Proteogenomic-based discovery of minor histocompatibility antigens with suitable features for immunotherapy of hematologic cancers.

Pre-clinical studies have shown that injection of allogeneic T cells primed against a single minor histocompatibility antigen (MiHA) could cure hematologic cancers (HC) without causing any toxicity to the host. However, translation of this approach in humans has been hampered by the paucity of molecularly defined human MiHAs. Using a novel proteogenomic approach, we have analyzed cells from 13 volunteers and discovered a vast repertoire of MiHAs presented by the most common HLA haplotype in European Americans: HLA-A*02:01;B*44:03. Notably, out of >6000 MiHAs, we have identified a set of 39 MiHAs that share optimal features for immunotherapy of HCs. These 'optimal MiHAs' are coded by common alleles of genes that are preferentially expressed in hematopoietic cells. Bioinformatic modeling based on MiHA allelic frequencies showed that the 39 optimal MiHAs would enable MiHA-targeted immunotherapy of practically all HLA-A*02:01;B*44:03 patients. Further extension of this strategy to a few additional HLA haplotypes would allow treatment of almost all patients.

[Urethral cancer: report of three cases and review of the literature]. / Cancer de l'urètre féminin, à propos de trois cas et revue de la littérature.

OBJECTIVE: To review the various clinical forms of female urethral cancer in the light of three clinical cases with a review of the corresponding treatment guidelines. METHOD: The authors report three cases of female urethral cancer. Case 1 consisted of squamous cell carcinoma in a 56-year-old woman with no particular history. Case 2 was a urothelial tumour arising in a urethral diverticulum in a 60-year-old smoker. Case 3 was a 69-year-old woman patient with invasive urothelial carcinoma. RESULTS: Case 1 was treated by segmental urethrectomy with no adjuvant therapy and a favourable course. Case 2 was treated by anterior pelvic exenteration with no adjuvant therapy. This patient relapsed in the form of peritoneal carcinomatosis two years later and died. Case 3 was initially treated by anterior pelvic exenteration followed by a chemoradiotherapy combination after local recurrence with a favourable course. CONCLUSION: There are many clinical presentations and histological forms of female urethral cancer. Localized distal lesions can be treated by simple circumferential resection. The treatment of other lesions comprises anterior pelvic exenteration and platinum- or M-VAC-based chemoradiotherapy. The main prognostic factors for these tumours are their size, histological type, site and the presence of pelvic lymph node extension.

[Laparoscopic partial nephrectomy: urology resident learning curve on a porcine model]. / Néphrectomie partielle laparoscopique: courbe d'apprentissage d'un interne en urologie sur un modèle porcin.

INTRODUCTION: Several teams have recently confirmed the technical feasibility of laparoscopic partial nephrectomy (LPN). However, this procedure is not widely performed because it is technically difficult and associated with a high rate of bleeding complications, even for experienced teams. The authors studied the LPN learning curve for urology residents using a porcine model based on analysis of the following criteria: operating time, warm ischaemia time and intraoperative and postoperative bleeding. MATERIALS AND METHODS: Forty LPN were performed by the same operator. All operations were performed after arterial clamping and heparinisation of the animal. The renal section was always the same, removing 40% of the kidney and always comprised the excretory tract. A continuous running suture on the excretory tract and interrupted sutures on the parenchyma were performed. Operating time and warm ischaemia time were recorded. Animals were monitored for ten days. Intraoperative and postoperative bleeding via drains was recorded and retrograde urography was performed on the 10th day to confirm the absence of excretory tract leaks. Linear regression statistical tests investigated a correlation between these various criteria and the number of cases performed. RESULTS: The mean total operating time, warm ischaemia time and total bleeding (intraoperative and postoperative) were 108 minutes (70-140 minutes), 38 minutes (22-50 minutes) and 95 ml (10-300 ml), respectively. Linear regression analysis revealed a direct correlation between the number of cases performed and intraoperative bleeding (p<0.001) and warm ischaemia time (p<0.001). These parameters became stable after the 10th operated case. Two cases of urine leaks were observed on D10 out of a series of 40 operations, with no correlation with the number of cases performed. CONCLUSION: The operating time and warm ischaemia time are directly correlated with the number of cases performed. Training on a porcine model appears to be a good way to reduce the learning curve in man. Ten operations are necessary to acquire the various steps of the procedure.

Malignant epithelioid renal angiomyolipoma involving the inferior vena cava in a patient with tuberous sclerosis.

Renal angiomyolipomas (AMLs) are mesenchymal tumors that occur either sporadically or are associated with tuberous sclerosis, and are generally considered to be benign. Malignant AML is extremely rare, and most are found to be epithelioid histopathologically. The authors report the case of a patient followed for renal AML. On CT surveillance, this lesion developed features of a malignant tumor involving the renal vein and inferior vena cava. The patient was treated by nephrectomy and tumor thrombectomy with retroperitoneal lymph node dissection. Histological examination demonstrated renal AML with a malignant epithelioid contingent. The various aspects of this histological and radiological variant are discussed.

[Metastasis to trocar site after lymphadenectomy with retroperitoneal laparoscopy]. / Métastase sur site de trocart après lymphadénectomie par laparoscopie rétropéritonéale.

The authors report a case of trocar site metastasis, one year after laparoscopic retroperitoneal lymphadenectomy. This is the seventh published case of trocar site metastasis in laparoscopic urological surgery. The lymph node resection procedure and the histological type of the tumour appear to be risk factors for the development of wall metastases.

Transcription factor HIF-1 is a necessary mediator of the pasteur effect in mammalian cells.

The ability to respond to differential levels of oxygen is important to all respiring cells. The response to oxygen deficiency, or hypoxia, takes many forms and ranges from systemic adaptations to those that are cell autonomous. Perhaps the most ancient of the cell-autonomous adaptations to hypoxia is a metabolic one: the Pasteur effect, which includes decreased oxidative phosphorylation and an increase in anaerobic fermentation. Because anaerobic fermentation produces far less ATP than oxidative phosphorylation per molecule of glucose, increased activity of the glycolytic pathway is necessary to maintain free ATP levels in the hypoxic cell. Here, we present genetic and biochemical evidence that, in mammalian cells, this metabolic switch is regulated by the transcription factor HIF-1. As a result, cells lacking HIF-1alpha exhibit decreased growth rates during hypoxia, as well as decreased levels of lactic acid production and decreased acidosis. We show that this decrease in glycolytic capacity results in dramatically lowered free ATP levels in HIF-1alpha-deficient hypoxic cells. Thus, HIF-1 activation is an essential control element of the metabolic state during hypoxia; this requirement has important implications for the regulation of cell growth during development, angiogenesis, and vascular injury.

Early detection of pancreatic cancer in patients with chronic pancreatitis: diagnostic utility of a K-ras point mutation in the pancreatic juice.

OBJECTIVE: Point mutations of the K-ras oncogene at codon 12 have been described several months before the onset of pancreatic cancer in isolated cases of chronic pancreatitis (CP). The aim of this study was to evaluate the interest of a prospective follow-up of patients with CP and K-ras mutations at codon 12 in the detection of early pancreatic cancer. METHODS: From February 1996 to March 1998, 36 patients (mean age 52.6 yr, 31 men, five women) with CP (alcoholic: 61.1%, pancreas divisum: 5.6%, autoimmune: 5.6%, unknown origin: 27.7%) were included and then prospectively monitored (median duration of 22 months) for detection of pancreatic carcinoma. K-ras point mutations were examined by two-step polymerase chain reaction combined with restriction enzyme digestion in pancreatic juice collected during endoscopic retrograde pancreatography. RESULTS: Ten patients (27.8%) were positive for K-ras mutation. Patients with and without the mutation were not different with respect to age and sex ratio. K-ras mutations were homogeneously distributed according to the etiology (alcoholic vs nonalcoholic) and morphological characteristics (ductal stricture or mass vs none) of CP. A pancreatic carcinoma was discovered at an invasive stage in two patients, respectively at 7 and 17 months after disclosure of a K-ras mutation, versus none in patients without the mutation (p < 0.02). CONCLUSIONS: Presence of a K-ras gene mutation is not rare in patients with CP and represents an increased risk of developing pancreatic cancer. However, its utility for the detection of early pancreatic cancer remains doubtful in clinical practice.

Immunofluorometric assay of human kallikrein 10 and its identification in biological fluids and tissues.

BACKGROUND: The human kallikrein 10 gene [KLK10, also known as normal epithelial cell-specific 1 gene (NES1)] is a member of the human kallikrein gene family. The KLK10 gene encodes for a secreted serine protease (hK10). We hypothesize that hK10 is secreted into various biological fluids and that its concentration changes in some disease states. The aim of this study was to develop a sensitive and specific immunoassay for hK10. METHODS: Recombinant hK10 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK10 antisera. A sandwich-type immunofluorometric assay was then developed using these antibodies. RESULTS: The hK10 immunoassay has a detection limit of 0.05 microg/L. The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6). The assay was linear from 0 to 20 microg/L with within- and between-run CVs <10%. hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum. CONCLUSIONS: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts. This assay can be used to examine the value of hK10 as a disease biomarker.

The impact on clinical practice of endoscopic ultrasonography used for the diagnosis and staging of pancreatic adenocarcinoma.

CONTEXT: Endoscopic ultrasonography is considered a highly accurate procedure for diagnosing small pancreatic tumors and assessing their locoregional extension. OBJECTIVE: To evaluate the impact of endoscopic ultrasonography on the management of pancreatic adenocarcinoma in clinical practice. PATIENTS: Sixty-four consecutive patients (mean age 70.5 plus/minus 11.9 years) hospitalized for staging or diagnosis of pancreatic adenocarcinoma were retrospectively (from January 1995 to November 1997) or prospectively studied (from December 1997 to August 1999). SETTING: Group 1 consisted of 52 patients with pancreatic adenocarcinoma which was discovered using computerized tomography scanning and/or ultrasound. Endoscopic ultrasonography was utilized for staging purposes only in patients who were considered to be operable and the tumor to be resectable based on computerized tomography scanning criteria. Group 2 consisted of 12 patients who were diagnosed as having a pancreatic adenocarcinoma using endoscopic ultrasonography whereas computerized tomography scanning and ultrasound was negative. MAIN OUTCOME MEASURES: The impact of endoscopic ultrasonography was analyzed on the basis of the number of patients requiring endoscopic ultrasonography as a staging procedure (Group 1) and by evaluating the performance of endoscopic ultrasonography in determining resectability (Groups 1 and 2) based on the surgical and anatomopathological results. RESULTS: Endoscopic ultrasonography was performed in 20 out of 64 patients (31.3%): 8/52 in Group 1 (15.4%) and all 12 patients of Group 2. Endoscopic ultrasonography correctly assessed an absolute contraindication to resection in 11 cases. Resection was confirmed in 8 of the 9 cases selected by endoscopic ultrasonography. The positive predictive value, negative predictive value and overall accuracy of endoscopic ultrasonography for determining resection were 89%, 100%, and 95%, respectively. CONCLUSIONS: The impact of endoscopic ultrasonography seems especially relevant for the detection of pancreatic tumors after negative computerized tomography scanning, and for the prevention of unnecessary laparotomies as complementary staging after ultrasonography and computerized tomography scanning.

[Hemorrhagic cystitis: etiology and treatment]. / Cystite hémorragique: étiologie et traitement.

Haemorrhagic cystitis consists of acute or insidious diffuse bleeding from the bladder mucosa. Although the aetiology of haemorrhagic cystitis is varied, the two predominant causes are chemical and irradiation. The chemical compounds most frequently responsible for haemorrhagic cystitis are oxaphosphorines (cyclophosphamide, ifosfamide). Haemorrhagic cystitis may also be due to toxic or infectious causes (bacterial, fungal, parasitic or viral). Finally, haemorrhagic cystitis can occur in the context of a systemic disease or may be isolated and idiopathic. Prevention of haemorrhagic cystitis, based on general measures and specific measures, is essential, but is not always effective. In the case of proven haemorrhagic cystitis, various treatment options are available, including and can associate clot extraction, continuous bladder irrigation, bladder instillations of haemostatic factors, formalin, hyperbaric oxygen therapy, arterial embolization or salvage surgery. Although therapeutic management is usually effective, it can sometimes be difficult due to the severity of the bleeding and its repercussions on the patient's general state. Deaths are not exceptional, emphasizing the seriousness of haemorrhagic cystitis. Based on a review of the literature, the authors review the aetiology and treatment of haemorrhagic cystitis.

Galectin-3-mediated adherence of Proteus mirabilis to Madin-Darby canine kidney cells.

Proteus mirabilis is an important cause of urinary tract infections (UTIs) and can result in acute pyelonephritis. Proteus mirabilis expresses several, morphologically distinct, fimbrial species, and previous studies have shown that the nonagglutinating fimbriae (NAF) mediate bacterial adherence to a number of cell lines, including Madin-Darby canine kidney (MDCK) cells. Immunoblot overlay analysis of the plasma membrane fraction from MDCK cells with purified NAF revealed a 34-kDa band, which has been analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Database search identified galectin-3 as a potential protein candidate. Immunocytochemical assay of MDCK cells with a galectin-3-specific monoclonal antibody, anti-Mac-2, confirmed its presence on the plasma membrane extracellular surface. Preincubation of P. mirabilis with anti-Mac-2 monoclonal antibodies, specific for galectin-3, resulted in the inhibition of bacterial binding to MDCK cells. These data suggest a role for galectin-3, interacting with appropriately glycosylated surface receptors and P. mirabilis fimbriae, as a mediator of bacterial adherence in vitro.

Results of the tension-free vaginal tape procedure for the treatment of type II stress urinary incontinence at a minimum followup of 1 year.

PURPOSE: We evaluated the safety and efficacy of the tension-free vaginal tape procedure for treating type II stress urinary incontinence in females. MATERIALS AND METHODS: Between April 1998 and April 1999, 62 women 28 to 86 years old (mean age 62.8) were treated consecutively for stress urinary incontinence with the tension-free vaginal tape procedure. Preoperative evaluation included history, physical examination and multichannel video urodynamics. All patients had type II stress urinary incontinence, none had preoperative detrusor instability or significant pelvic prolapse and in 16 previous surgery for stress incontinence had failed. RESULTS: All patients were followed at least 12 months after the procedure (median 16.2). A total of 42 and 20 women received spinal and local anesthesia, respectively. We noted 6 bladder perforations, including 5 in patients with a history of surgery for stress urinary incontinence. Blood loss was less than 200 cc in all cases. We observed no prolonged postoperative pain, infection or sling rejection. Post-void residual urine was less than 100 cc the day after surgery in 59 cases. Only 3 patients self-catheterized a maximum of 4 days. At followup 54 women (87.1%) were cured of stress urinary incontinence, 6 were improved (9.6%) and 2 had failure (3. 3%), while 4 (6.4%) had new onset detrusor instability without evidence of bladder outlet obstruction. CONCLUSIONS: The tension-free vaginal tape procedure appears to be a minimally invasive, safe and effective treatment for type II stress urinary incontinence. A history of surgery for stress incontinence seems to be a risk factor for bladder perforation.

[Spontaneous intraperitoneal rupture of the bladder treated with laparoscopy]. / Rupture spontanée intrapéritonéale de la vessie traitée par coelioscopie.

The authors report the case of a 53-year-old patient admitted for spontaneous bladder rupture occurring during an episode of alcoholic intoxication. Laparoscopic treatment consisted of peritoneal toilet with bladder suture and drainage of the abdominal cavity with a favourable postoperative course.

[Peroperative erection in lower urologic surgery]. / Erection peropératoire en chirurgie urologique basse.

Erection is a rare event during lower urinary tract surgery. When it is extremely refractory, endoscopy is impossible and the lower urinary tract surgery may need to be deferred. The development of erection during spinal anaesthesia is due to manipulations before complete installation of sensory block or incomplete blockade of sacral segments (S2 to S4) supplying the nervi erigentes. The mechanism of erection during general anaesthesia remains poorly elucidated. General anaesthesia may suppress central or peripheral sympathetic control of flaccidity. The peripheral target could be the smooth muscle of the cavernous tissue, either by a direct action or via alpha-adrenergic receptors. Psychogenic and reflexogenic stimuli have also been proposed, possibly facilitated by amplification of sensations during stage II anaesthesia. The various treatments are based on experience acquired in the field of priapism. Systemic treatments such as ketamine an beta 2 mimetics or benzodiazepines are not always effective and are associated with considerable adverse effects. Intracavernous injections of alpha-adrenergic drugs constitute the treatment of choice. Phenylephrine and etilefrine are preferred because of their rapid efficacy and particularly their only moderate cardiovascular adverse effects.

[Epididymal gaseous abscess after BCG treatment]. / Abcès caséeux épididymaire après BCG-thérapie.

The authors report a case of unilateral epididymitis with caseous abscess after BCG-therapy for superficial bladder tumour. The severity of the lesions required orchidectomy, followed by four-agent tuberculostatic therapy.

[Radiotherapy of localized prostatic cancer: follow-up of 48 patients. G.C.C.P. (Cooperative Group for the Study of Prostatic Cancer)]. / Radiothérapie des cancers de prostate localisés: 48 malades suivis plus de dix ans. G.C.C.P. (Groupe Coopératif d'Etude du Cancer de la Prostate).

OBJECTIVE: To evaluate the safety and long-term efficacy of curative-intent radiation therapy in patients with apparently localized prostate cancer. METHOD: 48 patients with T < 3 M0 prostate cancer recruited between 1981 and 1985 received regular clinical follow-up for at least ten years or until their death. Radiation therapy was given according to the protocols established by Ray and Bagshaw. RESULTS: Radiation therapy was safe and effective in most patients. The rate of escape phenomenon was less than 10% after two years. Tumor control rates were greater than 80% after five years and 50% after ten years. Unfortunately, local remission, even when prolonged, did not necessarily indicate a complete cure: 20% of local recurrences developed five to 11 years after radiation therapy. CONCLUSION: Radiation therapy may be the best first-line treatment in men older than 75 years of age and in those whose life expectancy seems shorter than ten years, but should probably not be considered curative.

Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry.

The coupling of microfabricated devices to nanoelectrospray mass spectrometers using both a triple quadrupole and a quadrupole time-of-flight mass spectrometer (QqTOF MS) is presented for the analysis of trace-level membrane proteins. Short disposable nanoelectrospray emitters were directly coupled to the chip device via a low dead volume connection. The analytical performance of this integrated device in terms of sensitivity and reproducibility was evaluated for standard peptide mixtures. A concentration detection limit ranging from 3.2 to 43.5 nM for different peptides was achieved in selected ion monitoring, thus representing a 10-fold improvement in sensitivity compared to that of microelectrospray using the same chip/mass spectrometer. Replicate injections indicated that reproducibility of migration time was typically less than 3.1% RSD whereas RSD values of 6-13% were observed on peak areas. Although complete resolution of individual components is not typically achieved for complex digests, the present chip capillary electrophoresis (chip-CE) device enabled proper sample cleanup and partial separation of multicomponent samples prior to mass spectral identification. Analyses of protein digests were typically achieved in less than 1.5 min with peak widths of 1.8-2.5 s (half-height definition) as indicated from individual reconstructed ion electropherograms. The application of this chip-CE/QqTOF MS system is further demonstrated for the identification of membrane proteins which form a subset of the Haemophilus influenzae proteome. Bands first separated by 1D-gel electrophoresis were excised and digested, and extracted tryptic peptides were loaded on the chip without any further sample cleanup or on-line adsorption preconcentration. Accurate molecular mass determination (< 5 ppm) in peptide-mapping experiments was obtained by introducing an internal standard via a postseparation channel. The analytical potential of this integrated device for the identification of trace-level proteins from different strains of H. influenzae is demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.

Post-translational modification of p53 protein in response to ionizing radiation analyzed by mass spectrometry.

The p53 tumor suppressor protein promotes cell cycle arrest or apoptosis in response to DNA damage and other forms of stress. p53 protein functions as a transcription factor by binding to specific DNA sequences and regulating the transcription of target genes. This activity of p53 is reported to be regulated by phosphorylation and acetylation occuring at various sites on the molecule. Here, we have used a direct and non-radioactive approach involving mass spectrometric analysis of p53 protein to identify sites that are covalently modified in vivo, either constitutively or in response to ionizing radiation. Following partial purification by immuno-affinity chromatography and enzymatic in-gel digestion, the resulting p53 peptides were analyzed by MALDI-TOF and nanoelectrospray mass spectrometry. Mass spectrometry analyses identified four sites at the N terminus that were phosphorylated in response to irradiation, a single constitutive phosphorylation site at serine 315 and several acetylation sites.