[Genital gender affirming surgery in trans men]. / Chirurgie génitale de masculinisation des hommes trans.

OBJECTIVE: Genital surgery for masculinisation in the context of gender incongruence is characterised by the reconstruction of male genitalia using multiple surgical techniques - free flap phalloplasty, pedicled flap phalloplasty or metaoidioplasty - which this article aims to describe. MATERIAL AND METHODS: A narrative review of the literature on these trans surgeries was carried out. RESULTS: Each technique has specific advantages and disadvantages, and does not produce the same results in terms of surgical risks, size of the reconstructed phallus, sensitivity, sexual function and urinary function. CONCLUSION: This surgical decision must therefore be shared with the person concerned, based on a clear understanding of their expectations and objectives. Long-term follow-up is recommended.

[Genital gender affirming surgery in trans women: Vulvo-vaginoplasty review]. / Chirurgie génitale d'affirmation de genre pour les femmes trans : revue des vulvo-vaginoplasties.

INTRODUCTION: A trans woman is a woman who was assigned male at birth, and who has a female gender identity. The majority are requesting a gender affirming genital surgery by vulvo-vaginoplasty. The objective is to review this surgery based on its history, then by presenting the different surgical techniques and their success and complication rates. MATERIAL AND METHODS: A narrative review was performed, based on a bibliography search with keywords from 2000 to 2022 on Pubmed. RESULTS: Vulvo-vaginoplasty for trans women began in 1931, and the first case series date from 1969. The procedure includes excision of scrotal skin, orchiectomy, clitoroplasty, urethroplasty, labioplasty, recto-vesico-prostatic dissection and creation of a vaginal cavity (performed by penile skin inversion and graft, intestine, or peritoneum). Vulvo-vaginoplasty by penile skin inversion (VPPI) is today the reference surgical technique. It represents the vast majority of surgeries performed with the longest follow-up. The majority of trans women are satisfied with the procedure aesthetically (90%) and functionally (80%), with an active sexuality. Major complications are rare (< 5%), they correspond to fistulas or vaginal stenosis. CONCLUSION: VPPI is the gold standard technique with satisfactory overall results, but long-term follow-up is requested.

Quality of life of patients treated for kidney stones 10-20mm in diameter in terms of the type of operation performed: A qualitative study.

INTRODUCTION: Urolithiasis is a common chronic disease whose effect on patients' quality of life (QOL) is considerable but depends on the treatment received, differing between types of surgery. Intrarenal stones can be treated with different techniques: extracorporeal shock wave lithotripsy (ESWL), flexible ureteroscopy (fURS), and mini percutaneous nephrolithotomy (mini-PCNL), with proportional success and complication rates. The aim of this study was to qualitatively explore the impact of the different techniques on patients' QOL and understand their experiences of treatment choices. METHODS: Patients treated for medium-sized kidney stones (10-20mm in diameter) were interviewed in a semi-structured manner. The interview data were transcribed and analyzed by theme according to consolidated criteria for reporting qualitative research (COREQ) guidelines. RESULTS: Data saturation was achieved after interviewing 15 patients. The mean interview time was 34min (standard deviation (SD), 6.8min). The mean patient age was 54 years (SD, 9.5 years). Eight patients underwent ESWL, 10 were treated with fURS, and 8 underwent mini-PCNL. Twenty-seven subthemes were coded and regrouped into eight major themes, namely: no sense of choice in the decision-making process for eleven patients; extremely negative experiences of double-J stents for fourteen patients; concern about the risk of recurrence or treatment failure for thirteen patients; complicated hygiene and dietary recommendations for nine patients; technique-dependent postoperative outcomes; relatively well-tolerated operations for thirteen patients; a poor experience of sick leave, often because of a double-J stent; different views regarding future operations. In fact, a third of patients would choose the most effective treatment, a third would choose the simplest procedure and the last third would trust their urologist. Patients' experiences of these operations are variable. CONCLUSION: Urologists must support their patients by presenting the different treatment options with clear, appropriate, and unbiased information. This should ensure patients take part in treatment decisions as part of a personalized treatment plan.

French AFU Cancer Committee Guidelines - Update 2022-2024: penile cancer.

OBJECTIVE: To update French oncology guidelines concerning penile cancer. METHODS: Comprehensive Medline search between 2020 and 2022 upon diagnosis, treatment and follow-up of testicular germ cell cancer to update previous guidelines. Level of evidence was evaluated according to AGREE-II. RESULTS: Epidermoid carcinoma is the most common penile cancer histology. Physical examination is mandatory to define local and inguinal nodal cancer stage. MRI with artificial erection can help to assess deep infiltration in cases of organsparing intention. Node negative patients (defined by palpation and imaging) will present micro nodal metastases in up to 25% of cases. Invasive lymph node assessment is thus advocated except for low risk patients. Sentinel node dynamic biopsy is the first line technique. Modified bilateral inguinal lymphadenectomy is an option with higher morbidity. 18-FDG-PET is recommended in patients with palpable nodes. Chest, abdominal and pelvis computerized tomography is an option. Fine needle aspiration (when positive) is an easy way to assess inguinal palpable node pathological involvement. Its results determine the type of lymphadenectomy to be performed (for diagnostic or curative purposes). Treatment is mostly surgical. Free margins status is essential, but it also has to be organ-sparing when possible. Brachytherapy and topic agents can cure in selected cases. Lymph node assessment should be synchronous to the removal of the tumour when possible. Limited inguinal lymph node involvement (pN1 stage) can be cured with the only lymphadenectomy. In case of larger lymph node stage, one should consider multidisciplinary treatment including chemotherapy and inclusion in a trial. CONCLUSIONS: Penile cancer needs demanding surgery to be cured, surrounded by chemotherapy in node positive patients. Lymph nodes involvement is a major prognostic factor. Thus, inguinal node assessment cannot be neglected.

French AFU Cancer Committee Guidelines Update 2022-2024: Adrenal tumor - Assessment of an adrenal incidetaloma and oncological management.

INTRODUCTION: The objective of this publication is to recall the initial work-up when faced with an adrenal incidentaloma and, if necessary, to establish the oncological management of an adrenal malignant tumor. MATERIAL AND METHODS: The multidisciplinary working group updated French urological guidelines about oncological assessment of the adrenal incidentaloma, established by the CCAFU in 2020, based on an exhaustive literature review carried out on PubMed. RESULTS: Although the majority of the adrenal masses are benign and non-functional, it is important to investigate them, as a percentage of these can cause serious endocrine diseases or be cancers. Malignant adrenal tumors are mainly represented by adrenocortical carcinomas (ACC), malignant pheochromocytomas (MPC) and adrenal metastases (AM). The malignancy assessment of an adrenal incident includes a complete history, a physical examination, a biochemical/hormonal assessment to look for subclinical hormonal secretion. Diagnostic hypotheses are sometimes available at this stage, but it is the morphological and functional imaging and the histological analysis, which will make it possible to close the malignancy assessment and make the oncological diagnosis. CONCLUSIONS: ACC and MPC are mainly sporadic but a hereditary origin is always possible. ACC is suspected preoperatively but the diagnosis of certainty is histological. The diagnosis of MPC is more delicate and is based on clinic, biology and imagery. The diagnosis of certainty of AM requires a percutaneous biopsy. At the end, the files must be discussed within the COMETE - adrenal cancer network (Appendix 1).

French AFU Cancer Committee Guidelines - Update 2022-2024: testicular germ cell cancer.

OBJECTIVE: Updated Recommendations for the management of testicular germ cell cancer. MATERIALS AND METHODS: Comprehensive review of the literature on PubMed since 2020 concerning the diagnosis, treatment and follow-up of testicular germ cell cancer (TGCT), and the safety of treatments. The level of evidence of the references was evaluated. RESULTS: The initial work-up for patients with testicular germ cell cancer is based on a clinical examination, biochemical (AFP, total hCG and LDH serum markers) and radiological assessment (scrotal ultrasound and thoracic-abdominal-pelvic [TAP] CT). Inguinal orchiectomy is the first therapeutic step whereby the histological diagnosis can be made, and the local stage and risk factors for stage I non-seminomatous germ cell tumours (NSGCT) can be determined. For patients with pure stage-I seminoma, the risk of progression is 15 to 20%. Therefore, surveillance in compliant patients is preferable; adjuvant chemotherapy with carboplatin AUC 7 is an option; and indications for para-aortic radiotherapy are limited. For patients with stage I NSGCT, there are various options between surveillance and a risk-adapted strategy (surveillance or 1 cycle of BEP [Bleomycin Etoposide Cisplatin] depending on the absence or presence of vascular emboli within the tumour). Retroperitoneal lymph node dissection for staging has a very limited role. The treatment for metastatic TGCT is BEP chemotherapy in the absence of any contraindication to bleomycin, for which the number of cycles is determined according to the prognostic risk group of the International Germ Cell Cancer Consortium Group (IGCCCG). Para-aortic radiotherapy is still a standard in stage IIA seminomatous germ cell tumours (SGCT). After chemotherapy, the size of residual masses should be assessed by TAP scan for NSGCT: retroperitoneal lymph node dissection is recommended for any residual mass of more than 1 cm, and all other metastatic sites should be excised. For SGCT, reassessment by 18F-FDG PET is required to specify the surgical indication for residual masses>3cm. Surgery is still rare in these situations. CONCLUSION: By adhering to TGCT management recommendations, excellent disease-specific survival rates are achieved; 99% for stage I and over 85% for metastatic stages.

[Isolated continent urinary diversion: An interesting or often regretted indication?] / Cystostomie continente isolée : une indication intéressante ou que l'on regrette souvent ?

INTRODUCTION: The aim of this study is to evaluate the percentage of patients developing vesical overactivity after the realization of a continent stoma without bladder enlargement. MATERIAL AND METHOD: This is a retrospective monocentric study between January 2007 and April 2021. Patients undergoing an isolated continent urinary diversion for neurological or non-neurological reasons were included. The data collected concerned the clinical symptoms of bladder overactivity and their treatment as well as the pre- and postoperative urodynamic information. RESULTS: During the period, 9 patients were included. The main indications for continent urinary diversion were patients with spinal cord injury. Clinical bladder overactivity was found postoperatively in 78% (7) of the patients, and detrusor overactivity in 57% (4) of them. The main symptoms were urinary incontinence (67%) and pollakiuria (56%, 5 patients). Most of the patients benefited from associated treatments (78% pre- and postoperative), mainly anticholinergic drugs. No patient required botulinum toxin injections preoperatively, compared with 6 (67%) postoperatively. The time of initiation of these injections varied between 3 and 8 months postoperatively. CONCLUSION: Isolated continent urinary diversion seems to induce or increase bladder overactivity. A larger multicentric study is necessary to validate our hypothesis.

Feasibility of EXIME® temporary prosthesis placement and removal in men with acute or chronic urinary retention after failure or inability to selfcatheterize.

INTRODUCTION: Urological emergencies represent 7% of admissions, 29% of which are acute urine retention. We report the first results of a protocol evaluating a new device in case of failure of self-catheterization, replacing a permanent catheter: the urethral device EXIME®. METHOD: Intention-to-treat study on the feasibility of inserting the EXIME® prosthesis in a day hospital after simple urethral gel instillation in men with urine retention. EXIME® was proposed to all patients after failure of Foley catheter removal and refusal or inability to learn self-catheterization. The protocol was referenced (NCT04218942) after obtaining the agreement of the committee for the protection of individuals. RESULTS: Among 278 patients admitted for a trial of Foley catheter removal, 15 patients with failed voiding resumption and refusal or failure of self-catheterization were offered the prosthesis. The median age was 73 years with a median retention volume of 700mL. The median prostatic volume was 60g. Fourteen patients had their prosthesis inserted in good conditions of comfort for the practitioner and the patient. One failed placement was noted. The difficulty of insertion was estimated by the practitioner at 0 on median (VAS from 0 to 10), and for its removal at 0. The pain during the insertion of the device was evaluated by the patients at 2.00 and for the removal at 0 (VAS from 0 to 10). 6 patients had satisfactory voiding recovery at D0. DISCUSSION: We proposed the placement of EXIME to patients who had failed the trial of Foley removal and were unable and/or unwilling to self-catheterize. These were patients with poor bladder contractility and a high risk of retention recurrence. Despite this mixed result, the simplicity of the device and the comfortable expectation of an endoscopic procedure seem promising. CONCLUSION: Insertion and retrieval of EXIME®prostatic prosthesis were easy and well tolerated in our population. Insertion failed in one patient. A comparative prospective study with self catheterization is necessary to determine its effectiveness.

[Penile prosthesis for erectile dysfunction in the neurological patient, indication, complications and satisfaction: Retrospective study on 27 patients]. / Implant pénien pour dysfonction érectile chez le patient neurologique, indication, complications et satisfaction : étude rétrospective sur 27 patients.

INTRODUCTION: Penile prosthesis for erectile dysfunction in patients with spinal cord injury or multiple sclerosis is sometimes discussed after failure of drug or instrumental treatments (vacuum). The objective of this study was to evaluate the complications, evolution and patient satisfaction after the implantation of a penile prosthesis in the neurological patient. MATERIALS AND METHODS: Multi-center retrospective study of 27 consecutive patients including 18 spinal cord injured patients and 9 patients with multiple sclerosis benefiting from the implantation of a penile prosthesis for erectile dysfunction purposes in two French centers between 2009 and 2019. Post-implantation complications, evolution of the use of the prosthesis and global patient satisfaction were evaluated using the standardized questionnaire Erectile Dysfunction Inventory of Treatment Satisfaction (EDITS) during a telephone call between March and May 2020. RESULTS: The average age of implantation was 46.4 years (±12.74). The length of follow-up to date of call was 6.05 years (±2.86). 8/27 patients (29.6 %) had at least one complication of any Clavien-Dindo grade included 2 infection. 2/27 (7,4 %) patients had a mechanical prosthesis injury during follow-up. The patient's dexterity with inflation of the prosthesis was perfect in 85 % of cases, and 75 % for deflation. The satisfaction rate for prosthesis use at the time of the call was 75.36/100pts for the patient and 66.88/100pts for the partner. CONCLUSION: This study found an increased rate of prothesis infection compared to the general population in the neurologic patient, but patient and partner satisfaction remain sustainable after more than 5 years of implantation. Dexterity was maintained over the long term, demonstrating a good selection of indications. These data invite to favorably consider the installation of a penile prosthesis in neurological patients who have failed first-line treatments. LEVEL OF EVIDENCE: 4.

[Anticoagulation impact on bleeding risk during HoLEP: Monocentric study of 156 patients]. / Impact des anticoagulants sur le risque hémorragique au cours de l'HoLEP : étude monocentrique de 156 patients.

OBJECTIVE: Compare the length of hospital stay and the complications after HoLEP between three groups of patients: a control group, a group with antiplatelet therapy, a group with anticoagulation therapy. MATERIALS: Retrospective cohort study that included all consecutive patients who underwent HoLEP for prostatic hyperplasia in our center from may 2013 to may 2016. Anticoagulated patients and patients under clopidogrel had respectively a relay with heparine and aspirine. Patients were seen after surgery at 1 and 3 months. RESULTS: A hundred and fifty six patients were analysed, mean age was 70.7 years (DS 6.8), mean prostate volume 88.8g (DS 34.1). 106 patients were in the control group, 34 had antiplatelet therapy and 16 had anticoagulation therapy. There were no difference between the 3 groups for mean age, mean prostatic volume, PSA. There was also no difference for length of intervention, irrigated volume and length of morcellation between the three groups. There were no difference between patients in the control group and patients with antiplatelet therapy for length of hospital stay (2.1 days vs 2.0 days), lenght of urethral catheterization (1.6 days vs 1.5 days). There was a statistical difference between patients in the control group and patients with anticoagulation therapy for lenght of hospital stay (2.0 days vs 4.4 days; P=0.01), length of bladder irrigation (0.9 day vs 1.8 days; P=0.01), lenght of urethral catheterization (1.6 days vs 3.5 days; P=0.01). Transfusion rate was 18.75% (n=3) for patients with anticoagulation, 2.9% (n=1) for patients under antiplatelet therapy and 0.9% (n=1) for patients in the control group. CONCLUSION: Anticoagulation during HoLEP is a valid option but need to be proceed with carefully management. LEVEL OF PROOF: 4.

The group III mGlu receptor agonist ACPT-I exerts anxiolytic-like but not antidepressant-like effects, mediated by the serotonergic and GABA-ergic systems.

Our earlier studies have demonstrated that (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid ACPT-I, a group III mGlu receptor agonist, produced anxiolytic-like and antidepressant-like actions after central administration. Here we describe the anxiolytic-like effects of ACPT-I after intraperitoneal administration in the stress-induced hyperthermia (SIH), elevated plus-maze (PMT) tests in mice and in the Vogel test in rats. However, the compound did not produce antidepressant-like effects in the tail suspension test (TST) or in the forced swim test (FST) in mice. The potential anxiolytic effect of ACPT-I (20 mg/kg) in the SIH test was inhibited by the benzodiazepine receptor antagonist flumazenil (given i.p., 10 mg/kg), and by a 5-HT(1A) receptor antagonist N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl) cyclohexane-carboxamide (WAY100635) (0.1 mg/kg s.c.). At the same time, ritanserin (0.5 mg/kg i.p.), the 5-HT2A/C receptor antagonist, did not change the anxiolytic-like effects of ACPT-I. The results of these studies indicate that the GABA-ergic and serotonergic systems are involved in the potential anxiolytic action of ACPT-I.

Peripheral administration of group III mGlu receptor agonist ACPT-I exerts potential antipsychotic effects in rodents.

Several lines of evidence implicate dysfunction of glutamatergic neurotransmission in the pathophysiology of schizophrenia. Previous behavioral studies have indicated that metabotropic glutamate (mGlu) receptors may be useful targets for the treatment of psychosis. It has been shown that agonists and positive allosteric modulators of group II mGlu receptors produce potential antipsychotic effects in behavioral models of schizophrenia in rodents. Group III mGlu receptors seem to be also promising targets for a variety of neuropsychiatric and neurodegenerative disorders. However, despite encouraging data in animal models, most ligands of group III mGlu receptors still suffer from weak affinities, incapacity to cross the blood-brain barrier or absence of full pharmacological characterization. These limitations slow down the validation process of group III mGlu receptors as therapeutic targets. In this work, we choose to study an agonist of group III mGlu receptors (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid (ACPT-I) using intraperitoneal administration in three animal behavioral models predictive of psychosis or hallucinations. The results of the present study show that ACPT-I, given at doses of 10 or 30mg/kg, decreased MK-801-induced hyperlocomotion and at a dose of 100mg/kg decreased amphetamine-induced hyperlocomotion in rats. Furthermore, ACPT-I dose-dependently decreased DOI-induced head twitches in mice and suppresses DOI-induced frequency and amplitude of spontaneous EPSPs in slices from mouse brain frontal cortices. These data demonstrate that ACPT-I is a brain-penetrating compound and illustrates its promising therapeutic role for the treatment of schizophrenia.

Regulation of alpha-smooth muscle actin and CRBP-1 expression by retinoic acid and TGF-beta in cultured fibroblasts.

We have reported that Cellular Retinol Binding Protein-1 (CRBP-1) is expressed de novo during skin wound healing by a proportion of fibroblastic cells which then differentiate into myofibroblasts and express alpha-smooth muscle actin. In fibroblasts cultured from different tissues we have shown that alpha-smooth muscle actin expression, mainly controlled by Transforming Growth Factor-beta (TGF-beta), is also regulated by retinoic acid and that CRBP-1, known to be a retinoic acid-responsive gene, is modulated by TGF-beta. The aim of the present study has been to investigate the relationships between retinoic acid and TGF-beta in regulating the expression of CRBP-1 and alpha-smooth muscle actin in cultured rat subcutaneous tissue fibroblasts. We have observed that the TGF-beta-induced, but not the retinoic acid-induced, alpha-smooth muscle actin expression is associated with a modulation of endogenous TGF-beta and TGF-beta receptors, suggesting that the action of retinoic acid on alpha-smooth muscle actin expression is not mediated by TGF-beta. The expression of CRBP-1 is regulated at the transcriptional level by TGF-beta and retinoic acid but not synergistically, suggesting a possible common pathway. However, retinoic acid, but not TGF-beta, increases the transcription of a transiently transfected chimeric construct containing the retinoic acid response element of the CRBP-1 promoter, indicating that TGF-beta does not influence CRBP-1 through the retinoic acid pathway. Our results indicate that distinct pathways regulate the genes involved in the appearance and evolution of the myofibroblastic cells. The characterization of these pathways will be helpful for the design of drugs influencing wound healing.

Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy.

Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22alpha promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22alpha promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-gamma expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-gamma driven either by the SM-myosin heavy chain enhancer/SM22alpha promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders.

Intraarterial beta irradiation induces smooth muscle cell apoptosis and reduces medial cellularity in a hypercholesterolemic rabbit restenosis model.

PURPOSE: Ionizing radiation has been shown to be a powerful inhibitor of neointimal hyperplasia following arterial injury in several animal models of post-percutaneous transluminal coronary angioplasty (post-PTCA) restenosis. This was previously shown to be associated with a reduction in smooth muscle cell (SMC) mitotic activity. This study evaluated the effect of intraarterial beta irradiation on the arterial wall SMC density and apoptosis. METHODS AND MATERIALS: Twenty-five carotid and 7 iliac arteries of hypercholesterolemic New Zealand white rabbits were injured using the Baumgartner technique. The impact of an 18 Gy beta radiation dose administered after balloon injury was studied and compared to a nonirradiated injured control group. The medial SMC density as well as the percentage of apoptotic cells were determined at 8 days, 21 days, and 6 weeks after injury using an automated computer-based software. Apoptotic cells were identified using in situ end-labeling of fragmented DNA. RESULTS: The values for medial apoptosis in control vs. irradiated arteries were: 0.014 +/- 0.023 vs. 0.23 +/- 0.28%, p = NS, at 8 days; 0.012 +/- 0.018 vs. 0.07 +/- 0.07%, p = 0.05, at 21 days; and 0 +/- 0 vs. 0.16 +/- 0.11%, p = 0.03, at 6 weeks. The overall incidence of medial apoptotic cells at all time points was 0.01 +/- 0.017 vs. 0.13 +/- 0.14% in controls and irradiated arteries respectively, p = 0.004. Medial SMC density was significantly decreased in irradiated arteries in comparison with controls (p < 0.01 at all time-points). CONCLUSIONS: Intraarterial beta irradiation stimulates medial SMC apoptosis in balloon-injured arteries. This, together with a decrease in SMC mitotic activity, contributes to a decrease in the arterial wall cellularity.

Isolation of Trypanosoma brucei CYC2 and CYC3 cyclin genes by rescue of a yeast G(1) cyclin mutant. Functional characterization of CYC2.

Two Trypanosoma brucei cyclin genes, CYC2 and CYC3, have been isolated by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G(1) cyclin function. CYC2 encodes a 24-kDa protein that has sequence identity to the Neurospora crassa PREG1 and the S. cerevisiae PHO80 cyclin. CYC3 has the most sequence identity to mitotic B-type cyclins from a variety of organisms. Both CYC2 and CYC3 are single-copy genes and expressed in all life cycle stages of the parasite. To determine if CYC2 is found in a complex with previously identified trypanosome cdc2-related kinases (CRKs), the CYC2 gene was fused to the TY epitope tag, integrated into the trypanosome genome, and expressed under inducible control. CYC2ty was found to associate with an active trypanosome CRK complex since CYC2ty bound to leishmanial p12(cks1), and histone H1 kinase activity was detected in CYC2ty immune-precipitated fractions. Gene knockout experiments provide evidence that CYC2 is an essential gene, and co-immune precipitations together with a two-hybrid interaction assay demonstrated that CYC2 interacts with CRK3. The CRK3 x CYC2ty complex, the first cyclin-dependent kinase complex identified in trypanosomes, was localized by immune fluorescence to the cytoplasm throughout the cell cycle.

GM-CSF expression by tumor cells correlates with aggressivity and with stroma reaction formation.

Granulation tissue involved in tissue repair and in the stroma reaction to epithelial tumors is characterized by the presence of myofibroblastic cells. It has been previously reported that granulocyte macrophage-colony stimulating factor (GM-CSF) induces a fibrotic reaction containing numerous myofibroblasts. This reaction results from a cascade of events, including stimulation of transforming growth factor-beta1 (TGF-beta1) production by macrophages which, in turn, promotes alpha-smooth muscle actin and collagen synthesis by fibroblasts. Moreover, GM-CSF is known to be expressed by many tumor cell types. In this study we have analyzed, by means of reverse transcription-polymerase chain reaction, GM-CSF mRNA expression in a progressive and a regressive rat colon carcinomas and in the corresponding cell lines, eliciting different degrees of desmoplastic reaction. We have also evaluated the expression of GM-CSF protein in selected cases. The expression of GM-CSF mRNA and, when tested, protein were higher in progressive compared to regressive cancer cells both in vivo and in vitro. We then investigated GM-CSF mRNA and protein expression in different human colon cancer cell lines known to exhibit different degrees of aggressivity in vivo. We found high levels of GM-CSF mRNA and protein in the most aggressive cell lines. Similar results were also obtained on human breast and cervical cancer cell lines. Our results are in agreement with the assumption that GM-CSF expression is correlated to tumor aggressivity. Conceivably, one of the GM-CSF actions affecting tumor progression is exerted through its influence on stroma reaction development.

Cultured porcine coronary artery smooth muscle cells. A new model with advanced differentiation.

Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage after enzymatic digestion of the media. The effects of heparin, transforming growth factor (TGF)-beta1 or TGF-beta2, and all-trans-retinoic acid (tRA) on proliferation, migration, and differentiation of these cells also were examined. Porcine arterial SMCs in culture not only express high levels of alpha-smooth muscle (SM) actin but, contrary to rodent SMCs, also maintain an appreciable expression of SM myosin heavy chain isoforms 1 and 2, desmin, and smoothelin, a recently described late differentiation marker of vascular SMCs. We demonstrate for the first time that smoothelin is colocalized with alpha-SM actin in these cells. Finally, we show that in the porcine model, heparin is more potent than TGF-beta1 or TGF-beta2 and tRA in terms of inhibition of proliferation and migration and of increasing the expression of differentiation markers. This model should be a useful complement to in vivo studies of SMC differentiation and of pathological situations such as restenosis and atheromatosis.

Retinoic acid regulates arterial smooth muscle cell proliferation and phenotypic features in vivo and in vitro through an RARalpha-dependent signaling pathway.

We have recently shown that all-trans retinoic acid (tRA) modulates arterial smooth muscle cell (SMC) morphologic features and biochemical composition in vitro. It has been proposed that different SMC phenotypes coexist in arteries, which may be retrieved in culture: hence, a differential action of tRA on distinct SMC subsets is conceivable. We have examined the effect of tRA on SMC proliferation, migration, plasminogen activator activity, and alpha-smooth muscle actin expression in 2 phenotypically different rat SMC populations, cultured respectively from the normal aortic media and from the intimal thickening (IT) after endothelial injury. tRA inhibited proliferation and increased migration and tissue-type plasminogen activator activity in both SMC populations, but decreased alpha-smooth muscle actin only in SMC cultured from the IT. The action of tRA is mediated by 2 families of nuclear receptors, RAR and RXR, each containing 3 isoforms, alpha, beta, and gamma. RAR and RAR-alpha agonists, but not RXR agonists, inhibited SMC proliferation in both cell populations and alpha-smooth muscle actin expression only in IT SMC. When administered intraperitoneally to balloon-injured rats, tRA and RAR-alpha agonists reduced the intimal hyperplasia in the carotid artery. Our results show that tRA and synthetic retinoids can affect the proliferation, migration, and differentiation of SMC in vitro. Furthermore, retinoids are able to reduce the IT induced by endothelial injury in vivo.