Catalytic properties of rabbit serum esterases hydrolyzing esterified monosaccharides.

Rabbit serum and one enzyme fraction isolated from rabbit serum by column chromatography (Fraction II) were used as catalysts in regioselective hydrolysis of radiolabelled pivaloylated monosaccharides (Piv = Me3CCO). The hydrolysis of 14C-labelled methyl 2-O-pivaloyl-(2-MP)-, 6-O-pivaloyl (6-MP)-, 2,6-di-O-pivaloyl-(2,6-DP) alpha-D- glucopyranosides and methyl 2-acetamido-2-deoxy-3,6- di-O-pivaloyl-(3,6-DPNAc) alpha-D-glucopyranosides, was studied, as well as that of the non-sugar substrates butyrylthiocholine, thiophenylbutyrate, phenylacetate and paraoxon. The specific activities of 2,6-DP, 3,6-DPNAc, butyrylthiocholine and thiophenylbutyrate were higher in Fraction II than in native sera, while those of phenylacetate and paraoxon were lower. Inhibition studies were done using the substrates mentioned and five different inhibitors, namely bis(p-nitrophenyl phosphate) (BNPP), eserine, paraoxon, HgCl2 and EDTA. The hydrolysis of 2,6-DP and 3,6-DPNAc was not inhibited by HgCl2 and only slightly by EDTA. Paraoxon, eserine and BNPP were progressive inhibitors of the hydrolysis of the two sugar substrates, and the pattern of inhibition resembled closely the inhibition of butyrylthiocholine and thiophenylbutyrate hydrolysis. This result applied to both, native serum and Fraction II. It was concluded that esterases in rabbit serum which hydrolyze pivaloylated sugar substrates belong to the category of serine esterases. Kinetic parameters (KM and Vmax), effects of temperature and pH on activity of esterases from Fraction II were also determined for the hydrolysis of sugar substrates.

Ion-exchange chromatography separation of the detergent and the solvent from immunoglobulins after solvent-detergent treatment.

For inactivation of lipid-enveloped viruses during the immunoglobulin production, the solvent-detergent (S/D) method was applied. Tri-n-butyl phosphate (solvent) and Triton X-100 (detergent) were removed from S/D treated immunoglobulins by ion-exchange chromatography on Q-Sepharose Fast Flow (FF). During the chromatographic procedure immunoglobulins remained bound on a Q-Sepharose FF, whereas solvent and detergent were eluted by washing with starting buffer. Elution of immunoglobulins was achieved by increasing the ionic strength of the starting buffer. The final immunoglobulin preparation contained less than 10 microg/ml of Triton X-100 and less than 2 microg/ml tri-n-butyl phosphate. It was confirmed that the S/D procedure did not cause a significant change in polymers and specific antibodies content. Immunoglobulin classes were also not affected by the same procedure.

Removal of detergent and solvent from solvent-detergent-treated immunoglobulins.

The solvent-detergent (S/D) method was applied for inactivation of lipid-enveloped viruses during the production of immunoglobulins. Amberlite XAD-7 resin was used for removal of solvent (tri-n-butyl phosphate, TnBP) and detergent (Triton X-100) after the performed S/D inactivation procedure. The S/D reagents from the immunoglobulin preparation were adsorbed on Amberlite XAD-7, while immunoglobulins passed through the column and retained their biological activity. Using the method developed here, the final immunoglobulin preparation contains less than 1 ppm of Triton X-100 and less than 2 ppm TnBP.

Partial purification of esterases from rabbit serum and their use in regioselective deacylations of sugars.

14C-Labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside was used as a substrate for the detection of esterase activity in the isolation of esterase II from rabbit serum. On treatment of methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside with rabbit serum and esterase II, the 6-O-pivaloyl group was removed selectively. Likewise, the 6-O-pivaloyl group was removed selectively from methyl 2-acetamido-2-deoxy-3,4,6-tri-O-pivaloyl-alpha-D-glucopyranoside.

Characterization of F VIII concentrates produced by two methods incorporating double virus inactivation.

The trend toward the production of high purity factor VIII concentrates for clinical use is still in progress. Although all plasma derivatives must undergo viral inactivation procedures, the possibility of transmission of viral diseases is not completely eliminated. In order to reduce such risk, we have included double virus inactivation in the procedure of factor VIII concentrate production. In a scale-up procedure for isolation of factor VIII cryoprecipitate, two methods were used. The first is based on the chromatographic purification of factor VIII after pasteurization of cryoprecipitate solution and solvent/detergent (S/D) inactivation of viruses. The second is based on multistep precipitation of factor VIII by sodium chloride and glycine. Viral inactivation was performed by combination of S/D treatment and heating of final freeze-dried product 30 min at 100 degrees C. The typical yield of factor VIII activity in the freeze-dried product was about 20% for the first method, and 25-30% for the second. Electrophoretic analyses of both factor VIII preparations by SDS-PAGE and IEF show very low content of contaminant proteins, in accordance with observed 400-650-fold increase of their specific activity over plasma. Both factor VIII products were stable in the liquid state for more than 24 h at room temperature. The final products, after double viral inactivation, are considered to be suitable for clinical evaluations.

Enzymatic deacylations of esterified saccharides. II. De-esterifications of radiolabelled O-acylglucopyranosides by mice serum and liver esterases.

1. 14C-Labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside (1) was used as a novel substrate for esterases from mouse serum and liver. 2. Stepwise de-esterification of the diester substrate 1 was achieved, and data on time-course experiments are reported. 3. Kinetic studies were undertaken to compare deacylation rates for the enzymatic de-esterification of the diester substrate 1 using both, mice sera and liver microsomal fractions. 4. Serum and liver esterase activities were studied in mice treated with an immunostimulating agent, peptidoglycan monomer (PGM), and a comparison made with esterases from untreated mice.

Comparison of immunogenicity of recombinant and plasma derived hepatitis B antigen in guinea-pigs.

Four different preparations of hepatitis B antigen (HBsAg) were tested in parallel with respect to their ability to elicit anti-HBs in guinea-pigs. We compared the effect of low doses (5 micrograms single dose and 30 micrograms total amount) of human plasma derived and recombinant HBsAg, in purified forms or prepared as vaccines, respectively. The highest titre of anti-HBs developed was detected in guinea-pigs immunized with plasma derived HBsAg, followed the by response in animals immunized with plasma derived vaccine. Purified recombinant antigen and recombinant vaccine exhibited poor immunogenicity compared with plasma derived antigen preparations. The increase in the dose of recombinant antigen (150 micrograms total amount) resulted in markedly improved response in two out of ten animals. Anti-HBs immunoglobulin fractions from pooled sera within each group of animals were isolated, partially purified, coupled to polystyrene beads and subsequently used in immunoradiometric assay with monoclonal 125I-anti-HBs. Only the anti-HBs isolated from pooled sera of animals immunized with purified plasma derived antigen met the requirements for high sensitivity and antigen specificity in such assay. Comparable properties were exhibited by the anti-HBs obtained from one guinea-pig immunized with a larger amount of recombinant antigen. With these two anti-HBs immunoglobulin preparations, amounts as low as 1 ng of HBsAg could be detected.

[Biological standardization of the allergenic extract of Ambrosia elatior]. / Bioloska standardizacija alergogenskog ekstrakta Ambrosia elatior.

Allergenic activity of allergen extract Ambrosia elatior (AE) was tested in fifteen volunteers extremely sensitive to the allergen. The research was performed by using the quantitative prick (P) and the intradermal (ID) test. The results were analyzed by the parallel line assay method. Allergenic activity of the specimen was estimated: by the number of the allergen units (AU) in 1 ml of the undiluted specimen according to the results of the P (prick) test; by the number of the allergen units (AU) in 1 ml of the undiluted specimen according to the results of the I. D. test, and by the in vitro standardized method of the RAST inhibition. These preliminary results show that allergen extract Ambrosio elatior has 100,000-130,000 Bu/ml and ca 1000 AU/ml while allergenic activity of the extract measured by the RAST inhibition method has ca 57,000 IU/ml.

Determination of foetal calf serum in vaccine preparations by immunoradiometric assay and enzyme-linked immunosorbent assay.

A simple and sensitive immunoradiometric assay (IRMA) for detection of foetal calf serum proteins in vaccine preparations has been developed. In this two-site sandwich assay the same preparation of polyclonal anti-FCS immunoglobulin (IgG) was used for coating the solid phase (as a capture antibody) and in a labelled form (I-125 labelled) for detection. The developed assay allows precise and accurate quantification of FCS proteins (down to 10 ng/ml) in vaccine preparations and has been used for screening of FCS residues (a) during production of measles vaccine, and (b) in various different commercial vaccine preparations. An enzyme-linked immunosorbent assay (ELISA) was also developed based on the same assay two-site sandwich principle, where anti-FCS was directly labelled with horse-radish peroxidase. ELISA was comparable in sensitivity and accuracy with IRMA.

Expression of lymphocytes Fc epsilon RII/CD23 in allergic children undergoing hyposensitization.

Owing to the proposed role of Fc epsilon RII/CD23 in allergic diseases, we analyzed the expression of this receptor on peripheral blood lymphocytes (pan-B, pan-T and CD4+ or CD8+ T cells) and its autoproteolytic product sCD23 in serum. This was done in 10 asthmatic children allergic to Dermatophagoides pteronyssinus (Dpt) before and 6 weeks after hyposensitization. FACS analysis of double, direct immunofluorescence staining of the whole blood revealed an elevated percentage of Fc epsilon RII/CD23+ lymphocytes in allergic children (10.29 +/- 5.0), a significantly higher percentage than in nonallergic children (5.7 +/- 2.4, p < 0.05). The majority of Fc epsilon RII/CD23+ were on B cells. A significant positive correlation between the percentages of CD23+ lymphocytes and serum IgE levels was found (Spearman rank = 0.63, p < 0.05). The percentage of CD20+CD23+ lymphocytes significantly decreased after 6 weeks of hyposensitization (6.2 +/- 3.6, p < 0.05), while the percentage of CD20+ lymphocytes remained unchanged. Similarly, hyposensitization was followed by a reduction of total serum IgE levels, but Dpt-specific IgG4 and IgE remained unchanged.

The effects of immunomodulating peptidoglycan monomer and muramyl dipeptide on hepatic microsomal UDP-glucuronyltransferase and beta-glucuronidase.

The effects of immunomodulating peptidoglycans, peptidoglycan monomer (PGM) and muramyl dipeptide (MDP), on hepatic microsomal UDP-glucuronyltransferase (uridine diphosphoglucuronate glucuronosyl transferase, EC 2.4.1.17) and beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.31) were tested in female C57Bl mice. 4-Methylumbelliferone and p-nitrophenol were used as representative substrates for one functional form of UDP-glucuronyltransferase (GT1) and testosterone for the second functional form (GT2) of the enzyme. Both PGM and MDP were found to transiently inhibit the activity of UDP-glucuronyltransferase. There was no significant difference in the magnitude of inhibition of the two functionally different enzyme forms. The activity of microsomal beta-glucuronidase was tested using 4-methylumbelliferyl glucuronide and p-nitrophenyl glucuronide as substrates. Time dependent transient inhibition of beta-glucuronidase activity was observed with both peptidoglycans. In addition, the effect of MDP on cytochrome P-450 was tested, since we have shown previously that PGM affected this system. MDP decreased the content of cytochrome P-450 and inhibited the activity of related enzymes.

The development of a latex agglutination inhibition assay for the determination of foetal calf serum in viral vaccines.

A simple but specific, sensitive and reproducible latex agglutination inhibition assay for the determination of foetal calf sera in viral vaccines has been developed and standardized. The detection limit was at nanogram level. The assay procedure requires two pipetting steps, a short centrifugation stage and the use of a spectrophotometer.

Characterization and partial purification of the Croatian national standard Dermatophagoides pteronyssinus allergen extract.

Lyophilized Dermatophagoides pteronyssinus (Der p) allergen extract (AE) and partially purified Der p extract (PAE) were prepared and characterized. Partial purification of AE was performed by gel filtration on Sephadex G-100 and Sephacryl S-300. Crossed immunoelectrophoresis (CIE) disclosed the same precipitating lines in AE and PAE preparations. The relative potencies of AE and PAE were determined and compared with the WHO International Standard for Der p by the RAST inhibition method. The potencies were 6.5 x 10(5) IU and 1.5 x 10(6) IU, respectively. Biologic standardization by quantitative skin testing was performed with AE (20 selected patients) and PAE (12 patients). Median Ch was calculated by linear regression analysis (log-log model). One ampoule of AE contained 65,300 BU and 1 ml (vial) of PAE contained 166,000 BU. Der p AE could serve as a croatian national standard for further production of Der p allergenic extracts.

Decrease in CD23+ B lymphocytes and clinical outcome in asthmatic patients receiving specific rush immunotherapy.

Rush immunotherapy (RIT) has been documented as useful in the treatment of patients with allergic bronchial asthma. To investigate the mechanisms of its action, we studied changes in the serum levels of total IgE, allergen-specific IgE and IgG4, and expression of CD23 on peripheral blood B cells in patients receiving RIT. Twenty patients with perennial bronchial asthma were evaluated before the beginning of RIT, as well as 6 weeks and 6 months later. Compared to pretreatment values, the level of Der-p-specific IgG4 and IgE significantly increased after 6 weeks and 6 months of RIT, while the total serum IgE remained unchanged. Furthermore, after 6 months of RIT, the percentage of CD23+B cells and its CD23 receptor density significantly decreased. Since the symptom score improved and the need for medication decreased, we evaluated RIT as a useful procedure. After 6 months, 30% of patients did not have an asthma attack, with no medication in the last month, while 10% of them were asthma free for the last 3 months. No significant correlation between the clinical improvement, and in vitro changes was found. Furthermore, the observed in vitro changes were not significantly different in patients who responded with clinical improvement, compared to those with unchanged intensity of asthma. In conclusion, during specific RIT we found a significant increase in Der-p-specific IgE and IgG4 antibodies, as well as a moderate decrease in CD23+ B cells and its CD23 receptor density. These findings suggest a change in the lymphokine profile of patients receiving specific immunotherapy, and that the inhibition of IL-4-induced B cell stimulation may be hypothesized as the most important mechanism.