Keiichi Miyashita

Development of the selective adsorbent for EDA containing fibronectin using heparin immobilized cellulose.

We have noticed that extra domain A containing fibronectin (EDA(+)FN) is closely related to inflammatory diseases and accordingly investigated a selective adsorbent to remove EDA(+)FN, which may increase the severity of the symptoms, from the plasma of rheumatoid arthritis (RA) patients. Three types of heparin immobilized cellulose (HC) were prepared and their adsorptive properties were evaluated by batchwise adsorption tests. The results showed that EDA(+)FN was adsorbed more efficiently than total FN on HC. In particular HC in which heparin was immobilized to amino residue had an excellent capacity. The adsorption rates of EDA(+)FN, total FN and antithrombin-III were 89, 15 and 17% respectively. This adsorbent is extremely useful in selectively removing EDA(+)FN from plasma.

Cryogelation in vitro

Cryogel is a physical gel formed by the heterophilic aggregation of extra domain A containing fibronectin (EDA(+)FN), plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep). Cryogelation is controlled by the interactions between each aggregate and the amount of aggregates. Therefore, the present study attempted to elucidate these properties by studying turbidity (tau). Although only Fbg formed a self-aggregate under low temperatures, from the temperature dependence of tau, the amount of aggregate in three-element (pFN/Fbg/Hep) solution surpassed that of the EDA(+)FN/Fbg/Hep system. The optimal condition for cryogelation was afforded by a solution with Fbg/EDA(+)FN/pFN/Hep expressed in the molar ratio of 12:0.04:0.79:1. This cryogel structure in solution was probably formed via structural changes induced by pFN in Fbg. The structural change in Fbg was examined by circular dichroism under optimal conditions. This concept was based on observations of the direct transmission scanning electron microscopy of a cryogel. The EDA(+)FN/pFN/Fbg/Hep aggregates displayed a network structure that manifested particulate crosslinkage. Cryogelation, a phenomenon related to induction of rheumatoid arthritis in humans, was facilitated by both the EDA(+)FN-Hep interaction and the structural changes of Fbg induced by pFN.

Different responses to surgical stress between extra domain A+ and plasma fibronectins.

1. Fibronectins (FN) are believed to have a role in haemorheological perturbation associated with tissue damage. Fibronectins exist in two antigenically related forms, plasma (p) and cellular fibronectin, which has the extra domain sequences A (EDA) or B (EDB). The present study was designed to determine changes in plasma p‐FN and EDA + FN under different types of surgical stress.

Increased extra domain-A containing fibronectin and hepatic dysfunction during septic response: an in vivo and in vitro study.

A massive inflammatory reaction resulting from systemic cytokine release is the common pathway underlying sepsis or multiple organ dysfunction. The role of extra domain sequence A-containing fibronectin (EDA+FN) formation during the septic response is not known. The present study investigates the role of EDA+FN during the septic response under in vitro and in vivo conditions. The direct effects of interleukin-1, interleukin-6, and tumor necrosis factor-alpha on EDA+FN production were evaluated in primary cultured human hepatocytes and fibroblasts. Serial plasma EDA+FN levels were measured using an enzyme-linked immunosorbent assay in 24 patients who developed postoperative sepsis following general abdominal surgery of which there were 17 survivors and 7 non-survivors. EDA+FN secretion was significantly increased in cultured hepatocytes but not fibroblasts at 24 and 48 h following exposure to IL-1 compared to controls. In the clinical setting plasma EDA+FN levels in non-survivors were significantly higher than in survivors. Moreover, the EDA+FN levels were correlated closely with liver function tests. EDA+FN levels may represent a specific marker of vascular injury or systemic inflammatory response syndrome that is associated with an adverse clinical outcome.

Development of a novel polycationic adsorbent for cryogel removal.

Cryogel, prevalent in the plasma of rheumatism patients, is a plasma fibronectin (pFN)-extra domain A containing FN (EDA(+)FN)-fibrinogen (Fbg) complex formed by adding heparin (HP) at a low temperature (4 degrees C). Although EDA(+)FN does not usually exist in normal plasma, its prevalence in rheumatic patients causes cryogelation in plasma. Removal of cryogel is thus a promising and novel approach to treating rheumatism. As HP-EDA(+)FN aggregate, which is induced by the main component of cryogel, is considered to be an anion, cationic materials capable of eliminating this anionic conjugate were innovated in this study. We found that an amino group density of 100-130 micromol/g (dry weight) of adsorbents prompted selective adsorption of the EDA(+)FN-HP complex. Elimination of EDA(+)FN as high as 80% accompanied by removal of the components of total FN (pFN) (10%) and Fbg (10%) in the model patient plasma was established.