Appearance of prolactin-releasing peptide-producing neurons in the area postrema of adrenalectomized rats

Prolactin-releasing peptide (PrRP) was found to be a novel hypothalamic peptide that stimulates prolactin release in vitro and in vivo. In the normal adult rat brain, PrRP neurons are known to be located in only three areas, i.e. the dorsomedial hypothalamic nucleus, ventrolateral reticular formation; and nucleus of the tractus solitarius in the medulla oblongata. These PrRP neurons project neurites into various brain areas, including regions such as the paraventricular nucleus, supraoptic nucleus, and bed nucleus of the stria terminalis. Both PrRP nerve fibers and a high level of PrRP receptor, UHR-1, mRNA are observed in the area postrema (AP),but no PrRP neurons are detected in the AP of normal rats. In this study, we clearly demonstrated that PrRP-producing cells newly appeared in the AP of adrenalectomized rats by in situ hybridization and immunocytochemistry. Our results suggest that PrRP may have some important roles in the AP of adrenalectomized rats. This is the first report demonstrating the appearance of PrRP-positive cells in the AP.     

Differential IL-12 responsiveness of T cells but not of NK cells from tumor-bearing mice in IL-12-responsive versus -unresponsive tumor models

While IL-12 administration induces tumor regression through stimulating T cells in tumor-bearing mice, this IL-12 effect is observed in some but not all tumor models. The present study aimed to compare IL-12 responsiveness of T cells from tumor-bearing mice in IL-12-responsive (CSA1M and OV-HM) and -unresponsive (Meth A) tumor models. Tumor regression in IL-12-responsive tumor models required the participation of T cells, but not of NK1.1(+) cells. Because a NK1.1(+) cell population was the major producer of IFN-gamma, comparable levels of IFN-gamma production were induced in IL-12-responsive and -unresponsive tumor-bearing mice. This indicates that the amount of IFN-gamma produced in tumor-bearing individuals does not correlate with the anti-tumor efficacy of IL-12. In contrast, IL-12 responsiveness of T cells differed between the responsive and unresponsive models: purified T cells from CSA1M/OV-HM-bearing or Meth A-bearing mice exhibited high or low IL-12 responsiveness respectively, when evaluated by the amounts of IFN-gamma produced in response to IL-12. T cells from CSA1M- or OV-HM-bearing but not from Meth A-bearing mice exhibited enhanced levels of mRNA for the IL-12 receptor (IL-12R). These results indicate that a fundamental difference exists in IL-12 responsiveness of T cells between IL-12-responsive and -unresponsive tumor models, and that such a difference is associated with the expression of IL-12R on T cells.     

Cytoplasmic p21(Cip1/WAF1) enhances axonal regeneration and functional recovery after spinal cord injury in rats

p21(Cip1/WAF1), known as a cell-cycle inhibitory protein, facilitates neurite outgrowth from neurons when present in the cytoplasm. The molecular mechanism of this action is that p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity. As myelin-derived inhibitors of axonal outgrowth act on neurons by activating Rho, that is responsible for the lack of spontaneous regeneration of the injured central nervous system (CNS), Rho-kinase may be a good molecular target against injuries in the CNS. In this study, we delivered TAT-fusion protein of cytoplasmic p21(Cip1/WAF1) locally after dorsal hemisection of the thoracic spinal cord in rats. The treatment significantly stimulated axonal regeneration and recovery of hindlimb function, and inhibited the cavity formation in the spinal cord after the injury. Cytoplasmic p21(Cip1/WAF1) may provide a potential therapeutic agent that produces functional regeneration following CNS injuries.     

Redistribution of fibroblasts and macrophages as micrometastases develop into established liver metastases

Fibroblastic tissue is an important component of malignant tumors, involved in the establishment of metastatic foci from micrometastases, and thought to prevent invasion of metastatic tumor cells into surrounding tissue. However, experimental models of fibrosis during the growth of micrometastasis into established metastases were not previously available. In the present paper, we performed immunohistochemical studies on experimental hepatic metastasis with colon 38 mouse colon carcinoma cells injected into syngeneic C57BL/6 mice. Early and late stages of metastatic nodules were examined for the distribution of endothelial cells, fibroblasts, and macrophages by the use of markers of these cells. One week after intrasplenic injection of colon 38 cells, micrometastases mainly appeared in the region of sinusoids accompanied with invasion of F4/80-positive Kupffer cells. Transitional metastases can be defined based on the histological appearance and intensive infiltration of both macrophages and fibroblasts. These transitional metastases were connected by protrusions of fibroblast-rich tissues co-localized with collagen-rich matrix and CD31-positive cells. This protrusion preceded fibrosis formation characteristics to established metastases associated with angiogenesis and segregation of tumor cells from host cells. Three stages can thus be classified during the development of hepatic metastasis in this syngeneic experimental system: micrometastasis, transitional metastasis, and established metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

An improved fixation method for transmission electron microscopy for the histological study of the lens.

An improved fixation technique for transmission electron microscopic observation that enables good fixation of all areas of the rat lens was devised. Immersion fixation with 0.1 M phosphate-buffered 1.25% glutaraldehyde at 4 degrees C for 12 hours followed by postosmication produced good results for all areas of the lens--anterior, equatorial, and posterior zones. The technique was particularly suitable for maintenance of the shape of the lens since practically no irregularity, vacuolar degeneration, or expansion of the intercellular spaces was noted. This technique, which requires only a relatively short time, was also useful for the detection of early ultrastructural changes associated with cataract in spontaneously diabetic WBN/Kob rats. We anticipate that our procedure will be widely applied.     

Trehalose 6,6'-dimycolate (cord factor) of Mycobacterium tuberculosis induces foreign-body- and hypersensitivity-type granulomas in mice

Granulomatous inflammation is characterized morphologically by a compact organized collection of macrophages and their derivatives. It is classified as either a hypersensitivity type or a foreign-body type. Lipid components of the Mycobacterium tuberculosis cell wall participate in the pathogenesis of infection. Strains of M. tuberculosis have cord factor (trehalose 6,6'-dimycolate [TDM]) on their surface. To clarify host responses to TDM, including immunogenicity and pathogenicity, we have analyzed the footpad reaction, histopathology, and cytokine profiles of experimental granulomatous lesions in immunized and unimmunized mice challenged with TDM. In the present study, we have demonstrated for the first time that TDM can induce both foreign-body-type (nonimmune) and hypersensitivity-type (immune) granulomas by acting as a nonspecific irritant and T-cell-dependent antigen. Immunized mice challenged with TDM developed more severe lesions than unimmunized mice. At the active lesion, we found monocyte chemotactic, proinflammatory, and immunoregulatory cytokines. The level was enhanced in immunized mice challenged with TDM. This result implies that both nonimmune and immune mechanisms participate in granulomatous inflammation induced by mycobacterial infection. Taken together with a previous report, this study shows that TDM is a pleiotropic molecule against the host and plays an important role in the pathogenesis of tuberculosis.     

A mouse monoclonal antibody, S2n8, detects a 140 kDa protein on the surface of human endometrial stromal cells and decidual cells

We developed a mouse monoclonal antibody, S2n8, by immunizingmice i.p. with human decidual cells collected in the first trimesterof pregnancy. By indirect immunofluorescence staining of frozensections, S2n8 was found to react with decidual cells and endometrialstromal cells throughout the menstrual cycle, but not with endometrialglandular cells or with the endometrial surface epithelium.Judging from the fluorescence intensity, the antigen expressionon stromal cells was weak in the proliferative phase, and becamestronger in the secretory phase. Decidual cells in the firsttrimester of pregnancy and decidual cells at term showed strongexpression of this antigen. Indirect immunofluorescence stainingof enzymatically dispersed decidual tissue revealed that theS2n8 antigen was expressed on the decidual cell surface. Flowcytometric analysis of 12 freshly prepared stromal cell-enrichedcell suspensions showed that 74.8–94.2% (mean ±SD 86.1 ± 6.6%) of the cells carried the antigen. Theexpression of S2n8 antigen on cultured stromal cells was enhancedby the addition of oestradiol and/or progesterone. The antigenicmolecule was purified by immunoaffinity chromatography fromdecidua collected in the first trimester of pregnancy, and themolecular weight was estimated to be 140 kDa. These findingsindicate that the S2n8 antigen is a useful cell surface markerfor stromal cells/decidual cells and is associated with theirdifferentiation. cell surface antigen/decidual cells/endometrial differentiation/endometrial stromal cells/monoclonal antibody     

IMMUNOHISTOCHEMICAL LOCALIZATION OF TYPE I, II, III, AND IV COLLAGENS IN THE LUNG

Type specific rabbit antibodies to bovine type I, 11, 111, and IV (basement membrane) collagens showing no cross-reaction with other types of collagen were prepared by cross-adsorption and diethylamiuoethyl-cellulose romatography. The antibodies to bovine type I and I11 collagens showed a high cross-reaction with the corresponding human collagens, but those to type I1 and IV collagens did moderate and no cross-reactions with human type I1 and IV collagens, respectively. By using these antibodies, tissue distribution of various types of collagen in normal bovine lung was examined by indirect immunofluorescence microscopy. Both type I and I11 collagens were found to distribute widely in the interstitium of bronchial tree, bronchial
lamina propria and of interlobules as well as alveolar nipples and adventitia of pulmonary arteries. Type I1 collagen was located only in bronchial cartilage. The tissues mainly stained for type 11 collagen were the alveolar interstitium (also stained faintly for type I collagen) and the intima and media of the arteries. Type IV collagen was located in a membranous fashion in alveolar septa and bronchial smooth muscles and subepithelial layers as well as capillaries and the intima and media of arteries. ACTA PATHOL. JPN. 31: 601–610, 1981.