Hyaluronan (HA) effects on immune response are suggested to be dependent on HA molecular weight (MW), as low MW HA should activate immune cells in contrast to high MW HA. However, some current studies do not support this conception and emphasize the importance of the form of preparation of HA, particularly with respect to its purity and origin. We compared the activation of mouse immune cells by HA samples (100kDa, 500kDa, and 997kDa) prepared from HA originating from rooster comb, and HA samples (71kDa, 500kDa, and 1000kDa) prepared from pharmacological grade HA originating from Streptococcus equi. Interestingly, in contrast to established theory, only middle and high MW HA originating from rooster comb induced the production of tumor necrosis factor-α by macrophages and in whole blood. Further, all tested preparations of HA failed to induce the expression of inducible nitric oxide synthase, the production of nitric oxide, or the expression of cyclooxygenase 2 in macrophages and splenocytes. Importantly, all HA samples originating from rooster comb were found to be contaminated by endotoxin (up to 1.23EU/ml). Hence, low MW HA did not reveal itself to have significantly higher immunostimulatory activity compared to HA of higher MW.
- MeSH
- buněčná imunita účinky léků genetika MeSH
- cyklooxygenasa 2 genetika MeSH
- kur domácí MeSH
- kyselina hyaluronová farmakologie MeSH
- makrofágy účinky léků imunologie MeSH
- molekulová hmotnost MeSH
- myši MeSH
- oxid dusnatý genetika MeSH
- RAW 264.7 buňky MeSH
- regulace genové exprese účinky léků MeSH
- Streptococcus equi chemie MeSH
- synthasa oxidu dusnatého genetika MeSH
- TNF-alfa genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells.
- MeSH
- chromatografie iontoměničová MeSH
- intracelulární prostor chemie MeSH
- kyselina hyaluronová metabolismus MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Streptococcus equi chemie metabolismus MeSH
- uridindifosfátové cukry analýza MeSH
- Publikační typ
- časopisecké články MeSH
