Innate immune response of bovine mammary epithelial cells in Mycoplasma bovis mastitis using an in vitro model of bovine mammary gland infection.

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland's defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1ß,IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1ß concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.

Protective effects of probiotics against tannin-induced immunosuppression in broiler chickens.

Tannins (TAs) are an anti-nutritional substance commonly used as a natural feed additive for livestock. However, our previous study described the dose-dependent adverse effects of TA on immune responses and growth in chickens. In this study, we evaluated the protective effects of a probiotic preparation (BT) consisting of three different bacteria (Bacillus mesenteric, Clostridium butyricum, and Streptococcus faecalis) against TA-induced immunosuppression in chickens. Forty chicks were divided into 4 groups as follows: the CON group (basal diet), BT group supplemented with 3 g BT/kg diet, tannic acid (TA) group supplemented with 30 g TA/kg diet, and BT+TA group supplemented with 3 g BT/kg diet + 30 g TA/kg diet. The feeding trial lasted for 35 days. Lymphocyte subset, macrophage phagocytosis, cytokine mRNA expression, and primary and secondary IgY immune responses were evaluated. BT supplementation significantly improved TA-induced reductions in final body weight, body weight gain, feed intake, and relative weights of lymphoid organs compared with the TA group. Furthermore, in the spleen and cecal tonsil (CT), the relative populations of CD4+, CD8+, and CD4+CD8+ cells in the BT+TA group were significantly ameliorated compared with the TA group. Additionally, comparison with the TA group showed that the chickens in the BT+TA group had an improved relative population of B cells in the CT and that macrophage phagocytosis in the spleen was significantly increased. Chickens in the BT+TA group showed significant increases in IFN-γ and IL-4 mRNA expression in the spleen compared with the TA group. The primary and secondary IgY responses were significantly improved. These results revealed that supplementation with BT protects against TA-induced immunosuppression in chickens.

Effects of branched-chain amino acids on immune status of young racing horses.

High-intensity exercise and competition are associated with depressed immune function. Young horses, which participate in high-intensity exercise and competitions, are at increased risk for the development of infectious disease due to depression of immune function. The effects of branched-chain amino acid (BCAA) supplementation on the immune status of young racing horses were evaluated, determining whether BCAA might help to avoid or reduce immune suppression during exercise and competitions. Twenty horses (10 male and 10 female) were treated with BCAA supplementation; another twenty untreated horses (10 male and 10 female) constituted control group. Peripheral blood was collected from each animal and evaluated for lymphocyte subsets, phagocytosis analysis of monocytes and granulocytes, lymphocyte proliferative response, and expression of cytokine-encoding messenger ribonucleic acids (mRNAs). The numbers of CD4+, CD8+, and major histocompatibility complex (MHC) class II+ cells in females of the treated group were significantly higher than those in females of the control group. The lymphocyte proliferative response in female of the treated group also was significantly higher than that in females of the control group. In addition, expression of mRNAs encoding interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ) in females of the treated group was significantly higher than that in females of the control group. There were no significant differences between males of the treated and control groups. The results of this study indicated the positive effects of BCAA supplementation in counteracting immunosuppression in young female racing horses during and following high-intensity exercise.

Reproductive cycle and maturation of Swinhoe's tree lizard (Diploderma swinhonis (Günther, 1864)) in Hyuga City, Miyazaki Prefecture, Japan.

Swinhoe's tree lizard (Diploderma swinhonis) is an arboreal agamid that is native to Taiwan. The species has been introduced to some areas of Japan and is regarded as an invasive alien species. In 2016, a nonnative population of D. swinhonis was discovered in Hyuga City, Miyazaki Prefecture, Japan, but little information was available on the ecology of the population at the time. The main purpose of this study was therefore to investigate the reproductive cycle and maturation of this population. Field research was conducted from 2017 to 2019, and 764 lizards were collected. Euthanized lizards were dissected and the reproductive organs were examined to determine the reproductive period, clutch size, clutch frequency and size at sexual maturity. Females with oviductal eggs or vitellogenic ovarian follicles were observed from May to October. Clutch size ranged from 2 to 8, and clutch frequency was more than twice a year. In males, spermiogenesis started in early May and testicular regression was observed in September. Males with spermatozoa in the epididymides were found from May to November. Minimum snout-vent length at sexual maturity was 50.2 mm in females and 53.0 mm in males. Comparisons of the findings of this study and reports from Taiwan suggest that the nonnative population of D. swinhonis in Hyuga City has a higher fecundity than populations in Taiwan. It is therefore considered necessary to exterminate the population in Hyuga City before this species colonizes other areas.

TRPA1 Channels Modify TRPV1-Mediated Current Responses in Dorsal Root Ganglion Neurons.

The transient receptor potential vanilloid 1 (TRPV1) channel is highly expressed in a subset of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia of experimental animals, responsible for nociception. Many researches have revealed that some TRPV1-positive neurons co-express the transient receptor potential ankyrin 1 (TRPA1) channel whose activities are closely modulated by TRPV1 channel. However, it is less investigated whether the activities of TRPV1 channel are modulated by the presence of TRPA1 channel in primary sensory neurons. This study clarified the difference in electrophysiological responses induced by TRPV1 channel activation between TRPA1-positive and TRPA1-negative DRG. TRPV1 and TRPA1 channel activations were evoked by capsaicin (1 µM), a TRPV1 agonist, and allyl isothiocyanate (AITC; 500 µM), a TRPA1 agonist, respectively. Capsaicin perfusion for 15 s caused a large inward current without a desensitization phase at a membrane potential of -70 mV in AITC-insensitive DRG (current density; 29.6 ± 5.6 pA/pF, time constant of decay; 12.8 ± 1.8 s). The capsaicin-induced currents in AITC-sensitive DRG had a small current density (12.7 ± 2.9 pA/pF) with a large time constant of decay (24.3 ± 5.4 s). In calcium imaging with Fura-2, the peak response by capsaicin was small and duration reaching the peak response was long in AITC-sensitive neurons. These electrophysiological differences were completely eliminated by HC-030031, a TRPA1 antagonist, in an extracellular solution or 10 mM EGTA, a Ca2+ chelator, in an internal solution. Capsaicin perfusion for 120 s desensitized the inward currents after a transient peak. The decay during capsaicin perfusion was notably slow in AITC-sensitive DRG; ratio of capsaicin-induced current 60 s after the treatment per the peak current in AITC-sensitive neurons (78 ± 9%) was larger than that in AITC-insensitive neurons (48 ± 5%). The capsaicin-induced current in the desensitization phase was attenuated by HC-030031 in AITC-insensitive DRG. These results indicate that (1) TRPV1-mediated currents in TRPA1-positive neurons characterize small current densities with slow decay, which is caused by TRPA1 channel activities and intracellular Ca2+ mobilization and (2) desensitization of TRPV1-mediated current in TRPA1-positive neurons is apparently slow, due to appending TRPA1-mediated current.

Long-Term Activation of Group I Metabotropic Glutamate Receptors Increases Functional TRPV1-Expressing Neurons in Mouse Dorsal Root Ganglia.

Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1) in dorsal root ganglion (DRG) neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h) treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG) increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC), a transient receptor potential ankyrin type 1 (TRPA1) agonist. Increase in the proportion was suppressed by phospholipase C (PLC), protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia.

Effects of cyanamide, an aldehyde dehydrogenase type 2 inhibitor, on glyceryl trinitrate- and isosorbide dinitrate-induced vasodilation in rabbit excised aorta and in anesthetized whole animal.

The contribution of aldehyde dehydrogenase type 2 (ALDH2) to bioactivation of glyceryl trinitrate (GTN) and isosorbide dinitrate (ISDN) was systematically examined in excised rabbit aorta and anesthetized whole animal with cyanamide, an ALDH2 inhibitor. In excised aortic preparation, the degree of inhibition by cyanamide in GTN-induced vasorelaxation (concentration ratio, calculated as EC(50) in the presence of cyanamide/EC(50) in the absence of cyanamide; 5.61) was twice that in ISDN-induced relaxation (2.78). However, the degree of inhibition by cyanamide, as assessed by the dose ratio (as described above, but calculated with doses) in anesthetized rabbits was 2.29 in GTN-induced hypotension (assessed by area under the curve (AUC) of 50 mmHg·min) and 7.68 in ISDN-induced hypotension. Thus, the inhibitor was 3 times more potent in ISDN-induced hypotension, a finding in conflict with to that obtained in excised aortic preparation. The rate of increase in plasma nitrite (NO(2)(-)) concentration at certain hypotensive effect (50 mmHg·min of AUC) in the presence and absence of cyanamide (ΔNO(2)(-) ratio) was larger in ISDN-induced hypotension (15.01) than in GTN-induced hypotension (3.28). These results indicate that the bioactivation pathway(s) of GTN is ALDH2-dependent in aortic smooth muscle, while ADLH2-independent mechanism(s) largely take place in the whole body. In contrast, the activation mechanism(s) of ISDN is largely ALDH2-dependent in both aortic smooth muscle and whole body. Plasma NO(2)(-) may be derived from pathways other than the cyanamide-sensitive metabolic route.

Resveratrol improves oxidative stress and protects against diabetic nephropathy through normalization of Mn-SOD dysfunction in AMPK/SIRT1-independent pathway.

OBJECTIVE: Despite the beneficial effects of resveratrol (RSV) on cardiovascular disease and life span, its effects on type 2 diabetic nephropathy remain unknown. This study examined the renoprotective effects of RSV in db/db mice, a model of type 2 diabetes. RESEARCH DESIGN AND METHODS: db/db mice were treated with RSV (0.3% mixed in chow) for 8 weeks. We measured urinary albumin excretion (UAE), histological changes (including mesangial expansion, fibronectin accumulation, and macrophage infiltration), oxidative stress markers (urinary excretion and mitochondrial content of 8-hydroxy-2'-deoxyguanosine [8-OHdG], nitrotyrosine expression), and manganese-superoxide dismutase (Mn-SOD) activity together with its tyrosine-nitrated modification and mitochondrial biogenesis in the kidney. Blood glucose, glycated hemoglobin, and plasma lipid profiles were also measured. The phosphorylation of 5'-AMP-activated kinase (AMPK) and expression of silent information regulator 1 (SIRT1) in the kidney were assessed by immunoblotting. RESULTS: RSV significantly reduced UAE and attenuated renal pathological changes in db/db mice. Mitochondrial oxidative stress and biogenesis were enhanced in db/db mice; however, Mn-SOD activity was reduced through increased tyrosine-nitrated modification. RSV ameliorated such alterations and partially improved blood glucose, glycated hemoglobin, and abnormal lipid profile in db/db mice. Activation of AMPK was decreased in the kidney of db/db mice compared with db/m mice. RSV neither modified AMPK activation nor SIRT1 expression in the kidney. CONCLUSIONS: RSV ameliorates renal injury and enhanced mitochondrial biogenesis with Mn-SOD dysfunction in the kidney of db/db mice, through improvement of oxidative stress via normalization of Mn-SOD function and glucose-lipid metabolism. RSV has antioxidative activities via AMPK/SIRT1-independent pathway.

Cushing's syndrome and big IGF-II associated hypoglycaemia in a patient with adrenocortical carcinoma.

A 41-year-old woman had a general health examination and was diagnosed with a non-functioning adrenocortical carcinoma (ACC). Despite surgery and chemotherapy with mitotane, the ACC progressed with metastases to the lymph nodes, liver and lung. Initially, she developed adrenal insufficiency and was treated with hydrocortisone. As the ACC progressed, it produced superabundant cortisol, resulting in clinically overt Cushing's syndrome. As the liver metastases grew, the patient developed hypoglycaemia with suppression of endogenous insulin secretion. She had to be given large quantities of glucose intravenously to remain normoglycaemic. The serum insulin-like growth factor (IGF)-II/IGF-I ratio had increased to 84. We identified big IGF-II, a primary hormonal mediator of non-islet cell tumour hypoglycaemia (NICTH), in the serum and tumour using western blotting. This is the first case of ACC that showed both Cushing's syndrome and NICTH associated with big IGF-II.

Unilateral cone dysfunction as a manifestation of acute zonal occult outer retinopathy.

PURPOSE: To report a patient with unilateral cone dysfunction (UCD) who later developed acute zonal occult outer retinopathy (AZOOR) in the contralateral eye. METHODS: A 19-year-old Japanese woman was referred complaining of decreased vision and photopsia of the left eye. Static perimetry, full-field electroretinography (ERG), and multifocal ERGs (mfERGs) were performed to evaluate her visual functions. She returned 9 months later with visual field defect and photopsia in the right eye. RESULTS: The static visual field of the left eye demonstrated a scotoma that extended from the physiologic blind spot to the center of the visual field. The cone full-field ERGs were extinguished with preservation of rod function. The mfERGs were reduced throughout the posterior pole of the left eye. The patient was diagnosed with UCD. She revisited us 9 months later complaining of visual symptoms in the right eye and was found to have an arcuate scotoma in the upper visual field corresponding to decreased mfERGs. These findings were consistent with clinical signs of AZOOR. CONCLUSION: These findings indicate that UCD is one of the clinical manifestations of AZOOR.

Capacitative Ca2+ entries and mRNA expression for TRPC1 and TRPC5 channels in human epidermoid carcinoma A431 cells.

In human epidermoid carcinoma A431 cells, capacitative Ca2+ entries in response to intracellular Ca2+ store depletion with thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, and uridine 5'-triphosphate, a phospholipase C-linked agonist, were inhibited by trivalent cations such as Gd3+ and La3+, and by the store-operated Ca2+ channel inhibitor, 2-aminoethoxydiphenyl borate. Of the seven types of canonical transient receptor potential (TRPC) channels as molecular candidates for store-operated Ca2+ channels, mRNAs for TRPC1 and TRPC5 were detected in the cells with the reverse transcription-polymerase chain reaction. Western blotting confirmed the protein expressions of TRPC1 and TRPC5 in A431 cells. The present results suggest that TRPC1 and/or TRPC5 channels serve as store-operated Ca2+ channels in A431 cells, and may function as regulators for intracellular Ca2+ signaling.