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Litomosoides chagasfilhoiis a filariid nematode parasite of the abdominal cavity of the wild rodent Akodon cursor(Winge, 1887), that has been described and used in Brazil as a new model for human filariasis. The fine structure of the intestine of this nematode was analyzed based on observations made by light and transmission electron microscopies of serial sections along the body. Cytochemical analysis was carried out to investigate the composition of the intestinal wall. This structure consisted of a basal lamina and an epithelium of variable thickness, composed of cells that have an irregular shape. The cytoplasm of intestinal cells contains few organelles: vacuoles, lysosomal bodies, spheroid bodies, endoplasmic reticulum, and many large lipid droplets. In the anterior portion of the intestine, the lysosomal bodies, spheroid bodies, and vacuoles presented positive reaction for acid phosphatase, and carbohydrates were detected in lysosomal bodies. The midbody and posterior regions presented less organelles and lipid droplets, and nuclei were more abundant. Residues of l-fucose were detected by Ulex europaeuslectin binding in the midbody sections. Basic proteins were associated to lipid droplets, in the posterior region. In the whole extension of the intestine, carbohydrates were detected on tight junctions. These results indicate that the metabolized material in the epithelium can contribute to the microfilariae development and also probably can be involved with the excretory/secretory mechanism of these nematodes.Litomosoides chagasfilhoiis a filariid nematode parasite of the abdominal cavity of the wild rodent Akodon cursor(Winge, 1887), that has been described and used in Brazil as a new model for human filariasis. The fine structure of the intestine of this nematode was analyzed based on observations made by light and transmission electron microscopies of serial sections along the body. Cytochemical analysis was carried out to investigate the composition of the intestinal wall. This structure consisted of a basal lamina and an epithelium of variable thickness, composed of cells that have an irregular shape. The cytoplasm of intestinal cells contains few organelles: vacuoles, lysosomal bodies, spheroid bodies, endoplasmic reticulum, and many large lipid droplets. In the anterior portion of the intestine, the lysosomal bodies, spheroid bodies, and vacuoles presented positive reaction for acid phosphatase, and carbohydrates were detected in lysosomal bodies. The midbody and posterior regions presented less organelles and lipid droplets, and nuclei were more abundant. Residues of l-fucose were detected by Ulex europaeuslectin binding in the midbody sections. Basic proteins were associated to lipid droplets, in the posterior region. In the whole extension of the intestine, carbohydrates were detected on tight junctions. These results indicate that the metabolized material in the epithelium can contribute to the microfilariae development and also probably can be involved with the excretory/secretory mechanism of these nematodes.

A series of novel thienopyrimidine-based receptor tyrosine kinase inhibitors has been discovered. Investigation of structure−activity relationships at the 5- and 6-positions of the thienopyrimidine nucleus led to a series of N,N‘-diaryl ureas that potently inhibit all of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinases. A kinase insert domain-containing receptor (KDR) homology model suggests that these compounds bind to the “inactive conformation” of the enzyme with the urea portion extending into the back hydrophobic pocket adjacent to the adenosine 5‘-triphosphate (ATP) binding site. A number of compounds have been identified as displaying excellent in vivo potency. In particular, compounds 28 and 76 possess favorable pharmacokinetic (PK) profiles and demonstrate potent antitumor efficacy against the HT1080 human fibrosarcoma xenograft tumor growth model (tumor growth inhibition (TGI) = 75% at 25 mg/kg·day, per os (po)).

Castration on days 0, 5, 10, 20, 40, and 60 caused increases in an apoptotic index (% of apoptotic cells) in seminal vesicle (SV) epithelium, peaking 1-3 days after castration. The peak apoptotic indices after castration on days 0, 5, 10, and 20 were significantly lower than peak apoptotic indices observed after castration on days 40 and 60. DNA extracted from mouse SVs 2 days after castration on days 0, 5, 10, and 60 showed a ladder pattern on agarose gel electrophoresis. The secretion of androgen by testes was confirmed by the growth retardation of the SVs after castration on days 0, 5, 10, and 20. It would appear that a proportion of SV epithelial cells dependent on testicular androgens for survival is smaller before day 20 than after day 20.

Purpose: We have previously shown that mesenchymal-epithelial interactions are necessary for the development of bladder smooth muscle. Specifically without fetal or adult urothelium embryonic rat bladder mesenchyma does not differentiate into smooth muscle. The mechanism responsible for this interaction is not known, although it is postulated that diffusable growth factors have a role. Our hypothesis is that diffusable factors within adult rat bladders influence smooth muscle differentiation. Materials and Methods: Chimeric bladders were created by surgically implanting 14-day embryonic rat bladder mesenchyma before smooth muscle differentiation into the detrusor space of adult syngeneic hosts to test whether the host urothelium would induce smooth muscle differentiation without being in direct contact with fetal bladder mesenchymal tissue. Sub-detrusor pockets were created between the serosa and smooth muscle layer, between the smooth muscle layer and lamina propria, and between the lamina propria and urothelium in direct contact with urothelium. Controls consisted of intact 14-day embryonic rat bladders with the urothelium not removed, and 14-day embryonic bladder mesenchyma recombined with urothelium (direct contact) placed within the sub-detrusor space of the bladder and under the renal capsule. Results: Immunohistochemical staining with antibodies directed against smooth muscle @a-actin and urothelium (cytokeratin 7) revealed smooth muscle differentiation in intact embryonic bladders and bladder mesenchyma plus urothelium recombinants in contrast to bladder mesenchyma alone, which had no @a-actin staining (morphometric smooth muscle analysis p = 0). There was no @a-actin staining in chimeric bladders even when bladder mesenchymal grafts were placed directly in contact with host urothelium. In addition, bladder mesenchyma plus urothelial recombinants within the host bladder had less @a-actin staining than their counterparts placed under the renal capsule (p = 0.001). Conclusions: A diffusable factor most likely exists within adult rat bladders that inhibits smooth muscle differentiation.

The objective of this study was to determine whether neonatal rat seminal vesicle mesenchyme (rSVM) can reprogram epithelial differentiation in a fully differentiated adult human bladder epithelium. For this purpose neonatal rSVM was isolated from newborn (0-day) Sprague-Dawley rats, and normal adult human bladder epithelium (hBLE) was isolated from radical cystoprostatectomy specimens to prepare rSVM+hBLE tissue recombinants in vitro. After overnight culture the tissue recombinants were grafted beneath the renal capsule of male athymic rodent hosts and allowed to grow in vivo for 6 months. As controls, rSVM and hBLE were grafted separately and allowed to grow for the same period. Tissue recombinants and control tissue grafts were harvested, and secretions were collected for biochemical studies. Tissues were fixed both for histologic as well as immunohistochemical staining. Neonatal rSVM induced normal adult human bladder urothelium to form glandular structures resembling prostate. The induced prostatic acini were filled with secretions that expressed human prostate-specific secretory proteins. These findings demonstrate that adult human urothelial cells retain a responsiveness to neonatal prostatic mesenchymal inductors. Change in urothelial histodifferentiation was associated with change in functional activity. The ability of the neonatal rat mesenchymal tissues to induce morphologic as well as biochemical changes in normal adult human urothelium provides a basis for human tissue engineering and organ reconstruction.

We have examined the role of Fibroblast Growth Factor 10 (FGF10) during the growth and development of the rat ventral prostate (VP) and seminal vesicle (SV). FGF10 transcripts were abundant at the earliest stages of organ formation and during neonatal organ growth, but were low or absent in growth-quiescent adult organs. In both the VP and SV, FGF10 transcripts were expressed only in a subset of mesenchymal cells and in a pattern consistent with a role as a paracrine epithelial regulator. In the neonatal VP, FGF10 mRNA was expressed initially in mesenchymal cells peripheral to the peri-urethral mesenchyme and distal to the elongating prostatic epithelial buds. At later stages, mesenchymal cells surrounding the epithelial buds also expressed FGF10 transcripts. During induction of the SV, FGF10 mRNA was present in mesenchyme surrounding the lower Wolffian ducts and, at later stages, FGF10 transcripts became restricted to mesenchymal cells subadjacent to the serosa. We investigated whether the FGF10 gene might be regulated by androgens by analysing the levels of FGF10 transcripts in SV and VP organs grown in serum-free organ culture. While FGF10 transcript levels increased after treatment with testosterone in the SV (but not VP), these changes were not sensitive to anti-androgen treatment, and thus it is likely that FGF10 mRNA was not directly regulated by testosterone. Also, FGF10 mRNA was observed in the embryonic female reproductive tract in a position analogous to that of the ventral prostate in males suggesting that FGF10 is not regulated by androgens in vivo. Recombinant FGF10 protein specifically stimulated growth of Dunning epithelial and BPH1 prostatic epithelial cell lines, but had no effect on growth of Dunning stromal cells or primary SV mesenchyme. Furthermore, FGF10 protein stimulated the development of ventral prostate and seminal vesicle organ rudiments in serum-free organ culture. When both FGF10 and testosterone were added to organs in vitro, there was no synergistic induction of development. Additionally, development induced by FGF10 was not inhibited by the addition of the anti-androgen Cyproterone Acetate demonstrating that the effects of FGF10 were not mediated by the androgen receptor. Taken together, our experiments suggest that FGF10 functions as a mesenchymal paracrine regulator of epithelial growth in the prostate and seminal vesicle and that the FGF10 gene is not regulated by androgens

Stromal-epithelial interactions are critical in determining patterns of growth, development and ductal morphogenesis in the mammary gland, and their perturbations are significant components of tumorigenesis. Growth factors such as epidermal growth factor (EGF) contribute to these reciprocal stromal-epithelial interactions. To determine the role of signaling through the EGF receptor (EGFR) in mammary ductal growth and branching, we used mice with a targeted null mutation in the Egfr. Because Egfr-/- mice die perinatally, transplantation methods were used to study these processes. When we transplanted neonatal mammary glands under the renal capsule of immuno-compromised female mice, we found that EGFR is essential for mammary ductal growth and branching morphogenesis, but not for mammary lobulo-alveolar development. Ductal growth and development was normal in transplants of mammary epithelium from Egfr-/- mice into wild-type (WT) gland-free fat pads and in tissue recombinants prepared with WT stroma, irrespective of the source of epithelium (StromaWT/Epi-/-, StromaWT/EpiWT). However, ductal growth and branching was impaired in tissue recombinants prepared with Egfr-/- stroma (Stroma-/-/EpiWT, Stroma-/-/Epi-/-). Thus, for ductal morphogenesis, signaling through the EGFR is required only in the stromal component, the mammary fat pad. These data indicate that the EGFR pathway plays a key role in the stromal-epithelial interactions required for mammary ductal growth and branching morphogenesis. In contrast, signaling through the EGFR is not essential for lobulo-alveolar development. Stimulation of lobulo-alveolar development in the mammary gland grafts by inclusion of a pituitary isograft under the renal capsule as a source of prolactin resulted in normal alveolar development in both Egfr-/- and wild-type transplants. Through the use of tissue recombinants and transplantation, we have gained new insights into the nature of stromal-epithelial interactions in the mammary gland, and how they regulate ductal growth and branching morphogenesis.

A procedure for the rapid preparation of autoradiograms from tissues incubated in vitro with 3H-estradiol is described. Slices of tissue were incubated in culture medium containing 17 nM 3H-estradiol, washed to remove unbound steroid, then processed for thaw-mount autoradiography. Exposure times were generally 3 to 4 weeks. Simultaneous in vitro competition with unlabeled progesterone, dihydrotestosterone, or hydrocortisone had no effect on the distribution or intensity of exposed silver grains, while competition with unlabeled estradiol or moxestrol abolished nuclear localization of silver grains. The exchange of labeled estradiol for bound endogenous estradiol during in vitro incubation of the tissues was demonstrated by a comparison of the pattern of incorporation of 3H-estradiol in tissues previously treated in vivo with unlabeled estradiol versus those not primed. A similar distribution and intensity of silver grains was observed in both the treated and untreated tissue groups. The rationale for the advantages of in vitro steroid autoradiography versus the in vivo technique is discussed.

Ventral and dorsolateral prostatic lobes (VP and DLP), obtained from mice at different ages and at different intervals after castration or treatment of castrated males with testosterone propionate (TP), were microdissected into two-dimensional arrays and incubated in vitro with 14C-thymidine. Labeled whole-mount specimens were fixed and dried onto glass slides, dipped into photographic emulsion, and processed autoradiographically. The morphological pattern of DNA synthetic activity was similar in the VP and DLP. During early postnatal periods (10–15 days after birth), DNA synthetic activity was highest at the distal ductal tips (near the capsule) and considerably lower in proximal ducts (near the urethra). At 30 days of age, DNA synthesis was almost totally confined to the distal ducts, with exceedingly low labeling in the proximal ductal areas. In the prostate of the intact or castrated adult, DNA synthesis was nearly absent throughout the gland, but silver grains were still observed on the ductal tips. During androgen-induced prostatic regeneration, DNA synthesis was detectable only in distal ducts 24 h after TP was administered. Labeling intensity reached a maximum on the third day of TP treatment in both distal and proximal ductal areas, thereafter, it subsided to focal labeling confined mostly to distal ducts. These results demonstrate that levels of DNA synthetic activity vary considerably within the prostate on a regional basis. Explanation of this heterogeneity in DNA synthetic activity within the prostate gland is fundamental to understanding the mechanism of androgenic regulation of prostatic growth and development.

Degenerative and regenerative changes in the ductal architecture of the ventral and dorsolateral prostates (VP and DLP) of the adult mouse were investigated in microdissected specimens over a time-course of 14 days following castration and subsequently during 14 days of administration of testosterone propionate. After castration, about 35% of the ductal tips and branch-points were lost in distal regions (usually near the capsule) in both prostatic lobes. By contrast, in more proximal regions of the prostate (closer to the urethra), the ducts survived in an atrophic condition. The ductal morphology that had been lost in the distal regions completely regenerated after testosterone propionate was administered to the castrated males. In the VP, androgen replacement simply returned the gland to its former size with moderate ductal distension; in the DLP, excessive epithelial infoldings and ductal distension were elicited in the distal regions of the ducts after 14 days of treatment with testosterone propionate. These results suggest that androgenic responsiveness and dependency are different in distal versus proximal ducts. Distal ducts are exquisitely androgen-dependent and androgen-sensitive; in proximal regions, androgen-dependency is not as strict.

Postnatal growth and development of the glandular architecture of the ventral and dorsolateral lobes of the mouse prostate (VP and DLP) were investigated by microdissection techniques that permitted precise quantification of the numbers of primary ducts emerging from the urethra, the terminal ductal tips, and ductal branch-points. At birth both the right and left lobe of the VP consisted of 1–3 main ducts that had already undergone secondary and tertiary branching. In contrast, at birth the more complex DLP had 9–12 unbranched main ducts per lobe on both the right and left sides. During the first 15 days after birth, 80.7% of tips and 76.4% of branch-points of the adult gland formed in the VP, and 70.4% of tips and 53.6% of the branch-points formed in the DLP. Ductal branching was completed by 60 to 90 days. The DLP developed in three stages: first, formation of unbranched main ducts (first 10 days); second, distal branching of each main duct resulting in 3–5 terminal branches per main duct (10–15 days after birth); third, elaboration of intraductal mucosal infolding in distal ducts after 30 days of age. Ducts of the lateral prostate (LP), a ventrolateral subdivision of the DLP, initiated branching morphogenesis between 1 to 5 days after birth. The LP grew into and became embedded within the capsule of the VP, which may explain why the ductal architecture of these two lobes are similar. These heretofore unrecognized differences in the organogenesis and morphology of the mouse VP and DLP and the striking morphological heterogeneity both between and within the lobes of the mouse prostate may be morphological manifestations of functional heterogeneities within the prostate.

We have investigated the capacity of vaginal and uterine epithelia (VE and UE) to reexpress normal morphology and hormone responsiveness following cell culture. VE and UE from adult ovariectomized mice were grown in a collagen gel matrix with serum-free medium for 7-10 days. Proliferation of these cells occurs in the absence of 17 beta-estradiol and is not stimulated by 17 beta-estradiol; the VE usually does not keratinize or stratify in vitro. Cultured VE and UE were recombined with homologous vaginal or uterine stroma (VS and US, respectively) and these recombinants were grown under the renal capsule of female hosts for 4 weeks. The epithelium of the VS + VE recombinants cycled, proliferated and stratified in response to estrogen and mucified normally in response to progesterone. The UE that was grown in vivo with cultured US also showed normal morphology and estrogen responsiveness. These changes in the UE and VE were not simply a result of return to the in vivo environment, as epithelia alone in collagen gels transplanted under the renal capsule did not survive. These results indicate that both VE and UE, which are not estrogen-dependent in vitro, reexpress their estrogen dependency and normal morphology when recombined with homologous stroma and grown in vivo. Thus, the changes these cells show when grown in culture are the result of altered conditions in vitro rather than an irreversible alteration in the cells themselves or the selection of specific subpopulations that are not mitogenically or morphologically responsive to estrogen under any condition.

Mesenchymal-epithelial interactions are essential for the development of the male reproductive tract. Tissue recombination experiments have been used to define the characteristics of these interactions. When mesenchyme, embryonic connective tissue, is recombined with epithelium from another organ an instructive induction may occur in which the developmental fate of the epithelium is altered. Instructive inductions are most common when the epithelium that is removed from the mesenchyme and the epithelium that is recombined with the mesenchyme are from the same germ layer. All of the mesenchyme of the male reproductive tract is of mesodermal origin. The epithelia of these organs are derived from either the mesodermal Wolffian duct epithelium or the endodermal urogenital sinus epithelium. Urogenital sinus mesenchyme can instructively induce bladder and urethral epithelium to form prostate (Donjacour, A. A. and Cunha, G. R. (1993) Endocrinol. 132, 2342-2350) and seminal vesicle mesenchyme can instructively induce epithelium from the ductus deferens and ureter (Cunha, G. R., Young, P., Higgins, S. J. and Cooke, P. S. (1991) Development 111, 145-158) to form seminal vesicle. To see whether inductive interactions could occur across germ layers in this system, seminal vesicle mesenchyme, normally associated with a mesodermal epithelium, was recombined with epithelium from neonatal or adult bladder or urethra, which are of endodermal origin. The resulting tissue recombinants were analyzed histologically and by immunocytochemistry and western blotting with antibodies to prostatic and seminal vesicle secretory proteins. Full prostatic differentiation was observed in tissue recombinants made with seminal vesicle mesenchyme plus either adult or neonatal bladder or urethral epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of the mammalian male accessory sexual organs requires both androgens and mesenchymal/epithelial interactions. Paracrine acting factors whose expression is mesenchymal and androgen dependent have been proposed to regulate development of these organs, although the identity of these paracrine mediators is unknown. Keratinocyte growth factor (Kgf) has been shown to play an important role in the development of the mouse seminal vesicle and rat ventral prostate. Also, Kgf is expressed in mesenchymal cells and has been shown to be regulated by androgens in prostatic cells grown in vitro. Thus Kgf has been proposed as a mediator of androgen action. We have investigated the expression of Kgf mRNA during development of the rat seminal vesicle and prostate, both in vitro and in vivo. Additionally we have examined mRNAs for Kgf receptor (KgfR), transforming growth factor alpha (Tgf alpha), epidermal growth factor receptor (EgfR) and cytokeratin 19 (CK19). The levels of growth factor and receptor mRNAs fluctuated during androgen-regulated development; however, these changes reflected variations in the mesenchymal/epithelial ratio rather than regulation by testosterone. Expression of Kgf is mesenchymal, while KgfR is epithelial and Tgf alpha is predominantly epithelial. The changes in the levels of mRNAs for these factors correlated well with changes in the level of an epithelial marker, CK19, suggesting they were due to alterations in the relative abundance of tissue compartments in which they were expressed. Kgf has been shown to mimic androgen action in explant cultures of seminal vesicle and prostate. We demonstrate here that anti-androgens are able to block Kgf stimulated development, suggesting that Kgf and androgen receptor signalling pathways may interact. Taken together our data suggest that, in vivo, Kgf may interact with androgen receptor signalling but it is not a direct target of androgen action.

Tissue recombinants of embryonic urogenital sinus mesenchyme (UGM) and epithelium of the urinary bladder (urothelium, BLE) of adult rats and mice were grown for 3-30 d in male syngeneic hosts. Short-term in vivo growth indicated that prostatic morphogenesis is initiated as focal outgrowths from the basal aspect of the adult urothelium. The solid epithelial buds elongate, branch, and subsequently canalize, forming prostatic acini. After 30 d of growth in the male hosts, prostatic acini exhibit secretory activity. The marked changes in urothelial morphology induced by the UGM are accompanied by the expression of fine-structural features indicative of secretory function (rough endoplasmic reticulum, Golgi apparatus, and secretory granules). During this process, urothelial cells express prostatic histochemical markers (alkaline phosphatase, nonspecific esterase, glycosaminoglycans) and prostate-specific antigens. The expression within BLE of prostatic characteristics is associated with the loss of urothelial characteristics. These data indicate that adult urothelial cells retain a responsiveness to embryonic mesenchymal inductors. Furthermore, mesenchyme-induced changes in urothelial cytodifferentiation appear to be coupled to changes in functional activity.

Adult bladder epithelium (BLE) is induced to differentiate into glandular epithelium after association with urogenital sinus mesenchyme (UGM) and subsequent in vivo growth in syngeneic male hosts. Alteration of epithelial cytodifferentiation is associated with the expression of prostate-specific antigens, histochemical and steroid metabolic activities. These observations suggest that the inductive influence of the UGM has reprogrammed both the morphological and functional characteristics of the urothelium. In this report, differences regarding the mechanisms and effects of androgenic stimulation of prostate and bladder are exploited to determine the extent to which UGM plus BLE recombinants express a prostatelike, androgen-dependent phenotype. Results from cytosolic and autoradiographic binding studies suggest that androgen binding is induced in UGM plus BLE recombinants and that this activity is accounted for by the induced urothelial cells. In UGM plus BLE recombinants, androgen-induced [3H]thymidine or [35S]-methionine uptake analyzed by two-dimensional gel electrophoresis was qualitatively and quantitatively similar to that of prostate as opposed to bladder. These studies indicate that expression within BLE of prostatic phenotype is associated with a loss of urothelial characteristics and that androgen sensitivity is presumably a function of the inductive activities of the stroma.

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.