Cell-based therapies have recently been proposed for the treatment of degenerative articular pathologies, such as early osteoarthritis, with an emphasis on autologous mesenchymal stem cells (MSCs), ...as an alternative to terminally differentiated cells. In this study, we performed a donor-matched comparison between infrapatellar fat pad MSCs (IFP-MSCs) and knee subcutaneous adipose tissue stem cells (ASCs), as appealing candidates for cell-based therapies that are easily accessible during surgery. IFP-MSCs and ASCs were obtained from 25 osteoarthritic patients undergoing total knee replacement and compared for their immunophenotype and differentiative potential. Undifferentiated IFP-MSCs and ASCs displayed the same immunophenotype, typical of MSCs (CD13+/CD29+/CD44+/CD73+/CD90+/CD105+/CD166+/CD31-/CD45-). IFP-MSCs and ASCs showed similar adipogenic potential, though undifferentiated ASCs had higher LEP expression compared to IFP-MSCs (p<0.01). Higher levels of calcified matrix (p<0.05) and alkaline phosphatase (p<0.05) in ASCs highlighted their superior osteogenic commitment compared to IFP-MSCs. Conversely, IFP-MSCs pellets showed greater amounts of glycosaminoglycans (p<0.01) and superior expression of ACAN (p<0.001), SOX9, COMP (p<0.001) and COL2A1 (p<0.05) compared to ASCs pellets, revealing a superior chondrogenic potential. This was also supported by lower COL10A1 (p<0.05) and COL1A1 (p<0.01) expression and lower alkaline phosphatase release (p<0.05) by IFP-MSCs compared to ASCs. The observed dissimilarities between IFP-MSCs and ASCs show that, despite expressing similar surface markers, MSCs deriving from different fat depots in the same surgical site possess specific features. Furthermore, the in vitro peculiar commitment of IFP-MSCs and ASCs from osteoarthritic donors towards the chondrogenic or osteogenic lineage may suggest a preferential use for cartilage and bone cell-based treatments, respectively.
We analyzed the use of isavuconazole (ISA) as treatment or prophylaxis for invasive fungal disease (IFD) in children with hemato-oncologic diseases. A multicentric retrospective analysis was ...performed among centers belonging to the Italian Association for Pediatric Hematology and Oncology (AIEOP). Pharmacokinetic (PK) monitoring was applied by a high-performance liquid chromatography-tandem mass spectrometry (HLPC-MS/MS) assay. Twenty-nine patients were studied: 10 during chemotherapy and 19 after allogeneic hematopoietic stem cell transplantation (HSCT). The patients consisted of 20 males and 9 females with a median age of 14.5 years (age range, 3 to 18 years) and a median body weight of 47 kg (body weight range, 15 to 80 kg). ISA was used as prophylaxis in 5 patients and as treatment in 24 cases (20 after therapeutic failure, 4 as first-line therapy). According to European Organization for Research and Treatment of Cancer (EORTC) criteria, we registered 5 patients with proven IFD, 9 patients with probable IFD, and 10 patients with possible IFD. Patients with a body weight of <30 kg received half the ISA dose; the others received ISA on the adult schedule (a 200-mg loading dose every 8 h on days 1 and 2 and a 200-mg/day maintenance dose); for all but 10 patients, the route of administration switched from the intravenous route to the oral route during treatment. ISA was administered for a median of 75.5 days (range, 6 to 523 days). The overall response rate was 70.8%; 12 patients with IFD achieved complete remission, 5 achieved partial remission, 5 achieved progression, and 3 achieved stable IFD. No breakthrough infections were registered. PK monitoring of 17 patients revealed a median ISA steady-state trough concentration of 4.91 mg/liter (range, 2.15 to 8.54 mg/liter) and a concentration/dose (in kilograms) ratio of 1.13 (range, 0.47 to 3.42). Determination of the 12-h PK profile was performed in 6 cases. The median area under the concentration-time curve from 0 to 12 h was 153.16 mg·h/liter (range, 86.31 to 169.45 mg·h/liter). Common Terminology Criteria for Adverse Events grade 1 to 3 toxicity (increased transaminase and/or creatinine levels) was observed in 6 patients, with no drug-drug interactions being seen in patients receiving immunosuppressants. Isavuconazole may be useful and safe in children with hemato-oncologic diseases, even in the HSCT setting. Prospective studies are warranted.
Quantitative gene expression analysis is widely used to evaluate the expression of specific tissue markers. To obtain reliable data it is essential to select stable housekeeping genes whose ...expression is not influenced by the anatomical origin of cells or by the culture conditions. No studies have evaluated housekeeping gene stability in intervertebral disc (IVD) cells and only few studies using cartilaginous endplate (CEP) and articular cartilage (AC) cells are present in the literature. We analysed the stability of four candidate housekeeping genes (GAPDH, TBP, YWHAZ and RPL13A) in human cells isolated from nucleus pulposus (NP) and annulus fibrosus (AF), CEP and AC. Cell isolation, expansion, cryoconservation, and differentiation in 3D pellets were tested. GeNorm, NormFinder, BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability. In each cell population, TBP alone or combined with YWHAZ was identified as the best normaliser in both monolayer and 3D pellets. GAPDH was the best performer only for AC cells in monolayer. In most culture conditions considering groups of two or more cell types, TBP was the most stable and YWHAZ was the second choice. GAPDH was the best performer only in 3D pellets with factors for AC and AF combined with CEP cells. RPL13A was the most stable only for AF with CEP cells at isolation. Our findings will be useful to properly design the experimental set-up of studies involving IVD, CEP or AC cells in different culture conditions, in order to obtain accurate and high quality data from quantitative gene expression analysis.
Degenerative processes of the intervertebral disc (IVD) and cartilaginous endplate lead to chronic spine pathologies. Several studies speculated on the intrinsic regenerative capacity of degenerated ...IVD related to the presence of local mesenchymal progenitors. However, a complete characterisation of the resident IVD cell populations, particularly that isolated from the endplate, is lacking. The purpose of the present study was to characterise the gene expression profiles of human nucleus pulposus (NPCs), annulus fibrosus (AFCs) and endplate (EPCs) cells, setting the basis for future studies aimed at identifying the most promising cells for regenerative purposes. Cells isolated from NP, AF and EP were analysed after in vitro expansion for their stemness ability, immunophenotype and gene profiles by large-scale microarray analysis. The three cell populations shared a similar clonogenic, adipogenic and osteogenic potential, as well as an immunophenotype with a pattern resembling that of mesenchymal stem cells. NPCs maintained the greatest chondrogenic potential and shared with EPCs the loss of proliferation capability during expansion. The largest number of selectively highly expressed stemness, chondrogenic/tissue-specific and surface genes was found in AFCs, thus representing the most promising source of tissue-specific expanded cells for the treatment of IVD degeneration.
Inflammation represents an important factor leading to metabolic imbalance within the intervertebral disc (IVD), conducive to degenerative changes. Therefore, a thorough knowledge of the IVD and ...endplate (EP) cell behaviour in such pathological environments is essential when designing regenerative therapeutic strategies. The present study aimed at assessing the molecular response of the IVD constitutive nucleus pulposus (NPCs)-, annulus fibrosus (AFCs)- and endplate (EPCs)-derived cells to interleukin (IL)-1β treatment, through large-scale, high-throughput microarray and protein analysis, identifying the differentially expressed genes and released proteins. Overall, the inflammatory stimulus downregulated stemness genes while upregulating pro-inflammatory, pro-angiogenic and catabolic genes, including matrix metalloproteases, which were not balanced by a concomitant upregulation of their inhibitors. Upregulation of anti-inflammatory and anabolic tumour necrosis factor inducible gene 6 protein (TNFAIP6), of IL-1 receptor antagonist (IL-1Ra) (at gene and protein levels) and of trophic insulin-like growth factor 1 (IGF1) was also observed in all cell types; IGF1 particularly in AFCs. An overall inhibitory effect of tumour necrosis factor alpha (TNFα) signal was observed in all cell types; however, EPCs showed the strongest anti-inflammatory behaviour. AFCs and EPCs shared the ability to limit the activation of the signalling mediated by specific chemokines. AFCs showed a slightly senescent attitude, with a downregulation of genes related to DNA repair or pro-mitosis. Results allowed for the identification of specific molecular targets in IVD and EP cells that respond to an inflammatory environment. Such targets can be either silenced (when pathological targets) or stimulated to counteract the inflammation.
Muscle traction and bone metabolism are functionally linked and co‐regulated by a series of factors. Although a role for steroid hormones was hypothesized, a clear definition of the bone–muscle ...interconnection still lacks. To investigate this relationship, we studied bone metabolism, muscle activity, and salivary steroid hormones profile in relation with the physical effort across a cycling stage race, a model of effort in absence of load. Nine pro‐cyclists were recruited; body weight and power output/energy expenditure were recorded. Diet was kept constant. Saliva was collected at days −1, 4, 8, 12, 14, 19, and 23; blood and urine were collected at days −1, 12, and 23. Salivary steroid hormones cortisol, dehydroepiandrosterone (DHEA), testosterone, and estradiol, serum lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and creatine kinase (CK) activities, plasma sclerostin, and urinary calcium and phosphorous were measured. Cortisol remained constant, testosterone decreased at day 4, and estradiol and DHEA firstly increased and then returned to basal levels. Hormone concentrations were not correlated with plasma volume shifts. LDH, CK, AST, sclerostin, and urinary calcium and phosphorous increased. DHEA and estradiol correlated with the physical effort and the bone‐muscular markers. A relationship between muscle activity, in absence of load, and bone resorption emerged under a putative regulation by DHEA and estradiol.
For the past decades, Cultural Heritage (CH) is commonly documented by digital-based imaging and analytical techniques. This documentation is used as a support by heritage scientists to study and ...help the preservation of CH objects. Multiple techniques or modalities are usually required and applied to complete the documentation and the possible diagnostic from it. In this paper we explored multimodal imaging strategies to survey, analyse and share semantically enriched digital replicas. Three challenging case-studies from the SUMUM research project aims to illustrate efficient multi-source approaches in multi-scalar, multi-temporal and multi-spectral contexts. From multimodal data acquisitions, a photogrammetric-based registration method (TACO) has been developed in order to exploit a 2D/3D semantic annotation process implemented into a CH oriented collaborative web platform (AIOLI). In the exemples showed, the structure and the content of the annotations work is based from condition reports provided by conservation and restoration experts. To this end, all the documentation gathered on CH objects are either directly merged by image based registration while complementary analysis can be spatially anchored to annotations as linked resources. The dissemination part is explored by built-in AIOLI’s collaborative features or external Potree-based viewer, to enhance the accessibility of the final 3D annotated scenes for further expertises or wide-public events and purposes.
► Rapid composting process of wet olive husk through the use of a selected microbial starter. ► Improved germination index and detoxification of humid olive husks. ► This compost from wet olive husks ...can substitute for turf as cultivation substrate. ► This compost influences positively the nutraceutical traits of tomato.
The production of a compost from olive wet husks is described. The process is enhanced through the use of starters prepared with virgin husks enriched with selected microbial cultures. This approach, with respect to composting without the use of starters, allows to achieve faster start of the process (10 vs. 45days), deeper humification (humification rate 19.2 vs. 12.2), shorter maturation time (2 vs. 4–5months) and better detoxification of the starting material. Furthermore, the compost produced can effectively substitute for turf as a cultivation substrate in horticulture at greenhouse level, with beneficial effects on nutraceutical traits of tomato fruits.
The impact of a soccer match on parameters related to protein catabolism and renal function was evaluated in male players.
Blood was collected before and immediately after a 90 minutes soccer match ...from 19 athletes of two first division teams in Brazil. Red blood cells (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), ammonia, uric acid, urea and creatinine were analyzed. The modification of plasma volume was calculated, and biochemical values were corrected for this change. Urea/creatinine ratio and equations to estimate the glomerular filtration rate (eGFR) were used to assess kidney function.
Plasma volume decreased from pre- to post-match. Post-match values higher than the pre-match ones were observed for RBC, Hb and Ht, as a consequence of plasma volume decrease. An increase in ammonia and creatinine concentrations post-match in comparison with pre-match values was registered, without changes in uric acid and urea levels. A reduction in urea/creatinine ratio and in eGFR was observed post-match, suggesting a decrease of renal function.
A soccer match induced alterations in parameters linked to renal function and protein metabolism in male athletes. Particular attention should be paid in the monitoring of the ammonia concentration as an indicator of metabolic activity and energy requirement during prolonged exercise.
Low frequency pulsed electromagnetic field (PEMF) has proven to be effective in the modulation of bone and cartilage tissue functional responsiveness, but its effect on tendon tissue and tendon cells ...(TCs) is still underinvestigated. PEMF treatment (1.5 mT, 75 Hz) was assessed on primary TCs, harvested from semitendinosus and gracilis tendons of eight patients, under different experimental conditions (4, 8, 12 h). Quantitative PCR analyses were conducted to identify the possible effect of PEMF on tendon-specific gene transcription (scleraxis, SCX and type I collagen, COL1A1); the release of pro- and anti-inflammatory cytokines and of vascular endothelial growth factor (VEGF) was also assessed. Our findings show that PEMF exposure is not cytotoxic and is able to stimulate TCs’ proliferation. The increase of SCX and COL1A1 in PEMF-treated cells was positively correlated to the treatment length. The release of anti-inflammatory cytokines in TCs treated with PEMF for 8 and 12 h was significantly higher in comparison with untreated cells, while the production of pro-inflammatory cytokines was not affected. A dramatically higher increase of VEGF-A mRNA transcription and of its related protein was observed after PEMF exposure. Our data demonstrated that PEMF positively influence, in a dose-dependent manner, the proliferation, tendon-specific marker expression, and release of anti-inflammatory cytokines and angiogenic factor in a healthy human TCs culture model.
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