A comparative analysis of heterogeneity in commercially available recombinant factor VIII products.

INTRODUCTION: Advances in analytical technologies enable investigation of possible correlations between molecular structure, aggregation and subvisible particle content. Regulatory agencies place increasing attention on potential risks associated with protein aggregates in the micron range in biological therapeutics. AIM: Assess the heterogeneity, high-molecular-weight protein (HMWP) species, subvisible particle content and posttranslational modifications in six commercially available recombinant FVIII (rFVIII) products. METHODS: Three B-domain-deleted (BDD) or B-domain truncated rFVIII products (turoctocog alfa, simoctocog alfa and moroctocog alfa) and three full-length rFVIII products (octocog alfa FS and two octocog alfa) were analysed. HMWP content, amount of micron range subvisible particles, tyrosine-1680 sulphation and N-glycan analysis were investigated. RESULTS: The B-domain-modified products had more protein size homogeneity vs the full-length products. Size exclusion-high-performance liquid chromatography data indicated no association between B-domain structure and aggregate content or size of the products tested. The rFVIII products showed large variation in subvisible particle concentration, with turoctocog alfa and simoctocog alfa having the lowest numbers (1000-1600 and 1800-2400 particles/100 IU, respectively). Turoctocog alfa and simoctocog alfa displayed the most complete tyrosine sulphation (>99.5%). CONCLUSION: Overall, there was no association between molecular structure (full-length B-domain, BDD or truncated) and subvisible particle or HMWP content. Dissimilarities may be related to production and product handling differences. In this study, turoctocog alfa, such as simoctocog alfa, had one of the lowest levels of subvisible particles and HMWP content, and high protein size homogeneity.

Occupational rhinoconjunctivitis caused by the common indoor plant, Hoya compacta.

BACKGROUND: Allergic reactions to the common house plant Hoya compacta (HC) have not previously been described. AIMS: To confirm HC as the cause of rhinoconjunctivitis in three horticultural workers. METHODS: Greenhouse working conditions were reproduced in our challenge chamber. RESULTS: All three cases developed rhinoconjunctivitis when working with HC plants. A control challenge was performed in two cases with iceberg lettuce causing no symptoms. Nasal volume measured by acoustic rhinometry (AR) fell after all three active challenges, but also after one of the control challenges. CONCLUSIONS: Our study confirms that HC may cause occupational rhinoconjunctivitis and asthma through a Type I hypersensitivity reaction. Specific inhalation challenges, nasal nitric oxide measurement and AR may be useful additional tools in supporting such diagnoses for occupational physicians to consider.

Relative tachycardia in patients with sepsis: an independent risk factor for mortality.

BACKGROUND: Excess activation of the sympathetic nervous system may be a risk factor for mortality in patients with the systemic inflammatory response syndrome (SIRS) or sepsis. AIM: To examine whether excessive tachycardia, relative to the degree of fever is an independent risk factor for death in patients with SIRS. DESIGN: Prospective observational study. SETTING: Departments of medicine in three university hospitals in Israel, Germany and Italy. METHODS: We collected data for 3382 patients with SIRS, whether community- or hospital-acquired, 91% with sepsis, as part of an ongoing trial. RESULTS: Overall 30-day mortality was 12% (408/3382). The pulse/temperature ratio was significantly higher in patients who died than in survivors: mean +/- SD 2.55 +/- 0.57 vs. 2.40 +/- 0.48 bpm/ degrees C (p < 0.0001). Excessive tachycardia was significantly associated with a mortality in a logistic model accounting for other strong predictors of mortality (OR 1.54, 95%CI 1.10-2.17). Patients with septic shock were the only group for whom this association did not hold. DISCUSSION: Our data are compatible with the hypothesis that some patients with sepsis experience an excess activation of the sympathetic nervous system, leading to a fatal outcome.

The need for macrolides in hospitalised community-acquired pneumonia: propensity analysis.

The present study compared beta-lactam macrolide ("combination") therapy versus beta-lactam alone ("monotherapy") for hospitalised community-acquired pneumonia, using propensity scores to adjust for the differences between patients. A prospective multinational observational study was carried out. Baseline patient and infection characteristics were used to develop a propensity score for combination therapy. Patients were matched by the propensity score (three decimal point precision) and compared with 30-day mortality and hospital stay. The propensity score was used as a covariate in a logistic model for mortality. Patients treated with monotherapy (n = 169) were older (mean+/-sd age 70.6+/-17.3 versus 65.0+/-19.6 yrs), had a higher chronic diseases score and a different clinical presentation compared with patients treated with combination therapy (n = 282). Unadjusted mortality was significantly higher with monotherapy (37 (22%) out of 169 versus 21 (7%) out of 282). Only 27 patients in the monotherapy group could be matched to 27 patients in the combination group using the propensity score. The mortality in these groups was identical, with three (11%) demises each. The multivariable odds ratio for mortality associated with combination therapy, adjusted for the propensity score and the Pneumonia Severity Index, was 0.69 (95% confidence interval 0.32-1.48). The benefit of combination therapy versus monotherapy cannot be reliably assessed in observational studies, since the propensity to prescribe these regimens differs markedly.

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin.

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.