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Try out PMC Labs and tell us what you think. Learn More. Performed the experiments: CS. Analyzed the data: CS SV. Human filarial infection is characterized by downregulated parasite-antigen specific T cell responses but distinct differences exist between patients with longstanding infection endemics and those who acquired infection through temporary residency or visits to filarial-endemic regions expatriates. Functional analysis showed a differential increase in genes associated with NFkB both groups and caspase activation endemics. Infection with the filarial parasite Loa loa causes a parasite-specific downregulation of T cell responses.

However, differences exist clinical and immunologic between patients born and living in filarial endemic regions endemics and those who become infected during travel or short-term residency expatriates. In this study, we showed that these differences reflect transcriptional differences within the T cell compartment. Our study describes potential transcriptional mechanisms for the variability seen in patients with different levels of exposure to and chronicity of filarial infection. Infection with the pathogenic filariae, Loa loaBrugia malayiWuchereria bancroftiand Onchocerca volvuluscauses an enormous disease burden throughout tropical and sub-tropical regions of the world.

Interestingly, however, the clinical manifestations of infection are often markedly different in those with lifelong exposure i. Indeed, filarial infections are less likely to be subclinical in expatriates [3] or transmigrants [4] compared to those with lifelong exposure.

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With availability of more sensitive assays for the definitive diagnosis of filarial infections, it is now known that infection occurs at much earlier ages than once believed [6]although relatively intense exposure to the vectors that transmit these infections is typically required for acquisition of infection.

However, expatriates who acquire infection are not subject to many of the environmental and familial factors that affect those born in endemic regions, the most notable being the alteration of immune responses specific for filarial antigens that occurs early in life [7][8][9]and that can persist long-term decades [10] as a consequence of in utero exposure to filarial antigens. Moreover, polyparasitism is much more frequent among patients from filarial-endemic regions than in expatriates. That individuals living in an endemic area are exposed continually to the parasite, irrespective of the infection status, is evidenced by the Ag-specific antibody responses seen among filarial-uninfected endemic individuals [11][12].

Indeed, both susceptibility to infection and the nature of the immune response has a ificant genetic component in helminth- and filarial-endemic populations [13][14][15][16]. Several studies have also demonstrated differences in immune responses to filarial antigens among filarial-infected travelers expatriates and those from filarial-endemic regions [1][2]. Filarial-infected individuals from endemic countries, while having increased antifilarial IgG4 antibodies [17]have more profoundly diminished parasite-specific T cell responses [12][18] than those seen in expatriates [1].

This parasite-specific hyporesponsiveness is reflected not only in diminished proliferative and cytokine responses [12][18][19]but also in the increased expression of molecules e. In addition, filarial Ags and live filarial parasites have themselves been shown to induce proliferative defects [22]apoptosis of T cells [23]and impairment of antigen presenting cell and function [24][25][26]that cumulatively may alter T cell responses.

A of studies have directly examined specific or candidate pathways in the cells of filarial-infected [24][25] individuals. Our findings demonstrate a striking difference in gene expression between endemic and expatriate patients with the same filarial infection and demonstrate that these differences manifest not only in T cells ex vivo but also in response to both parasite and nonparasite Ag.

Three Loa loa -infected patients who had lived most or all of their lives in a region endemic for loiasis and 3 expatriate L. All patients were examined at the NIH, and all had demonstrable microfilariae in their circulation at midday. None of the patients tested positive for HIV. One expatriate patient but no others had intestinal parasites as well. PBMCs from all patients were collected prior to any treatment, cryopreserved using standard techniques and stored in liquid nitrogen until used.

Wells with media alone were run for each antigen Free phone sex in Ke Loa for each cell type i. Following a 16 hr. After selection, cells were immediately homogenized in 1 ml Trizol Invitrogen followed by phase separation with chloroform. Technical replicates were done for each sample. The probe set for the microarrays was based on 70 mer oligonucleotides from the Human Genome Oligo Set V2.

Each array contained 21, oligonucleotides. Intensity values less than were given a threshold offollowed by conversion to the log base 2; each sample was then centered about the median. By this analysis, the negative log of the p-value for a given gene increases with increasing statistical ificance of the gene i. However, since the p-value does not give an indication of the direction of fold change, the negative log of the p-value was multiplied by the of the fold change between endemic and expatriate samples for a given gene.

Functional groups of genes whose ranks were ificantly lower or higher than would be expected by chance were derived in GSEA; a group of genes with common function and high ranking was evidence that the function was up-or-down-regulated in one patient group over the other. The Canonical Pathways gene sets version 2. Genes were divided into several clusters according to consistent positive or negative regulation across the samples and the Fisher Exact test was used to calculate the ificance of the of genes belonging to a particular Canonical Pathway.

A small p-value denotes that for a given pathway, the of genes belonging to that pathway within a cluster was greater than would be expected by chance. For Mf- and SLO-stimulated samples, genes that were ificantly either over- or under-expressed in comparison with paired unstimulated samples were analyzed; the ratio of stimulated to unstimulated expression was Free phone sex in Ke Loa calculated as the fold change for a particular gene. Functional gene sets that were enriched overall with small p-values were detected by means of GSEA using the Canonical Pathways gene sets similar to that done for unstimulated data.

All samples were run in triplicate and normalized to their own 18S ribosomal RNA. To establish if there was a correlation between the expression of a given gene using RT-PCR and the expression determined by microarray, the 3 patient values for each gene within each group were averaged for both techniques and then plotted against each other. To determine differences between s of genes expressed between the two patient groups, a statistical test for one proportion was done using the Normal approximation.

For each category, the of genes ased to that category in the two classes endemic and expatriate was compared to each other. Assuming a null proportion of 0. Genes not ificantly different are symbolized by black dots with each dot denoting a single gene. The x-axis is the fold difference log 2 between patient groups and the y-axis represents the log10 of the p-value. Each point illustrates the average value of the 3 individuals within each patient group for qRT-PCR x-axis vs microarray y-axis for the same sample. The Spearman Rank Correlation was used to calculate r- and p-values.

Data associated with these plots are shown in Table S1. When the genes expressed differentially between patient groups were analyzed further, the differences between endemic and expatriate patients could be inferred through functional assessments of these gene sets Figure 3. Annotation for cell functions was extracted from www. Both groups had a large of differentially expressed genes related to cell death Figure 4. As can be seen in this representative network, the endemic patients upregulated many of the genes involved in caspase activation whereas the expatriates were more likely to have relatively upregulated activation of the NFkB complex, including MAPK8, part of the p38 MAPK activation complex.

Network of differentially expressed molecules associated with cell death www. Lines represent direct solid lines and indirect dashed lines relationships between molecules and molecules within double circles represent complexes of genes. Hierarchical clustering and Gene Set Enrichment Analysis GSEA was utilized to determine whether patient groups could be distinguished by their canonical pathway profiles. As can be seen, the response to either Ag in general was different between the two patient groups.

Venn diagrams representing the of genes either upregulated upward facing arrows or downregulated downward facing arrows to parasite microfilarial Ag; MfAg, top panels and nonparasite streptolysin O; SLO, bottom panels antigens in endemic pink circles and expatriate blue circles filarial-infected patients. To establish an overall profile of genes upregulated in loiasis patients in response to MfAg, composite networks were identified representative networks shown in Figure 6. Figure 6A Free phone sex in Ke Loa that the majority of genes with altered expression from that seen in unstimulated cells were those associated with the NFkB both patient groups and Caspase endemic group complexes.

A second composite network Figure 6B consisted chiefly of those genes upregulated in the T cells from endemic patients.

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Two major networks Figures A and B; www. Lines represent direct solid lines and indirect dashed lines relationships between molecules, and molecules within double circles represent Free phone sex in Ke Loa of genes. As indicated in Figure 5because of the differences in s of genes with increased expression to MfAg, the contribution by expatriates to the overall composite profile was far less than that by the endemic patients.

The most clear-cut contribution by expatriates to the overall response Figure 6A was an upregulation of genes associated with the calcium-binding protein calmodulin. To establish the profile of downregulated genes in response to MfAg in loiasis patients, composite networks were again created representative network shown in Figure 7.

Major network www. Genes that were ificantly up- or down-regulated to MfAg with respect to unstimulated cells were also examined by GSEA. In contrast to what was seen in GSEA analysis for endemic patient cells, expatriate patients' cells in response to MfAg had enriched pathways associated with the innate and adaptive inflammatory response Figure S4. Of note, the response to SLO in both groups consisted of increased expression of several chemokine ligands including CCL20, CCL4 and CCL8interferon regulatory factors, genes associated with inflammatory cytokines, and importantly, the aling molecule STAT1, necessary for the production of interferons data not shown.

It has been felt that these disparities may reflect differences in immunologic responsiveness to the parasite in these patients. work has demonstrated that cells of filarial-infected endemic patients have markedly diminished parasite-specific T cell responses when compared to filarial-infected expatriate patients and even to uninfected endemic individuals [1][12]. In a study of transmigrants to an O. Similar findings have been seen in patients with acute or subacute schistosomiasis infection who had higher parasite-specific proliferative responses than did those with longstanding, chronic infection [31].

In addition, filarial-infected patients from endemic regions of the world cured of filarial infection following treatment continue to show diminished T cell responses to filarial antigens [32] while expatriate patients cured of infection and not re-exposed recover many of their Ag-specific T cell responses [33]. A major finding in this study was the importance of the inflammatory and cell death networks in the ex vivo unstimulated cells of filarial-infected patients in both patient groups though individual genes within these networks segregated by patient group.

For example, within cell death networks the expatriates were more likely to express genes associated with activation induced cell death whereas the endemic patients expressed genes associated with apoptosis. That increased cellular activation, cell death, and inhibition of cell death is occurring at a steady-state, suggests that under conditions of long-term Ag stimulation, a balance between pro- and anti-apoptotic transcriptional events e.

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Indeed, the finding that chronic filarial infection is associated with increased s of memory but decreased s of effector T cells [36] may support the idea of activation and T cell survival being tightly regulated through pro- and anti-apoptotic mechanisms. Moreover, the interference of IRF2 and MAF with the IFNs strongly suggests an anti-inflammatory role for these molecules that, at the very least, impairs Th1 differentiation in chronically infected patients. Taken together, the increased expression of all of these molecules in endemic patients may reflect the lack of clinical symptoms [1][2] and parasite-specific in vitro T cell responses [1][12] frequently observed in chronically infected patients with filarial infections and suggest possible mechanisms for the regulation of inflammatory activity.

To examine the larger relationships between genes that were differentially expressed by either patient group GSEA was used. Two highly enriched pathways in endemic patients were associated with T cell-B cell interactions, the ASBCell Pathway involved in Ag dependent B cell activation and the BBCell pathway involved in the induction of apoptosis in Fas-expressing inactive B cellssuggesting a possible role for T cells in the Ag-induced activation and cell death of B cells in filarial infection.

The enhancement of these latter pathways, might serve to explain the augmented pathology associated with infection in expatriates with Loa infection [5][45] seen to a much lesser degree in those with long-term infection.

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Indeed, although the of altered genes in response to MfAg was far fewer in expatriates in comparison to endemic patients, those genes that were upregulated were closely tied to pathways of the innate and adaptive inflammatory response i. This increase in inflammatory pathways was further supported by the upregulation of molecules associated with NFKB activation and calmodulin-mediated responses Figure 6 and data not shown as well as by a downregulation of the apoptotic DNA fragment pathway Figure S4.

In endemic patients with long-term infection, the response to MfAg was difficult to synthesize fully.

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Compared with expatriates, where the recall response to MfAg was clearly of an inflammatory nature, the T cell response to MfAg by endemic patients appeared to be balanced between activation and regulation of the immune response. This balance was perhaps most clearly seen in the transcriptional regulation of molecules associated with cell death and apoptosis, similar to the findings in unstimulated cells. Numerous other examples of the opposing nature of certain functions associated with differentially regulated genes in endemic patients were also in evidence.

Of particular interest is the interaction of CCR5 with its ligand CCL5 which induces the release of histamine from basophils and activates eosinophils, two common features associated with filarial infection [50]. Several other multi-functional complexes upregulated in the parasite Ag-driven cells of endemic patients may offer clues to mechanisms underlying the depressed T cell responses typically seen in these patients.

Ubiquitin and its associated molecules Figure 6B function in a wide variety of cellular processes including Ag presentation and apoptosis and, through a post-translational modification, mark proteins for degradation. Recently, GRAIL the E3-ubiquitin ligase gene related to anergy in lymphocytes was shown to be responsible for the Th2 hyporesponsiveness in a mouse model of chronic schistosomiasis [51]a finding suggested by data from human filarial infections [12][20][52]. In addition, several genes linked to the histone family of molecules were also upregulated, including the gene for histone h3.

This molecule, through an ERK dependent mechanism, allows the binding of transcription factors to the IL promoter and subsequent expression of the IL gene, which, as mentioned ly, is a prominent regulatory molecule associated with chronic filarial infection [53][54]. The present study thus demonstrates that the clinical and immunological differences ly observed between endemic and expatriate patients can be demonstrated at the transcriptional level in unstimulated ex vivo cells, during early recall responses to parasite Ag, and even to nonparasite Ag.

Indeed, the transcriptional differences between the two groups Free phone sex in Ke Loa filarial-infected patients reflect many of the differences that are seen between acute and chronic viral infection [65]. While the microarray findings in this study by no means constitute the final analysis of the differences between patients with long-standing infection and those with more recently acquired infection, they do suggest several mechanisms that warrant further investigation.

It must be argued, therefore, that chronicity and possibly in utero or neonatal exposure to filarial antigens helps define the disparities between these two groups of patients. Further characterization of the differences seen between long-term and newly acquired infection could help to define the natural progression of filarial infection and the responses that underlie this progression. Genes in brown represent those upregulated in one patient group with respect to the other while genes in blue are downregulated.

The heat map represents the values of differentially expressed genes in each of the 3 patients to MfAg on the right and to the corresponding media values on the left. Genes in brown represent those upregulated to MfAg with respect to media while genes in blue are downregulated. The data correspond to graphs illustrated in Figure 2 and represent the average values for the three individuals within each patient group. Timothy G.

Myers and Qin Su. The authors have declared that no competing interests exist. The funders had no role in study de, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology InformationU. Published online Feb Nutman 1. Thomas B. Thomas R. Unnasch, Editor.

Author information Article notes Copyright and information Disclaimer. Received Sep 13; Accepted Jan 1. Copyright This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which Free phone sex in Ke Loa that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

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