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Histochemical distribution of phosphodiesterase isoenzymes 3, 4 and 5 in the human prostate
65
66
SELECTIVE GROWTH OF EPITHELIAL BASAL CELLS OF THE HUMAN PROSTATE IN A THREE DIMENSIONAL ORGAN CULTURE
INTRACELLULAR CYTOKINE PROSTATIC T-LYMPHOCYTES
Sclli C.‘. Papini S.‘. Cl‘mpani 11.‘. DeMatteis A.‘. Revoltella R.’
Kramer C.‘. Steiner G.‘. Lee C.‘. Marberger
sp,sa University, Urology. Pisa. Italy, ‘National Research Council (CNR). Biomedical Technologies, Piss. Italy, Pisa University, Oncology. Piss, Italy
‘Unlverslty of Vienna. Urology, Vienna. Austria, Urology. Chicago, lJnited States of America
lNTRODUCTlON
& OBJECTIVES: The prostatic epithelinm consists of two defined compartments, the basal layer and the secretory OX. A small snbset of undifferentiated cells express the immunophenotype of both. and a putative stem cell role has been postulated. In this study primary cultures of human explants histolOgiCally
on gelatine sponges were used, which provide a useful model for growing tissue in 3. dimensions maintaimng its organ architecture, stromal and eplthelial organiration and cellular interactions present in viva for prolonged periods. We evaluated with this culture system the survival and expansion of cpithelial basal cells of benign adult prostates for rclati\ely
long periods.
)lATERIAL & METHODS: Prostallc fragmenta were obtained from 17 cystoprostatcctomy specimens. and histology rcvcalcd normal bcmgn prostate in X
and BPH in 9. Cultured fragments (I -3 mm. cubes, >3/sponge) were maintained in lnedia containmg 10% fetal calf scrnm. After 7. I4 and 21 -day intervals fragments
,ncludmg rhe sponge were fixed in 3.7% buffered formaldehyde and paraffinembedded. Sections were analyred by histology and immunohlstochemlstry. To demonstrate basal cells. mAb 34bctaEl2 and antI ~03 mb 4A4 (raised against the amino terminus of DNp63) were used. MAb MIB-I (Ki-67) was used to idcntifl cycling cells. Functional market-s included PSA. ET-l and GM-CSF.
RESULTS: Compared to the ongmal explant (tnne 0). after 7 days WC observed a progressive hypcrplasia of the basal cells with and without prcscrvatlon of the wcretorv or comnletc loss of I_. , cell lavcr. After I4 and 2 I davs a more .nroeressivc secretory cells (PSA and ET-I positive) ua, normally seen. Houwer, PSA-positike secretory cells were maintamed in media enriched wth androgens. In contrast, even without androgens. basal cells actively continued to proliferate with retention of the lumen or wit6 solid nests, often presenting squamous differentiation. Basal cells rcmxtantlv 34betaEl2. n63 and often GM-CSF nositivcj lined the surface of the entire fragments and &Id migrate to the unde;lying sponge and be isolated by affinity sorting. I-.----
i
CONCLUSIONS:
By considering the maintenance of stromal-epithelial interactions, prostate organotyplc cuhurcs in 3 dimensions represent a suitable dynamic organ culture system for investigating the proliferative function of basal cells in both benign and ncoplastic prostate. In addinon. rhe posslbillty to Isolate and purify basal cells by affinity sorting (nnmunocapture) offers the opportunity to flirther characterize then role. (Supported m part by grants from MIUR ProJcct SlEMZOOl. MIUR-PNR 200183. FIRB DMl9).
EXPRESSION
PATTERN
OF
M.’ ~Northwestem
University.
INTRODUCTION 81 OBJECTIVES: Previously, it has been demonstrated that T-ceil derived cytokines can induce hyperproliferation of BPH-derived stromal cells (Prostate, 52:43, 2002). However, it was also shown that IL-4, a type 2 cytokine, can partially inhibit stromal cell growth. Which type of cytokine and immune response may act on stromal cell growth in BPH was analysed by determining the intracellular cytokine profile of BPII T-cells. MATERIAL& METHODS: T-cells were freshly isolated from IO BPH-tissues obtained from transurethral resection and stimulated with phorbol 12.myristate l3-acetate and ionomycin m the presence of monensin. BPH-T-cells were grouped into CD4+/CDX- T-helper (Th), CDX+/CD4- T-cytotoxic/suppressor (Tc), CD4+/CDX+ double positive (DP) and CD4-/CD8- double negative (DN) T-cells and analyscd for cocxprcssion of intcrfcron-gamma (IFN-g). interleukin (IL)-2. -4. -5. -6. -10. -13. -tumour necrosis factor-alpha (TNF-a). and TNF-I3 by four colour flop cytometry. 2X=18% of CD4t T cells (55 4: of BPH T-cells) wcrc IFN-g+ I ILtype 2 cells and I2+6”C were IFN-g+.!IL41 type 0 cells. In relation. cyrotoxic and double negative T-cells showed a further dccrcasc m type I and type 0 cells in favour of a type 2. In double pos’tive I’-lymphocytes type 2 cells (35115%) were predominant (type 1: 12+7%, type 0: 514%). The mean anti-IL-6 reactivity ranged between I% in the case of T-helper and 5% of double positive T-cells. Anti-IL-5 staining recealed a similar pattern: X0/ anti-IL-5 reactive double positive T cells followed by CDX+ cytotoxic and double negative T-cells (3% and 2%, respectively). 4% of double positive T cells were IL-IO+ followed by CDX+ cytotoxic cells with 3°K Anti-TNF-a reactivity was almost ubiquitous and that of TNF-R was restricted to a very small T-cell population (less than 5%). RESULTS:
3- type
I
cells. IO+2% IFN-g-/[L-4+
CONCLUSIONS: In normal T-cells less than 5% of cells express type 2 cytokines. Therefore the finding that up to 35% of T-cells cxprcss type 2 cytokmes is a clear indication for a predominance of a type 2 or type 0 mlmune response in BPH tissue. A similar pattern of cytokine expression has been shown for autolmmune and chronic inflammatory diseases.
67
68
HISTOCHEMICAL DISTRIBUTION OF PHOSPHODIESTERASE ISOENZYMES 3,4 AND 5 IN THE HUMAN PROSTATE
PROSTATIC EPITHELIAL AND LUMINAL COMPONENTS IN THE TRANSITION ZONE ACINI. MORPHOMETRIC ANALYSIS IN CONTROLS AND PATIENTS WITH BENIGN PROSTATIC II\‘PERPI.ASIA
‘Hannover Medical School. Urology, llnnnover. llospital, Clinical Pharmacology. 1 und. Swcdcn
Unbmskl M
Germany.
-Lund University
INTRODUCTION & OBJECTIVES: With the introductmn of \ildenalil. the conccpl ofphosphodicstcrasc (PDE) inhibition has gained tremendous interest in the field of urology. Recently. the presence of mRNA transcripts encoding for I2 different PDE isoforms as well as the hydrolytic activity of PDE isoenzymes 4 and 5 were detcctcd in the human prostate. Moreover. the ability of inhibitors of PDE4 and 5 to reverse the adrenergic tension of prostatic strips isolated from the transitional zone was described (J. Ural. 166: 24X4 2490; 200 I). The aim of our study was to evaluate the prcscncc of PDEs 3, 4 and 5 in the anatomy of the human prostate by means of histochemical methods. MATERIAL & METHODS: Cryostat sectIons (IO pM) prepared from formaldehyde-fixated tissue scgmcnts. which had been excised from the transitional zone, were incubated for 4Xh with primary antibodies (Dilution 11250) directed against PDE isoenTyme\ 3, 4 and 5. Then. sectlons were incubated with either lluorescein iaothiocyanate(FITC) or Texas Red- (TR) labelled secondary amibochea for 2 h. VlsualiLation was commenced by means of a confocal laser microscope using a magnification of 10. to 10O-fold. Antibodies against PDE 3 and 4 were obtained from FabGcnnix Inc.. Shreveport, USA, PDES antibodies were kindly supplied by Tanabe Seiyaku Pharmaceutical Co., Osaka Japan. RESULTS: Positive immunostaining (TR) indicating the prescncc of PDE4 (CAMP PDE) was abundantly observed in the fibromuscular stroma, in glandular structures and in small vessels of the transitional zone. In contrast, PDE5 (cGMP PDE) itnmunostaining was seen in glandular and subglandular structures. No Positive FITC-reaction for PDE3 (cGMP-inhibited PDE) was detected. CONCLUSIONS: Our results confirm the presence of PDEs 4 and 5 in the transitional zone of the human prostate and presented evidence that these isoenzymes are not evenly distributed. The results from our study are in support the hypothesis that there might be a rationale for the use of PDE inhibitors in the pharmacotherapy of BPH and LUTS.
of
. C‘hagas M., Costa W.. SampaIn F
State Unlvcrsiry Bra/l1
of Klo dc Janewo. Urogenital
Research Unit.
Rio dc Janeiro,
INTRODUCTION & OBJECTIVES: Perform a quantitalibc analysis on the acini and the moditicatlons of the acmar epithelium of the transitional zone in normal and hyperplaqtic human prostates. MATERIAL & METHODS: BPH tissue samples of the transiflonal Lone were obtained from 20 patients with clmlcal symptoms of bladder outlet obstruction that were submitted to open prostatcctomy. The controls consisted of 20 transitional Lone of prostates obtained during necropsy of adults under 30 years old. The quantitative analysis consisted of the determination of the following parameters: number of acini. total acinar arca, arca of the lumen, epithclial arca. and the minimum, maximum and mean cpithelial height, by using computed hlstomorphomelric techniques. RESULTS: The mot-phomctric analysis showed the l‘ollowing results, in mm2, for normal and hyperplastic prostates, respectively: total area of the acini=0.04 I *0.0076 and 0.056+0.0160 (p=O.O005). area of the lumen=0.016t0.0034 and 0.036~0.0131 (p=O.OOOl), area of the epithelium=0.025*0.0046 and 0.019*0.003X (p=O.OOOS). The evaluation of the cpithelial height showed the following results, in mm. for normal and hyperplastic prostates, respectively: minimum height = 9.92 i I .67 and 6.45 i I I4 (p=O.OOOl), maximum height=54.38*4.09 and 41.5214.5 1 (p=O.OOOl), median height=27.89&2.48 and 19.96i2.20 (p=O.OOOl). CONCLUSIONS: There 1s no statistically significant diffcrcncc in the number of acini between controls and BPH patients. The total area of the acini as well as Ihe luminal area presented a statistically significant increase in BPH. On the other hand, the area and the height of the acinar epithelium presented a statistically significant decrease in BPH. ** Supported by Grants from the National Council of Scientific and Technological Development (CNPq-Brazil), and from the Foundation for Research Support of Rio de Janeiro (FAPERJ). European
Urology
Supplements
2 (2003)
No. 1, pp. 19
66
SELECTIVE GROWTH OF EPITHELIAL BASAL CELLS OF THE HUMAN PROSTATE IN A THREE DIMENSIONAL ORGAN CULTURE
INTRACELLULAR CYTOKINE PROSTATIC T-LYMPHOCYTES
Sclli C.‘. Papini S.‘. Cl‘mpani 11.‘. DeMatteis A.‘. Revoltella R.’
Kramer C.‘. Steiner G.‘. Lee C.‘. Marberger
sp,sa University, Urology. Pisa. Italy, ‘National Research Council (CNR). Biomedical Technologies, Piss. Italy, Pisa University, Oncology. Piss, Italy
‘Unlverslty of Vienna. Urology, Vienna. Austria, Urology. Chicago, lJnited States of America
lNTRODUCTlON
& OBJECTIVES: The prostatic epithelinm consists of two defined compartments, the basal layer and the secretory OX. A small snbset of undifferentiated cells express the immunophenotype of both. and a putative stem cell role has been postulated. In this study primary cultures of human explants histolOgiCally
on gelatine sponges were used, which provide a useful model for growing tissue in 3. dimensions maintaimng its organ architecture, stromal and eplthelial organiration and cellular interactions present in viva for prolonged periods. We evaluated with this culture system the survival and expansion of cpithelial basal cells of benign adult prostates for rclati\ely
long periods.
)lATERIAL & METHODS: Prostallc fragmenta were obtained from 17 cystoprostatcctomy specimens. and histology rcvcalcd normal bcmgn prostate in X
and BPH in 9. Cultured fragments (I -3 mm. cubes, >3/sponge) were maintained in lnedia containmg 10% fetal calf scrnm. After 7. I4 and 21 -day intervals fragments
,ncludmg rhe sponge were fixed in 3.7% buffered formaldehyde and paraffinembedded. Sections were analyred by histology and immunohlstochemlstry. To demonstrate basal cells. mAb 34bctaEl2 and antI ~03 mb 4A4 (raised against the amino terminus of DNp63) were used. MAb MIB-I (Ki-67) was used to idcntifl cycling cells. Functional market-s included PSA. ET-l and GM-CSF.
RESULTS: Compared to the ongmal explant (tnne 0). after 7 days WC observed a progressive hypcrplasia of the basal cells with and without prcscrvatlon of the wcretorv or comnletc loss of I_. , cell lavcr. After I4 and 2 I davs a more .nroeressivc secretory cells (PSA and ET-I positive) ua, normally seen. Houwer, PSA-positike secretory cells were maintamed in media enriched wth androgens. In contrast, even without androgens. basal cells actively continued to proliferate with retention of the lumen or wit6 solid nests, often presenting squamous differentiation. Basal cells rcmxtantlv 34betaEl2. n63 and often GM-CSF nositivcj lined the surface of the entire fragments and &Id migrate to the unde;lying sponge and be isolated by affinity sorting. I-.----
i
CONCLUSIONS:
By considering the maintenance of stromal-epithelial interactions, prostate organotyplc cuhurcs in 3 dimensions represent a suitable dynamic organ culture system for investigating the proliferative function of basal cells in both benign and ncoplastic prostate. In addinon. rhe posslbillty to Isolate and purify basal cells by affinity sorting (nnmunocapture) offers the opportunity to flirther characterize then role. (Supported m part by grants from MIUR ProJcct SlEMZOOl. MIUR-PNR 200183. FIRB DMl9).
EXPRESSION
PATTERN
OF
M.’ ~Northwestem
University.
INTRODUCTION 81 OBJECTIVES: Previously, it has been demonstrated that T-ceil derived cytokines can induce hyperproliferation of BPH-derived stromal cells (Prostate, 52:43, 2002). However, it was also shown that IL-4, a type 2 cytokine, can partially inhibit stromal cell growth. Which type of cytokine and immune response may act on stromal cell growth in BPH was analysed by determining the intracellular cytokine profile of BPII T-cells. MATERIAL& METHODS: T-cells were freshly isolated from IO BPH-tissues obtained from transurethral resection and stimulated with phorbol 12.myristate l3-acetate and ionomycin m the presence of monensin. BPH-T-cells were grouped into CD4+/CDX- T-helper (Th), CDX+/CD4- T-cytotoxic/suppressor (Tc), CD4+/CDX+ double positive (DP) and CD4-/CD8- double negative (DN) T-cells and analyscd for cocxprcssion of intcrfcron-gamma (IFN-g). interleukin (IL)-2. -4. -5. -6. -10. -13. -tumour necrosis factor-alpha (TNF-a). and TNF-I3 by four colour flop cytometry. 2X=18% of CD4t T cells (55 4: of BPH T-cells) wcrc IFN-g+ I ILtype 2 cells and I2+6”C were IFN-g+.!IL41 type 0 cells. In relation. cyrotoxic and double negative T-cells showed a further dccrcasc m type I and type 0 cells in favour of a type 2. In double pos’tive I’-lymphocytes type 2 cells (35115%) were predominant (type 1: 12+7%, type 0: 514%). The mean anti-IL-6 reactivity ranged between I% in the case of T-helper and 5% of double positive T-cells. Anti-IL-5 staining recealed a similar pattern: X0/ anti-IL-5 reactive double positive T cells followed by CDX+ cytotoxic and double negative T-cells (3% and 2%, respectively). 4% of double positive T cells were IL-IO+ followed by CDX+ cytotoxic cells with 3°K Anti-TNF-a reactivity was almost ubiquitous and that of TNF-R was restricted to a very small T-cell population (less than 5%). RESULTS:
3- type
I
cells. IO+2% IFN-g-/[L-4+
CONCLUSIONS: In normal T-cells less than 5% of cells express type 2 cytokines. Therefore the finding that up to 35% of T-cells cxprcss type 2 cytokmes is a clear indication for a predominance of a type 2 or type 0 mlmune response in BPH tissue. A similar pattern of cytokine expression has been shown for autolmmune and chronic inflammatory diseases.
67
68
HISTOCHEMICAL DISTRIBUTION OF PHOSPHODIESTERASE ISOENZYMES 3,4 AND 5 IN THE HUMAN PROSTATE
PROSTATIC EPITHELIAL AND LUMINAL COMPONENTS IN THE TRANSITION ZONE ACINI. MORPHOMETRIC ANALYSIS IN CONTROLS AND PATIENTS WITH BENIGN PROSTATIC II\‘PERPI.ASIA
‘Hannover Medical School. Urology, llnnnover. llospital, Clinical Pharmacology. 1 und. Swcdcn
Unbmskl M
Germany.
-Lund University
INTRODUCTION & OBJECTIVES: With the introductmn of \ildenalil. the conccpl ofphosphodicstcrasc (PDE) inhibition has gained tremendous interest in the field of urology. Recently. the presence of mRNA transcripts encoding for I2 different PDE isoforms as well as the hydrolytic activity of PDE isoenzymes 4 and 5 were detcctcd in the human prostate. Moreover. the ability of inhibitors of PDE4 and 5 to reverse the adrenergic tension of prostatic strips isolated from the transitional zone was described (J. Ural. 166: 24X4 2490; 200 I). The aim of our study was to evaluate the prcscncc of PDEs 3, 4 and 5 in the anatomy of the human prostate by means of histochemical methods. MATERIAL & METHODS: Cryostat sectIons (IO pM) prepared from formaldehyde-fixated tissue scgmcnts. which had been excised from the transitional zone, were incubated for 4Xh with primary antibodies (Dilution 11250) directed against PDE isoenTyme\ 3, 4 and 5. Then. sectlons were incubated with either lluorescein iaothiocyanate(FITC) or Texas Red- (TR) labelled secondary amibochea for 2 h. VlsualiLation was commenced by means of a confocal laser microscope using a magnification of 10. to 10O-fold. Antibodies against PDE 3 and 4 were obtained from FabGcnnix Inc.. Shreveport, USA, PDES antibodies were kindly supplied by Tanabe Seiyaku Pharmaceutical Co., Osaka Japan. RESULTS: Positive immunostaining (TR) indicating the prescncc of PDE4 (CAMP PDE) was abundantly observed in the fibromuscular stroma, in glandular structures and in small vessels of the transitional zone. In contrast, PDE5 (cGMP PDE) itnmunostaining was seen in glandular and subglandular structures. No Positive FITC-reaction for PDE3 (cGMP-inhibited PDE) was detected. CONCLUSIONS: Our results confirm the presence of PDEs 4 and 5 in the transitional zone of the human prostate and presented evidence that these isoenzymes are not evenly distributed. The results from our study are in support the hypothesis that there might be a rationale for the use of PDE inhibitors in the pharmacotherapy of BPH and LUTS.
of
. C‘hagas M., Costa W.. SampaIn F
State Unlvcrsiry Bra/l1
of Klo dc Janewo. Urogenital
Research Unit.
Rio dc Janeiro,
INTRODUCTION & OBJECTIVES: Perform a quantitalibc analysis on the acini and the moditicatlons of the acmar epithelium of the transitional zone in normal and hyperplaqtic human prostates. MATERIAL & METHODS: BPH tissue samples of the transiflonal Lone were obtained from 20 patients with clmlcal symptoms of bladder outlet obstruction that were submitted to open prostatcctomy. The controls consisted of 20 transitional Lone of prostates obtained during necropsy of adults under 30 years old. The quantitative analysis consisted of the determination of the following parameters: number of acini. total acinar arca, arca of the lumen, epithclial arca. and the minimum, maximum and mean cpithelial height, by using computed hlstomorphomelric techniques. RESULTS: The mot-phomctric analysis showed the l‘ollowing results, in mm2, for normal and hyperplastic prostates, respectively: total area of the acini=0.04 I *0.0076 and 0.056+0.0160 (p=O.O005). area of the lumen=0.016t0.0034 and 0.036~0.0131 (p=O.OOOl), area of the epithelium=0.025*0.0046 and 0.019*0.003X (p=O.OOOS). The evaluation of the cpithelial height showed the following results, in mm. for normal and hyperplastic prostates, respectively: minimum height = 9.92 i I .67 and 6.45 i I I4 (p=O.OOOl), maximum height=54.38*4.09 and 41.5214.5 1 (p=O.OOOl), median height=27.89&2.48 and 19.96i2.20 (p=O.OOOl). CONCLUSIONS: There 1s no statistically significant diffcrcncc in the number of acini between controls and BPH patients. The total area of the acini as well as Ihe luminal area presented a statistically significant increase in BPH. On the other hand, the area and the height of the acinar epithelium presented a statistically significant decrease in BPH. ** Supported by Grants from the National Council of Scientific and Technological Development (CNPq-Brazil), and from the Foundation for Research Support of Rio de Janeiro (FAPERJ). European
Urology
Supplements
2 (2003)
No. 1, pp. 19