El caseinato de sodio y la caseína α inhiben la proliferación de la línea celular mieloide de ratón 32D clone 3 (32Dcl3) mediante el TNF-α / Sodium caseinate and alfa-casein inhibit proliferation of the mouse myeloid cell line 32D clone 3 (32Dcl3) via TNF-α

Resumen Introducción. Se ha demostrado que el caseinato de sodio y sus componentes (caseínas α, β y κ) inhiben la proliferación de la línea celular hematopoyética de ratón 32D clone 3 (32Dcl3) e inducen su diferenciación hacia macrófagos. Se sabe que la caseína α induce la producción de IL-1β y que esta última citocina inhibe la proliferación celular mediante la producción del factor de necrosis tumoral alfa (TNF-α), pero se desconoce si el caseinato de sodio y las caseínas inducen la producción de TNF y si este es el responsable de la inhibición de la proliferación. Objetivo. Evaluar si el caseinato de sodio y las caseínas α, β y κ inhiben la proliferación de la línea celular 32Dcl3 mediante la producción de TNF-α. Materiales y métodos. Se usaron diferentes concentraciones de caseinato de sodio y de las caseínas α, β y κ en las células 32Dcl3. Posteriormente, se evaluaron la viabilidad celular mediante una prueba con el MTT [3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol], la inducción de apoptosis con la citometría de flujo y la síntesis del TNF-α con el ELISA. Además, se hicieron pruebas de neutralización con anti-TNF-α en células 32Dcl3 tratadas con caseinato de sodio y caseína α, y se evaluó la proliferación celular. Resultados. Se encontró que el caseinato de sodio y las caseínas α, β y κ reducían la proliferación de la línea celular 32Dcl3 sin afectar la viabilidad, y que solo el caseinato y la caseína α inducían la apoptosis y la liberación al medio de TNF-α. La proliferación de células 32Dcl3 tratadas con caseinato y caseína α se restableció al usar anticuerpos anti-TNF-α. Conclusión. El TNF-α fue el principal responsable de la inhibición de la proliferación en las células 32Dcl3 tratadas con caseinato de sodio o caseína α.

Abstract Introduction: Sodium caseinate (CS) and its components (alpha-casein, beta-casein, and kappa-casein) have been shown to inhibit the proliferation of the mouse hematopoietic 32D clone 3 (32Dcl3) cell line and induce its differentiation into macrophages. It is well-known that alpha-casein induces IL-1β production and that this cytokine inhibits the proliferation via the production of tumor necrosis factor alpha (TNF-alpha), but it is not known if CS and the caseins inhibit the proliferation via TNF-alpha production. Objective: To evaluate if CS and alpha-casein, beta-casein and kappa-casein inhibit the proliferation on 32Dcl3 cell line via TNF-alpha. Materials and methods: We used different concentrations of CS, alpha-casein, beta-casein and kappa-casein in 32Dcl3 cells to evaluate cell proliferation. We assessed cell viability by MTT, induction to apoptosis by flow cytometry, and TNF-alpha synthesis by ELISA. Additionally, we performed anti-TNF-alpha neutralization assays on 32Dcl3 cells treated with CS and alpha-casein and we evaluated proliferation. Results: The results showed that CS, alpha-casein, beta-casein, and kappa-casein reduced proliferation of the 32Dcl3 cell line without affecting the viability and that only CS and alpha-casein induced apoptosis and the release of TNF-alpha. The 32Dcl3 cells treated with CS and alpha-casein reestablished their proliferation by using anti-TNF-alpha antibodies. Conclusion: TNF-alpha was the main responsible for the inhibition of proliferation in 32Dcl3 cells treated with CS or alpha-casein.

Skewed Dendritic Cell Differentiation of MyD88-Deficient Donor Bone Marrow Cells, Instead of Massive Expansion as Myeloid-Derived Suppressor Cells, Aggravates GVHD

Graft-versus-host disease (GVHD), a life-threatening complication after bone marrow transplantation (BMT), is induced by activation of alloreactive donor T cells. Our previous study demonstrated that transplantation of myeloid differentiation factor 88 (MyD88)-deficient knockout (KO) bone marrow (BM) resulted in aggravation of GVHD. Here, to understand the cellular mechanism, we performed longitudinal in vivo imaging and flow cytometric analyses followed by transcriptome and functional examination of donor MyD88-KO BM progenies in GVHD hosts, using a major histocompatibility complex-matched but minor histocompatibility antigen-mismatched C57BL/6→BALB.B model. In GVHD hosts with MyD88-KO BMT, donor BM-derived CD11b+Gr-1+ cells were found to undergo cell death, a fate significantly different from the explosive expansion shown by the wild type (WT) counterparts, and also from the moderate expansion of the WT or MyD88-KO BM-derived cells in non-GVHD hosts. It was also revealed that MyD88-KO CD11b+Gr-1+ cells preferred differentiation into CD11c+ dendritic cells (DCs) to expansion as myeloid-derived suppressor cells in GVHD hosts or in high inflammatory in vitro conditions. These CD11c+ DCs comprised the majority of MyD88-KO CD11b+Gr-1+ apoptotic cells in GVHD hosts. Their ability to cross-present alloantigens of host origin contributed to the enhancement of T cell alloreactivity, causing GVHD aggravation and eventually death through the killing function of activated T cells. These results provide insights into the roles of MyD88 in myelopoiesis of donor BM and the protective effects in GVHD hosts, helpful information for development of a strategy to control GVHD.

Prognóstico de crianças com síndrome de Down e leucemia mielóide aguda ou mielopoese anormal transitória / [Prognosis of children with Down syndrome and acute myeloid leukemia or transient abnormal myelopoiesis]

Introdução: Crianças com síndrome de Down (SD) apresentam elevada frequência de mielopoese anormal transitória (TAM) e leucemia megacarioblástica aguda (LMA-M7). ATAM é exclusiva em neonatos com SD, e tem um importante potencial para transformação leucêmica. TAM e LMA-M7 carregam mutações somáticas no éxon 2 do GATA1 resultando na exclusiva expressão da proteína truncada (GATA1s). O objetivo principal desta pesquisa foi analisar os fatores associados ao prognóstico de crianças ≤ 4 anos de idade com SD diagnosticadas com TAM ou LMA atendidas em instituições oncológicas brasileiras. Material e método: foi realizado um estudo retrospectivo, de coorte, de crianças com TAM ou LMA. O fluxo de trabalho seguiu as seguintes etapas: 1º) seleção dos casos com SD e suspeita clínica de leucemia; 2º) re-análise para confirmação diagnóstica; 3º) seleção dos casos confirmadosde TAM e LM-SD; 4º) Rastreamento de mutações GATA1 (éxon2) por sequenciamento direto e classificação da mutação: I- perda da primeira metionina; II- erro de splicing; IIIcódon de terminação precoce (PTC 1, antes da Met-84, e PTC 2 após Met-84); 5º) Detecção dos transcritos alternativos por transcriptase reversa e reação em cadeia da polimerase (RTPCR) e análises densiométricas por meio do programa Quantity-One (Version 4.5.2; Bio-Rad Laboratories); Finalmente, 6º) Análise e interpretação dos dados considerando os objetivos doestudo por meio do pacote estatístico SPSS18, Chicago...

Introduction: Children with Down syndrome (DS) have a high frequency of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia (AML-M7). TAM is unique in neonates with Down syndrome, and has an important potential for leukemictransformation. TAM and AML-M7 carry somatic mutations in exon 2 of GATA1 resulting in exclusive expression of a truncated protein (GATA1s). The main objective of this research was to analyze the factors associated with the prognosis of children ≤ 4 years of age with DS diagnosed with AML or TAM served in Brazilian oncological institutions. Material and method: was performed a retrospective study, in a cohort of children with TAM or AML. Theworkflow followed the following steps: 1º) selection of cases with DS and clinical suspicion of leukemia; 2º) re-analysis for diagnostic confirmation; 3º) selection of confirmed cases ofTAM and AML; 4º) GATA1 mutation screening (exon2) by direct sequencing and classification of mutation: I- loss of the first methionine; II- splicing errors; III- premature termination codon (PTC 1, before the Met-84, and PTC 2 after the Met-84); 5º) detection of alternative transcripts by Reverse transcription polymerase chain reaction (RT-PCR) and densitometry analysis using the program Quantity-One (Version 4.5.2; Bio-RadLaboratories); Finally, 6º) Analysis and interpretation of data considering the objectives of the study using statistical packages (SPSS18, Chicago). Results: 97 cases, 63 diagnosed with AML, of these, 31 had a history of prior TAM, and 34 diagnosed with TAM were included in this analysis. GATA1 mutations were found in 77.8% of cases. It was observed that the type ofmutation affects ...

Acute Megakaryoblastic Leukemia with CD41a-/CD61-/CD42a+ Blasts in an Infant with Down Syndrome

Infants with Down syndrome have increased incidences of transient abnormal myelopoiesis (TAM) and acute leukemia, which are usually associated with acute megakaryoblastic leukemia (AMKL). A 5-day-old girl with Down syndrome was diagnosed with TAM; 4 months later, acute leukemic transformation was suspected. Bone marrow (BM) examination was performed, and the infant was diagnosed with acute leukemia (80% blasts). Although BM aspirates showed the presence of megakaryocytic blasts with cytoplasmic blebs, flow cytometry analysis revealed that they were negative for cells with CD41a and CD61 immunophenotypes. Further analysis revealed that the megakaryocyte-related marker CD42a was positive in 57% of blasts. Morphologic and immunophenotypic features are required to establish the lineage of megakaryocytic blasts, which are necessary for diagnosing AMKL. As most cases of AMKL were positive for CD41 and/or CD61 markers, their presence was evaluated during routine analysis. In order to identify the immunophenotypic features of AMKL in an infant with Down syndrome, we performed additional flow cytometry for CD42a, one of the megakaryocytic markers, and were able to assist in the early diagnosis of AMKL, as well as to use CD42a as an effective follow-up marker.

Therapeutic effects and prognostic factors for the limited-stage small cell lung cancer treated with multidisciplinary therapy / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the differences of objective response rate (ORR), side effects and survival among patients with limited-stage small cell lung cancer (LD-SCLC), who received concurrent chemoradiotherapy, sequential chemoradiotherapy or chemotherapy alone, and to analyze the influencing factors on their survival.</p><p><b>METHODS</b>One hundred and sixty-six patients diagnosed as LD-SCLC in Peking Union Medical College Hospital from January 2000 to December 2009 were included in this study. The differences of objective response rates, side effects and survival rates were analyzed by χ2 test. Kaplan-Meier test was used to calculate the overall survival (OS) and progress-free survival (PFS). Cox regression was used to detect the influencing factors on survival time of the patients.</p><p><b>RESULTS</b>The patients were divided into three groups: concurrent chemoradiotherapy (49 cases), sequential chemoradiotherapy (62 cases) and chemotherapy alone (55 cases). The chemotherapy was based on CE/EP regimen, with an average cycle of 5.2. Radiotherapy was of a common or 3-dimensional conformal technology, for regular segmentation irradiation with an average dose of 49.6 Gy. The total ORR was 73.4%, OS and PFS were 22.9 months and 10.8 months, 1, 3, 5-year survival rates were 82.7%, 31.8%, 18.6%, respectively. For the concurrent group, sequential group and chemotherapy alone group, the ORR was 89.4%, 67.2% and 66.0%, respectively. Compared the chemotherapy alone group and concurrent group with the sequential group, there were significant differences (P<0.05). For the concurrent group, sequential group and chemotherapy alone group, the median OS was 29.7 months, 22.6 months, and 19.5 months; the median PFS was 12.7 months, 10.8 months, and 9.8 months, respectively, with a non-significant difference between each two groups (P>0.05). For the concurrent group, sequential group and chemotherapy alone group, the 1-year survival rates were 91.1%, 86.3%, and 65.6%, the 3-year survival rates were 44.2%, 28.3% and 22.8%, and the 5-year survival rates were 24.2%, 21.4% and 11.1%, respectively, with significant differences among them (P<0.05). The major side effects were myelosuppression, gastrointestinal reactions, radiation pneumonia and radiation esophagitis. For the concurrent group, sequential group and chemotherapy alone group, the incidence of myelosuppression were 84.4%, 76.8% and 60.0%, respectively, with a significant difference (P=0.008) between the concurrent group and chemotherapy alone group. For the concurrent group and sequential group, the incidences of radiation pneumonia were 22.2% and 22.9%, with a non-significant difference (P=0.940). The incidences of radiation esophagitis were 47.2% and 16.7%, respectively, with a significant difference (P=0.002). Multivariate analysis showed that OS was significantly associated with gender (P=0.018) and ECOG score (P=0.009), and PFS was significantly associated with gender (P=0.050).</p><p><b>CONCLUSIONS</b>For LD-SCLC, concurrent chemoradiotherapy can significantly increase the objective response rate. Concurrent chemoradiotherapy and sequential chemoradiotherapy compared with chemotherapy alone can extend survival, and concurrent chemoradiotherapy is better, but the differences among the three regimens are not significant. Gender and ECOG score are important influencing factors of survival.</p>

Study of a new zebrafish mutant defective in primitive myelopoiesis / 南方医科大学学报

<p><b>OBJECTIVE</b>To perform phenotypic identification and characteristic analysis of a new zebrafish mutant 1276 defective in primitive myelopoiesis.</p><p><b>METHODS</b>The AB strain male zebrafish were mutagenized with N-ethyl N-nitrosourea (ENU) to induce mutations in the spermatogonial cells, and the mutations were transmitted to the offsprings. The F3 embryos were screened by neutral red staining for identifying the mutants defective in primitive myelopoiesis. One of the myeloid mutants 1276 was further studied by cytochemistry and whole mount in stiu hybridization (WISH) with different lineage markers.</p><p><b>RESULTS</b>A total of 2140 mutagenized genomes from the 1296 F2 families were analyzed, and 12 mutants were identified to show abnormal signal by neutral red staining. In the primitive hematopoiesis stage, the mutant 1276 showed the absence of neutral red staining-positive cells in the whole body. The expression of microglia marker apoe was totally lost in the head of the mutant, and the expression of the macrophage marker l-plastin was slightly decreased in the head and remained normal in the ventral dorsal aorta region, but the granulocytes and erythrocytes developed normally. in the definitive hematopoiesis stage, the mutant 1276 still showed abnormal macrophages as found in the primitive hematopoiesis stage, but the granulocytes, erythrocytes and lymphocytes appeared normal.</p><p><b>CONCLUSION</b>The zebrafish mutant 1276 shows abnormalities in the function, development and migration of the macrophages in the primitive hematopoiesis stage, which can not be compensated in the definitive hematopoiesis stage.</p>

Forward genetic screening for zebrafish mutants defective in myelopoiesis / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify zebrafish mutants with myelopoiesis defects by ENU mutagenesis and large-scale forward genetic screening.</p><p><b>METHODS</b>Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in the spermatogonial cells to generate the founders, which were outcrossed with AB to raise F1 fish. The F1 fish from different founders were mated to generate the F2 families. The F3 embryos from F2 sibling crosses were screened by Sudan black B staining and neutral red staining.</p><p><b>RESULTS</b>A total of 350 F2 families from F1 sibling crosses were screened, and 1424 F2 crosses were analyzed. Six mutations were identified resulting in abnormal Sudan black B staining and neutral red staining, indicating the involvement of neutrophil deficiency or macrophage abnormalities.</p><p><b>CONCLUSION</b>It is simple and cheap to induce and screen myelopoiesis deficiency in zebrafish by ENU chemical mutagenesis and Sudan black B staining and neutral red staining. These mutants shed light on the identification of the genes important to myelopoiesis in zebrafish.</p>

Prenatal Diagnosis of Transient Abnormal Myelopoiesis in a Down Syndrome Fetus

We report a case of transient abnormal myelopoiesis in a Down syndrome fetus diagnosed at 28(+3) weeks of gestation that rapidly progressed to intrauterine death 10 days later. Fetal hepatosplenomegaly with cerebral ventriculomegaly, although not specific, may be a suggestive finding of Down syndrome with transient abnormal myelopoiesis. Prompt fetal blood sampling for liver function test and chromosomal analysis are mandatory for early detection and management.

Insuficiencia medular / Bone marrow insufficiency

La insuficiencia medular es un síndrome hematológico; de pronóstico siempre reservado; que reconoce dos mecanismos etiopatogénicos: la aplasia medular y la mieloptisis. La primera es de naturaleza benigna y recuperable (50 por ciento) y la segunda maligna. Ambas se manifiestan clínicamente por una pancitopenia de la sangre periférica, la que se traduce por la asociación de un síndrome anémico normocítico y normocrómico arregenerativo, uno infeccioso febril y uno purpúrico. El diagnóstico clínico de insuficiencia medular es fácil, no así su tratamiento, especialmente en las mieloptisis.

Down Syndrome with Transient Myeloproliferative Disorder, Hepatic Fibrosis, and Hemochromatosis

Transient myeloproliferative disorder (TMD), which may mimic acute leukemia, occurs in neonates with Down syndrome along with hepatic fibrosis. TMD is recognized shortly after birth or in the neonatal period and is characterized by leukocytosis and thrombocytopenia, which resolve spontaneously in four to six weeks. And hepatic fibrosis is characterized by diffuse intralobular sinusoidal fibrosis, extramedullary hematopoiesis and hemochromatosis. A newborn male infant with Down syndrome, atrial septal defect and ventricular septal defect is reported. He showed abnormal myelopoiesis accompanying characteristic hepatic sinusoidal fibrosis. Knowing the cellular mechanism of hepatic fibrosis and its modulation by growth factors, a pathogenetic link between transient myeloproliferative disorder and the development of liver fibrosis in Down syndrome neonates, association of this triad no longer appears to be accidental.

A Case of Pure Red Cell Anemia Complicated by Antiepileptic Drugs

A 12-year-old boy developed pure red cell anemia(PRCA) during a combination therapy of antiepileptic drugs(AEDs) for epilepsy. His complex partial seizure was intractable to monotherapy. During 7 months of treatment, he was treated with Vigabatrin, Carbamazepine and Valproate. While switching from Carbamazepine to Valproate, he presented anemia but with no jaundice. His hemoglobin was 4.1g/dl bone-marrow biopsy revealed erythroid hypoplasia with normal myelopoiesis and megakaryocytopoiesis, indicating PRCA. Rapid recovery from PRCA was observed 1 month after discontinuation of Valproate, without immunosuppressive therapy. Although the hematologic toxicity of AEDs is well documented, isolated cessation of red cell production is uncommon. Our observation suggests that the synergistic toxicity caused by Valproate and Carbamazepine may induce PRCA through the inhibitory effect beyond the differentiation stage of BFU-E and CFU-E.

Linfopoese B associada ao granuloma hepático da esquistossomose: presença de precursores jovens e inibição da diferenciação / B lymphopoiesis associated with hepatic granulomas of schistosomiasis: youth presence of precursors and inhibition of differentiation

A reação inflamatória granulomatosa induzida, no fígado pelos ovos do Schistosoma mansoni é um sítio ativo de mielopoese extramedular potencialmente capaz de produzir todas as linhagens lielóides. O aumento da população de linfócitos B, ao longo da doença, associado à presença de precursores multipotentes, nos fez questionar se, em paralelo a mielopoese, haveria também linfopoese B. Foi demonstrado, por imunofenotipagem, a presença de células com característica de precursor B jovem (pro B) no granuloma. A expressão de RAG 1 e 5 foi demonstrada através da técnica de RT-PCR, confirmando a presença de precursosres B no granuloma. No entanto, colônias pré B não foram observadas, indicando um bloqueio na diferenciação de pro B para pre B. As células do estroma foram capazes de sustentar precursores B e a expressão de IL7 e SCF foi demonstrada. Porém, o meio condicionado de granuloma mostrou atividade inibidora da linfopoese B ao mesmo tempo que estimulava a mielopoese. Além disso, aproximadamente 50% dos precursores B do granuloma expressavam Mac 1. O estudo permitiu concluir que, há o desenvolvimento de um microambiente hematopoietico no granuloma, que permite a migração de progenitores e o multipotentes e o comprometimento para a linhagem B. No entanto, a produção extramedular de linfócitos B é bloqueada por fatores solúveis produzidos no granuloma