Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

Altered tear composition in smokers and patients with graves ophthalmopathy.

OBJECTIVES: To analyze, compare, and contrast tear composition in healthy nonsmokers, smokers who were otherwise healthy, and patients with Graves disease (GD) accompanied by Graves ophthalmopathy (GO) of varying severity. METHODS: Reflex tears were collected using Schirmer strips from 37 healthy nonsmokers, 33 otherwise healthy smokers, 51 patients with GD, and 85 patients with GO. Thyrotropin receptor-stimulating activity and serum thyroid-stimulating antibodies were measured. Pooled tear samples from healthy nonsmokers, healthy smokers, and patients with GO were separated by means of electrophoresis. Proteins expressed in healthy smokers, patients with GO, and healthy nonsmokers were separated by means of electrophoresis and analyzed by mass spectrometry. RESULTS: Based on the 97th percentile of findings from healthy nonsmokers, specific thyrotropin receptor-stimulating activity was detected in 25% of the tear samples from healthy smokers, 32% of those from patients with GD, and 41% of those from patients with GO. Clinical activity scores correlated with serum thyroid-stimulating antibody levels but not tear thyrotropin receptor-stimulating activity. Electrophoresis revealed additional proteins of 30 to 41 kDa in the tear samples from patients with GO and healthy smokers compared with samples from healthy nonsmokers. These proteins were identified as zinc-alpha2-glycoprotein and lactoferrin but have no thyrotropin receptor-stimulating activity. CONCLUSIONS: We demonstrate similar changes in tear composition in patients with GO and healthy smokers compared with healthy nonsmokers. Expression of zinc-alpha2-glycoprotein and lactoferrin is increased and their molecular weights are modified, suggesting degradation and/or changes during glycosylation, which may affect the bioactivities of zinc-alpha2-glycoprotein and lactoferrin. Clinical Relevance Smoking, which is a significant risk factor for the development of GO, modifies tear composition.

Erythropoietin induces changes in gene expression in PC-12 cells.

Erythropoietin (EPO) is the primary modulator of red blood cell production. Recently EPO has received considerable attention for its functions outside of hematopoiesis, including its effects in the nervous system where it has been shown to act as a neuroprotectant. To understand the function of EPO in the nervous system and to determine if EPO functions through the same signaling pathways identified in hematopoietic cells, we used cDNA array hybridization and RT-PCR to investigate the changes in gene expression induced by EPO in the neuronal-like PC-12 cell line. PC-12 cells cultured in the presence of EPO (10 U/ml) showed significant changes in gene expression by 3 h with a return to basal expression levels for the vast majority of genes by 24 h. The genes influenced by EPO included genes with known functions in cell proliferation, differentiation and apoptosis. Semi-quantitative RT-PCR confirmed that 24 h pre-treatment with EPO (10 pM) resulted in a 2.5-fold increase in the expression of the anti-apoptotic gene bcl(XL) and a 4-fold decrease in the expression of the pro-apoptotic gene bak. In addition to supporting the current models of EPO function these results suggest previously unidentified mechanisms by which EPO may function in neurons.