scholarly journals Transcriptional heterogeneity ofCryptococcus gattiiVGII compared with non-VGII lineages underpins key pathogenicity pathways

2018 ◽  
Author(s):  
Rhys A. Farrer ◽  
Christopher B. Ford ◽  
Johanna Rhodes ◽  
Toni Delorey ◽  
Robin C. May ◽  
...  
Keyword(s):  
Stress Response ◽  
Differential Expression ◽  
Ex Vivo ◽  
Early Time ◽  
Regulatory Proteins ◽  
Differentially Expressed ◽  
Transcriptional Profiles ◽  
Over Expression ◽  
Time Points ◽  
Mouse Macrophages

AbstractCryptococcus gattiiis a pathogenic yeast of humans and other animals, which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual RNA-seq experiment for four lineages ofC. gattii(VGI-IV) interacting with mouse macrophages at 1hr, 3hr and 6hr post infection. Comparison ofin vitrotoex vivogene expression indicates lineage VGII is transcriptionally divergent to non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment and ergosterol production. Various paralogs demonstrate sub-functionalisation between lineages including an upregulation of capsule biosynthesis-related geneCAP2, and downregulation ofCAP1in VGIII. Isolates also compensate for lineage-specific gene-losses by over-expression of genetically similar paralogs, including an over-expression of capsule geneCAS3in VGIV having lostCAS31. Differential expression of one in fiveC. gattiigenes was detected following co-incubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and a downregulation of capsule attachment genes. We also show that VGII switches expression of two laccase paralogs (fromLAC1toLAC2) during co-incubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages ofC. gattiiby upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This study highlights the evolutionary breadth of expression profiles amongst the lineages ofC. gattiiand the diversity of transcriptional responses at this host-pathogen interface.ImportanceThe transcriptional profiles of related pathogens and their response to host induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages ofC. gattiiin rich media. Our analyses identified key processes including cell capsule, ergosterol production and melanin that are differentially expressed between lineages, and we find that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone sub-functionalisation for various paralogs including capsule biosynthesis and attachment genes. Most genes appeared down-regulated during co-incubation with macrophages, with the largest decrease observed for capsule attachment genes, which appears coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched expression of the laccaseLAC1toLAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow derived macrophages, marking a role in the host response toC. gattii. This work highlights the dynamic roles of keyC. gattiivirulence genes in response to macrophages.

scholarly journals Transcriptional Heterogeneity ofCryptococcus gattiiVGII Compared with Non-VGII Lineages Underpins Key Pathogenicity Pathways

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Rhys A. Farrer ◽  
Christopher B. Ford ◽  
Johanna Rhodes ◽  
Toni Delorey ◽  
Robin C. May ◽  
...  
Keyword(s):  
Stress Response ◽  
Differential Expression ◽  
Ex Vivo ◽  
Early Time ◽  
Regulatory Proteins ◽  
Differentially Expressed ◽  
Content Type ◽  
Transcriptional Profiles ◽  
Time Points ◽  
Mouse Macrophages

ABSTRACTCryptococcus gattiiis a pathogenic yeast of humans and other animals which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual-transcriptome sequencing (RNA-seq) experiment for four lineages ofC. gattii(lineages VGI to IV) interacting with mouse macrophages at 1, 3, and 6 h postinfection. Comparisons ofin vitrotoex vivogene expression levels indicated that lineage VGII is transcriptionally divergent from non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment, and ergosterol production. Several paralogous genes demonstrated subfunctionalization between lineages, including upregulation of capsule biosynthesis-related geneCAP2and downregulation ofCAP1in VGIII. Isolates also compensate for lineage-specific gene losses by overexpression of genetically similar paralogs, including overexpression of capsule geneCAS3in VGIV, which have lost theCAS31gene. Differential expression of one in fiveC. gattiigenes was detected following coincubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and downregulation of capsule attachment genes. We also found that VGII switches expression of two laccase paralogs (fromLAC1toLAC2) during coincubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages ofC. gattiiby upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This report highlights the evolutionary breadth of expression profiles among the lineages ofC. gattiiand the diversity of transcriptional responses at this host-pathogen interface.IMPORTANCEThe transcriptional profiles of related pathogens and their responses to host-induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages ofC. gattiiin rich media. Our analyses identified key processes, including those involving cell capsule, ergosterol production, and melanin, that are differentially expressed between lineages, and we found that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone subfunctionalization for several paralogs, including capsule biosynthesis and attachment genes. Most genes appeared downregulated during coincubation with macrophages, with the largest decrease observed for capsule attachment genes, which appeared to be coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched from expression of the laccaseLAC1to expression ofLAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow-derived macrophages, marking a role in the host response toC. gattii. This work highlights the dynamic roles of keyC. gattiivirulence genes in response to macrophages.

scholarly journals Leishmania infection induces a limited differential gene expression in the sand fly midgut

2020 ◽  
Author(s):  
Iliano V. Coutinho-Abreu ◽  
Tiago D. Serafim ◽  
Claudio Meneses ◽  
Shaden Kamhawi ◽  
Fabiano Oliveira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Stages ◽  
De Novo ◽  
Early Time ◽  
Differentially Expressed ◽  
Sand Fly ◽  
Leishmania Infection ◽  
Lutzomyia Longipalpis ◽  
Late Time ◽  
Time Points

Abstract Background: Phlebotomine sand flies are the vectors of Leishmania worldwide. To develop in the sand fly midgut, Leishmania multiplies and undergoes multiple stage differentiations leading to the infective form, the metacyclic promastigotes. To gain a better understanding of the influence of Leishmania infection on midgut gene expression, we performed RNA-Seq comparing uninfected and Leishmania infantum -infected Lutzomyia longipalpis midguts at seven time points which cover the various Leishmania developmental stages including early time points when blood digestion is taking place, and late time points when the parasites are undergoing metacyclogenesis. Results: Out of over 13,841 transcripts assembled de novo , only 113 sand fly transcripts, about 1%, were differentially expressed. Further, we observed a low overlap of differentially expressed sand fly transcripts across different time points suggesting that each Leishmania stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> -32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 4 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by small fold changes (< 32 fold). The molecular function of these genes has been associated with the metabolism of lipids and detoxification of xenobiotics. Conclusion: Overall, it appears that Leishmania modulates sand fly gene expression early on in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points, where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

scholarly journals Leishmania infection induces a limited differential gene expression in the sand fly midgut

2019 ◽  
Author(s):  
Iliano V. Coutinho-Abreu ◽  
Tiago D. Serafim ◽  
Claudio Meneses ◽  
Shaden Kamhawi ◽  
Fabiano Oliveira ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Early Time ◽  
Differentially Expressed ◽  
Sand Fly ◽  
Leishmania Infection ◽  
Lutzomyia Longipalpis ◽  
Late Time ◽  
Blood Digestion ◽  
Time Points

Abstract Background: Phlebotomine sand flies are the vectors of Leishmania worldwide. To develop in the sand fly midgut, Leishmania multiplies and undergoes multiple stage differentiations leading to the infective form, the metacyclic promastigotes. To gain a better understanding of the influence of Leishmania infection in midgut gene expression, we performed RNA-Seq comparing uninfected Lutzomyia longipalpis midguts and Leishmania infantum-infected Lutzomyia longipalpis midguts at seven time points which cover the various developmental Leishmania stages including early time points when blood digestion is taking place and late time points when the parasites are undergoing metacyclogenesis. Results: Out of over 13,841 transcripts assembled de novo, only 113 sand fly transcripts, about 1%, were differentially expressed. Further, we observed a low overlap of differentially expressed sand fly transcripts across different time points suggesting a specific influence of each Leishmania stage on midgut gene expression. Two main patterns of sand fly gene expression modulation were noticed. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> -32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 4 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by small fold changes (< 32 fold), and the molecular function of such genes are associated with the metabolism of lipids and detoxification of xenobiotics (P450). Conclusion: Overall, it appears that Leishmania modulates sand fly gene expression early on in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points, when modest midgut gene expression changes correlate with a massive amount of parasites in the anterior midgut.

scholarly journals Leishmania infection induces a limited differential gene expression in the sand fly midgut

2020 ◽  
Author(s):  
Iliano V. Coutinho-Abreu ◽  
Tiago D. Serafim ◽  
Claudio Meneses ◽  
Shaden Kamhawi ◽  
Fabiano Oliveira ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Early Time ◽  
Differentially Expressed ◽  
Sand Fly ◽  
Leishmania Infection ◽  
Developmental Cycle ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Time Points

Abstract Background: Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages. Results: The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics. Conclusion: Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

scholarly journals Leishmania infection induces a limited differential gene expression in the sand fly midgut

2019 ◽  
Author(s):  
Iliano V. Coutinho-Abreu ◽  
Tiago D. Serafim ◽  
Claudio Meneses ◽  
Shaden Kamhawi ◽  
Fabiano Oliveira ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Early Time ◽  
Differentially Expressed ◽  
Sand Fly ◽  
Leishmania Infection ◽  
Lutzomyia Longipalpis ◽  
Late Time ◽  
Blood Digestion ◽  
Time Points

AbstractBackgroundPhlebotomine sand flies are the vectors of Leishmania worldwide. To develop in the sand fly midgut, Leishmania multiplies and undergoes multiple stage differentiations leading to the infective form, the metacyclic promastigotes. To gain a better understanding of the influence of Leishmania infection in midgut gene expression, we performed RNA-Seq comparing uninfected Lutzomyia longipalpis midguts and Leishmania infantum-infected Lutzomyia longipalpis midguts at seven time points which cover the various developmental Leishmania stages including early time points when blood digestion is taking place and late time points when the parasites are undergoing metacyclogenesis.ResultsOut of over 13,841 transcripts assembled de novo, only 113 sand fly transcripts, about 1%, were differentially expressed. Further, we observed a low overlap of differentially expressed sand fly transcripts across different time points suggesting a specific influence of each Leishmania stage on midgut gene expression. Two main patterns of sand fly gene expression modulation were noticed. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> −32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 4 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by small fold changes (< 32 fold), and the molecular function of such genes are associated with the metabolism of lipids and detoxification of xenobiotics (P450).ConclusionOverall, it appears that Leishmania modulates sand fly gene expression early on in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points, when modest midgut gene expression changes correlate with a massive amount of parasites in the anterior midgut.

scholarly journals Transcriptomic dissection reveals wide spread differential expression in chickpea during early time points of Fusarium oxysporum f. sp. ciceri Race 1 attack

PLoS ONE ◽  
2017 ◽  
Vol 12 (5) ◽  
pp. e0178164 ◽  
Author(s):  
Sumanti Gupta ◽  
Anirban Bhar ◽  
Moniya Chatterjee ◽  
Amartya Ghosh ◽  
Sampa Das
Keyword(s):  
Fusarium Oxysporum ◽  
Differential Expression ◽  
Early Time ◽  
Wide Spread ◽  
Time Points ◽  
Race 1

scholarly journals Leishmania infection induces a limited differential gene expression in the sand fly midgut

2020 ◽  
Author(s):  
Iliano V. Coutinho-Abreu ◽  
Tiago D. Serafim ◽  
Claudio Meneses ◽  
Shaden Kamhawi ◽  
Fabiano Oliveira ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Early Time ◽  
Differentially Expressed ◽  
Sand Fly ◽  
Leishmania Infection ◽  
Developmental Cycle ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Time Points

Abstract Background: Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages. Results: The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics. Conclusion: Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

Activation Status of Residual Host Dendritic Cells (DCs) but Not Their Number Governs Donor Lymphocyte Infusion (DLI)-Mediated Responses after Major Histocompatibility Complex (MHC)-Matched AlloBMT.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3043-3043
Author(s):  
Nadira Durakovic ◽  
Karl B. Bezak ◽  
Ephraim J. Fuchs ◽  
Luznik Leo
Keyword(s):  
T Cells ◽  
Differential Expression ◽  
Ex Vivo ◽  
Mesenteric Lymph Nodes ◽  
Time Points ◽  
Post Transplant ◽  
Donor Chimerism ◽  
Activation Status

Abstract The role of host antigen presenting cells (APCs), particularly DCs in governing DLI-mediated alloresponses post MHC-matched alloBMT is unclear. To evaluate whether the amount of residual host-derived APCs determines the alloresponse of DLI we constructed MHC-matched B10.D2 (H2d)→BALB/c (H2d) chimeras with varying degrees of residual host APCs. After 8 weeks of rest, chimeras received DLI in the form of 2 x 107 splenocytes from B10.D2 (Thy1.1+) mice that differ from BM donors by the expression of Thy 1.1+ marker on the lymphocytes. The differential expression of the Thy 1.1+ marker on DLI-derived T cells allowed us to precisely monitor their alloresponses in vivo. We found that in the MHC-matched model of alloBMT, contrary to the MHC-mismatched model, the amount of residual host APCs does not influence DLI-mediated T-cell alloresponses measured as the alloantigen-driven expansion of DLI-derived T cells and changes in donor chimerism. However, the early administration of DLI, 4 weeks after the conditioning resulted in statistically significant expansion of DLI-derived CD8+ and CD4+ T cells (p&lt;0.01). Since these data suggest the importance of timing of DLI administration on the DLI-mediated alloresponses in MHC-matched setting, we next evaluated whether the augmented alloresponses may be reflective of kinetics of donor DC reconstitution, changes in DC populations or their qualitative characteristics at different time points post-transplant. To test these variables we used C3H.SW (H-2Kb, CD45.2+)→B6Ly5.2 (H-2Kb, CD45.1+) MHC-matched model that enabled us to dissociate the origin of DCs at different time points post-transplant based on the differential expression of CD45 marker on all host and donor hematopoietic tissues. We found that host to donor DC turnover is rapid, results in near full conversion to donor DC chimerism in the spleen (&gt;99 %) and mesenteric lymph nodes (LN) (&gt;99%), but not in the subcutaneous LNs ( 90%). Comprehensive phenotypic analysis showed that donor-derived DC populations in the spleen and LNs do not differ in the chimeras early or late after conditioning, however, residual host-derived DCs express significantly higher levels of costimulatory molecules, CD80, CD40 and especially CD86 at early time points after conditioning (3 weeks vs 8 weeks). These finding suggest that residual host DC activation status may be the critical factor influencing the potency of DLI-mediated responses. To further investigate the role of DC activation status, we co-administered DLI with ex vivo-matured DCs or systemically activated them with agonistic anti-CD40 antibody or CpG immunostimulatory oligonucleotides that stimulate Toll-like receptor (TLR)-9. While both approaches augmented the in vivo proliferation of DLI-derived T cells, only CpGs augmented effector function of DLI-derived T cells, particularly the production of interferon-γ (p&lt;0.005) and conversion to full donor chimerism. Additionally, in the post-transplant C1498 leukemia relapse model survival of C3H.SW→C57BL/6 chimeras that received DLI and CpG immunostimulatory molecules was superior to the chimeras that received DLI only or DLI + ex vivo-matured host DCs (10/10 vs 2/10 vs 2/10; p&lt;0.01). These data indicate that DLI-mediated alloresponses in the setting of MHC-matched alloBMT are critically influenced by the activation status of residual host DCs and can be further manipulated by in vivo administering TLR-9 ligands but not by administering ex vivo-matured host DCs.

scholarly journals Differential MicroRNA Analyses of Burkholderia pseudomallei- and Francisella tularensis-Exposed hPBMCs Reveal Potential Biomarkers

2017 ◽  
Vol 2017 ◽  
pp. 1-13
Author(s):  
Regina Z. Cer ◽  
J. Enrique Herrera-Galeano ◽  
Kenneth G. Frey ◽  
Kevin L. Schully ◽  
Truong V. Luu ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Burkholderia Pseudomallei ◽  
Bacterial Infections ◽  
Mononuclear Cells ◽  
Early Time ◽  
Differentially Expressed ◽  
Infectious Agents ◽  
Time Points ◽  
Potential Biomarker ◽  
Mirna Species

Increasing evidence that microRNAs (miRNAs) play important roles in the immune response against infectious agents suggests that miRNA might be exploitable as signatures of exposure to specific infectious agents. In order to identify potential early miRNA biomarkers of bacterial infections, human peripheral blood mononuclear cells (hPBMCs) were exposed to two select agents, Burkholderia pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the nonpathogenic control Escherichia coli DH5α. RNA samples were harvested at three early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq analyses identified 87 miRNAs to be differentially expressed (DE) in a linear fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay. Through RNAseq identification and qPCR validation, we identified differentially expressed miRNA species that may be involved in the early response to bacterial infections. Based upon its upregulation at early time points postexposure in two different individuals, hsa-mir-30c-5p is a miRNA species that could be studied further as a potential biomarker for exposure to these gram-negative intracellular pathogens. Gene ontology functional analyses demonstrated that programmed cell death is the first ranking biological process associated with miRNAs that are upregulated in F. tularensis-exposed hPBMCs.

scholarly journals Suite of clinically relevant functional assays to address therapeutic efficacy and disease mechanism in the dystrophic mdx mouse

2017 ◽  
Vol 122 (3) ◽  
pp. 593-602 ◽  
Author(s):  
Yafeng Song ◽  
Shira T. Rosenblum ◽  
Leon Morales ◽  
Mihail Petrov ◽  
Christopher Greer ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Ex Vivo ◽  
Early Time ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
End Points ◽  
Time Points ◽  
Muscular Exertion ◽  
Physiological Tests ◽  
And Control

Duchenne muscular dystrophy (DMD) is a progressive primary myodegenerative disease caused by a genetic deficiency of the 427-kDa cytoskeletal protein dystrophin. Despite its single-gene etiology, DMD’s complex pathogenesis remains poorly understood, complicating the extrapolation from results of preclinical studies in genetic homologs to the design of informative clinical trials. Here we describe novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. By coupling force transduction, high-precision motion tracking, and respiratory measurements, we have achieved a suite of integrative physiological tests that provide novel insights regarding normal and pathological responses to muscular exertion. A common feature of these physiological assays is the precise tracking and analysis of volitional movement, thereby optimizing the relevance to clinical tests. Unexpectedly, the measurable biological distinction between dystrophic and control mice at early time points in the disease process is better resolved with these tests than with the majority of previously used, labor-intensive studies of individual muscle function performed ex vivo. For example, the dramatic loss of volitional movement following a novel, standardized grip test distinguishes control mice from mdx mice by a 17.4-fold difference of the means (3.5 ± 2.2 vs. 60.9 ± 12.1 units of activity, respectively; effect size 1.99). The findings have both mechanistic and translational implications of potential significance to the fields of basic myology and neuromuscular therapeutics. NEW & NOTEWORTHY This study uses novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. A measurable distinction between dystrophic and control mice was seen at early time points in vivo compared with invasive muscle studies performed ex vivo. These assays shed light on normal and pathological responses to muscular exertion and have significant mechanistic and translational implications for the fields of basic myology and neuromuscular therapeutics.

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