Parvalbumin gates chronic pain through the modulation of firing patterns in inhibitory neurons.

Spinal cord dorsal horn inhibition is critical to the processing of sensory inputs, and its impairment leads to mechanical allodynia. How this decreased inhibition occurs and whether its restoration alleviates allodynic pain are poorly understood. Here, we show that a critical step in the loss of inhibitory tone is the change in the firing pattern of inhibitory parvalbumin (PV)-expressing neurons (PVNs). Our results show that PV, a calcium-binding protein, controls the firing activity of PVNs by enabling them to sustain high-frequency tonic firing patterns. Upon nerve injury, PVNs transition to adaptive firing and decrease their PV expression. Interestingly, decreased PV is necessary and sufficient for the development of mechanical allodynia and the transition of PVNs to adaptive firing. This transition of the firing pattern is due to the recruitment of calcium-activated potassium (SK) channels, and blocking them during chronic pain restores normal tonic firing and alleviates chronic pain. Our findings indicate that PV is essential for controlling the firing pattern of PVNs and for preventing allodynia. Developing approaches to manipulate these mechanisms may lead to different strategies for chronic pain relief.

Immunogenicity of Non-Mutated Ovarian Cancer-Specific Antigens.

Epithelial ovarian cancer (EOC) has not significantly benefited from advances in immunotherapy, mainly because of the lack of well-defined actionable antigen targets. Using proteogenomic analyses of primary EOC tumors, we previously identified 91 aberrantly expressed tumor-specific antigens (TSAs) originating from unmutated genomic sequences. Most of these TSAs derive from non-exonic regions, and their expression results from cancer-specific epigenetic changes. The present study aimed to evaluate the immunogenicity of 48 TSAs selected according to two criteria: presentation by highly prevalent HLA allotypes and expression in a significant fraction of EOC tumors. Using targeted mass spectrometry analyses, we found that pulsing with synthetic TSA peptides leads to a high-level presentation on dendritic cells. TSA abundance correlated with the predicted binding affinity to the HLA allotype. We stimulated naïve CD8 T cells from healthy blood donors with TSA-pulsed dendritic cells and assessed their expansion with two assays: MHC-peptide tetramer staining and TCR Vß CDR3 sequencing. We report that these TSAs can expand sizeable populations of CD8 T cells and, therefore, represent attractive targets for EOC immunotherapy.

Breast cancer immunopeptidomes contain numerous shared tumor antigens.

Hormone receptor-positive breast cancer (HR+) is immunologically cold and has not benefited from advances in immunotherapy. In contrast, subsets of triple-negative breast cancer (TNBC) display high leukocytic infiltration and respond to checkpoint blockade. CD8+ T cells, the main effectors of anticancer responses, recognize MHC I-associated peptides (MAPs). Our work aimed to characterize the repertoire of MAPs presented by HR+ and TNBC tumors. Using mass spectrometry, we identified 57,094 unique MAPs in 26 primary breast cancer samples. MAP source genes highly overlapped between both subtypes. We identified 25 tumor-specific antigens (TSAs) mainly deriving from aberrantly expressed regions. TSAs were most frequently identified in TNBC samples and were more shared among The Cancer Genome Atlas (TCGA) database TNBC than HR+ samples. In the TNBC cohort, the predicted number of TSAs positively correlated with leukocytic infiltration and overall survival, supporting their immunogenicity in vivo. We detected 49 tumor-associated antigens (TAAs), some of which derived from cancer-associated fibroblasts. Functional expansion of specific T cell assays confirmed the in vitro immunogenicity of several TSAs and TAAs. Our study identified attractive targets for cancer immunotherapy in both breast cancer subtypes. The higher prevalence of TSAs in TNBC tumors provides a rationale for their responsiveness to checkpoint blockade.

The ever wider clinical spectrum of RMND1-related disorders and limitedness of phenotype-based classifications.

RMND1 has been identified as a mitochondriopathy-associated gene less than 12 years ago. The most common phenotype related to this gene is an early onset, severe form of encephalomyopathy that leads to death in a medium time of three years after birth. However, milder and later onset presentations have been reported in some individuals, including two in whom the mitochondriopathy was identified at ~ 40 years of age, and the early onset presentations have been the object of no reports in those who survived beyond age 10. It is thus unclear how lethal RMND1-related conditions really are. We herein describe the oldest case to have been identified hitherto with this condition, i.e., that of a white female who was 61 at the time of diagnosis but was still active in her everyday life. The gene defect identified was nonetheless associated with many manifestations including ovarian insufficiency and sensorineural hearing loss (two features of what is currently designated as Perrault syndrome) as well as chronic renal failure, asymptomatic myopathy, leukopenia, and a few others. In our opinion, this case is of great translational interest for at least three reasons. First, it hints towards the possibility of near-normal life expectancies in some if not many individuals with RMND1 insufficiency. Second, it underlines the wide clinical spectrum associated with this gene. Third, it brings us to question the use of eponyms and syndromic features to identify the true etiology of multisystemic phenotypes. KEY MESSAGES: RMND1-related conditions typically manifest at an early age with a progressive and lethal form of encephalomyopathy. More benign presentations have been described with some being categorized as Perrault syndrome but none have been diagnosed after the age of 45. The clinical spectrum and presenting age of RMND1-related mitochondriopathies are probably much more varied than implied in the current literature. The case reported in this manuscript illustrates the limitedness of phenotype-based classifications of genetic disorders to identify the defect at cause.

Induced pluripotent stem cells display a distinct set of MHC I-associated peptides shared by human cancers.

Previous reports showed that mouse vaccination with pluripotent stem cells (PSCs) induces durable anti-tumor immune responses via T cell recognition of some elusive oncofetal epitopes. We characterize the MHC I-associated peptide (MAP) repertoire of human induced PSCs (iPSCs) using proteogenomics. Our analyses reveal a set of 46 pluripotency-associated MAPs (paMAPs) absent from the transcriptome of normal tissues and adult stem cells but expressed in PSCs and multiple adult cancers. These paMAPs derive from coding and allegedly non-coding (48%) transcripts involved in pluripotency maintenance, and their expression in The Cancer Genome Atlas samples correlates with source gene hypomethylation and genomic aberrations common across cancer types. We find that several of these paMAPs were immunogenic. However, paMAP expression in tumors coincides with activation of pathways instrumental in immune evasion (WNT, TGF-ß, and CDK4/6). We propose that currently available inhibitors of these pathways could synergize with immune targeting of paMAPs for the treatment of poorly differentiated cancers.

The cell adhesion molecule Sdk1 shapes assembly of a retinal circuit that detects localized edges.

Nearly 50 different mouse retinal ganglion cell (RGC) types sample the visual scene for distinct features. RGC feature selectivity arises from their synapses with a specific subset of amacrine (AC) and bipolar cell (BC) types, but how RGC dendrites arborize and collect input from these specific subsets remains poorly understood. Here we examine the hypothesis that RGCs employ molecular recognition systems to meet this challenge. By combining calcium imaging and type-specific histological stains, we define a family of circuits that express the recognition molecule Sidekick-1 (Sdk1), which include a novel RGC type (S1-RGC) that responds to local edges. Genetic and physiological studies revealed that Sdk1 loss selectively disrupts S1-RGC visual responses, which result from a loss of excitatory and inhibitory inputs and selective dendritic deficits on this neuron. We conclude that Sdk1 shapes dendrite growth and wiring to help S1-RGCs become feature selective.

Atypical acute myeloid leukemia-specific transcripts generate shared and immunogenic MHC class-I-associated epitopes.

Acute myeloid leukemia (AML) has not benefited from innovative immunotherapies, mainly because of the lack of actionable immune targets. Using an original proteogenomic approach, we analyzed the major histocompatibility complex class I (MHC class I)-associated immunopeptidome of 19 primary AML samples and identified 58 tumor-specific antigens (TSAs). These TSAs bore no mutations and derived mainly (86%) from supposedly non-coding genomic regions. Two AML-specific aberrations were instrumental in the biogenesis of TSAs, intron retention, and epigenetic changes. Indeed, 48% of TSAs resulted from intron retention and translation, and their RNA expression correlated with mutations of epigenetic modifiers (e.g., DNMT3A). AML TSA-coding transcripts were highly shared among patients and were expressed in both blasts and leukemic stem cells. In AML patients, the predicted number of TSAs correlated with spontaneous expansion of cognate T cell receptor clonotypes, accumulation of activated cytotoxic T cells, immunoediting, and improved survival. These TSAs represent attractive targets for AML immunotherapy.

Major multilevel molecular divergence between THP-1 cells from different biorepositories.

The THP-1 cell line is broadly used as a model for acute myeloid leukemia (AML) with MLL fusion and to study monocyte differentiation and function. We studied THP-1 cells obtained from two major biorepositories. The two cell lines were closely related with a percentage match of short tandem repeat (STR) profiles ranging from 93.75% to 100%, depending on the algorithm used. Nevertheless, we found that the two cell lines presented discordant HLA type, cytogenetic aberrations and AML-related gene expression (including critical targets of MLL fusion). These discrepancies resulted mainly from loss of heterozygosity (LOH) involving five chromosomal regions. In view of their aberrant expression of key "leukemia" genes (e.g., LIN28B, MEIS1 and SPARC), we argue that one of the THP-1 cell lines may not be a reliable model for studying leukemia. Their defective expression of HLA molecules and abnormal adhesion properties is also a caveat for studies of antigen presentation. In a more general perspective, our findings show that seemingly minor discrepancies in STR profiles among cell lines may be the sign of major genetic drift, of sufficient magnitude to affect the reliability of cell line-based research.

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages.

Characterization of a putative Plasmodium falciparum SAC1 phosphoinositide-phosphatase homologue potentially required for survival during the asexual erythrocytic stages.

Despite marked reductions in morbidity and mortality in the last ten years, malaria still takes a tremendous toll on human populations throughout tropical and sub-tropical regions of the world. The absence of an effective vaccine and resistance to most antimalarial drugs available demonstrate the urgent need for new intervention strategies. Phosphoinositides are a class of lipids with critical roles in numerous processes and their specific subcellular distribution, generated through the action of kinases and phosphatases, define organelle identity in a wide range of eukaryotic cells. Recent studies have highlighted important functions of phosphoinositide kinases in several parts of the Plasmodium lifecycle such as hemoglobin endocytosis and cytokinesis during the erythrocytic stage however, nothing is known with regards to the parasite's putative phosphoinositide phosphatases. We present the identification and initial characterization of a putative homologue of the SAC1 phosphoinositide phosphatase family. Our results show that the protein is expressed throughout the asexual blood stages and that it localises to the endoplasmic reticulum and potentially to the Golgi apparatus. Furthermore, conditional knockdown and knockout studies suggest that a minimal amount of the protein are likely required for survival during the erythrocytic cycle.

MUC16 (CA125) regulates epithelial ovarian cancer cell growth, tumorigenesis and metastasis.

OBJECTIVES: MUC16 (CA125) protein is a high molecular weight mucin overexpressed in the majority of epithelial ovarian cancers (EOC) but not in the epithelium of normal ovaries suggesting that it might play a role in EOC pathogenesis. Here, we explored the phenotypic consequences of MUC16 knockdown and expression of its C-terminal domain with the aim of establishing a role for MUC16 in tumorigenesis. METHODS: MUC16 was down-regulated by stably expressing an anti-MUC16 endoplasmic reticulum-targeted single-chain antibody which prevented MUC16 cell surface localization in NIH:OVCAR3 cells. In addition, we generated epitope tagged, N-terminal region-deleted MUC16 constructs with (MUC16TMU) and without (MUC16CTD) cytoplasmic tail deletions and stably expressed them in SKOV3 cells. RESULTS: Although MUC16 knockdown did not affect the cell growth rate, knockdown cells reached a stationary growth phase after 4 days whereas control cells continued to grow for up to 7 days. Colony formation assays in soft agar demonstrated that MUC16 knockdown cells had >8-fold reduction in their ability to form colonies. Importantly, MUC16 knockdown completely prevents the formation of subcutaneous tumors in nude mice. Conversely, we show that ectopic expression of the MUC16CTD enhances SKOV3 tumor cell growth, colony formation in soft agar and enhances tumor growth and metastases in SCID mice. In addition, MUC16CTD expression increases cell motility, invasiveness, and metastatic property. Deletion of the cytoplasmic tail from the MUC16CTD completely abolished its ability to enhance tumor cell growth, cell motility and invasiveness. Furthermore, the increased invasive properties of MUC16CTD-expressing cells correlated with decreased expression of E-cadherin and increased expression of N-cadherin and vimentin. CONCLUSION: These findings provide the first evidence for a critical role of MUC16 in tumor cell growth, tumorigenesis and metastases.

Effects of milk fermented by Lactobacillus helveticus R389 on immune cells associated to mammary glands in normal and a breast cancer model.

Antitumour activity is an effect attributed to probiotics and fermented foods. Here, the immune cells in mammary glands and cytokine concentration in serum were analyzed using mice fed with milk fermented by Lactobacillus helveticus R389 or L89 (proteolytic-deficient variant), injected or not with breast tumour cells. Mice were fed 7 days with fermented milk, injected with breast tumour cells and 4 days post-injection, they received fermented milk. IgA, CD4, CD8, cytokines and Bcl-2 positive cells in mammary glands and cytokine in serum were determined. Mice fed with L. helveticus R389 fermented milk and injected with tumour cells increased IgA and CD4 positive cells in mammary glands (tumour control increased CD8 + cells). Mice from fermented milk control groups (without tumour cell injection) did not show changes in immune cell or cytokine positive cell numbers. IL-10 increases and IL-6 decreases were more pronounced in mice fed with milk fermented by L. helveticus R389 than in the other groups. This study demonstrated the immunoregulatory capacity of milk fermented by L. helveticus R389 on the immune response in mammary glands in presence of a local pathology (breast tumour). Orally administered fermented products could be used to modify the immune cell activation in distant mucosal sites and maintain these cells alert, but local stimulus was necessary to produce the activation of a local immune response in mammary glands, which could modulate the immune-endocrine relationship in these glands.