Fumihito Ohashi

Distribution of Cells Immunopositive for AM-3K, a Novel Monoclonal Antibody Recognizing Human Macrophages, in Normal and Diseased Tissues of Dogs, Cats, Horses, Cattle, Pigs, and Rabbits

The monoclonal antibody AM-3K, which was developed using human pulmonary macrophages as the immunogen, immunocytochemically labels most human macrophages except for blood monocytes and dendritic cell populations. AM-3K also shows cross-reactivity in some animal species. To evaluate the usefulness of AM-3K, the present study investigated the detailed distribution of AM-3K–immunopositive macrophages in normal and diseased tissues of dogs, cats, horses, cattle, pigs, and rabbits. Zambonis solution-fixed, paraffinembedded sections were the most available for the immunocytochemistry with AM-3K. In all animal species examined, AM-3K labeled most macrophages in splenic red pulp, lymph node sinuses and thymus, and tissue macrophages in the interstitium of various organs and sites such as the kidneys, lungs, heart, pancreas, intestines, and skin. Alveolar macrophages and perivascular microglial cells were also immunoreactive for AM-3K. Interestingly, Kupffer cells of dogs, cats, and horses were labeled for AM-3K, but those of cattle, pigs, and rabbits were not. Furthermore, in tumor tissues and inflammatory lesions such as liver fibrosis and encephalomalacia that were obtained from dogs, infiltrating macrophages were stained with AM-3K, but not all infiltrating macrophages reacted to AM-3K. In addition, only 30–50% of pulmonary and peritoneal macrophages obtained from cats and dogs were reactive for AM-3K. AM-3K did not react with blood monocytes, dendritic cell populations, and osteoclasts. These observations indicate that AM-3K specifically labels most exudate and tissue macrophages in the animal species examined. However, the expression of antigens recognized by AM-3K on macrophages may be dependent on differential maturation stages or different functions evoked by some conditions. AM-3K immunoreaction products were seen on the cytoplasmic membrane of macrophages by immunoelectron microscopy. AM-3K would be useful for detection of macrophage populations in the animal species examined here.

Immunohistochemical Analysis of Macrophages, Myofibroblasts, and Transforming Growth Factor-β Localization during Rat Renal Interstitial Fibrosis Following Long-Term Unilateral Ureteral Obstruction

Renal interstitial fibrosis was induced in rats by chronic unilateral ureteral obstruction (UUO). To identify the mechanisms behind the fibrosis, macrophage influx, myofibroblast involvement, and the localization of transforming growth factor-β (TGF-β, a fibrogenic cytokine) were investigated immunohistochemically in rats euthanatized at 0 (controls), 3, 6, 9, 12, and 15 days after UUO. The number of α-smooth muscle actin-positive myofibroblasts began to increase significantly in the medulla from day 3, and the development of medullary fibrosis was confirmed from day 6 by morphometric analysis. From day 9, papillary fibrosis also developed in association with an increased number of myofibroblasts. These myofibroblasts showed a parallel orientation to the mucosal surface of the pelvis. In the medulla and papilla, from day 6 the number of ED1 (primary antibody)-positive macrophages began to increase significantly. There appeared to be a relationship between macrophage influx and myofibroblast involvement. By contrast, in the cortex there was no marked increase in myofibroblasts nor development of fibrotic tissues, regardless of increased number of macrophages from day 6. Immunohistochemically, no staining for TGF-β was found in infiltrating macrophages or myofibroblasts. However, TGF-β was localized on some cortical proximal renal tubules both of normal control and obstructed kidneys in the early stages on days 3, 6, and 9, suggesting that the possible origin of TGF-β may be renal epithelia. However, the staining intensity for TGF-β on the renal epithelia tended to be weakened in advanced obstructed kidneys on days 12 and 15. The likely contribution of TGF-β to the advanced stages of UUO-induced renal fibrosis remains to be determined.

In Vivo Evaluation of Irinotecan-Loaded QuadraSphere Microspheres for Use in Chemoembolization of VX2 Liver Tumors

PURPOSE To investigate the pharmacokinetics and chemoembolization efficacy of irinotecan-loaded QuadraSphere microspheres (QSMs) in a rabbit VX2 liver tumor model. MATERIALS AND METHODS Fourteen rabbits with VX2 liver tumors were divided into two groups. In the irinotecan-loaded QSM group (n = 7), 3 mg of QSMs (30-60 μm) containing 12 mg of irinotecan (0.6 mL; 20 mg/mL) were injected into the left hepatic artery. In the control group (hepatic arterial infusion [HAI] and QSMs; n = 7), 3 mg of QSMs suspended in ioxaglic acid were injected following a bolus injection of 0.6 mL of irinotecan solution (20 mg/mL). Sequential irinotecan, SN-38, and SN-38G concentration changes were measured in plasma within 24 hours and at 1 week and in tissues at 1 week. The VX2 tumor growth rates at 1 and 2 weeks were calculated from computed tomographic images. RESULTS All rabbits underwent successful embolization. Plasma irinotecan, SN-38, and SN-38G concentrations in the irinotecan-loaded QSM group showed significantly sustained release compared with the control group (P = .01). Compared with the control group, the irinotecan-loaded QSM group had significantly higher irinotecan concentration in liver tumors (P = .03) and a tendency toward higher SN-38 concentration in liver tumors (P = .29). The SN-38G tissue concentrations were below the limits of quantification. The tumor growth rate was significantly lower and the tumor necrosis rate significantly higher in the irinotecan-loaded QSM group (P = .02 and P = .01, respectively). CONCLUSION Chemoembolization via irinotecan-loaded QSMs more effectively suppresses tumor growth than chemoembolization with unloaded QSMs after HAI. A clinical feasibility study is warranted.

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.

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.

Embolic Effects of Transcatheter Mesenteric Arterial Embolization with Microspheres on the Small Bowel in a Dog Model

PURPOSE To determine the arterial distribution and ischemic effects of various particle sizes after transcatheter embolization of the small bowel in a dog model. MATERIALS AND METHODS In 10 dogs, selective microsphere embolization was performed in six branches of the superior mesenteric artery. Microspheres were allocated into three size ranges (100-300 μm, 300-500 μm, and 500-700 μm) and four volume concentrations (0.625%, 1.25%, 2.5%, and 5%). For each size and volume concentration, embolization was performed of five branches at the origin of the last arcade. The distribution of microspheres and the range of ischemic changes of mucosa were evaluated histologically. Angiograms were categorized into two groups: group A, only the vasa recta nonopacified; group B, the last arcade or more proximal branches nonopacified. RESULTS Microspheres sized 100-300 μm penetrated into intramural arteries and 500-700 μm microspheres mainly blocked arteries in the mesentery. There was a significant difference among three sizes in terms of the locations within the vasculature (P < .0001). The larger volume and the smaller size resulted in more ischemia. The range of ischemic changes among three sizes and among four volume concentrations was significantly different (P = .004 and P < .0001, respectively). The range of ischemic changes with 500-700 μm microspheres in group B was significantly greater than in group A (0% in group A vs 83% in group B, P = .001). CONCLUSIONS In a dog model, embolization of the small bowel limited to the vasa recta with the use if 500-700 μm microspheres reduced the range of ischemic changes.

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.