Design of Targeted Flurbiprofen Biomimetic Nanoparticles for Management of Arthritis: In Vitro and In Vivo Appraisal

Flurbiprofen (FLUR) is a potent non-steroidal anti-inflammatory drug used for the management of arthritis. Unfortunately, its therapeutic effect is limited by its rapid clearance from the joints following intra-articular injection. To improve its therapeutic efficacy, hyaluronic acid-coated bovine serum albumin nanoparticles (HA-BSA NPs) were formulated and loaded with FLUR to achieve active drug targeting. NPs were prepared by a modified nano-emulsification technique and their HA coating was proven via turbidimetric assay. Physicochemical characterization of the selected HA-BSA NPs revealed entrapment efficiency of 90.12 ± 1.06%, particle size of 257.12 ± 2.54 nm, PDI of 0.25 ± 0.01, and zeta potential of −48 ± 3 mv. The selected formulation showed in-vitro extended-release profile up to 6 days. In-vivo studies on adjuvant-induced arthritis rat model exhibited a significant reduction in joint swelling after intra-articular administration of FLUR-loaded HA-BSA NPs. Additionally, there was a significant reduction in CRP level in blood as well as TNF-α, and IL-6 levels in serum and joint tissues. Immunohistochemical study indicated a significant decrease in iNOS level in joint tissues. Histopathological analysis confirmed the safety of FLUR-loaded HA-BSA NPs. Thus, our results reveal that FLUR loaded HA-BSA NPs have a promising therapeutic effect in the management of arthritis.

suPAR cut-offs for stratification of low, medium, and high-risk acute medical patients in the emergency department

Background Soluble urokinase plasminogen activator receptor (suPAR) levels have previously been associated with readmission and mortality in acute medical patients in the ED. However, no specific cut-offs for suPAR have been tested in this population. Methods Prospective observational study of consecutively included acute medical patients. Follow-up of mortality and readmission was carried out for 30- and 90 days stratified into baseline suPAR < 4, 4–6 and > 6 ng/ml. suPAR levels were measured using suPARnostic® Turbilatex assay on a Cobas c501 (Roche Diagnostics Ltd) analyser. Results A total of 1747 acute medical patients in the ED were included. Median age was 70 (IQR: 57–79) and 51.4% were men. Adjusted linear regression analysis showed that suPAR, independently of age, sex and C-reactive protein levels, predicted 30- and 90-day mortality (Odds ratio for doubling in suPAR 1.96 (95% confidence intervals: 1.42–2.70) Among patients with suPAR below 4 ng/ml (N = 804, 46.0%), 8 (1.0%) died within 90-day follow-up, resulting in a negative predictive value of 99.0% and a sensitivity of 94.6%. Altogether 514 (29.4%) patients had suPAR of 4–6 ng/ml, of whom 43 (8.4%) died during 90-day follow-up. Among patients with suPAR above 6 ng/ml (N = 429, 24.6%), 87 patients (20.3%) died within 90-day follow-up, resulting in a positive predictive value of 20.1% and a specificity of 78.7%. Conclusions suPAR cut-offs of below 4, between 4 and 6 and above 6 ng/ml can identify acute medical patients who have low, medium or high risk of 30- and 90-day mortality. The turbidimetric assay provides suPAR results within 30 min that may aid in the decision of discharge or admission of acute medical patients.

Higher levels of IgA and IgG at sepsis onset are associated with higher mortality: results from the Albumin Italian Outcome Sepsis (ALBIOS) trial

Background The role of intravenous immunoglobulins (IVIG) during sepsis is controversial, as different trials on IVIG have observed inconsistent survival benefits. We aimed to elucidate the possible association and clinical significance between circulating levels of immunoglobulins. Methods In a subset of 956 patients with severe sepsis and septic shock of the multicentre, open-label RCT ALBIOS, venous blood samples were serially collected 1, 2, and 7 days after enrolment (or at ICU discharge, whichever came first). IgA, IgG and IgM concentrations were assayed in all patients on day 1 and in a subgroup of 150 patients on days 2 and 7. Ig concentrations were measured employing a turbidimetric assay, OSR61171 system. Results IgA on day 1 had a significant predictive value for both 28-day and 90-day mortality (28-day mortality, HR: 1.50 (95% CI 1.18–1.92); 90-day mortality, HR: 1.54 (95% CI 1.25–1.91)). IgG, but not IgM, on day 1 showed similar results for 28-day (HR 1.83 (95% CI 1.33–2.51) and 90-day mortality HR: 1.66 (95% CI 1.23–2.25)). In addition, lower levels of IgG but not of IgA and IgM, at day 1 were associated with significantly higher risk of secondary infections (533 [406–772] vs 600 [452–842] mg/dL, median [Q1–Q3], p = 0.007). Conclusions In the largest cohort study of patients with severe sepsis or septic shock, we found that high levels of IgA and IgG on the first day of diagnosis were associated with a decreased 90-day survival. No association was found between IgM levels and survival. As such, the assessment of endogenous immunoglobulins could be a useful tool to identify septic patients at high risk of mortality. Trial registration #NCT00707122, Clinicaltrial.gov, registered 30 June 2008

EXPRESS: The effect of paraprotein polymerisation on quantitation by capillary zone electrophoresis and Hevylite®

Objectives Up to 3% of patients with monoclonal gammopathies have multiple serum paraproteins. This article investigates whether multiple isotype-matched paraproteins, as seen on capillary zone electrophoresis (CZE), are truly biclonal. Methods Sera containing multiple isotype-matched paraproteins were treated with the reducing agent dithiothreitol (DTT) and CZE performed pre- and post-treatment. Band resolution and effect of resolution on quantitation of paraprotein burden was assessed. The Hevylite® turbidimetric assay was also evaluated for ability to quantify such paraproteins. Results Among patients with biclonal isotype-matched (BIM) paraproteins, 23/24 (96%) IgA paraproteins resolved into a single band following treatment with DTT compared with only 1/12 (8%) IgG paraproteins. Daratumumab therapy accounted for the second band in 5/9 non-resolving IgGκ paraproteins. Where initially quantified as a single IgA “complex” (multiple bands in close proximity), the single post-DTT band averaged 2.8 g/L less (P=<0.001), likely due to inclusion of lower amounts of underlying serum proteins (y=0.97x-2.03, R2=0.993). Quantitating IgA BIM (n=58) using the Hevylite® assay gave higher results (P=0.002) than CZE (y=1.48x–7.13, R2=0.959). In contrast, single IgA paraprotein results (n=48) did not differ between the two methods (P=0.466; y=1.24x – 2.74, R2=0.898), suggesting that polymerisation enhances Hevylite® quantitation. Conclusions These results suggest that disulphide-mediated polymerisation of IgA paraproteins is more common than true biclonal gammopathy and support DTT treatment of samples with isotype-matched IgA bands before quantifying by CZE. The Hevylite® assay should be utilised with caution where polymerisation is likely. Where IgGκ BIM paraproteins appear on CZE, Daratumumab therapy should be considered.

Comparative Growth Inhibition of Bread Spoilage Fungi by Different Preservative Concentrations Using a Rapid Turbidimetric Assay System

Bread and intermediate moisture bakery products are mainly spoiled by yeasts and filamentous fungi. The inoculum load and preservation system used determines their shelf life. To extend the shelf life of such commodities, the use of chemical preservatives is the most common way to try and control the initiation of mold spoilage of bread. This study has utilized a rapid turbidimetric assay system (Bioscreen C) to examine the temporal efficacy of calcium propionate (CP) and potassium sorbate (PS) for controlling the growth of important bread spoilage fungi. The objectives were to compare the temporal growth of strains of three important spoilage fungi Hyphopichia burtonii (HB17), Paecilomyces variotii (PV11), and Penicillium roqueforti (PR06) isolated from visibly molded bread to (a) different concentrations of CP and PS (0–128 mM), (b) temperatures (25°C, 30°C), (c) water activity (aw; 0.95, 0.97), and (d) pH (5.0, 5.5). All three abiotic factors, pH, aw, and temperature, and preservative concentrations influenced the relative growth of the species examined. In general, PS was more effective than CP in inhibiting the growth of the strains of these three species. In addition, the Time to Detection (TTD) for the efficacy of the preservatives under the interacting abiotic factors was compared. The strain of Paecilomyces variotii (PV10) was the most tolerant to the preservatives, with the shortest TTD values for both preservatives. P. roqueforti was the most sensitive with the longest TTD values under all conditions examined. These results are discussed in the context of the evolution of resistance to food-grade preservatives by such spoilage fungi in bakery products.

Prospective comparison of (1,3)-beta-D-glucan detection using colorimetric and turbidimetric assays for diagnosing invasive fungal disease

Serum (1→3)-β-D-glucan (BDG), an pan fungal antigen, is detected in some invasive fungal diseases (IFDs). We compared two commercial kits, the Fungitell assay (FA) (colorimetric) and the Wako assay (WA) (turbidimetric) over a 4-month period to prospectively test 171 patients who mainly had hematological conditions (62%) and experienced episodes (n = 175) of suspected invasive fungal infection. Twenty-three episodes due to BDG-producing fungi were diagnosed (pneumocystosis, n = 12; invasive aspergillosis, n = 5; candidemia, n = 3; invasive fusariosis, n = 2; hepato-splenic candidiasis, n = 1). Both assays provided similar areas under the curves (AUC = 0.9). Using the optimized positivity thresholds (≥120 pg/ml for FA and ≥ 4 pg/ml for WA), the sensitivity and specificity were 81.8% (CI95: 61.5–92.7), 94.8% (90.1–97.3) for FA and 81.8% (61.5–92.7), 95.4% (90.9–97.8) for WA. Negative predictive value was 97.3% (93.3–99.0) for both tests. If the manufacturer's positivity threshold (≥11 pg/ml) was applied, the WA sensitivity decreased to 50%. Among 71 patients with bacterial infections, 21.1% were FA-positive and 5.6% were WA-positive (p &lt; 10−2). The WA performed similarly as compared to the FA with an optimized cutoff value. The WA is a single sample test that is clinically relevant when a prompt therapeutic decision is required. Lay Summary Serum (1→3)-β-D-glucan testing is dominated by two kits including Fungitell colorimetric assay (FA) and the Wako turbidimetric assay (WA). We compared them prospectively and observed that they both perform similarly when selecting their optimal threshold (≥120 pg/ml for FA and ≥ 4 pg/ml for WA).

Efficient Production of Human Hyaluronidase PH20 in HEK293T Cells Using Site Specific Integration

PH20 is hyaluronidase that hydrolyze the glycosidic bond of hyaluronic acid as a major proteoglycan found in extracellular matrices. PH20 is used in the subcutaneous space to increase the dispersion and absorption of co-administered drugs. PH20 is also injected against solid tumors for to better penetration of anticancer agents into the tumor tissue and inhibiting the tumor cell growth. In the present study, we have developed HEK293T stable cell lines secreting His-tagged human recombinant PH20 (rhPH20) in the culture supernatant through the PhiC31 integrase system. The produced rhPH20 was quantify using ELISA and turbidimetric assay and its activity was assessed through treatment of mouse cumulus-oocyte-complex (COCs). Furthermore, we have characterized genomic integration of PH20-containing vectors in one of the isolated clone with the highest levels of rhPH20 production. Our results demonstrated that the secreted rhPH20 in the culture supernatant contained a specific activity of approximately 3.5 IU/ml and it was properly able to denote all mouse oocytes. Consequently, it was revealed that PH20-expressing vectors integrated site-specifically in the PhiC31 pseudo attP sites in the host genome. Taken together, these results confirmed successful application of PhiC31 integrase as a robust approach for production of soluble, active rhPH20 in HEK293T cells.

Comparison of β-D-Glucan and Galactomannan in Serum for Detection of Invasive Aspergillosis: Retrospective Analysis with Focus on Early Diagnosis

The early diagnosis of invasive aspergillosis (IA) relies mainly on computed tomography imaging and testing for fungal biomarkers such as galactomannan (GM). We compared an established ELISA for the detection of GM with a turbidimetric assay for detection of the panfungal biomarker β-D-glucan (BDG) for early diagnosis of IA. A total of 226 serum specimens from 47 proven and seven probable IA cases were analysed. Sensitivity was calculated for samples obtained closest to the day of IA-diagnosis (d0). Additional analyses were performed by including samples obtained during the presumed course of disease. Most IA cases involved the respiratory system (63%), and Aspergillus fumigatus was the most frequently isolated species (59%). For proven cases, sensitivity of BDG/GM analysis was 57%/40%. Including all samples dating from –6 to +1 weeks from d0 increased sensitivities to 74%/51%. Sensitivity of BDG testing was as high as or higher than GM testing for all subgroups and time intervals analysed. BDG testing was less specific (90–93%) than GM testing (99–100%). Combining BDG and GM testing resulted in sensitivity/specificity of 70%/91%. Often, BDG testing was positive before GM testing. Our study backs the use of BDG for diagnosis of suspected IA. We suggest combining BDG and GM to improve the overall sensitivity.