1151
IMMUNE COMPLEXES IN MYELOPROLIFERATIVE DISORDERS G. D. PEGRUM
C. M. LEWIS
Hœmatology Department, Charing Cross Hospital Medical School, Fulham Palace Road, Hammersmith, London W6 8RF A fluorescein-labelled anti-human im-
Summary
munoglobulin was used to demonstrate that peripheral blood from patients with myelofibrosis had a high proportion of phagocytic cells containing fluorescent immune complexes. Cells from patients with other myeloproliferative diseases (either chronic myeloid leukæmia or polycythæmia rubra vera) did not show similar intracellular immune complexes. Serum from patients with myelofibrosis incubated with polymorphs from healthy subjects caused the appearance of inclusions similar to those found when the patients’ own cells were used, the healthy phagocytes apparently engulfing complexes from the patients’ sera. The presence of platelets or complement did not alter the incidence of intracellular fluorescence. These tests may help in the diagriosis of myelofibrosis and may also be valuable in recognising the onset of this condition in patients with polycythæmia rubra vera.
(P.B.S.) (Dulbecco A) containing 0-10% azide. Cells were resuspended in the P.B.S. at a concentration of about 107/ml. Direct Immunofluorescence with Cells from Patients and Healthy Subjects About 0.1 ml of the cell suspension was incubated at 4°C
saline
for 60 min with a 1/10 dilution of fluorescein-labelled-isothiocyanate (F.I.T.c.)-conjugated anti-human immunoglobulin (Wellcome). The cells were washed thoroughly, and slides were prepared and examined under incident ultraviolet light with a Leitz-Ortholux microscope. Incubation
of Leucocytes
with Serum
before Immunofluores-
cence
0.1ml of a suspension of healthy leucocytes was incubated 370C with 20 P of patients’ serum for 90 min. The cells were then washed three times and stained with the diluted F.I.T.C. anti-human immunoglobulin as described above. at
Experiments with Platelet-free Preparations Venous blood was defibrinated by swirling
with glass beads for 10-15 min. 5 ml of haemaccel was added, and the red cells were allowed to sediment out. Subsequent procedures were identical to those described above. Sera and Decomplementation
Samples of clotted blood were obtained at the same time as samples of heparinised blood, and serum was collected after centrifugation. Decomplementation was carried out by heating at 56°C for 30 min.
Introduction WE have recently demonstrated intracellular immune complexes in phagocytic cells from patients with myelofibrosis.’ We have now studied 14 such patients, and complexes were found in all. In several patients immunosuppressive chemotherapy led to both an improvement in their clinical condition and a reduction in the numbers of cells with large intracellular immune com-
plexes. complexes have been think that splenic Felty’s syndrome,2 itself is enlargement unlikely to be a crucial factor. One of the myelofibrotic patients who had undergone splenectomy showed exactly the same type of immunecomplex inclusion as the other patients. In addition, other conditions which give rise to massive splenomegaly
Although
similar intracellular
described in
we
Results Fluorescent Studies with Patients’
own Cells (see Figure) In leucocyte-rich preparations from healthy controls immune complexes were found in very few phagocytic cells-usually less than 1/100 cells. Among the myeloproliferative disorders studied, a similar proportion of cells containing fluorescent inclusions was found in the peripheral blood in chronic myeloid leukaemia and poly-
associated with immune-complex-bearing cells. We have now examined cells from a number of patients with myelofibrosis, chronic myeloid leukaemia, and polycythaemia rubra vera in order to determine whether or not these complexes are peculiar to myelofibrosis or can be found in other myeloproliferative disorders. We have also carried out a number of experiments with patients’ sera and healthy leucocytes to determine some of the characteristics of the immune complexes. We believe their presence in patients’ cells could result either from an inability of the phagocytes in myelofibrosis patients to handle complexes or from continuous production and removal from the plasma. are not
Patients and Methods We studied 23
patients with
a
definite
diagnosis
of
myelo-
proliferative disorders and 7 healthy subjects. Separation of White Blood-cells About 10 ml fresh heparinised blood was mixed with 5 ml polygeline (’Ha’maccel’, Hoechst) and allowed to stand for 15-30 min. The supernatant buffy-coat cells were removed, centrifuged, and washed three times in phosphate-buffered
Polycythaemia MyelofibrosIs Rubra vera
Chronic
Healthy
myeloid
controls
leukaemia
Frequency patients trols.
of immune complexes (log. scale) in leucocytes from with myeloproliferative disorders and healthy con-
1152 TABLE I-FLUORESCENT STUDIES WITH
PATIENTS’
LEUCOCYTES FROM HEALTHY
SUBJECTS
whether or not platelets were necessary for the formation of fluorescent inclusions. When sera from myelofibrotic patients were incubated with healthy plateletfree leucocytes obtained from defibrinated blood-samples there was no change in the proportion or appearance of cells bearing fluorescent inclusions (table n).
SERA AND
see
Role of Complement in Phagocytosis of Immune Complexes Decomplemented sera from myelofibrosis patients were as effective as whole sera in their ability to induce healthy leucocytes to phagocytose immune complexes (table n). Complement therefore seems unnecessary for the formation of the intracellular inclusions.
Discussion
’(+)>5‘-
inclusions, (++)>
10% inclusions, (-) < 1 % inclusions.
cythxmia rubra vera, but in myelofibrosis the range was much higher (5-50%). Fluorescent Studies with Patients’ Sera and Normal
Leucocytes (Table I) Immune complexes
in normal polymorphs after incubation in sera from patients with myelofibrosis. Negative results were obtained with sera from patients with polycythxmia rubra vera and chronic myeloid leukaemia. Healthy control sera were also uniformly
negative. Role of Platelets
were seen
in Formation
of Fluorescent Inclusions bind immune complexes by virtue of
Platelets can their Fc receptors, and
we were
therefore interested
to
TABLE II-INCUBATION OF HEALTHY POLYMORPHS WITH SERA FROM VARIOUS PATIENTS
*
(+)>5% fluorescent inclusions, (-) < 1 %. t Cells obtained from heparinised peripheral present. t Cells obtained from defibrinated blood. N.D.=not done. were
blood in which
platelets
Previously we demonstrated the presence of intracellular immune complexes in the phagocytic cells of patients with myelofibrosis. This suggested an autoimmune basis to the marrow fibrosis, and immunosuppressive therapy produced a significant remission of the accompanying refractory anxmia.1 The results presented in this paper indicate that immune complexes are present both in the patients’ phagocytic cells and also in their serum. There are therefore two tests which may be valuable for following the course of disease in these patients-(i) direct fluorescence of patients’ own leucocytes and (ii) indirect fluorescence of patients’ serum and healthy neutrophils. Any cell which has the necessary Fc receptors is capable of binding immune complexes, and the presence of these receptors on human platelets3 suggests that they too might be affected by the complexes in patients’ serum. There is, in fact, good evidence that platelet function is changed in many myelofibrotic patients (C. P. Tsagos and S. Wolffe, personal communication). In our experiments platelets did not appear to be important for the phagocytosis of complexes, for the large intracellular inclusions were evidently not produced from platelets coated with complexes. That complement was not required was expected, because phagocytosis is known to take place in the absence of complement.4 Our observations in myelofibrosis support other authors’ findings of various immunological abnormalities in this condition .5,6 It also adds weight to the suggestion, put forward many years ago, that bone-marrow fibrosis in myelofibrosis may have an autoimmune origin, analogous to cirrhosis of the liver or fibrosis in the thyroid. Our present results do not allow us to decide where the autoimmune antibody is directed, for measurement of the presence and uptake of complexes is clearly the endnoint in a reaction. Immunosuppressive therapy has produced a pronounced clinical improvement in some of our patients, and this is associated with a fall in the proportion of cells containing intracellular inclusions. We have not been able to demonstrate any improvement in bone-marrow trephine samples from the iliac crest, although haemoglobin levels have returned to normal. This is not surprising in view of the site and size of the sample obtained. The contention that it may be possible to reverse the fibrosisg in the marrow must await further evidence. A significant proportion of patients with polycythasmia rubra vera proceed to myelofibrosis,9,10 and it is
1153 sometimes difficult to decide when this change is taking place, For this reason we studied patients with established polycythaemia to see if any immunoglobulin cellinclusions could be found. So far, our examinations both of patients’ own leucocytes and patients’ sera plus healthy polymorphonuclear cells have been negative. None of these patients were considered to be "converting" to myelofibrosis. Among those whom we have classified as having myelofibrosis, 2 had established fibrosis yet still retained an increased red-cell mass. Both our techniques showed that these patients had immune complexes, which suggests that the immunological abnormalities associated with myelofibrosis may form at a relatively early stage in the disease. Clearly we need to study more patients with myeloproliferative disorders to determine when this change takes place. It may well be important for management, especially since we believe that myelofibrosis is better not treated with busulphan or phosphorus-32 at the stage of imminent myelofibrosis, because these agents may well aggravate the fibrosis in the bone-marrow. Examination of a patient’s white cells or serum for the presence of immune complexes might enable us to make an earlier diagnosis in the transition from polycythasmia rubra vera to myelofibrosis, so that more appropriate treatment can be instituted. Requests
for
reprints
should be addressed
to
G.D.P.
cebo-treated patients. Thus, (+)-cyanidanol-3 be of benefit in acute viral hepatitis.
Introduction AT present, there is no effective treatment for acute viral hepatitis. A new substance, the flavonoid, (+)cyanidanol-3,’ has been shown to decrease the hepatotoxicity of ethanol and other compounds in laboratory animals,2-4 and to be a powerful free-radical scavenger.s In addition, (+)-cyanidanol-3 has been reported to decrease serum-bilirubin in patients with acute viral hepatitiS.6-8 In order to establish the value of (+)-cyanidanol-3 in the treatment of acute viral hepatitis, a multicentre, double-blind trial was carried out. The results are
encouraging. Patients and Methods
A prospective, double-blind, placebo-controlled trial of cyanidanol-3 was carried out in patients with acute viral hepatitis in five departments of medicine. 106 consecutive adult inpatients with acute viral hepatitis were randomised. Patients with an alcohol consumption of more than 100 g/day and/or jaundice for more than 4 weeks before admission were excluded. Viral hepatitis was diagnosed on the history, clinical
examination and liver-function tests. If there had been jaundice in the previous year, or if the diagnosis was doubtful, a liver biopsy was carried out. All 11 biopsies were read by one
pathologist (H.P.). Treatment
REFERENCES 1. Lewis, C. M., Pegrum, G. D. Unpublished. 2. Hurd, E. R., Andreis, M., Ziff, M. Clin. exp. Immun. 1977, 28, 413. 3. Pfueller, S. L., Luscher, E. F. Immunochemistry, 1972, 9, 1151. 4. Mantovani, B., Rabinowitch, M., Nussenweig, V. J. exp. Med. 1972, 135, 780. 5. Boivin, P., Bernard, J. F., Hakim, J., Woroclans, M. Acta hœmat. 1974, 51, 91. 6. Lang, J. M., Obeiling, F., Mayer, S., Heid, E. Biomedicine, 1976, 25, 39. 7. Wyatt, J. P., Sommers, S. C. Blood, 1950, 5, 329. 8. Kelemen, E., Kraszanai, G., Endes, P., Szinay, G. Acta hœmat. 1972, 57, 171. 9. Minok, G. R., Buckman, T. E. Am. J. med. Sci. 1923, 166, 469. 10. Ikkala, E., Rapola, J., Kotilainen, M. Scand. J. Hœmat. 1967, 4, 453.
(+)-CYANIDANOL-3
A. L. BLUM W. DOELLE K. KORTÜM P. PETER G. STROHMEYER
P. H. S. H.
BERTHET GOEBELL
PELLONI
POULSEN N. TYGSTRUP
Departments of Medicine, Triemli Hospital, Zürich, Switzerland; University Hospital, Tübingen; University Hospital, Ulm; University Hospital, Düsseldorf, Federal
Republic of Germany; Rigshospitalet, Copenhagen and Hospital, University of Copenhagen, Denmark
Hvidorre
Summary
A double-blind trial of
(+)-cyanidanol-3 (2 g/day) versus placebo tablets was carried out in 100 patients with acute viral hepatitis. 51 received the drug and 49 placebo. (+)-Cyanidanol-3 accelerated the disappearance of HBsAg from the blood, lowered serum-bilirubin, and relieved symptoms such
was
started within 24 hours of randomisation.
(+)-cyanidanol-3 was given in a dose of 2 g daily for 4 weeks, and placebo in a dose of two tablets four times daily. No dietary restrictions were imposed, but patients with severe anorexia, nausea, and vomiting (8 on [+]-cyanidanol-3, 7 on placebo) received intravenous glucose. Likewise, bed rest was not imposed. Patients with prothrombin concentrations
IMMUNE COMPLEXES IN MYELOPROLIFERATIVE DISORDERS G. D. PEGRUM
C. M. LEWIS
Hœmatology Department, Charing Cross Hospital Medical School, Fulham Palace Road, Hammersmith, London W6 8RF A fluorescein-labelled anti-human im-
Summary
munoglobulin was used to demonstrate that peripheral blood from patients with myelofibrosis had a high proportion of phagocytic cells containing fluorescent immune complexes. Cells from patients with other myeloproliferative diseases (either chronic myeloid leukæmia or polycythæmia rubra vera) did not show similar intracellular immune complexes. Serum from patients with myelofibrosis incubated with polymorphs from healthy subjects caused the appearance of inclusions similar to those found when the patients’ own cells were used, the healthy phagocytes apparently engulfing complexes from the patients’ sera. The presence of platelets or complement did not alter the incidence of intracellular fluorescence. These tests may help in the diagriosis of myelofibrosis and may also be valuable in recognising the onset of this condition in patients with polycythæmia rubra vera.
(P.B.S.) (Dulbecco A) containing 0-10% azide. Cells were resuspended in the P.B.S. at a concentration of about 107/ml. Direct Immunofluorescence with Cells from Patients and Healthy Subjects About 0.1 ml of the cell suspension was incubated at 4°C
saline
for 60 min with a 1/10 dilution of fluorescein-labelled-isothiocyanate (F.I.T.c.)-conjugated anti-human immunoglobulin (Wellcome). The cells were washed thoroughly, and slides were prepared and examined under incident ultraviolet light with a Leitz-Ortholux microscope. Incubation
of Leucocytes
with Serum
before Immunofluores-
cence
0.1ml of a suspension of healthy leucocytes was incubated 370C with 20 P of patients’ serum for 90 min. The cells were then washed three times and stained with the diluted F.I.T.C. anti-human immunoglobulin as described above. at
Experiments with Platelet-free Preparations Venous blood was defibrinated by swirling
with glass beads for 10-15 min. 5 ml of haemaccel was added, and the red cells were allowed to sediment out. Subsequent procedures were identical to those described above. Sera and Decomplementation
Samples of clotted blood were obtained at the same time as samples of heparinised blood, and serum was collected after centrifugation. Decomplementation was carried out by heating at 56°C for 30 min.
Introduction WE have recently demonstrated intracellular immune complexes in phagocytic cells from patients with myelofibrosis.’ We have now studied 14 such patients, and complexes were found in all. In several patients immunosuppressive chemotherapy led to both an improvement in their clinical condition and a reduction in the numbers of cells with large intracellular immune com-
plexes. complexes have been think that splenic Felty’s syndrome,2 itself is enlargement unlikely to be a crucial factor. One of the myelofibrotic patients who had undergone splenectomy showed exactly the same type of immunecomplex inclusion as the other patients. In addition, other conditions which give rise to massive splenomegaly
Although
similar intracellular
described in
we
Results Fluorescent Studies with Patients’
own Cells (see Figure) In leucocyte-rich preparations from healthy controls immune complexes were found in very few phagocytic cells-usually less than 1/100 cells. Among the myeloproliferative disorders studied, a similar proportion of cells containing fluorescent inclusions was found in the peripheral blood in chronic myeloid leukaemia and poly-
associated with immune-complex-bearing cells. We have now examined cells from a number of patients with myelofibrosis, chronic myeloid leukaemia, and polycythaemia rubra vera in order to determine whether or not these complexes are peculiar to myelofibrosis or can be found in other myeloproliferative disorders. We have also carried out a number of experiments with patients’ sera and healthy leucocytes to determine some of the characteristics of the immune complexes. We believe their presence in patients’ cells could result either from an inability of the phagocytes in myelofibrosis patients to handle complexes or from continuous production and removal from the plasma. are not
Patients and Methods We studied 23
patients with
a
definite
diagnosis
of
myelo-
proliferative disorders and 7 healthy subjects. Separation of White Blood-cells About 10 ml fresh heparinised blood was mixed with 5 ml polygeline (’Ha’maccel’, Hoechst) and allowed to stand for 15-30 min. The supernatant buffy-coat cells were removed, centrifuged, and washed three times in phosphate-buffered
Polycythaemia MyelofibrosIs Rubra vera
Chronic
Healthy
myeloid
controls
leukaemia
Frequency patients trols.
of immune complexes (log. scale) in leucocytes from with myeloproliferative disorders and healthy con-
1152 TABLE I-FLUORESCENT STUDIES WITH
PATIENTS’
LEUCOCYTES FROM HEALTHY
SUBJECTS
whether or not platelets were necessary for the formation of fluorescent inclusions. When sera from myelofibrotic patients were incubated with healthy plateletfree leucocytes obtained from defibrinated blood-samples there was no change in the proportion or appearance of cells bearing fluorescent inclusions (table n).
SERA AND
see
Role of Complement in Phagocytosis of Immune Complexes Decomplemented sera from myelofibrosis patients were as effective as whole sera in their ability to induce healthy leucocytes to phagocytose immune complexes (table n). Complement therefore seems unnecessary for the formation of the intracellular inclusions.
Discussion
’(+)>5‘-
inclusions, (++)>
10% inclusions, (-) < 1 % inclusions.
cythxmia rubra vera, but in myelofibrosis the range was much higher (5-50%). Fluorescent Studies with Patients’ Sera and Normal
Leucocytes (Table I) Immune complexes
in normal polymorphs after incubation in sera from patients with myelofibrosis. Negative results were obtained with sera from patients with polycythxmia rubra vera and chronic myeloid leukaemia. Healthy control sera were also uniformly
negative. Role of Platelets
were seen
in Formation
of Fluorescent Inclusions bind immune complexes by virtue of
Platelets can their Fc receptors, and
we were
therefore interested
to
TABLE II-INCUBATION OF HEALTHY POLYMORPHS WITH SERA FROM VARIOUS PATIENTS
*
(+)>5% fluorescent inclusions, (-) < 1 %. t Cells obtained from heparinised peripheral present. t Cells obtained from defibrinated blood. N.D.=not done. were
blood in which
platelets
Previously we demonstrated the presence of intracellular immune complexes in the phagocytic cells of patients with myelofibrosis. This suggested an autoimmune basis to the marrow fibrosis, and immunosuppressive therapy produced a significant remission of the accompanying refractory anxmia.1 The results presented in this paper indicate that immune complexes are present both in the patients’ phagocytic cells and also in their serum. There are therefore two tests which may be valuable for following the course of disease in these patients-(i) direct fluorescence of patients’ own leucocytes and (ii) indirect fluorescence of patients’ serum and healthy neutrophils. Any cell which has the necessary Fc receptors is capable of binding immune complexes, and the presence of these receptors on human platelets3 suggests that they too might be affected by the complexes in patients’ serum. There is, in fact, good evidence that platelet function is changed in many myelofibrotic patients (C. P. Tsagos and S. Wolffe, personal communication). In our experiments platelets did not appear to be important for the phagocytosis of complexes, for the large intracellular inclusions were evidently not produced from platelets coated with complexes. That complement was not required was expected, because phagocytosis is known to take place in the absence of complement.4 Our observations in myelofibrosis support other authors’ findings of various immunological abnormalities in this condition .5,6 It also adds weight to the suggestion, put forward many years ago, that bone-marrow fibrosis in myelofibrosis may have an autoimmune origin, analogous to cirrhosis of the liver or fibrosis in the thyroid. Our present results do not allow us to decide where the autoimmune antibody is directed, for measurement of the presence and uptake of complexes is clearly the endnoint in a reaction. Immunosuppressive therapy has produced a pronounced clinical improvement in some of our patients, and this is associated with a fall in the proportion of cells containing intracellular inclusions. We have not been able to demonstrate any improvement in bone-marrow trephine samples from the iliac crest, although haemoglobin levels have returned to normal. This is not surprising in view of the site and size of the sample obtained. The contention that it may be possible to reverse the fibrosisg in the marrow must await further evidence. A significant proportion of patients with polycythasmia rubra vera proceed to myelofibrosis,9,10 and it is
1153 sometimes difficult to decide when this change is taking place, For this reason we studied patients with established polycythaemia to see if any immunoglobulin cellinclusions could be found. So far, our examinations both of patients’ own leucocytes and patients’ sera plus healthy polymorphonuclear cells have been negative. None of these patients were considered to be "converting" to myelofibrosis. Among those whom we have classified as having myelofibrosis, 2 had established fibrosis yet still retained an increased red-cell mass. Both our techniques showed that these patients had immune complexes, which suggests that the immunological abnormalities associated with myelofibrosis may form at a relatively early stage in the disease. Clearly we need to study more patients with myeloproliferative disorders to determine when this change takes place. It may well be important for management, especially since we believe that myelofibrosis is better not treated with busulphan or phosphorus-32 at the stage of imminent myelofibrosis, because these agents may well aggravate the fibrosis in the bone-marrow. Examination of a patient’s white cells or serum for the presence of immune complexes might enable us to make an earlier diagnosis in the transition from polycythasmia rubra vera to myelofibrosis, so that more appropriate treatment can be instituted. Requests
for
reprints
should be addressed
to
G.D.P.
cebo-treated patients. Thus, (+)-cyanidanol-3 be of benefit in acute viral hepatitis.
Introduction AT present, there is no effective treatment for acute viral hepatitis. A new substance, the flavonoid, (+)cyanidanol-3,’ has been shown to decrease the hepatotoxicity of ethanol and other compounds in laboratory animals,2-4 and to be a powerful free-radical scavenger.s In addition, (+)-cyanidanol-3 has been reported to decrease serum-bilirubin in patients with acute viral hepatitiS.6-8 In order to establish the value of (+)-cyanidanol-3 in the treatment of acute viral hepatitis, a multicentre, double-blind trial was carried out. The results are
encouraging. Patients and Methods
A prospective, double-blind, placebo-controlled trial of cyanidanol-3 was carried out in patients with acute viral hepatitis in five departments of medicine. 106 consecutive adult inpatients with acute viral hepatitis were randomised. Patients with an alcohol consumption of more than 100 g/day and/or jaundice for more than 4 weeks before admission were excluded. Viral hepatitis was diagnosed on the history, clinical
examination and liver-function tests. If there had been jaundice in the previous year, or if the diagnosis was doubtful, a liver biopsy was carried out. All 11 biopsies were read by one
pathologist (H.P.). Treatment
REFERENCES 1. Lewis, C. M., Pegrum, G. D. Unpublished. 2. Hurd, E. R., Andreis, M., Ziff, M. Clin. exp. Immun. 1977, 28, 413. 3. Pfueller, S. L., Luscher, E. F. Immunochemistry, 1972, 9, 1151. 4. Mantovani, B., Rabinowitch, M., Nussenweig, V. J. exp. Med. 1972, 135, 780. 5. Boivin, P., Bernard, J. F., Hakim, J., Woroclans, M. Acta hœmat. 1974, 51, 91. 6. Lang, J. M., Obeiling, F., Mayer, S., Heid, E. Biomedicine, 1976, 25, 39. 7. Wyatt, J. P., Sommers, S. C. Blood, 1950, 5, 329. 8. Kelemen, E., Kraszanai, G., Endes, P., Szinay, G. Acta hœmat. 1972, 57, 171. 9. Minok, G. R., Buckman, T. E. Am. J. med. Sci. 1923, 166, 469. 10. Ikkala, E., Rapola, J., Kotilainen, M. Scand. J. Hœmat. 1967, 4, 453.
(+)-CYANIDANOL-3
A. L. BLUM W. DOELLE K. KORTÜM P. PETER G. STROHMEYER
P. H. S. H.
BERTHET GOEBELL
PELLONI
POULSEN N. TYGSTRUP
Departments of Medicine, Triemli Hospital, Zürich, Switzerland; University Hospital, Tübingen; University Hospital, Ulm; University Hospital, Düsseldorf, Federal
Republic of Germany; Rigshospitalet, Copenhagen and Hospital, University of Copenhagen, Denmark
Hvidorre
Summary
A double-blind trial of
(+)-cyanidanol-3 (2 g/day) versus placebo tablets was carried out in 100 patients with acute viral hepatitis. 51 received the drug and 49 placebo. (+)-Cyanidanol-3 accelerated the disappearance of HBsAg from the blood, lowered serum-bilirubin, and relieved symptoms such
was
started within 24 hours of randomisation.
(+)-cyanidanol-3 was given in a dose of 2 g daily for 4 weeks, and placebo in a dose of two tablets four times daily. No dietary restrictions were imposed, but patients with severe anorexia, nausea, and vomiting (8 on [+]-cyanidanol-3, 7 on placebo) received intravenous glucose. Likewise, bed rest was not imposed. Patients with prothrombin concentrations
