Peter Petrusz

The Androgen Axis in Recurrent Prostate Cancer

Purpose. Prostate cancer that recurs during androgen deprivation therapy is referred to as androgen-independent. High levels of expression of androgen receptor and androgen receptor-regulated genes in recurrent prostate cancer suggest a role for androgen receptor and its ligands in prostate cancer recurrence. Experimental Design. Recurrent prostate cancer specimens from 22 men whose prostate cancer recurred locally during androgen deprivation therapy and benign prostate specimens from 48 men who had received no prior treatment were studied. Androgen receptor expression was measured using monoclonal antibody and automated digital video image analysis. Tissue androgens were measured using radioimmunoassay. Results. Epithelial nuclei androgen receptor immunostaining in recurrent prostate cancer (mean optical density, 0.284 ± SD 0.115 and percentage positive nuclei, 83.7 ± 11.6) was similar to benign prostate (mean optical density, 0.315 ± 0.044 and percentage positive nuclei, 77.3 ± 13.0). Tissue levels of testosterone were similar in recurrent prostate cancer (2.78 ± 2.34 pmol/g tissue) and benign prostate (3.26 ± 2.66 pmol/g tissue). Tissue levels of dihydrotestosterone, dehydroepiandrosterone, and androstenedione were lower (Wilcoxon, P = 0.0000068, 0.00093, and 0.0089, respectively) in recurrent prostate cancer than in benign prostate, and mean dihydrotestosterone levels, although reduced, remained 1.45 nm. Androgen receptor activation in recurrent prostate cancer was suggested by the androgen-regulated gene product, prostate-specific antigen, at 8.80 ± 10.80 nmol/g tissue. Conclusions. Testosterone and dihydrotestosterone occur in recurrent prostate cancer tissue at levels sufficient to activate androgen receptor. Novel therapies for recurrent prostate cancer should target androgen receptor directly and prevent the formation of androgens within prostate cancer tissue.

The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system

Abstract Somatostatin-containing neurons in the rat central nervous system were localized by immunocytochemical methods. The detection of somatostatin-like immunoreactivity was facilitated by (1) the use of brains from colchicine-treated rats, (2) the proteolytic pretreatment of sections with pronase and (3) a ‘double-bridge’ immunoperoxidase staining technique. In addition to the known distribution of somatostatin-like immunoreactivity we also observed immunoreactive perikarya in the following regions: the anterior olfactory nucleus, some areas of the preoptic and hypothalamic regions, the claustrum, the periaqueductal gray, the locus ceruleus, the central gray substance, the lateral parabrachial nucleus, the nucleus of the lateral lemniscus, the nucleus ambiguus, the spinal trigeminal nucleus, the nucleus of the solitary tract and various areas of the reticular formation. Immunoreactive neuronal processes were also observed in several major tracts of the brain, including the stria terminalis, the fornix and the medial forebrain bundle. Our results indicate that somatostatin-containing neurons may occur both as interneurons in some areas of the central nervous system and as projection neurons in others. The widespread but selective distribution of these neurons suggests that somatostatin is not only a hypothalamic regulator of neuroendocrine function, but may also function as a major neuromodulator mediating a variety of functions throughout the central nervous system.

Immunocytochemical Localization of β-Endorphin-Containing Neurons in the Rat Brain

β-Endorphin-containing neurons in the rat central nervous system were localized using three improvements of the unlabelled antibody-enzyme bridge immunocytochemical technique. These improvements were

Characterization of antisera to glutamate and aspartate.

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.

Gonadotropin-releasing hormone (GnRH) neurons and pathways in the rat brain

SummaryGonadotropin-releasing hormone (GnRH) neurons and their pathways in the rat brain were localized by immunocytochemistry in 6-to 18-day-old female animals, by use of thick frozen or vibratome sections, and silver-gold intensification of the diaminobenzidine reaction product. GnRH-immunoreactive perikarya were observed in the following regions: olfactory bulb and tubercle, vertical and horizontal limbs of the diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and different regions of the hippocampus (indusium griseum, Ammons horn). In addition to the known GnRH-pathways (preoptico-terminal, preoptico-infundibular, periventricular), we also observed GnRH-immunopositive processes in several major tracts and areas of the brain, including the medial and cortical amygdaloid complex, stria terminalis, stria medullaris thalami, fasciculus retroflexus, medial forebrain bundle, indusium griseum, stria longitudinalis medialis and lateralis, hippocampus, periaqueductal gray of the mesencephalon, and extracerebral regions, such as the lamina cribrosa, nervus terminalis and its associated ganglia. By use of the silver-gold intensification method we present Golgi-like images of GnRH perikarya and their pathways. The possible distribution of efferents from each GnRH cell group is discussed.

Immunocytochemical localization of proenkephalin-derived peptides in the central nervous system of the rat

Most of the early studies on the immunohistochemical distribution of enkephalin pentapeptide-like immunoreactivity used antisera that stained both proenkephalin- and prodynorphin-containing neurons. The present study used the peroxidase-antiperoxidase method, thick Vibratome sections and antisera specific for the carboxyl termini of [Met]enkephalin, [Met]enkephalyl-Arg6-Phe7, [Met]enkephalyl-Arg6-Gly7-Leu8, and metorphamide and for BAM 22P in order to obtain a detailed description of the distribution of authentic proenkephalin-containing perikarya and nerve processes. The peroxidase-antiperoxidase reaction product was intensified by the selective deposition of silver crystals in order to display the morphology of proenkephalin-containing neurons with great fidelity. The results indicate that the magnocellular perikarya in the supraoptic and paraventricular nuclei contain prodynorphin rather than proenkephalin as had been suggested by earlier investigators. The coarse fibers in the internal zone of the median eminence and the granule cell-mossy fiber pathway in the hippocampus also contain prodynorphin rather than proenkephalin. The number of proenkephalin-containing perikarya and/or the density of proenkephalin-containing nerve terminals in several other areas of the brain, e.g. the substantia nigra, the central amygdaloid nucleus, the periaqueductal gray and the parabrachial nuclei, were overestimated by earlier investigators. The distribution of authentic proenkephalin-containing perikarya and nerve processes is, despite these errors, similar to the distribution of enkephalin pentapeptide-like immunoreactivity described by earlier investigators. Proenkephalin-containing perikarya were identified for the first time in the medial and lateral habenular nuclei of the adult rat. Antisera specific for [Met]enkephalin, [Met]enkephalyl-Arg6-Phe7, [Met]enkephalyl-Arg6-Gly7-Leu8 and BAM 22P stain perikarya and nerve terminals with a similar distribution. The metorphamide antiserum also stains the same perikarya and nerve terminals; however, it also stains magnocellular perikarya in the zona incerta and the lateral hypothalamus that are not stained by any of the other proenkephalin-specific antisera.

The paraventriculo-infundibular corticotropin releasing factor (CRF) pathway as revealed by immunocytochemistry in long-term hypophysectomized or adrenalectomized rats

The immunocytochemical localization of corticotropin releasing factor (CRF)-containing pathways projecting from the paraventricular nucleus (PVN) to the external layer of the median eminence (ME) in long-term hypophysectomized or adrenalectomized rats is described. Immunocytochemistry was followed by silver intensification of the diaminobenzidine end-product. In comparison with untreated control rats, both hypophysectomy and adrenalectomy resulted in a dramatic increase in immunostaining of the CRF-containing perikarya and fibers, particularly those originating from the PVN and terminating in the ME. The staining was more intense in adrenalectomized than in hypophysectomized rats. The CRF-positive fibers emerging from the PVN form a medial, an intermediate and a lateral fiber pathway. The lateral and intermediate CRF tracts leave the dorsolateral part of the PVN and course laterally and medially of the fornix, respectively, then ventrally toward the optic tract. Just dorsal to the optic tract they turn in caudal direction and run parallel with and very close to the basal surface of the hypothalamus; individual fibers then turn medially to terminate in the external layer of the ME. Only a few fibers originate from the medial-ventral part of the PVN (medial pathway). These fibers run in ventral direction along the walls of the 3rd ventricle and terminate in the ME. Thus the majority of CRF fibers, similarly to other peptidergic systems, reach the medial basal hypothalamus from the anterolateral direction.

Improved immunoglobulin-enzyme bridge method for light microscopic demonstration of hormone-containing cells of the rat adenohypophysis

SummaryAn improved procedure of high efficiency is described for light microscopic localization of tissue antigens by the unlabeled antibody-enzyme bridge technique with unpurified antiserum to horseradish peroxidase followed by horseradish peroxidase. The method incorporates blocking of nonspecific tissue binding sites with normal serum derived from the species in which the second component of the bridge was made, incubation for extended periods of time (42–65 hrs) with high dilutions (up to 1∶1,000,000) of the primary antibody, and optimal conditions for the histochemical reaction with diaminobenzidine and H2O2. Hormone-containing cells of the rat adenohypophysis can be stained with this technique using dilutions of the primary antisera that may approach, or exceed, those used in radio-immunoassay or in electron microscopic immunocytochemistry with preformed peroxidase-antiperoxidase complexes. Based partly on analysis of experimental data, it is suggested that the term “efficiency” should be used to describe the ability of immunohistochemical methods to work at high dilutions of the primary antisera, while the term “sensitivity” should be reserved to describe the ability of such methods to detect a specified amount or concentration of tissue antigen.

Criteria of reliability for light microscopic immunocytochemical staining

SummaryThe following criteria of reliability are defined and discussed for immunocytochemical staining at the light microscopical level: efficiency, accuracy, precision, sensitivity, and specificity. Whenever practical, tests are suggested for obtaining information on the extent to which these criteria are fulfilled in a given system, and procedures are outlined for improving immunocytochemical staining in terms of these criteria. It is suggested that consideration of reliability criteria will help investigators in their choice of methodology, design of experimental strategy, and valid interpretation of the results.

Immunocytochemical localization of corticotropin releasing factor (CRF) in the rat spinal cord.

The presence of corticotropin releasing factor (CRF)-immunoreactive nerve fibers and cell bodies in the spinal cord is demonstrated. Immunopositive fibers were found in the lateral column of the white matter, in laminae I, V-VII, X, and in the intermediolateral column of the spinal cord. Complete transection of the spinal cord showed that the majority of the fibers in the lateral funiculus formed an ascending pathway; however, a few descending fibers were also detected. Hypophysectomy resulted in enhanced immunoreactivity of the fibers and staining of CRF-immunoreactive cell bodies in laminae V-VII, X, and in the intermediolateral sympathetic column. The results suggest that CRF is not merely an ACTH releasing factor, but also a regulatory peptide which may be involved in several stress-related neural responses.