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1.
Ann Rheum Dis ; 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33762264

RESUMO

INTRODUCTION: In light of the SARS-CoV-2 pandemic, protecting vulnerable groups has become a high priority. Persons at risk of severe disease, for example, those receiving immunosuppressive therapies for chronic inflammatory cdiseases (CIDs), are prioritised for vaccination. However, data concerning generation of protective antibody titres in immunosuppressed patients are scarce. Additionally, mRNA vaccines represent a new vaccine technology leading to increased insecurity especially in patients with CID. OBJECTIVE: Here we present for the first time, data on the efficacy and safety of anti-SARS-CoV-2 mRNA vaccines in a cohort of immunosuppressed patients as compared with healthy controls. METHODS: 42 healthy controls and 26 patients with CID were included in this study (mean age 37.5 vs 50.5 years). Immunisations were performed according to national guidelines with mRNA vaccines. Antibody titres were assessed by ELISA before initial vaccination and 7 days after secondary vaccination. Disease activity and side effects were assessed prior to and 7 days after both vaccinations. RESULTS: Anti-SARS-CoV-2 antibodies as well as neutralising activity could be detected in all study participants. IgG titres were significantly lower in patients as compared with controls (2053 binding antibody units (BAU)/mL ±1218 vs 2685±1102). Side effects were comparable in both groups. No severe adverse effects were observed, and no patients experienced a disease flare. CONCLUSION: We show that SARS-CoV-2 mRNA vaccines lead to development of antibodies in immunosuppressed patients without considerable side effects or induction of disease flares. Despite the small size of this cohort, we were able to demonstrate the efficiency and safety of mRNA vaccines in our cohort.

2.
J Bone Miner Res ; 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33534144

RESUMO

After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (µCT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours co-cultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1ß were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies. © 2021 American Society for Bone and Mineral Research (ASBMR).

3.
Int J Mol Sci ; 21(23)2020 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-33256266

RESUMO

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) signaling pathway serves as an important downstream mediator for a variety of cytokines, hormones, and growth factors. Emerging evidence suggests JAK/STAT signaling pathway plays an important role in bone development, metabolism, and healing. In this light, pro-inflammatory cytokines are now clearly implicated in these processes as they can perturb normal bone remodeling through their action on osteoclasts and osteoblasts at both intra- and extra-articular skeletal sites. Here, we summarize the role of JAK/STAT pathway on development, homeostasis, and regeneration based on skeletal phenotype of individual JAK and STAT gene knockout models and selective inhibition of components of the JAK/STAT signaling including influences of JAK inhibition in osteoclasts, osteoblasts, and osteocytes.

4.
ALTEX ; 37(4): 561-578, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32521037

RESUMO

The aim of the study was to establish an in vitro fracture hematoma (FH) model that mimics the in vivo situation of the human fracture gap in order to assess drug efficacy and effectiveness for the treatment of fracture healing disorders. Human peripheral blood and mesenchymal stromal cells (MSCs) were coagulated to produce in vitro FH models, which were incubated in osteogenic medium under normoxia/hypoxia and analyzed for cell composition, gene expression and cytokine/chemokine secretion. To evaluate the model, we studied the impact of dexamethasone (impairing fracture healing) and deferoxamine (promoting fracture healing). Under hypoxic conditions, MSCs represented the predominant cell population, while the frequencies of leukocyte populations decreased. Marker gene expression of osteogenesis, angiogenesis, inflammation, migration and hypoxic adaptation increased significantly over time and compared to normoxia, while cytokine/chemokine secretion remained unchanged. Dexamethasone favored the frequency of immune cells compared to MSCs, suppressed osteogenic and pro-angiogenic gene expression, and enhanced the secretion of inflammatory cytokines. Conversely, deferoxamine favored the frequency of MSCs over that of immune cells and enhanced the expression of the osteogenic marker RUNX2 and markers of hypoxic adaptation. In summary, we demonstrate that hypoxia is an important factor for modeling the initial phase of fracture healing in vitro and that both fracture-healing disrupting and promoting substances can influence the in vitro model comparable to the in vivo situation. Therefore, we conclude that our model is able to mimic in part the human FH and could reduce the number of animal experiments in early preclinical studies.

6.
Int J Mol Sci ; 21(3)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-32013232

RESUMO

Both inflammatory diseases like rheumatoid arthritis (RA) and anti-inflammatory treatment of RA with glucocorticoids (GCs) or non-steroidal anti-inflammatory drugs (NSAIDs) negatively influence bone metabolism and fracture healing. Janus kinase (JAK) inhibition with tofacitinib has been demonstrated to act as a potent anti-inflammatory therapeutic agent in the treatment of RA, but its impact on the fundamental processes of bone regeneration is currently controversially discussed and at least in part elusive. Therefore, in this study, we aimed to examine the effects of tofacitinib on processes of bone healing focusing on recruitment of human mesenchymal stromal cells (hMSCs) into the inflammatory microenvironment of the fracture gap, chondrogenesis, osteogenesis and osteoclastogenesis. We performed our analyses under conditions of reduced oxygen availability in order to mimic the in vivo situation of the fracture gap most optimal. We demonstrate that tofacitinib dose-dependently promotes the recruitment of hMSCs under hypoxia but inhibits recruitment of hMSCs under normoxia. With regard to the chondrogenic differentiation of hMSCs, we demonstrate that tofacitinib does not inhibit survival at therapeutically relevant doses of 10-100 nM. Moreover, tofacitinib dose-dependently enhances osteogenic differentiation of hMSCs and reduces osteoclast differentiation and activity. We conclude from our data that tofacitinib may influence bone healing by promotion of hMSC recruitment into the hypoxic microenvironment of the fracture gap but does not interfere with the cartilaginous phase of the soft callus phase of fracture healing process. We assume that tofacitinib may promote bone formation and reduce bone resorption, which could in part explain the positive impact of tofacitinib on bone erosions in RA. Thus, we hypothesize that it will be unnecessary to stop this medication in case of fracture and suggest that positive effects on osteoporosis are likely.


Assuntos
Inibidores de Janus Quinases/farmacologia , Janus Quinases/metabolismo , Osteogênese/efeitos dos fármacos , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Janus Quinases/antagonistas & inibidores , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo
7.
Arthritis Res Ther ; 21(1): 209, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533820

RESUMO

BACKGROUND: Fluorescence optical imaging (FOI) enables visualization of inflammation in the hands in rheumatic joint diseases with currently a lack of long-term follow-up studies. OBJECTIVE: To investigate FOI for treatment monitoring in a homogenous cohort of patients with early (disease duration < 2 years) and active (DAS28 > 3.2) RA over a period of 12 months. METHODS: Thirty-five RA patients (24 (68.6%) females, mean age 53.3 years (SD 13.6)) were investigated clinically by DAS28, tender joint count (TJC) and swollen joint count (SJC) and by FOI in phases 1-3 and PrimaVistaMode (PVM) before therapy change and after 12 months. The FOI activity score (FOIAS) was calculated based on individual joint scores from 0 to 3 in 30 joints per patient, adding up to a sum score (0-90). RESULTS: We found a statistically significant reduction of FOIAS in phase 1 from baseline (median 5.0, IQR 24.96) to follow-up (median 1.0, IQR 4.0) in all patients (p = 0.0045), both in responders and non-responders according to EULAR response criteria by DAS28. Statistically significant reductions over 12 months were found for median DAS28(ESR) 5.61 to 3.31, TJC 7.0 to 1.0, and SJC 5.0 to 1.0 (each p <  0.001). No statistically significant correlations were detected between the FOIAS change in phase 1 and DAS28(ESR), TJC, or SJC. Correlations between the other phases and clinical outcomes were weak to moderate. CONCLUSION: Reduced early enhancement in FOI phase 1 can be observed in clinically responding and non-responding early RA patients under treatment. Regarding potential marker performance, FOI probably shows a reduction of inflammation more objectively.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/diagnóstico , Imagem Óptica/métodos , Idoso , Artrite Reumatoide/tratamento farmacológico , Feminino , Fluorescência , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Fatores de Tempo
8.
J Back Musculoskelet Rehabil ; 32(6): 897-903, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958330

RESUMO

BACKGROUND AND OBJECTIVES: Bone marrow oedema is a multifactorial conditioned illness. Alongside any strain relief of an affected joint, treatment with Iloprost also belongs to the choice of cures. In past studies, a modulatory effect on bone could be shown. The hypothesis of the present work is that Iloprost has a growth-stimulating effect on osteoblasts in vitro. METHODS: Human osteoblasts were isolated and cultivated. Subsequently, the cells were treated with Iloprost in bioavailable concentrations. Alterations of the cell structure were examined by means of light microscopy. A regulation of the number of vital cells was carried out by using a CASY cell counter. Possible cell impairment after Iloprost treatment was analysed by means of XTT Elisa as well as FDA and PI staining via fluorescence microscopy. RESULTS: Using light microscopy, no changes in cell structure could be observed. With the CASY cell counter, no increase in the numbers of osteoblasts appeared after Iloprost treatment. Also, XTT Elisas and fluorescence microscopy did not reveal any cell impairment due to Iloprost. CONCLUSION: Our results could not confirm a modulatory effect in mature osteoblasts. On the basis of the present work we could not verify any growth-stimulating effect by Iloprost in mature osteoblasts in vitro. Admittedly, effects had been shown previously during osteogenesis, but we do exclude an effect on mature osteoblasts which have already differentiated.


Assuntos
Iloprosta/farmacologia , Osteoblastos/efeitos dos fármacos , Vasodilatadores/farmacologia , Células Cultivadas , Humanos , Microscopia , Osteoblastos/patologia
9.
PLoS One ; 14(4): e0214276, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30947253

RESUMO

Fractures in horses-whether simple fractures with just one clean break, or incomplete greenstick with stress fractures, or complications such as shattered bones can all be either minimal or even catastrophic. Thus, improvement in fracture healing is a hallmark in equine orthopedics. The fracture healing process implements a complex sequence of events including the initial inflammatory phase removing damaged tissue, re-establishment of vessels and mesenchymal stromal cells, a soft and hard callus phase closing the fracture gap as well as the remodeling phase shaping the bone to a scar-free tissue. Detailed knowledge on processes in equine fracture healing in general and on the initial phase in particular is apparently very limited. Therefore, we generated equine in vitro fracture hematoma models (FH models) to study time-dependent changes in cell composition and RNA-expression for the most prominent cells in the FH model (immune cells, mesenchymal stromal cells) under conditions most closely adapted to the in vivo situation (hypoxia) by using flow cytometry and qPCR. In order to analyze the impact of mesenchymal stromal cells in greater detail, we also incubated blood clots without the addition of mesenchymal stromal cells under the same conditions as a control. We observed a superior survival capacity of mesenchymal stromal cells over immune cells within our FH model maintained under hypoxia. Furthermore, we demonstrate an upregulation of relevant angiogenic, osteogenic and hypoxia-induced markers within 48 h, a time well-known to be crucial for proper fracture healing.


Assuntos
Consolidação da Fratura , Fraturas Ósseas/patologia , Fraturas Ósseas/terapia , Hematoma/terapia , Hipóxia/patologia , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Animais , Biomarcadores/metabolismo , Biópsia , Sobrevivência Celular/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Hematoma/patologia , Cavalos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Oxigênio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos
10.
Acta Biomater ; 86: 171-184, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30616076

RESUMO

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.


Assuntos
Colágeno Tipo I/farmacologia , Consolidação da Fratura/efeitos dos fármacos , Osteotomia , Tecidos Suporte/química , Animais , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/patologia , Calcificação Fisiológica/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/ultraestrutura , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fator de Necrose Tumoral alfa/metabolismo , Microtomografia por Raio-X
11.
Cartilage ; 10(3): 364-369, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-29468902

RESUMO

OBJECTIVE: In orthopedic joint injection, the most frequently used local anesthetics are ropivacaine, bupivacaine, and 1% or 2% lidocaine. The aim of this study was to examine effects of these various anesthetics on the viability of human chondrocytes. Our hypothesis was that all local anesthetics tested damage human chondrocytes in vitro. METHODS: Primary human chondrocytes were isolated and cultured from 6 donated human knee joints (mean age of donors 61.2 years). Local anesthetics were added to these cultures. Toxicity analysis was performed by visualization of cell structure using light microscopy. Determination of vital chondrocytes was performed by use of a Casy cell counter. Chondrocytes' cell death was examined by fluorescence microscopy and an XTT ELISA assay. RESULTS: Light microscope and fluorescence microscope data revealed a defect cell structure and increased number of dead cells after addition of 1% or 2% lidocaine and bupivacaine but not ropivacaine. We were able to show an increased level of XTT activity after treatment with bupivacaine, 2% lidocaine or ropivacaine. The count of vital chondrocytes was significantly decreased after treatment with bupivacaine, 1% or 2% lidocaine, and ropivacaine. CONCLUSIONS: The data show that treatment with local anesthetics induces cell damage of human chondrocytes in vitro. Ropivacaine seems to be a local anesthetic with the lowest toxic potential on human chondrocytes, a feature that may favor its preference for use in joint injection.


Assuntos
Anestésicos Locais/toxicidade , Bupivacaína/toxicidade , Células Cultivadas/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Lidocaína/toxicidade , Ropivacaina/toxicidade , Morte Celular , Células Cultivadas/ultraestrutura , Condrócitos/ultraestrutura , Humanos , Injeções Intra-Articulares , Microscopia de Fluorescência/métodos , Pessoa de Meia-Idade , Doadores de Tecidos
12.
Int J Mol Sci ; 19(8)2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-30087255

RESUMO

Mesenchymal stem/stromal cells (MSCs) are stem cells of the connective tissue, possess a plastic phenotype, and are able to differentiate into various tissues. Besides their role in tissue regeneration, MSCs perform additional functions as a modulator or inhibitor of immune responses. Due to their pleiotropic function, MSCs have also gained therapeutic importance for the treatment of autoimmune diseases and for improving fracture healing and cartilage regeneration. However, the therapeutic/immunomodulatory mode of action of MSCs is largely unknown. Here, we describe that MSCs express the inhibitory receptor CTLA-4 (cytotoxic T lymphocyte antigen 4). We show that depending on the environmental conditions, MSCs express different isoforms of CTLA-4 with the secreted isoform (sCTLA-4) being the most abundant under hypoxic conditions. Furthermore, we demonstrate that the immunosuppressive function of MSCs is mediated mainly by the secretion of CTLA-4. These findings open new ways for treatment when tissue regeneration/fracture healing is difficult.


Assuntos
Antígeno CTLA-4/imunologia , Células-Tronco Mesenquimais/imunologia , Adipogenia , Antígeno CTLA-4/análise , Células Cultivadas , Humanos , Tolerância Imunológica , Células-Tronco Mesenquimais/citologia , Osteogênese
13.
Microvasc Res ; 116: 34-44, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28993199

RESUMO

Hypoxia driven angiogenesis is a prominent feature of tissue regeneration, inflammation and tumor growth and is regulated by hypoxia-inducible factor (HIF)-1 and -2. The distinct functions of HIFs in the hypoxia-induced angiogenesis and metabolic switch of endothelial cells are still unknown and therefore aim of this study. We investigated the role of HIF-1 and -2 in the adaptation of immortalized human microvascular endothelial cells (HMEC-1) to hypoxic conditions (1% O2) in terms of angiogenesis, cytokine secretion, gene expression and ATP/ADP-ratio using shRNA-mediated reduction of the oxygen sensitive α-subunits of either HIF-1 or HIF-2 or the combination of both. Reduction of HIF-1α diminished cellular energy, hypoxia-induced glycolytic gene expression, and angiogenesis not altering pro-angiogenic factors. Reduction of HIF-2α diminished hypoxia-induced pro-angiogenic factors, enhanced anti-angiogenic factors and attenuated angiogenesis not altering glycolytic gene expression. Reduction of both HIFs reduced cell survival, gene expression of glycolytic enzymes and pro-angiogenic factors as compared to the corresponding control. Finally, we identified the macrophage migration inhibitory factor (MIF) to be redundantly regulated by HIF-1 and HIF-2 and to be essential in the process of hypoxia-driven angiogenesis. Our results demonstrate a major impact of HIF-1 and HIF-2 on hypoxia-induced angiogenesis indicating distinct but also overlapping functions of HIF-1 and HIF-2. These findings open new possibilities for therapeutic approaches by specifically targeting the HIF-1 and HIF-2 or their target MIF.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Endoteliais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Microvasos/metabolismo , Neovascularização Fisiológica , Adaptação Fisiológica , Antígenos de Diferenciação de Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular , Linhagem Celular , Microambiente Celular , Regulação Enzimológica da Expressão Gênica , Glicólise , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Microvasos/citologia , Neovascularização Fisiológica/genética , Transdução de Sinais
14.
Int J Mol Sci ; 18(3)2017 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-28282868

RESUMO

Immunologically restricted patients such as those with autoimmune diseases or malignancies often suffer from delayed or insufficient fracture healing. In human fracture hematomas and the surrounding bone marrow obtained from immunologically restricted patients, we analyzed the initial inflammatory phase on cellular and humoral level via flow cytometry and multiplex suspension array. Compared with controls, we demonstrated higher numbers of immune cells like monocytes/macrophages, natural killer T (NKT) cells, and activated T helper cells within the fracture hematomas and/or the surrounding bone marrow. Also, several pro-inflammatory cytokines such as Interleukin (IL)-6 and Tumor necrosis factor α (TNFα), chemokines (e.g., Eotaxin and RANTES), pro-angiogenic factors (e.g., IL-8 and Macrophage migration inhibitory factor: MIF), and regulatory cytokines (e.g., IL-10) were found at higher levels within the fracture hematomas and/or the surrounding bone marrow of immunologically restricted patients when compared to controls. We conclude here that the inflammatory activity on cellular and humoral levels at fracture sites of immunologically restricted patients considerably exceeds that of control patients. The initial inflammatory phase profoundly differs between these patient groups and is probably one of the reasons for prolonged or insufficient fracture healing often occurring within immunologically restricted patients.


Assuntos
Consolidação da Fratura/imunologia , Hospedeiro Imunocomprometido , Inflamação/imunologia , Indutores da Angiogênese/metabolismo , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Estudos de Casos e Controles , Citocinas/metabolismo , Feminino , Fraturas Ósseas/imunologia , Fraturas Ósseas/patologia , Hematoma/imunologia , Hematoma/patologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Neovascularização Fisiológica , Fenótipo
16.
Int J Nanomedicine ; 11: 5883-5896, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27877036

RESUMO

Combined individually tailored methods for diagnosis and therapy (theragnostics) could be beneficial in destructive diseases, such as rheumatoid arthritis. Nanoparticles are promising candidates for theragnostics due to their excellent biocompatibility. Nanoparticle modifications, such as improved surface coating, are in development to meet various requirements, although safety concerns mean that modified nanoparticles require further review before their use in medical applications is permitted. We have previously demonstrated that iron oxide nanoparticles with amino-polyvinyl alcohol (a-PVA) adsorbed on their surfaces have the unwanted effect of increasing human immune cell cytokine secretion. We hypothesized that this immune response was caused by free-floating PVA. The aim of the present study was to prevent unwanted immune reactions by further surface modification of the a-PVA nanoparticles. After cross-linking of PVA to nanoparticles to produce PVA-grafted nanoparticles, and reduction of their zeta potential, the effects on cell viability and cytokine secretion were analyzed. PVA-grafted nanoparticles still stimulated elevated cytokine secretion from human immune cells; however, this was inhibited after reduction of the zeta potential. In conclusion, covalent cross-linking of PVA to nanoparticles and adjustment of the surface charge rendered them nontoxic to immune cells, nonimmunogenic, and potentially suitable for use as theragnostic agents.


Assuntos
Células Sanguíneas/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Compostos Férricos/química , Nanopartículas de Magnetita/administração & dosagem , Álcool de Polivinil/química , Adsorção , Células Sanguíneas/metabolismo , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Nanopartículas de Magnetita/química
17.
Immunol Res ; 64(5-6): 1195-1206, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27629117

RESUMO

The initial inflammatory phase of fracture healing is of great importance for the clinical outcome. We aimed to develop a detailed time-dependent analysis of the initial fracture hematoma. We analyzed the composition of immune cell subpopulations by flow cytometry and the concentration of cytokines and chemokines by bioplex in 42 samples from human fractures of long bones <72 h post-trauma. The early human fracture hematoma is characterized by maturation of granulocytes and migration of monocytes/macrophages and hematopoietic stem cells. Both T helper cells and cytotoxic T cells proliferate within the fracture hematoma and/or migrate to the fracture site. Humoral immunity characteristics comprise high concentration of pro-inflammatory cytokines such as IL-6, IL-8, IFNγ and TNFα, but also elevated concentration of anti-inflammatory cytokines, e.g., IL-1 receptor antagonist and IL-10. Furthermore, we found that cells of the fracture hematoma represent a source for key chemokines. Even under the bioenergetically restricted conditions that exist in the initial fracture hematoma, immune cells are not only present, but also survive, mature, function and migrate. They secrete a cytokine/chemokine cocktail that contributes to the onset of regeneration. We hypothesize that this specific microenvironment of the initial fracture hematoma is among the crucial factors that determine fracture healing.


Assuntos
Osso e Ossos/imunologia , Fraturas Ósseas/imunologia , Hematoma/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Movimento Celular , Proliferação de Células , Separação Celular , Células Cultivadas , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Granulócitos , Humanos , Macrófagos , Masculino , Pessoa de Meia-Idade
18.
Clin Exp Rheumatol ; 34(5): 848-856, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27385076

RESUMO

OBJECTIVES: To compare sleep quality, disease activity and patient-reported outcomes such as fatigue and immune parameters in patients with rheumatoid arthritis treated with etanercept (ETA) or methotrexate (MTX). METHODS: Of 36 patients (28-joint Disease Activity Score, DAS28CRP≥3.2) in this 16-week (w), open, prospective study, 19 (11 women) received MTX 12.5-17 mg/w, and 17 (14 women) received ETA 25 mg x 2/w, alone or in combination with MTX. Clinical (DAS28CRP, visual analogue scale), laboratory (C-reactive protein [CRP]), sleep (polysomnography), functional (Multidimensional Fatigue Inventory; Health Assessment Questionnaire-Disability Index (HAQ-DI); 36-item Short-Form Health Survey (SF-36), immunological (humoral/cellular) and neuroendocrine (hormonal) parameters were recorded at baseline (BL), w8 and w16. RESULTS: BL characteristics did not differ significantly between the ETA and MTX groups except disease duration: mean age (years): 48.6±8.8 vs. 49.4±16.6; mean disease duration (months): 19.6±46.3 vs. 81.2±79.2; and DAS28CRP: 4.4±0.9 vs. 4.4±1.7, respectively. DAS28CRP, SF-36, and HAQ-DI improved significantly in both groups from BL to w16 (p≤0.05). The DAS28CRP improvements at w16 (mean changes -1.8 in the ETA group, and -1.4 in MTX group), were not statistically significant from each other. The absolute values of sleep efficiency, total sleep time, and stage 2 sleep duration increased significantly in the ETA group, but no significant changes were reported in the MTX group. CONCLUSIONS: Both therapies improved disease activity, CRP, SF-36 and HAQ-DI, with faster, more pronounced changes in DAS28CRP in the ETA group, which alone had significantly improved sleep parameters.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Etanercepte/uso terapêutico , Fadiga/prevenção & controle , Metotrexato/uso terapêutico , Transtornos do Sono-Vigília/prevenção & controle , Sono/efeitos dos fármacos , Adolescente , Adulto , Idoso , Antirreumáticos/efeitos adversos , Artrite Reumatoide/complicações , Artrite Reumatoide/imunologia , Artrite Reumatoide/fisiopatologia , Avaliação da Deficiência , Quimioterapia Combinada , Etanercepte/efeitos adversos , Fadiga/imunologia , Fadiga/fisiopatologia , Feminino , Humanos , Masculino , Metotrexato/efeitos adversos , Pessoa de Meia-Idade , Medidas de Resultados Relatados pelo Paciente , Projetos Piloto , Polissonografia , Valor Preditivo dos Testes , Estudos Prospectivos , Indução de Remissão , Transtornos do Sono-Vigília/imunologia , Transtornos do Sono-Vigília/fisiopatologia , Inquéritos e Questionários , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
19.
Immunol Res ; 64(3): 665-76, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26721805

RESUMO

The synovial tissue in rheumatoid arthritis (RA) represents a hypoxic environment with up-regulated pro-inflammatory cytokines and cellular infiltrates including neutrophils. Although inhibition of the interleukin (IL)6 receptor pathway by tocilizumab is a potent treatment option for RA, it may also cause adverse effects such as an occasionally high-grade neutropenia. We analysed the impact of tocilizumab on survival, mediator secretion, oxidative burst, phagocytosis and energy availability of high-dose toll-like receptor (TLR)2/4-stimulated neutrophils (to mimic an arthritis flare) under normoxic versus hypoxic conditions. Human neutrophils were purified, pre-treated with varying doses of tocilizumab, dexamethasone or human IgG1 and high-dose-stimulated with lipopolysaccharide (LPS) alone-triggering TLR2/4-, LPS plus IL6, or left unstimulated. Cells were then incubated under normoxic (18 % O2) or hypoxic (1 % O2) conditions and subsequently analysed. Neutrophil survival and energy availability were significantly decreased by tocilizumab in a dose-dependent manner in high-dose TLR2/4-stimulated cells, but to a greater extent under normoxia as compared to hypoxia. We also found high-dose LPS-stimulated oxidative burst and phagocytosis of neutrophils to be higher under hypoxic versus normoxic conditions, but this difference was reduced by tocilizumab. Finally, we observed that tocilizumab affected neutrophil mediator secretion as a function of oxygen availability. Tocilizumab is known for both beneficial effects and a higher incidence of neutropenia when treating RA patients. Our results suggest that both effects can at least in part be explained by a reduction in neutrophil survival, a dose-dependent inhibition of hypoxia-induced NADPH oxidase-mediated oxidative burst and phagocytosis of infiltrating hypoxic neutrophils and an alteration of mediator secretion.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Hipóxia/tratamento farmacológico , Imunoterapia/métodos , Neutrófilos/efeitos dos fármacos , Respiração Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Metabolismo Energético , Humanos , Subunidade alfa de Receptor de Interleucina-5/imunologia , Lipopolissacarídeos/imunologia , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Fagocitose , Explosão Respiratória
20.
Int J Nanomedicine ; 10: 3429-45, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26056442

RESUMO

Nanotechnology provides new opportunities in human medicine, mainly for diagnostic and therapeutic purposes. The autoimmune disease rheumatoid arthritis (RA) is often diagnosed after irreversible joint structural damage has occurred. There is an urgent need for a very early diagnosis of RA, which can be achieved by more sensitive imaging methods. Superparamagnetic iron oxide nanoparticles (SPION) are already used in medicine and therefore represent a promising tool for early diagnosis of RA. The focus of our work was to investigate any potentially negative effects resulting from the interactions of newly developed amino-functionalized amino-polyvinyl alcohol coated (a-PVA) SPION (a-PVA-SPION), that are used for imaging, with human immune cells. We analyzed the influence of a-PVA-SPION with regard to cell survival and cell activation in human whole blood in general, and in human monocytes and macrophages representative of professional phagocytes, using flow cytometry, multiplex suspension array, and transmission electron microscopy. We found no effect of a-PVA-SPION on the viability of human immune cells, but cytokine secretion was affected. We further demonstrated that the percentage of viable macrophages increased on exposure to a-PVA-SPION. This effect was even stronger when a-PVA-SPION were added very early in the differentiation process. Additionally, transmission electron microscopy analysis revealed that both monocytes and macrophages are able to endocytose a-PVA-SPION. Our findings demonstrate an interaction between human immune cells and a-PVA-SPION which needs to be taken into account when considering the use of a-PVA-SPION in human medicine.


Assuntos
Artrite Reumatoide/sangue , Nanopartículas de Magnetita/química , Álcool de Polivinil/química , Sobrevivência Celular/efeitos dos fármacos , Citocinas/sangue , Endocitose/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Nanopartículas de Magnetita/efeitos adversos , Microscopia Eletrônica de Transmissão , Monócitos/efeitos dos fármacos , Testes de Toxicidade/métodos
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