Human seminal fibronectin fragmentation patterns and their domain immunoreactivities in leucocytospermic patients.

The aim of the work was to analyse fibronectin (FN) domain immunoreactivities and profiles of FN fragmentation in seminal plasmas of fertile normozoospermic and infertile leucocytospermic male patients. ELISA with domain-specific monoclonal antibodies and immunoblotting were used in these measurements. Immunoblotting of normal and leucocytospermic seminal plasmas revealed the presence of twelve FN bands of ~70-196kDa with nearly identical FN profiles under reducing and non-reducing conditions. The epitopes of the cell-, fibrin-, collagen-binding FN domains and the extra domain A (EDA) FN segment retained the ability to bind their specific monoclonal antibodies, whereas the fibrin-heparin domain (N-terminal end) and the area around the disulfide bridges (C-terminal end) of the FN polypeptide did not show any reactivities with their respective specific antibodies. The mean values of cell- (338.4±138.4 and 398.3±310mgL(-1)), fibrin- (79.1±38.5 and 145.2±188.8mgL(-1)) and collagen-binding (19±19.8 and 50.9±73.4mgL(-1)) FN domain immunoreactivities and the relative amount of (EDA)FN did not show any significant differences between the normal and leucocytospermic groups. The high values of standard deviations for the FN domain immunoreactivities in the leucocytospermic group probably results from different aetiology of leucocytospermia. The profile of FN fragmentation and alterations of FN domain immunoreactivities in seminal plasma may influence their engagement in the fertilisation process. The analysis of seminal FN molecular status would be helpful for selecting the highest quality spermatozoa for use in assisted reproduction techniques.

Fibronectin molecular status determination useful to differentiate between rheumatoid arthritis and systemic lupus erythematosus patients.

To find whether the plasma fibronectin (FN) molecular status can be useful to differentiate between rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The expression of plasma FN domains was determined by ELISA using monoclonal domain-specific antibodies. FN molecular forms were revealed by immunoblotting and analyzed by densitometry. The following findings were found: (1) Mean values of (Fibrin-Heparin)FN concentration were lower in SLE and RA patients than in normal plasmas. The cut off points at 31 mg/l in SLE and at 45 mg/l in RA showed a sensitivity and specificity of 54, 55 and 75%, respectively. (2) Mean values of concentrations of (CBD)FN and (Ct)FN were lower in SLE than those in normal and RA plasmas. Quantified data showed the cut off points of (CBD)FN and (Ct)FN at 200 mg/l (58% of sensitivity, 56% of specificity) and 350 mg/l (58% of sensitivity, 58% of specificity) in SLE, as well as at 295 mg/l (52% of sensitivity, 51% of specificity) and 460 mg/l in RA (70% of sensitivity, 73% of specificity). (3) The plasma FN immunopatterns, characterized by the presence of high-molecular (260-310 kDa) and/or low-molecular (158-209 kDa) FN bands, were specific only for SLE samples. The analysis of plasma FN status revealed by its Fibrin-Heparin-, CBD- and Ct-domain reactivity with monoclonal antibody and immunoblotting can be helpful to differentiate the SLE in respect to RA and normal plasmas.

Differences between the early and advanced stages of rheumatoid arthritis in the expression of EDA-containing fibronectin.

In the present study the expression of alternatively spliced synovial EDA-containing FN (EDA-FN) was analyzed in plasma and synovial fluid samples of rheumatoid arthritis (RA) patients with from 2 months to 20 years disease duration. The patient samples were divided into groups: those with early, established, and late progressive radiographic changes as well as those with different disease activity established by a CRP concentration. The expression of EDA-FN was determined by sandwich-type ELISA using a specific anti-EDA monoclonal antibody. The relative amount of EDA-FN in synovial fluid, but not in plasma samples, was significantly lower in the early RA group than in established and late RA. In contrast, its level did not correlate with the CRP concentration. Synovial EDA-FN might be used as a supplemented marker of early RA.

Relative sialylation and fucosylation of synovial and plasma fibronectins in relation to the progression and activity of rheumatoid arthritis.

The expressions of terminal sugars in synovial and plasma fibronectins were studied in relation to rheumatoid arthritis (RA) progression defined according to the early, established and late radiological changes in the patients' hands. The relative amounts of sialic acid and fucose were analyzed by lectin-ELISA using appropriate sialic acid-linked alpha2-3 (Maackia amurensis) and alpha2-6 (Sambucus nigra) lectins as well as fucose-linked alpha1-6 (Aleuria aurantia), alpha1-2 (Ulex europaeus), and alpha1-3 (Tetragonolobus purpureus). In the early RA group, the synovial fibronectin reactivities were the lowest with the all lectins used. In the established and late groups, relative sialylation and fucosylation significantly increased. However, sialylation negligibly decreased, whereas fucosylation remained at nearly the same level in the late group. Moreover, the expression of alpha1-6-linked fucose was found to be related to disease activity. In contrast, plasma fibronectin reactivity with lectins showed different dynamic alterations. In the early RA group, the reactivity of fibronectin with the lectins used was similar to that of healthy individuals, whereas it increased significantly in the established RA group compared with the early and normal plasma groups. In the late RA group it decreased to a level similar to that of the normal group. The lower expressions of terminal sugars in synovial fibronectin were mainly associated with the early degenerative processes of RA. In conclusion, such alterations may be applicable as a stage-specific marker for diagnosis and therapy of RA patients. The higher expression of terminal sugars in fibronectin could be associated with repair and adaptation processes in longstanding disease.

Fibronectin fragments in human seminal plasma.

The study has revealed the presence of fibronectin (FN) fragments and a lack of intact FN in 72 seminal plasma samples. The FN fragmentation was examined by immunoblotting with a monoclonal antibody specific to the central cellular FN domain and was confirmed with a monoclonal antibody directed to the C-terminal domain of FN. Nine FN fragments between 60 and 200 kDa and five fragments of 60-150 kDa were identified in seminal plasma samples of normozoospermic and of terato-, oligoterato-, and oligoasthenoterato-spermic groups, respectively. The relative amounts of the 60, 90 and 100 kDa FN fragments were 2-3 times higher in seminal plasmas with abnormal semen characteristics than in the normozoospermic group. The results suggest that seminal plasma FN fragments may contribute to fertilization and the analysis of FN fragmentation may have a diagnostic value in andrological investigations.