Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens

Pneumocystis jirovecii is an unculturable fungus and the causative agent of Pneumocystis pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of mtLSU rRNA mitochondrial gene, the number of copies of mitochondrial genes had not been investigated. We developed and optimized six real-time PCR assays in order to determine the copy number of four mitochondrial genes (mtSSU rRNA, mtLSU rRNA, NAD1, and CYTB) in comparison to nuclear genome (DHPS and HSP70) and tested 84 bronchoalveolar fluids of patients at different stages of the infection. Unexpectedly, we found that copy number of mitochondrial genes varied from gene to gene with mtSSU rRNA gene being more represented (37 copies) than NAD1 (23 copies), mtLSU rRNA (15 copies) and CYTB (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (p = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of mtLSU rRNA, NAD1, and CYTB but not mtSSU rRNA differed according to P. jirovecii physiological state with a decreased number of copies when the fungal load is low. This suggests the existence of a mixture of various subspecies of mtDNA that can harbor different amplification rates. Overall, we revealed here an unexpected variability of P. jirovecii mtDNA copy number that fluctuates according to P. jirovecii's physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene.This work was supported by research project PI14CIII/00045 from the Spanish Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III. CV is supported by research fellowships from the Fondo de Investigaciones Sanitarias of the Spanish Ministry of Economy and Competitiveness (FI12/00095).S

Tracking the Genetic Susceptibility Background of B-Cell Non-Hodgkin's Lymphomas from Genome-Wide Association Studies

International audienceB-cell non-Hodgkin's lymphoma (NHL) risk associations had been mainly attributed to family history of the disease, inflammation, and immune components including human leukocyte antigen (HLA) genetic variations. Nevertheless, a broad range of genome-wide association studies (GWAS) have shed light into the identification of several genetic variants presumptively associated with B-cell NHL etiologies, survival or shared genetic risk with other diseases. The present review aims to overview HLA structure and diversity and summarize the evidence of genetic variations, by GWAS, on five NHL subtypes (diffuse large B-cell lymphoma DLBCL, follicular lymphoma FL, chronic lymphocytic leukemia CLL, marginal zone lymphoma MZL, and primary central nervous system lymphoma PCNSL). Evidence indicates that the HLA zygosity status in B-cell NHL might promote immune escape and that genome-wide significance variants can give biological insight but also potential therapeutic markers such as WEE1 in DLBCL. However, additional studies are needed, especially for non-DLBCL, to replicate the associations found to date

Neurological Syndromes Associated with Anti-GAD Antibodies

Glutamic acid decarboxylase (GAD) is an intracellular enzyme whose physiologic function is the decarboxylation of glutamate to gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter within the central nervous system. GAD antibodies (Ab) have been associated with multiple neurological syndromes, including stiff-person syndrome, cerebellar ataxia, and limbic encephalitis, which are all considered to result from reduced GABAergic transmission. The pathogenic role of GAD Ab is still debated, and some evidence suggests that GAD autoimmunity might primarily be cell-mediated. Diagnosis relies on the detection of high titers of GAD Ab in serum and/or in the detection of GAD Ab in the cerebrospinal fluid. Due to the relative rarity of these syndromes, treatment schemes and predictors of response are poorly defined, highlighting the unmet need for multicentric prospective trials in this population. Here, we reviewed the main clinical characteristics of neurological syndromes associated with GAD Ab, focusing on pathophysiologic mechanisms

Quantitative brain imaging analysis of neurological syndromes associated with anti-GAD antibodies

International audienceNeurological disorders associated with anti-glutamic acid decarboxylase (GAD) autoimmunity are rare and include a variety of neurological syndromes: stiff-person syndrome, cerebellar ataxia or limbic encephalitis. The diagnosis remains challenging due to the variety of symptoms and normal brain imaging. The morphological MRI of 26 patients (T1-weighted and Fluid-attenuated inversion recovery (FLAIR)weighted images) was analyzed at the initial stage of diagnosis, matched by age and sex to 26 healthy subjects. We performed a vertex-wise analysis using a generalized linear model, adjusting by age, to compare the brain cortical thickness of both populations. In addition, we used a voxel-based morphometry of cerebellum thickness obtained by CEREbellum Segmentation (CERES), as well as the hippocampus volumetry comparison using HIPpocampus subfield Segmentation (HIPS). Finally, we extracted 62 radiomics features using LifeX to assess the classification performance using a random forest model to identify an anti-GAD related MRI. The results suggest a peculiar profile of atrophy in patients with anti-GAD, with a significant atrophy in the temporal and frontal lobes (adjusted p-value < 0.05), and a focal cerebellar atrophy of the V-lobule, independently of the anti-GAD phenotype. Finally, the MRIs from anti-GAD patients were correctly classified when compared to the control group, with an area under the curve (AUC) of 0.98. This study suggests a particular pattern of cortical atrophy throughout all anti-GAD phenotypes. These results reinforce the notion that the different neurological anti-GAD phenotypes should be considered as a continuum due to their similar cortical thickness profiles

A human beta cell line with drug inducible excision of immortalizing transgenes.

International audienceAccess to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen.METHODS:We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion.RESULTS:We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture.CONCLUSIONS:EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture

Evaluation of a new Histoplasma spp. reverse transcriptase quantitative PCR assay

International audienceLaboratory diagnosis of histoplasmosis is based on various methods including microscopy, culture, antigen and DNA detection of Histoplasma capsulatum var. capsulatum (Hcc) or H. capsulatum var. duboisii (Hcd). To improve sensitivity of existing quantitative PCR assays, we developed a new reverse transcriptase qPCR (RTqPCR) assay allowing amplification of whole nucleic acids of Histoplasma spp.. and validated on suspected cases.The limit of detection was 20 copies and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp.. Whole nucleic acids of 1,319 prospectively collected consecutive samples from 907 patients suspected of histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. 44 had proven histoplasmosis due to Hcc (n=40) or Hcd (n=4) infections. RTqPCR was positive in 43/44 patients (97.7% sensitivity), in at least one specimen. Nine out of 863 cases (99% specificity) were RTqPCR positive and therefore classified as possible cases. RTqPCR was positive in 13/30 (43.3%) blood tested in proven cases. A positive RTqPCR in blood was significantly associated with Hcc progressive disseminated histoplasmosis with a positive RTqPCR in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RTqPCR assay enabling amplification of hcc and hcd is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and BAL

Longitudinally Extensive Myelitis Associated With Immune Checkpoint Inhibitors

International audienceObjective: To define the characteristics and the outcome of myelitis associated with immune checkpoint inhibitors (ICIs).Methods: We performed a retrospective research in the databases of the French Pharmacovigilance Agency and the OncoNeuroTox network for patients who developed myelitis following treatment with ICIs (2011-2020). A systematic review of the literature was performed to identify similar cases.Results: We identified 7 patients who developed myelitis after treatment with ICIs (anti-PD1 [n = 6], anti-PD1 + anti-CTLA4 [n = 1]). Neurologic symptoms included paraparesis (100%), sphincter dysfunction (86%), tactile/thermic sensory disturbances (71%), and proprioceptive ataxia (43%). At the peak of symptom severity, all patients were nonambulatory. MRI typically showed longitudinally extensive lesions, with patchy contrast enhancement. CSF invariably showed inflammatory findings. Five patients (71%) had clinical and/or paraclinical evidence of concomitant cerebral, meningeal, caudal roots, and/or peripheral nerve involvement. Despite the prompt discontinuation of ICIs and administration of high-dose glucocorticoids (n = 7), most patients needed second-line immune therapies (n = 5) because of poor recovery or early relapses. At last follow-up, only 3 patients had regained an ambulatory status (43%). Literature review identified 13 previously reported cases, showing similar clinical and paraclinical features. All patients discontinued ICIs and received high-dose glucocorticoids, with the addition of other immune therapies in 8. Clinical improvement was reported for 10 patients.Conclusion: Myelitis is a rare but severe complication of ICIs that shows limited response to glucocorticoids. Considering the poor functional outcome associated with longitudinally extensive myelitis, strong and protracted immune therapy combinations are probably needed upfront to improve patient outcome and prevent early relapses

Human Pancreatic β Cell lncRNAs Control Cell-Specific Regulatory Networks

Recent studies have uncovered thousands of long non-coding RNAs (lncRNAs) in human pancreatic β cells. β cell lncRNAs are often cell type specific and exhibit dynamic regulation during differentiation or upon changing glucose concentrations. Although these features hint at a role of lncRNAs in β cell gene regulation and diabetes, the function of β cell lncRNAs remains largely unknown. In this study, we investigated the function of β cell-specific lncRNAs and transcription factors using transcript knockdowns and co-expression network analysis. This revealed lncRNAs that function in concert with transcription factors to regulate β cell-specific transcriptional networks. We further demonstrate that the lncRNA PLUTO affects local 3D chromatin structure and transcription of PDX1, encoding a key β cell transcription factor, and that both PLUTO and PDX1 are downregulated in islets from donors with type 2 diabetes or impaired glucose tolerance. These results implicate lncRNAs in the regulation of β cell-specific transcription factor networks