Gene expression profiling unveils the temporal dynamics of CIGB-300-regulated transcriptome in AML cell lines.

BACKGROUND: Protein kinase CK2 activity is implicated in the pathogenesis of various hematological malignancies like Acute Myeloid Leukemia (AML) that remains challenging concerning treatment. This kinase has emerged as an attractive molecular target in therapeutic. Antitumoral peptide CIGB-300 blocks CK2 phospho-acceptor sites on their substrates but it also binds to CK2α catalytic subunit. Previous proteomic and phosphoproteomic experiments showed molecular and cellular processes with relevance for the peptide action in diverse AML backgrounds but earlier transcriptional level events might also support the CIGB-300 anti-leukemic effect. Here we used a Clariom S HT assay for gene expression profiling to study the molecular events supporting the anti-leukemic effect of CIGB-300 peptide on HL-60 and OCI-AML3 cell lines. RESULTS: We found 183 and 802 genes appeared significantly modulated in HL-60 cells at 30 min and 3 h of incubation with CIGB-300 for p < 0.01 and FC > = │1.5│, respectively; while 221 and 332 genes appeared modulated in OCI-AML3 cells. Importantly, functional enrichment analysis evidenced that genes and transcription factors related to apoptosis, cell cycle, leukocyte differentiation, signaling by cytokines/interleukins, and NF-kB, TNF signaling pathways were significantly represented in AML cells transcriptomic profiles. The influence of CIGB-300 on these biological processes and pathways is dependent on the cellular background, in the first place, and treatment duration. Of note, the impact of the peptide on NF-kB signaling was corroborated by the quantification of selected NF-kB target genes, as well as the measurement of p50 binding activity and soluble TNF-α induction. Quantification of CSF1/M-CSF and CDKN1A/P21 by qPCR supports peptide effects on differentiation and cell cycle. CONCLUSIONS: We explored for the first time the temporal dynamics of the gene expression profile regulated by CIGB-300 which, along with the antiproliferative mechanism, can stimulate immune responses by increasing immunomodulatory cytokines. We provided fresh molecular clues concerning the antiproliferative effect of CIGB-300 in two relevant AML backgrounds.

A co-formulation of interferons alpha2b and gamma distinctively targets cell cycle in the glioblastoma-derived cell line U-87MG.

BACKGROUND: HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model. METHODS: Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry. RESULTS: The three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG. CONCLUSIONS: We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.

Growth hormone secretagogue peptide-6 enhances oreochromicins transcription and antimicrobial activity in tilapia (Oreochromis sp.).

Growth Hormone-Releasing Peptide 6 (GHRP-6) (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2) is an agonist of the growth hormone secretagogue receptor. GHRP-6 mimics the effect of ghrelin. The present study focuses on the immunomodulatory effects of GHRP-6 in tilapia with and without the presence of Pseudomonas aeruginosa infection. GHRP-6 up-regulated the transcription levels of three piscidin-like antimicrobial peptides (Oreochromicins I, II, and III) and granzyme in a tissue-dependent manner. Antimicrobial activity stimulation in serum (lysozyme and anti-protease activity) was also confirmed. Besides, GHRP-6 enhanced the in vitro antimicrobial activity against P. aeruginosa in tilapia gills mucus and serum samples and decreased the bacterial load in vivo after infection with this Gram-negative bacterium. Our results evidenced, for the first time, a direct link between a growth hormone secretagogue ghrelin mimetic in fish and the enhancement of antimicrobial peptides transcription, which suggests that this secretagogue is capable to lead the activation of microbicidal activity in tilapia. Thus, these results open new possibilities for GHRP-6 application in aquaculture to stimulate the teleost immune system as an alternative treatment against opportunistic bacteria.

Selection of reference genes for use in quantitative reverse transcription PCR assays when using interferons in U87MG.

Relative gene quantification by quantitative reverse transcription PCR (qRT-PCR) is an accurate technique only when a correct normalization strategy is carried out. Some of the most commonly genes used as reference have demonstrated variation after interferon (IFN) treatments. In this work we evaluated the suitability of seven reference genes (RGs) [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), ß-2Microglobulin (B2M), ribosomal RNA subunits 18S and 28S, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) and the RNA helicase (DDX5)] for use in qRT-PCR assays in the glioblastoma-derived cell line U87MG treated with IFNα, IFNγ or a co-formulated combination of both IFNs (HeberPAG); untreated cell lines were included as control. Data was analyzed using geNorm and NormFinder softwares. The expression stability of the seven RGs decreased in order of DDX5/GAPDH/HMBS, 18S rRNA, YWHAZ, 28S rRNA and B2M. qRT-PCR analyses demonstrated that DDX5, GAPDH and HMBS were among the best stably expressed markers under all conditions. Both, geNorm and NormFinder, analyses proposed same RGs as the least variables. Evaluation of the expression levels of two target genes utilizing different endogenous controls, using REST-MCS software, revealed that the normalization method applied might introduce errors in the estimation of relative quantities. We concluded that when qRT-PCR is designed for studies of gene expression in U87MG cell lines treated with IFNs type I and II or their combinations, the use of all three GAPDH, HMBS and DDX5 (or their combinations in pairs) as RGs for data normalizations is recommended.

Regulation by IFN-α/IFN-γ co-formulation (HerberPAG®) of genes involved in interferon-STAT-pathways and apoptosis in U87MG.

Interferons (IFNs) are proteins of the family of cytokines. Their antiproliferative function has been taken into account for several clinical therapies against malignant diseases. In this family, IFNs α and γ have demonstrated the highest antitumor effects. HerberPAG® is a new co-formulation with IFNs, α2b and γ. It has been obtained to increase the antiproliferative effect of individual IFNs and decrease their associated toxicity. Glioblastoma multiforme (GBM) is the most common primary brain tumor and one of the most deadly forms of cancer. The objective of the present work is to obtain insights into the regulation of Interferon-STAT-pathways and apoptosis in U87MG, at the transcriptional level. As a pharmacogenomic strategy we quantified mRNAs levels in vitro by quantitative PCR, using the cell line U87MG as a model. Some of the genes involved in the first steps of IFNs signaling pathways (stat1 and stat3) and apoptosis events (tp53, bax, bcl-2, bad, caspase3 (casp3), caspase8 (casp8) and caspase9 (casp9)) were studied. The detected mRNAs expression pattern for stat1and stat3 indicates a higher tumor suppressor activity of HerberPAG® compared to individuals IFNs. The up-regulation of tp53, bax, bad, casp3, casp8 and casp9 genes and the down regulation of bcl-2 gen, after the treatment with HerberPAG® show a pro-apoptotic function. HerberPAG® gene-induced profile shows an advantage in relation to IFN α2b and γ with a higher stat1 expression and a downregulation of bcl-2 which increases bax:bcl-2 ratio. The regulation of genes involved in IFN-STAT-pathways and apoptosis may be the first evidences to explain the increased antiproliferative properties of this co-formulation.

Silent celiac disease in a cohort of healthy adults.

BACKGROUND: Recent studies suggest that celiac disease (CD) is common in many developing countries. Because the disease may be under diagnosed in Cuba, we studied the presence of the disease in a group of apparently healthy adult. AIMS/HYPOTHESIS: It was to assess for the first time, the presence of silent CD in a cohort of healthy Cuban adults individuals and to evaluate the tools for diagnosis of CD in this group. METHODS: A total of 200 healthy Cuban adult from Havana City were evaluated. Tissue transglutaminase antibodies (tTGA) were determined by one-step immunochromatographic assay and by commercial ELISA kit. CD specific human leukocyte antigen (HLA) typing was performed by polymerase chain reaction amplification, using sequence-specific primers. In the subject positive for tTGA, the CD was confirmed by intestinal biopsy. RESULTS: From the 200 studied individuals, only one subject was identified as positive by both assays, being submitted to duodenal biopsy. Morphological changes consistent with CD were found and also supported by HLA-DQ2 (HLA-DQA1*0501-DQB1*02). In the follow-up after one year, histological recovery was assessed by a second intestinal biopsy and the serological marker became negative. CONCLUSIONS: This study confirms the existence of silent CD among healthy adult in Cuba and highlights the importance of mass screening for this disease among them. The one-step immunochromatographic assay is a good tool for this purpose.

Diagnosis of dengue virus infection by the visual and simple AuBioDOT immunoglobulin M capture system.

The Dengue IgM Capture ELISA (MAC-ELISA) is the immunoenzymatic system recommended by the Pan American Health Organization and the World Health Organization for the serological diagnosis of dengue virus infection due to its high sensitivity, ease of performance, and use of a single acute-phase serum sample. However, tests with this enzyme-linked immunosorbent assay (ELISA) system are time-consuming and require equipment for washing, incubation, and reading of the results. AuBioDOT is a multistep visual diagnostic immunoassay that uses technology based on the immunoglobulin M (IgM) capture ELISA principle. This system uses white polyethylene opaque plates as the solid phase, colloidal gold as the marker, and silver ion amplification. It does not require special equipment, it is totally manually operated, and it can be performed in less than 1 h. The sensitivity and specificity of AuBioDOT for the detection of anti-dengue virus IgM antibodies were studied with a panel of 336 serum samples (150 serum samples from patients with suspected or serologically confirmed dengue virus infection, 186 serum samples from healthy blood donors and patients without dengue virus infection). The results were compared with those obtained by the MAC-ELISA. A sensitivity of 97.7% and a specificity of 97.1% were obtained. The concordance of the two tests was 97.3%, with a kappa index of 0.94. The application of AuBioDOT for the detection of anti-dengue virus IgM antibodies is recommended as an alternative method for the diagnosis of dengue virus infection, both for clinical diagnosis and for seroepidemiological surveillance. The system is useful under field conditions and in laboratories and requires little equipment.

Expression of a single-chain Fv antibody fragment specific for the hepatitis B surface antigen in transgenic tobacco plants.

An anti-Hepatitis B virus surface antigen (HBsAg) single chain Fv (scFv) antibody fragment was expressed in Nicotiana tabacum transgenic plants. The 6-histidine tagged scFv was targeted to either the cytosol, apoplast, and vacuole, or for retention in the endoplasmic reticulum. Expression of active scFv was detected by ELISA in fresh leaf material from F I transgenic plant lines representative of the genetic constructs targeting the antibody fragment to the apoplastic fluid (AF-12, 0.031% of the total soluble protein), vacuole (V-20, 0.032% of the total soluble protein), and endoplasmic reticulum (ER-52, 0.22% of the total soluble protein). No scFv was detected by ELISA or western blot in the plants transformed with the cytosol construct. The biologically active scFv was easily purified (to 94-95% purity) from ER-52 and AF-12 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the ER-52 plant line indicates that 15-20 microg of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale.

Purificación del antígeno recombinante del core de la hepatitis B y su uso potencial en el diagnóstico de la infección con el virus de la hepatitis B / Purification of the recombinant hepatitis B core antigen and its potential use for the diagnosis of hepatitis B virus infection

El gen del HBcAg del VHB fue clonado en el vector de expresión pRIV-2 y usado para la síntesis en E. coli de la proteína de 22 kD. La proteína expresada fue capaz de ensamblarse en forma de partícula con un nivel de expresión del 7(por ciento) con respecto al total de proteínas de E. coli presentes en una SDS-PAGE del 15 (por ciento). El HBcAg fue purificado hasta un 90 (por ciento) utilizando una combinación de dos pasos de precipitación con sulfato de amonio y una cromatrografía de filtración en gel de sepharose CL-4B. El recobrado total del método propuesto fue del 47(por ciento). El uso en el diagnóstico de la infección por el VHB fue evaluado por ELISA mediante un panel de sueros positivos y negativos. Los valores obtenidos de 99,3(por ciento) de especificidad y 97,9(por ciento) de sensibilidad demostrarón el uso potencial del HBcAg purificado en el serodiagnóstico de la infección con el VHB(AU)