Gene-expression profiling of laser-dissected islets and studies in deficient mice reveal chemokines as differential driving force of type 1 diabetes.

Although type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing ß-cells, its treatment is largely restricted to exogenous insulin administration. Only few therapies targeting the autoaggressive immune system have been introduced into clinical practice or are considered in clinical trials. Here, we provide a gene expression profile of the islet microenvironment obtained by laser-dissection microscopy in an inducible mouse model. Thereby, we have identified novel targets for immune intervention. Increased gene expression of most inflammatory proteins was apparent at day 10 after T1D induction and largely paralleled the observed degree of insulitis. We further focused on genes involved in leukocyte migration, including chemokines and their receptors. Besides the critical chemokine CXCL10, we found several other chemokines upregulated locally in temporary or chronic manner. Localization of the chemokine ligand/receptor pairs to the islet microenvironment has been confirmed by RNAscope. Interference with the CXCL16-CXCR6 and CX3CL1-CX3CR1 axes, but not the CCL5-CCR1/3/5 axis, resulted in reduced insulitis and lower T1D incidence. Further, we found that the receptors for the differentially expressed chemokines CXCL10, CXCL16 and CX3CL1 are distributed unevenly among islet autoantigen-specific T cells, which explains why the interference with just one chemokine axis cannot completely abrogate insulitis and T1D.

[Interferon-related gene array in predicting the efficacy of interferon therapy in chronic hepatitis B].

This study aims to clarify host factors of IFN treatment in the treatment of chronic hepatitis B (CHB) patients by screening the differentially expressed genes of IFN pathway CHB patients with different response to interferon (IFN) therapy. Three cases were randomly selected in IFN-responding CHB patients (Rs), non-responding CHB patients (NRs) and healthy participants, respectively. The human type I IFN response RT 2 profiler PCR array was used to detect the expression levels of IFN-related genes in peripheral blood monocytes (PBMCs) from healthy participants and CHB patients before and after Peg-IFN-α 2a treatment. The results showed that more differentially expressed genes appeared in Rs group than NRs group after IFN treatment. Comparing with healthy participants, IFNG, IL7R, IRF1, and IRF8 were downregulated in both Rs and NRs group before IFN treatment; CXCL10, IFIT1, and IFITM1 were upregulated in the Rs; IL13RA1 and IFI35 were upregulated in the NRs, while IFRD2, IL11RA, IL4R, IRF3, IRF4, PYHIN1, and ADAR were downregulated. The expression of IL15, IFI35 and IFI44 was downregulated by 4.09 ( t = 10.58, P < 0.001), 5.59 ( t = 3.37, P = 0.028) and 10.83 ( t = 2.8, P = 0.049) fold in the Rs group compared with the NRs group, respectively. In conclusion, IFN-response-related gene array is able to evaluate IFN treatment response by detecting IFN-related genes levels in PBMC. High expression of CXCL10, IFIT1 and IFITM1 before treatment may suggest satisfied IFN efficacy, while high expression of IL13RA1, IL15, IFI35 and IFI44 molecules and low expression of IFRD2, IL11RA, IL4R, IRF3, IRF4, PYHIN1 and ADAR molecules may be associated with poor IFN efficacy.

Cold acclimation diversity in Arabidopsis thaliana: CRISPR/Cas9 as a tool to fine analysis of Tandem Gene Arrays, application to CBF genes.

In this period of climate change, it is of major importance to increase knowledge about the mechanisms by whose plants adapt to their environment. Tandem gene arrays (TAG) are overrepresented in the pool of tandem duplicates involved in stress response and are consequently of special interest. Nevertheless, until recently, addressing questions about individual genes or fine regulations in such structures was very difficult due to the close arrangement of the genome, almost preventing the production of targeted simple or multiple mutants. The CRISPR/Cas9 new tool offers new opportunities as the setting of gene deletion strategies in various genetic backgrounds. Here, we used this technology on the cold acclimation CBF pathway in different accessions of Arabidopsis thaliana with the same set of guide RNAs. Deleted lines free of T-DNA have been produced for simple or multiple copies of CBF genes and evaluated for cold tolerance after acclimation. Expression levels of CBF genes and five COR genes have also been analyzed. Our data suggest first that two or three missing CBF genes are necessary to induce a strong reduction in cold tolerance and secondly that most deletions have a low impact on the expression of remaining CBF copies which contradicts the previous hypothesis in the literature. Our results thus show that the CRISPR/Cas9 deletion strategy is a useful performance tool to investigate how tandem gene arrays work.

The molecular phenotypes of injury, steatohepatitis, and fibrosis in liver transplant biopsies in the INTERLIVER study.

To extend previous molecular analyses of rejection in liver transplant biopsies in the INTERLIVER study (ClinicalTrials.gov #NCT03193151), the present study aimed to define the gene expression selective for parenchymal injury, fibrosis, and steatohepatitis. We analyzed genome-wide microarray measurements from 337 liver transplant biopsies from 13 centers. We examined expression of genes previously annotated as increased in injury and fibrosis using principal component analysis (PCA). PC1 reflected parenchymal injury and related inflammation in the early posttransplant period, slowly regressing over many months. PC2 separated early injury from late fibrosis. Positive PC3 identified a distinct mildly inflamed state correlating with histologic steatohepatitis. Injury PCs correlated with liver function and histologic abnormalities. A classifier trained on histologic steatohepatitis predicted histologic steatohepatitis with cross-validated AUC = 0.83, and was associated with pathways reflecting metabolic abnormalities distinct from fibrosis. PC2 predicted histologic fibrosis (AUC = 0.80), as did a molecular fibrosis classifier (AUC = 0.74). The fibrosis classifier correlated with matrix remodeling pathways with minimal overlap with those selective for steatohepatitis, although some biopsies had both. Genome-wide assessment of liver transplant biopsies can not only detect molecular changes induced by rejection but also those correlating with parenchymal injury, steatohepatitis, and fibrosis, offering potential insights into disease mechanisms for primary diseases.

Molecular diagnosis of ABMR with or without donor-specific antibody in kidney transplant biopsies: Differences in timing and intensity but similar mechanisms and outcomes.

We studied the clinical, histologic, and molecular features distinguishing DSA-negative from DSA-positive molecularly defined antibody-mediated rejection (mABMR). We analyzed mABMR biopsies with available DSA assessments from the INTERCOMEX study: 148 DSA-negative versus 248 DSA-positive, compared with 864 no rejection (excluding TCMR and Mixed). DSA-positivity varied with mABMR stage: early-stage (EABMR) 56%; fully developed (FABMR) 70%; and late-stage (LABMR) 58%. DSA-negative patients with mABMR were usually sensitized, 60% being HLA antibody-positive. Compared with DSA-positive mABMR, DSA-negative mABMR was more often C4d-negative; earlier by 1.5 years (average 2.4 vs. 3.9 years); and had lower ABMR activity and earlier stage in molecular and histology features. However, the top ABMR-associated transcripts were identical in DSA-negative versus DSA-positive mABMR, for example, NK-associated (e.g., KLRD1 and GZMB) and IFNG-inducible (e.g., PLA1A). Genome-wide class comparison between DSA-negative and DSA-positive mABMR showed no significant differences in transcript expression except those related to lower intensity and earlier time of DSA-negative ABMR. Three-year graft loss in DSA-negative mABMR was the same as DSA-positive mABMR, even after adjusting for ABMR stage. Thus, compared with DSA-positive mABMR, DSA-negative mABMR is on average earlier, less active, and more often C4d-negative but has similar graft loss, and genome-wide analysis suggests that it involves the same mechanisms. SUMMARY SENTENCE: In 398 kidney transplant biopsies with molecular antibody-mediated rejection, the 150 DSA-negative cases are earlier, less intense, and mostly C4d-negative, but use identical molecular mechanisms and have the same risk of graft loss as the 248 DSA-positive cases.

Evolving the surveillance and workup of heart transplant rejection: A real-world analysis of the Molecular Microscope Diagnostic System.

The Molecular Microscope Diagnostic System (MMDx) analyzes RNA transcripts of transplanted heart tissue to differentiate among T cell-mediated rejection (TCMR), antibody-mediated rejection (AMR), injury, and healthy tissue. However, little is known about its performance in relation to other modalities in a real-world heart transplant population. We evaluated whether MMDx performs in agreement with other validated modalities. Two hundred and twenty-eight corresponding endomyocardial biopsies (EMBx) and MMDx specimens from 135 adult heart transplant patients were retrospectively reviewed with correlating donor-derived cell-free DNA (dd-cfDNA). Rejection was classified on EMBx in 29 specimens (TCMR ≥ 2R and/or AMR ≥ 1), on MMDx in 56 specimens, and in 74 values with dd-cfDNA ≥0.20%. Despite moderate agreement between EMBx and MMDx (84% agreement, Cohen's kappa, 0.48, p < .001), systematic differences were observed (McNemar's test, p < .001) where MMDx classified 32 of 37 discordant cases as rejection. MMDx and dd-cfDNA demonstrated slight agreement (72% agreement, Cohen's kappa, 0.39, p < .001); however, systematic differences were also apparent where MMDx classified 12 of 50 discordant specimens as rejection when dd-cfDNA was <0.20% (McNemar's test, p < .001). Our findings provide insight on the performance of MMDx relative to other modalities in a heart transplant cohort and have implications on the surveillance and workup of allograft rejection in heart transplantation.

Utilizing Two Borrelia bavariensis Isolates Naturally Lacking the PFam54 Gene Array To Elucidate the Roles of PFam54-Encoded Proteins.

Lyme borreliosis is the most common vector-borne disease in the Northern Hemisphere, caused by spirochetes belonging to the Borrelia burgdorferi sensu lato species complex, which are transmitted by ixodid ticks. B. burgdorferi sensu lato species produce a family of proteins on the linear plasmid 54 (PFam54), some of which confer the functions of cell adhesion and inactivation of complement, the first line of host defense. However, the impact of PFam54 in promoting B. burgdorferi sensu lato pathogenesis remains unclear because of the hurdles to simultaneously knock out all PFam54 proteins in a spirochete. Here, we describe two Borrelia bavariensis strains, PBN and PNi, isolated from patients naturally lacking PFam54 but maintaining the rest of the genome with greater than 95% identity to the reference B. bavariensis strain, PBi. We found that PBN and PNi less efficiently survive in human serum than PBi. Such defects were restored by introducing two B. bavariensis PFam54 recombinant proteins, BGA66 and BGA71, confirming the role of these proteins in providing complement evasion of B. bavariensis. Further, we found that all three strains remain detectable in various murine tissues 21 days post-subcutaneous infection, supporting the nonessential role of B. bavariensis PFam54 in promoting spirochete persistence. This study identified and utilized isolates deficient in PFam54 to associate the defects with the absence of these proteins, building the foundation to further study the role of each PFam54 protein in contributing to B. burgdorferi sensu lato pathogenesis. IMPORTANCE To establish infections, Lyme borreliae utilize various means to overcome the host's immune system. Proteins encoded by the PFam54 gene array play a role in spirochete survival in vitro and in vivo. Moreover, this gene array has been described in all currently available Lyme borreliae genomes. By investigating the first two Borrelia bavariensis isolates naturally lacking the entire PFam54 gene array, we showed that both patient isolates display an increased susceptibility to human serum, which can be rescued in the presence of two PFam54 recombinant proteins. However, both isolates remain infectious to mice after intradermal inoculation, suggesting the nonessential role of PFam54 during the long-term, but may differ slightly in the colonization of specific tissues. Furthermore, these isolates show high genomic similarity to type strain PBi (>95%) and could be used in future studies investigating the role of each PFam54 protein in Lyme borreliosis pathogenesis.

Excitotoxic stimulation activates distinct pathogenic and protective expression signatures in the hippocampus.

Excitotoxic events underlying ischaemic and traumatic brain injuries activate degenerative and protective pathways, particularly in the hippocampus. To understand opposing pathways that determine the brain's response to excitotoxicity, we used hippocampal explants, thereby eliminating systemic variables during a precise protocol of excitatory stimulation. N-methyl-d-aspartate (NMDA) was applied for 20 min and total RNA isolated one and 24 h later for neurobiology-specific microarrays. Distinct groups of genes exhibited early vs. delayed induction, with 63 genes exclusively reduced 24-h post-insult. Egr-1 and NOR-1 displayed biphasic transcriptional modulation: early induction followed by delayed suppression. Opposing events of NMDA-induced genes linked to pathogenesis and cell survival constituted the early expression signature. Delayed degenerative indicators (up-regulated pathogenic genes, down-regulated pro-survival genes) and opposing compensatory responses (down-regulated pathogenic genes, up-regulated pro-survival genes) generated networks with temporal gene profiles mirroring coexpression network clustering. We then used the expression profiles to test whether NF-κB, a potent transcription factor implicated in both degenerative and protective pathways, is involved in the opposing responses. The NF-κB inhibitor MG-132 indeed altered NMDA-mediated transcriptional changes, revealing components of opposing expression signatures that converge on the single response element. Overall, this study identified counteracting avenues among the distinct responses to excitotoxicity, thereby suggesting multi-target treatment strategies and implications for predictive medicine.

Donor genetic variants as risk factors for thrombosis after liver transplantation: A genome-wide association study.

Thrombosis after liver transplantation substantially impairs graft- and patient survival. Inevitably, heritable disorders of coagulation originating in the donor liver are transmitted by transplantation. We hypothesized that genetic variants in donor thrombophilia genes are associated with increased risk of posttransplant thrombosis. We genotyped 775 donors for adult recipients and 310 donors for pediatric recipients transplanted between 1993 and 2018. We determined the association between known donor thrombophilia gene variants and recipient posttransplant thrombosis. In addition, we performed a genome-wide association study (GWAS) and meta-analyzed 1085 liver transplantations. In our donor cohort, known thrombosis risk loci were not associated with posttransplant thrombosis, suggesting that it is unnecessary to exclude liver donors based on thrombosis-susceptible polymorphisms. By performing a meta-GWAS from children and adults, we identified 280 variants in 55 loci at suggestive genetic significance threshold. Downstream prioritization strategies identified biologically plausible candidate genes, among which were AK4 (rs11208611-T, p = 4.22 × 10-05 ) which encodes a protein that regulates cellular ATP levels and concurrent activation of AMPK and mTOR, and RGS5 (rs10917696-C, p = 2.62 × 10-05 ) which is involved in vascular development. We provide evidence that common genetic variants in the donor, but not previously known thrombophilia-related variants, are associated with increased risk of thrombosis after liver transplantation.

Discovering novel injury features in kidney transplant biopsies associated with TCMR and donor aging.

We previously characterized the molecular changes in acute kidney injury (AKI) and chronic kidney disease (CKD) in kidney transplant biopsies, but parenchymal changes selective for specific types of injury could be missed by such analyses. The present study searched for injury changes beyond AKI and CKD related to specific scenarios, including correlations with donor age. We defined injury using previously defined gene sets and classifiers and used principal component analysis to discover new injury dimensions. As expected, Dimension 1 distinguished normal vs. injury, and Dimension 2 separated early AKI from late CKD, correlating with time posttransplant. However, Dimension 3 was novel, distinguishing a set of genes related to epithelial polarity (e.g., PARD3) that were increased in early AKI and decreased in T cell-mediated rejection (TCMR) but not in antibody-mediated rejection. Dimension 3 was increased in kidneys from older donors and was particularly important in survival of early kidneys. Thus high Dimension 3 scores emerge as a previously unknown element in the kidney response-to-injury that affects epithelial polarity genes and is increased in AKI but depressed in TCMR, indicating that in addition to general injury elements, certain injury elements are selective for specific pathologic mechanisms. (ClinicalTrials.gov NCT01299168).

Factors associated with kidney graft survival in pure antibody-mediated rejection at the time of indication biopsy: Importance of parenchymal injury but not disease activity.

We studied the relative association of clinical, histologic, and molecular variables with risk of kidney transplant failure after an indication biopsy, both in all kidneys and in kidneys with pure antibody-mediated rejection (ABMR). From a prospective study of 1679 biopsies with histologic and molecular testing, we selected one random biopsy per patient (N = 1120), including 321 with pure molecular ABMR. Diagnoses were associated with actuarial survival differences but not good predictions. Therefore we concentrated on clinical (estimated GFR [eGFR], proteinuria, time posttransplant, donor-specific antibody [DSA]) and molecular and histologic features reflecting injury (acute kidney injury [AKI] and atrophy-fibrosis [chronic kidney disease (CKD)] and rejection. For all biopsies, univariate analysis found that failure was strongly associated with low eGFR, AKI, CKD, and glomerular deterioration, but not with rejection activity. In molecular ABMR, the findings were similar: Molecular and histologic activity and DSA were not important compared with injury. Survival in DSA-negative and DSA-positive molecular ABMR was similar. Multivariate survival analysis confirmed the dominance of molecular AKI, CKD, and eGFR. Thus, at indication biopsy, the dominant predictors of failure, both in all kidneys and in ABMR, were related to molecular AKI and CKD and to eGFR, not rejection activity, presumably because rejection confers risk via injury.

The Diversity of Nitrogen-Cycling Microbial Genes in a Waste Stabilization Pond Reveals Changes over Space and Time that Is Uncoupled to Changing Nitrogen Chemistry.

Nitrogen removal is an important process for wastewater ponds prior to effluent release. Bacteria and archaea can drive nitrogen removal if they possess the genes required to metabolize nitrogen. In the tropical savanna of northern Australia, we identified the previously unresolved microbial communities responsible for nitrogen cycling in a multi-pond wastewater stabilization system by measuring genomic DNA and cDNA for the following: nifH (nitrogen fixation); nosZ (denitrification); hzsA (anammox); archaeal AamoA and bacterial BamoA (ammonia oxidation); nxrB (nitrite oxidation); and nrfA (dissimilatory NO3 reduction to NH3). By collecting 160 DNA and 40 cDNA wastewater samples and measuring nitrogen (N)-cycling genes using a functional gene array, we found that genes from all steps of the N cycle were present and, except for nxrB, were also expressed. As expected, N-cycling communities showed daily, seasonal, and yearly shifts. However, contrary to our prediction, probes from most functional groups, excluding nosZ and AamoA, were different between ponds. Further, different genes that perform the same N-cycling role sometimes had different trends over space and time, resulting in only weak correlations between the different functional communities. Although N-cycling communities were correlated with wastewater nitrogen levels and physico-chemistry, the relationship was not strong enough to reliably predict the presence or diversity of N-cycling microbes. The complex and dynamic response of these genes to other functional groups and the changing physico-chemical environment provides insight into why altering wastewater pond conditions can result an abundance of some gene variants while others are lost.

TNMplot.com: A Web Tool for the Comparison of Gene Expression in Normal, Tumor and Metastatic Tissues.

Genes showing higher expression in either tumor or metastatic tissues can help in better understanding tumor formation and can serve as biomarkers of progression or as potential therapy targets. Our goal was to establish an integrated database using available transcriptome-level datasets and to create a web platform which enables the mining of this database by comparing normal, tumor and metastatic data across all genes in real time. We utilized data generated by either gene arrays from the Gene Expression Omnibus of the National Center for Biotechnology Information (NCBI-GEO) or RNA-seq from The Cancer Genome Atlas (TCGA), Therapeutically Applicable Research to Generate Effective Treatments (TARGET), and The Genotype-Tissue Expression (GTEx) repositories. The altered expression within different platforms was analyzed separately. Statistical significance was computed using Mann-Whitney or Kruskal-Wallis tests. False Discovery Rate (FDR) was computed using the Benjamini-Hochberg method. The entire database contains 56,938 samples, including 33,520 samples from 3180 gene chip-based studies (453 metastatic, 29,376 tumorous and 3691 normal samples), 11,010 samples from TCGA (394 metastatic, 9886 tumorous and 730 normal), 1193 samples from TARGET (1 metastatic, 1180 tumorous and 12 normal) and 11,215 normal samples from GTEx. The most consistently upregulated genes across multiple tumor types were TOP2A (FC = 7.8), SPP1 (FC = 7.0) and CENPA (FC = 6.03), and the most consistently downregulated gene was ADH1B (FC = 0.15). Validation of differential expression using equally sized training and test sets confirmed the reliability of the database in breast, colon, and lung cancer at an FDR below 10%. The online analysis platform enables unrestricted mining of the database and is accessible at TNMplot.com.

Transcriptomic Changes Associated with Loss of Cell Viability Induced by Oxysterol Treatment of a Retinal Photoreceptor-Derived Cell Line: An In Vitro Model of Smith-Lemli-Opitz Syndrome.

Smith-Lemli-Opitz Syndrome (SLOS) results from mutations in the gene encoding the enzyme DHCR7, which catalyzes conversion of 7-dehydrocholesterol (7DHC) to cholesterol (CHOL). Rats treated with a DHCR7 inhibitor serve as a SLOS animal model, and exhibit progressive photoreceptor-specific cell death, with accumulation of 7DHC and oxidized sterols. To understand the basis of this cell type specificity, we performed transcriptomic analyses on a photoreceptor-derived cell line (661W), treating cells with two 7DHC-derived oxysterols, which accumulate in tissues and bodily fluids of SLOS patients and in the rat SLOS model, as well as with CHOL (negative control), and evaluated differentially expressed genes (DEGs) for each treatment. Gene enrichment analysis and compilation of DEG sets indicated that endoplasmic reticulum stress, oxidative stress, DNA damage and repair, and autophagy were all highly up-regulated pathways in oxysterol-treated cells. Detailed analysis indicated that the two oxysterols exert their effects via different molecular mechanisms. Changes in expression of key genes in highlighted pathways (Hmox1, Ddit3, Trib3, and Herpud1) were validated by immunofluorescence confocal microscopy. The results extend our understanding of the pathobiology of retinal degeneration and SLOS, identifying potential new druggable targets for therapeutic intervention into these and other related orphan diseases.

Molecular phenotyping of rejection-related changes in mucosal biopsies from lung transplants.

Diagnosing lung transplant rejection currently depends on histologic assessment of transbronchial biopsies (TBB) with limited reproducibility and considerable risk of complications. Mucosal biopsies are safer but not histologically interpretable. Microarray-based diagnostic systems for TBBs and other transplants suggest such systems could assess mucosal biopsies as well. We studied 243 mucosal biopsies from the third bronchial bifurcation (3BMBs) collected from seven centers and classified them using unsupervised machine learning algorithms. Using the expression of a set of rejection-associated transcripts annotated in kidneys and validated in hearts and lung transplant TBBs, the algorithms identified and scored major rejection and injury-related phenotypes in 3BMBs without need for labeled training data. No rejection or injury, rejection, late inflammation, and recent injury phenotypes were thus scored in new 3BMBs. The rejection phenotype correlated with IFNG-inducible transcripts, the hallmarks of rejection. Progressive atrophy-related changes reflected by the late inflammation phenotype in 3BMBs suggest widespread time-dependent airway deterioration, which was especially pronounced after two years posttransplant. Thus molecular assessment of 3BMBs can detect rejection in a previously unusable biopsy format with potential utility in patients with severe lung dysfunction where TBB is not possible and provide unique insights into airway deterioration. ClinicalTrials.gov NCT02812290.

Discrepancy analysis comparing molecular and histology diagnoses in kidney transplant biopsies.

Discrepancy analysis comparing two diagnostic platforms offers potential insights into both without assuming either is always correct. Having optimized the Molecular Microscope Diagnostic System (MMDx) in renal transplant biopsies, we studied discrepancies within MMDx (reports and sign-out comments) and between MMDx and histology. Interpathologist discrepancies have been documented previously and were not assessed. Discrepancy cases were classified as "clear" (eg, antibody-mediated rejection [ABMR] vs T cell-mediated rejection [TCMR]), "boundary" (eg, ABMR vs possible ABMR), or "mixed" (eg, Mixed vs ABMR). MMDx report scores showed 99% correlations; sign-out interpretations showed 7% variation between observers, all located around boundaries. Histology disagreed with MMDx in 37% of biopsies, including 315 clear discrepancies, all with implications for therapy. Discrepancies were distributed widely in all histology diagnoses but increased in some scenarios; for example, histology TCMR contained 14% MMDx ABMR and 20% MMDx no rejection. MMDx usually gave unambiguous diagnoses in cases with ambiguous histology, for example, borderline and transplant glomerulopathy. Histology lesions or features associated with more frequent discrepancies (eg, tubulitis, arteritis, and polyomavirus nephropathy) were not associated with increased MMDx uncertainty, indicating that MMDx can clarify biopsies with histologic ambiguity. The patterns of histology-MMDx discrepancies highlight specific histology diagnoses in which MMDx assessment should be considered for guiding therapy.

The molecular diagnosis of rejection in liver transplant biopsies: First results of the INTERLIVER study.

Molecular diagnosis of rejection is emerging in kidney, heart, and lung transplant biopsies and could offer insights for liver transplant biopsies. We measured gene expression by microarrays in 235 liver transplant biopsies from 10 centers. Unsupervised archetypal analysis based on expression of previously annotated rejection-related transcripts identified 4 groups: normal "R1normal " (N = 129), T cell-mediated rejection (TCMR) "R2TCMR " (N = 37), early injury "R3injury " (N = 61), and fibrosis "R4late " (N = 8). Groups differed in median time posttransplant, for example, R3injury 99 days vs R4late 3117 days. R2TCMR biopsies expressed typical TCMR-related transcripts, for example, intense IFNG-induced effects. R3injury displayed increased expression of parenchymal injury transcripts (eg, hypoxia-inducible factor EGLN1). R4late biopsies showed immunoglobulin transcripts and injury-related transcripts. R2TCMR correlated with histologic rejection although with many discrepancies, and R4late with fibrosis. R2TCMR , R3injury , and R4late correlated with liver function abnormalities. Supervised classifiers trained on histologic rejection showed less agreement with histology than unsupervised R2TCMR scores. No confirmed cases of clinical antibody-mediated rejection (ABMR) were present in the population, and strategies that previously revealed ABMR in kidney and heart transplants failed to reveal a liver ABMR phenotype. In conclusion, molecular analysis of liver transplant biopsies detects rejection, has the potential to resolve ambiguities, and could assist with immunosuppressive management.

T cell exhaustion is associated with antigen abundance and promotes transplant acceptance.

Exhaustion of T cells limits their ability to clear chronic infections or eradicate tumors. Here, in the context of transplant, we investigated whether T cell exhaustion occurs and has a role in determining transplant outcome. A peptide/MHC tetramer-based approach was used to track exhausted CD8+ T cells in a male-to-female skin transplant model. Transplant of large whole-tail skins, but not small tail skins (0.8 cm × 0.8 cm), led to exhaustion of anti-male tetramer+ CD8+ T cells and subsequently the acceptance of skin grafts. To study CD4+ T cell exhaustion, we used the TCR-transgenic B6 TEa cells that recognize a major transplant antigen I-Eα from Balb/c mice. TEa cells were adoptively transferred either into B6 recipients that received Balb/c donor skins or into CB6F1 mice that contained an excessive amount of I-Eα antigen. Adoptively transferred TEa cells in skin-graft recipients were not exhausted. By contrast, virtually all adoptively transferred TEa cells were exhausted in CB6F1 mice. Those exhausted TEa cells lost ability to reject Balb/c skins upon further transfer into lymphopenic B6.Rag1-/- mice. Hence, T cell exhaustion develops in the presence of abundant antigen and promotes transplant acceptance. These findings are essential for better understanding the nature of transplant tolerance.

The skeletal muscles of mice infected with Plasmodium berghei and Plasmodium chabaudi reveal a crosstalk between lipid mediators and gene expression.

BACKGROUND: Malaria is one of the most prevalent infectious disease in the world with 3.2 billion humans at risk. Malaria causes splenomegaly and damage in other organs including skeletal muscles. Skeletal muscles comprise nearly 50% of the human body and are largely responsible for the regulation and modulation of overall metabolism. It is essential to understand how malaria damages muscles in order to develop effective preventive measures and/or treatments. Using a pre-clinical animal model, the potential molecular mechanisms of Plasmodium infection affecting skeletal muscles of mice were investigated. METHODS: Mouse Signal Transduction Pathway Finder PCR Array was used to monitor gene expression changes of 10 essential signalling pathways in skeletal muscles from mice infected with Plasmodium berghei and Plasmodium chabaudi. Then, a new targeted-lipidomic approach using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to profile 158 lipid signalling mediators (LMs), mostly eicosanoids derived from arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), was applied. Finally, 16 key LMs directly associated with inflammation, oxidative stress, and tissue healing in skeletal muscles, were quantified. RESULTS: The results showed that the expression of key genes altered by Plasmodium infection is associated with inflammation, oxidative stress, and atrophy. In support to gene profiling results, lipidomics revealed higher concentrations of LMs in skeletal muscles directly related to inflammatory responses, while on the levels of LMs crucial in resolving inflammation and tissue repair reduced significantly. CONCLUSION: The results provide new insights into the molecular mechanisms of malaria-induced muscle damage and revealed a potential mechanism modulating inflammation in malarial muscles. These pre-clinical studies should help with future clinical studies in humans aimed at monitoring of disease progression and development of specific interventions for the prevention and mitigation of long-term chronic effects on skeletal muscle function.

Discovery and cross-validation of peripheral blood and renal biopsy gene expression signatures from ethnically diverse kidney transplant populations.

We determined peripheral blood (PB) and biopsy (Bx) RNA expression signatures in a Brazilian and US cohort of kidney transplant patients. Phenotypes assigned by precise histology were: acute rejection (AR), interstitial fibrosis/tubular atrophy/chronic rejection (CR), excellent functioning transplants (TX), and glomerulonephritis recurrence (GN). Samples were analyzed on microarrays and profiles from each cohort were cross-validated on the other cohort with similar phenotypes. We discovered signatures for each tissue: (1) AR vs TX, (2) CR vs TX, and (3) GN vs TX using the Random Forests algorithm. We validated biopsies signatures of AR vs TX (area under the curve [AUC] 0.97) and CR vs TX (AUC 0.87). We also validated both PB and Bx signatures of AR vs TX and CR vs TX with varying degrees of accuracy. Several biological pathways were shared between AR and CR, suggesting similar rejection mechanisms in these 2 clinical phenotypes. Thus, we identified gene expression signatures for AR and CR in transplant patients and validated them in independent cohorts of significantly different racial/ethnic backgrounds. These results reveal that there are strong unifying immune mechanisms driving transplant diseases and identified in the signatures discovered in each cohort, suggesting that molecular diagnostics across populations are feasible despite ethnic and environmental differences.