Hideo Akiyoshi

Binding of DC-HIL to dermatophytic fungi induces tyrosine phosphorylation and potentiates antigen presenting cell function.

APCs express receptors recognizing microbes and regulating immune responses by binding to corresponding ligands on immune cells. Having discovered a novel inhibitory pathway triggered by ligation of DC-HIL on APC to a heparin/heparan sulfate-like saccharide of syndecan-4 on activated T cells, we posited DC-HIL can recognize microbial pathogens in a similar manner. We showed soluble recombinant DC-HIL to bind the dermatophytes Trichophyton rubrum and Microsporum audouinii, but not several bacteria nor Candida albicans. Dermatophyte binding was inhibited completely by the addition of heparin. Because DC-HIL contains an ITAM-like intracellular sequence, we questioned whether its binding to dermatophytes can induce tyrosine phosphorylation in dendritic cells (DC). Culturing DC with T. rubrum (but not with C. albicans pseudohyphae) induced phosphorylation of DC-HIL, but not when the tyrosine residue of the ITAM-like sequence was mutated to phenylalanine. To examine the functional significance of such signaling on DC, we cross-linked DC-HIL with mAb (surrogate ligand), which not only induced tyrosine phosphorylation but also up-regulated expression of 23 genes among 662 genes analyzed by gene-array, including genes for profilin-1, myristoylated alanine rich protein kinase C substrate like-1, C/EBP, LOX-1, IL-1β, and TNF-α. This cross-linking also up-regulated expression of the activation markers CD80/CD86 and heightened APC capacity of DC to activate syngeneic T cells. Our findings support a dual role for DC-HIL: inhibition of adaptive immunity following ligation of syndecan-4 on activated T cells and induction of innate immunity against dermatophytic fungi.

The DC-HIL/Syndecan-4 Pathway Regulates Autoimmune Responses through Myeloid-Derived Suppressor Cells

Having discovered that the dendritic cell (DC)–associated heparan sulfate proteoglycan–dependent integrin ligand (DC-HIL) receptor on APCs inhibits T cell activation by binding to syndecan-4 (SD-4) on T cells, we hypothesized that the DC-HIL/SD-4 pathway may regulate autoimmune responses. Using experimental autoimmune encephalomyelitis (EAE) as a disease model, we noted an increase in SD-4+ T cells in lymphoid organs of wild-type (WT) mice immunized for EAE. The autoimmune disease was also more severely induced (clinically, histologically, and immunophenotypically) in mice knocked out for SD-4 compared with WT cohorts. Moreover, infusion of SD-4−/− naive T cells during EAE induction into Rag2−/− mice also led to increased severity of EAE in these animals. Similar to SD-4 on T cells, DC-HIL expression was upregulated on myeloid cells during EAE induction, with CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) as the most expanded population and most potent T cell suppressor among the myeloid cells examined. The critical role of DC-HIL was supported by DC-HIL gene deletion or anti–DC-HIL treatment, which abrogated T cell suppressor activity of MDSCs, and also by DC-HIL activation inducing MDSC expression of IFN-γ, NO, and reactive oxygen species. Akin to SD-4−/− mice, DC-HIL−/− mice manifested exacerbated EAE. Adoptive transfer of MDSCs from EAE-affected WT mice into DC-HIL−/− mice reduced EAE severity to the level of EAE-immunized WT mice, an outcome that was precluded by depleting DC-HIL+ cells from the infused MDSC preparation. Our findings indicate that the DC-HIL/SD-4 pathway regulates autoimmune responses by mediating the T cell suppressor function of MDSCs.

A Rhabdomyosarcoma Arising in the Larynx of a Dog

A neoplastic nodular lesion, 2 × 3 cm in diameter, was found in the larynx of a 6-year-old spayed female dog. The tumor was ill-circumscribed, consisting histologically of large round cells with abundant cytoplasm interspersed with small round cells with less cytoplasm and occasional multinucleated cells (myotubes). Immunohistochemically, tumor cells were positive for myoglobin, desmin and vimentin in varying degrees, but negative for S-100 protein, GFAP or cytokeratin. Cytoplasmic myofilaments/myofibrils with a dense Z-line-like structure were seen, the fine structures of which were complemented by PTAH stain. Based on these findings, the tumor was diagnosed as a rhabdomyosarcoma, a very rare tumor in the larynx of dogs.

Surgical resection of twenty-three cases of brain meningioma.

ABSTRACT The aim of this study was to report the effectiveness of MRI scanning during brain meningioma resection surgery in the canine and feline. The subjects were 23 cases from 2006 to 2008 in canines and feline diagnosed with meningioma. All were between 8 and 16 years of ages. There were 12 males and 11 females. An appropriate craniotomy was performed for each case according to the initial MRI taken to diagnose meningioma prior to the surgery. Once the dura mater was exposed, an MRI biopsy needle was placed in the tumor as a guide. The first MRI sequence during the surgery was performed with this needle to confirm the location of the tumor. This MRI image was also processed and displayed by multi planar reconstruction to reveal the tumor extent in three dimensions. A Sonopet was applied to the middle of the tumor to destroy the inner part and release pressure from the entire tumor. Creating some space between the brain tissue and tumor, we treated blood vessels and carefully resected them. This procedure was repeated until complete removal of the tumor was confirmed by MRI. Sixteen of the 23 cases survived for more than 2 years postoperatively. The other seven died due to other disorders within 2 years. Our method with MRI navigation during the surgery improved our surgical performance and contributed to a prolonged survival time for the patients. In order to perform multiple MRI procedures smoothly during the surgery, it is necessary to have skillful assistants.

Profiling of Serum Metabolites in Canine Lymphoma Using Gas Chromatography Mass Spectrometry

ABSTRACT Canine lymphoma is a common cancer that has high rates of complete remission with combination chemotherapy. However, the duration of remission varies based on multiple factors, and there is a need to develop a method for early detection of recurrence. In this study, we compared the metabolites profiles in serum from 21 dogs with lymphoma and 13 healthy dogs using gas chromatography mass spectrometry (GC-MS). The lymphoma group was separated from the control group in an orthogonal projection to latent structure with discriminant analysis (OPLS-DA) plot using ions of m/z 100–600, indicating that the metabolites profiles in lymphoma cases differed from those in healthy dogs. The lymphoma group was also separated from the control group on OPLS-DA plot using 29 metabolites identified in all serum samples. Significant differences were found for 16 of these metabolites with higher levels in the lymphoma group for 15 of the metabolites and lower levels for inositol. An OPLS-DA plot showed separation of the lymphoma and healthy groups using these 16 metabolites only. These results indicate that metabolites profile with GC-MS may be a useful tool for detection of potential biomarker and diagnosis of canine lymphoma.

Development and validation of a sandwich ELISA for use in measuring concentrations of canine surfactant protein A in serum of dogs

OBJECTIVE To develop and evaluate a sandwich ELISA incorporating rabbit antiserum specific for canine surfactant protein A (SP-A) for use in measuring concentrations of SP-A in serum of dogs. SAMPLE Serum samples obtained from 6 healthy dogs and 3 dogs with pulmonary disease. PROCEDURES Rabbit antiserum was prepared against purified canine SP-A. The IgG fraction was isolated via protein G affinity chromatography and was then biotinylated. The sandwich ELISA was performed by use of anti-SP-A antibody (IgG) preabsorbed with sera from healthy dogs. Validity of the ELISA was confirmed by determination of the detection limit, precision, reproducibility, and accuracy. Serum SP-A concentrations were measured in 6 healthy dogs and 3 dogs with pulmonary disease. RESULTS Detection limit of the ELISA was 2.0 ng/mL. Within- and between-assay coefficients of variation ranged from 3.8% to 14.1% and from 15.5% to 35.6%, respectively. The observed-to-expected recovery ratio ranged from 77.1% to 89.9%. Serum SP-A concentrations measured by use of the ELISA were ≤ 2.3 ng/mL in the 6 healthy dogs, 25.6 ng/mL in a dog with severe cardiac pulmonary edema, 8.3 ng/mL in a dog with pneumonia, and 10.1 ng/mL in a dog with lung lobe torsion. CONCLUSIONS AND CLINICAL RELEVANCE The sandwich ELISA was found to be useful for measuring purified canine SP-A concentrations and canine SP-A concentrations in serum samples. The ELISA was precise, reproducible, and accurate. The ELISA may be beneficial in assessing serum concentrations of canine SP-A as a potential biomarker of pulmonary diseases in dogs.

Change in peripheral blood lymphocyte count in dogs following adoptive immunotherapy using lymphokine-activated T killer cells combined with palliative tumor resection

We evaluated changes in peripheral blood lymphocyte (PBL) count in dogs following adoptive immunotherapy using lymphokine-activated T killer cells (T-LAK) in combination with surgery. Fifteen tumor-bearing dogs treated with T-LAK therapy combined with palliative resection of tumors were enrolled in the present study. T-LAK were generated from autologous peripheral blood mononuclear cells (PBMC) by culture with recombinant human interleukin -2 (rhIL-2) and solid phase anti-canine cluster of differentiation (CD)3 antibody. T-LAK were administrated intravenously at 2-4-week intervals. After the first administration of T-LAK, counts of PBL and T lymphocyte subsets (CD3(+), CD4(+) and CD8(+) cells) increased and the CD4/CD8 ratio decreased, with significant increases in CD8(+) cells (P<0.05). In 8 tumor-bearing dogs that were administered sequential T-LAK, available data on changes in PBL and T lymphocyte phenotypes until the fifth administration were also analyzed. In tumor-bearing dogs administered 5 rounds of T-LAK, CD8(+) cell counts were maintained high until the fifth administration of T-LAK. Moreover, the CD4/CD8 ratio remained low until the fifth administration of T-LAK. These results indicate that T-LAK therapy combined with surgery may increase peripheral blood T lymphocytes, particularly CD8(+) cells, in tumor-bearing dogs.

Treatment of a unicameral bone cyst in a dog using a customized titanium device

ABSTRACT A 4-year-old Shih-Tzu, referred for an enlarged left carpus, was diagnosed with a unicameral bone cyst. A customized titanium device was inserted into cystic lesion and fixed by titanium screws. Sufficient strength of the affected bone with the device inserted to maintain limb function was established after resection of contents of cystic lesion. There was no deterioration of the lesion of bone cyst, and acceptable function of the affected limb with no clinical signs of lameness was maintained during 36 months follow-up. The results of this study demonstrated that bone cyst curettage and use of a customized titanium device could provide an effective alternative treatment of huge lesion of unicameral bone cysts with the intent of preventing pathologic fractures.

Elevation of serum surfactant protein-A with exacerbation in canine eosinophilic pneumonia

A 7-year-old female spayed Labrador Retriever was admitted to our hospital, because of cough with sputum. She was diagnosed as having canine eosinophilic pneumonia (CEP) based on blood eosinophilia, bronchial pattern and infiltrative shadow observed on thoracic radiography, bronchiolar obstruction and air-space consolidation predominantly affecting the right caudal lung lobe, as revealed by computed tomography (CT), predominant eosinophils in CT-guided fine needle aspiration and the clinical course. She exhibited a good response to steroid therapy, and the cough disappeared. The serum surfactant protein (SP)-A level increased with the aggravated symptom and decreased markedly with improvement compared with the C-reactive protein level and the number of eosinophils. We propose that serum SP-A level is a good biomarker in CEP.

The influences of hyperbaric oxygen therapy with a lower pressure and oxygen concentration than previous methods on physiological mechanisms in dogs

Recently, hyperbaric oxygen therapy with a lower pressure and oxygen concentration (L-HBOT) than previous methods has been used for dogs in Japan; however, the influences of L-HBOT on dogs have not been clarified. To verify the influences of L-HBOT on physiological mechanism in dogs, we investigated blood gas parameters, glutathione peroxidase (GPx) activity, heart rate variability, stress-related hormones and skin conductance (SC) in 4 clinically normal beagle dogs with catheters in their carotid arteries and jugular veins when they were quiet, after running, after receiving L-HBOT (30% oxygen concentration, 1.3 atmospheres absolute, 30 min) or after not receiving L-HBOT. The results showed there were no changes in blood gas parameters, heart rate variability and catecholamine levels after L-HBOT. GPx activity was significantly higher, and the SC and cortisol level were lower in dogs that received L-HBOT than those when they were quiet. These results suggested that L-HBOT may have a small influence on oxygenation dynamics, activate antioxidant enzymes such as GPx, restrain autonomic nervous activity and control the balance between oxidation and antioxidation inside the body.