Protective role of protease-activated receptor-2 in anaphylaxis model mice.

Anaphylaxis is a severe life-threatening hypersensitivity reaction induced by mast cell degranulation. Among the various mediators of mast cells, little is known about the role of tryptase. Therefore, we aimed to elucidate the role of protease-activating receptor-2 (PAR-2), a receptor activated by tryptase, in murine anaphylactic models using PAR-2-deficient mice and newly generated tryptase-deficient mice. Anaphylaxis was induced by IgE-dependent and IgE-independent mast cell degranulation in mice. PAR-2 deficiency exacerbated the decrease in body temperature and hypotension during anaphylaxis; however, the number of skin mast cells, degree of mast cell degranulation, and systemic and local vascular hyperpermeability were comparable in PAR-2 knockout and wild-type mice. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), is an indispensable vasodilator in anaphylaxis. In the lungs of anaphylactic mice, PAR-2 deficiency promoted eNOS expression and phosphorylation, suggesting a protective effect of PAR-2 against anaphylaxis by downregulating eNOS activation and expression. Based on the hypothesis that the ligand for PAR-2 in anaphylaxis is mast cell tryptase, tryptase-deficient mice were generated using CRISPR-Cas9. In wild-type mice, the PAR-2 antagonist exacerbated the body temperature drop due to anaphylaxis; however, the effect of the PAR-2 antagonist was abolished in tryptase-deficient mice. These results suggest that tryptase is a possible ligand of PAR-2 in anaphylaxis and that the tryptase/PAR-2 pathway attenuates the anaphylactic response in mice.

Lapatinib as first-line treatment for muscle-invasive urothelial carcinoma in dogs.

Epidermal growth factor receptors 1 and 2 (EGFR and HER2) are frequently overexpressed in various malignancies. Lapatinib is a dual tyrosine kinase inhibitor that inhibits both EGFR and HER2. Although a phase III trial failed to show the survival benefits of lapatinib treatment after first-line chemotherapy in patients with EGFR/HER2-positive metastatic urothelial carcinoma, the efficacy of lapatinib for untreated urothelial carcinoma is not well defined. Here, we describe the therapeutic efficacy of lapatinib as a first-line treatment in a canine model of muscle-invasive urothelial carcinoma. In this non-randomized clinical trial, we compared 44 dogs with naturally occurring urothelial carcinoma who received lapatinib and piroxicam, with 42 age-, sex-, and tumor stage-matched dogs that received piroxicam alone. Compared to the dogs treated with piroxicam alone, those administered the lapatinib/piroxicam treatment had a greater reduction in the size of the primary tumor and improved survival. Exploratory analyses showed that HER2 overexpression was associated with response and survival in dogs treated with lapatinib. Our study suggests that lapatinib showed encouraging durable response rates, survival, and tolerability, supporting its therapeutic use for untreated advanced urothelial carcinoma in dogs. The use of lapatinib as a first-line treatment may be investigated further in human patients with urothelial carcinoma.

Antigen-driven autoantibody production in lungs of interstitial lung disease with autoimmune disease.

Interstitial lung disease (ILD) sometimes becomes a life-threatening complication of systemic autoimmune diseases; however, little is known about the immune response in lung lesions. We aimed to investigate humoural immunity in ILD associated with rheumatoid arthritis (RA), sjögren's syndrome (SjS), and mixed connective tissue disease (MCTD), using bronchoalveolar fluid (BALF) and serum samples from 15 patients with autoimmune disease associated-ILD. We first showed that BALF contained higher titers of disease-related autoantibodies than serum, suggesting the possibility of autoantibody production in lungs. Next, we produced 326 monoclonal antibodies from antibody-secreting cells in BALF, and the reactivity and their revertants, in which somatic hypermutations were reverted to germline, were analyzed. Among 123 antibodies from RA-ILD, 16 disease-related antibodies (anti-modified protein antibodies and rheumatoid factors) were identified, of which one antibody had both properties. The revertant antibodies changed their target modification in a complicated manner, suggesting that the antibodies were selected against various modifications in lungs. Among 146 antibodies from SjS-ILD and/or MCTD-ILD, seven anti-SSA/Ro60 antibodies and 15 anti-RNP antibodies were identified. Some of the anti-RNP antibodies recognized multiple RNP constituent proteins simultaneously, indicating that epitope spreading may progress in lungs. Our results revealed the existence of an active autoimmunity in the lungs of autoimmune disease associated-ILD.

Efficacy of chemotherapy and palliative hypofractionated radiotherapy for cats with nasal lymphoma.

Nasal lymphoma (NL) is the most common nasal tumor in cats, and radiotherapy, chemotherapy, or a combination of these treatments have been described as the treatment for this disease. However, the previous studies included various machines and protocols of radiotherapy. Therefore, we aimed to retrospectively compare the prognosis among cases treated with palliative hypofractionated radiotherapy, chemotherapy, and a combination of them with united machine and protocol of radiotherapy. When compared overall survival and progression free survival, there was no significant difference among these three groups. The data of this study suggested that similar efficacy could be achieved by palliative hypofractionated radiotherapy, chemotherapy, or a combination of them.

Distinct Expression of Coinhibitory Molecules on Alveolar T Cells in Patients With Rheumatoid Arthritis-Associated and Idiopathic Inflammatory Myopathy-Associated Interstitial Lung Disease.

OBJECTIVE: To identify immunologic factors in the lungs of patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD) and patients with idiopathic inflammatory myopathy-associated ILD (IIM-ILD) and to examine their pathologic mechanisms. METHODS: Eleven patients with RA-ILD, 16 with IIM-ILD, 6 with drug-induced ILD (DI-ILD), and 8 healthy controls were enrolled. Peripheral blood (PB) and bronchoalveolar lavage (BAL) fluid were immunophenotyped by flow cytometry. Alveolar macrophages (AMs) were analyzed by coculture assay with PB naive CD4+ T cells from healthy individuals and RNA sequencing. RESULTS: Several coinhibitory molecules were coexpressed on BAL fluid T cells (CTLA-4, programmed death 1 [PD-1], T cell immunoglobulin and mucin domain-containing protein 3 [TIM-3], and lymphocyte activation gene 3 protein, from most to least), whereas only PD-1 was expressed on PB T cells. CTLA-4+PD-1+CD4+ T cells were characteristic of RA-ILD, whereas CTLA-4+PD-1+TIM-3+CD8+ T cells were characteristic of IIM-ILD. BAL fluid PD-1+CD4+ T cells rarely expressed CXCR5, but their levels correlated with levels of plasmablasts and plasma cells (ρ = 0.57, P = 0.006), indicating that most of them would be considered peripheral helper T cells. In coculture experiments, AMs from patients with RA-ILD and IIM-ILD induced more PD-1 and TIM-3 on T cells (P < 0.05), suggesting that coinhibitory molecule expression on BAL fluid T cells was partly due to AMs. RNA sequencing showed significant down-regulation of PD ligand 1/2 genes in AMs from patients with RA-ILD compared to those with DI-ILD. CONCLUSION: We have identified differences in coinhibitory molecule expression between patients with RA-ILD and those with IIM-ILD. PD-1 on T cells in RA-ILD and TIM-3 on CD8+ T cells in IIM-ILD might be key factors in the disease process. Evaluation of coinhibitory molecules on BAL fluid T cells could be clinically useful.

Foxp3+ Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs.

Regulatory T cells (Tregs) can be targeted in cancer immunotherapy. A previous study has shown that the chemokine CCL17 and the receptor CCR4 play a role in Treg recruitment in canine urothelial carcinoma. Here, we describe the association of tumor-infiltrating Tregs with CCL17/CCR4 expression in dogs with other carcinomas. In this study, we investigated 23 dogs with mammary carcinoma, 14 dogs with oral squamous cell carcinoma, 16 dogs with pulmonary adenocarcinoma, and 8 healthy control dogs. Immunohistochemistry showed that Foxp3+ Tregs and CCR4+ cells were increased in the tumor tissues of mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma, when compared with the healthy tissues. The number of CCR4+ cells was associated with that of Foxp3+ Tregs. Double immunofluorescence labeling confirmed that most tumor-infiltrating Foxp3+ Tregs expressed CCR4. In vitro, canine carcinoma cell lines expressed CCL17 mRNA. Quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) showed that CCL17 mRNA expression in canine carcinomas was increased approximately 10- to 25-fold relative to that of healthy tissues. These results suggest that the CCL17/CCR4 axis may drive Treg recruitment in a variety of canine carcinomas. CCR4 blockade may be a potential therapeutic option for tumor eradication through Treg depletion.

Sphingosine-1-phosphate (S1P) signaling regulates the production of intestinal IgA and its potential role in the pathogenesis of canine inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a common gastrointestinal disease in dogs. Decreased production of intestinal immunoglobulin A (IgA) has been suggested as a possible pathogenesis in a subset of canine IBD; however, the underlying cause remains unclear. Sphingosine-1-phosphate (S1P) is a lipid mediator that regulates intestinal IgA production by controlling lymphocyte trafficking in mice. The objectives of this study were to clarify the role of S1P in IgA production in dogs and to evaluate the expression of S1P-related molecules in dogs with IBD. First, an S1P receptor antagonist was administrated to five healthy dogs. The S1P receptor antagonist significantly decreased the IgA concentration in sera and feces but did not affect the IgG concentration. Moreover, the immunoreactivity of intestinal IgA was significantly decreased by S1P signal blockade. These results indicate that S1P signaling specifically regulates the intestinal IgA production in dogs. Subsequently, the intestinal S1P concentration and the expression of S1P-related molecules were measured in dogs with IBD and healthy dogs. The intestinal concentration of S1P was significantly lower in dogs with IBD than in healthy dogs. In addition, the gene expression levels of S1P receptor (S1P1) and S1P synthase (SK1) were significantly lower in dogs with IBD than in healthy dogs. Taken together, these observations suggest that decreased S1P production, likely caused by a lower expression of S1P synthetase, leads to attenuation of S1P/S1P1 signaling pathway and the production of intestinal IgA in dogs with IBD.

Usefulness of serum Krebs von den Lungen-6 for the management of myositis-associated interstitial lung disease.

OBJECTIVE: To identify biomarkers for assessing myositis-associated interstitial lung disease (ILD). METHODS: We reviewed consecutive patients from our institution who had been newly diagnosed with PM, DM, or clinically amyopathic DM during the years 2002-2017. The patients were divided into two groups according to the presence of ILD, and the ILD group was further subdivided into three groups according to the clinical courses of induction failure, relapse and non-relapse. Baseline and time-course changes in the parameters were compared between groups. RESULTS: Among 110 patients enrolled, 75 (68%) had ILD. Baseline serum Krebs von den Lungen-6 (KL-6) was significantly higher in the ILD group than in the non-ILD group (1120 vs 236 U/ml; P < 0.001). In the ILD group consisting of the induction failure cases (n = 3), the relapse group (n = 24) and the non-relapse group (n = 48), baseline serum KL-6 was significantly different between the three groups [1971 vs 1870 vs 935 U/ml, respectively; P = 0.003 (relapse group vs non-relapse group)]. The time-course changes in serum KL-6 revealed that KL-6 significantly increased along with relapse, with the increase of 625 U/ml relevant to relapse. CONCLUSION: Serum KL-6 is a useful biomarker for assessing the disease activity of myositis-associated ILD.

Successful rituximab treatment of granulomatosis with polyangiitis with cranial neuropathies.

BACKGROUND: In granulomatosis with polyangiitis (GPA), peripheral nerve involvement is common but central nervous system (CNS) involvement is extremely rare and treatment strategy has not been established. We report a case of intravenous cyclophosphamide (IVCY)-resistant GPA with associated cranial neuropathies that was successfully treated with rituximab (RTX). CASE PRESENTATION: A 37-year-old man with intractable sinusitis had several months of headache, hoarseness, and dysphagia; a month of right-sided deafness and nasal bleeding; and a week of dysarthria, steppage gait, and numbness in the right L5 distribution. A magnetic resonance imaging (MRI) examination of the head showed an infiltrative lesion in the right skull base encasing the carotid sheath. Computed tomography (CT) scan of the chest revealed a 23 mm nodule in the left upper lobe. Histology was inconclusive. Therefore, the patient was diagnosed as GPA. He was treated with glucocorticoids (GC) and IVCY. Three months later, he was readmitted for recurrence of headache and new left-sided hearing loss. He was treated with GC and RTX, and a 1-year remission followed. The molecular mechanism of RTX is not fully understood. In this case, RTX was more effective at rapidly and strongly suppressing B cells than CY. Since the B cell count was proportional to the patient's clinical manifestations, B cells might represent a suitable target for the treatment of GPA with cranial neuropathies. CONCLUSIONS: GPA with cranial neuropathies might be useful with RTX as induction therapy.

Risk factors for the recurrence of interstitial lung disease in patients with polymyositis and dermatomyositis: a retrospective cohort study.

To identify risk factors for the recurrence of interstitial lung disease (ILD) in patients with polymyositis (PM)/dermatomyositis (DM). Forty-four PM/DM-ILD patients who had been treated with glucocorticoid and/or immunosuppressive agents as a remission induction therapy were enrolled. The patients were first classified into two groups: the early recurrence group that recurred within 52 weeks, and the non-early recurrence group, which was further classified into the late recurrence group that recurred after 52 weeks, and the non-recurrence group. The characteristics and treatment regimen between the groups were compared. Recurrence was experienced by 15 of 44 patients. The pulmonary vital capacity of the early recurrence group was significantly lower than the non-early recurrence group (46 vs 76%, p = 0.0003), and 60% of the early recurrence group was treated with glucocorticoid alone as a maintenance therapy in contrast to 10% in the non-early recurrence group (p = 0.004). The late recurrence was only related with a positivity for autoantibodies against aminoacyl-tRNA synthetases (anti-ARS antibodies, odds ratio 8.4, p = 0.02), but calcineurin inhibitors tended to decrease the relapse incidence in patients with anti-ARS antibodies. Low pulmonary vital capacity at disease onset and anti-ARS antibodies positivity are the risk factors for the recurrence of ILD with PM/DM. Calcinuerin inhibitors are important in preventing relapse.