Tyrosine hydroxylase and serotonin containing cells in embryonic rat rhombencephalon: a whole-mount immunocytochemical study

Rhombencephala from rat embryos were processed as whole-mounts for immunocytochemical detection of monoaminergic cell populations, using antibodies to tyrosine hydroxylase (TH) and serotonin (5-HT). Specific advantages of the whole-mount technique over the classical serial-section method were that even isolated immunoreactive (IR) cells could be detected easily, and three-dimensional relationships could be ascertained without the need for serial reconstruction. Embryos between embryonic days (E) 12 and 16 (the day following nocturnal mating being considered as E1) were used in this study. Both TH and 5-HT immunoreactivities were already detectable at E12, even in the smallest embryos (crown-rump length: 6 mm), but there was a striking difference in the number and regional distribution of these two types of IR cells. TH was expressed in several cell groups located in the rostral rhombencephalon (the presumed anlage of the A4-7 complex) as well as in the caudal rhombencephalon (the presumed anlagen of groups A1-2 and C1-3), whereas 5-HT was expressed in very few cells located near the rostral border of the rhombencephalon (presumed anlage of the B4-9 complex). Although the three-dimensional distribution of the TH-IR cell groups underwent some modifications during the period studied, its general pattern remained relatively stable after E12. This contrasted with the sequential appearance of the 5-HT-IR cell groups and their spatial transformations during this period. Using the rhombencephalic isthmus as a landmark, we found that conspicuous 5-HT-IR fibre bundles penetrated into the mesencephalon from E13 onwards, but that the 5-HT IR cell bodies were exclusively located caudal to the borderline between the mesencephalon and the rhombencephalon (the rhombencephalic isthmus). We therefore suggest the term "rostral rhombencephalic raphe nuclei" for the rostral 5-HT cell groups instead of "mesencephalic raphe nuclei," which is a misnomer. Close spatial association between TH and 5-HT-IR elements was observed mainly in the caudal rhombencephalon, where 5-HT-IR fibres coursed through an area containing numerous TH-IR cell bodies (the presumed anlagen of groups A1-2 and C1-3).     

Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.     

A functionally active complement system is present in uterine secretion of the mouse prior to implantation.

Sephadex beads were placed carefully in the uterus on days 2 and 3 and left for 6 to 8 h to absorb uterine secretion. The beads were then removed with volatile silicon oil and mounted on small pieces of nitrocellulose paper. Immuno-staining of these bead blots showed they contained the complement components C1q, C3, C4, and C5. We demonstrated that complement component C3 in the uterine secretion could be activated and deposited on model immune complexes, and also that antibody-coated erythrocytes were lysed in utero, that is, a membrane attack complex was produced. Thus, the mouse uterine secretion at the preimplantation stage contains a functionally active complement system.     

A complex component modulating immune-deficient cells in leprosy patients leading to loss of viability of Mycobacterium leprae--a possible vaccine.

Macrophages from peripheral blood of leprosy patients, both multi-bacillary and paucibacillary are unable to kill phagocytosed Mycobacterium leprae due to their inability to produce superoxide (O2-) and hydroxyl radicals (OH.). The macrophages from healthy individuals are able to kill M. leprae along with release of O2- and OH. radicals. The deficiency in the macrophages of both types of leprosy patients is removed by activation of these cells when exposed to a culture supernatant obtained after stimulation of peripheral blood mononuclear cells from the same patients with delipidified cell components of M. leprae which are most likely cell wall proteins. The activation of macrophages also leads to recognition of whole live M. leprae as an antigen by cells from lepromatous patients. This activation of the phagocytes by delipidified cell components is blocked by cyclosporin A, indicating the possible role of several steps involved in immune activation of cells. The observations thus indicate the significant ability of delipidified cell components to eliminate the deficiencies in the macrophages from leprosy patients and restore them to behave like the cells from healthy individuals. Considering all these, it is suggested that delipidified cell components could be potential modulators, and are probably capable of functioning as a vaccine for leprosy.     

Analysis of allergenic components of Bermuda grass pollen by monoclonal antibodies

A panel of 16 monoclonal antibodies (MoAbs) directed against Bermuda grass (Cynodon dactylon) pollen (BGP) were generated for identification and purification of the major allergenic components of the eliciting antigen (Ag). Radioimmunoprecipitation (RIP) analysis revealed that there were at least eight antigenic components with molecular weights (MW) ranging from 12 kilodalton (12 kDa) to 200 kDa. Each of these components has distinct biochemical characteristics based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Among them, Cyn d Bd67K and Cyn d Bd58K were basic proteins, Cyn d Bd35K consisted of at least four isomeric components with isoelectric points ranging from 6.2 to 7.2. The other antigens (Cyn d Bd68K, 48K, 38K, Cyn d Bd200K, Cyn d Bd46K, Cyn d Bd25K and Cyn d Bd12K) were all acidic proteins. The IgE binding capacity of all these antigens was determined with sera from 11 BGP-allergics by using a modified radioallergosorbent test. All but one of the antigens (Cyn d Bd200K) were found to react with human IgE from sera of BGP-allergic patients. Among those human IgE-binding molecules, Cyn d Bd35K reacted with allergic sera most frequently (10 of 11), followed by Cyn d Bd58K (8 of 11) and Cyn d Bd46K (7 of 11) respectively. Our results suggest that Cyn d Bd35K, Cyn d Bd58K, and Cyn d Bd46K are major allergens of BGP, and the MoAbs we obtained should be valuable tools for further purification of these allergens.     

Mechanisms of the CYNAP phenomenon: evidence in the Bw49/Bw50 model for epitopes with different spatial orientation of antibody

The mechanism of the cytotoxic-negative, absorption-positive (CYNAP) phenomenon was studied using the model of the Bw49/Bw50 split of the BW21 antigen. Anti-Bw49 antibody bound 60% as well to Bw 50 as to Bw49 cells; however, even at a cytotoxic titer of 64 against Bw49 cells, the antibody was not cytotoxic to Bw50 Cells. At equal numbers of antibody molecules bound, the anti-Bw49 antibody activated C4 and C3, and induced lysis for Bw49 but not for the Bw50 cells. These data are consistent with a model in which different spatial orientations for shared epitopes can account for CYNAP reactivity for at least some selected Bw4/Bw6-associated splits of B locus antigens.     

Ascending and descending components of the medial forebrain bundle in the rat as demonstrated by the horseradish peroxidase-blue reaction

Summary The ascending and descending components of the medial forebrain bundle (MFB) were investigated by means of horseradish peroxidase (HRP) with a sensitive substrate. The HRP was injected iontophoretically into the MFB at various levels from the anterior commissure to the posterior hypothalamus. In order to prevent the diffusion of HRP to other brain areas, a double micropipette system was used. The descending components of the MFB are derived from (1) the anterior cingulate area, infra- or prelimbic area, and sulcal cortex, (2) the lateral septal nucleus and diagonal band, (3) the bed nucleus of the stria terminalis, (4) the paraventricular nucleus (5) the substantia innominata, (6) the amygdaloid complex (AM), (7) the ventromedial (VM) and dorsomedial (DM) hypothalamic nuclei, (8) the entopeduncular nucleus and (9) nucleus periventricularis stellatocellularis. The ascending components of the MFB originate in: (1) the medial preoptic nucleus, (2) the nucleus periventricularis stellatocellularis and rotundocellularis, (3) the posterior hypothalamic nucleus, (4) the parafascicular nucleus, (5) the ventral premammillary nucleus, (6) the substantia grisea periventricularis, (7) the lateral habenular nucleus, (8) the VM and DM, (9) the paratenial nucleus, (10) the AM and (11) the arcuate nucleus.Abbreviations used in Figures and Tables a nucleus accumbens - abl nucleus amygdaloideus basalis, pars lateralis - abm nucleus amygdaloideus basalis, pars medialis - ac nucleus amygdaloideus centralis - AC anterior cingulate area - al nucleus amygdaloideus lateralis - am nucleus amygdaloideus medialis - ar nucleus arcuatus - CC tractus corporis callosi - CSDV commissura supraoptica dorsalis, pars ventralis - DB diagonal band - DM nucleus dorsomedialis hypothalami - EP nucleus entopeduncularis - ha nucleus anterior hypothalami - hl nucleus lateralis hypothalami - hp nucleus posterior hypothalami - IL infralimbic area of frontal cortex - lh nucleus habenulae lateralis - LH₁ medial forebrain bundle (MFB) at the level of commissura anterior - LH₂ lateral preoptic area - LH₃ MFB at the level of the nucleus anterior hypothalami - LH₄ MFB at the level of the nucleus ventromedialis hypothalami - LH₅ MFB at the level of the nucleus posterior hypothalami - MFB medial forebrain bundle - pf nucleus parafascicularis - PL prelimbic area of frontal cortex - pol nucleus preopticus lateralis - pom nucleus preopticus medialis - posc nucleus preopticus, pars suprachiasmatica - pt nucleus parataenialis - pv nucleus premamillaris ventralis - PV nucleus paraventricularis - pvs nucleus periventricularis stellatocellularis - pvr nucleus periventricularis rotundocellularis - SC sulcal cortex - SGPV substantia grisea periventricularis - SI substantia innominata - SL lateral septal nucleus - ST bed nucleus of stria terminalis - sum nucleus supramamillaris - TO tractus opticus - tmm nucleus medialis thalami, pars medialis - VM nucleus ventromedialis hypothalami The nomenclature used in this paper is according to König and Klippel's Stereotaxic Atlas (1967).     

Bovine colostrum supplementation does not affect plasma buffer capacity or haemoglobin content in elite female rowers

It has been reported that bovine colostrum (BC) supplementation improves buffer capacity () during exercise, but whether the improvement results from changes in tissue and/or blood buffer systems has not been determined. The purpose of the present study was to examine the effect of supplementation with BC on blood buffer systems. Thirteen elite females rowers were supplemented with 60 g·day^–1 of either BC (n=6) or whey protein (WP, n=7) during 9 weeks of pre-competition training in a randomised, double-blind, placebo-controlled, parallel design. All subjects undertook the study as a group and completed the same training program. Resting haemoglobin (Hb) concentration and plasma buffer capacity (p) (determined by titration with HCl) were measured pre- and post-supplementation. There were no differences in macronutrient intakes (P>0.56) or training volumes (P>0.99) between BC and WP during the study period. There were no differences in Hb [BC 13.28 (0.28) mg·dl^–1, WP 13.70 (0.26) mg·dl^–1; P=0.45] or p [BC 14.8 (1.1) nmol HCl·ml^–1·pH^–1, WP 14.8 (0.5) nmol HCl·ml^–1·pH^–1; P=0.68] between groups at week 0. p increased in both groups during the study period (P<0.001), but the increases were not significantly different between groups (P=0.52). Hb did not change significantly in either group (P=0.35). These data indicate that supplementation with BC does not affect p or Hb. We therefore suggest that adaptations in tissue buffer systems are responsible for the previously reported increases in buffer capacity that result from BC supplementation.     

Gas transport during high-frequency ventilation: Theoretical model and experimental validation

We present a theoretical model of gas transport through the dead space during high-frequency ventilation (HFV) with volumes less than dead space volume. The analysis is based on the axial distribution of transit times of gas moving through the dead space. The model predicts that for tidal volumes (V) much less than dead space (V_d), gas exchange will be proportional to the product of frequency (f) and V². If gas transport is analyzed in terms of Fick's law, then the effective diffusion coefficient (D_eff) can be shown to be equal to fV² times a constant, whose value equals the square of the coefficient of dispersion of axial transit times through the dead space . Experimental results in straight tubes fit the predictions of this model quite well. A through the entire dead space of about 30% is more than sufficient to account for gas exchange during HFV in physical models or in intact animals. An axial dispersion of this magnitude can be measured directly from a typical Fowler dead space determination in healthy subjects.     

Computer Imaging Analysis of Glomerular Extracellular Components in Patients with IgA Nephropathy

Computer imaging analysis was used for quantitative evaluation of the extents, amounts and distributions of glomerular extracellular components, such as the 7S and NC 1 domains of type IV collagen, laminin (LN), fibronectin (FN) and IgA, in glomeruli from patients with IgA nephropathy. Renal biopsy specimens from 13 patients with IgA nephropathy were incubated with mouse monoclonal antibodies against the FN or non collagenous (NC 1) domain of type IV collagen or polyclonal antiserum against the LN or 7S domain of human type IV collagen, and then stained with appropriate dilutions of FITC labeled anti mouse Ig antisera. Marked staining of the 7S or NC 1 domain of type IV collagen, LN or FN was detected in the glomerular capillary walls and/or mesangial areas in patients with IgA nephropathy. In particular, a prominent increase of FN was observed in the subendothelial regions of glomerular capillary walls, i.e. mesangial interposition, in the moderate or advanced stage of IgA nephropathy. Therefore, computer imaging analysis was shown to be useful for the quantitative determination of such components distributed in glomeruli from patients with IgA nephropathy. Acta Pathol Jpn 39: 296 305, 1989.