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Macrophages and cathepsin A activity in multiple sclerosis brain
227
Journal of the neuroloyical Sciences, 1974, 21 : 227 231 ~ Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
Short Report
Macrophages and Cathepsin A Activity in Multiple Sclerosis Brain
INTRODUCTION
Cathepsin A catalyzes the release of tyrosine from benzyloxycarbonyl-Glu-Tyr, acts optimally at pH 5.25 and does not require thiols for activity. The enzyme appears to be a carboxypeptidase with limited endopeptidase activity (Bowen and Davison 1973). Studies in several laboratories, including our own (Cuzner and Davison, 1973; Bowen, Smith and Davison 1973) demonstrate that acid hydrolases, in particular those acting on proteins and peptides, play a role in degenerative brain diseases. It is of special interest that the finding of increased cathepsin D activity in the rim of active Multiple Sclerosis (MS) plaques, has been implicated in the immunopathological process seen in MS (Einstein, Csejtey, Dalal, Adams, Bayliss and Hallpike 1972). Although cerebral white matter contains cathepsin A, the concentration in spleen and hence possibly in macrophages, is very much higher. We have, therefore, proposed that in inflammatory conditions of the brain measurement of cathepsin A may serve as a useful index of cellular infiltration by leucocytes (Bowen and Davison 1973). One aim of the present study, therefore, was to establish whether the concentration of cathepsin A is changed in MS brain since as a result of perivascular cuffing both monocytes and lymphocytes are often associated with MS plaques. However, lymphocytes do not appear to account for the increase in hydrolase content seen in canine distemper, a demyelinating disease closely related to MS (McMartin, Koestner and Long 1972). Consequently, monocytic phagocytes rather than lymphocytes may be a particularly important source of the increased acid hydrolase activity observed in MS plaques. Since microglia may originate from blood monocytes (Langevoort, Cohn, Hirsch, Humphrey, Spector and Van Furth 1970) and as monocytes are transformed into macrophages (Cohn and Benson 1965) we also include in this report data on the benzyloxycarbonyl-GluTyr acid hydrolases present in peritoneal macrophages.
MATERIALS AND METHODS
Brains were obtained from clinically-established cases of MS confirmed at autopsy. The acute case (A) was a 26-year-old female while the chronic case (B) was a 53-yearold female. The control brain (C) was from a 29-year-old male who had died from burns. The time between death and post-mortem for A, B and C was 24, 44 and 4 hr, respectively. After dissection, weighed samples of tissue were stored at - 2 0 ° C
228
I). M. BOWEN, A. N. I)AVISON
until assayed.The time between death and assay forA, Band C was 3.5 and I months. respectively. Thawed samples were routinely prepared for enzyme assa5 by homogenizing in ice-cold 0.01M Tris-HCl buffer, pH 7.2, (measured at 4°C), containing 0.4 .... ,,, Triton X100 (buffer). Hog peritoneal macrophages were kindly prepared by Dr. B. Balfour, Medical Research Council Laboratories, Mill Hill, by dosing pigs intraperitoneally with 150 ml of light paraffin oil and killing them 3 days later. Tile contents of the peritoneal cavity were harvested and washed (2 to 3 times) by centrifugation using heparinized Tris-Eagles medium (McFarlin and Balfour !973). The cells were suspended (to give about 1 mg protein/ml) in buffer and tbllowing sonication (Bowen and Radin 1968) were maintained at - 20'~C for 1 month prior to assay. Protein was measured according to Lowry, Rosebrough, Farr and Randall (1951). Benzyloxycarbonyl-Glu-Tyr hydrolase activity was measured essentially as previously described (Bowen and Davison 1973). EDTA was omitted from the reaction mixtures while the final concentration of Triton X100 and where appropriate of sodium iodoacetate and mercaptoethanol were 0.2"., w/v, 2raM and 4ram respectively. The pH of the incubation mixtures was varied by changing the pH of the incubation buffer; the substrate solution was prepared at the appropriate pH value. The reaction was terminated after incubating at 37°C for up to 4 hr. RESULTS AND DISCUSSION
Both the brain tissue from the two cases of MS and the cerebral white matter from the patient who died of a non-neurological cause catalyzed the hydrolysis of the synthetic substrate in the presence of iodoacetic acid (Fig. 11. Since the time between both death and postmortem and death and assay was less in the control than diseased brains the changes in enzyme activity observed in the diseased brain may be an
"64 E
~3
<.2 /
I
B--
\m
0 I
3"5
4 "0
!
|
•
4,5
5 '0
5 "5
p~
a
6"0
Fig. 1. Effect o f p H on the thiol-independent benzyloxycarbonyl-Gtu-Tyr hydrotase activity ofunfractionmed homogenates prepared from normal and diseased human brain. O : non-neurological control (white matter); I : chronic MS (plaque tissue); []: acute MS (white matter adjacent to a plaque). Enzyme activity is expressed as pmoles of amino acid released from the substrate/hr/g original wet weight of tissue.
229
SHORT REPORT
underestimate. In the absence of iodoacetate the pH optimum for the reaction was at pH 4.5. The addition of iodoacetate resulted in both a marked inhibition of hydrolysis and shift in the optimum from pH 4.5 to between pH 5 to 5.5 Thus human brain, in addition to cathepsin A appears to contain a thiol-dependent catheptic carboxypeptidase (Bowen and Davison 1973) that is optimally active at about pH 4.5. Since cathepsin A does not require thiols for activity and has an optimum between pH to 5.5, hydrolase activity detected in unfractionated brain homogenates in the presence of iodoacetate and at pH 5.5 is referred to as cathepsin A activity. The mean cathepsin A activity measured in this way in cerebral white matter from 14 patients who had died of non-neurological causes was 1.14 units of enzyme activity (S.D._+0.34, range 0.62 to 1.69 units). The activity of the control brain used in the current study was close to this mean (Fig. 1). The results in Fig. 1 clearly show that in the acute case of MS the cathepsin A content of the sample of cerebral white matter (adjacent to a plaque) was increased over the normal level. Similarly, the activity (2.78 units of enzyme activity) of plaque tissue from the same brain, but not associated with the adjacent tissue described in Fig. 1, was also markedly elevated. However, cathepsin A activity in the plaque from the chronic case of MS was within the upper range of the normal brains. Activity of tissue adjacent to this plaque was 1.82 units of enzyme activity, while values for another plaque from the same brain and the surrounding adjacent tissue were 1.70 and 1.47 units of enzyme activity, respectively. These results show that cathepsin A activity was markedly increased in the case of acute MS and only slightly elevated in the case of chronic MS. Analysis of several acid hydrolases, including cathepsin A, in other cases of MS is now in progress. The results obtained so far show the mean cathepsin A activity of 7 plaques from 4 chronic cases of MS to be 1.56 units of enzyme activity (S.D. +_0.31, range 1.01 to 2.00 units) while the mean activity of 6 plaques from 3 cases of acute MS is 2.91 units of enzyme activity (S.D. _+0.95, range 1.44 to 4.70 units). Thus in comparison to the activity in cerebral white matter (1.14 units) in control subjects the mean cathepsin A activity is elevated 37 '~ and 155 °/oin chronic and acute cases, respectively. The results in Table 1 show that the hydrolysis of benzyloxycarbonyl-Glu-Tyr at pH 3.5, when catalyzed by hog peritoneal macrophages, is stimulated 3 to 4 times by TABLE 1 BENZYLOXYCARBONYL-GLU-TYR ACID HYDROLASE ACTIVITY IN HOG PERITONEAL MACROPHAGES
Incubation condition Enzyme activit.r a pH
mercaptoethanol
3.5 3.5 5.5 5.5
+ +
11.55; 50.39; 37.50; 37.25;
22.42 60.18 36.62 20.30
aExpressed as #moles amino acid released from the substrate/hr/100 mg protein. The results are for 2 different batches of macrophages. Assay of another batch, at various pH values between 3.5 to 6.0, demonstrated that 2 m M iodoacetate: (1) Inhibited hydrolysis of the substrate 99% at pH 3.5 but only 48 and 47°/,, at pH 5 and 5.5 respectively; (2) Shifts the pH optimum from pH 4.5 to between pH 5 to 5.5.
230
D . M . BOWEN, A. N. DAVISON
mercaptoethanol and is completely inhibited by iodoacetate. Furthermore, at pH 5.5 activity was not increased by mercaptoethanol while the addition of iodoacetate resulted in a shift in the pH optimum from pH 4.5 to between pH 5 to 55. Macrophages, therefore, appear to contain the thiol-dependent benzytoxycarbonyl-Gtu-Tyr hydrolase (catheptic carboxypeptidase) and a second enzyme (cathepsin A) insensitive to mercaptoethanol and iodoacetate with a pH optimum of between 5.0 to 5.5. Correcting for overlapping pH optima and loss of enzyme activity on storage at -20°C the specific activity of cathepsin A in freshly prepared macq-ophages is probably at least 30 to 35 units of enzyme activity/100 mg protein, while the specific activity of cathepsin A in normal cerebral white matter is very much (about 30 times) less. Therefore, since reactive microglia (or gitter cells) are a specialized type of tissue macrophage (that may originate from blood monocytes) the finding of increased cathepsin A activity in MS tissue is consistent with the view that macrophages are at least one source of the increased hydrolase activity seen in the plaque area in MS brain. Cathepsin D activity in areas of demyelination in MS and canine distemper always exceeds the more variable changes in /3-glucuronidase and aryl sulphatase (Cuzner and Davison 1973; McMartin, Koestner and Long 1972). Since spleen contains a population of lysosomes enriched in cathepsin D, relative to fi-glucuronidase and aryl sulphatase (Bowers 1971) this specialized type of leucocytic lysosome may be involved in the pathogenesis of both demyelinating diseases. ACKNOWLEDGEMENTS We would like to thank the Wellcome Trust for supporting this work. SUMMARY
The cathepsin A activity of plaque tissue from cases of multiple sclerosis is higher than in cerebral white matter from control patients. Since the specific activity of cathepsin A in white matter from control patients is at least 30-fold less than the activity in macrophages the finding of higher cathepsin A activity in plaques may be indicative of infiltration by monocytic phagocytes. Miriam Marks Department of Neurochemistry, Institute oj Neurology, The National Hospital, Queen Square,
D. M. BOWEN A, N. DAVISON
London, WCIN3BG (Great Britain :)
(Received 14 September, 1973)
REFERENCES BOWEN, D. M. AND A. N. DAVISON (1973) Cathepsin A in h u m a n brain and spleen, Biochem; J., 131: 417419. BOWEN, D. M. ANt) N. S. RADIN (1968) Purification of cerebroside galactosidase from rat brain, Biochim. Biophys. Acta, 152: 587-598.
SHORT REPORT
231
BOWEN, D. M., C. B. SMITH AND A. N. DAVISON 0973) Molecular changes in senile dementia, Brain, 96 : 849-856. BOWERS, W. E. (1971) Distribution of tissue proteinases in lymphoid tissues. In: A. J. BARRETT AND J. T. DINGLE (Eds.), Tissue Proteinases (Proceedings Royal Society Wates Symposium on Tissue Proteinases, Enzymology and Biology), North Holland Publishing Co., Amsterdam, pp. 221-239. COHN, Z. A. AND B. BENSON(1965) Differentiation of mononuclear fibrocytes, morphology, cytochemistry and biochemistry, J. exp. Med., 121:153 170. CUZNER, M. L. AND A. N. DAVISON (1973) Changes in cerebral lysosomal enzyme activity and lipids in multiple sclerosis, J. neurol. Sci., 19 : 29-36. EINSTEIN, E. R., J. CSEJTEY,K. B. DALAL, C. W. M. ADAMS, O. B. BAYLISSAND J. F. HALLPIKE(1972) Proteolytic activity and basic protein loss in and around M.S. plaques: Combined biochemical and histochemical observations, J. Neurochem., 19 : 653-662. LANGEVOORT, H. L., Z. A. COHN, J. G. HIRSCH, J. H. HUMPHREY, W. G. SPECTOR AND R. VAN FURTH The nomenclature of mononuclear phagocytic cells proposal for a new classification. In: R. VAN FURTH (Ed.), Mononuclear Phagoo'tes, Blackwell, Oxford, p. 5. LOWRY, O. H., N. J. ROSENBROUGH, A. L. FARR AND R. J. RANDALL (1951) Protein measurement with the phenol reagent, J. biol. Chem., 193 : 265 275. MCFARLIN,D. AND B. E. BALFOUR0973) Contact sensitivity in the pig, Immunology, 25 : 995 1009. MCMARTIN, D. M , A. KOESTNER AND J. F. LONG (1972) Enzyme activities associated with the demyelinating phase of canine distemper, Part 1 (fl-Glucuronidase, acid and neutral proteinase), Acta Neuropath. (Berl.), 22:275 287.
Journal of the neuroloyical Sciences, 1974, 21 : 227 231 ~ Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
Short Report
Macrophages and Cathepsin A Activity in Multiple Sclerosis Brain
INTRODUCTION
Cathepsin A catalyzes the release of tyrosine from benzyloxycarbonyl-Glu-Tyr, acts optimally at pH 5.25 and does not require thiols for activity. The enzyme appears to be a carboxypeptidase with limited endopeptidase activity (Bowen and Davison 1973). Studies in several laboratories, including our own (Cuzner and Davison, 1973; Bowen, Smith and Davison 1973) demonstrate that acid hydrolases, in particular those acting on proteins and peptides, play a role in degenerative brain diseases. It is of special interest that the finding of increased cathepsin D activity in the rim of active Multiple Sclerosis (MS) plaques, has been implicated in the immunopathological process seen in MS (Einstein, Csejtey, Dalal, Adams, Bayliss and Hallpike 1972). Although cerebral white matter contains cathepsin A, the concentration in spleen and hence possibly in macrophages, is very much higher. We have, therefore, proposed that in inflammatory conditions of the brain measurement of cathepsin A may serve as a useful index of cellular infiltration by leucocytes (Bowen and Davison 1973). One aim of the present study, therefore, was to establish whether the concentration of cathepsin A is changed in MS brain since as a result of perivascular cuffing both monocytes and lymphocytes are often associated with MS plaques. However, lymphocytes do not appear to account for the increase in hydrolase content seen in canine distemper, a demyelinating disease closely related to MS (McMartin, Koestner and Long 1972). Consequently, monocytic phagocytes rather than lymphocytes may be a particularly important source of the increased acid hydrolase activity observed in MS plaques. Since microglia may originate from blood monocytes (Langevoort, Cohn, Hirsch, Humphrey, Spector and Van Furth 1970) and as monocytes are transformed into macrophages (Cohn and Benson 1965) we also include in this report data on the benzyloxycarbonyl-GluTyr acid hydrolases present in peritoneal macrophages.
MATERIALS AND METHODS
Brains were obtained from clinically-established cases of MS confirmed at autopsy. The acute case (A) was a 26-year-old female while the chronic case (B) was a 53-yearold female. The control brain (C) was from a 29-year-old male who had died from burns. The time between death and post-mortem for A, B and C was 24, 44 and 4 hr, respectively. After dissection, weighed samples of tissue were stored at - 2 0 ° C
228
I). M. BOWEN, A. N. I)AVISON
until assayed.The time between death and assay forA, Band C was 3.5 and I months. respectively. Thawed samples were routinely prepared for enzyme assa5 by homogenizing in ice-cold 0.01M Tris-HCl buffer, pH 7.2, (measured at 4°C), containing 0.4 .... ,,, Triton X100 (buffer). Hog peritoneal macrophages were kindly prepared by Dr. B. Balfour, Medical Research Council Laboratories, Mill Hill, by dosing pigs intraperitoneally with 150 ml of light paraffin oil and killing them 3 days later. Tile contents of the peritoneal cavity were harvested and washed (2 to 3 times) by centrifugation using heparinized Tris-Eagles medium (McFarlin and Balfour !973). The cells were suspended (to give about 1 mg protein/ml) in buffer and tbllowing sonication (Bowen and Radin 1968) were maintained at - 20'~C for 1 month prior to assay. Protein was measured according to Lowry, Rosebrough, Farr and Randall (1951). Benzyloxycarbonyl-Glu-Tyr hydrolase activity was measured essentially as previously described (Bowen and Davison 1973). EDTA was omitted from the reaction mixtures while the final concentration of Triton X100 and where appropriate of sodium iodoacetate and mercaptoethanol were 0.2"., w/v, 2raM and 4ram respectively. The pH of the incubation mixtures was varied by changing the pH of the incubation buffer; the substrate solution was prepared at the appropriate pH value. The reaction was terminated after incubating at 37°C for up to 4 hr. RESULTS AND DISCUSSION
Both the brain tissue from the two cases of MS and the cerebral white matter from the patient who died of a non-neurological cause catalyzed the hydrolysis of the synthetic substrate in the presence of iodoacetic acid (Fig. 11. Since the time between both death and postmortem and death and assay was less in the control than diseased brains the changes in enzyme activity observed in the diseased brain may be an
"64 E
~3
<.2 /
I
B--
\m
0 I
3"5
4 "0
!
|
•
4,5
5 '0
5 "5
p~
a
6"0
Fig. 1. Effect o f p H on the thiol-independent benzyloxycarbonyl-Gtu-Tyr hydrotase activity ofunfractionmed homogenates prepared from normal and diseased human brain. O : non-neurological control (white matter); I : chronic MS (plaque tissue); []: acute MS (white matter adjacent to a plaque). Enzyme activity is expressed as pmoles of amino acid released from the substrate/hr/g original wet weight of tissue.
229
SHORT REPORT
underestimate. In the absence of iodoacetate the pH optimum for the reaction was at pH 4.5. The addition of iodoacetate resulted in both a marked inhibition of hydrolysis and shift in the optimum from pH 4.5 to between pH 5 to 5.5 Thus human brain, in addition to cathepsin A appears to contain a thiol-dependent catheptic carboxypeptidase (Bowen and Davison 1973) that is optimally active at about pH 4.5. Since cathepsin A does not require thiols for activity and has an optimum between pH to 5.5, hydrolase activity detected in unfractionated brain homogenates in the presence of iodoacetate and at pH 5.5 is referred to as cathepsin A activity. The mean cathepsin A activity measured in this way in cerebral white matter from 14 patients who had died of non-neurological causes was 1.14 units of enzyme activity (S.D._+0.34, range 0.62 to 1.69 units). The activity of the control brain used in the current study was close to this mean (Fig. 1). The results in Fig. 1 clearly show that in the acute case of MS the cathepsin A content of the sample of cerebral white matter (adjacent to a plaque) was increased over the normal level. Similarly, the activity (2.78 units of enzyme activity) of plaque tissue from the same brain, but not associated with the adjacent tissue described in Fig. 1, was also markedly elevated. However, cathepsin A activity in the plaque from the chronic case of MS was within the upper range of the normal brains. Activity of tissue adjacent to this plaque was 1.82 units of enzyme activity, while values for another plaque from the same brain and the surrounding adjacent tissue were 1.70 and 1.47 units of enzyme activity, respectively. These results show that cathepsin A activity was markedly increased in the case of acute MS and only slightly elevated in the case of chronic MS. Analysis of several acid hydrolases, including cathepsin A, in other cases of MS is now in progress. The results obtained so far show the mean cathepsin A activity of 7 plaques from 4 chronic cases of MS to be 1.56 units of enzyme activity (S.D. +_0.31, range 1.01 to 2.00 units) while the mean activity of 6 plaques from 3 cases of acute MS is 2.91 units of enzyme activity (S.D. _+0.95, range 1.44 to 4.70 units). Thus in comparison to the activity in cerebral white matter (1.14 units) in control subjects the mean cathepsin A activity is elevated 37 '~ and 155 °/oin chronic and acute cases, respectively. The results in Table 1 show that the hydrolysis of benzyloxycarbonyl-Glu-Tyr at pH 3.5, when catalyzed by hog peritoneal macrophages, is stimulated 3 to 4 times by TABLE 1 BENZYLOXYCARBONYL-GLU-TYR ACID HYDROLASE ACTIVITY IN HOG PERITONEAL MACROPHAGES
Incubation condition Enzyme activit.r a pH
mercaptoethanol
3.5 3.5 5.5 5.5
+ +
11.55; 50.39; 37.50; 37.25;
22.42 60.18 36.62 20.30
aExpressed as #moles amino acid released from the substrate/hr/100 mg protein. The results are for 2 different batches of macrophages. Assay of another batch, at various pH values between 3.5 to 6.0, demonstrated that 2 m M iodoacetate: (1) Inhibited hydrolysis of the substrate 99% at pH 3.5 but only 48 and 47°/,, at pH 5 and 5.5 respectively; (2) Shifts the pH optimum from pH 4.5 to between pH 5 to 5.5.
230
D . M . BOWEN, A. N. DAVISON
mercaptoethanol and is completely inhibited by iodoacetate. Furthermore, at pH 5.5 activity was not increased by mercaptoethanol while the addition of iodoacetate resulted in a shift in the pH optimum from pH 4.5 to between pH 5 to 55. Macrophages, therefore, appear to contain the thiol-dependent benzytoxycarbonyl-Gtu-Tyr hydrolase (catheptic carboxypeptidase) and a second enzyme (cathepsin A) insensitive to mercaptoethanol and iodoacetate with a pH optimum of between 5.0 to 5.5. Correcting for overlapping pH optima and loss of enzyme activity on storage at -20°C the specific activity of cathepsin A in freshly prepared macq-ophages is probably at least 30 to 35 units of enzyme activity/100 mg protein, while the specific activity of cathepsin A in normal cerebral white matter is very much (about 30 times) less. Therefore, since reactive microglia (or gitter cells) are a specialized type of tissue macrophage (that may originate from blood monocytes) the finding of increased cathepsin A activity in MS tissue is consistent with the view that macrophages are at least one source of the increased hydrolase activity seen in the plaque area in MS brain. Cathepsin D activity in areas of demyelination in MS and canine distemper always exceeds the more variable changes in /3-glucuronidase and aryl sulphatase (Cuzner and Davison 1973; McMartin, Koestner and Long 1972). Since spleen contains a population of lysosomes enriched in cathepsin D, relative to fi-glucuronidase and aryl sulphatase (Bowers 1971) this specialized type of leucocytic lysosome may be involved in the pathogenesis of both demyelinating diseases. ACKNOWLEDGEMENTS We would like to thank the Wellcome Trust for supporting this work. SUMMARY
The cathepsin A activity of plaque tissue from cases of multiple sclerosis is higher than in cerebral white matter from control patients. Since the specific activity of cathepsin A in white matter from control patients is at least 30-fold less than the activity in macrophages the finding of higher cathepsin A activity in plaques may be indicative of infiltration by monocytic phagocytes. Miriam Marks Department of Neurochemistry, Institute oj Neurology, The National Hospital, Queen Square,
D. M. BOWEN A, N. DAVISON
London, WCIN3BG (Great Britain :)
(Received 14 September, 1973)
REFERENCES BOWEN, D. M. AND A. N. DAVISON (1973) Cathepsin A in h u m a n brain and spleen, Biochem; J., 131: 417419. BOWEN, D. M. ANt) N. S. RADIN (1968) Purification of cerebroside galactosidase from rat brain, Biochim. Biophys. Acta, 152: 587-598.
SHORT REPORT
231
BOWEN, D. M., C. B. SMITH AND A. N. DAVISON 0973) Molecular changes in senile dementia, Brain, 96 : 849-856. BOWERS, W. E. (1971) Distribution of tissue proteinases in lymphoid tissues. In: A. J. BARRETT AND J. T. DINGLE (Eds.), Tissue Proteinases (Proceedings Royal Society Wates Symposium on Tissue Proteinases, Enzymology and Biology), North Holland Publishing Co., Amsterdam, pp. 221-239. COHN, Z. A. AND B. BENSON(1965) Differentiation of mononuclear fibrocytes, morphology, cytochemistry and biochemistry, J. exp. Med., 121:153 170. CUZNER, M. L. AND A. N. DAVISON (1973) Changes in cerebral lysosomal enzyme activity and lipids in multiple sclerosis, J. neurol. Sci., 19 : 29-36. EINSTEIN, E. R., J. CSEJTEY,K. B. DALAL, C. W. M. ADAMS, O. B. BAYLISSAND J. F. HALLPIKE(1972) Proteolytic activity and basic protein loss in and around M.S. plaques: Combined biochemical and histochemical observations, J. Neurochem., 19 : 653-662. LANGEVOORT, H. L., Z. A. COHN, J. G. HIRSCH, J. H. HUMPHREY, W. G. SPECTOR AND R. VAN FURTH The nomenclature of mononuclear phagocytic cells proposal for a new classification. In: R. VAN FURTH (Ed.), Mononuclear Phagoo'tes, Blackwell, Oxford, p. 5. LOWRY, O. H., N. J. ROSENBROUGH, A. L. FARR AND R. J. RANDALL (1951) Protein measurement with the phenol reagent, J. biol. Chem., 193 : 265 275. MCFARLIN,D. AND B. E. BALFOUR0973) Contact sensitivity in the pig, Immunology, 25 : 995 1009. MCMARTIN, D. M , A. KOESTNER AND J. F. LONG (1972) Enzyme activities associated with the demyelinating phase of canine distemper, Part 1 (fl-Glucuronidase, acid and neutral proteinase), Acta Neuropath. (Berl.), 22:275 287.