Peroxidase-antiperoxidase Method Research Articles

Glial fibrillary acidic protein (GFAP) immunochemical profile after Junin virus infection of rat cultured astrocytes

Cultured astrocytes derived from newborn rat brain were inoculated with Junin virus (JV) to characterize their response to infection by means of their glial fibrillary acidic protein (GFAP) immunochemical profile. Samples from 1 to 11 days post-inoculation (pi), as well as matched controls, were serially harvested for GFAP labeling by peroxidase-antiperoxidase (PAP) method. It was only at day 3 that significantly greater values of GFAP staining ( P < 0.05) were disclosed by three complementary approaches: image analysis, ELISA and immunoblot densitometry. Since such increase was abolished by Triton X-100 treatment, soluble GFAP fraction appeared responsible for the early though transient enhancement of GFAP immunoreactivity that followed viral inoculation.

Localization of corticotropin-releasing factor immunoreactivity in the brain of the teleost Sparus aurata

The distribution of perikarya and fibers containing corticotropin-releasing factor (CRF) was studied in the brain of the teleost Sparus aurata by immunocytochemistry using the peroxidase-antiperoxidase method. Antisera against rat CRF, arginine vasotocin, and human adrenocorticotropin (ACTH) were used. Most CRF-immunoreactive neurons were located in the nucleus lateralis tuberis, but they were absent from the nucleus preopticus, which only contained arginine vasotocin neurons. Few CRF perikarya were identified in the nucleus preopticus periventricularis and in the mesencephalic tegmentum. A conspicuous bundle of immunoreactive fibers ran along the diencephalic floor and pituitary stalk to end near the cells of the hypophysial pars intermedia. No CRF was seen near the adenohypophysial rostral pars distalis. Our results suggest that, in Sparus aurata, CRF is a releasing factor for melanotropic cells. Its role as a releasing factor for ACTH is discussed.

Immunophenotyping of Lymphocytes in Bronchoalveolar Lavage Fluid: A New Flow Cytometric Method vs Standard Immunoperoxidase Technique

Characterizing lymphocyte subsets in bronchoalveolar lavage fluid (BALF) by flow cytometry (FC) proper gating of the lymphocyte subpopulation being analyzed is crucial. In order to test lymphocyte gate quality for the first time we used a DNA-dye to evaluate plasmamembrane integrity and thus to mark off fluorescent but not DNA-containing particles (eg, debris). A comparative prospective study between this newly developed FC technique and a standard peroxidase anti-peroxidase (PAP) method was performed. Samples of BALF from 50 patients with various pulmonary diseases were examined. After determination of the total cell yield, a differential cell count was performed. Subsequently, the immunophenotype of pan T lymphocyte CD3-, T-helper lymphocyte CD4-, and T-suppressor lymphocyte CD8-positive lymphocyte subsets was assessed with FC as well as with the PAP method. Both methods showed excellent correlation (CD3: r = 0.81; CD4: r = 0.97; CD8: r = 0.96; p < 0.05, respectively). Comparing the mean +/- SEM, FC tends to overestimate CD3+ cells (90.6 +/- 1.0% vs 85.8 +/- 1.3%). For CD4 (45.0 +/- 3.4% vs 44.4 +/- 3.4%) and CD8 (48.1 +/- 3.5% vs 46.7 +/- 3.5%), there was good agreement. In a clinical setting, the reliability of both methods was equivalent, and FC using a DNA-dye to test lymphocyte gate quality offered a rapid and reliable determination of lymphocyte subsets in BAL.

An ultrastructural double-label immunohistochemical study of the enkephalinergic input to dopaminergic neurons of the substantia nigra in pigeons.

Electron microscopic immunohistochemical double-label studies were carried out in pigeons to characterize the ultrastructural organization and postsynaptic targets of enkephalinergic (ENK+) striatonigral projection. ENK+ terminals in the substantia nigra were labeled with antileucine-enkephalin antiserum by using peroxidase-antiperoxidase methods, and dopaminergic neurons were labeled with anti-tyrosine hydroxylase antiserum by using silver-intensified immunogold methods. ENK+ terminals on dopaminergic neurons were equal in abundance to ENK+ terminals on nondopaminergic neurons, although the former were typically somewhat smaller than the latter (mean size: 0.50 vs. 0.75 micron, respectively). ENK+ terminals were evenly distributed on the cell bodies and dendrites of dopaminergic neurons, and they were evenly distributed on dendrites but rare on perikarya of nondopaminergic neurons. Transection of the basal telencephalic output revealed that 75% of the nigral ENK+ terminals were of basal telencephalic origin. These telencephalic ENK+ terminals included over 80% of those smaller than 0.80 micron on dopaminergic neurons and smaller than 1.0 micron on nondopaminergic neurons, and none greater than this in size. Both telencephalic and the nontelencephalic ENK+ nigral terminals made predominantly symmetric synapses on nigral neurons. Although the basal telencephalic ENK+ terminals uniformly targeted dendrites and perikarya, nontelencephalic ENK+ terminals seemed to avoid perikarya. The results indicate that ENK+ striatonigral neurons in birds may directly influence both dopaminergic and nondopaminergic neurons of the substantia nigra. Based on similar data for substance P-containing striatonigral terminals, the roles of enkephalin and substance P in influencing nigral dopaminergic neurons may differ slightly, as they appear to target preferentially different portions of dopaminergic neurons. The overall results in pigeons are similar to those for ENK+ terminals in the ventral tegmental area in rats, suggesting that the synaptic organization of the ENK+ input to the tegmental dopaminergic cell fields is similar in mammals and birds.

Distribution of calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity in the trigeminal ganglion and mesencephalic trigeminal nucleus of the cat

The distribution of calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivity (LI) in the cat trigeminal ganglion (TrG) and mesencephalic trigeminal nucleus (MTN) was studied by the indirect immunofluorescent technique and unlabelled peroxidase-antiperoxidase method. A positive reaction for both CGRP and NPY was found only in the TrG primary afferent neurons. Between 40% and 50% of the perikarya displayed CGRP-LI whereas few of the trigeminal principal neurons (approximately 1%) showed NPY-LI. Most of the CGRP-immunoreactive cells were small to medium-sized (20-40 microns in diameter) and were distributed throughout the ganglion. NPY appeared specifically in medium-sized to large-sized trigeminal neurons (up to 60 microns). CGRP and NPY also occurred in varicose nerve fibres running among the cell bodies and in pericellular baskets around a small number of large unstained ganglion cells. In contrast, CGRP- and NPY-LI were present only in the nerve fibres and terminals of the cat MTN. The immunoreactive varicosities travelled towards the negative MTN neurons and formed basket-like arborizations around their cell bodies. Synaptic contact between the immunolabelled axonal profiles and perikarya of trigeminal primary afferent neurons could not be seen in the TrG and MTN of the cat. The results provide further evidence for the interrelation between sensory and autonomic systems.

Characterization and localization of gonadotropin-releasing hormone in the brain and pituitary of the three-spined stickleback, Gasterosteus aculeatus

Radioimmunoassay (RIA) studies on highperformance liquid chromatography (HPLC) fractions of brain extracts of the three-spined stickleback, Gasterosteus aculeatus, provided evidence for at least two forms of gonadotropin-releasing hormone (GnRH). One form showed chromatographic and immunological properties similar to that of synthetic salmon GnRH (sGnRH). A second, unidentified form of GnRH eluted in the same position as chicken GnRH I (cGnRH-I); however, it did not cross-react in a cGnRH-I RIA. Furthermore, it cannot be excluded that chicken GnRH II (cGnRH-II) and maybe one other unidentified form are present in the stickleback. The distribution of GnRH in the brain of breeding adult male sticklebacks was studied by use of immunohistochemistry. Two antisera against sGnRH and antisera against mGnRH and cGnRH-II were applied on cryosections and visualized using the peroxidase-antiperoxidase method. Staining patterns were similar after incubations with all four antisera. Immunoreactive fibers were found in most parts of the brain. Three distinct groups of GnRH-immunoreactive perikarya were found in the nucleus olfactoretinalis, in the nucleus anterior periventricularis, and in the nucleus lateralis tuberis. Moreover, weakly stained cells occurred in a periventricular position in the midbrain. The proximal pars distalis of the pituitary, housing the gonadotropic cells, was richly innervated by GnRH-positive fibers. In the pars intermedia and in the rostral pars distalis, immunoreactive fibers were absent.

Use of quantitative ultrastructural immunoperoxidase labeling for analysis of catecholamine neurotoxicity and plasticity

Levels of catecholamines and the synthesizing enzyme, tyrosine hydroxylase (TH) are markedly decreased in the dorsal striatum, caudate-putamen nuclei, following neurotoxic lesions with 6-hydroxydopamine (6-OHDA). We examined whether pre-embedding immunoperoxidase labeling of TH could be standardized for quantitatively examining the density and ultrastructure of spared dopaminergic terminals in the striatum of lesioned rats. The peroxidase-antiperoxidase (PAP) method was used to localize rabbit antiserum against TH in caudate-putamen nuclei of adult rats given unilateral nigral injections of either vehicle or 6-hydroxydopamine in the early postnatal period. Experimental differences in fixation and immunocytochemical labeling were minimized by limiting comparisons of immunoreactivity to co-processed sections from the same litters of animals. Imaging software and a Phillips CM-10 electron microscope were used to quantitatively examine immunoreactive profiles in a narrow zone of tissue in contact with the embedding resin. Under these conditions variables attributed to differences in penetration were minimized. There were no significant differences in numbers or mean-cross sectional diameter of immunoreactive terminals in striatum ipsilateral versus contralateral to vehicle injections. ipsilateral to the 6-OHDA injections, the density (numbers/area) of striatal TH-immunoreactive terminals was reduced by 50–90% in the majority of animals. In the most extensively lesioned rats, the cross-sectional areas of the remaining immunoreactive axons were significantly larger than in the contralateral striatum of the same animal or either hemisphere of vehicle injected controls. These results confirm and extend earlier findings on the plasticity of residual dopaminergic terminals in adult animals after neurotoxic damage. They also establish a quantitative method for ultrastructural analysis of the density of immunoreactivity in thick sections of tissue labeled prior to plastic embedding. The method has broad applicability to quantitative studies of neurotoxicity and plasticity in brain.

Somatostatin-immunoreactive cells in the gastrointestinal tract of the frog Rana esculenta

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.

Multilayer peroxidase‐labeled antibody method: Comparison with labeled streptavidin‐biotin method, avidin‐biotin‐peroxidase complex method, and peroxidase‐antiperoxidase method

A new, extremely sensitive, "multilayer peroxidase-labeled antibody method" is introduced. This method utilizes peroxidase-labeled antibodies that immuno-react with their antigen, but also can be an antigen to other peroxidase-labeled antibodies. An example of this method is: use rabbit IgG as the first antibody, peroxidase-labeled goat antirabbit IgG as the second antibody, peroxidase-labeled rabbit antigoat IgG as the third antibody, peroxidase-labeled goat antirabbit IgG as the fourth antibody, and peroxidase-labeled rabbit antigoat IgG as the fifth antibody in succession. We compared the sensitivities of this multilayer peroxidase-labeled antibody method with that of commercially available immuno-peroxidase staining kits, i.e., avidin-biotin-complex method and peroxidase-antiperoxidase method, both by immunostaining and enzyme-linked immunosorbent assay. The generated signal and background in immunostained sections were determined by a computer-assisted image analyzer and by enzyme-linked immunosorbent assay spectrophotometrically. It was found that a maximum signal/background ratio was obtained with those sections reacted with the first four antibodies in succession by the multilayer peroxidase-labeled antibody method, and those reacted with the five antibodies in succession by the multilayer peroxidase-labeled antibody method in the enzyme-linked immunosorbent assay. Thus, the multilayer peroxidase-labeled antibody method is more sensitive than the avidin-biotin-complex method and the peroxidase-antiperoxidase method.

Immunohistochemical and Radioimmunological Assessment of Thyrotrophs in the Pituitary of Aging Rats

Thyrotrophs were studied by quantitative immunohistochemistry in the pituitary gland of young (4 months), old (20 months) and very old (29 months) male rats. An attempt was also made to correlate morphometric parameters with serum levels of thyrotropin (TSH), thyroxine (T4) and triiodothyronine (T3). Cells were immunostained by the peroxidase-antiperoxidase method and hormones were measured in serum by specific radioimmunoassays. There was a marked age-related reduction in TSH cell number, volume density and surface density but a significant increase in TSH cell area and perimeter. Basal serum levels of TSH increased, T4 decreased and T3 remained unchanged with age. There was a highly significant (p < 0.001) negative correlation between serum TSH and T4, but no significant correlation was found between TSH and morphometric parameters. The present results suggest that aged rats possess a reduced but functionally preserved thyrotrophic cell population. The coexistence of high circulating levels of TSH with reduced serum T4 suggests that aging brings about a progressive desensitization of the thyroid to TSH.

Visualization of Acetylcholine in the Mouse Brain by a Combination of Immunohistochemistry with Ionic Fixation.

Although acetylcholine (Ach) is an important neurotransmitter, there is no adequate histochemical method to detect Ach in tissue sections. We therefore developed a method to visualize the localization of Ach in tissue sections by a combination of immunohistochemistry with ionic fixation. Brains of mice, perfused with 4% formaldehyde and 5% phosphomolybdic acid (PMA) in 0.1 M phosphate buffer (pH 7.4) under sodium pentobarbital anesthesia, were removed and immersed quickly in boiling PMA solution for 5 min. Frozen sections were cut and soaked in a buffer containing 5% PMA at 4°C for 30 min, and immersed in a buffer containing bovine serum albumin at 4°C for 1 hr. Then the sections were incubated with anti-Ach antibody at 4°C for 12 hr. Subsequently, the immunoreaction due to Ach was visualized by the peroxidase-antiperoxidase method. Nerve cells in the brain sections from the interpeduncular nucleus of the cerebrum and bipolar cells in the multiform layer of the cerebral cortex were immmunohistochemically stained with the anti-Ach antibody.

Cytochemical localization of the acetone-inducible cytochrome P-450 isoform, CYP2E1, in murine colonic epithelium.

Immunolocalization of a toxicologically important cytochrome P-450 isoform, the alcohol-inducible CYP2E1, was examined in mouse colon. Male CD-1 mice (30-40 g) were exposed to acetone (1% v/v), a potent inducer of hepatic CYP2E1, in drinking water for 14 days. Tissue sections were fixed in 1% paraformaldehyde and incubated with anti-rat CYP2E1 polyclonal antibody. Immunohistochemical staining on tissue sections was performed using the indirect peroxidase-antiperoxidase method. In acetone-exposed mice, immunoreactivity was localized exclusively within the cytoplasm of surface epithelial cells of the proximal colon. Infrequently, only very faint staining was evident in colons of untreated control mice. Using the same monospecific antibody, the presence of CYP2E1 was confirmed by Western blot analysis. A 2-to 3-fold elevation in immunoreactivity corresponding to cytochrome P-4502E1 was found in colon microsomes isolated from acetone-exposed form acetone-exposed mice. Further evidence for colonic CYP2E1 is provided by elevation (up to 2.5-fold) in levels of chlorzoxazone 6-hydroxylase, a CYP2E1 substrate, after acetone treatment. The significance of these finding is discussed in terms of the potential for proximal colonic epithelial cells to participate directly in bioactivation of dietary promutagenic substances.

Hamster supraoptic nucleus: Cytoarchitectural, Morphometric, and three-dimensional reconstruction

The present paper describes the cytoarchitectonic, morphometric, and three-dimensional characteristics of the golden hamster supraoptic nucleus (SON) in order to provide an anatomical basis for subsequent morphofunctional studies that use this species as an experimental animal. The dimensions (volume and length) and the number of cells of each part of the supraoptic nucleus were obtained, as well as morphometric parameters of their neurons (cross-sectional area and maximum and minimum diameters). A three-dimensional reconstruction of hamster SON has been made in order to know the spatial morphology of this nucleus and to reveal the structural differences between both parts. Ten male adult golden hamsters (Mesocricetus auratus) were used. Animals were anaesthetized and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.2. The hypothalamic area from seven animals was dissected out, dehydrated, and embedded in paraffin. Serial sections of 10 microns were cut in a coronal plane. Sections were stained with thionin, dehydrated, cleared in eucalyptol, and mounted with Eukitt. To prove the neurosecretory nature to the SON, every fourth section was immunostained against neurophysin by using the peroxidase-antiperoxidase method. To study the neuronal morphometric parameters, all magnocellular neurons of the SON were drawn in sections separated 80 microns with the aid of a camera lucida under 500x magnification. Serial 50 microns thick frozen sections of the hypothalamus from three animals were drawn with camera lucida to determine the volume of the two parts of the SON and to make the three-dimensional reconstruction. The SON extends rostrocaudally 1.98 +/- 0.03 mm from the preoptic area to the tuberal hypothalamic area. Two classical parts can be clearly delimited: principal (SONp) and retrochiasmatic (SONr). The neuronal population of the two parts of the SON appears constituted only by magnocellular neurons. The volume of the SONp is 0.039 +/- 0.03 mm3 and contains about 762 +/- 93 magnocellular cells, with a density of 19,151.8 cells/mm3. The volume of the SONr is 0.126 +/- 0.03 mm3 and contains about 1,296 +/- 132 neurons with a density of 10,536.6 cells/mm3. The three-dimensional reconstruction reveals that the SONp appears located in a more cephalic, lateral, and dorsal position than the SONr, and a clear discontinuity between the two parts is observed. The present study shows that the classically termed SON, in the hamster, clearly consists of two spatially separated neural populations. The SONr is longer than SONp and has the larger volume and higher number of neurons; however, the neurons of the SONr are smaller in cell area than those of the SONp.

Immunohistochemical studies on the intestinal nerve of remak in the male chicken

A peroxidase anti-peroxidase method was used to investigate and compare the distribution of neuropeptide and catecholamine synthesizing enzyme immunoreactive (IR) ganglion cells and nerve fibres in the intestinal nerve of Remak (INR) of male chickens. In the INR there were three kinds of ganglion cells: tyrosine hydroxylase (TH)-, aromatic l-amino acid decarboxylase (AADC)- and phenylethanolamine- N-methyltransferase (PNMT)-IR cells; AADC- and PNMT-IR but TH-immunonegative cells; and ganglion cells being immunoreactive for methionine enkephalin (mENK)- and somatostatin (SOM). The first one was distributed throughout the INR. The second was restricted in the ileojejunal region, and the last was localized in the rectal region. Substance P- and vasoactive intestinal polypeptide-IR nerve fibres were distributed in common but variable in number around three kinds of ganglion cells. Then TH-IR cells were characterized by the distribution of many calcitonin gene related peptide- and a few cholecystokinin-IR fibres. mENK and SOM-IR cells, and TH-immunonegative cells were distinguished by the distribution of SOM- and galanin-IR fibres. In addition, TH-immunonegative cells were characterized by the distribution of mENK- and neuropeptide Y-IR nerve fibres which were very few in number. Fig. 21 summarizes the connections described in the present study.

Changes in laminin immunoreactivity as a marker for the state of liver preservation.

In order to examine the role of the extracellular matrix glycoprotein laminin as a marker for the preservation of liver tissue, dog livers were perfused and then preserved for 5 min, 1, 2, 4, 6, 8, 10, 12, 22 and 26 hours with HTK (histidine-tryptophan-ketoglutarate) solution at 5 degrees C and at 25 degrees C and with UW (University of Wisconsin) solution at 5 degrees C. The tissue was processed for the immunohistochemical demonstration of laminin using an anti-P1 and an anti-E8 antibody. The peroxidase-antiperoxidase method was used for the visualization of the immunohistochemical reaction. At the beginning of the preservation, immunostaining was observed for both fragments of laminin around bile ducts and blood vessels of the portal spaces, under all preservation conditions. Clear immunostaining was also visible in the wall of the terminal arterioles located between the liver lobules. In the 5 degrees C-preserved tissues, immunostaining for both laminin fragments occurred for preservation times between 4 and 6 hours in the form of isolated perisinusoidal deposits at the transition point where the sinusoids sprout from the terminal venules. In the 25 degrees C-preserved tissue, such a staining pattern was already visible after 1 to 2 hours, preservation time. Our results show that the occurrence of laminin immunoreactivity in the sinusoids can be taken as a marker for the state of liver preservation. A hypothesis for the presence and the role of this glycoprotein in the perisinusoidal space is presented.

Tooth pulp stimulation induces c-fos expression in the lateral habenular nucleus of the cat.

Detection of Fos protein expression by the peroxidase-antiperoxidase method was used to determine the area in the habenular (Hb) complex responding to electrical stimulation of the tooth pulp in the cat anaesthetized with pentobarbital. In the anaesthetic-injected group, the Fos-positive neurones were found bilaterally in the lateral Hb nucleus (HbL). Tooth pulp stimulation (intensity: 3 times the threshold for jaw-opening reflex) increased the number of positive neurones within the HbL by up to 300%, but did not induce any expression in the medical Hb nucleus. The increase in HbL was inhibited by morphine (2 mg kg-1, i.p.). These findings and the results of previous research suggest that HbL neurones are involved in defensive mechanisms by means of antinociception following noxious stimulation.

Immunoelectron microscopy of enkephalinergic innervation of GABAergic neurons in the periaqueductal gray

The pre-embedding double immunoreaction method was used to study synaptic relations of enkephalinergic and GABAergic neuronal elements in the ventrolateral part of the periaqueductal gray of the Wistar albino rat. The enkephalin-like neuronal elements were immunoreacted by the silver-gold intensified peroxidase-antiperoxidase method and the GABA-like immunoreactive neurons were immunoreacted by the unintensified peroxidase-antiperoxidase method. GABA-like immunoreactive neuronal somata were post-synaptic to both the enkephalin-like immunoreactive and the non-immunoreactive axon terminals. Enkephalin-like immunoreactive axon terminals were found to make synapses with GABA-like immunoreactive and non-immunoreactive dendrites. The synapses between the two kinds of chemically characterized neurons appeared to be both asymmetrical and symmetrical. Possible functional activity related to pain modulation, and synaptic relations between the enkephalinergic and GABAergic neurons in the periaqueductal gray and the dorsal raphe nucleus, are discussed.

PROGNOSTIC INDICATORS FOR EARLY-STAGE BREAST-CARCINOMA .2. VALUE OF CATHEPSIN-D EXPRESSION, DETECTED BY IMMUNOCYTOCHEMISTRY - A MULTIPARAMETRIC STUDY

Cathepsin D is a lysosomal aspartyl protease which seems to be involved in invasiveness and metastasis in breast carcinoma. Cathepsin D expression detected by immunocytochemistry is a new potentially useful prognostic marker. Additional research is warranted because: (i) few immunocytochemical studies have been performed to correlate cathepsin D expression and prognosis; (ii) a consensus has not yet been achieved on methodology and quality control issues; (iii) the association between cathepsin D expression and other new biological markers is poorly known and (iv) few studies have assessed whether cathepsin D expression is an independent prognostic marker when compared with other biological and clinico-pathological features using multivariate analysis. This study was undertaken in the same series of 165 stage I-II breast cancer patients (median follow-up of 5 years) already reported (Int J Oncol 4: 155-162, 1994) in whom cathepsin D was assessed using the D7E3 monoclonal antibody by immunocytochemistry in frozen specimens, using a standard peroxidase-antiperoxidase complex method. Cathepsin D was compared with another 7 biological markers, as well as with the conventional clinico-pathological features. Forty-two carcinomas of the 165 analyzed (25.5%) were cathepsin D-positive. This marker was significantly associated with c-erbB-2 expression (p=0.004) and tumour vascularization (p=0.02). Yet, a weak association was observed between cathepsin D and S-phase fraction (p=0.06). In univariate analysis cathepsin D was significantly associated both with relapse-free survival (p<0.01) and overall survival (p=0.02). In multivariate analysis, for relapse-free survival the variables cathepsin D/tumour vascularization, as well as S-phase fraction and EGFR, were significant and independent. For overall survival the variables cathepsin D/c-erbB-2 protein, as well as tumour vascularization and S-phase fraction were significant and independent. Immunocytochemical determination of cathepsin D, using the D7E3 antibody, seems to be a useful prognostic tool in early-stage breast cancer. In particular, cathepsin D adds prognostic information to tumour vascularization (in predicting relapse-free survival) and to c-erbB-2 expression (in predicting overall survival).

Information processing within the motor cortex. II. Intracortical connections between neurons receiving somatosensory cortical input and motor output neurons of the cortex

Connections between motor cortical neurons receiving somatosensory inputs from area 2 and large pyramidal cells in layer V were examined in the cat via intracellular injection of biocytin and immunohistochemistry of nonphosphorylated neurofilament proteins (npNFP). Biocytin was injected into pyramidal cells in layers II/III of the motor cortex that responded monosynaptically and polysynaptically to microstimulation of the somatosensory cortex and subsequently stained black by the avidin-biotinylated peroxidase complex method with diaminobenzidine (DAB) and nickel. By using a monoclonal antibody SMI-32 and a modified peroxidase-antiperoxidase method with Tris-aminophenyl-methane (TAPM) and p-cresol as a chromogen, pyramidal cells in layers III and V of the motor cortex were stained red for npNFP. In particular, all the large pyramidal cells in layer V, Betz cells, displayed intense npNFP immunoreactivity not only in the perikarya but also in the dendrites. Double staining with DAB/nickel and TAPM/p-cresol showed that biocytin-filled axon varicosities of the pyramidal cells, which were thought to receive monosynaptic inputs from area 2, made contacts with npNFP-positive dendrites in layers I-III around the biocytin-injected cell and in layers V-VI beneath the cell. The present results suggest that the corticocortical input from area 2 to pyramidal cells in layers II/III of the motor cortex is transferred to layer V pyramidal cells, including Betz cells, as well as to neighboring layer II/III pyramidal cells. Since tetanic stimulation of the somatosensory cortex reportedly produces long-term potentiation in layer II/III cells of the motor cortex, it seems reasonable to assume that a given area of the somatosensory cortex can produce a long-lasting change in the activity of a given group of output cells in the motor cortex.

Expression of tissue kallikrein in human kidney.

1. We studied the distribution of human tissue kallikrein mRNA in normal and diseased kidney, using in situ hybridization, together with immunohistochemical localization of renal kallikrein protein. Materials studied were (a) normal tissue from kidneys removed because of localized renal carcinoma, (b) kidneys removed because of post-traumatic haemorrhage and (c) renal biopsy specimens from patients with membranous glomerulonephritis and nephrotic syndrome. 2. A 1.35 kb EcoRI fragment of human tissue kallikrein cDNA was labelled with [32P]dCTP using the random-primer technique, and used for in situ hybridization. A specific rabbit antibody to active human urinary kallikrein was employed for immunocytochemistry, using a peroxidase-antiperoxidase method. 3. By in situ hybridization, no tissue kallikrein gene expression was seen in the carcinoma nephrectomy specimens. Positive expression was seen in the trauma nephrectomy tissue, and in four of five nephrotic syndrome biopsies. In all kidneys, expression was confined to the renal cortex. The dominant site of gene expression was the distal tubule. Apart from one area of positive signal related to an epithelial cell of Bowman's capsule, expression was not observed in glomeruli. Expression was also seen in the walls of large- and medium-sized blood vessels. 4. By immunohistochemistry, the dominant site of immunoreactivity was the distal tubule. Dense staining was also seen in granular peripolar cells and in isolated parietal epithelial cells close to the vascular pole. Isolated immunoreactive cells were seen in the media of large- and medium-sized arteries. 5. The tissue kallikrein gene in the kidney may not be constitutively expressed, but is expressed in response to physiological or pathological stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)