Actualización en el diagnóstico y estratificación de pacientes con Leucemia Mieloide Aguda: una necesidad país imperiosa

La leucemia mieloide aguda es una neoplasia con una elevada letalidad, con resultados inferiores en nuestro país respecto a la experiencia internacional publicada, posicionándola como una prioridad desde el punto de vista de salud pública oncológica. Actualmente, para su diagnóstico y estratificación se dispone de citología, inmunofenotipo, cariograma y escasas traslocaciones/mutaciones por biología molecular. Esta aproximación diagnóstica es insuficiente, ya que nos permite clasificar menos del 50% de los pacientes en un grupo específico y, por lo tanto, la elección de la terapia de consolidación se realiza con escasa información biológica. El rol de la morfología y de la citogenética progresivamente pierden relevancia pronóstica con respecto a la biología molecular, y la secuenciación de siguiente generación se ha posicionado como un elemento clave para el diagnóstico y estratificación de riesgo de estos pacientes. Además, la pesquisa de mutaciones germinales ha ido adquiriendo mayor relevancia, aumentando su frecuencia de detección e influyendo en la toma de decisiones respecto al tratamiento y en la selección de donante emparentado para un trasplante alogénico. En esta revisión se realiza una actualización del diagnóstico integrado de pacientes con leucemia mieloide aguda, a la luz de las nuevas clasificaciones diagnósticas (OMS 2022 e ICC 2022) y pronósticas (ELN 2022) y se propone un algoritmo a considerar para su implementación. Es perentorio como país invertir en nuevas tecnologías diagnósticas para mejorar el pronóstico de nuestros pacientes.

Acute myeloid leukemia is a neoplasm with a high lethality, with alarming results in our country, positioning it as a priority from the point of view of oncological public health. Cytology, immunophenotype, karyogram, and a few translocations/mutations by molecular biology are currently available for diagnosis and stratification. This diagnostic approach is insufficient since it allows classifying less than 50% of patients in a specific group. Therefore, consolidation therapy is selected with little biological information. The role of morphology and cytogenetics is progressively losing prognostic weight with respect to molecular biology, and next-generation sequencing has positioned itself as a key element for diagnosing our patients. In addition, the investigation of germline mutations is acquiring greater relevance, increasing its detection frequency and influencing decision-making regarding treatment and selecting a related donor for an allogeneic transplant. In this review, an update of the integrated diagnosis of patients with acute myeloid leukemia is carried out in light of the new diagnostic (WHO 2022 and ICC 2022), and prognostic classifications (ELN 2022). We propose an algorithm for integrated diagnosis to be considered for its implementation. It is imperative as a country to invest in new diagnostic technologies to improve the prognosis of our patients.

First report of pediatric ehrlichiosis in Mexico / Primer reporte de ehrlichiosis pediátrica en México

Abstract Background: Ehrlichia chaffeensis is responsible for most cases of human ehrlichiosis, an acute febrile tick-borne disease. This clinical entity is more commonly reported in adults from the United States. Therefore, it is of special interest to characterize this disease in children, given that very few cases in children have been reported outside of this country. Case report: We describe the case of a 15-year-old female from northeastern Mexico with a five-day history of myalgias, arthralgias, fever, abdominal pain, rash, and somnolence. The possibility of tick-borne disease was suspected considering that she lived with three tick-infested dogs that had recently died and a neighbor with similar symptoms who deteriorated rapidly and died a week earlier. Ehrlichia spp. was detected in blood samples by polymerase chain reaction. The patient completed a seven-day course of doxycycline and was discharged with complete resolution of symptoms. Conclusions: This case is the first report of ehrlichiosis in a pediatric patient in Mexico, illustrating the importance of considering tick-borne diseases as a differential diagnosis in patients with rash, fever, and altered level of consciousness. This initial clinical presentation may be indistinct from other conditions such as dengue, meningococcemia, and multisystem inflammatory syndrome in children (MIS-C), among others.

Resumen Introducción: Ehrlichia chaffeensis es responsable de la mayoría de los casos de ehrlichiosis humana, una enfermedad febril aguda transmitida por garrapatas. Esta entidad clínica se reporta con mayor frecuencia en adultos de Estados Unidos. Por lo tanto, es de especial interés caracterizarla en niños, dado que se han reportado muy pocos casos en niños fuera de este país. Caso clínico: Se describe el caso de una paciente de sexo femenino de 15 años, originaria y residente del noreste de México con una historia de cinco días de mialgias, artralgias, fiebre, dolor abdominal, erupción cutánea y somnolencia. Se sospechó la posibilidad de una enfermedad transmitida por garrapatas considerando que convivió con tres perros infestados de garrapatas que habían muerto recientemente y una vecina con síntomas similares, quien se deterioró rápidamente y murió una semana antes. Ehrlichia spp. se detectó en una muestra sérica mediante reacción en cadena de la polimerasa. La paciente completó un curso de siete días de doxiciclina y fue dada de alta con resolución de los síntomas. Conclusiones: Este caso es el primer reporte de ehrlichiosis en un paciente pediátrico en México que ilustra la importancia de considerar enfermedades transmitidas por garrapatas dentro del diagnóstico diferencial de pacientes con exantema, fiebre y alteración del estado de conciencia. Esta presentación clínica inicial puede ser indistinguible de otras entidades como dengue, meningococcemia y síndrome multisistémico inflamatorio, entre otras.

New sensitive real-time PCR targeting p28 gene for detection of Ehrlichia canis in blood samples from dogs / Novo PCR em tempo real sensível que visa o gene p28 para a detecção de Ehrlichia canis em amostras de sangue de cães

ABSTRACT: This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the 28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene, in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs' whole blood samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by an established cPCR and the new qPCR to compare each technique's performances. This new qPCR method for the molecular detection of E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R2) equivalent to 99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%). The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring during the disease treatment.

RESUMO: Este estudo tem como objetivo descrever um novo método de detecção de uma reação em cadeia da polimerase quantitativa em tempo real (qPCR) visando o gene da proteína da membrana externa de 28 kDa (p28), bem como comparar este método com um PCR convencional (cPCR), que visa o mesmo gene, a fim de avaliar o desempenho da técnica desenhada neste estudo na detecção de Ehrlichia canis (E. canis). As concentrações ideais de oligonucleotídeos foram alcançadas e a sensibilidade analítica e a especificidade do qPCR foram determinadas. Um total de 218 amostras de sangue total de cães foram coletadas convencionalmente para este estudo. O DNA foi extraído de cada amostra. Posteriormente, as amostras foram testadas por um cPCR estabelecido e o novo qPCR para comparar os desempenhos entre cada técnica. A curva padrão revelou 100% de eficiência e coeficiente de determinação (R2) equivalente a 99,8%. Dentre as amostras examinadas por qPCR, 24,31% foram consideradas positivas, percentual significativamente maior do que as detectadas por cPCR (15,13%). A técnica qPCR atingiu uma sensibilidade maior do que a cPCR na detecção de E. canis. A qPCR padronizada neste estudo é um método eficiente para a confirmação do diagnóstico de erliquiose monocítica canina (EMC) e pode fornecer o monitoramento de níveis de parasitemia ao longo do tratamento da doença.

Hemophagocytosis by blasts in a child with acute monocytic leukemia after chemotherapy / Hemofagocitose por células blásticas em paciente pediátrico com leucemia monocítica aguda após quimioterapia

ABSTRACT Objective: To describe the case of a child who presented hemophagocytic lymphohistiocytosis (HLH) associated with acute monocytic leukemia after chemotherapy, with hemophagocytosis caused by leukemic cells. Case description: In a university hospital in Southern Brazil, a 3-year-old female was diagnosed with acute monocytic leukemia with normal karyotype. The chemotherapy regimen was initiated, and she achieved complete remission six months later, relapsing after four months with a complex karyotype involving chromosomes 8p and 16q. The bone marrow showed vacuolated blasts with a monocytic aspect and evidence of hemophagocytosis. The child presented progressive clinical deterioration and died two months after the relapse. Comments: HLH is a rare and aggressive inflammatory condition characterized by cytopenias, hepatosplenomegaly, fever, and hemophagocytosis in the bone marrow, lymph nodes, spleen, and liver. Although rare, malignancy-associated HLH (M-HLH) is fatal. The patient in this case report met five out of the eight established criteria for HLH. The evolution of the patient's karyotype, regardless of the diagnostic profile, seemed secondary to the treatment for acute monocytic leukemia. In this case, the cytogenetic instability might have influenced the abnormal behavior of leukemic cells. This is a rare case of HLH in a child with acute monocytic leukemia.

RESUMO Objetivo: Descrever um caso de um paciente pediátrico que apresentou linfo-histiocitose hemofagocítica (LHH) associada à leucemia monocítica aguda pós-quimioterapia, com hemofagocitose causada pelas próprias células leucêmicas. Descrição do caso: Em um hospital universitário do Sul do Brasil, uma menina de três anos foi diagnosticada com leucemia monocítica aguda com cariótipo normal. Após receber protocolo quimioterápico, atingiu remissão seis meses depois do início do tratamento, recaíndo quatro meses após com um cariótipo complexo envolvendo ambos os cromossomos, 8p e 16q. A medula óssea mostrava-se infiltrada por células blásticas vacuolizadas com aspecto monocítico, com evidências de hemofagocitose. A criança apresentou um declínio clínico progressivo e dois meses após a recaída foi a óbito. Comentários: A LHH é uma condição inflamatória rara e agressiva caracterizada por citopenias, hepatoesplenomegalia, febre e hemofagocitose na medula óssea, linfonodos, baço e fígado. A LHH associada a doenças malignas, embora seja uma condição rara, é potencialmente fatal. A paciente deste caso apresentou cinco dos oito critérios estabelecidos para o diagnóstico de LHH. A evolução do cariótipo do paciente, independentemente do perfil do diagnóstico, pareceu ser secundária ao tratamento da leucemia monocítica aguda, sendo que a instabilidade citogenética pode ter influenciado o comportamento atípico observado nas células leucêmicas. Este é um dos raros casos de LHH em uma criança com leucemia monocítica aguda.

Progress of traditional Chinese medicine in treatment of acute mononuclear leukemia / 白血病·淋巴瘤

Acute mononuclear leukemia is a type of disease with a high incidence in acute myeloid leukemia. With the continuous deepening of traditional Chinese medicine research, some progress has been made in the research on its clinical efficacy and mechanism of action. This article reviews the research progress in the treatment of acute mononuclear leukemia with traditional Chinese medicine in recent years.

Ehrlichiosis monocítica canina en la provincia de Entre Ríos, Argentina: confirmación molecular de casos en la ciudad de Concordia / Canine monocytic ehrlichiosis in Entre Ríos province, Argentina: molecular confirmation of cases in Concordia city

Resumen El objetivo del siguiente trabajo fue confirmar la presencia de Ehrlichia canis por diagnóstico molecular, a partir de muestras de perros con diagnósticos presuntivos, oriundos de la ciudad de Concordia, provincia de Entre Ríos. ADN fue extraído de 14 muestras de sangre de perros con diagnóstico presuntivo de EMC. Para la detección de ADN deE. canis se amplificó un fragmento del gen dsb, específico de este género, y su confirmación específica se realizó mediante secuenciación. Doce de las 14 muestras mostraron bandas del tamaño esperado. Las secuencias obtenidas mostraron un 98-100% de identidad con secuencias registradas en GenBank para E. canis. Además, se amplificó un fragmento del gen ARNr 16S mitocondrial de garrapatas obtenidas de un perro positivo. Las secuencias demostraron que corresponden a R. sanguineus sensu stricto (linaje templado). En este estudio se confirma la presencia de E. canis en la ciudad de Concordia, Entre Ríos, resultando en una alerta para los médicos veterinarios de la región, a fin de incentivar estrategias de prevención de la enfermedad y control del vector.

Abstract The aim of this work was to confirm the presence of Ehrlichia canis in blood samples of dogs from Concordia, Entre Ríos Province, Argentina. DNA was extracted in 14 blood samples of dogs with presumptive diagnosis of CME. A PCR protocol targeting dsb gene, specific for genus Ehrlichia was carried out for detection and species confirmation was carried out by sequencing. Twelve out of 14 samples shown bands of the expected size. The obtained sequences revealed 98-100% identity with E. canis sequences registered in GenBank. Moreover, ticks were retrieved from an E. canis positive dog and a fragment of mitochondrial ARNr 16S gene was amplified. The obtained sequences were identified as R. sanguineus sensu stricto (temperate linage). This work confirmed the presence of E. canis in Concordia city, Entre Ríos province resulting in an alert for veterinary clinicians in this region aiming to encourage prevention strategies of this disease and vector control.

Primer reporte de un caso de ehrlichiosis monocítica canina en la provincia de Santa Fe, Argentina

La ehrlichiosis monocitica canina (EMC) es una enfermedad causada por la bacteria Ehrlichia canis, de distribución mundial, alta mortalidad en caninos doméstico y síntomas inespecíficos, lo que dificulta su diagnóstico clínico. Ehrlichia canis es transmitida por la garrapata Rhipicephalus sanguineus sensu lato a un hospedador, en Argentina se reconocen dos linajes (tropical y sensu stricto) de dicha especie. El objetivo del presente trabajo es reportar el primer caso confirmado de EMC por E. canis en un canino de la ciudad de Rafaela, Santa Fe, área endémica de R. sanguineus s.s. El 18/02/2019 llegó a la consulta privada un canino con síntomas inespecíficos como hipertermia tarde/noche, depresión, letargia, aplasia, inapetencia y pérdida de peso; más antecedente de parasitismo por garrapatas. La mascota presentaba anemia leve (4.730.000/mm³), enzimas hepáticas aumentadas (AST/ASA/GOT=72 U/l) y esplenomegalia. El test serológico in vitro y PCR para amplificar ADN E. canis fueron positivos, por lo que la mascota recibe tratamiento para EMC con doxiciclina 10 mg/kg/día durante 30 días, antiinflamatorios durante 5 días y protector hepático. A los 60 días de iniciado el tratamiento la mascota recuperó su peso normal y a los 120 días se realiza PCR como monitoreo de la eficacia del tratamiento dando resultado negativo.

Canine monocytic ehrlichiosis (CME) is a disease caused by Ehrlichia canis bacteria. It has a globally distributed and cause high mortality in domestic canines with nonspecific symptoms, which makes clinical diagnosis difficult. Eherlichia canis is transmitted to a host by the tick Rhipicephalus sanguineus sensu lato. In Argentina two lineages of this species are recognized (tropical and sensu stricto). The objective of this work is to report the first confirmed case of CME by E. canis in endemic area of R. sanguineus s.s from Rafaela, Santa Fe. On February 2, 2019 a canine arrived at the private clinic with non-specific symptoms such as late / night hyperthermia, depression, lethargy, aplasia, loss of appetite and weight loss, as well as, antecedent of tick parasitism. The pet had mild anemia (4,730,000/ m³), increased liver enzymes (AST/ASA/GOT = 72 U/l) and splenomegaly. The in vitro serological test and PCR to amplify E. canis DNA were positive, so the dog received treatment for CME with doxycycline 10 mg/kg/day for 30 days, anti-inflammatory for 5 days and liver protector. After 60 days of starting the treatment, the animal regained its normal weight and after 120 days the PCR have given negative result, checking the effectiveness of the treatment.

Canine monocytic ehrlichiosis in Buenos Aires, Argentina: comparison of serological and molecular assays / Ehrlichiose monocítica canina em Buenos Aires: comparação de testes serológicos e moleculares

Canine monocytic ehrlichiosis (CME) is an infectious disease caused by the bacterium Ehrlichia canis and transmitted by Rhipicephalus sanguineus sensu lato, a tick with worldwide distribution. When not diagnosed and treated early, disease can be severe. Currently, the disease is confirmed by serological or molecular assays. The objective of this study was to compare a serological assay based on immunochromatography (SPEED® EHRLI immunochromatographic test; BVT, France) and a molecular assay (a screening PCR followed by a nested PCR specific for E. canis) for the diagnosis of E. canis in suspected dogs from Buenos Aires city and southern Greater Buenos Aires, Argentina. Blood samples from 20 clinically healthy dogs (Control Group) and from 80 sick dogs suspected of having CME (Groups 1 to 4) were tested in parallel. Neither the immunochromatographic test nor the PCR assay was able to detect the presence of E. canis in the Control Group. In the group which had been previously tested by serology, the agreement between the tests was low (kappa: 0.200), whereas in the group which had been previously tested by PCR, the concordance between the tests was adequate (kappa: 0.650). The concordance between the tests evaluated in the total population studied was moderate (kappa: 0.496). The results of our study suggest that the use of rapid serological tests as a first approach, together with subsequent confirmation by PCR, will improve the diagnosis of CME.(AU)

A ehrlichiose monocítica canina (CME) é uma doença infecciosa transmitida pelo carrapato Rhipicephalus sanguineus sensu lato com distribuição mundial causada por Ehrlichia canis, que pode produzir uma doença grave se não foi diagnosticada e tratada precocemente. A confirmação da doença é feita diretamente pela detecção do DNA fazendo a reação em cadeia da polimerase (PCR) ou indiretamente por métodos sorológicos. O objetivo deste estudo foi comparar o método sorológico baseado na imunocromatografia e a técnica de PCR para o diagnóstico de E. canis em cães suspeitos da Cidade de Buenos Aires e da região sul da Grande Buenos Aires. As amostras de sangue de 20 cães clinicamente saudáveis (Grupo Controle) e de 80 cães com suspeita clínica de CME (Grupo 1-4) foram avaliadas em paralelo. O diagnóstico serológico foi feito pelo teste imunocromatográfico SPEED® EHRLI (BVT, França). Para a detecção molecular, foi utilizada uma PCR de triagem para amplificar um fragmento de 345 pb do gene que codifica a subunidade 16S do rRNA da família Anaplasmataceae. As amostras positivas depois foram processadas pela PCR aninhada específica para E. canis. No Grupo Controle, a presença de E. canis não foi detectada por PCR ou anticorpos específicos com o teste imunocromatográfico. No grupo em que a sorologia foi solicitada inicialmente (1 e 2), a concordância entre os testes foi baixo (kappa: 0,200) enquanto que no grupo onde o teste inicialmente solicitado foi a PCR, a concordância entre os testes era adequado (kappa: 0,650). A concordância entre os testes avaliados na população total estudada foi moderada (kappa: 0,496). Em conclusão, os resultados do nosso estudo sugerem que o uso de testes serológicos rápidos inicialmente, juntamente com a confirmação subsequente por PCR, permitirá melhorar o diagnóstico de CME.(AU)

A rare morphological and immunophenotypic presentation of adult B-cell acute lymphoblastic leukemia with blasts in the monocytic zone on CD45 side scatter

We present a case of B-acute lymphoblastic leukemiain an elderly patient who presented with severe weakness and pancytopenia. The patient was a 75 year old Female whose blasts had an unusual morphology in form of coarse azurophilic granules and cytoplasmic blebs and on flow cytometry the blasts were present in the bright CD45 zone with a high side scatter. Bone marrow aspirate sample was subjected to multicolour flow cytometry using Beckman Coulter Navios® which is an 8 colour flow cytometer.Flow cytometricanalysis of the bone marrow aspirate showed blasts in the monocytic zone with a precursor B cell immunophenotype. Complete blood counts showed pancytopenia with peripheral blood film not showingany blasts. Bone marrow aspirate smears showed 20% blasts with coarse azurophilic granules and cytoplasmic blebs.The position of the blasts in this case which were in monocytic zone giving them a bright expression of CD45 and a high side scatter on the CD45 side scatter. This is not the usual positionfor blasts in B-acute lymphoblastic leukemia as these blasts are less complex. A bright expression of CD45 by blasts in B-acute lymphoblastic leukemia is known to be associated with a poor prognosis but the clinical significance of blasts being bright CD45 with a high side scatter is a very rare occurrence and more number of cases with a similar presentation are required to determine a prognostic significance.

Molecular detection of Ehrlichia canis and Anaplasma platys in dogs from municipality of Belém, State of Pará, Brazil / Detecção molecular de Ehrlichia canis e Anaplasma platys em cães do município de Belém, Estado do Pará, Brasil

ABSTRACT: The occurrence of diseases transmitted by ticks in dogs is very frequent in Brazil, among these diseases we can highlight the ehrlichiosis and anaplasmosis, which are caused by Ehrlichia canis and Anaplasma platys, respectively. The objective of this study was to survey the occurrence of these pathogens in blood samples from domiciled and stray dogs from the city of Belém, Pará. Two hundred and seventy six dogs were sampled for convenience, and the DNA extracted from the blood of these animals was submitted to nested-PCR for research of E. canis and A. platys. E. canis DNA was detected in 39.4% (109/276) and A. platys DNA in 23.1% (64/276) of the samples, there was a statistically significant difference between the frequency of these agents (P<0.0001), and there was coinfection in 13.4% (37/276) of animals. The frequency of detection of these parasites was higher in stray dogs than in those domiciled for both E. canis (OR=2.84) and A. platys (OR=10.5). Considering the results, it was possible to conclude that E. canis and A. platys are present in the studied population, with stray dogs being more affected by these parasites.

RESUMO: A ocorrência de doenças transmitidas por carrapatos em cães é muito frequente no Brasil, dentre estas enfermidades podemos destacar a erliquiose e a anaplasmose, que são causadas por Ehrlichia canis e Anaplasma platys, respectivamente. O objetivo deste trabalho foi fazer um levantamento da ocorrência destes patógenos em amostras de sangue de cães domiciliados e errantes do município de Belém, Pará. Foram amostrados 276 cães por conveniência, sendo que o DNA extraído do sangue desses animais foi submetido à nested-PCR para a pesquisa de E. canis e A. platys. O DNA de E. canis foi detectado em 39,4% (109/276), e o DNA de A. platys em 23,1% (64/276) dos cães amostrados. Houve diferença estatisticamente significante entre a frequência desses agentes (p<0,0001), pois foi encontrada coinfecção entre os agentes em 13,4% (37/276) dos animais. A frequência de detecção desses parasitos foi maior em cães errantes do que nos domiciliados tanto para E. canis (OR=2,84) quanto para A. platys (OR=10,5). Diante dos resultados, foi possível concluir que E. canis e A. platys estão presentes na população canina estudada, sendo os cães errantes mais acometidos por esses parasitos.

Anti-tumorimmune effects of WT1 polypeptide vaccine on SCID mice with xenografted human monocytic leukemia / 西安交通大学学报(医学版)

Objective: To study the anti-tumor immune effects of WT1 peptide vaccine in SCID mice with xenografted human monocytic leukemia. Methods: Twenty-four hours after intraperitoneal injection of human peripheral blood lymphocytes, the xenograft human monocytic leukemia model in SCID mice was established by subcutaneous inoculation of THP1 cells. The mice were randomly divided into three groups with eight mice in each. Blank control group was vaccinated with incomplete Freund's adjuvant (IAF). Helper T cell epitope group was vaccinated with helper T cell epitopes and IAF. WT1 group was vaccinated with WT1 peptide, helper T cell epitopes and IAF. When the tumor volume grew to 100 mm3, intraperitoneal injection of vaccine components started. The SCID mice were killed 14 days after vaccination. LDH release method was adopted to detect the specific CTL killing activity of spleen cells. Histological characteristics of tumor tissue were observed under microscope after HE staining. Flow cytometry was used to test the levels of peripheral blood CD3+/CD4+T cells, CD3+/CD8+T cells and CD4+CD25+ Treg cells. ELISA method was applied to detect the levels of serum immunoglobulin, IL-2, γ-interferon, TGF-β and IL-10. Results: The xenograft human monocytic leukemia model was successfully established in SCID mice and tumor developed in all the SCID mice. In WT1 group, the activity of mouse spleen cells on THP1 cells was significantly higher than that in helper T cell epitope group and control group (P<0.05). The mean weight and volume of tumor were significantly lower in WT1 group than in helper T cell epitope group and control group (P<0.05). A large amount of tumor cell degeneration and necrosis was observed under the microscope in WT1 group mice and few tumor cells survived. Peripheral blood levels of CD3+/CD4+T cells, CD3+/CD8+T cells, IgG, IFN-γ and IL-2 were all higher in WT1 group than in helper T cell epitope group and control group (P<0.05). However, peripheral blood levels of CD4+/CD25+Treg cells, TGF-β and IL-10 were all lower in WT1 group than in helper T cell epitope group and control group (P<0.05). Conclusion: WT1 polypeptide vaccine can effectively produce anti-tumor immunity and kill leukemia cells in SCID mice with exnografted human monocytic leukemia.

Bioactive A-ring rearranged limonoids from the root barks of

Screening active natural products, rapid identification, and accurate isolation are of great important for modern natural lead compounds discovery. We hereby reported the isolation of seven new neotecleanin-type limonoids (-), seven new limonoids with 5-oxatricyclo[5.4.0.11., 4.]hendecane ring system (-), and two new precursors (-) together with four known limonoids (-) from the root barks of . Their structures, including their absolute configurations, were elucidated based on analyses of HR-ESI-MS, 1D/2D NMR, ECD spectrum calculations and single-crystal X-ray diffraction techniques. Compounds , , , , , , showed significant anti-inflammatory activities in LPS-induced RAW 264.7 cell line, BV2 microglial cells, and -stimulated THP-1 human monocytic cells. Walrobsin M () exhibited anti-inflammatory activity with IC value of 7.96±0.36 μmol/L, and down-regulated phosphorylation levels of ERK and p38 in a dose-dependent manner.

Clinical features and prognosis of acute monocytic leukemia with abnormality of chromosome 8 / 中国实用内科杂志

OBJECTIVE: To investigate the clinical characteristics and prognosis of patients with acute monocytic leukemia(AML-M5)with abnormality of chromosome 8.METHODS: The clinical features of 143 patients with AML-M5 were analyzed retrospectively,and the prognosis factors were analyzed.RESULTS: Out of 143 AML-M5 newly diagnosed patients,37 cases with chromosome 8 aberrations including t(8;21)accounting for 6.99%(10/143),trisomy 8 16.08%(23/143),and other 8 aberrations 2.80%(4/143);73 cases had normal karyotype,and 33 cases possessed non chromosome 8 abnormality.Statistically significant differences did not exist among age,sex,hemogram and bone marrow blasts(P>0.05).However,with chromosome 8 abnormality were predisposed to lower initial white blood cell count(P<0.05).Among 131 patients of receiving chemotherapy,the remission rate after the first course of inducible chemotherapy was 63.36%(83/131)and the one-year survival rate was 61.1%.Analysis of prognostic factors showed that age,the remission after the first induction of chemotherapy(complete remission or no remission),trisomy 8 chromosomal karyotype and treatment regimen(chemotherapy alone or plus hematopoietic stem cell transplantation) had effects on overall survival(P<0.05).Multivariate analysis revealed two independent risk factors:age≥60 years(P<0.05,HR=2.134,95% CI 1.204～3.784)and the complete remission after the first induction of chemotherapy(P<0.05,HR=0.408,95% CI 0.227～0.733).CONCLUSION: Chromosome 8 is easily involved in AML-M5.The patients with involvement of this aberration have lower initial white blood cell count and a poor prognosis.Patients after complete remission have hematopoietic stem cell transplantation is beneficial to prolong survival.

Ehrlichiosis monocítica humana y babesiosis en Venezuela / Human monocytic ehrlichiosis and babesiosis in Venezuela

Resumen La babesiosis o "Fiebre de garrapatas" es una enfermedad febril y anemizante, producida en animales domésticos y salvajes y ocasionalmente en humanos por especies del genero Babesia, las cuales son protozoarios intraeritrocíticos. Se denomina ehrlichiosis y anaplasmosis a un grupo de infecciones bacterianas transmitidas por garrapatas duras (Ixodidae), que afectan al ser humano y a los animales. Son de distribución universal, y están relacionadas con varias especies de los géneros Anaplasma y Ehrlichia (familia Anaplasmataceae). La fiebre de origen desconocido en pacientes con historia de viajes a zonas endémicas de Ixodes resulta con alto índice de sospecha para la búsqueda de infecciones por Babesia, Borrelia y Ehrliquia, ya que pueden ocurrir simultáneamente, particularmente con estos dos últimos géneros. Se reporta un caso de paciente femenina de 49 años de edad, procedente de Tumeremo estado Bolívar, ocupación minera, quien consultó por fiebre con escalofríos, cefalea y mialgias predominantes en miembros inferiores. Ingresó a Terapia intensiva por cuadro de distres respiratorio y shock séptico. Se descartó malaria por gota gruesa seriada negativas, el hemocultivo y urocultivo reportaron ausencia de crecimiento bacteriano. Se realizó frotis de capa blanca siendo positiva para Ehrliquia monocítica y Babesia bigemina. La paciente evolucionó satisfactoriamente luego del tratamiento con Doxiciclina, clindamicina más meropenem. El fin de este reporte es concientizar a la comunidad médica de la existencia de la ehrlichiosis como entidad clínica emergente en nuestro país y la posibilidad de coexistir con otros microorganismos que comparten el mismo vector, con el fin de considerar tratamiento empírico oportuno en pacientes con factores de riesgo en las zonas rurales.

Abstract Babesiosis or "Tick fever" is a feverish and anemic disease, produced in domestic and wild animals and occasionally in humans by species of the genus Babesia, which are intra-erythrocytic protozoans. Ehrlichiosis and anaplasmosis are called a group of bacterial infections transmitted by hard ticks (Ixodidae), which affect humans and animals. They are of universal distribution, and are related to several species of the genera Anaplasma and Ehrlichia (family Anaplasmataceae). Fever of unknown origin in patients with a history of travel to endemic areas of Ixodes results in a high index of suspicion for the search for Babesia, Borrelia and Ehrliquia infections, since they can occur simultaneously, particularly with these last two genera. We report a case of female patient, 49 years old, from Tumeremo Bolívar state, mining occupation, who consulted for fever with chills, headache and myalgias predominant in lower limbs. He entered intensive therapy for respiratory distress and septic shock. Malaria was ruled out by gross negative strains, blood culture and urine culture showed no bacterial growth. White-coat smears were positive for monocytic Ehrliquia and Babesia bigemina. The patient progressed satisfactorily after treatment with Doxycycline, clindamycin plus meropenem. The purpose of this report is to make the medical community aware of the existence of ehrlichiosis as an emerging clinical entity in our country and the possibility of coexisting with other microorganisms that share the same vector in order to consider timely empirical treatment in patients with factors risk in rural areas.

Synsepalum dulcificum extracts exhibit cytotoxic activity on human colorectal cancer cells and upregulate c-fos and c-jun early apoptotic gene expression

Objective:To explore cytotoxicity of Synsepalum dulcificum (S.dulcificum) Daniell (Sapotaceae) on human colon cancer (HCT-116 and HT-29),human monocytic leukemia (THP-1) and normal (HDFn) cell lines,and its effect on the expression of early apoptotic genes,c-fos and c-jun.Methods:Leaf,stem and berry of S.dulcificum were separately extracted by using 2 solvents,10％ ethanol (EtOH) and 80％ methanol (MeOH).PrestoBlue(R)cell viability assay and qRT-PCR assay were conducted to examine the above objectives respectively.Results:Stem MeOH,stem EtOH,and berry EtOH extracts of S.dulcificum were cytotoxic to HCT-116 and HT-29 human colon cancer cells.For HCT-116,IC50 values of these 3 extracts were not significantly different (P＞0.05) from that of the positive control bleomycin (IC5o of 33.57 μg/mL),while for HT-29,IC5o values of these 3 extracts were significantly lower (P＜0.05) than that of bleomycin (IC50 of 25.24 μg/mL).None of the extracts were cytotoxic to the THP-1 monocytic leukemia cells and HDFn normal human dermal fibroblasts.For both HCT-116 and HT-29,these extracts significantly up-regulated (P＜0.05) the expression of c-fos and c-jun compared to the untreated negative control.Conclusions:The results of this study suggest that cytotoxicity of stem MeOH,stem EtOH,and berry EtOH extracts of S.dulcificum on HCT-116 and HT-29 colon cancer cells is due to the induced apoptosis which is caused by the up-regulation of the expression of early apoptotic genes,c-fos and c-jun.

Effect of down-regulation of MLAA-22 gene on proliferation and differentiation of U937 cells / 西安交通大学学报(医学版)

Objective To explore the effect of down-regulation of MLAA-22 gene on proliferation and differentiation of U937 cells.Methods MLAA-22 gene was down-regulated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)system in U937 cells.The activity of single guide RNA (sgRNA)was detected by CruiserTMenzyme digestion assay.The mutation rate of MLAA-22 gene was analyzed by TA cloning and sequencing assay of PCR products of the gene mutation region.Cell proliferation was evaluated by CCK-8 assay.Expression of CD11b was tested by flow cytometry to evaluate cell differentiation. Results CruiserTMenzyme digestion assay showed the sgRNA of the CRISPR/Cas9 system identified the target spot efficiently.TA cloning and sequencing assay displayed the mutation rate of MLAA-22 gene was 61.3%.CCK-8 assay demonstrated that the proliferation was obviously inhibited in MLAA-22-knockdown U937 cells.In addition, flow cytometry assay indicated CD11b-positive cells significantly increased in MLAA-22-knockdown U937 cells. Conclusion MLAA-22 gene regulates the proliferation of U937 cells probably by regulating their differentiation, thus promoting the occurrence and development of acute monocytic leukemia.

Analysis of prognostic factors in children with acute myeloid leukemia(M4/M5) / 中华实用儿科临床杂志

Objective To investigate the clinical efficacy and prognostic factors for M4/M5subtypes in chil-dren with acute myeloid leukemia(AML).Methods A retrospective analysis of the clinical data of M4/M5subtypes in Shanghai Children′s Hospital Affiliated to Shanghai Jiaotong University,from January 2009 to December 2014 was carried out.The long-term efficacy,prognosis and relapse factors were analyzed.Results The clinical data of 46 ca-ses were collected,among which 38 cases were treated with more than 2 courses,including 22 male,16 female,19 cases M4and 19 cases M5.The median age was 5 years.5-year overall survival(OS)rate and 5-year event-free survival (EFS)rate were(57.7 ± 9.3)% and(47.2 ± 8.9)%,and 5-year EFS of M4and M5were(52.4 ± 12.7)% and (45.4 ± 11. 9)%. Compared with the international risk stratification:5-year EFS rate of favorable-risk, intermediate-risk and poor-risk were(77.2 ± 12.4)%,(49.5 ± 14.9)% and(25.0 ± 19.8)%(χ2=6.305,P=0.043).Single factor analysis showed that extramedullary infiltration(χ2=4.828,P=0.028),Chromosome karyotype (χ2=10.178,P=0.017),the eighth day assessment(χ2=5.382,P=0.020)and course of treatment(χ2=4.771, P=0.029)were prognostic factors;multivariate analysis showed extramedullary infiltration(HR =5.323,95%CI:1.620-17.490,P=0.006)and less-than-6 courses of treatment(HR=6.186,95%CI:1.726-22.176,P=0.005)were the independent risk factors of affecting survival.Conclusions (1)Strengthening treatment and ade-quate courses of treatment are the critical to improve the overall curative effect in children with M4/M5subtypes.(2) Extramedullary infiltration was the risk factor for survival and recurrence in M4/M5subtypes.(3)It is suggested that the children who have the initial symptoms and molecular biology with poor prognostic factors choose hematopoietic stem cell transplantation as early as possible.

Mechanism of HIF-1α-PDK1 signaling system involved in glucose metabolism and drug resistance in acute monocytic leukemia / 上海交通大学学报（医学版）

Objective • To investigate the mechanism of HIF-1α-PDK1 signaling system mediated glucose metabolism and drug resistance in acute monocytic leukemia cells. Methods • The expression of pyruvate dehydrogenase kinase 1 (PDK1) mRNA in U937, U937/DNR and acute monocytic leukemia cells was detected by quantitative polymerase chain reaction (qPCR). siRNA HIF-1α plasmid was constructed and transferred to U937 and U937/ DNR cells for 24 h by gene silencing. Cell proliferation inhibition was examined by MTT assay. The level of PDK1 mRNA was detected by qPCR, and the expression of PDK1 and multi-drug resistance gene 1 (MDR1) proteins was detected by Western blotting. Cell membrane potential was measured by flow cytometry using JC-1. Lactic acid level in the culture fluid was determined by blood gas analyzer. Dichloroacetate (DCA) and daunorubicin (DNR) were added to treat U937/DNR and acute monocytic leukemia cells for 24 h, MTT was used to calculate cell proliferation inhibition and Western blotting was used to estimate the expression of PDK1 and MDR1 proteins. Results • PDK1 mRNA was highly expressed in U937, U937/DNR and acute monocytic leukemia cells. Silencing hypoxia-inducible factor-1α (HIF-1α) significantly inhibited the proliferation activity, PDK1 and MDR1 expression and lactic acid production in U937 and U937/DNR cells. DCA could reverse the resistance to DNR in U937/DNR and relapsed acute monocytic leukemia cells. Conclusion • HIF-1α-PDK1 signaling system may regulate glucose metabolism and participate in the drug resistance of acute monocytic leukemia.

Synsepalum dulcificum extracts exhibit cytotoxic activity on human colorectal cancer cells and upregulate c-fos and c-jun early apoptotic gene expression

Objective: To explore cytotoxicity of Synsepalum dulcificum (S. dulcificum) Daniell (Sapotaceae) on human colon cancer (HCT-116 and HT-29), human monocytic leukemia (THP-1) and normal (HDFn) cell lines, and its effect on the expression of early apoptotic genes, c-fos and c-jun. Methods: Leaf, stem and berry of S. dulcificum were separately extracted by using 2 solvents, 10% ethanol (EtOH) and 80% methanol (MeOH). PrestoBlue® cell viability assay and qRT-PCR assay were conducted to examine the above objectives respectively. Results: Stem MeOH, stem EtOH, and berry EtOH extracts of S. dulcificum were cytotoxic to HCT-116 and HT-29 human colon cancer cells. For HCT-116, IC

Roles of Dectin-1 in phagocytosis of Candida albicans by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1 / 中华皮肤科杂志

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C.albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells served as the target cells,and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group).THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group.After transfection,the THP-1 macrophage-like cells in the above 2 groups were cocultured with heat-killed C.albicans separately.And then,fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C.albicans,and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR)-123 fluorescent cells.Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software.Results After transfection with siRNA-Dectin-1,the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t =26.163,P ＜ 0.001).After 1-,2-,4-hour co-culture of THP-1 macrophagelike cells with C.albicans,fluorescence microscopy showed that the phagocytosis rates of C.albicans by THP -1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5％ vs.22.1％,18.6％ vs.24.3％,39.2％ vs.59.1％,respectively,all P ＜ 0.05),so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C.albicans cells (2.2％ vs.4.7％,2.5％ vs.5.4％,5.1％ vs.8.3％,respectively,all P ＜ 0.05).After 30-minute,1-,2-and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C.albicans,flow cytometry showed that the MFI of C.albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs.45.7,54.3 vs.62.4,72.1 vs.84.9,93.6 vs.116.7,respectively,all P ＜ 0.05).Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C.albicans by macrophages.