Expression Of Soluble Factors Research Articles

Impact of the body mass index on perioperative immunological disturbances in patients with hip and knee arthroplasty

BackgroundObesity increases the risk for knee and hip joint implantation and negatively contributes to wound healing. In this study, in 52 patients undergoing hip and knee arthroplasty the amount of peripheral immune effector cells pre- and post-operative, as well as the expression of certain soluble factors affecting the functions of immune effector cells were investigated.MethodsThe peripheral immune cells and the expression of the soluble factors were determined by flow cytometry and correlated to each other in dependency of the BMI, the sex, and the kind of arthroplasty.ResultsThe pre-operative amounts of peripheral NK cells and cytotoxic T cells significantly decreased with increasing BMI. Furthermore, the expression of the immunomodulatory adipokine leptin nicely correlated to the BMI. These effects were stronger in males than in females. Furthermore, the correlation of the activation marker sTNF-R and peripheral T cells strongly decreased with increasing BMI. While IL-6, CD40L, and MPO were significantly induced after surgery, there were no correlations to the BMI.ConclusionsThe known wound-healing problems in obese patients and the osteoarthritis per se can be linked to the BMI. While obese patients exerted reduced peripheral NK cells and cytotoxic T cells (CTLs), IL-6 showed no involvement. However, the adipokine leptin strongly increased with the BMI strengthening its role as immunomodulatory molecule negatively interfering the functions of immune effector cells.

Comparative evaluation of aggressiveness traits in staphylococcal strains from severe infections versus nasopharyngeal carriage.

Despite their commensal status, staphylococci can become problematic pathogens expressing multiple and redundant virulence factors. This study aimed to evaluate aggressiveness markers comparatively in staphylococcal strains isolated from severe infections versus asymptomatic carriage in order to identify clinically relevant bacterial traits that could easily be detected in clinical practice and could be suggestive for particular host-pathogen interactions such as cyto-adhesion or biofilm formation, ultimately orienting the clinical decision-making process. We have used invitro phenotypic methods to assess adhesion to and invasion of eukaryotic cells, biofilm development, and expression of soluble virulence factors in 92 Staphylococcus spp. strains. The adhesion index, invasion capacity, biofilm formation and expression of soluble factors did not differ significantly between clinical and commensal strains. The major bacterial traits we found to be significantly more prevalent in clinical staphylococci were the aggregative adhesion pattern (P=0.012), cluster adhesion (P=0.001) and tetrad morphology (P=0.018). The aggregative adhesion pattern was correlated with higher cyto-adhesion (P<0.001), higher invasion capacity (P=0.003) and lower Carmeli scores (P=0.002). Three major bacterial traits, namely tetrad morphology, aggregative adhesion pattern, and resistance to methicillin (acronym: TAM), can be used to compute an aggressiveness score (SAS) predictive of the staphylococcal strain's virulence and capacity to initiate and develop a biofilm-driven chronic infectious process versus a fulminant acute infection, in a susceptible host.

Abstract B035: CXCL8/CXCR1 signaling promotes angiogenesis and hematopoietic stem and progenitor cell function

Abstract CXCL8 (IL-8) is a chemokine with pleiotropic roles in host defense, angiogenesis and tumor metastasis. CXCL8 and its specific receptor, CXCR1, are broadly expressed within the hematopoietic and vascular systems. In an effort to identify novel secreted factors with effects on hematopoietic stem and progenitor cell (HSPC) function, we have recently identified CXCL8/CXCR1 signaling as a positive regulator of HSPC colonization of the zebrafish caudal hematopoietic territory (CHT). The CHT is a vascular niche that serves as the primary site of hematopoiesis from 36 hours post fertilization (hpf) to 6 days post fertilization (dpf). This observation raised the question whether CXCR1 signaling might induce dynamic changes in the CHT that favor HSPC colonization. CXCR1 was expressed at high levels in endothelial cells using a kdrl(VEGFR2):CXCR1;kdrl:mCherry double transgenic line. The CHT was imaged by fluorescence confocal microscopy, reconstructed in 3 dimensions and the volume measured using digital image analysis software. Overexpression of CXCR1 within the endothelial cells of these animals increased the volume of the CHT by 28% (p = 0.02). To understand how CXCR1 affects the dynamics of niche development, we globally overexpressed CXCR1 beginning at 36 hpf using a heat shock induction system and performed time lapse confocal microscopy from 52 to 72 hpf. This revealed that overexpression of CXCR1 consistently increased the CHT volume from 53 to 72 hpf compared to control (21% increase at 72 hpf, p = 0.004). These studies did not show whether CXCR1 acted directly on the vascular niche or whether CXCR1 expression in endothelial cells might induce expression of soluble factors or activate circulating cells that then cause expansion of the niche through indirect mechanisms. To address this, we created parabiotic zebrafish by fusing kdrl:mCherry embryos at 4 hpf. One half of each parabiotic animal was modified by DNA microinjection to globally overexpress CXCR1 or GFP as a control via heat shock induction at 36 and 48 hpf. The volume of the CHT was measured in each half of each parabiotic animal at 72 hpf. In control parabiotics overexpressing GFP, there was no difference in CHT volume between modified and unmodified sides of the organism. However, in parabiotics overexpressing CXCR1, the CHT of the modified side was 27% larger compared with the unmodified side (p = 0.012), consistent with our previous results and suggesting that CXCR1 acts directly on the niche in this system. We then asked whether this volume change could affect HSPC engraftment. Parabiotic animals were created using Runx1:mCherry embryos that carry an HSPC-specific reporter transgene as “donors” and WT embryos as “recipients”. The recipient niche was modified as before to overexpress CXCR1 or GFP as a control. At 72 hpf there was no difference in HSPC colonization of donor and recipient niches when the recipient niche expressed GFP. However, when the recipient niche expressed CXCR1, there was a significant increase in HSPC colonization of the recipient niche compared to the donor niche (11.4+/-2.4 vs 19.8+/-3.5 HSPCs per CHT, p = 0.02). Taken together, these results identify a novel role for CXCL8/CXCR1 signaling in angiogenesis and HSPC biology and they provide a new example of how innate immune signaling pathways are important for stem cell function. Administration of CXCL8 to hematopoietic stem cell transplant recipients may therefore improve HSPC engraftment and clinical outcomes in patients who are being treated for hematologic malignancies. Citation Format: Bradley W. Blaser, Jessica L. Moore, Elliott Hagedorn, Brian Li, Vera Binder, Owen Tamplin, Leonard I. Zon. CXCL8/CXCR1 signaling promotes angiogenesis and hematopoietic stem and progenitor cell function [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B035.

Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC.In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL.We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2).Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2).In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. DisclosuresNo relevant conflicts of interest to declare.

Noradrenaline acting on astrocytic β2-adrenoceptors induces neurite outgrowth in primary cortical neurons

The neurotransmitter noradrenaline (NA) has anti-inflammatory properties and promotes expression of neurotrophic factors in the central nervous system (CNS) via activation of glial adrenoceptors. Here we examined the ability of conditioned media (CM) from NA-treated glial cells to impact upon neuronal complexity. Primary rat cortical neurons were treated either directly with NA (1–10 μM), or treated with CM from NA-stimulated primary mixed glial cells. Neuronal complexity was assessed using Sholl analysis. Exposure of neurons to CM from NA-stimulated glial cells increased all indices of neuronal complexity, whereas direct exposure of neurons to NA did not. CM from NA-stimulated astrocytes, but not microglia, also increased neuronal complexity indicating a key role for astrocytes. The β-adrenergic subtype was implicated in this response as the increase was blocked by the β-adrenoceptor antagonist propanolol, but not by the α-adrenoceptor antagonist phentolamine. CM from glial cells treated with the β2-adrenoceptor agonists salmeterol and clenbuterol, but not the β1-adrenoceptor agonist xamoterol, mimicked the ability of NA to increase neuronal complexity. NA induced expression of a range of growth factors (BDNF, NGF-β, GDNF, FGF-2 and IL-6) in glial cells. In addition to this, the phosphatidylinositol 3-kinase (PI3K), mitogen activated protein kinase (MAPK) and JAK-STAT signalling pathways are implicated in NA CM-induced neuritic growth as inhibition of these pathways attenuated NA CM-induced neuritic growth. In conclusion, this study indicates a novel role for NA acting at glial β2-adrenoceptors to induce neuritic growth through the expression of soluble factors that elicit a neurotrophic action and increase neuronal complexity.

Circulating mediators of bone remodeling in psoriatic arthritis: implications for disordered osteoclastogenesis and bone erosion

IntroductionDiverse bone pathologies are observed in patients with psoriatic arthritis (PsA). Uncoupling of bone remodeling with disordered osteoclastogenesis has been implicated in the pathogenesis of PsA. The aim of this study was to examine the role of soluble mediators of bone remodeling within the circulation of patients with PsA.MethodsPatients with PsA (n = 38), with psoriasis (n = 10), and healthy controls (n = 12) were studied. Serum was obtained for testing of Dikkopf-1 (Dkk-1), macrophage-colony stimulating factor (M-CSF), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) with ELISA. Patients with PsA also had bone densitometry, plain radiographs of the hands and feet, and assessment of peripheral blood osteoclast precursors. Radiographs were scored for erosion, joint-space narrowing, osteolysis, and new bone formation.ResultsCompared with those with psoriasis and healthy controls, patients with PsA had higher circulating concentrations of Dkk-1 and M-CSF. In patients with PsA, M-CSF and RANKL, but not Dkk-1, concentrations positively correlated with radiographic erosion, joint-space narrowing, and osteolysis scores. Mediators of bone remodeling did not correlate with the number of joints with new bone formation or with total hip-bone mineral density. Peripheral blood CD14+/CD11b+ cells, and the number of osteoclast-like cells and resorptive pits after culture with RANKL and M-CSF also correlated with radiographic damage scores. Circulating M-CSF concentrations correlated with the percentage of peripheral blood CD14+/CD11b+ cells.ConclusionsSystemic expression of soluble factors that promote osteoclastogenesis is disordered in patients with PsA and may contribute to periarticular bone loss in this disease.

T-cell exhaustion in HIV infection

Generation of memory T cells, which mediate immunity against microbes and cancers, relies, for optimal activity, on the interactions of multiple cell types that are highly regulated through the expression of soluble factors and negative and positive receptors. Their disruption will lead to aberrant immune responses, which can result in the invasion of the host by foreign pathogens. In chronic viral infections including HIV and hepatitis C virus, persistence of antigen and lack of CD4 help (HIV) disrupt memory T-cell function and induce defects in memory T-cell responses, which have been defined as T-cell exhaustion. In this review, we examine the molecular mechanisms involved in such T-cell dysfunction. Better understanding of these mechanisms will assist in the development of novel therapies to prevent the immune damage mediated by HIV infection.

Olfactory ensheathing cells promote neurite sprouting of injured axons in vitro by direct cellular contact and secretion of soluble factors.

Olfactory ensheathing cells (OECs) represent an exciting possibility for promoting axonal regeneration within the injured spinal cord. A number of studies have indicated the ability of these cells to promote significant reactive sprouting of injured axons within the injured spinal cord, and in some cases restoration of functional abilities. However, the cellular and/or molecular mechanisms OECs use to achieve this are unclear. To investigate such mechanisms, we report for the first time the ability of OECs to promote post-injury neurite sprouting in an in vitro model of axonal injury. Using this model, we were able to differentiate between the direct and indirect mechanisms underlying the ability of OECs to promote neuronal recovery from injury. We noted that OECs appeared to act as a physical substrate for the growth of post-injury neurite sprouts. We also found that while post-injury sprouting was promoted most when OECs were allowed to directly contact injured neurons, physical separation using tissue culture inserts (1 mm pore size, permeable to diffusible factors but not cells) did not completely block the promoting properties of OECs, suggesting that they also secrete soluble factors which aid post-injury neurite sprouting. Furthermore, this in vitro model allowed direct observation of the cellular interactions between OECs and sprouting neurites using live-cell-imaging techniques. In summary, we found that OECs separately promote neurite sprouting by providing a physical substrate for growth and through the expression of soluble factors. Our findings provide new insight into the ability of OECs to promote axonal regeneration, and also indicate potential targets for manipulation of these cells to enhance their restorative ability.