Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium

We investigated the relationship between mouse taste bud development and innervation of the soft palate. We employed scanning electron microscopy and immunohistochemistry using antibodies against protein gene product 9.5 and peripherin to detect sensory nerves, and cytokeratin 8 and α-gustducin to stain palatal taste buds. At E14, nerve fibers were observed along the medial border of the palatal shelves that tracked toward the epithelium. At E15.5, primordial stages of taste buds in the basal lamina of the soft palate first appeared. At E16, the taste buds became large spherical masses of columnar cells scattered in the soft palate basal lamina. At E17, the morphology and also the location of taste buds changed. At E18–19, some taste buds acquired a more elongated shape with a short neck, extending a variable distance from the soft palate basal lamina toward the surface epithelium. At E18, mature taste buds with taste pores and perigemmal nerve fibers were observed on the surface epithelium of the soft palate. The expression of α-gustducin was demonstrated at postnatal day 1 and the number of pored taste buds increased with age and they became pear-shaped at 8 weeks. The percent of pored fungiform-like papillae at birth was 58.3% of the whole palate; this increased to 83.8% at postnatal day 8 and reached a maximum of 95.7% at 12 weeks. The innervation of the soft palate was classified into three types of plexuses in relation to taste buds: basal nerve plexus, intragemmal and perigemmal nerve fibers. This study reveals that the nerve fibers preceded the development of taste buds in the palate of mice, and therefore the nerve fibers have roles in the initial induction of taste buds in the soft palate.     

A kinetic assay for eosinophil peroxidase activity in eosinophils and eosinophil conditioned media.

The activity of eosinophil peroxidase (EPO) is commonly employed as a measure of eosinophil activation in biologic fluids. Determination of product formation by this enzyme by end-point measurement may be affected profoundly by substrate concentrations, reaction time and degradation of end-product and enzyme. To determine more accurately EPO concentrations in media conditioned by isolated, purified eosinophils, we have developed a kinetic, colorimetric assay to measure EPO concentration as a function of maximum velocity of reaction (Vmax). An automated method for determining Vmax in a 96-well microplate colorimetric assay was utilized over a wide range of substrate concentrations. Concentrations greater than or equal to 3 x 10(-8) g/ml could be determined reliably with this assay. Peroxidase activity was inhibited in a concentration-dependent manner by the addition of 3-amino-1,2,4-triazole (AMT). The EPO concentration in eosinophils determined by this kinetic method was approximately 1.1 x 10(-5) g/10(6) eosinophils. Eosinophil activation with 10(-6) M f-Met-Leu-Phe (fMLP) caused substantial EPO secretion (9.0 +/- 1.7% vs. 2.9 +/- 0.6% total EPO content for control, P = 0.05) and decrease in eosinophil EPO concentration (92.3 +/- 4.2% of control, P = 0.038). Secretion was enhanced by the addition of 5 micrograms/ml cytochalasin B to 10(-6) M fMLP (25.9 +/- 12.7% total EPO content, P = 0.043 vs. control); similar decreases were noted in eosinophil EPO concentration (71.7 +/- 16.1% of control, P = 0.043). These data demonstrate that determination of EPO secretion by measurement of Vmax is a reliable, accurate method for precise quantification of this enzyme in media containing purified eosinophils or eosinophil products in the absence of other forms of peroxidase activity.     

Lysophospholipase activity of Ascaris suum-induced mouse peritoneal neutrophils and eosinophils.

Lysophospholipase activity of mouse peritoneal neutrophils and eosinophils was studied to determine if neutrophils and eosinophils have lysophospholipase activity when treated with Ascaris suum whole worm extract, if zymosan activated complement can induce increased lysophospholipase activity, or if the immune status of the host has an effect on lysophospholipase activity. Neutrophils from noninfected or infected (immunized) mice were found to have increased lysophospholipase activity when treated with A. suum whole worm extract or zymosan activated complement demonstrating neutrophils as a source of lysophospholipase activity in the presence or absence of an immune response. Eosinophils from immunized mice had increased lysophospholipase activity when treated with either A. suum whole worm extract or zymosan activated complement.     

Induction of estrogen receptor, peroxidase activity, and epithelial abnormalities in the mouse uterovaginal epithelium after neonatal treatment with diethylstilbestrol

Neonatal female NMRI mice were treated with daily doses of 10(-2) or 5 micrograms diethylstilbestrol (DES) on one or more of days 1-5 after birth. Using immunohistochemical techniques and a monoclonal antibody to the estrogen receptor, we demonstrated an estrogen-induced precocious appearance of receptor protein in the nuclei of the uterovaginal epithelium. High levels of peroxidase activity and a pronounced stromal infiltration with peroxidase positive cells occurred in the uterine cervix and upper vagina after estrogen treatment. This regional restriction in peroxidase activity was similar to the regional restriction for estrogen-induced epithelial abnormalities (heterotopic columnar epithelium, adenosis). A combined treatment with DES and corticosterone depressed peroxidase activity but not to the control level. The distribution of abnormal epithelium was similar in DES- and DES-corticosterone-treated females. The conclusion is that neonatal estrogen treatment induces an epithelial receptor for estrogen and a high level of cervical peroxidase activity, but the relationship between these parameters and the appearance of abnormal cervicovaginal epithelial changes could not be settled in the present study.     

Transient appearance of fibrinolytic activity at the epithelium of the rat uterus

The observation by Todd (1964) of a diffuse fibrinolytic activity related to the human endometrium in its secretory stage initiated a reinvestigation of the development of fibrinolytic activity in the uterus and vagina of the rat using the histochemical fibrin slide technique. In addition to the plasminogen activator known to be related to uterine vessels, in the rat in particular to myometrial arteries, diffuse fibrinolytic activity appeared fleetingly at the endometrial surface epithelium in relation to the oestrous cycle. Endometrial glandular epithelium remained inactive. The fibrinolytic activity of the endometrial epithelium is presumably released during cellular degeneration in the secretory phase. The vaginal epithelium produced fibrinolytic activity at an earlier phase of the ovarian cycle than did the endometrial epithelium.     

Ontogeny of macrophage function. I. Phagocytic activity and A-cell activity of newborn and adult mouse peritoneal macrophages.

Phagocytic activity and immune-participating A-cell activity of newborn and adult mouse macrophages were investigated under in vitro conditions. Thioglycollate medium-stimulated newborn (SNB) or adult (SA), and nonstimulated adult (NA) peritoneal macrophages were used. Immune complexes of sheep erythrocytes (SRBC), aldehyde-fixed SRBC, and latex beads were employed in phagocytosis tests. A-cell activity was estimated as the capacity to support primary or secondary responses of macrophage-deprived adult spleen cells to SRBC. Results obtained were: 1) phagocytic activity of NA macrophages was highest and that of SNB macrophages was higher than SA macrophages, 2) no difference was observed in A-cell activity for secondary response among SNB, SA, and NA macrophages, and 3) SNB macrophages were lacking in A-cell activity for primary response. These results suggest that the substantial A-cell function should be evaluated in primary responses, and that the A-cell and pahgocytic functions do not necessarily accompany each other.     

Distribution of peroxidase and granulocytes in the human uterus

A variety of uterine cell types demonstrate endogenous peroxidase activity. Ultracytochemical localization, biochemical assays, and uterine granulocyte counts were used to characterize peroxidase activity in various regions of the human uterus and cervix during the menstrual cycle and during the postmenopausal period. Previous studies of rat uteri, using electron microscopy and biochemical assays, have shown that endometrial peroxidase is induced by estrogenic stimulation (Anderson, De Sombre, and Kang, J Cell Biol 64:668, 1975; and Biol Reprod 16:409, 1977). Tissue samples from four regions of the human uterus and one sample from the endocervix were processed for ultrastructural cytochemistry, biochemical assay, and histology. Endogenous peroxidase activity was identified with electron microscopy in the endoplasmic reticulum of endometrial epithelial cells lining four regions of the uterine cavity; the isthmus, body (2), and fundus, of some proliferative phase (2 of 6), all secretory phase (4 of 4) and all postmenopausal (3 of 3) endometria. Peroxidase activity was not demonstrable in endocervical epithelial cells. Endogenous peroxidase activity was also identified in the cytoplasmic granules of uterine eosinophils and neutrophils and in the endoplasmic reticulum of mast cells. These uterine granule-containing cells, identified with special stains in the histologic sections, were quantitated. Approximately 80% of these "uterine granulocytes" from normal uteri without intrauterine devices were neutrophils. In women of reproductive age the uterine granulocytes, although present throughout the menstrual cycle, were most numerous in the endocervix and lower uterine segment. The highest biochemical assays of peroxidase activity were also obtained in the cervix and lower uterine segment. Uterine granulocyte counts varied directly with biochemical assays of peroxidase activity indicating that they were a major determinant of biochemical peroxidase activity. Endometrial epithelial peroxidase is anatomically and temporally well placed to function as an important adjunct in maintaining a mucosal barrier to microorganisms.     

Leukocyte distribution in the pseudopregnant mouse uterus.

Pseudopregnancy, induced by mating females with vasectomized males, is a frequently used model for studying pregnancy-like uterine changes in the absence of an embryo. Leukocytes make a significant contribution to uterine cellularity during pregnancy. The present study was designed to determine whether changes in numbers and distribution of leukocytes in the uterus during pseudopregnancy and following intraluminal injection of a deciduogenic stimulus parallel changes observed during the first eight days of pregnancy. Common leukocyte antigen positive (CLA+) cells, macrophages (F4/80+ cells), and granulocytes were assessed between days 1 and 8 of pseudopregnancy using qualitative and quantitative immunohistochemistry. High numbers of CLA+ leukocytes were present on days 1 and 2. Those were comprised primarily by macrophages, polymorphonuclear leukocytes, and eosinophils. High concentrations of leukocytes were detected in the endometrium, and some granulocytes were observed migrating through the luminal epithelium. Leukocytes, principally macrophages, were reduced in number and were distributed throughout the endometrium on days 3 and 4. Introduction of oil to the uterine lumen on day 4 stimulated primary decidualization. Decidual cells were CLA- and F4/80-, and, as decidualization proceeded, CLA+ and F4/80+ cells decreased in number in the anti-mesometrial uterus and were detected primarily in the deep endometrium. Later, a secondary decidualization zone developed in the mesometrial aspect of the uterus. Unlike the initial decidual reaction, which was relatively free of leukocytes, the secondary decidual zone contained very high numbers of CLA+ and F4/80+ cells. The uninjected uterine horn remained relatively unchanged from days 3 through 8.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-activating factor induces eosinophil peroxidase release from purified human eosinophils.

The degranulation response of purified human eosinophils to platelet-activating factor (PAF) has been studied. PAF induced release of eosinophil peroxidase (EPO) and beta-glucuronidase from highly purified human eosinophils with an EC50 of 0.9 nM. The order of release was comparable with that induced by phorbol myristate acetate (PMA). The new specific PAF antagonist 3-[4-(2-chlorophenyl)-9-methyl-H-thieno[3,2-f] [1,2,4]triazolo-[4,3a][1,4]-diazepin-2-yl](4-morpholinyl)- 1-propane-one (WEB 2086) inhibited the PAF-induced enzyme release by human eosinophils in a dose-dependent manner. The viability of eosinophils were unaffected both by PAF and WEB 2086. The results suggest that PAF may amplify allergic and inflammatory reactions by release of preformed proteins from eosinophil granules.     

Ontogeny and development of extrathymic T cells in mouse liver.

We previously demonstrated that the liver may be a major site of extrathymic T-cell differentiation in mice. In the present study, the ontogeny and subsequent development of such T cells in the liver and other organs were investigated. This study was possible because these T cells have T-cell receptors (TcR) of intermediate intensity (i.e. intermediate TcR cells) and constitutively express a high level of interleukin-2 receptor beta chain (IL-2R beta). Therefore the two-colour staining for CD3 (or alpha beta TcR) and IL-2R beta identifies even a small proportion of intermediate TcR cells. The total numbers of mononuclear cells obtained from the liver, thymus and spleen varied from foetal to adult life. Especially in the liver, many haematopoietic cells were present in the parenchymal space at the foetal stage. There were no lymphocytes in the sinusoidal lumen at this period. In contrast, lymphocytes appeared in the hepatic sinusoids after birth and increased with ageing. Phenotypic analysis revealed that intermediate TcR cells appeared in the liver and spleen on Day 4 after birth. Bright TcR cells of thymic origin were also present in the peripheral organs on Day 4. Thereafter, intermediate TcR cells increased in the liver, whereas bright TcR cells increased in the periphery as a function of age. Similarly, thymectomized and congenitally athymic mice had mainly intermediate TcR cells in the liver and, to some extent, periphery. It is concluded that intermediate TcR cells, possibly of extrathymic origin, are generated only after birth and expand with ageing.     

Ontogeny of rat thymic epithelium defined by monoclonal anticytokeratin antibodies.

Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti-CK monoclonal antibodies (mAbs) using the streptavidin-biotin immunoperoxidase method. Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated by double immunofluorescence labeling. The obtained results showed that the expression of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC, respectively, whereas CK19 is mainly a pan-TEC marker. In the adult animals, they are localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and some medullary TEC (CK19), or in a subset of medullary TEC and Hasall's corpuscles (HC) (CK10). The switch in their expression in the cortex was observed during the first two weeks of postnatal life.     

Extracellular release of peroxidase from eosinophils by interaction with immune complexes.

Much peroxidase is released from eosinophils that ingest complexes formed of human immunoglobulins with specific rabbit antibody. The complex formed of IgE with rabbit antibody, was particularly effective. The amount and rate of release of peroxidase was closely related to the amounts of complex ingested by the eosinophils, and degree of lysis of the cell granules. It is proposed that eosinophils attracted to an allergic lesion ingest complexes of IgE, show lysis of granules with release of peroxidase, and that the peroxidase reduces the allergic reaction.     

IgG and complement receptors on purified mouse eosinophils and neutrophils.

Mouse eosinophil and neutrophil receptors for IgG and complement have been examined by means of rosette formation, phagocytosis and 51Cr release assays, using mouse monoclonal antibodies and complement-coated sheep erythrocytes. Mouse eosinophils and neutrophils form a high number of rosettes in the presence of mouse complement but eosinophils show a higher requirement for complement molecules. Both types of granulocyte phagocytose complement-coated sheep erythrocytes very actively, although low levels of 51Cr release are obtained. Eosinophils and neutrophils show higher activity in the presence of IgG2b than in the presence of IgG1, and while both cell types are similarly active when the former antibody is used, neutrophils are the more active when IgG1 is used. However, it remains uncertain whether this is a result of the higher binding obtained with the IgG2b monoclonal antibody. Both cell types behave similarly at high antibody concentrations but neutrophils are the more active at high antibody dilutions. The 51Cr release assay is shown to be superior to the rosette assay at it allows comparisons between eosinophils and neutrophils at high antibody concentrations. A time course study indicates that highest values of phagocytosis of opsonized red cells are obtained after 5 minutes rather than the half to one hour incubation periods normally used.