Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.

The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

Spleen route accelerates engraftment of human hematopoietic stem cells.

Intravenous injections of human hematopoietic stem cells (hHSCs) is routinely used in clinic and for modeling hematopoiesis in mice. However, unspecific dilution in vascular system and non-hematopoietic organs challenges engraftment efficiency. Although spleen is capable of extra medullar hematopoiesis, its ability to support human HSC transplantation has never been evaluated. We demonstrate that intra-splenic injection results in high and sustained engraftment of hHSCs into immune-deficient mice, with higher chimerisms than with intravenous or intra-femoral injections. Our results support that spleen microenvironment provides a niche for HSCs amplification and offers a new route for efficient HSC transplantation.

[Acute renal failure of the donor in encephalic death: A real contraindication to kidney transplantation?] / L'insuffisance rénale aiguë du donneur en mort encéphalique : une réelle contre-indication à la transplantation rénale ?

INTRODUCTION: The shortage of kidney transplants encourages the expansion of the limits of eligibility criteria for donation. Many donors who are brain dead display acute renal failure at the time of death; is this a real contraindication to harvesting? The aim of this study was to assess kidney graft survival from donors after brain death with confirmed acute renal failure, with or without anuria previous donation. MATERIALS AND METHODS: All of the transplants performed in two university hospitals between 2010 and 2017 were analyzed retrospectively. All patients who underwent single kidney transplant from a brain-dead donor with acute renal failure (ARF) were included in this study. ARI was defined here by a decrease over 50 % of glomerular filtration rate (GFR) to a threshold below 45mL/min/1.73 m2 at the time of kidney procurement. Kidney graft survival, incidence of delayed graft function (DGF) and the GFR at 12 months were analyzed. Analysis of kidney transplant survival based on pre-implantation biopsies was additionally done. RESULTS: One hundred and sixty four patients were transplanted with a kidney from donor with ARF during the selected period. At the admission in ICU the average GFR was 67,7±19mL/min/1,73m2. At the time of donation, the average age of donors was 56.4±17.7 years, the GFR was 33.7±8.0mL/min/1.73 m2 16 % of donors were anuric. Cold ischemia time (CIT) was 16.8±5.0hours. The average age of recipients was 55.6±14.1 years. 81 % of the cases were primary transplants. Graft function took place within 7.8±9.4 days after transplantation. There were two non-primary functions (PNF). One hundred and fifty two patients (93 %) had a functional graft at 12 months. The mean GFR at 12 months was 46.8±20.1mL/min/1.73 m2 and 122 patients (73 %) had a GFR greater than 30mL/min/1.73 m2. Seventy-one percent of preimplantation biopsies revealed acute tubular necrosis (ATU); no cortical necrosis was observed. Survival of theses grafts was 85 %, comparable to the total population of study (P=0,21) CONCLUSION: The acute renal failure of the brain-dead donor should not alone be systematically a contraindication to harvesting and kidney transplantation.

[Renal cell carcinoma in candidates for renal transplantation and recipients of a kidney transplant: The French guidelines from CTAFU]. / Recommandations françaises du Comité de transplantation de l'association française d'urologie (CTAFU) : carcinome rénal sur reins natifs chez le patient transplanté rénal ou en attente de transplantation.

OBJECTIVE: To define guidelines for the management of renal cell carcinoma of the native kidney (NKRCC) in kidney transplant (KTx) recipients and renal cell carcinoma (RCC) in end-stage renal disease (ESRD) patients candidates for renal transplantation. METHOD: A review of the literature following a systematic approach (Medline) was conducted by the CTAFU to report renal cell carcinoma epidemiology, screening, diagnosis and management in KTx candidates and recipients. References were assessed according to a predefined process to propose recommendations with the corresponding levels of evidence. RESULTS: ESRD patients are at higher risk of RCC with a standardized incidence ratio of approximately 4,5 as compared with general population. NKRCC tumors occur in 1 to 3 % of KTx recipients with a 10 to 15-fold increased risk as compared with general population, especially in patients with acquired multicystic kidney disease. Most authors suggest yearly monitoring of the native kidneys using ultrasound imaging. Radical nephrectomy (either open or laparoscopic approach) is the preferred treatment of NKRCC in KTx recipients and RCC in ESRD. Surveillance in a valid option in small or cystic renal masses. In the localized setting, change in immunosuppressive therapy is not recommended besides perioperative avoidance of mTOR inhibitor to limit morbidity. CTAFU does not recommend a mandatory waiting time after nephrectomy for RCC in ESRD patients candidates for renal tranplantation when tumor stage

[Urothelial carcinoma in kidney transplant recipients and candidates: The French guidelines from CTAFU]. / Recommandations françaises du Comité de transplantation de l'association française d'urologie (CTAFU) : carcinome urothélial chez le patient transplanté rénal et le candidat à la transplantation rénale.

OBJECTIVE: To propose surgical recommendations for urothelial carcinoma management in kidney transplant recipients and candidates. METHOD: A review of the literature (Medline) following a systematic approcah was conducted by the CTAFU regarding the epidemiology, screening, diagnosis and treatment of urothelial carcinoma in kidney transplant recipients and candidates for renal transplantation. References were assessed according to a predefined process to propose recommendations with levels of evidence. RESULTS: Urothelial carcinomas occur in the renal transplant recipient population with a 3-fold increased incidence as compared with general population. While major risk factors for urothelial carcinomas are similar to those in the general population, aristolochic acid nephropathy and BK virus infection are more frequent risk factors in renal transplant recipients. As compared with general population, NMIBC in the renal transplant recipients are associated with earlier and higher recurrence rate. The safety and efficacy of adjuvant intravesical therapies have been reported in retrospective series. Treatment for localized MIBC in renal transplant recipients is based on radical cystectomy. In the candidate for a kidney transplant with a history of urothelial tumor, it is imperative to perform follow-up cystoscopies according to the recommended frequency, depending on the risk of recurrence and progression of NMIBC and to maintain this follow-up at least every six months up to transplantation whatever the level of risk of recurrence and progression. Based on current data, the present recommendations propose guidelines for waiting period before active wait-listing renal transplant candidates with a history of urothelial carcinoma. CONCLUSION: The french recommendations from CTAFU should contribute to improve the management of urothelial carcinoma in renal transplant patients and renal transplant candidates by integrating both oncologic objectives and access to transplantation.

Robot-assisted renal transplantation using the retroperitoneal approach (RART) with more than one year follow up: Description of the technique and results.

OBJECTIVES: To describe the technique and report our first experience of robotic-assisted renal transplantation (RART) with more than one year follow up. PATIENTS AND METHODS: In our center the first case of RART was realized in October 2013 with a cadaveric graft. We used the combined extra- and intraperitoneal robot assisted laparoscopic route with extraperitoneal positioning of the graft and intraperitoneal transplantation. The patient was placed in the supine position with arms along the body; the robot came from the right inferior part of the patient. Access to the retroperitoneal space was obtained using an Alexis trocar that permitted the insertion of the kidney with ice without losing the pneumoperitoneum. Ports included a 12-mm camera port (placed under the ombilicus), two 8-mm robotic ports (placed 6cms laterally from the previous port) and a 12-mm assistant port (placed between the upper port and the ombilic). All the pre-, per- and postoperative data were prospectively included in a database. We report the results of the initial experience of RART, performed with more than one year follow-up. RESULTS: This technique is the first described using the retroperitoneal approach that is the routine approach for conventional open renal transplantation. This approach permitted to perform excellent arterial, veinous and ureteral anastomosis. Eight cases of RART were conducted between October 2013 and November 2015 (five men and three women). The average age was 58 years (range 39-75years). The average body mass index was 28 (range 22-38). Five patients had history of abdominal surgery and were dialyzed for 30 months on average (range 3-63months). Three left and five right cadavers kidneys were transplanted in the right iliac fossa. The mean graft size was 109mm (range 90-130). The mean length of the incision for insertion of the graft was 60 mms (40-100mms). Mean warm ischemia time was 63minutes (range 46-84). The total operative time was 200minutes (149-245). No patient was transfused during surgery and two were transfused postoperatively. Median length of hospital stay was 14 days (range 10-30 days). Only one patient needed postoperative morphine, the pain visual analogic scale 12hours postoperatively was 2 (0-5). Mean serum creatinine at seven days, at three months and at one year was 400 (98-639micromol/L), 151 (80-235micromol/L) and 129 (86-194micromol/L) respectively. At one year follow-up, no patient had a wound infection or incisional hernia. One patient was re-operated for ureteral anastomosis stricture. CONCLUSION: The retroperitoneal approach for RART permits the kidney to be cooled and a direct access to the iliac vessels and bladder. This initial series with more than a year of post-monitoring RART shows promising results despite some initial technical difficulties. The procedure can still be improved and hoped to see an improvement in the results. A comparison to the results of the conventional route is necessary before diffusing the robot-assisted technique. LEVEL OF PROOF: 3.

[Impact of learning curve in renal transplantation]. / Impact de la courbe d'apprentissage dans la transplantation rénale.

OBJECTIVES: Renal transplantation is performed only in university hospital centres, in accredited transplanting centres. The aim of this study is to analyse the learning curve of this operation and its impact on the graft survival. PATIENTS-METHODS: Monocentric retrospective study in which 3 groups have been defined: Juniors 1, Juniors 2 and Seniors corresponding respectively to the first thirty transplantations and to the last thirty transplantations of 5 clinical leaders, and 30 transplantation graft of referent seniors. Data have been registered in a database. Operation times, lukewarm ischemic times and postoperative complications have been compared within the 3 groups. RESULTS: A clear difference of operation time has been noted within the 3 groups with an average time of 202 minutes for Juniors 1, 173 minutes for Juniors 2 and 140 minutes for Seniors (P<0.0001). Likewise, concerning lukewarm ischemic time and vascular anastomosis time respectively with an average time of 72, 59 and 40 min (P<0.0001). Vascular complications occurred in 20% of cases in Juniors 1, 44.3% of cases in Juniors 2 and 17% of cases in Seniors (P=0.65). There were no significant differences of survival without urinary complications: 20% of complications for Juniors 1, 10% for Juniors 2 and 17% for Seniors (P=0.63). Similarly results have been obtained with analysing complications following Clavien's order. CONCLUSION: This study reveals that renal transplantations operated by young surgeons require longer operation and lukeward ischemic time but without significant repercussions on the surgical complication rate and the global survival. This stresses on the importance of surgical training during medicine internship.

De novo kidney graft tumors: results from a multicentric retrospective national study.

De novo tumors in renal allografts are rare and their prevalence is underestimated. We therefore analyzed renal cell carcinomas arising in renal allografts through a retrospective French renal transplant cohort. We performed a retrospective, multicentric survey by sending questionnaires to all French kidney transplantation centers. All graft tumors diagnosed after transplantation were considered as de novo tumors. Thirty-two centers participated in this study. Seventy-nine tumors were identified among 41 806 recipients (Incidence 0.19%). Patients were 54 men and 25 women with a mean age of 47 years old at the time of diagnosis. Mean tumor size was 27.8 mm. Seventy-four (93.6%), 53 (67%) and 44 tumors (55.6%) were organ confined (T1-2), low grade (G1-2) and papillary carcinomas, respectively. Four patients died of renal cell carcinomas (5%). The mean time lapse between transplantation and RCC diagnosis was 131.7 months. Thirty-five patients underwent conservative surgery by partial nephrectomy (n = 35, 44.3%) or radiofrequency (n = 5; 6.3%). The estimated 5 years cancer specific survival rate was 94%. Most of these tumors were small and incidental. Most tumors were papillary carcinoma, low stage and low grade carcinomas. Conservative treatment has been preferred each time it was feasible in order to avoid a return to dialysis.

[Kidney transplantation in obese recipients: review of the Transplantation Committee of the French Association of Urology]. / Transplantation rénale et receveurs obèses : revue du comité de transplantation de l'Association française d'urologie.

INTRODUCTION: Transplantation Committee of the French Association of Urology (CTAFU) conducted a review of the complication of kidney transplantation in obese recipients. MATERIAL AND METHODS: A bibliographic research in French and English using Medline with the keywords "obesity", "body mass index", "kidney transplantation", "graft function", "survival", "wound complications", "graft rejection" and "graft survival" was performed. We limited the review for the last fifteen years because of the change in immunosuppressive treatment area. Only studies with more than 20 obese patients were selected. RESULTS: Wound or infectious postoperative complications and delayed graft function are more frequent in obese patients than in non-obese recipients. Similarly, transplant survival at 5 years is lower in obese patients. On the other hand, patient survival and acute rejection are the same between the two groups if recipient selection is carefully made, particularly with regard to heart complication. CONCLUSION: Kidney transplantation in obese patients is not an easy surgery with known complication. Obese patients will take time before transplantation to explain all the risk and a regular heart follow-up is crucial if we don't want to reduce patient survival. But obese survival is better if we proceed to kidney transplantation than if they stay on dialysis, arguing for a non-exclusion of the waiting list. So there is the need for a national study concerning obese patients on waiting list to enact future guidelines.

[Pelvic lymphadenectomy in prostate cancer: Should it be realized by laparoscopy?]. / Le curage ilio-obturateur dans le cancer de la prostate doit-il être réalisé par voie coelioscopique ?

AIM: Laparoscopic pelvic lymphadenectomy in localized prostatic cancer is performed since the 1990s, lessens the postoperative complications and respects carcinologic's principles (No. lymph nodes removed and lymph nodes metastasis). In order to verify that these objectives are achieved, we compared our results of pelvic lymphadenectomy by laparotomy and by laparoscopy for the past 12 years. PATIENTS AND METHODS: Between January 1997 and June 2008, 36 (23.8%) patients underwent open pelvic lymphadenectomy and 76.16% (115 cases) laparoscopic pelvic lymphadenectomy. We did a retrospective and comparative analysis of data including the preoperative characteristics, per- and postoperative complication as well pathologic results. RESULTS: Preoperative data were comparable between both groups. The comparison of the peroperative data showed an increased bleeding volume in the open group (105.6±420.9mL; 12.1±96.1mL: P=0.001) and longer operative time in the laparoscopic group (103.7±83.9min; 132.8±40.9min: P=0.006). Postoperative complications were similar. Pathologic results showed a significantly more important number of lymph nodes removed in the open group (7.2±3.5; 5.7±3.2: P=0.022), but the positive rate similar in both groups (13.9%; 22.6%: P=0.258). In order to remove "the learning curve effect", we compared 36 open pelvic lymphadenectomy to the last 36 laparoscopic pelvic lymphadenectomy. In the laparoscopic group the patients showed an upper Gleason score (6.3±1.1; 7±1: P=0.005); but there was no difference for the operative time, number of lymph nodes removed and the complications rates. CONCLUSIONS: After training, laparoscopic pelvic lymphadenectomy was similar to open pelvic lymphadenectomy.

Is urine culture routinely necessary before prostate biopsy?

The objective of this study was to assess the value of a urine bacterial culture performed before prostate biopsy. We performed a prospective study on 353 patients who underwent prostate biopsy. All patients had a urine bacterial culture performed before biopsy. We compared the outcomes of patients with bacteriuria (left untreated) with those of patients without bacteriuria. Of the 353 men, 12 had a pre-biopsy-positive bacterial culture and underwent prostate biopsy without any infectious complication. Fifteen patients with a negative pre-biopsy culture developed a post-biopsy-positive bacterial culture, but remained asymptomatic without any treatment. Only four men from the group without pre-biopsy bacteriuria developed an infectious complication, requiring 3 weeks of antibiotic therapy. The complication rates were similar for both groups. Our results suggest that routine urine bacterial culture before prostate biopsy is not useful when antibiotic prophylaxis and enema are performed. We do, however, suggest performing a urine bacterial culture before prostate biopsy for patients with a previous history of urinary tract infections.

[Management of perianastomotic stenoses complicating vascular accesses for haemodialysis]. / Prise en charge des sténoses périanastomotiques compliquant les abords vasculaires pour hémodialyse.

OBJECTIVE: Perianastomotic stenoses (PAS) complicating native arteriovenous fistulas (AVF) of the forearm can be treated by angioplasty or surgery. The objective of this study was to report primary patency (PP) and primary assisted patency (PAP) rates of surgery and angioplasty of these stenoses. The secondary objective was to identify factors influencing the patency rates of these reoperations. MATERIAL AND METHODS: Seventy-three patients with a mean age, 65 years were treated for PAS between January 1999 and December 2005 in two centres (Tours and Le Mans), which were retrospectively included. PAS were treated by surgery (n=21) or angioplasty (n=52). The two groups were comparable. The mean follow-up was 39 months for the angioplasty group and 49 months for the surgery group (p=0.088). RESULTS: The PP rate was 71+/-10% for surgery and 41+/-6% for angioplasty (p<0.0175). The PAP rate was not significantly different (p=0.462) between angioplasty (92+/-4%) and surgery (95+/-4%). In the endovascular group, the site of stenosis on the anastomosis was a risk factor for early recurrence (95% CI between 0.006 and 0.392; p=0.047). CONCLUSION: These results suggest that anastomotic stenoses should be treated surgically rather than by angioplasty. Angioplasty and surgery give identical patency rates in other types of perianastomotic stenoses at the cost of a higher reoperation rate for angioplasty.

Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake".

Zhang et al. (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein-coupled receptor GPR39. However, we found that I125-obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.

Potential involvement of adipocyte insulin resistance in obesity-associated up-regulation of adipocyte lysophospholipase D/autotaxin expression.

AIMS/HYPOTHESIS: Autotaxin is a lysophospholipase D that is secreted by adipocytes and whose expression is substantially up-regulated in obese, diabetic db/db mice. The aim of the present study was to depict the physiopathological and cellular mechanisms involved in regulation of adipocyte autotaxin expression. METHODS: Autotaxin mRNAs were quantified in adipose tissue from db/db mice (obese and highly diabetic type 2), gold-thioglucose-treated (GTG) mice (highly obese and moderately diabetic type 2), high-fat diet-fed (HFD) mice (obese and moderately diabetic type 2), streptozotocin-treated mice (thin and diabetic type 1), and massively obese humans with glucose intolerance. RESULTS: When compared to non-obese controls, autotaxin expression in db/db mice was significantly increased, but not in GTG, HFD, or streptozotocin-treated mice. During db/db mice development, up-regulation of autotaxin occurred only 3 weeks after the emergence of hyperinsulinaemia, and simultaneously with the emergence of hyperglycaaemia. Adipocytes from db/db mice exhibited a stronger impairment of insulin-stimulated glucose uptake than non-obese and HFD-induced obese mice. Autotaxin expression was up-regulated by treatment with TNFalpha (insulin resistance-promoting cytokine), and down-regulated by rosiglitazone treatment (insulin-sensitising compound) in 3T3F442A adipocytes. Finally, adipose tissue autotaxin expression was significantly up-regulated in patients exhibiting both insulin resistance and impaired glucose tolerance. CONCLUSIONS/INTERPRETATION: The present work demonstrates the existence of a db/db-specific up-regulation of adipocyte autotaxin expression, which could be related to the severe type 2 diabetes phenotype and adipocyte insulin resistance, rather than excess adiposity in itself. It also showed that type 2 diabetes in humans is also associated with up-regulation of adipocyte autotaxin expression.

[Clinical overview of New World tegumentary leishmaniasis]. / Panorama clinique des leishmanioses tégumentaires du Nouveau Monde.

This richly illustrated article (80 color photographs) based on the authors' experience in French Guyana documents the clinical diversity of American tegumentary leishmaniasis. Main highlights include the often outstanding aspect of lesions, the high frequency of forms not associated with ulceration or scab formation that must be recognized to achieve diagnosis in travellers returning from endemic zones, and the special prognosis of clinical forms associated with intradermic, lymphatic or hematogenous spread. The article also reviews an original diagnostic method based on culture of cutaneous biopsy specimens on specific nutrient mediums that provides isolates in a high percentage of cases (80%) and thus allows identification of offending parasite.

[Military Bioforce, example of governmental humanitarian action]. / La Bioforce militaire, exemple de moyen humanitaire gouvernemental.

Governmental humanitarian action is an old concept in France, that is a topic of current interest. To conduct humanitarian action, the French government has deployed various facilities. The military biological risk team was established to control epidemics, provide expertise during epidemiological emergencies, and conduct mass vaccination programs. The author describe the missions carried out by the biological risk team during the 1990s as an illustration of governmental humanitarian action.

Aberrant membrane hormone receptors in incidentally discovered bilateral macronodular adrenal hyperplasia with subclinical Cushing's syndrome.

Cortisol secretion in adrenal Cushing's syndrome can be regulated by the aberrant adrenal expression of receptors for gastric inhibitory polypeptide, vasopressin, catecholamines, LH/human CG (LH/hCG), or serotonin. Four patients with incidentally discovered bilateral macronodular adrenal hyperplasia without clinical Cushing's syndrome were evaluated for the possible presence of aberrant adrenocortical hormone receptors. Urinary free cortisol levels were within normal limits, but plasma cortisol levels were slightly elevated at nighttime and suppressed incompletely after dexamethasone administration. Plasma ACTH was partially suppressed basally but increased after administration of ovine CRH. A 51-yr-old woman had ACTH-independent increases of plasma cortisol after 10 IU AVP im (292%), 100 microg GnRH iv (184%), or 10 mg cisapride orally (310%); cortisol also increased after administration of NaCl (3%), hCG, human LH, and metoclopramide. In a 61-yr-old man, cortisol was increased by AVP (349%), GnRH (155%), hCG (252%), and metoclopramide (191%). Another 53-yr-old male increased plasma cortisol after AVP (171%) and cisapride (142%). Cortisol secretion was also stimulated by vasopressin in a 54-yr-old female. This study demonstrates that subclinical secretion of cortisol can be regulated via the aberrant function of at least V1-vasopressin, LH/hCG, or 5-HT4 receptors in incidentally identified bilateral macronodular adrenal hyperplasia.

SLC-1 receptor mediates effect of melanin-concentrating hormone on feeding behavior in rat: a structure-activity study.

Several studies have shown that melanin-concentrating hormone (MCH) is an orexigenic peptide in rat. In the present study, a structure-activity relationship with MCH analogs was performed in rat, both in vitro and in vivo. On rat recombinant SLC-1 receptor, both cAMP inhibition and [(125)I]S36057 binding were measured. In vivo, these analogs were injected intracerebroventricularly in rats and their effects were evaluated upon food intake. First, data obtained with the rat recombinant receptor were highly correlated with those obtained from its human counterpart. Second, agonist potencies in the cAMP assay were also highly correlated with binding affinities. These peptides could be classified into several groups according to their potency at the SLC-1 receptor (from subnanomolar activity to complete inactivity). Indeed, there was a strong correlation between their effects upon food intake and the results obtained at the rat SLC-1 receptor. The present report describes for the first time the rat SLC-1 receptor pharmacology and clearly establishes the relevance of the SLC-1 receptor in feeding behavior.

Cloning and molecular characterization of the novel human melanin-concentrating hormone receptor MCH2.

Using a genomics-based approach for screening orphan G-protein-coupled receptors, we have identified and cloned a novel high-affinity, melanin-concentrating hormone (MCH) receptor. This receptor, named S643b, displays the greatest overall identity (32%) with the previously reported human SLC-1 receptor (MCH1) and to a lesser extent with the somatostatin receptor subtypes. The gene encoding the S643b receptor spans more than 23 kilobase pairs (kb) and was mapped, by radiation hybrid experiments, on chromosome 6q14.3-q15. Comparison of the S643b cDNA with human genomic sequence reveals that the 340-amino-acid receptor is encoded by five exons. Its tissue distribution, as determined by Northern blot and reverse transcription-polymerase chain reaction analysis, indicates that a 4-kb transcript is predominantly expressed in the brain. When expressed in Chinese hamster ovary (CHO) cells, the S643b receptor displays a strong, dose-dependent, transient elevation of intracellular calcium in response to MCH (EC(50) = 9.5 nM). During the present study, we isolated a splice variant, designated S643a, encoding for a receptor that was not activated by MCH in a cellular calcium mobilization assay. Comparative pharmacological studies using CHO cells stably expressing either SLC-1 or S643b receptors demonstrated that similar structural features of MCH are required to stimulate intracellular Ca(2+) mobilization at both receptors. The identification and localization of this new MCH receptor (MCH2) provides further insight into the physiological implication of MCH in modulating behavioral responses, including food intake.