Posttraumatic stress disorder is characterized by functional dysregulation of dermal fibroblasts.

Incidence of mental health disorders are rising in modernity, with psychological stress linked to a propensity for developing various chronic diseases due to a relative inability of the body to counter the allostatic load on cellular level. Despite these high rates of comorbidities associated with posttraumatic stress disorder (PTSD), there is still a lack of understanding in terms of the peripheral effects of PTSD on tissue level. Therefore, the purpose of this study was to profile basal dermal fibroblast functional status in PTSD using a wide range of markers involved in the cell-to-cell communication facilitated by fibroblasts. Primary dermal fibroblasts derived from patients diagnosed with PTSD (n = 11) and matched trauma exposed controls (i.e. who did not develop PTSD, n = 10) were cultured using standard techniques. The patients and controls were matched based on age, sex, body-mass index (BMI) and lifestyle. The growth rate, population doubling time, cell surface marker expression (CD31, FNDC5) (flow cytometry), secretome (TIMP-2, MMP-9) (ELISAs), intracellular signalling capacity (Fluo-4 Ca2+ flux) and gene expression (IL-6, IL-10, PTX-3, iNOS, Arg1) were compared between groups. The data illustrated significant PTSD-associated fibroblast conditioning resulting in a blunted signalling capacity. This observation highlights the importance of including tissue-specific investigations in future studies focused on elucidating the association between PTSD and subsequent risk for somatic disease.

Establishment of fibroblast and myofibroblast phenotypes for use in in vitro co-culture models.

Fibroblasts function to secrete and modify components of the extracellular matrix. During wound healing, fibroblasts migrate to the site of injury and differentiate into contractile myofibroblasts; this differentiation is characterised by an increased contractile capacity. Fully differentiated myofibroblasts can be distinguished from fibroblasts via the higher expression of α-smooth muscle actin as well as a denser cytoskeleton. Impaired wound healing has been characterised by a lack of myofibroblasts; as a result, tissue does not fully regain its strength and function. Under pathological conditions, this may be associated with the effect that a pro-inflammatory microenvironment has on fibroblast and skeletal muscle progenitor cell migration and differentiation. Given their distinct roles in tissue maintenance and repair, the communication between fibroblasts versus myofibroblasts with other cellular mediators of repair is likely to influence cell behaviour and the outcome of wound repair. An in vitro test model is required to investigate this intercellular influence, but the establishment of such a model is hampered by the difficulty in retaining the dedifferentiated fibroblastic phenotype under regular serum-containing cell culture conditions. We present a model that supports the establishment and retention in culture of fibroblast and myofibroblast phenotypes for use in a simple, inexpensive, yet relevant in vitro 2D assay. This model is then applied in a co-culture setting to determine whether the presence of myoblasts affects the ability of fibroblasts versus myofibroblasts to close an in vitro wound. Our results emphasize the importance of considering the impact of paracrine communication between all cells during wound healing.

A regenerative approach to the pharmacological management of hard-to-heal wounds.

A wound is considered hard-to-heal when, despite the appropriate clinical analysis and intervention, the wound area reduces by less than a third at four weeks and complete healing fails to occur within 12 weeks. The most prevalent hard-to-heal wounds are associated with underlying metabolic diseases or vascular insufficiency and include arterial, venous, pressure and diabetic foot ulcers. Their common features include an abnormal immune response and extended inflammatory phase, a subdued proliferation phase due to cellular insufficiencies and finally an almost non-existent remodeling phase. Advances in wound care technology, tested in both pre-clinical models and clinical trials, have paved the way for improved treatment options, focused on regeneration. These interventions have been shown to limit the extent of ongoing inflammatory damage, decrease bacterial load, promote angiogenesis and deposition of granulation tissue, and stimulate keratinocyte migration thereby promoting re-epithelialization in these wounds. The current review discusses these hard-to-heal wounds in the context of their underlying pathology and potential of advanced treatment options, which if applied promptly as a standard of care, could reduce morbidity, promote quality of life, and alleviate the burden on a strained health system.

Ex vivo tolerization and M2 polarization of macrophages dampens both pro- and anti-inflammatory cytokine production in response to diabetic wound fluid stimulation.

Monocytes/macrophages play a prominent role in cutaneous wound healing. Persistent inflammation in diabetic wounds is associated with the inability of monocytic cells to switch from a phagocytic M1 (classically activated) to an anti-inflammatory, pro-regenerative M2 (alternatively activated) phenotype and as consequence, the proliferative phase of healing does not commence. A targeted cell therapy approach could potentially restore the pathological wound microenvironment through paracrine signalling to enable healing. This study investigated whether in vitro pre-treatment of monocytic (J774.1 A) cells - using a combination of endotoxin-induced immune tolerance (Pam3CSK4) and M2 polarization (IL-4) - could make these cells impervious to the pathological wound microenvironment and enhance the release of anti-inflammatory cytokines/growth factors. The effect of Pam3CSK4-induced tolerance and IL-4-associated polarization was assessed independently and in combination, on the expression of intracellular (flow cytometry) and secreted (ELISA) cytokines (TNF-ɑ, IL-6, IL-10, TGF-ß) with and without re-stimulation to define the optimal pre-treatment conditions. Successive pre-treatment approach consisting of endotoxin tolerance followed by IL-4 priming, dampened TNF-ɑ release and induced intracellular TGF-ß production upon re-stimulation. To mimic a chronic wound microenvironment, the J774A.1 monocytes were differentiated into macrophages using GM-CSF prior to pre-treatment (optimal condition) and subsequently exposed to diabetic wound fluid. The data demonstrated that in the presence of wound fluid, the successive pre-treatment, promoted M2 polarization (CD206) of monocytic cells and significantly dampened the intracellular production of both pro-inflammatory (TNF-ɑ, IL-6) and anti-inflammatory (IL-10, TGF-ß) cytokines.

Rheumatoid cachexia: the underappreciated role of myoblast, macrophage and fibroblast interplay in the skeletal muscle niche.

Although rheumatoid arthritis affects 1% of the global population, the role of rheumatoid cachexia, which occurs in up to a third of patients, is relatively neglected as research focus, despite its significant contribution to decreased quality of life in patients. A better understanding of the cellular and molecular processes involved in rheumatoid cachexia, as well as its potential treatment, is dependent on elucidation of the intricate interactions of the cells involved, such as myoblasts, fibroblasts and macrophages. Persistent RA-associated inflammation results in a relative depletion of the capacity for regeneration and repair in the satellite cell niche. The repair that does proceed is suboptimal due to dysregulated communication from the other cellular role players in this multi-cellular environment. This includes the incomplete switch in macrophage phenotype resulting in a lingering pro-inflammatory state within the tissues, as well as fibroblast-associated dysregulation of the dynamic control of the extracellular matrix. Additional to this endogenous dysregulation, some treatment strategies for RA may exacerbate muscle wasting and no multi-cell investigation has been done in this context. This review summarizes the most recent literature characterising clinical RA cachexia and links these features to the roles of and complex communication between multiple cellular contributors in the muscle niche, highlighting the importance of a targeted approach to therapeutic intervention.

Cellular regenerative therapy for acquired noncongenital musculoskeletal disorders.

Stem cells have an inherent capacity to facilitate regeneration; this has led to unprecedented growth in their experimental use in various clinical settings, particularly in patients with diseases with few alternative treatment options. However, their approved clinical use has to date been restricted largely to haematological diseases and epidermal transplantation to treat severe burns. After thorough searching of two databases, this review illuminates the role of stem cell therapy for treatment of musculoskeletal diseases. Research suggests that successful application of stem cells as regenerative mediators is in all likelihood dependent on the ability of endogenous tissue-resident reparative mediators to respond to paracrine signals provided by the applied stem cells. Therefore, an understanding of how the pathological environment influences this process is crucial for the ultimate success of stem cell therapies. The current review presents both the progress and limitations of stem cells as regenerative mediators in the context of musculoskeletal disorders.

A comparison between point-of-care testing and venous glucose determination for the diagnosis of diabetes mellitus 6-12 weeks after gestational diabetes.

AIM: To evaluate point-of-care-testing (POCT) for the diagnosis of Type 2 diabetes mellitus 6-12 weeks post-partum in women with gestational diabetes (GDM). METHODS: Post-partum glucose assessment (75-mg oral glucose tolerance test, OGTT) was performed prospectively in 122 women with GDM (1 November 2015 to 1 November 2017) at Tygerberg Hospital, Cape Town, South Africa. Individuals with known pre-existing diabetes were excluded. The accuracy and clinical utility of POCT (capillary finger-prick) were compared with laboratory plasma glucose (hexokinase and glucokinase methods). The OGTT consisted of two time points (fasting and 2 h) during which concurrent glucose samples (POCT and laboratory) were obtained. Bland-Altman plots and paired analysis were used to assess the analytical accuracy of POCT, whereas its diagnostic performance was determined using positive and negative predictive values to calculate specificity and sensitivity. RESULTS: Spearman's ranked correlation analysis indicated a strong association between POCT and laboratory glucose values at both OGTT time points (fasting, r = 0.95, P < 0.0001; 2 h, r = 0.88, P < 0.0001). Thirty-six women were diagnosed with Type 2 diabetes based on gold standard laboratory glucose levels (fasting > 7 mmol/l; 2 h > 11.1 mmol/l). POCT correctly identified Type 2 diabetes in 78% of women (28 of 36) with a positive predictive value of 89.3% and a negative predictive value of 96.7% at the fasting time point. The sensitivity and specificity of POCT to diagnose Type 2 diabetes were 89% (fasting), 85.7% (2 h) and 96.7% (fasting), 98.5% (2 h) respectively. POCT proved less sensitive to diagnose pre-diabetes (69%) but displayed satisfactory specificity (92%) at both time points assessed. CONCLUSION: POCT accurately identifies women with Type 2 diabetes 6-12 weeks after GDM.

A Direct Comparison of the Effects of the Antiretroviral Drugs Stavudine, Tenofovir and the Combination Lopinavir/Ritonavir on Bone Metabolism in a Rat Model.

Antiretroviral (ARV) treatment may induce metabolic complications in HIV patients on long-term therapy that can affect bone health. In this study, the effects of the ARVs Stavudine (d4T), Tenofovir (TDF) and Lopinavir/ritonavir (LPV/r) on bone metabolism and lipodystrophy were directly compared in rats to negate the consequences of HIV-associated confounding factors. Healthy 12-14-week-old male Wistar rats (n = 40) were divided into four treatment groups and received an oral animal equivalent dose of either Stavudine (6.2 mg/kg/day), TDF (26.6 mg/kg/day), LPV/r (70.8 mg/kg/day) or water (Control 1.5 mL water/day) for a period of 9 weeks. Whole-body DXA measurements, a biomechanical three-point breaking test and histomorphometric analysis were performed on the femurs and tibias at the end of the treatment period. Stavudine monotherapy was found to be associated with decreased femoral bone mineral density that translated into reduced bone strength, whereas histomorphometric analysis demonstrated that Stavudine induces an imbalance in bone metabolism at tissue level, evident in higher resorption (eroded surfaces, osteoclast surfaces and osteoclast number) and lower formation parameters (osteoblast surfaces and osteoid surfaces). This was less clear in the rats treated with either TDF or LPV/r. Furthermore, both Stavudine and TDF treatment resulted in significant bone marrow adiposity, although no significant redistribution of body fat was noted in the treated rats compared to controls. The data from this study suggest that in the absence of HIV-associated factors, LPV/r is less detrimental to bone metabolism compared to Stavudine and TDF.

Delayed wound healing and dysregulation of IL6/STAT3 signalling in MSCs derived from pre-diabetic obese mice.

Metabolic dysfunction that occurs in obesity and Type 2 diabetes results in a low-level inflammatory state which impacts on mesenchymal stem cells (MSCs) capacity to promote wound healing. The ability of either recombinant Interleukin-6 (rIL6) or pioglitazone to modulate MSC migration, essential for wound healing, by targeting the inflammation-modulated IL6/STAT3 signalling pathway was therefore investigated in bone marrow-derived MSCs from control (C57BL/6J) and pre-diabetic obese mice (B6. Cg-Lepob/J). The population doubling time, in vitro wound closure and mRNA expression profile of 84 genes involved in the IL6/STAT3 signalling pathway were assessed. IL6/STAT3 signalling dysregulation, caused by IL6 deficiency, resulted in skewing of the immune modulatory properties of MSCs to favour a pro-inflammatory profile. This could be nullified by addition of either rIL6 or conventional diabetes treatment. Therapies to improve diabetic wound healing should therefore focus on the cellular changes induced by the pathological inflammatory micro-environment.

Neutrophil and monocyte responses to downhill running: Intracellular contents of MPO, IL-6, IL-10, pstat3, and SOCS3.

High-intensity exercise results in immune activation. This study determined whether (a) there is concordance between serum MPO and neutrophil and/or monocyte intracellular MPO content; (b) peripheral blood mononuclear cells respond to inflammatory interleukins (ILs) by increasing intracellular signaling. Healthy male (n = 12) volunteers participated in high-intensity running (12 × 5 min, 10% decline, 15 km/h). Blood sample (pre, post, 4 h) analyses included serum concentrations of IL-1ß, IL-1ra, IL-4, IL-6, IL-8, IL-10, matrix metalloprotease-9 (MMP-9) and creatine kinase (CK). Intracellular IL-6, IL-10, MPO and STAT3/SOCS3 signaling were assessed in mononuclear cells. CK (1573 ± 756 u/L), MMP-9 (101 ± 27 ng/mL), neutrophil (9.89 ± 0.76 × 10(9) cells/L) and monocyte counts (1 ± 0.08 × 10(9) cells/L) increased at 4 h. At 4 h serum (7.1 ± 1.3 ng/mL) and monocyte MPO (1.7-fold) increased, whereas neutrophil MPO decreased (0.8-fold). Intracellular monocyte IL-10 and IL-6 decreased by 15% and 20-30%, respectively, coinciding with elevations in serum IL-10 of 14.5 ± 4.7 pg/mL and IL-6 of 5.4 ± 2.9 pg/mL, suggesting immune cell cytokine release in response to exercise. Intracellular PBMC p-STAT3 to total STAT3 ratio increased from pre to 4 h. Circulating monocytes are responsive to increased serum IL-6 suggesting a negative feedback loop via STAT3 signaling.

Thiazolidinedione-induced lipid droplet formation during osteogenic differentiation.

Chronic administration of the insulin-sensitising drugs, thiazolidinediones (TZDs), results in low bone mineral density and 'fatty bones'. This is thought to be due, at least in part, to aberrant differentiation of progenitor mesenchymal stem cells (MSCs) away from osteogenesis towards adipogenesis. This study directly compared the effects of rosiglitazone, pioglitazone, and netoglitazone treatment on osteogenesis and adipogenesis in MSCs derived from subcutaneous (SC) or visceral (PV) white adipose tissue. MSCs were isolated from adipose tissue depots of male Wistar rats and characterised using flow cytometry. The effects of TZD treatment on osteogenic and adipogenic differentiation were assessed histologically (day 14) and by quantitative PCR analysis (Pparγ2 (Pparg2), Ap2 (Fabp4), Adipsin (Adps), Msx2, Collagen I (Col1a1), and Alp) on days 0, 7, and 10. Uniquely, lipid droplet formation and mineralisation were found to occur concurrently in response to TZD treatment during osteogenesis. Compared with SC MSCs, PV MSCs were more prone to lipid accumulation under controlled osteogenic and adipogenic differentiation conditions. This study demonstrated that the extent of lipid accumulation is dependent on the nature of the Ppar ligand and that SC and PV MSCs respond differently to in vitro TZD treatment, suggesting that metabolic status can contribute to the adverse effects associated with TZD treatment.

Variable inflammation and intramuscular STAT3 phosphorylation and myeloperoxidase levels after downhill running.

Individual responses in creatine kinase (CK) release after eccentric exercise are divergent. This study aimed to identify whether this could be related to selected humoral or intramuscular inflammatory factors. Twenty-three subjects were divided into non-exercising (n = 5) and downhill run (DHR; n = 18) groups (12 × 5 min, 10% decline at 15 km/h). Blood samples were analyzed for white blood cell differential count, CK, myoglobin, tumor necrosis factor-α, granulocyte colony-stimulating factor, interleukin (IL)-1ß, IL-6, and IL-10. Muscle biopsies were analyzed for signal transducer and activator of transcription-3 (STAT3), IκBα, and myeloperoxidase (MPO). DHR participants clustered as early (DHR1) recovery, biphasic response (DHR2), or classic delayed exaggerated CK response (DHR3), with a delayed CK peak (4784 ± 1496 U/L) on day 4. For DHR1 and DHR2, CK peaked on day 1 (DHR1: 1198 ± 837 U/L) or on day 1 and day 4 (DHR2: 1583 ± 448 U/L; 1878 ± 427 U/L), respectively. Immediately post-DHR, IL-6 increased in DHR2 and DHR3 whereas IL-10 increased in all DHR groups. STAT3 signaling increased for DHR1 and DHR2 at 4 h, but MPO at day 2 only in DHR2. Objective cluster analysis uncovered a group of subjects with a characteristic biphasic CK release after DHR. The second elevation was related to their early cytokine response. The results provide evidence that early responses following eccentric exercise are indicative of later variation.

Cytokine and satellite cell responses to muscle damage: interpretation and possible confounding factors in human studies.

It is plausible that multiple muscle biopsies following a muscle damaging intervention can exacerbate the inflammatory and subsequent satellite cell responses. To elucidate confounding effects of muscle biopsy procedure on satellite cell number, indirect markers of damage and the inflammatory response following acute downhill running (DHR) were investigated. 10 healthy male participant were divided into a non-exercising control (n = 4) and DHR (12 × 5min bouts, 10 % decline at 85 % VO(2)max) (n = 6) group. Blood samples were taken pre, post and every 24 h for 9 days. Serum was analysed for creatine kinase (CK), myoglobin (Mb), lactate dehydrogenase (LDH), TNF-α, IL-6 and IL-10. Muscle biopsies taken on days 1 and 2 post intervention from opposing legs were analysed for Pax7(+) satellite cells. In the DHR group, Mb (536 ± 277 ng mL(-1)), IL-6 (12.6 ± 4.7 pg mL(-1)) and IL-10 (27.3 ± 11.5 pg mL(-1)) peaked immediately post DHR, while CK (2651 ± 1911 U L(-1)), LDH (202 ± 47 U L(-1)) and TNF-α (25.1 ± 8.7 pg mL(-1)) peaked on day 1. A 30 % increase in Pax7(+) satellite cells on day 1 in the DHR group was no longer apparent on day 2. H&E staining show evidence of phagocytosis in the DHR group. No significant changes over time were observed in the control group for any of the variables measured. Events observed in the DHR group were as a result of the intervention protocol and subsequent muscle damage. The relationship between SC proliferation and pro-inflammatory cytokine release appears to be complex since the IL-6/IL-10 response time differs significantly from the TNF-α response.

Satellite cell count, VO(2max) , and p38 MAPK in inactive to moderately active young men.

Satellite cells (SCs) are responsible for muscle repair following strenuous exercise or injury. SC responses to intervention have been studied, but most studies do not discuss or take into account the substantial variability in SC number among young individuals. We hypothesized that an active lifestyle reflected in higher VO(2max) may be associated with greater SC number. As training alters basal p38-mitogen-activated protein kinase (MAPK) activity, which is associated with SC proliferation, SC count may also correlate with this stress signaling kinase. Muscle biopsies from vastus lateralis of eight male participants were analyzed for fiber type, myogenin, and p38/phospho-p38 MAPK using SDS-PAGE and Western blotting. Immunofluorescence was used to detect Pax7(+) SCs. Two weeks following the biopsy, subjects underwent an incremental treadmill test to determine VO(2max) . A strong positive correlation (P = 0.0087) was found between the number of Pax7(+) nuclei and VO(2max) . Pax7(+) cell number correlated negatively with phospho-p38/p38 MAPK (P = 0.0006), but had no correlation with fiber type or myogenin. SC number is proportional to VO(2max) , and hence it can be postulated that higher levels of physical activity activate SC proliferation but not fusion, underlining the relevance of exercise in stimulating SC pool size even without injury.