OBJECTIVE:Anions in tears may be related to the pathogenesis of ocular surface diseases. A comprehensive method that can detect small quantities of anions in human tears has been developed. However, the differences in individual anion concentrations between tears and plasma remain unclear. This study investigated anion concentrations in dog tears. METHODS:To compare anion concentrations between tear and plasma samples in canines, 12 eyes of six healthy purpose-bred research Beagles (unneutered males, 3 years old) were investigated. Additionally, the tears of canines with various diseases were examined. PROCEDURE:Anion concentrations were assessed using a commercially available ion chromatography system. For this, at least 10 μL of tears were collected in a glass capillary tube and diluted with 300 μL of distilled water; plasma was obtained by centrifuging collected blood samples, which were then diluted 100-fold before chromatography. RESULTS:Anion concentrations differed significantly between the tears and plasma of Beagles. The average concentrations (mg/L) of anions in tears of Beagles were as follows: Cl-, 5374.06 ± 157.06; Br-, 27.84 ± 1.43; NO3 -, 0.70 ± 0.65; HPO4 2-, 77.54 ± 14.36; and SO4 2-, 16.41 ± 8.17. In plasma, the values were as follows: Cl-, 4603.81 ± 542.52; Br-, 18.34 ± 2.78; NO3 -, 1.31 ± 0.87; HPO4 2-, 83.63 ± 21.04; and SO4 2-, 105.14 ± 21.86. No statistically significant differences in anion concentrations were observed between tears from diseased and non-diseased eyes of canines. CONCLUSIONS:Herein, we observed that certain anions, such as Br- and Cl-, were consistently elevated in tear fluid than in the plasma.

Candida albicans can cause two major types of infections: superficial infection and systemic candidiasis. C. albicans infects diverse host niches, owing to a wide range of virulence factors and attributes, such as morphological transitions and phenotypic switching. C. albicans uses glycolysis, followed by alcoholic fermentation or mitochondrial respiration to rapidly generate ATP under aerobic conditions. In this study, we quantified the mRNA expression of several glycolysis-related enzymes associated with the initial phase of environmental changes using two strains: a type strain, NBRC 1385, and a strain from a patient with auto-brewery syndrome, LSEM 550. Additionally, we analyzed the regulation of a rate-limiting enzyme in glycolysis, phosphofructokinase 1 (PFK1). Our results showed that the mRNA expression of enzymes in the middle and last stages of glycolysis and alcoholic fermentation increased, and that of mitochondrial respiration enzymes decreased under short-term anaerobic conditions. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) administration showed similar results under anaerobic conditions. Moreover, PFK1 maintained its regulatory effect under different conditions; no significant change was observed in its mRNA expression. Our results suggest that C. albicans obtains energy via carbohydrate catabolism in the early phase of environmental change and survives in various parts of the host.

Previous studies on dolphin electrocardiograms have shown that they are mainly composed of increased negative waves, similar to ungulates. The electrocardiogram waveform was determined by the distribution of the Purkinje fibers. Based on the waveform of the dolphin electrocardiogram, Hamlin predicted that the Purkinje fibers would be distributed within the ventricular muscle, as in ungulates. The purpose of this study was to confirm the histological distribution of Purkinje fibers in dolphins. In the present study, bottlenose dolphin hearts were observed both grossly and histologically, and the effects of Purkinje fiber distribution and cardiac morphology on electrocardiogram waveforms were examined. This study showed that the Purkinje fibers of dolphins run just below the endocardium, as in humans, dogs, and cats, whose electrocardiograms mainly show positive waves. When the cardiac morphology of dolphins was observed carefully, the right ventricle was found to be extremely dilated compared to that of terrestrial mammals. In human recreational divers, right ventricular dilatation is induced by diving. We hypothesized that the dolphin's heart is in a state similar to that of the right heart dilatation in terrestrial animals. The dolphin electrocardiogram waveform was considered to be due to right axis deviation. Based on the above, we concluded that the dolphin electrocardiogram waveform was due to its ability to live in water. We found that the dolphins are genetically related to ungulates, particularly the hippopotamus, but that their hearts have evolved differently.

健常な27歳男性.発熱と全身痙攣のため受診.COVID-19抗原検査で陽性であり,胸部CTで肺炎像を認め,中等症IのCOVID-19の診断で入院となった.入院後中等症IIに増悪したが,ステロイド内服で軽快し退院した.しかし,発症後20日目に左上下肢の脱力が出現し,その2日後に再受診.精査の結果,右中大脳動脈領域の脳梗塞と左S状静脈洞血栓症の診断で再入院となった.その際,D-dimerは1.32 µg/mlと高度上昇は認めず,その他の凝固異常や血栓素因は認めなかった.脳梗塞の原因として,頭部MRIの経時的な変化から,脳動脈解離によるものと判断し,脳静脈洞血栓症と併せ,抗凝固療法と降圧管理を行い,粗大な麻痺なく退院となった.COVID-19感染により,脳動脈解離と血栓症という異なる機序の脳血管障害を同時に呈した,非常に稀な症例を報告する.

We share our experiences of instructing three patients with severe upper limb dysfunction on how to self-adjust CPAP/NPPV masks. In Case 1, we simplified the procedure by suturing a part of the headband as the left forearm was amputated. In Case 2, the patient had congenitally short limbs with short stature; thus, we provided an additional belt to the headband to maintain the headband's configuration while wearing the mask. In Case 3, the patient had left hemiplegia due to stroke and, repetitive coaching was conducted during the recovery phase rehabilitation program. Difficulties with self-adjusting NPPV/CPAP masks can occur whenever there is limited hand mobility above the head, including upper limb dysfunction. Simplifying procedures and providing sufficient time for instruction could help achieve independence. There have been no previous reports describing similar training details. We believe that sharing this knowledge will be helpful to both patients and healthcare professionals.

Background. Pleural mesothelioma is difficult to diagnose at an early stage and has a poor prognosis. This tumor may occasionally metastasize to other organs in the terminal stage. However, intrapulmonary miliary metastases are rare. We herein report a case of epithelioid pleural mesothelioma that presented with miliary metastases during chest imaging and an autopsy. Case. The patient was a 92-year-old woman without a history of asbestos exposure. The initial sign of mesothelioma was left pleural effusion. The patient lived for one year after the definitive diagnosis without specific treatment other than pleurodesis. Two months prior to death, chest radiography and computed tomography showed intrapulmonary miliary metastases. An autopsy confirmed this pattern of metastasis. The reason for the occurrence of miliary metastases remains unclear. However, the invasive characteristics of the tumor were speculated based on the histological and immunohistochemical findings. For example, a homozygous deletion of p16, which is related to a poor prognosis, was noted. In addition, the mesothelioma possessed micropapillary components, which indicated its aggressive characteristic. Thus, we speculated that these invasive characteristics contributed to hematogenous metastasis and lymphangitic spread and ultimately resulted in miliary metastases. Conclusion. We presented a case of epithelioid pleural mesothelioma with a rare metastatic pattern. We speculate that the pathogenesis of intrapulmonary miliary metastasis was related to the aggressive behavior, characterized by p16 deletion or micropapillary components.

Objective: Secretory immunoglobulin A (sIgA) is important for mucosal immunity due to the inhibition of pathogen infection. The submandibular gland is known to secrete more sIgA than the parotid and sublingual glands. In this study, we focused on the relationship between the secretion of accumulated intracellular sIgA and beta-adrenergic receptor stimulation, and clarified the autonomic regulatory mechanism of sIgA secretion in sub mandibular gland cells using dispersed gland cells. Design: Sprague-Dawley rats (male, 6 weeks old, 200-250 g) were euthanized and their submandibular glands were removed. Dispersed submandibular gland cells placed in Krebs-Ringer-Bicarbonate solution were stimulated by autonomic nerve agonists. The concentration of secreted sIgA was measured using a rat IgA ELISA kit. The results were analysed using ANOVA and Tukey's test. Results: Cells stimulated with the non-selective beta-adrenoreceptor agonist, isoprenaline, secreted significantly more sIgA compared with the unstimulated control. The beta 2-adrenoreceptor agonist, fenoterol, caused significantly more sIgA secretion than the control, and more sIgA secretion than the beta 1-adrenoreceptor agonist, xamoterol. sIgA secretion by isoprenaline stimulation was dose dependent. Inhibition of the beta receptor by propranolol completely blocked sIgA secretion following isoprenaline stimulation. Conclusion: Stimulation of beta receptors could result in more secretion of intracellularly accumulated sIgA compared with stimulation of other autonomic receptors in the autonomic modulation of mucosal immunity.

━━ Background. Most cases of non-small-cell lung cancer with skeletal muscle metastases show a poorer prognosis than those without skeletal muscle metastases. This is speculated to be partly due to the effect of epithelial-mesenchymal transition on tumor metastatic mechanisms. Although neither conventional chemotherapy nor radiotherapy has shown efficacy in cases of skeletal muscle metastases, little is known about the effec-tiveness of immune checkpoint inhibitors. Case. A 77-year-old man was injured in a motorbike accident, and he be-came aware of pain in his back and the back of his thighs and legs. The pain prevented him from remaining stand-ing. We found a tumor shadow in his right hilar region on chest X-ray at the first visit. We diagnosed him with large-cell lung cancer with multiple metastases to the mediastinum and abdominal lymph nodes, liver, bilateral adrenal glands, skin, and muscles (cT4N3M1c Stage IVB according to the UICC TNM classification, 8th edition). Immunohistochemical staining was positive for vimentin, and the programmed cell death 1 ligand 1 tumor proportion score was 95%. Therefore, we started treatment with pembrolizumab as immune checkpoint therapy. By the end of the first course of this regimen, the primary tumor as well as the metastatic lesions had markedly con-tracted, and his performance status had improved. Conclusion. A favorable response was obtained using the immune checkpoint inhibitor pembrolizumab for the treatment of non-small-cell lung cancer with skeletal muscle metastases. 2021;61:396-401) F igu re 2. Abdom i nal pelv i s contrast CT i mages obta i ned at basel i ne of mult i ple skeletal muscle metastases ( A, B, C ), after one course of pembrol i zumab ( D, E, F ), show i ng a remarkable reduct i on i n the tumor s i ze. F igu re 3. Immunoh i stochem i str y revealed that the mal i gnant cells were pos i t i ve for c y tokerat i n 7 ( A ) and v i ment i n ( B ).

Neutrophils are essential for innate immunity as the first line of defence. Neutrophils act as phagocytic white blood cells to kill bacteria and other microorganisms. A strong respiratory burst of neutrophils, dependent on reactive oxygen species, is produced during phagocytosis. Platelet-activating factor (PAF) is a signalling molecule with several prominent roles in tissue injury, inflammation, and platelet aggregation. However, the detailed mechanisms and intracellular signalling pathways involved in PAF-mediated neutrophil activation remain unclear. Here, we investigated the effect of PAF on changes in calcium concentration ([Ca2+](i)) and oxygen radical (O-2(-)) generation in activating canine neutrophils. We further evaluated these effects of PAF with inhibition of G protein-coupled receptors using the specific inhibitor suramin. Blood samples were collected from a total of five dogs and neutrophils were isolated. PAF stimulation of canine neutrophils caused an increase in [Ca2+](i) as well as the generation of O-2(-), and the PAF receptor was sensitive to suramin. The results suggested that PAF stimulation of canine neutrophils may cause Ca2+ influx from the endoplasmic reticulum into the cytoplasm (as the first wave) and then trigger store-operated Ca2+ entry (as the second wave), which is an important intracellular signal transduction pathway for neutrophil activation. Furthermore, O-2(-) generation by PAF stimulation may depend on the intracellular signalling pathway, with increasing inositol trisphosphate levels and [Ca2+](i) via G protein-coupled receptors. The finding that PAF-activating platelet aggregation is involved in canine neutrophil activation suggests a close relationship between haemostasis and neutrophil activation in dogs, offering new insight into the response to infection.

OBJECTIVE:The physiological expression of cell division cycle 42 (cdc42) in major salivary glands, and paracellular transport of fluorescein isothiocyanate-dextran (FITC-dextran) in SMIE cells, which regulate cdc42 expression, was investigated to clarify the involvement of cdc42 in salivary production.DESIGN:The physiological expression of cdc42 in the rat submandibular gland, parotid gland, sublingual gland, and SMIE cells was detected using SDS-PAGE and western blotting. The paracellular transport of FITC-dextran in transwells was compared in transfected SMIE cells, exhibiting up- or downregulated cdc42 expression.RESULTS:Cdc42 was expressed in all major salivary glands and SMIE cells. SMIE cells transfected with the cdc42 plasmid had an increase efflux. In addition, SMIE cells transfected with the cdc42 siRNA showed decreased efflux.CONCLUSION:We suggest that cdc42 enhances paracellular transport in salivary glands without any morphological changes, including cell-cell adhesion.

This study was undertaken to establish a method for measuring mRNA expression by using real-time RT-PCR in the diagnosis of canine meningiomas. When performing real-time RT-PCR, it is essential to include appropriate control tissues and to select appropriate housekeeping genes as an internal standard. Based on the results of our study, RPS18 constitutes a suitable internal standard for the comparison of mRNA expression between normal meninges and meningiomas. The results showed increased mRNA expression of VEGFA and EGFR; however, mRNA expression of KDR was reduced. Measuring mRNA expression by using real-time RT-PCR with appropriate control tissues and internal standards can provide useful information to understanding the pathogenesis of canine meningiomas, which corresponds with immunohistochemical findings.

In healthy individuals, plasma glucose levels are maintained within a normal range. During fasting, endogenous glucose is released either through glycogenolysis or gluconeogenesis. Gluconeogenesis involves the formation of glucose-6-phosphate from a variety of precursors followed by its subsequent hydrolysis to glucose. Gluconeogenesis occurs in the liver and the kidney. In order to compare gluconeogenesis in canine liver and kidney, the activity and expression of the rate limiting enzymes that catalyze the fructose-6-phosphate and fructose 1,6-bisphosphate steps, namely, phosphofructokinase-1 (PFK-1) (glycolysis) and fructose bisphosphatase-1 (FBP-1) (gluconeogenesis), were examined. Healthy male and female beagle dogs aged 1-2 years were euthanized humanely, and samples of their liver and kidney were obtained for analysis. The levels of PFK-1 and FBP-1 in canine liver and kidney were assessed by enzymatic assays, Western blotting, and RT-qPCR. Enzyme assays showed that, in dogs, the kidney had higher specific activity of PFK-1 and FBP-1 than the liver. Western blotting and RT-qPCR data demonstrated that of the three different subunits (PFK-M, PFK-L, and PFK-P) the PFK-1 in canine liver mainly comprised PFK-L, whereas the PFK-1 in the canine kidney comprised all three subunits. As a result of these differences in the subunit composition of PFK-1, glucose metabolism might be regulated differently in the liver and kidney.

Phosphofructokinase-1 (EC: 2.7.1.11, PFK-1) catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate using adenosine triphosphate and is a key regulatory enzyme of glycolysis. Mammalian PFK-1 isozymes are composed of three kinds of subunits (PFK-M, -L, and -P), with different properties. It has been suggested that the proportion of PFK-1 subunits in different organs is based on the organ energy metabolism. In this study, we analyzed the activity and subunit composition of canine PFK-1. We found that, in dogs, the skeletal muscle only has PFK-M, the liver mainly has PFK-L, and the brain expresses all of them. The knowledge of the composition of PFK-1 could provide useful information for determination of the differences in glycolysis in various organs of dogs.

Phosphofructokinase-1 (PFK-1) is the most important rate-controlling enzyme for glycolysis in both prokaryotes and eukalyotes. PFK-1 activity is regulated by multiple cellular metabolites, including nucleotides. Intracellular nucleotides are produced through various metabolic processes, including energy metabolism and intracellular signaling pathways. The activity of PFK-1, purified from canine skeletal muscle, was evaluated in the presence of various concentrations of adenine nucleotides to examine the regulation of glucose catabolism in canine skeletal muscle. Although UTP did not inhibit PFK-1 activity to the same extent as ATP, it substituted for ATP as a phosphate donor. cAMP functioned in a similar manner as AMP, as an activating effector of the PFK-1 reaction. ADP activated PFK-1 at low concentrations, but slightly inhibited PFK-1 at higher concentrations. The results suggested that canine PFK-1 had three different binding sites for adenine nucleotides acting as phosphate donors, activating effectors, and inhibitory effectors. Moreover, PFK-1 was shown to be activated by AMP and inhibited by ATP, while UTP and cAMP regulated PFK-1 activity. The results also suggested that ADP binds to the allosteric sites (for ATP and AMP) of PFK-1. Each adenine nucleotide functions as either an activating or inhibitory effector of PFK-1 reaction in canine skeletal muscle. Therefore, intracellular nucleotides may play an important role in regulating glucose metabolism by binding to the allosteric site of the enzyme.

The proinflammatory cytokine interleukin 1β (IL-1β) induces prostaglandin E 2 (PGE 2 ) production via upregulation of cyclooxygenase-2 (COX-2) expression in synovial fibroblasts. This effect of IL-1β is involved in osteoarthritis. We investigated MAPK signaling pathways in IL-1β-induced COX-2 expression in feline synovial fibroblasts. In the presence of MAPK inhibitors, IL-1β-induced COX-2 expression and PGE 2 release were both attenuated. IL-1β induced the phosphorylation of p38, JNK, MEK, and ERK1/2. A JNK inhibitor prevented not only JNK phosphorylation but also MEK and ERK1/2 phosphorylation in IL-1β-stimulated cells, but MEK and ERK1/2 inhibitors had no effect on JNK phosphorylation. A p38 inhibitor prevented p38 phosphorylation, but had no effect on MEK, ERK1/2, and JNK phosphorylation. MEK, ERK1/2, and JNK inhibitors had no effect on p38 phosphorylation. We also observed that in IL-1β-treated cells, phosphorylated MEK, ERK1/2, and JNK were co-precipitated with anti-phospho-MEK, ERK1/2, and JNK antibodies. The silencing of JNK1 in siRNA-transfected fibroblasts prevented IL-1β to induce phosphorylation of MEK and ERK1/2 and COX-2 mRNA expression. These observations suggest that JNK1 phosphorylation is necessary for the activation of the MEK/ERK1/2 pathway and the subsequent COX-2 expression for PGE 2 release, and p38 independently contributes to the IL-1β effect in synovial fibroblasts.

Acute kidney injury (AKI) is characterized by a sudden loss of renal function. Early recognition of AKI, especially in critically ill patients, is essential for adequate therapy. Currently, neutrophil gelatinase-associated lipocalin (NGAL) is considered to be an effective biomarker of AKI; however, the regulation of its expression and function in renal tubular cells remains unclear. In this study, we investigated the regulation of the expression and function of NGAL in IL-1β-treated Madin-Darby canine kidney (MDCK) cells as a model of renal tubular cells. IL-1β induced a disturbance in the localization of E-cadherin and zonaoccludin-1 (ZO-1). The transepithelial electrical resistance (TER) also decreased 5 days after IL-1β treatment. IL-1β induced NGAL mRNA expression and protein secretion in a time- and dose-dependent manner, which occurred faster than the decrease in TER. In the presence of ERK1/2 and p38 inhibitors, IL-1β-induced NGAL mRNA expression and protein secretion were significantly attenuated. In the presence of recombinant NGAL, IL-1β-induced disturbance in the localization of E-cadherin and ZO-1 was attenuated, and the decrease in TER was partially maintained. These results suggest that NGAL can be used as a biomarker for AKI and that it functions as a protector from AKI.

The proinflammatory cytokine interleukin-1β (IL-1β) induced cyclooxygenases-2 (COX-2) mRNA expression and lipid mediator prostaglandin E2 release and in a time- and dose-dependent manner in canine dermal fibroblasts. The MEK inhibitor U0126 and the ERK inhibitor FR180204 clearly inhibited IL-1β-induced prostaglandin E2 release and COX-2 mRNA expression. IL-1β enhanced ERK1/2 phosphorylation, which was attenuated by inhibitors of MEK and ERK. The NF-κB inhibitor BAY 11-7082 also suppressed IL-1β-induced prostaglandin E2 release and COX-2 mRNA expression. Treatment of fibroblasts with IL-1β led to the phosphorylation of p65 and degradation of IκBα occurred, indicating that IL-1β treatment activated NF-κB. MEK and ERK1/2 inhibitors had no effect on the phosphorylation of p65 subunit induced by IL-1β, whereas the NF-κB inhibitor completely blocked IL-1β-induced phosphorylation of ERK1/2. We also observed that IκBα-knockdown enhanced the phosphorylation of p65 and ERK1/2. These findings suggest that stimulation of MEK/ERK signaling pathway by NF-κB activation regulates IL-1β-induced COX-2 expression and subsequent prostaglandin E2 release in canine dermal fibroblasts.

Bone marrow stromal cells (BMSCs) are considered as candidates for regenerative therapy and a useful model for studying neuronal differentiation. The role of basic fibroblast growth factor (bFGF) in neuronal differentiation has been previously studied; however, the signaling pathway involved in this process remains poorly understood. In this study, we investigated the signaling pathway in the bFGF-induced neuronal differentiation of canine BMSCs. bFGF induced the mRNA expression of the neuron marker, microtubule associated protein-2 (MAP2) and the neuron-like morphological change in canine BMSCs. In the presence of inhibitors of fibroblast growth factor receptors (FGFR), phosphatidylinositol 3-kinase (PI3K) and Akt, i.e., SU5402, LY294002, and MK2206, respectively, bFGF failed to induce the MAP2 mRNA expression and the neuron-like morphological change. bFGF induced Akt phosphorylation, but it was attenuated by the FGFR inhibitor SU5402 and the PI3K inhibitor LY294002. In canine BMSCs, expression of FGFR-1 and FGFR-2 was confirmed, but only FGFR-2 activation was detected by cross-linking and immunoprecipitation analysis. Small interfering RNA-mediated knockdown of FGFR-2 in canine BMSCs resulted in the attenuation of bFGF-induced Akt phosphorylation. These results suggest that the FGFR-2/PI3K/Akt signaling pathway is involved in the bFGF-induced neuronal differentiation of canine BMSCs.