Immunochemical Studies Research Articles

Cranial Nerves Paralysis Revealing Two Cases of Histiocytic Sarcoma.

Pathological, immuno-chemical and cytogenetic studies have allowed in the two last decades to distinguished real histiocytic sarcoma (HS) from hemophagocytic activation syndrome secondary to lymphoma, Langerhans histiocytosis, benign histiocytic proliferation or dendritic cells tumor. Strict immunophenotypic criteria have been defined by the International Lymphoma Study Group (ILSG) in 2002. Published cases are rare as most cases have been reclassified into lymphoma. Clinical presentation is bad known. We report two cases with initial neurological involvement. Case 1: A 32-year-old black patient was admitted in hospital in May 2002 for rapidly progressive blurred vision of the right eye, bilateral deafness and marked gait disturbance. Physical examination demonstrated hepatosplenomegaly, ataxia, bilateral deafness and loss of visual acuity mainly in the right eye. MR imaging of the brain demonstrated white matter abnormalities. Cerebrospinal fluid (CSF) study showed mild protein level (0.43g/l), low glucose level and 16/mm3 histiocytic cells. Bone marrow biopsy, liver biopsies, bone scan, thoracic CT scan were all normal. A diagnostic splenectomy was performed. Pathological study was consistent with HS. Despite high dose systemic chemotherapy containing cytosine-arabinoside, methotrexate, methylprednisolone and intrathecal therapy, he developed kidneys' tumoral infiltration. After two courses of cladribine the patient died with intra cranial hypertension symptoms. Bone marrow smears demonstrated tumoral infiltration. Case 2: A 63-year-old white female patient presented in november 2002 a one month history of paresthesias in the left trigeminal nerve. CSF study demonstrated high protein level (0.5 g/l), normal glucose, increased cells (21/mm3). Brain MR imaging showed nodular lesions of both trigeminal nerves and protuberance. After 5 days course of intravenous high dose methylprednisolone her state worsened. A left trigeminal nerve chirurgical biopsy was performed. Histologic study diagnosed HS. Abdominal and thoracic CT scan and bone marrow biopsy were normal. Despite high dose chemotherapy including ifosfamide, cytosine-arabinoside and methotrexate, she died with progressive neurological symptoms. Pathological study performed on spleen (case1) and on trigeminal nerve (case2) demonstrated diffuse infiltration by non-cohesive large cells with abundant eosinophilic cytoplasm. Hemophagocytosis in tumoral cells were noted in the 2 cases. Immunophenotyping asserted HS (Table1). Cytogenetic study performed in bone marrow of patient 1 demonstrated clonal complexe karyotype. We report the first 2 cases of HS with initial cranial nerve involvement and complete immunophenotypic study. Only 5 other cases with neurological involvement, either cerebral or spinal cord mass are described to date. The two cases fulfil the WHO and ILSG criteria for HS and immunostaining for CD163 (hemoglobin scavenger receptor), the most histiocyte specific marker, is strongly positive in the two cases. Clonality is demonstrated in case1.Table 1HMB45,CD20CD3,CD30, ALKCD35CD68CD1alysosymeCD 163case 1−−−+−−+case 2−−−+−weak+

Aerobiological and immunochemical studies on Fungal aerosol of North Calcutta, India

Aerobiological and immunochemical studies on Fungal aerosol of North Calcutta, India

Aerobiological and Immunochemical studies in Domjur, Howrah District, West Bengal, India

Aerobiological and Immunochemical studies in Domjur, Howrah District, West Bengal, India

The Chaotrope-soluble Glycoprotein GP2 Is a Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.

Naris occlusion alters the electro-olfactogram: Evidence for compensatory plasticity in the olfactory system

Unilateral naris occlusion (NO) has been widely used as a method of olfactory deprivation to study the role of stimulus-driven activity in olfactory development [P.C. Brunjes, Unilateral naris closure and olfactory system development, Brain Res. Rev. 19 (1994) 146-160]. Recent immunochemical studies of the olfactory epithelium (OE) following NO provide evidence for a previously unknown compensatory response to deprivation [D.M. Coppola, A. Waguespack, M. Reems, M.L. Butman, J. Cherry, Naris occlusion alters transductory protein immunoreactivity in olfactory epithelium, Histol. Histopathol. 21 (2006) 487-501; A. Waguespack, M. Reems, M.L. Butman, J. Cherry, D.M. Coppola, Olfactory receptor neurons have enhanced olfactory marker protein immunoreactivity in the nasal cavity ipsilateral to naris occlusion, Brain Res. 1044 (2005) 1-7]. To further investigate this phenomenon we measured electro-olfactograms (EOG) from the open and occluded OE of adult mice that had undergone NO as newborns. EOG waveforms from the open side OE of NO mice were indistinguishable from those obtained from untreated animals. However, amplitudes of EOGs from the occluded OE of NO mice were greater, on average, than those recorded at matched locations from the open side. This result was consistent across turbinates, odors, and all but the highest odor concentration. In addition, EOGs recorded from the occluded OE had significantly slower onset and recovery kinetics. Responses in a double-pulse protocol confirmed that the kinetics of the cellular or population processes that underlie the EOG are slowed by NO. These results provide the most direct support, to date, for compensatory plasticity in the olfactory system.

Immunochemical and Immunohistochemical Studies on Distribution of Elastin Fibres in Human Atherosclerotic Lesions using a Polyclonal Antibody to Elastin-derived Hexapeptide Repeat

A polyclonal antibody to elastin-derived hexapeptide repeat, H-(Val-Gly-Val-Ala-Pro-Gly)(3)-NH(2), was prepared in order to investigate the differences between elastin fibres in intimal hyperplasia and media in human atheroscleroic lesions. The hexapeptide repeat and alpha-elastin were recognized by this polyclonal antibody in enzyme-linked immunosorbent assay (ELISA), but other elastin-derived peptides such as tetrapeptide repeat, pentapeptide repeat and nonapeptide were not. In the series of hexapeptide repeats, H-(VGVAPG)(n)-NH(2) where n is 1-7, the polyclonal antibody reacted strongly with oligomers (n = 3-7) and weakly with dimer (n = 2), but not with monomer (n = 1). CD measurements suggested that the beta-turn structure is important for recognition by the polyclonal antibody. In an immunohistochemical study, elastin was stained more strongly in intimal hyperplasia than in media, suggesting that newly synthesized elastin in intimal hyperplasia is morphologically distinct from that in media.

Une cause rare de diminution de l'acuité visuelle : la tumeur à cellules granuleuses neurohypophysaire

A neurohypophyseal granular cell tumor was found in a 60-year-old man who presented diminished visual acuity. Neuronavigation resection via a fronto-pteronial approach removed split up material. The diagnosis was established by histology and immunochemical studies. Granulous cell tumors, which are common, are rarely located in the neurohypophysis. Their origin remains a subject of debate, but unlike the other localizations, these tumors in the sellar and/or supra-sellar area are always considered benign. The clinical course depends on the quality of the surgical resection; there is no consensus on appropriate management.

Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteria

Soluble inorganic pyrophosphatases (inorganic diphosphatases, EC 3.6.1.1) were isolated and characterized from three phylogenetically diverse cyanobacteria--Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Pseudanabaena sp. PCC 6903--and one anoxygenic photosynthetic bacterium, Rhodopseudomonas viridis (purple nonsulfur). These enzymes were found to be family I soluble inorganic pyrophosphatases with c. 20 kDa subunits with diverse oligomeric structures. The corresponding ppa genes were cloned and functionally validated by heterologous expression. Cyanobacterial family I soluble inorganic pyrophosphatases were strictly Mg(2+)-dependent enzymes. However, diverse cation cofactor dependence was observed for enzymes from other groups of photosynthetic bacteria. Immunochemical studies with antibodies to cyanobacterial soluble inorganic pyrophosphatases showed crossreaction with orthologs of other main groups of phototrophic prokaryotes and suggested a close relationship with the enzyme of heliobacteria, the nearest photosynthetic relatives of cyanobacteria. A slow-growing Escherichia coli JP5 mutant strain, containing a very low level of soluble inorganic pyrophosphatase activity, was functionally complemented up to wild-type growth rates with ppa genes from diverse photosynthetic prokaryotes expressed under their own promoters. Overall, these results suggest that the bacterial family I soluble inorganic pyrophosphatases described here have retained functional similarities despite their genealogies and their adaptations to diverse metabolic scenarios.

Neuropathological and Immunochemical Studies of Brain Parenchyma in Acetylcholinesterase Knockout Mice: Implications in Alzheimer's Disease

The 'cholinergic hypothesis', based on the correlation of the reduction of cholinergic activity in Alzheimer's disease (AD) with cognition and memory, is currently the most widely-held view for AD. Drug treatments for AD focus mainly on inhibition of acetylcholinesterase (AChE), and to some extent butyrylcholinesterase (BChE). In addition to changes in AChE in AD, there is a rise in the level of the sister enzyme BChE. However, the role of the two cholinesterases is poorly understood in vivo. We characterized several proteins immunohistochemically in brain sections from AChE nullizygote (AChE-/-) and wild type AChE+/+ mice. Previous studies had shown that AChE-/- mouse tissues are devoid of AChE activity and that the overall cholinesterase activity is significantly decreased in the knockout group [16]. Despite the differences of cholinesterase activity, we found no significant structural alterations between the experimental groups. Immunohistochemical examination revealed no neuronal, dendritic, astrocytic, synaptic, microglial, and endothelial differences between AChE-/- and AChE+/+ mice. Similarly, the histochemical examination showed no morphologic alterations between AChE-/- and AChE+/+ mice. Our studies show that neither the absence of AChE nor the presence exclusively of BChE is associated with neuroglial and vascular pathology.

Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide an insight into their structure

The extracellular domains (ECDs) of human nicotinic acetylcholine receptors (nAChRs) are of major pharmacological interest as drug targets in the autoimmune disease myasthenia gravis and in various neurological disorders. We have previously expressed and purified the human muscle α1-, β1-, γ- and ɛ-nAChR-ECDs, as well as the wild type and a mutant of neuronal α7-ECD, in yeast Pichia pastoris. The far-UV circular dichroism (CD) studies of these ECDs, presented here, revealed a major prevalence of β-sheet (∼40%) and a small proportion of α-helical (∼5%) structure for all ECDs, in good agreement with the secondary structure composition of the Torpedo muscle-type nAChR-ECDs and in less, but considerable, agreement with that of the homologous invertebrate acetylcholine-binding proteins (AChBPs). The near-UV CD studies of these nAChR-ECDs indicated well-defined tertiary structures, as was previously suggested by biochemical and immunochemical studies. Furthermore, the binding of cholinergic ligands to the mutant of α7-ECD resulted in no changes in its secondary structure, but revealed significant local conformational changes. Our present studies probe the structure of human nAChR-ECDs for the first time and further suggest that our expressed proteins fold to a near-native conformation, thus being suitable for further structural studies.

Protein kinase C ε phosphorylates keratin 8 at Ser8 and Ser23 in GH4C1 cells stimulated by thyrotropin‐releasing hormone

Protein kinase C epsilon (PKCepsilon) is activated by thyrotropin-releasing hormone (TRH), a regulator of pituitary function in rat pituitary GH(4)C(1) cells. We analyzed the downstream mechanism after PKCepsilon activation. Exposure of GH(4)C(1) cells to TRH or a phorbol ester increased the phosphorylation of three p52 proteins (p52a, p52b and p52c) and decreased the phosphorylation of destrin and cofilin. GF109203X, an inhibitor of protein kinases including PKC, inhibited phosphorylation of the p52 proteins by TRH stimulation. Peptide mapping, amino-acid sequencing, and immunochemical studies indicated that p52a, p52b, and p52c are the differentially phosphorylated isoforms of keratin 8 (K8), an intermediate filament protein. The unphosphorylated K8 (p52n) localized exclusively to the cytoskeleton, whereas the phosphorylated forms (especially p52c), which are increased in TRH-stimulated cells, localized mainly to the cytosol. K8 phosphorylation was enhanced in PKCepsilon-overexpressing clones, and purified recombinant PKCepsilon directly phosphorylated K8 with a profile similar to that observed in TRH-stimulated cells. PKCepsilon and K8 colocalized near the nucleus under basal conditions and were concentrated in the cell periphery and cell-cell contact area after TRH stimulation. MS analyses of phospho-K8 and K8-synthesized peptide (amino acids 1-53) showed that PKCepsilon phosphorylates Ser8 and Ser23 of K8. Phosphorylation of these sites is enhanced in TRH-stimulated cells and PKCepsilon-overexpressing cells, as assessed by immunoblotting using antibodies to phospho-K8. These results suggest that K8 is a physiological substrate for PKCepsilon, and the phosphorylation at Ser8 and Ser23 transduces, at least in part, TRH-PKCepsilon signaling in pituitary cells.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Predominant expression of lysosomal N-acylethanolamine-hydrolyzing acid amidase in macrophages revealed by immunochemical studies

Bioactive N-acylethanolamines, including anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory substance), and N-oleoylethanolamine (an anorexic substance) are enzymatically hydrolyzed to fatty acids and ethanolamine. Fatty acid amide hydrolase plays a major role in this reaction. In addition, we cloned cDNA of an isozyme termed “ N-acylethanolamine-hydrolyzing acid amidase (NAAA)” [K. Tsuboi, Y.-X. Sun, Y. Okamoto, N. Araki, T. Tonai, N. Ueda, Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase, J. Biol. Chem. 280 (2005) 11082–11092]. Previous biochemical analyses suggested the expression of NAAA in macrophage cells and various rat tissues including lung and brain. To clarify the physiological significance of NAAA, here we immunochemically studied NAAA for the first time. We developed an antibody specific for rat NAAA, and by Western blotting revealed that NAAA is glycosylated and subjected to specific proteolysis. In alveolar macrophages isolated from rat lung, NAAA was immunocytochemically localized in lysosomes. In the whole lung tissue, only alveolar macrophages were immunostained for NAAA. Conformably, the mRNA and protein levels and activity of NAAA in alveolar macrophages were much higher than those in the whole lung tissue. In brain, intraventricular macrophages were positively stained with anti-NAAA antibody, while microglia appeared to be negative. These results strongly suggested the importance of macrophages as an expression site of NAAA in rat tissues.

Identification of Lanthionine Synthase C-like Protein-1 as a Prominent Glutathione Binding Protein Expressed in the Mammalian Central Nervous System

Proteomic experiments were performed to identify novel glutathione (GSH) binding proteins expressed in the mammalian central nervous system. Bovine brain lysate was affinity purified using an immobilized glutathione-Sepharose column. Proteins that bound the immobilized glutathione were eluted with free glutathione and identified by one- and two-dimensional electrophoresis coupled with mass spectrometric analysis of tryptic fragments. Major proteins purified by this technique were glutathione S-transferase-mu (GST-mu) and GST-pi and lanthionine synthase C-like protein-1 (LanCL1). LanCL1 is a mammalian homologue of a prokaryotic enzyme responsible for the synthesis of thioether (lanthionine) cross-links within nascent polypeptide chains, yielding macrocyclic proteins with potent microbicidal activity. An antibody against LanCL1 was generated and applied to immunochemical studies of spinal cord tissue from SOD1G93A transgenic mice, a model for amyotrophic lateral sclerosis (ALS), wherein LanCL1 expression was found to be increased at presymptomatic stages of the disease. These results indicate LanCL1 is a glutathione binding protein possibly significant to neurodegenerative disease.

Contributions of host and symbiont pigments to the coloration of reef corals

For a variety of coral species, we have studied the molecular origin of their coloration to assess the contributions of host and symbiont pigments. For the corals Catalaphyllia jardinei and an orange-emitting color morph of Lobophyllia hemprichii, the pigments belong to a particular class of green fluorescent protein-like proteins that change their color from green to red upon irradiation with approximately 400 nm light. The optical absorption and emission properties of these proteins were characterized in detail. Their spectra were found to be similar to those of phycoerythrin from cyanobacterial symbionts. To unambiguously determine the molecular origin of the coloration, we performed immunochemical studies using double diffusion in gel analysis on tissue extracts, including also a third coral species, Montastrea cavernosa, which allowed us to attribute the red fluorescent coloration to green-to-red photoconvertible fluorescent proteins. The red fluorescent proteins are localized mainly in the ectodermal tissue and contribute up to 7.0% of the total soluble cellular proteins in these species. Distinct spatial distributions of green and cyan fluorescent proteins were observed for the tissues of M. cavernosa. This observation may suggest that differently colored green fluorescent protein-like proteins have different, specific functions. In addition to green fluorescent protein-like proteins, the pigments of zooxanthellae have a strong effect on the visual appearance of the latter species.

Cyclic AMP signaling contributes to neural plasticity and hyperexcitability in AH sensory neurons following intestinal Trichinella spiralis-induced inflammation

Trichinella spiralis infection causes hyperexcitability in enteric after-hyperpolarising (AH) sensory neurons that is mimicked by neural, immune or inflammatory mediators known to stimulate adenylyl cyclase (AC)/cyclic 3′,5′-adenosine monophosphate (cAMP) signaling. The hypothesis was tested that ongoing modulation and sustained amplification in the AC/cAMP/phosphorylated cAMP related element binding protrein (pCREB) signaling pathway contributes to hyperexcitability and neuronal plasticity in gut sensory neurons after nematode infection. Electrophysiological, immunological, molecular biological or immunochemical studies were done in T. spiralis-infected guinea-pigs (8000 larvae or saline) after acute-inflammation (7 days) or 35 days p.i., after intestinal clearance. Acute-inflammation caused AH-cell hyperexcitability and elevated mucosal and neural tissue levels of myeloperoxidase, mast cell tryptase, prostaglandin E2, leukotrine B4, lipid peroxidation, nitric oxide and gelatinase; lower level inflammation persisted 35 days p.i. Acute exposure to blockers of AC, histamine, cyclooxygenase or leukotriene pathways suppressed AH-cell hyperexcitability in a reversible manner. Basal cAMP responses or those evoked by forskolin (FSK), Ro-20-1724, histamine or substance P in isolated myenteric ganglia were augmented after T. spiralis infection; up-regulation also occurred in AC expression and AC-immunoreactivity in calbindin (AH) neurons. The cAMP-dependent slow excitatory synaptic transmission-like responses to histamine (mast cell mediator) or substance P (neurotransmitter) acting via G-protein coupled receptors (GPCR) in AH neurons were augmented by up to 2.5-fold after T. spiralis infection. FSK, histamine, substance P or T. spiralis acute infection caused a 5- to 30-fold increase in cAMP-dependent nuclear CREB phosphorylation in isolated ganglia or calbindin (AH) neurons. AC and CREB phosphorylation remained elevated 35 days p.i.. Ongoing immune activation, AC up-regulation, enhanced phosphodiesterase IV activity and facilitation of the GPCR-AC/cAMP/pCREB signaling pathway contributes to T. spiralis-induced neuronal plasticity and AH-cell hyperexcitability. This may be relevant in gut nematode infections and inflammatory bowel diseases, and is a potential therapeutic target.

Does PGA external stenting reduce compliance mismatch in venous grafts?

BackgroundAutogenous vein grafting is widely used in regular bypassing procedures. Due to its mismatch with the host artery in both mechanical property and geometry, the graft often over expands under high arterial blood pressure and forms a step-depth where eddy flow develops, thus causing restenosis, fibrous graft wall, etc. External stents, such as sheaths being used to cuff the graft, have been introduced to eliminate these mismatches and increase the patency. Although histological and immunochemical studies have shown some positive effects of the external stent, the mechanical mismatch under the protection of an external stent remains poorly analyzed.MethodsIn this study, the jugular veins taken from hypercholesterolemic rabbits were transplanted into the carotid arteries, and non-woven polyglycolic acid (PGA) fabric was used to fabricate the external stents to study the effect of the biodegradable external stent. Eight weeks after the operation, the grafts were harvested to perform mechanical tests and histological examinations. An arc tangent function was suggested to describe the relationship between pressure and cross-sectional area to analyse the compliance of the graft.ResultsThe results from the mechanical tests indicated that grafts either with or without external stents displayed large compliance in the low-pressure range and were almost inextensible in the high-pressure range. This was very different from the behavior of the arteries or veins in vivo. The data from histological tests showed that, with external stents, collagen fibers were more compact, whilst those in the graft without protection were looser and thicker. No elastic fiber was found in either kind of grafts. Furthermore, grafts without protection were over-expanded which resulted in much bigger cross-sectional areas.ConclusionThe PGA external extent contributes little to the reduction of the mechanical mismatch between the graft and its host artery while remodeling develops. For the geometric mismatch, it reduces the cross-section area, therefore matching with the host artery much better. Although there are some positive effects, conclusively the PGA is not an ideal material for external stent.

Engineering flax with increased flavonoid content and thus Fusarium resistance

Flavonoids are a group of secondary plant metabolites important for plant growth and development, and thus the regulation of their biosynthesis is of special interest. We used a transgenic approach for flavonoid content manipulation. The multigene construct contained the cDNAs for chalcone synthase (CHS), and chalcone isomerase (CHI) and dihydroflavonol reductase (DFR) were prepared. Following flax plants transformation, the levels of the products of the enzyme overproduction were assessed in leaves and seeds. The simultaneous expression of genes resulted in a significant increase in the levels of flavanones, flavones, flavonols and anthocyanins, suggesting those three overproducing enzymes efficiently control the flavonoid route of the phenylpropanoid pathway. The increase in the flavonoid content in the transgenic flax plants might be the reason for observed, enhanced antioxidant capacity of those plants. The increased antioxidative properties of transgenic plants lead to improved resistance to Fusarium, the main pathogen of flax. The changes in phenylpropanoids accumulation in transgenic plants affect cell wall carbohydrate content. Immunochemical studies revealed significant increase in carbohydrates, constituents of pectin and hemicellulose. Since pectins contribute to flax stem retting, the compounds increase might affect fibre production. An increase in pectin and hemicellulose content leads to enhanced disease resistance of those plants.

The effect of long-term exposure to combinations of growth promoters in Long Evans rats: Part 2. Adrenal morphology (histopathology and immunochemical studies)

The aim of the study was to investigate the effects of long-term exposure (45 days) to growth promoters: clenbuterol (CB: 1mgkg−1bw) and/or dexamethasone (DEX: 0.1mgkg−1bw), in adrenal gland morphology, and the possibility of recovery after the withdrawal of drug treatment. Animals were sacrificed at different days of withdrawal (W0, W5, W10, W15 and W20), and adrenal glands processed for histopathology and immunohistochemistry. Adrenals of CB treatment showed typical features of long-term administration of β-agonists at W0 such as capillary dilatation in the fasciculata-reticularis zone, and this feature was also presented at W20. Adrenals of CB+DEX treatments showed the same results of CB treatment at days W0 and W20. However, DEX treatment presented the typical results of the exposure to corticoids with the atrophy of adrenal cortex. Immunohistochemistry of adrenal cortex steroidogenic enzymes (P450: scc, 3β-HSD, aromatase) denoted that neither positive staining nor localization was affected by treatments. Aromatase enzyme was immunolocalized in adrenal medulla cells in controls as well as in treated groups. The immunolocalization of glucocorticoid receptors showed an increase in CB (+++) and CB+DEX (++) treatments compared to the control group (0) and DEX treatment (0). Histopathological and immunohistochemical results are closely related to those found for adrenal endocrine function. We can conclude that chronic administration of growth promoters influence adrenal morphology and glucocorticoid receptor expression.

Cushing's Syndrome Due to Ectopic CRH Secretion by Adrenal Pheochromocytoma Accompanied by Renal Infarction

Ectopic production of corticotropin-releasing hormone (CRH) by a pheochromocytoma is an infrequent cause of Cushing's syndrome. We report the case of a 43-year-old man with Cushing's syndrome due to a CRH-producing adrenal pheochromocytoma. The patient had clinical and biochemical evidence of hypercortisolism in conjunction with high ACTH levels and non-suppressible serum cortisol levels on low-dose and high-dose dexamethasone suppression testing. In addition to these clinical features of one month's duration, the patient developed symptoms of pheochromocytoma including headache, hypertension that was resistant to conventional therapy and excessive sweating. Biochemical testing confirmed elevated 24-hour urinary catecholamines and metabolites. Abdominal CT revealed a 4.5 x 4 x 3.5 cm mass in the left adrenal gland. He underwent elective left adrenalectomy. Light microscopic and immunochemical studies revealed a pheochromocytoma that contained immunoreactive CRH and was negative for ACTH. Plasma ACTH and dexamethasone supression tests normalized after surgery. This is an unusual case of a CRH-secreting pheochromocytoma. This was complicated by renal infarction, illustrating further the complexity of Cushing's syndrome in a patient with pheochromocytoma caused by CRH hypersecretion.