Adhesive properties of plasma-circulating and platelet-derived microvesicles from healthy individuals.

BACKGROUND: Microvesicles (MVs) produced by platelets upon activation possess high procoagulant activity and represent a possible thrombotic risk marker. However, direct experimental evaluation of the adhesive properties of MVs and their potential role in thrombus growth is lacking. OBJECTIVES: We investigated integrin αIIbß3 status and adhesive properties of plasma-circulating and platelet-derived MVs from healthy individuals. METHODS: MVs were isolated from whole blood or produced from activated platelets. Flow cytometry was used for quantification of fluorescently labeled PAC-1 and fibrinogen binding to MVs. Confocal microscopy was used for evaluation of MVs adhesion to fibrinogen and for estimation of their involvement in whole blood thrombus formation in a parallel-plate flow chambers under arterial shear conditions. RESULTS AND CONCLUSIONS: Neither circulating plasma MVs, nor platelet-activation-produced MVs bound PAC-1. However, both types of MVs specifically and weakly bound fibrinogen (about 400 molecules of bound fibrinogen per MV versus >100,000 per non-procoagulant activated platelet). Still, the MVs did not adhere stably to the immobilized fibrinogen. Both types of MVs were weakly incorporated into a thrombus and did not affect thrombus formation: average thrombus height in the recalcified whole blood in the presence of platelet-activation-produced MVs was 4.19 ± 1.38 µm versus 4.87 ± 1.72 µm (n = 6, p > 0.05) in the control experiments. This suggests that MVs present in plasma of healthy individuals are not likely to be directly involved in thrombus formation under arterial flow conditions.

Rituximab and Abatacept Are Effective in Differential Treatment of Interstitial Lymphocytic Lung Disease in Children With Primary Immunodeficiencies.

Background: Interstitial lymphocytic lung disease (ILLD), a recently recognized complication of primary immunodeficiencies (PID), is caused by immune dysregulation, abnormal bronchus-associated lymphoid tissue (BALT) hyperplasia, with subsequent progressive loss of pulmonary function. Various modes of standard immunosuppressive therapy for ILLD have been shown as only partially effective. Objectives: To retrospectively evaluate the safety and efficacy of abatacept or rituximab in treatment of ILLD in children with PID. Methods: 29 children (median age 11 years) with various forms of PID received one of the two therapy regimens predominantly based on the lesions' immunohistopathology: children with prevalent B-cell lung infiltration received rituximab (n = 16), and those with predominantly T-cell infiltration received abatacept (n = 17). Clinical and radiological symptoms were assessed using a severity scale developed for the study. Results: The targeted therapy with abatacept (A) or rituximab (R) enabled long-term control of clinical (A 3.4 ± 1.3 vs. 0.6 ± 0.1; R 2.8 ± 1 vs. 0.7 ± 0.05, p < 0.01) and radiological (A 18.4 ± 3.1 vs. 6.0 ± 2.0; R 30 ± 7.1 vs. 10 ± 1.7, p < 0.01) symptoms of ILLD in both groups and significantly improved patients' quality of life, as measured by the total scale (TS) score of 57 ± 2.1 in treatment recipients vs. 31.2 ± 1.9 before therapy (p < 0.01). Conclusions: ILLD histopathology should be considered when selecting treatment. Abatacept and rituximab are effective and safe in differential treatment of ILLD in children.

Free-breathing T2* mapping for MR myocardial iron assessment at 3 T.

BACKGROUND: Timely diagnosis of cardiac iron overload is important for children with transfusion-dependent anaemias and requires modern measure methods. Nowadays, myocardial iron quantification is performed by magnetic resonance (MR) breath-hold techniques, sensitive to respiratory motion and unfeasible in patients who are unable to hold their breath. Free-breathing T2* mapping sequences would allow to scan children who cannot hold their breath for a specified duration. Our aim was to test a free-breathing T2* mapping sequence, based on motion correction by multiple signal accumulation technique. METHODS: We used an electrocardiographically gated T2* mapping sequence based on multiple gradient echo at 3-T in 37 paediatric patients with haematologic disorders aged from 2 to 16. We compared T2* values of myocardium and signal-to-noise ratio of this new sequence with standard breath-holding T2* mapping sequence. T2* values were measured in the interventricular septum for both methods in studies with adequate image quality. RESULTS: All children were scanned without complications. Five patients were excluded from analysis because of the presence of respiratory artefacts on the T2* images with breath-holding technique due to patient's inability to hold their breath. Breath-holding T2* was 19.5 ± 7.7 ms (mean ± standard deviation), free-breathing T2* was 19.4 ± 7.6 ms, with positive correlation (r = 0.99, R2 = 0.98; p < 0.001). The free-breathing sequence had a higher signal-to-noise ratio (median 212.8, interquartile range 148.5-566.5) than the breath-holding sequence (112.6, 71.1-334.1) (p = 0.03). CONCLUSION: A free-breathing sequence provided accurate measurement of myocardial T2* values in children.

Alterations in the parameters of classic, global, and innovative assays of hemostasis caused by sample transportation via pneumatic tube system.

BACKGROUND: Pneumatic tube system (PTS) is an integral part of large medical facilities providing rapid interconnection between units within the hospital and often used to transport blood samples. The aim of our study was to compare a wide variety of hemostasis assays to identify assays sensitive to this transport method and diagnostic relevance of the alterations. METHODS: Routine coagulation and platelet tests (APTT, PT, TT, fibrinogen, light transmission aggregometry (LTA) with ADP, collagen, ristomycin and epinephrine), whole blood flow cytometry platelet function test (levels of CD42b, CD61, CD62P, PAC1, annexin V binding and mepacrine release) and global coagulation tests (thromboelastography (TEG), thrombin generation (TGT), thrombodynamics (TD), thrombodynamics-4D (TD-4D)) were determined in PTS- and manually transported samples of 10 healthy volunteers. RESULTS: There were no significant differences between the values of APTT, PT, TT or fibrinogen between the samples transported by PTS or manually. The results for LTA demonstrated increase in the collagen-induced aggregation (84 ±â€¯7% versus 73 ±â€¯5%), while the response to epinephrine was decreased (58 ±â€¯20% versus 72 ±â€¯7.4%). Flow cytometry-based platelet function test showed a pre-activation of platelets by PTS-transportation while all integral assays of coagulation tested in the present study (TEG, TGT, TD, TD-4D) demonstrated a hypercoagulation shift. CONCLUSIONS: Transportation by PTS caused significant shifts in parameters of functional and integral assays that exceeded parameter variation values and sometimes even were comparable to normal ranges. The results obtained in this study indicate that using of PTS for such assays may cause sufficient alterations of results and can lead to patient's mistreatment.

The role of nelarabine in the treatment of T-cell acute lymphoblastic leukemia: literature review and own experience.

AIM: The analysis of experience of nelarabine use in refractory/relapsed T-cell acute lymphoblastic leukemia (T-ALL) depending on the immunophenotype and the line of therapy. MATERIALS AND METHODS: All the patients with relapsed or refractory T-ALL aged from 0 to 18 years who received treatment with nelarabine as a part of the therapeutic element R6 were included in the study. For all patients a detailed immunological analysis of leukemia cells with discrimination of immunological variants TI, TII, TIII or TIV was performed. Patients administered with nelarabine as a first therapeutic element were referred to the first-line therapy group, other patients were referred to the second-line therapy group. Nelarabine was ad- ministered as intravenous infusion at a dose of 650 mg/m2, on days 1-5. Allogeneic hematopoietic stem cells transplantation (allo-HSCT) was considered for all patients. RESULTS: From 2009 to 2017, 54 patients with refractory/relapsed T-ALL were treated with nelarabine. Five-year event-free survival (EFS) and overall survival (OS) was 28% for all patients, cumulative risk of relapse (CIR) was 27%. EFS was significantly higher in nelarabine first-line therapy group in comparison with second-line therapy group (34±8% vs 8±8%, p=0,05). In patients after allo-HSCT EFS, OS and CIR were 51±10%, 50±10% and 39,1±9,5% accordingly. The best results were achieved in patients with TI immunophenotype. No toxicity-related mortality as well as severe neurologic complications or discontinuation of therapy associated with use of nelarabine were reported. CONCLUSION: The use of nelarabine is an effective strategy for the treatment of relapsed and refractory T-ALL. The best treatment outcomes were obtained in patients with TI immunophenotype and in the first-line therapy group. Optimal dosage regimens can be established dur- ing controlled clinical trials.

Hematopoietic stem cell transplantation with alpha/beta T-lymphocyte depletion and short course of eculizumab in adolescents and young adults with paroxysmal nocturnal hemoglobinuria.

AIM: The main goal is to optimize hematopoietic stem cell transplantation (HSCT) approach among adolescents and young adults with paroxysmal nocturnal hemoglobinuria (PNH) by means of Graft-versus-host disease (GVHD) and post-transplant complications risk lowering. MATERIALS AND METHODS: We report our experience of HSCT from HLA-matched unrelated donors using TCR alfa/beta and CD19 depletion in 5 pts (1M/4F) with PNH, developed after successful immunosuppressive therapy (IST) of acquired aplastic anemia (AA). Median age of pts at the moment of transplantation was 17,8 years (range 14,5-22,7), median interval from IST to PNH was 4 years (5mo - 6,5 y). In all patients non-severe pancytopenia was present: granulocytes 0,8х109/l (0,8-1,8 х109/l) platelets 106 х109/l (27-143 х109/l) and Hb -78 g/l, median PNH clone size in granulocytes was 94 (range 75-99)%. One pts previously developed sinus thrombosis. Conditioning consisted of thoraco-abdominal irradiation 4-6 Gy, cyclophosphamide 100 mg/kg, fludarabine 150 mg/m2 and anti-thymocyte globulin (ATG) or alemtuzumab. Eculizumab was given from day (-7) till day (+14) (every 7 days, only 4 times). GVHD prophylaxis was tacrolimus ± methotrexate. RESULTS: Infusedgraft characteristics were: CD34+ - 8,1х106/kg, CD3TCRab·150х103/kg, CD3gd+ - 7,3х106/kg, СD19+ - 221х103/kg, NK -6,4х108/kg. Engraftment was achieved in all 5 pts with a median of 15(12-18) и 13(10-18) days for granulocytes and platelets, respectively. Skin acute GVHD grade I developed in only 1 pt, and subsided with short course of glucocorticoids. CMV reactivation occurred in 1 pt; there were no episodes of Epstein-Barr Virus (EBV) o rAdenovirus (AdV) reactivation. Full donor myeloid chimerism was established in all pts by day +30. Immune reconstitution was delayed until 6 months after transplant but no severe infections occurred. All pts are alive 1,7-5,5 years (med 4 years) after HSCT with normal hematopoiesis and immune function, full donor chimerism and no late sequelae. CONCLUSION: Transplantation of TCRalfa/beta and CD19 depleted hematopoietic cells from matched unrelated donor after immunoablative conditioning and supported with short course of eculizumab is perfectly safe and efficient technology leading to cure in young patients with PNH.

TCR-alpha/beta and CD19 depletion and treosulfan-based conditioning regimen in unrelated and haploidentical transplantation in children with acute myeloid leukemia.

We evaluated the depletion of TCR-alpha/beta cells from the graft of children with high-risk AML, who received transplantation from unrelated (n=20) and haploidentical donors (n=13). The preparative regimen included treosulfan, melphalan, fludarabine and anti-thymocyte globulin. Grafts were PBSC engineered by TCR-alpha/beta and CD19 depletion. The graft contained a median of 9 × 10(6)/kg of CD34+ and 20 × 10(3)/kg of αß-T cells. Post-transplant immune suppression included tacrolimus till day +30 and Mtx in 21 patients, tacrolimus in 5, Mtx in 2 and no prophylaxis in 5 patients. Sixteen patients received native or TCR-alpha/beta-depleted donor lymphocytes at a median of 47 (40-204) days. Median follow-up is 1.76 years. Primary engraftment was achieved in 33 patients (100%). Cumulative incidence of acute GvHD (aGvHD) grade 2-3 was 39 (26-60)%, half of them had skin-only aGvHD. Cumulative incidence of chronic GvHD was 30(18-50)%. Transplant-related mortality is 10(4-26)%. Event-free survival (EFS) is 60(43-76)% and overall survival (OS) is 67(50-84)% at 2 years. In a subgroup of patients, who received transplantation in CR, EFS is 66(48-84)% and OS-72(53-90)% at 2 years. Our data suggest that TCR-alpha/beta and CD19 depletion is a robust method of graft manipulation, which can be used to engineer grafts for children with AML.

[Age-related characteristics of the efficacy of different glucocorticosteroids in the therapy of acute lymphoblastic leukemia].

AIM: To determine predictors for decision-making on a differential approach to choosing glucocorticosteroids (GCS) for children and adolescents with acute lymphoblastic leukemia (ALL). SUBJECTS AND METHODS: The analysis covered 1064 primary patients aged to 1 to 18 years with ALL who had been registered at the clinics of Russia and Belorussia in April 2002 to November 2006. Before induction therapy, the patients were randomized into a dexamethasone (DEXA) 6 mg/m2 group (n=539) and a methylprednisolone (MePRED) 60 mg/m2 one (n=525). RESULTS: The entire group showed no statistically significant differences in survival rates between the patients receiving DEXA or MePRED. However, an analysis of age groups revealed the benefits of DEXA in children younger than 14 years (the event-free survival (EFS) was 76±2 and 71±2%, respectively (p=0.048); the overall survival (OS) was 81±2 and 77±2%, respectively (p=0.046); therapy-induced mortality was 6.4% (DEXA) andl 1.1% (MePRED) (p=0.01 4); the rate of isolated extramedullary relapses was 1.5% (DEXA) and 4.4% (MePRED) (p=0.009). At the same time, EFS and OS in 14-to-18-year-old adolescents were statistically significantly higher than in those who used MePRED (EFS, 65±6 and 52±6%, respectively (p=0.087); OS, 72±6 and 61±6%, respectively; (p=0.l 7). CONCLUSION: The findings suggest that it is possible that the choice of a GCS for ALL therapy must be also based on a patient's age. There is a need for further studies of this matter in prospective randomized multicenter trials in children and adolescents.

Efficacy of plerixafor in children with malignant tumors failing to mobilize a sufficient number of hematopoietic progenitors with G-CSF.

Mobilization of suitable amounts of PBSCs with granulocyte-CSF (G-CSF) can be difficult in children. We report the results of using plerixafor in pediatric patients who failed to mobilize progenitors with G-CSF-based regimens. Thirty-three patients, median age 9 years (range 1-18 years) and median weight 29 kg (range 10-85 kg) were enrolled into the study. After 4 days of G-CSF stimulation, the median CD34+ cell count in peripheral blood was 10.4 per µL (range 0.27-23.0 per µL). Plerixafor was administered subcutaneously (0.24 µg/kg in 30 patients and 0.3 µg/kg in 3 patients) 11-12 h before apheresis. At the time of apheresis, CD34+ cell counts increased to a median of 44.1 per µL (range 8.4-357.0 per µL), a median 4.4-fold increase. Two patients (6%) failed to mobilize. Thirty-one patients underwent apheresis and in 27 >2 × 10(6) CD34+ cells per kg of body weight were collected after one procedure. In total, 31 of 33 patients mobilized successfully and the median number of cryopreserved CD34+ cells was 5.6 × 10(6) /kg body weight (2.7 × 10(6)-27.4 × 10(6)). Twenty-four patients underwent transplantation. Engraftment was achieved in all but one patient, who died on day +9 after hematopoietic stem cell transplantation. The median time of neutrophil and platelet recovery was day +12 and +16, respectively. Our study confirms that plerixafor has impressive efficacy and very modest toxicity in children.

[Transplantation of the bone marrow from a HLA-compatible unrelated donor after immunoablative conditioning in children with acquired aplastic anemia unresponsive to combined immunosuppressive therapy: preliminary results].

AIM: To analyze the efficiency of transplantation of the bone marrow from a HLA-compatible unrelated donor and continued immunosuppressive therapy (IST) in children with aplastic anemia (AA) unresponsive to 2 courses of IST. SUBJECTS AND METHODS: The study enrolled 14 children aged 2-16 years (median 9 years). A control group comprised 26 patients in whom IST was continued. The median interval between the diagnosis of AA and transplantation was 26 months (9-156 months). The conditioning regimen consisted of thoracoabdominal irradiation in a dose of 2 Gy, fludarabin (Flu) 100-150 mg/m2, cyclophosphamide (Cy) 100-200 mg/kg, antithymocyte globulin (ATG) in 11 patients and Flu, Cy, and ATG in 3. A graft-versus-host reaction was prevented with mycophenolate mefetil in all the patients, tacrolimus in 11, and cyclosporin A in 3. Donors were compatible for high-resolution typing of 10/10 and 9/10 alleles in 8 and 6 patients, respectively; the source of a transplant was bone marrow in 13 patients and granulocyte colony-stimulating factor-mobilized peripheral blood precursors in one case. RESULTS: Thirteen patients achieved primary engraftment after single transplantation; one patient did after repeat transplantation. Grades I to II graft-versus-host reaction (GVHR) developed in 9 patients; postengraftment life-threatening infections in 3, extensive chronic GVHR in 2, circumscribed GVHR in 7. All fourteen hemopoietic cell transplant recipients followed for a median 17.5 months (range 1-71 months) were survivors. CONCLUSION: The likelihood of good survival after unrelated transplantations in AA is much higher than that after continued IST: 100% versus 15 +/- 11%.

[Clinico-laboratory variants of the course and results of therapy of hepatitis-associated aplastic anemias in children].

AIM: To study clinical and laboratory characteristics of hepatitides and evaluate efficacy of immunosuppressive therapy and transplantation of the bone marrow in hepatitis-associated aplastic anemia (HAAA). MATERIAL AND METHODS: A retrospective analysis of case histories of children with HAAA was made. For all the patients standard tests for detection of aquired aplastic anemia and hepatitis were conducted. Transplantation of hemopoietic stem cells (THSC) from HLA-identical donors was made in 4 patients, 25 patients were treated with combined immunosuppressive therapy (antithymocytic globulin--ATG plus cyclosporin A -CsA), one patients received monotherapy with CsA, two--prednisolone and a short course of CsA, one child was untreated. RESULTS: Of 260 children admitted to hospital from April 1989 to July 2005 for aquired aplastic anemia, 33 (12.7%) met diagnostic criteria of HAAA. Boys to girls ratio was 267. Hepatitides were severe: median of alaninaminotransferase concentration was 1215 IU/l, aspartataminotransferase--789 IU/l, bilirubin--152.5 mcmol/l. Median of the interval from hepatitis symptoms to documentation of pancytopenia was 66 days (0-204 days). All four patients after THSC are alive for 30-72 months. Probability of complete remission after the first course of ATG+CsA is 0.72 +/- 0.09, probability of survival 0.81 +/- 0.07, median of the interval to transfusion independence--50 days. CONCLUSION: HAAA prognosis is good only in administration of up-to-date therapy. After seronegative hepatitis it is necessary to control hemogram parameters and in the presence of minimal cytopenia patients should be directed to hematological hospital.

Alkylator-free conditioning regimen for patients with acquired aplastic anemia, transplanted from genetically identical twins.

Allogeneic stem cell transplantation remains the best option for young patients with SAA. With genetically identical twin as an ideal donor, the majority of SAA patients require appropriate immunosuppression before and after stem cell transplantation to obtain long-term hematopoietic reconstitution. Alkylating agents, used during conditioning, are associated with short- and long-term toxic effects that lead to poor compliance of treatment and could compromise the quality of future life. Three SAA patients, transplanted from genetically identical twins without using alkylating agents during conditioning, showed rapid and sustained hematological reconstitution without any evidence of conditioning-related toxicity.

[On fluoroquinolones treatment safety in children (clinical, morphological and catamnesis data)]. / O bezopasnosti primeneniia ftorkhinolonov u detei (kliniko-morfologicheskie i katamnesticheskie dannye).

Results of prospective comparative investigation of monofluoroquinolones (ciprofloxacin, ofloxacin, pefloxacin) arthropathy are presented. The trial was performed at 144 children with mucoviscidosis (aged 0.5-16) and at 37 children with aplastic anemia (aged 1.75-15). Two groups differ by necessary antibacterials regimes and hence by different abilities for arthropathy development: patients with mucoviscidosis were treated with fluoroquinolones followed by repeated short courses in combination with other antibacterials; patients with aplastic anemia--were treated permanently for a long time with low doses as monotherapy for autoinfection prophylaxis. Analysis was performed on the base of catamnesis, year growth rate, postmortal morphological investigation of the right knee joint. It was shown that quinolone arthropathy development didn't depend on treatment duration, as it developed during the first three weeks of the fluoroquinolone use, but depended on the drug, patient age and nosology. Arthropathy has favourable prognosis and was fully resolved at the period from 7 days to 3 month according to the arthropathy form (arthrologic, arthritic). Quinolones arthropathy at the children has specific features, the main one is absence of cartilage damage confirmed by morphological analysis.

[Time course changes in the count of peripheral granulocytes in children with acquired aplastic anemias treated with antithymocyte globulin, cyclosporin A and granulocytic colony stimulating factor]. / Dinamika kolichestva granulotsitov v perifericheskoi krovi u detei s priobretennymi aplasticheskimi anemiiami, poluchivshikh lechenie antitimotsitarnym globulinom, tsiklosporinom A i granulotsitarnym koloniestimuliruiushchim faktorom.

AIM: To examine the pattern of changes in the count of peripheral granulocytes in children with aplastic anemias (AA), receiving a combined immunosuppressive therapy with antithymocytic globulin (ATG) and cyclosporin A in combination with granulocytic colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 31 children (17 boys and 14 girls) aged 2-15 years (median 9 years) with newly diagnosed severe and very severe acquired AA took a combined immunosuppressive therapy with ATG and cyclosporin A in combination with G-CSF in an initial dose of 10 micrograms/kg a day. RESULTS: A three-linear and response was recorded in 19 (61%) children, an isolated granulocytic response was in 26 (84%). The interval median before the recovery of granulocytes to 1.5 x 10(9)/l and 5 x 10(9)/l was 19 and 38 days, respectively. CONCLUSION: Use of G-CSF may increase the count of granulocytes in the vast majority of patients with AA, without dramatic influence on the frequency of a three-linear response. Intermittent use of G-CSF may maintain the count of granulocytes long at the safe level and reduce the cost of treatment.