Nerve replacement strategies for cavernous nerves.

OBJECTIVE: This article reviews novel restorative therapies for cavernous nerves that may be used to replace resected cavernous nerves at the time of pelvic surgery. METHODS: A literature-based presentation (Medline search) on current nerve replacement strategies was conducted with emphasis on neurobiological factors contributing to the restoration of erectile function after cavernous nerve injuries. RESULTS: A promising alternative to autologous nerve grafts for extending the length of successful nerve regeneration are artificial nerve guides. The addition of neurotrophic factors, extracellular matrix components and Schwann cells has been shown to promote cavernous nerve regeneration. Neurotrophic factors can be incorporated in the scaffold or can be supplied by cells seeded into the stroma. The regenerative capacity of these cells can be further enhanced by genetic modification with neurotrophic factor encoding genes. CONCLUSIONS: Artificial nerve guides, especially biodegradable ones containing growth-promoting factors or cells, are a promising option for the repair of cavernous nerve lesions.

[Tissue engineering of erectile nerves]. / Tissue Engineering erektiler Nerven.

Dissection of the cavernous nerves eliminates spontaneous erections and may lead to irreversible erectile dysfunction due to degeneration of cavernous tissue. Novel procedures to reconstruct penile innervation include cavernous nerve interposition grafting and neurotrophic treatments to revitalize penile neural input, evaluated thus far in various preclinical models of cavernous nerve injury. Schwann cells crucially contribute to successful axonal regeneration by mechanical and paracrine mechanisms in the injured nerve, and Schwann cells seeded into guidance channels have been successfully employed to support regeneration in animal models of cavernous nerve injury. Gene therapy, tissue engineering, and reconstructive techniques have been combined to deliver neurotrophic factors and recover erectile function.

Schwann cell seeded guidance tubes restore erectile function after ablation of cavernous nerves in rats.

PURPOSE: Dissection of the cavernous nerves eliminates spontaneous erections. We evaluated the ability of Schwann cell seeded nerve guidance tubes to restore erections after bilateral cavernous nerve resection in rats. MATERIALS AND METHODS: Sections (5 mm) of the cavernous nerve were excised bilaterally, followed by immediate bilateral microsurgical reconstruction. In 10 animals per group (20 study nerves) reconstruction was performed by genitofemoral nerve interposition, interposition of silicone tubes or interposition of silicone tubes seeded with homologous Schwann cells. As the control 10 animals (20 study nerves) underwent sham operation (positive control) and bilateral nerve ablation (without reconstruction) was performed in a further 10 (negative control). Erectile function was evaluated 3 months postoperatively by relaparotomy, electrical nerve stimulation and intracavernous pressure recording. RESULTS: After 3 months neurostimulation resulted in an intact erectile response in 90% (18 of 20) of Schwann cell grafts, while treatment with autologous nerves (30% or 6 of 20) or tubes only (50% or 10 of 20) was less successful (p <0.01). Whereas untreated ablated rats showed no inducible erections (0% or 0 of 20), all sham operated animals had an intact erectile response (100% or 20 of 20). Maximum intracavernous pressure upon electrostimulation was significantly elevated using Schwann cell grafts compared to results in the other treatment groups (p <0.001). Morphological evaluation revealed advanced regeneration within Schwann cell grafts. CONCLUSIONS: Schwann cell seeded guidance tubes restore erectile function after the ablation of cavernous nerves in rats and they are superior to autologous nerve grafts.

New ultradeformable drug carriers for potential transdermal application of interleukin-2 and interferon-alpha: theoretic and practical aspects.

Transfersomes (TFs) are highly deformable hydrophilic lipid vesicles that are able to penetrate the skin barrier spontaneously because of their characteristics. Transfersomes are able to transport noninvasively low- and high-molecular-weight molecules into the body. We describe the formulation and several biologic characteristics of interleukin-2 (IL-2)- and interferon-alpha (IFNalpha )-containing TFs. TFs contain natural phosphatidylcholine and sodium cholate. Recombinant human IL-2 and human hybrid IFNalpha were added to TFs and incubated for 24 hours at 4 degrees C. Immunotransfersomes were isolated from free IL-2 and IFNalpha by filtration (Centrisart, Sartorius). The biologic activity of immunotransfersomes was measured by a cytotoxic lymphoid line assay for IL-2 and by an A549-encephalomyocarditis virus assay for IFN; concentrations of proteins were determined by the enzyme-linked immunosorbent assay (ELISA). It was possible to incorporate a large amount of IL-2 and IFN in TFs (75-80%), and the incorporated IL-2, and IFN were biologically active. The increased lipid/protein ratio (90.9/1.0) led to a growing probability of association. We were thus able to show that IL-2 and IFN are trapped by transfersomes in a biologically active form and in sufficient concentrations for immunotherapy. In upcoming experiments these IL-2- and IFN-containing TFs will be used for a transdermal approach in the murine RENCA cell line model.

Formulation of interleukin-2 and interferon-alpha containing ultradeformable carriers for potential transdermal application.

INTRODUCTION: Transfersomes (TF) are new highly deformable hydrophilic lipid vesicles, which are able to spontaneously penetrate the skin barrier because of their characteristics. Transfersomes are able to transport non-invasively low as well as high molecular weight molecules into the body. We describe the formulation and several biological characteristics of Interleukin-2 and Interferon-a containing TF. MATERIAL AND METHODS: TF contain natural phosphatidylcholine and sodium cholate. Recombinant human IL-2 and human hybrid interferon-alpha A/D were added to TF and incubated for 24 hours at 4 degrees C. Immunotransfersomes were isolated from free IL-2 and IFN by filtration (Centrisart, Sartorius). Biological activity of immunotransfersomes was measured by CTLL-cell-assay for IL-2 and by A549--EMCV-assay for IFN, concentrations of proteins by ELISA. RESULTS: It has been possible to incorporate a high amount of IL-2 and IFN in TF (75-80%). Incorporated IL-2 and IFN were biological active. The increase of the proportion of lipid to protein to 90.9/1 led to growing probability of association. CONCLUSION: We were able to show, that IL-2 as well as IFN is trapped by transfersomes in biological active form and in sufficient concentrations for immunotherapy. In upcoming experiments these IL-2 and IFN-containing TF are used for a transdermal approach in the murine RENCA cell line model.

Clinical relevance of urokinase plasminogen activator, its receptor, and its inhibitor in patients with renal cell carcinoma.

BACKGROUND: Urokinase plasminogen activator (u-PA) plays a key role in the metastatic process by promoting plasmin mediated tissue degradation. Metastatic cell invasion requires localized proteolysis, which may be directed by u-PA receptor. The binding of u-PA and PAI-1 to the u-PA-receptor may cause internalization of the trimeric complex into the cell and activate a tyrosine-kinase. In a prospective study the u-PA, u-PA-R, and PAI-1 content in patients with renal cell carcinoma (RCC) and benign renal tissue were correlated with traditional prognostic factors such as the TNM staging, histologic grading, ploidy, and the clinical outcome of the patients. METHODS: One hundred fifty-two patients who underwent transperitoneal tumor nephrectomy for RCC were followed up for a mean of 23.9 months. u-PA, u-PA-R, and PAI-1 from the tumor tissue and corresponding benign renal tissue were quantified from detergent extracted tissue samples (1% Trinton-X-100 in triethanolamine-buffered saline) and measured with an enzyme-linked immunoadsorbent assay. RESULTS: PAI-1 significantly correlated with the prevalence of distant metastasis (M0: 10.04 vs. M1 23.79, P=0.02) and the development of new metastasis postoperatively (M0: 10.85 vs. M1 27.36, P=0.001). A cut-off level of 12 ng/mg protein for PAI-1 selected a group of patients at high risk for relapse. Forty-one patients had PAI-1 > 12 ng/mg with 6 relapses compared with 55 patients with PAI-1 < 12 ng/mg with 1 relapse during the follow-up. Content of mu-PA correlated with the development of distant metastases (log rank 4.32, P=0.037). A cut-off value of 0.84 ng/mg selected 2 groups: a group at high risk for metastasizing (u-PA > 0.84, n=11 with 9 events and a group at low risk (u-PA < 0.84 with 94 patients and 5 events). Applying a cut-off value of 0.85 for u-PA-R 2 groups could be discriminated: 31 patients had no relapse with u-PA-R < 0.85 and 18 had 3 recurrences with u-PA-R > 0.85 g/ml. CONCLUSIONS: u-PA, u-PA-R, and PAI-1 are strong and independent prognostic factors for predicting early relapse for RCC. Especially with PAI-1, a high and low risk group for disease free survival can be discriminated.

Prognostic value of urokinase plasminogen activator and plasminogen activator inhibitor-1 in renal cell cancer.

PURPOSE: Urokinase type plasminogen activator and its inhibitor plasminogen activator inhibitor are associated with invasion and formation of metastases in tumors. In a prospective study urokinase and plasminogen activator inhibitor-1 content in renal cell cancer and benign renal tissue was correlated with the traditional factors of TNM staging and grading as well as ploidy and actual clinical outcome of the patients. MATERIAL AND METHODS: A total of 152 patients, who underwent transperitoneal tumor nephrectomy for renal cell cancer, was followed for a mean of 23.9 months. Urokinase and plasminogen activator inhibitor-1 from the tumor tissue and corresponding benign renal tissue were quantified from detergent extracted tissue samples (1% triton-X = 100 in TBS) and measured with an enzyme-linked immunosorbent assay. RESULTS: Urokinase content correlated with the development of distant metastases (log rank 4.32, p = 0.037). Cutoff value was 0.84 ng/mg. A group of 11 patients were considered to be at high risk for metastases (9 events) based on urokinase greater than 0.84 ng/mg., while 94 patients were considered to be at low risk (5 events) with urokinase less than 0.84 ng/mg. Plasminogen activator inhibitor-1 significantly correlated with the prevalence of distant metastases (log rank 5.17, MO, 10.04 versus M1, 23.79, p = 0.02) and the development of new metastases postoperatively (MO, 10.85 versus M1, 27.36, p = 0.001). Cutoff value was 12 ng/mg. protein. A group of 41 patients were considered at high risk for relapse (6) based on plasminogen activator inhibitor-1 greater than 12 ng/mg. protein compared to 55 patients with plasminogen activator inhibitor-1 less than 12 ng/mg. protein with only 1 relapse during followup. CONCLUSIONS: Plasminogen activator inhibitor-1, the specific inhibitor of urokinase is a strong and independent prognostic factor in predicting early relapse of renal cell carcinoma. High and low risk groups for disease-free survival can be discriminated by plasminogen activator inhibitor-1 antigen content in the tumor tissue.

Activity of NK 611, a new epipodophyllotoxin derivative, against colony forming units from freshly explanted human tumours in vitro.

NK 611 is a new semisynthetic analogue of etoposide, which presumably also acts through inhibition of topoisomerase II, and has been found to be more potent against several cancer cell lines in vitro than etoposide. The objectives of our study were to determine the activity of NK 611 against freshly explanted clonogenic cells from human tumours and compare this agent with etoposide and other clinically useful agents. After exposure for 1 h in 45 evaluable tumour specimens, NK 611 showed clear concentration-dependent antitumour activity. At 51 microM, 49% of specimens were markedly inhibited. Using a long-term (21-28 day) exposure at 6.8 microM, 58% of 50 evaluable specimens were profoundly inhibited. At equimolar concentrations, NK 611 was as active as etoposide. Across all tumour types studied, NK 611 was as active as vinblastine, bleomycin, doxorubicin, 5-fluorouracil, mitomycin-C and cisplatin. Our results showed cross resistance to etoposide in the majority of specimens. Activity of NK 611 was greater with long-term exposure than with short-term exposure indicating schedule dependency. We conclude that NK 611 has a wide spectrum of in vitro antitumour activity. Since preliminary clinical information suggests that this drug is well tolerated at high doses, further development of this agent in Phase II trials with multiple dosing schedules is warranted.

Patterns of ploidy in renal cell carcinoma.

In a prospective study the patterns of ploidy in 119 kidneys from patients with renal cell carcinoma were analyzed by flow cytometry. A rate of DNA aneuploidy of 53% was observed in the investigated tumors. However, in 4% of the kidneys aneuploid cell distributions were also proven in macroscopically intact tissue far away from the tumor. Although we do not know at present, if these were benign, premalignant or malignant cell alterations, this observation should be considered for decisions regarding conservative renal surgery.

Adenosine triphosphate and adenosine diphosphate in human semen: correlation with sperm count and motility.

Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) have been correlated with traditional semen parameters such as forward motility and sperm count. ATP and ADP were determined by a bioluminescence assay using the luciferin-luciferase reaction. Short boiling of the ejaculate was performed to inactivate phosphatases (ATPases) in the seminal plasma and the sperm tails. Fresh and deep-frozen semen samples from 45 men with oligozoospermia (n = 22) and normal sperm count (n = 23) were evaluated. Freezing of the sperm for 12 weeks did not effect the ATP or ADP content in the spermatozoa as compared to fresh semen. ATP and ADP concentration was in the range of picomoles/microliter and showed a significant correlation with the number of normal sperm per milliliter and a less marked correlation with the sperm motility. ATP and ADP concentration in the spermatozoa can be measured relatively easy and is reproducible. Unlike the monotonous evaluation of sperm motility by a technician, this biochemical method provides an objective parameter for semen quality. These qualities suggest that this method could be a way to determine the fertilizing potential of semen and to relate this to actual pregnancy rates.

Activity of phagocytic granulocytes in patients with prostatic cancer.

Chemilumenescence (CL) occurs due to the phagocytosis of bacteria and of tumor cells by polymorphonuclear neutrophils (PMN). Levels of CL were measured in patients with prostatic cancer and from normal subjects. Patients with advanced disease (stage C, D) showed no elevated CL levels as compared to healthy individuals or patients with minimal disease (stage A, B). Following external radiation therapy in patients with stage A-C prostatic carcinoma high levels of CL were recorded. Estrogen medication also resulted in increased CL levels, while estramustine did not affect phagocytic activity. Intradermal BCG vaccination caused increased PMN activity. Progressive prostatic cancer in hormone treated patients was associated with increased CL as compared to patients with stable or regressive disease.

Natural killer cells in patients with renal cell cancer.

Cytotoxic activity of natural killer (NK) cells in peripheral blood was studied in 60 patients with renal cell carcinoma (RCC) by means of a 51Cr release assay against K 562 cells, homologous and autologous RCC. All patients were tested prior to surgery and thereafter followed up for 1 year. Immunostimulating agents as interferon, levamisole, OMPI, and various bacterial extracts were tested for their boosting capacity on NK cells. Patients with localized tumor showed increased activity in comparison to age-matched controls, whereas NK cell activity and interferon-boosted activity in patients with advanced disease and tumor spread was decreased. Augmentation of NK activity could only be achieved by interferon, while application of levamisole, OMPI, and bacterial extracts resulted rather in suppression of cytotoxic activity.

Simultaneous determination of six tumor markers in patients with prostatic carcinoma and bladder tumors.

Serum levels of fucosyltransferase (FT), phosphohexoseisomerase (PHI), tissue polypeptide antigen (TPA), Tennessee antigen (TAG), carcinoembryonic antigen (CEA) and prostatic acid phosphate (PAP) were determined in 75 healthy individuals and in 86 patients with prostatic carcinoma and 38 patients with bladder tumors. The discrimination capacities of the different markers were compared by using inverse distribution plots. At a rate of 5% false positive values the sensitivities for bladder tumors were: FT 30%, TPA 24%, CEA 16%, TAG 15%. The sensitivities for prostatic carcinoma were: PAP 63%, PHI 36%, TPA 18%, CEA 14%, TAG 14%.

Natural killer cell activity in patients with prostatic carcinoma and its in vivo boosting with bacillus Calmette-Guérin.

In patients with prostatic carcinoma, natural killer (NK) cell activity was monitored in a chromium release assay. Cells from the human myeloid cell line K 562 and from a human prostatic cancer cell line (PC 3) were used as target cells. Additionally, in vitro stimulation with beta-interferon was done in each sample. NK activity in patients with localized prostatic cancer was increased as compared to age-matched controls. Patients with advanced disease showed reduced levels of NK activity. Furthermore, hormonally treated patients in relapse had significantly lower activity than patients in remission or with stable disease. Hormone therapy was without major influence on NK activity. BCG vaccination as an adjuvant immunotherapy was done in a small group of patients. Baseline NK activity and interferon-stimulated activity were enhanced by BCG in most cases.