Feasibility of quantitative analysis of colonic perfusion using indocyanine green to prevent anastomotic leak in colorectal surgery.

BACKGROUND: The aim of this study was to quantify Fluorescence angiography with indocyanine green (ICG) in colorectal cancer anastomosis, determine influential factors in its temporary intensity and pattern, assessing the ability to predict the AL, and setting the cut-off levels to establish high- or low-risk groups. METHODS: Retrospective analysis of prospectively managed database, including 70 patients who underwent elective surgery for colorectal cancer in which performing a primary anastomosis was in primary plan. In all of them, ICG fluorescence angiography was performed as usual clinical practice with VisionSense™ VS Iridium (Medtronic, Mansfield, MA, USA), in Elevision™ IR Platform (Medtronic, Mansfield, MA, USA). Parameters measured at real time or calculated were T0, Tmax, ∆T, Fmax, %pos, Fpos, and Slope. RESULTS: 70 patients were included, 69 anastomosis were performed and one end colostomy. Arterial hypertension demonstrated higher Fmax, as well as the location of the anastomosis (the nearest to rectum, the most intensity detected). A statistical relationship was found between AL and the lower Fpos and Slope. The decision of changing the subjectively decided point of division did not demonstrate statistical difference on the further development of AL. All parameters were analyzed to detect the cut-off related with AL. Only in case of Fpos lower than 158.3 U and Slope lower than 13.1 U/s p-value were significant. The most valuable diagnostic parameter after risk stratification was the Negative Predictive Value. CONCLUSION: Quantitative analysis of ICG fluorescence in colorectal surgery is safe and feasible to stratify risk of AL. Hypertension and location of anastomosis influence the intensity of fluorescence at the point of section. A change of division place should be considered to avoid AL related to vascular reasons when intensities of fluorescence at the point of section is lower than 169 U or slopes lower than 14.4 U/s.

Expression and purification of the surface proteins from Andes virus.

Andes virus is the main causative agent of Hantavirus cardiopulmonary syndrome in South America. There are currently no vaccines or treatments against Andes virus. However, there are several evidences suggesting that antibodies against Andes virus envelope glycoproteins may be enough to confer full protection against Hantavirus cardiopulmonary syndrome. The goal of the present work was to express, purify and characterize the extracellular domains of Andes virus glycoproteins Gn and Gc. We generated two adenoviral vectors encoding the extracellular domains of Andes virus glycoproteins Gn and Gc. Both molecules were expressed by adenoviral transduction in SiHa cells. We found that sGc ectodomain was mainly secreted into the culture medium, whereas sGn was predominantly retained inside the cells. Both molecules were expressed at very low concentrations (below 1 µg/mL). Treatment with the proteasome inhibitor ALLN raised sGc concentration in the cell culture medium, but did not affect expression levels of sGn. Both ectodomains were purified by immobilized metal ion affinity chromatography, and were recognized by sera from persons previously exposed to Andes virus. To our knowledge, this is the first work that addresses the expression and purification of Andes virus glycoproteins Gn and Gc. Our results demonstrate that sGn and sGc maintain epitopes that are exposed on the surface of the viral envelope. However, our work also highlights the need to explore new strategies to achieve high-level expression of these proteins for development of a vaccine candidate against Andes virus.

Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor.

The yeast Pichia pastoris is one of the most robust cell factories in use for the large-scale production of biopharmaceuticals with applications in the fields of human and animal health. Recently, intracellular high-level expression of rabbit hemorrhagic disease virus (RHDV) capsid protein (VP1) as a self-assembled multipurpose antigen/carrier was established as a production process from P. pastoris. Since recovery of VP1 from the culture media implies technological and economic advantages, the secretion of VP1 variants was undertaken in this work. Conversely, extensive degradation of VP1 was detected. Variations to culture parameters and supplementation with different classes of additives were unable to diminish degradation. Strategies were then conducted during fermentations using a recombinant variant of a non-specific BPTI-Kunitz-type protease inhibitor (rShPI-1A) isolated from the sea anemone Stichodactyla helianthus. The presence of the inhibitor in the culture medium at the recombinant protein induction phase, as well as co-culture of the yeast strains expressing VP1 and rShPI-1A, led to VP1 protection from proteolysis and to production of ordered virus-like particles. A yeast strain was also engineered to co-express the rShPI-1A inhibitor and intact VP1. Expression levels up to 116 mg L(-1) of VP1 were reached under these approaches. The antigen was characterized and purified in a single chromatography step, its immunogenic capacity was evaluated, and a detection test for specific antibodies was developed. This work provides feasible strategies for improvements in P. pastoris heterologous protein secretion and is the first report on co-expression of the ShPI-1A with a recombinant product otherwise subjected to proteolytic degradation.