Publications

In studies of the mechanism of lysis of red blood cells by washed streptococci with hemolytic activity (cell-bound hemolysin, CBH) no components released spontaneously by RBC or streptococci, or by interaction between these cells, could be found to induce the formation of soluble hemolysin by the streptococci. It was also found that separation of RBC from streptococci even by Millipore filter or a very thin layer of agar could prevent their hemolysis. By means of cellulose columns it was possible to separate RBC from streptococci after a short incubation. RBC thus separated from streptococci with which they had been incubated underwent hemolysis on subsequent incubation at 37°C. By varying the period of incubation prior to separation it was possible to demonstrate the transfer of increasing amounts of hemolysin from streptococci to RBC with increasing periods of incubation. A considerable part of this appeared to be at a constant rate. A theory is presented on the relationship between the streptococcal cell-bound hemolysin and the group of oxygen-stable streptococcal hemolysins, in terms of a transferable hemolytic moiety and binding sites for this moiety on the streptococcal cell, on various molecular species which can act as inducers of the oxygen-stable hemolysins, and on the RBC, with the affinity of the respective binding sites for the hemolytic moiety increasing in that order.

The relationship of the streptococcal hemolysin which is recognized on incubation of RBC with streptococcal cells (cell-bound hemolysin, CBH), to RNA hemolysin, a representative of oxygen-stable hemolysin (streptolysin S) has been studied. A number of similarities have been found in the conditions for optimal production of each of these hemolysins, a requirement for cysteine, Mg++, and glucose; maximal production by streptococci in the stationary phase; similar curves of pH-dependence. In both systems, the production of hemolysin was inhibited by certain antibiotics, by ultraviolet irradiation, and by sonic disruption and was absent in the same streptococcal mutant strain. The hemolytic activity of both systems was inhibited by lecithin, trypan blue, and papain. Similarities were also found in relative susceptibilities to the two hemolytic systems of erythrocytes of a number of animal species. These data support a suggestion advanced in an earlier study that a streptococcal hemolytic moiety, which can be induced by, and carried on, a number of diverse agents to comprise the group of oxygen-stable hemolysins, serves, in its original attachment to a component of the streptococcal cell, to produce the hemolytic effect recognized as the cell-bound hemolysin.

Cysteine and other sulfhydryl compounds markedly enhance the formation of streptolysin S by 4 strains of group A streptococci. In the presence of cysteine (0.001 M) 10,000-30,000 hemolytic units per ml were obtained from strain S 84 type 3 in comparison with 1,000-3,000 units in the absence of cysteine. The role of cysteine in formation of hemolysin is not clear but it probably acts as a donor of SH compounds essential for an unknown process, rather than as a reducing agent. The inhibition of hemolysin formation by 3 amino acid analogues phenylserine, phenylglycine and acetyl-pro-line is reversed to a large extent by sulfhydryl compounds. Thus 0.002 M of cysteine reverses to a large extent the inhibition of hemolysin formation caused by as much as 0.1 M of phenylserine.

The production of oxygen-stable hemolysin in growing and resting Group A streptococci has been induced by RNA, by detergents, and by mammalian blood serum proteins, in the presence of glucose, Mg(++), and cysteine. Of the serum proteins, albumin and alpha lipoprotein could act as inducers. In the case of both these serum proteins treatment with trypsin did not affect the capacity to induce hemolysin production, but removal of the bound lipids by alcohol-ether or chloroform-methanol destroyed this property. In comparisons of the conditions of production and of activity between the hemolysin produced by RNA on one hand and albumin and detergents on the other, some data indicated similarities among the hemolysins, and others, differences. The similarities included similar degrees of temperature dependence for production and equal degrees of inhibition by serum beta lipoprotein. Differences found among these hemolysins included differences between, the rate of production of the RNA hemolysin from that of albumin or detergent hemolysin by both resting and growing streptococci, and the failure of utilization of glucosamine as an energy source for the production of albumin hemolysin, in contrast with that of RNA hemolysin. The fact that the data have in some cases indicated similarities and in other cases differences among the hemolysins raises the question of whether these are different molecular species, or a single hemolysin synthesized by the streptococci via different pathways of metabolism, or complexes of a single hemolytic moiety with various molecular carriers.

The oxygen-stable streptococcal hemolysins, which can be induced by a number of diverse substances, have been studied. Differences among these hemolysins have been found in electrophoresis, chromatography, pH stability, and susceptibility to some organic solvents and to an enzyme, RNAase. These properties have in each case been found to characterize the inducing substances as well. In a number of instances it has been found possible to incubate one inducer with the hemolysin induced by another of these agents and then, after appropriate fractionation, to find hemolytic activity in the fraction containing the fresh inducer. These observations suggest that the oxygen-stable streptococcal hemolysins are constituted as carrier-hemolysin complexes, the carriers being the set of molecular species effective as inducers, and the prosthetic group being transferred from one carrier to another under appropriate conditions. After transfer of the hemolytic moiety from a hemolysin molecule which is susceptible to inactivation by a given agent or set of conditions to a carrier which is not itself significantly affected by this agent, the new, derived, hemolysin has been found not to be inactivated by the agent. The hemolysins of this group can thus be inactivated by enzymatic attack on the prosthetic group, or by hydrolysis or deformation of the postulated carrier molecule.

A heat stable factor (SF) in normal human serum which is cytotoxic to Landschutz ascites tumor cells has been described. The cytotoxic action requires the presence of human or rabbit complement but guinea pig complement is ineffective. The substance is found in the beta-globulin fraction and has characteristics similar to C′4 of human complement. It is selectively absorbed on human and mouse tumor cells and human placenta but not by normal human liver, kidney or heart. SF inhibits the action of heterologous antibody on ascites and HeLa cells. We are indebted to Dr. A. Rimon, Marcus Memorial Blood Bank Institute, Jaffa, for the Cohn fractions, and to Dr. Esther Tenenbaum of this Department for a generous supply of HeLa cells and Chang cells.

THE mechanism of cell destruction in allergic inflammation is not well understood. Two explanations for production of cell damage have, however, been proposed: (a) that the antigen-antibody reaction causes, in some non-specific manner, permeability changes in cells which result in the release of active mediators of inflammation (histamine, etc.) and that the latter are directly involved in the allergic process1; (b) that the antigen-antibody reaction activates proteolytic enzymes (plasmin, proteases) that injure the cells2–4.

STUDIES on the cytotoxic action of antibodies, plasmin and streptococcal haemolysins on Ehrlich ascites tumour cells (E-cells) have been recently described (GCinsburg, 1959; Ginsburg and Grossowicz, 1960; Ginsburg and Ram, 1960). It was found that the cytopathogenic changes induced in the tumour cells by streptococcal haemolysins were morphologically very similar to those caused by anti-Ehrlich ascites tumour rabbit serum (AE), the action of the latter is known to be complement dependent (Flax, 1956; Goldberg and Green, 1959). It is known that both the haemolytic as well as the cytopathogenic effect caused by streptococcal haemolysins can be inhibited by antihaemolytic agents such as cholesterol, lecithin, congo red and trypan blue (Van Heyningen, 1950; Bernheimer, 1954; Ginsburg, 1959; Ginsburg and Grossowicz, 1960). On the other hand, the cytotoxic and haemolytic effect of antibodies can be inhibited by agents known to destroy or inhibit complement action, chelating agents, plasmin, heparin etc. (Gorrill and Hobson, 1952; Pillemer, Ratnoff, Blum and Lepow, 1953; Mayer, 1 958; Ginsburg and Ram, 1960). The present study shows that the cytotoxic action of antibodies produced in the rabbit against Ehrlich ascites tumour cells can be inhibited by various phospholipids and some of their constituents as well as by agents known to inhibit streptococcal haemolysins. In addition the inhibition of immune haemolysis by phospholipids will be described.

SEVERAL studies were undertaken in order to reproduce in laboratory animals cardiac lesions which would resemble those appearing in human beings suffering from the sequelae of streptococcal infections (Schultz, 1936 ; Smith, Morgan and Mudd, 1940; Gross, Cooper and Philips, 1941; Robinson, 1947; Schultz, 1947; Murphy and Swift, 1949; Murphy, 1949; Clawson, 1950; Robinson, 1951; Murphy, 1952; Glaser, Thomas, Morse and Darnell, 1956). These studies describe the histopathological changes obtained in laboratory animals following inoculation by various routes of haemolytic streptococci, of their products and of a number of other micro-organisms. As to the mechanism involved in the production of cardiac lesions, two main theories have been proposed: (a)allergic phenomena,or production ofa uto- antibodies to the heart muscle (Rich and Gregory,1944; Rich,1946; Cavelty, 1947a and 1947b), (b) direct action of haemolytic streptococci and their toxins on the heart muscle (McLeod, 1953; Kellner and Robertson, 1954a and 1954b). Although both theories have been supported by some experimental evidence, the exact mechanism has not yet been clarified. This study describes the histo-pathological changes obtained in rabbits as a result of single intramyocardial injections of haemolytic streptococci and their cell-free extract, as well as of other bacterial species, both related and not related to haemolytic streptococci. The possible mechanism involved, and especially the role of trauma to the myocardium, in inducing cardiac lesions, is described.

IT has been recently shown that various strains of Streptococcus pyoqenes group A possess a cell-bound haemolysin (CBH) which can be released from the streptococcal cells into the surrounding medium by some surface active materials (Tween 40, Tween 80, Triton) by crystalline albumin, but not by sonlic energy (Ginsburg and Grossowicz, 1957 ; Ginsburg, 1958; Ginsburg and Grossowicz, 1959). This haemolytic factor was designated as streptolysin " D " (SLD) D signifying detergent. SLD is distinguished from streptolysin 0 (SLO) streptolysin S (SLS) (Todd, 1938) and from the intracellular haemolysin (IH) described by Schwab (Schwab, 1956) on the bases of different substrate requirements for formation, sensitivity to U.N. irradiation and to sonic energy (Ginsburg, 1958; Ginsburg and Gros- sowicz, unpublished). Besides haemolysing RBC of various animal species both cell-free and cell- bounid SLD, are also capable of injuring and killing various mamimalian cells in vitro (Ehrlich ascites tumour cells, fibroblasts, amnion cells, leucocytes) (Gins- buirg, 1958 ; Ginsburg and Grossowicz, 1959). The purpose of the present study is to show that Ehrlich ascites tuilour cells damaged by different streptococcal haemolysins may be disintegrated bv various proteolytic enzymes which by themselves are not lethal