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

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.

Monitoring methotrexate in clinical samples from cancer patients during chemotherapy with a LSPR-based competitive sensor.

A competitive binding assay based on localized surface plasmon resonance (LSPR) of folic acid-functionalized gold nanoparticles (FA-AuNPs) and human dihydrofolate reductase enzyme (hDHFR) was developed to detect nanomolar to micromolar concentrations of the widely applied anti-cancer drug, methotrexate (MTX). By the nature of the competitive assay for MTX, the LSPR shift from specific binding between FA-AuNPs and the free enzyme was inversely proportional to the concentration of MTX. In addition, the dynamic range for MTX was tuned from 10(-11) to 10(-6) M by varying the concentration of hDHFR from 1 to 100 nM. Inter-day reproducibility and recovery of MTX spiked in phosphate buffer saline (PBS) were excellent. Potential interferents such as FA, trimethoprim (TMP) and 4-amino-4-deoxy-N-methylpteroic acid (DAMPA) did not occur in the concentration range of interest for MTX. Clinical samples of human serum from patients undergoing MTX chemotherapy were analyzed following a simple solid-phase extraction step to isolate MTX from the serum matrix, with a limit of detection of 155 nM. Validation of the LSPR method was carried out in comparison to Fluorescence Polarization Immunoassay (FPIA), a commonly used method in clinical settings, and LC-MS/MS, a reference technique. The results of the LSPR competitive assay compared well to FPIA and LC-MS/MS, with a slope of 2.4 and 1.1, respectively, for the correlation plots. The method established herein is intended for therapeutic drug monitoring (TDM) of MTX levels in patients undergoing chemotherapy to ensure safety and efficacy of the treatment.

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 B(12) 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.

An in vitro comparison of four different immunoassays for the monitoring of Infliximab biosimilars drug levels.

BACKGROUND: SB2 (Renflexis®, Merck) and CT-P13 (Inflectra®, Pfizer) are biosimilars of the reference Infliximab (Remicade®, Janssen) and are approved in Canada for use in indications for which Infliximab is approved, including inflammatory bowel disease. These biosimilars are structurally different but exhibit comparable physicochemical characteristics, pharmaceutical effectiveness and immunogenicity compared to Infliximab. Optimal Infliximab therapy currently relies on therapeutic drug monitoring offered by several reference laboratories. OBJECTIVE: Because the appropriate dosing depends on accurate determination of drug levels and anti-drug antibodies, the ability of current Infliximab assays to measure the biosimilars and corresponding antibodies needs to be demonstrated. METHODS: The correlation between Infliximab and the biosimilars measured with four different enzyme-linked immunosorbent assays for Infliximab detection was evaluated. Spiked serum samples were assayed with kits from (A) Immunodiagnostik/ALPCO Diagnostics, (B) R-Biopharm, (C) Theradiag and (D) Progenika Biopharma. The impact of various concentrations of antibodies to Infliximab on the quantification of biosimilars was also tested. RESULTS: A good correlation of SB2, CT-P13 and reference Infliximab spiked serum samples was observed with the four assays. The observed bias between the original drug and biosimilars is clinically insignificant and less than the usual analytical variability observed with these methods. The quantification of the biosimilars and Infliximab was equally impacted in serums containing antibodies to Infliximab. The recovery of the drugs was inversely correlated with the concentration of anti-Infliximab antibodies, suggesting common immunodominant epitopes for SB2, CT-P13 and Infliximab. CONCLUSION: The ability of these assays to properly quantify the biosimilars Renflexis® and Inflectra® has been demonstrated. The therapeutic drug monitoring required for Infliximab therapy can be adequately performed with the biosimilars using the kits currently in use or available in clinical laboratories.

Whole blood titanium metal ion measurement reproducibility of two laboratories.

BACKGROUND: Metal ion blood concentrations evaluation can be useful in monitoring wear and corrosion of orthopedic implants. Elevated metal ion level may help detecting defective hip arthroplasty implants and serve as an indicator for revision surgery. Our objective was to evaluate the reproducibility of titanium metal ion level measurements by two different laboratories. METHODS: Seventy-one whole blood samples were collected from 64 patients with unilateral ceramic-on-ceramic hip arthroplasty. For each patient, two whole blood samples were collected and analyzed in two different laboratories. RESULTS: For each case, laboratory 1 had significantly higher values than laboratory 2. There was a clinically significant absolute difference between the two laboratories, above the predetermined threshold, for 90% of samples. A mean variation ratio of 410% between the two laboratories was found. CONCLUSION: Not all laboratories use the same techniques and calibrations to perform these measurements. Therefore, their results should be interpreted with caution and clinical decision should rely on metal ion trends provided by the same laboratory.

Easy-to-use equations for the estimation of urine relative saturation in the assessment of risk of recurrence in urinary stones formers.

It is a fact that recurrence of urinary stones is a common medical problem. One of the key factors used in determining the risk of urinary stone-formation is the urine relative saturation in the major constituents of lithiasis. Nomograms were developed in the 1970's to estimate the relative saturation of urine. We present here easy-to-use mathematical equations derived from these nomograms. These equations can be integrated directly in the LIS of any laboratories, and can be used as a tool in the treatment and prevention of recurrent stone-formation.

Inflammatory response to peritoneal implantation of alginate-poly-L-lysine microcapsules.

A thorough understanding of the mechanisms involved in the host reaction to alginate-poly-L-lysine microcapsules (HRM) is important to design methods for the evaluation, selection, and development of biocompatible biomaterials and microcapsules or treatments to control this reaction. The objective of this study was to identify those immune cells and cytokines involved in the pathogenesis of the HRM. The total and differential cell counts were evaluated, and the mRNA expression of TNF-alpha, IL-1beta, IL-6 and TGF-beta1 was measured in peritoneal washings at 3, 17, 48, 96 and 168 h after saline or microcapsule injections. Neutrophil number and IL-1beta and IL-6 m-RNA expression presented an early transient increase, with no differences between saline and microcapsule injections, suggesting a reaction to the procedure. Macrophages, lymphocytes and TNF-alpha were significantly more activated over a longer period of time, after microcapsule implantation than saline injection. They are likely involved in transforming the reaction into a chronic inflammatory process. TGF-beta1 and IL-1beta presented a late (day 7) significant increase after microcapsule but not saline injections. They are likely involved in transforming the reaction into a fibrogenic process. These results suggest that macrophages, lymphocytes, TNF-alpha, IL-1beta and TGF-beta1 play a role in the pathogenesis of the HRM.

Microencapsulation of living cells in semi-permeable membranes with covalently cross-linked layers.

Microencapsulation in semi-permeable membranes protects transplanted cells against immune destruction. Microcapsule strength is critical. We describe a method to microencapsulate living cells in alginate-poly-L-lysine (PLL)-alginate membranes with covalent links between adjacent layers of microcapsule membranes, while preserving the desired membrane molecular weight cut-off (MWCO) and microencapsulated cell viability. A heterobifunctional photoactivatable cross-linker, N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) was used. The N-hydroxysuccinimide ester group of ANB-NOS was covalently linked to PLL. Islets of Langerhans were immobilized in alginate beads, incubated in PLL-ANB-NOS and again in alginate. Upon illumination with UVA, covalent links were created between the phenyl azide residue of ANB-NOS and alginate from both the core bead and the outer coating. Covalently linked microcapsules remained intact after 3 years in a strong alkaline buffer (pH 12), whereas standard microcapsules disappeared within 45 s in the same solution. A standardized mechanical stress broke 22-fold more standard than covalently linked microcapsules. The MWCO and microencapsulated cell viability were similar with standard and covalently linked microcapsules. These microcapsules, extremely resistant to chemical and mechanical stresses, will be useful in numerous applications.

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 patient's 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).

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

STUDY OBJECTIVE: 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). DESIGN: Prospective, observational, pharmacokinetic study. SETTING: Intensive care unit of a tertiary care hospital in Montréal, Canada. PATIENTS: Twenty critically ill adults who were undergoing continuous venovenous hemodiafiltration and receiving a 4-hour infusion of piperacillin 4 g-tazobactam 0.5 g every 8 hours for a documented or suspected infection. INTERVENTION: Blood samples were collected every hour over an 8-hour dosing interval. Prefilter and postfilter blood samples, and effluent and urine samples were also collected. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the proportion of patients who achieved an unbound piperacillin plasma concentration above a target minimum inhibitory concentration (MIC) of 64 mg/L (MIC that inhibits 90% of isolates for Pseudomonas aeruginosa) for at least 50% of the dosing interval; 18 (90%) of the 20 patients achieved this outcome. In all patients, the free piperacillin concentrations were above the Pseudomonas aeruginosa breakpoint of 16 mg/L for the entire time interval. Regarding piperacillin pharmacokinetic parameters, the median (interquartile range) minimum unbound plasma concentration was 65.15 mg/L (51.30-89.30), maximum unbound plasma concentration was 141.3 mg/L (116.75-173.90), sieving coefficient was 0.809 (0.738-0.938), total clearance was 65.82 ml/minute (53.79-102.87), and renal clearance was 0.16 ml/minute (0.05-3.04). The median CRRT dose was 32.0 ml/kg/h (25.0-39.8). CONCLUSIONS: Administration of a 4-hour infusion of piperacillin-tazobactam was associated with a favorable pharmacodynamic profile in patients undergoing CRRT. Concentrations associated with maximal activity were attained in our patients.

Insulin-like growth factor II allows prolonged blood glucose normalization with a reduced islet cell mass transplantation.

IGF-II has been reported to decrease neonatal islet cell apoptosis and in vitro adult islet cell necrosis and apoptosis, but the usefulness of IGF-II in a transplantation setting is unknown. We evaluated the effect of in vitro IGF-II incubations on microencapsulated rat islet survival both in vitro and in minimal mass transplantations into diabetic mice. After 6 d in culture, fresh examinations, histology, fluorescence microscopy, sodium 3'-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)-benzene sulfonic acid hydrate assay, and apoptosis studies all indicated that IGF-II significantly improves islet cell viability in a dose-dependent fashion. IGF-II 100 ng/ml and 500 ng/ml induced a 51% and 83% increase of viable islets (P = 0.052, P < 0.01). A 20%, 29%, and 33% reduction of the apoptotic index was observed with 50, 100, and 500 ng/ml incubations respectively (P < 0.05; P < 0.005; P < 0.001). Ten weeks after transplantation of 150 encapsulated rat islet equivalents incubated with IGF-II 500 ng/ml, 80% of diabetic mice were normoglycemic. Without IGF-II preincubation, only 8% of the recipients remained normoglycemic with the transplantation of 150 islets and 42% with 300 islets (P < 0.05). In conclusion, IGF-II promotes islet cell survival, and allows successful transplantation using a smaller number of islets.

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 (>$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.

Altered laboratory findings associated with end-stage renal disease.

Several laboratory parameters can be altered in advanced renal failure. Results may be difficult to interpret and may become misleading and unreliable in such a context. On the other hand, some of the alterations may reflect real abnormalities. Thus sufficient knowledge and careful judgment are required by the clinician. We reviewed different publications related to biochemical anomalies in renal failure and report some of the main findings. The sections are divided as follows: cardiovascular risk factors and markers, inflammation markers, pancreatic and liver function tests, hormones, bone turnover indices and parathyroid hormone assays, tumor markers, carbohydrate metabolism indicators, and others. The information provided should be useful to clinicians involved in the care of renal failure patients.