Estudos estruturais e funcionais de Tsa1 de Saccharomyces cerevisiae: um modelo biológico para o estudo de inibidores de crescimento celular para leucemia linfoide aguda / Functional and structural evaluation of Tsa1 from Saccharomyces cerevisiae: a biological model for study of cellular growth inhibitors for acute lymphocytic leukemia

As peroxirredoxinas (Prx) são enzimas antioxidantes que se destacam pela capacidade de decompor uma grande variedade de hidroperóxidos com elevada eficiência (106-108M-1s-1), mantendo essas moléculas em níveis adequados à homeostase celular. Entretanto, já foi demonstrado que em diversos tipos tumorais os níveis de Prx são extremamente aumentados e experimentos envolvendo sua inativação resultam na diferenciação ou apoptose de células tumorais. Recentemente, foi descoberto um diterpenóide denominado adenantina que seria o primeiro inibidor para as Prx1 e Prx2 de humanos e foi demonstrada que sua aplicação em células de leucemia mieloide aguda promoveu diferenciação ou apoptose dessas células. Nesse contexto, o presente trabalho apresenta duas vertentes: 1) A caracterização das alterações estruturais e funcionais promovidas pela ligação da adenantina ao sítio ativo das Prx utilizando Tsa1 de Saccharomyces cerevisiae como modelo biológico, em função da sua alta similaridade com Prx2 de humanos; 2) Avaliação da atividade antitumoral dose dependente de adenantina sobre as linhagens celulares REH e MOLT-4 de leucemia linfoide aguda. No que concerne à primeira linha de investigação, nossos resultados revelam que Tsa1 é suscetível à inibição por adenantina, uma vez que o tratamento reduziu em ~66 % a velocidade de decomposição de peróxido de hidrogênio. Adicionalmente, a mutação da Thr44 de Tsa1, pertencente à chamada tríade catalítica, por uma Ser resultou em uma proteína mais suscetível a alterações na estrutura secundária e à inibição da atividade peroxidásica em função da ligação com adenantina, apresentando uma diminuição de ~85% na velocidade de reação. Características semelhantes foram observadas para a proteoforma Tsa2 de S. cerevisiae, que carreia naturalmente a substituição da Thr44 pela Ser. Análises de sequências de Prx em bancos de dados revelaram que majoritariamente proteínas contendo Ser são encontradas em organismos procariotos, muitos deles patogênicos. Finalmente, demonstramos por meio de ensaios citotoxicidade que as bactérias Staphylococcus aureus e Staphylococcus epidermidis, que possuem uma Ser na tríade catalítica, têm seu crescimento inibido pelo tratamento com adenantina (IC50 de 460µM e 77µM, respectivamente), enquanto que para Escherichia coli, que possui Thr nessa posição, a toxicidade da adenantina foi bastante baixa (não foi possível determinar o IC50 nas condições utilizadas). Dessa forma, os dados apresentados neste trabalho demonstram o potencial da utilização da adenantina tanto como antibiótico quanto como antileucêmico

Peroxiredoxins (Prx) are antioxidant enzymes which stand out due the ability to decompose a wide variety of hydroperoxides with high efficiency (106-108M-1s-1) maintaining these molecules at suitable levels to cellular homeostasis and participating in several signaling events. However, it has been shown that, in many tumor types, Prx levels are extremely increased and experiments involving its inactivation have resulted in differentiation or apoptosis of tumor cells. It was recently found a diterpenoid, called adenanthin, that would be the first human Prx1 and Prx2 inhibitor and it was demonstrated that its application in acute myeloid leukemia cells was able to promote differentiation or apoptosis. In this context, this work presents two lines of research: 1) Characterization of structural and functional changes promoted by adenanthin binding to Prx active site using Tsa1 from Saccharomyces cerevisiae as biological model, due to its high similarity to human Prx2. 2) Evaluation of adenanthin dose-dependent antitumor activity over the acute lymphoid leukemia cell lines REH and MOLT-4. As regards the first line of research, our result reveal that Tsa1 is susceptible to inhibition by adenanthin, since the treatment with this binder reduced the hydrogen peroxide decomposition velocity in ~ 66%. In addition, the replacement of Thr44 from Tsa1, aminoacid belonging to the so-called catalytic triad, by a Ser resulted in a protein more susceptible to alterations in secondary structure and to peroxidase activity inhibition in function of adenanthin binding, presenting ~85% of decrease in reaction velocity. Similar characteristics were observed for Tsa2 proteoform from S. cerevisiae, which naturally carries the substitution of Thr44 by Ser. Prx sequences analyzes in databases revealed that mostly Ser-containing proteins are found in prokaryotic organisms, many of them pathogenic ones. Finally, we demonstrate through cytotoxicity assays that the bacteria Staphylococcus aureus and Staphylococcus epidermidis, which have a Ser in catalytic triad, have their growth inhibited by adenanthin treatment (IC50 of 460µM and 77µM, respectively), whereas for Escherichia Coli, which has Thr at that position, the tocyxicity of adenanthin was quite low (it was not possible to determine the IC50 under the used conditions). Regarding the second line of investigation, we found that adenanthin is able to induce the death of leukemic cell lines REH and MOLT-4, and for the last one, there was an unexpected proliferation of cells treated by the longest incubation period (72 hours), characterizing a possible indication of differentiation process. In this sense, the data presented here demonstrate the potential of adenanthin use in both antibiotic and antileukemic treatment

Classification of Acute Leukemias – Past, Present,and Future.

The perspective of the classification of any disease is to treat them according to their biologic behavior. Standard criteria to distinguish between myeloid and lymphoid acute leukemias were laid down as the first of its kind, by the French-American-British (FAB) working group. The FAB classification had a cursory correlation with clinical outcome, poor concordance owing to inter-observer variation, and failure to incorporate cytogenetic data. Hence, the World Health Organization (WHO) classification of leukemias evolved in 1997 with the goal of improving the objectivity and reproducibility, which had incorporated cytogenetic abnormalities and immunology as principal designating criteria, other than the morphology. Major changes were made in the subsequent editions of WHO classification, incorporating newer genetic abnormalities such as mutations of nucleophosmin member 1, CCAAT/enhancer-binding protein alpha; renaming of the existing classes, etc. The role of the genes encoding guanine nucleotide-binding protein gamma 11, amphiregulin, and ceruloplasmin; the biomarkers platelet factor 4 and connective tissue-activating peptide III, complement fragment C3a; the mRNA coding for plexin C1, leukotriene B4 receptor 1, and Immunoglobulin superfamily member 2; mixed lineage leukemia gene rearrangement in the prognosis of leukemias is proven. Thus, the approach of diagnostics using cytogenetics and immunophenotyping may further be modified.

Citarabina y reacciones cutáneas en leucemia aguda mieloide / Cytarabine and skin reactions in acute myeloid leukemia

La citarabina es un antimetabolito utilizado en el tratamiento de las leucemias agudas mieloides (LAM). Esta droga presenta numerosos efectos adversos (mielosupresión, toxicidad en sistema nervioso central, hepática, gastrointestinal, ocular y cutánea). La toxicidad dermatológica es habitualmente descrita como rara, sin embargo existen diferencias en la incidencia comunicada. Se realizó un estudio retrospectivo donde se incluyeron todas las LAM tratadas con quimioterapia que incluía citarabina, entre el 1º de julio 2006 y el 1° de julio 2012. Se incluyeron 46 pacientes con una mediana de edad de 55 años. La incidencia global de reacciones cutáneas fue de 39% (n = 18). La presencia de lesiones cutáneas no se asoció con sexo, edad, antecedentes de atopía, de reacciones medicamentosas, tipo de LAM ni dosis de citarabina utilizada. Las lesiones se observaron entre 2 a 8 días de iniciado el tratamiento. En cuanto al grado lesional, 27.8% presentaron grado 1, 38.9% grado 2 y 33.3% grado 3. No existieron lesiones grado 4 ni muerte vinculada a toxicidad cutánea. En cuanto al tipo de lesiones, 55.6% se presentaban con máculas, 22.2% con pápulas y 22.2% con eritema. Con respecto a la distribución de las lesiones, 52% de los pacientes presentaron una distribución difusa, 39.3% acral y 8.7% a nivel flexural. Las reacciones adversas cutáneas con la administración de citarabina son frecuentes en nuestro medio, en algunos casos con afectación grave. Si bien suelen resolverse espontáneamente, pueden determinar mayor riesgo de infección, así como comprometer la calidad de vida.

Cytarabine is an antimetabolite used in the treatment of acute myeloid leukemia (AML). It has many adverse effects as: myelosuppression, toxic reactions involving central nervous system, liver, gastrointestinal tract, eyes or skin. Dermatologic toxicity is often described as rare; nevertheless there are differences in the reported frequency. We performed a retrospective study including all AML treated with chemotherapy that involved cytarabine between 1st July of 2006 and 1st July of 2012; 46 patients were included with a median age of 55 years. The overall incidence of skin reactions was 39% (n = 18). Sex, age, history of atopy, history of drug reactions, or dose of cytarabine used, were not associated with them. Skin reactions were observed from 2 to 8 days after treatment started. Considering injury degree: 27.8% had grade 1, 38.9% grade 2 and 33.3% grade 3. We did not find any injury grade 4 or death associated with skin toxicity. As for the type of injury: 55.6% presented macules, 22.2% papules and 22.2% erythema. Lesions distribution was diffuse in 52% of patients, acral in 39.3%, and at flexural level in 8.7%. Adverse cutaneous reactions secondary to the administration of cytarabine are frequent in our service and include some cases with severe involvement. Although these reactions usually resolve spontaneously, they determine an increased risk of infection and a compromise of the patient quality of life.

Clinical outcome of managment of acute myeloid leukemia with idarubicin plus cytarabine

A prospective study was conducted at Department of Clinical Oncology, King Edward Medical College / Mayo Hospital, Lahore from July 2003 to June 2004 to evaluate the effect of Idarubicin plus Cytarabine in chemo naive Acute Myeloid Leukemia [AML] patients. A total of 15 consecutive patients were enrolled with age group 15-58 years. Patients were classified according to French American British [FAB] classification. Induction therapy with Cytarabine as continuous infusion for 7 days and Idarubicin was given on day 1-3. For assessment of response, all patients were subjected to bone marrow examination fifteen days after completion of Induction chemotherapy. Consolidation Therapy with high dose Cytarabine was given on days 1, 3 and 5. Cytarabine was repeated after 28 days for 4 cycles in patients with complete remission after induction therapy. A remission induction rate of 66.7% was observed. Four patients died because of complications. One patient lost to follow up. Idarubicine and cytarabine is effective r egimen for achieving complete remission in AML Chemo-naive patients

Leucemias agudas na infância / Childhood acute leukemias

As leucemias constituem a neoplasia mais freqüente na infância. São responsáveis por cerca de um terço dos cânceres pediátricos. A leucemia linfocítica aguda (LLA) representa cerca de 75% dos casos de leucemias infantis. As leucemias mielóides agudas (LMA) representam 15% a 20% das leucemias em pacientes com idade inferior a 15 anos. As manifestações clínicas das leucemias agudas são decorrentes da inibição da hematopoiese pelas células leucêmicas e dos efeitos da infiltração leucêmica em diversos órgãos e sistemas. Observa-se anemia devido à diminuição dos eritrócitos, sangramentos decorrentes da trombocitopenia e infecções conseqüentes à neutropenia. A infiltração dos diferentes tecidos resulta em hepatomegalia, esplenomegalia e linfadenomegalia. O diagnóstico das leucemias é firmado pela presença de mais de 25% de células leucêmicas na punção aspirativa de medula óssea (mielograma). O tratamento da LLA é feito com regimes quimioterápicos de longa duração. A indicação do trans- plante de medula óssea durante uma primeira remissão permanece controversa. A probabilidade de cura da LLA pode chegar a 80%. Na LMA, com o uso exclusivo de quimioterapia, a probabi- lidade de sobrevida livre de doença (SLD) por períodos prolonga- dos é de 30% a 40%. Quando se emprega o transplante de medula óssea alogênico, a probabilidade estimada de SLD a longo prazo é de 50% a 60%...(AU)

The leukemias are the most common malignant neoplasm in child- represents 15% to 20% of cases in patients less than 15 years old. The clinical manifestations of acute leukemias are secondary to inhibition of hematopoiesis by leukemic cells and to the effects of leukemic infiltration in different organs and systems. Anemia is caused by reduction of erythrocytes, bleeding is secondary to thrombocytopenia, and infections are related to neutropenie. The infiltration of tissues results in hepatomegaly, splenomegaly and lymphadenopathy Acute leukemias are diagnosed by abone mar- row aspirate that demonstrates more than 25% of the bone mar- row cells as a relatively homogeneous population of blasts. The treatment of ALL consists of prolonged chemotherapy regimens. The role of allogeneic bone marrow transplantation during first remission remains controversiet. The probability of cure in ALL reaches 80%, In AML, with chemotherapy alone, the probability of long term disease free survival is from 30% to 40%. When bone marrow transplantation is used the estimated probability of prolonged disease free survival is 50% to 60%...

Immunophenotyping of acute leukemia in northwestern Iran

The significance of immunophenotyping is growing day by day. It provides basic information in regard to classification and prognosis of acute leukemia which helps the management of patients. This study was conducted to Identify CD markers in leukemic patients admitted to Tabriz, in northwestern Iran. Immunophenotyping of 60 patients with acute leukemia was determined. Patients with acute myelogenous leukemia [AML] were 42% of M2 type, 23.6% M3, 15.7% M4, 13% M1 and 5.7% M5. CD13 and CD33 were the most prevalent myeloid markers. T-lymphoid markers consisted mainly of CD7 and its occurrence was mostly in M2 and M4, and least in M3 subtypes. The most common lymphoid markers in patients with Tcell acute lymphoblastic leukemia [ALL] were CD2, CD3, CD7 and in those with B-cell ALL were CD10, CD19 and HLA-DR. The most prevalent myeloid markers in T-ALL were CD14, CD33 and CD13

Classificação FAB das leucemias mielóides agudas / The FAB classification of acute myeloid leukemias

No relato é apresentada uma sinopse sobre a classificação das leucemias mielóides agudas baseada na sugestão do grupo francoamericano-britânico.

Leucemies aigues myeloblastiques de l'enfant. A propos de 21 cas

Between 1989 and 1996,21 cases with acute non lymphoblastic leukemia [11 males and 10 females] were diagnosed in our institution. Median age was 9 years [range, 2-15 years]. leukocyte count was more than 50.109/1 in 47% of cases. According to the French-American-British [FAB] criteria, 7 cases were classified M 1, 10 cases were classified M2, 1 classified M4Eo and 3 classified M5. All patients were treated with" 7+3 " protocol and complete remission was achieved in 17 cases [80%], 2 cases [10%] Failed to respond and 2 [10%] died during induction. Relapse was observed in 15 cases. The 3-year survival rate was 20% and the relapse-free survival rate was 12% confirming the worse prognosis of this leukemia when treated with standard chemotherapy

Immunofenotipos celulares en 97 adultos con leucemia aguda / Immunophenotypes in 97 adults con leucemia aguda

Objetivo. Analizar la expresión de los antígenos de superficie de las células hematopoyécticas en pacientes con leucemia aguda (LA) empleando citofluorografía y criterios actualizados de clasificación de leucemias agudas de linaje mixto (LALM). Material y métodos. Se estudiaron 97 pacientes con LA de novo. Se emplearon anticuerpos monoclonales dirigidos contra antígenos de superficie de linaje: linfoide B (CD10, CD19, CD20, CD21, y CD22), linfoide T (CD2, CD3, CD5, CD7) y mieloide (CD13, CD14, CD15, CD33, y CD41). Además se incluyeron el CD34, HLA-DR y TdT como marcadores de maduración. Resultados. Doce casos inclasificables por citomorfología pudieron ser clasificados mediante inmunofenotipo. Empleando los datos citomorfológicos, citoquímicos e inmunofenotípicos, 54 casos correspondieron a leucemia aguda linfoide (LAL) y 43 a leucemia aguda mieloide (LAM). De las LAL, 63 por ciento fueron de estirpe B, 15 por ciento de linaje T, 7 por ciento T/B, 6 por ciento indiferenciadas y 9 por ciento de linaje mixto (dos o más antígenos mieloides asociados). De las LAM, en el 86 por ciento el inmunofenotipo fue compatible con estirpe mieloide, en el 2 por ciento de indiferenciadas y en el 12 por ciento de linaje mixto (dos o más antígenos linfoides asociados). Además, el 26 por ciento de las LAL y el 12 por ciento de las LAM expresaron sólo un antígeno aberrante mieloide y linfoide, respectivamente. El CD13 y el CD7 fueron los antígenos aberrantes expresados con mayor frecuencia en las LAL y en las LAM, respectivamente. La frecuencia de expresión más alta del antígeno CD34 se observó en las LALM (90 por ciento). Conclusiones. El análisis del fenotipo inmunológico de las LA permitió: a) establecer el diagnóstico en los casos inclasificables por citomorfología; b) identificar las LALM cuya frecuencia es semejante a la informada en la literatura; y c) confirmar la heterogenidad de las LA

Intraobserver and interobserver reproducibility of the FAB classification in acute leukaemia.

Intraobserver and interobserver reproducibility of FAB classification for acute leukaemia was assessed using the modified criteria of the FAB classification. Leishman stained peripheral smear and May Grunwald Giemsa stained bone marrow smears from 72 cases of acute leukaemia were used for this purpose. Cytochemical stains used were peroxidase, PAS and Sudan black B. Intraobserver and interobserver concordance/discordance was calculated. Kappa statistic was used to correct the chance expected agreement. Intraobserver and interobserver concordance was 76% which improved to 91% when cytochemistry was included. Lymphocytic/Nonlymphocytic concordance was 87.5% and 90% respectively for intraobserver and interobserver groups.

Utilidad de las esterasas combinadas en la diferenciación de las leucemias agudas no linfoblásticas / Usefulness of combined esterases in the differentiation of acute nonlymphoblastic leukemias

En este trabajo se proponen algunas modificaciones al método combinado para esterasas específicas y no específicas (alfa - naftil acetato y cloroacetato), descrito originalmente por Yam et al, para una mejor identificación y diferenciación de los subtipos de las leucemias agudas no linfoblásticas (LANL). De 54 estudios citoquímicos realizados a pacientes con diagnóstico probable de leucemia aguda, 25 reaccionaron positivamente a las esterasas, los cuales según la clasificación FAB, correspondieron a LAM2, LAM4, LAM5 y a Síndromes Mielodisplásticos

Estudo das leucemias mieloides agudas e síndromes linfoproliferativas à microscopia eletrônica / Acute myeloid leukemias and lymphoproliferative syndromes by electron microscopy

Foram estudados o sangue periférico e ou a medula óssea de 24 portadores de hemopatias malignas por meio da microscopia eletrônica, sendo 12 casos de leucemia mielóide aguda e 12 casos de síndromes linfoproliferativas. Nos 15 casos de leucemias agudas fizemos uma correlaçäo com a classificaçäo FAB (Frech-American British Group), 1978, baseada na morfologia e citoquímica na microscopia de luz, com o estudo morfológico e a reaçäo citoquímica da mieloperoxidase, através do microscópio eletrónico. Assim, dois casos de LMA, que apresentavam dúvidas no microscópio de luz, com o estudo ao microscópio eletrônico confirmamos os tipos morfológicos: um caso de LMA que apresentava dúvidas do ML, com o estudo ao ME foi reclassificado. Todos os outros casos de leucemias agudas e todos de síndromes linfoproliferativas foram confirmados à microscopia eletrônica, corroborando no diagnóstico final. Os nossos resultados, de um modo geral, foram concordantes com a literatura

Determinación del fenotipo inmunológico de las leucemias agudas en la ciudad de puebla, México: identificación de 19 casos de leucemia aguda megacarioblástica, la variedad M7 de la classificación FAB / Immunological phenotype determination of acute leukemias in the city of Puebla, México: identification of 19 cases of acute megakaryoblastic leukemia, variety M7 of FAB classification

Se presentan los resultados de la clasificación inmunologica de 147 casos de leucemias agudas sin morfologia mieloide definitiva. Entre ellos, se identificaron 128 casos de leucemia aguda linfoblástica y 19 casos de leucemia aguda megacarioblástica, la variedad M7 de la clasificación FAB. De los casos de leucemia linfoblástica, la variedad mas frecuente fue la leucemia linfoblástica "comun", con antigeno CALLA CD10, seguida de la leucemia linfoblástica de linfócitos "nulos". Las leucemias linfoblásticas de linfócitos B ocuparón el 11% de los casos y las de linfócitos T el 54%, siendo estas ultimas prevalencias diferentes de las observadas en otros sitios del mundo. La clasificación inmunológica fue util para establecer el prognóstico de los pacientes, ya que los enfermos con leucemias linfóides de fenotipo inmunológico "favorable" tuvieron mejor evolución que aquellos con fenotipo inmunológico T o B. Se describen tambien los datos clínicos y de laboratorio de las leucemias megacrioblásticas, hasta hace poco consideradas como infrecuentes, que ocuparón el 8% de los casos de leucemia aguda en esta serie