Since high levels of hyperthermia induce immunosuppression to a certain extent (i.e., granulocytosis and lymphocytopenia) in patients, we applied mild hyperthermia in volunteers using equipment enabling well-controlled hyperthermia. Restricted control of rectal temperature at 39.4 (± 0.2)°C for 30 min was conducted and various parameters of the body were examined. The most prominent change observed during exposure to hyperthermia was elevated levels of pH and PO₂ in the blood, even in the venous blood. A transient elevation of ACTH, cortisol and growth hormone in the blood was also seen during this time. In parallel with this phenomenon, the number of total lymphocytes and those of its subsets (especially CD57⁺ or CD56⁺ NK cells and NKT cells) increased. More interestingly, the proportion of HLA-DR (MHC class II antigens) increased in NK and NKT cells, and their intensity on the surface of CD20⁺ B cells increased. These results suggest that mild hyperthermia is important for modulation of the functions of the circulatory, endocrine and immune systems.

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The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteoclasts in tumoral lesions of mammary carcinoma metastasized to bone of non-immunocompromised mice. Mouse mammary carcinoma cells (BALB/c-MC) were injected through the left cardiac ventricle into 5-week-old female wild-type Balb/c mice. The femora and tibiae of mice with metastasized cancer were extracted, and thereafter processed for histochemical analyses. The foci of metastasized tumor cells occupied the metaphyseal area, and the cell death zones could be identified within the tumor mass. Abundant tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were found among the alkaline phosphatase (ALP)-reactive osteoblastic cell layer that covered the bone surface neighboring the metastatic lesion. In contrast, F4/80-positive macrophages/monocytes were localized adjacent to, or invading the metastatic tissue. In addition, some F4/80-positive cells were found in the aforementioned cell death zones. Unlike F4/80-positive cells, CD45R-positive B-lymphocytes did not accumulate at the surfaces of the tumor lesions, nor infiltrate into them, but were found scattered over bone marrow. Interestingly, some CD45R-positive cells were observed close to TRAP-positive osteoclasts in the stromal tissue surrounding the tumor lesion. Our findings suggest that, in the bone metastatic lesions of non-immunocompromised mice, F4/80-positive macrophages/monocytes accumulated on and/or infiltrated into the tumor nests, while CD45R-positive B-lymphocytes were associated with osteoclasts, rather than attacking metastatic tumor cells.

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We showed earlier that neurite outgrowth of rat pheochromocytoma PC12 cells was stimulated by royal jelly extract (PERJ) or its unique component, AMP N₁-oxide, via adenosine A2a receptors. In this study, we found that stimulated neurite outgrowth occurred in medium supplemented with serum, but not in serum-free medium. The pentapeptide GRGDS, which includes the RGD sequence commonly shared by extracellular matrix (ECM) components, could attenuate the effect of serum, suggesting that integrin receptor signaling was essential for the neurite outgrowth induced by PERJ or AMP N₁-oxide. PERJ or AMP N₁-oxide also activated extracellular signal-regulated kinases 1 or 2 (ERK1/2); however, this activation was not associated with the neurite outgrowth. As it is known that Mn²⁺ induces neurite outgrowth from PC12 cells and activates ERK1/2 through integrin signals and that activation of ERK1/2 is essential for Mn2+-induced neurite outgrowth, a difference in the mechanism between Mn²⁺-induced and PERJ- or AMP N₁-oxide-induced neurite outgrowth is suggested. Furthermore, we demonstrated that PERJ contained no ECM component-like substances. These results demonstrate that AMP N₁-oxide and its analogues were the only entities in PERJ with neurite outgrowth-inducing activity and that they required integrin signaling in addition to activation of A2a receptors to induce neurite outgrowth.

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Xerostomia frequently arises in patients with head and neck malignancies that are treated by radiation. However, the mechanisms responsible for the destruction of the salivary gland remain unknown. We previously established a xerostomia model of mice and identified the pathway through which nitric oxide (NO) affects the pathogenesis of radiation-induced salivary gland dysfunction. Although the toxicity of NO alone is modest, NO with superoxide anion (O₂^•‾) rapidly forms peroxynitrite (ONOO‾), a more powerful toxic oxidant. In this study, we used the experimental model to examine: 1) when NO and O₂^•‾ production is maximum in the salivary gland after irradiation;2) whether peroxynitrite, as assessed by nitrotyrosine production, is responsible for salivary gland dysfunction; and 3) the effect of the iNOS selective inhibitor, aminoguanidine (AG), on nitrotyrosine formation. The increases in production of NO and O₂^•‾ in the salivary gland peaked on day 7 after irradiation. Nitrotyrosine detected immunohistochemically was significantly reduced by AG in the salivary gland. On the basis of these results, we concluded that NO together with O₂^•‾ forms the more reactive ONOO‾, which might be an important pathogenic factor in radiation-induced salivary gland dysfunction.

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Effects of isolation and vitrification protocols on follicular survival after warming were examined. Mouse preantral follicles enzymatically or mechanically isolated from ovaries of 12-day-old mice were exposed either to 2 M ethylene glycol (EG) for 2 or 5 min, or to ascending concentrations (0.15 then 0.3 M) of raffinose for 2 or 5 min each (2-2 and 5-5 min). They were then exposed to a vitrification solution (VS) composed of 6 M EG and 0.3 M raffinose for 0.5, 1, or 2 min before vitrification. Mechanically isolated follicles showed higher survival than enzymatically isolated follicles, regardless of periods of exposure to EG or raffinose and subsequent exposure to VS. After 10 days of culture, follicular growth and maturational ability of oocytes derived from vitrified follicles exposed to 2 M EG for 5 min and to VS for 1 min were higher than those from follicles exposed to raffinose solutions for 2-2 min and to VS for 1 min. Histological evaluation revealed that exposure of preantral follicles to raffinose solutions caused cytoplasmic vacuolation in granulosa cells which could be due to cellular shrinkage during dehydration; whereas, exposure to 2 M EG induced morphological alterations in follicles only to a lesser extent.

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Nerve growth factor (NGF) is a well-known neurotrophic factor and the NGF signaling through the receptor, TrkA, plays important roles in regulating neuronal differentiation and survival. A recent study has demonstrated that the TrkAs expressed in undifferentiated PC12 cells were associated with caveolae, which were invaginated small pits on the plasma membrane. Caveolae are frequently seen in many cell types such as endothelial cells, fibroblasts and hepatocytes, but few in neurons. In the present study, we performed immunocytochemistry of TrkA in differentiated PC12 cells and analyzed the ultrastructural localization of TrkA by conventional electron microscopy and high-voltage electron microscopic (HVEM) tomography. The TrkA immunoreactivities were mainly associated with the cytoplasmic vesicles (10—30 nm in diameter) and a part of the plasma membrane. The HVEM tomography showed that the TrkA immunoreactivities were often assembled into ring-like structures (400—800 nm in diameter) near the plasma membrane, unlike typical flask-shaped invaginations of caveolae (50—100 nm in diameter). These results suggest that TrkA are not localized in the caveolae, at least in differentiated PC12 cells, but other invaginations are involved in a novel process of internalization of ligand-bound TrkA.

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