Online Electrochemical Reduction of Both Inter- and Intramolecular Disulfide Bridges in Immunoglobulins.

Electrochemical reduction of intermolecular disulfide bridges has previously been demonstrated in immunoglobulins but failed to achieve reduction of intramolecular bonds. We now report an improved method that achieves the full reduction of both intermolecular and intramolecular disulfide bridges in a set of monoclonal antibodies based on their intact mass and on MS/MS analysis. The system uses an online electrochemical flow cell positioned online between a chromatography system and a mass spectrometer to give direct information on pairs of heavy and light chains in an antibody. The complete reduction of the intramolecular disulfide bridges is important, as the redox state affects the intact mass of the antibody chain. Disulfide bonds also hamper MS/MS fragmentation of protein chains and thus limit the confirmation of the amino acid sequence of the protein of interest. The improved electrochemical system and associated protocols can simplify sample processing prior to analysis, as chemical reduction is not required. Also, it opens up new possibilities in the top-down mass spectrometry analysis of samples containing complex biomolecules with inter- and intramolecular disulfide bridges.

Hydrogen/deuterium exchange mass spectrometry with improved electrochemical reduction enables comprehensive epitope mapping of a therapeutic antibody to the cysteine-knot containing vascular endothelial growth factor.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has become a popular method for analysis of the conformational dynamics and interactions of proteins. Disulfide-bonded proteins, however, present a challenge to HDX-MS as they require efficient disulfide bond reduction prior to enzymatic proteolysis. Electrochemical reduction (ER) provides an attractive solution to tackle disulfide-bonded proteins that are resistant to conventional chemical reduction during HDX-MS. However, ER-enabled HDX-MS has been limited by technical challenges including partial unwanted protein oxidation side-reactions, incompatibility with certain buffer components and most importantly, a lack of overall method robustness. In this study, we have sought to address these challenges. We perform a systematic screening of the compatibility of ER to buffers commonly used in HDX-MS samples by using a reliable and simple system suitability test (SST). Furthermore, we demonstrate the benefits of a new design of the electrochemical cell (EC) for ER-enabled HDX-MS, which include a) high repeatability and robustness over large sample batches without the need for electrode polishing and b) high reduction efficiency of disulfide-bonded proteins without unwanted oxidation side-reactions. We show the real-world applicability of the optimized ER-enabled HDX-MS workflow by performing an epitope mapping of a Fab fragment of a therapeutic monoclonal antibody (mAb) to the cysteine knot-containing vascular endothelial growth factor (VEGF). The results allow us to comprehensively map sites in VEGF involved in mAb binding. Overall, our findings show how ER and HDX-MS can be combined to enable analysis of the conformation and interactions of challenging disulfide-rich proteins.

Clinical performance of serum prostate-specific antigen isoform [-2]proPSA (p2PSA) and its derivatives, %p2PSA and the prostate health index (PHI), in men with a family history of prostate cancer: results from a multicentre European study, the PROMEtheuS project.

OBJECTIVES: To test the sensitivity, specificity and accuracy of serum prostate-specific antigen isoform [-2]proPSA (p2PSA), %p2PSA and the prostate health index (PHI), in men with a family history of prostate cancer (PCa) undergoing prostate biopsy for suspected PCa. To evaluate the potential reduction in unnecessary biopsies and the characteristics of potentially missed cases of PCa that would result from using serum p2PSA, %p2PSA and PHI. PATIENTS AND METHODS: The analysis consisted of a nested case-control study from the PRO-PSA Multicentric European Study, the PROMEtheuS project. All patients had a first-degree relative (father, brother, son) with PCa. Multivariable logistic regression models were complemented by predictive accuracy analysis and decision-curve analysis. RESULTS: Of the 1026 patients included in the PROMEtheuS cohort, 158 (15.4%) had a first-degree relative with PCa. p2PSA, %p2PSA and PHI values were significantly higher (P < 0.001), and free/total PSA (%fPSA) values significantly lower (P < 0.001) in the 71 patients with PCa (44.9%) than in patients without PCa. Univariable accuracy analysis showed %p2PSA (area under the receiver-operating characteristic curve [AUC]: 0.733) and PHI (AUC: 0.733) to be the most accurate predictors of PCa at biopsy, significantly outperforming total PSA ([tPSA] AUC: 0.549), free PSA ([fPSA] AUC: 0.489) and %fPSA (AUC: 0.600) (P ≤ 0.001). For %p2PSA a threshold of 1.66 was found to have the best balance between sensitivity and specificity (70.4 and 70.1%; 95% confidence interval [CI]: 58.4-80.7 and 59.4-79.5 respectively). A PHI threshold of 40 was found to have the best balance between sensitivity and specificity (64.8 and 71.3%, respectively; 95% CI 52.5-75.8 and 60.6-80.5). At 90% sensitivity, the thresholds for %p2PSA and PHI were 1.20 and 25.5, with a specificity of 37.9 and 25.5%, respectively. At a %p2PSA threshold of 1.20, a total of 39 (24.8%) biopsies could have been avoided, but two cancers with a Gleason score (GS) of 7 would have been missed. At a PHI threshold of 25.5 a total of 27 (17.2%) biopsies could have been avoided and two (3.8%) cancers with a GS of 7 would have been missed. In multivariable logistic regression models, %p2PSA and PHI achieved independent predictor status and significantly increased the accuracy of multivariable models including PSA and prostate volume by 8.7 and 10%, respectively (P ≤ 0.001). p2PSA, %p2PSA and PHI were directly correlated with Gleason score (ρ: 0.247, P = 0.038; ρ: 0.366, P = 0.002; ρ: 0.464, P < 0.001, respectively). CONCLUSIONS: %p2PSA and PHI are more accurate than tPSA, fPSA and %fPSA in predicting PCa in men with a family history of PCa. Consideration of %p2PSA and PHI results in the avoidance of several unnecessary biopsies. p2PSA, %p2PSA and PHI correlate with cancer aggressiveness.

Angioplasty and stent treatment of transplant renal artery stenosis.

Transplant renal artery stenosis is a major complication that requires a therapeutic approach involving surgery or angioplasty. The aim of this study was to analyse the evolution of renal transplant patients with renal allograft artery stenosis treated by angioplasty and stent placement. Thirteen patients were diagnosed with transplant renal artery stenosis. Clinical suspicion was based on deterioration of renal function and/or poorly controlled hypertension with compatible Doppler ultrasound findings. The diagnosis was confirmed by arteriography, performing an angioplasty with stent placement during the same operation. A progressive improvement in renal function was observed during the first 3 months after the angioplasty, and renal function then remained stable over 2 years. In addition, blood pressure improved during the first 2 years, and as a consequence there was no need to increase the average number of anti-hypertensive drugs administered (2.5 drugs per patient). In conclusion, angioplasty with stent placement is a safe and effective procedure for the treatment of transplant renal artery stenosis.

Use of multidetector CT in presurgical evaluation of potential kidney transplant recipients.

Kidney transplantation is the treatment of choice for end-stage renal disease. Optimal presurgical evaluation of the potential kidney transplant recipient is important for the success of the transplantation. Multidetector computed tomography (CT) allows assessment of the feasibility of kidney transplantation; detection of coexisting illnesses that may affect survival of the graft and that must be treated before transplantation; and evaluation of possible peripheral vascular disease, which is present in a significant number of potential kidney transplant recipients. Multidetector CT provides a wide range of information in these patients. Vascular and extravascular systems can be evaluated, allowing one to determine whether kidney transplantation is possible, whether presurgical procedures are necessary, and which is the best surgical technique for each candidate. Knowledge of the surgical techniques, use of an optimal multidetector CT technique, and the ability to identify common and uncommon radiologic findings are essential for correct evaluation of potential kidney transplant recipients.

CT findings in urinary diversion after radical cystectomy: postsurgical anatomy and complications.

Numerous surgical procedures have been developed for urinary diversion in patients who have undergone a radical cystectomy for bladder cancer or, less frequently, a benign condition. Because urinary diversion procedures are complex, early and late postsurgical complications frequently occur. Possible complications include alterations in bowel motility, anastomotic leaks, fluid collections (abscess, urinoma, lymphocele, and hematoma), fistulas, peristomal herniation, ureteral strictures, calculi, and tumor recurrence. Computed tomography (CT) is an accurate method for evaluating such events. Multiplanar reformatting and three-dimensional volume rendering of multidetector CT image data are particularly useful for achieving an accurate and prompt diagnosis of complications and obtaining information that is essential for adequate surgical management. In addition, knowledge of urinary diversion procedures, normal postsurgical appearances, and optimal CT technique for postsurgical evaluations is essential for detecting complications and avoiding misdiagnosis.

Embolization of a bleeding aneurysm in a patient with spontaneous perirenal haematoma due to Polyarteritis nodosa.

OBJECTIVE: To report a rare case of a Spontaneous perirenal haematoma due to Polyarteritis nodosa treated with a selective embolización of the bleeding aneurysm. Polyarteritis nodosa (PAN) is one of a spectrum of diseases thet belongs to the pathologic category of necrotizing vasculitis. Spontaneous perirenal haematoma (SPH) is an unusual complication of PAN. METHODS: We are introducing a patient with SPH due to PAN. RESULTS: The patient was treated with embolization of a left renal bleeding aneurysm of the upper interlobar artery. Pan is the most frequent vascular disease associated with spontaneous renal hematoma. Therefore the diagnosis may be difficult to determine. CONCLUSIONS: A selective embolization of the bleeding aneurysm is a therapeutical maneuver to be considered.

Protective effect of carnosol on CCl(4)-induced acute liver damage in rats.

BACKGROUND: We recently reported that (Lamiaceae) may alleviate CCl(4)-induced acute hepatotoxicity in rats, possibly blocking the formation of free radicals generated during CCl(4) metabolism. Carnosol, one of the main constituents of Rosmarinus, has been shown to have antioxidant and scavenging activities. Therefore, it is plausible to expect that carnosol may mediate some of the effects of Rosmarinus on oxidative stress consequences induced by CCl(4) in the liver. DESIGN: We evaluated the effectiveness of carnosol to normalize biochemical and histological parameters of CCl(4)-induced acute liver injury. METHODS: Male Sprague Dawley rats (n = 5) injured by CCl(4) (oral dose 4 g/kg of body weight) were treated with a single intraperitoneal dose (5 mg/kg) of carnosol. Twenty-four hours later, the rats were anaesthetized deeply to obtain the liver and blood, and biochemical and histological parameters of liver injury were evaluated. RESULTS: Carnosol normalized bilirubin plasma levels, reduced malondialdehyde (MDA) content in the liver by 69%, reduced alanine aminotransferase (ALT) activity in plasma by 50%, and partially prevented the fall of liver glycogen content and distortion of the liver parenchyma. CONCLUSIONS: Carnosol prevents acute liver damage, possibly by improving the structural integrity of the hepatocytes. To achieve this, carnosol could scavenge free radicals induced by CCl(4), consequently avoiding the propagation of lipid peroxides. It is suggested that at least some of the beneficial properties of Rosmarinus officinalis are due to carnosol.

Tumor de células germinales del ovario: estudio retrospectivo en SOLCA-Quito / Tumor of germinal cells of the ovary: retrospective study in Solca-Quito

Analiza que los Tumores de Células Germinales del Ovario (TGO) representan el 20xciento de todos los tumores en Europa y Norte América; 5xciento al 8xciento de estos son malignos, ocurren en mujeres jóvenes, son de crecimiento rápido y su incidencia aumenta en la Disgenesia gonadal. El presente es un estudio retrospectivo realizado en el Hospital de SOLCA Núcleo de Quito, donde se registraron 18 casos de Tumores Malignos de Células Germinales del Ovario (TGOM) reportados entre 1985 y 1994, y confirmados por histopatología. La edad media fue de 20 años (rango: 7-38 años); la sintomatología más frecuente reportada: masa abdominal 72.5xciento, dolor abdominal 66.6xciento. El estudio mayor realizado es la Ultrasonografía abdomino-pélvica, en la que se identificaron tumores sólidos (52xciento), quísticos (17,6xciento) y mixtos (17,6xciento). El tumor más frecuente es el Disgerminoma (44xciento), siendo el estadío IA el más frecuente, seguido del IIIC. Todas las pacientes recibieron quimioterapia adyuvante, y fueron controladas cada 6 meses con marcadores tumorales, con una sobrevida global del 53xciento (Teratomas inmaduros 100xciento, tumores mixtos 75xciento y Digerminomas 50xciento). Se concluye que el comportamiento de estos tumores en el presente estudio es similar al reportado en la literatura mundial, a excepción de la baja sobrevida, posiblemente debido a la falta de un análisis más prolijo de los factores pronósticos en el manejo terapéutico.

Tumor de células gigantes multicéntrico sincrónico: presentación de un caso y revisión de la literatura / Tumor of giant cells multicentric synchronous: presentation of a case and overhaul of Literature

Presenta un caso de tumor de células gigantes (TCG) de la muy rara variedad multicéntrico sincrónico localizado en húmero proximal y fémur distal derechos, en un paciente mestizo de 20 años. Se comenta el cuadro clínico, procedimiento diagnóstico, estudio histopatológico y discusión de la bibliografía sobre las diversas opciones de tratamiento quirúrgico. El tumor de células gigantes tiene una incidencia del 4xciento al 7xciento de las neoplasias óseas, representando el tipo multicéntrico menos del 1xciento de estos. Se reportan en la literatura mundial apenas 46 casos, todos ellos metacrónicos, con lo cual el tipo sincrónico es todavía más raro. El sinnúmero de procedimientos quirúrgicos y la variedad de resultados terapéuticos ponen de manifiesto el difícil manejo de esta neoplasia...