Disruption of PPARgamma signaling results in mouse prostatic intraepithelial neoplasia involving active autophagy.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) regulates the interface between cellular lipid metabolism, redox status and organelle differentiation. Conditional prostatic epithelial knockout of PPARgamma in mice resulted in focal hyperplasia which developed into mouse prostatic intraepithelial neoplasia (mPIN). The grade of PIN became more severe with time. Electron microscopy (EM) showed accumulated secondary lysosomes containing cellular organelles and debris suggestive of autophagy. Consistent with this analysis the autophagy marker LC-3 was found to be upregulated in areas of PIN in PPARgamma KO tissues. We selectively knocked down PPARgamma2 isoform in wild-type mouse prostatic epithelial cells and examined the consequences of this in a tissue recombination model. Histopathologically grafted tissues resembled the conditional PPARgamma KO mouse prostates. EM studies of PPARgamma- and PPARgamma2-deficient epithelial cells in vitro were suggestive of autophagy, consistent with the prostatic tissue analysis. This was confirmed by examining expression of beclin-1 and LC-3. Gene expression profiling in PPARgamma-/gamma2-deficient cells indicated a major dysregulation of cell cycle control and metabolic signaling networks related to peroxisomal and lysosomal maturation, lipid oxidation and degradation. The putative autophagic phenotypes of PPARgamma-deficient cells could be rescued by re-expression of either gamma1 or gamma2 isoform. We conclude that disruption of PPARgamma signaling results in autophagy and oxidative stress during mPIN pathogenesis.

Alterations in lipoxygenase and cyclooxygenase-2 catalytic activity and mRNA expression in prostate carcinoma.

Recent studies in prostate tissues and especially cell lines have suggested roles for arachidonic acid (AA) metabolizing enzymes in prostate adenocarcinoma (Pca) development or progression. The goal of this study was to more fully characterize lipoxygenase (LOX) and cyclooxygenase-2 (COX-2) gene expression and AA metabolism in benign and malignant prostate using snap-frozen tissues obtained intraoperatively and mRNA analyses and enzyme assays. Formation of 15-hydroxyeicosatetraenoic acid (15-HETE) was detected in 23/29 benign samples and 15-LOX-2 mRNA was detected in 21/25 benign samples. In pairs of pure benign and Pca from the same patients, 15-HETE production and 15-LOX-2 mRNA were reduced in Pca versus benign in 9/14 (P=.04) and 14/17 (P=.002), respectively. Under the same conditions, neither 5-HETE nor 12-HETE formation was detectable in 29 benign and 24 tumor samples; with a more sensitive assay, traces were detected in some samples, but there was no clear association with tumor tissue. COX-2 mRNA was detected by nuclease protection assay in 7/16 benign samples and 5/16 tumors. In benign and tumor pairs from 10 patients, COX-2 was higher in tumor versus benign in only 2, with similar results by in situ hybridization. Paraffin immunoperoxidase for COX-2 was performed in whole mount sections from 87 additional radical prostatectomy specimens, with strong expression in ejaculatory duct as a positive control and corroboration with in situ hybridization. No immunostaining was detected in benign prostate or tumor in 45% of cases. Greater immunostaining in tumor versus benign was present in only 17% of cases, and correlated with high tumor grade (Gleason score 8 and 9 vs. 5 to 7). In conclusion, reduced 15-LOX-2 expression and 15-HETE formation is the most characteristic alteration of AA metabolism in Pca. Increased 12-HETE and 5-HETE formation in Pca were not discernible. Increased COX-2 expression is not a typical abnormality in Pca in general, but occurs in high-grade tumors.

Clinical under staging of high risk nonmuscle invasive urothelial carcinoma treated with radical cystectomy.

PURPOSE: The role of radical cystectomy in patients with nonmuscle invasive urothelial carcinoma of the bladder remains controversial. The risk of overtreatment must be balanced against the potential benefit of aggressive therapy. We reviewed our results in these patients with a particular emphasis on clinical under staging. MATERIALS AND METHODS: We reviewed the records of 214 consecutive patients who underwent radical cystectomy for urothelial carcinoma between April 1995 and August 1999, focusing on those with nonmuscle invasive, stages T1 or less disease. We assessed clinical and pathological data as well as outcomes based on pathological disease extent. RESULTS: A total of 78 patients (36%) underwent radical cystectomy for clinical stages T1 or less disease. Indications included disease refractory to intravesical therapy in 29 cases (37%), pathological findings reflective of high grade stage T1 or multifocal disease in 26 (33%), radiographic suspicion of invasive disease in 15 (20%) and severe symptoms in 8 (10%). Cancer was clinically under staged with stages pT2 or greater disease in 31 patients (40%) according to final pathology results. Under staging was most pronounced in the 10 patients (67%) with suspicious radiography and in the 18 (64%) with absent muscle in the biopsy specimen. Of the 78 patients with pathological stages pT1 disease or less 98% had no evidence of disease compared to 65% with stages pT2 or greater disease (p <0.01). CONCLUSIONS: Despite the intent to perform early cystectomy a significant percent of patients harbored occult muscle invasive and/or metastatic disease. In clinical and pathological, superficial stages T1 or less cases disease-free survival was excellent. Due to these results, the selection of high risk superficial transitional cell carcinoma cases for continued bladder sparing treatment should include uninvolved muscle on biopsy and absent radiographic suspicion of invasion.

Small intestinal submucosa as a urethral coverage layer.

PURPOSE: Urethrocutaneous fistula is the most common complication of hypospadias surgery. Numerous techniques have been used to decrease the incidence of this complication and the use of biocompatible materials in surgery has expanded the options in difficult situations. We hypothesized that porcine small intestinal submucosa may be used as a coverage layer after urethral surgery. We evaluated the histological changes associated with small intestinal submucosa when used as a coverage layer over the urethra in a rabbit model. METHODS AND METHODS: We performed urethral surgery in 16 New Zealand White rabbits divided into 4 animals each in groups 1-sham operation with penile degloving only, 2-penile degloving and small intestinal submucosa patch placement, 3-urethrotomy without a patch and 4-urethrotomy with a small intestinal submucosa patch. The graft edges were marked with permanent suture at surgery for later identification. All rabbits were maintained for 6 weeks before sacrifice. The urethra of each animal was then serially sectioned and examined histologically. RESULTS: Histological examination of animals with an small intestinal submucosa patch revealed a foreign body tissue reaction with an infiltrate of histiocytes, giant cells and lymphocytes in the area of graft placement. There was no histological evidence of remaining small intestinal submucosa patch in any sections. The urethral mucosa healed normally in all cases in which it was disrupted. There was no evidence of acute or chronic inflammation in any group 1 or 2 nonsmall intestinal submucosa animals and none in the animals with a small intestinal submucosa graft in areas other than the former graft site. There were also no urethrocutaneous fistulas in any of the 8 rabbits that underwent urethrotomy. CONCLUSIONS: Small intestine submucosa provides an adequate coverage layer in the rabbit penis after urethrotomy. Histologically the foreign material did not alter normal healing of the urethral mucosa, although it did appear to cause an infiltration of histiocytes, giant cells and lymphocytes. Small intestinal submucosa has previously been studied as a scaffold on which tissue may be remodeled or may regenerate. Our study shows that small intestinal submucosa did not interfere with normal tissue healing in this animal model. When used as a urethral coverage layer, it appears to provide extra tissue between the urethra and skin. Small intestinal submucosa may potentially decrease the incidence of urethrocutaneous fistula after urethral surgery.

15-Lipoxygenase-2 expression in benign and neoplastic sebaceous glands and other cutaneous adnexa.

15-Lipoxygenase-2 has a limited tissue distribution in epithelial tissues, with mRNA detected in skin, cornea, lung, and prostate. It was originally cloned from human hair rootlets. In this study the distribution of 15-lipoxygenase-2 was characterized in human skin using immunohistochemistry and in situ hybridization. Strong uniform 15-lipoxygenase-2 in situ hybridization (n = 6) and immunostaining (n = 16) were observed in benign cutaneous sebaceous glands, with expression in differentiated secretory cells. Strong 15-lipoxygenase-2 immunostaining was also observed in secretory cells of apocrine and eccrine glands. Variable reduced immunostaining was observed in skin-derived sebaceous neoplasms (n = 8). In the eyelid, Meibomian glands were uniformly negative for 15-lipoxygenase-2 in all cases examined (n = 9), and sebaceous carcinomas apparently derived from Meibomian glands were also negative (n = 12). The mechanisms responsible for differential expression in cutaneous sebaceous vs eyelid Meibomian glands remain to be established. In epidermis, positive immunostaining was observed in the basal cell layer in normal skin, whereas five examined basal cell carcinomas were negative. Thus, the strongest 15-lipoxygenase-2 expression is in the androgen regulated secretory cells of sebaceous, apocrine, and eccrine glands. This compares with the prostate, in which 15-lipoxygenase-2 is expressed in differentiated prostate secretory cells (and reduced in the majority of prostate adenocarcinomas). The product of 15-lipoxygenase-2, 15-hydroxyeicosatetraenoic acid, may be a ligand for the nuclear receptor peroxisome proliferator activated receptor-gamma, which is expressed in sebocytes, and contribute to secretory differentiation in androgen regulated tissues such as prostate and sebaceous glands.

A probasin-large T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential.

Neuroendocrine (NE) cells may be involved not only in growth and differentiation of the normal prostate but also in carcinogenesis and progression of prostate adenocarcinoma (Pca), including development of androgen resistance. However, the exact pathophysiology of NE cells in Pca remains poorly understood. Here we describe a transgenic model of Pca with progressive NE differentiation. Seven lines of transgenic mice with the rat prostate-specific large probasin promoter linked to the SV40-large T antigen (Tag) that develop prostatic neoplasia have been established. In this study, one of the seven lines (12T-10) was characterized by examination of 52 mice aged from 2-12 months. With advancing age, low-grade prostatic intraepithelial neoplasia, high-grade prostatic intraepithelial neoplasia, microinvasion, invasive carcinoma, and poorly or undifferentiated carcinoma with NE differentiation appeared in the prostates in sequential order. Whereas Tag is expressed uniformly in prostate epithelium, only an increasing subset of cells in prostatic intraepithelial neoplasia showed NE differentiation by chromogranin immunostaining. Frankly invasive carcinoma developing subsequently showed occasional definitive glandular differentiation (adenocarcinoma) and particularly undifferentiated carcinoma with NE histological features similar to those observed in NE carcinomas in humans. The NE carcinomas occurred in the dorsolateral and ventral lobes and were generally androgen receptor negative. Twenty-one of 32 (66%) mice aged > or = 6 months and 15 of 17 (88%) mice aged > or = 9 months developed metastatic tumors, as confirmed by histology and/or Tag immunohistochemistry. Metastases occurred at the later time points, with metastasis to regional lymph nodes, liver, and lung being particularly common. Metastases showed histological features of NE differentiation, as confirmed by chromogranin immunostaining and electron microscopy. An athymic nude mouse that received a s.c. implant of a primary NE tumor developed Tag-positive metastatic tumors with similar NE differentiation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified identical protein profiles between the primary NE tumor and lesions in the extraprostatic organs. Hence, in the 12T-10 large probasin promoter-Tag mouse, high-grade prostatic intraepithelial neoplasia develops progressively greater NE differentiation and progresses to invasive adenocarcinoma and NE carcinoma, with a high percentage of metastases. The predictable progression through these stages will allow testing of therapeutic interventions as well as possible further delineation of the role of NE cells in Pca progression.

Ratio of free-to-total prostate specific antigen correlates with tumor volume in patients with increased prostate specific antigen.

PURPOSE: We evaluated the relationship between the ratio of free-to-total prostate specific antigen (PSA) and prostate pathology, including grade, stage and tumor volume, among patients with prostate cancer who underwent radical prostatectomy. MATERIALS AND METHODS: We prospectively analyzed 54 consecutive patients with prostate cancer who underwent radical prostatectomy and in whom frozen serum was available for assessment of free-to-total PSA ratio. Pathological review was done with whole mount sections, and total tumor volume was determined by planimetry. Comparison between free-to-total PSA ratio and pathological parameters was performed using the Pearson correlation coefficient. RESULTS: Among the 54 patients mean total and free-to-total PSA ratio were 5.81 and 14.2 ng./ml., respectively, and free-to-total PSA ratio directly correlated with prostate volume (p = 0.037), and inversely correlated with Gleason score (p = 0.012) and extracapsular disease (p = 0.0074). Furthermore, there was a significant relationship between free-to-total PSA ratio and pathological stage pT2a/b in 39 cases versus pT3a/b in 15 (p = 0.005). Overall, there was no correlation between free-to-total PSA ratio and tumor volume. However, among 37 patients with an increased PSA, defined as greater than 4.0 ng./ml., a significant inverse relationship between free-to-total PSA ratio and tumor volume was identified (p = 0.01). Among this subset there was only a weak correlation with prostate volume (p = 0.049). CONCLUSIONS: Our findings suggest that free-to-total PSA ratio may be predictive of tumor biology among those patients with a total PSA of greater than 4 ng./ml. as evidenced by good correlation with tumor grade and volume. This finding appears to be independent of prostate volume. These preliminary results suggest the need for additional studies among patients with an increased PSA designed to evaluate the potential role of free-to-total PSA ratio in combination with traditional clinical variables in the prediction of prostate cancer pathology.

15S-Hydroxyeicosatetraenoic acid activates peroxisome proliferator-activated receptor gamma and inhibits proliferation in PC3 prostate carcinoma cells.

15-Lipoxygenase (15-LOX)-2 is expressed in benign prostate secretory cells and benign prostate produces 15S-hydroxyeicosatetraenoic acid (15S-HETE) from exogenous arachidonic acid (AA). In contrast, 15S-LOX-2 and 15S-HETE formation are reduced in prostate carcinoma (Pca). The mechanisms whereby reduced 15-LOX-2 may contribute to Pca development or progression are not known. We investigated the expression of peroxisome proliferator-activated receptor (PPAR) gamma in benign and malignant prostate tissues and the ability of 15S-HETE to activate PPARgamma-dependent transcription and modulate proliferation of the Pca cell line PC3. In contrast to benign prostate and similar to most Pca tissues, 15-LOX-2 mRNA was not detected in PC3 cells, and they did not produce detectable 15-HETE from [14C]AA. By reverse transcription-PCR, PPARgamma mRNA was present in 18 of 18 benign and 9 of 9 tumor specimens. The PPARgamma ligand BRL 49653 and 15S-HETE caused a dose-dependent inhibition of PC3 proliferation in a 14-day soft agar colony-forming assay (IC50 of 3 and 30 microM, respectively). 15S-HETE (10 microM) caused greater inhibition than 10 microM 15R-HETE. At 3 days, BRL 49653 and 15S-HETE caused a slight increase in cells in G0-G1 and a corresponding decrease in cells in S phase. In PC3 cells transiently transfected with a luciferase reporter linked to a PPAR response element, 1 microM BRL 49653 and 10 microM 15S-HETE caused approximately threefold and greater than twofold induction of PPAR-dependent transcription, respectively. By quantitative real-time reverse transcription-PCR and Northern analysis, 3-day treatment with BRL 49653 and 15S-HETE caused a reduction of PPARgamma expression but a marked up-regulation of the PPAR response element containing adipocyte type fatty acid binding protein. These results support the hypothesis that 15-LOX-2-derived 15S-HETE may constitute an endogenous ligand for PPARgamma in the prostate and that loss of this pathway by reduced expression of 15-LOX-2 may contribute to increased proliferation and reduced differentiation in prostate carcinoma.

Prediction of tumour volume and pathological stage in radical prostatectomy specimens is not improved by taking more prostate needle-biopsy cores.

OBJECTIVE: To determine what, if any, additional prognostic information is available from the prostate needle biopsy by comparing the number of biopsy cores obtained with the pathology assessed from the radical retropubic prostatectomy (RRP) specimen. PATIENTS AND METHODS: The results from 135 consecutive patients who underwent RRP at a single institution were reviewed. Needle biopsy information (number of cores, percentage of positive cores, laterality of the positive cores, and Gleason sum) were compared with the pathological data of the RRP specimen, including stage, Gleason sum and tumour volume. Patients were further stratified into those with six or fewer cores (96 men) or more than six cores (39 men). Clinical data, including biopsy information and pathological findings, were compared using univariate and multivariate models. RESULTS: Overall, univariate analysis showed that the total prostate-specific antigen (PSA) level, number of positive cores, bilateral positive cores and percentage of positive cores were directly correlated with tumour volume (P=0.01). Also, PSA and percentage of positive cores were directly correlated with extracapsular extension (P=0.008 and P=0.01, respectively). In the multivariate model, the most important independent predictors of RRP tumour volume and pathological stage were the preoperative PSA level and percentage of cancer in the biopsy (P<0.01). There was no significant relationship between the number of cores obtained and the predicted pathology of the RRP specimen. There were no differences in the number of positive cores, bilateral positive cores or percentage tumour in the cores between men with more or less than six biopsies. In men with more than six core biopsies, there was no significant increase in prognostic information for tumour volume and extracapsular extension, or a correlation between the Gleason sum on biopsy and the RRP specimen. Taking more than six biopsies did not result in a significantly greater detection of potentially indolent tumours (defined as a tumour volume of <0.5 mL). CONCLUSIONS: While taking more prostate needle biopsy cores seems to improve the detection of prostate cancer, there appears to be no major improvement in prognostic information over that gained from traditional sextant biopsies. Furthermore, the results suggest that the percentage of positive cores is the best predictor of both pathological stage and tumour volume, from among the information readily available from prostate needle biopsy. Given the variability in the number of cores obtained for diagnosis in clinical practice, these results add credence to the use of the percentage of positive cores in the biopsy set, with known predictors such as PSA and Gleason score, into future models that attempt to predict tumour biology.

Reduced 15-lipoxygenase-2 immunostaining in prostate adenocarcinoma: correlation with grade and expression in high-grade prostatic intraepithelial neoplasia.

Arachidonic acid (AA) metabolites are implicated in the oncogenesis of several tumors, including prostate cancer. 15-Lipoxygenase-2 (15-LOX-2) is a novel AA-metabolizing enzyme with a limited tissue distribution, which includes prostate, lung, skin, and cornea. Previous studies have shown that 15-LOX-2 is present in benign prostate secretory cells and reduced in prostate adenocarcinoma and that production of the 15-LOX-2 metabolite 15S-hydroxyeicosatetraenoic acid is reduced in malignant compared with benign prostate. The objective of this study was to determine the frequency with which 15-LOX-2 immunostaining is reduced in prostate carcinoma and to correlate reduced expression with tumor differentiation (grade) and other pathologic parameters in radical prostatectomy specimens. Paraffin immunoperoxidase with a polyclonal antibody specific for 15-LOX-2 was performed on tumors and benign portions from 70 cases, and the percentage of tumor immunostaining for 15-LOX-2 was assessed. Whereas uniform 15-LOX-2 immunostaining was observed in secretory cells of benign glands, it was markedly reduced or absent in most adenocarcinomas: 23 of 70 tumors showed completely absent 15-LOX-2 immunostaining, and 45 of 70 cases showed negative immunostaining in more than 50% of the tumor. The extent of reduced 15-LOX-2 immunostaining correlated with tumor differentiation, with retained expression particularly in Gleason score 5 tumors versus a significant reduction of 15-LOX-2 in higher-grade tumors (mean +/- SD tumor 15-LOX-2 positive: Gleason score 5 = 67%+/-30%, Gleason score 6 = 16%+/-30%, Gleason score 7 = 23%+/-28%, Gleason score > or =8 = 41%+/-46%). In 16 cases with multifocal tumors or different foci of the same tumor with different grades, the higher-grade foci had significantly reduced 15-LOX-2 expression compared with the lower-grade foci. In peripheral zone tumors without complete loss of 15-LOX-2 expression, there was a significant inverse relationship between 15-LOX-2 immunostaining and tumor volume. There was not a significant correlation between 15-LOX-2 immunostaining and serum PSA or pathologic stage. In a subset of 27 cases, 15-LOX-2 expression in high-grade prostatic intraepithelial neoplasia (HGPIN) glands was significantly reduced compared with benign glands. These data show that in contrast to the uniform expression of 15-LOX-2 in differentiated secretory cells of benign prostate, reduced 15-LOX-2 is a common alteration in prostate carcinoma, and this correlates with tumor cell differentiation. That reduced expression is seen in HGPIN suggests that this may be an early alteration in carcinoma development.

Enhanced expression of cyclooxygenase-2 in high grade human transitional cell bladder carcinomas.

Studies in human and animal models have shown that cyclooxygenase (COX)-2 is up-regulated in several epithelial carcinomas including colon, breast, and lung. To elucidate the possible involvement of COX-2 in human bladder cancer we examined the expression of COX isoforms in benign tissue and in bladder carcinoma specimens. Paraffin embedded tissues from 75 patients with urothelial carcinomas were immunostained with specific antibodies raised against COX-1 and COX-2. COX-1 expression was detected in smooth muscle cells in both benign and malignant bladders. COX-2 immunoreactivity was absent in benign tissue and in specimens with low-grade urothelial carcinoma (0/23). In contrast, expression of COX-2 was detected in malignant epithelial cells in 38% (17/47) of specimens with high-grade urothelial carcinomas. Expression of COX-2 in high-grade bladder cancer was confirmed by radioactive in situ hybridization using a COX-2-selective riboprobe. Both immunohistochemistry and in situ hybridization showed COX-2 expression in a small subset of malignant cells. COX-2 mRNA was also expressed in three out of seven malignant urothelial cell lines. These data demonstrate elevated expression of COX-2 in a high percentage of high-grade bladder carcinomas, suggesting a possible role of COX-2 in the progression of bladder urothelial carcinoma and supporting its potential as a therapeutic target in human bladder carcinoma.

Expression of adhesion molecules in kidney with experimental chronic obstructive uropathy: the pathogenic role of ICAM-1 and VCAM-1.

BACKGROUND: Chronic obstructive uropathy induced by maintained unilateral ureter ligation in the rat is characterized morphologically by interstitial inflammation, interstitial fibrosis, and tubular atrophy. Infiltrating mononuclear inflammatory cells, particularly T lymphocytes and macrophages, may contribute to the progression of this lesion by mediating tubular injury and by the activation of interstitial fibroblasts, with resultant tubular atrophy and interstitial fibrosis, respectively. Altered expression and activation of adhesion molecules by leukocytes, vascular endothelial cells, and parenchymal cells likely contributes both to the infiltration of inflammatory cells into the tubulointerstitial compartment and to the interaction of activated inflammatory cells with parenchymal cells. METHODS: In the current study, we examined changes in the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in a 90-day model of maintained unilateral ureter ligation in male Sprague-Dawley rats. RESULTS: Rat kidneys showed constitutive expression of ICAM-1 mRNA and constitutive immunostaining for ICAM-1 in peritubular capillaries, glomeruli, and a small percentage of cortical tubules. Ureter ligation resulted in a rapid increase in ICAM-1 mRNA, which was almost 2-fold greater than those of the contralateral and control kidneys as early as 3 h and which was maintained at a 4- to 6-fold higher level in the ligated vs. contralateral kidneys throughout the entire 90-day time course. There was a marked increase in ICAM-1 immunostaining within the tubular epithelium, with up to 80% of both cortical and medullary tubular cross-sections showing strong apical immunostaining from day 6 to 25, with a subsequent decrease throughout the remainder of the experiment. ICAM-1 immunostaining in the expanding interstitium in the ligated kidneys showed a gradual increase throughout the duration of the experiment. In contrast, glomerular immunostaining for ICAM-1 was decreased in the ligated compared to the contralateral kidneys throughout the entire experiment. There was a later but prominent increase in VCAM-1 mRNA in ligated kidneys, which was first evident at 2 days and which was maintained 2- to 10-fold greater than the contralateral kidneys throughout the entire time course. VCAM-1 immunostaining increased in the expanding interstitium, but decreased in glomeruli in obstructed vs. contralateral kidneys. Tubular staining for VCAM-1 did not change after ureter ligation. CONCLUSION: Increased ICAM-1 and VCAM-1 may contribute to the prominent inflammatory cell infiltration in the chronic tubulointerstitial nephritis accompanying maintained unilateral ligation. Tubule expression of ICAM-1, which occurs during a similar time course as previously documented for tubular cell proliferation and especially tubular cell apoptosis in this model, may contribute to injurious interactions of activated inflammatory cells with tubular epithelium.

15-lipoxygenase-2 (15-LOX-2) is expressed in benign prostatic epithelium and reduced in prostate adenocarcinoma.

Human 15S-lipoxygenase-2 (15-LOX-2) is a recently identified lipoxygenase that has approximately 40% sequence identity to the known human 5S-, 12S-, and 15S-lipoxygenases. 15-LOX-2 has a limited tissue distribution, with mRNA detected in prostate, lung, skin, and cornea, but not in numerous other tissues, including peripheral blood leukocytes. In the current study, we have characterized the distribution of 15-LOX-2 in the human prostate by immunohistochemistry, demonstrated the ability of benign prostate tissue to form 15S-hydroxyeicosatetraenoic acid (15S-HETE) from exogenous arachidonic acid (AA), and begun characterizing possible alterations in 15-LOX-2 in prostate adenocarcinoma. Incubation of benign prostate tissue with [14C]AA resulted in formation of [14C]15-HETE, as determined by reverse- and straight-phase high-performance liquid chromatography. 15-HETE was the major AA metabolite formed. By immunohistochemistry, 15-LOX-2 is located in secretory cells of peripheral zone glands and large prostatic ducts and somewhat less uniformly in apical cells of transition and central zone glands. 15-LOX-2 was not detected in the basal cell layer, stroma, ejaculatory ducts, seminal vesicles, or transitional epithelium. Immunostaining of 18 radical prostatectomy specimens showed a loss of 15-LOX-2 in the majority of prostate adenocarcinomas; 14 of 18 cases showed loss of 15-LOX-2 in >25% of the tumor (mean, 74.9% negative for 15-LOX-2; range, 38.9% to 100%). Incubation of paired pure benign and pure malignant prostate tissue from the same radical prostatectomies showed that 15-HETE formation was markedly reduced (>90%) or undetectable in incubations of prostate adenocarcinoma. 15-LOX-2 is a novel human lipoxygenase with a limited tissue distribution that is strongly expressed in benign prostate glandular epithelium and lost to a variable degree in the majority of prostate adenocarcinomas.

Chronic obstructive uropathy in severe combined immunodeficient (SCID) mice: lymphocyte infiltration is not required for progressive tubulointerstitial injury.

Progressive renal injury in humans and experimental animal models is characterized by tubular atrophy, infiltration of mononuclear inflammatory cells, and interstitial fibrosis. Permanent unilateral ureter ligation represents a reproducible model for investigating mechanisms of progressive kidney injury, and in the rat is characterized by tubular epithelial cell proliferation followed by apoptosis and progressive infiltration of monocytes and lymphocytes. Nevertheless, whether monocytes or lymphocytes play a dominant role in causing tubulointerstitial damage remains to be elucidated. In the current study, a model of chronic obstructive uropathy in the mouse is established and the role of lymphocyte infiltration in the evolution of the tubule and interstitial alterations is investigated. Permanent ligation of the left ureter in wild-type (C3H/HeJ) mice resulted in progressive atrophy of tubules and interstitial fibrosis compared with the contralateral kidney over a 30-d period. Immunoperoxidase studies on frozen sections taken from kidneys at 0, 3, 10, 20, and 30 d after ureter ligation showed that the tubulointerstitial injury was accompanied by a marked and progressive increase in interstitial macrophages and T lymphocytes, with no appreciable increase in B lymphocytes. No increase in inflammatory cells was detected in contralateral kidneys over the same time frame. The significance of T lymphocyte infiltration was examined by comparing the degree of tubular atrophy and interstitial fibrosis and the nature and quantity of the inflammatory infiltrate in wild-type mice and C3HSMn.C-Scid/J (SCID) mice subjected to permanent left ureter ligation. SCID mice have genetic defects in immunoglobulin and T cell receptor gene rearrangements and are devoid of circulating mature B and T lymphocytes. Wild-type and SCID mice developed tubular atrophy and interstitial volume expansion in the ligated kidney to the same degree and at the same rate. SCID mice developed a prominent and marked monocyte/macrophage infiltrate in the ligated kidney, which was essentially equal to that in wild-type mice. In contrast, consistent with the known absence of mature lymphocytes in SCID mice, there was essentially no T lymphocyte infiltration into the ligated kidney of SCID mice. These results demonstrate the effective establishment of the model of maintained unilateral ureter ligation in mice, which is readily applicable to genetic mutant strains thus allowing for specific investigation of the role of individual components of the inflammatory response in progressive tubulointerstitial injury. These studies further demonstrate that lymphocyte infiltration is not required for progressive tubular atrophy and increased interstitial fibrosis after maintained unilateral ureter ligation.

LFA-1 is sufficient in mediating neutrophil emigration in Mac-1-deficient mice.

To better define the specific function of Mac-1 (CD11b) versus LFA-1 (CD11a) and the other CD11 integrins in vivo, we have disrupted murine CD11b by targeted homologous recombination in embryonic stem cells and generated mice which are homozygous for a mutation in CD11b. A null mutation was confirmed by Southern blotting, RNase protection assay, immunohistochemistry, and flow cytometry. Neutrophils isolated from mice deficient in Mac-1 were defective in adherence to keyhole limpet hemocyanin-coated glass, iC3b-mediated phagocytosis, and homotypic aggregation. When challenged by thioglycollate intraperitoneally, Mac-1-deficient mice had similar levels of neutrophil accumulation in the peritoneal cavity at 1, 2, and 4 h. Treatment with mAb to LFA-1 blocked 78% of neutrophil accumulation in Mac-1-deficient mice and 58% in wild-type mice. Neutrophil emigration into the peritoneal cavity 16 h after the implantation of fibrinogen-coated disks was not reduced in Mac-1-deficient mice whereas neutrophil adhesion to the fibrinogen-coated disks was reduced by > 90%. Neutrophils from Mac-1-deficient mice also showed reduced degranulation. Our results demonstrate that Mac-1 plays a critical role in mediating binding of neutrophils to fibrinogen and neutrophil degranulation, but is not necessary for effective neutrophil emigration, which is more dependent upon LFA-1.

Susceptibility to local and systemic bacterial infections in intercellular adhesion molecule 1-deficient transgenic mice.

The contribution of intercellular adhesion molecule 1 (ICAM-1) during systemic and local bacterial infections was studied in transgenic ICAM-1-deficient and control mice that were injected intraperitoneally (ip) or intradermally (id) with Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus. Mortality rates, blood cultures, white blood cell (WBC) counts and absolute neutrophil counts (ANCs) were obtained daily until cultures were sterile. Six and 24 h after injections, autopsies were done on randomly selected ip-inoculated mice and biopsies were done on randomly selected id-inoculated mice. Survival rates were similar. In ICAM-1-deficient mice, ip P. aeruginosa resulted in higher incidences of bacteremia at 24 h (P = .003) and 48 h (P = .002); id S. aureus resulted in larger skin lesions (P = .026). Leukocytosis persisted in ICAM-1-deficient mice 6 h after ip injection of E. coli; however, WBC counts and ANCs in peritoneal fluid did not differ. Although the inflammatory responses were similar histologically in ICAM-1-deficient and normal mice, differences in site- and stimulus-specific susceptibilities were noted.

Neutrophil induced oxidative injury of cardiac myocytes. A compartmented system requiring CD11b/CD18-ICAM-1 adherence.

We have previously shown that cytokines and postischemic cardiac lymph induce expression of intercellular adhesion molecule-1 (ICAM-1, CD54) on canine adult cardiac myocytes. ICAM-1 expression allows adherence of activated neutrophils to myocytes that is blocked by anti-CD18 mAb, R15.7, or anti-ICAM-1 mAb, CL18/6. Interleukin 1, tumor necrosis factor-alpha, or interleukin 6-stimulated cardiac myocytes were loaded with 2',7'-dichlorofluorescin, and oxidation to the fluorescent dichlorofluorescein was monitored. Fluorescence and neutrophil/myocyte adherence followed the same time course, and both were blocked by monoclonal antibodies to CD18, CD11b, and ICAM-1, but mAb R7.1, recognizing a functional epitope on CD11a, was not inhibitory. The iron chelator, desferroxamine, and the hydroxyl radical scavenger, dimethylthiourea, did not inhibit neutrophil adherence, but completely inhibited fluorescence. In contrast, the extracellular oxygen radical scavengers superoxide dismutase and catalase, and the extracellular iron chelator, starch-immobilized desferroxamine, did not affect either fluorescence or adherence. Under the experimental conditions used, no superoxide production could be detected in the extracellular medium. Fluorescence microscopy demonstrated that fluorescence began within 5 min after neutrophil adherence to an individual myocyte, and myocyte contracture followed rapidly. Fluorescent intensity was highest initially at the site of myocyte-neutrophil adherence. When only neutrophils were loaded with 2',7'-dichlorofluorescein, fluorescence was observed only in those neutrophils adhering to the cardiac myocytes. Thus, adherence dependent on Mac-1 (CD11b/CD18) and ICAM-1 (CD54) activates the neutrophil respiratory burst resulting in a highly compartmented iron-dependent myocyte oxidative injury.

Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18-dependent mechanism.

Cardiac myocytes were isolated from adult dogs and incubated with isolated canine neutrophils (PMN). Intercellular adhesion was low and unchanged by stimulation of the PMN with zymosan activated serum or platelet activating factor (PAF) at concentrations that significantly enhance PMN adhesion to protein-coated glass and canine endothelial cell monolayers. Intercellular adhesion was significantly increased only when both myocytes and PMN were stimulated (e.g., myocytes incubated with IL-1, tumor necrosis factor, or phorbol myristate acetate, and PMN were chemotactically stimulated). Inhibitors of protein synthesis diminished the IL-1 beta-induced effect by greater than 80%. The IL-1 beta, PAF-stimulated PMN-myocyte adhesion was associated with substantial H2O2 production. Under conditions with low PMN-myocyte adhesion (i.e., IL-1 beta alone, PAF alone, or no stimulus) H2O2 production was generally less than 5% of that occurring with high adhesion. An anti-CD18 monoclonal antibody (R15.7) inhibited stimulated PMN-myocyte adhesion by greater than 95% and reduced H2O2 production by greater than 90%. Control isotype-matched, binding, and nonbinding antibodies were without effect on adherence or H2O2 production. The results indicate that cytokine stimulation of adult myocytes induces expression of a ligand involved in CD18-dependent adherence of canine neutrophils.