Cytoplasmic TDP-43 aggregation is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Here we investigated the role of exosomes in the secretion and ...propagation of TDP-43 aggregates. TDP-43 was detected in secreted exosomes from Neuro2a cells and primary neurons but not from astrocytes or microglia. Evidence is presented that protein aggregation and autophagy inhibition are factors that promote exosomal secretion of TDP-43. We also report that levels of exosomal TDP-43 full length and C-terminal fragment species are upregulated in human amyotrophic lateral sclerosis brains. Exposure of Neuro2a cells to exosomes from amyotrophic lateral sclerosis brain, but not from control brain, caused cytoplasmic redistribution of TDP-43, suggesting that secreted exosomes might contribute to propagation of TDP-43 proteinopathy. Yet, inhibition of exosome secretion by inactivation of neutral sphingomyelinase 2 with GW4869 or by silencing RAB27A provoked formation of TDP-43 aggregates in Neuro2a cells. Moreover, administration of GW4869 exacerbated the disease phenotypes of transgenic mice expressing human TDP-43
mutant. Thus, even though results suggest that exosomes containing pathological TDP-43 may play a key role in the propagation of TDP-43 proteinopathy, a therapeutic strategy for amyotrophic lateral sclerosis based on inhibition of exosome production would seem inappropriate, as in vivo data suggest that exosome secretion plays an overall beneficial role in neuronal clearance of pathological TDP-43.
J. Neurochem. (2010) 113, 1188-1199. The finding of a secretion pathway and toxicity for mutant superoxide dismutase 1 (SOD1) raised up the possibility of using immunization approaches to reduce or ...neutralize the burden of toxic SOD1 species in the nervous system. Here we tested a passive immunization approach based on intracerebroventricular infusion in G93A-SOD1 mice of monoclonal antibodies specific to misfolded forms of SOD1 (mSOD1). We tested two monoclonal antibodies that bind distinct epitopes in mSOD1 and that do not bind to intact wild-type (WT) SOD1. One antibody succeeded in reducing the level of mSOD1 by 23% in the spinal cord and in prolonging the lifespan of G93A-SOD1 mice in proportion to the duration of treatment. However, another monoclonal antibody binding to a different SOD1 epitope failed to confer protection indicating that not all anti-SOD1 antibodies might be suitable for immunotherapy. Interestingly, the variable Fab fragment of an anti-SOD1 antibody was sufficient to confer some protection in G93A-SOD1 mice. The partial dispensability of Fc region should offer some advantages for development of immunotherapy with antibodies of smaller molecular size and low immunogenicity. From these results, we propose that passive immunization strategies should be considered as potential avenues for treatment of familial amyotrophic lateral sclerosis caused by SOD1 mutations.
After resection, colorectal cancer liver metastases (CRLM) surrounded by a desmoplastic rim carry a better prognosis than the metastases replacing the adjacent liver. However, these histopathological ...growth patterns (HGPs) are insufficient to guide clinical decision-making. We explored whether the adaptive immune features of HGPs could refine prognostication.
From 276 metastases resected in 176 patients classified by HGPs, tissue microarrays were used to assess intratumoral T cells (CD3), antigen presentation capacity (MHC class I) and CD73 expression producing immunosuppressive adenosine. We tested correlations between these variables and patient outcomes.
The 101 (57.4%) patients with dominant desmoplastic HGP had a median recurrence-free survival (RFS) of 17.1 months compared to 13.3 months in the 75 patients (42.6%) with dominant replacement HGP (p = 0.037). In desmoplastic CRLM, high vs. low CD73 was the only prognostically informative immune parameter and was associated with a median RFS of 12.3 months compared to 26.3, respectively (p = 0.010). Only in dominant replacement CRLM, we found a subgroup (n = 23) with high intratumoral MHC-I expression but poor CD3
T cell infiltration, a phenotype associated with a short median RFS of 7.9 months.
Combining the assessments of HGP and adaptive immune features in resected CRLM could help identify patients at risk of early recurrence.
There is emerging evidence that the misfolding of superoxide dismutase 1 (SOD1) may represent a common pathogenic event in both familial and sporadic amyotrophic lateral sclerosis (ALS). To reduce ...the burden of misfolded SOD1 species in the nervous system, we have tested a novel therapeutic approach based on adeno-associated virus (AAV)–mediated tonic expression of a DNA construct encoding a secretable single-chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the AAV encoding the single-chain antibody in SOD1G93A mice at 45 days of age resulted in sustained expression of single-chain antibodies in the spinal cord, and it delayed disease onset and extension of life span by up to 28%, in direct correlation with scFv titers in the spinal cord. The treatment caused attenuation of neuronal stress signals and reduction in levels of misfolded SOD1 in the spinal cord of SOD1G93A mice. From these results, we propose that an immunotherapy based on intrathecal inoculation of AAV encoding a secretable scFv against misfolded SOD1 should be considered as potential treatment for ALS, especially for individuals carrying SOD1 mutations.
The cytoplasmic aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of degenerating neurons in amyotrophic lateral sclerosis (ALS) and subsets of frontotemporal dementia (FTD). In order ...to reduce TDP-43 pathology, we generated single-chain (scFv) antibodies against the RNA recognition motif 1 (RRM1) of TDP-43, which is involved in abnormal protein self-aggregation and interaction with p65 NF-κB. Virus-mediated delivery into the nervous system of a scFv antibody, named VH7Vk9, reduced microgliosis in a mouse model of acute neuroinflammation and mitigated cognitive impairment, motor defects, TDP-43 proteinopathy, and neuroinflammation in transgenic mice expressing ALS-linked TDP-43 mutations. These results suggest that antibodies targeting the RRM1 domain of TDP-43 might provide new therapeutic avenues for the treatment of ALS and FTD.
Finding a noninvasive radiomic surrogate of tumor immune features could help identify patients more likely to respond to novel immune checkpoint inhibitors. Particularly, CD73 is an ectonucleotidase ...that catalyzes the breakdown of extracellular AMP into immunosuppressive adenosine, which can be blocked by therapeutic antibodies. High CD73 expression in colorectal cancer liver metastasis (CRLM) resected with curative intent is associated with early recurrence and shorter patient survival. The aim of this study was hence to evaluate whether machine learning analysis of preoperative liver CT-scan could estimate high vs low CD73 expression in CRLM and whether such radiomic score would have a prognostic significance.
We trained an Attentive Interpretable Tabular Learning (TabNet) model to predict, from preoperative CT images, stratified expression levels of CD73 (CD73
vs. CD73
) assessed by immunofluorescence (IF) on tissue microarrays. Radiomic features were extracted from 160 segmented CRLM of 122 patients with matched IF data, preprocessed and used to train the predictive model. We applied a five-fold cross-validation and validated the performance on a hold-out test set.
TabNet provided areas under the receiver operating characteristic curve of 0.95 (95% CI 0.87 to 1.0) and 0.79 (0.65 to 0.92) on the training and hold-out test sets respectively, and outperformed other machine learning models. The TabNet-derived score, termed rad-CD73, was positively correlated with CD73 histological expression in matched CRLM (Spearman's ρ = 0.6004; P < 0.0001). The median time to recurrence (TTR) and disease-specific survival (DSS) after CRLM resection in rad-CD73
vs rad-CD73
patients was 13.0 vs 23.6 months (P = 0.0098) and 53.4 vs 126.0 months (P = 0.0222), respectively. The prognostic value of rad-CD73 was independent of the standard clinical risk score, for both TTR (HR = 2.11, 95% CI 1.30 to 3.45, P < 0.005) and DSS (HR = 1.88, 95% CI 1.11 to 3.18, P = 0.020).
Our findings reveal promising results for non-invasive CT-scan-based prediction of CD73 expression in CRLM and warrant further validation as to whether rad-CD73 could assist oncologists as a biomarker of prognosis and response to immunotherapies targeting the adenosine pathway.
The drug targets IL23 and IL12 regulate pathogenicity and plasticity of intestinal Th17 cells in Crohn's disease (CD) and ulcerative colitis (UC), the two most common inflammatory bowel diseases ...(IBD). However, studies examining Th17 dysregulation in mesenteric lymph nodes (mLNs) of these patients are rare. We showed that in mLNs, CD could be distinguished from UC by increased frequencies of CCR6
CXCR3
RORγ
Tbet
CD4
(Th17) memory T cells enriched in CD62L
effector memory T cells (T
), and their differentially expressed molecular profile. Th17 T
cells (expressing
, and
) displayed a higher pathogenic/cytotoxic (
, and
) gene signature in CD relative to UC, while non-pathogenic/regulatory genes (
) were more elevated in UC. In both CD and UC, IL12 but not IL23, augmented IFNγ expression in Th17 T
and switched their molecular profile toward an ex-Th17 (Th1
)-biased transcriptomic signature (increased
, and decreased
), suggesting that Th17 plasticity occurs in mLNs before their recruitment to inflamed colon. We propose that differences observed between Th17 cell frequencies and their molecular profile in CD and UC might have implications in understanding disease pathogenesis, and thus, therapeutic management of patients with IBD.
Inflammatory immune cells can modulate activation of hepatic stellate cells (HSCs) and progression of liver fibrosis. Type 3 inflammation characterized by production of interleukin-17A (IL-17) and ...IL-22 by innate and adaptive immune cells is implicated in many inflammatory conditions of the gut and can be counteracted by regulatory T cells (T
), but its contribution to liver fibrosis is still poorly understood. Here, we evaluated the contribution of type 3 inflammation in liver fibrosis using clinical liver biopsies, in vitro stimulation of primary HSCs, and in vivo mouse models. We report dysregulated type 3 responses in fibrotic lesions with increased IL-17
CD4
/FOXP3
CD4
ratio and increased IL-17 and IL-22 production in advanced liver fibrosis. Neutrophils and mast cells were the main sources of IL-17 in situ in humans. In addition, we demonstrate a new profibrotic function of IL-22 through enhancement of transforming growth factor-β signaling in HSCs in a p38 mitogen-activated protein kinase-dependent manner. In vivo, IL-22RA1 knockout mice exhibited reduced fibrosis in response to thioacetamide and carbon tetrachloride. Blocking either IL-22 or IL-17 production using aryl hydrocarbon receptor or RAR-related orphan receptor gamma-t antagonists resulted in reduced fibrosis. Together, these data have identified a pathogenic role for type 3 immune response mediated by IL-22 in driving liver fibrosis during chronic liver injury.
In developed countries, colorectal cancer is the second cause of cancer-related mortality. Chemotherapy is considered a standard treatment for colorectal liver metastases (CLM). Among patients who ...develop CLM, the assessment of patient response to chemotherapy is often required to determine the need for second-line chemotherapy and eligibility for surgery. However, while FOLFOX-based regimens are typically used for CLM treatment, the identification of responsive patients remains elusive. Computer-aided diagnosis systems may provide insight in the classification of liver metastases identified on diagnostic images. In this paper, we propose a fully automated framework based on deep convolutional neural networks (DCNN) which first differentiates treated and untreated lesions to identify new lesions appearing on CT scans, followed by a fully connected neural networks to predict from untreated lesions in pre-treatment computed tomography (CT) for patients with CLM undergoing chemotherapy, their response to a FOLFOX with Bevacizumab regimen as first-line of treatment. The ground truth for assessment of treatment response was histopathology-determined tumor regression grade. Our DCNN approach trained on 444 lesions from 202 patients achieved accuracies of 91% for differentiating treated and untreated lesions, and 78% for predicting the response to FOLFOX-based chemotherapy regimen. Experimental results showed that our method outperformed traditional machine learning algorithms and may allow for the early detection of non-responsive patients.
Although the neuroprotective effects of estrogens are well recognized, the exact mechanisms involved in the ability of these sex steroids to protect the cerebral tissue still remain unclear. We ...tested in our study the hypothesis that estradiol (E(2)) modulates the innate immune response and expression of genes encoding proteins that a provide survival signal to neurons during infection. Mice received a single systemic or cerebral injection of LPS to trigger a robust but transient inflammatory reaction in the brain. The endotoxin increased transcriptional activation of genes encoding TLR2, TNF-alpha, and IL-12 in microglial cells. Expression of these transcripts was largely inhibited in the brain of ovariectomized mice at time 24 h postchallenge. E(2) replacement therapy totally rescued the ability of the endotoxin to trigger microglial cells and these permissive effects of E(2) are mediated via the estrogen receptor (ER)alpha. Indeed, ERalpha-deficient mice exhibited an inappropriate reaction to LPS when compared with ERbeta-deficient and wild-type mice. This defective innate immune response was also associated with a widespread viral replication and neurodegeneration in ovariectomized mice inoculated intranasally with HSV-2. These data provide evidence that interaction of E(2) with their nuclear ERalpha plays a critical role in the control of cytokines involved in the transfer from the innate to adaptive immunity. This transfer is deviant in mice lacking E(2), which allows pathogens to hide from immune surveillance and exacerbates neuronal damages during viral encephalitis.
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