Modifications in the distribution of met-enkephalin in the cat spinal cord after administration of clonidine. An immunocytochemical study

The purpose of this study was to demonstrate the utility of a T2-weighted single shot turbo spin-echo technique--the so-called "Local Look" (LoLo) and more recently renamed "Zoom Imaging" technique--for MR-guided percutaneous interventions. We performed 28 procedures on 22 patients using a 1.5-T system for MR guidance. All procedures were controlled with the LoLo technique, which acquires T2-weighted images in 600 msec. This is achieved by using a small field of view (250 x 125 mm) along with a maximum echo train length, the so-called "single shot method." To prevent backfolding artifacts, the 90 degrees and 180 degrees pulses were oriented orthogonally to each other. Because signal is created only in the region in which the pulses overlap, no backfolding can occur from outside this area. Half of the biopsies were additionally monitored using a fast gradient-echo sequence, which was compared with the LoLo technique. All of the procedures were technically successful, and there were no procedural complications. The LoLo technique produced images that had good contrast between the lesion and the needle artifact, and the artifact size was smaller than that produced by the gradient-echo technique. Subjective judgment of the ability to accurately delineate the needle tip indicated that the LoLo technique was either superior to (73%) or equal to (27%) the gradient-echo sequence in all cases. The LoLo technique is an accurate and effective method for MR guidance of percutaneous procedures, because it shows good lesion contrast and small needle artifacts. The additional use of a gradient-echo sequence during the procedure planning stage is advisable in more difficult cases, particularly when adjacent blood vessels are a concern. Monitoring of the needle tip is best performed with the LoLo technique.     

Diversity of pituitary cells in primary cell culture. An immunocytochemical study

A bioassay system for rapid detection of carcinogenic agents has been developed using male Fischer 344 rats to bridge the gap between long-term carcinogenicity tests and short-term screening assays. The system, called the medium-term liver bioassay, is fundamentally based on the 2-stage hypothesis of tumor production, employing initiation by diethylnitrosamine (200 mg/kg, i.p.) in the first stage and test chemical administration during the second, in combination with two-thirds partial hepatectomy. It requires only 8 wk for animal experimentation and a further few weeks for quantitative analysis of immunohistochemically demonstrated glutathione S-transferase placental form positive hepatic foci. A total of 291 chemicals/substances have already been analyzed in our laboratory. Among 63 chemicals that were proved to be carcinogenic in the liver of rat and/or mouse, 57 (90%) gave positive results irrespective of their mutagenicity. Negative compounds include peroxisome proliferators and tamoxifen. Even nonhepatocarcinogens were positive at a rate of 24%. Eighty-six percent (12/14) of mouse liver carcinogens were also positive. On the other hand, only 2 out of 45 noncarcinogens showed very weak positivity. Thus, the efficacy of the system for hepatocarcinogens has been well established. This bioassay is increasingly regarded as an appropriate alternative test for carcinogenicity risk assessment and is practically used for a rapid evaluation of hepatocarcinogenicity of chemicals.     

An immunocytochemical study of the FMRFamide neuropeptide gene products in Drosophila

We have mapped protein expression of the FMRFamide neuropeptide gene in Drosophila with polyclonal antisera against three small peptides whose sequences were derived from the Drosophila proFMRFamide precursor. One antiserum was affinity-purified and extensively characterized. The enriched antibodies labeled 15-21 bilaterally symmetric pairs of neurons in a pattern that corresponded very closely to the pattern of in situ hybridization that was determined previously (Schneider et al. [1991] J. Comp. Neurol. 304:608-622; O'Brien et al. [1991] J. Comp. Neurol. 304:623-638). The other antisera produced complementary results. These findings suggest that the antisera specifically label cells that express the FMRFamide gene. In larvae we consistently observed strong staining in identified interneurons and neuroendocrine cells, and moderate to weak staining in neurons of unknown function. The adult pattern of expression included both larval neurons whose immunoreactivity persisted through metamorphosis and adult-specific neurons. During metamorphosis, we observed transient staining in a small number of neurons and in specific neuropil regions that included the central body, the protocerebral bridge, and the optic ganglia. Based on these morphological features, we suggest that the FMRFamide-like neuropeptides in Drosophila play a number of functional roles, perhaps affecting both physiological and developmental phenomena. Such roles include general modulation throughout all post-embryonic stages, via the blood, and also more stage- and region-specific modulation within the CNS.     

HMGI(Y) expression in human uterine leiomyomata. Involvement of another high-mobility group architectural factor in a benign neoplasm

Chromosomal rearrangements involving 6p21 have been observed in uterine leiomyomata and a variety of other benign tumors. The gene for HMGI(Y), a member of the high-mobility group (HMG) family of proteins, has been localized to 6p21. To determine whether rearrangements observed in this area alter HMGI(Y) expression, we analyzed HMGI(Y) DNA-binding activity in protein extracts from uterine leiomyoma and normal myometrium tissues. This report describes a uterine leiomyoma specimen with an inv(6)(p21q15). A genomic P1 clone that contains the HMGI(Y) region of chromosome 6 is found to span the inversion breakpoint by fluorescent in situ hybridization of metaphase chromosomes. Expression of HMGI(Y) protein in this leiomyoma specimen is increased dramatically as compared with the matching normal myometrial tissue. Elevated HMGI(Y) expression was also found in 8 of 16 leiomyomas without cytogenetically detectable chromosome 6p21 aberrations but not in any of the 9 matching myometrial tissues. Analysis of the genetic events involved in the pathobiology of these benign tumors will provide a basis for understanding the process of improper cellular growth and might be important in deciphering the multistep pathway of tumorigenesis.     

Segmental up-regulation of transforming growth factor-beta in the pathogenesis of primary megaureter. An immunocytochemical study

OBJECTIVE: To examine the maturational-delay hypothesis of primary megaureter (PM), i.e. that the condition arises by a segmental maturational delay of the ureteric wall that can resolve spontaneously within the first year of life, using comparative immunocytochemistry of ureters resected from infants and from homologous pre-natal ureters. MATERIALS AND METHODS: Seventeen distal urinary tracts were obtained from children with PM who were referred for surgery (aged 6 months to 8 years, mean 2.1 years). These were compared with ureteric buds obtained from 11-week-old human and 11- to 38-week-old calf fetuses. The samples were immunostained using a monoclonal antibody specific for transforming growth factor beta (TGF-beta). RESULTS: The histological appearances of the narrowed ureteric segments from patients under 18 months old were like the fetal ureteric buds at 26-38 weeks of gestation. Positive TGF-beta immunoreactions were detected in the longitudinal muscle layer in the ureter from patients 6-12 months old. Such reactions weakened progressively in those patients older than 1 year, becoming negative in all children older than 3 years. The TGF-beta immunolabelling in resected ureters was closely similar to that in fetal ureters from 20 to 26-week old calves. CONCLUSIONS: From these results, PM should be ascribed to a segmental developmental delay of the terminal ureter arising at about 20 weeks of gestation, with a possible pathogenetic involvement of autocrine TGF-beta overexpression.     

Renal immunocytochemical distribution and pharmacological properties of the dual angiotensin II/AVP receptor

There is evidence that sympathetic nerve activity leads to endothelium-derived nitric oxide release, which in turn attenuates neurogenic vasoconstriction. Here we tested in vivo (1) whether the magnitude of the vasoconstriction induced by N(G)-nitro-L-arginine methyl ester given systemically is altered when ongoing sympathetic activity is abolished by sectioning the lumbar sympathetic trunk, and (2) whether hindlimb sympathetic vasoconstriction elicited by electrical stimulation of the lumbar sympathetic trunk is enhanced after inhibition of nitric oxide synthesis. Blood flow in the microvascular beds of hairless skin and skeletal muscle of the rat hindlimb was measured with laser Doppler flowmetry. Sectioning the lumbar sympathetic trunk resulted in an increase of blood flow in both tissues, indicating that tonic neurogenic vasoconstriction was abolished. Inhibition of nitric oxide synthesis resulted in vasoconstriction in both vascular beds. This vasoconstriction was more pronounced after abolition of sympathetic activity than with intact sympathetic supply in skin but was smaller in skeletal muscle. The vasoconstriction elicited by graded electrical stimulation of the centrally sectioned lumbar sympathetic trunk with frequencies less than 5 Hz was significantly enhanced after blockade of nitric oxide in skeletal muscle but not in skin microvasculature. These findings suggest that under physiological conditions, sympathetic nerve impulses directly promote the release of nitric oxide in skeletal muscle but not in cutaneous blood vessels. Therefore, basal nitric oxide release is probably in part dependent on sympathetic activity in skeletal muscle, whereas it appears to be mainly due to flow-dependent shear stress in hairless skin microvasculature.     

Cocaine adversely affects development of cortical embryonic neurons in vitro: immunocytochemical study of calcium-binding proteins

Neurons of cerebral cortex from 15-16 day old embryos of white rats (Sprague-Dawley) were cultured in MEM enriched with 5% horse serum. On the 7th day after plating the cultures were divided into three experimental and one control groups (6-8 Petri dishes in each group). In group 1, cultures were grown without additives. In group 2, cocaine chloride was added at concentrations 0.3, 0.6 and 1 mg/ml of culture. In group 3, a monoclonal antibody against calcium-binding proteins, parvalbumin (APV) or calbindin (ACB) was added at a concentration 25 microl/ml. In group 4, a combination of cocaine +APV was added at a concentration 1 mg+25 microl/ml of culture media. On the 10th day cultures were immunostained using APV and ACB antibodies. In developing GABAergic neurons of group 2 cocaine produced cytotoxic effects that were expressed in drastic decrease in number of neurons and in degeneration of their processes. The lower concentrations of cocaine caused milder cytotoxity and their effects were reversible. The highest concentration of cocaine caused irreversible degeneration of neurons. Similar cytotoxity was caused by APV or ACB in group 3. The most severe cytotoxic effects were seen in group 4, where a mixture of cocaine and APV was used. Overall, it can be concluded that cocaine in higher concentrations directly affects development of GABAergic neurons in vitro.     

Anatomical distribution of beta-endorphin (1-27) in the cat brainstem: an immunocytochemical study

IL-4 is a pleiotropic cytokine which exerts its actions on various lineages of hematopoietic and nonhematopoietic cells. This cytokine is one of the central regulators of immunity in health and disease states. An alternative splice variant, in which the second of four exons is omitted, has been recently described and designated as IL-4delta2. The variant has been previously described as a potential naturally occurring antagonist of human IL-4 (hIL-4)-stimulated T cell proliferation. In this study, we investigated the effects of recombinant human (rh) IL-4delta2 on monocytes and B cells. In monocytes, rhIL-4delta2 blocked inhibitory action of hIL-4 on LPS-induced cyclooxygenase-2 expression and subsequent prostaglandin E2 secretion. In B cells, rhIL-4delta2 was an antagonist of the hIL-4-induced synthesis of IgE and expression of CD23. Our results broaden the spectrum of hIL-4-antagonistic activities of rhIL-4delta2, thus creating the background for the potential use of rhIL-4delta2 as a therapeutic anti-hIL-4 agent.     

Differential distribution of nicotinamide adenine dinucleotide phosphate-diaphorase and neural nitric oxide synthase in the rat choroid plexus. A histochemical and immunocytochemical study

This study used NADPH diaphorase (NADPHd) histochemistry and neuronal nitric oxide synthase immunocytochemistry to examine the localization of nitric oxide synthase in the choroid plexus of the lateral ventricles and the fourth ventricle of rat brain. That the NADPHd reaction product in choroid plexus was specific to nitric oxide synthase was evaluated: (i) by comparison to immunocytochemical labelling for nitric oxide synthase; and (ii) by comparing NADPHd histochemical staining in choroid plexus and brain (rich in nitric oxide synthase-positive and NADPHd-positive neurons) in the presence or absence of iodonium diphenyl or dichlorophenolindophenol, two potent albeit non-selective inhibitors of nitric oxide synthase activity. In brain, NADPHd histochemistry homogeneously stained neuronal cell bodies, axons and dendrites, while it produced particulate cytoplasmic staining of all epithelial cells in the choroid plexuses of the lateral and fourth ventricles. Within the choroid plexus of the lateral ventricles, NADPHd-positive nerve fibres were also observed around blood vessels and coursing among the epithelial cells. The distribution of immunoreactivity for nitric oxide synthase in brain and in nerve fibres in the choroid plexuses of the lateral ventricles resembled the distribution of histochemical labelling for NADPHd. Choroid plexus epithelial cells were, however, devoid of nitric oxide synthase immunoreactivity. Consistent with this, iodonium diphenyl and dichlorophenolindophenol (0.1 mM) inhibited NADPHd histochemical staining in brain neurons and in choroid plexus nerve fibres, but not in choroid plexus epithelial cells. These results demonstrate that the choroid plexus of the lateral ventricles in rat brain is innervated by nitric oxide synthase-positive nerve fibres. These nitric oxide synthase-positive nerve fibres may have an important role in the regulation of cerebrospinal fluid balance. Although choroid plexus epithelial cells contain an enzyme with NADPHd activity, this enzyme is not nitric oxide synthase.     

Lung capillary albumin leak in oxygen toxicity. A quantitative immunocytochemical study

Uterine leiomyomas or fibroids are common among women of reproductive age, but their biology is poorly understood. The PTH-related protein (PTHrP) has been identified in a number of sites throughout the reproductive tract. We, therefore, examined whether fibroids express PTHrP mRNA and compared their level of expression with that in normal myometrium. Total RNA prepared from fibroid tissue and corresponding normal myometrium from seven patients was examined by RNase protection analysis. In all cases, fibroid and myometrial tissue expressed PTHrP, and in six of seven cases, PTHrP expression was higher in fibroids than in normal myometrium. Cultured fibroid cells from four patients also expressed higher levels of PTHrP mRNA than corresponding cultured normal myometrial cells. Tissue extracts from eight patients and conditioned medium from cultured cells from nine patients were examined for PTHrP immunoreactivity using a two-site immunoradiometric assay. In tissue extracts and conditioned medium, the mean PTHrP concentration was significantly higher in fibroids than normal myometrium. Immunohistochemical staining of fibroid and myometrial tissue was positive for PTHrP. Finally, PTHrP-(1-34) induced a dose-dependent increase in cAMP in fibroid and myometrial cells in vitro. These findings suggest that PTHrP may have an autocrine/paracrine function in regulating myometrial physiology and may play a role in regulating fibroid growth or differentiation.     

The Purkinje cell in olivopontocerebellar atrophy. A Golgi and immunocytochemical study

Purkinje cells were examined in three familial cases of olivopontocerebellar atrophy (OPCA) by means of the Golgi method, and neurofilament and calcium-binding protein immunocytochemistry. Reduced dendritic arborizations, as seen with different techniques, early formation of axonal spheroids, and abnormal accumulation of phosphorylated neurofilament epitopes in dendrites, somata and axonal spheroids, together with limited formation of proximal spine-like protrusions were the main changes in Purkinje cells. These lesions are unlikely to be the consequence of anterograde degeneration secondary to olivary atrophy, as postulated by some investigators, but probably represent primary damage to Purkinje cells in patients with OPCA. Reduced dendritic arborizations result in a decrease of receptor sites for parallel fibres and deprive granule cells of their main targets. Abnormal accumulation of neurofilaments in somata, dendrites and axonal spheroids may contribute to an abnormal transport and may impair protein turnover in the distal regions of Purkinje cells.     

The effects of prenatal exposure to cocaine on the dopaminergic cells in the rat retina. An immunocytochemical and neurochemical study

5-Hydroxytryptamine1A (5-HT1A) receptors have been visualized at the electron microscopic level in selected areas (dorsal raphe nucleus, hippocampus, septum) of the rat brain using specific anti-peptide antibodies. 5-HT1A receptor immunoreactivity was found almost exclusively in the somatodendritic compartment of neurons and was very rarely observed within processes possibly belonging to glial cells. The immunoenzymatic reaction product was associated exclusively with dendritic spines in the dorsal hippocampus, whereas in the dorsal raphe nucleus and the septal complex, immunoreactivity was found in both dendritic processes and somata. Although some immunolabeling was observed within the cytoplasm of cell bodies, 5-HT1A receptor immunoreactivity was essentially confined to the plasma membrane where it was unevenly distributed. It was frequently associated with synapses (except in the dorsal raphe nucleus), but was also found extrasynaptically in both somata and dendrites. These data suggest that the action of serotonin via 5-HT1A receptor could occur through junctional as well as nonjunctional transmission.     

Distribution of cholecystokinin immunoreactivity in the BALB/c mouse forebrain: an immunocytochemical study

The present study describes cholecystokinin (CCK) immunoreactivity (CCK-IR) distribution in the brains of control and colchicine-treated mice. In the brains of control mice, the CCK-IR strongly revealed numerous axons and terminals. Perikarya exhibiting a faint to moderate immunoreactivity were also observed in areas such as cortices, hippocampus, amygdala, septum, and thalamus. The colchicine treatment did not seem to notably affect the brain CCK-IR innervation, but resulted in profound changes of the perikaryal staining. Indeed, the regions, which contained numerous moderately stained perikarya in the control animals, exhibited after colchicine treatment immunoreactive perikarya intensely stained but only in moderate number. This feature obviously appeared in the cortex in which, in addition to strongly stained perikarya, colchicine induced the appearance of numerous CCK-IR hillocks. In the lateral amygdala and thalamus of colchicine-treated animals, the somatic immunoreactivity was considerably decreased. The regions, such as paraventricular hypothalamic nucleus and bed nucleus of the stria terminalis, which in the control animals did not exhibit any stained perikaryon, showed a high number of strongly stained cell bodies after colchicine treatment. This study, mapping the mouse forebrain CCK-IR, demonstrated a wide distribution of this peptide. Moreover, CCK-IR is spontaneously visible in neurons of untreated mouse in some brain areas previously shown in the rat to exhibit CCK mRNA, but no clear perikaryal CCK-IR even after colchicine treatment.     

Aromatase immunoreactivity in axon terminals of the vertebrate brain. An immunocytochemical study on quail, rat, monkey and human tissues

Correct detection of premature contractions and incompetent uterine cervix is still a challenging obstetrical problem since these factors remain a major cause of perinatal loss. Ultrasonography offers additional important data for the prediction of these pathologies which have common sonographic patterns; shortening of the cervical length, funneling of the membranes and dilatation of the endocervical canal. The first section of this review highlights sonography of normal cervical anatomy, while the second section focuses on recent advances in sonographic detection of premature contractions and incompetent cervix. It is believed that due to its accuracy and reproducibility, this noninvasive technique should become more integrated into this aspect of antenatal care.     

Defective proximal tubule lysosomal acidification by Bence Jones proteins. An immunoelectron microscopy study

AIMS: To determine their significance, we examined the expression pattern of the four epidermal growth factor receptor (EGFR) family members as well as the phosphotyrosine kinase activity in breast tumour tissues. METHODS AND RESULTS: Fifty-three malignant breast tumours, four breast cancer cell lines, and 10 benign breast tumours were investigated. Fifty-three per cent (28/53) of the malignant tumours expressed EGFR protein, and the majority of these positive tumours were strongly positive. Eighty per cent (8/10) of the benign tumours also expressed EGFR protein, but all in a lower or moderate level. An association between EGFR expression and increasing malignancy grade was found in the group of infiltrating ductal carcinomas. Of the malignant tumours, 35.8% (19/53) expressed c-erbB-2 protein and 17% (9/53) c-erbB-3 protein, while no expression of c-erbB-2 and c-erbB-3 proteins was found in the benign tumours. Contrary to previous reports, we observed c-erbB-4 receptor protein to be less expressed in the malignant breast tumours. The 'normal' breast epithelial cells adjacent to the malignant tumours and the benign tumours demonstrated intensified membrane staining for c-erbB-4, while a number of the malignant tumours demonstrated a weak cytoplasmic staining or were negative. However, several malignant tumours with strong membrane staining for the c-erbB-4 protein were also found. No simple association between the expression of the four receptors and phosphotyrosine kinase activity was found. CONCLUSION: Our study has revealed a complex expression pattern of the EGFR family members in breast tumour cells. While the data about EGFR, c-erbB-2, c-erbB-3 and phosphotyrosine are largely in line with what has been reported, we found the c-erbB-4 protein expression to be decreased in the malignant tumours.     

An immunohistochemical study of neuronal microtubule-associated proteins in Hirschsprung's disease

Neuronal microtubule-associated proteins (MAPs) are important components of neurons and are believed to regulate neuronal function and development by controlling the assembly of microtubules and the interaction of microtubules with other cytoplasmic organelles. We studied the immunohistochemical localization of MAPs 1, 2, 5, and tau in the intestinal tissues of five patients with Hirschsprung's disease and in five normal controls using monoclonal antibodies. Microtubule-associated proteins 5 and tau proved to be excellent enteric neuronal markers; they were specifically located in the nerve cell bodies and processes of normal intestine as well as in the abnormal hypertrophied nerve fibers of aganglionic colon. Fine fibrillar structures in the neuroplasm were revealed in greater detail than were those obtained from studies with conventional markers, including neuron-specific enolase, S-100 protein, and neurofilament protein. A slight reduction of MAPs 5 and tau immunoreactivity was observed in the aganglionic colon compared with normal colon. Microtubule-associated proteins 1 and 2 were absent from the nerve fibers in both normal and aganglionic colon. This study suggests that immunostaining for MAPs 5 and tau may be superior to other immunohistochemical methods for diagnosing Hirschsprung's disease; however, in view of its limited retrospective nature these findings need to be corroborated by a large prospective evaluation.     

Cortical dysplasia: an immunocytochemical study of three patients

The role that T and B lymphocytes play in the clearance of Giardia muris in the mouse model is well known, but the cytokines produced by CD4+ T cells in response to Giardia antigenic stimulation are unknown. In this study, we have determined how Giardia trophozoite antigenic crude extract and T cell mitogens can trigger the production of cytokines by Peyer's patch and spleen cells removed from infected animals. When Giardia trophozoite proteins were used to challenge the cells in vitro, IL-4, IL-5 and IFN-gamma were not detected in the culture supernatant. When the cells were challenged with Con-A, all three cytokines were released in vitro. However, the level of each cytokine released by the spleen or Peyer's patch cells varied with the latent, acute and elimination phases of the infection. The high levels of IL-4 and IL-5 released by Peyer's patch cells confirm the importance of IgA in the control of the infection. However, we propose that the relative success of G. muris in completing its life cycle in a primary infection might be due, in part, to the stimulation of a Th2-type response (IL-4, IL-5). A stronger Th1 response (IFN-gamma) may lead to a better control of the primary infection.