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FIBRINOLYTIC ACTIVITY OF LUNG TISSUE IN RENAL FAILURE
191
relapses. In most cases more than 10 mg. daily was required to delay or prevent periods of active disease judged biochemically. Presuming that relapses are deleterious, prolonged treatment with prednisone in cirrhosis of the liver is iustified and advisable. REFERENCES
Harvald, B., Madsen, S. (1959) Nord. Med. 61, 431. (1961) Acta med. scand. 169, 381. Keith, S. H., Pollard, M. H. (1955) J. Lab. clin. Med. 46, 785. Kind, P. R. N., King, E. J. (1954) J. clin. Path. 7, 322. Owren, P. A., Aas, K. (1951) Scand. J. clin. Lab. Invest. 3, 201. Wells, R. (1960) Lancet, ii, 1416. With, T. K. (1942) Nord. Med. 13, 721. — —
FIBRINOLYTIC ACTIVITY OF LUNG TISSUE IN RENAL FAILURE M. MACLEOD Aberd., F.R.C.P.E.
M.D.
SENIOR LECTURER IN MEDICINE
A. L. STALKER M.D. Aberd.
showed
no
macroscopic
D. OGSTON M.A., M.B. Aberd. GARDEN RESEARCH FELLOW, DEPARTMENT OF MEDICINE
UNIVERSITY OF ABERDEEN
THE ability of most tissues to lyse fibrin has been known for many years (Fleisher and Loeb 1915). It has been shown that such activity is due to an activator of plasminogen-the precursor of the active proteolytic enzyme plasmin (Astrup and Permin 1947). Tissues vary in their
plasminogen-activator content : large amounts are normally present in uterus, lymph-nodes, prostate, thyroid, and lung; and smaller amounts in kidney, muscle, and testis (Albrechtsen 1957). The presence of plasminogen activator in tissue suggests that it may have a role in the removal of unwanted fibrin. Patients who die from renal failure may show not only pulmonary features due to oedema but also a fibrinous intra-alveolar exudate. This exudate becomes hyalinised to form membranes lining the sacs, and dense plugs in the alveolar ducts. Fibroblastic organisation occurs, and may be seen in all degrees. No convincing mechanism for this organisation has yet been suggested. We have therefore investigated the fibrinolytic activity of lung tissue from patients who have died in renal failure. ,
Methods
Portions of lungs were obtained at necropsy from patients who died in renal failure, and, as controls, from others who died of conditions not associated with renal failure. The specimens, taken from areas of the upper lobe which
were
stored
Estimation
of Fibrinolysis Fibrinolytic activity of lung-tissue fragments, and of extracts, was determined by a modification of the fibrin-plate method of Astrup and Mullertz (1952). Pooled human oxalated plasma, diluted 1/1 with veronal-acetate/hydrochloric-acid buffer at pH -7-4, was clotted in plastic petri plates by the addition of thrombin containing calcium chloride. Because the activity on unheated fibrin plates measures not only the plasminogen activator but also other proteolytic enzymes, glass plates heated to 85°C to destroy the plasminogen were also used. Such plates become insensitive to plasminogen activator. Fibrinolytic Activity of Tissue Duplicate samples of lung tissue, weighing 20-25 mg. after thawing, were placed on the fibrin plates and incubated at 37°C for twenty-four hours. The fibrinolytic activity of the tissue fragments was estimated in both heated and unheated plates by measuring the mean width of lysis from the edge of the tissue. A zone of lysis of less than 2 mm. from the tissue border was recorded as ++.
SENIOR LECTURER IN PATHOLOGY
evidence of infection,
at -20°C until used.
as a
trace, 2-4
mm. as
+, and
over
4
mm.
Fibrinolytic Activity of Potassium-thiocyanate extracts Astrup and Stage (1952) claimed that quantitative extraction of tissue activator could be obtained with potassiumthiocyanate. The method we used is a modification of that of Astrup and Albrechtsen (1957). Weighed samples of lung tissue were homogenised in 10 volumes of 2M potassiumthiocyanate. After standing for three hours at 4°C the extracts were filtered through Whatman no. 1 filterpaper, and 0 05 ml. aliquots were applied to fibrin plates which were incubated at 37°C for twenty-four hours. Two diameters of the zone of lysis were measured at right-angles to each other. Their product, in square millimetres, expressed the fibrinolytic activity of the extract.
Fibrinolytic Activity of Saline Homogenates Samples of lung were homogenised in 10 volumes of 0-85% saline, and the homogenate was allowed to stand for three hours at 4°C. After being filtered through cotton gauze to remove gross particles, it was treated as described by Lieberman (1961) for the detection of inhibitor in both the sediment and supernatant fractions. The plates were incubated at 370C for twentyfour hours, and the fibrinolytic activity was recorded as for the thiocyanate extracts. A significant reduction in the fibrinolytic activity of the control sediment fraction was taken to indicate the presence of an inhibitor. Results Tissue Lung
Table i summarises the clinical and histological features of nine patients who died in renal failure (" urxmic "). All showed much glomerular damage. Four of these lungs had no demonstrable plasminogen activator, and the other
TABLE I-THE NINE CASES OF RENAL FAILURE
192 TABLE II-PLASMINOGEN-ACTIVATOR ACTIVITY OF CONTROL AND URaeMIC LUNGS
This defect may be important in the formation of the excessive fibrinous exudate commonly seen in renal failure. REFERENCES
Albrechtsen, O. K. (1957) Brit. J. Hœmat. 3, 284. Astrup, T., Albrechtsen, O. K. (1957) Scand. J. clin. Lab. Invest. 9, 233. Müllertz, S. (1952) Arch. Biochem. 40, 346. Permin, P. M. (1947) Nature, Lond. 159, 681. — Stage, A. (1952) ibid. 170, 929. Fleisher, M. S., Loeb, L. (1915) J. biol. Chem. 21, 477. Lieberman, J. (1961) New Engl. J. Med. 265, 363. —
—
a trace. Fourteen patients who died of associated with renal failure were studied also (" control "). All showed the plasminogen activator in samples of lung tissue (table 11). These tests were repeated on numerous occasions with the same result. Samples of five control and six urxmic lungs were tested for proteolytic activity on heated plates. None of these showed any activity.
five had
only
causes not
Potassium-thiocyanate Extracts Potassium-thiocyanate extracts from six control lungs and seven ur2emic lungs were tested for activity on unheated fibrin plates. The six controls gave lytic values ranging from 150 to 256 sq. mm., with a mean of 197; the range of the seven uraemic lungs was from 56 to 203 sq. mm., with a mean of 128.
Saline Homogenates The sediment and supernatant fractions of saline homogenates from three uraEmic lungs (previously found to have no whole-tissue fibrinolytic activity) were tested for inhibitor. An inhibitor was detected in both of these fractions in two of the samples, and in the supernatant fraction of the third. Discussion
Tissue fragments were used to detect " available" tissue plasminogen activator. The results from control and uraemic samples indicate a virtual absence of " available " activator in the lungs of patients who died in renal failure. KSCN was used to extract activator completely from lung tissue. As compared with tissue fragments, the difference between urxmic and control potassiumthiocyanate extracts was smaller. It thus follows that, while the amount of activator may be reduced in uraemic lungs, significant amounts are still present; and the defect" in fibrinolytic activity is associated with " unavailability rather than with an absolute deficiency of tissue activator. The evident activity of ureamic potassium-thiocyanate extracts may be the result of the splitting of an activatorinhibitor complex. In a preliminary study, using saline homogenates, inhibitor was found in uraemic lungs. It seems likely that the tissue activator may be "unavailable" because an inhibitor is present, but this conclusion must be tentative until more is known about the nature and precise measurement of the inhibitors of fibrinolysis. This defect in the fibrinolytic enzyme system of lung tissue may be important in the pathogenesis of the fibrinous intra-alveolar exudate which appears in renal failure.
Summary
fibrinolytic activity of lung tissue has been studied by means of tissue fragments, potassium-thiocyanate extracts, and saline homogenates, in a series of lungs from patients who died in renal failure, and from a control The
group without renal failure. In renal failure there is a defect in the fibrinolytic enzyme system of lung tissue, and this seems more likely to be due to inhibition of activator than to its absolute
deficiency.
THE RECOGNITION OF
HUMAN BLOOD CHIMÆRAS M. F. A. WOODRUFF M.D., M.S. Melb., F.R.C.S. PROFESSOR
MILES Fox Ch.M. Manc., F.R.C.S. M.R.C. RESEARCH FELLOW
DEPARTMENT OF SURGICAL SCIENCE AND THE M.R.C. RESEARCH GROUP ON TRANSPLANTATION, UNIVERSITY OF EDINBURGH
KARIN A. BUCKTON
PATRICIA A.
B.Sc. St. And.
JACOBS
B.Sc. St. And.
OF THE M.R.C. CLINICAL EFFECTS OF RADIATION RESEARCH
WESTERN GENERAL
HOSPITAL,
UNIT,
EDINBURGH
A BLOOD chimasra may be defined as an individual who has in his blood-stream cells which are the lineal descendants of cells transplanted from another individual. Sometimes the foreign cells are derived from a twin, and the transplantation occurs spontaneously during intrauterine life because of anastomoses between the placental circulations of the developing fcetuses-in which event the individuals may be termed natural chimaeras. Chimxras may also be produced artificially under certain conditions by deliberate transplantation of haemopoietic tissue (for review see Woodruff 1960). Natural chimserism, though common among dizygotic cattle twins, is rare in man; but five cases have been recognised and reported (Dunsford et al. 1953, Booth et al. 1957, Nicholas et al. 1957). Artificial chimasrism is commonly produced in laboratory animals, especially mice, by transplanting ha:mopoietic tissue from an animal of the same species either to a normal immature (fcetal or newborn) recipient or to an adult which has been exposed to ionising radiation in high dosage or treated with various cytotoxic drugs. In man homotransplantation of haemopoietic tissue has now been performed in many patients, either after accidental irradiation or after radiotherapy or chemotherapy for the treatment of cancer, and some of the recipients have certainly become temporary chimxras; but so far there seems to have been only one case, reported by Beilby et al. (1960), in which the state of chimgerism was shown to have persisted for more than a few weeks. The methods which have been used in the diagnosis and investigation of human chimaeras are: Differential red-cell grouping. Nuclear sexing of polymorphonuclear Experimental skin grafting.
leucocytes.
To these we have now added another method: Chromosomal sexing of peripheral-blood leucocytes grown in tissue culture.
Differential Red-cell
Grouping
The natural human chimxras have all been recognised by finding red cells of two different kinds in their blood. All in fact possessed a mixture of group-A and group-0 cells, and it was possible to separate the two components and determine how they differed in respect of other blood-group systems (Booth et al. 1957). All, moreover, normally had no anti-A agglutinin in their serum, though
relapses. In most cases more than 10 mg. daily was required to delay or prevent periods of active disease judged biochemically. Presuming that relapses are deleterious, prolonged treatment with prednisone in cirrhosis of the liver is iustified and advisable. REFERENCES
Harvald, B., Madsen, S. (1959) Nord. Med. 61, 431. (1961) Acta med. scand. 169, 381. Keith, S. H., Pollard, M. H. (1955) J. Lab. clin. Med. 46, 785. Kind, P. R. N., King, E. J. (1954) J. clin. Path. 7, 322. Owren, P. A., Aas, K. (1951) Scand. J. clin. Lab. Invest. 3, 201. Wells, R. (1960) Lancet, ii, 1416. With, T. K. (1942) Nord. Med. 13, 721. — —
FIBRINOLYTIC ACTIVITY OF LUNG TISSUE IN RENAL FAILURE M. MACLEOD Aberd., F.R.C.P.E.
M.D.
SENIOR LECTURER IN MEDICINE
A. L. STALKER M.D. Aberd.
showed
no
macroscopic
D. OGSTON M.A., M.B. Aberd. GARDEN RESEARCH FELLOW, DEPARTMENT OF MEDICINE
UNIVERSITY OF ABERDEEN
THE ability of most tissues to lyse fibrin has been known for many years (Fleisher and Loeb 1915). It has been shown that such activity is due to an activator of plasminogen-the precursor of the active proteolytic enzyme plasmin (Astrup and Permin 1947). Tissues vary in their
plasminogen-activator content : large amounts are normally present in uterus, lymph-nodes, prostate, thyroid, and lung; and smaller amounts in kidney, muscle, and testis (Albrechtsen 1957). The presence of plasminogen activator in tissue suggests that it may have a role in the removal of unwanted fibrin. Patients who die from renal failure may show not only pulmonary features due to oedema but also a fibrinous intra-alveolar exudate. This exudate becomes hyalinised to form membranes lining the sacs, and dense plugs in the alveolar ducts. Fibroblastic organisation occurs, and may be seen in all degrees. No convincing mechanism for this organisation has yet been suggested. We have therefore investigated the fibrinolytic activity of lung tissue from patients who have died in renal failure. ,
Methods
Portions of lungs were obtained at necropsy from patients who died in renal failure, and, as controls, from others who died of conditions not associated with renal failure. The specimens, taken from areas of the upper lobe which
were
stored
Estimation
of Fibrinolysis Fibrinolytic activity of lung-tissue fragments, and of extracts, was determined by a modification of the fibrin-plate method of Astrup and Mullertz (1952). Pooled human oxalated plasma, diluted 1/1 with veronal-acetate/hydrochloric-acid buffer at pH -7-4, was clotted in plastic petri plates by the addition of thrombin containing calcium chloride. Because the activity on unheated fibrin plates measures not only the plasminogen activator but also other proteolytic enzymes, glass plates heated to 85°C to destroy the plasminogen were also used. Such plates become insensitive to plasminogen activator. Fibrinolytic Activity of Tissue Duplicate samples of lung tissue, weighing 20-25 mg. after thawing, were placed on the fibrin plates and incubated at 37°C for twenty-four hours. The fibrinolytic activity of the tissue fragments was estimated in both heated and unheated plates by measuring the mean width of lysis from the edge of the tissue. A zone of lysis of less than 2 mm. from the tissue border was recorded as ++.
SENIOR LECTURER IN PATHOLOGY
evidence of infection,
at -20°C until used.
as a
trace, 2-4
mm. as
+, and
over
4
mm.
Fibrinolytic Activity of Potassium-thiocyanate extracts Astrup and Stage (1952) claimed that quantitative extraction of tissue activator could be obtained with potassiumthiocyanate. The method we used is a modification of that of Astrup and Albrechtsen (1957). Weighed samples of lung tissue were homogenised in 10 volumes of 2M potassiumthiocyanate. After standing for three hours at 4°C the extracts were filtered through Whatman no. 1 filterpaper, and 0 05 ml. aliquots were applied to fibrin plates which were incubated at 37°C for twenty-four hours. Two diameters of the zone of lysis were measured at right-angles to each other. Their product, in square millimetres, expressed the fibrinolytic activity of the extract.
Fibrinolytic Activity of Saline Homogenates Samples of lung were homogenised in 10 volumes of 0-85% saline, and the homogenate was allowed to stand for three hours at 4°C. After being filtered through cotton gauze to remove gross particles, it was treated as described by Lieberman (1961) for the detection of inhibitor in both the sediment and supernatant fractions. The plates were incubated at 370C for twentyfour hours, and the fibrinolytic activity was recorded as for the thiocyanate extracts. A significant reduction in the fibrinolytic activity of the control sediment fraction was taken to indicate the presence of an inhibitor. Results Tissue Lung
Table i summarises the clinical and histological features of nine patients who died in renal failure (" urxmic "). All showed much glomerular damage. Four of these lungs had no demonstrable plasminogen activator, and the other
TABLE I-THE NINE CASES OF RENAL FAILURE
192 TABLE II-PLASMINOGEN-ACTIVATOR ACTIVITY OF CONTROL AND URaeMIC LUNGS
This defect may be important in the formation of the excessive fibrinous exudate commonly seen in renal failure. REFERENCES
Albrechtsen, O. K. (1957) Brit. J. Hœmat. 3, 284. Astrup, T., Albrechtsen, O. K. (1957) Scand. J. clin. Lab. Invest. 9, 233. Müllertz, S. (1952) Arch. Biochem. 40, 346. Permin, P. M. (1947) Nature, Lond. 159, 681. — Stage, A. (1952) ibid. 170, 929. Fleisher, M. S., Loeb, L. (1915) J. biol. Chem. 21, 477. Lieberman, J. (1961) New Engl. J. Med. 265, 363. —
—
a trace. Fourteen patients who died of associated with renal failure were studied also (" control "). All showed the plasminogen activator in samples of lung tissue (table 11). These tests were repeated on numerous occasions with the same result. Samples of five control and six urxmic lungs were tested for proteolytic activity on heated plates. None of these showed any activity.
five had
only
causes not
Potassium-thiocyanate Extracts Potassium-thiocyanate extracts from six control lungs and seven ur2emic lungs were tested for activity on unheated fibrin plates. The six controls gave lytic values ranging from 150 to 256 sq. mm., with a mean of 197; the range of the seven uraemic lungs was from 56 to 203 sq. mm., with a mean of 128.
Saline Homogenates The sediment and supernatant fractions of saline homogenates from three uraEmic lungs (previously found to have no whole-tissue fibrinolytic activity) were tested for inhibitor. An inhibitor was detected in both of these fractions in two of the samples, and in the supernatant fraction of the third. Discussion
Tissue fragments were used to detect " available" tissue plasminogen activator. The results from control and uraemic samples indicate a virtual absence of " available " activator in the lungs of patients who died in renal failure. KSCN was used to extract activator completely from lung tissue. As compared with tissue fragments, the difference between urxmic and control potassiumthiocyanate extracts was smaller. It thus follows that, while the amount of activator may be reduced in uraemic lungs, significant amounts are still present; and the defect" in fibrinolytic activity is associated with " unavailability rather than with an absolute deficiency of tissue activator. The evident activity of ureamic potassium-thiocyanate extracts may be the result of the splitting of an activatorinhibitor complex. In a preliminary study, using saline homogenates, inhibitor was found in uraemic lungs. It seems likely that the tissue activator may be "unavailable" because an inhibitor is present, but this conclusion must be tentative until more is known about the nature and precise measurement of the inhibitors of fibrinolysis. This defect in the fibrinolytic enzyme system of lung tissue may be important in the pathogenesis of the fibrinous intra-alveolar exudate which appears in renal failure.
Summary
fibrinolytic activity of lung tissue has been studied by means of tissue fragments, potassium-thiocyanate extracts, and saline homogenates, in a series of lungs from patients who died in renal failure, and from a control The
group without renal failure. In renal failure there is a defect in the fibrinolytic enzyme system of lung tissue, and this seems more likely to be due to inhibition of activator than to its absolute
deficiency.
THE RECOGNITION OF
HUMAN BLOOD CHIMÆRAS M. F. A. WOODRUFF M.D., M.S. Melb., F.R.C.S. PROFESSOR
MILES Fox Ch.M. Manc., F.R.C.S. M.R.C. RESEARCH FELLOW
DEPARTMENT OF SURGICAL SCIENCE AND THE M.R.C. RESEARCH GROUP ON TRANSPLANTATION, UNIVERSITY OF EDINBURGH
KARIN A. BUCKTON
PATRICIA A.
B.Sc. St. And.
JACOBS
B.Sc. St. And.
OF THE M.R.C. CLINICAL EFFECTS OF RADIATION RESEARCH
WESTERN GENERAL
HOSPITAL,
UNIT,
EDINBURGH
A BLOOD chimasra may be defined as an individual who has in his blood-stream cells which are the lineal descendants of cells transplanted from another individual. Sometimes the foreign cells are derived from a twin, and the transplantation occurs spontaneously during intrauterine life because of anastomoses between the placental circulations of the developing fcetuses-in which event the individuals may be termed natural chimaeras. Chimxras may also be produced artificially under certain conditions by deliberate transplantation of haemopoietic tissue (for review see Woodruff 1960). Natural chimserism, though common among dizygotic cattle twins, is rare in man; but five cases have been recognised and reported (Dunsford et al. 1953, Booth et al. 1957, Nicholas et al. 1957). Artificial chimasrism is commonly produced in laboratory animals, especially mice, by transplanting ha:mopoietic tissue from an animal of the same species either to a normal immature (fcetal or newborn) recipient or to an adult which has been exposed to ionising radiation in high dosage or treated with various cytotoxic drugs. In man homotransplantation of haemopoietic tissue has now been performed in many patients, either after accidental irradiation or after radiotherapy or chemotherapy for the treatment of cancer, and some of the recipients have certainly become temporary chimxras; but so far there seems to have been only one case, reported by Beilby et al. (1960), in which the state of chimgerism was shown to have persisted for more than a few weeks. The methods which have been used in the diagnosis and investigation of human chimaeras are: Differential red-cell grouping. Nuclear sexing of polymorphonuclear Experimental skin grafting.
leucocytes.
To these we have now added another method: Chromosomal sexing of peripheral-blood leucocytes grown in tissue culture.
Differential Red-cell
Grouping
The natural human chimxras have all been recognised by finding red cells of two different kinds in their blood. All in fact possessed a mixture of group-A and group-0 cells, and it was possible to separate the two components and determine how they differed in respect of other blood-group systems (Booth et al. 1957). All, moreover, normally had no anti-A agglutinin in their serum, though