Haematological complications in polycythaemia vera and thrombocythaemia patients treated with radiophosphorus (32P).

We have evaluated 230 patients with myeloproliferative disorders treated in the last 15 years with 32P. None of the patients affected by essential thrombocythaemia developed haematological complications. In the larger group of polycythaemia patients (214 subjects) only 38 patients (17 males and 21 females) developed complications. 60.5% of these subjects had a minor complications: 1.8% showed a thrombocytopenia lower than 100.10e9/lt, 2.3% anaemia with Hb lower than 10 g%, 2.6% leukopenia lower than 40.10e9/lt and 2.3% a pancytopenia. All these complications were transient and eventually treated with limited blood transfusions. We could not identify a correlation between the dose used and the development of such complications. We noted only that the occurrence of anaemia, given a similar dose, was more frequent in females. Only 7% of all patients presented a major complication after 32P administration. In this case too, there was no correlation with the dose administered. Myelofibrosis and chronic myeloid leukaemia resulted to be the more frequent complication (9 out of 15) but we could not clarify if they represented a natural evolution of polycythaemia vera or were due to the treatment with 32P. Acute leukaemia developed only in 5 patients and again we could not recognized a correlation with the dose administered. Moreover, the time from the diagnosis of polycythaemia vera the onset of acute leukaemia ranged widely. 32P has a definite effect on the prevention of thrombotic and haemorrhagic complications in polycythaemia patients since it prolongs their life but it also increases the incidence of acute leukaemia.     

Pruritus and severe iron deficiency in polycythaemia vera.

Six patients diagnosed as having polycythaemia vera had severe pruritus that persisted despite adequate haematological control. Iron supplementation was given when iron deficiency was noted in all six patients. The pruritus began to improve two to 10 days after the start of treatment and had completely disappeared after two to three weeks. In three patients the iron treatment was stopped because of unacceptably high haemoglobin concentrations; the pruritus recurred. Since chronic iron treatment may result in increases in red cell mass indiscriminate use of iron in patients with polycythaemia vera and pruritus is not advocated. Nevertheless, in patients with severe symptoms and evidence of iron deficiency treatment with iron, continuing for two to three weeks after the symptoms have abated, may be beneficial.     

Familial Polycythaemia Caused by a New Haemoglobin Variant: Hb Heathrow, β 103 (G5) Phenylalanine Leucine

A new variant of haemoglobin A (Hb A) with a high affinity for oxygen has been found in an English family. Five members are affected and all are polycythaemic. This variant (Hb Heathrow) is the first of this class to be found in this country and has the same electrophoretic mobility as Hb A. It was discovered only by measuring the oxygen affinity of the patients'' red cells. This emphasizes the need for measuring the oxygen affinity of haemoglobin in patients with polycythaemia if other clinical and haematological features associated with polycythaemia rubra vera are absent.     

Hepatogenic polyglobulinemias and polycythemias]

The authors distinguish three kinds of hepatogenous polyglobulia: Polycythaemia caused by Budd-Chiari syndrome, polycythaemia caused by a Mosse syndrome (cirrhosis without liver venous thrombosis) and polyglobulia caused by liver tumours. In all three cases the same mechanism is likely to induce polycythaemia or polyglobulia respectively. In addition to the three cases of the Mosse syndrome published in 1966, the present paper deals with three cases of Budd-Chiari syndrome. Twice the Budd-Chiari syndrome was followed by a polycythaemia, once a Budd-Chiari syndrome was developed in the course of a polycythaemia vera.     

Lacunar infarcts in polycythaemia with raised packed cell volumes.

Lacunar (small deep cerebral infarcts) infarction is described in association with raised packed cell volumes. Two patients had polycythaemia vera, one stress polycythaemia. They presented with transient ischaemic episodes and were shown by computed tomography to have lacunes deep in the basal ganglia and internal capsule. Such lesions may be caused by small vessel occlusions related to increased viscosity and impaired oxygen consumption by adjacent tissues. Finding a raised packed cell volume in patients with lacunes and transient ischaemic attacks offers a further possibility of treatment.     

On the existence of a myeloproliferative factor in patients with a myeloproliferative syndrome (author's transl)]

Serum of patients suffering from a chronic myeloproliferative disorder (polycythaemia, era, osteomyelofibrosis, chronic myeloid leukaemia) and serum of lethally irradiated rats injected before application of a single doses of erythropoietin did not enhance the effect of erythropoietin -- measured with the iron incorporation rate of polycythemic mice. The rationale for these experiments is to try to find a "myeloproliferative factor", which augments the number of stem cells as described in sera of patients with polycythaemia vera, osteomyelofibrosis, and lethally irradiated mice.     

Decreased bone marrow iron in chronic granulocytic leukaemia: a consistent finding not reflecting iron deficiency

The iron status of 50 patients with Ph'-positive chronic granulocytic leukaemia (CGL) was evaluated at diagnosis by means of bone marrow and blood studies. A decreased or absent iron in semiquantitative estimation on bone marrow smears was observed in 92% of patients, and 88% had a low sideroblast score. In contrast, normal Hb and serum iron concentrations were found in the majority of cases, and only two out of the 50 patients displayed a decreased serum ferritin. To ascertain whether the bone marrow pattern of iron depletion could be due to an expansion of the red cell mass, the latter parameter was measured by isotopic methods in a subgroup of 11 patients. Normal or slightly increased values were obtained in all cases. We conclude that absent or decreased marrow iron is a common feature in the chronic phase of CGL, that generally does not reflect true iron deficiency. Since such a finding is also usual in polycythaemia vera and idiopathic myelofibrosis, it should be included among the features shared by the chronic myeloproliferative disorders.     

Simultaneous occurrence of Hb C trait and polycythaemia vera

A slow abnormal haemoglobin was found in a 27 year-old Negro man who had polycythaemia vera. Chemical and structural analysis showed it to be Hb C. The oxygen affinity showed a normal P50 value. Clinical and haematological investigations are described and discussed.     

Increased fibroblast colony stimulating activity (F-CSA) in serum of myeloproliferative disorders

Sera of patients with primary myelofibrosis (PMF), primary thrombocythemia (PT), polycythaemia vera (PV) and chronic myeloid leukemia (CML) contained a significantly increased F-CSA (or F-CSAs) compared to those of normal subjects and patients with secondary thrombocytosis (ST). This F-CSA was heat sensitive and had the capacity to promote both proliferation and maturation of normal marrow fibroblast colony-forming cells (CFU-F). This F-CSA seemed to be different from human platelet derived growth factor (PDGF), tumor necrosis factor (TNF) and fibroblast growth factor (FGF) from bovine brain. This F-CSA might be of importance in the pathogenesis of bone marrow fibrosis in myeloproliferative disorders.     

Low incidence of familial occurrence of thrombocythaemia and/or thrombocytosis

Several reports have been published about familial polycythaemia vera (PV) but no information is available about the incidence of thrombocytosis in the same family. In our population of thrombocytosic patients, both with primary thrombocytosis (133 cases) and secondary (37 cases), we found only two family related subjects. One of them had PV and the other essential thrombocytosis (ET). Our results seem to indicate that familial thrombocytosis is a rare phenomenon, much less frequent then familial thrombocytopenia.     

5'-AMP-activated protein kinase activation prevents postischemic leukocyte-endothelial cell adhesive interactions

Preconditioning (PC) with nitric oxide (NO) donors or agents that increase endothelial NO synthase (eNOS) activity 24 h before ischemia-reperfusion (I/R) prevents postischemic leukocyte rolling (LR) and stationary leukocyte adhesion (LA). Since 5'-AMP-activated protein kinase (AMPK) phosphorylates eNOS at Ser1177, resulting in activation, we postulated that AMPK activation may trigger the development of a preconditioned anti-inflammatory phenotype similar to that induced by NO donors. Wild-type (WT) C57BL/6J and eNOS(-/-) mice were treated with the AMPK agonist 5-aminoimidazole-4-carboxamide 1-beta-d-furanoside (AICAR) 30 min (early AICAR PC) or 24 h (late AICAR PC) before I/R; LR and LA were quantified in single postcapillary venules in the jejunum using intravital microscopy. I/R induced comparable marked increases in LR and LA in WT and eNOS(-/-) mice relative to sham-operated (no ischemia) animals. Late AICAR PC prevented postischemic LR and LA, whereas early AICAR PC prevented LA in WT mice. Late AICAR PC was ineffective in preventing I/R-induced LR but not LA in the eNOS(-/-) mice, and the same pattern was seen in WT animals treated with the NOS inhibitor N(omega)-nitro-l-arginine. Early AICAR PC remained effective in preventing LA in eNOS(-/-) mice. Our results indicate that both early and late PC with an AMPK agonist produces an anti-inflammatory phenotype in postcapillary venules. Since the protection afforded by late AICAR PC on postischemic LR was prevented by NOS inhibition in WT mice and absent in eNOS-deficient mice, it appears that eNOS triggers this protective effect. In stark contrast, antecedent AMPK activation prevented I/R-induced LA by an eNOS-independent mechanism.     

Thiamphenicol induced bone marrow suppression as a therapy of myeloproliferative diseases

Sixteen patients, suffering from myeloproliferative diseases (9 polycythaemia vera, 7 primary thrombocythaemia) and 20 control subjects were treated for 14 days with 1 g of thiamphenicol per day. The effect of 40 treatment cycles was studied. The regimen resulted in lowering the haemoglobin values by 4.8% in controls (p greater than or equal to 0.001) and patients (p greater than or equal to 0.01); the reticulocyte count dropped 43% in the patient group (p greater than or equal to 0.01) and 32% in the controls (p greater than or equal to 0.05); the thrombocyte count was decreased 42% (p greater than or equal to 0.0001) vs 29% (p greater than or equal to 0.0001) in the control group. The administration of 1 g/day of thiamphenicol for a 14 day period reduced the myelopoietic activity by 40%. The decrease of all values was statistically significant. After discontinuation of thiamphenicol therapy the haematological parameters returned to the initial values within 1-2 months. The myelodepressant activity of thiamphenicol may therefore be applied to the therapy of the myeloproliferative diseases in order to reduce the risk of spontaneous haemorrhages and thrombosis.     

Chemiluminescence (CL) in blood samples reflects leukocyte activation (LA) during anaphylactoid reactions in dogs

Inflammation is accompanied by leukocyte activation (LA). We decribe a simple ex vivo technique for studying LA that might help to find new LA inhibitors for the treatment of pathologic events related to LA. Arterial and venous blood samples obtained from six permanently catheterized beagle dogs ?60, 0, +15 min and +23 h after i.v. challenge with C 48/80, and also blood samples from six normal beagles, were minimally diluted 1:2.5 with buffer. Total leukocyte counts (LC), and luminol amplified CL, induced by opsonized zymosan (C₃-Z), were estimated. Blood samples from dogs elicited CL responses of almost 1/10 the magnitude of erythrocyte-free human leukocytes, whereas blood samples from rats reacted three orders of magnitude less. Obviously quenching of CL by accompanying erythrocytes in blood samples from dogs is not important, for CL correlated almost linearly with the CL in differently diluted samples. In arterial, but not in venous samples from catheterized dogs, absolute CL and LC, both were significantly depressed (p < 0.05) 15 min after C 48/80 challenge. CL/10⁶ leukocytes was augmented twofold. All leukocyte deviations returned to pre-values 23 h post-challenge.     

Circulating lupus-type anticoagulant, a risk factor for thrombosis by inhibition of protein C activation

The lupus anticoagulant is a risk factor of thrombosis. The non thrombogenic endothelial surface could be a target for the lupus anticoagulant. We have investigated the effect of purified immunoglobulins G of five patients with LA on the thrombomodulin activity of cultured human endothelial cells from umbilical cord vein. The rate of activation of purified protein C (PC) (30 nM) by the endothelial cells in the presence of thrombin (0.1 U/dish) has been measured by hydrolysis of substrate S 2366. Activated PC has been 7.37 +/- 0.78 pmoles X ml-1 X h-1 in the presence of buffer and 7.2 +/- 0.78 pmoles X ml-1 X h-1 in the presence of control IgG (2 mg/dish). Heat aggregated IgG did not induce any significant change. Patient's IgG lowered significantly the rate of PC activation (4.86 +/- 1.04 pmoles X ml-1 X h-1, p less than 0.001). Fab fragment from two of these patient's IgG displayed the same inhibition. Moreover neutralization of this effect was obtained by addition of phospholipids (70% phosphatidylcholine, 30% phosphatidylserine) in excess to patient's IgG. Activation of PC has been also performed using purified rabbit thrombomodulin and a similar inhibition by patient's IgG was found. These results seem to indicate that antibodies present in the IgG fractions containing LA could be directed against phospholipids associated to thrombomodulin activity. Reduction of PC activation if present in the patients with LA could play a role in the occurrence of thrombosis.     

Changes in erythropoiesis during the course of polycythaemia vera.

Using 58Fe, 51Cr and cytological parameters, the authors have examined erythropoiesis in 44 polycythaemia vera patients diagnosed as such on the basis of the usual parameters (exept for determination of the erythropoietin level). In the patients divided into four types the following characteristica were observed. In type I, increased erythropoiesis is evident by accelerated plasma iron clearance, greater PIT and EIT as well as enhanced iron utilization and production indices. In type II, in addition to the former signs of increased erythropoiesis moderately shortened red cell life-span and hyposideraemia characteristic of splenic sequestration and resulting from bleeding and blood letting seem to be accompanied by microcytosis. There is a metaplastic erythropoiesis in type III, bone marrow activity decreases, but the increased erythropoiesis is indicated by several parameters already observed earlier. At the time the iron utilization indicative of effective erythropoiesis is decreased, thus ineffective erythropoiesis and considerably shortened red cell life-span are responsible for the enhanced iron turnover. This is also shown by the regression calculations. In type IV effective erythropoiesis was considerably decreased in the patients with severe anaemia. Sings which are indicative of metaplastic erythropoiesis are absent. In one of the patients the morphological changes characteristic of dyserythropoiesis were found. Although all our patients were given treatment. We believe that these alterations in the character of erythropoiesis are not likely to be the consequences of therapy.     

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.     

Restoration of Dioxin-Induced Damage to Fetal Steroidogenesis and Gonadotropin Formation by Maternal Co-Treatment with α-Lipoic Acid

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disruptor, causes reproductive and developmental toxic effects in pups following maternal exposure in a number of animal models. Our previous studies have demonstrated that TCDD imprints sexual immaturity by suppressing the expression of fetal pituitary gonadotropins, the regulators of gonadal steroidogenesis. In the present study, we discovered that all TCDD-produced damage to fetal production of pituitary gonadotropins as well as testicular steroidogenesis can be repaired by co-treating pregnant rats with α-lipoic acid (LA), an obligate co-factor for intermediary metabolism including energy production. While LA also acts as an anti-oxidant, other anti-oxidants; i.e., ascorbic acid, butylated hydroxyanisole and edaravone, failed to exhibit any beneficial effects. Neither wasting syndrome nor CYP1A1 induction in the fetal brain caused through the activation of aryl hydrocarbon receptor (AhR) could be attenuated by LA. These lines of evidence suggest that oxidative stress makes only a minor contribution to the TCDD-induced disorder of fetal steroidogenesis, and LA has a restorative effect by targeting on mechanism(s) other than AhR activation. Following a metabolomic analysis, it was found that TCDD caused a more marked change in the hypothalamus, a pituitary regulator, than in the pituitary itself. Although the components of the tricarboxylic acid cycle and the ATP content of the fetal hypothalamus were significantly changed by TCDD, all these changes were again rectified by exogenous LA. We also provided evidence that the fetal hypothalamic content of endogenous LA is significantly reduced following maternal exposure to TCDD. Thus, the data obtained strongly suggest that TCDD reduces the expression of fetal pituitary gonadotropins to imprint sexual immaturity or disturb development by suppressing the level of LA, one of the key players serving energy production.     

Intermittent hypoxia in patients with unexplained polycythaemia.

The aetiology of polycythaemia is unclear in up to 30% of patients. Twenty patients with unexplained polycythaemia were investigated to see whether they had an intermittent hypoxic stimulus to erythropoiesis that was undetected by conventional investigations for hypoxic secondary polycythaemia. Overnight polygraphic sleep studies showed that five patients had prolonged nocturnal hypoxaemia. Their arterial oxygen saturation was below 92%, the level at which appreciable hypoxic stimulation of erythropoiesis occurs, for 26-68% of the time for which they were studied. Considerable evidence is accumulating that intermittent hypoxia is a potent stimulus to erythropoiesis, and clinicians should consider the possibility of nocturnal hypoxia in patients with unexplained polycythaemia. Appropriate investigation will lead to the correct diagnosis of polycythaemia secondary to hypoxia in some cases previously regarded as idiopathic, and treatment may then be planned accordingly.     

Identity of the abnormal F-group chromosome associated with polycythaemia vera

Summary A technique for the identification of individual chromosomes has been applied to bone marrow cells from 4 patients with polycythaemia vera. These patients were already known to have the abnormal F-group chromosomes which occur in about 20% of cases of this disease. In each individual the particular chromosome involved was found to be a number 20, from which some material had been deleted, probably from the long arm.

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Zusammenfassung Eine Technik für die Identifikation einzelner Chromosomen wurde auf Knochenmarkzellen von 4 Patienten mit Polycythaemia Vera angewandt. Von diesen Patienten wußte man bereits, daß sie die abnormen F-Chromosomen besitzen, die in ungefähr 20% der Fälle dieser Krankheit vorkommen. Bei allen 4 Patienten stellete sich heraus, daß das spezielle Chromosom die Nr. 20 hat; es bestand jeweils eine Deletion, wahrscheinlich des langen Armes.

     

Lipoic acid affects cellular migration into the central nervous system and stabilizes blood-brain barrier integrity

Reactive oxygen species (ROS) play an important role in various events underlying multiple sclerosis (MS) pathology. In the initial phase of lesion formation, ROS are known to mediate the transendothelial migration of monocytes and induce a dysfunction of the blood-brain barrier (BBB). In this study, we describe the beneficial effect of the antioxidant alpha-lipoic acid (LA) on these phenomena. In vivo, LA dose-dependently prevented the development of clinical signs in a rat model for MS, acute experimental allergic encephalomyelitis (EAE). Clinical improvement was coupled to a decrease in leukocyte infiltration into the CNS, in particular monocytes. Monocytes isolated from the circulation of LA-treated rats revealed a reduced migratory capacity to cross a monolayer of rat brain endothelial cells in vitro compared with monocytes isolated from untreated EAE controls. Using live cell imaging techniques, we visualized and quantitatively assessed that ROS are produced within minutes upon the interaction of monocytes with brain endothelium. Monocyte adhesion to an in vitro model of the BBB subsequently induced enhanced permeability, which could be inhibited by LA. Moreover, administration of exogenous ROS to brain endothelial cells induced cytoskeletal rearrangements, which was inhibited by LA. In conclusion, we show that LA has a protective effect on EAE development not only by affecting the migratory capacity of monocytes, but also by stabilization of the BBB, making LA an attractive therapeutic agent for the treatment of MS.