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.

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.

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.

[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.

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.

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.

Fetal pancreas transplantation in miniature swine. IV. Suppression of DTH and MLR responses by treatment with ultraviolet light-irradiated peripheral blood lymphocytes.

Irradiation of peripheral blood lymphocytes of miniature swine with ultraviolet light prevented them from initiating proliferative responses in allogeneic mixed lymphocyte reactions and also reduced IL-2 production in these MLRs. When pigs were injected in a series of 4-5 weekly transfusions with UV-irradiated allogeneic PBL differing at the MHC, PBL of recipient pigs progressively responded less strongly to donor PBL in MLRs over the treatment period. These pigs also gave negligible delayed-type hypersensitivity responses to donor PBL at the end of the treatment period. Of the seven UV-irradiated PBL-treated pigs, four produced no antidonor PBL antibody and three produced antibody. Serum from the three antibody-producing pigs also suppressed MLRs of unrelated PBL. By contrast, pigs that received a series of injections of untreated allogeneic PBL gave strong DTH responses to donor PBL and heightened proliferation in MLRs with donor PBL, and all produced antidonor PBL antibody.

A comparative study of germinal center: fowls and mammals.

The mammalian germinal center is organized into a dark zone containing proliferating centroblasts and a light zone filled with nondividing B cells (called centrocytes), follicular dendritic cells and a few scattered T cells. We clarified these two zones in the chicken germinal center with immunohistology. Proliferating cells and immunoglobulin negative cells were detected in the circumference ring of the chicken germinal center. The central part of the chicken germinal center contained B cells expressing immunoglobulin, follicular dendritic cells and a few T cells. Most of the B cells in the central part of the chicken germinal center did not enter into the S phase. These results suggest that the chicken germinal center is also organized into the dark zone (the circumference ring of germinal center) and the light zone (the central part of germinal center).

Development of maternal IgG-free chick obtained from surgically bursectomized hen.

IgG-free eggs and chicks were developed, so as to study the role of maternal IgG in the development of the immune system. Surgical bursectomy on the 18th day of incubation deprived chickens of B cells and eliminated IgG synthesis. Bursectomized chickens are usually dead before sexual maturity under conventional conditions. When surgically bursectomized chickens were housed in an isolated clean room and antibiotics were administered to them, they could survive to sexual maturity. Finally, we succeeded in obtaining IgG-free fertilized eggs and maternal IgG-free chicks from surgically bursectomized hens. The amount of yolk IgG in IgG-free eggs was one-ten thousandth less than that in normal eggs. The level of IgM in the serum of maternal IgG-free chicks reached six times higher than that of normal chicks 5 days after hatching.

Two cases of accidental transmission of hepatitis C to medical staff.

Two medical doctors (a 38-year-old male and a 25-year-old female) were admitted to our department with jaundice. They had injured themselves with, respectively, a scalpel and a needle contaminated with anti-hepatitis C virus antibody-positive blood, at 2 and 1.5 months, respectively, prior to admission. They were both suffering from clinically typical acute hepatitis. The polymerase chain reaction showed that hepatitis C RNA and anti-hepatitis C virus antibody were transiently positive in the male doctor, while serum transaminase levels were transiently abnormal. In the female doctor, hepatitis C RNA was initially negative, but became strongly positive, and abnormal serum transaminase levels persisted. Interferon therapy resulted in the hepatitis C RNA's becoming negative again, and the transaminase levels returned to normal. The clinical and other findings strongly suggested that in both patients hepatitis C developed as a result of the respective in-hospital accidents.

Prevalence of hepatitis C virus antibodies in hemodialysis patients and dialysis staff.

To estimate the prevalence of hepatitis C virus (HCV) infection in dialysis patients, serum anti-HCV antibodies were evaluated in 489 Japanese patients undergoing hemodialysis, and 152 members of the hospital dialysis staff by enzyme-linked immunosorbent assays for anti-C100, anti-KCL-163 (HCV nonstructural protein), and anti-JCC (translation product of the presumptive HCV core gene). Of the 489 hemodialysis patients, 100 (20.4%) were positive for anti-C100, 107 (21.9%) for anti-KCL-163, and 168 cases (34.4%) for anti-JCC. These rates were significantly higher than those for either the hospital staff or the healthy blood donors. Forty-two per cent of the dialysis patients were anti-HCV positive by at least one assay, suggesting that HCV infection is more common among this population than previously thought. Positivity for anti-HCV was related to the duration of hemodialysis. Elevated alanine aminotransferase levels were present in 12.5% of the dialysis patients, 77% of whom were also anti-HCV positive. The positivity rates among the 152 members of the hospital staff were 0.7% for anti-C100, 2.6% for anti-KCL-163, and 8.6% for anti-JCC, with the anti-JCC rate of positivity exceeding that of the healthy blood donors.

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.

Abscess of bovine brain stem diagnosed by contrast MRI examinations.

A 3-month-old, female Japanese Black calf that showed signs of neurological dysfunction soon after birth was twice examined by magnetic resonance imaging (MRI). Survey MR images showed changes in a hydrocephalus from mild to severe and the existence of a mass above the brain stem that could be distinguished from the surrounding cerebral parenchyma. Contrast MRI examinations using Gd-DO3A-butriol showed the mass to have a doughnut-like form. As the mass changed, the clinical signs aggravated. We diagnosed a brain stem abscess, which we confirmed pathologically. To our knowledge there are no other reports of the use of contrast MRI to examine cattle.

A retrospective study of ventral fenestration for disk diseases in dogs.

Sixty-six dogs suffering from intervertebral disk diseases on cervical and/or thoracolumbar disks were treated by a ventral fenestration. The breeds affected with disk diseases were Dachshund (65.1%), Beagle (15.2%), Shih Tsu (4.5%), Pekingese (4.5%), and others (18.2%). Sites suffered from disk diseases and received disk fenestration most frequently were from T10-11 to L2-3. However, other disks were also needed the operation in some cases. An average number of disks received fenestration was 6.0. Days to walk after the fenestration in 51 cases varied 1 to 120 days (average: 14.9 +/- 22.0 days, mean +/- S.D.) depending on their severity. The recovery rates from the follow-up study were as follows: In grade I (3 cases, able to walk with back pain) and grade II (14 cases, slight ataxia and paresis) it was 100%, respectively. The rates in grade III (23 cases, paraplegia with positive deep pain) and grade IV (26 cases, paraplegia with lack of deep pain) were 73.9% and 65.4%, respectively. A few recurrence after the operation were observed in this study. As a result, the ventral fenestration was extremely effective as a treatment for disk diseases in the dog.

Immunosuppression in dogs by pretransfusions with ultraviolet (UV)-irradiated whole blood.

Ultraviolet (UV)-irradiated whole dog blood prevented the initiation of proliferative responses in allogeneic mixed lymphocyte reactions (MLR). When dogs were given 4 weekly intravenous transfusions of UV-irradiated allogeneic donor whole blood, peripheral blood lymphocytes (PBL) of the recipients responded less significantly to donor PBL in MLR for over one week after the final transfusion. Red blood cell (RBC)-crossmatching of these dogs was negative. Dogs treated with UV-irradiated blood did not produce anti-donor PBL antibody, or IgG, IgM and C3 determined by the indirect Coombs test. These dogs also had negligible delayed type hypersensitivity (DTH) responses to donor PBL at the end of the treatment period, recognized as slightly suppression of skin graft rejection. In contrast, six dogs receiving injections of untreated allogeneic whole blood evidenced high proliferation in MLR, agglutination in RBC-crossmatching, acute rejection of skin grafts and three of them produced C3 in the Coombs test.

Effects of surgical orthodontic treatment for malalignment due to the prolonged retention of deciduous canines in young dogs.

This study was conducted to clarify the effects of surgical orthodontic treatment for malalignment caused by the prolonged retention of deciduous canines in young dogs. Fifty-five dogs with malocclusion or pre-malocclusion were divided into two groups. Group 1 received surgical orthodontic treatment (32 dogs) using modification of Ross and Yamane's method of treating for malalignment due to prolonged retention of deciduous canine teeth; Group 2 received no treatment (23 dogs). Occurrence of malocclusion 1-3 weeks after treatment of the group receiving surgery was only 3.8%. However, malocclusion was seen in 73.4% of the non-treatment group approximately 2 years after the first examination. Surgical orthodontic treatment was very useful in treating malalignment due to prolonged retention of deciduous canines in young dogs.

Effect of directly ultraviolet-irradiated allografts of fetal pancreas in experimental diabetic dogs.

The proliferative responses of UV-irradiated islets from fetal pancreas decreased to 53.8 +/- 4.7% (mean +/- SEM) compared to that of UV-irradiated islets by allogeneic mixed islet-cell lymphocyte culture. In 5 pancreatectomized dogs, UV irradiated fetal dog pancreas was transplanted either into omentum pouches or the spleen without immunosuppressive agents. The diabetic status (daily insulin requirement for hyperglycemia, decrease in body weight, urine glucose) improved in dogs after allo-transplantation. The survival time after total pancreatectomy was significantly longer in allografted dogs than those treated only with daily insulin injections.

Histopathological findings on ulcerative lesions of carpal and tarsal joints in Japanese black cattle.

In order to determine the pathogenesis of ulcerative lesions of the articular cartilages in Japanese Black cattle, tissue samples of the ulcerative lesion, marginal portion of the ulcer, macroscopically normal portions and synovial membranes were histopathologically examined by light microscopy, scanning electron microscopy and contact microradiography. The results are summarized as follows: (1) In the ulcerative lesions, degeneration and complete destruction of articular cartilage and its replacement with a proliferation of myelogenic connective tissue were observed. (2) In macroscopically normal portions, fissures of the articular surface and changes of the trabecular pattern in subchondral bone were present. (3) In the marginal portions of the ulcerative lesions, evidences of the repair process such as connective tissue growth from subchondral bones and articular cartilages were seen. (4) In synovial membranes, no pathological findings were observed. And (5) among the above mentioned changes, no inflammatory findings were seen. In conclusion, the ulcerative lesions of bovine articular cartilage may be regarded as the early stage of osteochondrosis to osteoarthrosis since the findings such as noninflammatory destruction or degeneration and remodeling of the joints are characteristics of the latter.

Intrauterine transplantation of isogenic pancreatic islets in experimental diabetic rats.

The effect of intrauterine transplantation (IU group) as a potential immunologically privileged site on the diabetic state of the recipient was compared with that of conventional intraperitoneal transplantation (IP group) using Fisher 344 rats. Islets were isolated from the pancreata of normal rats and transplanted into the uterus and peritoneal cavity of the isogenic rats with experimental diabetes, which were treated with estradiol benzoate and progesterone. Although all the rats in both groups became normoglycemic within 4 days after transplantation, all of those in the IU group relapsed into a diabetic state up to the 20th day after transplantation. On the other hand, 6 of 8 rats in the IP group remained normoglycemic throughout the experimental period. Weight gain and diminution of urinary glucose excretion in the IU group were significantly lower than those in the IP group (P < 0.01). The glycosylated hemoglobin level in the IU group did not differ significantly from that in the IP group, but the serum level of fructosamine in the IU group was significantly higher than that in the IP group (P < 0.01). These results indicate that the response to fluctuations of blood glucose of islets in the uterine cavity is less than that of islets in the peritoneal cavity. Histologically, islets were observed to be aggregated in the uterine cavity, however the number of cells decreased markedly with time. Although this study demonstrated that blood glucose was normalized by transplantation of islets into the uterine cavity of diabetic rats, long-term survival of the islets in this location was not obtained.

Early in vitro detection of interleukin-2-like activities by pretreatment with allogeneic blood lymphocytes in miniature swine.

In order to determine the means of monitoring the immunological status of allograft recipients in miniature swine, an assay was developed to measure interleukin (IL)-2 production in vitro by pretreatment of donor peripheral blood lymphocytes (PBL). Miniature swine were given 0 to 4 weekly intravenous transfusions of 5-10 X 10(7) donor PBL incompatible at major histocompatibility complex (MHC) and assayed in vitro for donor specific immune IL-2-like activities. The results are summarized as follows: (1) IL-2-like activity in 24 hr and 48 hr supernatants from mixed lymphocyte cultures (MLC) with MHC-incompatible PBL was detected without pretreatment. The 48 hr MLC supernatant exhibited a high IL-2-like activity compared with the 24 hr; (2) IL-2-like activity after only one transfusion with MHC-incompatible PBL was higher than that without pretreatment; (3) IL-2-like activity in 4 weekly transfusions was detectable slightly earlier than that without pretreatment or three transfusions with MHC-incompatible PBL.