Francois Leblond

Studies on small (< 300 microns) microcapsules: II--Parameters governing the production of alginate beads by high voltage electrostatic pulses.

The size of microcapsules is a critical parameter in the immunoisolation of islets of Langerhans by microencapsulation. The use of smaller capsules decreases the total implant volume and improves insulin kinetics and oxygen supply. A high voltage electrostatic pulse system was used for the production of small (<300 μm) alginate beads, the first step of the encapsulation technique. However, islets often protruded from capsules that were too small, further emphasizing the need for a method to control bead size. A study of 7 parameters [electrostatic pulse amplitude (A), duration (D) and wavelength (λ), pump flow rate (P), needle gauge, alginate viscosity and distance between electrodes] showed that P (r = 0.981, p = 0.003) and λ (r = 0.988, p = 0.0002) were the principal determinants of bead size. To detect potential interactions between parameters, 270 combinations of different levels of A, D, λ, and P were studied. A multivariate regression analysis of these data confirmed that P and λ are the prime determinants of bead size, and showed that a 2-parameter (P, λ) model could be used to precisely predict bead size (R2 = 0.84), while keeping the application simple. The precision of the predictive model is only slightly improved by the use of additional parameters. The reliability of the data used to elaborate this model was demonstrated (p = 0.6226) by comparing them with a second data set obtained under the same conditions. A third set of experiments confirmed the applicability of the model. This work has major implications on the preclinical application of microencapsulation since it showed that it is possible to predetermine the bead size.

Peroxynitrite production by human neutrophils, monocytes and lymphocytes challenged with lipopolysaccharide

To assess peroxynitrite formation in lipopolysaccharide (LPS)‐stimulated human blood, we have measured nitric oxide (NO)‐dependent intracellular oxidation of dihydrorhodamine 123 (DHR 123) to rhodamine. LPS increased DHR 123 oxidation in neutrophil granulocytes, monocytes and lymphocytes in a time‐dependent fashion. Greater extent of DHR 123 oxidation was detected in neutrophils and monocytes than in lymphocytes. These changes were accompanied by accumulation of rhodamine in the plasma. While intracellular DHR 123 oxidation and rhodamine accumulation in the plasma were not affected by inhibition of constitutive NO synthase at 30 and 60 min after addition of LPS, they were markedly attenuated by inhibition of inducible NO synthase at 4, 8, 16 and 24 h after addition of LPS. These results demonstrate that human leukocytes can produce high amounts of peroxynitrite in response to LPS, and may contribute to the elevated plasma peroxynitrite levels observed during endotoxic shock.

Protection of islets of Langerhans from antibodies by microencapsulation with alginate poly-l-lysine membranes.

Microencapsulation of islets has been proposed to prevent their immune destruction following transplantation. An indirect immunofluorescence technique has been developed and used to study the permeability of the alginate-poly-L-lysine microcapsules to antibodies. Wistar rat islets were incubated with the R2D6 monoclonal mouse IgM antibody against rat islets, microen-capsulated, and incubated with fluorescein-labeled goat IgG antibodies against mouse IgG and IgM. For the negative controls, the first antibody was omitted or both antibodies were omitted. The positive controls included islets incubated with both antibodies before they were encapsulated. Our study demonstrated that the alginate-poly-L-lysine membranes are not permeable to IgG when poly-L-lysine of molecular weights ranging from 21,000 to 390,000 are used. This simple immunofluorescence technique demonstrated the nonpermeability of the microcapsules to IgG, and could be useful for the initial evaluation of new types of membranes.

Regulation of the proprotein convertase subtilisin/kexin type 9 in intestinal epithelial cells

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally promotes the degradation of the low-density lipoprotein receptor (LDLr) in hepatocytes and increases plasma LDL cholesterol. It is not clear, however, whether PCSK9 plays a role in the small intestine. Here, we characterized the patterns of variations of PCSK9 and LDLr in fully differentiated Caco-2/15 cells as a function of various potential effectors. Cholesterol (100 microM) solubilized in albumin or micelles significantly downregulated PCSK9 gene (30%, P<0.05) and protein expression (50%, P<0.05), surprisingly in concert with a decrease in LDLr protein levels (45%, P<0.05). Cells treated with 25-hydroxycholesterol (50 microM) also displayed significant reduction in PCSK9 gene (37%, P<0.01) and protein (75% P<0.001) expression, whereas LDLr showed a decrease at the gene (30%, P<0.05) and protein (57%, P<0.01) levels, respectively. The amounts of PCSK9 mRNA and protein in Caco-2/15 cells were associated to the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein-2 (SREBP-2) that can transcriptionally activate PCSK9 via sterol-regulatory elements located in its proximal promoter region. On the other hand, depletion of cholesterol content by hydroxypropyl-beta-cyclodextrin upregulated PCSK9 transcripts (20%, P<0.05) and protein mass (540%, P<0.001), in parallel with SREBP-2 protein levels. The addition of bile acids (BA) taurocholate and deoxycholate to the apical culture medium lowered PCSK9 gene expression (25%, P<0.01) and raised PCSK9 protein expression (30%, P<0.01), respectively, probably via the modulation of farnesoid X receptor. Furthermore, unconjugated and conjugated BA exhibited different effects on PCSK9 and LDLr. Altogether, these data indicate that intestinal PCSK9 is highly modulated by sterols and emphasize the distinct effects of BA species.

Age-Dependent Demethylation of Sod2 Promoter in the Mouse Femoral Artery

We studied the age-dependent regulation of the expression of the antioxidant enzyme manganese superoxide dismutase (MnSOD encoded by Sod2) through promoter methylation. C57Bl/6 mice were either (i) sedentary (SED), (ii) treated with the antioxidant catechin (CAT), or (iii) voluntarily exercised (EX) from weaning (1-month old; mo) to 9 mo. Then, all mice aged sedentarily and were untreated until 12 mo. Sod2 promoter methylation was similar in all groups in 9 mo but decreased (p < 0.05) in 12 mo SED mice only, which was associated with an increased (p < 0.05) transcriptional activity in vitro. At all ages, femoral artery endothelial function was maintained; this was due to an increased (p < 0.05) contribution of eNOS-derived NO in 12 mo SED mice only. CAT and EX prevented these changes in age-related endothelial function. Thus, a ROS-dependent epigenetic positive regulation of Sod2 gene expression likely represents a defense mechanism prolonging eNOS function in aging mouse femoral arteries.

Rationale for the administration of systemic 5-FU in combination with heated intraperitonal oxaliplatin

BACKGROUND Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) with oxaliplatin (OX) is the standard of care for selected patients with peritoneal carcinomatosis of colorectal origin. Because 5-FU is mandatory to improve efficacy of OX when used by systemic route, several teams now empirically combine intravenous (IV) 5-FU with HIPEC OX, but this practice has yet to be supported by preclinical data. Using a murine model, we studied the impact of IV 5-FU on peritoneal absorption of HIPEC OX. METHODS Under general anesthesia, 24 Sprague-Dawley rats were submitted to 4 different doses of IV 5-FU (0, 100, 400 and 800 mg/m2) and a fixed dose of HIPEC OX (460 mg/m2) perfused at 40 °C during 25 min. At 25 min, samples in different compartments were harvested (peritoneum, portal vein and systemic blood) and the concentrations of 5-FU and OX were measured by high performance liquid chromatography. RESULTS Peritoneal absorption of OX was significantly higher (17.0, 20.1, 34.9 and 38.1 nmol/g, p < 0.0001) with increasing doses of 5-FU (0, 100, 400 and 800 mg/m2, respectively). Peritoneal absorption of OX reached a plateau between 400 and 800 mg/m2 of IV 5-FU. CONCLUSION IV 5-FU enhances peritoneal absorption of HIPEC OX. The most efficient dose of IV 5-FU to be used in combination with HIPEC OX seems to be 400 mg/m2.

Electrocautery effect on intestinal vascularisation in a murine model

Abstract Background: The use of electrocautery devices is associated with complications such as perforation or fistulisation when used near intestinal structures. This is likely due to its effect on vascularisation of the bowel wall. To test this hypothesis we established a murine model to quantify the effect of electrocautery injury on the intestinal microvascularisation. Methods: Sprague-Dawley rats were subjected to five electrocautery injuries on the small bowel in coagulation mode (30 W intensity) and in cut mode (40 W, 80 W and 200 W intensities) for durations of 1, 2 and 5 s. 5 mg/kg of fluorescein was injected intravenously, the injured bowel segments harvested and the rat sacrificed. The segments were analysed to measure the fluorescence of injured bowel compared to adjacent unharmed tissue. Results: A significant decrease in bowel wall microvascularisation occurred with increasing intensity (coag 30 W/cut 40 W versus cut 200 W 1 s: p < 0.05) and duration of electrocautery injury (cut 40 W 1/2 s versus 5 s: p < 0.05). There was a 40% perforation rate when decreased bowel wall microvascularisation was 25% or more. Despite similar electrocautery injury, a significantly greater microvascularisation decrease was observed in jejunum compared to ileum (p < 0.05). Conclusion: We successfully established a murine model to quantify the decrease of bowel wall microvascularisation associated with electrocautery use. Unsurprisingly, the decrease in microvascularisation is greater with higher intensity and duration of electrocautery and is associated with more perforations in the experimental model. The jejunum seems more vulnerable to electrocautery injury than the ileum. These observations support caution when using electrocautery devices near intestinal structures.

The Anti-Hypercholesterolemic Effect of Low p53 Expression Protects Vascular Endothelial Function in Mice

Aims To demonstrate that p53 modulates endothelial function and the stress response to a high-fat western diet (WD). Methods and Results Three-month old p53+/+ wild type (WT) and p53+/− male mice were fed a regular or WD for 3 months. Plasma levels of total cholesterol (TC) and LDL-cholesterol were significantly elevated (p<0.05) in WD-fed WT (from 2.1±0.2 mmol/L to 3.1±0.2, and from 0.64±0.09 mmol/L to 1.25±0.11, respectively) but not in p53+/− mice. The lack of cholesterol accumulation in WD-fed p53+/− mice was ass–ociated with high bile acid plasma concentrations (p53+/− =  4.7±0.9 vs. WT =  3.3±0.2 μmol/L, p<0.05) concomitant with an increased hepatic 7-alpha-hydroxylase mRNA expression. While the WD did not affect aortic endothelial relaxant function in p53+/− mice (WD =  83±5 and RD =  82±4% relaxation), it increased the maximal response to acetylcholine in WT mice (WD =  87±2 vs. RD =  62±5% relaxation, p<0.05) to levels of p53+/−. In WT mice, the rise in TC associated with higher (p<0.05) plasma levels of pro-inflammatory keratinocyte-derived chemokine, and an over-activation (p<0.05) of the relaxant non-nitric oxide/non-prostacyclin endothelial pathway. It is likely that in WT mice, activations of these pathways are adaptive and contributed to maintain endothelial function, while the WD neither promoted inflammation nor affected endothelial function in p53+/− mice. Conclusions Our data demonstrate that low endogenous p53 expression prevents the rise in circulating levels of cholesterol when fed a WD. Consequently, the endothelial stress of hypercholesterolemia is absent in young p53+/− mice as evidenced by the absence of endothelial adaptive pathway over-activation to minimize stress-related damage.