Robert Robitaille

Studies on small (<350 μm) alginate‐poly‐L‐lysine microcapsules. III. Biocompatibility of smaller versus standard microcapsules

Microencapsulation of islets of Langerhans has been proposed as a means of preventing their immune destruction following transplantation. Microcapsules of diameters <350 microm made with an electrostatic pulse system present many advantages relative to standard microcapsules (700-1500 microm), including smaller total implant volume, better insulin kinetics, better cell oxygenation, and accessibility to new implantation sites. To evaluate their biocompatibility, 200, 1000, 1120, 1340, or 3000 of these smaller microcapsules (<350 microm) or 20 standard microcapsules (1247+/-120 microm) were implanted into rat epididymal fat pads, retrieved after 2 weeks, and evaluated histologically. The average pericapsular reaction increased with the number of small microcapsules implanted (p<0.05; 3000 vs. 200, 3000 vs. 1000, and 1000 vs. 200 microcapsules). At equal volume and alginate content, standard microcapsules caused a more intense fibrosis reaction than smaller microcapsules (p<0.05). In addition, 20 standard microcapsules elicited a stronger pericapsular reaction than 200 and 1000 smaller microcapsules (p<0.05) although the latter represented a 3.4-fold larger total implant surface exposed. We conclude that microcapsules of diameters <350 microm made with an electrostatic pulse system are more biocompatible than standard microcapsules.

Miniature multi-channel SPR instrument for methotrexate monitoring in clinical samples.

A multi-channel fully integrated SPR biosensor was applied for the analysis of an anti-cancer drug, methotrexate (MTX) as a potential analytical tool used in clinical chemistry laboratories for therapeutic drug monitoring (TDM). MTX concentrations in a patients serum undergoing chemotherapy treatments can be determined by surface plasmon resonance (SPR) sensing using folic acid-functionalized gold nanoparticles (FA-AuNP) in competition with MTX for the bioreceptor, human dihydrofolate reductase (hDHFR) immobilized on the SPR sensor chip. To validate this biosensor, 13 nm FA-AuNP were shown to interact with immobilized hDHFR in the absence of MTX and this interaction was inhibited in the presence of MTX. The sensor was calibrated for MTX in phosphate buffer at different dynamic range by varying nanoparticle sizes (5, 13, 23 nm) and by modifying the Kd of the bioreceptor using wild-type and mutant hDHFR. Furthermore, initial binding rate data analyzes demonstrated quantitative and fast sensor response under 60s. This MTX assay was subsequently adapted to a fully integrated multi-channel SPR system built in-house and calibrated in human serum with a dynamic range of 28-500 nM. The SPR system was applied to analyzes of actual clinical samples and the results are in good agreement with fluorescence polarization immunoassay (FPIA) and LC-MS/MS. Finally, the prototype system was tested by potential clinical users in a hospital setting at the biochemistry laboratory of a Montreal hospital (Hôpital Maisonneuve-Rosemont).

Oral Ferrous Sulfate Does Not Increase Preoperative Hemoglobin in Patients Scheduled for Hip or Knee Arthroplasty

BACKGROUND: Low hemoglobin (Hb) concentrations before lower limb joint replacement are associated with the need for blood transfusions and increased mortality. To optimize preoperative Hb, blood conservation protocols often recommend oral iron supplements, even in nonanemic patients. OBJECTIVE: To investigate the impact of ferrous sulfate on the change in Hb prior to hip or knee arthroplasty and evaluate the effect of oral iron on hematocrit, mean corpuscular volume (MCV), ferritin, and transferrin saturation, as well as its tolerability and treatment adherence. METHODS: We conducted a prospective, observational cohort study of adults with Hb concentrations between 10 and 15 g/dL who received iron supplementation prior to hip or knee arthroplasty. Systemic inflammatory diseases, vitamin B12 or folate deficiency, and current use of iron supplements, intravenous iron, or erythropoietin were exclusion criteria. All participants were prescribed ferrous sulfate 300 mg 3 times daily for a minimum of 3 weeks. Complete blood cell counts and iron studies were performed before therapy and surgery. RESULTS: Eighty-seven patients with a mean (SD) Hb of 13.47 (0.84) g/dL were included in the study. Preoperative Hb decreased after treatment with iron (-0.14 [0.53] g/dL, p = 0.015). Hematocrit also declined (-0.6% [1.8%], p = 0.002), whereas ferritin increased (25.8 [38.6] ng/mL, p < 0.001). No significant change was seen in MCV and transferrin saturation. The most common adverse effects were constipation (33.3%), heartburn (13.8%), and abdominal pain (12.6%). The adherence rate was 67.1%. CONCLUSIONS: Oral ferrous sulfate supplementation is not an effective method to increase preoperative Hb in patients scheduled for hip or knee arthroplasty, and its use is associated with adverse effects.

Studies on small (<350 ?m) alginate-poly-L-lysine microcapsules. V. Determination of carbohydrate and protein permeation through microcapsules by reverse-size exclusion chromatography

Membrane molecular weight (MW) cut-off is a critical factor for immunoprotection of transplanted microencapsulated cells as well as for graft survival. Our goal was to study dextran and protein permeation through small (<350 microm in diameter) alginate-poly-L-lysine microcapsules made with an electrostatic system. Microcapsules were packed into a column, and gel-sieving chromatography was performed with proteins and dextrans of known MW. The objectives of this study were (1) to validate this approach for the assessment of the MW cut-off of <350 microm-in-diameter microcapsules and (2) to evaluate the effect on MW cut-off of changes in experimental conditions. Elution profiles of proteins suggest that the MW cut-off of our small microcapsules lies between 14,500 and 44,000 Da whereas dextrans > or =19,000 Da were excluded. The increase in poly-L-lysine (PLL) concentration from 0.02 to 0.08% reduced the MW cut-off. Increasing the PLL MW from 11.6 to 69.6 kDa induced no change in the MW cut-off. The results also show that the method can be used to discriminate between adsorption and absorption and that insulin diffuses freely across the microcapsule membrane. This method will be useful in establishing the ideal MW cut-off, in optimizing microcapsule characteristics, and in performing routine quality controls.

Alginate‐poly‐L‐lysine microcapsule biocompatibility: A novel RT‐PCR method for cytokine gene expression analysis in pericapsular infiltrates

Transplantation of microencapsulated islets of Langerhans is impaired by a pericapsular host reaction that eventually induces graft failure. We are studying the role of cytokines in the pathogenesis of this reaction, using the model of alginate-polylysine microcapsule implantation in rat epididymal fat pads. The objectives were: (1) to develop a method to measure, by semiquantitative PCR, TGF-beta1 gene expression in fat pad pericapsular infiltrates, and (2) to use this method to evaluate TGF-beta1 gene expression 14 days after microcapsule implantation. TGF-beta1 mRNA level was significantly higher in pericapsular infiltrate cells than in nonimplanted tissue cells and saline-injected tissue cells (p < 0.0001 and p < 0.01, respectively). There was no significant difference between the TGF-beta1 mRNA levels of the two types of controls (p = 0.0945). These results suggest that TGF-beta1 plays a role in the pathogenesis of the pericapsular reaction. The method developed can be used to study the role of other fibrogenic cytokines potentially involved. This will shed light on the mechanisms underlying the pericapsular reaction and will serve as a basis for the development of strategies to control this reaction.

Successful and cost-efficient replacement of immunoassays by tandem mass spectrometry for the quantification of immunosuppressants in the clinical laboratory.

The purpose of this paper is to describe the implantation of mass spectrometry in replacement of immunoassays for the measurement of immunosuppressant drugs in the clinical setting, from scientific and financial perspectives. A straightforward, rapid, and economical method was developed for the simultaneous quantification of tacrolimus, sirolimus, and cyclosporine. Following a simple protein precipitation step, supernatants are injected on a small C(18) guard cartridge and gradient elution of the immunosuppressants is performed in a total chromatographic run time of 2.25 min. Sodium adducts of the compounds and internal standards are quantified by electrospray tandem mass spectrometry. The method shows inter-assay impression of less than 10-15% for all compounds with good extraction efficiency (89-104%) and minimal matrix effects, except for sirolimus where ion suppression is more pronounced. The method correlates well with chemiluminescent microparticle immunoassays (on the Abbott Architect analyzer), although the immunoassay results are significantly higher than those obtained by HPLC-MS/MS. The transition from immunoassays to mass spectrometry was well received by the laboratory staff, and significant reductions in reagent costs have been realized (>

Time course of transforming growth factor-β1 (TGF-β1) mRNA expression in the host reaction to alginate-poly-L-lysine microcapsules following implantations into rat epididymal fat pads

250,000 CAD per year). With these savings, the purchase and installation of two complete HPLC-MS/MS systems was completely financed in less than three years.

Posaconazole-Loaded Leukocytes as a Novel Treatment Strategy Targeting Invasive Pulmonary Aspergillosis.

Microencapsulation of islets of Langerhans within semipermeable membranes has been proposed to prevent their immune destruction after transplantation. However, the successful application of this method is impaired by a pericapsular reaction, which eventually induces graft failure. Our goal is to study the role of cytokines in the pathogenesis of this reaction, using the model of alginate-poly-L-lysine microcapsule implantation into Wistar rat epididymal fat pads (EFP). The specific objective of this study was to determine the time course of transforming growth factor (TGF)-beta(1) mRNA expression by semi-quantitative reverse transcriptase-polymerase chain reaction. Microcapsules induced an increase of TGF-beta(1) mRNA expression that reached a maximum 14 days after implantation. Seven, 14, 30, and 60 days after microcapsule implantation, the expression of TGF-beta(1) mRNA was significantly higher in pericapsular infiltrate cells than in nonimplanted EFP cells (p<0.05, p<0.0001, p<0.005, and p<0.01, respectively). Injection of physiological saline induced a small and gradual augmentation of TGF-beta(1) mRNA expression with a maximum 30 days after injection (p<0.01 vs. nonimplanted EFP cells). These results demonstrated that microcapsule implantation, in comparison with saline injection, induce an early, extended, and amplified TGF-beta(1) mRNA expression. This suggests that TGF-beta(1) plays a role in the pathogenesis of the pericapsular host reaction.

Pharmacokinetics of an extended 4-hour infusion of piperacillin-tazobactam in critically ill patients undergoing continuous renal replacement therapy.

Background Impaired delivery of antifungals to hyphae within necrotic lesions is thought to contribute to therapeutic failure in invasive pulmonary aspergillosis (IPA). We hypothesized that transfusion of leukocytes loaded ex vivo with the lipophilic antifungal posaconazole could improve delivery of antifungals to the sites of established infection and improve outcome in experimental IPA. Methods The HL-60 leukemia cell line was differentiated to a neutrophil-like phenotype (differentiated HL-60 [dHL-60] cells) and then exposed to a range of posaconazole concentrations. The functional capacity and antifungal activity of these cells were assessed in vitro and in a mouse model of IPA. Results Posaconazole levels in dHL-60 cells were 265-fold greater than the exposure concentration. Posaconazole-loaded cells were viable and maintained their capacity to undergo active chemotaxis. Contact-dependent transfer of posaconazole from dHL-60 cells to hyphae was observed in vitro, resulting in decreased fungal viability. In a neutropenic mouse model of IPA, treatment with posaconazole-loaded dHL-60 cells resulted in significantly reduced fungal burden in comparison to treatment with dHL-60 cells alone. Conclusions Posaconazole accumulates at high concentrations in dHL-60 cells and increases their antifungal activity in vitro and in vivo. These findings suggest that posaconazole-loading of leukocytes may hold promise for the therapy of IPA.

Hemodialysis effects on phenytoin pharmacokinetics.

To evaluate the pharmacokinetic and pharmacodynamic profiles of piperacillin‐tazobactam administered as a 4‐hour infusion in critically ill patients undergoing continuous renal replacement therapy (CRRT).