C16ORF70/Mytho promotes healthy ageing in C. elegans and prevents cellular senescence in mammals.

The identification of genes that confer either extension of lifespan or accelerate age-related decline was a step forward in understanding the mechanisms of ageing and revealed that it is partially controlled by genetics and transcriptional programs. Here we discovered that the human DNA sequence C16ORF70 encoded for a protein, named MYTHO (Macroautophagy and YouTH Optimizer), which controls life- and health-span. MYTHO protein is conserved from C. elegans to humans and its mRNA was upregulated in aged mice and elderly people. Deletion of the ortholog myt-1 gene in C. elegans dramatically shortened lifespan and decreased animal survival upon exposure to oxidative stress. Mechanistically, MYTHO is required for autophagy likely because it acts as a scaffold that binds WIPI2 and BCAS3 to recruit and assemble the conjugation system at the phagophore, the nascent autophagosome. We conclude that MYTHO is a transcriptionally regulated initiator of autophagy that is central in promoting stress resistance and healthy ageing.

Anti SARS-CoV-2 Monoclonal Antibodies in Pre-Exposure or Post-Exposure in No- or Weak Responder to Vaccine Kidney Transplant Recipients: Is One Strategy Better than Another?

Background: Kidney transplant recipients (KTRs) are likely to develop severe COVID-19 and are less well-protected by vaccines than immunocompetent subjects. Thus, the use of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) to confer a passive immunity appears attractive in KTRs. Methods: This retrospective monocentric cohort study was conducted between 1 January 2022 and 30 September 2022. All KTRs with a weak antibody response one month after three doses of mRNA vaccine (anti spike IgG < 264 (BAU/mL)) have received tixagevimab-cilgavimab in pre-exposure (group 1), post-exposure (group 2) or no specific treatment (group 3). We compared COVID-19 symptomatic hospitalizations, including intensive care unit hospitalizations, oxygen therapy, and death, between the three groups. Results: A total of 418 KTRs had SARS-CoV-2 infection in 2022. During the study period, we included 112 KTRs in group 1, 40 KTRs in group 2, and 27 KTRs in group 3. The occurrence of intensive care unit hospitalization, oxygen therapy, and COVID-19 death was significantly increased in group 3 compared to group 1 or 2. In group 3, 5 KTRs (18.5%) were admitted to the intensive care unit, 7 KTRs (25.9%) needed oxygen therapy, and 3 KTRs (11.1%) died. Patients who received tixagevimab-cilgavimab pre- or post-exposure had similar outcomes. Conclusions: This retrospective real-life study supports the relative effectiveness of tixagevimab-cilgavimab on COVID-19 infection caused by Omicron, used as a pre- or post-exposure therapy. The continued evolution of Omicron variants has made tixagevimab-cilgavimab ineffective and reinforces the need for new therapeutic monoclonal antibodies for COVID-19 active on new variants.

Human adipose tissue as a major reservoir of cytomegalovirus-reactive T cells.

Introduction: Cytomegalovirus (CMV) is a common herpesvirus with a high prevalence worldwide. After the acute infection phase, CMV can remain latent in several tissues. CD8 T cells in the lungs and salivary glands mainly control its reactivation control. White adipose tissue (WAT) contains a significant population of memory T cells reactive to viral antigens, but CMV specificity has mainly been studied in mouse WAT. Therefore, we obtained blood, omental WAT (oWAT), subcutaneous WAT (sWAT), and liver samples from 11 obese donors to characterize the human WAT adaptive immune landscape from a phenotypic and immune receptor specificity perspective. Methods: We performed high-throughput sequencing of the T cell receptor (TCR) locus to analyze tissue and blood TCR repertoires of the 11 donors. The presence of TCRs specific to CMV epitopes was tested through ELISpot assays. Moreover, phenotypic characterization of T cells was carried out through flow cytometry. Results: High-throughput sequencing analyses revealed that tissue TCR repertoires in oWAT, sWAT, and liver samples were less diverse and dominated by hyperexpanded clones when compared to blood samples. Additionally, we predicted the presence of TCRs specific to viral epitopes, particularly from CMV, which was confirmed by ELISpot assays. Remarkably, we found that oWAT has a higher proportion of CMV-reactive T cells than blood or sWAT. Finally, flow cytometry analyses indicated that most WAT-infiltrated lymphocytes were tissue-resident effector memory CD8 T cells. Discussion: Overall, these findings postulate human oWAT as a major reservoir of CMV-specific T cells, presumably for latent viral reactivation control. This study enhances our understanding of the adaptive immune response in human WAT and highlights its potential role in antiviral defense.

PHAF1/MYTHO is a novel autophagy regulator that controls muscle integrity.

Skeletal muscles play key roles in movement, posture, thermogenesis, and whole-body metabolism. Autophagy plays essential roles in the regulation of muscle mass, function and integrity. However, the molecular machinery that regulates autophagy is still incompletely understood. In our recent study, we identified and characterized a novel Forkhead Box O (FoxO)-dependent gene, PHAF1/MYTHO (phagophore assembly factor 1/macro-autophagy and youth optimizer), as a novel autophagy regulator that controls muscle integrity. MYTHO/PHAF1 is upregulated in multiple conditions leading to muscle atrophy, and downregulation of its expression spares muscle atrophy triggered by fasting, denervation, cachexia and sepsis. Overexpression of PHAF1/MYTHO is sufficient to induce muscle atrophy. Prolonged downregulation of PHAF1/MYTHO causes a severe myopathic phenotype, which is characterized by impaired autophagy, muscle weakness, myofiber degeneration, mammalian target of rapamycin complex 1 (mTORC1) hyperactivation and extensive ultrastructural defects, such as accumulation of proteinaceous and membranous structures and tubular aggregates. This myopathic phenotype is attenuated upon administration of the mTORC1 inhibitor rapamycin. These findings position PHAF1/MYTHO as a novel regulator of skeletal muscle autophagy and tissue integrity.

MYTHO is a novel regulator of skeletal muscle autophagy and integrity.

Autophagy is a critical process in the regulation of muscle mass, function and integrity. The molecular mechanisms regulating autophagy are complex and still partly understood. Here, we identify and characterize a novel FoxO-dependent gene, d230025d16rik which we named Mytho (Macroautophagy and YouTH Optimizer), as a regulator of autophagy and skeletal muscle integrity in vivo. Mytho is significantly up-regulated in various mouse models of skeletal muscle atrophy. Short term depletion of MYTHO in mice attenuates muscle atrophy caused by fasting, denervation, cancer cachexia and sepsis. While MYTHO overexpression is sufficient to trigger muscle atrophy, MYTHO knockdown results in a progressive increase in muscle mass associated with a sustained activation of the mTORC1 signaling pathway. Prolonged MYTHO knockdown is associated with severe myopathic features, including impaired autophagy, muscle weakness, myofiber degeneration, and extensive ultrastructural defects, such as accumulation of autophagic vacuoles and tubular aggregates. Inhibition of the mTORC1 signaling pathway in mice using rapamycin treatment attenuates the myopathic phenotype triggered by MYTHO knockdown. Skeletal muscles from human patients diagnosed with myotonic dystrophy type 1 (DM1) display reduced Mytho expression, activation of the mTORC1 signaling pathway and impaired autophagy, raising the possibility that low Mytho expression might contribute to the progression of the disease. We conclude that MYTHO is a key regulator of muscle autophagy and integrity.

In Obese Patients With Type 2 Diabetes, Mast Cells in Omental Adipose Tissue Decrease the Surface Expression of CD45, CD117, CD203c, and FcϵRI.

The paradigm of mast cells in type 2 diabetes is changing. Although they were first considered deleterious inflammatory cells, now they seem to be important players driving adipose tissue homeostasis. Here we have employed a flow cytometry-based approach for measuring the surface expression of 4 proteins (CD45, CD117, CD203c, and FcϵRI) on mast cells of omental (o-WAT) and subcutaneous white adipose tissue (s-WAT) in a cohort of 96 patients with morbid obesity. The cohort was split into three groups: non-T2D, pre-T2D, and T2D. Noteworthy, patients with T2D have a mild condition (HbA1c <7%). In o-WAT, mast cells of patients with T2D have a decrease in the surface expression of CD45 (p=0.0013), CD117 (p=0.0066), CD203c (p=0.0025), and FcϵRI (p=0.043). Besides, in s-WAT, the decrease was seen only in CD117 (p=0.046). These results indicate that T2D affects more to mast cells in o-WAT than in s-WAT. The decrease in these four proteins has serious effects on mast cell function. CD117 is critical for mast cell survival, while CD45 and FcϵRI are important for mast cell activation. Additionally, CD203c is only present on the cell surface after granule release. Taking together these observations, we suggest that mast cells in o-WAT of patients with T2D have a decreased survival, activation capacity, and secretory function.

Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants.

BACKGROUND: Lipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments. RESULTS: So far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction. CONCLUSIONS: The expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery.

Prognostic Value of C4d Immunolabelling in Adult Patients With IgA Vasculitis.

Background and Objectives: Glomerular C4d deposits are associated the severity and outcomes of IgA nephropathy. Whether this holds true in immunoglobulin A vasculitis (IgAV) is not known. The main objective of the study was to analyze the prognostic value of glomerular C4d immunolabelling on kidney impairment in adults with IgAV. Design, Setting, Participants, Measurements: This retrospective cohort study included 120 adults with IgAV and a kidney biopsy performed between 1995 and 2018 in two French university hospital centers. All paraffin-embedded biopsies were reassessed according to Oxford classification. Immunofluorescence for C4d was performed in all cases. For analysis, patients were grouped according to positivity for C4d in the glomerular area. The main outcome was a composite endpoint of 50% increase in 24 h-proteinuria, or eGFR decrease by 50%, or kidney replacement therapy. Results: The median follow-up was 28.3 months. Twenty-three patients met the composite endpoint, 12 for kidney replacement therapy, 6 for an eGFR decrease >50% and 5 for a >50% increase in proteinuria. At time of biopsy, the median proteinuria was 1.9 g/24 h and the median eGFR 73.5 mL/min/1.73 m2. Among the 102 patients evaluable for C4d, 24 were positive on >30% glomeruli, mainly with a parieto-mesangial pattern. In this group, the initial proteinuria was more frequently nephrotic than in the C4d- group (60% vs. 33%, P = 0.039). Mesangial hypercellularity was more frequent in the C4d+ group (42% vs. 13%; P = 0.006) whereas macroscopic hematuria was more frequent in the C4d- group (18% vs. 0%; P = 0.03). After a median follow-up of 28 months, kidney survival did not differ according to C4d status. Conclusion: In a population of adult IgAV patients, glomerular positivity for C4d was associated with the severity of the kidney disease at presentation, but not with subsequent renal function deterioration.

The Autophagy, Inflammation and Metabolism Center international eSymposium - an early-career investigators' seminar series during the COVID-19 pandemic.

The Autophagy, Inflammation and Metabolism (AIM) Center organized a globally accessible, virtual eSymposium during the COVID-19 pandemic in 2020. The conference included presentations from scientific leaders, as well as a career discussion panel, and provided a much-needed platform for early-career investigators (ECIs) to showcase their research in autophagy. This Perspective summarizes the science presented by the ECIs during the event and discusses the lessons learned from a virtual meeting of this kind during the pandemic. The meeting was a learning experience for all involved, and the ECI participants herein offer their thoughts on the pros and cons of virtual meetings as a modality, either as standalone or hybrid events, with a view towards the post-pandemic world.

Role of autophagy in muscle disease.

Beside inherited muscle diseases many catabolic conditions such as insulin resistance, malnutrition, cancer growth, aging, infections, chronic inflammatory status, inactivity, obesity are characterized by loss of muscle mass, strength and function. The decrease of muscle quality and quantity increases morbidity, mortality and has a major impact on the quality of life. One of the pathogenetic mechanisms of muscle wasting is the dysregulation of the main protein and organelles quality control system of the cell: the autophagy-lysosome. This review will focus on the role of the autophagy-lysosome system in the different conditions of muscle loss. We will also dissect the signalling pathways that are involved in excessive or defective autophagy regulation. Finally, the state of the art of autophagy modulators that have been used in preclinical or clinical studies to ameliorate muscle mass will be also described.

In Patients With Obesity, the Number of Adipose Tissue Mast Cells Is Significantly Lower in Subjects With Type 2 Diabetes.

Type 2 diabetes (T2D) is a rising global health problem mainly caused by obesity and a sedentary lifestyle. In healthy individuals, white adipose tissue (WAT) has a relevant homeostatic role in glucose metabolism, energy storage, and endocrine signaling. Mast cells contribute to these functions promoting WAT angiogenesis and adipogenesis. In patients with T2D, inflammation dramatically impacts WAT functioning, which results in the recruitment of several leukocytes, including monocytes, that enhance this inflammation. Accordingly, the macrophages population rises as the WAT inflammation increases during the T2D status worsening. Since mast cell progenitors cannot arrive at WAT, the amount of WAT mast cells depends on how the new microenvironment affects progenitor and differentiated mast cells. Here, we employed a flow cytometry-based approach to analyze the number of mast cells from omental white adipose tissue (o-WAT) and subcutaneous white adipose tissue (s-WAT) in a cohort of 100 patients with obesity. Additionally, we measured the number of mast cell progenitors in a subcohort of 15 patients. The cohort was divided in three groups: non-T2D, pre-T2D, and T2D. Importantly, patients with T2D have a mild condition (HbA1c <7%). The number of mast cells and mast cell progenitors was lower in patients with T2D in both o-WAT and s-WAT in comparison to subjects from the pre-T2D and non-T2D groups. In the case of mast cells in o-WAT, there were statistically significant differences between non-T2D and T2D groups (p = 0.0031), together with pre-T2D and T2D groups (p=0.0097). However, in s-WAT, the differences are only between non-T2D and T2D groups (p=0.047). These differences have been obtained with patients with a mild T2D condition. Therefore, little changes in T2D status have a huge impact on the number of mast cells in WAT, especially in o-WAT. Due to the importance of mast cells in WAT physiology, their decrease can reduce the capacity of WAT, especially o-WAT, to store lipids and cause hypoxic cell deaths that will trigger inflammation.

Cytometric analysis of adipose tissue reveals increments of adipocyte progenitor cells after weight loss induced by bariatric surgery.

Obesity-related comorbidities are, in large part, originated from the dysfunction of adipose tissue. Most of them revert after the normalization of body mass. Adipose tissue is essentially occupied by adipocytes. However, different populations of immunological cells and adipocyte precursor cells (AdPCs) are the main cellular components of tissue. During obesity, body fat depots acquire a low-level chronic inflammation and adipocytes increase in number and volume. Conversely, weight loss improves the inflammatory phenotype of adipose tissue immune cells and reduces the volume of adipocytes. Nevertheless, very little is known about the evolution of the human AdPCs reservoir. We have developed a flow cytometry-based methodology to simultaneously quantify the main cell populations of adipose tissue. Starting from this technical approach, we have studied human adipose tissue samples (visceral and subcutaneous) obtained at two different physiological situations: at morbid obesity and after bariatric surgery-induced weight loss. We report a considerable increase of the AdPCs reservoir after losing weight and several changes in the immune cells populations of adipose tissue (mast cells increase, neutrophils decrease and macrophages switch phenotype). No changes were observed for T-lymphocytes, which are discussed in the context of recent findings.