Peyronie's disease fibroblasts demonstrate tumorigenicity in the severe combined immunodeficient (SCID) mouse model.

Peyronie's disease is a localized connective tissue disorder, caused by trauma to the erect penis, which results in cellular proliferation and excess extracellular matrix production within the tunica albuginea of the penis. We have previously demonstrated that cells derived from Peyronie's disease plaque tissue demonstrate increased cell growth, increased S-phase on flow cytometry, stabilization and inactivation of p53, and consistent morphologic transformation, all suggesting that these cells are biologically transformed. Severe combined immunodeficient (SCID) mice have been used extensively to study the pathobiology of malignant and benign tissue and cells. This study was undertaken to determine if Peyronie's derived fibroblasts had the potential to demonstrate tumorigenicity in the SCID mouse model, thus confirming their biologically transformed nature. Cultured fibroblasts were derived from three sources, namely, plaque tissue excised from men with Peyronie's disease, tunical tissue excised from young men with congenital penile curvature and neonatal foreskins. BALB/C SCID mice were divided into three groups and each group was inoculated with cultured fibroblasts from each of the three different sources. All animals were evaluated regularly and maintained in isolation for a period of 3 months following inoculation. All SCID mice inoculated with cells derived from Peyronie's disease plaque tissue (n=10) developed subcutaneous nodules at a mean time period of 2.5+/-0.5 months following injection. The mean maximum dimension and weight of the nodules at the time of killing the animal was 1.1+/-0.2 cms and 0.6+/-0.2 g, respectively. Histologically, the nodules were composed of large pleomorphic epithelioid cells with a high mitotic activity, which were negative for cytokeratin but positive for vimentin. None of the SCID mice inoculated with cells cultured from either normal tunica (n=5) or foreskin (n=5) developed subcutaneous nodules. In conclusion, the tumorigenic nature of Peyronie's disease plaque-derived fibroblasts sheds further light on the pathobiologic characteristics of these cells. Specifically, these data confirm that cells cultured from Peyronie's disease plaque are biologically transformed. Future refinement and study of this animal model may permit a more complete understanding of the pathophysiology of Peyronie's disease and fibromatoses in general. Furthermore, such an animal model may, in the future, allow a more ready evaluation of the therapeutic interventions for Peyronie's disease.

Chromosomal instability is demonstrated by fibroblasts derived from the tunica of men with Peyronie's disease.

Peyronie's disease is a fibrotic disorder, a condition characterized by cellular proliferation and excess extracellular matrix production. Previous work in related conditions has demonstrated chromosomal instability. This investigation was undertaken to analyze fibroblasts derived from Peyronie's disease tunical tissue for abnormalities of chromosome number and progression of cytogenetic aberrations during cell culture. Tunical tissue was excised from men with Peyronie's disease from both plaque and nonplaque tissue and cells were explanted in culture. Control cells were derived from both neonatal foreskins and normal tunica from men with congenital penile curvature. Fluorescent in situ hybridization was used to probe for chromosomes 7, 8, 17, 18, X and Y. Control cells demonstrated normal copy number for all chromosomes analyzed. In contrast, Peyronie's disease plaque-derived fibroblasts demonstrated frequent aneusomies in chromosomes 7, 8, 17, 18 and X and recurrent deletions of chromosome Y. Peyronie's disease nonplaque tunica-derived fibroblasts demonstrated infrequent chromosomal changes early in culture; however, with repeated passaging the majority of cell cultures demonstrated aneusomies in at least one chromosome. These data indicate that Peyronie's disease plaque-derived fibroblasts have consistent aneusomies even at early passage and that nonplaque tunica-derived cells from men with Peyronie's disease also demonstrate chromosomal instability. This suggests that the tunica albuginea of men with Peyronie's disease may be predisposed to undergoing unregulated fibrosis. These findings confirm the transformed nature of the Peyronie's disease tunical fibroblasts studied in this analysis. While the etiology of these findings is not clear, it is likely that these pathobiological characteristics contribute to the pathophysiology of this disease process.

Peyronie's disease cell culture models: phenotypic, genotypic and functional analyses.

Peyronie's disease is a fibromatosis of the tunica albuginea. While trauma is believed to be the inciting event, the exact pathophysiology of this condition is unknown. In vitro analysis of cell biology can shed light on the pathogenesis of medical conditions and has been used for many decades as a research tool. We have established a cell culture model, which we have used to study the pathobiology of cells derived from Peyronie's disease plaque tissue. In 10 separate cell cultures derived from different individuals, these cells have demonstrated consistent phenotypic, genotypic and functional alterations. In neither of the control cell cultures, neonatal foreskin fibroblasts and normal tunica-derived fibroblasts have any of the above aberrations been demonstrated. The cells studied have been shown to be fibroblasts in nature with a sub-population of myofibroblasts present in culture. The Peyronie's disease plaque tissue-derived fibroblasts have demonstrated (i) consistent morphologic transformation (ii) increased S-phase on flow cytometry (iii) decreased dependence on culture medium (iv) cytogenic instability (v) excess production of fibrogenic cytokines and (vi) stabilization and dysfunctionalization of p53. Further refinement of this model and future analyses may permit an increased understanding of the pathogenesis of this condition and allow the development of therapeutic strategies.

Basic fibroblast growth factor expression in Peyronie's disease.

PURPOSE: Peyronie's disease is a fibromatosis resulting in scarring of the tunica albuginea. While the inciting event is believed to be trauma to the erect penis, little is understood about the cascade of cellular events that leads to the formation of the plaque. Dysregulated wound healing serves as a paradigm for the study of this condition. Previous work has demonstrated a role for fibrogenic cytokines in wound healing, fibromatoses, including Peyronie's disease. We analyze the expression of the fibrogenic cytokine, basic fibroblast growth factor (FGF), by fibroblasts derived from Peyronie's disease tissue. MATERIALS AND METHODS: Patients with Peyronie's disease undergoing either penile prosthesis insertion or Nesbit penile plication surgery had biopsy specimens removed from the plaque and from normal tunical tissue remote from the plaque. Cell cultures were derived from these specimens. Cultured cells were characterized using immunofluorescence staining and immunosorbent digital imaging. The cell culture supernatants were analyzed using an enzyme-linked immunosorbent assay for the production of basic FGF. Foreskin tissue from men without Peyronie's disease was used as control cells. RESULTS: Five independent cell lines were established from plaque tissue and 4 independent cell lines were established from normal tunica from the same subjects. Intracellular antigen expression was consistent with the cells being myofibroblasts. Production of basic FGF by the plaque derived myofibroblasts was significantly greater compared to production by normal tunical myofibroblasts and foreskin fibroblasts. CONCLUSIONS: These data demonstrate the successful establishment of cell lines from plaque tissue and normal tunica from men with Peyronie's disease. The findings indicate a potential role for basic FGF over expression in the tunical fibrosis that occurs in this condition. This information may allow a better understanding of the basic mechanisms involved in the development of this disease. Furthermore, it may permit the elaboration of therapeutic strategies to prevent or reduce tunical scarring and plaque formation.

Perturbation of cell cycle regulators in Peyronie's disease.

Peyronie's disease is a fibromatosis of the tunica albuginea, characterized by development of a plaque consisting primarily of collagen. It has been suggested that trauma to the erect penis is the inciting event. More recent research has focused on the cellular events leading to the dysregulated wound healing and plaque formation. Previous work has shown chromosomal aneusomies and this combined with an increased S-phase in plaque derived cell cultures suggests a perturbation in the cell cycle in this condition. The p53 protein has been shown to be an important cell cycle regulator and pro-apoptotic factor. Aberrant p53 function leading to cell immortalization and proliferation has been implicated in several human malignancies. We hypothesized that abnormal p53 function may explain the high proliferative ability of fibroblasts derived from Peyronie's plaques. This study was undertaken to study the presence and function of p53 and its downstream elements (p21, mdm-2) in Peyronie's disease cell cultures. Plaque-derived fibroblasts have been established in culture and characterized. These cells and control neonatal foreskin fibroblasts were subjected to 5 Gy of gamma radiation to induce DNA damage. After fixation, antibodies to p53 and its transcriptional elements were used to stain irradiated and non-irradiated cells and levels of p53, p21 and mdm-2 were quantified using combined immunofluorescence and flow cytometry. Non-irradiated plaque fibroblasts demonstrated the presence of p53, p21 and mdm-2 at baseline. In control foreskin fibroblasts no p53 or mdm-2 were detectable at baseline. In irradiated foreskin-derived cells significant changes in all elements were demonstrated indicating a fully functional p53 pathway and cell cycle checkpoint system in these cells. In contrast, plaque-derived cells showed no such alterations in levels of cell cycle regulators following irradiation. This is highly suggestive of an aberration of the p53 pathway in plaque-derived fibroblasts. Peyronie's plaque-derived fibroblasts demonstrated stabilization and defunctionalization of p53 protein combined with appropriate responses of its transcriptional elements. These findings may explain the high cell proliferation rates in these cells and suggests a role for perturbation of the p53 pathway in the pathogenesis of Peyronie's disease.

Inhibition of Peyronie's plaque fibroblast proliferation by biologic agents.

Peyronie's disease is a fibromatosis of the tunica albuginea which affects up to 2% of men. Plaque development is believed to result, at least in part, from fibroblast proliferation and excess collagen deposition. Numerous oral and intralesional therapies have been used, including verapamil, colchicine and steroids. The purpose of this study was to investigate the in vitro effects of prostaglandin-E1 (PGE1), verapamil and colchicine on the proliferation rates of fibroblasts derived from Peyronie's disease tissue. Using tissue culture, multiple cell lines comprising fibroblasts from Peyronie's plaque, normal tunica and foreskin were established. Cells of low passage were removed from the parent culture and incubated with varying concentrations of PGE1 (0.1-10 mg/ml), verapamil (10-1000 mg/ml), and colchine (2.5 mg/ml). Proliferation was assessed at 48, 72 and 96 hours using the Vybrant MTT cell proliferation and then compared to control cells. Six plaque lines and 5 normal tunical cell lines were established. These cell lines exhibited excellent linear growth in culture media alone. Co-culture wih PGE1 resulted in no significant inhibition at 0.1 and 1 mg/ml, but a mean inhibition of 60.6+/-11.5% at a concenrtation of 10 mg/ml was noted. Similar inhibition was noted with verapamil at 100 and 1000 mg/ml with a mean inhibition of 65.2+/-10.6%. Colchicine resulted in a mean inhibition of 28% at a concentration of 2.5 mg/ml. Maximum inhibition occurred at 96 hours in all cases. There was no statisitically significant difference in proliferation rates between plaque and normal tunical cell lines. We have developed an in vitro model to assess the effects of biologically active agents on the growth of fibroblasts derived from Peyronie's disease tissue. Our data suggests that PGE1, verapamil, and colchicine inhibit in vitro proliferation of fibroblasts at specific concentrations. Refinement and application of this knowledge may allow the development of useful pharmacologic strategies for men with PD.

[The surgical treatment of strangulated inguinal-crural hernias in geriatric patients]. / Il trattamento chirurgico delle ernie inguino-crurali strozzate nei pazienti in età geriatrica.

The authors describe their own experience relative to 79 patients, aged 70 years or older, who underwent, during three years, emergency surgical intervention for inguinal or crural strangulated hernia. They report a postoperative mortality rate of 8.8% and a postoperative morbidity rate of 40%. They stress the need for timely diagnosis and a meticulous surgical treatment. Therefore, they underline the great importance of the anesthesiologic approach and perioperative intensive care.

[Inguinal hernia containing bladder and ureter. A clinical case]. / L'ernia inguinale a contenuto vescico-ureterale. Contributo clinico.

The authors describe an uncommon case of inguinal hernia with bladder and ureter content. Bladder herniation preoperative diagnosis has been achieved by means of clinical history, objective and instrumental examination (cystography). As usually happens, ureteral herniation was a chance finding; this could involve a trick in surgery setting up and doubts in the treatment methods.

[Acute intestinal ischemia. Diagnosis and surgical treatment]. / Ischemia intestinale acuta. Diagnosi e trattamento chirurgico.

The authors report their experience of 32 patients operated for acute intestinal ischemia. A massive intestinal infarction was diagnosed in 24 cases. The overall postoperative mortality rate was 72%. The postoperative survival rate was 20% after mesenteric infarction and 42% in patients with limited acute intestinal ischemia. The need for early specific diagnosis is stressed, because the therapeutic options vary widely in relation to different types of acute intestinal ischemia. Furthermore, the basic role of parenteral nutrition in postoperative treatment of short bowel syndrome is underlined.

Identification of two different point mutations associated with the fluoride-resistant phenotype for human butyrylcholinesterase.

The fluoride variant of human butyrylcholinesterase owes its name to the observation that it is resistant to inhibition by 0.050 mM sodium fluoride in the in vitro assay. Individuals who are heterozygous for the fluoride and atypical alleles experience about 30 min of apnea, rather than the usual 3-5 min, after receiving succinyldicholine. Earlier we reported that the atypical variant has a nucleotide substitution which changes Asp 70 to Gly. In the present work we have identified two different point mutations associated with the fluoride-resistant phenotype. Fluoride-1 has a nucleotide substitution which changes Thr 243 to Met (ACG to ATG). Fluoride-2 has a substitution which changes Gly 390 to Val (GGT to GTT). These results were obtained by DNA sequence analysis of the butyrylcholinesterase gene after amplification by PCR. The subjects for these analyses were 4 patients and 21 family members.

Lack of [3H]quinuclidinyl benzylate binding to biologically relevant binding sites on mononuclear cells.

We analyzed the binding characteristics of [3H]quinuclidinyl benzylate ([3H]QNB), a muscarinic cholinergic ligand, to rat and human mononuclear cells (MNC). Under various assay conditions, atropine-sensitive, saturable binding occurred with an apparent Kd of 10 nM. Conditions which disrupted the MNC membrane reduced total binding and eliminated specific binding. Muscarinic agonists were unable to inhibit [3H]QNB binding to MNC at concentrations up to 10(-2) M. Stereoisomers dexetimide and levetimide were equipotent inhibitors of binding (IC50 2 x 10(-5) M). We conclude that, although atropine-sensitive binding of [3H]QNB to MNC occurs, the binding is not consistent with the presence of a biologically relevant muscarinic cholinergic receptor.

DNA mutation associated with the human butyrylcholinesterase K-variant and its linkage to the atypical variant mutation and other polymorphic sites.

Genomic DNA from two families exhibiting the K-variant phenotype of serum butyrylcholinesterase was amplified by PCR and sequenced to determine the molecular basis of this variant. The K-variant phenotype was found to be associated with a DNA transition from guanine to adenine at nucleotide 1615, which caused an amino acid change from alanine 539 to threonine (GCA----ACA; Ala539----Thr). There was a 30% reduction of serum butyrylcholinesterase activity associated with this mutation. Amplification and sequencing of DNA from a random sample of 47 unrelated people gave a frequency of .128 for the K-variant allele. Thus, 1 person in 63 should be homozygous for the K-variant, making the K-variant the most common butyrylcholinesterase variant. The K-variant mutation was also found to be present in 17 (89%) of 19 butyrylcholinesterase genes containing the point mutation which causes the atypical phenotype of butyrylcholinesterase (GAT----GGT; Asp70----Gly). The presence of the K-variant in the same molecule as the atypical variant does not contribute to the qualitative change in the atypical enzyme, but it most likely accounts for the approximately one-third reduction in Vmax of butyrylcholinesterase activity in atypical serum. Two additional point mutations located in noncoding regions of the gene were also observed to be in linkage disequilibrium with the K-variant mutation. As many as four different point mutations have been identified within a single butyrylcholinesterase gene. Inhibition tests of the enzyme in plasma are usually used to distinguish the K-variant from the usual enzyme when the former is present with the heterozygous atypical variant (AK phenotype vs. UA phenotype). Inhibition tests were performed on plasma enzyme from the four possible genotypic combinations of the heterozygous atypical mutation with or without the K-variant mutation on either allele; we found that the AK phenotype was caused by three genotypes (A/K, AK/K, and U/A) and that the UA phenotype was caused by two genotypes (U/A and U/AK).

Subcellular distribution of hypothalamic prolactin-like immunoreactivity.

Prompted by interest in immunohistochemical reports of prolactin-like immunoreactivity (PLI) in the rat hypothalamus, we investigated and have reported that an immunoreactive and bioactive prolactin-like material can be extracted from the rat hypothalamus. In the present communication the subcellular distribution of this protein is reported. Using a sensitive and specific radioimmunoassay for rat prolactin and a standardized procedure for subcellular fractionation of neuronal tissue, we have found that 90% of hypothalamic PLI is particulate-bound with only 10% remaining in the S4 or cytosolic fraction. Almost 80% of the particulate-bound PLI is found in the P2 fraction containing myelin, synaptosomes and mitochondria. When P2 is further fractioned on a discontinuous sucrose density gradient, approximately 66% of the P2-associated PLI was found in subfractions rich in synaptosomes and poor in myelin and mitochondria. Such findings support the probability that hypothalamic PLI functions trans-synaptically as a neuromodulator in the brain.

Acetylcholinesterase in cultured human leukemia/lymphoma cell lines.

Fifty-two cultured leukemia/lymphoma cell lines were studied for their acetylcholinesterase activity. There was a striking effect of maturity on enzyme activity, only the most mature cells showing significant activity. Mature T cells exhibited far more enzyme activity than mature B cells, paralleling results on normal T and B cells.