Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces.

Epithelial tissues continually undergo apoptosis. Commensal organisms that inhabit the epithelium influence tissue homeostasis, in which regulatory T cells (Treg cells) have a central role. However, the physiological importance of epithelial cell apoptosis and how the number of Treg cells is regulated are both incompletely understood. Here we found that apoptotic epithelial cells negatively regulated the commensal-stimulated proliferation of Treg cells. Gut commensals stimulated CX3CR1(+)CD103(-)CD11b(+) dendritic cells (DCs) to produce interferon-ß (IFN-ß), which augmented the proliferation of Treg cells in the intestine. Conversely, phosphatidylserine exposed on apoptotic epithelial cells suppressed IFN-ß production by the DCs via inhibitory signaling mediated by the cell-surface glycoprotein CD300a and thus suppressed Treg cell proliferation. Our findings reveal a regulatory role for apoptotic epithelial cells in maintaining the number of Treg cell and tissue homeostasis.

Cutting Edge: Involvement of the Immunoreceptor CD300c2 on Alveolar Macrophages in Bleomycin-Induced Lung Fibrosis.

Idiopathic pulmonary fibrosis is a chronic, progressive, and irreversible fibrotic lung disease. Although inflammation plays a central role in the pathogenesis of idiopathic pulmonary fibrosis, how inflammatory responses are regulated remains unclear. In this article, we show that mice deficient in the immunoreceptor CD300c2 (also called MAIR-II, LMIR2, and CLM-4) showed longer survival; less collagen deposition in the lung; lower levels of neutrophil chemoattractants, such as TNF-α, CXCL1, and CCL2; and fewer neutrophils in the bronchoalveolar fluid than wild-type mice after intratracheal administration of bleomycin (BLM). We also found that BLM administration induced the release of the danger-associated molecular pattern HMGB-1, which caused CD300c2-deficient alveolar macrophages, via TLR4, to produce lower levels of neutrophil chemoattractants than wild-type alveolar macrophages. Our findings demonstrate that CD300c2 contributes to BLM-induced inflammatory responses mediated by alveolar macrophages.

Correlations of mRNA Levels among Efflux Transporters, Transcriptional Regulators, and Scaffold Proteins in Non-Small-Cell Lung Cancer.

Multidrug resistance (MDR) due to enhanced drug efflux activity of tumor cells can severely impact the efficacy of antitumor therapies. We recently showed that increased activity of the efflux transporter P-glycoprotein (P-gp) associated with activation of Snail transcriptional regulators may be mediated mainly by moesin in lung cancer cells. Here, we aimed to systematically evaluate the relationships among mRNA expression levels of efflux transporters (P-gp, breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2)), scaffold proteins (ezrin (Ezr), radixin (Rdx), and moesin (Msn); ERM proteins), and SNAI family members (Snail, Slug, and Smac) in clinical lung cancer and noncancer samples. We found high correlations between relative (cancer/noncancer) mRNA expression levels of Snail and Msn, Msn and P-gp, Slug and MRP2, and Smuc and BCRP. These findings support our previous conclusion that Snail regulates P-gp activity via Msn and further suggest that Slug and Smuc may contribute to the functional regulation of MRP2 and BCRP, respectively, in lung cancer cells. This trial is registered with UMIN000023923.

Retrospective study of 540 cats with respiratory diseases in Japan (2003-2020).

BACKGROUND: Few epidemiological studies on respiratory medicine and the relationship between clinical signs and various respiratory diseases in cats have been reported. OBJECTIVES: This retrospective study aimed to investigate the prevalence and breed predisposition to feline respiratory diseases in Japan and determine the association between clinical signs, duration and type of respiratory diseases. METHODS: The medical records of cats with feline respiratory diseases were examined to obtain information on age, sex, breed, final diagnosis, clinical signs and duration. The odds ratios (ORs) and 95% confidence intervals were calculated to evaluate breed predispositions. Mann-Whitney U, Kruskal-Wallis and Dunn's tests were used to assess the duration of clinical signs. RESULTS: This study included 540 cats with 615 respiratory diagnoses. The American Shorthair breed was predisposed to bronchopneumonia (BP; OR: 5.0) and pulmonary tumour (PT; OR: 3.6), while the Russian Blue breed exhibited a predisposition to inflammatory lower airway diseases (OR: 3.4), BP (OR: 6.1) and interstitial lung diseases (OR: 11.1). Similarly, the Scottish Fold breed displayed predisposition to PTs (OR: 5.8). The duration of clinical signs among nasal diseases, nasopharyngeal diseases and lower tracheal/bronchial and pulmonary diseases differed significantly (p = 0.001, p = 0.012, p < 0.0001, respectively). CONCLUSIONS: The results suggest that some popular breeds in Japan are predisposed to feline respiratory diseases, especially the American Shorthair, Russian Blue and Scottish Fold breeds. The characteristics of occurrence, clinical signs and duration of each disease will aid in diagnosing, treating, preventing and elucidating the pathophysiology of feline respiratory disease.

Relationship between Respiratory Rate, Oxygen Saturation, and Blood Test Results in Dogs with Chronic or Acute Respiratory Disease: A Retrospective Study.

This study aimed to investigate the association of respiratory rate (RR), oxygen saturation (SpO2), and blood findings with respiratory disease in dogs and to compare the examination findings in the chronic and acute phases. Dogs that visited a veterinary referral hospital with respiratory symptoms were classified into the chronic disease group (GC), and those that visited the emergency veterinary hospital were classified into the acute disease group (GA). In total, 704 and 682 dogs were included in GC and GA, respectively. The RR and SpO2 were significantly higher and lower, respectively, in patients with lung disease compared to other disease sites in both groups. White blood cell counts were significantly increased in patients with lung and pleural diseases in both groups. Respiratory alkalosis and respiratory acidosis were most common in GC and GA, respectively. The C-reactive protein levels were elevated in both groups, primarily in patients with lung disease. Associations between the results of several tests for understanding and diagnosing respiratory conditions and diseases were recognized, and differences in the trends of the chronic and acute phases were clarified. These tools may be used as adjuncts to other tests for the diagnosis and monitoring of treatment responses.

Retrospective study of 1050 dogs with respiratory symptoms in Japan (2005-2020).

BACKGROUND: Few studies have investigated the incidence of respiratory diseases based on anatomical sites or the relationship between breed and these diseases. OBJECTIVE: The objective of this study was to investigate the prevalence of canine respiratory diseases among dogs in Japan, with relationship to the breed. METHODS: We retrospectively reviewed the medical records of dogs with respiratory symptoms and calculated the odds ratio (OR) to evaluate the relationship between breed and disease. RESULTS: A total of 1050 dogs with respiratory symptoms were included in this study. Miniature dachshunds were the most common breed affected by respiratory diseases. Among tracheobronchial diseases, there was a significant association between some small breeds and tracheobronchial collapse, miniature dachshunds (OR: 4.44, 8.43, 95% confidence interval [CI]: 3.17-6.22, 4.33-16.0) and chronic bronchitis and bronchiectasis. Among nasal diseases, miniature dachshunds (OR: 27.2, 95% CI: 16.8-44.8) and golden retrievers (OR: 21.0, 95% CI: 6.43-69.3) were the most affected by non-infectious rhinitis and nasal aspergillosis, respectively. Brachycephalic obstructive airway syndrome was the most common disease among pharyngeal and laryngeal diseases, with a relationship with breed being found in some brachycephalic breeds, and Pomeranians (OR: 2.7, 95% CI: 1.42-5.17). CONCLUSIONS: Respiratory diseases in dogs are strongly correlated with popular breeds in Japan. Miniature dachshunds, in particular, are associated with many respiratory diseases, which may differ from international reports. Thus, this result may help in the early detection, prevention, treatment, and elucidation of the pathophysiology of canine respiratory diseases.

Construction of diagnostic prediction model for canine nasal diseases using less invasive examinations without anesthesia.

Advanced imaging techniques under general anesthesia are frequently employed to achieve a definitive diagnosis of canine nasal diseases. However, these examinations may not be performed immediately in all cases. This study aimed to construct prediction models for canine nasal diseases using less-invasive examinations such as clinical signs and radiography. Dogs diagnosed with nasal disease between 2010 and 2020 were retrospectively investigated to construct a prediction model (Group M; GM), and dogs diagnosed between 2020 and 2021 were prospectively investigated to validate the efficacy (Group V; GV). Prediction models were created using two methods: manual (Model 1) and LASSO logistic regression analysis (Model 2). In total, 103 and 86 dogs were included in GM and GV, respectively. In Model 1, the sensitivity and specificity of neoplasia (NP) and sino-nasal aspergillosis (SNA) were 0.88 and 0.81 in GM and 0.92 and 0.78 in GV, respectively. Those of non-infectious rhinitis (NIR) and rhinitis secondary to dental disease (DD) were 0.78 and 0.88 in GM and 0.64 and 0.80 in GV, respectively. In Model 2, the sensitivity and specificity of NP and SNA were 0.93 and 1 in GM and 0.93 and 0.75 in GV, respectively. Those of NIR and DD were 0.96 and 0.89 in GM and 0.80 and 0.79 in GV, respectively. This study suggest that it is possible to create a prediction model using less-invasive examinations. Utilizing these predictive models may lead to appropriate general anesthesia examinations and treatment referrals.

Slug Mediates MRP2 Expression in Non-Small Cell Lung Cancer Cells.

Transcriptional factors, such as Snail, Slug, and Smuc, that cause epithelial-mesenchymal transition are thought to regulate the expression of Ezrin, Radixin, and Moesin (ERM proteins), which serve as anchors for efflux transporters on the plasma membrane surface. Our previous results using lung cancer clinical samples indicated a correlation between Slug and efflux transporter MRP2. In the current study, we aimed to evaluate the relationships between MRP2, ERM proteins, and Slug in lung cancer cells. HCC827 cells were transfected by Mock and Slug plasmid. Both mRNA expression levels and protein expression levels were measured. Then, the activity of MRP2 was evaluated using CDCF and SN-38 (MRP2 substrates). HCC827 cells transfected with the Slug plasmid showed significantly higher mRNA expression levels of MRP2 than the Mock-transfected cells. However, the mRNA expression levels of ERM proteins did not show a significant difference between Slug-transfected cells and Mock-transfected cells. Protein expression of MRP2 was increased in Slug-transfected cells. The uptake of both CDCF and SN-38 was significantly decreased after transfection with Slug. This change was abrogated by treatment with MK571, an MRP2 inhibitor. The viability of Slug-transfected cells, compared to Mock cells, significantly increased after incubation with SN-38. Thus, Slug may increase the mRNA and protein expression of MRP2 without regulation by ERM proteins in HCC827 cells, thereby enhancing MRP2 activity. Inhibition of Slug may reduce the efficacy of multidrug resistance in lung cancer.

Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a.

Although tumor-infiltrating regulatory T (Treg) cells play a pivotal role in tumor immunity, how Treg cell activation are regulated in tumor microenvironments remains unclear. Here, we found that mice deficient in the inhibitory immunoreceptor CD300a on their dendritic cells (DCs) have increased numbers of Treg cells in tumors and greater tumor growth compared with wild-type mice after transplantation of B16 melanoma. Pharmacological impairment of extracellular vesicle (EV) release decreased Treg cell numbers in CD300a-deficient mice. Coculture of DCs with tumor-derived EV (TEV) induced the internalization of CD300a and the incorporation of EVs into endosomes, in which CD300a inhibited TEV-mediated TLR3-TRIF signaling for activation of the IFN-ß-Treg cells axis. We also show that higher expression of CD300A was associated with decreased tumor-infiltrating Treg cells and longer survival time in patients with melanoma. Our findings reveal the role of TEV and CD300a on DCs in Treg cell activation in the tumor microenvironment.

CD300a blockade enhances efferocytosis by infiltrating myeloid cells and ameliorates neuronal deficit after ischemic stroke.

Immune responses contribute to tissue injury and repair during and after ischemic stroke. However, the spatiotemporal and initiating molecular events remain incompletely understood. Here, we show that mice deficient in the phosphatidylserine receptor CD300a, which is highly expressed on brain myeloid cells including Ly6Chi monocytes, exhibited ameliorated neurological deficit after middle cerebral artery occlusion (MCAO). CD300a inhibited signaling through the CD300b-DNAX-activation protein 12 (DAP12) signaling pathway to prevent efferocytosis of apoptotic cells. Deficiency of CD300a enhanced efferocytosis by myeloid cells infiltrating the brain as early as 1 hour after MCAO and reduced release of damage-associated molecular patterns from dead cells, resulting in milder inflammation in the penumbral region. Treatment with an anti-CD300a neutralizing antibody ameliorated the neurological deficit after MCAO. These findings reveal an important role of efferocytosis in the super-acute phase of ischemic stroke pathology and identified CD300a as a target for immunotherapy in treating ischemic stroke.

Acid hydrolysis of native and annealed starches and branch-structure of their Naegeli dextrins.

Eight commercial starches, including common corn, waxy corn, wheat, tapioca, potato, Hylon V, Hylon VII, and mung bean starch, were annealed by a multiple-step process, and their gelatinization characteristics were determined. Annealed starches had higher gelatinization temperatures, reduced gelatinization ranges, and increased gelatinization enthalpies than their native starches. The annealed starches with the highest gelatinization enthalpies were subjected to acid hydrolysis with 15.3% H2SO4, and Naegeli dextrins were prepared after 10 days' hydrolysis. Annealing increased the acid susceptibility of native starches in the first (rapid) and the second (slow) phases with potato starch showing the greatest and high amylose starches showing the least changes. Starches with a larger shift in onset gelatinization temperature also displayed a greater percent hydrolysis. The increase in susceptibility to acid hydrolysis was proposed to result from defective and porous structures that resulted after annealing. Although annealing perfected the crystalline structure, it also produced void space, which led to porous structures and possible starch granule defects. The molecular size distribution and chain length distribution of Naegeli dextrins of annealed and native starches were analyzed. The reorganization of the starch molecule during annealing occurred mainly within the crystalline lamellae. Imperfect double helices in the crystalline lamellae improved after annealing, and the branch linkages at the imperfect double helices became protected by the improved crystalline structure. Therefore, more long chains were observed in the Naegeli dextrins of annealed starches than in native starches.

Polyethylenimine combined with liposomes and with decreased numbers of primary amine residues strongly enhanced therapeutic antiviral efficiency against herpes simplex virus type 2 in a mouse model.

The development of antiviral agents that have novel mechanisms of action is urgently required in the topical therapy of herpes simplex virus type 2 (HSV-2) infections. We reported previously that topical application of branched 3610-Da polyethylenimine (PEI) exhibited preventative antiviral activity. In this study, to develop therapeutic anti-HSV-2 agents, the most potent PEI combined with ~200 nm-sized liposomes with or without oleic acid (liposomes/PEI) was selected in vitro and further evaluated using in vivo studies. The mechanism of action in vivo was elucidated using PEIs with decreased numbers of primary amine residues, resulting from ethylene carbonate treatment, and polyallylamine, a linear polyamine consisting of primary amines. Cytotoxicity and antiviral activity in vitro, and the appearance of acute herpetic disease and virus yields in mice intravaginally administered with liposomes/PEI were evaluated in cell culture assays and a mouse genital herpes model, respectively. In addition, the cellular association of liposome/PEI was examined by flow cytometry and confocal microscopy. PEI showed higher antiviral activity postinfection than preinfection in vivo. Liposome/PEI and PEI with decreased numbers of primary amine residues at a dose of 0.2 mg PEI/mouse exhibited more potent therapeutic antiviral activity than acyclovir and PEI alone without acute lesion appearance or toxicity pre- or postinfection, but polyallylamine was moderately effective only preinfection. Liposome concentrations were important for the effectiveness of liposome/PEI. This finding suggests that PEI combined with liposomes and with slightly decreased numbers of primary amines may be an effective vaginally administrated antiviral drug, and secondary and tertiary amine residues of PEI may contribute to the inhibitory efficiency against viral infection.