Enhancement of natural killer cell activity by oral administration of a fermented soybean product in dogs.

Background: Probiotics are known for their ability to enhance cellular immunity, including the activation of macrophages, natural killer (NK) cells, and cytotoxic T lymphocytes. Natto is a Japanese traditional probiotic food made by fermenting soybean with bacteria Bacillus subtilis var. natto. Components of natto include spores of B. subtilis natto, poly-γ-glutamic acid, and levan, which have demonstrated their immunoadjuvant and anti-allergic effects through various in vitro and in vivo studies. However, it remains unclear whether oral administration of natto can modulate the immune activity in animals. Aim: This study aimed to investigate the effects of oral administration of natto on the immune system of dogs. Methods: Eight dogs were randomly divided into two groups: a natto-treated group and an untreated group. The dogs in the natto-treated group were fed with 10 g/head/day of a freeze-dried natto product in addition to a usual amount of regular dry food for 14 days, whereas the dogs in the untreated group were fed with the regular dry food alone. To determine cellular immune activity, the cell surface antigen analysis of peripheral blood lymphocytes and cytotoxicity analysis of peripheral blood mononuclear cells were carried out before and after the natto administration period. Additionally, a relative expression of inflammatory cytokines in peripheral blood monocytes after the introduction of antigen-stimulation was also examined. Results: At the end of the administration period, a proportion of NK cells (CD3- CD5- CD21- cells and CD3+ CD5dim CD8+ cells) in peripheral blood lymphocytes were found to be significantly increased, and the cytotoxic effect of the peripheral blood mononuclear cells on canine tumor cells were greatly enhanced in the natto-treated group, but not in the untreated group. The expression of TNF-α in peripheral blood mononuclear cells following an antigen-stimulation was increased considerably in the dogs after administration of natto. Conclusion: We conclude that oral administration of natto activated the cytotoxic activity of peripheral NK cells in dogs, and a daily intake of natto might be helpful in augmenting cellular immune activity.

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs.

Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.

Evaluation of monitoring methods in asymptomatic dogs with high serum cystatin C concentrations.

This study evaluated the monitoring methods in asymptomatic dogs with high serum cystatin C (Cys-C) concentrations. Ten dogs with high serum Cys-C were divided into two groups based on the owner's choice; one receiving clinical pathology-based monitoring at an animal hospital specialised in chronic kidney disease, and the other receiving symptom-based monitoring at home, partly because they showed no clinical symptoms. The dogs that received the clinical pathology-based monitoring led to an early treatment intervention, resulted in a longer survival period than dogs received the symptom-based monitoring (P<0.05). It became clear that early treatment intervention by clinical pathology-based monitoring extends the renal survival period even in asymptomatic dogs with increased serum Cys-C concentrations.

Concentrations of leptin, adiponectin, and resistin in the serum of obese cats during weight loss.

We monitored changes in serum leptin, adiponectin, and resistin concentrations in obese cats during weight loss. Six naturally developed obese cats were fed low-fat, high-fiber dry food during a 9-week experimental period. Serum leptin, adiponectin, and resistin concentrations were measured at week 0, 4, 8, and 9. Body weight became significantly lower week 4 onward than that at week 0 (P<0.05 or 0.01). At week 9, serum leptin concentrations were significantly lower than those at week 0 (P<0.05). Contrarily, serum adiponectin and resistin concentrations did not significantly differ within the 9 weeks. While serum leptin levels were strongly positively correlated with body weight (r=0.923, P<0.001), serum adiponectin levels were moderately negatively correlated with it (r=-0.529, P<0.01), with serum resistin having a no correlation with body weight. Serum leptin levels might be more closely related with pathogenesis of adiposity than serum adiponectin or resistin in cats.

Serum cystatin C concentration measured routinely is a prognostic marker for renal disease in dogs.

This study examined the predictive value of serum cystatin C (Cys-C) concentration, measured during routine periodic health examinations, in the renal prognosis of dogs. A cohort of 140 dogs weighing <15 kg whose serum Cys-C concentrations were measured during periodic health examinations from December 2013 to March 2016 were prospectively studied, with renal disease-related death the predicted end point. Of the 140 dogs, nine died from renal diseases during the follow-up period (539 ±â€¯249 days). Serum Cys-C concentrations were higher in the dogs that subsequently died of renal disease than in the censored group (0.8 ±â€¯0.25 vs. 0.3 ±â€¯0.1 mg/dl, respectively; P < .01). Dogs with high serum Cys-C concentrations (>0.55 mg/dl) had a shorter (P < .01) renal disease-specific survival period than those with low serum Cys-C concentrations (≤0.55 mg/dl). In conclusion, high serum Cys-C concentrations in periodic health examinations in dogs <15 kg predicted poorer prognosis for renal function.

Molecular cloning of feline resistin and the expression of resistin, leptin and adiponectin in the adipose tissue of normal and obese cats.

Resistin, one of the adipokines that has a cycteine-rich C-terminus, is considered to relate to the development of insulin resistance in rats. However, in cats, there is little knowledge regarding resistin. In this study, we cloned the feline resistin cDNA from adipose tissue by RT-PCR. The feline resistin clone contained an entire open reading frame encoding 107 amino acids that had 72.8%, 75.4%, 50.9% and 51.8% homology with bovine, human, mouse and rat homologues, respectively. In both subcutaneous and visceral adipose tissues, the transcription levels of feline resistin mRNA were significantly higher in obese cats than normal cats, and those of feline adiponectin mRNA were significantly lower in obese cats than normal cats. However, there was no difference in the expression of feline leptin between normal and obese cats. On the other hand, in both normal and obese cats, there were no significant differences in resistin, leptin and adiponectin mRNA levels between subcutaneous and visceral adipose tissues. In cats, the altered expression of resistin and adiponectin mRNA with obesity may contribute to the pathogenesis of insulin resistance and subsequent diabetes mellitus. In addition to feline adiponectin, the feline resistin cDNA clone obtained in this study will be useful for further investigation of the pathogenesis of obesity in cats.

Magnetic Resonance Imaging of Ovarian Activity in Microminipigs Showing Normal Estrous Cycles.

We investigated whether magnetic resonance imaging (MRI) might be applicable to evaluation of the ovarian activity of microminipigs. Firstly, using three mature microminipigs, we confirmed ovarian position and morphology by laparotomy or laparoscopy, and then acquired MRI images in various patterns to determine the most suitable condition for the acquisition. Next, using four microminipigs, we performed daily MRI, starting 10 days after ovulation and ending 10 days after the subsequent ovulation, as the starting day of standing estrus was taken as day 0. While the ovarian structure could not be discriminated on T1-weighted imaging, it was possible to confirm the follicles during estrus as hyperintense regions on T2-weighted imaging. With chronological MRI, 3-5 follicles were visible on T2-weighted imaging during the interval from day -2 to day 1, and their size immediately prior to ovulation was 3-5 mm. However, confirmation of the presence of small follicles and the corpus luteum was difficult.

Gallbladder lymphoma in a miniature dachshund.

A 7-year-old, miniature dachshund was referred for examination and treatment of persistent anorexia, deep yellow-coloured urine and leucocytosis. The clinical sign of jaundice, results from a serum biochemistry profile and ultrasonographic images suggested a biliary tract obstruction. A cholecystectomy was performed to remove the obstruction. Histopathological assessment of the resected gallbladder and partial common bile duct indicated diffuse large B-cell lymphoma. Twelve days after the initial operation, a second procedure was performed due to bile leakage into the abdominal cavity. Chemotherapy was administered twice after the second operation but discontinued, because the dog showed adverse effects. The dog is still alive 24 months after the surgery. To the authors' knowledge, this is the first description of canine gallbladder lymphoma.

Differentiation ability of multipotent hematopoietic stem/progenitor cells detected by a porcine specific anti-CD117 monoclonal antibody.

CD117 is a cytokine receptor expressed on the surface of hematopoietic stem cells with a likely role in cell survival, proliferation and differentiation. In order to study the differentiation activity of porcine CD117 hematopoietic cells in vitro and in vivo we prepared an anti-swine CD117 Mab (2A1) with high specificity for flow-cytometrical analysis. The 2A1 Mab did not recognize mouse or human mast cells suggesting that 2A1 is species-specific. Swine bone marrow (BM) CD117+ cells differentiated in vitro mainly into erythroid and monocyte lineages in the methylcellulose-based colony assay. When the swine BM CD117+ cells were transplanted in vivo into immunodeficient NOG (NOD/SCID/IL-2gc-null) mice, a significant amount of swine CD45+ leukocytes, including CD3 positive T cells, were developed in the mice. These results revealed that the swine BM CD117+ cells possess hematopoietic stem/progenitor activity and when monitored in immunodeficient mice or in vitro they can develop into lymphoid, erythroid, and myeloid cells efficiently with the new monoclonal antibody.

Computer tomography diagnosis of meningoencephalocele in a calf.

A 24-day-old female Holstein calf had a soft, painless fluctuating swelling on the median plane in the frontal region, but did not show any clinical symptoms including neurological signs. Computer tomography (CT) distinctly showed the cyst filled with fluid and part of the encephalon. Hence, this swelling was diagnosed as meningoencephalocele, but not meningocele. The meningoencephalocele was successfully repaired surgically. Meningoencephalocele can thus be easily recognised by CT in a calf.

Expression analysis of CCL27 and CCL28 mRNA in lesional and non-lesional skin of dogs with atopic dermatitis.

Chemokines are important regulators of the selective recruitment of inflammatory cells into sites of allergic inflammation. Since canine atopic dermatitis (AD) shares many clinical features of human AD, patterns of chemokine production in dogs may also be similar with those in humans. The aim of this study was to examine mRNA expression of CCL27 and CCL28 in lesional skin of dogs with AD to demonstrate similarity of chemokine production with human counterparts. RNA was extracted from skin biopsy specimens of 12 dogs with AD. The mRNA expression of CC chemokines (CCL4, CCL19, CCL20, CCL21, CCL24, CCL27 and CCL28) was analyzed by quantitative real-time PCR and was compared between lesional and non-lesional skin. Seven types of chemokines examined were constitutively expressed in both lesional and non-lesional skin. It was found that mRNA expression levels of CCL27 and CCL28 among the chemokines were significantly different between lesional and non-lesional skin (P<0.05). Expression level of CCL27 mRNA in lesional skin was significantly lower than that in non-lesional skin. On the other hand, CCL28 mRNA expression in lesional skin was found to be higher than that in non-lesional skin. These results suggest that CCL28 but not CCL27 may play important roles in immunopathogenesis of canine AD, indicating that experimental canine study may provide additional information that can be extrapolated to human AD.

Japanese black cattle with orotic aciduria detected by gas-chromatography/mass-spectrometry.

A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectrometry (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.

Pericardial mesothelioma in a neonatal calf.

We encountered an extremely rare tumor, a pericardial mesothelioma, in a neonatal calf. The patient calf showed severe abdominal distention, and died immediately after birth. The thoracic cavity was contained a huge heart with a large amount of pericardial fluid. A number of granular and cobblestone-like nodules were dispersed over the epicardium and pericardium. The nodules consisted of papillary proliferations of neoplastic cells, and the neoplasm occasionally showed mesenchymal proliferations. Immunohistochemistry revealed that they had the characteristics of mesothelial cells (cytokeratin-and vimentin-positive), and the neoplasm was diagnosed as mesothelioma.

Serum growth hormone and insulin-like growth factor-1 concentrations in Japanese black cattle with renal tubular dysplasia.

To elucidate the cause of growth retardation, 14 cattle with renal tubular dysplasia (RTD) were investigated by determining serum growth hormone (GH) and insulin-like growth factor (IGF)-1 concentrations. Compared with 6 healthy cattle, the baseline, maximum, area under the curve and peak amplitude of serum GH concentrations were higher or tended to be higher. Serum IGF-1 concentrations were lower normal or lower in cattle with RTD than in healthy cattle. Serum IGF-1 concentrations correlated significantly with body weight and chest girth, but not with serum GH and creatinine concentrations. Growth retardation in RTD might be associated partially with GH resistance.

Truncation and mutagenesis analysis of the human X-arrestin gene promoter.

X-arrestin (arrestin-3) is an arrestin present specifically in the outer segments of red-, green-, and blue-cone photoreceptors. The X-arrestin gene is on Xcen-q22, and consists of 17 exons with a promoter containing a TATA box and elements important for photoreceptor expression, including three CRX and one PCE-1-like element. In order to delineate the promoter structure necessary for the pan-cone-specific expression of X-arrestin, the expression of the gene in retinoblastoma cell lines was investigated, and a structure-function analysis of the promoter was conducted in the appropriate cellular substrate. Expression of X-arrestin was detected at a low level in the Y79 retinoblastoma cell line but not in the WERI retinoblastoma cell line. Truncation and expression analysis of the X-arrestin promoter in Y79 showed maximal activity in the proximal 378-bp region containing the CRX and PCE-1-like elements upstream of the TATA and CAAT boxes and a negative regulator in the distal 1-2-kbp region. Mutagenesis of the three CRX and PCE-1-like elements and expression analysis demonstrated complete elimination of the promoter activity. Mutagenesis of the TATA box and PCE-1-like element individually resulted in similar decrease in promoter activity, but the decrease in the promoter activity was greater when the CRX elements were mutagenized with a 5' to 3' spatial gradient in the negative effect, suggesting a cooperative effect of the three CRX elements. The regulation of expression from this promoter may involve the binding of a multi-protein enhanceosome complex at the CRX triplet and the PCE-1-like element, resulting in the recruitment and activation of the RNA polymerase II complex at the downstream TATA box.

Penetrating keratoplasty surgery combined with vitrectomy after failing previous corneal surgery.

PURPOSE: The preoperative and postoperative factors influencing visual outcome were analyzed in 15 eyes of 15 patients with graft opacity after keratoplasty associated with vitreoretinal disease who underwent combined surgery of fresh corneal retransplantation and vitrectomy. MATERIALS AND METHODS: The data obtained consisted of diagnosis, preoperative visual acuity, corneal and vitreoretinal findings at the time of surgery, interval between the first visit and surgery, intraocular pressure before surgery, gonioscopic findings, results of bacterial culture of surgical specimens, surgical procedure for vitrectomy, corneal and vitreous findings after surgery, visual acuity 6 months after surgery, intraocular pressure after surgery, and additional surgical techniques. RESULTS: The cause of corneal opacity was graft rejection in all patients, who had been treated with high-dose administrations of steroids and cyclosporin. The preoperative diagnosis was proliferative vitreoretinopathy (PVR) in eight eyes and fungal endophthalmitis in seven eyes. Corneal transparency was achieved in 7 eyes (46.6%) 6 months after surgery. Vitreoretinal findings improved in 9 eyes (60%) and PVR recurred in 6 eyes (40%). Visual acuity improved in 7 eyes (46.6%), did not change in 2 eyes (13.3%), and deteriorated in 6 eyes (40%). The six eyes with decreased visual acuity developed phthisis bulbi. Preoperative intraocular pressure was 2.1 mmHg on average in the phthisis bulbi group, significantly lower than in the group with superior prognosis. Goniosynechia was noted before surgery and did not improve after surgery in all six eyes. CONCLUSIONS: The outcome was poor in eyes with goniosynechia and ocular hypotony, and combined surgery is not indicated for either anatomic or visual preservation in such cases. Care should be taken not to overlook intraocular infection in patients undergoing immunosuppressive therapy against graft rejection. The early detection of retinal detachment is also important in eyes exhibiting hypotony after surgery.