Alteraciones hematológicas como consecuencia de COVID-19 y sus vacunas / Hematological changes as a consequence of COVID-19 and its vaccines

El virus SARS-CoV-2 continúa infectando a millones de individuos en el mundo. Aunque los síntomas más frecuentes observados en los pacientes con COVID-19 son fiebre, fatiga y tos, en los casos severos la hipercoagulabilidad y la inflamación son dos condiciones que pueden producir complicaciones y causar daño en órganos, poniendo en riesgo la vida del paciente. Con el fin de clasificar a los pacientes durante el triaje, se han explorado diferentes marcadores hematológicos, incluidos el recuento de plaquetas, linfocitos y eosinófilos, y la relación neutrófilos/ linfocitos, entre otros. Por su parte, para la evaluación de las coagulopatías, se vienen determinando marcadores como el dímero D y el fibrinógeno. En esta revisión se abordan las coagulopatías y los parámetros hematológicos en pacientes con COVID-19, al igual que las anormalidades en la coagulación como la trombocitopenia trombótica inmune inducida por las vacunas contra el SARS-CoV-2

The SARS-CoV-2 virus continues to infect millions of individuals around the world. Although the most frequent symptoms observed in patients with COVID-19 are fever, fatigue and cough, in severe cases hypercoagulability and inflammation are two conditions that can cause complications and organ failure, putting the patient's life at risk. In order to classify patients during triage, different hematological markers have been explored, including platelet, lymphocyte, and eosinophil counts, and the neutrophil/lymphocyte ratio, among others. Furthermore, for the evaluation of coagulopathies, markers such as D-dimer and fibrinogen are being evaluated. This review addresses the coagulopathies and hematological parameters in patients with COVID-19, as well as coagulation abnormalities such as immune thrombotic thrombocytopenia induced by SARS-CoV-2 vaccines

TFAP2C increases cell proliferation by downregulating GADD45B and PMAIP1 in non-small cell lung cancer cells

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of death in the world. NSCLC diagnosed at an early stage can be highly curable with a positive prognosis, but biomarker limitations make it difficult to diagnose lung cancer at an early stage. To identify biomarkers for lung cancer development, we previously focused on the oncogenic roles of transcription factor TFAP2C in lung cancers and revealed the molecular mechanism of several oncogenes in lung tumorigenesis based on TFAP2C-related microarray analysis. RESULTS: In this study, we analyzed microarray data to identify tumor suppressor genes and nine genes downregulated by TFAP2C were screened. Among the nine genes, we focused on growth arrest and DNA-damage-inducible beta (GADD45B) and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) as representative TFAP2C-regulated tumor suppressor genes. It was observed that overexpressed TFAP2C resulted in inhibition of GADD45B and PMAIP1 expressions at both the mRNA and protein levels in NSCLC cells. In addition, downregulation of GADD45B and PMAIP1 by TFAP2C promoted cell proliferation and cell motility, which are closely associated with NSCLC tumorigenesis. CONCLUSION: This study indicates that GADD45B and PMAIP1 could be promising tumor suppressors for NSCLC and might be useful as prognostic markers for use in NSCLC therapy.

Glomerulonefritis colapsante con marcadores de desdiferenciación podocitaria en síndrome hemofagocítico secundario a linfoma hepatoesplénico de células T / Collapsing glomerulonephritis with podocyte markers in hemophagocytic syndrome secondary to hepatosplenic T-cell lymphoma

El síndrome hemofagocítico es una condición clínica e histológica grave, secundaria a diferentes procesos. La glomerulonefritis colapsante es una podocitopatía proliferativa, generalmente de pronóstico desfavorable para la función renal. Se presenta un caso en el que las dos condiciones aparecieron asociadas, lo cual es una forma infrecuente de presentación del linfoma hepatoesplénico de células T. Se discute, asimismo, el papel de los marcadores de desdiferenciación podocitaria en esta glomerulopatía, y se revisan la fisiopatología y el tratamiento.

The hemophagocytic syndrome is a serious clinical-histological entity secondary to different diseases. Collapsing glomerulonephritis is a proliferative podocytopathy that usually has an unfavorable renal prognosis. We present a case in which both entities were associated, which is an infrequent form of hepatosplenic T-cell lymphoma. In addition, we review the role of the markers of podocyte dedifferentiation in this glomerulopathy and its pathophysiology and treatment.

Effects of Syzygium aromaticum, Cinnamomum zeylanicum, and Salvia triloba extracts on proliferation and differentiation of dental pulp stem cells

Abstract Hypersensitivity, local irritative and cytotoxic effects are known for the chemical components of Syzygium aromaticum and Cinnamomum zeylanicum contained in dental materials. However, there is no intimate data in dentistry using the whole extracts of these plants and introducing new ones. Salvia triloba is a well-known anti-inflammatory plant that correspondingly could be used in several dental traumas. Objectives: We aimed to show and compare the effect of S. aromaticum, C. zeylanicum, and S. triloba extracts on dental pulp stem cells (DPSCs) proliferation, differentiation, and immune responses. Material and Methods: Using xCELLigence, a real time monitoring system, we obtained a growth curve of DPSCs with different concentrations of the Extracts. A dose of 10 μg/mL was the most efficient concentration for vitality. Osteogenic differentiation and anti-inflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. Results: The level of osteonectin (ON, early osteogenic marker) decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation) differed among the extracts, in which S. aromaticum presented the highest value, followed by S. triloba and C. zeylanicum. Surprisingly, the determined calcium granules were reduced in S. aromaticum and S. triloba. In response to tumor necrosis factor alpha (TNF-α), S. triloba-treated DPSCs showed the most reduced level of IL-6 cytokine level. We suggest C. zeylanicum as a promising osteogenic inducer and S. triloba as a potent anti-inflammatory agent, which could be used safely in biocomposite or scaffold fabrications for dentistry. Conclusions: Because calcium granule formation and cell viability play a critical role in hard tissue formation, S. aromaticum in dentistry should be strictly controlled, and the mechanism leading to reduced calcium granule formation should be identified.

CDX-2, MUC-2 and B-catenin as intestinal markers in pure mucinous carcinoma of the breast

BACKGROUND: Pure mucinous adenocarcinoma of the breast is a rare entity characterized by the production of variable amounts of mucin comprising 1% to 6% of breast carcinomas. Some mucinous adenocarcinomas have shown expression of intestinal differentiation markers such as MUC-2. This study examines the expression of intestinal differentiation markers in this type of breast carcinoma. RESULTS: Twenty-two cases of pure mucinous adenocarcinoma of the breast were assessed. Immunochemistry was performed for beta-catenin, CDX-2 and MUC-2. All cases were positive for B-catenin. MUC-2 positivity was observed in all cases; 63. 6% were 3 plus positive. All cases were negative for CDX-2. CONCLUSIONS: These results suggest that mucinous breast carcinomas express some markers of intestinal differentiation, such as MUC-2 and beta-catenin; however, future studies with a larger series of cases and using molecular techniques that help affirm these results are needed.

Induced maturation of hepatic progenitor cells in vitro

Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin, and ALPL gene expression

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.

Mecanismos de ação da bradicinina na diferenciação neural in vitro / Mechanisms of bradykinin in neural differentiation

Durante o desenvolvimento do sistema nervoso, as células têm a tarefa de proliferar, migrar, diferenciar, morrer ou amadurecer de modo altamente preciso para formar estruturas complexas. Tal precisão é alcançada em decorrência da interação perfeita entre as células que se comunicam por meio de mensageiros químicos no ambiente extracelular. Nesse contexto, nosso grupo tem reportado o envolvimento da bradicinina (BK) em processos do desenvolvimento neural. Recentemente, observou-se que a BK desempenha um papel importante na determinação do destino neural, favorecendo a neurogênese em detrimento da gliogênese em diversos modelos de diferenciação, além de potencializar a migração celular observada no modelo de neuroesferas de rato (Trujillo et al, 2012). Essas descobertas motivaram, como objetivo geral dessa tese, a investigação dos mecanismos subjacentes à BK que determinam seus efeitos. Dessa forma, o principal modelo de diferenciação utilizado foi as células precursoras neurais (CPNs) isoladas do telencéfalo de embriões de camundongos. Estas células proliferam na presença dos fatores de crescimento (GFs) EGF + FGF2, mantendo-se multipotentes e formando as neuroesferas, ao passo que migram e diferenciam em neurônios e glias pela remoção desses GFs, com boa proximidade aos eventos do desenvolvimento do cortex in vivo. Como resultados do presente trabalho, observou-se, inicialmente, que a BK também influencia efetivamente na diferenciação neural no modelo de CPNs murinas. Ao término da diferenciação, observou-se que esta cinina favoreceu a migração e promoveu o enriquecimento neuronal, evidenciado pelo aumento da expressão das proteínas ß3-Tubulina e MAP2. Constatou-se também, que se observa uma baixa taxa de proliferação ao término da diferenciação na presença de BK (Trujillo et al, 2012), em consequência da grande proporção de neurônios em cultura estimulada por esta cinina. Esta relação causal foi evidenciada pelo ensaio de incorporação de EdU e concomitante imuno-detecção dos marcadores ß3-Tubulina, GFAP e Nestina. Fatores que promovem a neurogênese podem promovê-la suprimindo a proliferação celular em CPNs indiferenciadas, mais especificamente, alongando a fase G1 do ciclo celular que resulta na divisão de diferenciação. Assim, investigou-se também se a BK influencia nesse processo. Análises por citometria de fluxo demonstraram que esta cinina suprimiu a proliferação estimulada pelos GFs, levando ao acúmulo de células na fase G1 do ciclo celular. Esse acúmulo não provém do bloqueio do ciclo, uma vez que se observam grandes proporções de células nas fases subsequentes à G1, indicando que essa fase foi apenas prolongada pela BK e, assim, corroboraria no favorecimento da neurogênese. Outra face dos mecanismos adjacentes à BK para seus efeitos na diferenciação neural se refere às vias de sinalização disparadas por esta cinina. Observou-se que a BK induz a produção de AMPc por intermédio de proteínas G sensíveis à toxina pertussis (TP) (provavelmente através da subunidade ßγ de proteínas Gi) e promove a mobilização de cálcio dos estoques intracelulares, evidenciando o envolvimento da família de proteínas Gq. Esses resultados sugerem que o receptor B2 de cinina acopla-se tanto às proteínas Gi quanto às proteínas Gq em CPNs. A exposição dessas células à BK também ativou as vias da PI3K/Akt e da MAPK p38, mas não influenciou na ativação de STAT3 e JNK. Destaca-se o potencial da rota da MAPK ERK como uma das principais cascatas responsáveis por decodificar sinais de mensageiros externos em respostas celulares. O tratamento com BK em CPNs ativou a ERK por tempo prolongado e estimulou sua translocação para o núcleo. O efeito de BK na glio- e neurogênese de CPNs foi dependente da atividade de ERK, porque o bloqueio farmacológico dessa enzima impediu esse efeito de BK. Por outro lado, o favorecimento da migração induzido por esta cinina foi dependente da atividade da p38, enquanto, o seu efeito antiproliferativo foi condicionado à atividade das suas duas MAPKs, ERK e p38. Além disso, a via da PI3K/Akt ativada por BK não influenciou nos três eventos avaliados. Finalmente, utilizou-se nessa tese uma abordagem reducionista da diferenciação, porém amplamente utilizada por estudos mecanísticos de neurogênese, as células PC12. Assim, observou-se que a BK também ativa a ERK por tempo prolongado e com translocação nuclear, sendo que tal forma de ativação dessa quinase é proposta na literatura como necessária e suficiente para induzir a neurogênese dessas células. Demonstrou-se ainda que o bloqueio apenas da ativação sustentada de ERK, pela inibição das atividades das PKCs clássicas, impede o favorecimento da neurogênese por BK em células PC12. Juntos, esses resultados contribuem para elucidação dos mecanismos de ação da BK na regulação da diferenciação neural, colaborando para melhor entender esse processo e prevendo possíveis aplicações em terapias de reparo neuronal em pacientes com doenças, por exemplo, de Parkinson, Alzheimer, Esclerose Múltipla e lesões isquêmicas

During CNS development cells perform the task of proliferating, migrating, differentiating, dying or maturing in highly accurate patterns. Such accuracy is reached as a result of the perfect interaction among the cells that constantly communicate with each other through cell-cell contact or through chemical messengers present in the extracellular medium. In this context, our group has reported the involvement of bradykinin (BK) in neural differentiation of stem cell models (Trujillo et al, 2012). Recently, it has been observed that BK plays an important role in determining neural destination, favoring neurogenesis over gliogenesis in several models of differentiation, besides potentializing cell migration observed in the model of rat neurospheres. These discoveries have motivated, as the general objective of this thesis, the investigation of the mechanisms underlying BK-promoted effects on neural differentiation using neural precursor cells (NPCs) isolated from the telencephalon of mice embryos. These cells proliferate in the presence of growth factors (GFs) EGF + FGF2, remaining multipotent and forming neurospheres, while they migrate and differentiate in neurons and glias following removal of these GFs, resembling in simplified conditions events of the development of the cortex in vivo. As results of the present thesis, it was initially observed that BK also effectively influences neural differentiation fate of the mouse NPC model. This kinin favored migration and promoted neuronal enrichment, evidenced by increased expression of ß3-Tubulin and MAP2 marker proteins. Moreover, proliferation rates were largely decreased following differentiation in the presence of BK (Trujillo et al, 2012), due to the large proportion of neurons in the culture stimulated by this kinin. This causal relation was evidenced by the EdU incorporation assay and the concomitant immunodetection rates of ß3- Tubulin, GFAP and Nestin markers. Factors which promote neurogenesis can promote it by suppressing cell proliferation in undifferentiated NPCs, more specifically, prolonging the G1 phase of the cell cycle that result in the division of differentiation. Thus, it was further investigated whether BK influences this process. Flow cytometry analyses showed that this kinin suppressed the proliferation stimulated by GFs, resulting in the accumulation of cells in the G1 phase of the cell cycle. This accumulation is not caused by a cycle block, since wide proportions of cells are observed in phases subsequent to the G1, indicating that this phase was only prolonged by BK, thus corroborating for favoring neurogenesis. Another aspect of the mechanisms adjacent to BK for its effects on neural differentiation refers to the signaling pathways triggered by this kinin. Here, we show that the kinin B2 receptor couples to both Gi and Gq proteins in NPCs. BK induced the production of intracellular cAMP by activation of G proteins sensitive to pertussis toxin (PT) (probably through ßγ subunit of Gi proteins) and promoted the mobilization of calcium from intracellular stocks, demonstrating the involvement of YM-254890-sensitive Gq proteins. Exposure of these cells to BK also activated PI3K/Akt and MAPK p38 pathways, but did not affect the activation of STAT3 and JNK. It is important to note the potential MAPK-ERK route as one of the main cascades responsible for decoding signals from external messengers into cellular responses. NPC treatment with BK activated ERK for prolonged time and stimulated its translocation into the nucleus. The effect of BK on glio- and neurogenesis of NPCs depended plainly on ERK activity, because the pharmacological blockade of this enzyme prevented the BK-exerted effects. On the other hand, the favoring of migration induced by this kinin was dependent on p38 activity, while its antiproliferative effect was conditioned to the activity of both the MAPKs ERK and p38. In addition, the PI3K/Akt pathway activated by BK did not affect any of the three evaluated events. Finally, we used in this thesis a reductionist approach of differentiation based on the use of PC12 cells, which has been widely used for mechanistic studies of neurogenesis. Thus, it was observed that BK also activated ERK for prolonged time and with nuclear translocation, considering that such form of kinase activation is proposed in the literature as necessary and sufficient to induce neurogenesis in these cells. This study also demonstrated that blockade only of the sustained ERK activation, through the inhibition of the activity of classic PKCs, prevents the favoring of neurogenesis by BK in PC12 cells. Together, these results compose novel mechanisms of action of BK on events of neural development in vitro, contributing to the better understanding of this process and foreseeing possible applications in the future for neuronal repair strategies

Therapeutic monoclonal antibodies: scFv patents as a marker of a new class of potential biopharmaceuticals / Terapêuticas de anticorpos monoclonais: patentes scFv como um marcador de uma nova classe de produtos biofarmacêuticos potenciais

Monoclonal antibodies represent the fastest growing class of biopharmaceutical products and have a host of applications in medical research, diagnosis, therapy, and basic science. The production of recombinant monoclonal antibodies has revolutionized the generation of immunoglobulins, and their use represents a strategic breakthrough, affecting the global pharmaceutical market for therapeutic proteins. In the present work, a review of scFv, and the number of related patents, has been carried out. The results show that several countries have scFv patents, most notably the United States, China and United Kingdom. The target of these scFv antibodies was also assessed and the results demonstrate that most are directed toward cancer therapy.

Anticorpos monoclonais representam a classe de maior crescimento em produtos de biofármacos e possuem várias aplicações em pesquisa médica, diagnóstico, terapias e ciência básica. A produção de anticorpos monoclonais recombinantes revolucionou a geração de imunoglobulinas e sua utilização implica em avanço estratégico, afetando o mercado farmacêutico global de proteínas terapêuticas. No presente trabalho, uma revisão sobre scFv e a relação do seu número de patentes foi analisada. Os resultados mostram que vários países apresentam patentes de scFv com destaque para os Estados Unidos, China e Reino Unido. Os alvos desses anticorpos também foram avaliados e as análises revelaram que a maioria é destinado a terapias contra o câncer.

Leucemia de células dendríticas: a propósito de un caso / Dendritic leukemia cells: a purpose of a case

La leucemia de células dendríticas son patologías poco frecuentes, que involucran fundamentalmente a la piel, pero con una alta tendencia de metástasis. La inmunohistoquímica es una herramienta valiosa junto con la biopsia para el diagnóstico definitivo. Se presenta el caso de una paciente joven, quien acudió por presentar una lesión en la pierna izquierda de dimensiones y características únicas, cuyo diagnóstico requirió de la utilización de marcadores monoclonales específicos en la identificación de esta entidad.

The dendritic cell leukemias are pathologies very uncommon which involve mainly the skin, but with a high tendency of metastasis. The flow cytometric is a valuable tool together with tissue biopsy for a definitive diagnosis. We present the case of a young female who complained for a left leg lesion with unique dimensions and characteristics, in where the diagnostic process required the use of the specific monoclonal markers in the identification of this particular disease.

Cáncer pulmonar: avances en el siglo XXI / lung cancer: advances in the XXI century

En la presente década, el cáncer pulmonar ha producido más de 4.800.000 muertes cada año, consolidándose como la primera causa de mortalidad por cáncer en todo el mundo, para ambos sexos. Según la Organización Mundial de la Salud, no hay esperanzas de soluciones para los próximos cincuenta años. La enfermedad es compleja, grave, mortal, epidémica, tabaco dependiente, formando parte del 6% de la mortalidad general por tabaquismo. El objeto principal de esta revisión es informar el avance que ha logrado la ciencia médica en el presente siglo. La contextualización del tema nos permite hacer una selección de los hallazgos más relevantes en: epidemiología como es, la incipiente introducción del comportamiento molecular gracias al uso de marcadores tumorales; en patología, las biopsias pueden informar sobre la variedad histológica y el perfil genético de la misma a objeto de evaluar el alto riesgo de morir de acuerdo a los genes identificados. Desde el punto de vista clínico, destaca a la séptima edición de la estadificación del cáncer de pulmón de células no pequeñas, por medio del sistema TNM, la cual ayuda a crear un mejor pronóstico y una mejor selección terapéutica; los procedimientos diagnósticos de última generación como: tomografía helicoidal, tomografía por emisión de positrones, ultrasonido transbronquial, broncoscopia autofluorescente, broncoscopia virtual, citometría cuántica automatizada y otros, han sido de gran valor para el diagnóstico temprano de la enfermedad. El tratamiento curativo está reservado a lesiones incipientes y el mejor esquema terapéutico para carcinoma de células no pequeñas es la cirugía y quimioterapia adyuvante. La genoterapia e inmunoterapia continúan en fase experimental, la quimioterapia con resultados controversiales y la prevención primaria la consideramos clave en el control definitivo de la enfermedad. Al final del trabajo se hacen proyecciones para el futuro y consideraciones preliminares...

In this decade. the pulmonary cancer has produced more than 4.800.000 dealths each year, consolidating as the first cause of mortality from cancer in the world, for both sexes. according to the World Health Organization, there is no hope of solution for the next fifty years. The disease is complex, serious, fatal, epidemic, tobacco depending and part of 6% of overall mortality by smoking. The main purpose of this review is to report the progress of science medical in this century. the contextualization of the them let to us to make a selection from the findings more relevant in: epidemiology as it is the early introduction of the molecular component through the use of tumor markers; in pathology, biopsies studies can report on histological variety and genetic profile to order to report on the high risk of dealth, according to the identified genes. From the clinical point of view, is the seventh edition of the standing of the lung cancer non small cell, in the TNM system, which helps create a better prognosis and better therapeutic: targeting cancer stading diagnosis procedures of last generation as: helical scans, tomography by emission of positrons, transbronchial ultrasound, autofluorescense bronchoscopy, virtual bronchoscopy, automated quantum citometry and others, have been of great value to the early disease diagnosis. Curative treatment has been reserved to emerging injuries and the best therapeutic scheme for non-small cell carcinoma is surgery and adjuvant chemotherapy. Genotherapy and immunotherapy continue in experimental phase, quimioprevention with vontroversial result and primary prebention mean the key in the final control of the disease. At the end of the work, there are proyection for the future and preliminary consideration with emphasis on the state of uncertainty that we have before the epidemic, the challenge it represents for the medical community and the need to futher study of the tumor genome with the prospect of a effective geno...

Detección de anticuerpos contra los antígenos de diferenciación tumoral proteinasa 3 (PR3) y mieloperoxidasa (MPO) en la leucemia promielocítica / Detection of antibodies to antigens of proteinase 3 (PR3) tumor differentiations and myeloperoxidase (MPO) in cases of promyelocytic leukemia

La leucemia promielocítica (LPM) subtipo M3 representa del 5-15 por ciento en la clasificación FAB de las leucemias mieloides agudas (LMA). Está asociada con características genéticas únicas que incluyen la translocación recíproca t(15;17)(q22;q12). El mecanismo por el que ocurre la t(15;17) no se conoce. Las leucemias de estirpe mieloide expresan diversos antígenos de diferenciación tumoral como son la proteinasa 3 (PR 3) y la mieloperoxidasa (MPO) que se encuentran sobreexpresados en el promielocito. Se plantea que participan en la maduración y en la regulación de la división celular. Existe poca información acerca de la respuesta inmune de pacientes con LPM dirigida contra las células tumorales. En nuestro trabajo se detectó la presencia de anticuerpos contra los antígenos de diferenciación tumoral PR3 y MPO en diferentes fases del tratamiento de la enfermedad, mediante inmunofluorescencia indirecta. Los anticuerpos anti PR3 y anti MPO se detectaron en aquellos pacientes sin tratar y en fase de inducción, no así en la consolidación y mantenimiento, de ahí su posible utilidad como marcadores de diferenciación celular.

ABSTRACT Promyelocytic leukemia (PML) subtype M3 represents the 5-15 percent in the FAB classification of acute myeloid leukemias (AML). It is associated with the unique genetic features including the reciprocal t-translocation (15;17) (q22;q12). The mechanism of t is unknown. The myeloid leukemias express different tumoral differentiation antigens such as the proteinase 3 (PR 3) and myeloperoxidase (MPO) which are over-expressed in promyelocyte. It is involved in maturation and regulation of cell division. There is scarce information on the immune response of patients with PLM against tumor cells. In our paper we detected presence of antibodies to RP3 and MPO tumor differentiation antigens in different phases of disease treatment by indirect immunofluorescence. Anti-MPO and anti-PR3 antibodies were detected in those patients without treatment and in induction phase but not in the consolidation and maintenance, thus its potential usefulness as cellular differentiation markers.

Procalcitocina en pacientes con neutropenia y fiebre secundarias a quimioterapia / Procalcitocina in patients with neutropenia and fever secondary to chemotherapy

Los pacientes con malignidades hematológicas frecuentemente se complican por infecciones. La procalcitonina (PCT) puede diferenciar la sepsis de la inflamación. Establecer la utilidad de PCT para determinar complicaciones infecciosas en adultos con malignidades hematológicas y neutropenia febril (NF) provenientes de la Ciudad Hospitalaria “Dr. Enrique Tejera” (CHET) y Centro Policlínico Valencia (CPV), Valencia, Venezuela. Estudio prospectivo que incluyó 30 pacientes neutropénicos febriles (H:M, 20:10), edad 49,4±21 años. En 5ml de sangre venosa, extraidos en las primeras 48 horas siguientes a la fiebre, se midió mediante ensayo semicuantitativo PCT-Q®. PCT<0.5ng/ml considerada negativa, >0.5 <2ng/ml infección sistémica, >2 <10ng/ml posible sepsis severay >10ng/ml: posible shock séptico. Se utilizó estadística descriptiva y probabilidad de asociación mediante test de Fisher, con p<0,05 considerada estadísticamente significativa. La asociación entre PCT y hallazgos clínicos de infección no fue estadísticamente significativa (p = 0,1); la asociación entre PCT y hemocultivos positivos fue estadísticamente significativa (p = 0,006) y también entre PCT y mortalidad (p = 0,034). La PCT elevada se asocia a bacteriemia y mortalidad en adultos neutropéniucos febriles, empeorando su pronóstico

Frequently patients with haematologic malignancies (HM) are complicated with infections. Procalcitonin (PCT) allows to differentiate sepsis from inflammation. To assess the usefulness of PCT in infectious complications in adult patients with HM and febrile neutropenia (FN). This prospective study, included 30 FN patients (M:F,20:10), age 49,4±21 years. A blood sample in the first 48 hours after the start of fever PCT was processed by semi-quantitative assay (PCT-Q®). Values of PCT<0.5ng / ml were considered negative, >0.5<2ng/ml suggested systemic infection, >2<10ng/ml, possible severe sepsis and >10ng/ml possible septic shock. Descriptive statistics and probability of association by fisher test were performed and p < 0,05 considered statistically significant. There was no stastical correlation between PCT and clinical findings of infection (p= 0,1) but between PCT and positive blood cultures it was statistically significant (p = 0,006) and also between PCT and mortality (p = 0,034). Elevated PCT is associated with bacteremia and mortality among adult FN patients with HM, worsening their prognosis

Modulación de la diferenciación de células dendríticas por antígenos tumorales pulmonares / Modulation of dendritic cell differentiation by lung tumor antigens

El cáncer es, en la actualidad, un serio problema de salud pública en el ámbito mundial. Una de las aproximaciones más exploradas desde hace cerca de veinte años, es la inmunoterapia con células dendríticas (CD), células presentadoras de antígenos (CPA) profesionales. No obstante, los resultados de los estudios clínicos que emplean CD autólogas pulsadas con antígenos tumorales no han sido tan exitosos como se proyectaba. Una de las hipótesis propuestas que justifique la eficacia tan variable en los protocolos terapéuticos, es la inducción de tolerancia en las CD por los antígenos tumorales y la subsecuente baja actividad de las células efectoras (linfocitos T CD8+, CD4+ y Natural Killer). En este trabajo se evidencia que los antígenos tumorales provenientes de un adenocarcinoma pulmonar favorecen la reaparición de co-receptores como el CD14, conocido marcador de inmadurez en CD. Esto sugiere que en algunos aspectos las CD expuestas a tales antígenos, muestran defectos en la diferenciación. Como se sabe, las CD inmaduras tienen una potencia estimuladora mucho menor sobre las células efectoras, comparativamente con las CD de fenotipo maduro. Sin embargo, antígenos provenientes de la línea comercial A549 (adenocarcinoma) no produjeron cambios similares en la maduración de CD. Ello resalta la necesidad de caracterizar la respuesta individual in vitro al planear una terapia basada en CD, a fin de tener mayor probabilidad de éxito en el tratamiento.

Cancer is currently a very serious public health problem worldwide. Dendritic cell (DC) based immunotherapy has been one of the most active research fields during the last 20 years. However, results of several studies, mainly involving tumor antigen pulsed autologous DCs, have not been as successful as expected. Induction of a tolerant behavior of DCs upon the contact with tumor derived antigens is one of the hypotheses proposed since other effector cells like CD8+, CD4+ T lymphocytes and Natural Killers, would be rendered to a non-responsive state seriously impairing tumor growth control. In this work we found additional evidence about a likely DC differentiation defect caused by lung adenocarcinoma specific antigens freshly obtained from a patient, as suggested by the presence of the co-receptor CD14 on the surface of mature DCs (CD14 is a known marker for initial stages of DC differentiation). Our results confirm that some aspects in tumor antigen treated DCs resemble their immature phenotype, largely less efficient presenting cells, and very weak immune response inducers. Interestingly, tumor antigens obtained from the lung adenocarcinoma derived A549 cell line were unable to cause the same effect. This highlights the requirement for a complete characterization of the functional nd phenotypic individual conditions when planning a DC based immunotherapy in order to have good success likelihood at cancer patient’s treatment.

Caracterização da expressão dos genes da via de sinalização WNT e do processo de transição epitélio-mesênquima durante a embriogênese de rim e suas implicações em tumores de Wilms / Gene expression characterization of WNT signaling pathway and epithelial-mesenchymal transition during kidney embryogenesis and their implications in Wilms tumors

O desenvolvimento do rim envolve interações complexas entre as células epiteliais e mesenquimais. Uma interrupção em qualquer passo crucial da progressão morfogenética pode levar a má formação renal e doenças, incluindo câncer. O tumor de Wilms (TW) ou nefroblastoma é originado pela interrupção da diferenciação das células do blastema metanéfrico resultando em um tumor composto por três componentes histológicos, blastema, epitélio e estroma... Nesse estudo, para identificar as alterações moleculares do início da diferenciação do rim e que são recapituladas no TW, a expressão de genes pertencentes às vias de transdução de sinal foi avaliada em células do blastema metanéfrico de quatro estágios temporais da diferenciação em camundongos (de 15,5 dias pós-coito até o rim completamente diferenciado), e em células do blastema de TWs e de rins diferenciados em humanos... O RNA total das células capturadas a laser foi amplificado e hibridado. Foram selecionados 18 genes, cuja expressão no início da diferenciação do rim foi recapitulada no TW, dos quais 11 foram menos expressos no tumor e apresentaram expressão crescente durante a nefrogênese e 7 foram mais expressos no tumor e apresentaram expressão decrescente durante a nefrogênese ... As alterações da expressão desses genes devem ser eventos precoces da interrupção da diferenciação que leva a transformação maligna. Adicionalmente, combinações de trios de genes foram avaliadas como marcadores preditivos de recaída em TW. O melhor trio composto por IGF2, TESK1 e PAX2, foi capaz de discriminar corretamente 85,71% das amostras quanto a recaída e não recaída. Esse estudo contribuiu para a identificação de novos genes associados com o aparecimento do TW, e como são alterações moleculares precoces, provavelmente são desencadeadoras do aparecimento do TW. Dessa forma, são candidatos apropriados para serem testados como alvos terapêuticos e marcadores moleculares para auxiliar a prática diária do TW.

Kidney development involves complex interactions between epithelial and mesenchymal cells. A disruption at any crucial step of the morphogenetic progression may lead to kidney malformations and diseases, including cancer. Wilms tumor (WT) or nephroblastoma originates from the metanephric blastema cells, which were unable to complete the differentiation process, resulting in a tumor composed by three histological components, blastema, epithelia and stroma. Blastemal component presents molecular characteristics which are highly similar to the earliest stages of kidney development, suggesting that it retains the key molecular events responsible for WT onset. Deregulation of signal transduction pathways has showed to be an important factor for interruption of renal differentiation, and probably for the WT onset. In this study, aiming to identify the early molecular alterations potentially involved in WT arising, the expression pattern of genes belonging to the transduction signaling pathways was evaluated in cells of the metanephric blastema in four temporal differentiation stages from 15,5 days post-coitum to the fully differentiated kidney in mice, and in blastemal cells of WT and human differentiated kidneys. To evaluate nephrogenesis in mice and WT in humans, we developed a model that allows the hybridization of target molecules of both species in the same cDNA microarray platform. Three hundred and twenty-six stretches of orthologous genes from humans and mice that share high similarity at nucleotide level were immobilized on a glass platform. Wnt signaling pathway, phosphatidylinositol (PI3K) and genes related to epithelial-mesenchymal transition and epithelial-mesenchymal (EMT/MET), associated to the embryonic development, were among the evaluated pathways. Blastemal cells were laser captured from 24 WT samples at four stages of nephrogenesis. Total RNA from these samples was amplified and hybridized in the platform. Eighteen genes were selected, whose expression in the initial of kidney differentiation were also observed in WT, where 11 were down-regulated in WT and reported an increasing expression during nephrogenesis and 7 were up-regulated in WT and presented a decreasing expression during nephrogenesis. Non-supervised hierarchical clustering based on the expression of 18 genes grouped differentiated kidneys from both species, human and murine, and discriminated both from WT samples, which were grouped with the earliest kidney stages, validating the model proposed by this study. The 18 genes were assessed in independent groups of samples at mRNA and protein levels. Twelve out of 18 genes were selected for validation by quantitative RT-PCR in the initial sample set and the expression of 9 (75%) genes was confirmed. From the 9 genes, 5 (55.6%) were validated in an independent group of samples. At protein level, 8 genes were evaluated by immunohistochemistry in WT, during nephrogenesis and in differentiated kidneys in humans. Quantitative evaluation of protein expression was consistent with the results of mRNA expression for 5 (62.5%) genes. This study identified WNT5B, PI3KCA, FZD2, GRK7, TESK1 and TIMP3, which had not been previously associated to WT. Gene expression alterations may be the early events of the interruption of cell differentiation that leads to malignant transformation. Additionally, combinations of trios of genes were tested as predictors of WT relapse revealing IGF2, TESK1 and PAX2 as the best trio because it was able to correctly discriminate 85.71% of the samples. This study contributes to the identification of new genes associated to the WT onset and to the early molecular alterations, which are likely to trigger the WT arising. In this way, these genes are candidates to be tested for therapeutic targets and also for molecular markers to assist the daily WT practice.

Imuno-histoquímica para o diagnóstico precoce de vitiligo / Immunohistochemistry for early diagnosis of vitiligo

O vitiligo é uma doença de pele freqüente que acomete 1% da população e é caracterizada por máculas despigmentadas conseqüentes à perda progressiva e localizada dos melanócitos da epiderme. Na maioria dos pacientes, o diagnóstico é feito por exame clínico. A biópsia da pele é realizada quando há necessidade de diagnóstico diferencial com doenças hipocromiantes. O diagnóstico histopatológico de vitiligo é difícil nos preparados corados por hematoxilina e eosina (HE). Há poucos estudos sobre a melhoria da qualidade diagnóstica no vitiligo. OBJETIVO: Avaliar a utilidade dos marcadores imuno-histoquímicos proteína S-100, human melanoma black-45 (HMB-45) e Melan-A para o diagnóstico precoce em casos clinicamente suspeitos ou duvidosos de vitiligo. Material e métodos: Lâminas histológicas de biópsias de pele sã e lesada de 10 pacientes com suspeita clínica de vitiligo coradas pelos métodos de HE, proteína S-100, HMB-45 e Melan-A. Utilizou-se contracoloração com Giemsa como modificação técnica para diferenciar a melanina da imunomarcação. RESULTADOS: Seis casos, com manifestação clínica recente, apresentaram infiltrado linfocitário, do tipo dermatite de interface, na pele lesada na HE. As colorações por S-100, HMB-45 e Melan-A marcaram os melanócitos da camada basal da pele sã, e a proteína S-100 evidenciou as células de Langerhans. Na pele lesada, os melanócitos estavam ausentes ou diminuídos quando comparados com a pele normal. A proteína S-100 demonstrou maior número de células de Langerhans, o que é característico das lesões de vitiligo. CONCLUSÃO: A imuno-histoquímica pode ser utilizada como método auxiliar no diagnóstico dos casos duvidosos de vitiligo.

Vitiligo is a frequent skin disease that affects 1% of the population. It presents depigmented macules resulting from a gradual loss of melanocytes in the epidermis. In most cases, the diagnosis is made by clinical examination. Skin biopsies are performed when it is necessary to compare it with other hypomelanosis. Histopathological diagnosis of vitiligo is often difficult in hematoxylin-eosin (H&E) stained sections. There are a few studies on the improvement of diagnostic quality in vitiligo. OBJECTIVE: To evaluate the use of immunohistochemical markers, such as S-100 protein, human melanoma black-45 (HMB-45) and Melan-A, in the early diagnosis of clinically suspected or doubtful cases of vitiligo. Materials and methods: Histological sections of biopsies from healthy and affected skin areas from 10 patients clinically suspected of vitiligo. The samples were stained with H&E, S-100 protein, HMB-45 and Melan-A methods. Counterstaining with Giemsa was applied as a technical modification to differentiate melanin from immunolabelling. RESULTS: Six cases with recent clinical manifestation showed lymphocyte infiltrates, such as interface dermatitis, in the affected skin in the H&E staining technique. S-100 protein, HMB-45 and Melan-A staining marked the basal layer melanocytes of the healthy skin and S-100 protein antigen evidenced Langerhans cells. Melanocytes were absent or less frequent in affected skin areas in comparison with normal skin. S-100 protein showed a larger number of Langerhans cells, what is a common feature of vitiligo lesions. Conclusion: Immunohistochemistry may be used as an auxiliary technique for the diagnosis of suspected vitiligo cases.

Estudio inmunohistoquímico del carcinoma de glándula mamaria: en el Hospital Vargas de Caracas / Immunohistochemical study of mammary gland carcinoma: in Hospital Vargas de Caracas

Estudiar la asociación entre diferentes elementos morfológicos con aspectos inmunohistoquímicos y su posible utilidad pronóstica. Estudio descriptivo-retrospectivo, de 651 casos de carcinoma mamario con marcaje inmunohistoquímico en el Hospital Vargas de Caracas desde mayo 2001 a julio 2004. Se analizaron características morfológicas de los diferentes tipos tumorales, aplicando la clasificación de Scarff-Bloom-Richardson modificada, relacionándolos con la expresión de diferentes marcadores inmunohistoquímicos: receptores de estrógeno, receptores de progesterona, c-erbB-2 y Ki-67, realizados mediante la técnica de biotina-estreptavidina. La edad promedio fue 51,72 años. El tumor más frecuente fue el carcinoma ductal infiltrante (88,7 por ciento), seguido del carcinoma lobulillar infiltrante (4,6 por ciento). El 5,5 por ciento de los carcinomas ductales eran bien diferenciados, 42,3 por ciento moderadamente diferenciados, 52,2 por ciento poco diferenciados; de estos, el 77 por ciento tenían receptores de estrógeno y de progesterona ≤ 10 por ciento; los carcinomas bien diferenciados mostraron positividad variable. Los tumores poco diferenciados presentaron c-erbB-2 positivo en 51,36 por ciento, entre los bien diferenciados el 81,84 por ciento fueron negativos, el carcinoma intraductal fue positivo en un 42,86 por ciento, generalmente asociado a comedocarcinoma. El 94,37 por ciento de los tumores poco diferenciados fueron Ki-67 positivos. El carcinoma ductal infiltrante de tipo clásico es el tumor maligno más frecuente de la glándula mamaria, con una edad promedio de 51,5 años, generalmente es poco diferenciado, implicando posiblemente tumores con conducta biológica agresiva. Esto se evidencia por la negatividad para receptores hormonales y la expresión aumentada de c-erbB-2 y Ki-67.

To study and assess the association between different morfhological and pathologic features and immunohistochemistry as a prognostic factor. A retrospective-descriptive study of 651 cases of breast cancer by immunohistochemistry markers was analyzed in the Vargas Hospital of Caracas since may 2001 until july 2004. We evaluate the pathologic features of the different tumor subtypes, and relation this with the expression of immunohistochemistry markers: the estrogen receptor, progesterone receptor, c-erbB-2 and Ki-67, made by the biotin-streptavidin technique. The median patient age was 51.72 years. The most frequent type of tumor was the invasive duct carcinoma (88.7 %), follow by the invasive lobular carcinoma (4.6 %). The 5.5 % of the duct carcinomas were well differentiated tumors, 42.3 % moderate differentiated, and 52.2 % poorly differentiated; in which 77 % had estrogen receptor and progesterone receptor ≤ 10 %, well differentiated tumors show a variable positivity. The poorly differentiated tumors show positive c-erbB-2 in 51.36 %, in the well-differentiated group 81.84 % were negative; the c-erbB-2 on intraductal carcinoma was positive in 42.86 %, generally associated with the comedocarcinoma; the 94.7 % of the poorly differentiated tumors were Ki-67 positives. Classic invasive duct carcinoma is the most frequent malignant tumor, with a median age of 51.5 years, and the poorly differentiated tumors generally show an aggressive biologic conduct, associated to patients with estrogen receptors and progesterone receptors negative tumors and a high expression of the c-erbB-2 y Ki-67.

Estudo da expressão de galectina-3 em carcinomas de língua de camundongos / Study of the galectin-3 expression in tongue carcinomas of mice

INTRODUÇÃO: A galectina-3 (Gal-3) é uma lectina de mamíferos ligante de resíduos b-galactosídeos. Numerosos estudos têm mostrado que a Gal-3 apresenta importantes papéis na biologia tumoral, atuando em fenômenos como apoptose, metástase e transformação maligna. No entanto, em carcinomas de cabeça e pescoço, a real significância da sua expressão ainda é pouco compreendida. OBJETIVO: O objetivo deste estudo foi avaliar a expressão de Gal-3 em tumores de língua induzidos experimentalmente em camundongos desafiados com o carcinógeno 4-nitroquinolona-1-óxido (4NQO). MATERIAL E MÉTODOS: Quarenta e dois camundongos C57BL/6, machos, foram desafiados com 4NQO na água de beber por 16 semanas e sacrificados em diferentes períodos depois do tratamento. Após o sacrifício, as línguas foram removidas, processadas, coradas por hematoxilina e eosina (HE) e microscopicamente analisadas quanto à presença de carcinoma. Ensaio imuno-histoquímico para detecção do antígeno Gal-3 e análise descritiva da sua expressão nos tumores induzidos foram realizados. RESULTADOS: Ao final do experimento, foram produzidos 15 tumores. No tecido epitelial não-neoplásico, forte imunorreatividade foi observada apenas na camada parabasal. Nas camadas mais superficiais a intensidade de marcação foi mais fraca, e na camada basal variou de ausente a fraca. Todos os carcinomas bem diferenciados exibiram fraca marcação, exceto nas áreas queratinizantes. No único caso de carcinoma pouco diferenciado, forte imunorreatividade para Gal-3 foi observada. CONCLUSÃO: Nossos resultados descritivos mostram que a transformação maligna é acompanhada de redução da intensidade de expressão da Gal-3 e que o aumento da sua expressão com a perda da diferenciação neoplásica sugere a sua vinculação com agressividade tumoral.

BACKGROUND: Galectin-3 (Gal-3) is a b-galactoside-binding mammalian lectin. Numerous studies have demonstrated that Gal-3 plays an important role in tumor biology, acting in some events such as apoptosis, metastasis and malignant transformation. However, in carcinomas of head and neck, the real significance of Gal-3 expression requires a better understanding. OBJECTIVES: The aim of this paper was to evaluate Gal-3 expression in tongue carcinomas experimentally induced in mice challenged with carcinogen 4-nitroquinoline-1-oxide (4NQO). MATERIAL AND METHOD: Forty-two C57BL/6 male mice were challenged with 4NQO in drinking water for 16 weeks and killed at different periods after induction. In each period, their tongues were removed, routinely processed, stained with hematoxylin and eosin (H&E) and microscopically analyzed as to the presence of carcinoma. Immunohistochemical test for Gal-3 and a descriptive analysis of its expression in induced tumors were performed. RESULTS: By the end of the experiment, 15 tumors had been induced. In the non-neoplastic lingual epithelium, strong and weak immunoreactivity for Gal-3 was noted in parabasal and superficial layers, respectively. In the basal layer, Gal-3 expression varied from absent to weak. All the well-differentiated carcinomas showed weak immunoreactivity for Gal-3, except in keratinized areas. The only case of poorly differentiated squamous cell carcinoma indicated strong immunoreactivity for Gal-3. CONCLUSION: Our results show that malignant transformation is associated with reduced expression of Gal-3, whereas its increased expression in poorly differentiated carcinoma seems to be connected with tumor aggressiveness.