Protection by a polyvalent vaccine against challenge infection and pyelonephritis.

The protective effect of immunization with a polyvalent vaccine (SolcoUrovac) was studied in the mouse and the rat. The i.m. immunization increased the resistance of mice to challenge infection with all homologous strains of bacteria. The LD50 values for E. coli, Proteus mirabilis and Streptococcus were 3.5-4.5 times and that of Klebsiella as much as 600 times that in nonimmunized mice. Protection against challenge with heterologous E. coli was also achieved and persisted for about 20 weeks. Immunization with the vaccine also provided marked protection against pyelonephritis in rats. Kidneys with abscesses were seen only one-third as often as in controls, and the size of the individual abscesses was substantially smaller in the vaccinated animals. Based on the quantity of bacteria in the kidneys it was postulated that the vaccination increased the clearance of bacteria.

Urinary antibody response after immunisation with a vaccine against urinary tract infection.

Immunisation of mice with SolcoUrovac vaccine induced an approximately 10-fold increase of the total amount of IgG and a 2-fold increase of IgA immunoglobulins in urine. IgG antibodies to SolcoUrovac antigens appeared in urine after the first injection, and booster injections caused a further increase of the titer. IgA antibodies appeared in the urine after the second injection, and the third injection doubled the titer. IgM immunoglobulins and specific IgM class antibodies to SolcoUrovac were not found in any urine tested. The exact origin of the immunoglobulins in the urine as well as the specificity of immune response is discussed.

Purification and some properties of collagenase proenzyme activator from rheumatoid synovial fluid.

1. An activator of leucocyte latent collagenase has been extracted from rheumatoid synovial fluid by a preparative method consisting of six steps including precipitation by ammonium sulphate and chromatography on Sephadex G-100, QAE-Sephadex and SP-Sephadex C-50. The purification factor was nearly 1000 and the activator isolated could be shown to have a high degree of homogeneity.--2. Gel chromatography indicated a molecular weight of ca. 60 000.--3. Kinetic studies of the activation and inactivation of the activator during incubation at higher temperatures demonstrated its enzymic nature.--4. Activation of latent collagenase was partially inhibited by iPr2P-F and KCN. Soybean trypsin inhibitor, iodoacetamide, TosLysCH2Cl and TosPheCH2Cl had no effect.--5. Leucocyte latent collagenase was also activated by an excess of trypsin and p-hydroxymercuribenzenesulphonic acid, but only to the extent of about 40% of its activation capacity. Purified neutral protease from human leucocyte granules had no effect on latent collagenase.--6. Several typical substrates for proteases, peptidases, esterases and glycosidases were not attacked by the activator. The possibility that the activator is a known enzyme, such as kallikrein, urokinase or cathepsin B1, could be excluded.

Effect of antirheumatic drugs on neutral protease from human leucocyte granules.

The inhibitory effect of 38 antirheumatic and other agents on purified neutral protease from human polymorphonuclear leucocytes has been studied by determining the decrease in enzyme activity on Z-Ala-NPH as substrate. Analgesics, salicylates, cytostatic agents and steroids, as well as D-penicillamine, colchicine, allopurinol, chlorzoxazone and chlorpromazine, either had no effect on neutral protease or inhibited it only to a very small extent. Typical antirheumatic agents like gold and pyrazolone derivatives suppressed the activity of the enzyme at a concentration of 10(-5)M. The two sulphonated polysaccharides Arteparon and pentosan polysulphate (SP 54) were the most potent inhibitors of neutral protease (inhibition down to 10(-8)M). Increasing concentrations of various inorganic salts gradually suppressed the effect of some otherwise effective drugs on neutral protease. The drugs were completely ineffective at a salt concentration of 0.5 M. At physiological concentrations, however, this effect was insignificant. Inhibition of neutral protease may be one way in which some antirheumatic drugs exert a therapeutic effect in rheumatic diseases.

Purification and some properties of a neutral protease from human leukocyte granules and its comparison with pancreatic elastase.

1. A cationic protease has been purified from the granule fraction of blood-donor leukocytes by a preparative method including precipitation by acetone and chromatography on Bio-Gel A 1.5 m, CM-Sephadex C-50 and Sephadex G-G-75. 2. The pH optimum against denatured bovine hemoglobin is 7.4. Gel chromatography indicated a molecular weight close to 23 000. 3. This neutral protease (EC 3.4.-.-) is able to split the synthetic esters Z-Ala-NPh and AcAla3OMe, its activity on the former substrate being 2.2 times greater than that of pancreatic elastase, on the latter the same. It differs crucially from pancreatic elastase in having small elastinolytic activity. 4. In cationic disk electrophoresis, neutral protease resolves into three protein bands with lower mobility than lysozyme: all bands exhibit esterolytic activity against 2-acetoxy-3-naphthoic acid o-toluidide, strongly suggesting that they represent isoenzymes. 5. The enzyme is completely inhibited by iPr2P-F, partially so by soybean trypsin inhibitor and Trasylol. Cysteine, EDTA and TosLysCH2Cl have no effect. 6. During chromatography on CM-Sephadex C-50 a more positively charged enzyme(s) was identified. This had hemoglobinolytic activity at pH 7.4 but only a small esterolytic effect on Z-Ala-NPh; it showed only traces of activity against AcAla3OMe.

Hydroxyproline levels and collagenolytic activity in synovial fluids of patients with rheumatic diseases.

Free, total, and peptide hydroxyproline levels were determined in synovial fluid obtained from the knee joints of 60 patients with theumatoid arthritis (RA), and 26 patients with degenerative joint disease. In addition, in 160 synovial fluid samples obtained from 121 patients including 50 with degenerative joint disease, 60 with RA, 3 with Reiter's syndrome, 3 with hydarthrosis intermittens and 5 with ankylosing spondylitis, the collagenolytic activity was determined. The mean values of free and peptide hydroxyproline in the inflammatory and degenerative fluids were the same, but slight differences were found in the mean values of total hydroxyproline. No effect on the level of free and bound hydroxyproline was observed after treatment with intra-articular hydrocortisone and gold salts. The collagenolytic activity of synovial fluid was registered in 38% of cases of RA and in some cases of Reiter's syndrome and hydrarthrosis intermittens, but it was not found in 50 cases of degenerative joint disease or in cases of ankylosing spondylitis. During a longer observation of patients with inflammatory forms of RA a variability in the collagenolytic activity was observed in repeated examinations of the fluid obtained from the same patient; this activity appeared and disappeared. The incidence of collagenolytic activity and its values were higher in patients with active rheumatoid process and this activity was present more frequently in patients with a short history of the disease (up to 3 years). The collagenolytic activity of rheumatoid fluids was, to a high degree, inhibited by normal human serum. The problem of presence or lack of collagenolytic activity in rheumatoid fluids is discussed.