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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

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the 'classical' (M1) and 'alternative' (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

The diversity of T cell subpopulations demands a growing number of biomarkers to be studied from small samples. To bridge the gap between multicolor flow cytometry or classical qPCR and RNA-Seq, we applied highly multiplexed whole transcriptome AmpliSeq-RNA with 20,803 genes to perform sensitive and specific gene expression profiling (GEP). This study analyzed untouched primary human naïve and memory T cells and compared results to microarrays. Method 8 T cell subsets were isolated by FACS from 4 healthy donors without restimulation: CD4+ or CD8+ naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA−), effector memory TEM (CCR7−CD45RA−) and TEMRA (CCR7−CD45RA+). AmpliSeq transcriptome human gene expression kit (Thermo Fisher) generated barcoded libraries from total RNA. 8–10 samples multiplexed per Ion 540 chip were sequenced on an Ion S5 System. Samples were also analyzed by Affymetrix HG-U133 Plus 2 genechips. Results The resulting Excel matrix of normalized reads [RPM = reads per million reads] had a dynamic range of more than 5 log units. Biological replicates of T cell subsets showed high correlation with r ≥ 0.925. Technical replicates (same libraries) demonstrated high reproducibility with r ≥ 0.995. A high concordance with genechip data were found for expression pattern of analyzed markers. For several very high or low abundant genes the AmpliSeq-RNA data were more sensitive and more specific than the hybridization-based genechip data. Conclusion AmpliSeq-RNA (counting PCR products by NGS) is an interesting alternative to perform straightforward GEP. The analysis of phenotypically well-defined T cell subsets without artificial in vitro stimulation allows more precise determination of in vivo relevant functions.

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.

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.

The activity of endo-beta-mannanase ([1--4]-beta-mannan endohydrolase EC 3.2.1.78) is likely to be central to the metabolism of cell wall mannans during the germination of grains of coffee (Coffea spp.). In the present paper, we report the cloning and sequencing of two endo-beta-mannanase cDNAs (manA and manB) by different strategies from Coffea arabica L.. The manA cDNA was obtained by the use of oligonucleotides homologous to published sequences of other endo-beta-mannanases and manB by the use of oligonucleotides deduced from a purified enzyme from coffee. ManA and B proteins share about 56% sequence homology and include highly conserved regions found in other mannan endohydrolases. Purification of the activity by chromatography followed by separation by two-dimensional electrophoresis and amino acid sequencing demonstrated the existence of at least seven isomers of the ManB form. The existence of multiple manB genes was also indicated by Southern analysis, whereas only one or two gene copies were detected for manA. Northern hybridizations with manA- and manB-specific probes showed that mRNA transcripts for both cDNAs were present at the same periods of bean germination with transcript peaks at 20 days after imbibition of water (DAI). Transcripts were not detected during grain maturation or in the other tissues such as roots, stems, flowers and leaves. The peak endo-beta-mannanase activity occurred at approximately 28 DAI and was not detected in grains prior to imbibition. Activity and mRNA levels appeared to be tightly co-ordinated. Tests of substrate specificity with the purified ManB enzyme showed that activity required a minimum of five mannose units to function efficiently.

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.