Absence of JAK2V617F mutation in patients with beta-thalassemia major and thrombocytosis due to splenectomy.

The report of Janus Kinase 2 (JAK2) mutations in myeloid malignancies with high frequency in myeloproliferative neoplasms has been well known since 2005. By monitoring allele burden, it is found that the expression of JAK2V617F mutation is increasing significantly from essential thrombocytosis to polycythemia vera. Furthermore, JAK2 abnormalities are reported in the majority of unexplained thrombotic episodes. Thalassemic syndromes are characterized by ineffective erythropoiesis and thrombocytosis, mainly due to splenectomy. The high incidence of thromboembolic events has led to the identification of a prothrombotic state in these patients. The contribution of JAK2 mutations to the hypercoagulable state of thalassemic patients is still unknown. Furthermore, the potential role of Janus Kinase mutations in hepcidin expression and consequently in ineffective erythropoiesis is still under investigation. This study was scheduled to determine whether the presence of JAK2V617F mutation in thalassemic patients is associated with thrombocytosis. We studied 20 patients DNA with beta-thalassemia for JAK2V617F mutation by using RG-PCR method. None of the patients were positive for this particular mutation. More studies are needed to prove the role of JAK2 in ineffective erythropoiesis, iron metabolism and thrombocytosis and to determine if using JAK2 inhibitors in thalassemic patients can be a potential therapeutic option.

Cardiomyogenic potential of human adult bone marrow mesenchymal stem cells in vitro.

BACKGROUND: The aim of this study was to investigate the ability of adult human bone marrow mesenchymal stem cells to differentiate towards a cardiomyogenic phenotype IN VITRO. METHODS: Bone marrow samples were aspirated from 30 patients undergoing open heart surgery from the anterior iliac crest. Second passaged cells were treated with 10 microM 5-azacytidine. As control groups we used cells not expanded in culture and cells untreated with 5-azacytidine. Morphologic characteristics were analysed by confocal and electron microscopy. The expression of the cytoskeletal protein vimentin and muscle-specific myocin heavy chain was analysed by immunohistochemistry. The expression of the cardiomyocyte specific genes alpha-cardiac actin, beta-myocin heavy chain and cardiac troponin-T was detected by reverse transcriptase polymerase chain reaction. RESULTS: Mesenchymal stem cells were spindle-shaped with irregular processes. Cells treated with 5-azacytidine assumed a stick-like morphology. They connected with adjoining cells to form myotube-like structures. Numerous myofilaments were detected in induced cells which were immunohistochemically positive for myosin heavy chain and vimentin. The mRNAs of all specific cardiac genes were expressed in both induced and uninduced cells. CONCLUSION: These results indicate that adult human bone marrow mesenchymal stem cells treated with 5-aza can differentiate towards a cardiomyogenic lineage IN VITRO.

Pulmonary and myocardial manifestations due to Toxocara canis infection.

We report a case of Toxocara canis infection manifested by persistent eosinophilia, pulmonary nodules on CT scan, and myocardial manifestations in a 38-year-old man who was a dog trainer. Toxocariasis is not common in adults and rarely affects the heart.

Alterations in the fibrinolytic system components during acute myocardial infarction.

The purpose of this study was to evaluate the changes in tissue-plasminogen activator (t-PA), plasminogen activator inhibitor - type 1 (PAI-1) and D-dimer (DD) antigen plasma levels in acute myocardial infarction (AMI) patients after thrombolytic therapy with two different thrombolytic agents, rt-PA or acetyl-streptokinase and to find out any correlation between the plasma t-PA, PAI-1 and DD levels with the infarct size as it is estimated from the peak of serum CPK levels. The plasma antigen levels of t-PA, PAI-1 and DD were measured by the enzyme immunoassay method (Stago), in 57 consecutive patients (M = 46, F = 11, mean age 55.6 +/- 8.8 years) and in 25 normal subjects (M = 18, F = 7, mean age 54.0 +/- 5.5 years). In 47 out of the 57 patients who were treated successfully with 100 mg of rt-PA (26 patients) or with 1.5 MU 21 of acetyl-streptokinase, as well as in 10 patients who were not treated, samples were obtained again 4 and 24 hours after the end of thrombolytic therapy or admission, respectively. During the acute phase of myocardial infarction the t-PA, PAI-1 and DD antigen plasma levels were significantly higher than in healthy people. There were no significant changes in the t-PA, PAI-1 and DD plasma levels of the patients who were not treated with a thrombolytic agent. We found a significant elevation of t-PA (p < 0.001), PAI-1 (p < 0.05) and DD (p < 0.001) after 4 hours in comparison with the baseline (at presentation, before therapy). After 24 hours the t-PA and DD plasma levels remained significantly higher (p < 0.001) while the PAI-1 plasma levels returned to the pre-therapy levels. There were no significantly different changes in the t-PA, PAI-1 and DD plasma levels of either group of patients, treated with rt-PA or acetyl-streptokinase while the t-PA and PAI-1 levels were positively correlated with infarct size as estimated from peak serum CPK levels.

Responsiveness to recombinant human erythropoietin (rh-Epo) of marrow erythroid progenitors (CFU-E and BFU-E) from B-chronic lymphocytic leukemia (B-CLL).

The responsiveness of bone marrow erythroid progenitors (CFU-E and BFU-E) to various concentrations of recombinant human erythropoietin (rh-Epo) (2,5,20,40,100,200 and 500 U/ml) was investigated in vitro in 18 patients with B-chronic lymphocytic leukemia to assess the clinical usefulness of rh-Epo in this disease. Bone marrow mononuclear cells were cultured by methylcellulose methods for CFU-E and BFU-E assays. The B-chronic lymphocytic leukemia patients were divided into two groups according to the percentage of lymphocytes in the bone marrow (under 70% and over 70%). Among the patients with few lymphocytes, more than one third demonstrated some degree of response to rh-Epo. Among the patients with a high percentage of lymphocytes in the bone marrow, some revealed no response to rh-Epo, but there were patients who showed a good response to rh-Epo. Because erythroid progenitors from B-chronic lymphocytic leukemia appeared sensitive to rh-Epo in vitro, we propose that high doses of this drug may be clinically effective in some patients with this disease, regardless of the degree of lymphocytic inflitration of the bone marrow.

Psittacosis and arthritis.

Recent findings justify the opinion that Chlamydia psittaci is the reappearance of a forgotten pathogen. The clinical manifestation and the course of psittacosis are extremely variable, whereas the clinical spectrum of the infection with the different strains of C. psittaci is not known. Reactive arthritis during the course of psittacosis has been rarely described in humans. However, it has been stated that C. psittaci could be added to the list of infectious agents able to induce reactive arthritis. We describe a patient who presented with clinical signs consistent with reactive arthritis during the course of psittacosis, and we emphasize the good therapeutical results with ceftriaxone in the treatment of psittacosis.

Elution of 111Indium from reticuloendothelial cells.

Measurement of isotope accumulation in an organ is often used to assess that organ's removal of blood cells labelled with the isotope. This technique is only valid if the isotope does not elute from the organ. Elution of 111In from the liver and spleen has been investigated in 14 subjects following intravenous injection of heat-damaged erythrocytes labelled with 111In. The elution rate from the spleen was found to be low, about 2% of the initial activity per day. The liver accumulated activity with respect to its initial uptake at a rate of about 5% per day. Bone marrow was not visualised except in two patients in whom it was identifiable in the initial scan.

Kinetics of heat damaged autologous red blood cells. Mechanism of clearance from blood.

The kinetics of radiolabelled heat damaged red cell (HDRBC) distribution have been studied in humans using a gamma camera, and compared with the kinetics of other blood cells. Liver uptake of 111In labelled HDRBC was completed within about 10 min of injection; splenic uptake was biphasic with a half time of about 5 min over the first 20 min following injection, and a later half time much longer than this. Activity initially present in the lung fields cleared within 24 h. The rate constants of liver uptake of 99mTc labelled HDRBC and of 111In labelled platelets were very similar; the rate constants of splenic uptake of these 2 particles were also very similar up to about 20 min following injection when the splenic platelet levels became constant and the HDRBC level continued to slowly rise. Splenic uptake and blood clearance of red cells coated with IgG (IgG-RBC), in contrast to HDRBC, were monoexponential. It was concluded that: (1) the blood clearance of HDRBC was due to pooling within, and to irreversible extraction by, the spleen; (2) liver uptake of HDRBC, which was irreversible, was completed within 10 min of injection; (3) IgG-RBC clearance was due to irreversible extraction by the spleen; (4) HDRBC uptake in the lung was unrelated to reticuloendothelial function, and represented prolonged transit through the lung microvasculature.

Analysis of heat-damaged erythrocyte clearance curves.

The rate of clearance from the blood of heat-damaged erythrocytes (HDE) is used routinely as a quantitative assessment of splenic function. The time taken for the value at 3 min to fall by 50% (t0.5)is usually taken as the index of function. The clearance of HDE is dependent on three processes: splenic blood flow, splenic HDE extraction ratio and intrasplenic transit time of "unextracted' HDE, returning to the circulation. Exponential analysis of the clearance curve can resolve these three functions. Simple methods of analysis, however, such as t0.5, which are applied directly to the curve, may be weighted in favour of any one of them. In this paper, a large number of clearance curves have been analysed and the components of splenic function resolved. The t0.5, the percentage fall in HDE between 8 and 28 min (C20), the rate constant at 8 min (K8) and the rate constant of the tail of the curve (alpha 2) have been correlated with these components. K8 showed a close correlation with splenic blood flow, and alpha 2 with the rate of HDE phagocytosis. In general, the correlation between the various components of splenic function was better with C20 than with t0.5. This is explained predominantly by the fact that the t0.5 includes liver clearance. The t0.5 should therefore be used with caution as an estimate of splenic function, which can be usefully assessed by applying alternative simple methods of analysis described.

Measurement of splenic function in humans using heat damaged autologous red blood cells.

Functional hyposplenism, a clinical entity which has only recently been recognised, is usually assessed quantitatively by the rate of clearance of radiolabelled heat damaged erythrocytes (HDE) from the circulation. Based on recent observations on the kinetics of HDE, we have developed a 3 compartmental model of HDE distribution between the spleen and blood, and calculated, in a large series of clearance curves, splenic blood flow, HDE intrasplenic transit time and splenic HDE extraction ratio. Transit time (about 15 min) was of the same order as platelet transit time and extraction ratio (about 35%) was similar to that recorded in animals. Thr relationships between blood flow, transit time and extraction ratio provided evidence in support of 2 separate functional compartments, of which only one was engaged in phagocytosis, present within the spleen.

Spleen function and platelet kinetics.

In patients suffering from various platelet abnormalities, quantitative scanning after injection of indium-111 (111In) labelled platelets showed three different patterns of platelet destruction and distribution. In patients with a normal platelet life span but with evidence of increased splenic pooling, the spleen tended to be the main site of destruction. In patients with a moderately reduced platelet life span, the distribution of destruction in the system and variable destruction in the marrow. However, because of its rapidity this destruction was difficult to quantify, and it was difficult in these cases to distinguish reliably between spleen pool, sequestration, and destruction. Destruction of platelets on the liver appeared to be unimportant in all three groups. 111In, because of its physical characteristics, is preferable to chromium-51 as a platelet label in the assessment of abnormal platelet kinetics.

Radionuclide distribution following injection of 111Indium-labelled platelets.

Platelets labelled with 111In displayed similar survival curves after incubation with 111In-oxine in plasma, plasma-saline and dextrose saline media. The use of autologous red cells to cushion platelets during high-speed centrifugation facilitated platelet resuspension without greatly affecting the duration of the labelling procedure. Quantitative scanning after reinjection of labelled platelets in haematologically normal subjects showed that, initially, splenic indium amounted to about 35% of the injected dose and hepatic indium about 12%; these levels rose only slightly over the subsequent duration of the platelet life span. Subtraction of the signal from indium in platelets thought to be normally pooled within the spleen from the total indium signal gave splenic indium uptake curves which reflected splenic platelet destruction. Initially, the sum of indium levels in spleen, liver and blood equalled 100% of the dose. Thereafter, the sum fell progressively at a rate thought to be approximately equal to the rate of bone marrow uptake.

Use of 111Indium-labeled platelets to measure spleen function.

The distribution of 111In-labelled platelets following intravenous bolus injection has been studied using a gamma camera and computer system. Liver uptake, which accounted for about 10% of the dose, was completed between 6 and 10 min after injection. Blood pool and splenic 111In, which accounted for the remainder of the dose, reached constant levels simultaneously about 20 min after injection. The kinetics of splenic uptake are consistent with a two compartmental model in which circulating and splenic platelets are in dynamic equilibrium with each other. From analysis of the kinetics, splenic blood flow and the mean transit time of platelets through the spleen have been calculated in normal subjects and in patients with haematological disorders. Blood flow, which was about 200 ml per min in normals, tended to increase with increasing spleen size. Transit time was not dependent on spleen size; it was about 10 min in all but one of the subjects.