Variations in Expression of Markers by Populations of Adherent Cells from Trypanosoma cruzi-Infected Mice1

The kinetics of differentiation and maturation of phagocytic cells during the acute and chronic stages of experimental Chagas' disease was examined by monitoring changes in expression of peroxidase (PO), nonspecific esterase (NSE), C3b receptors (CR), Fc receptors (FcR), and phagocytic ability of cells in the blood, spleen, and peritoneal cavity. The significant changes recorded in the blood were: marked increases in the percentages of CR- and FcR-positive adherent cells during both the acute and chronic phase; Ia-positive cells increased two-fold in the acute period and remained elevated in the chronic stage. In the spleen, the major alterations recorded during both the acute and chronic stages were: two- to three-fold increases in the percentages of NSE- and PO-positive adherent cells and three- to four-fold increases in the proportions of CR- and FcR-positive cells. In addition, Ia-positive cells increased from 70% to approximately 90% of the adherent cell population. In the peritoneal cavity, a two- to four-fold elevation in the percentages of both PO- and NSE-positive cells was observed. The number of Ia-positive cells increased from 10% before infection to 85–90% during the acute phase and to 96–98% during the chronic period. All of the changes described above occurred in the absence of noticeable increases in phagocytic ability except for an elevation in the percentage of circulating latex-ingesting cells seen during chronicity. These results indicate that infection with Trypanosoma cruzi alters the pathways of differentiation of cells of the mononuclear phagocyte lineage.     

MicroRNA Profiling of Pericardial Fluid Samples from Patients with Heart Failure

Aims

Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate biomarkers due to their relative stability and easy quantification from clinical samples. Pericardial fluid contains hormones secreted by the heart and is known to reflect the cardiac function. In this study, we sought to investigate whether pericardial fluid contains microRNAs and if so, whether they could be used to distinguish between different cardiovascular pathologies and disease stages.

Methods and Results

Pericardial fluid was collected from heart failure patients during open-heart surgery. MicroRNA profiles of altogether 51 patients were measured by quantitative real-time PCR (qPCR) using Exiqon human panels I and II. On the average, 256 microRNAs were detected per sample, and 70 microRNAs out of 742 profiled microRNAs were detected in every sample. The five most abundant microRNAs in pericardial fluid were miR-21-5p, miR-451a, miR-125b-5p, let-7b-5p and miR-16-5p. No specific signatures for cardiovascular pathologies or clinically assessed heart failure stages could be detected from the profiles and, overall, microRNA profiles of the samples were found to be very similar despite the heterogeneity in the study population.

Conclusion

Measured microRNA profiles did not separate the samples according to the clinical features of the patients. However, several previously identified heart failure marker microRNAs were detected. The pericardial fluid microRNA profile appeared to be a result of an active and selective secretory process indicating that microRNAs may act as paracrine signalling factors by mediating the local crosstalk between cardiac cells.     

Frequent House Invasion of Trypanosoma cruzi-Infected Triatomines in a Suburban Area of Brazil

Background

The demographic transition of populations from rural areas to large urban centers often results in a disordered occupation of forest remnants and increased economic pressure to develop high-income buildings in these areas. Ecological and socioeconomic factors associated with these urban transitions create conditions for the potential transmission of infectious diseases, which was demonstrated for Chagas disease.

Methodology/Principal Findings

We analyzed 930 triatomines, mainly Triatoma tibiamaculata, collected in artificial and sylvatic environments (forests near houses) of a suburban area of the city of Salvador, Bahia State, Brazil between 2007 and 2011. Most triatomines were captured at peridomiciles. Adult bugs predominated in all studied environments, and nymphs were scarce inside houses. Molecular analyses of a randomly selected sub-sample (n=212) of triatomines showed Trypanosoma cruzi infection rates of 65%, 50% and 56% in intradomestic, peridomestic and sylvatic environments, respectively. We detected the T. cruzi lineages I and II and mixed infections. We also showed that T. tibiamaculata fed on blood from birds (50%), marsupials (38%), ruminants (7%) and rodents (5%). The probability of T. cruzi infection was higher in triatomines that fed on marsupial blood (odds ratio (OR) = 1.95, 95% confidence interval (CI) = 1.22-3.11). Moreover, we observed a protective effect against infection in bugs that fed on bird blood (OR = 0.43, 95% CI = 0.30-0.73).

Conclusions/Significance

The frequent invasion of houses by infected triatomines indicates a potential risk of T. cruzi transmission to inhabitants in this area. Our results reinforce that continuous epidemiological surveillance should be performed in areas where domestic transmission is controlled but enzootic transmission persists.     

Parasitological and immunological aspects of Trypanosoma cruzi infection in nude rats

The role of the thymus on immunity of rats against Trypanosoma cruzi infection was investigated in vivo. The athymic (nu/nu) rats were shown to be significantly more susceptible to the acute phase of the infection than the control nu/+ rats, as measured by increased parasitemia and mortality. Specific anti-T. cruzi antibodies, complement, IgM and IgG2a serum levels were determined. The results would indicate the essential role of antibodies in immunity to acute Chagas' disease through T-dependent immune response.     

Biomarkers in Trypanosoma cruzi-Infected and Uninfected Individuals with Varying Severity of Cardiomyopathy in Santa Cruz,Bolivia

Background

Twenty to thirty percent of persons with Trypanosoma cruzi infection eventually develop cardiomyopathy. If an early indicator were to be identified and validated in longitudinal studies, this could enable treatment to be prioritized for those at highest risk. We evaluated cardiac and extracellular matrix remodeling markers across cardiac stages in T. cruzi infected (Tc+) and uninfected (Tc−) individuals.

Methods

Participants were recruited in a public hospital in Santa Cruz, Bolivia and assigned cardiac severity stages by electrocardiogram and echocardiogram. BNP, NTproBNP, CKMB, troponin I, MMP-2, MMP-9, TIMP-1, TIMP-2, TGFb1, and TGFb2 were measured in specimens from 265 individuals using multiplex bead systems. Biomarker levels were compared between Tc+ and Tc− groups, and across cardiac stages. Receivers operating characteristic (ROC) curves were created; for markers with area under curve>0.60, logistic regression was performed.

Results

Analyses stratified by cardiac stage showed no significant differences in biomarker levels by Tc infection status. Among Tc+ individuals, those with cardiac insufficiency had higher levels of BNP, NTproBNP, troponin I, MMP-2, TIMP-1, and TIMP-2 than those with normal ejection fraction and left ventricular diameter. No individual marker distinguished between the two earliest Tc+ stages, but in ROC-based analyses, MMP-2/MMP-9 ratio was significantly higher in those with than those without ECG abnormalities.

Conclusions

BNP, NTproBNP, troponin I, MMP-2, TIMP-1, and TIMP-2 levels rose with increasing severity stage but did not distinguish between Chagas cardiomyopathy and other cardiomyopathies. Among Tc+ individuals without cardiac insufficiency, only the MMP-2/MMP-9 ratio differed between those with and without ECG changes.     

小鼠肝脏MicroRNA的提取与分离

本文对小鼠肝脏microRNA进行分离制备及相关性功能研究.采用poly(A)加尾方法,分离提取小鼠肝脏中的microRNA;利用T4 RNA连接酶,在microRNA两端加上连接引物,通过RT PCR制备microRNA的cDNA文库;经过对cDNA文库的质粒连接,克隆测序获得microRNA. 结果显示：获得4条有效序列,其中包括2条已知序列miR 122和2条未知小分子RNA序列.经查证, 2条microRNA片段在miRBase库中未有记录,在二级结构分析中可形成茎环结构,符合microRNA的特征. BLAST比对显示： 2个序列位于小鼠载脂蛋白B基因和小鼠28S核糖体RNA上,可能与基因调控有关,其功能有待进一步研究.     

MicroRNA Expression and Virulence in Pandemic Influenza Virus-Infected Mice

The worst known H1N1 influenza pandemic in history resulted in more than 20 million deaths in 1918 and 1919. Although the underlying mechanism causing the extreme virulence of the 1918 influenza virus is still obscure, our previous functional genomics analyses revealed a correlation between the lethality of the reconstructed 1918 influenza virus (r1918) in mice and a unique gene expression pattern associated with severe immune responses in the lungs. Lately, microRNAs have emerged as a class of crucial regulators for gene expression. To determine whether differential expression of cellular microRNAs plays a role in the host response to r1918 infection, we compared the lung cellular “microRNAome” of mice infected by r1918 virus with that of mice infected by a nonlethal seasonal influenza virus, A/Texas/36/91. We found that a group of microRNAs, including miR-200a and miR-223, were differentially expressed in response to influenza virus infection and that r1918 and A/Texas/36/91 infection induced distinct microRNA expression profiles. Moreover, we observed significant enrichment in the number of predicted cellular target mRNAs whose expression was inversely correlated with the expression of these microRNAs. Intriguingly, gene ontology analysis revealed that many of these mRNAs play roles in immune response and cell death pathways, which are known to be associated with the extreme virulence of r1918. This is the first demonstration that cellular gene expression patterns in influenza virus-infected mice may be attributed in part to microRNA regulation and that such regulation may be a contributing factor to the extreme virulence of the r1918.H1N1 influenza A viruses continue to pose serious threats to public health, as exemplified by the ongoing 2009 H1N1 influenza pandemic. The 1918-1919 H1N1 influenza pandemic was even deadlier in comparison, causing more than 20 million deaths worldwide. The keys to unlocking the mystery of the extreme virulence of the 1918 virus were provided with the reconstruction of the virus (reconstructed 1918 influenza virus [r1918]) by reverse genetics (37). The lethality of r1918 has since been examined in both mouse and macaque models (17, 18). Unlike the nonlethal infections of some other H1N1 influenza virus strains, such as A/Texas/36/91 (Tx/91) or A/Kawasaki/173/01 (K173), the r1918 causes severe and lethal pulmonary disease. We subsequently conducted functional genomics analyses that revealed that the extreme virulence of r1918 was correlated with atypical expression of immune response-related genes, including massive induction of cellular genes related to inflammatory response and cell death pathways (17, 18). In spite of these findings, the mechanistic basis for these atypical gene expression patterns remains unknown.Cellular gene expression is a complicated process and is subject to regulation by many cellular factors. As a group of newly identified cellular regulators, microRNAs are known to regulate the expression of a large number of targets, mainly cellular genes. Through mRNA degradation or translational repression of their targets, microRNAs regulate a wide range of crucial physiologic and pathological processes. For example, miR-34a acts as a tumor suppressor by inhibiting the expression of sirt1 (40), whereas miR-21 contributes to myocardial disease by inhibiting the expression of spry1 (36). By targeting zeb1/2, the miR-200 family members play roles in maintaining the epithelial phenotype of cancer cells (27). Furthermore, Let-7s regulates the expression of hbl-1, which drives the developmental progression of epidermal stem cells (5). Cellular microRNAs also play critical roles in virus-host interactions. The cellular microRNA miR-122 is an indispensable factor in supporting hepatitis C virus (HCV) replication (16), whereas miR-196 and miR-296 substantially attenuate viral replication through type I interferon (IFN)-associated pathways in liver cells (28). Furthermore, miR-125b and miR-223 directly target human immunodeficiency virus type 1 (HIV-1) mRNA, thereby attenuating viral gene expression in resting CD4⁺ T cells (14), and miR-198 modulates HIV-1 replication indirectly by repressing the expression of ccnt1 (34), a cellular factor necessary for HIV-1 replication. More importantly, viruses may promote their life cycles by modulating the intracellular environment through actively regulating the expression of multiple cellular microRNAs. For example, human T-cell lymphotropic virus type 1 (HTLV-1) modulates the expression of a number of cellular microRNAs in order to control T-cell differentiation (3). Similarly, human cytomegalovirus (HCMV) selectively manipulates the expression of miR-100 and miR-101 to facilitate its own replication (38). In contrast, the involvement of microRNAs during influenza A virus infection or pathogenesis is largely unknown.To determine whether cellular microRNAs play a role in the host response to influenza virus infection, we performed a systematic profiling of cellular microRNAs in lung tissues from mice infected with r1918 or a nonlethal seasonal influenza virus, Tx/91 (17). We identified a group of microRNAs whose expression patterns differentiated the host response to r1918 and Tx/91 infection. We assessed the potential functions of differentially expressed microRNAs by analyzing the predicted target genes whose expression was inversely correlated with the expression of these microRNAs. Our report provides a new perspective on the contribution of microRNAs to the pathogenesis of lethal 1918 influenza virus infection.     

MicroRNA Expression Profiling in Mild Asthmatic Human Airways and Effect of Corticosteroid Therapy

Background

Asthma is a common disease characterised by reversible airflow obstruction, bronchial hyperresponsiveness and chronic inflammation, which is commonly treated using corticosteroids such as budesonide. MicroRNAs (miRNAs) are a recently identified family of non-protein encoding genes that regulate protein translation by a mechanism entitled RNA interference. Previous studies have shown lung-specific miRNA expression profiles, although their importance in regulating gene expression is unresolved. We determined whether miRNA expression was differentially expressed in mild asthma and the effect of corticosteroid treatment.

Methodology/Principal Findings

We have examined changes in miRNA using a highly sensitive RT-PCR based approach to measure the expression of 227 miRNAs in airway biopsies obtained from normal and mild asthmatic patients. We have also determined whether the anti-inflammatory action of corticosteroids are mediated through miRNAs by determining the profile of miRNA expression in mild asthmatics, before and following 1 month twice daily treatment with inhaled budesonide. Furthermore, we have analysed the expression of miRNAs from individual cell populations from the airway and lung.We found no significant difference in the expression of 227 miRNAs in the airway biopsies obtained from normal and mild asthmatic patients. In addition, despite improved lung function, we found no significant difference in the miRNA expression following one month treatment with the corticosteroid, budesonide. However, analysis of bronchial and alveolar epithelial cells, airway smooth muscle cells, alveolar macrophages and lung fibroblasts demonstrate a miRNA expression profile that is specific to individual cell types and demonstrates the complex cellular heterogeneity within whole tissue samples.

Conclusions

Changes in miRNA expression do not appear to be involved in the development of a mild asthmatic phenotype or in the anti-inflammatory action of the corticosteroid budesonide.