Immunofluorescence signal intensity measurements as a semi-quantitative tool to assess sarcoglycan complex expression in muscle biopsy

Abstract

Sarcoglycanopathies are highly heterogeneous in terms of disease progression, muscular weakness, loss of ambulation and cardiac/respiratory involvement. Their clinical severity usually correlates with the residual protein amount, which makes protein quantification extremely relevant. Sarcoglycanopathy diagnosis is genetic, but skeletal muscle analysis - by both immunohistochemistry and Western blot (WB) - is still mandatory to establish the correct diagnostic process. Unfortunately, however, WB analysis cannot be performed if the bioptic specimen is scarce. This study provides a sensitive tool for semi-quantification of residual amount of sarcoglycans in patients affected by sarcoglycanopathies, based on immunofluorescence staining on skeletal muscle sections, image acquisition and software elaboration. We applied this method to eleven sarcoglycanopathies, seven Becker muscular dystrophies and four age-matched controls. Fluorescence data analysed in patients and compared to age-matched controls showed a significant reduction of the mutated sarcoglycan expression and a variable reduction of the other sarcoglycans. Fluorescence normalized data analysed in relation to the age of onset of the disease, showed a negative correlation of α-sarcoglycan fluorescent signal versus fibrosis in patients with an early age of onset and a negative correlation between δ-sarcoglycan signal and fibrosis in both intermediate and late age of onset groups. The availability of a method that allows objective quantification of the sarcolemmal proteins, faster and less consuming than WB analysis and able to detect low residual sarcoglycan expression with great sensitivity, proves useful to better define both patient prognosis and expected disease evolution. The proposed method could be employed also to monitor the efficacy of therapeutic interventions and during clinical trials.

Enterohemorrhagic Escherichia coli (EHEC) Disrupts Intestinal Barrier Integrity in Translational Canine Stem Cell-Derived Monolayers

“…Fluorescence images were acquired under 63× objective, with excitation laser sources of 405 nm, 499 nm, 553 nm, and 653 nm and high efficiency Leica HyD detector, and processed using LAS X software (Leica). Intensity of fluorescence signal for E-cadherin and ZO-1 was quantified by performing line scan using ImageJ 1.54d (39). Mean of maximum intensity value of each peak was determined by taking the peak intensity value from at least four peaks per line scan and normalizing it by the value obtained from non-pathogenic E. coli infected monolayers.…”