Hypoxic hepatitis with marked elevation of serum ferritin probably due to activation of intrahepatic macrophages: another form of hypoxic hepatitis hitherto not reported?

BACKGROUND AND STUDY AIMS: Hypoxic hepatitis (HH) is an acute liver injury that develops in patients with underlying diseases, such as heart failure, respiratory failure, septic/toxic shock. However, some patients do not have underlying diseases or episodes which are known to result in HH. Here, we analyzed the clinical characteristics of this particular patient group (called 'unknown HH' hereafter) to understand its pathogenesis. PATIENTS AND METHODS: Between October 2010 and January 2016, 157 consecutive patients with acute liver injury were admitted to our hospital. Among these patients, 15 patients were categorized as unknown HH. Medical histories and blood test results of unknown HH were analyzed. RESULTS: Among 15 patients of unknown HH, 11 were habitual drinkers and all experienced one of digestive symptoms which might result in mild hypovolemia such as vomiting, diarrhea, appetite loss, and epigastralgia. All patients of unknown HH presented marked elevation of serum ferritin concentration paralleled with aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase (LDH) concentrations. The serum levels of ferritin, ALT, LDH, and prothrombin time-international normalized ratio (PT-INR) were rapidly decreased during hospitalization and all 15 patients of unknown HH recovered without any complication. CONCLUSIONS: We found the particular group of HH with marked elevation of serum ferritin probably due to intrahepatic macrophage activation. Anti-inflammatory treatments might be effective for this group of hypoxic hepatitis.

Application of sonoelastography for evaluating the stiffness of equine superficial digital flexor tendon during healing.

Sonoelastography can assess the inner stiffness of tissues. Sonoelastographic evaluation of injured equine superficial digital flexor tendons (SDFTs) is considered to be useful for assessing the stiffness of a lesion even during late-stage rehabilitation. The purpose of this study was to investigate and compare the sonoelastographic appearance of injured SDFTs over time from the onset of the injury. Eighteen horses were classified into three groups according to the length of time from injury onset: group A, within two weeks after injury; group B, approximately five months after injury; and group C, approximately nine months after injury. Longitudinal and transverse images of all injured SDFTs were obtained using grey-scale ultrasonography and sonoelastography. Grey-scale and sonoelastographic images were evaluated by two observers using echogenicity-grading and colour-grading systems, respectively. The authors evaluated the interobserver agreement and compared the grades among the three groups. The results indicated almost perfect interobserver agreement. Significant differences were found in the sonoelastography among the three groups, whereas no significant difference was found in the grey-scale ultrasonography between groups B and C. Sonoelastography is a feasible and useful modality to evaluate the equine injured SDFTs in vivo and to distinguish between them among the different phases even during the chronic phase.

Meticillin-resistant Staphylococcus aureus colonisation and infection in Thoroughbred racehorses and veterinarians in Japan.

Meticillin-resistant Staphylococcus aureus (MRSA) infections have been confirmed in hospitalised Thoroughbred racehorses at the hospitals of two training centres in Japan since 2009. To investigate the source of infection, the authors examined the rate of nasal MRSA colonisation in 600 healthy Thoroughbred racehorses, 53 veterinarians and 16 office staff at the racehorse hospitals of the two training centres. MRSA was not isolated from healthy Thoroughbred racehorses or hospital office staff. However, MRSA was isolated from 16 veterinarians (30.1 per cent), and the colonisation rate was significantly higher in veterinarians than in the office staff of the same hospitals. Also, 10 of the 16 MRSA strains (62.5 per cent) isolated from veterinarians were classified as type II by staphylococcal cassette chromosome mec (SCCmec) typing and ST5 by multilocus sequence typing. Pulsed-field gel electrophoresis analysis demonstrated that these 10 MRSA strains of SCCmec type II and ST5 were genetically identical or very similar to 9 MRSA strains isolated from infected horses hospitalised at these hospitals between 2009 and 2013. These results indicate that SCCmec type II and ST5 MRSA strains were probably transmitted between veterinarians and infected horses.

Epidemiological survey of the hoof wall cavity ('Gidoh' in Japanese) in racehorses.

In October 2001, a survey was conducted about cavities formed within the hoof wall (called GIDOH in Japanese) of racehorses at the Ritto and Miho Training Centres, which are managed by the Japan Racing Association (JRA). Gidoh is defined as a progressive cavity within the deep layers between the stratum medium and stratum internum. A total of 148 out of 5386 surveyed horses (2.75 per cent) were affected. Out of 244 affected feet, fore hooves (84.02 per cent) were more susceptible than hind hooves, and the site most affected was midline dead centre of the toe (59.62 per cent) which tends to place extra stress at the break-over point in a straight-line exercise. Logistic regression analysis revealed that prevalence was significantly related with horse affiliation (OR 0.65, 95 per cent CI 0.46 to 0.91) and age (OR 1.43 per one year, 95 per cent CI 1.27 to 1.61). We hypothesised that the primary cause of Gidoh development in JRA stables was mechanical deformation of the hoof wall during exercise, and secondary exciting causes can subsequently lead to the spread of the lesion over the entire hoof wall.

Relationships between myonuclear domain size and fibre properties in the muscles of Thoroughbred horses.

REASONS FOR PERFORMING STUDY: The myonuclear domain (MND) is the region of cytoplasm governed by a single myonucleus. Myonuclear domain size is an important factor for muscle fibre plasticity because each myonucleus has limitations in the capacity of protein synthesis. Previous studies have demonstrated that differences in MND size exist in different fibre types in several species, including horses. OBJECTIVES: To understand the basic mechanism of muscle plasticity, the relationships between MND size, muscle fibre type population and metabolic properties of skeletal muscles throughout the whole body in Thoroughbred horses were examined. METHODS: Post mortem samples were taken from 20 muscles in 3 Thoroughbred horses aged 3-5 years of age. Fibre type population was determined on serial cross sections of each muscle sample, stained for monoclonal antibodies to each myosin heavy chain isoform. Oxidative (succinic dehydrogenase; SDH) and glycolytic (phosphofructokinase; PFK) enzyme activities were determined spectrophotometrically in each muscle sample. Furthermore, 30 single fibres were isolated from each muscle under stereomicroscopy and then fibre volume and myonuclear number for a given length analysed under confocal microscopy. The MND size of each single fibre was measured after normalisation of sarcomere length to 2.8 µm by staining with membrane-specific dye. RESULTS: Immunohistochemical staining indicated that soleus, vastus lateralis and gluteus medius muscles had the highest percentage of type I, IIa and IIx muscle fibre, respectively. Biochemical analysis indicated highest activities of SDH and PFK in diaphragm and longissimus lumborum muscles, respectively. MNDs were largest in the splenius muscle and smallest in the soleus and masseter muscles. Myonuclear domain size is significantly related to type I muscle fibre population, but not to SDH activities of the muscles. CONCLUSION: The MND size of muscle fibre depends on fibre type population rather than mitochondrial enzyme activities.

Quantities and types of ceramides and their relationships to physical properties of the horn covering the claws of clinically normal cows and cows with subclinical laminitis.

Quantities and types of ceramides and their relationships to physical properties of the horn covering the claws of clinically normal cows and cows with subclinical laminitis were investigated. Total ceramide content of the horn covering the sole and wall from cows with subclinical laminitis was 872.2 +/- 146.6 microg/g and 528.6 +/- 61.3 microg/g, respectively, and was significantly (P < 0.01, 0.05) lower than that from clinically normal cows. The mean moisture content in the claws from cows with subclinical laminitis (43.5% +/- 4.3%) was significantly (P < 0.05) higher than that in the claws from clinically normal cows. The hardness of claws from cows with subclinical laminitis (35.2 +/- 3.5) was significantly (P < 0.05) less than that of claws from clinically normal cows. Significant correlations between ceramides and moisture content (P < 0.001) and between ceramide and hardness (P < 0.001) were found in clinically normal cows and cows with subclinical laminitis. Our results indicate that decreases in ceramide contents may be related to changes in physical properties of the horn covering the claw in cows with subclinical laminitis.

Effects of biotin supplementation on serum biotin levels and physical properties of samples of solar horn of Holstein cows.

Effects of dietary biotin supplementation on serum biotin levels and physical properties of sole horn of 40 Holstein cows were evaluated. The mean serum biotin level in biotin-supplemented cows after 10 mo of biotin supplementation (1163.2 +/- 76.2 pg/mL) was significantly higher (P = 0.007) than that in control cows (382.0 +/- 76.2 pg/mL). The sole horn of biotin-supplemented cows was significantly harder (P = 0.026) and had a significantly lower moisture content (P = 0.021) than that of control cows. No morphologic differences in horn tubules or intertubular horn were found between the biotin-supplemented and control cows. The total lipid content of sole horn was significantly higher (P = 0.030) in the biotin-supplemented cows than in the control cows. These results suggest that dietary biotin supplementation causes increases in serum biotin levels and changes in physical properties and fat content of sole horn.

Prevalence of superficial digital flexor tendonitis and suspensory desmitis in Japanese Thoroughbred flat racehorses in 1999.

REASONS FOR PERFORMING STUDY: Overstrain injuries to the superficial digital flexor tendon (SDFT) and suspensory ligament (SI) are among the most common musculoskeletal injuries which contribute to the considerable wastage of racing Thoroughbreds. Many epidemiological studies have demonstrated the prevalence of and risk factors for tendon injury when racing but have not included those injuries sustained during training. However, since tendon injury during training is seen commonly in clinical practice, it is appropriate to determine the overall prevalence of tendon injury sustained during both training and racing. OBJECTIVE: To determine the prevalence of overstrain injury to the SDFT and SL during training and racing among Thoroughbred flat racehorses in Japan in 1999. METHODS: A retrospective study was performed using a sample population of 10,262 Thoroughbred racehorses. The medical information database of Thoroughbred racehorses registered by the Japan Racing Association (JRA) in 1999 was analysed for SDFT and SL overstrain injury diagnosed by a veterinarian employed by JRA during training and racing. Jump racehorses were excluded from this study. RESULTS: The prevalence of forelimb SDFT tendonitis and SL desmitis was 11.1% (1130 cases) and 3.61% (370 cases) of the population, respectively. In the hindlimb, there were 0.06% (6 cases) and 0.14% (14 cases), respectively. Risks of SDF tendonitis in the forelimb in 3-year-olds or older horses were significantly higher than in 2-year-olds. In contrast, the risk of SL desmitis in the forelimb at age 3 and 4 years was 2.23 and 2.11 times higher, respectively, than in 2-year-olds, but this increased to 5.07 times in those age > or = 5 years. Entire males were at greater risk in comparison to females and geldings. CONCLUSIONS: The results suggest that the prevalence of SDF tendonitis and SL desmitis in the forelimb was associated with the horse's age and sex. The prevalence of SL desmitis increased further with age compared with SDF tendonitis, possibly reflecting a more rapid accumulation of degeneration in this structure. POTENTIAL RELEVANCE: The age-related risk demonstrated in this study provides further support that overstrain injuries are associated with accumulated degeneration. These data provide a valuable resource for further research into the aetiology of tendon injury in the racehorse.

Physiological changes in the concentrations of biotin in the serum and milk and in the physical properties of the claw horn in Holstein cows.

Physiological changes in the concentrations of biotin in the serum and milk and in the physical properties of the claw horn were examined in Holstein cows. A lower concentration of biotin in the serum and a higher concentration of biotin in milk were found during early and late lactation and during the dry period, and a significant (p<0.05) inverse correlation was found between serum and milk biotin concentrations. A high moisture content and a low level of hardness of the claw horn were found during mid-lactation. Our results indicate that change in the serum biotin concentration probably results from the loss of biotin in the milk of cows during each stage of lactation and also confirm that the moisture content and hardness of the claw horn undergo physiological changes.

Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system.

The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels revealed titers which correlated highly with respective hemagglutinin inhibition (HI) antibody titers which ranged from <1:8 to 1:256 (correlation coefficient among them was 0.850). ELISA OD levels and HI titers increased at 5 and 7 days post-inoculation, respectively, in a horse inoculated intranasally with LP/93. Respective antibody levels were observed to change in an essentially parallel manner during a period of 1 month. Similarly, ELISA OD levels correlated with HI titers in horses during a period of 6 weeks following intramuscular inoculation with inactivated single-strain vaccines containing LP/93, A/equine/Kentucky/1/81 (H3N8) or A/equine/Rome/5/91 (H3N8). A similar pattern was also observed in eight horses throughout a 10-week period following inoculation with a commercially available inactivated trivalent vaccine containing A/equine/Newmarket/1/77(H7N7), A/equine/Kentucky/81 and LP/93. From these results, it is suggested that this ELISA system could be used for disease diagnosis and surveillance of HI antibody titers among vaccinated horses.

Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells.

Histoplasmosis in the lung of a race horse with yersiniosis.

A 4-year-old female thoroughbred race horse died of acute peritonitis caused by necrotizing granulomatous duodenitis. Yersinia enterocolitica was immunohistochemically demonstrated in macrophages in granulomas developed in the duodenum, lung, liver and abdominal lymph nodes. The yeast-like fungi were found in the cytoplasmic vacuoles of macrophages in the lung that infiltrated into the granulomas and surrounding alveoli with congestive edema. The yeast-like fungi were positively stained by Gomori-Grocott chromic acid methenamine silver stain and immuno-histochemically stained with anti-histoplasma antibody. In this case, it was considered that granulomas formed in the duodenum, lung, liver and abdominal lymph nodes were primarily caused by Yersinia enterocolitica due to idiopathic weakening of the immune system. Yeast-like fungi immunohistochemically identified as histoplasmas secondarily infected the lung. This is the first case regarded as equine histoplasmosis capsulati in Japan.

Histological and morphometrical studies on the mucosa of the equine guttural pouch (auditory tube diverticulum).

The present study attempted to clarify the characteristics of the guttural pouch mucosa in equines and to evaluate its foreign substance clearance ability. The specimens were collected from nine regions (eight in the guttural pouch mucosa, and one in the nasopharynx mucosa). We first examined the pouch mucosa by light and electron microscopy. We then measured the frequency of goblet cells per 200 epithelial cells, the length of the cilia, the thickness of the epithelial cell layer and lamina propria and statistically analyzed the data. The guttural pouch mucosa consisted of stratified columnar epithelia with brush-like cilia, and there were almost no histological differences between adults and foals. The morphometrical study revealed significant differences in goblet cell frequency (p < 0.001) and the thickness of lamina propria (p < 0.05). By contrast, no statistically significant difference was detected in the length of the cilia or the thickness of the epithelial cell layer. These findings suggest that the guttural pouch mucosa provides foreign substance clearance ability, but that its ability varies among different regions of the epithelium.

A survey of white line disease in Japanese racehorses.

A survey was carried out into white line disease in 1781 Thoroughbred racehorses kept in stables at the Japan Racing Association (JRA) Miho Training Center (MTC) September-October 1996. The survey was conducted while horses were being shod by farriers. The horses that still exhibited damaged white lines after regular trimming were diagnosed as having white line disease. The factors recorded were age, sex, number of diseased horses, number of diseased hooves, number of lesions by region over the bearing border of the hoof and the classified length of such lesions. The percentage of total diseased horses was 11.5% (204 animals), with incidence increasing significantly with age (P< or =0.01). Occurrence was independent of sex (P>0.05) was more frequent in the fore- than in the hindhoof and developed more frequently at the toe than at any other region of the forehoof-bearing border. Most lesions ranged from 20 to 30 mm in length.

Translocation t(X;21)(q13.3; p11.1) in a girl with Menkes disease.

A girl with a 46,X,t(X;21) (q13.3;p11.1) karyotype presented with skin redundancy, especially in the neck, prominent occiput and micrognathia, and later developed hypotonia, hypopigmentation, sparse scalp hair, and profound mental retardation characteristic of Menkes disease. Her serum copper (14 microg/dl) and ceruloplasmin (9 mg/dl) levels were extremely low. Fluorescent in situ hybridization analysis with a 100-kb P1-derived artificial chromosome probe containing the Menkes disease gene demonstrated three twin-signals, one on the normal X chromosome and one each on derivative chromosomes X and 21, indicating that the Xq13.3 breakpoint was located within the gene. Replication pattern analysis showed that the normal X chromosome was late replicating, whereas the derivative X chromosome was selectively early replicating. These results indicated that Menkes disease in our patient resulted from a de novo translocation that disrupts the disease gene.

Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes.

BACKGROUND: Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. RESULTS: Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, approximately 120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed the both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. CONCLUSION: Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

Onychomycosis in white line disease in horses: pathology, mycology and clinical features.

This paper describes onychomycosis in horses and reports the pathological findings, associated fungi and incidence of concurrent white line disease. In addition to these observations, relevance between post mortem and clinical findings of onychomycosis are discussed in 3 necropsied horses. Samples were collected from 100 hooves from a total of 51 Thoroughbreds suffering from white line disease. Of these, 15 hooves from 13 horses were also complicated with severe hoof wall fissure formation. Preparations from the same samples were used both for histopathology and for culture to identify the associated fungi. Onychomycosis was diagnosed when it could be confirmed histologically. Fungal identification was based on morphological characteristics in culture and the associated fungi were determined by comparison with culture morphology. As a result, 10 samples collected from horses were diagnosed as suffering from onychomycosis. Seven of these showed complicating severe inner hoof wall fissures and the soil fungus Scedosporium apiospermum or the teleomorph of this fungus, i.e., Pseudallescheria boydii, was isolated. The fungus was found to be ubiquitous in the fissure cavities, the terminal horn of the white line and the terminal horn-like laminae of the metaplastic white line-like tissue. It can be concluded that onychomycosis frequently causes white line disease and/or makes it worse. Associated with deterioration of the submural condition, the main associated fungus for onychomycosis in this series, was Genus Scedosporium and the most susceptible region was the terminal horn of the hypertrophied white line and/or the terminal horn-like laminae of the metaplastic white line-like tissue.

Cloning and characterization of NE-dlg: a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein interacts with the APC protein.

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dig) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dig). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.

Effect of transportation on the composition of bronchoalveolar lavage fluid obtained from horses.

OBJECTIVE: To study the effects of extended transportation on the composition of bronchoalveolar lavage fluid (BALF) obtained from horses. ANIMALS: 30 horses (14 males, 16 females: 25 Thoroughbreds and 5 Thoroughbred-Arabian cross-breds; 27 to 30 months old) without a history or clinical signs of respiratory tract disease. Bronchoalveolar lavage was performed on nontransported control horses (groups 1 and 2) and transported horses (group 3). PROCEDURE: 20 horses were used to determine the effect of 41 hours of transportation on the composition of BALF (group 3). Bronchoalveolar lavage fluid was analyzed for recovered volume, number and distribution of nucleated cells, total protein and phospholipid concentrations, and phospholipid composition. RESULTS: Total number of nucleated cells in BALF from group-3 horses increased by approximately four-fold after transportation. Total protein concentration in BALF from group-3 horses also increased by approximately fivefold after transportation. Total phosphorus concentrations in group-3 horses decreased significantly from time 0 to immediately after transportation. In group-3 horses, the most characteristic change in composition of BALF after transport was a significant decrease in the concentration of phosphatidylglycerol. CONCLUSION AND CLINICAL RELEVANCE: The decrease in phosphatidylglycerol concentration in BALF after transportation indicates a reduction in the quantity of surfactant. This change may reflect either a decreased production of surfactant by alveolar type II epithelial cells or an increased removal of surfactant from the alveolar region. It is likely that extended transportation resulted in a decreased concentration of surfactant in BALF. Such a decrease may reduce the pulmonary defence mechanisms in the alveolar region, possibly resulting in infection.