Reduced non-switched memory B cell subsets cause imbalance in B cell repertoire in systemic sclerosis.

OBJECTIVES: Analysis of peripheral blood B lymphocytes in patients with systemic sclerosis (SSc) has provided evidence for specific alterations in naive and memory B cell balance. However, memory B cell subsets in SSc have not been thoroughly investigated. This study sought to identify phenotypic abnormalities and activation markers in peripheral blood memory B cells in SSc subtypes. METHODS: Blood samples were obtained from 28 SSc patients with early form of disease (9 limited (lcSSc), 19 diffuse cutaneous SSc (dcSSc)) and 15 healthy controls. After magnetic bead separation of CD19+ B cells, multiparametric flow cytometry was performed and CD19+CD27- IgD+ naive, CD19+CD27+ memory, CD19+CD27+IgD+ non-switched memory CD19+CD27+IgD- switched memory, CD19+CD27-IgD- double negative (DN) memory, CD80+ or CD95+ activated cells were identified. RESULTS: The proportion of naive B cells was higher (p=0.046) in SSc than in controls, with a decreased percentage of memory (p=0.018), especially non-switched memory B cells (p=0.015). The dcSSc patients had a significantly higher frequency of switched memory and DN memory B cells compared to lcSSc patients (p=0.025 and p=0.031). Percentage of CD95+CD27+ memory and CD95+ DN memory B cells was also significantly elevated in dcSSc compared to lcSSc patients (p=0.038 and p=0.045). CONCLUSIONS: We conclude that the decreased proportion of memory B cells in SSc is due to reduction of non- switched memory B cells, resulting in an imbalance between the tolerogenic and activated memory B cell types. Elevated switched and activated CD95+ DN memory B cells may serve as a biomarker for dcSSc and can have a pathogenic potential by cytokine and autoantibody production.

Revising lysenin expression of earthworm coelomocytes.

Lysenin is a species-specific bioactive molecule of Eisenia andrei earthworms. This protein is a potent antimicrobial factor; however its cellular expression and induction against pathogens are still not fully understood. We developed a novel monoclonal antibody against lysenin and applied this molecular tool to characterize its production and antimicrobial function. We demonstrated by flow cytometry and immunocytochemistry that one subgroup of earthworm immune cells (so called coelomocytes), the chloragocytes expressed the highest amount of lysenin. Then, we compared lysenin expression with earlier established coelomocyte (EFCC) markers. In addition, we determined by immunohistology of earthworm tissues that lysenin production is only restricted to free-floating chloragocytes. Moreover, we observed that upon in vitro Staphylococcus aureus but not Escherichia coli challenged coelomocytes over-expressed and then secreted lysenin. These results indicate that among subpopulations of coelomocytes, lysenin is mainly produced by chloragocytes and its expression can be modulated by Gram-positive bacterial exposure.