126 Proc. roy. Soc. Med. Volume 70 1977 Supplement 3
A Prelimiinary Report on the Effects of D-penicillamine and Gold on Certain Proteolytic Enzyme Systems
A
by Dr J W Czekalowski, Dr J Frazer and Dr D A Hall (Departments of Microbiologyl Virology and Medicine, School of Medicine, University of Leeds, Leeds, LS2 9NL) This preliminary communication is based on the results of our work aimed at an explanation of the sequential development of the pathology of arthritic lesions and of the inflammatory process and immunological responses associated with them, all of which are dependent on tissue degradation. We postulate that the release of degradation products into the tissues - thus exposing them to the host's defence mechanism is mediated through the action of proteolytic enzyme systems originating either from endogenous or exogenous sources (Evanson 1970). A study of the effect of substances active in the treatmenit of rheumatoid arthritis on such enzyme systems could, therefore, provide a useful method for screening newly developed drugs for their potential antiarthritic activity. In pursuing another line of research we observed that enzyme systems isolated from Mycoplasma arthritidis are capable of digesting a partially degraded collagen, namely gelatin (Czekalowski et al. 1973). We have accomplished an appreciable purification of these enzymes (Woolcock et al. 1973, Frazer et al., in preparation), and while investigating their characteristics it has been observed that they are affected by
Fig 1 The effect ofgold (-A-A-) and penicillamine (s--) on the gelatinolytic activity of an enzyme system derived from Mycoplasma arthritidis. The results are expressed as percentages of the activity of the system in the absence of either drug. Substrate concentration 0.4 %
I'
1
*2
%Gelatin in substrate 5 6 .3 .4
Fig 2 The effect ofsubstrate concentration on the activity of the gelatinolytic enzyme system by (A) two concentrations ofgold (- Q-Q- 1.15 millimolar and -A-A- 4.6 millimolar) and (B) two concentrations of penicillamine (- 0- O- I millimolar and -A-A5 millimolar). Activity is expressed in arbitrary units related to the optical density at 600 nm of the colour formed with ninhydrin by the amino groups liberated by the enzyme. The two relatively straight lines (44 ) in both A andB represent the activity of the enzyme in the absence of either drug
certain antiarthritic drugs, thus suggesting that these enzymes may well prove to be a useful tool in the assessment of such substances. It will be shown in this paper that the kinetic behaviour of these enzymes is such as would appear to correlate directly with clinical observations on the effect of the same compounds (Eastmond 1976). The in vitro studies were carried out in experimental systems consisting of mycoplasmal enzymes, gelatin substrate and an investigated substance (Woolcock et al. 1973). In this study penicillamine (Distamine) and gold (in the form of sodium aurothiomalate: Myocrisin) were investigated either separately or together.
127
Penicillamine
Fig 3 Lineweaver/Burk plots (the reciprocal ofactivity, lIv, against the reciprocal ofsubstrate concentration, I/[S])for the datapresented in Fig 2. The mean valuefor the Michaelis-Menten constant, calculatedfrom the intercept of the extrapolated lines on the x-axis-Km=o0.976. A--A and * represent the effects of * lower and higher concentrations respectively ofgold and penicillamine (for details see legendfor Fig 2). *--* represents the activity of the enzyme in the absence of either drug
We observed that when either penicillamine or gold is added at concentrations between 1 and 10 millimolar to the experimental system (1.15 millimolar gold is equivalent to 0.5 mg sodium aurothiomalate/ml), the number of amino groups liberated through the digestion of the substrate (a measure of the enzyme activity) increases to between 200 % and 260 % of the value obtained in controls (Fig 1). The activation of the enzyme system by penicillamine is greater than that by gold, and an increase in concentration of either substance above 3-4 millimolar results in a reduction of the activation. The effect of both substances can be demonstrated very clearly when varying amounts of substrate are employed, and the activity is plotted against substrate concentration (Fig 2). Moreover, when the above findings are presented in the form of the reciprocal Lineweaver-Burk plots (Fig 3), an analysis confirms that both substances activate the enzyme. This is shown by the fact that the lines representing systems containing various levels of gold and penicillamine lie below and are progressively less steep than the lines for the control system. The fact that the extrapolated lines meet at a single point on the x-axis indicates that the activation occurs by the attachment of the reacting substance to a site on the enzyme molecule other than its 'active centre'. The failure of the plots for those systems which contain either penicillamine or gold to retain their linearity at substrate concentrations above 0.4%0 (1/s=2.5) indicates that when they modify the enzyme system, the overall reaction is increasingly inhibited by substrate at concentrations above this level, whereas in the control system it does not have this effect, at least up to concentrations of 0.6 % (I /s =about 1 .7) of the substrate. The simultaneous administration of both substances to the enzyme system has a very striking effect. When expressed in terms of the effect of
gold on the response of the enzyme to penicillamine (Fig 4) it can be shown that both high and low concentrations of gold lower the activation induced by penicillamine, so that the optimum degree of activation never rises above 800%, as opposed to the 150 % elevation of activity resulting from penicillamine alone. At concentrations of penicillamine above 2 to 3 millimolar the rate at which the activation is reduced with increasing amounts of penicillamine is three times greater than that which occurs at these levels of penicillamine alone. When the changes in activity Activity
(arbitrary values 6
-
il
1
millimolar penicillamine 2 4 3
5
Fig 4 The effect ofgold on the response of the gelatinolytic enzyme system to penicillamine: (a) penicillamine alone (4-); (b) penicillamine and 1.15 millimolar gold (-A -A-); (c) penicillamine and 4.6 millimolar gold (-----). Substrate concentration 0.4 %
128 Proc. roy. Soc. Med. Volume 70 1977 Supplement 3
Fig 5 The effect ofpenicillamine on the response ofthe gelatinolytic enzyme system to gold. (a) gold alone, (b) gold and 1 millimolar penicillamine, (c) gold and 2 millimolar penicillamine, (d) gold and 3 millimolar penicillamine, (e) gold and 4 millimolar penicillamine, (f) gold andS millimolar penicillamine
are plotted as the effect of penicillamine on the response of the enzyme to gold (Fig 5), an even more marked dependence on concentration is observable. Increasing concentrations of penicillamine progressively depress the enhanced response of the enzyme to gold until at penicillamine concentrations above 0.003 molar, the activation is more than 50 % repressed at all gold concentrations.
On the basis of these data it may be concluded that gold and penicillamine simultaneously present at concentrations above 2 millimolar each, induce an appreciable reduction in the activation of the enzyme system, whereas at lower concentrations, especially of penicillamine, the activity may be increased two to three fold. The results obtained from our in vitro research indicate clearly that the magnitude of activation of this model enzyme is related to the concentration of penicillamine and gold, and is also dependent on whether these substances are added individually or in combination. Figs 4 and 5, although recording the actual changes observed when gold and penicillamine are added simultaneously, are somewhat difficult to interpret, since the first points of the various lines are all different, referring as they do to activities in the presence of varying amounts of gold (Fig 4) and penicillamine (Fig 5). If these lines are replotted as changes in activity rather than absolute values (Fig 6), much of this confusion is removed. From Fig 6A it can be seen that the effect of gold at both the tested concentrations is to lower the amount by which penicillamine activates the enzyme at all concentrations of the latter, but especially at penicillamine concentrations above 2 millimolar. In these preliminary studies only a limited range of concentrations has been examined. It may well be that at higher concentrations of penicillamine, 4 millimol/l gold would be adequate to remove completely the activation due to the penicillamine. Fig 6B demonstrates quite clearly the progressive erosion by increasing concentrations of penicillamine of the activation induced by gold. Activa-
Fig 6 The comparative effects ofgold and penicillamine respectively, on the activation of the gelatinolytic enzyme by the other substance. In this pair ofgraphs the effects of the two drugs are expressed in terms of the change in activity resulting from the addition of increasing amounts of the drug as listed on the abscissa; the value for zero concentration of this drug at each recorded concentration of the other drug being taken as the origin for each individual curve. A: (a) penicillamine alone, (b) penicillamine plus 1.15 millimolar gold, (c) penicillamine plus 4.6 millimolar gold. B: (a) gold alone, (b) goldplus 1 millimolarpenicillamine, (c) gold plus 2 millimolar penicillamine, (d) goldplus 3 millimolarpenicillamine, (e) goldplus 4 millimolar penicillamine, (f) goldplus 5 millimolar penicillamine
Penicillamine tion continues when 1 or 2 millimolar penicillamine is present, but at higher levels of penicillamine the degree of activation is reduced progressively. At concentrations of 4 or 5 millimolar penicillamine the reduction in activation is considerable but is independent of the gold level. We accept, as a working hypothesis, that our model enzyme system isolated from Mycoplasma arthritidis (Czekalowski et al. 1973, Woolcock et al. 1973) is functionally closely related to endogenous enzymes which may be involved in degradation of components of the connective tissues, collagen in particular. It is suggestive to us, therefore, that our findings offer an explanation of the inconsistency of the in vivo response resulting from differing doses and modes of administration of the investigated substances. It has, for example, been observed that treatment with gold, the simultaneous administration of both drugs in high dosage, or of penicillamine after previous gold therapy (while gold is presumably still present in the tissue) can result in a remission of the arthritic symptoms, but may be complicated by the development of fibrotic lesions elsewhere in the body (Geddes & Brostoff 1976, Eastmond 1976). The suggestion that the degradation of collagen plays an important role in the development of the pathology of rheumatoid arthritis (Glynn 1969) has gained considerable support from the observation that specific proteolytic enzymes can be isolated from the tissues of affected joints (Evanson 1976). Activation of these enzymes will not only result in an enhanced destruction of the joint tissue, but also in the release into the surroundings of degradation products which will not only act as stimuli for the generalized immunological responses of the body, but will also initiate localized foci of inflammation. Similar, but not necessarily identical, enzyme systems with the power to degrade collagen or its partially denatured derivatives have been identified throughout the body where collagen fibres exist (Weiss 1976). Here their function is to counterbalance the synthetic activities of the fibroblasts, thus maintaining the required amounts of collagen in the various tissues. It is, therefore, apparent that similar collagenolytic enzymes may have opposite functions in different tissues: for instance, being pathogenic in the case of the arthritic joint, but normally physiological in other tissues. Activation or inhibition of enzymes in these two sites will, therefore, have opposite functional effects, and if we assume that gold and penicillamine react in vivo in a similar fashion to that which they have been shown to do in vitro, activation by low concentrations of either drug
129
will result in the exacerbation of the joint lesions. However, there will be a concomitant enhancement of the catabolic activity elsewhere which will prevent the deposition of collagen in locations in which it is not required in higher concentrations. Similarly the inhibition which results from high concentrations of both drugs, although repressing pathological changes in the joints, will simultaneously permit fibrosis elsewhere in the body owing to a disturbance in the balance between anabolic and catabolic activities in these tissues. It has been reported that the administration of penicillamine to subjects who have previously been receiving gold therapy, results not only in a remission of the arthritic symptoms, but also in the development of respiratory trouble following alveolar fibrosis (Eastmond 1976). It has been shown that gold will bind to connective tissue (Adam et al. 1964) and will remain in the body long after gold therapy has been terminated. The subsequent administration of penicillamine will release the gold from the tissues and the two drugs, then being present together, may inhibit collagenolytic systems in more than one centre in the body, with the result that complications such as those mentioned above occur. Concluding, we express the view that our mycoplasmal enzymes might prove to be of considerable potential value in the evaluation of antiarthritic substances and may help in the elucidation of the mechanisms involved in the development of certain pathological conditions of connective tissues. REFERENCES Adam M, Bartl P, Deyl Z & Rosmus J (1964) Experientia 20,203 Czekalowski J W, Hall D A & Woolcock P R (1973) Journal of General Microbiology 75, 125 Eastmond C J (1976) British MedicalJournal i, 1506 Evanson J M (1970) In: Chemistry and Molecular Biology of the Intracellular Matrix. Ed. E A Balazs. Academic Press, New York; p 1637 (1976) In: Methodology of Connective Tissue Research. Ed. D A Hall. Joynson-Bruvvers, Oxford; p 175 Geddes D M & Brostoff J (1976) British Medical Journal i, 1444 Glynn L E (1969) Annals of the Rheumatic Diseases Suppl. 28, 3 Weiss J B (1976) In: International Review of Connective Tissue Research. Ed. D A Hall & D S Jackson. Academic Press, New York; p 101 Woolcock P R, Czekalowski J W & Hall D A (1973) Journal of General Microbiology 78, 23
DISCUSSION
Dr F Wollheim (Malmo): Has penicillamine been tested alone, without gold in the system? Dr Czekalowski: No, we have not done that. We have carried out only the experiments of which I presented the results.
A Prelimiinary Report on the Effects of D-penicillamine and Gold on Certain Proteolytic Enzyme Systems
A
by Dr J W Czekalowski, Dr J Frazer and Dr D A Hall (Departments of Microbiologyl Virology and Medicine, School of Medicine, University of Leeds, Leeds, LS2 9NL) This preliminary communication is based on the results of our work aimed at an explanation of the sequential development of the pathology of arthritic lesions and of the inflammatory process and immunological responses associated with them, all of which are dependent on tissue degradation. We postulate that the release of degradation products into the tissues - thus exposing them to the host's defence mechanism is mediated through the action of proteolytic enzyme systems originating either from endogenous or exogenous sources (Evanson 1970). A study of the effect of substances active in the treatmenit of rheumatoid arthritis on such enzyme systems could, therefore, provide a useful method for screening newly developed drugs for their potential antiarthritic activity. In pursuing another line of research we observed that enzyme systems isolated from Mycoplasma arthritidis are capable of digesting a partially degraded collagen, namely gelatin (Czekalowski et al. 1973). We have accomplished an appreciable purification of these enzymes (Woolcock et al. 1973, Frazer et al., in preparation), and while investigating their characteristics it has been observed that they are affected by
Fig 1 The effect ofgold (-A-A-) and penicillamine (s--) on the gelatinolytic activity of an enzyme system derived from Mycoplasma arthritidis. The results are expressed as percentages of the activity of the system in the absence of either drug. Substrate concentration 0.4 %
I'
1
*2
%Gelatin in substrate 5 6 .3 .4
Fig 2 The effect ofsubstrate concentration on the activity of the gelatinolytic enzyme system by (A) two concentrations ofgold (- Q-Q- 1.15 millimolar and -A-A- 4.6 millimolar) and (B) two concentrations of penicillamine (- 0- O- I millimolar and -A-A5 millimolar). Activity is expressed in arbitrary units related to the optical density at 600 nm of the colour formed with ninhydrin by the amino groups liberated by the enzyme. The two relatively straight lines (44 ) in both A andB represent the activity of the enzyme in the absence of either drug
certain antiarthritic drugs, thus suggesting that these enzymes may well prove to be a useful tool in the assessment of such substances. It will be shown in this paper that the kinetic behaviour of these enzymes is such as would appear to correlate directly with clinical observations on the effect of the same compounds (Eastmond 1976). The in vitro studies were carried out in experimental systems consisting of mycoplasmal enzymes, gelatin substrate and an investigated substance (Woolcock et al. 1973). In this study penicillamine (Distamine) and gold (in the form of sodium aurothiomalate: Myocrisin) were investigated either separately or together.
127
Penicillamine
Fig 3 Lineweaver/Burk plots (the reciprocal ofactivity, lIv, against the reciprocal ofsubstrate concentration, I/[S])for the datapresented in Fig 2. The mean valuefor the Michaelis-Menten constant, calculatedfrom the intercept of the extrapolated lines on the x-axis-Km=o0.976. A--A and * represent the effects of * lower and higher concentrations respectively ofgold and penicillamine (for details see legendfor Fig 2). *--* represents the activity of the enzyme in the absence of either drug
We observed that when either penicillamine or gold is added at concentrations between 1 and 10 millimolar to the experimental system (1.15 millimolar gold is equivalent to 0.5 mg sodium aurothiomalate/ml), the number of amino groups liberated through the digestion of the substrate (a measure of the enzyme activity) increases to between 200 % and 260 % of the value obtained in controls (Fig 1). The activation of the enzyme system by penicillamine is greater than that by gold, and an increase in concentration of either substance above 3-4 millimolar results in a reduction of the activation. The effect of both substances can be demonstrated very clearly when varying amounts of substrate are employed, and the activity is plotted against substrate concentration (Fig 2). Moreover, when the above findings are presented in the form of the reciprocal Lineweaver-Burk plots (Fig 3), an analysis confirms that both substances activate the enzyme. This is shown by the fact that the lines representing systems containing various levels of gold and penicillamine lie below and are progressively less steep than the lines for the control system. The fact that the extrapolated lines meet at a single point on the x-axis indicates that the activation occurs by the attachment of the reacting substance to a site on the enzyme molecule other than its 'active centre'. The failure of the plots for those systems which contain either penicillamine or gold to retain their linearity at substrate concentrations above 0.4%0 (1/s=2.5) indicates that when they modify the enzyme system, the overall reaction is increasingly inhibited by substrate at concentrations above this level, whereas in the control system it does not have this effect, at least up to concentrations of 0.6 % (I /s =about 1 .7) of the substrate. The simultaneous administration of both substances to the enzyme system has a very striking effect. When expressed in terms of the effect of
gold on the response of the enzyme to penicillamine (Fig 4) it can be shown that both high and low concentrations of gold lower the activation induced by penicillamine, so that the optimum degree of activation never rises above 800%, as opposed to the 150 % elevation of activity resulting from penicillamine alone. At concentrations of penicillamine above 2 to 3 millimolar the rate at which the activation is reduced with increasing amounts of penicillamine is three times greater than that which occurs at these levels of penicillamine alone. When the changes in activity Activity
(arbitrary values 6
-
il
1
millimolar penicillamine 2 4 3
5
Fig 4 The effect ofgold on the response of the gelatinolytic enzyme system to penicillamine: (a) penicillamine alone (4-); (b) penicillamine and 1.15 millimolar gold (-A -A-); (c) penicillamine and 4.6 millimolar gold (-----). Substrate concentration 0.4 %
128 Proc. roy. Soc. Med. Volume 70 1977 Supplement 3
Fig 5 The effect ofpenicillamine on the response ofthe gelatinolytic enzyme system to gold. (a) gold alone, (b) gold and 1 millimolar penicillamine, (c) gold and 2 millimolar penicillamine, (d) gold and 3 millimolar penicillamine, (e) gold and 4 millimolar penicillamine, (f) gold andS millimolar penicillamine
are plotted as the effect of penicillamine on the response of the enzyme to gold (Fig 5), an even more marked dependence on concentration is observable. Increasing concentrations of penicillamine progressively depress the enhanced response of the enzyme to gold until at penicillamine concentrations above 0.003 molar, the activation is more than 50 % repressed at all gold concentrations.
On the basis of these data it may be concluded that gold and penicillamine simultaneously present at concentrations above 2 millimolar each, induce an appreciable reduction in the activation of the enzyme system, whereas at lower concentrations, especially of penicillamine, the activity may be increased two to three fold. The results obtained from our in vitro research indicate clearly that the magnitude of activation of this model enzyme is related to the concentration of penicillamine and gold, and is also dependent on whether these substances are added individually or in combination. Figs 4 and 5, although recording the actual changes observed when gold and penicillamine are added simultaneously, are somewhat difficult to interpret, since the first points of the various lines are all different, referring as they do to activities in the presence of varying amounts of gold (Fig 4) and penicillamine (Fig 5). If these lines are replotted as changes in activity rather than absolute values (Fig 6), much of this confusion is removed. From Fig 6A it can be seen that the effect of gold at both the tested concentrations is to lower the amount by which penicillamine activates the enzyme at all concentrations of the latter, but especially at penicillamine concentrations above 2 millimolar. In these preliminary studies only a limited range of concentrations has been examined. It may well be that at higher concentrations of penicillamine, 4 millimol/l gold would be adequate to remove completely the activation due to the penicillamine. Fig 6B demonstrates quite clearly the progressive erosion by increasing concentrations of penicillamine of the activation induced by gold. Activa-
Fig 6 The comparative effects ofgold and penicillamine respectively, on the activation of the gelatinolytic enzyme by the other substance. In this pair ofgraphs the effects of the two drugs are expressed in terms of the change in activity resulting from the addition of increasing amounts of the drug as listed on the abscissa; the value for zero concentration of this drug at each recorded concentration of the other drug being taken as the origin for each individual curve. A: (a) penicillamine alone, (b) penicillamine plus 1.15 millimolar gold, (c) penicillamine plus 4.6 millimolar gold. B: (a) gold alone, (b) goldplus 1 millimolarpenicillamine, (c) gold plus 2 millimolar penicillamine, (d) goldplus 3 millimolarpenicillamine, (e) goldplus 4 millimolar penicillamine, (f) goldplus 5 millimolar penicillamine
Penicillamine tion continues when 1 or 2 millimolar penicillamine is present, but at higher levels of penicillamine the degree of activation is reduced progressively. At concentrations of 4 or 5 millimolar penicillamine the reduction in activation is considerable but is independent of the gold level. We accept, as a working hypothesis, that our model enzyme system isolated from Mycoplasma arthritidis (Czekalowski et al. 1973, Woolcock et al. 1973) is functionally closely related to endogenous enzymes which may be involved in degradation of components of the connective tissues, collagen in particular. It is suggestive to us, therefore, that our findings offer an explanation of the inconsistency of the in vivo response resulting from differing doses and modes of administration of the investigated substances. It has, for example, been observed that treatment with gold, the simultaneous administration of both drugs in high dosage, or of penicillamine after previous gold therapy (while gold is presumably still present in the tissue) can result in a remission of the arthritic symptoms, but may be complicated by the development of fibrotic lesions elsewhere in the body (Geddes & Brostoff 1976, Eastmond 1976). The suggestion that the degradation of collagen plays an important role in the development of the pathology of rheumatoid arthritis (Glynn 1969) has gained considerable support from the observation that specific proteolytic enzymes can be isolated from the tissues of affected joints (Evanson 1976). Activation of these enzymes will not only result in an enhanced destruction of the joint tissue, but also in the release into the surroundings of degradation products which will not only act as stimuli for the generalized immunological responses of the body, but will also initiate localized foci of inflammation. Similar, but not necessarily identical, enzyme systems with the power to degrade collagen or its partially denatured derivatives have been identified throughout the body where collagen fibres exist (Weiss 1976). Here their function is to counterbalance the synthetic activities of the fibroblasts, thus maintaining the required amounts of collagen in the various tissues. It is, therefore, apparent that similar collagenolytic enzymes may have opposite functions in different tissues: for instance, being pathogenic in the case of the arthritic joint, but normally physiological in other tissues. Activation or inhibition of enzymes in these two sites will, therefore, have opposite functional effects, and if we assume that gold and penicillamine react in vivo in a similar fashion to that which they have been shown to do in vitro, activation by low concentrations of either drug
129
will result in the exacerbation of the joint lesions. However, there will be a concomitant enhancement of the catabolic activity elsewhere which will prevent the deposition of collagen in locations in which it is not required in higher concentrations. Similarly the inhibition which results from high concentrations of both drugs, although repressing pathological changes in the joints, will simultaneously permit fibrosis elsewhere in the body owing to a disturbance in the balance between anabolic and catabolic activities in these tissues. It has been reported that the administration of penicillamine to subjects who have previously been receiving gold therapy, results not only in a remission of the arthritic symptoms, but also in the development of respiratory trouble following alveolar fibrosis (Eastmond 1976). It has been shown that gold will bind to connective tissue (Adam et al. 1964) and will remain in the body long after gold therapy has been terminated. The subsequent administration of penicillamine will release the gold from the tissues and the two drugs, then being present together, may inhibit collagenolytic systems in more than one centre in the body, with the result that complications such as those mentioned above occur. Concluding, we express the view that our mycoplasmal enzymes might prove to be of considerable potential value in the evaluation of antiarthritic substances and may help in the elucidation of the mechanisms involved in the development of certain pathological conditions of connective tissues. REFERENCES Adam M, Bartl P, Deyl Z & Rosmus J (1964) Experientia 20,203 Czekalowski J W, Hall D A & Woolcock P R (1973) Journal of General Microbiology 75, 125 Eastmond C J (1976) British MedicalJournal i, 1506 Evanson J M (1970) In: Chemistry and Molecular Biology of the Intracellular Matrix. Ed. E A Balazs. Academic Press, New York; p 1637 (1976) In: Methodology of Connective Tissue Research. Ed. D A Hall. Joynson-Bruvvers, Oxford; p 175 Geddes D M & Brostoff J (1976) British Medical Journal i, 1444 Glynn L E (1969) Annals of the Rheumatic Diseases Suppl. 28, 3 Weiss J B (1976) In: International Review of Connective Tissue Research. Ed. D A Hall & D S Jackson. Academic Press, New York; p 101 Woolcock P R, Czekalowski J W & Hall D A (1973) Journal of General Microbiology 78, 23
DISCUSSION
Dr F Wollheim (Malmo): Has penicillamine been tested alone, without gold in the system? Dr Czekalowski: No, we have not done that. We have carried out only the experiments of which I presented the results.
