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Background: During COVID-19 pandemic, health professionals have been working in an extreme uncertainty context. Affected patients needed to be cared at home as long as possible to avoid virus spreading and hospital resources saturation. The Veneto Regional Administration (North-east of Italy) released Regional guidelines about it. The Western Healthcare District of the Local Health Authority of the city of Vicenza (180,000 inhabitants) implemented a healthcare pathway following them. Aim of the study is to describe the results and outcomes of such implementation. Methods: In the implemented health care pathway, a new service called "Special Unit of continuity of care" (USCA) with physicians and nurses has been dedicated to the prise en charge at home of patients suffering from Sars-CoV-2. They were referred to the USCA by general practitioners or by hospital specialists, and managed through a daily clinical monitoring by regular home visits and phone calls, specialist consultations and therapy management. In order to prevent hospital admission, an oxygen concentrator when possible has been employed and managed at home by the members of the USCA when the oxygen saturation was below 93%. An observational retrospective study has been conducted using anonymized data from different databases: the USCA activity database (from 12/01/20 to 21/31/21), the hospital and Emergency Department discharge databases, and the "healthcare co-payments exemptions database". The latter database refers to the people excluded - because of their chronicity - from the co-payment of a list of medical exams and services. Descriptive and multivariate logistic regression analyses have been implemented. Results: 1,419 patients suffering from Sars-CoV-2 have been cared and managed by the USCA in the considered period of time (mean 11.4 days), of whom 787 (55.5%) with at least one chronic condition (described in the above quoted "healthcare co-payments exemption database") and 261 provided with oxygen concentrator. 275 (19.4%) needed a hospital admission, 39 (2.8%) in intensive unit; 53 died during hospitalization (3.8%). Out of the 261 patients utilizing oxygen concentrator, 103 have been admitted to hospital (39.5%), 7.3% in intensive unit and 8.0% died. In implemented multivariate analyses, the use of oxygen concentrator, proxy measure of the severity of the condition, is the major determinant for the risk of hospital admission (adj OR: 3.2, CI 2.3-4.3) and of dying within 30 days (adj OR: 2.8 CI 1.5-5.1). Among the 261 patients provided with oxygen concentrator, 158 (60,5%) have been managed at home without any admission to emergency department and/or hospitalization. Conclusions: In an uncertain context such as COVID-19 pandemic, the already-implemented home care model has been modified by integrating the USCA physicians and nurses and specialist care networks to prevent hospitalization and the sense of isolation and abandonment of people as much as possible. Almost 1,500 patients suffering from COVID-19 have been cared for at home over 13 months by such new service with complex and multidisciplinary activities. The risk of hospitalization and death appears determined by the severity of the pathology with high and significant OR 60% of patients with oxygen concentrators who, despite an initial high hyposaturation were not hospitalized, represent, partly, the group of patients who would have been requiring hospital care in the absence of a home care pathway in a standard situation.
Subject(s)
COVID-19 , Home Care Services , Humans , Italy/epidemiology , COVID-19/epidemiology , COVID-19/therapy , Retrospective Studies , Home Care Services/organization & administration , Continuity of Patient Care/organization & administration , Hospitalization/statistics & numerical data , Pandemics , Oxygen Inhalation Therapy/statistics & numerical dataABSTRACT
BACKGROUND AND AIM: Lung ultrasound (LUS) is a convenient imaging modality in the setting of coronavirus disease-19 (COVID-19) because it is easily available, can be performed bedside and repeated over time. We herein examined LUS patterns in relation to disease severity and disease stage among patients with COVID-19 pneumonia. METHODS: We performed a retrospective case series analysis of patients with confirmed SARS-CoV-2 infection who were admitted to the hospital because of pneumonia. We recorded history, clinical parameters and medications. LUS was performed and scored in a standardized fashion by experienced operators, with evaluation of up to 12 lung fields, reporting especially on B-lines and consolidations. RESULTS: We included 96 patients, 58.3% men, with a mean age of 65.9 years. Patients with a high-risk quick COVID-19 severity index (qCSI) were older and had worse outcomes, especially for the need for high-flow oxygen. B-lines and consolidations were located mainly in the lower posterior lung fields. LUS patterns for B-lines and consolidations were significantly worse in all lung fields among patients with high versus low qCSI. B-lines and consolidations were worse in the intermediate disease stage, from day 7 to 13 after onset of symptoms. While consolidations correlated more with inflammatory biomarkers, B-lines correlated more with end-organ damage, including extrapulmonary involvement. CONCLUSIONS: LUS patterns provide a comprehensive evaluation of patients with COVID-19 pneumonia that correlated with severity and dynamically reflect disease stage. LUS patterns may reflect different pathophysiological processes related to inflammation or tissue damage; consolidations may represent a more specific sign of localized disease, whereas B-lines seem to be also dependent upon generalized illness due to SARS-CoV-2 infection.
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BACKGROUND: There is a significant variability in survival rates for cardiopulmonary resuscitation (CPR) in out of-hospital cardiac arrest (OHCA), and some data indicate that ultrasound improves CPR. OBJECTIVES: We evaluated the feasibility of ultrasound for monitoring chest compressions in OHCA. METHODS: We planned a prospective study in patients with an ultrasound-integrated CPR for OHCA. Chest compressions were performed on the intermammillary line (IML), but the position was changed according to the quality of the heart squeezing, evaluated by ultrasound. End-tidal carbon dioxide (ETCO2) was used as the control parameter. Then we compared the area with the highest squeezing with the position of the heart in the chest computed tomography (CT) scans of 20 hospitalized patients. RESULTS: Chest compressions were good, partial, and inadequate on the IML in 58.4%, 48.9%, and 2.8% of cases, respectively. These percentages were 75%, 25%, and 0% after these modifications: none (47.2%), increased depth (8.3%), hands moved on the lower third of the sternum (27.8%), on left parasternal line of the lower part of the sternum (13.9%), and on the center of the sternum (1 case). Accordingly, ETCO2 improved significantly (20.37 vs. 37.10, p < 0.0001). The CT scans showed that the larger biventricular area (BVA) was under the parasternal line of the lower third of the sternum, and the mean distance IML-BVA was 5.7 cm. CONCLUSIONS: Our study has demonstrated that CPR in OHCA can be improved using ultrasound and changing the position of the hands. This finding was connected with the ETCO2 and confirmed by chest CT scans.
Subject(s)
Cardiopulmonary Resuscitation , Out-of-Hospital Cardiac Arrest , Carbon Dioxide , Humans , Out-of-Hospital Cardiac Arrest/diagnostic imaging , Out-of-Hospital Cardiac Arrest/therapy , Prospective Studies , Ultrasonography, InterventionalSubject(s)
HIV Infections , Liver/diagnostic imaging , Multiple Organ Failure , Pneumonia, Pneumocystis , Respiratory Insufficiency , Ultrasonography/methods , Adult , CD4 Lymphocyte Count/methods , Clinical Deterioration , Fatal Outcome , HIV Infections/complications , HIV Infections/immunology , Humans , Immunocompromised Host , Male , Multiple Organ Failure/diagnosis , Multiple Organ Failure/etiology , Pneumonia, Pneumocystis/diagnosis , Pneumonia, Pneumocystis/physiopathology , Pneumonia, Pneumocystis/therapy , Radiography, Thoracic/methods , Respiration, Artificial/methods , Respiratory Insufficiency/diagnosis , Respiratory Insufficiency/microbiology , Respiratory Insufficiency/physiopathology , Respiratory Insufficiency/therapy , Tomography, X-Ray Computed/methodsABSTRACT
Nerium oleander is an evergreen small tree, very used in some countries such as India and in Sri Lanka as a self-medication method. We report a case of a 55 year-old man with intermittent, advanced AV block secondary to oleander intoxication.
Subject(s)
Atrioventricular Block , Nerium , Atrioventricular Block/chemically induced , Atrioventricular Block/diagnosis , Electrocardiography , Humans , India , Male , Middle Aged , Self MedicationABSTRACT
Lung ultrasound (LUS) is one of the most important and innovative applications in emergency and critical care medicine for the management of critically ill patients. Ultrasound has been widely used in the COVID-19 pandemic as an extremely reliable technique and has proved to have a key role in the diagnosis and monitoring of patients with acute respiratory failure. The diagnostic accuracy of LUS is higher than chest X-ray and similar to computed tomography, which is considered the gold standard. COVID-19 pneumonia has some distinctive ultrasonographic signs but not pathognomonic, and LUS significantly improves the management of COVID-19 patients speeding up the diagnostic path. The examination is bedside; reduces the risk of contamination, avoiding mobilization of the patients; cuts down the amount of radioactive exposure; and gives real-time answers to many diagnostic and therapeutic doubts. Finally, the instruments are small and the scanner and the probes can be protected from contamination easily.
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BACKGROUND: Pre-hospital ultrasound is a new challenge and lung ultrasound could be an interesting opportunity in the pre-hospital medical service. The aim of our study was to evaluate the efficacy of lung ultrasound in out-of-hospital non-traumatic respiratory insufficiency. METHODS: We planned a case-controlled study in the ULSS 5 ovest vicentino area (Vicenza-Italy) enrolling subjects with severe dyspnea caused by cardiac heart failure or acute exacerbation of chronic obstructive pulmonary disease. We compared drugs administration, oxygen delivery, and laboratory tests between those patients with ultrasound integrated management and those without ultrasound. RESULTS: Pre-hospital lung ultrasound had a high specificity (94.4%) and sensitivity (100%) for the correct identification of alveolar interstitial syndrome using B lines, whereas the percentages obtained with pleural effusion were lower (83.3, 53.3%, respectively). The patients with ultrasound integrated management received a more appropriate pharmacological therapy (p 0.01), as well as non-invasive ventilation (CPAP) was used more frequently in those with an acute exacerbation of chronic obstructive pulmonary disease (p 0.011). Laboratory tests and blood gases analysis were not significant different between the two study groups. In a sub-analysis of the patients with an A profile, we observed a significant lower concentration of PCO2 in those with an ultrasound integrated management (PCO2: 42.62 vs 52.23 p 0.049). According with physicians' opinion, pre-hospital lung ultrasound gave important information or changed the therapy in the 42.3% of cases, whereas it just confirmed physical examination in the 67.7% of cases. CONCLUSIONS: Pre-hospital lung ultrasound is easy and feasible, and learning curve is rapid. Our study suggests that cardiac heart failure and acute exacerbation of chronic obstructive pulmonary disease can be considered two indications for pre-hospital ultrasound, and can improve the management of patient with acute respiratory insufficiency.
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BACKGROUND/AIM: Osteosarcoma originates from mesenchymal stem cells with impaired bone differentiation. In the present study we investigated the effect of ascorbic acid (AsA) on osteogenic differentiation and apoptosis of the MG-63 osteosarcoma cell line. MATERIALS AND METHODS: We evaluated the expression of runt-related transcription factor-2 (RUNX2) and secreted phosphoprotein 1 (SPP1) genes by real-time Polymerase Chain Reaction (PCR) and of endogenous bone morphogenetic protein-2 (BMP2) and osteocalcin proteins by immunohistochemistry. We analyzed osteoblast maturation by phosphatase alkaline synthesis and calcium deposition, and apoptosis by (TUNEL) test and Annexin staining. RESULTS: Our results showed that RUNX2 and SPP1 gene expression was increased in cells treated with low concentrations of AsA with respect to untreated cells. At higher concentrations, AsA induced apoptosis of osteosarcoma cells, possibly with the involvement of p21. CONCLUSION: Our findings support the ability of AsA to induce both differentiation, by affecting the target involved in early and late phases of osteogenic maturation, and apoptosis in poorly-differentiated osteosarcoma cells.
Subject(s)
Apoptosis/drug effects , Ascorbic Acid/pharmacology , Bone Neoplasms/pathology , Cell Differentiation/drug effects , Osteosarcoma/pathology , Antineoplastic Agents/pharmacology , Antioxidants/pharmacology , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Cell Line, Tumor , Cell Lineage/drug effects , Cell Survival/drug effects , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Dose-Response Relationship, Drug , Gene Expression Regulation, Neoplastic/drug effects , Humans , Osteopontin/genetics , Osteopontin/metabolism , Osteosarcoma/genetics , Osteosarcoma/metabolismABSTRACT
Bone mineral density (BMD) and peak bone mass (PBM) are important determinants of skeletal resistance. The development of bone densitometry improved the possibility of studying BMD and the influence of genetic and environmental factors on bone. Heredity factors are important for BMD, and Runx-2 is accepted as a regulator of osteoblasts and bone formation. The aim of our study was to evaluate the behavior of Runx-2 during skeletal maturity in the healthy young-adult population. We analyzed spine and hip BMD in 153 volunteers, 98 women and 55 men, using dual-energy X-ray absorptiometry. In a subgroup of these volunteers, a sample of peripheral blood was taken to perform gene expression analysis of Runx-2 both in peripheral mesenchymal stem cells (MSCs; 28 subjects) and in peripheral mononuclear cells (PBMCs; 140 subjects). In our work BMD was comparable in both genders after puberty, then became higher in men than women during the third and fourth decades. PBM was achieved in the third decade in women and in the fourth in men. More interestingly, Runx-2 gene expression highly correlated with BMD in both genders. MSCs and PBMCs showed the same gene expression profile of Runx-2. In conclusion, PBM is reached earlier in females, BMD becomes higher in males later in life, and BMD and PBM are strictly associated with Runx-2. In addition, PBMC should be considered an important source for gene expression analysis in bone diseases.
Subject(s)
Aging/physiology , Bone Density/genetics , Core Binding Factor Alpha 1 Subunit/genetics , Absorptiometry, Photon , Adolescent , Adult , Bone Density/physiology , Female , Gene Expression , Humans , Leukocytes, Mononuclear/metabolism , Male , Mesenchymal Stem Cells/metabolism , Pelvic Bones/physiology , Spine/physiologyABSTRACT
BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes and conditions causing bone loss may induce a switch from the osteoblast to adipocyte lineage. In addition, the expression of Runx2 and the PPARγ2 transcription factor genes is essential for cellular commitment to an osteogenic and adipogenic differentiation, respectively. Modified lipoproteins derived from the oxidation of arachidonate-containing phospholipids (ox-PAPCs: POVPC, PGPC and PEIPC) are considered important factors in atherogenesis. METHODOLOGY: We investigated the effect of ox-PAPCs on osteogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). In particular, we analyzed the transcription factor Runx2 and the PPARγ2 gene expression during osteogenic and adipogenic differentiation in absence and in presence of ox-PAPCs. We also analyzed gene expression level in a panel of osteoblastic and adipogenic differentiation markers. In addition, as circulating blood cells can be used as a "sentinel" that responds to changes in the macro- or micro-environment, we analyzed the Runx2 and the PPARγ2 gene expression in MSCs-like and ox-PAPC levels in serum of osteoporotic patients (OPs). Finally, we examined the effects of sera obtained from OPs in hMSCs comparing the results with age-matched normal donors (NDs). PRINCIPAL FINDINGS: Quantitative RT-PCR demonstrated that ox-PAPCs enhanced PPARγ2 and adipogenic gene expression and reduced Runx2 and osteoblast differentiation marker gene expression in differentiating hMSCs. In OPs, ox-PAPC levels and PPARγ2 expression were higher than in NDs, whereas Runx2 was lower than in ND circulant MSCs-like. CONCLUSIONS: Ox-PAPCs affect the osteogenic differentiation by promoting adipogenic differentiation and this effect may appear involved in bone loss in OPs.
Subject(s)
Cell Differentiation/drug effects , Core Binding Factor Alpha 1 Subunit/genetics , Gene Expression Regulation/drug effects , Mesenchymal Stem Cells/drug effects , Osteoporosis, Postmenopausal/pathology , PPAR gamma/genetics , Phosphatidylcholines/pharmacology , Adipogenesis/drug effects , Case-Control Studies , Densitometry , Female , Humans , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Middle Aged , Osteogenesis/drug effects , Osteoporosis, Postmenopausal/blood , Osteoporosis, Postmenopausal/genetics , Phenotype , Phosphatidylcholines/blood , Serum/metabolismABSTRACT
BACKGROUND: Bone disease is one of the major complications of solid organ transplantation, causes considerable morbidity, and most patients are treated with immunosuppressant drugs after graft. The majority of studies reported rapid bone loss and an increased incidence of fractures after transplantation. The aim of our study was to evaluate osteoporosis and fracture prevalence, bone metabolism, and the effect of immunosuppressant agents on bone after heart transplantation. METHODS: We planned a cross-sectional study in 180 heart transplant patients recruited from 3 different centers with a less than 10 years from graft. Each patient underwent a densitometric scan, and in 157 of them, an x-ray of the spine was performed to evaluate fractures. Biochemical assessment of bone metabolism was made at the time of the visit. Physical activity, diet, and calcium intake were evaluated using a specific questionnaire. RESULTS: Vertebral fractures were diagnosed in 40% of subjects, but densitometric osteoporosis was observed only in 13% of spine and in 25% of hip scans. Interestingly, increasing T-score threshold up to -1.5 standard deviation, the prevalence of fractured patient improved significantly, reaching 60% in both genders. Bone content was inversely correlated with glucocorticoids, while a positive correlation was found with cyclosporine A. Almost all subjects had vitamin D deficiency. CONCLUSIONS: Standard densitometric criteria are unreliable to identify bone fragility after transplantation, and a different threshold (-1.5 standard deviation) should be considered. Transplanted patients should be adequately supplemented with vitamin D, and the effects of immunosuppressant agents on bone need further investigation.
Subject(s)
Heart Transplantation/adverse effects , Spinal Fractures/etiology , Absorptiometry, Photon , Adult , Aged , Bone Density , Bone Diseases, Metabolic/diagnosis , Bone Diseases, Metabolic/drug therapy , Bone Diseases, Metabolic/etiology , Cross-Sectional Studies , Female , Humans , Immunosuppressive Agents/adverse effects , Male , Middle Aged , Osteoporosis/diagnosis , Osteoporosis/drug therapy , Osteoporosis/etiology , Risk Factors , Vitamin D/therapeutic use , Vitamin D Deficiency/complications , Vitamin D Deficiency/drug therapyABSTRACT
INTRODUCTION: The purpose of this study was to evaluate the effects of risedronate (Ris) in the modulation of bone formation in rats with glucocorticoid (GC)-induced osteoporosis by histomorphometric, immunohistochemical and gene expression analyses. METHODS: We analyzed structure, turnover and microarchitecture, cyclooxygenase 2 (COX-2) levels and osteocyte apoptosis in 40 female rats divided as follows: 1) vehicle of methylprednisolone (vGC) + vehicle of risedronate (vRis); 2) Ris 5 µg/Kg + vGC; 3) methylprednisolone (GC) 7 mg/Kg + vRis; 4) GC 7 mg/Kg +Ris 5 µg/Kg. In addition, we evaluated cell proliferation and expression of COX-2 and bone alkaline phosphatase (b-ALP) genes in bone marrow cells and MLO-y4 osteocytes treated with Ris alone or in co-treatment with the selective COX-2 inhibitor NS-398 or with dexametasone. RESULTS: Ris reduced apoptosis induced by GC of osteocytes (41% vs 86%, P < 0.0001) and increased COX-2 expression with respect to controls (Immuno-Hystochemical Score (IHS): 8.75 vs 1.00, P < 0.0001). These positive effects of Ris in bone formation were confirmed by in vitro data as the viability and expression of b-ALP gene in bone marrow cells resulted increased in a dose dependent manner. CONCLUSIONS: These findings suggest a positive effect of Ris in bone formation and support the hypothesis that the up-regulation of COX-2 could be an additional mechanism of anabolic effect of Ris.
Subject(s)
Bone Density Conservation Agents/pharmacology , Cyclooxygenase 2/genetics , Etidronic Acid/analogs & derivatives , Osteocytes/drug effects , Osteogenesis/drug effects , Animals , Apoptosis/drug effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/enzymology , Cell Line/drug effects , Cell Survival/drug effects , Cells, Cultured , Etidronic Acid/pharmacology , Female , Gene Expression Regulation, Enzymologic/drug effects , Glucocorticoids/pharmacology , Osteocytes/cytology , Osteocytes/enzymology , Rats , Rats, Sprague-Dawley , Risedronic Acid , Up-Regulation/drug effectsABSTRACT
The acute-phase response (APR) is the most frequent side effect after the first dose of intravenous nitrogen-containing bisphosphonates (N-BPs). It has been demonstrated in vitro that N-BPs stimulate gammadelta T-cell proliferation and production of cytokines and that vitamin D is able to modulate them. Therefore, we have studied the relationship between bone metabolism parameters, particularly for 25-hydroxyvitamin D [25(OH)D], and APR in patients treated with 5 mg zoledronic acid intravenously. Ninety N-BP-naive osteoporotic women (63.7 +/- 10.6 years of age) were stratified for the occurrence of APR (APR(+)) or not (APR(-)) and quantified by body temperature and C-reactive protein (CRP). The APR(+) women had significantly lower 25(OH)D levels than the APR(-) women. Levels of 25(OH)D were normal (>30 ng/mL) in 31% of APR(+) women and in 76% of APR(-) women. The odds ratio (OR) to have APR in 25(OH)D-depleted women was 5.8 [95% confidence interval (CI) 5.30-6.29; p < .0002] unadjusted and 2.38 (95% CI 1.85-2.81; p < .028) after multiple adjustments (for age, body mass index, CRP, calcium, parathyroid hormone, and C-telopeptide of type I collagen). Levels of 25(OH)D were negatively correlated with postdose body temperature (r = -0.64, p < .0001) and CRP (r = -0.79, p < .001). An exponential increase in fever and CRP has been found with 25(OH)D levels lower than 30 ng/mL and body temperature less than 37 degrees C, whereas normal CRP was associated with 25(OH)D levels above 40 ng/mL. The association between post-N-BPs APR and 25(OH)D suggests an interesting interplay among N-BPs, 25(OH)D, and the immune system, but a causal role of 25(OH)D in APR has to be proven by a randomized, controlled trial. However, if confirmed, it should have some practical implications in preventing APR.
Subject(s)
Acute-Phase Reaction/physiopathology , Amines , Bone Density Conservation Agents , Bone and Bones/metabolism , Diphosphonates , Osteoporosis/drug therapy , Vitamin D/analogs & derivatives , Acute-Phase Reaction/complications , Aged , Amines/therapeutic use , Bone Density Conservation Agents/adverse effects , Bone Density Conservation Agents/therapeutic use , Diphosphonates/adverse effects , Diphosphonates/therapeutic use , Female , Humans , Middle Aged , Vitamin D/bloodABSTRACT
Bisphosphonates (BPs) are widely used in the treatment of postmenopausal osteoporosis and other metabolic bone diseases. They bind strongly to bone matrix and reduce bone loss through inhibition of osteoclast activity. They are classified as nitrogen- and non-nitrogen-containing bisphosphonates (NBPs and NNBPs, respectively). The former inhibit farnesyl diphosphate synthase while the latter induce the production of toxic analogs of adenosine triphosphate. These mechanisms of action are associated with different antifracture efficacy, and NBPs show the most powerful action. Moreover, recent evidence indicates that NBPs can also stimulate osteoblast activity and differentiation. Several randomized control trials have demonstrated that NBPs significantly improve bone mineral density, suppress bone turnover, and reduce the incidence of both vertebral and nonvertebral fragility fractures. Although they are generally considered safe, some side effects are reported (esophagitis, acute phase reaction, hypocalcemia, uveitis), and compliance with therapy is often inadequate. In particular, gastrointestinal discomfort is frequent with the older daily oral administrations and is responsible for a high proportion of discontinuation. The most recent weekly and monthly formulations, and in particular the yearly infusion of zoledronate, significantly improve persistence with treatment, and optimize clinical, densitometric, and antifracture outcomes.
ABSTRACT
OBJECTIVE: While the role of osteoclasts in bone loss has been well investigated, the involvement of osteoblast-lineage cells has not been completely elucidated. Several genes contribute to normal osteoblastic differentiation from mesenchymal stem cells (MSCs), but an understanding of their role in the pathogenesis of osteoporosis is still lacking. The present study was undertaken to evaluate a possible alteration of osteogenic gene expression as a mechanism contributing to bone loss. METHODS: We studied the osteogenic differentiation process in MSCs obtained from the peripheral blood of 31 patients with osteoporosis and 20 normal donors. The cells were evaluated by colony-forming unit-fibroblastic assay and cultured in osteogenic medium to analyze the transcription factors runt-related transcription factor 2 (RUNX-2) and Sp7 and the bone-related genes COL1A1, SPARC, and SPP1 after 3, 8, and 15 days of differentiation. In addition, to determine possible differences between the 2 groups in terms of osteoclastic and osteoblastic activation, we quantified the osteoprotegerin (OPG) and RANKL levels in the supernatants of osteoblastic culture. RESULTS: Circulating MSCs were increased in osteoporosis patients compared with normal donors. In contrast, gene expression analysis revealed down-regulation of RUNX2, Sp7, COL1A1, SPARC, and SPP1 in patients with osteoporosis, associated with a lower OPG:RANKL ratio. CONCLUSION: These results suggest that an alteration of osteoblastic differentiation may contribute to the pathogenesis of osteoporosis. The noninvasive approach used in the present study could be proposed as a useful tool for studying mesenchymal involvement in bone diseases.
Subject(s)
Cell Differentiation/physiology , Mesenchymal Stem Cells/pathology , Osteoblasts/pathology , Osteogenesis/physiology , Osteoporosis, Postmenopausal/pathology , Osteoporosis, Postmenopausal/physiopathology , Aged , Bone Resorption/physiopathology , Case-Control Studies , Cells, Cultured , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Core Binding Factor Alpha 1 Subunit/metabolism , Female , Humans , Mesenchymal Stem Cells/metabolism , Middle Aged , Osteoblasts/metabolism , Osteonectin/metabolism , Osteopontin/metabolism , Osteoporosis, Postmenopausal/metabolism , Osteoprotegerin/metabolism , RANK Ligand/metabolism , Sp7 Transcription Factor , Transcription Factors/metabolismABSTRACT
MSCs are known to have an extensive proliferative potential and ability to differentiate in various cell types. Osteoblastic differentiation from mesenchymal progenitor cells is an important step of bone formation, though the pattern of gene expression during differentiation is not yet well understood. Here, to investigate the possibility to obtain a model for in vitro bone differentiation using mesenchymal stem cells (hMSCs) from human subjects non-invasively, we developed a method to obtain hMSCs-like cells from peripheral blood by a two step method that included an enrichment of mononuclear cells followed by depletion of unwanted cells. Using these cells, we analyzed the expression of transcription factor genes (runt-related transcription factor 2 (RUNX2) and osterix (SP7)) and bone related genes (osteopontin (SPP1), osteonectin (SPARC) and collagen, type I, alpha 1 (COLIA1)) during osteoblastic differentiation. Our results demonstrated that hMSCs can be obtained from peripheral blood and that they are able to generate CFU-F and to differentiate in osteoblast and adipocyte; in this study, we also identified a possible gene expression timing during osteoblastic differentiation that provided a powerful tool to study bone physiology.
Subject(s)
Cell Differentiation , Gene Expression Profiling , Mesenchymal Stem Cells/metabolism , Osteoblasts/metabolism , Cells, Cultured , Humans , Immunohistochemistry , Mesenchymal Stem Cells/cytology , Osteoblasts/cytology , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/geneticsABSTRACT
BACKGROUND: RT-PCR has been widely used for the analysis of gene expression in many systems, including tumor samples. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) has been frequently considered as a constitutive housekeeping gene and used to normalize changes in specific gene expression. However, GAPDH has been shown to be up-regulated in many cancers and down-regulated by chemotherapic drugs. Bisphosphonates, potent inhibitors of bone resorption, have recently shown a direct and indirect antitumor effect in vitro and in animal models. They exert their effects mainly by inhibiting the mevalonate pathway but also by modulating the expression of many genes not only in osteoclasts but also in cancer cells. METHODS: We evaluated GAPDH gene expression by real time RT PCR in breast (MCF-7 and T47D) and prostate (PC3 and DU-145) cancer cell lines treated with amino and non-amino bisphosphonates. RESULTS: Our results showed that amino-bisphosphonates significantly decrease in a dose-dependent manner the expression of GAPDH gene. CONCLUSION: Therefore, GAPDH is inaccurate to normalize mRNA levels in studies investigating the effect of bisphosphonates on gene expression and it should be avoided. On the other hand, this gene could be considered a potential target to observe the effects of bisphosphonates on cancer cells.