IntroductionIn 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.ObjectiveHere 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.Methods42 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.ResultsAnti-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.ConclusionWe 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.

Bone healing commences with an inflammatory reaction which initiates the regenerative healing process leading in the end to reconstitution of bone. An unbalanced immune reaction during this early bone healing phase is hypothesized to disturb the healing cascade in a way that delays bone healing and jeopardizes the successful healing outcome. The immune cell composition and expression pattern of angiogenic factors were investigated in a sheep bone osteotomy model and compared to a mechanically-induced impaired/delayed bone healing group. In the impaired/delayed healing group, significantly higher T cell percentages were present in the bone hematoma and the bone marrow adjacent to the osteotomy gap when compared to the normal healing group. This was mirrored in the higher cytotoxic T cell percentage detected under delayed bone healing conditions indicating longer pro-inflammatory processes. The highly activated periosteum adjourning the osteotomy gap showed lower expression of hematopoietic stem cell markers and angiogenic factors such as heme oxygenase and vascular endothelial growth factor. This indicates a deferred revascularization of the injured area due to ongoing pro-inflammatory processes in the delayed healing group. Results from this study suggest that there are unfavorable immune cells and factors participating in the initial healing phase. In conclusion, identifying beneficial aspects may lead to promising therapeutical approaches that might benefit further by eliminating the unfavorable factors.

BackgroundBone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages.Methodology/Principal FindingsBone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis.Conclusions/SignificanceHypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches.

During hematoma formation following injury, an inflammatory reaction ensues as an initial step in the healing process. As granulation tissue matures, revascularization is a prerequisite for successful healing. The hypothesis of this study was that scarless tissue reconstitution in the regenerative bone healing process is dependent on a balanced immune reaction that initiates revasculatory steps. To test this hypothesis, cellular composition and expression profiles of a bone hematoma (regenerative, scarless) was compared with a muscle soft tissue hematoma (healing with a scar) in a sheep model. Upregulation of regulatory T helper cells and anti-inflammatory cytokine expression (IL-10) coincided with an upregulation of angiogenic factors (HIF1α and HIF1α regulated genes) in the regenerative bone hematoma but not in the soft tissue hematoma. These results indicate that the timely termination of inflammation and early onset of revascularization are interdependent and essential for a regenerative healing process. Prolonged pro-inflammatory signaling occurring in a delayed bone-healing model supports the finding that timely termination of inflammation furthers the regenerative process. Differing cellular compositions are due to different cell sources invading the hematoma, determining the ensuing cytokine expression profile and thus paving the path for regenerative healing in bone or the formation of scar tissue in muscle injury.

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.

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Objective. Glucocorticoids (GCs) exert their antiinflammatory and immunosuppressive effects in humans primarily via the cytosolic GC receptor (cGR) but also via rapid, nongenomic mechanisms. Most likely, membrane-bound GRs (mGR) are involved in nongenomic GC signaling. The aim of this study was to investigate the origin and functional activity of mGR.Methods. We analyzed the origin of mGR using mGR-expressing HEK 293T cells, by transient and stable RNA interference-mediated GR reduction. GR messenger RNA (mRNA) and cGR and mGR protein levels were analyzed by real-time quantitative polymerase chain reaction, immunoblotting, and highsensitivity immunofluorescence staining. Furthermore, we analyzed the functional activity of mGR, using membrane-impermeable bovine serum albumin (BSA)-bound dexamethasone (DEX-BSA) in human monocytes. Membrane-bound GR-expressing monocytes were treated with DEX, DEX-BSA, or BSA. Cell lysates were analyzed using PepChip arrays in order to identify kinases triggered by DEX-BSA, with validation using Bio-Plex assays and immunoblotting.Results. Our data showed that transient reduction of GR mRNA in HEK 293T cells decreased cGR protein levels but not mGR protein levels. However, stably transfected cells showed reduced cGR protein expression and significantly reduced mGR protein expression. Furthermore, 51 kinase substrates were identified for which phosphorylation was either reduced or increased. We observed p38 MAP kinase (MAPK) as one possible upstream kinase. Validation of these data by Bio-Plex phosphoprotein assay and immunoblotting showed increased phosphorylation of p38 MAPK after treatment with DEX-BSA.Conclusion. Our data demonstrate that the human GR gene encodes for both cGR and mGR. Membrane-bound GR retains functional activity, as indicated by induced phosphorylation of p38 MAPK due to DEX-BSA treatment. Membrane-bound GRmediated cellular signaling needs to be investigated further in order to clarify its therapeutic potential.Glucocorticoids (GCs) are the most commonly used drugs in the treatment of a wide range of rheumatic diseases. These substances exert their immunosuppressive, antiinflammatory, and antiallergic effects on primary and secondary immune cells, tissues, and organs (1). Unfortunately, GCs have the potential to exert unwanted effects on metabolism, bone tissue, muscles, eyes, and skin, especially when the dose and duration of treatment are increased (2-5). These disadvantages drive the need for an improved risk/benefit ratio for GCs, for instance by understanding the mechanisms of GC action in greater detail. The therapeutic effects of GCs are primarily mediated by cytosolic GC receptor ␣ (cGR␣), the classic genomic mechanism of GC action Dr. Buttgereit's work was supported by grants from the DFG (Bu 1015/7-1) and the Deutsche Sparkassenstiftung Medizin. Mr. Hahne's work was supported by the DFG Graduate School GSC 203 Project at the Berlin-Brandenburg School for Regenerative Therapies.

The initial inflammatory phase of bone fracture healing represents a critical step for the outcome of the healing process. However, both the mechanisms initiating this inflammatory phase and the function of immune cells present at the fracture site are poorly understood. In order to study the early events within a fracture hematoma, we established an in vitro fracture hematoma model: we cultured hematomas forming during an osteotomy (artificial bone fracture) of the femur during total hip arthroplasty (THA) in vitro under bioenergetically controlled conditions. This model allowed us to monitor immune cell populations, cell survival and cytokine expression during the early phase following a fracture. Moreover, this model enabled us to change the bioenergetical conditions in order to mimic the in vivo situation, which is assumed to be characterized by hypoxia and restricted amounts of nutrients. Using this model, we found that immune cells adapt to hypoxia via the expression of angiogenic factors, chemoattractants and pro-inflammatory molecules. In addition, combined restriction of oxygen and nutrient supply enhanced the selective survival of lymphocytes in comparison with that of myeloid derived cells (i.e., neutrophils). Of note, non-restricted bioenergetical conditions did not show any similar effects regarding cytokine expression and/or different survival rates of immune cell subsets. In conclusion, we found that the bioenergetical conditions are among the crucial factors inducing the initial inflammatory phase of fracture healing and are thus a critical step for influencing survival and function of immune cells in the early fracture hematoma.