Applications and challenges of marker-assisted selection in the Western Australian Wheat Breeding Program

Interactions with the extracellular matrix (ECM) through integrin adhesion receptors provide cancer cells with physical and chemical cues that act together with growth factors to support survival and proliferation. Antagonists that target integrins containing the beta1 subunit inhibit tumor growth and sensitize cells to irradiation or cytotoxic chemotherapy in preclinical breast cancer models and are under clinical investigation. We found that the loss of beta1 integrins attenuated breast tumor growth but markedly enhanced tumor cell dissemination to the lungs. When cultured in three-dimensional ECM scaffolds, antibodies that blocked beta1 integrin function or knockdown of beta1 switched the migratory behavior of human and mouse E-cadherin-positive triple-negative breast cancer (TNBC) cells from collective to single cell movement. This switch involved activation of the transforming growth factor-beta (TGFbeta) signaling network that led to a shift in the balance between miR-200 microRNAs and the transcription factor zinc finger E-box-binding homeobox 2 (ZEB2), resulting in suppressed transcription of the gene encoding E-cadherin. Reducing the abundance of a TGFbeta receptor, restoring the ZEB/miR-200 balance, or increasing the abundance of E-cadherin reestablished cohesion in beta1 integrin-deficient cells and reduced dissemination to the lungs without affecting growth of the primary tumor. These findings reveal that beta1 integrins control a signaling network that promotes an epithelial phenotype and suppresses dissemination and indicate that targeting beta1 integrins may have undesirable effects in TNBC

Checkpoint inhibition reduces the threshold for Drug-Specific T-Cell priming and increases the incidence of sulfasalazine hypersensitivity

An emerging clinical issue associated with immune-oncology agents is the collateral effects on the tolerability of concomitant medications. One report of this phenomenon was the increased incidence of hypersensitivity reactions observed in patients receiving concurrent immune checkpoint inhibitors (ICIs) and sulfasalazine (SLZ). Thus, the aim of this study was to characterize the T cells involved in the pathogenesis of such reactions, and recapitulate the effects of inhibitory checkpoint blockade on de-novo priming responses to compounds within in vitro platforms. A regulatory competent human dendritic cell/T-cell coculture assay was used to model the effects of ICIs on de novo nitroso sulfamethoxazole- and sulfapyridine (SP) (the sulfonamide component of SLZ) hydroxylamine-specific priming responses. The role of T cells in the pathogenesis of the observed reactions was explored in 3 patients through phenotypic characterization of SP/sulfapyridine hydroxylamine (SPHA)-responsive T-cell clones (TCC), and assessment of cross-reactivity and pathways of T-cell activation. Augmentation of the frequency of responding drug-specific T cells and intensity of the T-cell response was observed with PD-1/PD-L1 blockade. Monoclonal populations of SP- and SPHA-responsive T cells were isolated from all 3 patients. A core secretory effector molecule profile (IFN-γ, IL-13, granzyme B, and perforin) was identified for SP and SPHA-responsive TCC, which proceeded through Pi and hapten mechanisms, respectively. Data presented herein provides evidence that drug-responsive T cells are effectors of hypersensitivity reactions observed in oncology patients administered ICIs and SLZ. Perturbation of drug-specific T-cell priming is a plausible explanation for clinical observations of how an increased incidence of these adverse events is occurring

Amoxicillin and Clavulanate Form Chemically and Immunologically Distinct Multiple Haptenic Structures in Patients

Amoxicillin-clavulanate (AC) is one of the most common causes of drug induced liver injury (DILI). The association between AC-DILI and HLA alleles and the detection of drug-specific T cells in patients with AC-DILI indicate that the adaptive immune system is involved in the disease pathogenesis. In this study, mass spectrometric methods were employed to characterize the antigen formed by AC in exposed patients and the antigenic determinants that stimulate T cells. Amoxicillin formed penicilloyl adducts with lysine residues on human serum albumin (HSA) <i>in vitro</i>, with K190 and K199 being the most reactive sites. Amoxicillin-modified K190 and K199 have also been detected in all patients, and more extensive modification was observed in patients exposed to higher doses of amoxicillin. In contrast, the binding of clavulanic acid to HSA was more complicated. Multiple adducts were identified at high concentrations <i>in vitro</i>, including those formed by direct binding of clavulanic acid to lysine residues, novel pyrazine adducts derived from binding to the degradation products of clavulanic acid, and a cross-linking adduct. Stable adducts derived from formylacetic acid were detected in all patients exposed to the drug. Importantly, analysis of hapten–protein adducts formed in the cell culture medium revealed that the highly drug-specific T-cell responses were likely driven by the markedly different haptenic structures formed by these two drugs. In this study, the unique haptenic structures on albumin in patients formed by amoxicillin and clavulanic acid have been characterized and shown to function as chemically distinct antigens which can stimulate separate, specific T-cell clones

SHANK3 controls maturation of social reward circuits in the VTA.

Haploinsufficiency of SHANK3, encoding the synapse scaffolding protein SHANK3, leads to a highly penetrant form of autism spectrum disorder. How SHANK3 insufficiency affects specific neural circuits and how this is related to specific symptoms remains elusive. Here we used shRNA to model Shank3 insufficiency in the ventral tegmental area of mice. We identified dopamine (DA) and GABA cell-type-specific changes in excitatory synapse transmission that converge to reduce DA neuron activity and generate behavioral deficits, including impaired social preference. Administration of a positive allosteric modulator of the type 1 metabotropic glutamate receptors mGluR1 during the first postnatal week restored DA neuron excitatory synapse transmission and partially rescued the social preference defects, while optogenetic DA neuron stimulation was sufficient to enhance social preference. Collectively, these data reveal the contribution of impaired ventral tegmental area function to social behaviors and identify mGluR1 modulation during postnatal development as a potential treatment strategy

SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism

Genetic mutations of SHANK3 have been reported in patients with intellectual disability, autism spectrum disorder (ASD) and schizophrenia. At the synapse, Shank3/ProSAP2 is a scaffolding protein that connects glutamate receptors to the actin cytoskeleton via a chain of intermediary elements. Although genetic studies have repeatedly confirmed the association of SHANK3 mutations with susceptibility to psychiatric disorders, very little is known about the neuronal consequences of these mutations. Here, we report the functional effects of two de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in patients with ASD. We show that Shank3 is located at the tip of actin filaments and enhances its polymerization. Shank3 also participates in growth cone motility in developing neurons. The truncating mutation (STOP) strongly affects the development and morphology of dendritic spines, reduces synaptic transmission in mature neurons and also inhibits the effect of Shank3 on growth cone motility. The de novo mutation in the ankyrin domain (Q321R) modifies the roles of Shank3 in spine induction and morphology, and actin accumulation in spines and affects growth cone motility. Finally, the two inherited mutations (R12C and R300C) have intermediate effects on spine density and synaptic transmission. Therefore, although inherited by healthy parents, the functional effects of these mutations strongly suggest that they could represent risk factors for ASD. Altogether, these data provide new insights into the synaptic alterations caused by SHANK3 mutations in humans and provide a robust cellular readout for the development of knowledge-based therapies

Diclofenac Hypersensitivity: Antibody Responses to the Parent Drug and Relevant Metabolites

Background: Hypersensitivity reactions against nonsteroidal antiinflammatory drugs (NSAIDs) like diclofenac (DF) can manifest as Type I-like allergic reactions including systemic anaphylaxis. However, except for isolated case studies experimental evidence for an IgE-mediated pathomechanism of DF hypersensitivity is lacking. In this study we aimed to investigate the possible involvement of drug-and/or metabolite-specific antibodies in selective DF hypersensitivity. Methodology/Principal Findings: DF, an organochemically synthesized linkage variant, and five major Phase I metabolites were covalently coupled to carrier proteins. Drug conjugates were analyzed for coupling degree and capacity to crosslink receptor-bound IgE antibodies from drug-sensitized mice. With these conjugates, the presence of hapten-specific IgE antibodies was investigated in patients' samples by ELISA, mediator release assay, and basophil activation test. Production of sulfidoleukotrienes by drug conjugates was determined in PBMCs from DF-hypersensitive patients. All conjugates were shown to carry more than two haptens per carrier molecule. Immunization of mice with drug conjugates induced drug-specific IgE antibodies capable of triggering mediator release. Therefore, the conjugates are suitable tools for detection of drug-specific antibodies and for determination of their anaphylactic activity. Fifty-nine patients were enrolled and categorized as hypersensitive either selectively to DF or to multiple NSAIDs. In none of the patients' samples evidence for drug/metabolite-specific IgE in serum or bound to allergic effector cells was found. In contrast, a small group of patients (8/59, 14%) displayed drug/metabolite-specific IgG. Conclusions/Significance: We found no evidence for an IgE-mediated effector mechanism based on haptenation of protein carriers in DF-hypersensitive patients. Furthermore, a potential involvement of the most relevant metabolites in DF hypersensitivity reactions could be excluded