Assessment of the Composition and Biologic Activity of Platelet Rich Plasma and its Relationship to Clinical Outcomes in Patients with Knee Osteoarthritis

Recent studies suggest positive clinical outcomes associated with platelet-rich-plasma (PRP) administration to treat knee osteoarthritis (OA). However, the results remain inconclusive in part because of the high variability in PRP preparations and the limited information regarding the relevant biologically active components of PRP. We hypothesize that the variability in clinical response is driven by the heterogeneous composition of PRP. In this study we evaluated the composition and biological activity of PRP and further correlated our findings to clinical outcomes in patients receiving intra-articular injections for knee OA. After IRB approval and patient consent, we enrolled 51 patients (mean age: 57.9 ± 10.1; mean BMI: 26.0 ± 4.1) with mild-moderate knee OA (Kellgren Lawrence grades 1-3), eligible for intra-articular PRP injection. We obtained MRI at baseline and outcome measures (KOOS JR and PNS) at baseline, 6 weeks, 6 months, and 12 months after PRP injection. Patients were categorized as “good” and “poor” responders based on the outcome measures, corrected using published Minimally Clinically Important Difference (MCID) values. Aliquots of PRP and whole blood from the same patients were used to evaluate composition (CBC with differential and multiplex ELISA) and biologic activity, using a co-culture system of macrophages and fibroblast incubated with TNFa with and without PRP (10% v:v) for 24 hours. Total RNA from cells was used for RNAseq, Nanostring, and RTqPCR analysis. On average, we collected 4.07 ± 01.05 mL of PRP, and 3.24 ± 0.85 mL of PRP were injected intra-articularly. PRP preparations yielded mean fold-changes of 1.60 ± 0.37 platelets and 0.19 ± 0.08v leukocytes, relative to whole-blood from the same patients (set as 1). On average, all patients that reached the 6-month time-point (N = 32) reported improved outcomes at 6-weeks and 6-months after PRP administration (KOOS and PNS p\u3c0.05 vs. baseline). After MCID corrections, we identified “good” (N=17, positive response using both measures) and “poor” responders (N=15, poor response in one or both measures). RNAseq analyses showed PRP-dependent changes in the TNFa-induced modulation of a number of genes, including CXCL7 and CCL5. NanoString and RTqPCR analyses confirmed the RNAseq results. Comparisons of PRP from good and poor responders identified changes in the composition and biologic activity between these groups. This pilot study integrated clinical data with genomic approaches to evaluate variability in the composition and activity of PRP, and how this may influence outcomes in patients with knee OA. We uncovered subsets of genes differentially modulated by co-treatment of PRP with TNFa, in agreement with the concept that the reduced knee OA pain in patients treated with PRP is driven by the ability of PRP to modulate inflammation. Furthermore, we identified changes in the composition and biologic activity of PRP between “good” and “poor” responders

Transcription cofactor GRIP1 differentially affects myeloid cell-driven neuroinflammation and response to IFN-β therapy.

Macrophages (MФ) and microglia (MG) are critical in the pathogenesis of multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). Glucocorticoids (GCs) and interferon β (IFN-β) are frontline treatments for MS, and disrupting each pathway in mice aggravates EAE. Glucocorticoid receptor-interacting protein 1 (GRIP1) facilitates both GR and type I IFN transcriptional actions; hence, we evaluated the role of GRIP1 in neuroinflammation. Surprisingly, myeloid cell-specific loss of GRIP1 dramatically reduced EAE severity, immune cell infiltration of the CNS, and MG activation and demyelination specifically during the neuroinflammatory phase of the disease, yet also blunted therapeutic properties of IFN-β. MФ/MG transcriptome analyses at the bulk and single-cell levels revealed that GRIP1 deletion attenuated nuclear receptor, inflammatory and, interestingly, type I IFN pathways and promoted the persistence of a homeostatic MG signature. Together, these results uncover the multifaceted function of type I IFN in MS/EAE pathogenesis and therapy, and an unexpectedly permissive role of myeloid cell GRIP1 in neuroinflammation

Querying quantitative logic models (Q2LM) to study intracellular signaling networks and cell-cytokine interactions

Mathematical models have substantially improved our ability to predict the response of a complex biological system to perturbation, but their use is typically limited by difficulties in specifying model topology and parameter values. Additionally, incorporating entities across different biological scales ranging from molecular to organismal in the same model is not trivial. Here, we present a framework called “querying quantitative logic models” (Q2LM) for building and asking questions of constrained fuzzy logic (cFL) models. cFL is a recently developed modeling formalism that uses logic gates to describe influences among entities, with transfer functions to describe quantitative dependencies. Q2LM does not rely on dedicated data to train the parameters of the transfer functions, and it permits straight-forward incorporation of entities at multiple biological scales. The Q2LM framework can be employed to ask questions such as: Which therapeutic perturbations accomplish a designated goal, and under what environmental conditions will these perturbations be effective? We demonstrate the utility of this framework for generating testable hypotheses in two examples: (i) a intracellular signaling network model; and (ii) a model for pharmacokinetics and pharmacodynamics of cell-cytokine interactions; in the latter, we validate hypotheses concerning molecular design of granulocyte colony stimulating factor.National Institutes of Health (U.S.) (Grant P50-GM068762)National Institutes of Health (U.S.) (Grant R24-DK090963)United States. Army Research Office (Institute for Collaborative Biotechnologies Grant W911NF-09-0001

Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: Determinants of DNA Binding and Redox Regulation by Disulfide Bond Formation.

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors

Monitoring of high energy deuteron beams in the experiments with massive targets

The influence of massive uranium target (500 kg natU) of assembly "QUINTA" on the results of high energy deuteron beam monitoring with aluminum and copper foils was investigated. In order to increase the accuracy of deuteron beam off-line monitoring, the measurements of the cross sections of fragmentation reaction natCu(d,x)24Na were performed at 1.32, 2, 4 и 8 GeV deuteron energies. For the same deuteron energies the cross sections of residual nuclei 7 Be, 42K, 52Mn, 57Co and 58Co for natCu(d,x) reaction were measuredИсследовано влияние массивной урановой мишени (500 кг natU) установки «КВИНТА» на результаты мониторирования пучков дейтронов с помощью алюминиевых и медных фольг. Для улучшения точности off-line мониторирования пучков дейтронов, бомбардирующих протяженные мишени из тяжёлых элементов, были проведены измерения сечений реакции фрагментации natCu(d,x)24Na для дейтронов с энергией 1,32; 2; 4 и 8 ГэВ. Для этих же энергий дейтронов измерены сечения выхода изотопов 7 Be, 42K, 52Mn, 57Co и 58Co в реакциях natCu(d,x).Досліджено вплив масивної уранової мішені (500 кг natU) установки «КВІНТА» на результати моніторування пучків дейтронів за допомогою алюмінієвих і мідних фольг. Для покращення точності off-line моніторування пучків дейтронів, які бомбардують протяжні мішені з важких елементів, були проведені вимірювання перетинів реакції фрагментації natCu(d,x)24Na для дейтронів з енергією 1,32; 2; 4 і 8 ГеВ. Для цих же енергій дейтронів виміряні перетини виходу ізотопів 7 Be, 42K, 52Mn, 57Co в реакціях natCu(d,x)

JunB as a potential mediator of PTHrP actions: new gene targets Ephrin B1 and VCAM-1

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75431/1/j.1601-0825.2008.01489.x.pd

Elongator function in tRNA wobble uridine modification is conserved between yeast and plants

Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life

Measuring parameters of the deuteron beams in experiments with the target assembly QUINTA using solid-state track detectors

The results of measurements of the deuteron beams parameters with energies of 1, 4 and 8 GeV at the irradiation of the uranium subcritical assembly "QUINTA" are presented. The data obtained on the incident beam position relative to the axis of the target and on the real geometric parameters of the beam allow one to analyze correctly the spatial distribution of reaction rates within the target assembly and compare these measured in different irradiation runs as well as to simulate experiments by such codes as MCNPX, GEANT4, FLUKA et al. The investigation has been performed at the V.I. Veksler and A.M. Baldin Laboratory of High Energy Physics, JINR.Представлены результаты измерений параметров пучка на мишени при облучении подкритической урановой сборки «КВИНТА» дейтронами с энергиями 1, 4 и 8 ГэВ. Информация о положении пучка падающих частиц относительно оси сборки и о его реальных геометрических параметрах позволяет корректно анализировать данные о пространственных распределениях скоростей реакций внутри мишенной сборки и сравнивать их для различных сеансов облучения, а также моделировать эксперименты программами типа MCNPХ, GEANT4, FLUKA и другими. Работа выполнена в Лаборатории физики высоких энергий им. В.И. Векслера и А.М. Балдина ОИЯИ.Представлено результати вимірювань параметрів пучка на мішені при опроміненні підкритичної уранової збірки «КВІНТА» дейтронами з енергіями 1, 4 та 8 ГеВ. Інформація про положення пучка падаючих частинок щодо осі збірки та про його реальні геометричні параметри дозволяє коректно аналізувати дані про просторові розподіли швидкостей реакцій усередині мішеневої збірки та порівняти їх для різних сеансів опромінення, а також моделювати експерименти програмами типу MCNPX, GEANT4, FLUKA та іншими. Робота виконана в Лабораторії фізики високих енергій ім. В.І. Векслера і О.М. Балдіна ОІЯД

Plasma membrane receptor mediated MAPK signaling pathways are activated in human uterine cervix at parturition

BACKGROUND: Cervical ripening resembles an inflammatory reaction. Estrogens induce leukocyte migration into tissue and factors promoting cervical remodeling and labor, although the mechanisms are only partially known. The aim of this study was to investigate whether plasma membrane receptor mediated pathways, known to be activated by estrogens and proinflammatory compounds, are involved in cervical ripening before labor. METHODS: The expression and distribution of mitogen activated protein kinases (MAPK), which transduce extracellular signals into intracellular responses through phosphorylation, and their intracellular targets transcription factors c-Jun and c-Fos proteins (AP-1) were analysed in cervical biopsies from term pregnant women (TP), immediately after parturition (PP), and from non-pregnant women (NP). Immunohistochemistry and RT-PCR techniques were used. RESULTS: Cell-specific alterations in the immunostaining pattern for MAPK were observed. The expressions of activated, phosphorylated MAPK forms pERK1/2, pJNK and p38MAPK were significantly increased in cervical stroma until TP and pERK1/2 expression was significantly enhanced in PP group. c-Jun was significantly increased in cervical stroma and smooth muscle in TP as compared to NP group. c-Fos was significantly increased in stroma, squamous epithelium and glandular epithelium in PP as compared to TP group. CONCLUSION: We report, for the first time, cell-specific activation of pMAPKs and their targets transcription factors c-Fos and c-Jun (AP-1) proteins in human uterine cervix until term pregnancy, and immediately after parturition. These results suggest a role for MAPK activation in cervical ripening before labor

Identification of β-catenin binding regions in colon cancer cells using ChIP-Seq

Deregulation of the Wnt/β-catenin signaling pathway is a hallmark of colon cancer. Mutations in the adenomatous polyposis coli (APC) gene occur in the vast majority of colorectal cancers and are an initiating event in cellular transformation. Cells harboring mutant APC contain elevated levels of the β-catenin transcription coactivator in the nucleus which leads to abnormal expression of genes controlled by β-catenin/T-cell factor 4 (TCF4) complexes. Here, we use chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) to identify β-catenin binding regions in HCT116 human colon cancer cells. We localized 2168 β-catenin enriched regions using a concordance approach for integrating the output from multiple peak alignment algorithms. Motif discovery algorithms found a core TCF4 motif (T/A–T/A–C–A–A–A–G), an extended TCF4 motif (A/T/G–C/G–T/A–T/A–C–A–A–A–G) and an AP-1 motif (T–G–A–C/T–T–C–A) to be significantly represented in β-catenin enriched regions. Furthermore, 417 regions contained both TCF4 and AP-1 motifs. Genes associated with TCF4 and AP-1 motifs bound β-catenin, TCF4 and c-Jun in vivo and were activated by Wnt signaling and serum growth factors. Our work provides evidence that Wnt/β-catenin and mitogen signaling pathways intersect directly to regulate a defined set of target genes