Simon Reinke

Terminally Differentiated CD8+ T Cells Negatively Affect Bone Regeneration in Humans

A subset of T cells inhibits bone regeneration in humans. No Bones About It Sticks and stones may break your bones, but immune cells will not hurt you, at least if Reinke et al. have anything to say about it. The immune system seems to have a hand in everything these days, and bone repair is no exception. T cells have been implicated in modulating bone fracture repair, even in the absence of infection. Reinke et al. take these studies into patients and find that delayed fracture healing correlated with a subset of T cells—terminally differentiated effector memory CD8+ T (TEMRA) cells. The authors examined the number of CD8+ TEMRA cells over time and found that the difference in CD8+ TEMRA cell number in patients with delayed healing reflected the individual’s immune profile, or lifelong response to infection, rather than a more acute, fracture-related event. They specifically found these cells in fracture hematoma, one of the earliest stages of fracture healing. They then took these studies into mice and found that the absence of CD8+ T cells improved bone regeneration, whereas adding CD8+ T cells impaired fracture healing. This mechanistic link supported their association in patients and suggests that these CD8+ TEMRA cells may be targeted or serve as markers for intervention in patients with delayed bone fracture healing. There is growing evidence that adaptive immunity contributes to endogenous regeneration processes: For example, endogenous bone fracture repair is modulated by T cells even in the absence of infection. Because delayed or incomplete fracture healing is associated with poor long-term outcomes and high socioeconomic costs, we investigated the relationship between an individual’s immune reactivity and healing outcome. Our study revealed that delayed fracture healing significantly correlated with enhanced levels of terminally differentiated CD8+ effector memory T (TEMRA) cells (CD3+CD8+CD11a++CD28−CD57+ T cells) in peripheral blood. This difference was long lasting, reflecting rather the individual’s immune profile in response to lifelong antigen exposure than a post-fracture reaction. Moreover, CD8+ TEMRA cells were enriched in fracture hematoma; these cells were the major producers of interferon-γ/tumor necrosis factor–α, which inhibit osteogenic differentiation and survival of human mesenchymal stromal cells. Accordingly, depletion of CD8+ T cells in a mouse osteotomy model resulted in enhanced endogenous fracture regeneration, whereas a transfer of CD8+ T cells impaired the healing process. Our data demonstrate the high impact of the individual adaptive immune profile on endogenous bone regeneration. Quantification of CD8+ TEMRA cells represents a potential marker for the prognosis of the healing outcome and opens new opportunities for early and targeted intervention strategies.

Human memory T cells from the bone marrow are resting and maintain long-lasting systemic memory

Significance Memory T cells are essential components of immunological memory. In the apparent absence of antigen, numbers of recirculating antigen-specific memory T cells dwindle, provoking the question of whether there is immunological memory without memory T cells. Here we show that human memory T cells can reside in the bone marrow as resting cells in terms of proliferation, transcription, and mobility. The repertoire of bone marrow memory T cells is enriched for systemic pathogens representing persistent, recent, and childhood challenges. In terms of absolute numbers, memory T cells specific for systemic antigens are maintained predominantly in the bone marrow, in particular those representing historic encounters. In the bone marrow, a population of memory T cells has been described that promotes efficient secondary immune responses and has been considered to be preactivated, owing to its expression of CD69 and CD25. Here we show that human bone marrow professional memory T cells are not activated but are resting in terms of proliferation, transcription, and mobility. They are in the G0 phase of the cell cycle, and their transcriptome is that of resting T cells. The repertoire of CD4+ bone marrow memory T cells compared with CD4+ memory T cells from the blood is significantly enriched for T cells specific for cytomegalovirus-pp65 (immunodominant protein), tetanus toxoid, measles, mumps, and rubella. It is not enriched for vaccinia virus and Candida albicans-MP65 (immunodominant protein), typical pathogens of skin and/or mucosa. CD4+ memory T cells specific for measles are maintained nearly exclusively in the bone marrow. Thus, CD4+ memory T cells from the bone marrow provide long-term memory for systemic pathogens.

Absolute and functional iron deficiency in professional athletes during training and recovery

BACKGROUND Iron deficiency (ID) is one of the most important metabolic dysfunctions. Athletic performance depends on oxygen transport and mitochondrial efficiency, thus on optimal iron balance. We hypothesised that physical extremes result in ID in elite athletes and that the short recovery period may be insufficient to allow a lasting replenishment of iron reserves. METHODS Iron metabolism was examined in 20 elite rowing athletes and 10 professional soccer players at the end of a competitive season, after recuperation and during pre-season training. Absolute ID values were defined as ferritin <30 μg/L, functional ID as ferritin 30-99 μg/L or 100-299 μg/L+transferrin saturation <20%. RESULTS At the end of season, 27% of all athletes had absolute ID and 70% showed functional ID. Absolute iron depletion was not generally restored after recuperation and observed at all time points in 14% of the athletes. Although athletes with initially low ferritin levels showed a slight increase during recuperation (p<0.09), these increases remained within borderline levels. Furthermore, 10% showed borderline haemoglobin levels, suggestive of mild anaemia, as defined by the World Health Organisation. CONCLUSION A significant proportion of professional athletes have ID, independent of the training mode. Although recuperation seems to allow a certain recovery of iron storage, particularly in athletes with initially low ferritin levels, this retrieval was insufficient to fully normalise reduced iron levels. Therefore, iron status should be carefully monitored during the various training and competitive periods in elite athletes. An adequate iron supplementation may be needed to maintain balanced iron stores.

Synthetic niche to modulate regenerative potential of MSCs and enhance skeletal muscle regeneration

Severe injury to the skeletal muscle often results in the formation of scar tissue, leading to a decline in functional performance. Traditionally, tissue engineering strategies for muscle repair have focused on substrates that promote myogenic differentiation of transplanted cells. In the current study, the reported data indicates that mesenchymal stromal cells (MSCs) transplanted via porous alginate cryogels promote muscle regeneration by secreting bioactive factors that profoundly influence the function of muscle progenitor cells. These cellular functions, which include heightened resistance of muscle progenitor cells to apoptosis, migration to site of injury, and prevention of premature differentiation are highly desirable in the healing cascade after acute muscle trauma. Furthermore, stimulation of MSCs with recombinant growth factors IGF-1 and VEGF165 was found to significantly enhance their paracrine effects on muscle progenitor cells. Multifunctional alginate cryogels were then utilized as synthetic niches that facilitate local stimulation of seeded MSCs by providing a sustained release of growth factors. In a clinically relevant injury model, the modulation of MSC paracrine signaling via engineered niches significantly improved muscle function by remodeling scar tissue and promoting the formation of new myofibers, outperforming standalone cell or growth factor delivery.

The influence of recovery and training phases on body composition, peripheral vascular function and immune system of professional soccer players.

Professional soccer players have a lengthy playing season, throughout which high levels of physical stress are maintained. The following recuperation period, before starting the next pre-season training phase, is generally considered short but sufficient to allow a decrease in these stress levels and therefore a reduction in the propensity for injury or musculoskeletal tissue damage. We hypothesised that these physical extremes influence the body composition, blood flow, and endothelial/immune function, but that the recuperation may be insufficient to allow a reduction of tissue stress damage. Ten professional football players were examined at the end of the playing season, at the end of the season intermission, and after the next pre-season endurance training. Peripheral blood flow and body composition were assessed using venous occlusion plethysmography and DEXA scanning respectively. In addition, selected inflammatory and immune parameters were analysed from blood samples. Following the recuperation period a significant decrease of lean body mass from 74.4±4.2 kg to 72.2±3.9 kg was observed, but an increase of fat mass from 10.3±5.6 kg to 11.1±5.4 kg, almost completely reversed the changes seen in the pre-season training phase. Remarkably, both resting and post-ischemic blood flow (7.3±3.4 and 26.0±6.3 ml/100 ml/min) respectively, were strongly reduced during the playing and training stress phases, but both parameters increased to normal levels (9.0±2.7 and 33.9±7.6 ml/100 ml/min) during the season intermission. Recovery was also characterized by rising levels of serum creatinine, granulocytes count, total IL-8, serum nitrate, ferritin, and bilirubin. These data suggest a compensated hypo-perfusion of muscle during the playing season, followed by an intramuscular ischemia/reperfusion syndrome during the recovery phase that is associated with muscle protein turnover and inflammatory endothelial reaction, as demonstrated by iNOS and HO-1 activation, as well as IL-8 release. The data provided from this study suggest that the immune system is not able to function fully during periods of high physical stress. The implications of this study are that recuperation should be carefully monitored in athletes who undergo intensive training over extended periods, but that these parameters may also prove useful for determining an individuals risk of tissue stress and possibly their susceptibility to progressive tissue damage or injury.

Exercise capacity and body composition in living-donor renal transplant recipients over time

BACKGROUND Renal transplantation (RTx) restitutes the function of the failing organ and induces convalescence of the entire organism. Our study investigates whether this is accompanied by improvements in cardiovascular function and structural changes. METHODS A total of 25 Caucasian patients (14 male, median age 44.2 +/- 9.2 years, BMI 23.7 +/- 4.0 kg/m(2)) were assessed in a prospective trial before, 1, 3 and 12 months after RTx from living donors by clinical examination, cardiopulmonary exercise testing, dual X-ray absorptiometry (DEXA) and analysis of plasma indices. RESULTS Creatinine clearance improved from 8.0 +/- 3.1 to 60.9 +/- 18.1 mL/min at 1 month, but declined at 3 (51.6 +/- 16.3 mL/min) and 12 months (53.6 +/- 20.8 mL/min, P = 0.04 versus month 1). Body composition shifted from lean towards fat tissue (25.8 +/- 12.5-31.2 +/- 11.2% body fat content, P = 0.0001). Only baseline lean weight correlated with fat increase over time (r(2) = 0.28, P = 0.008). Patients with fat content above median (n = 13) had a 3-fold increased hazard ratio of infection (CI 1.04-9.41, P = 0.042) and overall hospitalization (hazard ratio 2.95, CI 1.10-7.93, P = 0.03). PeakVO(2) decreased over RTx (23.2 +/- 6.0- 17.6 +/- 5.1 mL/kg/min) and returned to baseline levels not until 1 year later (P < 0.001). After an initial decline, muscle oxidative capacity (peakVO(2)/lean mass) improved from 33.6 +/- 10.1 to 35.0 +/- 8.2 mL/kg/min at 12 months after RTx (P < 0.001). CONCLUSIONS After RTx, body composition shifted continuously towards fat tissue, and baseline lean weight significantly correlated with fat increase over time. Both severe infections and hospitalizations are associated with a higher fat content before RTx. Exercise capacity (peakVO(2)) worsened after RTx and restitutes during follow-up, with muscle quality (peakVO(2)/lean) even exceeding baseline levels after 12 months.

Qualifying stem cell sources: how to overcome potential pitfalls in regenerative medicine?

Regenerative medicine aims to replace lost cells and to restore damaged tissues and organs by either tissue‐engineering approaches or stimulation of endogenous processes. Due to their biological properties, stem cells promise to be an effective source for such strategies. Especially adult multipotent stem cells (ASCs) are believed to be applicable in a broad range of therapies for the treatment of multifactorial diseases or age‐related degeneration, although the molecular and cellular mechanisms underlying their regenerative function are often hardly described. Moreover, in some demanding clinical situations their efficiency remains limited. Thus, a basic understanding of ASCs regenerative function, their complex interplay with their microenvironment and how compromising conditions interfere with their efficiency is mandatory for any regenerative strategy. Concerning this matter, the impact of patient‐specific constraints are often underestimated in research projects and their influence on the study results disregarded. Thus, researchers are urgently depending on well‐characterized tissue samples or cells that are connected with corresponding donor information, such as secondary diseases, medication. Here, we outline principle pitfalls during experimental studies using human samples, and describe a potential strategy to overcome these challenges by establishing a core unit for cell and tissue harvesting. This facility aims to bridge the gap between clinic and research laboratories by the provision of a direct link to the clinical operating theatres. Such a strategy clearly supports basic and clinical research in the conduct of their studies and supplies highly characterized human samples together with the corresponding donor information. Copyright

CD31+ Cells From Peripheral Blood Facilitate Bone Regeneration in Biologically Impaired Conditions Through Combined Effects on Immunomodulation and Angiogenesis

Controlled revascularization and inflammation are key elements regulating endogenous regeneration after (bone) tissue trauma. Peripheral blood‐derived cell subsets, such as regulatory T‐helper cells and circulating (endothelial) progenitor cells, respectively, can support endogenous tissue healing, whereas effector T cells that are associated with an aged immune system can hinder bone regeneration. CD31 is expressed by diverse leukocytes and is well recognized as a marker of circulating endothelial (precursor) cells; however, CD31 is absent from the surface of differentiated effector T cells. Thus, we hypothesized that by separating the inhibitory fractions from the supportive fractions of circulating cells within the peripheral blood (PB) using the CD31 marker, bone regeneration in biologically compromised conditions, such as those observed in aged patients, could be improved. In support of our hypothesis, we detected an inverse correlation between CD31+ cells and effector T cells in the hematomas of human fracture patients, dependent on the age of the patient. Furthermore, we demonstrated the regenerative capacity of human PB‐CD31+ cells in vitro. These findings were translated to a clinically relevant rat model of impaired bone healing. The transplantation of rat PB‐CD31+ cells advanced bone tissue restoration in vivo and was associated with an early anti‐inflammatory response, the stimulation of (re)vascularization, and reduced fibrosis. Interestingly, the depletion or enrichment of the highly abundant CD31+/14+ monocytes from the mixed CD31+ cell population diminished tissue regeneration at different levels, suggesting combined effects within the PB‐CD31+ subsets. In summary, an intraoperative enrichment of PB‐CD31+ cells might be a novel option to facilitate endogenous regeneration under biologically impaired situations by supporting immunomodulation and vascularization.

In situ detection of CD73+ CD90+ CD105+ lineage: Mesenchymal stromal cells in human placenta and bone marrow specimens by chipcytometry: In Situ Detection of Human MSCs

Mesenchymal stromal cells (MSCs) support endogenous regeneration and present therefore promising opportunities for in situ tissue engineering. They can be isolated and expanded from various tissues, for example, bone marrow, adipose tissue, or placenta. The minimal consensus definition criteria of ex vivo expanded MSCs requires them to be positive for CD73, CD90, and CD105 expression, while being negative for CD34, CD45, CD14, CD19, and HLA‐DR. This study aimed to compare the in situ phenotype of MSCs with that of their culture‐expanded progeny. We report for the first time in situ detection of cells expressing this marker combination in human placenta cryosections as well as in bone marrow aspirates using multiplex‐immunohistology (Chipcytometry), a technique that allows staining of more than 100 biomarkers consecutively on the same cell.

Rapid detection of periprosthetic joint infection using a combination of 16s rDNA polymerase chain reaction and lateral flow immunoassay: A Pilot Study

Objectives The objective of this study was to develop a test for the rapid (within 25 minutes) intraoperative detection of bacteria from synovial fluid to diagnose periprosthetic joint infection (PJI). Methods The 16s rDNA test combines a polymerase chain reaction (PCR) for amplification of 16s rDNA with a lateral flow immunoassay in one fully automated system. The synovial fluid of 77 patients undergoing joint aspiration or primary or revision total hip or knee surgery was prospectively collected. The cohort was divided into a proof-of-principle cohort (n = 17) and a validation cohort (n = 60). Using the proof-of-principle cohort, an optimal cut-off for the discrimination between PJI and non-PJI samples was determined. PJI was defined as detection of the same bacterial species in a minimum of two microbiological samples, positive histology, and presence of a sinus tract or intra-articular pus. Results The 16s rDNA test proved to be very robust and was able to provide a result in 97% of all samples within 25 minutes. The 16s rDNA test was able to diagnose PJI with a sensitivity of 87.5% and 82%, and a specificity of 100% and 89%, in the proof-of-principle and validation cohorts, respectively. The microbiological culture of synovial fluid achieved a sensitivity of 80% and a specificity of 93% in the validation cohort. Conclusion The 16s rDNA test offers reliable intraoperative detection of all bacterial species within 25 minutes with a sensitivity and specificity comparable with those of conventional microbiological culture of synovial fluid for the detection of PJI. The 16s rDNA test performance is independent of possible blood contamination, culture time and bacterial species. Cite this article: V. Janz, J. Schoon, C. Morgenstern, B. Preininger, S. Reinke, G. Duda, A. Breitbach, C. F. Perka, S. Geissler. Rapid detection of periprosthetic joint infection using a combination of 16s rDNA polymerase chain reaction and lateral flow immunoassay: A Pilot Study. Bone Joint Res 2018;7:12–19. DOI: 10.1302/2046-3758.71.BJR-2017-0103.R2.