Identification and frequency of a new HLA-A allele, A*030104.

We here identified a novel HLA-A allele, A*030104, which was found in a Japanese family. The direct sequencing revealed that A*030104 was identical to A*030101 except for a nucleotide substitution of GAG to GAA at codon 63 without an amino acid replacement. The frequencies of A*030104 and A*0301 including A*030104 in Japanese population (n = 22,360) were approximately 0.013 and 0.40%, respectively.

Effects of topical nipradilol and timolol maleate on intraocular pressure, facility of outflow, arterial blood pressure and pulse rate in dogs.

Nipradilol is an alpha(1), beta-blocker with milder side effects than other beta-blockers used in humans. In this study the effects of nipradilol were compared with those of timolol maleate in dogs. Twelve clinically normal dogs (nine mongrels, two beagles, and one Akita) were used. We applied 0.25% nipradilol or 0.5% timolol maleate drops for a period of 28 days. Intraocular pressure (IOP) was measured before and after administration on the 2nd, 4th, 7th, 14th, 21st and 28th day. Blood pressure, pulse rate and coefficient of aqueous outflow (C-value) were also measured before and after administration on the 7th, 14th, 21st and 28th day. Both nipradilol and timolol maleate significantly lowered IOP from the 2nd day to the end of the study period. Nipradilol lowered IOP to an equivalent degree to timolol maleate. There was no significant change in blood pressure and pulse rate throughout the study period with administration of nipradilol. C-value showed a significant rise from the 14th day with administration of nipradilol, while it did not show any significant change during the study period with administration of timolol maleate. The reduction of IOP by nipradilol was similar to that by an existing beta-adrenergic antagonist, timolol maleate, but nipradilol was associated with fewer systemic side effects in dogs. Nipradilol appears to be a useful drug for treatment of glaucoma in dogs.

Detection of matrix metalloproteinases in canine mammary tumours: analysis by immunohistochemistry and zymography.

Expression of matrix metalloproteinases (MMPs) was examined immunohistochemically (MMP-9 and -2) and by gelatin zymography (MMP-9) in 12 benign and 12 malignant canine mammary tumours. In nine of 12 benign tumours, weak expression of MMP-9 was demonstrated immunohistochemically in the cytoplasm of neoplastic cells. In the remaining three benign tumours (mixed or complex) MMP-9 expression was observed in the neoplastic luminal epithelial cells and myoepithelial cells in some areas. The neoplastic luminal epithelial cells in 12 malignant tumours reacted strongly for MMP-9. MMP-2 immunolabelling was observed in both benign and malignant tumour tissues, but was stronger in the latter. Zymography and densitometry showed that expression of MMP-9 was higher in the benign mammary tumours than in normal canine mammary tissues, but highest in the malignant mammary tumours. In benign and malignant canine mammary tumours, the zymography and densitometry results for MMP-9 accorded well with the immunohistochemical results obtained with anti-human MMP-9 monoclonal antibody.

Horse lumbrical muscle: possible structural and functional reorganization in regressive muscle.

An anatomical study of horse lumbrical muscle (Lm) was carried out by light and electron microscopy in combination with immunochemical and cytochemical methods. Paraffin sections were subjected to haematoxylin and eosin (H & E) and Masson's trichrome staining for morphometric analysis. Paraffin sections were also used for immunostaining by anti-PGP 9.5 for reaction with nerve-protein associated-structures, anti-heat-shock protein 70 (hsp 70) for detection of gene expression changes, anti-fast myosin for the determination of muscle fibre types, and for detection of apoptotic gene expression of muscle fibres by the TUNEL method. The distribution of muscle fibre types on frozen sections was also examined by assaying ATPase (pH 4.2). We found that the proximal end of the tendon of the unipennate-shaped Lm binds to the deep digital flexor tendon, and the distal end of the Lm tendon connects to the medial surface of the palmar annular ligament. The Lm was not always present, but when found it varied in length greatly, up to 8 cm (muscle part alone), and weighed less than 1 g. The Lm was white, pale, or reddish in colour depending on the ratio of muscle fibre and connective tissue contents. The semi-tendinized regressive Lm was composed of rich vasculature, peripheral nerves, and nerve-like organs similar to the neuromuscular spindle (NMS). The extrafusal muscle fibres (e-lm) that surround the NMS were replaced with a thick outer capsule of connective tissues (CT) in the Lm nerve-like organ, which we named the neurotendinous capsule (NTC) organ. NTC organs exist alone or as multiple structures (up to eight) surrounded by a common outer capsule at the outermost CT ring. The NTC possesses several intrafusal muscle fibres (ifm) just as the NMS does. That the ifm was associated with nerve endings was confirmed by anti-PGP 9.5 and electron microscopic observation. Some muscle fibres in ifm and e-lm reacted with anti-fast twitch myosin and with anti-hsp 70. The e-lm exhibited at least two fibre types, determined by ATPase (pH 4.2) assay. The ifm exhibited mainly type I (slow twitch) fibres. No apoptotic gene expression was detected in either ifm or e-lm, suggesting the Lm is a vital organ. The degenerating fibres observed in ifm and e-lm indicate that the turnover rate of cytoplasmic components is accelerated. We attribute this phenomenon to the necessity for adaptation to new environmental demands. The surprising finding of tubular aggregates (TAs) in ifm of the NTC organ suggests that the Lm is continuously adapting. Some results related to variation in diameter of the collagen fibrils, isolation of the NTC organ and the myofibrillar protein constituents are also discussed. In conclusion, the so-called regressive Lm has rich vasculature, many peripheral nerves, and newly described NTC organs. The induction of heat-shock protein, lack of apoptotic gene expression in ifm and e-lm fibres, and TA formation in ifm suggest that horse Lm responds to environmental stress through reorganization and/or remodelling of cell constituents. We hypothesize that the horse Lm has lost its original role as a contractile element and changed to another function, likely as a vital nerve organ.

Cytogenetic studies of Hynobiidae (Urodela)XVIII. A ZZ/ZW sex-determining mechanism in a hynobiid salamander species, Hynobius tokyoensis Tago.

The karyotype of Hynobius tokyoensis (2n = 56) was analyzed using three kinds of banding methods to determine the morphological differentiation of the sex chromosomes of this species. Salamanders and egg sacs were collected from seven localities around Tokyo, Japan. Of 28 chromosome pairs, microchromosome No. 21 was identified as a ZZ/ZW-type sex chromosome. The Z chromosome was acrocentric, whereas the W chromosome was submetacentric, with a heterochromatic, elongated short arm. Interestingly, the W chromosome is of three distinct types, W(A), W(B), and W(C), based on R-banding and Ag-NOR patterns. W(A) was detected in five populations from southern habitats, whereas W(B) and W(C) were detected in one population each from northern habitats. W(A), W(B), and W(C) were all found to carry Ag-NORs on their heterochromatic short arms. Considering the karyotypes of other species belonging to the same genus, we discuss the evolution of the sex chromosomes of H. tokyoensis.

Immunohistochemical detection of inhibin-alpha, -betaB, and -betaA chains and 3beta-hydroxysteroid dehydrogenase in canine testicular tumors and normal testes.

Immunohistochemical detection of inhibin-alpha, -betaA and -betaB chains and 3beta-hydroxysteroid dehydrogenase (HSD) was carried out on primary testicular tumors from 15 dogs and normal testes from three adult dogs. Histopathologically, the tumors were composed of three types: Leydig cell tumors in five dogs, Sertoli cell tumors in five dogs, and seminoma in five dogs. In normal testes, immunostaining against inhibin-alpha, -betaA, and -betaB chains and 3beta-HSD revealed positive reactivity in the cytoplasm of Leydig cells. In testicular tumors, immunoreactive cells against inhibin-alpha, -betaA, and -betaB chains and 3beta-HSD were localized in all Leydig cell tumors but not in any Sertoli cell tumors or seminomas. The results of radioimmunoassay for plasma inhibin in dogs with Leydig cell tumors showed higher concentrations than those in dogs with Sertoli cell tumors and seminomas and those in normal dogs. The concentration of inhibin in the plasma was markedly decreased by the surgical removal of the Leydig cell tumor in one dog. Our findings suggest that inhibin is synthesized by normal and neoplastic Leydig cells in the canine testis, and the secreted inhibin may be inhibin A and inhibin B.

Immunohistochemical detection of apolipoprotein B-100 and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs.

Accumulation of lipids and hyalinosis in the splenic arteries of aged dogs are frequently detected by routine histopathologic examinations. The purpose of this study was to pinpoint the localization of canine apolipoprotein B-100 (CApoB-100) and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs (n = 80) through the use of immunohistochemical techniques. CApoB-100 deposits were found in the subendothelial space, extracellular matrix, and atheromatous lesions in the tunica media of the arteries in dogs > or = 6 years of age. Foamy cytoplasm of the infiltrated macrophages was also CApoB-100 immunopositive. In dogs > or = 10 years of age, almost all central arteries were CApoB-100 immunopositive. Hyaline deposits within the wall were characterized by immunopositivity against canine IgA, IgM, IgG, and albumin. Lipid accumulation in splenic arteries may be an age-related lesion and a precursor of the atheromatous plaques associated with splenic hemorrhage and infarcts later in life. In addition, deposition of immunoglobulins, probably mediated by immune complexes, may play an important role in the development of canine vascular diseases similar to human disease.

The effect of third eyelid gland removal on the ocular surface of dogs.

To evaluate the effect of third eyelid removal on the ocular surface of dogs, we operated on five young Beagle dogs and observed changes to tear function using the following tests: phenol red thread test (PRT), Schirmer tear test (STT-1), modified Schirmer tear test (STT-2), pH and tear break-up time (BUT). There was a significant decrease (37%) in STT-2 within 2 weeks after the excision and this declined further to 60% at 1 year. The pH value increased after excision. Presurgical pH was 7.17 +/- 0.20 (mean +/- SD), which increased to 7.55 +/- 0.24 in the 14-60 days following removal, and further increased to 7.77 +/- 0.65 at 1 year. The PRT and STT-1 decreased by 26% within 3-7 months compared to pre-excision values, but by 1 year the values recovered to near normal. The BUT pre-excision value was 24.0 +/- 8.1 s, which shortened to 13.5 +/- 4.5 s after 5 months and continued to decrease further during the study. There were no overt visual signs of KCS during the observational period. However, microinjury of the keratoconjunctival epithelium was observed for all operated eyes when vital staining was used at 1 year post surgery. Surgical excision of the third eyelid in Beagle dogs influenced tear quality level and affected the stability of the tear layer, and at 1 year there was evidence of microinjury to the keratoconjunctival epithelium.

Cardiovascular effects of medetomidine, detomidine and xylazine in horses.

The cardiovascular effects of medetomidine, detomidine, and xylazine in horses were studied. Fifteen horses, whose right carotid arteries had previously been surgically raised to a subcutaneous position during general anesthesia were used. Five horses each were given the following 8 treatments: an intravenous injection of 4 doses of medetomidine (3, 5, 7.5, and 10 microg/kg), 3 doses of detomidine (10, 20, and 40 microg/kg), and one dose of xylazine (1 mg/kg). Heart rate decreased, but not statistically significant. Atrio-ventricular block was observed following all treatments and prolonged with detomidine. Cardiac index (CI) and stroke volume (SV) were decreased with all treatments. The CI decreased to about 50% of baseline values for 5 min after 7.5 and 10 microg/kg medetomidine and 1 mg/kg xylazine, for 20 min after 20 microg/kg detomidine, and for 50 min after 40 microg/kg detomidine. All treatments produced an initial hypertension within 2 min of drug administration followed by a significant decrease in arterial blood pressure (ABP) in horses administered 3 to 7.5 microg/kg medetomidine and 1 mg/kg xylazine. Hypertension was significantly prolonged in 20 and 40 microg/kg detomidine. The hypotensive phase was not observed in 10 microg/kg medetomidine or detomidine. The changes in ABP were associated with an increase in peripheral vascular resistance. Respiratory rate was decreased for 40 to 120 min in 5, 7.5, and 10 microg/kg medetomidine and detomidine. The partial pressure of arterial oxygen decreased significantly in 10 microg/kg medetomidine and detomidine, while the partial pressure of arterial carbon dioxide did not change significantly. Medetomidine induced dose-dependent cardiovascular depression similar to detomidine. The cardiovascular effects of medetomidine and xylazine were not as prolonged as that of detomidine.

Combination of continuous intravenous infusion using a mixture of guaifenesin-ketamine-medetomidine and sevoflurane anesthesia in horses.

The anesthetic and cardiovascular effects of a combination of continuous intravenous infusion using a mixture of 100 g/L guaifenesin-4 g/L ketamine-5 mg/L medetomidine (0.25 ml/kg/hr) and oxygen-sevoflurane (OS) anesthesia (GKM-OS anesthesia) in horses were evaluated. The right carotid artery of each of 12 horses was raised surgically into a subcutaneous position under GKM-OS anesthesia (n=6) or OS anesthesia (n=6). The end-tidal concentration of sevoflurane (EtSEV) required to maintain surgical anesthesia was around 1.5% in GKM-OS and 3.0% in OS anesthesia. Mean arterial blood pressure (MABP) was maintained at around 80 mmHg under GKM-OS anesthesia, while infusion of dobutamine (0.39+/-0.10 microg/kg/min) was necessary to maintain MABP at 60 mmHg under OS anesthesia. The horses were able to stand at 36+/-26 min after cessation of GKM-OS anesthesia and at 48+/-19 minutes after OS anesthesia. The cardiovascular effects were evaluated in 12 horses anesthetized with GKM-OS anesthesia using 1.5% of EtSEV (n=6) or OS anesthesia using 3.0% of EtSEV (n=6). During GKM-OS anesthesia, cardiac output and peripheral vascular resistance was maintained at about 70% of the baseline value before anesthesia, and MABP was maintained over 70 mmHg. During OS anesthesia, infusion of dobutamine (0.59+/-0.24 microg/kg/min) was necessary to maintain MABP at 70 mmHg. Infusion of dobutamine enabled to maintaine cardiac output at about 80% of the baseline value; however, it induced the development of severe tachycardia in a horse anesthetized with sevoflurane. GKM-OS anesthesia may be useful for prolonged equine surgery because of its minimal cardiovascular effect and good recovery.

Systemic atherosclerosis in dogs: histopathological and immunohistochemical studies of atherosclerotic lesions.

Histopathological and immunohistochemical studies were carried out on five cases of canine systemic atherosclerosis. The five animals were male, and showed hypercholesterolaemia and hypertriglyceridaemia on biochemical analysis of plasma. Histopathologically, atherosclerotic lesions were seen in the aorta and muscular arteries in many organs, including the heart, spleen, kidneys, lungs, pancreas, alimentary tract, urogenital organs, eyes, prostate and urinary bladder. The lesions were characterized by the deposition of lipids and infiltration of lipid-laden foamy cells in the tunica intima and tunica media, sometimes forming fibrofatty plaques, containing abundant sudanophilic material, cholesterol clefts and mineralized material. The lesions started in the tunica intima and extended to the tunica media and tunica adventitia. Immunohistochemical examination with canine apolipoprotein B-100 (CApoB-100) antibody identified the lipids containing low density lipoprotein. Immunoreactivity to CApoB-100 antibody was recognized in the tunica intima, lipid-laden foamy cell cytoplasm and smooth muscle cells in the tunica media, and fibrofatty plaque. These histopathological and immunohistochemical features were similar to those of human atherosclerotic lesions.

In vitro cytotoxicity of imidazolyl-1,3,5-triazine derivatives.

We examined in vitro cytotoxic activity of imidazolyl-1,3,5-triazine derivatives using human breast cancer cell lines (MCF-7, R-27, T-47D and ZR-75-1) and murine leukemia cell line (P388). The percentage of viable cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazorium bromide (MTT) assay. Hexamethylmelamine (HMM), a 1,3,5-triazine derivative has previously been recognized as an antitumor agent effective against lung, ovarian and breast cancer, but failed to show a significant cytotoxic activity in the present study. In contrast, four imidazolyl-1,3,5-triazine derivatives, 2-(1-imidazolyl)-4,6-bis(morpholino)-1,3,5-triazine, 2-(1-imidazolyl)-4-morpholino-6-(3-thiazolidinyl)-1,3,5-triazine, 2-(4-cyano-4-phenylpiperidino)-4-(1-imidazolyl)-6-morpholino-1,3,5-triaz ine and 2-(1-imidazolyl)-4-(N-methyl-N-phenylamino)-6-morpholino-1,3,5-triazine showed cytotoxic activity for most cell lines, which was significantly greater than the activity of hydroxymethylpentamethylmelamine (HMPMM), a major metabolite of HMM.

Equine synovial villi: distinctive structural organization of vasculature and novel nerve endings.

The structural arrangement and cellular distribution of endothelial and lining cells of the synovial villi were studied in the equine palmar/plantar recess of the metacarpo- and metatarsophalangeal joints by light microscopy and electron microscopy. The extent and distribution of blood vessels varied with villous shape and length. The majority of vessels formed concentric circles in cross and longitudinal sections and probably are arranged in a convoluted, spiral or helical pattern. The villi do not contain smooth muscle cells or typical capillaries as observed in other organs. Under the electron microscope, the endothelium is surrounded by connective tissue and discontinuous circular cells, presumably fibroblasts. The outermost layer was sometimes surrounded by type A and/or B synovial cells. The lumen of the blood vessels at the top of villus appeared to be constricted in most cases, with a diameter of about 12 +/- 3 microns. Blood vessels formed by more than six endothelial cells in the middle portion of villus generally were not constricted. Well-developed cytoplasmic processes extended into the lumen of blood vessels. The constriction of blood vessels with no apparent smooth muscle presence and the observation of numerous intermediate filaments in the cytoplasm of the endothelial cells suggests that these villous blood vessels constrict through contraction of their own endothelial cells. Lining cells were distributed unevenly even within a single villus; the villous lining cells seemed to have directional preferences with domination of synovial type A cells. Surprisingly, structures resembling myelinated nerve ends (approximately 0.2 microns) were observed between juxtaposed endothelial cells as well as directly on an endothelial cell, suggesting that these nerve endings may be a sensor detector of either pressure or temperature or have a proprioceptive-like function. Synovial villi have a distinctive structural arrangement of vessels, lining cells, and nerve endings.

Antagonistic effects of atipamezole on medetomidine-induced sedation in horses.

The antagonistic effects of atipamezole (20, 40, 60, 80, and 100 micrograms/kg i.v.) on medetomidine (10 micrograms/kg i.v.)-induced sedation were evaluated in horses. Although 20 and 40 micrograms/kg of atipamezole were not sufficient to reverse the sedation, 60 micrograms/kg did effectively reverse the sedation. Atipamezole at 80 micrograms/kg was more potent, and significantly shortened the duration of sedation without any apparent side effects, but a higher dose of 100 micrograms/kg was not more effective than 80 micrograms/kg. The possible use of atipamezole as a reversal agent may enhance the value and availability of medetomidine as a chemical restraint agent in horses.

Radiographic and immunohistochemical analysis of leukocyte adhesion deficiency in Holstein heifers.

Radiographic and immunohistochemical analyses were performed in two Holstein heifers with leukocyte adhesion deficiency (BLAD). Severe bone resorption, osteolysis and severe progressive periodontitis in submandibula due to dysfunction of leukocytes in heifers affected with BLAD were demonstrated by radiographic examination. Immunohistochemical analysis of lymph nodes using anti-CD18 monoclonal antibody demonstrated that CD18-positive cells were not found on those from a heifer affected with BLAD, whereas CD18-positive cells were clearly present in lymph nodes from a clinically normal heifer. These characteristic findings support the importance of adherence-dependent leukocyte functions in host defense.

Hypertrophic Z-line observed in aged one-humped camel (Camelus dromedarius).

The ultrastructure of cardiac muscle from 3 aged one-humped camels (Camelus dromedarius) was examined to see if aged camels also develop hypertrophic Z-lines as observed in the heart of aged humans and other animals. The results proved that the aged camel heart develops hypertrophic Z-lines together with other age-associated alterations, including loss of myofibrils and myofilaments, increase of intermyofibrillar spaces and intercellular spaces, and disruption of mitochondrial cristae and intercalated disc structure. Hypertrophic Z-lines are somewhat smaller than those of other species, but occur both in the atria and ventricles. Although the majority of cardiac muscle samples revealed a normal structure, the finding of hypertrophic Z-lines in camel heart is the first such observation in a ruminant animal. The anomalous camel muscle resembles that of other species in terms of cell alterations commonly associated with aged hearts.

Canine acute phase response: relationship between serum cytokine activity and acute phase protein in dogs.

The changes in serum activity of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF) were studied in dogs with acute inflammation. Dogs with local inflammation induced by an intramuscular injection of turpentine oil showed clinically a typical progression in the inflammatory response, recovering on day 14 after the treatment. Serum concentrations of C-reactive protein (CRP) and alpha 1 acid glycoprotein (alpha 1AG) increased, and the albumin concentration decreased in all dogs during the acute phase response. These values each returned to the normal range from day 14 to 21. Serum IL-6-like activity was detected from 2 hr to day 6 after treatment. Serum TNF-like activity in the treatment group was detected at a low level from 3 to 24 hr after treatment, but there was no statistically significant difference compared with the control group. The temporal changes in serum IL-6 and TNF-like activities preceded those in serum concentrations of CRP, alpha 1 AG, and albumin. No dogs showed a detectable rise in serum IL-1-like activity after treatment.

Bioassay for interleukin-1, interleukin-6, and tumor necrosis factor-like activities in canine sera.

To measure interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-like activities in canine serum, bioassays were conducted using human melanoma A375S1, IL-6 dependent murine hybridoma MH60.BSF2, and WEHI 164 murine sarcoma subclone 28-4. Clinically normal adult beagles were experimentally induced endotoxic shock by an intravenous injection of lipopolysaccharide or local inflammation by an intramuscular injection of turpentine oil. IL-1-like activity was detected in sera from dogs with endotoxic shock. IL-6 and TNF-like activities were detected in sera from both dogs with endotoxic shock and local inflammation. IL-1-like activity in sera from the dogs with endotoxic shock declined after dilution with either medium or serum obtained before treatment (pre-serum), but the IL-1-like activity was maintained to a greater extent in samples diluted with pre-serum compared to those diluted with medium. TNF-like activity declined equally after dilution with either medium or pre-serum. On the other hand, IL-6-like activity was inhibited at low dilution. It was, therefore, necessary to dilute the serum samples to 1:180 from dogs with endotoxic shock or 1:60 from dogs with local inflammation, in order to minimize the effect of inhibitory factors on IL-6-like activity. IL-6-like activity was neutralized by monoclonal antibody against murine IL-6 receptors. TNF-like activity was neutralized by anti-mouse TNF alpha rabbit serum. However IL-1-like activity was not neutralized by either anti-mouse or anti-human IL-1 rabbit serum.

Two cases of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) (case report).

Two Holstein calves showing clinical signs such as ulcerative stomatitis, severe gingivitis, periodontitis, loss of teeth and stunted growth, associated with marked neutrophilia, were evaluated by clinicopathologic analysis, neutrophil functions and flow cytometric analysis of CD18 expression on neutrophils. Decreased CL response, chemotaxis, yeast phagocytosis, and deficient CD18 expression of neutrophils from affected animals were demonstrated. Pathological findings involved were ulcerative gingivitis, severe periodontitis, laryngitis, and multiple ulcers in forestomach. This study demonstrates that neutrophil functions are closely associated with impaired iC3b receptor, and these affected animals were diagnosed as bovine leukocyte adhesion deficiency (BLAD).