Benign Prostatic Hypertrophy (BPH, also known as benign prostatic hyperplasia or benign prostatic enlargement), is one of the most common benign proliferative conditions associated with aging in men and is pathologically characterized by the proliferation of fibroblast/myofibroblast and epithelial cell types in the prostate. Previous studies from our laboratory have shown that the CXC-type chemokines, CXCL5 and CXCL12, are secreted by aging prostate stroma and promotes both proliferative and transcriptional responses from prostate epithelial cells. Using array-based gene expression profiling and quantitative reverse-transcriptase polymerase chain reaction, we now show that the transcriptome of the aging prostate stroma is characterized by the up-regulation of several genes that encode secreted inflammatory mediators, including secreted CXC-type chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL12), interleukins (IL11, IL33), and transcripts with cytokine homology (CYTL1). At the protein level, ELISA experiments demonstrated that CXCL1, CXCL5, and CXCL6 were secreted by primary prostate stromal fibroblasts explanted from aging prostate stroma. Dose-response assays confirmed that, like CXCL5 and CXCL12, CXCL1 and CXCL6 promote low-level proliferative responses from both prostate stromal fibroblasts and epithelial cells. Taken together, these data suggest that inflammatory mediators are secreted by prostatic stroma consequent to aging, that the levels of these mediators are sufficient to promote lowlevel increases in the proliferative rate of both epithelial and stromal fibroblast cell types. Moreover, these processes may account for the low-level, but cumulative, proliferation of both epithelial and fibroblastic/myofibroblastic cell types that characterizes the aging-associated development of benign prostatic hypertophy.

CXCL5 is a proangiogenic CXC-type chemokine that is an inflammatory mediator and a powerful attractant for granulocytic immune cells. Unlike many other chemokines, CXCL5 is secreted by both immune (neutrophil, monocyte, and macrophage) and nonimmune (epithelial, endothelial, and fibroblastic) cell types. The current study was intended to determine which of these cell types express CXCL5 in normal and malignant human prostatic tissues, whether expression levels correlated with malignancy and whether CXCL5 stimulated biologic effects consistent with a benign or malignant prostate epithelial phenotype. The results of these studies show that CXCL5 protein expression levels are concordant with prostate tumor progression, are highly associated with inflammatory infiltrate, and are frequently detected in the lumens of both benign and malignant prostate glands. Exogenous administration of CXCL5 stimulates cellular proliferation and gene transcription in both nontransformed and transformed prostate epithelial cells and induces highly aggressive prostate cancer cells to invade through synthetic basement membrane in vitro. These findings suggest that the inflammatory mediator, CXCL5, may play multiple roles in the etiology of both benign and malignant proliferative diseases in the prostate.

Technetium-99m labeling of antibodies has been suboptimal because of low affinity adventitious binding, nonspecific labeling, and loss of immunoreactivity. The diamide dithiolate ligand system (N2S2) forms highly stable, well-defined tetradentate complexes with Tc(V). Antibodies and their fragments have been labeled by conjugation of preformed "mTc4,5-bis(thioacetamido)pentanoate active ester to protein amine groups to give a chemically known 99"Tc-N2S2 complex covalently linked to antibody. Evaluations of the "'Tc-N2S2-bound antibodies and their fragments have shown high stability and retained immunoreactivity.Successful targeting ofdiagnostic radionuclides to tumors not only provides a tool to diagnose and stage cancer but also demonstrates feasibility for therapy where ligand systems can be applied to therapy radionuclides. Early studies with radiolabeled antibodies utilized radioiodine (1231/1311) because of extensive experience in protein radioiodination, covalent attachment, and ready availability of the radionuclide (1,2). Improved tumor-to-nontumor ratios were achieved with 1"'In compared to 131I by using diethylenetriaminepentaacetate (DTPA) bifunctional chelating agent technology (3)(4)(5) MATERIALS AND METHODS Preparation of 99mTc-4,5-bis(thioacetamido)pentanoyl (N2S2)-Conjugated Anti-Melanoma 9.2.27 F(ab')2 Fragment. To a mixture of 25 1.l of 4,5-bis(benzoylthioacetamido)pentanoic acid (1.0 mg/ml solution in 90% CH3CN) and 100 ,ul of 1 M NaOH was added 100 mCi of sodium [99mTc]pertechnetate in 1.0 ml of saline (0.9% NaCl). Then 1.0 mg of sodium dithionite (0.10 ml of a freshly prepared 10 mg/ml solution) was added, and the mixture was heated at 750C for 15 min.The pH was brought to about 6 with 0.10 ml of 1 M HCI and 0.30 ml of 0.2 M sodium phosphate buffer (pH 6.0). Then 10.0 mg of 2,3,5,6-tetrafluorophenol (0.10 ml of a 100 mg/ml solution in 90% CH3CN) and 12.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.10 ml of a 125.0 mg/ml solution in 90% CH3CN) were added, and the solution was heated at 75°C for 30 min. The resulting tetrafluorophenyl active ester derivative of "mTc-4,5-bis(thioacetamido)pentanoate was purified by loading the reaction mixture on a conditioned C18 cartridge (J. T. Baker), washing with 2.0 ml of 20% (vol/vol) ethyl alcohol/0.01 M sodium phosphate, pH 7.0, eight times, and eluting with 100% CH3CN. The solvent was evaporated under a stream of N2. Then 0.5 ml of the 9.2.27 F(ab')2 fragment (16) at 2.5 mg/ml and 0.50 ml of 0.2 M sodium phosphate (pH 9.0) were added for conjugation. After 15 min at room temperature, 25 mg of lysine (0.25 ml of a 250-mg/ml solution at pH 9.0) was added to quench unreacted ester. The 99mTc-N2S2-9.2.27 F(ab')2 was purified by passage through a G-25 Sephadex column (Pharmacia) equilibrated with phosphate-buffered saline.Abbreviations: N2S2, diamide dithiolate chelating system; 99M Tc-N2S2-9.2.27 F(ab')2, 99mTc-4,5-bis(thioacetamido)pentanoyl-9.2.27 F(ab')2 fragments; DPTA, diethylenetriaminepentaacetate.

Recent studies from our group suggest that extracellular matrix (ECM) deposition and fibrosis characterize the peri-urethral prostate tissues of some men suffering from Lower Urinary Tract Symptoms (LUTS) and that fibrosis may be a contributing factor to the etiology of LUTS. Fibrosis can generally be regarded as an errant wound-healing process in response to chronic inflammation, and several studies have shown that the aging prostate tissue microenvironment is rich with inflammatory cells and proteins. However, it is unclear whether these same inflammatory proteins, particularly CXC-type chemokines, can mediate myofibroblast phenoconversion and the ECM deposition necessary for the development of prostatic tissue fibrosis. To examine this, immortalized and primary prostate stromal fibroblasts treated with TGF-β1, CXCL5, CXCL8, or CXCL12 were evaluated morphologically by microscopy, by immunofluorescence and qRT-PCR for αSMA, collagen 1, vimentin, calponin, and tenascin protein and transcript expression, and by gel contraction assays for functional myofibroblast phenoconversion. The results of these studies showed that that immortalized and primary prostate stromal fibroblasts are induced to express collagen 1 and 3 and αSMA gene transcripts and proteins and to undergo complete and functional myofibroblast phenoconversion in response to CXC-type chemokines, even in the absence of exogenous TGF-β1. Moreover, CXCL12-mediated myofibroblast phenoconversion can be completely abrogated by inhibition of the CXCL12 receptor, CXCR4. These findings suggest that CXC-type chemokines, which comprise inflammatory proteins known to be highly expressed in the aging prostate, can efficiently and completely mediate myofibroblast phenoconversion and may thereby promote fibrotic changes in prostate tissue architecture associated with the development and progression of male lower urinary tract dysfunction.