The in vitro efficacy of varidase versus streptokinase or urokinase for liquefying thick purulent exudative material from loculated empyema.

Patients with loculated parapneumonic effusion or empyema are sometimes treated with streptokinase or urokinase in an attempt to facilitate pleural fluid drainage by liquefying the pleural exudate and destroying the fibrin membranes producing the loculation. This study evaluated the effectiveness of streptokinase, urokinase, and Varidase (the combination of streptokinase and streptodornase) in liquefying gummy, purulent, exudative material from loculated empyemas. An empyema was created by injecting 10(8) Pasteurella multocida bacteria into the pleural space of New Zealand white rabbits. Twenty specimens, each containing 0.5 g of purulent material obtained 5 days after empyema induction, were placed in test tubes. Streptokinase (15,000 IU), urokinase (10,000 IU), Varidase (4,000-15,000 IU streptodornase + 15,000 IU streptokinase) or saline was added to five sets of four test tubes each. The amount of nonliquefied material that remained after incubation with the fibrinolytic agents was quantitated. Over the 6-h incubation period, the amount of nonliquefied material decreased from 0.5 g to 0.02 g in the Varidase group but never decreased to less than 0.4 g in any of the other three treatment groups. Liquefaction of thick pleural exudates from rabbits with empyema can be achieved with Varidase but not with streptokinase or urokinase.

Effect of Varidase (streptokinase) on biofilm formed by Staphylococcus aureus.

Staphylococcus aureus forms a fibrin-rich biofilm in the presence of plasma which is highly resistant to attack by the human immune system and to chemotherapy. Varidase, composed mainly of streptokinase, is used for hydrolyzing clots. In this study, we attempted to destroy the biofilm of S. aureus with Varidase and to apply this drug in the treatment of staphylococcal infections. Four clinical isolates were used in the experiments. These organisms formed a several-millimeter-thick biofilm on type IV collagen coated coverslips in trypticase soy broth containing 50% human plasma. The biofilm was composed of bacterial cell which adhered to fibrillar fibers and of sediment derived from plasma. 10,000 U/ml of Varidase, the dose which is used clinically, removed the sediment and reduced the number of live bacteria in biofilms to less than 20% of control. 200 U/ml of Varidase was also effective against biofilms of the organisms. An equal combination of Varidase and ofloxacin had an additive effect on the bacteria. The results of this study demonstrate that Varidase is highly effective in destroying biofilms of S. aureus in vitro and suggest that this drug would be useful for treating staphylococcal infections.

Varidase: the science behind the medicament.

Varidase is used throughout the world for the topical treatment of purulent and suppurating wounds. Its efficacy is centred on two enzymes, streptokinase and streptodornase. However, these represent only a small proportion of the bulked solid. This article gives an overview of the preparation and mode of action of Varidase, as well as showing some of the research and development that has gone into improving the assessment of its quality and composition.

Bee venom immunotherapy induces a shift in cytokine responses from a TH-2 to a TH-1 dominant pattern: comparison of rush and conventional immunotherapy.

BACKGROUND: The mechanism of immunotherapy is unclear. Allergic disease is known to involve enhanced TH-2 cytokine responses to allergen. OBJECTIVE: In order to investigate the mechanisms of immunotherapy, we have examined changes in cytokine secretion before (13 patients) and during (nine patients) both rush and conventional venom immunotherapy (VIT) in bee venom allergic patients. METHODS: Peripheral blood mononuclear cells were stimulated in vitro with bee venom, non-specific antigen or mitogen and secretion of IL-4 (TH-2) and IFN gamma (TH-1) over the culture period measured. RESULTS: Untreated patients had TH-2 responses to venom and TH-1 responses to antigen and strong proliferative responses to venom. Controls showed no response (proliferation or cytokines) to venom and the normal TH-1 response to antigen. VIT resulted in marked changes in cytokine secretion to venom, with reduction of the abnormal TH-2 response and induction of a TH-1 response. The pattern differed in rush and conventional VIT. One day after rush VIT there was a significant fall in IL-4 secretion (P < 0.01), which rose by 3 weeks then declined. In conventional VIT there was a gradual reduction of IL-4 production significant after 2 months and undetectable by 6 months. IFN gamma secretion was induced by VIT. Proliferative responses mirrored the IL-4 changes. One day after rush VIT there was a loss of T cells, monocytes and NK cells from peripheral blood. CONCLUSION: This study shows that immunotherapy shifted cytokine responses to allergen from a TH-2 to a TH-1 dominant pattern, suggesting direct effects on T cells. How these cytokine changes relate to clinical desensitization is not clear. In the longer term they would result in an isotype switch from IgE to IgG. Early changes in cytokine or chemokine production might downregulate mast cell or basophil reactivity and explain the rapid desensitization in rush VIT.

Effect of extracellular products of Streptococcus on macrophage Fc receptors for IgG.

The effect of the extracellular products of Streptococcus (EPS) on the receptors for Fc of IgG of dog alveolar macrophage (AM) was evaluated by the rosette test. The AM controls produced 80 per cent rosettes; those treated with EPS, a reduced number of rosettes, which was directly related with the concentration of EPS. The adhesion of EPS treated AM to glass was less than that of the AM controls. Incubation of AM with streptokinase-streptodornase did not modify the amount of rosettes. The data support the possibility that streptolysin-O is the substance which modifies, or destroys the Fc receptors, or the integrity of the cell membrane. Ouabain did not alter the capacity of the dog alveolar macrophages to form rosettes, suggesting that cellular energy is not required for the interaction between the Fc receptor and the IgG.

Enzymatic wound cleaning and absorbable sutures. An experimental study on Varidase and Dexon sutures.

The effect of an enzymatic preparation for wound cleaning (Varidase) on the mechanical properties of absorbable sutures (Dexon) was studied in vitro and in vivo in a rat model. In vitro the sutures demonstrated a significant decrease in strength (breaking strength and energy absorption) and extensibility after 12 days of incubation in saline. Incubation in Varidase, however, further decreased the mechanical properties significantly. The stiffness of the sutures was independent of treatment and time. In vivo changes of mechanical properties of the sutures resemble those of the in vitro study, except for a decrease in stiffness of the sutures. The sutures in a primary closed wound had the same strength (energy absorption) as the sutures of an open wound treated by saline, while the sutures of an open wound treated by Varidase tended to have a decreased strength (p = 0.08). This study supports the hypothesis that an enzymatic process may be involved in the degradation of Dexon. The continuous use of Varidase in Dexon-sutured wounds for a period longer than a few days is, therefore, questioned.

The skin inflammatory response of the badger (Meles meles).

Twenty-five badgers, captured in an area where they had been implicated in outbreaks of bovine tuberculosis, received intradermal inoculations of control medium, 150 micrograms phytohaemagglutinin (PHA), 40 units streptokinase/10 units streptodornase (SK/SD), 200 micrograms purified protein derivative of Mycobacterium bovis (PPD), Freund's incomplete adjuvant (IFA), and Freund's complete adjuvant (CFA), each in 0.1 ml of inoculum. The reactions were assessed by skinfold thickness and skin histology 30 min--7 days after inoculation. Control medium caused slight cellular reaction, mostly polymorphonuclear leucocytes (PMNs), but no significant increase in skinfold thickness. SK/SD provoked no reaction. PHA stimulated a marked increase in skinfold thickness; the cellular reaction was predominantly PMNs, with some macrophages occurring after several days. IFA and CFA promoted a long-lasting increase in skinfold thickness, and a mixed histological picture of PMNs and macrophages; later in the response, especially to CFA, giant cells and some lymphocytes occurred. PPD stimulated a small increase in skinfold thickness with a timing (2-3 days) consistent with delayed hypersensitivity (DTH); there was, however, no erythema or palpable oedema or induration. The histology was an initial multifocal reaction of PMNs with a later phase of lymphocytes and macrophages with some granuloma formation. Other cell types (eosinophils, basophils) were seen in varying proportions in all reaction sites. M. bovis was isolated from four badgers; the cellular reaction to PPD was stronger than in uninfected animals, but other aspects of the skin response were unaffected. This study shows the capacity of badgers for strong inflammatory responses, and is the first report of a DTH response to PPD in this species.

Histamine releasing lymphokines: a new approach to some old problems.

Studies carried out in recent years reveal that some endogenous substances can degranulate mast cells and basophils. Lymphokines produced in vitro by T lymphocytes in presence of an antigen or a mitogen, which are capable of provoking histamine liberation in basophils and mast cells have been described. Lastly the mechanisms for histamine release not mediated by IgE in bronchial asthma and which can be mediated by these factors, is discussed.

A basophil-activating factor from human T lymphocytes.

Human T lymphocytes stimulated with phytohaemagglutinin (PHA) or streptokinase-streptodornase (SK-SD) generate an activity which elicits non-cytotoxic histamine release from human basophils. Filtration of the T lymphocyte-derived activity on columns of Sephadex G-100 and Fractogel 55F sequentially revealed one predominant basophil-activating factor of mol. wt. 70,000-90,000, that was designated BAF-T. BAF-T was composed of two acidic proteins of approximate pI 4.4 and 5.2-5.5, as assessed by isoelectric focusing. The distinction of BAF-T from IgE was confirmed by the failure of BAF-T to bind to an anti-IgE affinity column and the capacity of BAF-T to release histamine maximally from basophils desensitized to IgE-dependent stimuli. The inability of BAF-T to release histamine from human lung mast cells and dog cutaneous mastocytoma cells suggests target cell specificity. The source and activity of BAF-T are consistent with a specific contribution of this mediator to human cellular immune and hypersensitivity responses involving T lymphocytes and basophils.

The skin test antigen stimulated killer (STAK) cell mediating NK like CMC is OKM1 positive and OKT3 negative.

Recently we demonstrated that candida antigen stimulated natural killer cell like cell-mediated cytolysis (NK like CMC) in peripheral blood mononuclear cells (PBMNC) isolated from normal individuals (Tartof et al., 1980). Utilizing monoclonal antibodies directed against human mononuclear cell subpopulations in conjunction with a fluorescence activated cell sorter (FACS) we determined that, similar to the previously described NK cell, the skin test antigen stimulated killer (STAK) cell is a larger OKM1 positive, OKT3 negative cell. We obtained similar results using two different skin test antigens. Thus, stimulation of NK like CMC in PBMNC by skin test antigens probably represents activation of NK or NK like cells.

Human alveolar macrophage support of lymphocyte responses to mitogens and antigens. Analysis and comparison with autologous peripheral-blood-derived monocytes and macrophages.

Observations from animal studies and investigations using human peripheral-blood-derived macrophages may not be representative of human alveolar macrophage functions. Thus, we extensively examined and compared accessory cell functions of autologous human alveolar macrophages and peripheral-blood-derived monocytes and macrophages obtained from healthy, nonsmoking volunteer subjects. The alveolar and peripheral-blood-derived cells differed in ability to enhance mitogen- and antigen-stimulated transformation responses of purified autologous lymphocytes. Peripheral-blood-derived cells supported greater lymphocyte responses to optimal concentrations of several mitogens, but alveolar macrophages supported much greater responses to suboptimal concentrations of the mitogen phytohemagglutinin. Peripheral-blood-derived macrophages supported, but alveolar macrophages did not support, lymphocyte responses to streptococcal and influenza virus antigens. Such relative accessory cell functions of the different macrophages and monocytes were not due to different kinetics of response, nor were they due to differences in the proportion of macrophages required to support the lymphocyte responses. These data indicate that human alveolar macrophages have functional characteristics different from autologous peripheral-blood-derived monocytes and macrophages. Such differences may be important in both pulmonary immune homeostasis and in the pathogenesis of pulmonary disease.

Non-specific suppression of antigen-induced lymphocyte blastogenesis in Onchocerca volvulus infection in man.

Lymphocyte blastogenic responses to O. volvulus antigen (Oncho Ag), SKSD, and the mitogen PHA were tested in three groups of persons: light to moderately infected persons (INF); previously exposed but uninfected persons (EXP) and normal controls (NC). The exposed group showed significant responsiveness to Oncho Ag (delta ct/min = 6,002 +/- 1,375), while the infected (delta ct/min = 943 +/- 418) and normal control (delta ct/min = 428 +/- 418) groups did not. The mean blastogenic response to SKSD were EXP, 8,644 +/- 5,249; NC 6,039 +/- 2,880; INF, 2,619 +/- 1,012. The reduced reactivity in the INF group to Oncho Ag showed a significant correlation with reactivity to SKSD (P less than 0.05). To elucidate the mechanism of hyporesponsiveness in the infected group rigorous adherent cell depletion, by adherence to plastic followed by a nylon wool column, was utilized. When 20% plastic adherent cells were added back to the T cells prepared in this fashion, the mean blastogenic response to SKSD was significantly augmented (P less than 0.01). In contrast, the responsiveness to Oncho Ag was not significantly altered. The addition of indomethacin (1 microgram/ml) or autologous plasma had no significant effect on reactivity to either SKSD or Oncho Ag. There were no significant differences in the mean reactivity of the three groups to PHA-M (delta ct/min EXP 78,514 +/- 12,564; INF 62,393 +/- 14,447; NC 61,423 +/- 4,465). These results suggest that O. volvulus infection is associated with decreased lymphocyte reactivity to both parasite related and unrelated antigens, and imply that the mechanism for the two types of hyporesponsiveness may be distinct. While a weakly adherent suppressor cell may account for non-specific hyporesponsiveness, the mechanism of parasite specific decreased reactivity remains unknown.

Prediction of sepsis in the multitraumatic patient by assays of lymphocyte responsiveness.

In vitro lymphocyte response to antigens, mitogens and in mixed lymphocyte culture were studied at intervals after injury in 31 patients with extensive trauma. Mean responses were significantly depressed up to 15 to 20 days. Responses were lower and the duration of suppression longer in those patients who become infected, and the suppression of response preceded the onset of infection. Extremely low responses were found in three patients who later died. This in vitro system is suitable for the serial monitoring of patients, as it reflects the extent of injury, infectious sequelae and prognosis. Its results are quantifiable and avoid the problems associated with repeated skin testing.

Collagens act as ligands to stimulate human monocytes to produce mononuclear cell factor (MCF) and prostaglandins (PGE2).

Blood mononuclear cells from patients with rheumatoid arthritis produce the lymphokine, leukocyte inhibitory factor (LIF) in response to collagens in vitro, and blood monocytes release prostaglandins (PGE2) and a factor, mononuclear cell factor (MCF) which stimulates collagenase and PGE2 production by cultured synovial cells. We therefore examined the effect of collagens on the production of PGE2 and MCF. Blood mononuclear cells from 6 patients with rheumatoid arthritis and 6 normal subjects were cultured in native human types I, II, or III collagen-coated tubes, or with streptokinase-streptodornase (SK-SD), and the supernatant media derived from these cultures analyzed for the presence of MCF, PGE2, and LIF. Types II and III collagens, as well as SK-SD, markedly stimulated MCF production by the cells from all 12 subjects (MCF activity, expressed as a mean stimulation index (SI) +/- SEM, was 43 +/- 12 for type II, 33 +/- 7 for type III, and 37 +/- 23 for SK-SD). Type I collagen was less stimulatory (mean SI 10 +/- 7). Cells from the patients with rheumatoid arthritis, but not the normal subjects, produced LIF in response to types II or III collagens but not to type I collagen. PGE2 production by blood mononuclear cells paralleled that of MCF, although abrogation of PGE2 release with indomethacin did not reduce MCF production. alpha chains purified from denatured collagens also stimulated MCF production. Using cells from patients with rheumatoid arthritis, type II collagen stimulated production of all three factors in the presence of polymyxin B or fibronectin-depleted serum, suggesting, respectively, that neither endotoxin nor fibronectin were responsible for their generation. Monocytes, purified from normal blood by an adherence technique, but not lymphocytes depleted of monocytes, released MCF and PGE2 when cultured with type II collagen. These results demonstrate that collagens can act as ligands to stimulate monocytes, as well as antigens to stimulate sensitized lymphocytes, to produce soluble factors that may contribute to the destruction of connective tissue.

Modulation of human lymphocyte function by C3a and C3a(70-77).

Human C3a and the synthetic octapeptide C3a (70-77), which retains the activities of an anaphylatoxin, inhibit in a concentration-dependent manner the generation of leukocyte inhibitory factor (LIF) activity by human mononuclear leukocytes and T lymphocytes cultured with the mitogens phytohemagglutinin (PHA) or concanavalin A (Con A) or the antigen streptokinase-streptodornase (SK-SD). The generation of LIF activity was inhibited by 50% by 10(-8) M C3a or C3a(70-77) with PHA or Con A as the stimulus, whereas a more than 10-fold higher concentration of C3a(70-77) than C3a was required to achieve the same level of suppression with SK-SD as the stimulus. Similar concentrations of C3a(70-77) inhibited to the same extent the migration of T lymphocytes stimulated by alpha-thioglycerol of Con A. Neither C3a nor C3a(70-77) altered significantly the uptake of [3H]thymidine by human mononuclear cells exposed to PHA, Con A, or SK-SD. The capacity of C3a(70-77)-Sepharose,m but not Sepharose alone, to adsorb or inactivate mononuclear leukocytes required for the generation of LIF activity established a direct interaction. Analysis of the lymphocytes in the effluent from C3a(70-77)-Sepharose columns, using monoclonal antibodies to surface antigens, showed a selective depletion of the helper/inducer population of lymphocytes. C3a might represent an important mediator of the functionally selective regulation of human T lymphocyte activities by the complement system.

Soluble suppressor supernatants elaborated by concanavalin A-activated human mononuclear cells. I. Characterization of a soluble suppressor T cell proliferation.

When activated with the mitogenic lectin, concanavalin A, a subset of human peripheral blood mononuclear cells differentiate into potent suppressor cells that negatively modulate both cellular and humoral immune reactions. In addition to inhibitory cell-cell interactions, these regulatory cells elaborate a soluble immune suppressor supernatant (SISS) containing at lest 2 distinct suppressor factors. One of these factors, SISS-T, inhibits mitogen- and antigen-stimulated T cell proliferation, whereas the other, SISS-B, inhibits B cell immunoglobulin production. Characteristics of the latter inhibitor are reported in the companion paper. Properties of the soluble suppressor of T cell proliferation (SISS-T) include: 1) a m.w. of 30 to 45,000, 2) inhibition by a noncytotoxic mechanism, 3) instability at 56 degrees C, 4) loss of activity in the presence of the monosaccharide N-acetyl-D-glucosamine and retention on N-acetyl-D-glucosamine affinity columns, 5) binding to the same surface glycoprotein receptors recognized by the lectins wheat germ agglutinin and Agaricus bisporus lectin, which produce similar inhibition of mitogen- or antigen-induced lymphocyte proliferation, 6) elaboration by cells irradiated with 500 and 2000 R but not 6000 R, 7) a minimum requirement for 24 hr of lectin stimulation for production, and 8) elaboration by adherent cells or alternatively cellular collaboration requiring the participation of adherent cells. These data indicate that human suppressor cells are capable of modulating T cell function via the production of a soluble saccharide-specific factor(s) that interacts with defined surface glycoprotein or glycolipid receptors. These same receptors recognized by the suppressive endogenous lectin are also activated by selected exogenous nonmitogenic lectins that produce similar inhibition of T cell metabolism.

Age dependence of subpopulations and functions of human peripheral lymphocytes.

The eventual dependency of a series of immune parameters on age has been studied in a material of 288 normal persons, divided into 2 sexes and with 16 persons in each 5-year age interval between 5 and 95 years, using a cryobiological freezing and storage system for lymphocytes with subsequent testing in large series. Lymphocyte transformation responses were studied after stimulation with a series of mitogens and specific antigens (PHA, PWM, PPD, Con A, SA, EC, CA and SK/SD), as well as with allogene cells in mixed lymphocyte culture. Values were found to decrease with increasing age. No age-dependency could be demonstrated for T and B lymphocytes using rosette formation tests. There was no difference between the two sexes. Thus, increasing age does not change the distribution between T and B lymphocytes, but does lead to a decrease in the function of the cell-mediated response.