.66, P<0.005); among prolactin-positive adenomas, those treated preoperatively with bromocriptine had more Ag-NORs than untreated tumors (1.75 vs 1.57, P<0.005). These results suggest that the Ki-67 and DNA polymerase PCIs and the BrdUdR LI predict the growth potential of individual pituitary adenomas, whereas the number of Ag-NORs appears to correlate with hormone production rather than with the proliferative potential.Supported by grants CA-13525 and CA-50210 from the National Cancer Institute, by a grant from the Phi Beta Psi Sorority, and by Grant-in-Aid A 63771083 from the Ministry of Education, Science, and Culture of Japan     

GD3 expression by cultured human tumor cells of neuroectodermal origin

Summary Seven monoclonal antibodies (mAbs) reactive with ganglioside II³(NeuAc)₂-LacCer (GD3) were generated; four of these mAbs (DMAb-21, DMAb-22, DMAb-23, and DMAb-24) by immunizing mice with GD3 adsorbed to Salmonella minnesota and the remaining three (DMAb-7, DMAb-8, and DMAb-17) with melanoma line SK-MEL 28, which contains 1.4 nmol sialic acid of GD3 per mg protein. The specificities of the mAbs were defined by high-performance thin-layer chromatography (HPTLC) immunostain and solid-phase radioimmunoassay (SP-RIA) with a panel of purified gangliosides. DMAb-7 and DMAb-8 reacted with GD3, IV³(NeuAc)₂nLcOse₄Cer(3,8-LD1), and very weakly with IV³(NeuAc)₂II³NeuAc-GgOse₄Cer (GTla), but not with II³NeuAc-LacCer (GM3), II³NeuAcGgOse₃Cer(GM2), II³NeuAc-GgOse₄Cer(GM1), II³NeuAc, IV³NeuAcGgOse₄Cer (GD1a), II³(NeuAc)₂GgOse₃(GD2), II³(NeuAc)₂GgOse₄Cer (GD1b), IV³NeuAcII³(NeuAc)₂, GgOse₄Cer(GT1b), suggesting the binding epitope to be a terminal tetrasaccharide NeuAc2-8NeuAc2-3Gal1-4(Glc or GlcNAc). DMAb-7 and DMAb-8 were used to investigate the expression of GD3 on cultured human tumor cells of neuroectodermal origin. Thirteen of 19 gliomas, 3 of 5 medulloblastomas, 5 of 5 neuroblastomas, 2 of 2 melanomas, and 1 of 3 teratomas were shown to react with DMAb-8 and/or DMAb-7 by cell surface-RIA (CS-RIA) and immunofluorescence (IF) assays. HPTLC and densitometric analysis confirmed these results, as positive immunostains in the GD3 region were obtained with oligoganglioside fractions from 9 glioma, 1 medulloblastoma, 2 neuroblastoma, 1 melanoma, and 1 teratoma cell line. Glioma cell line U-105 MG and medulloblastoma cell line Daoy contain GD3 as shown by HPTLC immunostain analysis of extracts, although GD3 was undetectable on the cell surface as determined by CS-RIA and IF. There was no detectable GD3 found in gangliosides isolated from cell lines U-373 MG, D-54 MG, TE-671, and PA-1, which were negative for both DMAb-7 and DMAb-8 by CS-RIA and IF assay. Our results provide evidence that GD3 is expressed extensively with significant quantitative heterogeneity on cultured human neuroectodermal tumor cells including glioma, medulloblastoma, neuroblastoma, and melanoma.Supported by NIH grants R37 CA11898, NS 20023, and CA32672 and by grants from the Swedish Medical Research Council (project no. 03X-627), Swedish Cancer Society (project no. 2260-B88-01X) and the National Swedish Board for Technical Development (project no. 84-4667)     

Phenotypic differences within the AB blood type of the feline AB blood group system

Flow cytometry immunolabeling, tube agglutination tests, and thin-layer chromatography immunostaining with two different anti-A monoclonal antibodies (anti-A mAb1 and anti-A mAb2) and one anti-B mAb were used to demonstrate differences in expression of the A and B antigens among erythrocytes from type A and four different type AB cats. Although the flow cytometric patterns of reactivity and agglutination scores for erythrocytes from types A and B cats detected with the anti-A and anti-B mAbs were consistent, reactivity among erythrocytes of different type AB cats was variable. By flow cytometric analysis, 99.9% of type A erythrocytes, no type B erythrocytes, 2.5–4.0% of erythrocytes from type AB cats 1, 3, and 4, and 60.7% of erythrocytes from type AB cat 2 had detectable A antigen when anti-A mAb1 was used. In contrast, 86.4% of type A erythrocytes, no type B erythrocytes, 20.2–38.0% of erythrocytes from type AB cats 1, 3, and 4, and 68.5% of erythrocytes from type AB cat 2 had detectable A antigen when anti-A mAb2 was used. In addition, 86.9% of type B erythrocytes, no type A erythrocytes, 83.1–96.8% of erythrocytes from type AB cats 1, 3, and 4, and 73.0% of erythrocytes from type AB cat 2 had detectable B antigen when the anti-B mAb was used. Agglutination scores of type AB cats were comparable to the percent binding on flow cytometry. Thin-layer chromatography immunostains confirmed differences in the amount of A antigen between erythrocyte glycolipids of type A and AB cats and those of type AB cats 1 and 2. These results suggest that at least two different phenotypes exist within the feline AB blood type, which differ in the amount of A antigen expressed on the erythrocyte surface.     

Variants of lymphoid lines produced with ricin A-chain monoclonal antibody conjugates

Conjugates of ricin A-chain with monoclonal anti-light chain antibodies specifically killed cells hearing kappa or lambda immunoglobulin (Ig) light chains. Exposure of cells from B-lymphoblastoid cell lines (B-LCL) to conjugate for less than 30 h had only a slight effect on cell growth, but on 48 h exposure a marked killing effect was achieved. After recovery of growth, cells were re-exposed to conjugate for 9-14 days. Treatment of cells from the EB4 line (sIgG lambda) in this way yielded 4 variants which showed a marked reduction in levels of surface Ig lambda and secreted Ig lambda with slight, or no, reduction in MHC class II expression and similar growth rates to the parent line. Variant lines retained their phenotype over long periods of culture.