Chain mAb Research Articles

Fate Discrimination of Normal B Cells When Cross-Linked with Anti-IgM Antibodies of Different Affinities and Valence.

The B-cell receptor (BCR) is a key signaling molecule that engages antigens and triggers pathways that induce survival, proliferation, differentiation, anergy, or apoptosis. The “choice” of which functional fate the B cell makes is decided upon by a series of receptor and cellular interactions. The potential for ligand-initiated signal transduction through the BCR is assessed in terms of ligand binding affinity for membrane Ig. We have used a series of anti-IgM H chain mAb that differ in their affinities (HB57 − Ka = 5x108 “high”, Mu53 − Ka = 2x107 “intermediate”, and P24 − Ka = ~2x106 M−1 “low”) in both soluble and immobilized forms as models for antigenic stimulation of the BCR, and assessed the response of normal B cells isolated by negative selection from peripheral blood. Proliferative responses were observed upon cross-linking the BCR with antibodies of all affinities at 10 ug/ml of immobilized antibodies for 72 hours, with a modest stimulation at 1ug/ml and no stimulation at either 0.01 or 0.1 ug/ml. Interestingly, the low affinity antibody P24 showed the highest proliferative response (3.3 fold), while stimulation with the moderate affinity antibody was intermediate (2.2 fold) and the response of the high affinity antibody, was negligible. Soluble antibodies did not stimulate proliferation at any concentration tested. When B cells were co-incubated with soluble anti-IgM antibodies plus IL 4,10ug/ml of either high or intermediate affinity induced proliferation with stimulation indices of 2.5 and 1.9, respectively, while the low affinity antibody still had no effect. Co-incubation with IL 4 and immobilized anti-IgM antibodies induced the same pattern of responses as seen with antibody alone, slightly increasing proliferation induced with 1 ug/ml low and intermediate and markedly increasing stimulation by all three antibodies at 10ug/ml. Our results suggest that even in the absence of T cell help, normal B cells can be activated via the BCR upon cross-linking and the efficiency at which this is accomplished is largely dependent on affinity for the BCR.

Administration of FFP Repletes and Sustains the Megakaryocyte/Platelet-Derived Factor V Pool To Confer Hemostatic Competence in a Factor V-Deficient Individual.

Recent studies by our laboratory have demonstrated unequivocally that factor V is endocytosed by megakaryocytes from plasma to form the platelet-derived factor V pool. In the current study, we have determined the time-dependent acquisition of factor V by platelets in a 67 year old factor V-deficient patient, previously shown to be completely devoid of plasma- and platelet-derived factor V. The patient now receives weekly tranfusions of two units of FFP to prevent gastrointestinal bleeding. Plasma and platelet lysate samples were prepared from fresh, whole blood drawn prior to (0 hrs), and following (2, 6, 24, 96, 168 hrs) patient transfusion. A factor V radioimmunoassay (RIA), which can detect factor V concentrations as low as 0.075 μg/mL, a standard factor V clotting-based activity assay, and/or western blotting analyses, which utilize a mixture of anti-human factor V light chain and heavy chain mAbs, were used to evaluate the appearance, and subsequent disappearance, of factor V in these two blood compartments. Prior to transfusion (t = 0 hr), the patient's plasma-derived factor V could not be detected by any of the three factor V assays. The patient's plasma-derived factor V level peaked immediately following transfusion (t = 2 hr) reaching a concentration of 1.3 +/− 0.08 μg/mL as measured by RIA, and declined until it reached an undetectable level at 96 hrs post transfusion. These data were confirmed by the results of both the clotting-based assays and western blotting analyses. As the patient's platelet-derived factor V levels were below the sensitivity of both the RIA and clotting assay, they were analyzed by western blotting. Such analyses confirmed that subsequent to its endocytosis, the patient's platelet-derived factor V is stored in a partially protelytically activated form similar to that of a control individual. Due to the partially proteolytically activated state of the platelet-derived cofactor, platelet lysates treated with thrombin to activate the factor V to factor Va were used for quantative western blotting. Interestingly, and perhaps consistent with the patient's receipt of weekly FFP transfusions, factor V/Va could still be detected in platelets immediately prior to transfusion. Subsequent to transfusion and in marked contrast to changes in the plasma-derived cofactor pool, a significant increase in the patient's platelet-derived factor V/Va level was not observed until 24 hrs post transfusion. These data are consistent with previous studies demonstrating that platelets do not endocytose factor V. Although the concentration of the platelet-derived factor V/Va pool decreased over the subsequent 6 days, antigen remained detectable even though the plasma-derived pool had been depleted 3 days earlier. These combined observations indicate that, subsequent to FFP administration, the patient's megakaryocytes acquire and proteolytically process plasma-derived factor V normally. Furthermore, the consistent presence of factor V/Va within the patient's platelets is in all likelihood preventing gastrointestinal bleeding in this individual, which supports the concept that platelet-derived factor V represents the hemostatically relevant factor V pool.

HLA Class I Antigen Down-Regulation in Primary Ovary Carcinoma Lesions: Association with Disease Stage

To investigate TAP1, TAP2, and HLA class I antigen expression in primary ovarian carcinoma lesions and to assess the clinical significance of defects in the expression of these molecules. Fifty-one formalin-fixed, paraffin-embedded primary ovarian carcinoma lesions were stained with affinity-purified rabbit anti-TAP1 and anti-TAP2 antibodies and with anti-HLA class I heavy chain monoclonal antibody (mAb) HC-10 using the immunoperoxidase reaction. The results of immunohistochemical staining were correlated with the histopathologic characteristics of the lesions and with patients' survival. Ovarian surface epithelium, thecal cells of follicles, and stromal cells were stained by anti-TAP1, anti-TAP2, and anti-HLA class I antigen xenoantibodies with a homogeneous pattern. In contrast, no staining of lutheinic cells by these antibodies was detected. Forty-one and 32 out of 51 primary ovarian carcinoma lesions were stained by anti-TAP1 and anti-TAP2 xenoantibodies and by anti-HLA class I antigen mAb HC-10, respectively. The staining patterns by anti-TAP1 and anti-TAP2 xenoantibodies were completely concordant, but did not correlate with that by anti-HLA class I heavy chain mAb HC-10. TAP1 and TAP2 expression was associated neither with the histopathologic characteristics of the lesions nor with clinical variables. On the other hand, HLA class I antigen down-regulation was associated with disease stage: the odds ratio of stage III for HLA class I antigen negative patients was 7.6 (95% confidence interval, 1.9-30.5; P= 0.007), whereas for TAP negative patients was 5.1 (95% confidence interval, 0.9-28.4; P = 0.07). Follow up was available for 39 out of the 51 patients. Multivariate analysis showed that both grading and staging were associated with a higher risk of death, whereas TAP and HLA class I antigen phenotypes were not. The lack of association between TAP and HLA class I antigen expression is compatible with the possibility that multiple mechanisms underlie HLA class I antigen down-regulation in primary ovarian carcinoma lesions. The potential role of immunologic events in the clinical course of ovarian carcinoma suggests that the association between HLA class I antigen down-regulation and disease progression may reflect the escape of tumor cells from immune recognition and destruction.

Prolactin affects both survival and differentiation of T-cell progenitors

We have analysed the in vitro effects of prolactin on thymocyte development concluding that PRL favours the survival and differentiation of T-cell progenitors. Fetal, adult thymocytes and CD45 + fetal liver lymphoid progenitors express PRL-R. PRL induces survival, proliferation and differentiation of lymphoid progenitors whereas both an anti-PRL antiserum and an anti-PRL-R mAb block T-cell development accumulating CD25 +DN (CD4 −CD8 −) cells. Furthermore, IL2 rescues the blockade of T-cell development in FTOC treated with anti-PRL antiserum but PRL does not recover cultures treated with an anti-IL2R α chain mAb, which drastically blocks the T-cell development. These results support IL2/IL2R mediation of PRL effects on developing thymocytes.

Kallikrein modification with affinity to IgG present in higher amounts than normal in plasma from patients with Crohn's disease

This study presents immunological and functional evidence to suggest that the activation of prekallikrein (PK) in human plasma yields two modifications of kallikrein. One of these modifications showed amidolytic properties strongly deviating from those registered for the main part of the enzyme. The substrates were S‐2302, Bz‐Pro‐Phe‐Arg‐pNA, S‐2366 and S‐2222. In PAGE immunoblots the PK heavy chain mAb 13G11 was found to detect the kallikrein 85 kD double band and bands with mol. weights of about 152 and 135 kD. Such a 152 kD band could be removed together with an IgG fraction on a Protein G column. In this study the kallikrein identity was confirmed by an estimation of the levels obtained in amidolytic assays of mixtures of normal plasma and plasma deficient in FXI, which in immunological assays showed a PK level of 140–150% of normal. A comparison in amidolytic assays of normal plasma and plasma from patients with Crohn's disease showed that patients' plasma contained a significantly higher level than normal of modified kallikrein. An IgG removal procedure removed all modified kallikrein, and did not affect ordinary kallikrein levels.

Monoclonal antibodies specific for Campylobacter fetus lipopolysaccharides

Four monoclonal antibodies (mAbs) (M1357, M1360, M1823 and M1825) which reacted with Campylobacter fetus lipopolysaccharide (LPS) core region epitopes were produced and characterized. Reactivity of these mAbs with C. fetus core LPS epitopes was determined by enzyme-linked immunosorbent assay (ELISA) with whole cell proteinase K digests and phenol–water extracted LPS, and by immunoblotting with proteinase K digests. The specificities of the four mAbs were evaluated using an indirect ELISA. One of the mAbs reacted with 42 and three of the mAbs reacted with 41 of the 42 C. fetus strains examined. No reaction was observed between the four mAbs and 32 non- C. fetus bacteria tested, with the exception of one mAb with one organism. The four mAbs reacted with serotype A and B strains indicating the presence of shared epitopes in C. fetus LPS core oligosaccharides. The specificities of three mAbs previously produced to C. fetus LPS O-antigens (M1177, M1183 and M1194) were also evaluated and no reaction was observed with these mAbs and the 32 non- C. fetus bacteria tested. Strong immunofluorescence reactions were observed with the anti-O chain mAbs and selected C. fetus strains of the homologous serotype. These anti-LPS core oligosaccharide and anti-LPS O chain mAbs are highly specific for C. fetus and are potentially useful as immunodiagnostic reagents for detection, identification and characterization of C. fetus.

Monoclonal antibody-based enzyme immunoassay for detection of ricin.

A sensitive and specific enzyme-linked immunoadsorbent assay (ELISA) was developed to detect ricin in biological fluids. The assay is based on the sandwich format using monoclonal antibodies (MAbs) of two distinct specificities. An affinity-purified anti-ricin B chain MAb (1G7) is utilized to adsorb ricin from solution and the second anti-ricin A chain MAb (5E11) conjugated with peroxidase is then used to form a sandwich, and peroxidase allows color development and measurement of optical density at 450 nm. Standard curves were linear over the range of 2.5-100 ng/mL ricin. The limit of detection was below 5 ng/mL in assay buffer as well as in a 1:10 dilution of urine or 1:50 dilution of human serum spiked with ricin.

Roles of NKT cells in resistance against infection with Toxoplasma gondii and in expression of heat shock protein 65 in the host macrophages

We investigated the roles of γδ T, NK, and NK1.1 + T-like (NKT) cells in protective immunity against infection with Toxoplasma gondii. γδ T cells, NKT and NK cells, and NK cells in BALB/c mice were depleted by treatment with anti-TCR-γδ monoclonal antibody (mAb), anti-interleukin-2 receptor beta chain (IL-2Rβ) mAb, and anti-asialoGM1 Ab, respectively, and these mice were infected with T. gondii. Treatment of mice with anti-TCR-γδ mAb aggravated toxoplasmosis, while treatment with anti-asialoGM1 Ab had no effects. Treatment with anti-IL-2Rβ mAb enhanced the expression of heat shock protein 65 (HSP65) and gamma interferon (IFN-γ) mRNA, while it inhibited interleukin-4 (IL-4) mRNA expression, ameliorating toxoplasmosis. In addition to NK cells, anti-IL-2Rβ mAb eliminated cells expressing IL-2Rβ and intermediate levels of CD3 (IL-2Rβ + CD3 int). Mice treated with anti-IL-2Rβ mAb decreased the number of DX5 + CD3 int cells, which are considered to be equivalent to NK1.1 +T cells in NK1.1 allele-negative strains. IL-2Rβ + CD3 int cells isolated from splenic and hepatic lymphoid cells were confirmed to express the TCR-Vα14 transcript. The magnitude of HSP65 induction in macrophages correlated with the protective potential against T. gondii infection after treatment with the antibodies, supporting our previous finding that γδ T cells play an essential role in the induction of HSP65 in host macrophages. Interestingly, NKT cells suppressed the expression of γδ T cell-induced HSP65 and IFN-γ. Furthermore, depletion of IL-2Rβ + CD3 int cells suppressed the IL-4 mRNA expression. These results suggest that NKT cells may be the cells responsible for suppression of protective immunity against T. gondii infection by interfering with the γδ T cell-induced HSP65 expression, possibly through the generation of IL-4.

Colloidal gold-based immunochromatographic assay for detection of ricin

A rapid immunochromatographic assay was developed to detect ricin. The assay was based on the sandwich format using monoclonal antibodies (Mabs) of two distinct specificities. One anti-ricin B chain Mab (1G7) was immobilized to a defined detection zone on a porous nitrocellulose membrane, while the other anti-ricin A chain Mab (5E11) was conjugated to colloidal gold particles which served as a detection reagent. The ricin-containing sample was added to the membrane and allowed to react with Mab (5E11)-coated particles. The mixture was then passed along the porous membrane by capillary action past the Mab (1G7) in the detection zone, which will bind the particles that had ricin bound to their surface, giving a red color within this detection zone with an intensity proportional to ricin concentration. In the absence of ricin, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of ricin was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to100 pg/ml.

Characterization of monoclonal antibodies recognizing immunoglobulin κ and λ chains in pigs by flow cytometry

The existence of two types of the immunoglobulin (Ig) light chain in pigs was documented>30 years ago and has been confirmed by the cloning of porcine light chain genes homologous to human and murine Ig kappa (Igκ) and Ig lambda (Igλ). However, immunochemical reagents defining these two light chain isotypes have not been characterized. Here, we show that rabbit antisera specific for human Igκ and Igλ and certain anti-porcine light chain monoclonal antibodies (mAb) are useful in distinguishing light chain isotypes by flow cytometry (FCM). Porcine B cell lines L23 and L35 stained positive only with anti-human Igλ antiserum and were negative when tested using anti-human Igκ antiserum. While mAbs K139.3E1, 1G6 and 27.7.1 also tested positive on these cell lines, mAb 27.2.1 did not. Therefore, FCM was used to examine the hypothesis that K139.3E1, 1G6 and 27.7.1 are Igλ-specific whereas mAb 27.2.1 recognizes the Igκ chain in pigs. Double staining of peripheral blood mononuclear cells (PBMC) with pairs of anti-light chain mAbs and using cocktails of anti-light chain mAbs and anti-human polyclonal antiserum, confirmed this hypothesis with the exception that mAb K139.3E1 appears to recognize only a subset of Igλ + B cells in most pigs. In summary, we identified two pan-specific anti-pig Igλ mAbs, one anti-λ mAb that recognizes a λ-light chain subset and one anti-pig Igκ mAb.

Cross-Talk between RANKL and FRP-1/CD98 Systems: RANKL-Mediated Osteoclastogenesis Is Suppressed by an Inhibitory Anti-CD98 Heavy Chain mAb and CD98-Mediated Osteoclastogenesis Is Suppressed by Osteoclastogenesis Inhibitory Factor

The two pathways to osteoclastogenesis, RANKL-mediated and CD98-mediated osteoclastogenesis, have recently been reported. RANKL, OCIF, and TIMP-3 mRNAs are not found in monocytes freshly isolated or incubated with anti-FRP-1/CD98hc antibody. RANK, TACE, and M-CSF mRNAs can be detected in these cells. Interestingly, the expressed amount of RANK mRNA increases by cultivation of monocytes with anti-CD98hc antibody and maximal expression is observed in osteoclast-like cells. CD98-mediated cell aggregation and multinucleated giant cell formation are blocked by OCIF. OCIF also suppressed the CD98-mediated induction of Sp1 and c-src mRNAs in monocytes. Soluble RANK shows no effect on CD98-mediated cell aggregation and multinucleated giant cell formation. When blood monocytes were incubated with RANKL and M-CSF, c-src and Sp1 mRNAs were first found in blood monocytes incubated with these cytokines for 7 days. On the contrary, c-src mRNA could be detected 3 h after treatment of blood monocytes with anti-CD98hc mAb. LAT-1 mRNA was not found, and the expression levels of Y+LAT-1 and Y+LAT-2 mRNAs were not changed in monocytes stimulated without or with anti-CD98hc mAb or RANKL and M-CSF. An inhibitory mAb directed against CD98hc, HBJ 127, shows a suppressive effect on RANKL-mediated cell aggregation and cell fusion. Thus, there is cross-talk between these two pathways.

Papers to Appear in Forthcoming Issues

(Received and Accepted Dates Follow Title)HIV-1 Tat Protects CD4+ Jurkat T Lymphoblastoid Cells from Apoptosis Mediated by TNF-Related Apoptosis-Inducing Ligand. Davide Gibellini, Maria Carla Re, Cristina Ponti, Claudia Maldini, Claudio Celeghini, Alessandra Cappellini, Michele La Placa, and Giorgio Zauli. (Received 5/23/00; accepted 11/21/00.)IgD Receptor-Mediated Signal Transduction in T Cells. Seetha M. Lakshmi Tamma, Yan Wu, Igor Toporovsky, Viera Lima, and Richard F. Coico. (Received 7/24/00; accepted 11/21/00.)Cross-Talk between RANKL and FRP-1/CD98 Systems: RANKL-Mediated Osteoclastogenesis Is Suppressed by an Inhibitory Anti-CD98 Heavy Chain mAb and CD98-Mediated Osteoclastogenesis Is Suppressed by Osteoclastogenesis Inhibitory Factor. Kouki Mori, Noriki Miyamoto, Yasumitsu Higuchi, Kazuyoshi Nanba, Morihiro Ito, Masato Tsurudome, Machiko Nishio, Mitsuo Kawano, Atsumasa Uchida, and Yasuhiko Ito. (Received 8/11/00; accepted 11/22/00.)Interleukin (IL)-4/IL-9 and Exogenous IL-16 Induce IL-16 Production by BEAS-2B Cells, a Bronchial Epithelial Cell Line. Nozomi Yoshida, Masafumi Arima, Gang Cheng, Fukiko Eda, Hirokuni Hirata, Kyoko Honda, Fumiya Fukushima, and Takeshi Fukuda. (Received 10/22/00; accepted 11/9/00.)

Induction of c-Src in Human Blood Monocytes by Anti-CD98/FRP-1 mAb in an Sp1-Dependent Fashion

Freshly isolated human blood monocytes expressed neither c-src mRNA nor c-Src. However, when monocytes were incubated with anti-CD98 heavy chain (HC) mAb, expression of c-src mRNA, c-Src, and activated c-Src was induced. Many binding sites for the ubiquitous transcription factor Sp1 were identified in the promoter region of the c-src gene. Surprisingly, Sp1 and Sp1 mRNA were not found in monocytes that were freshly isolated or incubated with control antibody. Stimulation with anti-CD98HC mAb also resulted in the expression of Sp1 and its translocation to the nucleus. Herbimycin A, genistein, manumycin A, PD-98059, SB203580, and HBJ127 suppressed CD98HC-mediated c-src and Sp1 mRNA induction. On the contrary, H-7, Wortmannin, HA1077, and Y-27632 showed no effect on c-Src and Sp1 induction. Furthermore, anti-CD98HC mAb induced activation of tyrosine kinases and ERK kinases. These findings suggest that the tyrosine kinase(s)-Ras-MAPK-Sp1 pathway(s) is involved in CD98HC-mediated induction of c-Src in human blood monocytes.

Vascular endothelial cells provide T cells with costimulatory signals via the OX40/gp34 system

We investigated whether gp34, the ligand of OX40, expressed on EC is involved in costimulation of T cells. Normal CD4+ T cells were stimulated with anti-CD3-coated beads, phytohemagglutinin (PHA), or concanavalin A (Con A) in the presence or absence of irradiated human umbilical vein endothelial cells (HUVEC). Stimulation of T cells with each of these mitogens results in significant T-cell proliferation only when HUVEC were present, and this proliferation was inhibited markedly by anti-OX40 or anti-gp34 monoclonal antibody (mAb). T cells cultured with HUVEC produced more interleukin (IL)-2 than those cultured without HUVEC. The addition of anti-IL-2R alpha chain and anti-IL-2R beta chain mAbs abolished the costimulatory effects of HUVEC. Thus, the augmentation of T-cell proliferation appears to be attributable to increased IL-2 production. These results suggest that gp34 expressed on HUVEC plays a role in potentiation of T-cell immune response by providing OX40+ T cells with costimulatory signals.

Epithelial cell-specific laminin 5 is required for survival of early thymocytes.

The gene LamC2 encoding the gamma2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the alpha3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75-90%) was seen in the CD25+CD44+ and CD25+CD44-subsets of the CD4-CD8- double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin alpha3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.

BLOCK THE COMMON γ CHAIN OF CYTOKINE RECEPTORS INDUCES PERMANENT ALLOGRAFT TOLERANCE

58 T cell growth factors (i.e. IL-2, IL-4, IL-7, IL-9, IL-15) are remarkably redundant in promoting acute allograft rejection due to at least in part the shared key receptor component. Indeed, the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 use the same IL-2 receptor γc chain as a critical signaling component. Hence targeting individual TCGFs has failed to induce allograft tolerance. In the present study, we examined the role of the γc chain signals in TCGF stimulated cell proliferation in vitro and rejection of islet allografts in vivo. Block the γc chain using noncytolytic mAbs (4G3/ 3E12) markedly inhibited IL-2, IL-4, IL-7, and IL-15 stimulated cell proliferation in vitro and promoted apoptotic cell death of responding cells due to marked downregulation of bcl-2. Crude DBA/ 2J (H-2d) islet allografts were then transplanted under the renal capsule into chemically induced diabetic B6AF1 (H-2b/ d.k) recipients treated with anti-γc chain mAbs or isotype control antibody on post transplant days 0, 1, 3, 7 (0.5mg/ day/ injection). Control Ab treated mice rejected the islet allograft with a MST of 17 days (n=6). In contrast, all anti-γc treated mice experienced prolonged engraftment (MST>150 days, n=8). Removal of the primary islet allografts 150 day after transplantation led to rapid hyperglycemia and the diabetic mice accepted a second DBA/ 2J islet allograft without any immunosuppression. Furthermore systemic administration of high doses of a long lived form of IL-2 to tolerized recipient mice failed to provoke allograft rejection, suggesting a state of stable allograft tolerance. Our data suggest that targeting the common γc chain of TCGF receptors may have important therapeutic potential in the clinic.

Part of prekallikrein removed from human plasma together with IgG?immunoblot experiments and functional tests

With the present study, evidence is provided that prekallikrein (PK) in human plasma might be present in two different states, one of them removed along with IgG on Protein G columns. At a plasma dilution of 1 + 2.5, small amounts of an IgG fraction were left in plasma along with all of the PK. At a dilution of 1 + 11, nearly all IgG was removed. The removal in parallel of part of the PK was shown in immunoblot experiments and confirmed in amidase assays. One monoclonal antibody against PK (13G11) and two preparations of polyclonal antibodies were used for the immunoblot experiments. Different peptide substrates (S-2302, S-2222, Bz-Pro-Phe-Arg-pNA), along with protease inhibitors (soybean trypsin inhibitor, corn trypsin inhibitor, lima bean trypsin inhibitor) were used for the amidase assays. The amidase assays indicated that factors XII and XI were reduced by Protein G columns. In all experiments with extensive removal of IgG, protein recognized by the factor XII light chain mAb C6B7 was removed at the same time. This antibody preparation did not detect purified contact factors, but it did recognize a preparation of purified beta-FXIIa, and also significant amounts of protein present in plasma deficient in factor XII and not detectable in plasma deficient in PK. This protein accordingly seems to be connected with the PK fraction removed with IgG.

Fibre type-specific expression of p94, a skeletal muscle-specific calpain.

Members of the calpain proteinase family are present in all mammalian cells, although a novel calpain 94 kDa isoform is found almost exclusively in skeletal muscle. p94 is difficult to purify from muscle and recombinant p94 autolyses rapidly when expressed in COS cells. However, in vivo the enzyme may be stabilised by interaction with titin, which has two well-characterised binding sites for p94 at the N2- and M-lines. Both these titin subdomains are subject to muscle-specific alternative splicing, which could be related to p94 expression level or stability in muscles of different fibre type. In this study, porcine longissimus dorsi (LD), trapezius (TZ) and adductor longus (AL) were characterised as fast, intermediate and slow using commercially available specific anti-human fast- and slow-myosin heavy chain mAbs and also by conventional histochemistry. p94 was quantified both in whole muscle preparations and single fibres by western blotting using an anti-p94 antiserum generated by expressing a recombinant p94 sequence as a GST fusion protein antigen. SDS PAGE and immunoblotting revealed a single band of approximately 94 kDa with identical mobility in all muscle and fibre preparations. The intensity of the 94 kDa band was greater in LD (22 +/- 1.7 densitometric units mean +/- SEM, n = 3) than TZ and AL (10 +/- 2.3 and 6 +/- 0.9 units, respectively). Expressed as a ratio relative to actin immunoreactivity, p94 is present in all types of single fibres isolated from TZ, but at a significantly lower level (P < 0.01) in slow type I (0.08 +/- 0.01, n = 9), compared to fast IIA/IIB fibres (0.22 +/- 0.02, n = 26). No evidence was seen for rapid or variable rate of p94 degradation in either type of fibre. These data suggest a positive correlation between p94 expression level and fast glycolytic characteristics in porcine muscle.

Interaction affinity between cytokine receptor components on the cell surface.

The anti-common gamma chain (gammac) mAb CP.B8 is shown to inhibit interleukin 4 (IL-4)-dependent proliferation of phytohemagglutinin (PHA) activated T cells noncompetitively with respect to cytokine by blocking the IL-4-induced heterodimerization of IL-4Ralpha and gammac receptor chains. Affinities for the binding of IL-4 to Cos-7 cells transfected with huIL-4Ralpha, and to PHA blasts expressing both IL-4Ralpha and gammac, were used to estimate the affinity of the key interaction between gammac and the binary IL-4Ralpha.IL-4 complex on the cell surface. This affinity was defined in terms of the dimensionless ratio [IL-4Ralpha.IL-4.gammac]/[IL-4Ralpha.IL-4], which we designate KR. The results show that on PHA blasts this interaction is relatively weak; KR approximately 9, implying that approximately 10% of the limiting IL-4Ralpha chain remains free of gammac even at saturating concentrations of IL-4. This quantitative treatment establishes KR as a key measure of the coupling between ligand binding and receptor activation, providing a basis for functional distinctions between different receptors that are activated by ligand-induced receptor dimerization.

Comparison of an anti-HSV-2 monoclonal IgG and its IgA switch variant for topical immunoprotection of the mouse vagina

An IgG 2a monoclonal antibody (Mab) directed against glycoprotein D of herpes simplex virus 2 (HSV-2) was compared with an IgA heavy chain Mab switch variant to investigate the effect of isotype for topical immunoprotection of the murine vagina. The IgA Mab, a mixture of monomeric and polymeric IgA, was indistinguishable from its IgG parent in an in vitro HSV-2 neutralization assay. When these class switched Mabs were delivered to the mouse vagina, we also found no significant difference between the IgG and IgA for preventing vaginal transmission of HSV-2 infection. The implications of these results for active and passive immunization strategies against vaginal transmission of genital herpes infections are discussed.