ABSTRACT
Tissue clearing is an old-fashioned method developed in the 1900's and used to turn an opaque biological object into a 3D visualizable transparent structure. Developed and diversified over the last decade, this method is most of the time applied to mammals' tissues, and especially mouse and human tissues for cytological, histological and pathophysiological studies. Through autofluorescence, immunofluorescence, in situ hybridization, intercalating agents, fluorescent transfection markers or fluorescent particle uptake, optically cleared samples can be monitored to discover new biological structures and cellular interactions through 3D-visualization, which can be more challenging in some extend through classical histological methods. Most of the tissue clearing procedures have been developed for specific applications like endogenous fluorescence visualization, immunolabeling or for revealing specific organs. Thus, choosing the adapted protocol may be empirical for non-model species, especially for mollusks for which very little related literature is available. Herein, we suggest an effective optical tissue clearing procedure for the freshwater snail Biomphalaria glabrata, known as the intermediate host of the human parasite Schistosoma mansoni. This clearing procedure involves solvents with a minimal toxicity, preserves the endogenous fluorescence of labeled parasites inside snail tissues and is compatible with an immunolabeling procedure.
ABSTRACT
BACKGROUND AND OBJECTIVES: The aim of this study was to identify novel biomarkers for multiple sclerosis (MS) diagnosis and prognosis, addressing the critical need for specific and prognostically valuable markers in the field. METHODS: We conducted an extensive proteomic investigation, combining analysis of (1) CSF proteome from symptomatic controls, fast and slow converters after clinically isolated syndromes, and patients with relapsing-remitting MS (n = 10 per group) using label-free quantitative proteomics and (2) oligodendrocyte secretome changes under proinflammatory or proapoptotic conditions using stable isotope labeling by amino acids in cell culture. Proteins exhibiting differential abundance in both proteomic analyses were combined with other putative MS biomarkers, yielding a comprehensive list of 87 proteins that underwent quantification through parallel reaction monitoring (PRM) in a novel cohort, comprising symptomatic controls, inflammatory neurologic disease controls, and patients with MS at various disease stages (n = 10 per group). The 11 proteins that passed this qualification step were subjected to a new PRM assay within an expanded cohort comprising 158 patients with either MS at different disease stages or other inflammatory or noninflammatory neurologic disease controls. RESULTS: This study unveiled a promising biomarker signature for MS, including previously established candidates, such as chitinase 3-like protein 1, chitinase 3-like protein 2, chitotriosidase, immunoglobulin kappa chain region C, neutrophil gelatinase-associated lipocalin, and CD27. In addition, we identified novel markers, namely cat eye syndrome critical region protein 1 (adenosine deaminase 2, a therapeutic target in multiple sclerosis) and syndecan-1, a proteoglycan, also known as plasma cell surface marker CD138 and acting as chitinase 3-like protein 1 receptor implicated in inflammation and cancer signaling. CD138 exhibited good diagnostic accuracy in distinguishing MS from inflammatory neurologic disorders (area under the curve [AUC] = 0.85, CI 0.75-0.95). CD138 immunostaining was also observed in the brains of patients with MS and cultured oligodendrocyte precursor cells but was absent in astrocytes. DISCUSSION: These findings identify CD138 as a specific CSF biomarker for MS and suggest the selective activation of the chitinase 3-like protein 1/CD138 pathway within the oligodendrocyte lineage in MS. They offer promising prospects for improving MS diagnosis and prognosis by providing much-needed specificity and clinical utility. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that CD138 distinguishes multiple sclerosis from other inflammatory neurologic disorders with an AUC of 0.85 (95% CI 0.75-0.95).