Search

Liquid-based cytology continues to be utilized as an adjunct to conventional cytology in most Australian laboratories, even though a direct-to-vial ThinPrep protocol has been introduced in many countries with established cervical screening programs. Manual screening of ThinPrep slides has been widely practiced for more than 10 years and the recent introduction of the ThinPrep Imaging System (TPI) has been reported as being more sensitive than the conventional smear (CS) in the identification of high-grade cervical disease.We report our experience with ThinPrep Imaging since its introduction into our routine gynecological cytology service. 87,284 split sample pairs reported using the Imaging System demonstrated a decrease in unsatisfactory reports (3.65% for CS and 0.87% for TPI) and an increase in possible high grade and definite high-grade squamous reports (1.57% for CS and 1.62% for TPI).For 1,083 biopsy confirmed high-grade lesions, the correct diagnosis of high grade or possible high-grade squamous disease was made on the ThinPrep imaged slide in 61.0% (661/1,083) of cases and on the CS in 59.4% (643/1,083). This was not statistically significant. When all abnormalities identified on cytology were considered, including possible low grade and definite low-grade abnormalities, the difference in sensitivity for Thinprep imaged slides of 96.0% (1,040/1,083) and CSs of 91.6% (992/1,083) was statistically significant.

Background: Doxazosin, an alpha-adrenergic antagonist, has been shown to induce apoptosis in prostatic stromal cells. The mechanism of this apoptotic action by Doxazosin remains undefined. The present study was carried out to demonstrate that the effect of Doxazosin on apoptosis of prostate stromal cells is mediated through an autocrine action of TGF-beta1., Methods: Primary cultures of human prostate cells were treated with varying concentrations of Doxazosin (0, 0.1, 1, 10, and 100 microM) for a period up to 3 days. At the end of the 3-day culture, cell numbers were counted. Apoptosis was assessed by a colorimetric terminal deoxyribonucleotide transferase labeling technique. TGF-beta1 was determined by enzyme-linked immunosorbent assay (ELISA)., Results: Compared to control cultures, cell numbers were significantly decreased as much as 68.4% in cultures treated with 10 microM of Doxazosin after 3 days incubation, while apoptosis increased by 64.7% in cultures treated with the same concentration of Doxazosin after 24 h. This decrease in cell number was reversed when antibody to TGF-beta1 was added to these cultures. Addition of TGF-beta1 (0, 1.0, and 10 ng/mL) to the cultures also decreased the cell numbers. Quantitation of TGF-beta1 in lysates of cells by ELISA revealed that the cells treated with Doxazosin (10 microM) produced as much as 62.5% more TGF-beta1 than in that of untreated cells., Conclusions: These results demonstrate that the apoptotic effect of Doxazosin on human prostatic stromal cells is mediated through an autocrine production of TGF-beta1., (Copyright 2001 Wiley-Liss, Inc.)

Simian virus 40 large T antigen (SVLTAg) has been used to immortalize cells; however, the mechanism leading to immortalization is still unclear. We hypothesize that DNA mismatch repair (MMR) activity is important during SVLTAg-induced immortalization. To test this hypothesis, we used the SVLTAg-immortalized cell line BPH-1 derived from human benign prostate epithelial cells to analyze MMR activity and the expression of MMR genes (hMLH1, hPMS1, hPMS2, hMSH2, hMSH3, and hMSH6). The results demonstrated that BPH-1 cells were deficient in repairing G:T, A:C, and G:G mispairs in bacteriophage M13mp2. Reverse-transcription polymerase chain reaction experiments indicated MMR genes (hMSH3, hMSH6, and hPMS1) were expressed at a low level in BPH-1 cells. In contrast, all six MMR genes were expressed in human benign prostate hyperplasia tissues. Downregulation of hMSH3, hMSH6, and hPMS1 genes is not a result of the hypermethylation mechanism because demethylation with 5-aza-2'-deoxycytidine did not restore expression of these genes. Although the hMLH1 gene is expressed in BPH-1 cells, western blotting and exon analyses demonstrated that hMLH1 was mutated and/or deleted in BPH-1 cells., (Copyright 2001 Wiley-Liss, Inc.)

We report an unbalanced translocation involving chromosomes 14 and 21 which presented as fetal ventriculomegaly at 33 weeks gestation. Second trimester ultrasound had indicated normal fetal anatomy, including normal intracranial structures. Parental karyotypes showed a paternal balanced translocation: 46,XY,t(14;21)(q12;q21). The unbalanced translocation in the fetus resulted in trisomy for 14pter-->q12 and monosomy for 21pter-->q21. Postnatal examination showed that the male infant had a cleft palate, but no cleft lip, and mild dysmorphic features. Postnatal MRI revealed bilateral and symmetric dilatation of the occipital horns, atria, and temporal horns of the lateral ventricles. Molecular cytogenetic techniques were used to delineate further the breakpoint on chromosome 14 to a site distal of the D14S1071 locus and the breakpoint on chromosome 21 to a region between D21S1918 and D21S1902. More precise definitions of chromosomal breakpoints in such clinical cases should provide more accurate prognosis for individuals with unbalanced karyotypes and assist in the identification of putative developmentally important genes., (Copyright 2001 Wiley-Liss, Inc.)

A preferred anti-cancer vaccine would be tumor-specific, simple to rapidly construct and safe to administer. It would permit immunization against a spectrum of the tumor's distinctive antigens, without requiring their prior identification. Toward these goals, we describe a modification of standard extracorporeal photopheresis (ECP) which initiates, within a single day, both monocyte-to-dendritic cell (DC) differentiation and malignant cell apoptosis. The transition of monocytes to immature DCs was identified by the expression of cytoplasmic CD83 and membrane CD36 in the absence of membrane CD14 staining, as well as induction of membrane CD83 expression. Differentiating DCs were avidly phagocytic and engulfed apoptotic malignant T cells. Differentiating DCs were capable of stimulating significant proliferation of normal alloreactive lymphocyte responders, indicting increased expression of membrane MHC class II molecules. This approach provides a clinically practical means of developing tumor-loaded cells that have initiated the transition to DCs without the requirement of exogenous cytokines, excessive cellular manipulation or isolation. Construction of DC vaccines using this methodology can be generalized to other diseases and may offer a novel approach for improved cancer immunotherapy., (Copyright 2001 Wiley-Liss, Inc.)

The action of prostaglandin E(2) (PGE(2)) in the preoptic area is thought to play an important role in producing fever. Pharmacologic evidence suggests that, among the four subtypes of E-series prostaglandin (EP) receptors, i.e., EP(1), EP(2), EP(3), and EP(4), the EP(1) receptor mediates fever responses. In contrast, evidence from mice with EP receptor gene deletions indicates that the EP(3) receptor is required for the initial (<1 hour) fever after intravenous (i.v.) lipopolysaccharide (LPS). To investigate which subtypes of EP receptors mediate systemic infection-induced fever, we assessed the coexpression of Fos-like immunoreactivity (Fos-IR) and EP(1-4) receptor mRNA in nuclei in the rat hypothalamus that have been shown to be involved in fever responses. Two hours after the administration of i.v. LPS (5 microg/kg), Fos-IR was observed in the ventromedial preoptic nucleus, the median preoptic nucleus, and the paraventricular hypothalamic nucleus. In these nuclei, EP(4) receptor mRNA was strongly expressed and the Fos-IR intensely colocalized with EP(4) receptor mRNA. Strong EP(3) receptor mRNA expression was only seen within the median preoptic nucleus but Fos-IR showed little coexpression with EP(3) receptor mRNA. EP(2) receptor mRNA was not seen in the PGE(2) sensitive parts of the preoptic area. Although approximately half of the Fos-immunoreactive neurons also expressed EP(1) receptor mRNA, EP(1) mRNA expression was weak and its distribution was so diffuse in the preoptic area that it did not represent a specific relationship. In the paraventricular nucleus, EP(4) mRNA was found in most Fos-immunoreactive neurons and levels of EP(4) receptor expression increased after i.v. LPS. Our findings indicate that neurons expressing EP(4) receptor are activated during LPS-induced fever and suggest the involvement of EP(4) receptors in the production of fever., (Copyright 2000 Wiley-Liss, Inc.)

Many malignant cells secrete transforming growth factor-beta (TGF-beta), a potent immunosuppresant, suggesting that TGF-beta production may represent a significant tumor escape mechanism from host immunosurveillance. Establishment of a leukocyte subpopulation with disrupted TGF-beta signaling in the tumor-bearing host offers a potential means for immunotherapy of cancer. Downregulation of TGF-beta secretion in tumor cells results in restoration of immunogenicity in the host, while T-cell insensitivity to TGF-beta results in accelerated differentiation and autoimmunity, elements of which may be required in order to combat self-antigen-expressing tumors in a tolerized host. The rationale, approaches, and potential pitfalls of this strategy will be discussed., (Copyright 2000 Wiley-Liss, Inc.)

Background: LNCaP cells are androgen-sensitive human prostate cancer cells. They are characterized by a bell-shaped growth curve in response to increasing doses of dihydrotestosterone (DHT) in culture. At a low concentration of DHT (0.1 nM), these cells show an increase in proliferation, but their growth is arrested at a high concentration (100 nM) of DHT. Results of our previous study demonstrated that the inhibitory effect of DHT at a high concentration was mediated through the action of TGF-beta1. The objective of the present study was to elucidate the mechanism of the proliferative effect of DHT in LNCaP cells. METHODS AND RESULTS DHT stimulated LNCaP proliferation only when cells were cultured in the presence of serum. In serum-free cultures, the characteristic DHT-induced proliferation was not observed. The addition of neutralizing antibody against FGF-2 (basic fibroblast growth factor) was able to inhibit this DHT-induced proliferation. These results suggest that the proliferative effect of DHT was mediated through the action of FGF-2. However, results of the reverse transcriptase polymerase chain reaction indicated that LNCaP cells did not express FGF-2 message. As a result, the source of FGF-2 in these cultures must be the serum supplemented in the culture media. FGF-2 can bind to heparin sulfate chains within the extracellular matrix (ECM). In cultures treated with exogenous heparin, the proliferative effect of DHT was abolished. These results led to the development of the hypothesis that DHT treatment mediates the release of FGF-2 entrapped in the ECM through increased heparinase activity. The addition of heparinase to cultures of LNCaP cells, in the absence of DHT, was able to stimulate cell proliferation. Moreover, 0.1 nM DHT caused a significant increase in heparinase activity., Conclusions: These results provide a possible mechanism for DHT action in LNCaP cells. In the absence of DHT, FGF-2 in culture was trapped in the extracellular matrix and was not available to interact with LNCaP cells. However, in the presence of 0.1 nM DHT, heparinase activity in the culture was elevated and, as a result, it liberated the trapped FGF-2 which, in turn, stimulated proliferation in LNCaP cells., (Copyright 2000 Wiley-Liss, Inc.)

During neonatal development, cardiac myocytes undergo a transition from hyperplastic to hypertrophic growth. Whether these cells are terminally differentiated and permanently withdrawn from the cell cycle shortly after birth is controversial. Nevertheless, the clinical observation that functionally significant myocardial regeneration has not been documented in cardiovascular disease or injury during adulthood seems to support the notion that the vast majority of cardiac myocytes do not proliferate once they differentiate. Regardless of the controversy, the elucidation on how mitosis is blocked in cardiac myocytes may facilitate development of new cardiovascular therapies, based on the regeneration of the adult myocardium. To better understand postnatal myocardial development, we performed suppression subtractive hybridization to isolate genes that are differentially expressed in day one or day seven postnatal rat ventricular myocardium. Here we report the down-regulated mRNA expression of the 40-kDa subunit of replication factor C (RFC p40 or RFC2), which is an essential processive factor for proliferating cellular nuclear antigen-dependent DNA replication during neonatal myocardial development., (Copyright 2000 Wiley-Liss, Inc.)

Background: Promoter elements within the 5' DNA region of the rat C(3)1 gene have been shown to direct prostate-specific expression of gene products when they are fused through recombinant DNA procedures and used to produce transgenic mice. In order to test the in vivo effects of chronic overexpression of the mouse c-myc protooncogene on the prostate glands of transgenic mice, we created several lines of C(3)1-c-myc transgenic mice and then examined the phenotype of males with this genetic alteration., Methods: The modified promoter and 5' region of the rat C(3)1 gene was fused to the coding region of the mouse c-myc gene using recombinant DNA techniques. This DNA was used to create three different founder lines of transgenic mice. Tissues from males and females heterozygous for the transgene were examined for expression of the recombinant mouse c-myc mRNA by an RNase protection assay. Prostates from males were examined for expression of recombinant c-myc mRNA by in situ hybridization. Thin sections of fixed ventral prostates from males were analyzed by microscopy for histological abnormalities., Results: Three different lines of transgenic mice were obtained from these procedures. These mice demonstrated expression of recombinant mouse c-myc mRNA in the testis and ventral prostates of males and in the uterus of females. In situ hybridization demonstrated that the epithelial cells were the source of recombinant c-myc expression in the ventral prostates of the transgenic lines. Microscopic analysis of the ventral prostates from these mice demonstrated abnormalities in epithelial cell morphology seemingly typical of an intraepithelial neoplasia-like phenotype. However, none of the males of any of the lines developed overt prostatic adenocarcinoma over their lifetimes., Conclusions: Chronic overexpression of c-myc in the ventral prostate epithelial cells of C3(1)-c-myc transgenic mice leads to the development of epithelial cell abnormalities similar to those seen in low-grade prostatic intraepithelial neoplasia in humans. These abnormalities were not found to progress to adenocarcinoma over the lifetimes of the transgenic mice, suggesting the need for additional oncogenic changes in the pathway to prostatic adenocarcinomas. Furthermore, our cumulative experience with the use of the C3(1) gene promoter in the generation of transgenic mice suggests that the probasin promoter element provides a much more specific and effective means to target transgenes to the prostate glands of mice., (Copyright 2000 Wiley-Liss, Inc.)

Background: Although normal prostatic development is androgen-dependent, the prostate continues to grow in the neonate despite castration. However, the manner in which neonatal growth of the prostate occurs, in the absence of the testis, remains largely unknown. The purpose of this study was to examine the differentiation of prostatic epithelial cells after neonatal castration., Methods: Immunohistochemistry was utilized to detect the expression of differentiation products: basal-cell cytokeratin (CK 5), luminal-cell cytokeratin (CK 18), and prostatic steroid-binding protein (PBP), a ventral prostate-specific marker indicative of secretory function in luminal cells. The reverse transcription-polymerase chain reaction was used to detect transcription products of the three polypeptide subunits of PBP, designated C1, C2, and C3. Rats were castrated on day 5 after birth, and ventral prostates were collected on day 14. Dihydrotestosterone was injected (100 microg/animal every 2 days) in castrated animals to determine if PBP expression could be initiated by androgen., Results: Although no major effects of castration were detected on the differentiation of stromal or basal cells (which differentiate prior to day 5), castration had a pronounced effect on luminal-cell differentiation. Castration inhibited PBP protein expression, but did not affect the expression of luminal-cell cytokeratin (CK 18) protein. Furthermore, castration reduced C1, C2, and C3 transcription. Androgen replacement to castrated animals allowed for the initiation of PBP expression, although its onset was delayed., Conclusions: These observations indicate that the testis is not necessary for prostatic luminal-cell differentiation, but is necessary for full expression of luminal-cell secretory phenotype. Furthermore, our study suggests that factors of testicular origin, in addition to androgen, are needed for proper timing of PBP expression. This investigation establishes that the cytological and the physiological differentiation of the rat prostate are differentially regulated., (Copyright 2000 Wiley-Liss, Inc.)

Background: Prostatic epithelial cells are sensitive to the inhibitory effects of TGF-beta. However, TGF-beta signaling in the prostate is dependent on androgenic status. Under the in vivo conditions, it is difficult to dissociate the effect of TGF-beta from that of androgen on the prostate., Methods: The objective of the present study was to create and verify a transgenic mouse system in which epithelial cells of the ventral prostate are insensitive to the actions of TGF-beta. By using a modified prostate-specific promoter, C3(1), the TGF-beta dominant negative receptor is only expressed in the epithelial cells of the ventral prostate, and these cells are resistant to TGF-beta. Morphology of transgenic animal prostates was compared to wild-type animal prostates by immunohistochemistry and microscopy., Results: The prostate of transgenic mice exhibited an abnormal morphology with multiple layers of epithelial cells lining the proximal ducts, in contrast to the simple cuboidal monolayer of cells seen in the normal prostate. This observation was accompanied by a loss of apoptosis in this region, as seen by TUNEL assay. There was no significant difference in serum levels of testosterone between the wild-type and transgenic animals., Conclusions: These results demonstrated that a loss of sensitivity to TGF-beta results in the accumulation of multiple layers of epithelial cells in the proximal region of the ventral prostate. This abnormal growth illustrates that TGF-beta plays an important role in regulating prostate growth. The current transgenic system can be used as an experimental model to study the functional role of TGF-beta in prostatic growth and function., (Copyright 2000 Wiley-Liss, Inc.)

Background: The extracellular matrix (ECM) is an intricate network composed of an array of molecules that play an integral role in the regulation of cell function, differentiation, and tissue-specific gene expression in various epithelia. In the present study, we examined the distribution of collagen type IV and laminin along the rat ventral prostatic duct before and after castration., Methods: Mature Sprague-Dawley rats were castrated and their prostates processed for immunocytochemistry of ECM proteins, laminin, and collagen type IV. Tissue sections were also processed for apoptosis staining, using the 3' end-labeling technique. To examine the effect of ECM proteins on epithelial growth, rat ventral epithelial cells were cultured on ECM-coated surfaces., Results: In the intact rat, laminin was localized in the basement membrane along all regions of the ventral prostate ductal system. Collagen type IV was found to be distributed evenly in the basement membrane of the distal and intermediate regions but was absent or poorly organized in the proximal region, where apoptosis in the epithelium occurs at a high rate. In the regressing prostate after castration, there was a shift in apoptosis from the proximal region to the distal intermediate regions of the prostatic duct. Associated with the shift was a remodeling of basement membrane proteins due to the specific loss of collagen type IV in the distal and intermediate regions. Collagen type IV reappeared underneath the epithelium 7 days after castration, when apoptosis in the epithelium stopped. In vitro, collagen type IV enhanced the growth of ventral prostatic epithelial cells, as assessed by cell number., Conclusions: Collagen basement membrane type IV mediates growth of rat ventral prostate epithelium, and its loss during tissue remodeling after castration is associated with cell death., (Copyright 2000 Wiley-Liss, Inc.)

Background: Although growth factors such as epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, and TGF-beta are important regulators of prostate cell growth in vitro and in animal models, evidence to support their role in human prostate cancer development remains sparse. We previously showed that men without prostate cancer have concentrations of EGF and TGF-alpha in expressed prostatic fluid (EPF) that are individually distinct and stable over time. This study addressed whether growth factor levels in EPF are associated with the presence or progression of prostate cancer., Methods: We measured levels of immunoreactive EGF, TGF-alpha, and TGF-beta1 in stored EPF samples from three age-matched groups: 19 men with untreated, histologically diagnosed prostate cancer (CaP), 38 with benign prostate hyperplasia (BPH), and 19 with normal prostate glands (NPD)., Results: Median TGF-alpha was lower in the BPH group (0.45 ng/ml) than in either CaP (0.63 ng/ml) or NPD (0.58 ng/ml) groups (P = 0.03 and 0.12, respectively). For EGF, the median was lowest in the CaP group and highest in the NPD group (92.5 ng/ml vs. 175.5 ng/ml, P = 0.006). For TGF-beta1, the median level in CaP was 2.7 times higher than the median level among all controls (6.65 ng/ml vs. 2.46 ng/ml, P = 0.002). Growth factor levels were not associated with tumor stage or Gleason score. However, the single case with distant metastases had TGF-beta1 levels 23-fold higher than the CaP median., Conclusions: The results suggest that at the time of CaP diagnosis, EGF levels in EPF are significantly lower, and TGF-beta1 levels significantly higher, than normal. Marked overexpression of TGF-beta1 in advanced CaP might be reflected in extremely high EPF levels., (Copyright 1999 Wiley-Liss, Inc.)

Background: The present review summarizes the cellular action of TGF-beta in benign and malignant growth of the prostate., Methods: TGF-beta is a pleiotropic growth factor. It plays an important role in the regulation of growth and differentiation in many cells. In benign prostatic epithelia, its action is mediated through a paracrine mechanism. It inhibits proliferation and induces apoptosis in prostatic epithelia. It provides a mechanism to maintain epithelial homeostasis in the prostate. In prostatic stroma, its continual action leads to smooth muscle differentiation. This effect of TGF-beta may regulate the development of prostatic smooth muscle nodules in benign prostatic hyperplasia., Results: As prostatic epithelial cells undergo malignant transformation, two major events occur regarding TGF-beta action. These include the loss of expression of functional TGF-beta receptors and overproduction of TGF-beta in malignant cells. The loss of expression of functional TGF-beta receptors provides a growth advantage to cancer cells over their benign counterparts. The overproduction of TGF-beta by cancer cells has a multitude of adverse consequences. TGF-beta can promote extracellular matrix production, induce angiogenesis, and inhibit host immune function. The biological consequence of these activities is an enhanced tumorigenicity in prostate cancer. Results of our recent studies with a rat prostate cancer model suggest that the immunosuppressive effect of TGF-beta seems to be the primary cause of tumor progression. This is because, if these cancer cells were engineered to reduce the production of TGF-beta, tumor growth was inhibited in syngeneic hosts but not in immune compromised hosts., Conclusions: Our future research should take advantage of this knowledge to devise therapeutic strategies aimed at eradicating prostate cancer.

Background: SGP-2 is a ubiquitous secreted glycoprotein that prevents cellular apoptosis. This study was carried out to determine the extracellular action of SGP-2 in a model of tumor necrosis factor-alpha (TNF)-induced cytotoxicity using two human prostatic cancer lines, LNCaP and PC3. These two lines were selected because LNCaP cells are highly sensitive to the cytotoxic effect of TNF, while PC3 cells are resistant to TNF at 24 hr., Methods: Cells were cultured in the presence or absence of TNF (10 ng/ml). LNCaP cells were treated with varying concentrations of exogenous SGP-2, while PC3 cells were treated with antisera to SGP-2 with and without exogenous SGP-2. Following a 24-hr treatment, cultures were assessed by counting of cell number and by the trypan blue exclusion assay., Results: Western blot analysis of conditioned media revealed that PC3 secreted more SGP-2 than did LNCaP. The sensitivity to TNF in LNCaP cells was reduced by the addition of exogenous SGP-2. PC3 cells became sensitive to TNF when SGP-2 antibody was added to the culture. The effect of SGP-2 antibody on PC3 cells was reversed by the addition of exogenous SGP-2 to the culture., Conclusions: These results suggest that SGP-2 can act as an extracellular mediator of anti-TNF-induced cytotoxicity.

The preliminary results of an international collaborative study examining premature menopause in fragile X carriers are presented. A total of 760 women from fragile X families was surveyed about their fragile X carrier status and their menstrual and reproductive histories. Among the subjects, 395 carried a premutation, 128 carried a full mutation, and 237 were noncarriers. Sixty-three (16%) of the premutation carriers had experienced menopause prior to the age of 40 compared with none of the full mutation carriers and one (0.4%) of the controls. Based on these preliminary data, there is a significant association between fragile X premutation carrier status and premature menopause.

Background: This study was undertaken to attempt to characterize changes in in vitro growth rates and cellular phenotypes of human prostatic stroma associated with aging and/or development of benign prostatic hyperplasia (BPH)., Methods: Prostate stromal cell strains were established from 12 tissue donors of varying age. Culture growth rate was determined by cell counts over a 6-day period. Cell phenotype was assessed by immunocytochemical staining for smooth muscle alpha-actin, smooth muscle myosin, and prolyl-4-hydroxylase., Results: Growth rates of prostate stromal strains in vitro varied. Stromal cells derived from aged males with BPH had significantly slower growth rates than cells from younger donors. A positive reaction for prolyl-4-hydroxylase, a mesenchymal cell marker, was present in all cell cultures regardless of donor age. Expression of smooth muscle-specific actin, a nonspecific smooth muscle cell marker, was present in 48-79% of prostate stromal cultures. Staining for smooth muscle myosin, a specific smooth muscle cell marker, was found to vary significantly with age. The percentage of smooth muscle myosin-positive cells derived from males aged 15, 45, 57, and 72 years were 0.70 +/- 0.14%, 2.12 +/- 0.30%, 4.20 +/- 0.89%, and 26.25 +/- 1.0%, respectively. The shape and size of actin- and/or myosin-positive stromal cells from a 72-year-old donor culture were also usually larger and polygonal in shape as compared to thin and elongated shapes in 15-year-old donor cultures. The shape of actin- and/or myosin-positive cells from a 45-year-old donor culture demonstrated both phenotypes., Conclusions: These results suggest that in human prostate stromal cells cultured as described, the growth rate decreases, the percent of smooth muscle cells increases, and the cellular shape changes with increasing donor age and/or development of BPH.

Recent studies have identified several neuropeptide systems in the hypothalamus that are critical in the regulation of body weight. The lateral hypothalamic area (LHA) has long been considered essential in regulating food intake and body weight. Two neuropeptides, melanin-concentrating hormone (MCH) and the orexins (ORX), are localized in the LHA and provide diffuse innervation of the neuraxis, including monosynaptic projections to the cerebral cortex and autonomic preganglionic neurons. Therefore, MCH and ORX neurons may regulate both cognitive and autonomic aspects of food intake and body weight regulation. The arcuate nucleus also is critical in the regulation of body weight, because it contains neurons that express leptin receptors, neuropeptide Y (NPY), alpha-melanin-stimulating hormone (alpha-MSH), and agouti-related peptide (AgRP). In this study, we examined the relationships of these peptidergic systems by using dual-label immunohistochemistry or in situ hybridization in rat, mouse, and human brains. In the normal rat, mouse, and human brain, ORX and MCH neurons make up segregated populations. In addition, we found that AgRP- and NPY-immunoreactive neurons are present in the medial division of the human arcuate nucleus, whereas alpha-MSH-immunoreactive neurons are found in the lateral arcuate nucleus. In humans, AgRP projections were widespread in the hypothalamus, but they were especially dense in the paraventricular nucleus and the perifornical area. Moreover, in both rat and human, MCH and ORX neurons receive innervation from NPY-, AgRP-, and alpha-MSH-immunoreactive fibers. Projections from populations of leptin-responsive neurons in the mediobasal hypothalamus to MCH and ORX cells in the LHA may link peripheral metabolic cues with the cortical mantle and may play a critical role in the regulation of feeding behavior and body weight.

Background and Objectives: Imaging monitored cryosurgery is emerging as an important minimally invasive surgical technique for treatment of cancer. Although imaging allows excellent control over the process of freezing itself, recent studies show that at high subzero temperatures cells survive freezing. Antifreeze proteins (AFP) are chemical compounds that modify ice crystals to needle-like shapes that can destroy cells in cellular suspensions. The goal of this study was to determine whether these antifreeze proteins can also destroy cells in frozen tissue and serve as chemical adjuvants to cryosurgery., Methods: Livers from six rats were excised, perfused with solutions of either phosphate-buffered saline (PBS) or PBS with 10 mg/ml AFP-I, and frozen with a special cryosurgery apparatus. Lobes were frozen with one or two freeze-thaw cycles and the cell viability was examined with a two stain fluorescent dye test and histological assessment., Results: A significant percentage of hepatocytes survive freezing on the margin of a frozen cryolesion. AFP significantly increase cellular destruction in that region apparently through formation of intracellular ice., Conclusions: This preliminary study demonstrates that antifreeze proteins may be effective chemical adjuvants to cryosurgery.

Background: Regional variations in stromal-epithelial interactions, mediated through soluble growth factors, may be responsible for differences in epithelial growth and death observed between regions of the rat prostatic ductal system. Since transforming growth factor-beta 1 (TGF-beta 1) can induce prostatic epithelial cell death in vitro and in vivo, we examined the localization and production of TGF-beta 1 with respect to the functional regions of the rat prostatic ductal system., Methods: The distribution of TGF-beta 1 in the rat ventral prostate was examined by immunohistochemistry. Cell type-specific expression of TGF-beta 1 was determined using RT-PCR analysis of prostate epithelial and stromal cell fractions separated by Percoll gradient centrifugation., Results: Immunohistochemical staining of normal prostate revealed regional variations in stromal TGF-beta 1 protein, which was most abundant in the stroma surrounding the degenerative proximal ducts. TGF-beta 1 staining was also tightly associated with the prostatic smooth muscle. Results of RT-PCR experiments confirmed the major source of TGF-beta 1 mRNA in normal rat prostate to be the stroma, with lesser expression by the epithelium., Conclusions: Stromal TGF-beta 1 was associated with cell death in the adjacent epithelial cell compartment in the prostatic ductal system, and alpha-smooth muscle actin-positive stromal cells may play a negative growth-regulatory role in the rat ventral prostate through production of TGF-beta 1.

In order to investigate the genomic instability associated with prostate cancer, 36 microsatellite marker loci on chromosomes 1p, 3p, 5q, 8p, 8q, 9p, 11q and 13q were analyzed using microdissected samples from prostate cancer and adjoining microscopically normal tissues from the same slide. DNA was extracted from the normal and tumor cells of 40 microdissected prostate-cancer samples, amplified by PCR, and analyzed for microsatellite instability (MSI) using 36 different polymorphic DNA markers. In the present study, we have utilized a highly refined technique of PCR product separation on a sequencing gel, developed in our laboratory, which clearly shows high-quality results for the microsatellite instability in prostate cancer. The results of this study suggest that 45% (18 out of 40) showed genomic instability at a minimum of 1 locus; 4 cases each showed MSI at one and 2 loci, 4 cases had MSI at 3 loci, 3 cases showed MSI at 5 loci, while one case each showed MSI at 7, 8 and 15 loci. There was no significant correlation between the MSI and stage or grade of the tumors. This extensive study on genomic instability in prostate cancer found the occurrence of MSI to be very high, which suggests a role of MSI in the pathophysiology of prostate cancer.

Background: Prostatic fluid (PF) provides a unique medium for noninvasive evaluation of critical growth and differentiation signals in the prostatic microenvironment. The purpose of this study was to establish the feasibility of measuring two prostatic mitogens, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) in PF, and specifically to quantify extraneous variability attributable to the assay itself, sample handling, or biological variation within an individual over time., Methods: PF was collected by transrectal massage from consecutive patients attending a urology clinic. Pooled PF and individual samples from 25 men with stable benign prostatic hyperplasia (BPH) were analyzed for EGF and TGF-alpha by radioimmunoassay and for total protein., Results: Reproducibility was adequate at dilutions as low as 1:50 (2-microliter pooled sample) and 1:5 (20 microliters) for EGF and TGF-alpha, respectively. Results were not affected by freeze-thaw cycles, time in storage, or protease inhibition in fresh PF. EGF and TGF-alpha were detectable in 100% and 92% of individual men, with respective means of 152 and 0.2 ng/ml. Correlations between two samples obtained from the same man within 12 months were highly significant (EGF r = 0.89, TGF-alpha r = 0.71). Protein concentrations were consistent over time; expression of either peptide per weight of protein rather than per volume did not improve within-man correlation. Between-man variability far exceeded within-man variability for both peptides, and was estimated to account for 84% and 61% of the total variability in EGF and TGF-alpha, respectively. There was no correlation between EGF and TGF-alpha in the same samples., Conclusions: We conclude that men with BPH secrete consistent and distinct levels of EGF-related peptides in PF, and that these levels can be detected with acceptable sensitivity and precision by radioimmunoassay (RIA). Measurement of TGF-alpha, which has not been reported previously, requires a relatively larger sample.

Loss of heterozygosity (LOH) on chromosome 11 is frequently altered in various epithelial cancers. The present study was designed to investigate LOH on chromosome 11 in microdissected samples of normal prostatic epithelium and invasive carcinoma from the same patients. For this purpose, DNA was extracted from the microdissected normal and tumor cells of 38 prostate cancers, amplified by polymerase chain reaction PCR and analyzed for LOH on chromosome 11 using 9 different polymorphic DNA markers (D11S1307, D11S989, D11S1313, D11S898, D11S940, D11S1818, D11S924, D11S1336 and D11S912). LOH on chromosome 11 was identified in 30 of 38 cases (78%) with at least one marker. Four distinct regions of loss detected were: 1) at 11p15, at loci between D11S1307 and D11S989; 2) at 11p12, on locus D11S131 (11p12); 3) at 11q22, on loci D11S898, D11S940 and D11S1818; and 4) at 11q23-24, on loci between D11S1336 and D11S912. We found 25% of the tumors with LOH at 11p15; 39% had LOH at 11p12; 66% had LOH at 11q22; and 47% had LOH at 11q23-24. These deletions at 11p15, 11p12, 11q22 and 11q23-24 loci were not related to the stage or grade of the tumor.

The molecular basis for cardiovirulence in the coxsackievirus B3 (CVB3) genome was examined in a murine model of acute myocarditis. Infectious cDNAs representing a highly cardiovirulent coxsackievirus B3 (CVB3m) and a noncardiovirulent (CVB30) virus were used to construct infectious chimeric cDNAs. Assays of the resulting recombinant viruses for cardiovirulence in adolescent male CD-1 mice showed that the 5' nontranslated region (5' NTR) of the CVB3m genome plays the major role in determining cardiovirulence and that the genomic region encoding the capsid proteins has a minor additive effect in increasing cardiovirulence. Nucleotide sequences in the 5' NTR of CVB3m and CVB30 differ at 23 positions; 14 are located in four stemloop motifs of the secondary structure and may influence the cardiovirulent phenotype by regulating RNA or protein synthesis. A comparison of predicted amino acid sequences of capsid proteins in CVB3m and CVB30 identified two amino acids as potential candidate contributors to cardiovirulence, i.e., amino acids at positions A207 (Asn-Asp) in the puff structure of the E-F loop of VP2 and A566 (Gln-Glu) in the C terminal of VP3 at the external surface. The data from this study and published literature support the conclusion that cardiovirulence of a CVB3 can depend on several regions of the genome.

Background: Recent hypotheses to explain the etiology of abnormal growth associated with prostate disease have invoked perturbations in the rate of apoptosis as an important contributor to the onset and progression of these diseases. For this reason, the apoptosis suppressing oncoprotein bcl-2 has come under scrutiny with regards to its role in prostate diseases. In order to evaluate the role of bcl-2 in human prostate disease and to develop an animal model to test anti-bcl-2 therapies, we generated transgenic mice in which bcl-2 expression is targeted to the mouse prostate gland., Methods: Mouse embryos were microinjected with recombinant DNA constructed by fusing a modified rat C3(1) promotor element to cDNA encoding human bcl-2. Presence of the C3(1)-bcl-2 transgene in progeny was identified by Southern blot and polymerase chain reaction (PCR) analysis. RNase protection assays were used to analyze RNA from 15 organs of these mice. Western blot assays and immunohistochemical staining were used to confirm the tissue-specific protein expression of human bcl-2 and its cellular localization., Results: Three lines of C3(1)-bcl-2 transgenic mice were established. Founder mice carried 2-20 copies of the transgene. Expression of human bcl-2 from the transgene was limited to the prostate gland and testis of males as well as the uterus of females. In the prostate gland, human bcl-2 protein was found only in prostatic epithelial cells. Microscopic analysis of prostate glands from individual males (three lines) showed that these glands were often abnormal, with increased accumulation of cells in the prostatic stroma as well as the epithelium., Conclusions: These transgenic mice appear to provide a novel animal model for studying neoplastic development of the prostate, with particular emphasis on the bcl-2 protein and the role of apoptosis regulation in such development.

This review will present a new concept on the etiology of the development of benign prostatic hyperplasia (BPH). Conventionally, two known etiological factors for the development of BPH have been aging and the presence of functional testes. Assignment of these two factors, although reasonable, has not been conducive to aid the research community to identify and isolate the patho-physiological agents that are directly responsible for the development of this disease. In the present review, we proposed a broadened concept of intrinsic and extrinsic factors for BPH. This concept offers identifiable research opportunities that will facilitate our quest in search for etiological agents for BPH. A brief description of various intrinsic and extrinsic factors and justifications for their selection will be discussed.

Identification of loss of heterozygosity on specific genetic loci is crucial for understanding the pathogenesis of prostate cancer at the molecular level. This is especially important because the deleted regions may contain putative tumor suppressor genes. Chromosome 3p loss appears to be frequently associated with various epithelial cancers. To our knowledge, there is no report on loss of heterozygosity (LOH) of chromosome 3 in human prostate cancer. The present study was designed to investigate the LOH on chromosome 3p in microdissected samples of delineated regions of normal and invasive carcinoma areas of prostatic epithelium from the same tumor sections. For this purpose, DNA was extracted from microdissected normal and tumor cells of 38 prostate cancers, amplified by PCR and analyzed for LOH on chromosome 3p using 6 different polymorphic DNA markers (D3S1560, THRB, D3S647, D3S1298, D3S1228 and D3S1296). Our results suggest that LOH was identified in 34 of 38 cases (89%) with at least one marker. Twelve of 30 informative cases showed LOH at D3S1560; 18 of 22 informative cases showed loss at THRB; 20 of 38 informative cases showed deletion at D3S647; 16 of 38 informative cases showed loss at D3S1298; 12 of 34 informative cases showed LOH at D3S1228; and 6 of 34 informative cases showed LOH at D3S1296 regions. Our results suggest that the LOH is on the 3p24-26 and 3p22-12 regions of the short arm of chromosome 3, indicating 2 discrete areas of deletion on chromosome 3p. The deletion at 3p24-26 and 3p22-12 was not related to the stage or grade of the tumor.

Volume measurements were performed on intact bull and mouse sperm heads and amembranous sperm nuclei, both in the fully hydrated (fluid cell) and dehydrated (air-dried on glass coverslips) states by atomic force microscopy (AFM). Data were obtained by analyzing a small population of cells/nuclei, as well as by performing repeated measurements on single cells imaged following the addition of increasing concentrations of propanol. Results show that the volume of fully hydrated, intact sperm heads and amembranous sperm chromatin particles are at least twice the volume of their air-dried counterparts. Dehydration occurs rapidly in air, and the reduction in volume of chromatin induced by water loss appears to be completely reversible. These studies demonstrate that both mouse and bull sperm chromatin are extensively hydrated in the native state, and are not as compact as previous studies have suggested.

The present study was conducted to isolate and to characterize stromal cells from the human prostate and to study the effects of androgen and different growth factors in this model system. Benign prostatic hyperplasia (BPH) tissue samples were obtained from transurethral resection of the prostate (TURP). Tissue specimens were mechanically and enzymatically dissociated by treatment with DNAse and collagenase. Epithelial cells were separated from stromal cells by discontinuous Percoll gradient centrifugation. The stromal cells obtained were cultured in phenol red-free RPMI-1640 supplemented with 10% fetal bovine serum. Immunocytochemical analysis revealed that the stromal cell cultures were composed of both smooth muscle cells and fibroblasts. The short and broad, smooth muscle cells wee identified by using an antibody directed against alpha-smooth muscle actin. The thin and elongated fibroblasts stained positively for prolyl 4-hydroxylase. Smooth muscle cells were the predominant cell type in the present investigation. Typical cultures contained up to 99% of cells staining positively for alpha-smooth muscle actin. The prostate smooth muscle cultures were treated with dihydrotestosterone (DHT), bovine pituitary extract (BPE), basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta). When cells were cultured in serum free RPMI-1640 supplemented with ITS+ (insulin, transferrin, and selenious acid) no significant (P > 0.05) mitogenic effect in medium supplemented with ITS+. In the presence of 10% charcoal-stripped fetal bovine serum (cFBS) DHT, at a concentration of 0.1 nM, was able to cause a slight but significant (P < 0.05) mitogenic effect on BPH smooth muscle cells growth. Basic FGF was able to stimulate BPH smooth muscle cells in a concentration-dependent fashion. The combination of DHT and 0.1 ng/ml bFGF was able to increase the proliferation of prostate smooth muscle cells above either agents alone. Addition of BPE to serum free RPMI-1640 caused a significant (P < 0.05) stimulation of cell proliferation in a concentration-dependent fashion. Addition to TGF-beta to serum or BPE containing RPMI-1640 caused a significant (P < 0.05) inhibition to cell proliferation in a concentration-dependent fashion. TGF-beta was cytostatic to the benign prostatic smooth muscle cells only in the presence of media containing growth stimulating factors found in charcoal-stripped serum or in bovine pituitary extract. These results demonstrated that stromal fraction isolated from BPH specimens was composed of both fibroblasts and smooth muscle cells. These cells could be cultured and were able to respond to various growth stimulatory and inhibitory agents.

The rat prostate is composed of a complex system of branching ducts which terminate proximally at the urethra. It has been recognized that epithelial cells lining the ducts respond differently to androgen in various regions of the ducts, with responses ranging from proliferation to apoptosis, but the cellular mechanisms underlying these effects are unclear. Interaction between prostatic stroma and epithelium is essential to normal prostate growth and development, and the prostatic stroma is thought to be the first site of androgen action. Therefore we have examined the organization and distribution of stromal cell types along the rat prostatic ductal system. Using immunohistochemical techniques, we observed abundant fibrous tissue surrounding the distal region of the ducts, with a sparse, discontinuous smooth muscle layer. The intermediate region was surrounded by a continuous layers of smooth muscle one to two cells thick, which increased to greater than four layers thick at the proximal region. Fibrous tissue was located in interductal spaces and occasionally interspersed within the muscle layers in both regions. These observations indicate that regional variations in the distribution of stromal cell types exist and suggest that their corresponding secretory products could be responsible for the various effects of androgen on the epithelium in the rat prostatic ductal system.

The effect of alpha-interferon on the hepatitis C genotypes was examined in 25 anti-HCV-positive haemophilic patients. The rate of multiple HCV genotypes in patients who are likely to have mixed infections was also studied. Pretreatment results showed that 3/25 (12%) patients had a change in genotypes, whereas posttreatment this rose to 10/25 (40%). Seven of 10 (70%) patients who showed a change in genotype had a clinical response to alpha-interferon. Six of 25 (24%) patients showed a complete clinical response to alpha-interferon, and the majority of these were either type 2 or 3. This study supports previous evidence that type 1 is less likely to respond to alpha-interferon, and that alpha-interferon may alter the concentrations of the various circulating genotypes present in multiply-infected patients. There are many difficulties in studying hepatitis C in haemophilic patients due to the sequence heterogeneity within each individual, and this study has shown that no ideal method exists as yet for looking at HCV genotypes in multiply-infected individuals.

In this preliminary study, we report that an enzyme-linked immunofluorescence assay (EFLA) was developed for the determination of PR92 antigen in prostatic fluid, utilizing anti-PR92 monoclonal antibody. Fluid samples from 64 patients were assayed. PR92 antigen was expressed as unit per microgram (U/microgram) of prostatic fluid proteins. One hundred percent of men (7 out of 7) less than 50 years of age demonstrated concentrations less than 25 U/micrograms; 91% of men (10 out of 11) with documented carcinoma, and only 9.5% of men (2 out of 21) with benign prostatic hyperplasia, demonstrated concentrations above 230 U/micrograms. The mean concentration of PR92 antigen in prostatic fluid of a group of patients suspected of having prostate cancer (high-risk group; 227 +/- 42 U/micrograms) was significantly greater than that of those with benign prostatic hyperplasia (87 +/- 23 U/micrograms; P = 0.05). Further evaluation of this potential marker and of other antigens within the prostatic fluid is warranted.

This study was undertaken to establish an androgen-sensitive model of human prostatic carcinoma in nude mice. The androgen-sensitive prostatic carcinoma cell line, LNCaP, was suspended in reconstituted basement membrane (Matrigel) and injected subcutaneously into nude mice. The LNCaP cell line was chosen for the study, because the cell line is androgen-sensitive and secretes prostate specific antigen (PSA) into culture media. Following injection of 1 x 10(6) LNCaP cells with 0.25 ml of Matrigel, 88% of mice exhibited palpable tumor burdens after 12 weeks of observation. In addition, significant levels of PSA were observed in the serum of LNCaP-bearing mice. Bilateral orchiectomy of mice resulted in tumor regression and stabilization of serum PSA levels, compared to testis-intact controls. A significant correlation of PSA to tumor volume and weight was observed. The castrate level of testosterone was confirmed by radioimmunoassay and was similar to testosterone levels in female nude mice. Matrigel allows for a conducive environment to propagate LNCaP cells in nude mice. Furthermore, the growth can be manipulated by castration, leading to involution of tumor and stabilization of serum PSA level. This in vivo model of hormone-sensitive human prostate cancer cell line will serve as a model for the study of prostate tumor biology and treatment.

Magnetic resonance (MR) angiography, although still in its infancy, is recognized as a valuable diagnostic tool. Investigation of the utility of contrast media as applied to MR angiography is, to no surprise, preliminary. In restricted instances, with present techniques, contrast media-enhanced MR angiography can provide additional valuable diagnostic information. Inspection of two-dimensional images (as opposed to three-dimensional projections) and comparison of MR images before and after administration of contrast agent are particularly important. Improved visualization of intracranial aneurysms, arteriovenous malformations, venous anomalies, and arterial occlusions has been demonstrated on three-dimensional time-of-flight MR angiograms after intravenous administration of a gadolinium chelate, relative to studies performed before administration of the contrast agent.

High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.

Prolactin and testosterone are synergistic in stimulating growth of the rat prostate. The lateral lobe is more sensitive to this synergism than the ventral and dorsal lobes. To investigate whether prolactin acts directly in the rat prostate or indirectly through another systemic mediator, anterior pituitary grafts (1 mm3) were implanted in the lateral prostate of castrated Sprague-Dawley rats in whom a 0.5 cm or 1.0 cm testosterone-filled silastic tubing was implanted subcutaneously at the same time. Rats were randomly assigned to receive either the pituitary or a muscle chip of similar size grafted beneath the fascia lateral to the lateral prostate. Twenty-one days later, serum prolactin levels were not elevated in pituitary-grafted animals and were not significantly different from those in muscle-grafted rats. The mean lateral prostate weight on the grafted side in pituitary-implanted rats with 1.0 cm testosterone tubing was 43% heavier than either that of the contralateral side or the corresponding weights in muscle-implanted rats. In pituitary-implanted rats with 0.5 cm testosterone tubing, the mean lateral prostate weight on the grafted side was 60% heavier than either that of the contralateral side or that of the corresponding weights in muscle-implanted rats. The weight of the ventral and dorsal lobes of the prostate was not significantly affected by the presence of pituitary grafts in one of the lateral lobes. The local effect of prolactin on the lateral prostate was further demonstrated by an overall decline in tissue concentrations of dihydrotestosterone in the grafted side. These results provided evidence to indicate that there was a direct effect of prolactin on growth of the lateral prostate in rats.

Parkinson's disease evolves slowly, and there is current interest in exploring the earliest stages of the disorder, because of new approaches to studying pathogenesis and developing potential neuroprotective treatment. Recognizing early Parkinson's disease is not easy. The certainty of diagnosis increases as the disease advances. To address the problem of identifying Parkinson's disease in its initial phases of clinical expression, we propose the following designated levels of confidence for the diagnosis: (1) clinically possible, (2) clinically probable, and (3) clinically definite. Laboratory support for the diagnosis may be applied to each category. Criteria are provided as a framework underpinning this classification.

Previous studies from our laboratory have shown that reconstituted basement membrane and stromal secretory products are important regulators of benign prostatic epithelial cell growth and differentiation. In the present study we evaluated the impact of extracellular matrix (ECM) and soluble stromal secretory products on the proliferation and secretory activity of the androgen-responsive prostatic carcinoma cell line LNCaP. In these studies, dihydrotestosterone (DHT) was a potent mitogen for LNCaP cells cultured on plastic or on type I collagen. The growth response to DHT was greatly attenuated when LNCaP cells were grown on prostatic stromal ECM. Cells grown on stromal ECM also exhibited clustered morphology compared to the monolayer growth observed on plastic and secreted elevated levels of prostate specific antigen (PSA) and prostatic acid phosphatase (PAP). These findings indicate that cultivation of LNCaP on stromal ECM will promote the expression of differentiated functions. In additional studies, stromal cell conditioned medium (SCM) significantly increased PSA/PAP secretion by LNCaP cells in the presence of 10 nM DHT. The enhancement of DHT-induced PSA/PAP secretion by SCM was most pronounced when LNCaP cells were grown on stromal ECM. SCM did not significantly alter LNCaP proliferation. These studies indicate that prostatic stromal ECM and soluble secretory products will promote differentiated function in cultured LNCaP cells. In addition, we show that DHT can act as either a growth or differentiation-promoting stimulus depending on the presence of stromal factors.

The present study was conducted to study the interaction between epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in benign human prostatic epithelial cells in culture. Primary cultures of human prostatic epithelial cells were grown in complete WAJC, which consisted of WAJC-404 medium and, in addition to other defined additives, EGF and bovine pituitary extract (BPE). Incomplete WAJC contained the same composition except EGF and BPE were deleted. TGF-beta was added into media at concentrations of 0, 0.1, and 1.0 ng/ml. When cells were grown in complete WAJC, they proliferated rapidly. Cell proliferation was greatly suppressed when incomplete WAJC was used. Addition of TGF-beta to these cultures caused a significant reduction in the final cell number when either complete WAJC or incomplete WAJC was used. In additional experiments, cells were prelabeled with 3H-thymidine for 72 hr prior to treatment with TGF-beta. The percentage of radioactivity released into the medium at the end of a 6-day culture was used as an indication of the extent of cell death. Trypan blue exclusion test was also used to assess the extent of cell death. Addition of TGF-beta into complete WAJC did not significantly affect the extent of cell death beyond what was considered as the result of normal cellular turnover. Addition of TGF-beta into incomplete WAJC, however, caused a significant increase in the percent of cell death in the culture. These results demonstrated an interaction between EGF and TGF-beta in proliferation and cell death in human prostatic epithelia in culture. In the presence of EGF alone in the culture medium, prostatic epithelial cells were stimulated to proliferate. The rate of proliferation was greatly diminished when EGF was deleted from the medium or when TGF-beta was added in the presence of EGF. Finally, cell death was induced when TGF-beta was added into the medium in the absence of EGF.

Twelve patients with a high suspicion of brain metastases by previous clinical or radiologic examinations were studied in a phase III investigation with magnetic resonance (MR) imaging at 1.5 T after a bolus intravenous injection of 0.1 mmol/kg gadoteridol followed at 30 minutes by a second bolus injection of 0.2 mmol/kg gadoteridol. All lesions were best demonstrated (showed greatest enhancement) at the 0.3-mmol/kg (cumulative) dose, with image analysis confirming signal intensity enhancement in the majority of cases after the second gadoteridol injection. More lesions were detected with the 0.3-mmol/kg dose than with the 0.1-mmol/kg dose, and more lesions were detected with the 0.1-mmol/kg dose than on precontrast images. In this limited clinical trial, high-dose gadoteridol injection (0.3-mmol/kg cumulative dose) provided improved lesion detection on MR images specifically in intracranial metastatic disease.

Protamines 1 and 2 have been isolated from the sperm of frozen and isopropanol preserved Syrian hamster (Mesocricetus auratus) epididymides and analyzed by gel electrophoresis, high-performance liquid chromatography (HPLC), and amino acid analysis and sequencing. The results show that alcohol preservation does not alter the primary structure of the two sperm nuclear proteins and that the preservation of mammalian reproductive organs in alcohol is a viable alternative to freezing tissues collected in the field. Sperm were isolated from tissues fixed in isopropanol for as long as 7 months without detectable adverse effects on either the isolation of sperm or the primary structure of the protamines.

To study the neuronal and hormonal control of prostatic secretion, the prostatic urethra was cannulated in urethane anesthetized rats. The volume of prostatic secretion was measured following infusion of cholinergic and adrenergic agonists intact animals. Prostatic secretion was elicited by norepinephrine, phenylephrine and carbachol; but not by clonidine, isoproterenol, pilocarpine, or acetylcholine. Phenylephrine and norepinephrine infusions caused a high initial rate of secretion, which then declined rapidly. Carbachol infusion, in contrast, produced a low but constant rate of secretion that was maintained for up to 1 hour. Histological examination of the prostate revealed contraction of smooth muscle surrounding prostatic ducts after infusion of phenylephrine and norepinephrine, but not carbachol. Prostatic secretion was also measured in castrated rats supplemented with various doses of testosterone. Testosterone exerted a dose dependent control of prostatic weight and secretory volume. These results indicate 1) alpha 1 receptor agonists can cause contraction of smooth muscle to expel fluid from the rat prostate, 2) carbachol induces prostatic secretion through a mechanism other than contraction of gland, and 3) testosterone has a primary role in controlling prostatic size.

As part of a phase III clinical trial, 25 patients with suspected intracranial or spinal disease underwent magnetic resonance (MR) imaging before and after intravenous injection of 0.1 mmol/kg Gd-HP-DO3A (1,4,7-tris[carboxymethyl]-10-[2' hydroxypropyl]-1,4,7,10- tetraazacyclododecane), a neutral (nonionic) gadolinium chelate. Laboratory analysis included a complete blood count; blood chemistry; measurement of electrolyte levels; hepatic function, clotting function, and iron metabolism panels; and urinalysis both before and 24 hours after contrast agent administration. No statistically significant changes related to contrast agent administration were noted in laboratory values after administration of the contrast agent. In this selected group of patients, the contrast agent-enhanced study provided greater diagnostic information than did the precontrast study in 69% of head cases and 67% of spine cases. These initial clinical trials demonstrate that Gd-HP-DO3A is a safe, efficacious agent for head and spine examinations.