High-soluble CGA levels are associated with poor survival in bladder cancer.

Recently, a neuroendocrine-like molecular subtype has been discovered in muscle-invasive urothelial bladder cancer (BC). Chromogranin A (CGA) is a widely used tissue and serum marker in neuroendocrine tumors. Our aim was to evaluate serum CGA (sCGA) concentrations and their associations with clinical and follow-up data in BC and renal cell carcinoma (RCC). sCGA concentrations were analyzed in the following cohorts: (1) BC training set (n = 188), (2) BC validation set (n = 125), (3) RCC patients (n = 77), (4) healthy controls (n = 97). CGA immunohistochemistry and RT-qPCR analyses were performed in 20 selected FFPE and 29 frozen BC tissue samples. Acquired data were correlated with clinicopathological parameters including comorbidities with known effect on sCGA as well as with patients' follow-up data. sCGA levels were significantly higher in BC but not in RCC patients compared to healthy controls. High sCGA levels were independently associated with poor overall and disease-specific survival both in the BC training (P < 0.001, P = 0.002) and validation set (P = 0.009, P = 0.017). sCGA levels were inversely correlated with glomerulus filtrating rate (GFR) and linearly correlated with creatinine clearance and urea concentrations. These correlations were not related to the prognostic value of sCGA. Tissue CGA levels were low to absent independently of sCGA concentrations. Our results demonstrate elevated levels and an independent prognostic value for sCGA in BC but not in RCC. Despite the significant correlation between sCGA and GFR, the prognostic relevance of sCGA seems not related to impaired renal function or other comorbidities.

[Prevalence of macrovascular arterial involvement of the 4 limbs in systemic sclerosis: About a case series of 14 patients]. / Prévalence de l'atteinte artérielle macrovasculaire des 4 membres dans la sclérodermie systémique : à propos de 14 patients.

INTRODUCTION: Trophic disorders of the extremities are a common complication of systemic sclerosis (SSc), mainly related to microvascular damage. However, SSc seems to be a risk factor for premature athero-thrombotic disease that can affect the peripheral arteries, participate in the occurrence of trophic disorders and promote the occurrence of infectious complications. The objective of this study was to assess the prevalence of arterial disease of the limbs in SSc patients. METHODS: Consecutive inclusions in the context of a multidisciplinary consultation centered on disability of the hand with collection of clinical data [cardiovascular risk factors (CVRF), history of trophic disorders of ischemic origin, peripheral pulse palpation, Allen maneuver the upper (UL) and lower limbs (LL)], and hemodynamic data (flow recorded by Doppler in radial, ulnar, anterior and posterior tibial arteries, and measurement of systolic indices ankles). RESULTS: Fourteen patients were included (11 right-handers, 2 left-handers, 1 ambidextrous). The sex-ratio male/female was 0.27 and the average age of 58.1±10.4 years. The main CVRF were age and smoking. In the UL, 42.8% of patients had a history of trophic disorders, Allen maneuver was abnormal for 35.7% of the superficial palmar arch, 42.9% of ulnar pulse were not perceived and there was no recordable flow in 25% of ulnar artery. In the LL, 14.3% of patients had already presented trophic disorders toes, Allen maneuver was abnormal for 15.4% of the posterior tibial artery, 25.6% of posterior tibial pulse were not perceived and flow of 15.4% of posterior tibial arteries was pathological. CONCLUSION: The distal macrovascular disease preferentially affecting the ulnar and posterior tibial arteries with a high frequency to the UL and two times less at LL. The pathophysiology is unclear but it could be a proper manifestation of SSc. It seems necessary that SSc patients have a strict balance of their CVRF and a screening of macrovascular arterial lesions. There is also the question of the place of an anti-atherosclerotic therapy in these patients.

Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation.

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M, 21,000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.

Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experiments.

Monoclonal antibodies are now widely used to measure the concentration of steroid hormones in human serum samples. The great development of molecular engineering techniques over the past 10 years has made possible the improvement of specificity and/or sensitivity of selected antibodies. We have obtained two monoclonal antibodies, 17E12E5 and 10G6D6, using estradiol-6-ethyl methoxy carbonyl (EMC)-bovine serum albumin (BSA) as immunogen. To tentatively improve their affinities for natural estradiol, we have initiated their structural and functional studies. For this purpose, we have cloned and sequenced the genes encoding the variable fragments of each antibody. Single chain variable fragments (scFv) were produced into the periplasmic space of E. coli using the pLIP6 expression vector. Mapping of the functional structures of both antibodies was obtained by combination of modelling and mutational analyses together with cross-reaction studies. The two binding pockets are described and models of estradiol complexed to 17E12E5 and 10G6D6 are proposed.

Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris: evidence of hyper O-glycosylation.

A truncated form of surface antigen 1 (SAG1t), the immunodominant surface antigen of Toxoplasma gondii, was expressed in the methylotrophic yeast, Pichia pastoris. The truncated protein lacked the C-terminal residues which, in native SAG1, encompass a glycosylphosphatidylinositol anchorage site. The single potential N-glycosylation site was mutated and a sequence encoding a hexahistidine tag was introduced at the C-terminal of the construction to aid purification by immobilized metal-chelate chromatography. Recombinant SAG1t was efficiently secreted into the culture medium as three protein species having molecular masses of 29, 38/45 and 50/60 kDa. This heterogeneity was dependent upon the composition of the medium used to grow the yeast transformants. Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAG1t heterogeneity was related to the presence of O-linked oligosaccharides containing alpha1-2-, alpha1-3- or alpha1-6-linked mannoses. The glycosylated and deglycosylated recombinant SAG1t were recognized by monoclonal and human-serum-derived antibodies, specific for the native SAG1, which suggested that the O-glycosylations had no major effect on the protein conformation. However, ELISA and Western-blot analysis with human sera showed that the O-carbohydrates added by P. pastoris could be recognized as antigenic structures. As a consequence, purification of the unglycosylated 29 kDa recombinant SAG1t species or deglycosylation is required in order to use recombinant SAG1t as a diagnostic reagent. Moreover, the presence of carbohydrates, not found on the native protein, suggests that addition of unnatural glycan structures by P. pastoris is a potential drawback that should be considered when using this expression system.