Vaccine-draining lymph nodes of cancer patients for generating anti-cancer antibodies.

BACKGROUND: Our research is focused on using the vaccine draining lymph node to better understand the immune response to cancer vaccines and as a possible source of anti-cancer reagents. We evaluated vaccine draining lymph nodes archived from a clinical study in melanoma patients and determined the reaction of B cells to the vaccine peptides. METHODS: Mononuclear cells (MNCs) were recovered from cryopreserved lymph nodes that were directly receiving drainage from multi-peptide melanoma vaccine. The patients were enrolled on a vaccine study (NCT00089219, FDA, BB-IND No. 10825). B cell responses in the vaccine-draining lymph nodes were studied under both stimulated and un-stimulated conditions. Cryopreserved cells were stimulated with CD40L, stained with multiple human cell-surface markers (CD19, CD27, IgM) to identify different categories of B cell sub populations with flow cytometry. Hybridomas were generated from the lymph node cells after CD40L-stimulation. Cells were fused to murine plasmacytoma P3X63.Ag8.653 using Helix electrofusion chamber. ELISA was used to evaluate hybridoma derived antibody binding to vaccine peptides. RESULTS: Viable MNCs were satisfactorily recovered from lymph nodes cryopreserved from six vaccine study patients 8-14 years previously. B cell ELISPOT demonstrated responses for each patient to multiple vaccine peptides. CD40L stimulation of lymph node cells increased the proportion of CD19+ CD27+ cells from 12 to 65% of the sample and increased the proportion of class-switched cells. Screening of IgG secreting clones demonstrated binding to melanoma vaccine peptides. CONCLUSIONS: B cells were successfully recovered and expanded from human cryopreserved vaccine-draining lymph nodes. Individual B cells were identified that secreted antibodies that bound to cancer vaccine peptides. The ability to reliably generate in vitro the same antibodies observed in the blood of vaccinated patients will facilitate research to understand mechanisms of human antibody activity and possibly lead to therapeutic antibodies.

Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase.

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 µg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays.

Modeling single cell antibody excretion on a biosensor.

We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates.

pSer40 tyrosine hydroxylase immunohistochemistry identifies the anatomical location of C1 neurons in rat RVLM that are activated by hypotension.

Identification of neurons, and their phenotype, that are activated in response to specific stimuli is a critical step in understanding how neural networks integrate inputs to produce specific outputs. Here, we developed novel mouse monoclonal antibodies of different IgG isotypes that are specific to tyrosine hydroxylase (TH), and to tyrosine hydroxylase activated at its serine 40 position (pSer40TH), in order to assess changes in the activity of phenotypically identified cardiovascular neurons using fluorescence immunohistochemistry. We find that the proportion of C1 pSer40TH-positive neurons in the central and medial region of the rat rostral ventrolateral medulla (RVLM) increases dramatically following hydralazine treatment, whereas phenylephrine treatment does not significantly change the pSer40TH/TH ratio in these regions compared to control. This finding suggests that there is a mediolateral topology associated with the activation of C1 neurons following baroreceptor loading or unloading. Overall, we conclude first, that our newly characterized monoclonal antibodies are specific, and selective, against TH and pSer40TH. Secondly, that they can be used to label TH and pSer40TH immunoreactive neurons simultaneously, and thirdly that that they can be used to identify the activation state of catecholamine synthetizing neurons after physiological stimuli. Finally, we find that there is basal level of activation of TH neurons in the lateral, central and medial regions (∼ 70%, 30% and 45%, respectively) of the C1 area, but that following unloading of the baroreceptors there is a marked increase in activation of central (∼ 80%) and medial (∼ 90%) C1 neurons in the RVLM.

Hybridoma Technology.

The generation of hybridoma cell lines by the fusion of splenocytes from immunized mice with immortal myeloma cells is a well-established method for the production of monoclonal antibodies. Although other methods have emerged as an effective alternative for the generation of monoclonal antibodies, the use of hybridoma technology remains a viable technique that is accessible to a wide number of laboratories that perform basic cell biological research. Hybridoma technology represents a relatively simple procedure at minimal cost for the continuous production of native whole immunoglobulins. This chapter will describe the materials and methodologies needed for the successful generation of monoclonal antibody (mAb)-producing hybridoma cell lines against target antigens.

Control of late apoptotic events by the p38 stress kinase in L-glutamine-deprived mouse hybridoma cells.

L-Glutamine (Gln) starvation rapidly triggers apoptosis in Sp2/0-Ag14 (Sp2/0) murine hybridoma cells. Here, we report on the role played by the stress-activated kinase p38 mitogen-activated protein kinase (MAPK) in this process. p38 activation was detected 2 h after Gln withdrawal and, although treatment with the p38 inhibitor SB203580 did not prevent caspase activation in Gln-starved cells, it reduced the occurrence of both nuclear condensation/fragmentation and apoptotic body formation. Similarly, transfection of Sp2/0 cells with a dominant negative p38 MAPK reduced the incidence of nuclear pyknosis and apoptotic body formation following 2 h of Gln starvation. Gln withdrawal-induced apoptosis was blocked by the overexpression of the anti-apoptotic protein Bcl-xL or by the caspase inhibitor Z-VAD-fmk. Interestingly, Bcl-xL expression inhibited p38 activation, but Z-VAD-fmk treatment did not, indicating that activation of this MAPK occurs downstream of mitochondrial dysfunction and is independent of caspases. Moreover, the anti-oxidant N-acetyl-l-cysteine prevented p38 phosphorylation, showing that p38 activation is triggered by an oxidative stress. Altogether, our findings indicate that p38 MAPK does not contribute to the induction of apoptosis in Gln-starved Sp2/0 cells. Rather, Gln withdrawal leads to mitochondrial dysfunction, causing an oxidative stress and p38 activation, the latter contributing to the formation of late morphological features of apoptotic Sp2/0 cells.

Label-free hybridoma cell culture quality control by a chip-based impedance flow cytometer.

Impedance flow cytometry (IFC) was evaluated as a possible alternative to fluorescence-based methods for on-line quality monitoring of hybridoma cells. Hybridoma cells were cultured at different cell densities and viability was estimated by means of IFC and fluorescence-based flow cytometry (FCM). Cell death was determined by measuring the impedance phase value at high frequency in low conductivity buffer. IFC data correlate well with reference FCM measurements using AnnexinV and 7-AAD staining. Hybridoma cells growing at different densities in cell culture revealed a density-dependent subpopulation pattern. Living cells of high density cultures show reduced impedance amplitudes, indicating particular cellular changes. Dead cell subpopulations become evident in cultures with increasing cell densities. In addition, a novel intermediate subpopulation, which most probably represents apoptotic cells, was identified. These results emphasize the extraordinary sensitivity of high frequency impedance measurements and their suitability for hybridoma cell culture quality control.

Inhibitory effect of drug-free hybrid liposomes on metastasis of human neuroblastoma.

PURPOSE: Hybrid liposomes composed of vesicular and micellar molecules have been used as drug-delivery systems. It has become clear that hybrid liposomes alone have an inhibitory effect against the growth of various tumor cells. The present study was designed to determine whether a drug-free hybrid liposome composed of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylenealkyl ether (EO) [90 mol% DMPC/10% C(12)(EO)(21) (HL21), 90 mol% DMPC/10% C(12)(EO)(23) (HL23), or 90 mol% DMPC/10% C(12)(EO)(25) (HL25)], inhibit the liver metastasis of human neuroblastoma cells and thus increases survival. METHODS: A human neuroblastoma cell, TNB9, and BALB/C-nu/nu athymic mice were used in this study. First, we determined the inhibitory effect of the hybrid liposomes on TNB9 cells in vitro. Next, to determine the inhibitory effect of the hybrid liposomes on metastasis of neuroblastoma cells to the liver, we made a murine hepatic metastasis model by implanting TNB9 cells (2 × 106) in the spleen of the mice and compared anatomic appearance, weights, and histological findings of the livers of treated mice and control mice 60 days after the beginning of a 7-day intraperitoneal injection of a hybrid liposome. We also compared survival rates using the Kaplan-Meier method. RESULTS: In mice implanted with TNB9 neuroblastoma cells and treated with HL21 or HL25, no histological evidence of metastasis was found, the weight of the liver was normal, and survival was a mean of 88 and 87.9 days, respectively. In contrast, mice treated with HL23 and control mice had countless tumor cell masses histologically, their liver weight was increased, and their survival was 73.0 and 68.6 days, respectively. CONCLUSIONS: Two kinds of hybrid liposomes, HL21 and HL25, inhibit metastasis of human neuroblastoma cells to the liver, and thus increase survival.

Inhibition of T cell response to native low-density lipoprotein reduces atherosclerosis.

Immune responses to oxidized low-density lipoprotein (oxLDL) are proposed to be important in atherosclerosis. To identify the mechanisms of recognition that govern T cell responses to LDL particles, we generated T cell hybridomas from human ApoB100 transgenic (huB100(tg)) mice that were immunized with human oxLDL. Surprisingly, none of the hybridomas responded to oxidized LDL, only to native LDL and the purified LDL apolipoprotein ApoB100. However, sera from immunized mice contained IgG antibodies to oxLDL, suggesting that T cell responses to native ApoB100 help B cells making antibodies to oxLDL. ApoB100 responding CD4(+) T cell hybridomas were MHC class II-restricted and expressed a single T cell receptor (TCR) variable (V) beta chain, TRBV31, with different Valpha chains. Immunization of huB100(tg)xLdlr(-/-) mice with a TRBV31-derived peptide induced anti-TRBV31 antibodies that blocked T cell recognition of ApoB100. This treatment significantly reduced atherosclerosis by 65%, with a concomitant reduction of macrophage infiltration and MHC class II expression in lesions. In conclusion, CD4(+) T cells recognize epitopes on native ApoB100 protein, this response is associated with a limited set of clonotypic TCRs, and blocking TCR-dependent antigen recognition by these T cells protects against atherosclerosis.

The diabetogenic mouse MHC class II molecule I-Ag7 is endowed with a switch that modulates TCR affinity.

Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-Ag7, typically have a small, uncharged amino acid residue at position 57 of their beta chain (beta57); this results in the absence of a salt bridge between beta57 and Argalpha76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Argalpha76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3beta. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 A) electrostatic interactions existed among Argalpha76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue beta57 of the I-Ag7 and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.

Fusion induced reversal of dendritic cell maturation: an altered expression of inflammatory chemokine and chemokine receptors in dendritomas.

Dendritic cell-mediated cancer immunotherapy employs several ways to engage tumor antigens. We have demonstrated in both pre-clinical animal studies and early clinical trials that dendritomas, highly purified hybrids between dendritic cells (DC) and tumor cells, are superior activators of anti-tumor immunity. It has been argued, however, that DC vaccines may be dysfunctional in lymph node migration. In the present study we examined inflammatory chemokine and chemokine receptor expression as well as other maturation induced genes in dendritomas produced from either immature or mature DCs in order to shed light on their capacity to migrate from injection sites to draining lymph nodes and elicit an appropriate immune response. RNA microarray analysis was used to identify gene expression profiles for inflammatory chemokines and receptors and other maturation induced genes within dendritomas, lysate-pulsed dendritic cells, immature DCs and mature DCs. Gene regulation was confirmed with relative quantification, real-time RT-PCR in a separate experiment. We found that fusion of immature DCs to tumor cells initiates maturation with respect to inflammatory chemokines, chemokine receptors and other maturation induced genes in a similar pattern as LPS matured DCs. Interestingly, we saw a reversed gene profile when mature DCs were fused to tumor cells. LPS matured DCs displayed the chemokine repertoire expected with DC maturation; however, once fused to tumor cells, these chemokines and other maturation induced genes reverted to levels comparable to immature DCs. It appears that mature DCs used for dendritoma production result in a de-mature genotype. Our results indicate that dendritomas from immature DC/tumor cell fusions may be more effective in migration from injection site to draining lymph nodes and, therefore, would be more effective in stimulating anti-tumor immunity.

Monoclonal antibodies against human aspartyl (asparaginyl) beta-hydroxylase developed by DNA immunization.

We newly cloned the gene encoding the human aspartyl (asparaginyl) beta-hydroxylase (HAAH) from the surgical tissue of a patient with hepatocellular carcinoma. This study was designed to generate HAAH-specific monoclonal antibody (MAb) for further exploration of its structure and function. Mice were co-immunized with naked plasmid DNA containing N-terminal domain of encoding HAAH gene and recombinant HAAH polypeptide. Hybridomas were developed by the electrofusion of the splenocytes from mice immunized with plasmid DNA to Sp2/0 myeloma cells in vitro. Three hybridoma cell lines (designated G3, G9, and F11, respectively) stably secreting HAAH-specific MAbs were obtained. The specificity and sensitivity of MAbs were assessed by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Results showed that the three MAbs belong to IgG1 kappa isotype, the titer of MAbs reached was 5 x 10(4) - 1 x 10(5), and the affinity constant (k(aff)) of MAbs ranged between 2.5 x 10(8) - 1.1 x 10(9). MAb G3 was preliminarily applied to detection expression of HAAH for seven tumor tissues, including hepatocellular carcinoma, lung cancer, kidney cancer, cholangiocarcinoma, prostate cancer, breast cancer, and glioblastoma by immunohistochemical stain. Our studies demonstrated that co-immunization of naked DNA containing encoding gene of target antigen and recombinant target protein, and combined with in vitro electrofusion, is an effective and simple method to raise MAbs.

The flavonoid rutin but not the alkaloid arborinine induces apoptosis in a B-cell hybridoma cell line.

The effects of arborinine, an alkaloid extracted from Erthela bahiensis and of rutin, a flavonoid obtained from Dimorphandra mollis (Benth.), Brazilian medicinal plants, on the viability and function of a murine B-cell hybridoma as a tumor model were investigated. The flavonoid rutin at 50 microM induced an increase in the number of apoptotic cells of one- to fivefold and reductions in cellular proliferation and monoclonal antibody production. Less but still significant necrosis was also induced by rutin under the same experimental conditions. On the other hand, the alkaloid arborinine exerted no significant effects on the studied parameters.

Synthesis of alpha-galactosyl ceramide (KRN7000) and analogues thereof via a common precursor and their preliminary biological assessment.

A new practical synthesis of alpha-GalCer and of its analogues is presented, opening the chance to easily modify the sphingosine chain. The common precursor is a disaccharide, obtained by coupling tetra-O-benzyl-D-galactose with allyl 2,3-O-isopropylidene-D-lyxofuranoside. Introduction of alkyl chains via Wittig reaction (for alpha-GalCer and OCH) or via Williamson reaction (for oxa analogues) followed by standard synthetic steps allows one to efficiently obtain such compounds. The analogues are able to activate iNKT cells when presented by CD1d expressing cells.

Genetic characterization of human Dsg3-specific B cells isolated by flow cytometry from the peripheral blood of patients with pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune bullous disease caused by anti-desmoglein 3 (Dsg3) IgG autoantibodies; however, the Dsg3-specific B cells that produce anti-Dsg3 IgG are not well characterized. OBJECTIVES: Our aims were to develop a flow cytometric method for the isolation of Dsg3-specific B cells from the peripheral blood of patients with active PV and to identify the variable regions within their heavy- and light-chain immunoglobulin genes. METHODS: Dsg3-specific B cells were isolated as CD3(-)IgD(-)PI(-)Dsg3E-tag+ cells using recombinant human Dsg3 with an E-tag (rDsg3-E-His) as a probe. Heavy- and light-chain cDNA was produced by PCR from single B cells; these were used to characterize the usage and CDR3 sequence in the variable region of each gene. RESULTS: Staining conditions were optimized using mouse hybridoma cells against human Dsg3 and peripheral blood mononuclear cells (PBMCs) from a PV patient. Individual Dsg3-specific B cells were isolated by FACS from four PV patients at a frequency of 1-18 per 10(5) PBMCs. CDR3 sequences and identical gene usage in the variable region were identified in several B cells from the same PV patients. Common gene usage was also found among several PV patients. CONCLUSIONS: These results suggested clonal expansion of autoreactive B cells and restricted gene usage for autoreactive B cells against Dsg3. Our method for the isolation of Dsg3-specific B cells will allow the systematic analysis of immunoglobulin gene usage in PV patients, which may elucidate the mechanism of immunopathogenesis.

[Growth and differentiation of cell hybrids obtained by fusing mouse PCC4azal teratocarcinoma cells and mouse spleen cells under different in vitro culture conditions].

Cell hybrids obtained by fusing mouse PCC4azal teratocarcinoma cells and spleen cells induced to proliferation and treated with the demethylating agent 5-azacytidine prior to fusion are described. The obtained hybrids demonstrated no expression of T lymphocyte marker genes CD11 and CD45, which indicates possible somatic nucleus reprogramming by factors present in teratocarcinoma cells. Irrespective of culture conditions, cell hybrids demonstrated a relatively stable chromosome number: they lost on average no more than four chromosomes after 30 passages. Culturing in medium containing hypoxanthine, aminopterin, and thymidine (selective conditions) decreased the differentiation capacity of cell hybrids compared to nonselective conditions, which is likely due to the inhibition of their metabolism. For the first time, teratocarcinoma cell hybrid differentiation into cardiomyocytes under the influence of DMSO has been demonstrated in vitro.

The metastatic T-cell hybridoma antigen/P-selectin glycoprotein ligand 1 is required for hematogenous metastasis of lymphomas.

Using variants of the murine BW5147 lymphoma cell-line, we have previously identified 3 monoclonal antibodies (MAbs) that discriminate between metastatic and nonmetastatic BW5147-derived T-cell hybridomas and lymphomas, as well as BW5147-unrelated T-lymphomas. These MAbs were reported to recognize an identical membrane-associated sialoglycoprotein, termed "metastatic T-cell hybridoma antigen" (MTH-Ag). Here, we document that the expression pattern of the MTH-Ag on metastatic and nonmetastatic BW5147 variants correlates with that of the P-selectin glycoprotein ligand 1 (PSGL-1), a sialomucin involved in leukocyte recruitment to sites of inflammation. Moreover, the MAbs against the MTH-Ag recognize PSGL-1 when it is transfected in MTH-Ag-negative BW5147 variants, suggesting that the MTH-Ag is PSGL-1. Overexpression of MTH-Ag/PSGL-1 in MTH-Ag-negative BW5147 variants did not affect their in vivo malignancy. Yet, down-regulation of MTH-Ag/PSGL-1 expression on metastatic, MTH-Ag-positive BW5147 variants, using an RNA interference (RNAi) approach, resulted, in a dose-dependent manner, in a significant reduction of liver and spleen colonization and a delay in mortality of the recipient mice upon intravenous inoculation. Collectively, these results demonstrate that, although MTH-Ag/PSGL-1 overexpression alone may not be sufficient for successful dissemination and organ colonization, MTH-Ag/PSGL-1 plays a critical role in hematogenous metastasis of lymphoid cancer cells.

Presentation of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes.

OBJECTIVE: To assess the ability of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) to function as antigen-presenting cells (APCs) for arthritogenic autoantigens found within inflamed joint tissues. METHODS: Human class II major histocompatibility complex (MHC)-typed FLS were used as APCs for murine class II MHC-restricted CD4 T cell hybridomas. Interferon-gamma (IFNgamma)-treated, antigen-loaded FLS were cocultured with T cell hybridomas specific for immunodominant portions of human cartilage gp-39 (HC gp-39) or human type II collagen (CII). T cell hybridoma activation was measured by enzyme-linked immunosorbent assay of culture supernatants for interleukin-2. Both synthetic peptide and synovial fluid (SF) were used as sources of antigen. APC function in cocultures was inhibited by using blocking antibodies to human class II MHC, CD54, or CD58, or to murine CD4, CD11a, or CD2. RESULTS: Human FLS could present peptides from the autoantigens HC gp-39 and human CII to antigen-specific MHC-restricted T cell hybridomas. This response required pretreatment of FLS with IFNgamma, showed MHC restriction, and was dependent on human class II MHC and murine CD4 for effective antigen presentation. Furthermore, FLS were able to extract and present antigens found within human SF to both the HC gp-39 and human CII T cell hybridomas in an IFNgamma-dependent and MHC-restricted manner. CONCLUSION: RA FLS can function as APCs and are able to present peptides derived from autoantigens found within joint tissues to activated T cells in vitro. In the context of inflamed synovial tissues, FLS may be an important and hitherto overlooked subset of APCs that could contribute to autoreactive immune responses.

Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation.

OBJECTIVE: Therapeutic anticoagulation is widely used, but limitations in efficacy and bleeding complications cause an ongoing search for new agents. However, with new agents developed it seems to be an inherent problem that increased efficiency is accompanied by an increase in bleeding complications. We investigate whether targeting of anticoagulants to activated platelets provides a means to overcome this association of potency and bleeding. METHODS AND RESULTS: Ligand-induced binding sites (LIBS) on fibrinogen/fibrin-binding GPIIb/IIIa represent an abundant clot-specific target. We cloned an anti-LIBS single-chain antibody (scFv(anti-LIBS)) and genetically fused it with a potent, direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). Specific antibody binding of fusion molecule scFv(anti-LIBS)-TAP was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. ScFv(anti-LIBS)-TAP prolonged occlusion time comparable to enoxaparine, recombinant TAP, and nontargeted mutant-scFv-TAP. ScFv(anti-LIBS)-TAP revealed antithrombotic effects at low doses at which the nontargeted mutant-scFv-TAP failed. In contrast to the other anticoagulants tested, bleeding times were not prolonged by scFv(anti-LIBS)-TAP. CONCLUSIONS: The novel clot-targeting approach of anticoagulants via single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa promises effective anticoagulation with reduced bleeding risk.

IgE basement membrane zone antibodies induce eosinophil infiltration and histological blisters in engrafted human skin on SCID mice.

Bullous pemphigoid (BP) is characterized by the deposition of IgG in the basement membrane zone, infiltration of eosinophils, and blister formation. The purpose of this study was to evaluate a potential role of IgE basement membrane antibodies in the histological findings of BP. LABD97 is a component of the shed ectodomain of bullous pemphigoid antigen 2. We have developed an IgE hybridoma to LABD97 antigen. This hybridoma was injected subcutaneously in SCID mice with engrafted human skin. A subcutaneous hybridoma secreting IgE antibodies developed. An IgE mouse hybridoma to trinitrophenyl was used as a control. Human grafts and mouse skin were examined grossly over 21 days, histologically, and immunopathologically at day 21 after injection of the hybridoma. A visible subcutaneous tumor developed in 10-14 days. Erythema and intense scratching developed 2-3 days before the tumor in test mice, but not in controls. At day 21, 16/16 test mice developed intense eosinophil infiltration and degranulation of the human mast cells within the grafts and 13/16 developed histological, but not clinically visible, basement membrane blisters. Human skin grafts of control mice and normal mouse skin on the test mice and control mice did not develop any histological abnormalities. IgE antibodies to LABD97 recapitulate the histological inflammatory process seen in BP.