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We recently described a new variant of endemic pemphigus foliaceus (EPF) in El Bagre, Colombia, that resembles Senear-Usher syndrome and identified autoantibodies to desmoglein 1 (Dsg1), as well as to multiple known and unknown antigens including plectins, in the serum of these patients. Here, we developed a cost-effective ELISA assay capable of detecting the heterogeneous antibody population observed in these EPF patients, and useful for serum epidemiological studies. A protein extract obtained from trypsin-digested fresh bovine skin and further purified on a concanavalin A matrix was used as antigen. This extract contains an important conformational epitope (a 45 kDa tryptic fragment of the Dsg1 ectodomain), which is recognized by antibodies in serum from patients with all varieties of pemphigus foliaceus (PF), and from half of those with pemphigus vulgaris with active clinical disease. The cut-off and threshold values were normalized using human serum obtained from both endemic and non-endemic areas for PF. The efficiency of this ELISA was tested using 600 serum samples from controls and patients diagnosed with EPF, non-endemic PF and other bullous diseases. The overall sensitivity and specificity of the assay were determined to be 95% and 72%, respectively, with reproducibilities of 98% (intraassay) and 95% (interassay). Comparing the ELISA with other tests to detect EPF autoantibodies, this ELISA was the most sensitive, followed by direct immunofluorescence (DIF), indirect immunofluorescence using anti-IgG4 monoclonal antibodies and immunoprecipitation (IP), respectively. The most specific assay was IP, followed by DIF. Immunoblotting to Dsg1 exhibited both poor sensitivity and poor specificity, although plectins were well visualized. We conclude that this ELISA is an excellent tool for field serological studies, allowing testing of multiple serum samples simultaneously and for detecting, with appropriate restriction and sensitivity, the heterogeneous antibody population seen in patients with this variant of EPF. Finally, autoantibody serum levels obtained with this ELISA correlated well with the clinical activity and extent of disease in patients with El Bagre EPF.

Flagellar beating drives sperm through the female reproductive tract and is vital for reproduction. Flagellar waves are generated by thousands of asymmetric molecular components; yet, paradoxically, forward swimming arises via symmetric side-to-side flagellar movement. This led to the preponderance of symmetric flagellar control hypotheses. However, molecular asymmetries must still dictate the flagellum and be manifested in the beat. Here, we reconcile molecular and microscopic observations, reconnecting structure to function, by showing that human sperm uses asymmetric and anisotropic controls to swim. High-speed three-dimensional (3D) microscopy revealed two coactive transversal controls: An asymmetric traveling wave creates a one-sided stroke, and a pulsating standing wave rotates the sperm to move equally on all sides. Symmetry is thus achieved through asymmetry, creating the optical illusion of bilateral symmetry in 2D microscopy. This shows that the sperm flagellum is asymmetrically controlled and anisotropically regularized by fast-signal transduction. This enables the sperm to swim forward.

Sperm motility is essential for fertilization. The asymmetry of flagellar beat in spermatozoa is finely regulated by intracellular calcium concentration ([Ca2+]i). Recently, we demonstrated that the application of high concentrations (10-20 μM) of the Ca2+ ionophore A23187 promotes sperm immobilization after 10 minutes, and its removal thereafter allows motility recovery, hyperactivation and fertilization. In addition, the same ionophore treatment overcomes infertility observed in sperm from Catsper1-/-, Slo3-/- and Adcy10-/-, but not PMCA4-/-, which strongly suggest that regulation of [Ca2+]i is mandatory for sperm motility and hyperactivation. In this study we found that prior to inducing sperm immobilization, high A23187 concentrations (10 μM) increase flagellar beat. While 5-10 μM A23187 substantially elevates [Ca2+]i and rapidly immobilizes sperm in a few minutes, smaller concentrations (0.5 and 1 μM) provoke smaller [Ca2+]i increases and sperm hyperactivation, confirming that [Ca2+]i increases act as a motility switch. Until now the [Ca2+]i thresholds that switch motility on and off were not fully understood. To study the relationship between [Ca2+]i and flagellar beating, we developed an automatic tool that allows the simultaneous measurement of these two parameters. Individual spermatozoa were treated with A23187 which is then washed to evaluate [Ca2+]i and flagellar beat recovery using the implemented method. We observe that [Ca2+]i must decrease below a threshold concentration range to facilitate subsequent flagellar beat recovery and sperm motility.

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