Protocol design and synopsis: Omalizumab as Monotherapy and as Adjunct Therapy to Multiallergen OIT in Children and Adults with Food Allergy (OUtMATCH).

Background: Food allergy is common and causes substantial morbidity and even mortality. Safe and effective treatments for food allergy would therefore be highly desirable, especially for individuals with multiple food allergies.

Objectives: Our aim was to describe a phase 3 study on treatment of patients with multiple food allergies with omalizumab.

Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma.

We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell-specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations.

Therapeutic control of B cell activation via recruitment of Fcgamma receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold.

Objective: To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications.

Methods: DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells.

Selective blockade of the inhibitory Fcgamma receptor (FcgammaRIIB) in human dendritic cells and monocytes induces a type I interferon response program.

The ability of dendritic cells (DCs) to activate immunity is linked to their maturation status. In prior studies, we have shown that selective antibody-mediated blockade of inhibitory FcgammaRIIB receptor on human DCs in the presence of activating immunoglobulin (Ig) ligands leads to DC maturation and enhanced immunity to antibody-coated tumor cells. We show that Fcgamma receptor (FcgammaR)-mediated activation of human monocytes and monocyte-derived DCs is associated with a distinct gene expression pattern, including several inflammation-associated chemokines, as well as type 1 interferon (IFN) response genes, including the activation of signal transducer and activator of transcription 1 (STAT1).

Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma-receptor IIB (CD32B) from the activating Fcgamma-receptor IIA (CD32A): biochemical, biological and functional characterization.

Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants.

CD32B, the human inhibitory Fc-gamma receptor IIB, as a target for monoclonal antibody therapy of B-cell lymphoma.

Human CD32B (FcgammaRIIB), the low-affinity inhibitory receptor for IgG, is the predominant Fc receptor (FcR) present on B cells. Immunohistochemical and expression studies have identified CD32B expression in a variety of B-cell malignancies, suggesting that CD32B is a potential immunotherapeutic target for B-cell malignancies. A high-affinity monoclonal antibody (mAb 2B6), from a novel panel of anti-human CD32B-specific mAbs, was chimerized (ch2B6) and humanized (hu2B6-3.

Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions.

Human monocyte-derived DCs (moDCs) and circulating conventional DCs coexpress activating (CD32a) and inhibitory (CD32b) isoforms of IgG Fcgamma receptor (FcgammaR) II (CD32). The balance between these divergent receptors establishes a threshold of DC activation and enables immune complexes to mediate opposing effects on DC maturation and function. IFN-gamma most potently favors CD32a expression on immature DCs, whereas soluble antiinflammatory concentrations of monomeric IgG have the opposite effect.

A new tyrosine phosphorylation site in PLC gamma 1: the role of tyrosine 775 in immune receptor signaling.

Phospholipase Cgamma (PLCgamma) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLCgamma is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLCgamma1 activation are not fully elucidated.

A dynamic constitutive and inducible binding of c-Cbl by PLCgamma1 SH3 and SH2 domains (negatively) regulates antigen receptor-induced PLCgamma1 activation in lymphocytes.

We investigated the structural requirements for c-Cbl-mediated inhibition of Ag receptor-induced PLCgamma1 activation. Analysis of site-specific c-Cbl mutants indicated that tyrosine phosphorylation of c-Cbl was required for down-regulation of the PLCgamma1/Ca2+ pathway. Coprecipitation experiments indicated that c-Cbl and PLCgamma1 constitutively interact through a PLCgamma1 SH3 domain-dependent mechanism and that c-Cbl and PLCgamma1 can inducibly interact through the SH2(C) domain of PLCgamma1.

70Z/3 Cbl induces PLC gamma 1 activation in T lymphocytes via an alternate Lat- and Slp-76-independent signaling mechanism.

The oncoprotein 70Z/3 Cbl signals in an autonomous fashion or through blockade of endogenous c-Cbl, a negative regulator of signaling. The mechanism of 70Z/3 Cbl-induced signaling was investigated by comparing the molecular requirements for 70Z/3 Cbl- and TCR-induced phospholipase C gamma 1 (PLC gamma 1) activation. 70Z/3 Cbl-induced PLC gamma 1 tyrosine phosphorylation required, in addition to the PLC gamma 1 N-terminal SH2 domain, the C-terminal SH2 and SH3 domains that were dispensable for TCR-induced phosphorylation.

Membrane raft-dependent regulation of phospholipase Cgamma-1 activation in T lymphocytes.

Numerous signaling molecules associate with lipid rafts, either constitutively or after engagement of surface receptors. One such molecule, phospholipase Cgamma-1 (PLCgamma1), translocates from the cytosol to lipid rafts during T-cell receptor (TCR) signaling. To investigate the role played by lipid rafts in the activation of this molecule in T cells, an influenza virus hemagglutinin A (HA)-tagged PLCgamma1 was ectopically expressed in Jurkat T cells and targeted to these microdomains by the addition of a dual-acylation signal.

Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cgamma-1 activity.

We demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca(2+)/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cgamma1 (PLCgamma1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCgamma1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCgamma1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCgamma1 phosphorylation and PI hydrolysis.

Functional independence and interdependence of the Src homology domains of phospholipase C-gamma1 in B-cell receptor signal transduction.

B-cell receptor (BCR)-induced activation of phospholipase C-gamma1 (PLCgamma1) and PLCgamma2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCgamma activation, the mechanism coupling PLCgamma to the BCR remains undefined. The role of PLCgamma1 SH2 and SH3 domains at different steps of BCR-induced PLCgamma1 activation was examined by reconstitution in a PLCgamma-negative B-cell line.

Unbalanced germ-line expression of hMLH1 and hMSH2 alleles in hereditary nonpolyposis colorectal cancer.

We analyzed the hMLH1 and hMSH2 genes in 30 unrelated hereditary nonpolyposis colorectal cancer (HNPCC) patients using mutational and immunohistochemical analyses combined whenever possible with primer extension assays, designed to estimate hMLH1 and hMSH2 transcript expression in peripheral blood lymphocytes. Single-strand conformational polymorphism screening and PCR-direct sequencing revealed seven hMLH1 and five hMSH2 sequence variants in 14 unrelated HNPCC patients, including three definite pathogenic mutations, four amino acid substitutions of uncertain pathogenic significance, and five polymorphisms. Immunohistochemistry indicated the lack of either hMLH1 or hMSH2 protein expression in tumors from 13 patients, and the absence of both hMLH1 and hMSH2 immunostaining was observed in the tumor from one additional case.

A role for neutral sphingomyelinase-mediated ceramide production in T cell receptor-induced apoptosis and mitogen-activated protein kinase-mediated signal transduction.

Studying apoptosis induced by T cell receptor (TCR) cross-linking in the T cell hybridoma, 3DO, we found both neutral sphingomyelinase activation and production of ceramide upon receptor engagement. Pharmacological inhibition of ceramide production by the fungal toxin, fumonisin B1, impaired TCR-induced interleukin (IL)-2 production and programmed cell death. Addition of either exogenous ceramide or bacterial sphingomyelinase reconstituted both responses.

Microsatellite instability in thyroid tumours and tumour-like lesions.

Fifty-one thyroid tumours and tumour-like lesions were analysed for instability at ten dinucleotide microsatellite loci and at two coding mononucleotide repeats within the transforming growth factor beta (TGF-beta) type II receptor (TbetaRII) and insulin-like growth factor II (IGF-II) receptor (IGFIIR) genes respectively. Microsatellite instability (MI) was detected in 11 out of 51 cases (21.5%), including six (11.

Transcripts with splicings of exons 15 and 16 of the hMLH1 gene in normal lymphocytes: implications in RNA-based mutation screening of hereditary non-polyposis colorectal cancer.

Germline mutations of the hMLH1 gene are estimated to account for a large fraction of kindreds affected by hereditary non-polyposis colorectal cancer (HNPCC). In a significant number of cases, hMLH1 mutations result in the expression of truncated proteins. We report here two novel alternatively spliced forms of hMLH1 mRNA in normal lymphocytes.

Optimized PCR labeling in mutational and microsatellite analysis.

To optimize the labeling and visualization of PCR products we tested different variables, including deoxynucleotide concentration and ratio, dilution of labeled product, number of PCR cycles, and use of one-step or nested labeling protocols. Labeling was achieved using a fixed amount of labeled dATP, whose relative specific activity was varied by adding increasing amounts of cold dATP. Optimal PCR-labeling intensity was reached at dATP concentrations between 0.

ErbB2 expression increases the spectrum and potency of ligand-mediated signal transduction through ErbB4.

Interleukin 3-dependent murine 32D cells do not detectably express members of the ErbB receptor family and do not proliferate in response to known ligands for these receptors. 32D transfectants were generated expressing human ErbB4 alone (32D.E4) or with ErbB2 (32D.

Microsatellite instability in early gastric cancer.

Microsatellite replication errors (RERs), consisting in random tumour-associated allele contractions or expansions, represent a frequent genetic alteration in gastric cancer and appear to be associated with important clinicopathologic parameters. To verify the role of microsatellite instability in the initial phases of gastric carcinogenesis, we analysed the status of II microsatellites in paired microdissected samples of tumour and unaffected mucosa from 30 cases of early gastric carcinoma. Fifteen tumours (50%) demonstrated RERs: these included 7 cases with RERs at one locus and 8 cases with RERs at 2 or more loci.

Novel allele of the hMLH1 gene bearing a TTC deletion in the 3' untranslated region.

SSCP analysis of the hMLH1 gene in two kindreds affected by hereditary nonpolyposis colorectal cancer (HNPCC) revealed the presence of unique conformers in all patients affected by colorectal cancer. Sequence analysis of the corresponding region of the gene revealed a 3 base pairs deletion within a TTC tandem repeat (G TTC TCC T-->G TTC T) beginning 29 base pairs downstream of the termination codon of the gene in the 3' untranslated region. This deletion causes the loss of an MboII restriction site.

Alterations of microsatellites in neurofibromas of von Recklinghausen's disease.

von Recklinghausen's disease, or type I neurofibromatosis, a common familial tumor syndrome, is characterized by the occurrence of multiple benign neoplasms of nerve sheath cells. The disease is caused by germ-line mutations of the NF1 gene, which encodes a member of the GTPase-activating superfamily of Ras regulatory proteins. We analyzed 5 dinucleotide repeat loci in DNAs from neurofibromas and matched normal skin from 16 NF1 patients.