Ana L. Gomes

Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR) protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA). The efficiency was 0.99 and the correlation coefficient (R(2)) was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83 degrees C). The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden) in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

Gene Expression Profiling during Early Acute Febrile Stage of Dengue Infection Can Predict the Disease Outcome

Background We report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data, validated by quantitative PCR and tested in independent samples. Methodology/Principal Findings The study was performed on patients of a well-characterized dengue cohort from Recife, Brazil. The samples analyzed were collected prospectively from acute febrile dengue patients who evolved with different degrees of disease severity: classic dengue fever or dengue hemorrhagic fever (DHF) samples were compared with similar samples from other non-dengue febrile illnesses. The DHF samples were collected 2–3 days before the presentation of the plasma leakage symptoms. Differentially-expressed genes were selected by univariate statistical tests as well as multivariate classification techniques. The results showed that at early stages of dengue infection, the genes involved in effector mechanisms of innate immune response presented a weaker activation on patients who later developed hemorrhagic fever, whereas the genes involved in apoptosis were expressed in higher levels. Conclusions/Significance Some of the gene expression signatures displayed estimated accuracy rates of more than 95%, indicating that expression profiling with these signatures may provide a useful means of DHF prognosis at early stages of infection.

Molecular approaches for the detection of Schistosoma mansoni: possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection

The detection of specific DNA sequences by polymerase chain reaction (PCR) has proved extremely valuable for the analysis of genetic disorders and the diagnosis of a variety of infectious disease pathogens. However, the application to the detection of Schistosoma mansoni is rare, despite a recommendation of the World Health Organization that a major focus of research on schistosomiasis should be on the development and evaluation of new strategies and tools for control of the disease. In this context, a few studies were published for the detection of the parasite in snails, monitoring of cercariae in water bodies, and diagnosis of human infection. The present minireview describes sensitive and specific PCR based systems to detect S. mansoni, indicating possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection.

Classification of Dengue Fever Patients Based on Gene Expression Data Using Support Vector Machines

Background Symptomatic infection by dengue virus (DENV) can range from dengue fever (DF) to dengue haemorrhagic fever (DHF), however, the determinants of DF or DHF progression are not completely understood. It is hypothesised that host innate immune response factors are involved in modulating the disease outcome and the expression levels of genes involved in this response could be used as early prognostic markers for disease severity. Methodology/Principal Findings mRNA expression levels of genes involved in DENV innate immune responses were measured using quantitative real time PCR (qPCR). Here, we present a novel application of the support vector machines (SVM) algorithm to analyze the expression pattern of 12 genes in peripheral blood mononuclear cells (PBMCs) of 28 dengue patients (13 DHF and 15 DF) during acute viral infection. The SVM model was trained using gene expression data of these genes and achieved the highest accuracy of ∼85% with leave-one-out cross-validation. Through selective removal of gene expression data from the SVM model, we have identified seven genes (MYD88, TLR7, TLR3, MDA5, IRF3, IFN-α and CLEC5A) that may be central in differentiating DF patients from DHF, with MYD88 and TLR7 observed to be the most important. Though the individual removal of expression data of five other genes had no impact on the overall accuracy, a significant combined role was observed when the SVM model of the two main genes (MYD88 and TLR7) was re-trained to include the five genes, increasing the overall accuracy to ∼96%. Conclusions/Significance Here, we present a novel use of the SVM algorithm to classify DF and DHF patients, as well as to elucidate the significance of the various genes involved. It was observed that seven genes are critical in classifying DF and DHF patients: TLR3, MDA5, IRF3, IFN-α, CLEC5A, and the two most important MYD88 and TLR7. While these preliminary results are promising, further experimental investigation is necessary to validate their specific roles in dengue disease.

Complement factor H gene (CFH) polymorphisms C-257T, G257A and haplotypes are associated with protection against severe dengue phenotype, possible related with high CFH expression

Four genetic polymorphisms located at the promoter (C-257T) and coding regions of CFH gene (exon 2 G257A, exon 14 A2089G and exon 19 G2881T) were investigated in 121 dengue patients (DENV-3) in order to assess the relationship between allele/haplotypes variants and clinical outcomes. A statistical value was found between the CFH-257T allele (TT/TC genotypes) and reduced susceptibility to severe dengue (SD). Statistical associations indicate that individuals bearing a T allele presented significantly higher protein levels in plasma. The -257T variant is located within a NF-κB binding site, suggesting that this variant might have effect on the ability of the CFH gene to respond to signals via the NF-κB pathway. The G257A allelic variant showed significant protection against severe dengue. When CFH haplotypes effect was considered, the ancestral CG/CG promoter-exon 2 SNP genotype showed significant risk to SD either in a general comparison (ancestral × all variant genotypes), as well as in individual genotypes comparison (ancestral × each variant genotype), where the most prevalent effect was observed in the CG/CG × CA/TG comparison. These findings support the involvement of -257T, 257A allele variants and haplotypes on severe dengue phenotype protection, related with high basal CFH expression.

Gemcitabine-Induced TIMP1 Attenuates Therapy Response and Promotes Tumor Growth and Liver Metastasis in Pancreatic Cancer

Gemcitabine constitutes one of the backbones for chemotherapy treatment in pancreatic ductal adenocarcinoma (PDAC), but patients often respond poorly to this agent. Molecular markers downstream of gemcitabine treatment in preclinical models may provide an insight into resistance mechanisms. Using cytokine arrays, we identified potential secretory biomarkers of gemcitabine resistance (response) in the transgenic KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) mouse model of PDAC. We verified the oncogenic role of the cytokine tissue inhibitor of matrix metalloproteinases 1 (TIMP1) in primary pancreatic tumors and metastases using both in vitro techniques and animal models. We identified potential pathways affected downstream of TIMP1 using the Illumina Human H12 array. Our findings were validated in both primary and metastatic models of pancreatic cancer. Gemcitabine increased inflammatory cytokines including TIMP1 in the KPC mouse model. TIMP1 was upregulated in patients with pancreatic intraepithelial neoplasias grade 3 and PDAC lesions relative to matched normal pancreatic tissue. In addition, TIMP1 played a role in tumor clonogenic survival and vascular density, while TIMP1 inhibition resensitized tumors to gemcitabine and radiotherapy. We observed a linear relationship between TIMP-1 expression, liver metastatic burden, and infiltration by CD11b+Gr1+ myeloid cells and CD4+CD25+FOXP3+ Tregs, whereas the presence of tumor cells was required for immune cell infiltration. Overall, our results identify TIMP1 upregulation as a resistance mechanism to gemcitabine and provide a rationale for combining chemo/radiotherapy with TIMP1 inhibitors in PDAC. Cancer Res; 77(21); 5952-62. ©2017 AACR.

An efficient and robust MRI-guided radiotherapy planning approach for targeting abdominal organs and tumours in the mouse

Introduction Preclinical CT-guided radiotherapy platforms are increasingly used but the CT images are characterized by poor soft tissue contrast. The aim of this study was to develop a robust and accurate method of MRI-guided radiotherapy (MR-IGRT) delivery to abdominal targets in the mouse. Methods A multimodality cradle was developed for providing subject immobilisation and its performance was evaluated. Whilst CT was still used for dose calculations, target identification was based on MRI. Each step of the radiotherapy planning procedure was validated initially in vitro using BANG gel dosimeters. Subsequently, MR-IGRT of normal adrenal glands with a size-matched collimated beam was performed. Additionally, the SK-N-SH neuroblastoma xenograft model and the transgenic KPC model of pancreatic ductal adenocarcinoma were used to demonstrate the applicability of our methods for the accurate delivery of radiation to CT-invisible abdominal tumours. Results The BANG gel phantoms demonstrated a targeting efficiency error of 0.56 ± 0.18 mm. The in vivo stability tests of body motion during MR-IGRT and the associated cradle transfer showed that the residual body movements are within this MR-IGRT targeting error. Accurate MR-IGRT of the normal adrenal glands with a size-matched collimated beam was confirmed by γH2AX staining. Regression in tumour volume was observed almost immediately post MR-IGRT in the neuroblastoma model, further demonstrating accuracy of x-ray delivery. Finally, MR-IGRT in the KPC model facilitated precise contouring and comparison of different treatment plans and radiotherapy dose distributions not only to the intra-abdominal tumour but also to the organs at risk. Conclusion This is, to our knowledge, the first study to demonstrate preclinical MR-IGRT in intra-abdominal organs. The proposed MR-IGRT method presents a state-of-the-art solution to enabling robust, accurate and efficient targeting of extracranial organs in the mouse and can operate with a sufficiently high throughput to allow fractionated treatments to be given.

An MRI-Compatible High Frequency AC Resistive Heating System for Homeothermic Maintenance in Small Animals

Purpose To develop an MRI-compatible resistive heater, using high frequency alternating current (AC), for temperature maintenance of anaesthetised animals. Materials and Methods An MRI-compatible resistive electrical heater was formed from narrow gauge wire connected to a high frequency (10–100 kHz) AC power source. Multiple gradient echo images covering a range of echo times, and pulse-acquire spectra were acquired with the wire heater powered using high frequency AC or DC power sources and without any current flowing in order to assess the sensitivity of the MRI acquisitions to the presence of current flow through the heater wire. The efficacy of temperature maintenance using the AC heater was assessed by measuring rectal temperature immediately following induction of general anaesthesia for a period of 30 minutes in three different mice. Results Images and spectra acquired in the presence and absence of 50–100 kHz AC through the wire heater were indistinguishable, whereas DC power created field shifts and lineshape distortions. Temperature lost during induction of anaesthesia was recovered within approximately 20 minutes and a stable temperature was reached as the mouse’s temperature approached the set target. Conclusion The AC-powered wire heater maintains adequate heat input to the animal to maintain body temperature, and does not compromise image quality.