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Protein aggregation, which takes place both in vivo and in vitro, is an important degradative pathway for all proteins. Protein aggregates have distinct physicochemical and biological properties that are important to study and characterize from the perspective of both fundamental and applied sciences. The size of protein aggregates varies across a huge range, spanning several orders of magnitude. Currently, protein aggregates larger than hundreds of nanometers in diameter are impossible to physically fractionate. Here, we present a new method to fractionate microscopic proteinaceous particles using preparative fluorescence-activated cell sorting technology. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2128–2135, 2013

Osteocytes are the terminally differentiated cells of the osteoblastic lineage embedded within the mineralized bone matrix. T: hey have been identified as key players in mechanotransduction and in mineral and phosphate homeostasis. In addition, they appear to have a role in mediating bone formation, since they secrete the bone formation inhibitor sclerostin. In contrast to osteoblasts and osteoclasts, which reside on the bone surface, it has been difficult to isolate and analyze cellular and molecular properties of osteocytes due to their specific location inside the "hard" mineralized bone compartment. This chapter describes a method to isolate osteocytes from newborn mouse calvaria and adult mouse long bone, followed by immediate total RNA extraction allowing to selectively study osteocytic versus osteoblastic gene expression by quantitative real-time polymerase chain reaction (qPCR). The osteocyte-enriched cell fraction isolated by this method can further be purified by FACS and selectively expresses osteocytic marker genes, such as Dmp1 and Sost.

The mechanism whereby IL-17 drives rheumatoid arthritis remains incompletely understood. We demonstrate that anti–IL-17 therapy in collagen-induced arthritis ameliorates bone damage by reducing the number of osteoclasts in joints. We found equal numbers of CD4+ Th17 and IL-17 producing γδ T cells in the joints of arthritic mice, and in vitro, both populations similarly induced osteoclastogenesis. However, individual depletion and adoptive transfer studies revealed that in vivo, Th17 cells dominated with regard to bone destruction. Unlike γδ T cells, Th17 cells were found in apposition to tartrate-resistant acid phosphatase positive osteoclasts in subchondral areas of inflamed joints, a pattern reproduced in patient biopsies. This localization was caused by Ag-specific retention, because OVA-primed Th17 cells showed a γδ T cell-like diffuse distribution. Because IL-23, as produced by osteoclasts, enhanced T cell-mediated osteoclastogenesis, we propose that Ag-specific juxtaposition is key to foster the molecular cross talk of Th17 cells and osteoclasts, thus driving arthritic bone destruction.

Objective: To determine the relative percentages of Th17 and Treg cells in CD3+ T cells isolated from the blood of healthy control subjects, untreated MS patients, and IFNb- or fingolimod-treated MS patients. Background Fingolimod has recently been approved as the first oral treatment for relapsing Multiple Sclerosis (MS). Fingolimod treatment reduces the blood CD4 T cell count by approximately 60-70%. As a consequence, the quantification of the small functional T helper (Th) and regulatory T cell (Treg) subsets in blood of fingolimod-treated patients requires prior enrichment of T cells. Design/Methods: The percentage of FOXP3+ Treg cells and of RORC2+ TH17 cells was determined by quantitative RT-PCR in CD3+ T cells purified by MACS. The percentages of CD4+ and CD8+ naive, central memory and effector memory T cells were determined by flow cytometry, using the markers CCR7 and CD45RA. Results: Compared to healthy controls, the percentage of both Tregs and Th17 cells was increased in MS patients, both untreated and those treated with IFNb. In fingolimod-treated MS patients, the percentages of both Treg and TH17 cells were reduced by approximately 70% compared to control groups, and this correlated with a reduction of the total CD4+ T cell count. The remaining blood CD4+ T cells were enriched in CCR7-CD45RA+/- effector memory T cells. Conclusions: The increase of Tregs and TH17 cells in blood of drug-untreated and IFNb-treated MS patients suggests activation of pro- and anti-inflammatory pathways. In fingolimod-treated MS patients, the reduction of Tregs and TH17 cells corresponds to reduction of CD4+CCR7+ T cells, suggesting that mechanisms leading to reduced counts involve the lymph node retention receptor CCR7, rather than specific functional helper/regulatory phenotypes. Supported by: We thank L Kappos and M Mehling for providing blood samples. Disclosure: Dr. Brinkmann has received personal compensation for activities with Novartis as an employee. Dr. Brinkmann has received research support from Novartis. Dr. Raulf has received personal compensation for activities with Novartis as an employee.Dr. Raulf has received research support from Novartis. Dr. Vedrine has received personal compensation for activities with Novartis. Dr. Vedrine has received research support from Novartis. Dr. Allard has received personal compensation for activities with Novartis as an employee. Dr. Allard has received research support from Novartis.