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SOME EFFECTS OF LONG-TERM PROGESTERONE TREATMENT IN RABBITS AND THEIR RELEVANCE TO PRE-ECLAMPSIA
170
these tumours were often larger than those in the unimmunised control group. In all the three criteria for tumour evaluation, the thymic A.L.S. showed a progressive increase in activity from 1 to 7 weeks, a slight decrease thereafter, and a significant rise again in the 12th and 15th weeks after the third injection of thymic cells in the rabbits at the 12th week. As compared with thymic A.L.S., the spleen A.L.s. and the brain antisera demonstrated a less potent, although significant and consistent immunosuppressive effect in this system. In contrast to thymic A.L.s., the spleen A.L.s. and the brain antisera activity reached a peak earlier and did not show the striking, subsequent rise at 12-15 weeks. Representative tumours in the N.R.s.-treated group, the unimmunised control group, and one of the thymic A.L.s.-treated groups are shown in fig. 2. The results of the entire experiment are summarised graphically in fig. 3 in which the tumourindex value for each antiserum is plotted against the time (week no.) of collection of that antiserum. Discussion
immunity to S180 developed in Swiss mice is primarily of the cellular variety, and from our results it seems that the treatment of these mice with A.L.S. during the period of immunisation prevents the development of such inimunity. Thymic A.L.S. was far more potent than spleen A.L.s. in this respect, but the reasons for this difference are not clear at present. The significant immunosuppressive activity caused by the anti-brain sera was indeed surprising. Although the presence of a few lymphocytes, not detectable on a slide-smear preparation, in the brain-cell suspension cannot be excluded as a possibility it seems unlikely that the A.L.s.-like effect of the anti-brain sera could be explained solely on that basis. Levey and Medawar (1966) reported that antiepidermal-cell sera had significant A.L.s.-like activity in the mouse skin-homograft system. The antigens responsible for production of A.L.s. may be more widely distributed than has previously been supposed, and may The
involve the brain cells.
Lymphoagglutinin titres and the immunosuppressive effect demonstrated by various A.L.S. preparations were A similar finding has been reported by not correlated. Jeejeebhoy (1967). This demonstrates, once again, that the leukoagglutinin titres, as they are used in evaluating the horse antihuman A.L.s. preparations (Starzl et al. 1967), probably do not correlate with their immunosuppressive activity. Although tumour allografts are not strictly comparable to normal organ allografts, A.L.S. seems to manifest its effect similarly in both systems. As in the organ allograft system, A.L.S. prevented development of immunity to S180, and from our preliminary results (not yet published), it seems that A.L.s. destroys pre-existing immunity also, in this system. The tumour system offers certain distinct advantages in the study of A.L.S. action: the graft is transplanted by a simple injection of tumour cells into the web of the hind foot; the growth of the tumour is then observed by simple external, gross examination; and the weight of the tumour or, the tumour index as defined by us, at the end of the experiment (12 to 13 days after the second tumour injection) seems to be correlated with the immunosuppressive effect of a specific preparation of A.L.S. Although the exact quantitative nature of this relationship has not yet been worked out, the experimental model as we have described, promises to be useful in a
study of the of A.L.S.
properties, purification,
and mode of action
We thank Mrs. Marianne Sandrock, Miss Jeri Corlett, Angela Fantauzzo for technical assistance. Requests for reprints should be addressed to S. D. D.
and Miss
REFERENCES
Abbot, W. M., Otherson, H. B., Monaco, A. P., Simmons, R. L., Wood, M. L., Russell, P. S. (1966) Surg. Forum, 17, 228. Chew, W. B., Lawrence, J. S. (1937) J. Immun. 33, 271. Gray, J. G., Monaco, A. P., Russell, P. S. (1964) Surg. Forum, 15, 142. Jeejeebhoy, H. F. (1967) Transplantation, 5, 273. Levey, R. H., Medawar, P. B. (1966) Proc. natn. Acad. Sci., U.S.A. 56, 1130.
Monaco, A. P., Wood, M. L., Gray, J. G., Russell, P. S. (1966) J. Immun. 96, 211. Starzl, T. E., Marchioro, T. L., Porter, K. A., Iwasaki, Y., Cerilli, G. J. (1967) Surgery Gynec. Obstet. 124, 301. Turk, J. L., Willoughby, D. A. (1967) Lancet, i, 249. Woodruff, M. F. A., Anderson, N. A. (1963) Nature, Lond. 200, 702. (1964) Ann. N.Y. Acad. Sci. 120, 119. —
—
SOME EFFECTS OF LONG-TERM PROGESTERONE TREATMENT IN RABBITS AND THEIR RELEVANCE TO PRE-ECLAMPSIA D. F. HORROBIN D.Phil. Oxon. OF THE DEPARTMENT OF ST.
MARY’S
HOSPITAL MEDICAL
PHYSIOLOGY,
SCHOOL, LONDON W.2
were given daily doses of progesterone over a period of many weeks. In all of them the arterial pressure rose. One rabbit died, the immediate cause being gross pulmonary oedema. When the other rabbits were killed there was evidence of impending left-ventricular failure. The renal glomeruli showed changes similar to those which have been described as specific to pre-eclampsia in women.
Summary
Rabbits
Introduction
PROGESTERONE stimulates appetite and promotes rapid weight-gain in animals and man (Brobeck et al. 1947, Gilbert and Gillman 1956, Dewar 1957 a, b, Southam and Gonzaga 1965). The relation between rapid weightgain and pre-eclampsia is well known. The experiments described here were carried out to investigate whether progesterone given in high dosage over a long period could raise arterial pressure. Methods Two male rabbits were used for a preliminary study, which was followed by a more complete experiment on six female New Zealand White rabbits. Arterial pressure was measured in the intact, unaneesthetised state by the ear-capsule method (Grant and Rothschild 1934). A black ring, 3 mm. in diameter, was painted on the rubber diaphragm of the capsule. The diastolic pressure was taken as the point when pressure within the capsule was just sufficient to exclude blood from the earartery beneath the ring once during every cardiac cycle. The systolic pressure was taken as the point when blood was excluded throughout the cycle. The relation of these readings to absolute pressures is uncertain, but there is little reason to doubt that changes in the readings reflect changes in the underlying absolute pressures. Initial difficulty was experienced on account of varying vasomotor activity and fluctuations of pressure due to fear or excitement (Stott 1966). The readings became much more consistent after (1) the rabbits were examined in the cage room itself; (2) they were restrained only loosely in an open-topped box; (3) at least 5 minutes were allowed to elapse between lifting the rabbits from their cages and taking the first reading;
171 and (4) all readings were taken after 5 P.M. when the animal house was quiet. During an initial control period, each morning each rabbit was weighed and given an intramuscular injection of ethyl oleate. Each evening five successive readings of diastolic and systolic pressures were taken and the means calculated. After the control period, (1) A and B (the two males) were given 5 mg. per day of progesterone (B.D.H., ’Progestin’) for 5 days, followed by 100 mg. daily for 6 days and ethyl oleate for 9 days; (2) C and D acted as controls, and received daily ethyloleate injections for the duration of the experiment; (3) E received 25 mg. progesterone per day for 10 days, ethyl oleate for 20 days, followed by 25 mg. progesterone for 13 days after which it died; (4) F received 25 mg. progesterone for 47 days, followed by 50 mg. for 15 days; (5) G received 25 mg. progesterone for 47 days, followed by ethyl oleate for 15 days; and (6) H received 25 mg. for 10 days, ethyl oleate for 20 days, 25 mg. progesterone for 46 days, and 50 mg. for 15 days. At the end of the experiment the five surviving New Zealand White rabbits were killed by an overdose of pentobarbitone sodium (’ Nembutal ’) and were examined post mortem. Tissue from brain, lung, kidney, liver, adrenal, muscle, and fat was immediately fixed in formol-saline solution, and prepared for histological examination. 50 µ sections were stained with hæmatoxylin and eosin and with periodic-acid/Schiff.
Arterial Pressure
Results
The pilot experiment on rabbits A and B revealed that 5 mg. progesterone per day tended to lower arterial pressure, and 100 mg. per day raised it to 10-25 mm. Hg above control levels. A dose of 25 mg. per day (6-10 mg. per kg.) was decided on. This is comparable to the amount of progesterone secreted by healthy women in the third trimester of pregnancy. In the control rabbits C and D (fig. la) changes in arterial pressure were not significant. Their weight-gain was smooth. In the remaining four rabbits, both diastolic and systolic pressures rose during periods of progesterone injection. The rises took 3-5 days to become apparent, and only reached plateau levels after 2-3 weeks. Cessation of progesterone treatment was followed by a slow fall to control levels. Each rabbit had a sharp but transient increase in the rate of weight-gain at the beginning of progesterone treatment. In rabbit E, during the first period of progesterone injection both systolic and diastolic pressures rose to 15-20 mm. Hg above control levels. During the second control period, arterial pressures fell to normal after 7 days. Pressure was again rising during the second period of progesterone injection when the rabbit died.
Fig. 2-Glomerulus from obliteration of capsular
rabbit F space
showing swelling of tuft with (haemotoxylin and eosin, x 350).
In rabbit F, after 3 weeks of progesterone injections, diastolic pressure reached a plateau about 15 mm. Hg above control and systolic pressure a plateau about 20 mm. Hg above control. These levels were maintained until 15 days before the rabbit was killed when the progesterone dose was increased: a further slight rise occurred during this period. In rabbit G, after 3 weeks’ progesterone treatment, diastolic pressure reached a plateau about 20 mm. Hg above control and systolic pressure a plateau about 35 mm. Hg above control. 15 days before the rabbit was killed, the progesterone treatment was stopped. During this time systolic pressure fell to within 10 mm. Hg of control and diastolic pressure to within 5 mm. Hg of control. The changes in arterial pressure in rabbit H are shown in fig. lb. Postmortem Examination
No abnormalities were found in the control rabbits C and D. E died of acute pulmonary cedema, bloodstained foam exuding from its mouth and nostrils just before death. The lungs themselves were oozing blood, and there was blood in the pleural cavity. There was a greyish exudate on the lung surface. The liver appeared congested. G showed no abnormalities, apart from some small grey patches of exudate on the lung surface. F and H had much larger patches of similar exudate; the lungs themselves were grossly congested.
Microscopy No abnormalities were detected in any organs other than the lungs, the kidneys, and the liver. In E. F. and H the lung
tissue was grossly congested, with red cells and much eosinophilic exudate in the alveoli. The lungs of G which had not received any progesterone for 15 days before death were relatively mildly affected. The most interesting and specific changes were seen in the kidneys. The glomerular tufts were enlarged, and in many cases the normal space between capsule and glomerulus was eliminated. Glomeruli throughout the kidneys were affected, those in the outer cortex being
severely damaged. The enlargement seemed mainly due to a swelling of the endothelial cells without proliferation.
most
Fig. 1—(a)
arterial pressures in the control rabbit D; (b) arterial pressure changes in rabbit H.
172
As a result, capillary lumens were narrowed or obliterated, and few red cells were present. In some glomeruli, clear vacuoles were seen (fig. 2) apparently within endothelial cells. In the periodic-acid/Schiff sections, the basement membrane appeared to be blurred in the treated rabbits. Of all these changes, the only one which can be quantitatively expressed is the relation between the diameter of the glomerulus and the diameter of its capsule. In each of the four progesterone-treated and two control rabbits, one hundred capsules and glomeruli were measured. In each case two diameter readings were taken at 90° to one another. The means of the two readings for the capsule and the two for the tuft were then calculated. For each malpighian corpuscle, the relation between capsular size and glomerular size was expressed as the ratio of the mean glomerular diameter to the mean capsular diameter. The mean of the hundred ratios was then calculated for each rabbit (see accompanying table). All the treated rabbits
(100 MALPIGHIAN RABBIT)
MEAN RATIOS OF GLOMERULAR TO CAPSULAR DIAMETER CORPUSCLES MEASURED IN EACH
could thus cause all the features of The oedema could be due to dilatation of arterioles with raised capillary pressure and increased filtration. If the high blood-pressure damages organs such as the placenta and kidney, other pressor agents may be released, maintaining the hypertension even if progesterone output falls owing to placental injury. The hypothesis can explain the puzzling features of pre-eclampsia and
Progesterone pre-eclampsia.
eclampsia. 1. A large and/or active placenta is associated with preeclampsia (Jeffcoate and Scott 1959, Jeffcoate 1966). There is a higher incidence of pre-eclampsia in twin pregnancies, in hydrops foetalis, in diabetes, and in hydatidiform mole. In each case, the placenta is large and/or secretes large amounts of human chorionic gonadotrophin (H.c.G.) (Hellman and Hertig 1938, Smith and Smith 1940, ten Berge and van Assen 1947, Eton and Short 1960, Chun et al. 1964). Since the placenta is the major source of progesterone and since H.C.G. stimulates progesterone output, the relation between these disorders is logical. Even in otherwise uncomplicated pregnancies, the teaching that in pre-eclampsia the placenta is small has been found to be untrue (Scott 1958, McClure Browne 1964,
Chakravarty 1967).
showed higher ratios than the two control rabbits. The difference between the mean in the control rabbit with the higher ratio and that in the treated rabbit with the lowest ratio was highly significant (p < 0-001). In each of the progesterone-treated rabbits, the liver was mildly congested but showed no other abnormalities, except in rabbit F, where the liver was grossly laden with glycogen, an appearance typical of that seen in a deliberately overfed animal. The progesterone may have overstimulated the rabbit’s appetite. Discussion
These experiments support the hypothesis that the initial rise in arterial pressure in pre-eclampsia is caused by progesterone. The mechanism of the pressure rise is uncertain. Progesterone antagonises aldosterone and promotes renal loss of sodium (Landau and Lugibihl 1961). Apparently in compensation, the aldosterone output rises (Gerrall et al. 1960), possibly mediated by the reninangiotensin mechanism. However, renin levels do not appear to be raised in pre-eclampsia above the levels in normal pregnancy (Brown et al. 1966). Patients with preeclampsia may be more responsive to normal angiotensin levels (Chesley 1965), but whether progesterone is implicated in this sensitisation is unknown. Another possibility is that progesterone could act by relaxing smooth-muscle tone in the carotid-sinus wall. Locally administered smooth-muscle relaxants raise blood-pressure in this way (Landgren et al. 1952) but as yet no parenterally administered agent has been found to operate thus. The renal lesions in the progesterone-treated rabbits are similar to those in human kidneys which have been described as specific to pre-eclampsia (Pollak and Nettles 1960)-namely, swelling and ischaemia of the glomerular tuft as a whole, swelling of the endothelial cells without multiplication, and the formation of vacuoles. In severe cases the basement membrane may be thickened. This description could be applied, virtually without alteration, to the changes seen in the treated rabbits.
2. In otherwise uncomplicated cases, pre-eclampsia is much commoner in first pregnancies. This may be because the liver is less able effectively to inactivate the large amounts of progesterone when it meets them for the first time. Macnaughton and Greig (1965) have demonstrated that the rate of conversion of tritiated progesterone to labelled pregnanediol is less in pre-eclampsia and in hydatidiform mole than in normal pregnancy. 3. Women who gain weight rapidly in pregnancy tend to become oedematous and hypertensive and to have large babies (Hytten et al. 1966, Lancet 1967, MacGillivray 1967, Thomson et al. 1967). This is logical if large placentas are associated with large babies and a high progesterone output. 4. The blood-volume is lower in pre-eclampsia than in normal pregnancy. This could be partly due to increased renal loss of sodium and water (Landau and Lugibihl 1961) and partly due to increased capillary filtration. 5. Many eclamptic fits occur after parturition. Progesterone can cause sleep and anaesthesia in animals and man (Selye 1941 a, b, Costa and Bonnycastle 1952, Merryman et al. 1954, Woolley and Timiras 1962, Heuser 1967). In epileptics, the luteal phase of the menstrual cycle and progesterone treatment are both associated with a low seizure-rate (Laidlan 1956, Logothetis et al. 1959). Menstruation and the days immediately preceding menstruation, when the blood progesterone concentration is falling (Woolever 1963) are accompanied by an increased seizure-rate. The depression brought about by progesterone seems to be followed by a rebound excitation. By contrast, in animals dehydroepiandrosterone has been found to precipitate convulsions (Heuser 1967). The concentration of dehydroepiandrosterone is low in the blood in pregnancy, because it is converted to oestrogens by the placenta (Gardner et al. 1954, Migeon 1954, Baulieu and Dray 1963, Siitterei and Macdonald 1963, Bolte et al. 1964). After expulsion of the placenta plasma concentrations return to normal. At parturition, convulsions are likely, because the concentration of the anxsthetic, progesterone, is falling while that of the convulsant, dehydroepiandrosterone, is rising. I should like to thank Prof. A. D. M. Greenfield and Prof. P. J. Huntingford for much advice and encouragement. Professor
173 Greenfield very
kindly
put
at my
disposal
the facilities of the
physio-
logy department at St. Mary’s Hospital; Dr. J. G. Bate of the Samaritan Hospital gave me much help with the interpretation of the histological changes and prepared the photograph; Miss Sylvia Legg
prepared the
sections. REFERENCES
Baulieu, E. E., Dray, F. (1963) J. clin. Endocr. 23, 1298. Bolte, E., Marcuso, S., Friksson, G., Wiqvist, N., Diczfalusy, E. (1964) Acta endocr. Copenh. 45, 560. Brobeck, J. R., Wheatland, M., Straminger, J. L. (1947) Endocrinology, 40, 65.
Brown, J. J., Davies, D. L., Doak, P. B., Lever, A. F., Robertson, J. I. S., Trust, P. (1966) J. Obstet. Gynœc. Br. Commonw. 73, 410. Chakravarty, A. P. (1967) ibid. 74, 247. Chesley, L. C. (1965) Bull. N.Y. Acad. Med. 41, 811. Chun, D., Braga, C., Chow, C., Lok, L. (1964) J. Obstet. Gynœc. Br. Commonw. 71, 180. Costa, P. J., Bonnycastle, D. D. (1952) Archs intern. Pharmacodyn. 91, 330. Dewar, A. D. (1957a) J. Endocr. 15, 216. (1957b) ibid. p. 230 Eton, B., Short, R. V. (1960) J. Obstet. Gynœc. Br. Commonw. 67, 785. Gardner, L. I., Wetton, R. L., Ellis, W., Hughes, E. C. (1954) Proc. Soc. exp. Biol. Med. 86, 804. Gerrall, A. G., Robertson, M. E., Laidlaw, J. C. (1960) Acta endocr. Copenh. suppl. 51, 157. Gilbert, C., Gillman, J. (1956) S. Afr. J. med. Sci. 21, 75. Grant, R. T., Rothschild, P. (1934) J. Physiol. 81, 265. Hellman, L. M., Hertig, A. (1938) Am. J. Path. 14, 111. Heuser, G. (1967) Anœsthesiology, 28, 173. —
Preliminary Communications INHIBITION OF ANTIPLASMIN, AND FIBRINOLYTIC EFFECT OF PROTEASE IN PATIENTS WITH CANCER
Hytten, F. E., Thomson, A. M., Taggart, N. R. (1966) J. Obstet. Gynœc. Br. Commonw. 73, 553. Jeffcoate, T. N. A. (1966) Proc. R. Soc. Med. 59, 397. Scott, J. S. (1959) Am. J. Obstet. Gynec. 77, 475. Laidlan, J. (1956) Lancet, ii, 1235. Lancet (1967) ii, 197. Landau, R. L., Lugibihl, K. (1961) Rec. progr. Hormone Res. 27, 249. Landgren, S., Neil, E., Zotterman, Y. (1952) Acta physiol. scand. 25, 24. Logothetis, J., Korner, R., Morrell, F., Torres, F. (1959) Neurology, 9, 352. McClure Browne, J. C. (1964). Cited in Lewis, T. L. T. (1964) Progress in Clinical Obstetrics and Gynæcology. London. MacGillivray, I. (1967) Scot. med. J. 12, 237. Macnaughton, M. C., Greig, M. (1965) J. Obstet. Gynœc. Br. Commonw. 72, 1029. Merryman, M., Boiman, R., Barnes, L., Rothchild, I. (1954) J. clin. Endocr. 14, 1567. Migeon, C. J. (1964). Cited in Gardner, L. I., Welton, R. L., Ellis, W., Hughes, E. C. (1954) Proc. Soc. exp. Biol Med. 86, 804. Pollak, V. E., Nettles, J. B. (1960) Medicine, 39, 469. Scott, J. S. (1958) J. Obstet. Gynœc. Br. Emp. 65, 689. Selye, H. (1941a) Proc. Soc. exp. Biol. Med. 46, 116. (1941b) J. Immunol. 41, 259. Siitterei, P. K., Macdonald, P. C. (1963) Steroids, 2, 713. Smith, O. W., Smith, G. V. (1940) Proc. Soc. exp. Biol. Med. 44, 104. Southam, A. L., Gonzaga, F. P. (1965) Am. J. Obstet. Gynec. 91, 142. Stott, F. D. (1966) Biomed. Eng. 1, 544. ten Berge, B. S., van Assen, F. J. J. (1947) Ned. Tijdschr. Geneesk. 91, 1229. Thomson, A. M., Hytten, F. E., Billewicz, W. J. (1967) J. Obstet. Gynœc. Br. Commonw. 74, 1. Woolever, C. H. (1963) Am. J. Obstet. Gynec. 85, 981. Woolley, D. E., Timiras, P. S. (1962) Endocrinology, 70, 196. —
—
endogenous fibrinolytic system: a fibrinolytic enzyme from Aspergillus oryzce fulfilled both these requirements. This enzyme has been called aspergillin O,9 and protease i.10 MATERIALS AND METHODS
Materials
CA-7, protease I.-The fibrinolytic enzymes of A.
oryzae
containing
C2 (Connaught) units per mg. dry substance, as CA-7 (Connaught Laboratories, Toronto, lot 1016-2) or protease I (Astra, Sweden, lot 17047). 1000
Protease
I (a fibrinolytic enzyme from Aspergillus oryzœ) inhibited thromboplastin and antiplasmin activity in vitro; and the antiplasmin inhibitory activity is potentiated in vitro by human thromboplastin. The in-vivo effects of this enzyme were examined in patients with cancer, since thromboplastin activity is raised in cancer tissue, and such patients have increased levels of antiplasmin. Thirteen patients
Summary
with
with occlusive vascular disease were of protease. Side-effects were infusions given mild and transient, except for pathological fibrinolysis (in three cases on a high dose) and a coagulative defect in one case. Subjective improvement was noted, but it is too early to judge the effects of therapy. cancer
and
one
one or more
INTRODUCTION
THE induction of
adequate anticoagulation or fibrinolysis by heparin, dicoumarol, or plasmin in animals decreases the metastatic spread of blood-borne cancer cells.1-5 The lowering of prothrombin levels by warfarin sodium inhibits the locomotion and growth of V2 carcinoma cells in rabbit tissues.6 The thromboplastin activity of cancer tissue is many times greater than that of normal tissue,and the inhibitors of fibrinolysis, the antiplasmins, are increased in patients with cancer.8 We have been looking for an agent which would inhibit this thromboplastic activity of human cancer tissue, and decrease the antiplasmins and thus enhance the 1. 2. 3.
4. 5. 6.
7. 8.
Terranova, T., Chissone, F. Boll. Soc. ital. Biol. spér. 1952, 23, 1224. Lacour, F., Oberling, C., Guerin, M. Bull. Ass. fr. Cancer, 1955, 42, 531. Wood, S., Jr., Holyoke, E. D., Yardley, J. M. Proc. Am. Ass. Cancer Res. 1956, 2, 157. Grossi, C. E., Agostino, D., Cliffton, E. E. Cancer Res. 1960, 20, 605. Fisher, B., Fisher, E. R. Surgery, St. Louis, 1961, 50, 240. Thornes, R. D. in Endogenous Factors Influencing Host Tumour Balance (edited by R. W. Wissler, T. L. Dao, S. Wood, Jr.); p. 255. Chicago, 1967. O’Meara, R. A. Q., Thornes, R. D. Ir. J. med. Sci. 1961, 423, 106. Thornes, R. D., O’Donnell, J. M., O’Brien, D. J. ibid. 1967, 494, 73.
Plasmin.-’ Kabi 1398 ’, human plasmin grade " b " measured in casein units (Sgouris) lot LmD 3. Reference serum.-Pooled sera from 20 healthy people stored at - 20°C and diluted 1/8 in saline solution at time of use. Bovine fibrinogen.-Kabi grade " B1"lot QdX 19, 0,2%. Stored as 0-6% in 2-8% w/v sodium chloride at ň20°C, thawed, and diluted 1 J3 with tris(hydroxymethyl) aminomethane (" tris ") buffer pH 7-4. Thrombin.-Thrombin, topical (bovine origin) (Parke Davis & Co.), 50 units per ml. In tris buffer at pH 7-4, stored at -20°C. " Normal saline ".-0.9% w/v sodium-chloride solution (clinical,
Antigen Ltd.). .Hfpat-tM.ň’ Pularin’(Evans Ltd., batch G15840). e-aminocaproic acid.-’ Epsikapron ’ (Kabi, lot 84850). Trasylol’.-Protease inhibitor (FBA Pharmaceuticals), 25,000 kallikrein-inactivating units per ampoule. ’
Methods Methods were as follows: antiplasmin activity, 8 dilute blood-clot lysis-time (B.L.T.)," and fibrinogen.12 Human thromboplastin was prepared from fresh frozen human chorion and titrated by the method of O’Meara and Thomes,7 and has similar activity to the thromboplastin extracted from cancer tissue (cancer coagulative factor, c.C.F.).13 Bedside test.-Measured by Thornes’ method.14 Protease resistance (serum-level of inhibitor to protease) was estimated by Roschlau’s method 15 modified to use 0-1ml. of whole blood instead of 1-0 ml. and 1/10 of the amount of protease (CA-7). To facilitate the reading of the end-point of titration, water is added to the tubes after 10 minutes incubation, and the percentage lysis can be easily assessed by inversion of the tubes. The results are expressed as " test tube requirement " (T.T.R.) units. 9. 10. 11. 12. 13.
14. 15.
Stefanini, M., Adamis, D. M., Soardi, F., Marin, H. M., Mele, R. Lancet, 1959, ii, 443. Bergkvist, R. Acta chem. scand. 1963, 17, 1521. Fearnley, G. R., Balmforth, G., Fearnley, E. B. Clin. Sci. 1957, 16, 645. Blombäck, B., Blombäck, M. Arkiv. Kemi. 1956, 10, 415. Boggust, W. A., O’Brien, D., O’Meara, R. A. Q., Thornes, R. D. Ir. J. med. Sci. 1963, 447, 131. Thornes, R. D. J. Ir. med. Ass. 1963, 53, 194. Roschlau, W. H. E. Can. J. Physiol. Pharmac. 1964, 42, 109.
these tumours were often larger than those in the unimmunised control group. In all the three criteria for tumour evaluation, the thymic A.L.S. showed a progressive increase in activity from 1 to 7 weeks, a slight decrease thereafter, and a significant rise again in the 12th and 15th weeks after the third injection of thymic cells in the rabbits at the 12th week. As compared with thymic A.L.S., the spleen A.L.s. and the brain antisera demonstrated a less potent, although significant and consistent immunosuppressive effect in this system. In contrast to thymic A.L.s., the spleen A.L.s. and the brain antisera activity reached a peak earlier and did not show the striking, subsequent rise at 12-15 weeks. Representative tumours in the N.R.s.-treated group, the unimmunised control group, and one of the thymic A.L.s.-treated groups are shown in fig. 2. The results of the entire experiment are summarised graphically in fig. 3 in which the tumourindex value for each antiserum is plotted against the time (week no.) of collection of that antiserum. Discussion
immunity to S180 developed in Swiss mice is primarily of the cellular variety, and from our results it seems that the treatment of these mice with A.L.S. during the period of immunisation prevents the development of such inimunity. Thymic A.L.S. was far more potent than spleen A.L.s. in this respect, but the reasons for this difference are not clear at present. The significant immunosuppressive activity caused by the anti-brain sera was indeed surprising. Although the presence of a few lymphocytes, not detectable on a slide-smear preparation, in the brain-cell suspension cannot be excluded as a possibility it seems unlikely that the A.L.s.-like effect of the anti-brain sera could be explained solely on that basis. Levey and Medawar (1966) reported that antiepidermal-cell sera had significant A.L.s.-like activity in the mouse skin-homograft system. The antigens responsible for production of A.L.s. may be more widely distributed than has previously been supposed, and may The
involve the brain cells.
Lymphoagglutinin titres and the immunosuppressive effect demonstrated by various A.L.S. preparations were A similar finding has been reported by not correlated. Jeejeebhoy (1967). This demonstrates, once again, that the leukoagglutinin titres, as they are used in evaluating the horse antihuman A.L.s. preparations (Starzl et al. 1967), probably do not correlate with their immunosuppressive activity. Although tumour allografts are not strictly comparable to normal organ allografts, A.L.S. seems to manifest its effect similarly in both systems. As in the organ allograft system, A.L.S. prevented development of immunity to S180, and from our preliminary results (not yet published), it seems that A.L.s. destroys pre-existing immunity also, in this system. The tumour system offers certain distinct advantages in the study of A.L.S. action: the graft is transplanted by a simple injection of tumour cells into the web of the hind foot; the growth of the tumour is then observed by simple external, gross examination; and the weight of the tumour or, the tumour index as defined by us, at the end of the experiment (12 to 13 days after the second tumour injection) seems to be correlated with the immunosuppressive effect of a specific preparation of A.L.S. Although the exact quantitative nature of this relationship has not yet been worked out, the experimental model as we have described, promises to be useful in a
study of the of A.L.S.
properties, purification,
and mode of action
We thank Mrs. Marianne Sandrock, Miss Jeri Corlett, Angela Fantauzzo for technical assistance. Requests for reprints should be addressed to S. D. D.
and Miss
REFERENCES
Abbot, W. M., Otherson, H. B., Monaco, A. P., Simmons, R. L., Wood, M. L., Russell, P. S. (1966) Surg. Forum, 17, 228. Chew, W. B., Lawrence, J. S. (1937) J. Immun. 33, 271. Gray, J. G., Monaco, A. P., Russell, P. S. (1964) Surg. Forum, 15, 142. Jeejeebhoy, H. F. (1967) Transplantation, 5, 273. Levey, R. H., Medawar, P. B. (1966) Proc. natn. Acad. Sci., U.S.A. 56, 1130.
Monaco, A. P., Wood, M. L., Gray, J. G., Russell, P. S. (1966) J. Immun. 96, 211. Starzl, T. E., Marchioro, T. L., Porter, K. A., Iwasaki, Y., Cerilli, G. J. (1967) Surgery Gynec. Obstet. 124, 301. Turk, J. L., Willoughby, D. A. (1967) Lancet, i, 249. Woodruff, M. F. A., Anderson, N. A. (1963) Nature, Lond. 200, 702. (1964) Ann. N.Y. Acad. Sci. 120, 119. —
—
SOME EFFECTS OF LONG-TERM PROGESTERONE TREATMENT IN RABBITS AND THEIR RELEVANCE TO PRE-ECLAMPSIA D. F. HORROBIN D.Phil. Oxon. OF THE DEPARTMENT OF ST.
MARY’S
HOSPITAL MEDICAL
PHYSIOLOGY,
SCHOOL, LONDON W.2
were given daily doses of progesterone over a period of many weeks. In all of them the arterial pressure rose. One rabbit died, the immediate cause being gross pulmonary oedema. When the other rabbits were killed there was evidence of impending left-ventricular failure. The renal glomeruli showed changes similar to those which have been described as specific to pre-eclampsia in women.
Summary
Rabbits
Introduction
PROGESTERONE stimulates appetite and promotes rapid weight-gain in animals and man (Brobeck et al. 1947, Gilbert and Gillman 1956, Dewar 1957 a, b, Southam and Gonzaga 1965). The relation between rapid weightgain and pre-eclampsia is well known. The experiments described here were carried out to investigate whether progesterone given in high dosage over a long period could raise arterial pressure. Methods Two male rabbits were used for a preliminary study, which was followed by a more complete experiment on six female New Zealand White rabbits. Arterial pressure was measured in the intact, unaneesthetised state by the ear-capsule method (Grant and Rothschild 1934). A black ring, 3 mm. in diameter, was painted on the rubber diaphragm of the capsule. The diastolic pressure was taken as the point when pressure within the capsule was just sufficient to exclude blood from the earartery beneath the ring once during every cardiac cycle. The systolic pressure was taken as the point when blood was excluded throughout the cycle. The relation of these readings to absolute pressures is uncertain, but there is little reason to doubt that changes in the readings reflect changes in the underlying absolute pressures. Initial difficulty was experienced on account of varying vasomotor activity and fluctuations of pressure due to fear or excitement (Stott 1966). The readings became much more consistent after (1) the rabbits were examined in the cage room itself; (2) they were restrained only loosely in an open-topped box; (3) at least 5 minutes were allowed to elapse between lifting the rabbits from their cages and taking the first reading;
171 and (4) all readings were taken after 5 P.M. when the animal house was quiet. During an initial control period, each morning each rabbit was weighed and given an intramuscular injection of ethyl oleate. Each evening five successive readings of diastolic and systolic pressures were taken and the means calculated. After the control period, (1) A and B (the two males) were given 5 mg. per day of progesterone (B.D.H., ’Progestin’) for 5 days, followed by 100 mg. daily for 6 days and ethyl oleate for 9 days; (2) C and D acted as controls, and received daily ethyloleate injections for the duration of the experiment; (3) E received 25 mg. progesterone per day for 10 days, ethyl oleate for 20 days, followed by 25 mg. progesterone for 13 days after which it died; (4) F received 25 mg. progesterone for 47 days, followed by 50 mg. for 15 days; (5) G received 25 mg. progesterone for 47 days, followed by ethyl oleate for 15 days; and (6) H received 25 mg. for 10 days, ethyl oleate for 20 days, 25 mg. progesterone for 46 days, and 50 mg. for 15 days. At the end of the experiment the five surviving New Zealand White rabbits were killed by an overdose of pentobarbitone sodium (’ Nembutal ’) and were examined post mortem. Tissue from brain, lung, kidney, liver, adrenal, muscle, and fat was immediately fixed in formol-saline solution, and prepared for histological examination. 50 µ sections were stained with hæmatoxylin and eosin and with periodic-acid/Schiff.
Arterial Pressure
Results
The pilot experiment on rabbits A and B revealed that 5 mg. progesterone per day tended to lower arterial pressure, and 100 mg. per day raised it to 10-25 mm. Hg above control levels. A dose of 25 mg. per day (6-10 mg. per kg.) was decided on. This is comparable to the amount of progesterone secreted by healthy women in the third trimester of pregnancy. In the control rabbits C and D (fig. la) changes in arterial pressure were not significant. Their weight-gain was smooth. In the remaining four rabbits, both diastolic and systolic pressures rose during periods of progesterone injection. The rises took 3-5 days to become apparent, and only reached plateau levels after 2-3 weeks. Cessation of progesterone treatment was followed by a slow fall to control levels. Each rabbit had a sharp but transient increase in the rate of weight-gain at the beginning of progesterone treatment. In rabbit E, during the first period of progesterone injection both systolic and diastolic pressures rose to 15-20 mm. Hg above control levels. During the second control period, arterial pressures fell to normal after 7 days. Pressure was again rising during the second period of progesterone injection when the rabbit died.
Fig. 2-Glomerulus from obliteration of capsular
rabbit F space
showing swelling of tuft with (haemotoxylin and eosin, x 350).
In rabbit F, after 3 weeks of progesterone injections, diastolic pressure reached a plateau about 15 mm. Hg above control and systolic pressure a plateau about 20 mm. Hg above control. These levels were maintained until 15 days before the rabbit was killed when the progesterone dose was increased: a further slight rise occurred during this period. In rabbit G, after 3 weeks’ progesterone treatment, diastolic pressure reached a plateau about 20 mm. Hg above control and systolic pressure a plateau about 35 mm. Hg above control. 15 days before the rabbit was killed, the progesterone treatment was stopped. During this time systolic pressure fell to within 10 mm. Hg of control and diastolic pressure to within 5 mm. Hg of control. The changes in arterial pressure in rabbit H are shown in fig. lb. Postmortem Examination
No abnormalities were found in the control rabbits C and D. E died of acute pulmonary cedema, bloodstained foam exuding from its mouth and nostrils just before death. The lungs themselves were oozing blood, and there was blood in the pleural cavity. There was a greyish exudate on the lung surface. The liver appeared congested. G showed no abnormalities, apart from some small grey patches of exudate on the lung surface. F and H had much larger patches of similar exudate; the lungs themselves were grossly congested.
Microscopy No abnormalities were detected in any organs other than the lungs, the kidneys, and the liver. In E. F. and H the lung
tissue was grossly congested, with red cells and much eosinophilic exudate in the alveoli. The lungs of G which had not received any progesterone for 15 days before death were relatively mildly affected. The most interesting and specific changes were seen in the kidneys. The glomerular tufts were enlarged, and in many cases the normal space between capsule and glomerulus was eliminated. Glomeruli throughout the kidneys were affected, those in the outer cortex being
severely damaged. The enlargement seemed mainly due to a swelling of the endothelial cells without proliferation.
most
Fig. 1—(a)
arterial pressures in the control rabbit D; (b) arterial pressure changes in rabbit H.
172
As a result, capillary lumens were narrowed or obliterated, and few red cells were present. In some glomeruli, clear vacuoles were seen (fig. 2) apparently within endothelial cells. In the periodic-acid/Schiff sections, the basement membrane appeared to be blurred in the treated rabbits. Of all these changes, the only one which can be quantitatively expressed is the relation between the diameter of the glomerulus and the diameter of its capsule. In each of the four progesterone-treated and two control rabbits, one hundred capsules and glomeruli were measured. In each case two diameter readings were taken at 90° to one another. The means of the two readings for the capsule and the two for the tuft were then calculated. For each malpighian corpuscle, the relation between capsular size and glomerular size was expressed as the ratio of the mean glomerular diameter to the mean capsular diameter. The mean of the hundred ratios was then calculated for each rabbit (see accompanying table). All the treated rabbits
(100 MALPIGHIAN RABBIT)
MEAN RATIOS OF GLOMERULAR TO CAPSULAR DIAMETER CORPUSCLES MEASURED IN EACH
could thus cause all the features of The oedema could be due to dilatation of arterioles with raised capillary pressure and increased filtration. If the high blood-pressure damages organs such as the placenta and kidney, other pressor agents may be released, maintaining the hypertension even if progesterone output falls owing to placental injury. The hypothesis can explain the puzzling features of pre-eclampsia and
Progesterone pre-eclampsia.
eclampsia. 1. A large and/or active placenta is associated with preeclampsia (Jeffcoate and Scott 1959, Jeffcoate 1966). There is a higher incidence of pre-eclampsia in twin pregnancies, in hydrops foetalis, in diabetes, and in hydatidiform mole. In each case, the placenta is large and/or secretes large amounts of human chorionic gonadotrophin (H.c.G.) (Hellman and Hertig 1938, Smith and Smith 1940, ten Berge and van Assen 1947, Eton and Short 1960, Chun et al. 1964). Since the placenta is the major source of progesterone and since H.C.G. stimulates progesterone output, the relation between these disorders is logical. Even in otherwise uncomplicated pregnancies, the teaching that in pre-eclampsia the placenta is small has been found to be untrue (Scott 1958, McClure Browne 1964,
Chakravarty 1967).
showed higher ratios than the two control rabbits. The difference between the mean in the control rabbit with the higher ratio and that in the treated rabbit with the lowest ratio was highly significant (p < 0-001). In each of the progesterone-treated rabbits, the liver was mildly congested but showed no other abnormalities, except in rabbit F, where the liver was grossly laden with glycogen, an appearance typical of that seen in a deliberately overfed animal. The progesterone may have overstimulated the rabbit’s appetite. Discussion
These experiments support the hypothesis that the initial rise in arterial pressure in pre-eclampsia is caused by progesterone. The mechanism of the pressure rise is uncertain. Progesterone antagonises aldosterone and promotes renal loss of sodium (Landau and Lugibihl 1961). Apparently in compensation, the aldosterone output rises (Gerrall et al. 1960), possibly mediated by the reninangiotensin mechanism. However, renin levels do not appear to be raised in pre-eclampsia above the levels in normal pregnancy (Brown et al. 1966). Patients with preeclampsia may be more responsive to normal angiotensin levels (Chesley 1965), but whether progesterone is implicated in this sensitisation is unknown. Another possibility is that progesterone could act by relaxing smooth-muscle tone in the carotid-sinus wall. Locally administered smooth-muscle relaxants raise blood-pressure in this way (Landgren et al. 1952) but as yet no parenterally administered agent has been found to operate thus. The renal lesions in the progesterone-treated rabbits are similar to those in human kidneys which have been described as specific to pre-eclampsia (Pollak and Nettles 1960)-namely, swelling and ischaemia of the glomerular tuft as a whole, swelling of the endothelial cells without multiplication, and the formation of vacuoles. In severe cases the basement membrane may be thickened. This description could be applied, virtually without alteration, to the changes seen in the treated rabbits.
2. In otherwise uncomplicated cases, pre-eclampsia is much commoner in first pregnancies. This may be because the liver is less able effectively to inactivate the large amounts of progesterone when it meets them for the first time. Macnaughton and Greig (1965) have demonstrated that the rate of conversion of tritiated progesterone to labelled pregnanediol is less in pre-eclampsia and in hydatidiform mole than in normal pregnancy. 3. Women who gain weight rapidly in pregnancy tend to become oedematous and hypertensive and to have large babies (Hytten et al. 1966, Lancet 1967, MacGillivray 1967, Thomson et al. 1967). This is logical if large placentas are associated with large babies and a high progesterone output. 4. The blood-volume is lower in pre-eclampsia than in normal pregnancy. This could be partly due to increased renal loss of sodium and water (Landau and Lugibihl 1961) and partly due to increased capillary filtration. 5. Many eclamptic fits occur after parturition. Progesterone can cause sleep and anaesthesia in animals and man (Selye 1941 a, b, Costa and Bonnycastle 1952, Merryman et al. 1954, Woolley and Timiras 1962, Heuser 1967). In epileptics, the luteal phase of the menstrual cycle and progesterone treatment are both associated with a low seizure-rate (Laidlan 1956, Logothetis et al. 1959). Menstruation and the days immediately preceding menstruation, when the blood progesterone concentration is falling (Woolever 1963) are accompanied by an increased seizure-rate. The depression brought about by progesterone seems to be followed by a rebound excitation. By contrast, in animals dehydroepiandrosterone has been found to precipitate convulsions (Heuser 1967). The concentration of dehydroepiandrosterone is low in the blood in pregnancy, because it is converted to oestrogens by the placenta (Gardner et al. 1954, Migeon 1954, Baulieu and Dray 1963, Siitterei and Macdonald 1963, Bolte et al. 1964). After expulsion of the placenta plasma concentrations return to normal. At parturition, convulsions are likely, because the concentration of the anxsthetic, progesterone, is falling while that of the convulsant, dehydroepiandrosterone, is rising. I should like to thank Prof. A. D. M. Greenfield and Prof. P. J. Huntingford for much advice and encouragement. Professor
173 Greenfield very
kindly
put
at my
disposal
the facilities of the
physio-
logy department at St. Mary’s Hospital; Dr. J. G. Bate of the Samaritan Hospital gave me much help with the interpretation of the histological changes and prepared the photograph; Miss Sylvia Legg
prepared the
sections. REFERENCES
Baulieu, E. E., Dray, F. (1963) J. clin. Endocr. 23, 1298. Bolte, E., Marcuso, S., Friksson, G., Wiqvist, N., Diczfalusy, E. (1964) Acta endocr. Copenh. 45, 560. Brobeck, J. R., Wheatland, M., Straminger, J. L. (1947) Endocrinology, 40, 65.
Brown, J. J., Davies, D. L., Doak, P. B., Lever, A. F., Robertson, J. I. S., Trust, P. (1966) J. Obstet. Gynœc. Br. Commonw. 73, 410. Chakravarty, A. P. (1967) ibid. 74, 247. Chesley, L. C. (1965) Bull. N.Y. Acad. Med. 41, 811. Chun, D., Braga, C., Chow, C., Lok, L. (1964) J. Obstet. Gynœc. Br. Commonw. 71, 180. Costa, P. J., Bonnycastle, D. D. (1952) Archs intern. Pharmacodyn. 91, 330. Dewar, A. D. (1957a) J. Endocr. 15, 216. (1957b) ibid. p. 230 Eton, B., Short, R. V. (1960) J. Obstet. Gynœc. Br. Commonw. 67, 785. Gardner, L. I., Wetton, R. L., Ellis, W., Hughes, E. C. (1954) Proc. Soc. exp. Biol. Med. 86, 804. Gerrall, A. G., Robertson, M. E., Laidlaw, J. C. (1960) Acta endocr. Copenh. suppl. 51, 157. Gilbert, C., Gillman, J. (1956) S. Afr. J. med. Sci. 21, 75. Grant, R. T., Rothschild, P. (1934) J. Physiol. 81, 265. Hellman, L. M., Hertig, A. (1938) Am. J. Path. 14, 111. Heuser, G. (1967) Anœsthesiology, 28, 173. —
Preliminary Communications INHIBITION OF ANTIPLASMIN, AND FIBRINOLYTIC EFFECT OF PROTEASE IN PATIENTS WITH CANCER
Hytten, F. E., Thomson, A. M., Taggart, N. R. (1966) J. Obstet. Gynœc. Br. Commonw. 73, 553. Jeffcoate, T. N. A. (1966) Proc. R. Soc. Med. 59, 397. Scott, J. S. (1959) Am. J. Obstet. Gynec. 77, 475. Laidlan, J. (1956) Lancet, ii, 1235. Lancet (1967) ii, 197. Landau, R. L., Lugibihl, K. (1961) Rec. progr. Hormone Res. 27, 249. Landgren, S., Neil, E., Zotterman, Y. (1952) Acta physiol. scand. 25, 24. Logothetis, J., Korner, R., Morrell, F., Torres, F. (1959) Neurology, 9, 352. McClure Browne, J. C. (1964). Cited in Lewis, T. L. T. (1964) Progress in Clinical Obstetrics and Gynæcology. London. MacGillivray, I. (1967) Scot. med. J. 12, 237. Macnaughton, M. C., Greig, M. (1965) J. Obstet. Gynœc. Br. Commonw. 72, 1029. Merryman, M., Boiman, R., Barnes, L., Rothchild, I. (1954) J. clin. Endocr. 14, 1567. Migeon, C. J. (1964). Cited in Gardner, L. I., Welton, R. L., Ellis, W., Hughes, E. C. (1954) Proc. Soc. exp. Biol Med. 86, 804. Pollak, V. E., Nettles, J. B. (1960) Medicine, 39, 469. Scott, J. S. (1958) J. Obstet. Gynœc. Br. Emp. 65, 689. Selye, H. (1941a) Proc. Soc. exp. Biol. Med. 46, 116. (1941b) J. Immunol. 41, 259. Siitterei, P. K., Macdonald, P. C. (1963) Steroids, 2, 713. Smith, O. W., Smith, G. V. (1940) Proc. Soc. exp. Biol. Med. 44, 104. Southam, A. L., Gonzaga, F. P. (1965) Am. J. Obstet. Gynec. 91, 142. Stott, F. D. (1966) Biomed. Eng. 1, 544. ten Berge, B. S., van Assen, F. J. J. (1947) Ned. Tijdschr. Geneesk. 91, 1229. Thomson, A. M., Hytten, F. E., Billewicz, W. J. (1967) J. Obstet. Gynœc. Br. Commonw. 74, 1. Woolever, C. H. (1963) Am. J. Obstet. Gynec. 85, 981. Woolley, D. E., Timiras, P. S. (1962) Endocrinology, 70, 196. —
—
endogenous fibrinolytic system: a fibrinolytic enzyme from Aspergillus oryzce fulfilled both these requirements. This enzyme has been called aspergillin O,9 and protease i.10 MATERIALS AND METHODS
Materials
CA-7, protease I.-The fibrinolytic enzymes of A.
oryzae
containing
C2 (Connaught) units per mg. dry substance, as CA-7 (Connaught Laboratories, Toronto, lot 1016-2) or protease I (Astra, Sweden, lot 17047). 1000
Protease
I (a fibrinolytic enzyme from Aspergillus oryzœ) inhibited thromboplastin and antiplasmin activity in vitro; and the antiplasmin inhibitory activity is potentiated in vitro by human thromboplastin. The in-vivo effects of this enzyme were examined in patients with cancer, since thromboplastin activity is raised in cancer tissue, and such patients have increased levels of antiplasmin. Thirteen patients
Summary
with
with occlusive vascular disease were of protease. Side-effects were infusions given mild and transient, except for pathological fibrinolysis (in three cases on a high dose) and a coagulative defect in one case. Subjective improvement was noted, but it is too early to judge the effects of therapy. cancer
and
one
one or more
INTRODUCTION
THE induction of
adequate anticoagulation or fibrinolysis by heparin, dicoumarol, or plasmin in animals decreases the metastatic spread of blood-borne cancer cells.1-5 The lowering of prothrombin levels by warfarin sodium inhibits the locomotion and growth of V2 carcinoma cells in rabbit tissues.6 The thromboplastin activity of cancer tissue is many times greater than that of normal tissue,and the inhibitors of fibrinolysis, the antiplasmins, are increased in patients with cancer.8 We have been looking for an agent which would inhibit this thromboplastic activity of human cancer tissue, and decrease the antiplasmins and thus enhance the 1. 2. 3.
4. 5. 6.
7. 8.
Terranova, T., Chissone, F. Boll. Soc. ital. Biol. spér. 1952, 23, 1224. Lacour, F., Oberling, C., Guerin, M. Bull. Ass. fr. Cancer, 1955, 42, 531. Wood, S., Jr., Holyoke, E. D., Yardley, J. M. Proc. Am. Ass. Cancer Res. 1956, 2, 157. Grossi, C. E., Agostino, D., Cliffton, E. E. Cancer Res. 1960, 20, 605. Fisher, B., Fisher, E. R. Surgery, St. Louis, 1961, 50, 240. Thornes, R. D. in Endogenous Factors Influencing Host Tumour Balance (edited by R. W. Wissler, T. L. Dao, S. Wood, Jr.); p. 255. Chicago, 1967. O’Meara, R. A. Q., Thornes, R. D. Ir. J. med. Sci. 1961, 423, 106. Thornes, R. D., O’Donnell, J. M., O’Brien, D. J. ibid. 1967, 494, 73.
Plasmin.-’ Kabi 1398 ’, human plasmin grade " b " measured in casein units (Sgouris) lot LmD 3. Reference serum.-Pooled sera from 20 healthy people stored at - 20°C and diluted 1/8 in saline solution at time of use. Bovine fibrinogen.-Kabi grade " B1"lot QdX 19, 0,2%. Stored as 0-6% in 2-8% w/v sodium chloride at ň20°C, thawed, and diluted 1 J3 with tris(hydroxymethyl) aminomethane (" tris ") buffer pH 7-4. Thrombin.-Thrombin, topical (bovine origin) (Parke Davis & Co.), 50 units per ml. In tris buffer at pH 7-4, stored at -20°C. " Normal saline ".-0.9% w/v sodium-chloride solution (clinical,
Antigen Ltd.). .Hfpat-tM.ň’ Pularin’(Evans Ltd., batch G15840). e-aminocaproic acid.-’ Epsikapron ’ (Kabi, lot 84850). Trasylol’.-Protease inhibitor (FBA Pharmaceuticals), 25,000 kallikrein-inactivating units per ampoule. ’
Methods Methods were as follows: antiplasmin activity, 8 dilute blood-clot lysis-time (B.L.T.)," and fibrinogen.12 Human thromboplastin was prepared from fresh frozen human chorion and titrated by the method of O’Meara and Thomes,7 and has similar activity to the thromboplastin extracted from cancer tissue (cancer coagulative factor, c.C.F.).13 Bedside test.-Measured by Thornes’ method.14 Protease resistance (serum-level of inhibitor to protease) was estimated by Roschlau’s method 15 modified to use 0-1ml. of whole blood instead of 1-0 ml. and 1/10 of the amount of protease (CA-7). To facilitate the reading of the end-point of titration, water is added to the tubes after 10 minutes incubation, and the percentage lysis can be easily assessed by inversion of the tubes. The results are expressed as " test tube requirement " (T.T.R.) units. 9. 10. 11. 12. 13.
14. 15.
Stefanini, M., Adamis, D. M., Soardi, F., Marin, H. M., Mele, R. Lancet, 1959, ii, 443. Bergkvist, R. Acta chem. scand. 1963, 17, 1521. Fearnley, G. R., Balmforth, G., Fearnley, E. B. Clin. Sci. 1957, 16, 645. Blombäck, B., Blombäck, M. Arkiv. Kemi. 1956, 10, 415. Boggust, W. A., O’Brien, D., O’Meara, R. A. Q., Thornes, R. D. Ir. J. med. Sci. 1963, 447, 131. Thornes, R. D. J. Ir. med. Ass. 1963, 53, 194. Roschlau, W. H. E. Can. J. Physiol. Pharmac. 1964, 42, 109.