Human lung microvascular endothelial cells as potential alternatives to human umbilical vein endothelial cells in bio-3D-printed trachea-like structures.

BACKGROUND: We have been trying to produce scaffold-free structures for airway regeneration using a bio-3D-printer with spheroids, to avoid scaffold-associated risks such as infection. Previous studies have shown that human umbilical vein endothelial cells (HUVECs) play an important role in such structures, but HUVECs cannot be isolated from adult humans. The aim of this study was to identify alternatives to HUVECs for use in scaffold-free structures. METHODS: Three types of structure were compared, made of chondrocytes and mesenchymal stem cells with HUVECs, human lung microvascular endothelial cells (HMVEC-Ls), and induced pluripotent stem cell (iPSC)-derived endothelial cells. RESULTS: No significant difference in tensile strength was observed between the three groups. Histologically, some small capillary-like tube formations comprising CD31-positive cells were observed in all groups. The number and diameters of such formations were significantly lower in the iPSC-derived endothelial cell group than in other groups. Glycosaminoglycan content was significantly lower in the iPSC-derived endothelial cell group than in the HUVEC group, while no significant difference was observed between the HUVEC and HMVEC-L groups. CONCLUSIONS: HMVEC-Ls can replace HUVECs as a cell source for scaffold-free trachea-like structures. However, some limitations were associated with iPSC-derived endothelial cells.

Transcatheter arterial embolisation in four dogs with hepatocellular carcinoma.

Four dogs with hepatocellular carcinoma were treated by transcatheter arterial embolisation. In all dogs, the tumour-supplying arteries were selectively embolised with gelatine sponge particles. Post-embolisation tumour volumes decreased relative to pre-embolisation volumes in all dogs. No adverse reactions were observed in three dogs after treatment but one dog showed pancreatitis. These results suggest that transcatheter arterial embolisation is a feasible treatment for dogs with hepatocellular carcinoma.

Assessment of canine ovaries autografted to various body sites.

The influence of graft site on the survival of canine follicles and oocytes after autografting was investigated. Hemi-ovaries were autografted to three locations (quadriceps femoris muscle fascia, kidney capsule, and gastrosplenic ligament), and grafted ovaries were recovered (under anesthesia) 28 to 31 d after transplantation. The grafted hemi-ovaries were bisected: one-quarter ovary was used for histological assessment and another quarter for evaluation of oocyte viability. As controls, the remaining fresh hemi-ovaries were used to assess the viability of follicles and oocytes in non-transplanted ovaries. Most follicles in the histological sections of the grafts were classified as primordial or primary follicles. Antral follicles were not observed in the grafts, irrespective of the graft site. The percentages of viable follicles in the sections from control ovaries, and the ovaries grafted to the kidney capsule, the quadriceps femoris muscle fascia, and the gastrosplenic ligament were 17.4, 22.9, 18.3, and 32.4%, respectively. A total of 12 oocytes was recovered from the 15 hemi-ovaries grafted in five bitches, of which five (41.7%) oocytes from the ovaries grafted to the quadriceps femoris muscle fascia and the kidney capsule were cultured for assessment of meiotic competence. Three oocytes were viable but remained in the germinal vesicle stage after 72 h of maturation culture. The quadriceps femoris muscle fascia might be useful for grafting like the kidney capsule, but improvement of follicle survival and meiotic competence of oocytes in the grafts is necessary.

Quantitative investigation and classification by MRI of residual cleft cysts in the pituitary glands of cows.

The application of MRI to 167 Holstein cows and 76 Japanese black cows made it possible to distinguish two types of residual cleft cysts (RCCs) in their pituitary glands on the basis of the differences in signal intensity: fluid-filled lesions with an intensity equal to that of cerebrospinal fluid (CSF) (hypointense on T1-weighted images and hyperintense on T2-weighted images); and solid lesions that were hypointense compared with cerebral parenchyma and isointense with CSF on T1-weighted images, and hypointense compared with cerebral parenchyma on T2-weighted images. Solid material was observed in lesions with a mean (sd) diameter of 2.09 (1.28) mm in 56 (33.5 per cent) of the Holstein cows, and in lesions with a mean diameter of 1.91 (0.89) mm in 20 (26.3 per cent) of the Japanese black cows. Four of the Holstein cows had RCCs more than 7 mm in diameter. Long-term development of the RCCs was suggested by histological findings of epithelial stratification and a structural shift from having a single to a stratified epithelial layer within the residual clefts of the affected pituitary glands.

[Bronchial schwannoma diagnosis by surgical treatment; report of a case].

18-year-old male was referred to our hospital due to persistent cough. The patient was admitted for the investigation of the abnormal shadow on a chest X-ray and chest computed tomography (CT). Chest CT showed a 2.5 cm nodular shadow in the right lower lobe. Bronchofiberscopy revealed the polypoid lesion at the right lower lobe bronchus obstructing the entire lumen of B8-10. The tumor surface was smooth and rich in small vessels. Right lower lobectomy was peformed. The diagnosis of schwannoma was confirmd with the S-100 positive immunohistochemical stain. Bronchial schwannoma is relatively rare disease; less than 90 cases have been reported with respect to schwannoma of case report in Japan.

Development of multidrug resistance in a canine lymphoma cell line.

New multidrug resistant cell lines developed from the canine B cell lymphoma cell line (GL-1) were characterized in terms of chemosensitivity to some antineoplastics and P-glycoprotein (Pgp) expression. GL-1 was continuously exposed to a culture medium containing gradually increasing levels of doxorubicin and the cells that could grow in the presence of doxorubicin were obtained. Chemosensitivity of these cells to various antineoplastics were investigated with or without verapamil, which reversed Pgp-mediated drug resistance. The expression of Pgp on the cells was also examined by Western blot analysis. As a result, three kinds of resistant cell lines, designated as GL-DOX60, 300, and 4000 were obtained. These cell lines showed stable proliferation in the medium containing 60, 300, and 4000 ng/ml, respectively. These cells were much more resistant to vincristine than doxorubicin. This resistance was strongly reversed by the presence of verapamil. On the other hand, cisplatin was effective enough in killing these derived cells. In the Western Blot analysis, some bands that reacted to the anti-human Pgp monoclonal antibodies were observed in GL-DOX4000. The cells derived from GL-1 have multidrug resistance potential mediated by canine Pgp. The cells produced in this experimental trial are considered to be useful models for various investigations on canine multidrug resistance.

Protein expression of Mdm2 and p53 in hyperplastic and neoplastic lesions of the canine circumanal gland.

Hyperplastic and neoplastic lesions of the circumanal gland in dogs were examined immunohistochemically for nuclear expression of Mdm2 and p53 proteins. Mdm2 nuclear labelling was detected in 64.3% of hyperplastic lesions and 70.7% of adenomas, the immunolabelling being stronger in the latter. Mdm2 reactivity in reserve-like cells in adenomas showed wide variation, the immunoreactive cells ranging from 4 to 80%. No reactivity was shown in 81.9% of adenocarcinomas. In contrast to Mdm2 reactivity, each of 14 hyperplastic lesions and 11 adenocarcinomas, and 60 of 65 adenomas (92.3%) failed to show nuclear p53 reactivity. These results suggest that increased expression of Mdm2 is an early event in circumanal gland tumorigenesis, and may be present in the absence of nuclear p53 protein accumulation.

Detection of centrosome amplification as a surrogate marker of dysfunction in the p53 pathway -p53 gene mutation or MDM2 overexpression.

Abstract In human and canine cancers, the inactivation of p53 protein as well as p53 gene mutation and MDM2 overexpression result in centrosome amplification that in turn contributes to chromosomal instability. To explore the usefulness of the detection of centrosome amplification as a surrogate marker of dysfunction in the p53 pathway, we systematically analysed centrosome amplification, p53 overexpression, p53 gene mutation and MDM2 overexpression in canine tumours. Centrosome amplification was detected in 16 of 51 (31%) naturally developing tumours in dogs. All the tumour specimens with aberrations in the p53 pathway, including p53 overexpression, p53 gene mutation or MDM2 overexpression, showed centrosome amplification, suggesting that the detection of centrosome amplification could serve as a preliminary surrogate marker of dysfunction in the p53 pathway.

Overexpression of lipoprotein lipase improves insulin resistance induced by a high-fat diet in transgenic rabbits.

AIMS/HYPOTHESIS: Dysfunctions of lipoprotein lipase (LPL) have been found to be associated with dyslipidaemias, atherosclerosis, obesity and insulin resistance. There are two conflicting hypotheses regarding the roles of LPL in glucose metabolism and insulin resistance. Whether systemically increased LPL activity would be beneficial or detrimental to insulin sensitivity is yet to be resolved. To address this issue, we studied transgenic rabbits overexpressing human LPL transgene. METHODS: LPL transgenic and control rabbits were fed a 10% high-fat diet (HFD) for 16 weeks. To evaluate glucose metabolism, we compared plasma levels of glucose and insulin in transgenic rabbits with control rabbits and performed an intravenous glucose tolerance test. In addition, we measured adipose tissue accumulation in HFD-fed rabbits. RESULTS: Increased LPL activity in transgenic rabbits resulted in a significant reduction of plasma triglycerides and non-esterified fatty acids, but not in basal levels of glucose and insulin. HFD feeding induced an elevation of plasma glucose levels accompanied by hyperinsulinaemia in control rabbits, but was significantly inhibited in transgenic rabbits. The intravenous glucose tolerance test showed that transgenic rabbits had faster glucose clearance associated with lower levels of insulin secretion than control rabbits. In addition, there was a significant reduction of body adipose tissue in transgenic rabbits compared with in control rabbits fed an HFD. Scanning electron microscopic examination revealed that adipocytes in transgenic rabbits were predominately small cells. CONCLUSIONS/INTERPRETATION: Our results showed that systemically increased LPL activity improves insulin resistance and reduces adipose accumulation in transgenic rabbits, indicating that systemic elevation of LPL may have potential benefits for the treatment of insulin resistance and obesity.

Quantitative electroencephalography of medetomidine, medetomidine-midazolam and medetomidine-midazolam-butorphanol in dogs.

The purpose of this study was to evaluate the effects of the administration of an alpha2-adrenoceptor agonist alone and in combination with other derivatives on brain wave activity. In addition, the diagnostic values of the electroencephalogram (EEG) for judging the depth of the balanced anaesthesia with an alpha2-adrenoceptor agonist was evaluated. The treatments comprised 20 microg/kg medetomidine (Me-20), 80 microg/kg medetomidine (Me-80), 20 microg/kg medetomidine and 0.5 mg/kg midazolam (Me-Mi) administered intramuscularly, and 20 microg/kg medetomidine with 0.5 mg/kg midazolam and 0.1 mg/kg butorphanol (Me-Mi-Bu). The EEG was recorded continuously at pre-administration, and at 7, 10, 20, 30, 45 and 60 min after administration. The recorded data were analysed by separating the power spectrum into 1-3, 4-7, 8-13 and 14-30 Hz bands. Spectral-edge analysis was used to calculate the spectral edge frequency 90 (SEF90) and the median edge frequency (MEF). Time-related changes in power spectrum analysis showed a significant increase in the Me-80 group in the 1-3 Hz band. The power for 1-3 Hz in the Me-80 group was significantly higher than in all the other groups. In the 14-30 Hz band, there was a significant reduction of power in all groups following administration of the agents. The SEF90 frequencies were significantly reduced in all groups except for the Me-20 group after administration of the agents. The SEF90 frequencies in the Me-20, Me-Mi and Me-Mi-Bu were all significantly higher than those in the Me-80 group. However, there was no significant difference between the Me-20, Me-Mi and Me-Mi-Bu groups in any analyses. Our results demonstrated that the changes in quantitative EEG made by the Me-Mi-Bu and Me-Mi groups were similar to those made by Me-20 groups. Present results suggest that the EEG should be interpreted with caution in assessing the anaesthetic level in balanced anaesthesia in dogs.

Expression of T helper 1 and T helper 2 cytokine mRNAs in freshly isolated peripheral blood mononuclear cells from dogs with atopic dermatitis.

Using reverse transcription-polymerase chain reaction (RT-PCR) and semi-quantitative PCR techniques, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-4, IL-5 and IL-10 in freshly isolated peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without atopic dermatitis (AD). The expression of IFN-gamma mRNA in dogs with AD was lower than that in dogs without AD (healthy control). The expression of IL-5 mRNA was higher in dogs with AD than in control dogs, but there were no significant differences in IL-4 mRNA and IL-10 mRNA expression between the groups. The number of circulating eosinophils was higher in dogs with AD than in control dogs, although eosinophilia was found in only one dog with AD. These results suggest that there is a tendency for the PBMCs of atopic dogs to express a type 2 cytokine pattern that is similar to the pattern observed in human AD patients.

Effect of medetomidine on electroencephalography and use of a quantitative electroencephalograph for evaluating sedation levels in dogs.

This study was designed to characterize the effect of medetomidine (Med) on canine electroencephalography (EEG), to evaluate the use of quantitative EEG for assessing sedation levels and to explore the correlation between the serum concentration of Med and the quantitative EEG. Four groups of dogs were given Med at doses of 20, 40, 80 and 160 microg/kg (Med-20, Med-40, Med-80 and Med-160 groups). Following Med administration, there was synchrony between each unipolar EEG lead. On EEG power spectrum analysis of the bipolar leads, all groups showed a significant depression of the 14-30 Hz components. The power of the 1-3 Hz component in the Med-80 and Med-160 groups was significantly increased, although there were few changes in the other groups. Similar results were obtained from raw data analysis. As a result of quantitative EEG analysis, spectrum edge frequency 90 analysis (SEP90) showed that the frequency was significantly reduced in all groups after Med administration. A dose-response effect was observed in all groups except for the Med-160 group. Both of these EEG analyses were significantly correlated with the serum concentration of Med. However, the result of the SPF90 analysis sugested a stronger correlation than that for median edge frequency analysis. In conclusion, care must be taken in veterinary clinical diagnoses when Med is used during EEG recording, as Med may cause increased activity in the low frequency band and a decrease in high frequency band activity. In addition, quantitative EEG analysis may be useful in assessing the depth of sedation and in further studies on Med administration.

Hydrocephalus and syringomyelia in a cat.

A 3-month-old male Japanese cat with feline parvovirus infection, showing central and cervical nerve abnormalities, was diagnosed as hydrocephalus and syringomyelia by use of magnetic resonance imaging (MRI). The cat was maintained clinically by medical treatment even though he could not stand. The MRI scans obtained about 5 months later showed that the ventricles had increased in size and the cervical syrinx had extended into the thoracic spinal cord. Ventriculoperitoneal (VP) shunt was performed. One week after surgery, neurological conditions had improved. At the postoperative MR images, the ventricles had decreased in size and the syrinx in the cervical and thoracic spinal cord could no longer be seen. The cat was still alive and was able to walk well.

Ivermectin treatment of demodicosis in 56 hamsters.

Fifty-six hamsters with demodicosis were treated with daily oral administration of ivermectin (0.3 mg/kg). Thirty-three cases (58.9%) were cured and 6 cases (10.7%) had improved clinically but needed to continue treatment. Of 5 cases (8.9%) who relapsed within 3 months and were retreated with ivermectin, 4 were cured and 1 needed further treatment. Five cases (8.9%) had improved clinically but died within 3 months. Seven cases (12.5%) had not improved and died within 3 months. Overall 49 (87.5%) hamsters had improved clinically. No significant differences in prognosis of demodicosis were detected according to sex, breed, age and clinical features, but the prognosis of demodicosis in hamsters with concurrent disease was poor.

[Two cases of intravascular lymphomatosis diagnosed antemortem by transbronchial lung biopsy].

Patient 1 was a 71-year-old man who had been admitted to our hospital with high-grade fever. Chest computed tomographic (CT) images revealed clear peripheral and high-density central areas. Abnormal laboratory findings included elevated LDH and hypoxemia. Interstitial pneumonia was suspected, and transbronchial lung biopsy (TBLB) confirmed the diagnosis of intravascular lymphomatosis (IVL). The patient succumbed before completion of chemotherapy. Patient 2 was a 65-year-old man admitted with high-grade fever. Abnormal laboratory findings included pancytopenia, hypoxemia, and elevated levels of LDH and soluble interleukin-2 receptor. Chest CT images revealed diffuse, mildly dense areas in the upper fields of both lungs. TBLB specimens yielded a diagnosis of IVL. Complete clinical remission was obtained with CHOP multiagent chemotherapy. Although IVL is usually diagnosed at autopsy, in these 2 cases an antemortem diagnosis was made on the basis of TBLB findings. Also, multiagent chemotherapy achieved a complete clinical remission in Patient 2.

Antemortem evaluation for magnetic resonance imaging of the equine flexor tendon.

In this study antemortem evaluation of equine flexor tendons--the superficial digital flexor tendon and the deep digital flexor tendon--using magnetic resonance (MR) images was performed. Postmortem flexor tendons were used to prepare the slice positions, coil and body positions for MR imaging. It was possible by this method to take antemortem MR images of equine limbs that distinguished features as well as postmortem images described in previous studies. The total time of antemortem scanning was about 40 min. This study is the first to report antemortem MR images in horses.

In-vitro anti-proliferative effects of some anti-tumour drugs on feline mammary tumour cell lines.

Six anti-tumour drugs namely; doxorubicin, mitoxantrone, vincristine, cisplatin, recombinant human tumour necrosis factor alpha (rh-TNFalpha) and recombinant feline interferon gamma (rf-IFNgamma) were singly evaluated for their anti-proliferative effects on two feline cell lines (FRM and NAC) derived from mammary adenocarcinoma and grown as monolayers. We obtained concentration response curves that enabled the determination of the concentration inhibiting growth by 50 per cent (IC50) for the chemotherapeutic agents with VCR exhibiting exponential-plateau curves. Differences in anti-proliferative effects of drugs to a given cell line and between the cell lines were also observed. NAC cells were relatively more resistant compared with FRM cells. The relative resistances for NAC cells were 4.19, 12.96, 0.05 and 2.10-fold to doxorubicin, mitoxantrone, vincristine and cisplatin, respectively. FRM cells were more resistant to VCR at lower concentrations compared with NAC cells. The cells appeared, at least in vitro, least sensitive to rh-TNFalpha and rf-IFNgamma. rh-TNFalpha and rf-IFNgamma were 23 and 29 per cent inhibitory to FRM cells and only 13 and 15 per cent inhibitory to NAC cells, respectively.