Blood and plasma selenium levels and GSH-Px activities in patients with arterial hypertension and chronic heart disease

Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 (No. 175). The new mAb MIL8 (No. 082) and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6.     

Comparison of workshop CD45R monoclonal antibodies with OvCD45R monoclonal antibodies in sheep

The reactivity of the antibovine monoclonal antibodies (mAbs) comprising temporary cluster TC1 was compared with that of two OvCD45R mAbs on sheep cells. Three of the mAbs--CC31, CC99 and CC103--did not cross-react with sheep cells. All the workshop mAbs precipitated two molecules of apparent molecular weight (MW) 200 kDa and 220 kDa while the antisheep CD45R mAb 20-96 precipitated a single band of 220 kDa. Cell surface expression was examined by single colour FACS (fluorescence activated cell sorting) analysis of efferent and afferent lymph cells and peripheral blood lymphocytes and the distribution of the antigens on CD4+, CD8+ and T19+ (WC1) and B cells was determined by two colour fluorescence staining. By cellular distribution and immunohistology the TC1 mAbs could be divided into four distinct groups which differed from a fifth group comprising the two OvCD45R antibodies.     

P-selectin mediates adhesion of the human melanoma cell line NKI-4: identification of glycoprotein ligands

Activated endothelial cells and stimulated platelets express the cell adhesion molecule P-selectin (CD62P), which mediates adhesion to various leukocytes and certain types of cancer cells. In this study, we show Ca2+-dependent binding of P-selectin to NKI-4 cells, a cell line derived from a human melanoma. The binding is inhibited by P7 (a leukocyte adhesion blocking mAb against P-selectin), but not by PL5 (a leukocyte adhesion blocking mAb against P-selectin glycoprotein ligand-1; PSGL-1). Further, expression of PSGL-1 could not be detected on NKI-4 cells by either PL5 mAb or an Ab against a synthetic peptide corresponding to a portion of the PSGL-1 sequence. P-selectin affinity chromatography of lysates from in vivo [3H]-glucosamine-labeled NKI-4 cells resulted in the isolation of three glycoproteins, with apparent molecular masses of approximately 250, approximately 110, and approximately 100 kDa under reducing conditions and approximately 230, approximately 105, and approximately 85 kDa under nonreducing conditions. These molecules could be precipitated by P-selectin, but not by E-selectin. EDTA and the P7 mAb, but not the PL5 mAb, inhibited the binding of P-selectin to the purified ligands. Surprisingly, we found that sodium chlorate, a sulfation inhibitor, did not inhibit the binding of P-selectin to NKI-4 cells and that [35S]-sulfate did not label the NKI-4 cell ligands. We conclude that P-selectin-dependent adhesion of the human melanoma cell line NKI-4 is mediated by a novel class of glycoprotein ligands.     

Evidence that the thyrotropin receptor ectodomain contains not one, but two, cleavage sites

TSH receptor (TSHR) cleavage into two subunits (A and B) was explored using two new mammalian cell lines expressing the recombinant receptor; 1) TSHR-10,000 CHO cells overexpressing the TSHR; 2) TSHRmyc cells with a c-myc epitope inserted at residues 338-349. Immunoprecipitation or immunoblotting of TSHR-10,000 cells with mAb to either the A subunit or the B subunit revealed multiple forms of the TSHR: 1) uncleaved receptors of approximately 115 kDa and approximately 100 kDa with complex carbohydrate and high mannose carbohydrate, respectively; 2) two subunit TSHR with an approximately 62 kDa A subunit containing complex carbohydrate. The A subunit was approximately 35 kDa after enzymatic deglycosylation (predicted C-terminus near residue 330). The nonglycosylated B subunit was evident primarily as an approximately 42 kDa band (predicted N terminus near residue 380). The sum of the A and B subunit polypeptide backbones was smaller than the predicted size of the TSHR, a polypeptide backbone (84.5 kDa), raising the possibility that an approximately 5-kDa polypeptide fragment was excised during intramolecular cleavage. This hypothesis was supported by data obtained with the TSHRmyc cells. Thus, mAb to the c-myc epitope and to amino acid residues 22-35 (mAb A10) were equally effective in detecting the single chain forms of the TSHR in these cells. However, the 35 kDa, deglycosylated A subunit was clearly visible on immunoprecipitation with mAb A10 to the TSHR amino terminus, but not with the anti-myc mAb, indicating loss of the c-myc epitope at residues 338-349. Further, even though the A subunit was not detected in TSHRmyc cells with anti-myc mAb, 125I-TSH cross-linking to the cell surface showed similar A subunit expression in TSHRmyc and wild-type TSHR expressing cells. In summary, our study provides a surprising and novel finding for G protein-coupled receptors. Contrary to the prevailing concept of one cleavage site in the TSHR, we present evidence that there are, in fact, two such sites. The TSHR, like insulin, may release a C peptide during intramolecular cleavage into two subunits.     

Tobacco use among multi-ethnic Latino populations

The equine homologue of the leucocyte integrin LFA-1 (CD11a/CD18) has been characterized using a panel of four monoclonal antibodies (mAbs). The antibodies labelled almost all leukocytes, thymocytes and lymph node cells from normal horses, and immunoprecipitated two noncovalently associated polypeptides with molecular weights of 180 kDa and 100 kDa, respectively. The antigen recognized by one mAb could be precipitated by another in this cluster in a sequential immunoprecipitation assay. The mAbs, however, did not block the activities on lymphocyte function of one another. A mAb to the beta subunit of human LFA-1 cross-reacted with equine LFA-1, but an antibody to its alpha subunit did not, suggesting that the beta subunit of the leukocyte integrin may be more highly-conserved. Functionally, H20A and a human CD18 antibody (MHM23) inhibited phorbol ester-mediated homotypic lymphocyte aggregation, whereas mAb CZ3.2 induced rather than inhibited the homotypic cell aggregation. The formation of lymphocyte aggregates induced by CZ3.2 was not blocked by the inhibitory antibodies H20A or MHM23. CZ3.1 seemed to have little inducible or inhibitory effects on homotypic cell aggregation. The mAb CZ3.1 defined a unique LFA-1 determinant present on granulocytes, but absent on lymphocytes in members of an extended horse family, in contrast to the other antibodies which labelled both granulocytes and lymphocytes from these animals. In all other horses tested, no differences in reactivity of CZ3.1 and the other LFA-1 antibodies were observed when the antibodies were tested on lymphocytes or granulocytes. Our results indicate that common epitopes are shared' between human and equine LFA-1, and that the described panel of monoclonal antibodies identifies distinct determinants present on the equine LFA-1 molecule. The following monoclonal antibodies used in this study were given official workshop designations at the Second International Workshop on Equine Leukocyte Antigens (Lunn et al., 1998) CZ3.1 (Cor) = W45; CZ3.2 (Cor) = W77.     

Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies

To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4-binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb.     

Inhibition of natural killer cell cytotoxicity by cell growth-related molecules

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho-1, we have found inhibitory non-MHC class I cell surface molecules that are noncovalently-associated with 200 kDa and 40 kDa antigens. Poly I-C-induced rat NK cells were not cytotoxic to rat fetus-derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H-ras oncogene-induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho-1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho-1 antigens were also expressed on other NK-resistant lines, such as mouse BALB3T3 fibroblast, EL-4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK-sensitive mouse YAC-1 and H-ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN-gamma clearly induced the cell surface expression of Cho-1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho-1 antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK-resistant WFB as well as HEPM cells with F(ab')2 fragments of mAb Cho-1 resulted in the acquisition of susceptibility to NK cytolysis. Cho-1 antigens may be novel molecules that regulate the NK resistance of cells.     

The fibrinogen RIBS-I epitope (gamma373-385) appears proximate to the gamma408-411 adhesive domain but is not involved in interaction between receptor-bound or surface-adsorbed fibrinogen and platelet GPIIbIIIa

The carboxyl terminus of the fibrinogen (Fg) gamma chain (gamma400-411) is necessary and sufficient to support platelet aggregation and adhesion. However, a monoclonal antibody (mAb) to the Fg RIBS-I epitope (gamma373-385), the anti-Fg-RIBS-I, which binds only to platelet-bound or surface-adsorbed Fg but not soluble Fg, inhibits platelet aggregation. In this study, we showed that this same antibody also inhibits the adhesion of platelets to Fg-coated polystyrene beads. We then investigated the mechanisms by which the anti-Fg-RIBS-I antibody inhibits platelet aggregation and adhesion. The Fg RIBS-I epitope does not interact with platelet GPIIbIIIa, since recombinant Fg missing the last four amino acids, the Ala-Gly-Asp-Val, on the carboxyl terminus of its gamma chains supports neither platelet aggregation nor adhesion to surfaces, nor GPIIbIIIa binding, while it binds anti-Fg-RIBS-I normally. Purified, soluble GPIIbIIIa (265 kDa) inhibits the binding of both the anti-Fg-RIBS-I and 4A5 (a mAb specific to gamma408-411 of Fg), however, peptide G13 (1.5 kDa), corresponding to the Fg gamma chain binding domain on GPIIba (GPIIb300-312), only inhibits the binding of 4A5, and does not affect the binding of the anti-Fg-RIBS-I to Fg. The anti-Fg-RIBS-I reduces the on-rate of the 4A5 binding to Fg with no measurable changes in the dissociation of the Fg-bound 4A5. These data indicate that the inhibition of platelet aggregation and adhesion by the anti-Fg-RIBS-I antibody is due to the steric hindrance of the Fg gamma400-411 to platelet GPIIbIIIa. Thus the Fg RIBS-I epitope (gamma373-385) does not appear to be involved in direct interaction with platelet GPIIbIIIa, leaving the gamma408-411 of Fg as the sole domain mediating platelet aggregation and adhesion.     

Compromised bone density 11.4 years after diagnosis of anorexia nervosa

In this study we present data on a novel cell surface antigen recognized by monoclonal antibody (mAb) VPM30, originally thought to recognize only bovine and ovine sIg+ B cells from peripheral blood. Here we show that the antigen, molecular mass 28 kDa, is not only found in B cell follicles in frozen sections, but when used on paraffin sections VPM30 specifically stains B cells in the light zone of germinal centers but not in the mantle or dark zones. In addition we show that the antigen is also expressed by 90% of T cells after activation, with kinetics of antigen expression mirroring those of proliferation. By both size and distribution, the antigen appears to be novel, corresponding to no known cluster of differentiation, and will be of great use in the study of ruminant cellular immune responses.     

Identification of two distinct populations of dendritic cells in afferent lymph that vary in their ability to stimulate T cells

Immunofluorescent staining and flow cytometric analysis of dendritic cells from cattle afferent lymph has established that within the afferent lymph veiled cells (ALVC) there are two phenotypically distinct, major populations. One is CD11a+, CD5+, CD21- and expresses the bovine WC10 (workshop cluster 10) molecule and the Ag recognized by mAb CC81 but is not recognized by mAbs CC149 and IL-A24. The second ALVC subpopulation is CD11a-, CD5-, CD21+/-, workshop cluster 10- and is not recognized by mAb CC81 but is recognized by mAb CC149. Thus, the two populations, which can be identified by staining for CD11a, are defined by the differential expression of a number of Ag. The ALVC populations had differing capacities to stimulate T cells. CD11a- ALVC were more effective at stimulating proliferative responses in allogeneic CD4+ T cells and CD8+ T cells. This was not related to binding of CTLA4Ig or CD40L fusion proteins, implying similar levels of expression of their ligands, CD80 and CD86 or CD40. Both subsets were able to present OVA to resting memory CD4+ T cells, indicating that both were able to take up and process soluble native protein. In contrast, the CD11a- ALVC were more effective in presenting respiratory syncytial virus Ag to resting CD4+ T cells. Considering the central role of dendritic cells in the initiation of immune responses in naive animals, the two cell types may have different roles in the induction of primary responses induced following infection or immunization.     

Antiinflammatory effect of tepoxalin: blood and synovial tissue studied in patients with knee arthrosis

The role of carbohydrates in embryo implantation in the mouse was investigated using an embryo transfer model and a blastocyst-uterine epithelial cell co-culture system. The monoclonal antibody (mAb) AH6 directed to LeY oligosaccharide (Fuc alpha1-2 Gal beta 1-4 [Fuc alpha1-3] GlcNAc) and other three mAbs directed to carbohydrates whose structures are closely related to LeY were used to show the effect of carbohydrate specificity on implantation. In the embryo transfer model, donor blastocysts (4 days post-coitus) were pretreated with mAb AH6 (experimental) or other mAbs (control) and transferred into one uterine horn of a recipient. The implantation rate was checked after 5 days. Implantation was significantly inhibited by mAb AH6 pretreatment, and inhibition was not observed in control groups. In the co-culture system, the attachment and outgrowth rate of blastocysts on the surface of uterine epithelial cells was significantly inhibited when monolayer epithelial cells or blastocysts were pretreated with mAb AH6. The most obvious effect of mAb AH6 was obtained during 2-4 h co-incubation. No inhibition was observed in the control groups. It was, therefore, concluded that oligosaccharide LeY recognized by mAb AH6 plays an essential role at the initial stage of implantation. It may act as a mediator molecule for adhesion between the surface of blastocyst and epithelial cell, and its function is carbohydrate-specific.     

Characterization of murine monoclonal anti-endothelial cell antibodies (AECA) produced by idiotypic manipulation with human AECA

The IgG fraction of human anti-endothelial cell antibodies (AECA) obtained from a patient with Wegener's granulomatosis was used as immunogen to raise AECA mAb in mice selected among those which developed vasculitis-like lesions after immunization. Three mAb (BGM, 3C8 and 7G2), selected by cyto-ELISA and flow cytometry analyses, featured a specific reactivity with human umbilical vein endothelial cells (HUVEC) and the mouse endothelial cell line H5V; on the contrary, HEp2 cells, the murine melanoma B16 cell line, the extracellular matrix as well as several other antigens tested were not recognized. BGM mAb, an IgG3 precipitating a 70 kDa structure from HUVEC, was able to induce endothelial cells to secrete amounts of IL-6 significantly higher than irrelevant controls or mAb binding different endothelial antigens (i.e. CD31, CD29, ICAM-1 and HLA class I). BGM mAb induced significant levels of antibody-dependent cell cytotoxicity (13 +/- 2.5 versus 0.6 +/- 0.03%). To the best of our knowledge, BGM is the first murine mAb specific for human endothelial cells generated by idiotypic manipulation; secondly, its biological properties further support the notion of a pathogenic role for AECA in autoimmune-mediated diseases.     

An anti-digoxin monoclonal antibody seems to express more than one functional paratope

An anti-digoxin monoclonal antibody (mAb 4G3) has been produced and characterized with respect to its fine specificity and affinity. In an independent series of experiments anti-idiotypic monoclonal antibody (mAb 7G9) was selected which reacted with the antigen-binding center of an anti-human chorionic gonadotropin monoclonal antibody (anti-hCG mAb 1B10). In detailed studies on its binding characteristics it has been shown that mAb 4G3 binds to an anti-idiotypic monoclonal antibody mAb 7G9 in solution. Western blotting experiments showed that mAb 4G3 reacted against antiidiotypic antibody under non-reducing conditions, only. Moreover, mAb 4G3 has been shown to express self-binding properties. Absorption with saturating amounts of its specific hapten, i.e. digoxin, did not change the binding of mAb 4G3 to anti-idiotypic antibody and its self-binding ability. It is speculated on the basis of these data that mAb 4G3 possesses more than one functional paratope.     

Long-lasting complete inhibition of human solid tumors in SCID mice by targeting endothelial cells of tumor vasculature with antihuman endoglin immunotoxin

In the present study, we developed an antitumor immunoconjugate that appears to be promising as a novel curative antitumor agent against a variety of human solid tumors. We generated a new antihuman endoglin (EDG) monoclonal antibody (mAb) K4-2C10 (or termed SN6f) that cross-reacts with mouse endothelial cells. Such cross-reactive anti-EDG mAbs have not been reported previously. This mAb was used to target tumor-associated vasculature in SCID mice inoculated with human tumors. No anti-EDG mAb or its immunoconjugates have previously been successfully used for targeting vasculature in vivo. In this study, MCF-7 human breast cancer cells were inoculated s.c. into SCID mice. K4-2C10 did not react with the MCF-7 cells but showed a weak reactivity with mouse endothelial cells. The mAb reacted with the proliferating endothelial cells more strongly than with the quiescent endothelial cells. The mAb exhibited much stronger reactivity (>10-fold) with human endothelial cells than with mouse endothelial cells and reacted strongly with vascular endothelium of tumor-associated blood vessels in a variety of human malignant tissues. Conjugates of K4-2C10 with ricin A chain (RA) and deglycosylated ricin A chain (dgRA) showed a weak but specific cytotoxic activity against murine endothelial cells in vitro; the 50% inhibitory dose of the RA and dgRA conjugates was 54 nm and 29 nm, respectively. Remarkable antitumor efficacy was observed when a small amount (a total of 60 microgram corresponding to 24% of the LD50 dose) of the dgRA conjugate was administered i.v. into SCID mice that had been inoculated s.c. with MCF-7. Unconjugated mAb K4-2C10 was not significantly effective in the inhibition of the tumor growth. The immunotoxin (IT) completely inhibited growth of the tumor in all of the treated mice (n = 8). Furthermore, similar antitumor efficacy was observed when the IT was administered i.v. into the tumor-inoculated SCID mice that had been pretreated with unconjugated K4-2C10 to block the potentially available weak binding sites of normal tissues. The strong therapeutic effects of the IT were reproduced in another set of therapeutic experiments. No significant side effects were observed in the mice. The differences in the tumor growth between the control group and the IT-treated groups were statistically significant. The IT showed antiangiogenic activity in the dorsal air sac method. The results indicate that K4-2C10 IT effectively treated the tumor-bearing mice by selectively inhibiting the tumor-associated blood vessels and by disrupting tumor-associated angiogenesis. The strong antitumor efficacy of the K4-2C10 IT is remarkable in view of the fact that K4-2C10 and its IT showed only a weak reactivity with mouse endothelial cells, and a relatively small amount of the IT was administered i.v. to treat s.c. tumors. We anticipate that the K4-2C10 IT will show much stronger antitumor efficacy and antiangiogenic activity in patients with solid tumors and other angiogenesis-associated diseases. The present results demonstrate for the first time that an anti-EDG mAb or its immunoconjugate can effectively target tumor-associated vasculature in vivo.     

Progressive hearing loss in hard-of-hearing children

Roles of CD8+ and CD4+ cells on lethal graft-versus-host disease (GVHD) were investigated. Injection of spleen cells from C57BL/6 (B6) female mice into (BALB/c x B6)F1 nu/nu female mice caused subacute lethal GVHD (survival: 10-50 days). Injection of anti-Lyt-2.2 (CD8) monoclonal antibody (mAb) on days zero, four and 14 into recipient mice prolonged their survival for at least the 200-day observation period. Injection of anti-L3T4 (CD4) mAb also prolonged survival of the mice for more than 70 days, but they eventually died by 150 days. Pretreatment of the donor B6 spleen cells with anti-Lyt-2.2 (CD8) mAb and complement (C) prevented the development of GVHD, and their pretreatment with anti-L3T4 (CD4) mAb and C markedly prolonged the survival of recipient mice. Injection of a mixture of donor spleen cells pretreated with anti-Lyt-2.2 (CD8) mAb and C and those pretreated with anti-L3T4 (CD4) mAb and C induced subacute lethal GVHD. Injection of anti-L3T4 (CD4) mAb, but not anti-Lyt-2.2 (CD8) mAb on days five, nine and 14 prolonged survival of the recipient mice. These results indicated that the collaboration of CD8+ cells and CD4+ cells was necessary for induction of subacute lethal GVHD. CD4+ cells but not CD8+ cells were involved in mediating subacute GVHD from the onset of the disease. CD8+ cells were, however, capable of inducing late-onset lethal GVHD. Direct phenotyping of T cells in the recipient mice revealed that the CD4+ cells were incapable of repopulating without CD8+ cells, but that CD8+ cells were capable of repopulating without CD4+ cells.     

Differential effects of antibodies to vascular cell adhesion molecule-1 and distinct epitopes of the alpha4 integrin in HgCl2-induced nephritis in Brown Norway rats

Four distinct epitopes (A, B1, B2, and C) have been functionally defined on the human alpha4 integrin. In this study, two cross-reactive antihuman alpha4 monoclonal antibodies (mAb) (HP2/1 and HP2/4 specific for epitopes B1 and B2, respectively) were used to functionally characterize the rat VLA-4 subunit and to define similar functional epitopes in this rodent species. It was found that B1 and B2 anti-alpha4 mAb completely block adhesion to fibronectin, but the inhibition of adhesion to vascular cell adhesion molecule-1 (VCAM-1) with HP2/1 mAb was lower than with HP2/4 mAb. It was also observed that epitope B2 HP2/4 mAb induced homotypic aggregation in rat lymphocytes, whereas epitope B1 HP2/1 mAb did not. Using the HgCl2 model of nephritis, this study shows the protective effect of both anti-alpha4 mAb against infiltration of the renal interstitium by leukocytes. Nevertheless, HP2/1 mAb, but not HP2/4 mAb, virtually abolished the anti-glomerular basement membrane antibody synthesis and glomerular deposits. These findings indicate the dual but independent role played by alpha4 integrins in both extravasation of leukocytes and in the production of antibodies. Finally, this study demonstrates that anti-rat VCAM-1 mAb showed a positive reactivity of the renal vascular endothelium and, most importantly, that administration of anti-VCAM-1 antibodies completely abrogated the interstitial cell infiltrates without affecting anti-glomerular basement membrane antibody production. These results confirm the important role played by VLA-4/VCAM-1 pathway in leukocyte infiltration, and further support the dual and independent role of alpha4 integrins in both renal infiltration and autoantibody synthesis in this model of renal disease.     

Expression of a recombinant human growth hormone binding protein in baculovirus/insect cell system

An eucaryotic recombinant human growth hormone binding protein (rGHBP) was expressed in baculovirus-infected insect cells and purified by affinity chromatography from culture supernatant. This mannose-rich 34-kDa protein specifically bound human growth hormone (hGH) with the same affinity (kDa = 0.42 x 10(-9) M) than the 51.5 kDa GHBP we purified and characterised from human plasma (kDa = 1.1 x 10(-9) M). A high molecular form of the rGHBP was detected by silver-stained SDS-PAGE, Western blot (mAb 263), affinity cross-linking and Western ligand blot with 125I-hGH. Reduction experiments with beta-mercaptoethanol suggested that this form involved a disulfide bound between two rGHBPs.     

Epitope mapping of T4 endonuclease VII with monoclonal antibodies reveals importance of both ends of the protein for target binding

Endonuclease VII (endo VII) of bacteriophage T4 is a Holliday-structure resolving enzyme that can also recognize many other defects in DNA via an altered secondary structure. The protein has a molecular mass of 18 kDa and exists as a dimer in solution. Here we report the production and characterization of monoclonal antibodies (mAbs) directed against the highly purified enzyme. From one fusion 15 hybrid cell lines producing mAbs with high affinity for endo VII could be established. The mAbs were used for epitope mapping of the protein by using N-terminal, C-terminal and internal peptides of endo VII as antigens in enzyme-linked immunoabsorbant assays. Three classes of mAbs were distinguished as follows: (1) the predominant class with 13 mAbs recognized a C-terminal epitope located between amino acid residues 115 and 145; (2) a second class, represented by one mAb, recognized an epitope located at the N terminus between amino acid residues 16 and 65; (3) a third class, represented by one mAb, recognized an epitope built from nearly the entire native protein including amino acid residues from the C and N terminus of endo VII. The latter finding suggests close proximity of the two ends, which are provided apparently by the same monomer, since the mAb from class III does also react with a mutant protein deficient in dimerization. Internal sequences of endo VII between amino acid residues 78 and 145 did not react with any of the mAbs.     

Definition of the specificity of monoclonal antibodies against porcine CD45 and CD45R: report from the CD45/CD45R and CD44 subgroup of the Second International Swine CD Workshop

Swine cell binding analyses of a set of 48 monoclonal antibodies (mAbs), including eleven standards, assigned to the CD44 and CD45 subset group of the Second International Swine CD Workshop yielded 13 clusters. Although none of these corresponded to CD44, seven mAbs formed a cluster which was identified as being specific for restricted epitopes of CD45 (CD45R). In addition, a T-cell subset specific cluster comprised of four mAbs was also identified. Two mAbs (STH106 and SwNL 554.1) reacted exclusively with CD8 bright lymphocytes, the other two (2B11 and F01G9) with a subset of CD4 lymphocytes. The other 10 clusters were either specific for MHC-class I like molecules or overlapped with clusters identified by the adhesion molecule subgroup and are therefore just briefly discussed in this report. The specificity of all the mAbs in the CD45R cluster was verified by their ability to immunoprecipitate distinct proteins and to react with CHO cells expressing individual isoforms of CD45. Three CD45R mAbs (3a56, MIL5, -a2) did react with a 210 kDa isoform(s), while another three (STH267, FG2F9, 6E3/7) only recognized a 226 kDa isoform(s). The remaining one (MAC326) precipitated both a 210 and 226 kDa protein. The specificity of all the mAbs in the CD45R cluster, and of the CD45 common mAbs, was confirmed by their reactivity with CHO cells transfected with cDNAs encoding the extracellular and transmembrane portions of distinct CD45R isoforms. Those mAbs recognizing a 210 kDa protein reacted with CHO cells expressing the CD45RC isoform, while those capable of precipitating a 226 kDa, but not the 210 kDa, polypeptide recognized CHO cells expressing either the CD45RAC and the relatively rare CD45RA isoform. MAC326 was unique in its inability to react with CHO cells engineered to produce the CD45RC and CD45RAC isoforms. Thus, three mAbs (6E3/7, STH267, and FG2F9) appear to be specific for an epitope(s) encoded by the A exon, while one (MAC326) recognizes a determinant encoded by the C exon. The remaining three mAbs (3a56, -a2, MIL5) are apparently specific for an epitope(s) which results from the fusion of the C exon to the invariant leader sequence and is destroyed by inclusion of the A exon. All three CD45 common mAbs, K252.1E4, MAC323 and 74.9.3, did react with the CHO cells lines expressing either the CD45RA, CD45RC, CD45RAC or CD45RO isoforms, but not with untransfected CHO cells. When the natural expression of CD45 isoforms was examined by reacting lymphocytes with CD45R mAbs, a high level expression of isoforms containing the A exon-generated domain was detected in all B cells while the majority of CD4+ T cells had undetectable or lower expression density of this protein than B cells. In contrast, the density of expression of the CD45 isoform(s) containing the C exon-generated domain ranged from undetectable to high in CD4+ T cells whereas the amounts were approximately ten-fold lower in B cells. Overall this panel of CD45 mAbs will be very useful in analyzing the maturation and differentiation of swine lymphoid cells subsets.     

Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody, LL2

LL2 is a murine monoclonal antibody IgG2a reactive with B cells and non-Hodgkin's B-cell lymphoma, which, in a radioiodinated form, induces responses in lymphoma patients [Goldenberg et al. (1991) J Clin Oncol 9:548-564]. In this report we identify LL2 as a member of the CD22 cluster. The molecular size of the antigen, its expression profile, and competitive blocking studies were used to establish this identification. By Western blot analysis and immunoprecipitation studies using the Raji Burkitt's lymphoma cell line metabolically labelled with [3H]leucine, the LL2 antigen was determined to correspond to a molecular mass of 140 kDa. The molecular mass of the LL2 antigen, and the B-cell-restricted reactivity of the LL2 antibody, were consistent with both the CD21 and CD22 clusters. To assess additional similarities and differences between LL2 and anti-CD22 and anti-CD21, the binding of these mAb to cultured cell lines, Nalm-6 and Molt-4, was compared by flow cytometry. The binding profile of LL2 on these cell lines was consistent with anti-CD22, but not anti-CD21. Sequential immunoprecipitation and cross-blocking studies with anti-CD22 monoclonal antibodies recognizing established CD22 epitopes were performed to confirm that LL2 reacts with CD22 and to determine which epitope LL2 recognizes. Binding of 131I-LL2 to Raji cells is inhibited over 90% by prior incubation of the target cells with unlabelled RFB4, indicating that LL2 belongs to the same epitope group as RFB4, i.e., epitope B.