[Intensity of processes of oxidative modification of proteins in women's and cow's milk].

Breast milk is a source of all the essential nutritional components necessary for the full growth and development of the child, therefore, it is necessary to study its composition and physical and chemical properties in order to adapt human milk substitutes. Adapted infant milk formulas are produced mainly from cow's milk, bringing formula nutrient composition closer to the composition of women's milk, adapting it in accordance with the requirements of the infant body. However, technological processes for the production of dairy products contribute to the activation of oxidative reactions, the violation of protein conformation. The purpose of the study was to compare the intensity of formation of carbonyl derivatives of human and cow's milk proteins during spontaneous and metal-catalyzed oxidation. Material and methods. The object of the study were samples of mature milk of healthy nursing mothers (n=12), and samples of drinking ultra-pasteurized milk for baby nutrition (n=8) which were used as a comparison material. The intensity of oxidative modification of milk proteins was determined spectrophotometrically by the interaction of carbonyl derivatives of amino acid residues with 2.4-dinitrophenylhydrazine to form 2.4-dinitrophenylhydrazone derivatives in a native sample of biological material and under induction of protein oxidation in vitro by the Fenton reaction by adding FeSO4 and hydrogen peroxide solutions. The content of nonprotein sulfhydryl groups was determined after protein precipitation spectrophotometrically with 5.5'-dithio-bis-2-nitrobenzoic acid. Results. The intensity of spontaneous (basic) oxidation doesn't have significant differences between the indicators of breast and cow's milk. Significant differences were established in the content of carbonyl derivatives of amino acid residues of human and cow's milk proteins during metal-catalyzed oxidation. Incubation with iron ions caused 1.5-2.5 fold more formation of both aldehyde and ketone derivatives of cow's milk proteins, recorded in the visible and ultraviolet spectrum. In cow's milk during spontaneous oxidation and induction of oxidation by a metal, the percentage of aldehyde-dinitrophenylhydrazones was lower than in breast milk and, conversely, the proportion of ketone-dinitrophenylhydrazones, late markers of oxidative degradation of proteins, was significantly higher. The content of non-protein sulfhydryl groups in cow's milk was 2 times less than in fresh human milk. A significant excessive content of aldehyde-dinitrophenylhydrazones (2 times) and ketone-dinitrophenylhydrazones (2.6 times) undet metal-catalyzed protein oxidation of cow's milk in comparison with breast milk indicates a lower level of antioxidant reserves of cow's milk. This is confirmed by the reduced level of non-protein sulfhydryl groups. The results obtained indicate the need to improve the antioxidant status of dairy products for infant nutrition.

[Repeated haploidentical allogeneic hematopoietic stem cell transplantation with TCR αß/CD19 depletion in patient with primary myelofibrosis. Case report].

Indications of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with primary myelofibrosis are intermediate-2 and high-risk group of DIPSS (Dynamic International Prognostic Scoring System), beginning of the disease in childhood. The other adverse factors affect engraftment and survival after allo-HSCT, example partialy matched donor. But the result of allo-HSCT from matched related donors and result of allo-HSCT from haploidentical donors are comparable. The method for haploidentical hematopoietic stem cell transplantation is T-cell-depletion. This is clinical case of T-cell-depleted haploidentical hematopoietic stem cell transplantation in patient with primary myelofibrosis, the diagnosis was established in childhood.

[Efficiency of interferon therapy in patients with essential thrombocythemia or polycythemia vera]. / Effektivnost' interferonoterapii u bol'nykh essentsial'noi trombotsitemiei i istinnoi politsitemiei.

AIM: To evaluate the efficiency of interferon (IFN) therapy in patients with essential thrombocythemia (ET) and polycythemia vera (PV). SUBJECTS AND METHODS: A total of 61 patients (41 with ET and 20 with PV) were examined. Prior to study enrolment, 44 (72%) patients with ET or PV received one or other therapy (aspirin was not taken into account). The mean Jak2V617F mutant allele at baseline was 23% (6-54%) in the patients with ET and 40% (11-88%) in those with PV. The median time from diagnosis to enrollment was 49 months. RESULTS: The paper presents the clinical and molecular findings of long-term INF-α therapy in patients with ET or PV. The median follow-up was 52 months. Recombinant IFN-α2 showed its ability to induce complete hematologic remission (ET (76%), PV (70%)) and a complete molecular response. 22 (69%) out of 32 patients were noted to have a smaller number of cells with the Jak2V617F mutation. In the patients with PV and in those with ET, the relative reduction in the proportion of cells with the Jak2V617F mutant gene averaged 85% and 56% of the baseline values, respectively. There was a reduction in the proportion of cells expressing the Jak2V617F mutation in both the ET (from 12 to 2.2%; p=0.001) and PV (from 32.7% to 3.2%) groups (р=0.001). Ten (31%) patients achieved a deep molecular remission (≤2% Jak2V617F allele); among them, 5 patients were not found to have Jak2V617F mutation. The obtained molecular response remained in 7 of the 10 patients untreated for 11 to 86 months. The long-term treatment with IFN-α led to normalization of the morphological pattern of bone marrow in 5 of the 7 PV or ET patients. CONCLUSION: Significant molecular remissions achieved by therapy with recombinant interferon-α2 confirm the appropriateness of this treatment option in in the majority of patients with ET or PV.

[Role of splenectomy in the treatment of myelofibrosis].

AIM: To evaluate the clinical and hematologic efficiency of splenectomy (SE) in patients with myelofibrosis (MF) resistant to conventional traditional treatment. SUBJECTS AND METHODS: Case histories were retrospectively analyzed in 52 MF patients who had been followed up at the Hematology Research Center, Ministry of Health of the Russian Federation, in 2004 to 2012 and undergone therapeutic SE (47 patients with primary myelofibrosis, 4 with postpolycythemia myelofibrosis, and 1 with postthrombocythemia myelofibrosis). The mean age was 47 years at diagnosis and 53 years before surgery. The patients younger than 50 years of age constituted 60%. Massive and giant splenomegaly was detected in 37 (71%) patients. The spleen weighing 0.9 to 2.9 and 3 to 7 kg was removed in 15 (29%) and 35 (67%) patients, respectively. In 2 cases, the weight of the removed spleen was as much as 10 and 11 kg. RESULTS: By the moment of SE, the disease duration averaged 76 (from 1 to 240) months. Twenty-one (40%) patients developed perioperative complications, including bleeding (15%), thrombosis (11.5%), and infectious complications (13.5%). There were no deaths from surgical interventions in the intra- and early postoperative periods. In more than 80% of the patients after SE, their general condition improved and the symptoms of intoxication disappeared; in the majority of patients, the therapeutic effect lasted about 2 years. In the follow-up period, 33 (63%) patients died; the time to death averaged 27 (1-84) months following SE. The causes of death were blast transformation in 27 (82%) patients and comorbidity in 6 (18%); 19 (37%) patients with an average post-SE follow-up of 37 (4-72) months continued hydroxyurea treatment. The median survival after SE was equal to 3 years; the median overall survival was 11 years. CONCLUSION: SE is effective palliative care with an acceptable level of occurring complications for individual patients with MF. Contraindications to SE as blast crisis and severe comorbidities should be strictly taken into account.

[Study of bone marrow microvessel density is one of the diagnostic approaches in patients with Ph-negative chronic myeloproliferative diseases].

AIM: To define an association of bone marrow microvessel density (MVD) with histological properties (the magnitude of fibrosis and quantification of megakaryocytes (MGKC)) in patients with Ph-negative chronic myeloproliferative diseases (CMPD). SUBJECTS AND METHODS: MVD was analyzed in 93 patients with different forms of CMPD, by estimating histological parameters. True polycythemia (TP) was present in 28 patients; 20 patients had essential thrombocythemia (ET), 36 had subleukemic myelosis, out them 6 were in a prefibrotic stage, and 9 with diagnosed post-TP (ET) myelofibrosis. The grade of myelofibrosis was estimated from the degree of bone marrow fibrosis as 0, 1, 2, and 3 and the clusters of MGKC were in accordance with degrees: 0, 1, and 2. MVD was studied from the absolute number of CD34-positive vascular structures. RESULTS: In patients with TP, fibrosis was defined as grade 0 and 1 in 23 (82%) and 5 (18%) cases, respectively. The content of reticulin fiber was in the normal range in 19 (95%) of the 20 patients with ET. The clusters of MGKC of grades 1 and 2 showed an even distribution among patients with ET and those with TP. Fibrosis was absent in all the patients (n = 6) with prefibrotic-stage primary myelofibrosis (PMF). The patients with PMF had high MVD values [6.5 (range 2.8-22)] than those with TP [4.0 (range 1.76-10.2)] or ET [3.7 (range 2-8.5)] and the controls [3.2 (range 2-4.1)] (p < 0.001) confirming that angiogenesis is uninvolved at the onset of disease in patients with ET and those with TP. The patients with prefibrotic-stage PMF had higher values [6.0 (range 4.8-10.6)] than those with ET [3. 7 (range 2-8.5)] (p < 0.001). This suggests that angiogenesis is an early sign preceding the development of fibrosis. CONCLUSION: Bone marrow angiogenesis assessment (from MVD measurements) may be an additional criterion for the diagnosis of disease evolution and an additional criterion between ET and PMF in a prefibrotic stage.

[Dasatinib treatment of imatinib-resistant and imatinib-intolerant patients with chronic myeloid leukemia in a chronic phase].

AIM: To analyse resistance to imatinib therapy, efficacy and safety of dasatinib. MATERIAL AND METHODS: A total of 18 patients with chronic myeloid leukemia (CML) in a chronic stage received dasatinib for 9-30 months (median 30 months) to September 2008. RESULTS: Lethal outcomes during dasatinib treatment were absent. To September 2008, 16 (89%) patients were alive, 2 (11%) patients died of the disease progression after dasatinib discontinuation. A complete clinicohematological response was observed in all the patients. Major cytogenetic, complete cytogenetic, major molecular, complete molecular responses were achieved in 12 (67%), 10 (55%), 7 (39%) and 5 (28%) patients, respectively. Hematological and non-hematological toxicity occurred in 9 (50%) patients. Now 12 (67%) patients continue dasatinib treatment, in 6 (33%) patients the drug was discontinued. CONCLUSION: The results from trials in Russian Hematological Research Center are the same as in the international study. Dasatinib is effective and well tolerated therapeutic option for imatinib-resistant patients with a chronic phase of CML.

[A comparative study of leptin content in diabetics and obese subjects].

AIM: To compare leptin content in morphologically heterogenic group of patients with type 2 diabetes mellitus and obese patients without family history of diabetes. MATERIAL AND METHODS: Leptin concentration was estimated with enzyme immunoassay (Leptin kit Sandwish ELISA, DRG diagnostics GmdH) in 21 patients with type 2 diabetes mellitus (2DM) and 12 obese patients without family history of diabetes. Distribution of patients with a documented diagnosis type 2 diabetes mellitus was conducted with application of instruments for antropometric examination (G.P. Gneupel, Switzerland). Statistic processing was made with the program Statistica 6 using Student's t-criterion. RESULTS: Patients with diabetes mellitus and phenotypical marker of 2DM had the lowest level of leptin (mean leptin level 1.99 ng/ml in males, 11.01 ng/ml in females versus 2DM patients without the marker (mean leptin level 9.16 n/mg in males, 23.56 n/mg in females) and control group (13.86 ng/ ml in males, 22.55 ng/ml in females). CONCLUSION: There is a correlation between leptin concentration and anthropological features of the patients.

[Recovery of polyclonial hemopoiesis in females with chronic myeloid leukemia with a complete cytogenetic response].

AIM: To study feasibility of hemopoiesis clonality determination in assessment of remission completeness in patients with chronic myeloid leukemia (CML) using polymerase chain reaction (PCR HUMARA). MATERIAL AND METHODS: We have examined 28 patients with newly diagnosed CML, 10 CML patients with a complete cytogenetic response (CCR) to therapy with imatinib mesilate and/or alpha-interferon, 24 healthy control females. Twelve patients with untreated CML were homozygous by HUMARA gene (human androgenic receptor gene) and were withdrawn from the study. Leukocytes of peripheral blood from all the patients were investigated with PCR HUMARA for mono- or polyclonal hemopoiesis. Clonality was defined as allele proportion (a/p) of polymorphic loci of HUMARA gene. Remission completeness was confirmed cytogenetically and by molecular methods. RESULTS: The value a/p in 10 patients with CCR varies from 0.69 to 1.33 and is similar to those in the control group. CONCLUSION: The PCR HUMARA technique adequately assesses reduction of Ph-positive clone in CML patients with CCR and points to polyclonal hemopoiesis.

[Prognosis factors in imatinib mesilate therapy in patients with a chronic phase of Ph-positive chronic myeloid leukemia: data from a multicenter non-randomized trial in Russia].

AIM: To reveal prognostically significant factors affecting efficacy of glivek therapy in untreated (duration of the disease < or = 6 months) and pretreated (duration of the disease > 6 months) patients with chronic myeloid leukemia (CML) in a chronic phase. MATERIAL AND METHODS: A total of 338 patients (64 untreated and 274 pretreated) with a chronic-phase CML on glivek therapy entered the trial. RESULTS: Five-year survival on glivek was high (89, 98 and 88% in untreated and pretreated patients, respectively). Incidence of transformation in the acceleration phase and blast crisis was low both in untreated and pretreated patients (1.6 and 11%, respectively) and correlated with the rate of a complete cytogenetic response (CCR). Untreated patients had no factors affecting treatment efficacy negatively, CCR probability was 96%. Blastemia, thrombocytosis and splenomegaly reduced CCR probability significantly in pretreated patients. Slow reduction of the tumor mass, late achievement of a complete hematological response and a cytogenetic response decreased probability of CCR. CONCLUSION: Glivek is a drug of choice for patients with chronic-phase CML. High probability of CCR both in untreated and pretreated patients lowers the risk of the disease transformation into the phase of acceleration/blast crisis and raises overall survival in both groups.

Application of PRV-1 mRNA expression level and JAK2V617F mutation for the differentiating between polycytemia vera and secondary erythrocytosis and assessment of treatment by interferon or hydroxyurea.

Increased PRV-1 mRNA expression and the presence of Jak2(V617F) mutation in peripheral blood granulocytes are specific markers for chronic myeloproliferative disorders (MPD), which facilitate the differential diagnosis between polycythemia vera (PV) and secondary erythrocytosis (SE) and may be helpful for monitoring treatment efficacy in MPD patients. We evaluated the presence of the Jak2V617F mutation and increased PRV-1 mRNA expression along with previously established markers - erythropoietin (EPO) independent colony formation (EEC) and erythropoietin level for diagnosis of PV and assessment of treatment efficiency. Increased PRV-1 expression was found in 37 out of 46 patients diagnosed with PV (80%), in 4 out of 15 patients diagnosed with essential thrombocythemia (ET) (27%) and in 4 out of 8 patients with chronic idiopathic myelofibrosis (CIMF) (50%), and increased PRV-1 expression plus EEC formation was observed in 19 of 36 examined MPD patients indicating the superiority of PVSG and WHO bone marrow criteria for the diagnosis of ET, PV and CIMF. We could confirm a very high sensitivity, specificity and utility of the Jak2(V617F) mutation for differential diagnosis between PV and SE. Spontaneous EEC, serum EPO levels, PRV-1 expression was evaluated in 22 PV patients who carried the Jak2(V617F) mutation. A concordance of increased PRV-1 expression and presence of Jak2(V617F) mutation in 19/22 (85%); of increased PRV-1/Jak2/EEC in 14/22 (63%); and of Jak2/PRV-1/EEC/low Epo level in 10/22 (45%) patients was found indicating the superiority of the presence of Jak2(V617F) mutation for the diagnosis of PV. IFN-alpha therapy in patients with PV was more effective then hydroxyurea treatment and significantly reduced increased PRV-1 expression together with higher levels of Jak2(V617F) mutation (50-100%) in PV patients treated with hydroxy urea (HU) and lower levels of Jak2(V617F) mutation (35-90%) in PV patients treated with IFN-alpha. Normal PRV-1 expression level was observed in 44% of PV patients who achieved clinical remission and only in 3% of patient who did not. These preliminary observations indicate that the Jak2(V617F) mutation in particular and PRV-1 overexpression appear to be suitable markers for monitoring treatment efficiency in prospective randomised clinical studies comparing pegylated interferon and hydroxyurea in well defined PV patients with a clear indication for cytoreductive therapy.

Early and late hemopoietic precursor cells in patients with chronic myeloproliferative diseases.

We evaluated the content of early and late cobblestone area-forming cells, immediate progeny of hemopoietic stem cells, and committed precursor cells in the bone marrow and peripheral blood of patients with chronic myeloproliferative diseases and healthy donors. In patients with essential thrombocythemia, the number of late cobblestone area-forming cells in the peripheral blood decreased, while other parameters did not differ from those in healthy donors. In patients with idiopathic myelofibrosis, we found a decreased number of late and early cobblestone area-forming cells in the bone marrow and late cobblestone area-forming cells in the peripheral blood, while the count of early cobblestone area-forming cells in the peripheral blood increased. In patients with chronic myeloid leukemia, the number of early cobblestone area-forming cells in the bone marrow decreased, but the count of late and early cobblestone area-forming cells in the peripheral blood increased. The number of endogenous committed precursor cells in the peripheral blood increased in all groups of patients with chronic myeloproliferative diseases and, particularly, in patients with idiopathic myelofibrosis and chronic myeloid leukemia. Functional characteristics of immediate descendants of hemopoietic stem cells probably reflect the level of damage and attest to the existence of various mechanisms underlying the defect of the hemopoietic stem cell during chronic myeloproliferative diseases.

[Hyperhomocysteinemia--one of the factors underlying thrombotic complications in patients with chronic myeloproliferative diseases].

AIM: To assess incidence of hyperhomocysteinemia (HHC) in patients with chronic myeloproliferative diseases (CMPD) and to analyse possible correlation between an elevated concentration of plasma homocystein (HC) and thrombotic complications. MATERIAL AND METHODS: The trial enrolled 61 patients: 39 CMPD patients with thrombotic complications and free of them, 22 nonhematological patients with thrombosis. The control group consisted of 40 healthy donors. The examination protocol included determination with standard methods of HC plasma concentration, platelet and plasma components of hemostasis, mutation of factor V Leiden gene, prothrombin and methylenetetrahydrofolate reductase (MTHFR). RESULTS: Mean HC concentration in the serum in CMPD patients was 19 +/- 1.7 mcmol/l which appeared higher than in healthy donors (12 +/- 1.3 mcmol/l). The highest HC was in patients with subleukemic myelosis (SLM)--23 +/- 2.3 mcmol). No difference in HC concentration in plasma was observed in CMPD carriers of homo- or heteroxygous mutation of C667T gene or CMPD patients without the mutation. In CMPD content of factor VIII was higher in HHC than in normal HC (222 +/- 26.5 and 116 +/- 20%, respectively, p = 0.002). For von Willebrand factor 202 +/- 15.6 and 120 +/- 14.6%, respectively (p < 0.003). HC reduction in response to vitamin therapy was the greater the higher its initial level was. CONCLUSION: There is correlation between HHC and thrombosis in CMPD patients. HC concentration may depend on the proliferative stage of CMPD. As HC is a significant independent factor of thrombotic complications risk, it is necessary to detect and treat HHC.

[Chronic myeloproliferative diseases and pregnancy].

AIM: To analyse the course of pregnancy in chronic myeloproliferative diseases (CMPD) with hyperthrombocytosis, primarily, essential thrombocytemia. MATERIAL AND METHODS: The analysis of thrombogenic risk factors covered literature data and 8 cases observed by the authors. RESULTS: Six pregnant women received long-term treatment with preparations of interferon-alpha in a dose 9-20 million IU a week (both before and during pregnancy). Rapid reduction of hyperthrombocytosis (1100-4000 x 10(9) l) and the absence of a negative effect on development of the fetus were seen in all the cases. Normal delivery on week 37-39 was in 4 patients, spontaneous abortion on week 24 was provoked by a car accident. Three gravidas (gestational week 28, 33 and 34) are still under observation. Lupus anticoagulant or elevation of anticardiolipin antibodies level was detected in 4 of 8 patients, 2 patients had heterozygous mutation of methylentetrahydrofolatereductase genes and factor V (Leiden). These patients were given lannacher, faxiparine, folic acid and discrete plasmapheresis (in 2 cases). CONCLUSION: Gravidas with hyperthrombocytosis, if not contraindicated, must be treated with aspirin and interferon-alpha preparations at any gestational term. Moreover, it is necessary to exclude additional most prevalent causes of thrombophilia for adequate prevention of thromboses.

[Prediction of interferon therapy efficacy in chronic myeloid leukemia according to data of histomorphological study]. / Prognozirovanie éffektivnosti interferonoterapii pri khronicheskom mieloleikoze po dannym gistomorfologicheskogo issledovaniia.

AIM: To investigate factors determining prognosis and efficacy of induction therapy including interferon-alpha-2b (intron-A, Schering Plough) in patients at an early chronic stage of Ph-positive chronic myeloid leukemia (CML) as shown by histomorphological examination. MATERIAL AND METHODS: The analysis covered 52 CML patients treated at an early chronic phase with intron-A in a standard daily dose 5 IU/m2 in combination with low-dose cytosinearabinoside (10 mg/m2, s.c. , daily for 10 days of each month). The treatment efficacy was assessed by the international criteria of complete and partial hematological remission and cytogenetic response. The cytogenetic study employed the direct method, even and G-differential staining, fluorescent hybridization in situ (FISH). The sections were stained with hematoxilin-eosine by Gomori, van Gieson. Histological samples were examined with histomorphometry. Immunohistochemical examination was made on paraffin sections using a panel of monoclonal antibodies CD3, CD4, CD8, CD20, NK, PCNA, Ki-67 (Dako, Denmark). RESULTS: Repeated assessment of histomorphological parameters such as erythroid lineage, degree of myelofibrosis and reduction of leukemic population indicate the treatment efficacy. Estimation of the level of leukemic population proliferation in trephine biopsies from CML patients with monoclonal antibodies PCNA and Ki-67 before the treatment is prognostically significant as it further correlates with the cytogenetic response (r = 0.821, p = 0.000000). CONCLUSION: It is valid to study histomorphological picture of CML to prognosticate and assess treatment efficacy with standard doses of interferon-alpha with high probability.

[Difficulties in forensic medical evaluation of some forms of craniocerebral injury]. / Trudnosti v sudebno-meditsinskoi otsenke nekotorykh form cherepno-mozgovoi travmy.

Conclusions of 41 repeated expert evaluations of craniocerebral injuries within the framework of criminal and civil cases investigation are analyzed. Some aspects of clinical and forensic medical diagnosis of lethal and nonlethal injuries to the head, evaluation of the quality of medical care, and qualification of the severity of harm to health are discussed. Causes of typical expert errors and approaches to their prevention are shown.

[Analysis of the suffix short retroelement copies and the encoded protein domain of Drosophila]. / Analiz kopii suffiksa--korotkogo retroélementa Drozofily--i kodiruemogo im belkovogo domena.

To analyze the copies of the suffix short retro-element, its homologs were sought in nucleic acid sequence databases of the Drosophila melanogaster genome. The search yielded several conserved (near identical in sequence) copies, which are indicative of recent suffix transposition, and numerous divergent copies, which suggest ancient suffix transposition. Analysis of the short suffix ORF revealed a conserved protein domain, which was also found as the eighth C-terminal domain in reverse transcriptases of certain long interspersed elements (LINEs). The suffix-encoded polypeptide proved to be homologous to DNA- and RNA-recognizing domains.