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Chapter 4: Differential diagnosis of primary hyperparathyroidism.
No full textInternational audienceThe differential diagnosis of primary hyperparathyroidism can be considered clinically, biologically and radiologically. Clinically, primary hyperparathyroidism should be suspected in case of diffuse pain, renal lithiasis, osteoporosis, repeated fracture, cognitive or psychiatric disorder, or disturbance of consciousness. Nevertheless, the differential diagnosis of primary hyperparathyroidism is mainly biological, particularly in atypical forms, which must be differentiated from hypercalcemia with hypocalciuria or non-elevated PTH on the one hand, and from normo-calcemia with elevated PTH, hypophosphatemia or hypercalciuria on the other. Any differential diagnosis must be preceded by an analysis of the factors likely to disturb phospho-calcium parameters: vitamin D deficiency (assay), renal insufficiency (eGFR measurement), malabsorption (inflammatory disease of the digestive tract, celiac disease, bariatric surgery, etc.), insufficient calcium intake (GRIO questionnaire) and iatrogenic causes (diuretics, anti-osteoporotic drugs, excessive vitamin D or calcium supplementation, lithium, corticosteroid therapy, phosphorus intake). Once these factors have been eliminated, hypercalcemia with hypocalciuria should suggest a genetic cause. Hypercalcemia with non-elevated PTH may be secondary to neoplasm, hypervitaminosis D (excessive intake, production or catabolism), immobilization or endocrine causes. Elevated PTH values without hypercalcemia must be differentiated from normo-calcemic hyperparathyroidism. High PTH levels are found in PTH-resistant patients, as well as in hypophosphatemic (especially X-linked) or hypercalciuric tubulopathies (certain rare diseases, immobilization, loop diuretics or idiopathic causes favored by a metabolic syndrome). Radiologically, brown tumor must be differentiated primarily from bone metastasis, chondrosarcoma and giant cell tumor
HIV-1 cell-to-cell infection of macrophages escapes type I interferon and host restriction factors, and is resistant to antiretroviral drugs
No full textInternational audienceHIV-1-infected macrophages participate in viral transmission, dissemination, and establishment of tissue virus reservoirs. Despite counteracting viral proteins (Vif, Vpu, Vpr and Nef), cell-free virus macrophage infection is restricted by host cell factors, including those induced by interferons. Here, we show that these viral proteins and type I interferon do not influence HIV-1 cell-to-cell transfer to macrophages by cell-cell fusion with infected T cells, still leading to the formation of multinucleated giant cells (MGCs). Accordingly, depletion of SERINC5 and APOBEC3G do not alter virus spreading and formation of virus-producing MGCs. We further show that the nuclei derived from infected T cells remains transcriptionally active in MGCs and may explain resistance to restriction factors and antiretroviral drugs. Unexpectedly, we detect viral DNA in myeloid nuclei shortly after the initial fusion with macrophages. Together, these findings unravel how HIV-1 macrophage infection by cell-cell fusion escapes type I interferon and cellular restriction factors independently of the viral auxiliary proteins, while displaying resistance to antiretroviral drugs
Bringing malaria diagnosis and treatment closer to the people: economic rationale for expanding malaria community case management to all ages in a rural district in Madagascar
No full textInternational audienceBackground Expanding malaria community case management (mCCM) to all ages could shift the point-ofcare to the community leading to improved healthcare access in underserved populations. This study assesses the economic viability of such an expansion in Farafangana district, Madagascar.Methods A cluster-randomized trial was conducted across 30 health centres and the 502 community health workers (CHW) in their catchment areas, with the intervention arm implementing the age-expanded mCCM intervention. CHWs across both arms received training, supplies, and supervision to manage malaria. An economic evaluation assessed cost-effectiveness from health sector and societal perspectives, measuring outcomes in disability-adjusted life years (DALYs) averted. The impact of CHW compensation and economic risks were evaluated using sensitivity analyses. ResultsWithout CHW compensation, annual costs were 21.86 to 135.64, and -1,172,283 in net economic benefits. Sensitivity analyses supported these findings.Conclusions Age-expanded mCCM is highly cost-effective and can enhance malaria treatment access in resourcelimited settings.</div
Monitoring molecular markers associated with antimalarial drug resistance in south-east Senegal from 2021 to 2023
No full textInternational audienceBackground Since 2006, artemisinin-based combination therapies (ACTs) have been introduced in Senegal in response to chloroquine resistance (CQ-R) and have shown high efficacy against Plasmodium falciparum. However, the detection of the PfKelch13R515K mutation in Kaolack, which confers artemisinin resistance in vitro, highlights the urgency of strengthening antimalarial drug surveillance to achieve malaria elimination by 2030. Objective To assess the proportion of P. falciparum parasites carrying molecular signatures associated with antimalarial resistance (PfKelch13, Pfmdr1, Pfcrt, dhfr and dhps) in isolates collected at Kédougou using multiplex amplicon deep sequencing. Methods Venous blood samples were collected from patients diagnosed with P. falciparum infection over a 3-year period (2021, 2022 and 2023). Parasite DNA was extracted, and multiplex amplicon sequencing was used to investigate gene polymorphisms. Results Analysis of PfKelch13 did not reveal any non-synonymous mutations. Pfcrt mutations were present in 45% of the samples, mainly K76T (44%) and I356T (36%). The dominant Pfmdr-1 allele was Y184F (62%). The sextuple mutant 51I/59R/108N + 436A/437G/613S dhfr/dhps was observed in 10% of the samples. Conclusion The absence of PfKelch13 mutants suggests that ACT efficacy remains uncompromised, although clinical outcome studies are required to confirm this. Analysis of Pfcrt and Pfmdr-1 shows that CQ-R alleles, probably from previous CQ use, are slowly decreasing. Likewise, the detection of the dhfr/dhps sextuple mutant highlights the need to monitor sulfadoxine-pyrimethamine resistance and the emergence of 581G. There is therefore a need for continued antimalarial resistance surveillance in Senegal
Potency of all-D amino acid antimicrobial peptides derived from the bovine rumen microbiome on tuberculous and non-tuberculous mycobacteria
No full textInternational audienceDespite the availability of antibiotics, tuberculosis (TB), caused by Mycobacterium tuberculosis, was once again declared the world's leading cause of death from a single infectious agent in 2023. Furthermore, the rising prevalence of drug-resistant strains of M. tuberculosis, coupled with the limitations of existing therapeutics, underscores the urgent need for new antimicrobial agents that act through different mechanisms, thereby providing novel therapeutic options. From this perspective, antimicrobial peptides (AMPs) derived from the bovine rumen microbiome have shown promise against many resistant pathogens and may therefore offer a promising alternative against TB. Here, we evaluated the efficacy of AMPs from bovine rumen microbiome, namely the Lynronne 1, 2 & 3 and P15s as well as their all-D amino acid enantiomers, against non-tuberculous (M. abscessus, M. marinum and M. smegmatis) and tuberculous (M. bovis BCG, M. tuberculosis) mycobacteria. In particular, their antimycobacterial activity was assessed against extracellularly and intracellularly replicating M. tuberculosis H37Rv pathogenic strain. Their innocuity was further studied by determining their respective cytotoxicity against human cell lines and hemolytic activity on human erythrocytes. Finally, their mechanism of action was investigated by a membrane permeabilization assay and a lipid insertion assay via surface pressure measurement. Although all-D enantiomers showed increased cytotoxicity to human cell lines, they still offer a good therapeutic window with improved activity compared to their L-form counterparts, especially Lynronne 2Dall and P15sDall which emerged as the best growth inhibitors of all mycobacteria. Remarkably, the all-D enantiomers also demonstrated activity against intramacrophagic replicating M. tuberculosis H37Rv, with very limited toxicity towards human cells and no hemolytic activity at their respective minimum inhibitory concentration. Membrane permeabilization and monolayer lipid insertion assays suggested that these peptides mostly act by insertion into the mycobacterial membrane resulting in a rapid membranolytic effect. These findings highlight the potential of the all-D enantiomers of Lynronne peptides, as attractive candidates for the development of new anti-TB drugs. Their effective antibacterial properties combined with low toxicity underscore Lynronne 2Dall and P15sDall as building blocks for the development of promising alternatives to conventional antibiotics in the treatment of mycobacterial infections, particularly against M. tuberculosis
Development of a permethylation-based detection method for oligo/ polysialic acid structures via tandem MALDI-TOF mass spectrometry
No full textInternational audienceOligo/polysialic acid (oligo/polySia) refers to a class of linear polymers composed of Sias, found extensively at the non-reducing ends of glycans across various organisms, from bacteria to vertebrates. Different types of oligo/polySia have been characterized, varying in Sia composition, α-ketosidic linkages to penultimate Sia residues, and degree of polymerization. Detection of oligo/polySia-capped glycans is typically achieved through western blotting or ELISA using specific antibodies or endo-N-acylneuraminidase. Additionally, a sensitive chemical approach involving the direct labeling of oligo/polySia with 1,2-diamino-4,5-methylenedioxybenzene, followed by separation and quantification via anion-exchange high-performance liquid chromatography, has been developed. However, detection through mass spectrometry (MS) has remained challenging due to the multiple negative charges and structural instability of these glycans. In this study, we applied a permethylation technique to convert carboxyl group of Sias into methyl esters, thereby neutralizing their negative charges, which allowed for improved MS analysis of sialylated glycans. We optimized both the permethylation and glycan purification processes. Consequently, we achieved the first successful detection of polySia structures using MALDI-TOF MS. There are more in-source decay ions in the MS profiling of permethylated α2,9-linked polySias compared to α2,8-linked polySias. Furthermore, we present the first examples of oligo/polySia sequencing by collision-induced dissociation on a TOF/TOF instrument, enabling us to differentiate between α2,8- and α2,9-linked polySias. We extended this approach to analyze oligo/polySia structures with O-linked glycans in polysialoglycoprotein from salmonid eggs. For the first time, the analysis of intact oligo/polySia structures with O-linked glycans were successfully detected using MALDI-TOF/TOF MS/MS with this optimized permethylation method
Conformation-Driven Phase Separation in the Linker Domain of Focal Adhesion Kinases
No full textInternational audienceProtein tyrosine kinase 2 (Pyk2), also known as focal adhesion kinase 2, and focal adhesion kinase 1 (Fak1) are two related non-receptor tyrosine kinases (hereafter referred to as FAKs). Here, we focused on characterizing a linker region of the FAK proteins (hereafter referred to as FAK KFL for Kinase FAT Linker), which in the case of Pyk2 has previously been shown to play a functional role in calcium sensing through its interaction with calmodulin. Using structural nuclear magnetic resonance spectroscopy, we provide chemical shift assignments for the FAK KFLs, defining their conformational properties. Analysis of the FAK KFL conformations revealed their predominantly disordered nature, except for well-defined segments with a significant tendency to form -helices, which were modeled to form homo-dimeric interfaces. In addition, we showed that the FAK KFL segments form condensates in vitro under high crowding conditions. By directly comparing the conformational properties of the Pyk2 and Fak1 KFL domains and providing structural data, this study provides valuable insights into the structural basis of FAK KFL interactions. Furthermore, the results show that disordered segments in proteins within the focal adhesion complex undergo phase separation, a process of potential biological significance due to protein clustering
Unveiling the Molecular Legacy of Transient Insulin Resistance: Implications for Hepatic Metabolic Adaptability
No full textInternational audienceBackground: Metabolic flexibility (MetF) is an organism's ability to adjust to changing metabolic supplies and energy demands. Insulin plays a central role in coordinating MetF through molecular mechanisms such as signaling pathways, transcriptional responses, and circadian regulation. Insulin resistance (IR) can impair MetF, contributing to type 2 diabetes and obesity, often stemming from continuous challenges such as sedentary lifestyles, poor diets, and circadian disruptions. Transient IR episodes, like gestational diabetes or stress-induced hyperglycemia, also heighten the risk of later diabetes development. Yet, the molecular processes post-transient IR remain poorly understood despite their health significance.Aims and Methods: Our aims were to characterize the hepatic response to a high fat diet challenge in mice previously exposed to a transient IR episode. We integrated transcriptomic, epigenomic, lipidomic, and molecular clock assessments to provide a molecular basis for the observed dysregulations.Results: Our study shows that temporarily blocking the insulin receptor in young mice leads to laterlife liver issues hindering PPARα-mediated adaptation to a high-fat diet. This is linked to decreased histone active marks at PPARα sites and reduced endogenous PPARα ligands. Transient insulin receptor blockade also altered the liver's molecular clock, particularly affecting PPARα transcriptional responsiveness.Conclusions: Seemingly reversible and unnoticed metabolic challenges in early adulthood may predispose the liver to exacerbated metabolic dysfunctions when confronted with chronic challenges later in life.</p
Hepatitis E Virus-induced antiviral response by plasmacytoid dendritic cells is modulated by the ORF2 protein
No full textInternational audienceType I and III interferons (IFN-I/III) are critical to protect the host during viral infection. IFN-mediated antiviral responses against hepatitis E virus (HEV) are suppressed and defeated by viral escape mechanisms at play in infected hepatocytes. Here, we studied the anti-HEV function of IFN secreted by plasmacytoid dendritic cells (pDCs), which are specialized producers of IFNs. We showed that pDCs co-cultured with HEV-infected cells secreted IFN in a cell-to-cell contact-dependent manner. Pharmacological inhibitor and antibodies targeting contact proteins revealed that pDC response against HEV required the endosomal nucleic-acid sensor TLR7 and adhesion molecules, such as ICAM-I and α L β 2 -integrin. IFNs secreted by pDCs reduced viral spread. Intriguingly, ORF2, the capsid protein of HEV, can be produced in various forms by the infected cells. During infection, a fraction of the intracellular ORF2 protein localizes into the nucleus while another ORF2 fraction packages viral genomes to produce infectious virions. In parallel, glycosylated forms of ORF2 are also massively secreted by infected cells. Using viral genome expressing ORF2 mutants, we showed that glycosylated ORF2 forms contribute to better recognition of infected cells by pDCs by regulating contacts between infected cells and pDCs. ORF2 forms may thus modulate pDC-mediated anti-HEV response. Together, our results suggest that liver-resident pDCs, which exhibit comparable IFN-producing ability as blood-derived pDCs, may be essential to control HEV replication
