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The National Institute for Medical Research, London, was asked by the WHO Expert Committee on Biological Standardization to take steps to set up an international standard of streptokinase-streptodornase. This paper describes the nature and handling of the material and its establishment as the International Standard for Streptokinase-Streptodornase, with a defined potency of 3100 International Units of Streptokinase and 2400 International Units of Streptodornase per ampoule. The International Unit of Streptokinase is defined as the activity contained in 0.002090 mg of the International Standard for Streptokinase-Streptodornase. The International Unit of Streptodornase is defined as the activity contained in 0.002700 mg of the International Standard for Streptokinase-Streptodornase.
The cleansing effects on pus and debris of streptokinase-streptodornase and of stabilized crystalline trypsin were compared in 40 patients (12 males, 28 females) with necrotic varicose or arteriosclerotic leg ulcers. Both preparations produced significant partial or total cleansing of the ulcers of necrotic material, and both enhanced the granulation and epithelialization of most of the ulcers. Streptokinase-streptodornase was significantly more effective in removing freshly formed damp and smearing pus and debris. Neither preparation prevented the formation of or removed very deep adherent necrotic areas, especially in arteriosclerotic leg ulcers. Trypsin caused significantly more pain than streptokinase-streptodornase during the treatment period and on changing dressings. Both enzymes indirectly reduced the bacterial flora of the ulcers, probably by removal of necrotic wet material.
Extracellular streptodornase was purified from fermentation media of Streptococcus equisimilis by stepwise carboxymethyl-Sepharose column chromatography. The active enzyme fraction was eluted with phosphate buffer containing 0.2 M NaCl. The purified enzyme showed a homogeneity on SDS-PAGE and had a subunit molecular weight of 35 kDa. Conformational change of streptodornase by binding to calf thymus DNA was examined by circular dichroism (CD). CD study clearly showed a DNA-induced conformational change in the secondary structure of streptodornase, resulting in a decrease of alpha-helical content of the enzyme.
The M1T1 strain remains the most frequently isolated strain from group A streptococcal (GAS) infection cases worldwide. We previously reported that M1T1 differs from the fully sequenced M1 SF370 strain. To better understand the reason for the persistence and increased virulence of M1T1, we analysed its secreted proteome and identified two virulence proteins that are not present in the sequenced M1 SF370 strain: streptococcal pyrogenic exotoxin A (SpeA) and a streptodornase D (SdaD) homologue. In the present study, we determined the nucleotide sequence of the M1T1 streptodornase and found that its deduced amino acid sequence is highly similar to other streptococcal streptodornases, and is most closely related to the SdaD of GAS strain M49. M1T1 Sda shares two highly conserved domains with several DNases and putative DNases in streptococci; however, it possesses a unique C-terminal amino acid sequence. Thus, we named the protein Sda1, and we detected the presence of the sda1 gene in 16 M1T1 clinical isolates. The cloned and expressed Sda1 degrades both streptococcal and mammalian DNA at physiological pH. Amino acid similarity analyses of known GAS deoxyribonucleases suggest that Sda1 may be a chimeric protein created through recombination events. Moreover, a natural mutation that resulted in longer Sda1 and SdaD as compared to other GAS DNases was found to confer increased activity on the protein. Analysis of the sequences flanking sda1 determined that it is carried by a prophage or a prophage-like element inserted in the tRNA-Ser gene of M1T1 GAS. Ongoing studies in our laboratory aim to determine the contribution of Sda1 to the virulence of this globally disseminated M1T1 strain.
In 31 patients with leg ulcers the wounds were randomly allocated to treatment either with a solution of streptokinase plus streptodornase in saline or physiological saline alone. The occurrence of pus and debris in the wounds was evaluated by a "blinded" investigator at the initiation of treatment and on Days 5, 10 and 15. In 12 out of 13 patients treated with the streptokinase-streptodornase solution and in 8 out of 16 saline-treated patients the wounds were clean by Day 10.
Necrotic materials most frequently found in leg ulcers, including crusts with fibrinogen, pus and blood clots, were exposed to solutions of streptokinase-streptodornase and stabilized crystalline trypsin respectively. The investigations were performed at the temperatures of 26 degrees and 33 degrees C, representing the extreme values of the temperature range found in leg ulcers (arteriosclerotic ulcers, stasis ulcers) and at the pH corresponding to that of the enzyme preparations in wet dressings. Streptokinase-streptodornase demonstrated in vitro a more potent proteolytic activity than crystalline trypsin on necroses, crusts of fibrinoid and purulent exudate, which were more rapidly and thoroughly broken up. Both enzyme preparations were, however, equally effective on three-day-old blood clots.
77 M- and 48 T-typed strains of A-streptococci were compared with respect to their extinction, acidification and nuclease-production as a function of the growth-time. Strains of the same serotype (M 12) showed a change in the distribution of the isoenzymes of nucleases with respect to the growth-time. Half of the strains produced nuclease activity after 4 h. Streptodornase B was produced by all strains. With the growth the number of the nucleases found at the same time diminished, after 96 h in 70% of the strains the nuclease activity was represented by streptodornase B only.
The clinical efficacy of dextranomer (Debrisan) and streptokinase-streptodornase (Varidase) was compared in a controlled randomized in-patient study. There were two patient groups: Group A consisting of 28 patients with a total of 31 venous leg ulcers and Group B consisting of 56 patients with other infected wounds (posttraumatic and postoperative wounds, amputation stumps, burn wounds, arteriosclerotic ulcers, decubital ulcers, ulcers due to bone disease, rheumatic ulcers, ulcers due to erysipelas and wounds of mixed aetiology). Both agents have a good cleansing effect as well as a good effect on infection and wet necrosis. Dextranomer stimulates the formation of granulation tissue faster than streptokinase-streptodornase.
Tests for delayed cutaneous hypersensitivity were carried out with Candida and streptokinase-streptodornase antigens in 245 normal pediatric subjects, ranging in age from 17 days to 5 10/12 years. The group comprised patients with a variety of acute illnesses and a series of control subjects. There was a progressive increase in the frequency of cutaneous responses with age. About one-third of infants under one year and four-fifths of one to five-year-old children had positive induration reactions to at least one antigen. Candida was the more reactive antigen in the first year of life; thereafter there was comparable responsiveness to both antigens. Our results showed no difference in percent reactivity between acutely ill and control subjects.
Delayed skin reactor (DSR), the material in streptokinase-streptodornase responsible for eliciting delayed hypersensitivity reactions in man, is stable as a lyophilized powder or when stored at -70 degrees C. Storage at a pH above or below 6.0 leads to a decrease in the ability of DSR to stimulate lymphocyte proliferation in vitro. Its molecular weight is 30,000 as estimated by sodium dodecyl sulfate gel electrophoresis and it is composed of approximately 260 amino acid residues without apparent sulfhydryl-bridged subunits. The extinction coefficient of DSR (A 1% 280 nm) is 2.6.
Using the 3H-thymidine incorporation test, the lymphocyte-stimulating activity of streptokinase-streptodornase (SK-SD, Varidase) and its fractions were evaluated. The dose-response curves of all the preparations except the third fraction had two peaks with the maximum around 5 and 40 mug. The dialyzable fraction of Sk-SD (D-SK-SD) occupying approximately half of the original sample had almost no lymphocyte-stimulating activity; and conversely, it had a potent inhibitory effect on the 3H-thymidine incorporation into lymphocytes that normally occurs with non-dialyzable SK-SD (ND-SK-SD), PPD and PHA-P. Therefore, it will be desirable to remove the dialyzable fraction from the commercial SK-SD preparation when it is used for the determination of the capacity of cellular immunity against tsk-sd.
Homocytotrophic antibodies (HCA) were produced in rats by primary injection of streptokinase-streptodornase (SK-SD) after the antigen was absorbed on alminum hydroxide gel. The properties of rat SK-SD-specific HCA were qualitatively similar to SK-SD skin-sensitizing antibodies found in sera of human beings, namely, homocytotropic activity (48 h homologous PCA reaction), heat lability at 56 degrees C, and inactivation by reduction with 2-mercaptoethanol followed by alkylation with iodoacetamide. In neutralization tests, D-SK-SD, a low molecular hapten-like substance contained in SK-SD preparation, absorbed the antibody against ND-SK-SD antigen-specifically. The neutralizing activity mostly resided in fraction 1 of D-SK-SD separated through DEAE-Sephadex A-25 ion-exchange column, opposing the inhibitory activity of fraction 3 on lymphocyte transformation. The possible implication of these findings in the immunological treatment of streptococcal disorders is discussed.
The immediate skin reaction (IR) with streptokinase-streptodornase (SK-SD) was found with a high incidence in children with renal disease and was confirmed to be mediated by a specific IgE antibody against SK-SD by means of the radioallergosorbent test (RAST). This test revealed high radiocounts in children with renal disease who showed the IR and there was a close correlation between the RAST counts and the intensity of the IR. Furthermore, the RAST counts in the severe nephrotic syndrome belonging to corticosteroid dependent and resistant group were far higher than those in the corticosteroid sensitive group. From these results, a determination of the RAST counts against SK-SD seemed to help evaluate the severity of some nephrotic syndromes and the specific IgE antibody may be involved in the pathogenesis of the disease.
To study antibody (Ab) biosynthesis in rheumatoid arthritis (RA), the immunoglobulin (Ig)M anti-Fc, anti-Fab', and antistreptokinase-streptodornase (SKSD) produced by peripheral blood lymphocytes (PBL) were measured at intervals from 1 to 19 d in culture. PBL from 17 seropositive patients with active RA and 30 age-matched controls were evaluated. Within the first 24 h, PBL from six of eight patients released >30 ng IgM anti-Fc, even in the absence of pokeweed mitogen (PWM). This early release of Ab was blocked by cycloheximide. With or without PWM, PBL from normal donors did not release IgM anti-Fc until after 3-5 d in vitro. By day 9, unstimulated PBL from seven patients made > 100 ng IgM anti-Fc. Un-stimulated PBL from normals never made >95 ng of this Ab. When PWM was added, PBL from normal donors released as much IgM anti-Fc as was found in RA donor cultures. Paradoxically, addition of PWM to PBL of RA patients suppressed release of IgM anti-Fc in 4 of 17 cases to levels significantly below those found in unstimulated cultures of the same cells. Without PWM, PBL from RA donors frequently failed to make IgM anti-SKSD (P < 0.05 compared with normal donors' cells). With PWM, the quantities of IgM anti-SKSD released were comparable. Fluctuations in IgM anti-Fab' levels during the life-time of these cultures were sufficient to suggest that these Ab may be taken up in immune complexes. This hypothesis was verified by acidifying (pH 3.1) culture supernatants to which (125)I-Fab' had been added previously. The samples were then neutralized (pH 7.6) and 12% polyethylene glycol was added to separate free from antibody-bound (125)I-Fab'. This procedure increased the quantity of (125)I-Fab' precipitated by > 10-fold in some cases. These studies suggest that there are a variety of abnormalities in Ab biosynthesis in RA. These include spontaneous synthesis of comparatively large quantities of IgM anti-Fc, relatively suppressed release of IgM anti-SKSD, and a paradoxical reduction, in some cases, in the biosynthesis of IgM anti-Fc after addition of PWM.
We have evaluated whether neutralising anti-streptokinase IgG antibodies are produced following streptokinase-streptodornase therapy of leg ulcer patients. Serum anti-streptokinase IgG concentrations in 10 leg ulcer patients were determined before, and 1 week, 2 weeks, and 3 weeks following the treatment. We observed only a negligible increase in neutralizing anti-streptokinase IgG concentrations during the observation period, which was probably of no therapeutical significance.
Leukotaxis of circulating polymorphonuclear leukocytes (PMN's) appears to be a critical step in the generation of local inflammatory reactions. The present studies demonstrate a reversible, dose-dependent inhibition of PMN chemotaxis by streptokinase-streptodornase (SK-SD). This inhibition does not appear to depend upon the presence of lymphocytes or the release of chemokinetic humoral factors and phagocytic and bactericidal capacities of PMN's are unaffected by the doses of SK-SD employed.
Streptokinase-streptodornase (SK-SD) induced immediate and delayed skin test reactions in a high proportion of patients with disorders in which beta-streptococcal infection may be involved. The P-K test and absorption experiments with antigen or anti-IgE suggested that the immediate reaction was mediated by specific IgE antibody against SK-SD. Furthermore, the ratio of specific IgE antibody against SK-SD to total IgE was roughly calculated to be 20 percent. Finally, the RAST technique was applied to detect specific IgE antibody against SK-SD which showed high radiocounts bound to SK-SD-coupled particles in the sera of nephrotic patients who had a strong immediate reaction against SK-SD and a severe disease state.