Ivan Krhen

The Transcriptional Co-Activator cAMP Response Element-Binding Protein-Binding Protein Is Expressed in Prostate Cancer and Enhances Androgen- and Anti-Androgen-Induced Androgen Receptor Function

Progression of human prostate cancer toward therapy resistance occurs in the presence of wild-type or mutated androgen receptors (ARs) that, in some cases, exhibit aberrant activation by various steroid hormones and anti-androgens. The AR associates with a number of co-activators that possess histone acetylase activity and act as bridging molecules to components of the transcription initiation complex. In previous reports, it was shown that the transcriptional co-activator CREB (cAMP response element-binding protein)-binding protein (CBP) enhances AR activity in a ligand-dependent manner. In the present study, we have investigated whether CBP modifies antagonist/agonist balance of the nonsteroidal anti-androgens hydroxyflutamide and bicalutamide. In prostate cancer DU-145 cells, which were transiently transfected with CBP cDNA, hydroxyflutamide enhanced AR activity to a greater extent than bicalutamide in the presence of either wild-type or the mutated AR 730 val-->met. In two sublines of LNCaP cells that contain the mutated AR 877 thr-->ala and overexpressed CBP, increase in AR activity was observed after treatment with hydroxyflutamide but not with bicalutamide. Anti-androgens did not influence AR expression in cells transfected with CBP cDNA, as judged by Western blot analysis. Endogenous CBP protein was detected by Western blot in nuclear extracts from the three prostate cancer cell lines, LNCaP, PC-3, and DU-145, all derived from therapy-resistant prostate cancer. In addition, CBP was expressed in both basal and secretory cells of benign prostate epithelium, high-grade prostate intraepithelial neoplasia, and prostate cancer clinical specimens, as evidenced by immunohistochemical staining. Taken together, our findings demonstrate the selective enhancement of agonistic action of the anti-androgen hydroxyflutamide by the transcriptional co-activator CBP, which is a new, potentially relevant mechanism contributing to the acquisition of therapy resistance in prostate cancer.

Loss of heterozygosity of thenm23-H1 gene in human renal cell carcinomas

This study evaluates the potential contribution of thenm23-H1 gene to malignant transformation in patients with renal cell carcinoma. Using specific oligonucleotide primers for thenm23-H1 microsatellite repetitive sequence, gene instability was followed by polymerase chain reaction/loss of heterozygosity assay on 54 tumor specimens and the corresponding normal tissue samples. We also determined, immunohistochemically, the relative concentration and localization of thenm23-H1 protein product. From 77.7% informative cases, DNA from 6 tumors exhibited loss of heterozygosity, regardless of the tumor stage (TNM). Out of 39 samples analyzed, 30 were negative for Nm23-H1 protein, while the others were only slightly positive. No correlation with tumor stage was found. Normal renal tissue was also negative for this protein. Our results provide the evidence for loss of heterozygosity, followed by means of microsatellite tandem-repeat polymorphism, at thenm23-H1 locus in renal cell carcinoma. However, since no correlation was found between the tumor stage or metastatic potential on the one hand, and allelic loss and specific protein expression on the other, it seems thatnm23-H1 does not play a key role in the invasiveness of this tumor type.

Laparoscopic partial nephrectomy with diode laser: a promising technique

OBJECTIVE The aim of this study was to evaluate application of diode laser in laparoscopic partial nephrectomy (LPN), and to question this technique in terms of ease of tumor excision and reduction of warm ischemia time (WIT). BACKGROUND DATA LPN is the standard operative method for small renal masses. The benefits of LPN are numerous, including preserving renal function and prolonging overall survival. However, reduction of WIT remains main challenge in this operation. In order to shorten WIT, many techniques have been developed, with variable results. PATIENTS AND METHODS We performed a prospective collection and analysis of health records for patients who were operated on between March 2011 and August 2012. Inclusion criteria were single tumor ≤ 4 cm, predominant exophytic growth and intraparenchymal depth ≤ 1.5 cm, with a minimum distance of 5 mm from the urinary collecting system. RESULTS We operated on 17 patients. Median operative time was 170 min. In all but two patients, we had to perform hilar clamping. Median duration of WIT was 16 min. Pathohistological evaluation revealed clear cell renal cancer and confirmed margins negative for tumor in all cases. Median size of the tumor was 3 cm. Median postoperative hospitalization was 5 days. Average follow up was 11.5 months. There were no intraoperative complications. One postoperative complication was noted: perirenal hematoma. CONCLUSIONS Laser LPN is feasible, and offers the benefit of shorter WIT, with effective tissue coagulation and hemostasis. With operative experience and technical advances, WIT will be reduced or even eliminated, and a solution to some technical difficulties, such as significant smoke production, will be found.

Trends in prostate cancer incidence and mortality in Croatia, 1988-2008.

Aim To describe and interpret prostate cancer incidence and mortality trends in Croatia between 1988 and 2008. Methods Incidence data for the period 1988-2008 were obtained from the Croatian National Cancer Registry. The number of prostate cancer deaths was obtained from the World Health Organization mortality database. We also used population estimates for Croatia from the Population Division of the Department of Economic and Social Affairs of the United Nations. Age standardized incidence and mortality rates were calculated by the direct standardization method. To describe time trends of incidence and mortality, joinpoint regression analysis was used. Results Average age-standardized incidence rate between the first and last five-year period doubled, from 19.0/100 000 in 1988-1992 to 39.1 per 100 000 in 2004-2008. Age-standardized mortality rate increased by 6.9%, from 14.5 to 15.5 per 100 000. Joinpoint analysis of incidence identified two joinpoints. The increasing incidence trend started from 1997, with the estimated annual percent of change (EAPC) of 12.9% from 1997-2002 and of 4.1% from 2002-2008. Joinpoint analyses of mortality identified one joinpoint. Mortality trend first decreased, with EAPC of -3.0% from 1988-1995 to increase later with EAPC of 2.0% from 1995-2008. Conclusion The incidence of prostate cancer in Croatia has been on the increase since 1997. Trend in mortality is increasing, contrary to the trends in some higher-income countries. An improvement in the availability of different treatment modalities as well as establishing prostate cancer units could have a positive impact on prostate cancer mortality in Croatia.

Antibodies to Tamm-Horsfall protein in patients treated with extracorporeal shock wave lithotripsy (ESWL)

The aim of this study was to determine antibodies to Tamm-Horsfall protein in patients with nephrolithiasis treated with extracorporeal shock wave lithotripsy (ESWL). The values of antibodies to Tamm-Horsfall protein were determined by direct enzyme immunoassay. No statistically significant differences (P > 0.05) were observed for the IgG and IgM classes of antibodies between the groups of healthy subjects and patients with nephrolithiasis before, and 30 and 60 days after ESWL. The values of IgA class determined 30 days after treatment were significantly higher (P < 0.05) in patients, which could be due to the stimulation of the immune system. The highest values of antibodies to Tamm-Horsfall protein were obtained in both groups in the test with secondary antibodies directed toward IgM class, implicated at the presence of cross-reactive antibodies. Determination of antibodies to THP subunits isolated form urine of patients with nephrolithiasis should be performed.

“Open” Biopsy of the Testis and Testicular Sperm Extraction

Modern reproductive medicine offers new possibilities for treating infertility in men, including, among other approaches, the freezing of gametes or parts of the male or female sex gonad. In fertility practice, clinical indications for cryopreservation include storage of spermatozoa, testicular and ovarian tissues, and early embryos. Apart from freezing spermatozoa, the introduction of intracytoplasmic sperm injection into the oocyte (ICSI) in the late 1980s also created a need to freeze the testicular parenchyma. In the ICSI procedure, an oocyte is fertilized by injecting a single spermatozoon. Men with a low spermatozoa count, low spermatozoa motility, or a lack of healthy spermatozoa must provide a sample with a few viable male gametes for ICSI. Several techniques for spermatozoa retrieval from the epididymis and testis have been described and may successfully be combined with ICSI, including microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), testicular spermatozoa aspiration (TESA), and testicular spermatozoa extraction (TESE). TESE has been used to treat cases of obstructive and nonobstructive azoospermia. Thus, frozen–thawed pieces of testicular tissue are usually used for TESE in the case of severe male infertility, including in men with azoospermia and testicular tumors. If the tumor is not too large and the surrounding testicular parenchyma is relatively preserved, one may obtain multiple testicular biopsy samples from that area and use them for TESE/ICSI.

OCT 4 immunohistochemistry in postpubertal cryptorchidism

Patients with cryptorchidism are at an increased risk for germ cell testicular cancer. OCT 4 has been shown to be a sensitive and specific marker for some types of germ cell testicular cancer. We undertook this study to establish whether OCT 4 immunohistochemistry is a useful tool in the pathohistologic evaluation of postpubertal patients with cryptorchidism. Seventeen postpubertal patients underwent orchidectomy for cryptorchidism at our center since 1997. Immunohistochemical staining with OCT 4 was performed on these samples. Characteristic OCT 4 nuclear staining was positive in two patients. One patient was correctly diagnosed on previous pathohistological evaluation, while OCT4 immunohistochemical staining revealed previously unidentified intratubular germ cell neoplasia in the other patient. OCT 4 immunohistochemistry can be useful in diagnosing a testicular germ cell tumor in patients with cryptorchidism. If we consider a low number of postpubertal patients with cryptorchidism a benefit of immunohistochemical staining with OCT4, this could favor the use of OCT 4 staining in work-up of cryptorchidism.

The role of chlamydia trachomatis in the etiology of chronic prostatitis

INTRODUCTION & OBJECTIVES: Basic factors for the classification of prostatitis syndrome are clinical symptoms and signs and the presence of bacteria and leukocytes in selectively collected urine samples and in EPS by Meares and Stamey localisation technique. The aim of this study was to investigate the etiology of chronic bacterial prostatitis and inflammatory chronic pelvis pain syndrome i.e. nonbacterial prostatitis. MATERIALS & METHODS: We examined a total of 388 patients, older than 18 years of age, with symptoms of chronic prostatitis. The inclusion criteria for chronic bacterial prostatitis was as follows: - a bacterial count of 103 cfu/ml or more (if only Gram-positive cocci are found in EPS, a bacterial count of 10 x 104 cfu/ml or more is required), and 10 or more WBCs/hpf (including macrophages) in EPS or VB3 -finding of 10 or many times greater number of bacteria in EPS and urine bladder sample collected immediately after prostatic massage, than in first voided urine or midstream urine. The inclusion criteria for Chlamydia trachomatis prostatitis was the presence of 10 or more WBCs/hpf in EPS or VB3, the presence of Chlamydia trachomatis in EPS or VB3, absence of Chlamydia trachomatis in urethral swabs and other possible pathogens of chronic prostatitis in VB1, VB2, EPS or VB3. The inclusion criteria for nonbacterial prostatitis or inflammatory chronic pelvis pain syndrome was the isolation of Ureaplasma urealyticum, Mycoplasma hominis or Trichomonas vaginalis and 10 or more WBCs/hpf in EPS of VB3. RESULTS: Disease etiology was determined in 276 (71, 13%) patients. Chlamydia trachomatis was proved to be causative in 109 patients, Trichomonas vaginalis in 52, Escherichia coli in 26, Enterococcus in 25, Proteus mirabilis in 14, Klebsiella pneumoniae in 6, Streptococcus agalactiae in 8, Ureaplasma urealyticum in 7patients with chronic prostatitis. Other patients with had mixed infection. CONCLUSION: Chlamydia trachomatis was proved to be the causative pathogen in one third of patients with chronic prostatitis. Since Chlamydia trachomatis is a bacterium, chlamydial prostatic infection should be defined as chronic bacterial prostatitis.

Gentamicin concentrations in urine, cortex and medulla in an acutely obstructed kidney animal model.

Abstract Gentamicin was administered intraperitoneally, three times in 12 h to Hartley type guinea-pigs which had undergone complete unilateral ureteral obstruction with normal contralateral ureteral function for either 24 hours, 7 days or 21 days. Two hours after the last drug dose urine samples were collected from urinary bladder and obstructed ureter. Healthy and obstructed kidneys were then surgically removed from all sacrificed animals. Gentamicin concentration in urine of healthy kidney was 112-266 μg/ml, and in obstructed kidney 18-53 μg/ml, with a tendency of linear decrease over a 3-week obstruction period. The gentamicin concentration in obstructed renal cortex never exceeded one-third of the gentamicin concentration in unobstructed renal cortex. The maximum gentamicin concentration in obstructed renal medulla was 75% of the gentamicin concentration in unobstructed renal medulla.