BACKGROUND: Bacillus Calmette-Guerin (BCG) therapy is an established immunotherapy for non-muscle invasive bladder cancer (NMIBC); however, the response variability of patients remains a challenge, necessitating insight into immune cell function. Previous studies established that a preexisting Th2 immune microenvironment correlates with a positive BCG therapy outcome. Therefore, in this study, we explored the role of mast cells (MCs) and eosinophils in bladder cancer as a potential predicting tool for BCG immunotherapy response. METHODS: We investigated the effect of MCs and eosinophils on bladder cancer cell viability together with their chemotactic migration towards cancer cells in vitro. The effect of BCG on these immune cells was also evaluated. Moreover, we performed an orthotopic model of bladder cancer in MC- and eosinophil-deficient mice. Finally, to evaluate whether these immune cells predict the therapy response, 26 patient biopsies pre-BCG treatment were analyzed for MC and eosinophil numbers in the tissue and sequenced for gene expression. RESULTS: Eosinophils, but not MCs, reduced bladder cancer cell viability in vitro and inhibited tumor growth in vivo. However, addition of BCG did not increase these effects in vitro. Patient biopsy analysis and mRNA sequencing showed that neither cell type predicted long-term therapy responsiveness. Gene expression analysis suggested that extracellular matrix and epithelial-to-mesenchymal transition factors could influence BCG therapy outcomes. CONCLUSION: Even though eosinophils exhibit anti-tumorigenic effects in bladder cancer, neither MCs nor eosinophils were predictive of the long-term BCG therapy response. However, our findings implicate that matrix-related factors may modulate BCG therapy responses.

BACKGROUND: ATAD3A, a nuclear gene encoding the ATAD3A protein, has diverse roles in mitochondrial processes, encompassing mitochondrial dynamics, mitochondrial DNA maintenance, metabolic pathways and inter-organellar interactions. Pathogenic variants in this gene cause neurological diseases in humans with recognizable genotype-phenotype correlations. Yet, gaps in knowledge remain regarding the underlying pathogenesis. METHODS: To further investigate the gene function and its implication in health and disease, we utilized CRISPR/Cas9 genome editing to generate a knockout model of the zebrafish ortholog gene, atad3. We characterized the phenotype of the null model, performed mitochondrial and functional tests, and compared the transcriptome of null embryos to their healthy siblings. RESULTS: Analysis of atad3-null zebrafish embryos revealed microcephaly, small eyes, pericardial edema and musculature thinning, closely mirroring the human rare disease phenotype. Larvae exhibited delayed hatching and embryonic lethality by 13 days post-fertilization (dpf). Locomotor activity, ATP content, mitochondrial content, and mitochondrial activity were all reduced in the mutant embryos. Transcriptome analysis at 3 dpf via RNA-sequencing indicated decline in most mitochondrial pathways, accompanied by a global upregulation of cytosolic tRNA synthetases, presumably secondary to mitochondrial stress and possibly endoplasmic reticulum (ER)-stress. Differential expression of select genes was corroborated in fibroblasts from an affected individual. CONCLUSIONS: The atad3-null zebrafish model emerges as a reliable representation of human ATAD3A-associated disorders, with similarities in differentially expressed pathways and processes. Furthermore, our study underscores mitochondrial dysfunction as the primary underlying pathogenic mechanism in ATAD3A-associated disorders and identifies potential readouts for therapeutic studies.

Triple-negative breast cancer (TNBC) is an aggressive subtype that accounts for 10-15 % of breast cancer. Current treatment of high-risk early-stage TNBC includes neoadjuvant chemo-immune therapy. However, the substantial variation in immune response prompts an urgent need for new immune-targeting agents. This requires a comprehensive understanding of TNBC's tumor microenvironment. We recently demonstrated that Galectin-3 (Gal-3) binding protein/Gal-3 complex secreted by TNBC cells induces immunosuppression, through inhibiting CD45 signaling in T cells. Here, we further investigated the interaction between secreted Gal-3 and T cells in TNBC. Using CRISPR/Cas9 gene editing of the TNBC MDA-MB-231 cell-line, we obtained Gal-3 negative((neg)) clones. We studied these in an in-vitro model, co-cultured with peripheral blood mononuclear cells (PBMC) to imitate immune-tumor interaction, and in an in-vivo model, when implanted in mice. Gal-3(neg) tumors in mice had decelerated tumor growth after PBMC inoculation. In contrast, the Gal-3 positive((pos)) tumors continued growing despite PBMC inoculation, and tumor T regulatory cell (CD4/FoxP3+) infiltration increased. RNA sequencing of T cells from women with TNBC with elevated plasma levels of Gal-3 revealed significantly lower expression of oxidative phosphorylation genes than in T cells from healthy women. Similarly, in our in-vitro model, the decreased expression of oxidative phosphorylation genes and mitochondrial dysfunction resulted in a significant increase in CD8 intracellular reactive oxygen species. Consequently, T exhausted cells (CD8/PD1/Tim3/Lag3+) significantly increased in PBMC co-cultured with Gal-3(pos) TNBCs. To conclude, we revealed a novel TNBC-related Gal-3 suppressor mechanism that involved upregulation of CD4 T regulatory and of CD8 T exhausted cells.

Demyelination and axonal injury in chronic-progressive Multiple Sclerosis (MS) are presumed to be driven by a neurotoxic bystander effect of meningeal-based myeloid infiltrates. There is an unmet clinical need to attenuate disease progression in such forms of CNS-compartmentalized MS. The failure of systemic immune suppressive treatments has highlighted the need for neuroprotective and repair-inducing strategies. Here, we examined whether direct targeting of CNS myeloid cells and modulating their toxicity may prevent irreversible tissue injury in chronic immune-mediated demyelinating disease. To that end, we utilized the experimental autoimmune encephalomyelitis (EAE) model in Biozzi mice, a clinically relevant MS model. We continuously delivered intracerebroventricularly (ICV) a retinoic acid receptor alpha agonist (RARα), as a potent regulator of myeloid cells, in the chronic phase of EAE. We assessed disease severity and performed pathological evaluations, functional analyses of immune cells, and single-cell RNA sequencing on isolated spinal CD11b+ cells. Although initiating treatment in the chronic phase of the disease, the RARα agonist successfully improved clinical outcomes and prevented axonal loss. ICV RARα agonist treatment inhibited pro-inflammatory pathways and shifted CNS myeloid cells toward neuroprotective phenotypes without affecting peripheral infiltrating myeloid cell phenotypes, or peripheral immunity. The treatment regulated cell-death pathways across multiple myeloid cell populations and suppressed apoptosis, resulting in paradoxically marked increased neuroinflammatory infiltrates, consisting mainly of microglia and CNS / border-associated macrophages. This work establishes the notion of bystander neurotoxicity by CNS immune infiltrates in chronic demyelinating disease. Furthermore, it shows that targeting compartmentalized neuroinflammation by selective regulation of CNS myeloid cell toxicity and survival reduces irreversible tissue injury, and may serve as a novel disease-modifying approach.

BACKGROUND: Chicken embryos emerge from their shell by the piercing movement of the hatching muscle. Although considered a key player during hatching, with activity that imposes a substantial metabolic demand, data are still limited. The study provides a bioenergetic and transcriptomic analyses during the pre-post-hatching period. METHODS: Weight and morphology alongside content determination of creatine and glycogen were analysed. Transcriptome identified differentially expressed genes and enriched biological processes associated with hatching muscle development, catabolism, and energy provision. Using gene set enrichment, we followed the dynamics of gene-sets involved in energy pathways of oxidative phosphorylation, protein catabolism, glycolysis/gluconeogenesis, and glycogen metabolism. RESULTS: Results show several significant findings: (A) Creatine plays a crucial role in the energy metabolism of the hatching muscle, with its concentration remaining stable while glycogen concentration is depleted at hatch and placement. (B) The hatching muscle has the capacity for de-novo creatine synthesis, as indicated by the expression of related genes (AGAT, GAMT). (C) Transcriptome provided insights into genes related to energy pathways under conditions of pre-hatch oxygen and post-hatch glucose limitations (oxidative phosphorylation: NDUF, MT-ND, SDH, UQCR, COX, MT-CO, ATP5, MT-ATP; glycolysis/gluconeogenesis: FBP,G6PC, PFKM; glycogen metabolism: PPP1, PYGL, GYG1). (D) The post-hatch upregulation of protein catabolic processes genes (PSMA, RNF, UBE, FBX), which align with the muscle's weight dynamics, indicates a functional shift from movement during hatching to lifting the head during feeding. CONCLUSIONS: There is a dynamic metabolic switch in the hatching muscle during embryo-to-hatchling transition. When glycogen concentration depletes, energy supply is maintained by creatine and its de-novo synthesis. Understanding the hatching muscle's energy dynamics is crucial, for reducing hatching failures in endangered avian species, and in domesticated chickens.

Due to its capacity to drive osteoclast differentiation, the receptor activator of nuclear factor kappa-β ligand (RANKL) is believed to exert a pathological influence in periodontitis. However, RANKL was initially identified as an activator of dendritic cells (DCs), expressed by T cells, and exhibits diverse effects on the immune system. Hence, it is probable that RANKL, acting as a bridge between the bone and immune systems, plays a more intricate role in periodontitis. Using ligature-induced periodontitis (LIP), rapid alveolar bone loss was detected that was later halted even though the ligature was still present. This late phase of LIP was also linked with immunosuppressive conditions in the gingiva. Further investigation revealed that the ligature prompted an immediate migration of RANK-expressing Langerhans cells (LCs) and EpCAM DCs, the antigen-presenting cells (APCs) of the gingival epithelium, to the lymph nodes, followed by an expansion of T regulatory (Treg) cells in the gingiva. Subsequently, the ligatured gingiva was repopulated by monocyte-derived RANK-expressing EpCAM DCs, while gingival epithelial cells upregulated RANKL expression. Blocking RANKL signaling with monoclonal antibodies significantly reduced the frequencies of Treg cells in the gingiva and prevented gingival immunosuppression. In addition, RANKL signaling facilitated the differentiation of LCs from bone marrow precursors. To further investigate the role of RANKL, we used K14-RANKL mice, in which RANKL is overexpressed by gingival epithelial cells. The elevated RANKL expression shifted the steady-state frequencies of LCs and EpCAM DCs within the epithelium, favoring LCs over EpCAM DCs. Following ligature placement, heightened levels of Treg cells were observed in the gingiva of K14-RANKL mice, and alveolar bone loss was significantly reduced. These findings suggest that RANKL-RANK interactions between gingival epithelial cells and APCs are crucial for suppressing gingival inflammation, highlighting a protective immunological role for RANKL in periodontitis that was overlooked due to its osteoclastogenic activity.

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

The MAPK p38α was proposed to be a prominent promoter of skeletal muscle aging. The skeletal muscle tissue is composed of various muscle types, and it is not known if p38α is associated with aging in all of them. It is also not known if p38α is associated with aging of other tissues. JNK and ERK were also proposed to be associated with aging of several tissues. Nevertheless, the pattern of p38α, JNK, and ERK activity during aging was not documented. Here, we documented the levels of phosphorylated/active p38α, Erk1/2, and JNKs in several organs as well as the soleus, tibialis anterior, quadriceps, gastrocnemius, and EDL muscles of 1-, 3-, 6-, 13-, 18-, and 24-month-old mice. We report that in most tissues and skeletal muscles, the MAPKs' activity does not change in the course of aging. In most tissues and muscles, p38α is in fact active at younger ages. The quadriceps and the lungs are exceptions, where p38α is significantly active only in mice 13 months old or older. Curiously, levels of active JNK and ERKs are also elevated in aged lungs and quadriceps. RNA-seq analysis of the quadriceps during aging revealed downregulation of proteins related to the extra-cellular matrix (ECM) and ERK signaling. A panel of mRNAs encoding cell cycle inhibitors and senescence-associated proteins, considered to be aging markers, was not found to be elevated. It seems that the pattern of MAPKs' activation in aging, as well as expression of known 'aging' components, are tissue- and muscle type-specific, supporting a notion that the process of aging is tissue- and even cell-specific.

The postnatal interaction between microbiota and the immune system establishes lifelong homeostasis at mucosal epithelial barriers, however, the barrier-specific physiological activities that drive the equilibrium are hardly known. During weaning, the oral epithelium, which is monitored by Langerhans cells (LC), is challenged by the development of a microbial plaque and the initiation of masticatory forces capable of damaging the epithelium. Here we show that microbial colonization following birth facilitates the differentiation of oral LCs, setting the stage for the weaning period, in which adaptive immunity develops. Despite the presence of the challenging microbial plaque, LCs mainly respond to masticatory mechanical forces, inducing adaptive immunity, to maintain epithelial integrity that is also associated with naturally occurring alveolar bone loss. Mechanistically, masticatory forces induce the migration of LCs to the lymph nodes, and in return, LCs support the development of immunity to maintain epithelial integrity in a microbiota-independent manner. Unlike in adult life, this bone loss is IL-17-independent, suggesting that the establishment of oral mucosal homeostasis after birth and its maintenance in adult life involve distinct mechanisms.

Recent studies have highlighted the therapeutic potential of small extracellular bodies derived from mesenchymal stem cells (MSC-sEVs) for various diseases, notably through their ability to alter T-cell differentiation and function. The current study aimed to explore immunomodulatory pathway alterations within T cells through mRNA sequencing of activated T cells cocultured with bone marrow-derived MSC-sEVs. mRNA profiling of activated human T cells cocultured with MSC-sEVs or vehicle control was performed using the QIAGEN Illumina sequencing platform. Pathway networks and biological functions of the differentially expressed genes were analyzed using Ingenuity pathway analysis (IPA) software, KEGG pathway, GSEA and STRING database. A total of 364 differentially expressed genes were identified in sEV-treated T cells. Canonical pathway analysis highlighted the RhoA signaling pathway. Cellular development, movement, growth and proliferation, cell-to-cell interaction and inflammatory response-related gene expression were altered. KEGG enrichment pathway analysis underscored the apoptosis pathway. GSEA identified enrichment in downregulated genes associated with TNF alpha and interferon gamma response, and upregulated genes related to apoptosis and migration of lymphocytes and T-cell differentiation gene sets. Our findings provide valuable insights into the mechanisms by which MSC-sEVs implement immunomodulatory effects on activated T cells. These findings may contribute to the development of MSC-sEV-based therapies.

Non-alcoholic steatohepatitis (NASH) is an aggressive form of fatty liver disease with hepatic inflammation and fibrosis for which there is currently no drug treatment. This study determined whether an indoline derivative, AN1284, which significantly reduced damage in a model of acute liver disease, can reverse steatosis and fibrosis in mice with pre-existing NASH and explore its mechanism of action. The mouse model of dietary-induced NASH reproduces most of the liver pathology seen in human subjects. This was confirmed by RNA-sequencing analysis. The Western diet, given for 4 months, caused steatosis, inflammation, and liver fibrosis. AN1284 (1 mg or 5 mg/kg/day) was administered for the last 2 months of the diet by micro-osmotic-pumps (mps). Both doses significantly decreased hepatic damage, liver weight, hepatic fat content, triglyceride, serum alanine transaminase, and fibrosis. AN1284 (1 mg/kg/day) given by mps or in the drinking fluid significantly reduced fibrosis produced by carbon tetrachloride injections. In human HUH7 hepatoma cells incubated with palmitic acid, AN1284 (2.1 and 6.3 ng/ml), concentrations compatible with those in the liver of mice treated with AN1284, decreased lipid formation by causing nuclear translocation of the aryl hydrocarbon receptor (AhR). AN1284 downregulated fatty acid synthase (FASN) and sterol regulatory element-binding protein 1c (SREBP-1c) and upregulated Acyl-CoA Oxidase 1 and Cytochrome P450-a1, genes involved in lipid metabolism. In conclusion, chronic treatment with AN1284 (1mg/kg/day) reduced pre-existing steatosis and fibrosis through AhR, which affects several contributors to the development of fatty liver disease. Additional pathways are also influenced by AN1284 treatment.

Universal Minicircle Sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind the single-stranded G-rich UMS sequence, conserved at the replication origins of minicircles in the kinetoplast DNA, the mitochondrial genome of kinetoplastids. Trypanosoma brucei UMSBP2 has been recently shown to colocalize with telomeres and to play an essential role in chromosome end protection. Here we report that TbUMSBP2 decondenses in vitro DNA molecules, which were condensed by core histones H2B, H4 or linker histone H1. DNA decondensation is mediated via protein-protein interactions between TbUMSBP2 and these histones, independently of its previously described DNA binding activity. Silencing of the TbUMSBP2 gene resulted in a significant decrease in the disassembly of nucleosomes in T. brucei chromatin, a phenotype that could be reverted, by supplementing the knockdown cells with TbUMSBP2. Transcriptome analysis revealed that silencing of TbUMSBP2 affects the expression of multiple genes in T. brucei, with a most significant effect on the upregulation of the subtelomeric variant surface glycoproteins (VSG) genes, which mediate the antigenic variation in African trypanosomes. These observations suggest that UMSBP2 is a chromatin remodeling protein that functions in the regulation of gene expression and plays a role in the control of antigenic variation in T. brucei.

Systemic light chain amyloidosis (AL) is a clonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL producing PC lines and to use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LCs from patients suffering from AL amyloidosis. The AL LC producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma (MM) LC producing cells. Using RNAsequencing the AL LC producing lines showed higher mitochondrial oxidative stress, decreased activity of the myc and cholesterol pathways. The neoplastic behavior of PCs is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate AL's unique cellular pathways, thus expediting the development of specific treatments for AL patients.

Obstructive sleep apnea (OSA) is a highly prevalent condition, characterized by intermittent hypoxia (IH), sleep disruption, and altered autonomic nervous system function. OSA has been independently associated with dyslipidemia, insulin resistance, and metabolic syndrome. Brown adipose tissue (BAT) has been suggested as a modulator of systemic glucose tolerance through adaptive thermogenesis. Reductions in BAT mass have been associated with obesity and metabolic syndrome. No studies have systematically characterized the effects of chronic IH on BAT. Thus, we aimed to delineate IH effects on BAT and concomitant metabolic changes. C57BL/6J 8-week-old male mice were randomly assigned to IH during sleep (alternating 90 s cycles of 6.5% FO followed by 21% FO) or normoxia (room air, RA) for 10 weeks. Mice were subjected to glucose tolerance testing and F-FDG PET-MRI towards the end of the exposures followed by BAT tissues analyses for morphological and global transcriptomic changes. Animals exposed to IH were glucose intolerant despite lower total body weight and adiposity. BAT tissues in IH-exposed mice demonstrated characteristic changes associated with "browning"-smaller lipids, increased vascularity, and a trend towards higher protein levels of UCP1. Conversely, mitochondrial DNA content and protein levels of respiratory chain complex III were reduced. Pro-inflammatory macrophages were more abundant in IH-exposed BAT. Transcriptomic analysis revealed increases in fatty acid oxidation and oxidative stress pathways in IH-exposed BAT, along with a reduction in pathways related to myogenesis, hypoxia, and IL-4 anti-inflammatory response. Functionally, IH-exposed BAT demonstrated reduced absorption of glucose on PET scans and reduced phosphorylation of AKT in response to insulin. Current studies provide initial evidence for the presence of a maladaptive response of interscapular BAT in response to chronic IH mimicking OSA, resulting in a paradoxical divergence, namely, BAT browning but tissue-specific and systemic insulin resistance. We postulate that oxidative stress, mitochondrial dysfunction, and inflammation may underlie these dichotomous outcomes in BAT.

Biological sex is a fundamental trait influencing development, reproduction, pathogenesis, and medical treatment outcomes. Modeling sex differences is challenging because of the masking effect of genetic variability and the hurdle of differentiating chromosomal versus hormonal effects. In this work we developed a cellular model to study sex differences in humans. Somatic cells from a mosaic Klinefelter syndrome patient were reprogrammed to generate isogenic induced pluripotent stem cell (iPSC) lines with different sex chromosome complements: 47,XXY/46,XX/46,XY/45,X0. Transcriptional analysis of the hiPSCs revealed novel and known genes and pathways that are sexually dimorphic in the pluripotent state and during early neural development. Female hiPSCs more closely resembled the naive pluripotent state than their male counterparts. Moreover, the system enabled differentiation between the contributions of X versus Y chromosome to these differences. Taken together, isogenic hiPSCs present a novel platform for studying sex differences in humans and bear potential to promote gender-specific medicine in the future.