The metabolism of hyaluronan (HA) relies on HA synthases and hyaluronidases, among which hyaluronidase-1 (HYAL1) and -2 (HYAL2) have been proposed as key actors. Congenital HYAL1 deficiency leads to mucopolysaccharidosis IX (MPS IX), a rare lysosomal storage disorder characterized by joint abnormalities. Knowledge of HYAL2 is limited. This protein displays weak in vitro hyaluronidase activity and acts as a receptor for oncogenic ovine retroviruses. We have generated HYAL2-deficient mice through a conditional Cre-lox system. Hyal2(-/-) mice are viable and fertile. They exhibit localized congenital defects in frontonasal and vertebral bone formation and suffer from mild thrombocytopenia and chronic, possibly intravascular, hemolysis. In addition, Hyal2(-/-) mice display 10-fold increases in plasma levels of HA and 2-fold increases in plasma hyaluronidase activity. Globally, there is no HA accumulation in tissues, including bones, but liver sinusoidal cells seem overloaded with undigested HA. Taken together, these elements demonstrate for the first time that murine HYAL2 has a physiological activity in vivo that is relevant for craniovertebral bone formation, maintenance of plasma HA concentrations, and erythrocyte and platelet homeostasis. In addition, the viability of HYAL2-deficient mice raises the possibility that a similar defect, defining a new MPS disorder, exists in humans.

No abstract

Many patients with primary immunodeficiency disease (PIDD) require lifelong immunoglobulin (Ig) replacement therapy. Home-based subcutaneous (SC) infusion provides advantages to patients with PIDD compared to hospital-based intravenous infusion. One limitation of current practice with SCIg infusion is the need for small-volume infusions at multiple injection sites on a frequent basis. A method was developed for large-volume SC infusion that uses preinfusion of recombinant human hyaluronidase (rHuPH20) to facilitate fluid dispersion. Miniature swine was used as a preclinical model to assess the effects of rHuPH20-facilitated infusions, of a single monthly dose, on fluid dispersion, infusion-related pressure, swelling, induration, and tissue damage. Preinfusion of vehicle (control) or rHuPH20 (75 U/g Ig) was performed simultaneously on contralateral abdominal sites on each animal, followed by infusion of 300 mL 10 % Ig (30 g) at each site. Compared to control infusions, rHuPH20 significantly reduced infusion pressure and induration (p < 0.05) and accelerated postinfusion Ig dispersion. Histological evaluation of infusion site tissue showed moderate to severe swelling for the control. Swelling after rHuPH20-facilitated infusion was mild on day 1 and had completely resolved shortly thereafter. Laser Doppler imaging of control infusion sites revealed local cutaneous hypoperfusion during Ig infusion, which was reduced almost 7-fold (p < 0.05) with the use of rHuPH20. These results demonstrate that rHuPH20-facilitated Ig infusion is associated with improved dispersion of Ig, resulting in reduced tissue pressure, induration, and reduced risk of tissue damage from mechanical trauma or local ischemia, thus enabling SC administration of large volumes of Ig at a single site.

SummaryTumor necrosis factor-Stimulated Gene 6 protein (TSG-6) is a hyaluronan (HA)-binding glycoprotein containing an HAbinding Link module. Because of its well-defined structure, HA binding properties and small size, TSG-6 is an excellent candidate as an alternative to animal-derived HA-binding protein (HABP) for the detection of HA. The present work describes the generation and characterization of a novel recombinant HA-binding probe obtained by fusion of a modified TSG-6 Link module with mutationally inactivated heparin-binding sequence and the Fc portion of human IgG1 (TSG-6-ΔHep-Fc) for tissue HA detection in histological samples. Direct binding assays indicated strong binding of TSG-6-ΔHep-Fc to HA, with little residual binding to heparin. Histolocalization of HA in formalin-fixed, paraffin-embedded tissue sections using biotin-TSG-6-ΔHep-Fc resulted in hyaluronidase-sensitive staining patterns similar to those obtained with biotin-HABP, but with improved sensitivity. HA was detected in many human tissues, and was most abundant in soft connective tissues such as the skin dermis and the stroma of various glands. Digital image analysis revealed a linear correlation between biotin-HABP and biotin-TSG-6-ΔHep-Fc staining intensity in a subset of normal and malignant human tissues. These results demonstrate that TSG-6-ΔHep-Fc is a sensitive and specific probe for the detection of HA by histological methods. (J Histochem Cytochem 62:672-683, 2014)

© F e r r a t a S t o r t i F o u n d a t i o nTechnologies) and analyzed using flow cytometry (FACS Calibur, BD Biosciences). 8RBC senescence was assessed by measuring the exposure of phosphatidylserine, 9 auto-immunoglobulin G (IgG), 10 and C3 on RBC after in vitro incubation with fluorescein isothiocyanate-(FITC)-annexin V (BD Biosciences), FITC-rabbit anti-mouse IgG (Dako), or rat monoclonal anti-mouse C3 antibody (Abcam) followed by Alexa-488-anti-rat antibody (Life Technologies), respectively.For the deformability studies, a pre-determined volume of blood (23 to 30 mL for hematocrit values of 33% to 44%, respectively) was added to 5 mL of isotonic polyvinylpyrrolidone solution. One milliter of this suspension was submitted to 12 different shear stress values at 37°C using a LORCA ektacytometer (Mechatronics Instruments). A decrease in elongation index indicates a loss of RBC deformability. 11,12 Transplantation of bone marrowMice were irradiated twice (2×6 Gy at a 3-h interval). One hour later, they were given an intravenous injection of either control or Hyal2 -/-bone marrow (5x10 6 cells). The mice were kept under sterile conditions for 12 weeks to allow bone marrow reconstitution and blood analyses. 13 Survival of transfused red blood cells RBC from control or Hya2-/-donor mice were labeled by intravenous injection of sulfo-NHS-LC-biotin, collected 1 h later, washed, and re-suspended to achieve a hematocrit of 25%. Two hundred microliters of this suspension were injected into the tail vein of control and Hya2 -/-recipient mice. The percentage of biotinylated erythrocytes remaining in the circulation at various time points was determined using FACS. 14 Incubation of red blood cellsBuffy coat-free plasma was collected by centrifugation. Complement was inactivated (56°C for 30 min). RBC were incubated for 24 h in either Hyal2 -/-plasma, control plasma, or the latter + 7.5 mg/mL high molecular mass (3.9×10 6 Da) hyaluronan, corresponding to plasma hyaluronan concentrations in Hyal2 -/-mice. RBC were then incubated with FITC-annexin-V and analyzed using FACS. Blood and plasma viscosities and blood pressureThe viscosity of blood and plasma was measured at 38°C under various shear rates using a rotation viscosimeter (LVDV-II+Pro C/P, Brookfield Engineering Laboratories).Blood pressure was measured using the tail-cuff method (CODA, Kent Scientific, Torrington, CT, USA). 15,16 Other measurementsVascular adhesion molecule-1, intercellular adhesion molecule-1, and P-selectin were measured using enzyme linked immunosorbent assays (R&D Systems), while the activity of A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13 (ADAMTS13) was determined using Lifecodes ® Activity Assay (Immucor). von Willebrand factor (vWF) multimers were assessed in the laboratory of Claudine Caron, Lille, France. Fibrin was detected using polyclonal rabbit anti-human fibrinogen antibody (Dako). 17 Statistical analysesUnpaired and Mann-Whitney tests were used for single comparisons; two-way ANOVA and the...