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Essential hypertension (EH) is a multifactorial, polygenic condition, and is one of the most important comorbidities that contributes to stroke, myocardial infarction, cardiac failure, and renal failure. The continuous increasing rate of morbidity and mortality associated with EH presents an unmet need of population-based studies to explore pathophysiology as well as newer strategies for better diagnosis, prognosis and treatment. This study aimed to determine genotype and allele frequencies of A1166C polymorphism of AT1R gene in Indian patients with EH and correlated with serum levels of Angiotensin II. A total of 200 patients with EH and 200 age- and gender-matched control individuals were included in this study from the General Medicine Department Outpatient at Narayana Medical College and Hospital, Nellore, Andhra Pradesh, India. Patients with systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg were considered as hypertensive. The findings of this study revealed significantly increased risk of C/A heterozygote and allele C in both men and women. Moreover, both men and women patients with EH showed higher serum levels of Angiotensin II with C/A as well as AA genotypes. These findings indicate a significant association of 1166 C/A polymorphism of the AT1R gene with increased risk of hypertension in Indian population.
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The magnitude of variations in the level of circulating mitochondrial (cir-mtDNA) and nuclear DNA (cir-ncDNA) in different diseases has indicated the need for investigating a discriminative approach for evaluating their diagnostic significance. This study reports a typical in-house process for extracting both types of cir-DNAs from a single plasma sample and assessed their usefulness in discriminating type 2 diabetes mellitus patients from healthy individuals to eliminate the prevailing dispute about their discriminative role and improve their diagnostic value. This approach offers a more precise and valuable tool for distinguishing the impact of cir-mtDNA from cir-ncDNA in diagnostic implications.
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Diabetes Mellitus Tipo 2 , Humanos , Diabetes Mellitus Tipo 2/diagnóstico , Patologia Molecular , Mitocôndrias/genética , DNA Mitocondrial/genéticaRESUMO
BACKGROUND: Chronic liver diseases (CLD) are the major public health burden due to the continuous increasing rate of global morbidity and mortality. The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option. However, identifying the fate of transplanted cells in vivo represents a crucial obstacle. AIM: To evaluate the potential applicability of DiD dye as a cell labeling agent for long-term, and non-invasive in vivo tracking of transplanted cells in the liver. METHODS: Magnetically sorted, epithelial cell adhesion molecule positive (1 × 106 cells/mL) fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency (SCID) mice. Near-infrared (NIR) imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d. The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing. RESULTS: NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7, 15, 30, 45, 60, and 80 post-transplantation. Furthermore, positive staining of cytokeratin, c-Met, and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells. Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase, glutamate-pyruvate transaminase, and bilirubin. The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80. CONCLUSION: DiD-labeling is promising for long-term and non-invasive in vivo cell tracking, and understanding the regenerative mechanisms incurred by the transplanted cells.
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Background and Aim: The major aim of this study was to identify the most common stemness genes across different stem cell types and further validate them in human fetal subventricular zone-derived primary and cultured neural precursor cells (NPCs). This study involved the use of a unique method of stemness meta-analysis (SMA) for investigating comprehensive upregulation and downregulation of differentially expressed genes (DEGs) among different stem cell populations. Materials and Methods: A total of 55 mouse and human data sets targeting crucial genes identified in seven different types of stem cells population were screened and subjected to independent DEGs analysis using SMA. Identified 30 meta-gene signatures were subjected to functional enrichment analysis based on their biological processes and molecular functions. Validation of enriched meta-gene signatures was performed using RT-qPCR. Cellular localization of ABCB1 and ABCG2 was identified using immunofluorescence staining, whereas functional assessment was performed using western-blot. Results: SMA analysis revealed that among 52 commonly expressed genes, 30 genes were either upregulated or downregulated in at least two stem cell populations. Further gene enrichment analysis showed nine genes (ABCB1, ABCG2, HSPA4, HSPA9, HSPA14, Nestin, Sox-2, Oct-4, and Notch-2) with the highest combined scores among 30 meta-gene signatures. RT-qPCR demonstrated that all the enriched gene signatures were significantly upregulated in primary NPCs and further downregulated during NPCs lineage differentiation in culture except HSPA4, HSPA9, and HSPA14 gene transcripts. Conclusions: The stemness meta-gene signatures were abundantly expressed in human NPCs population which categorically suggest the involvement of these genes/pathways in pluripotency maintenance and molecular switches for lineage differentiation while HSP-70 had a neuroprotective effect.
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Transportadores de Cassetes de Ligação de ATP , Células-Tronco Neurais , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Expressão Gênica , Humanos , Camundongos , Células-Tronco Neurais/metabolismoRESUMO
INTRODUCTION: Human mesenchymal stem cells (hMSCs) holds great promise for managing several clinical conditions. However, the low engraftment efficiency and obscurity to harvest these cells without compromising the cellular viability, structural and functional properties from the culture niche still remain major obstacles for preparing intact regenerative constructs. Although few studies have demonstrate different methods for generating cell-liberated amniotic scaffolds, a common method for producing completely cell-liberated amnion (D-HAM) and chorion (D-HCM) scaffolds and their cytocompatibility with hMSCs yet to be demonstrated. METHODS: A common process was developed for preparing D-HAM and D-HCM scaffolds for assessing hMSCs engraftment efficiency, proliferation and molecular shifts to generate cell-laden biological discs. The structural and functional integrity of D-HAM and D-HCM was evaluated using different parameters. The compatibility and proliferation efficiency of hMSCs with D-HAM and D-HCM was evaluated. RESULTS: Histological analysis revealed completely nucleic acid-free D-HAM and D-HCM scaffolds with intact extracellular matrix, mechanical and biological properties almost similar to the native membranes. Human MSCs were able to adhere and engraft on D-HCM better than D-HAM and expanded faster. Ultrastructural observations, crystal violet staining and expression studies showed better structural and functional integrity of hMSCs on D-HCM than D-HAM and control conditions. CONCLUSION: A common, simple and reliable process of decellularization can generate large number of cell-liberated amniotic scaffolds in lesser time. D-HCM has better efficiency for hMSCs engraftment and proliferation and can be utilized for preparing suitable cell-laden constructs for tissue engineering applications.
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Essential hypertension (EH) is a multifactorial and complex disease with high rate of incidence and associated co-morbidities. Previous studies do not provide unanimous results for the risk of hypertension and association with Fok I genotype frequency and serum vitamin D levels. Hence, this study was undertaken to determine the status of Fok I vitamin D receptor (VDR) gene polymorphism along with vitamin D levels and blood pressure in patients with EH. Four hundred (200 controls and 200 cases of essential hypertension) participants from general Indian population were enrolled in this study. Peripheral blood samples were collected for genotyping Fok I-VDR gene polymorphism using PCR-RFLP method whereas 25-OH vitamin D levels in serum were quantified using high performance liquid chromatography (HPLC). Significantly reduced 25-OH vitamin D levels were observed in patients with EH (24.04 ± 8.62 vs 50.46 ± 15.46) compared to control subjects (p = 0.0001). Homozygous recessive genotype 'ff' frequency was increased by 8.06 fold (CI: 3.71-17.47, p = 0.0001) in patients with EH compared to dominant 'FF' genotype frequency. In conclusion, recessive 'ff' genotype frequency correlates with reduced serum vitamin D levels and results in significantly increased systolic and diastolic blood pressures leading to predisposition of EH.
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INTRODUCTION: The reconstruction/regeneration of human bone injuries/defects represents a crucial challenge due to the lack of suitable bio/immune compatible and implantable biological grafts. The available strategies represent implications of several types of grafting materials in the form of metals, synthetic, and various kinds of biological scaffolds; however, the lack of appropriate biological components required for activating and enhancing repair mechanisms at the lesion-site limits their wider applicability. METHODS: In this study, a unique approach for generating human osteogenic implantable grafts was developed using biofabrication technology. Using a gradient change of detergents and continuous agitation, developed a unique technique to generate completely cell-free amnion and chorion scaffolds. The absence of cellular components and integrity of biological and mechanical cues within decellularized human amnion (D-HAM) and chorion (D-HCM) were evaluated and compared with fresh membranes. Allogenic bone grafts were prepared through induction of human mesenchymal stem cells (hMSCs) into osteogenic cells on D-HAM and D-HCM and evaluated for their comparative behavior at the cellular, histological and molecular levels. RESULTS: The common decellularization process resulted in an efficient way to generate D-HAM and D-HCM while retaining their intact gross-anatomical architecture, surface morphology, extracellular matrix components, and mechanical properties. Both these scaffolds supported better growth of human umbilical cord blood derived MSCs as well as osteogenic differentiation. Comparative investigation revealed better growth rate and differentiation on D-HCM compared to D-HAM and control conditions. CONCLUSION: D-HCM could be used as a better choice for producing suitable allogenic bone grafts for efficient bone healing applications.
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Âmnio/citologia , Transplante Ósseo , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Âmnio/ultraestrutura , Regeneração Óssea , Cálcio/metabolismo , Adesão Celular , Diferenciação Celular , Córion/citologia , Córion/ultraestrutura , Humanos , Imunofenotipagem , Ácidos Nucleicos/metabolismo , Osteogênese , Transplante HomólogoRESUMO
INTRODUCTION: Bladder dysfunction has been considered as one of the most critical health conditions with no proper treatment. Current therapeutic approaches including enterocystoplasty have several limitations. Hence, biofabrication of cell-laden biological allografts using decellularized Goat urinary bladder scaffolds for organ reconstruction/regeneration was major objective of this study. MATERIALS AND METHODS: An efficient method for decellularization of Goat urinary bladder (N = 3) was developed by perfusion of gradient change of detergents through ureter. The retention of organ architecture, extracellular matrix composition, mechanical properties and removal of cellular components was characterized using histological, cellular and molecular analysis. Further, mesenchymal stem cells (MSCs) from human umbilical cord blood (UCB) were used for preparing biological construct of decellularized urinary bladder (DUB) scaffolds to augment the urinary bladder reconstruction/regeneration. RESULTS: The decellularization method adopted in this study generated completely DUB scaffolds within 10 h at 100 mm Hg pressure and constant flow rate of 1 mL/min. The DUB scaffold retains organ architecture, ECM composition, and mechanical strength. No significant amount of residual nucleic acid was observed post-decellularization. Furthermore, MSCs derived from human UCB engrafted and proliferated well on DUB scaffolds in highly aligned manner under xeno-free condition. CONCLUSION: Biofabricated humanized urinary bladder constructs provides xeno-free allografts for future application in augmenting urinary bladder reconstruction/regeneration with further development.
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Aloenxertos/citologia , Microtecnologia , Regeneração/fisiologia , Alicerces Teciduais/química , Bexiga Urinária/citologia , Bexiga Urinária/fisiologia , Animais , Proliferação de Células , Colágeno/metabolismo , Glicosaminoglicanos/metabolismo , Cabras , Humanos , Imunofenotipagem , Teste de Materiais , Ácidos Nucleicos/análise , Imagem Óptica , Bexiga Urinária/ultraestruturaRESUMO
AIM: Aim of the study was to investigate the effect of PRP and MTA individually and combined on in vitro human bone marrow mesenchymal stem cells' (MSCs) proliferation and osteo/odontogenic differentiation potential. MATERIALS AND METHODS: MSCs were cultured in vitro with MTA, 5% PRP, 10% PRP, MTA with 5%PRP, and MTA with 10% PRP. Fetal calf serum (FCS) was used as control. Cell viability and proliferative efficiency were tested with cell adhesion and MTT assay. Osteo/odontogenic differentiation was assessed and quantified with alizarin red staining. RESULTS: MTA alone, MTA with 5% PRP, and MTA with 10% PRP showed significantly high proliferation at day 7 and 14 when compared to the control group. Enhanced differentiation and the highest calcium deposition was observed in MTA with the 10% PRP group. CONCLUSION: Within limitations of the in vitro environment, results imply an increased proliferation and induction of MSCs into osteo/odontogenic differentiation by the combination rather than a mere sealing of PRP by MTA. CLINICAL SIGNIFICANCE: PRP and MTA have the potential for true regeneration of the pulp tissue. Moreover, the combination of PRP and MTA can be utilized to expand the MSCs to generate adequate numbers for clinical applications, without xenogenic contamination. How to cite this article: Vanka A, Vishwakarma SK, Bhat MK, et al. Osteo/odontogenic Differentiation of Human Mesenchymal Stem Cells with Platelet-rich Plasma and Mineral Trioxide Aggregate. J Contemp Dent Pract 2019;20(10):1171-1178.
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Células-Tronco Mesenquimais , Plasma Rico em Plaquetas , Compostos de Alumínio , Compostos de Cálcio , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Combinação de Medicamentos , Humanos , Óxidos , SilicatosRESUMO
Spinal cord injury (SCI) is one of the most precarious conditions which have been one of the major reasons for continuous increasing mortality rate of SCI patients. Currently, there is no effective treatment modality for SCI patients posing major threat to the scientific and medical community. The available strategies don't mimic with the natural processes of nervous tissues repair/regeneration and majority of the approaches may induce the additional fibrotic or immunological response at the injury site and are not readily available on demand. To overcome these hurdles, we have developed a ready to use bioengineered human functional neurological construct (BHNC) for regenerative applications in SCI defects. We used cryopreserved meningeal tissues (CMT) for bioengineering these neurological constructs using acellularization and repopulation technology. The technology adopted herein generates intact neurological scaffolds from CMT and retains several crucial structural, biochemical and mechanical cues to enhance the regenerative mechanisms. The neurogenic differentiation on CMT scaffolds was almost similar to the freshly prepared meningeal scaffolds and mimics with the natural nervous tissue developmental mechanisms which offer intact 3D-microarchitecture and hospitable microenvironment enriched with several crucial neurotrophins for long-term cell survival and function. Functional assessment of developed BHNC showed highly increased positive staining for pre-synaptic granules of Synapsis-1 along with MAP-2 antibody with punctuate distribution in axonal regions of the neuronal cells which was well supported by the gene expression analysis of functional transcripts. Given the significant improvement in the field may enable to generate more such ready to use functional BHNC for wider applicability in SCI repair/regeneration.
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Materiais Biomiméticos/farmacologia , Criopreservação , Meninges/fisiologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Fenômenos Biomecânicos , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Humanos , Meninges/efeitos dos fármacos , Meninges/ultraestrutura , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Acute liver failure (ALF) is one of the most devastating fatal conditions which have posed crucial challenges to the clinicians and researchers for identifying permanent cure. Currently liver transplantation has been considered as the only managerial option. However it's wider applicability has been limited owing to non-availability of quality donor organs, cost-intensiveness, surgical hitches, life-long use of immunosuppressive drugs and long-term complications. Since last decades, several liver support systems have been developed for the management of failing liver in acute condition. However, the major limitation has been the lack of natural biological support and long-term survival of the grafts post-transplantation. Repopulation of decellularized xenogeneic organs is one of the emerging technologies for development of humanized neo-organs for demanding regenerative application. However, the earlier reported studies do not fulfil the insistence to provide immunologically tolerable humanized liver grafts for clinical applications. Here we demonstrate an efficient approach to generate transplantable humanized liver grafts which provides long-term support to the failing liver in Acute Liver Failure (ALF) animal models. These bioengineered humanized liver tissue grafts expresses several liver specific transcripts and performed crucial synthetic (albumin production) and detoxification (urea synthesis) functions at comparative level to normal liver. Intraperitoneal transplantation of these humanized liver grafts offered favourable microenvironment to exchange toxic substances across the barrier during ALF condition and provided long-term survival and function of the graft. In summary, the results of present study provide a first proof of concept in pre-clinical ALF animal model for the applicability of these bioengineered humanized livers in the management of failing liver on demand and may be considered as potential bridge to liver transplantation.
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Bioengenharia , Falência Hepática Aguda/terapia , Transplante de Fígado , Peritônio/cirurgia , Animais , Biomarcadores/metabolismo , Movimento Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Fígado/irrigação sanguínea , Fígado/cirurgia , Fígado/ultraestrutura , Masculino , Imagem Óptica , Ratos Wistar , Recuperação de Função Fisiológica , Esterilização , Alicerces Teciduais/química , Transplante HeterólogoRESUMO
Immune cells sense and programme its cellular machinery appropriately to the environmental changes through the activation of cytoprotective adaptive pathway so-called the "integrated stress response (ISR)". However, the mechanisms implicated in ISR-induced protective responses are poorly understood. Here, we show that ISR activation by arsenite (Ar) results in suppression of IL-1ß production in macrophages and inhibition of DSS-induced colitis in a murine model through a novel posttranscriptional and translation regulatory (PTR) mechanism. Ar triggers PTR events through eIF2α-phosphorylation, which results in the attenuation of active polysome formation leading to the accumulation of translationally stalled IL-1ß mRNAs. Translationally stalled IL-1ß mRNAs recruit RNA-binding proteins (TIA-1/TIAR), resulting in the formation of RBP-RNA complexes known as stress granules (SGs). The SGs bound IL-1ß mRNAs might undergo degradation through induction of autophagy. Also, we show that Ar posttranslationally impairs processing and secretion of IL-1ß by diminishing inflammasome activation. Altogether, this study unveils a novel mechanism of IL-1ß regulation and further suggests that pharmacological activation of cytoprotective ISR pathway might provide an effective therapeutic intervention against inflammatory diseases.
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Colite/imunologia , Interleucina-1beta/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Biossíntese de Proteínas/imunologia , Estabilidade de RNA/imunologia , Estresse Fisiológico/imunologia , Animais , Arsenitos/farmacologia , Linhagem Celular , Colite/induzido quimicamente , Colite/patologia , Sulfato de Dextrana/efeitos adversos , Sulfato de Dextrana/farmacologia , Inflamassomos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Estresse Fisiológico/efeitos dos fármacosRESUMO
End stage liver diseases (ESLD) represent a major, neglected global public health crisis which requires an urgent action towards finding a proper cure. Orthotropic liver transplantation has been the only definitive treatment modality for ESLD. However, shortage of donor organs, timely unavailability, post-surgery related complications and financial burden on the patients limits the number of patients receiving the transplants. Since last two decades cell-based therapies have revolutionized the field of organ/tissue regeneration. However providing an alternative organ source to address the donor liver shortage still poses potential challenges. The developments made in this direction provide useful futuristic approaches, which could be translated into pre-clinical and clinical settings targeting appropriate applications in specific disease conditions. Earlier studies have demonstrated the applicability of this particular approach to generate functional organ in rodent system by connecting them with portal and hepatic circulatory networks. However, such strategy requires very high level of surgical expertise and also poses the technical and financial questions towards its future applicability. Hence, alternative sites for generating secondary organs are being tested in several types of disease conditions. Among different sites, omentum has been proved to be more appropriate site for implanting several kinds of functional tissue constructs without eliciting much immunological response. Hence, omentum may be considered as better site for transplanting humanized bioengineered ex vivo generated livers, thereby creating a secondary organ at intra-omental site. However, the expertise for generating such bioengineered organs are limited and only very few centres are involved for investigating the potential use of such implants in clinical practice due to gap between the clinical transplant surgeons and basic scientists working on the concept evolution. Herein we discuss the recent advances and challenges to create functional secondary organs through intra-omental transplantation of ex vivo generated bioengineered humanized livers and their further application in the management of ESLD as a supportive bridge for organ transplantation.
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Spinal cord injury (SCI) is one of the most devastating conditions echoes with inflammation, enhanced fibrosis and larger axonal gaps due to destruction of neurological cells which has caused continuous increasing mortality rate of SCI patients due to absence of suitable treatment modalities. The restoration of structural and functional aspect of damaged neurological tissues at the lesion site in spinal cord has been challenging. Recent developments have showed tremendous potential of neural stem cell-based strategies to form a neuronal relay circuit across the injury gap which facilitates some levels of improvement in SCI condition. However, to provide better therapeutic responses, critical mass of grafted cells must survive for long-term and differentiate into neuronal cells with well-developed axonal networks. Hence, development of tissue specific biological neuronal constructs is highly desirable to provide mechanical and biological support for long-term survival and function of neurological cells within natural biological niche. In this study, we report development of a tissue specific neuronal constructs by culturing human neural precursor cells on decellularized meningeal scaffolds to provide suitable biological neuronal construct which can be used to support mechanical, structural and functional aspect of damaged spinal cord tissues. This particular tissue specific biological construct is immunologically tolerable and provides precisely orchestral three-dimensional platform to choreograph the long-distance axonal guidance and more organized neuronal cell growth. It passes sufficient mechanical and biological properties enriched with several crucial neurotrophins required for long-term survival and function of neurological cells which is required to form proper axonal bridge to regenerate the damaged axonal connectomes at lesion-site in SCI.
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Ulcerative colitis (UC) is a persistent inflammatory illness, which is clinically categorised as Inflammatory bowel disease (IBD), affecting millions of people worldwide. The precise cause behind the pathology of the disease remains unknown. However, the involvement of multiple factors including genetic predisposition, immunological deregulations, microbiota imbalance, and environmental triggers has been suggested. Amongst all these factors, the over-active immunological response reported in UC patients seems to be a promising target for therapy. Moreover, identification of gene signatures associated with disease onset and progression would help in better understanding of the molecular mechanisms involved in the disease pathogenesis. Here, we have conducted meta-analysis of gene expression profiles of UC patient microarray datasets accessible in public databases and further validated the in-silico findings in UC patients' blood samples. Our study reveals that UC pathogenesis perturbs expression of several inflammatory genes. In addition, we report a novel gene signature comprising of TIA1 (T cell restricted intracellular antigen) and TIAR (TIA1 related protein; also known as TIAL1), which were found to be significantly downregulated in UC patients. TIA1 and TIAR are RNA-binding proteins (RBPs), which function as a translational represser by binding to ARE sequences in the 3' UTR of mRNAs encoding inflammatory mediators including cytokines. Our findings demonstrate that deletion of TIAR using gene specific siRNAs in-vitro results in enhanced production of inflammatory cytokine IL-1ß. In conclusion, the findings of this study reveal that down regulation of TIA1/TIAR genes could be responsible for UC associated inflammation. This study highlights the usefulness of the meta-analysis approach in the identification of unique gene signatures that might deliver mechanistic insights into UC pathogenesis and possibly assist in discovery of prognostic markers and therapeutic interventions.
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Colite Ulcerativa/imunologia , Proteínas de Ligação a RNA/imunologia , Transcriptoma/imunologia , Regiões 3' não Traduzidas/imunologia , Regulação para Baixo/imunologia , Expressão Gênica/imunologia , Humanos , Inflamação/imunologia , Doenças Inflamatórias Intestinais/imunologia , Interleucina-1beta/imunologia , RNA Mensageiro/imunologia , Antígeno-1 Intracelular de Células T/imunologiaRESUMO
BACKGROUND: The present study has been aimed to identify molecular dynamics of pancreatic transcription factors (pTFs) during events of directed trans-differentiation of human hepatic progenitor cells (hHPCs) into insulin producing cells (InPCs) within bioengineered humanized neoorgan. The study demonstrates applicability of acellularized whole splenic scaffold (ASOS) to generate insulin producing humanized transplantable neoorgan through activation of pancreatic transcription factors. METHODS: An efficient acellularization process was developed for xenogeneic rat spleen using change in different gradients of reagents perfusion through splenic artery for varying time points. The acellularized xenogeneic spleen scaffold was characterized thoroughly for preservation of extra-cellular matrix and retention of organ specific vasculature and mechanical properties. Further scaffolds were sterilized and repopulated with hHPCs which were triggered using a stage wise induction with growth factors and hyperglycemic challenge for trans-differentiation into InPCs. Dynamics of pTFs alone or simultaneously during induction process was identified using gene expression analysis and immunological staining. RESULTS: The cells within the engineered neoorgan respond to growth factors and extrinsic hyperglycemic challenge and generate large number of InPCs under controlled dynamic regulation of pTFs. Highly controlled regulation of pTFs generates higher percentage of Nkx-6.1+/C-peptide+ cells within the engineered splenic scaffolds. Generation of high percentage of insulin and C-peptide positive cells in three-dimensional organ architecture responded better to hyperglycemic stimuli and produced higher quantity of insulin than 2D-culture system. CONCLUSION: The present study provides a novel platform for designing effective regenerative strategies using whole organ scaffolds to control hyperglycemia under tight regulation of pTFs using humanized neoorgan system.
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Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Engenharia Tecidual/métodos , Fatores de Transcrição/metabolismo , Animais , Peptídeo C/genética , Peptídeo C/metabolismo , Diferenciação Celular , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hiperglicemia/metabolismo , Simulação de Dinâmica Molecular , Ratos , Baço/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Técnicas de Cultura de Tecidos , Alicerces Teciduais , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Gold nanoparticles (GNPs) are key diagnostic and therapeutic agents in biomedical sciences. Several studies have been carried out in different therapeutic areas such as in cancer treatment, antibacterial topical agents, imaging agents etc. There is a necessity to evaluate the gold nanoparticles cytotoxicity at all fronts. Since blood is the first point of contact in any therapy, it is required to have a thorough in vitro investigation of gold nanoparticles to avoid any adverse effects. OBJECTIVE: The objective of the current study is to evaluate the effect of gold nanoparticles capped with lipase on blood clotting factors, platelets, coagulation time and blood clotting strength. METHODS: Whole blood samples were drawn from healthy volunteers. Plasma and plasma with platelets were isolated from the blood and all the samples were treated with lipase capped gold nanoparticles, except control. Plasma fibrinogen formed in the blood coagulation process after contacting with nanoparticles was quantitatively evaluated. In addition, platelet aggregation, blood clotting kinetics, strength of the blood clot and time were evaluated post nanoparticle treatment. RESULTS: The work primarily explores the effect of GNPs on blood with changing concentrations of lipase capping. Plasma fibrinogen levels of plasma samples were found to be moderately elevated, however, there is no significant effect on blood clotting kinetics, strength, and platelet aggregation. Also, the study showed that lipase capped GNPs did not result in aggregation upon interaction with plasma components and remained stable for 1 hour after incubation. CONCLUSIONS: Our study revealed that lipase capped GNPs synthesized using NaBH4 approach were stable and hemocompatible. There is an increase in fibrinogen levels after the exposure to nanoparticles, an observation which is consistent with other studies. However, the functional consequences of such increase are unknown. The results of no significant platelet aggregation, change in blood clotting time, kinetics, and clot strength revealed the non-toxic effect of lipase capped GNPs towards blood components, which is essential for any in vivo applications.
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Testes de Coagulação Sanguínea/métodos , Ouro/química , Lactobacillus plantarum/patogenicidade , Lipase/metabolismo , Nanopartículas Metálicas/química , Adolescente , Adulto , Voluntários Saudáveis , Humanos , Adulto JovemRESUMO
INTRODUCTION: Ethanol exposure to developing brain may alter the growth and differentiation of neurological cells resulting in unfavorable pathologies. Earlier studies have provided very limited mechanistic insights of cellular and molecular mechanisms which do not mimic with human situation due to varying cell types and poses potential challenges for investigation. Therefore, the present study was undertaken to evaluate the role of ABC transporters and heat shock proteins mediated response in human neural precursor cells (NPCs) and its lineages during proliferation and lineage differentiation against ethanol exposure. METHODS: Effect of ethanol exposure was examined for neuronal cell survival and variation in cellular phenotype during neurospheres development and lineage differentiation. Generation of reactive oxygen species, and variation in cell cycle was identified along with transcriptional profiling for pluripotent markers (Nestin, NCAM, Sox-2, and Notch-2), drug transporters (ABCB1 and ABCG2) and stress protein (HSP70) during ethanol exposure. RESULTS: ABC transporters as well as HSP70 mRNA expression was higher during proliferation as compared to differentiation with chronic ethanol (1â¯M) exposure (pâ¯<â¯0.01). Ethanol exposure resulted in higher variability in size and shape of developing neurospheres and decreased ability to form new neurosphere colonies. Significant changes were observed in dendrite development due to late ethanol exposure (pâ¯<â¯0.0001). CONCLUSION: The present study demonstrated significant role of ABC transporters and HSP70 proteins in providing defense against ethanol-induced damage in human neurological cells. However, the over-expression of ABC transporter and HSP-70 proteins during such pathological conditions do not provide complete defense and additional strategies are required to repair the damage.
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Transportadores de Cassetes de Ligação de ATP/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Depressores do Sistema Nervoso Central/toxicidade , Etanol/toxicidade , Proteínas de Choque Térmico HSP70/biossíntese , Células-Tronco Neurais/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Humanos , Células-Tronco Neurais/metabolismo , Oxirredução/efeitos dos fármacos , Transcriptoma/efeitos dos fármacosRESUMO
AIM: To develop appropriate humanized three-dimensional ex-vivo model system for drug testing. METHODS: Bioengineered humanized livers were developed in this study using human hepatic stem cells repopulation within the acellularized liver scaffolds which mimics with the natural organ anatomy and physiology. Six cytochrome P-450 probes were used to enable efficient identification of drug metabolism in bioengineered humanized livers. The drug metabolism study in bioengineered livers was evaluated to identify the absorption, distribution, metabolism, excretion and toxicity responses. RESULTS: The bioengineered humanized livers showed cellular and molecular characteristics of human livers. The bioengineered liver showed three-dimensional natural architecture with intact vasculature and extra-cellular matrix. Human hepatic cells were engrafted similar to the human liver. Drug metabolism studies provided a suitable platform alternative to available ex-vivo and in vivo models for identifying cellular and molecular dynamics of pharmacological drugs. CONCLUSION: The present study paves a way towards the development of suitable humanized preclinical model systems for pharmacological testing. This approach may reduce the cost and time duration of preclinical drug testing and further overcomes on the anatomical and physiological variations in xenogeneic systems.
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INTRODUCTION: Progress in understanding pathophysiological mechanisms and the development of targeted regenerative strategies have been hampered by the lack of predictive disease models, specifically for the conditions to which affected cell types are inaccessible. The present study has aimed to unearth the role of valproic acid (VPA) and mild hypothermia (MH) as promising strategy to enhance the neuroprotective mechanisms in undifferentiated and differentiated human neural precursor cells (hNPCs) against ethanol-induced damage. METHODS: 5mM VPA alone or in combination with MH (33°C) was used to prevent the damage in proliferating and differentiating hNPCs. CD133+ve enriched hNPCs were cultured in vitro and exposed to 1M chronic ethanol concentration for 72h and followed by VPA and MH treatment for 24h. Morphometric analysis was performed to identify changes in neurospheres development and neuronal cell phenotypes. Flow cytometry and RT-qPCR analysis was performed to investigate alterations in key molecular pathways involved in cell survival and signaling. RESULTS: Combination of VPA with MH displayed higher proportion of neuronal cell viability as compared to single treatment. Combination treatment was most effective in reducing apoptosis and reactive oxygen species levels in both the undifferentiated and differentiated hNPCs. VPA with MH significantly improved neuronal cell phenotype, active chromatin modeling, chaperon and multi-drug resistant pumps activity and expression of neuronal signaling molecules. CONCLUSION: The study provided an efficient and disease specific in vitro model and demonstrated that combined treatment with VPA and MH activates several neuroprotective mechanisms and provides enhanced protection against ethanol-induced damage in cultured undifferentiated and differentiated hNPCs.