The present study was performed to determine the morphologic change and selected molecular features of spontaneous lung tumors in cats examined at the North Carolina State University Veterinary Teaching Hospital. Thirty-nine primary lung carcinomas represented 0.69% of all feline cases admitted to the hospital. Most lung tumors were observed in aged cats (P < .0001), and no sex predilection was found (P < .4241). Persian cats with pulmonary carcinoma were overrepresented in the data set, at least 4 times more frequently than other breeds. The histologic tumor types included adenocarcinoma (64.1%), bronchioloalveolar carcinoma (20.5%), and adenosquamous carcinoma (15.4%). Metastasis was observed in about 80% of 39 cases, with decreasing order of intrapulmonary metastasis, intrathoracic carcinomatosis, regional lymph nodes, and distant organs, including digits. The size of the largest tumor mass was significantly associated with metastatic potential (P < .001). Based on immunohistochemistry, more than 80% (20 of 24) of feline lung tumors were positively labeled with either surfactant protein A or thyroid transcription factor 1. Epidermal growth factor receptor mutant and p53 proteins were detected in approximately 20% (5 of 24) and 25% (6 of 24) of the feline lung tumor cases, respectively. Limited sequencing analysis of K-ras and p53 genes in 3 selected normal and neoplastic lung tissues did not reveal any alteration. Results indicate that primary lung carcinomas are rare but aggressive tumors in cats, thereby warranting further studies on molecular carcinogenesis.
Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.
Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability. Permalink https://escholarship.org/uc/item/8t3660m4 Journal Abstract 33 34 Background: Psychological stress and heightened MC activation are linked with important 35 immunological disorders including allergy, anaphylaxis, asthma, and functional bowel 36diseases, but the mechanisms remain poorly defined. We have previously demonstrated that 37 activation of the corticotropin releasing factor (CRF) system potentiates MC degranulation 38 responses during IgE-mediated anaphylaxis and psychological stress, via CRF receptor 39 subtype 1 (CRF 1 ) expressed on MCs. 40 41 Objective: In this study, we investigated the role of CRF receptor subtype 2 (CRF 2 ) as a 42 modulator of stress-induced MC degranulation and associated disease pathophysiology. 43 44 Methods: In vitro MC degranulation assays were performed with bone marrow derived MCs 45 (BMMCs) derived from WT and CRF 2 -deficient (CRF 2 -/-) mice and RBL-2H3 MCs transfected 46 with CRF 2 -overexpressing plasmid or CRF 2 -siRNA. In vivo MC responses and associated 47 pathophysiology in IgE-mediated passive systemic anaphylaxis (PSA) and acute 48 psychological restraint stress were measured in WT, CRF 2 -/-, and MC-deficient Kit W-sh/W-sh 49 knock-in mice. 50 51 Results: Compared with WT mice, CRF 2 -/exhibited heightened serum histamine levels and 52 exacerbated PSA-induced anaphylactic responses and colonic permeability. In addition, 53 CRF 2 -/mice exhibited increased serum histamine and colonic permeability following acute 54 restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF 2 55 expressed on MCs suppresses store-operated Ca 2+ entry (SOCE) signaling and MC 56 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3 degranulation induced by diverse MC stimuli. Experiments with MC-deficient Kit W-sh/W-sh mice 57 systemically engrafted with WT and CRF 2 -/-BMMCs demonstrated the functional importance 58 of MC-CRF 2 in modulating stress-induced pathophysiology. 59 60 Conclusions: MC CRF 2 is a negative, global modulator of stimuli-induced MC degranulation 61 and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis. 62 63 Key messages 64 • Loss of CRF 2 function induces exacerbated MC degranulation, IgE-mediated 65 anaphylaxis and psychological stress-induced intestinal barrier dysfunction. 66 • MC-specific CRF 2 suppresses degranulation induced by diverse MC stimuli via 67 negative regulation of SOCE. 68 • Further characterization of the mechanisms by which CRF 2 negatively modulates MC 69 activation could lead to novel therapeutic approaches for stress-related immunological 70 disorders associated with MC hyperactivity. 71 72
Life stress is a major risk factor in the onset and exacerbation of mast cell-associated diseases, including allergy/anaphylaxis, asthma, and irritable bowel syndrome. Although it is known that mast cells are highly activated upon stressful events, the mechanisms by which stress modulates mast cell function and disease pathophysiology remains poorly understood. Here, we investigated the role of corticotropin-releasing factor receptor subtype 1 (CRF) in mast cell degranulation and associated disease pathophysiology. In a mast cell-dependent model of IgE-mediated passive systemic anaphylaxis (PSA), prophylactic administration of the CRF-antagonist antalarmin attenuated mast cell degranulation and hypothermia. Mast cell-deficient mice engrafted with CRF bone marrow-derived mast cells (BMMCs) exhibited attenuated PSA-induced serum histamine, hypothermia, and clinical scores compared with wild-type BMMC-engrafted mice. mice engrafted with CRF BMMCs also exhibited suppressed in vivo mast cell degranulation and intestinal permeability in response to acute restraint stress. Genetic and pharmacologic experiments with murine BMMCs, rat RBL-2H3, and human LAD2 mast cells demonstrated that although CRF activation did not directly induce MC degranulation, CRF signaling potentiated the degranulation responses triggered by diverse mast cell stimuli and was associated with enhanced release of Ca from intracellular stores. Taken together, our results revealed a prominent role for CRF signaling in mast cells as a positive modulator of stimuli-induced degranulation and in vivo pathophysiologic responses to immunologic and psychologic stress.
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