Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

BACKGROUND: Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES: The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS: The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS: We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Human Adipose-Derived Stem Cells Reduce Cellular Damage after Experimental Spinal Cord Injury in Rats.

Traumatic spinal cord injury (SCI) is a devastating condition without an effective therapy. Cellular therapies are among the promising treatment strategies. Adult stem cells, such as mesenchymal stem cells, are often used clinical research for their immunomodulatory and regenerative potential. This study aimed to evaluate the effect of human adipose tissue-derived stem cells (ADSC) infusion through the cauda equina in rats with SCI. The human ADSC from bariatric surgery was isolated, expanded, and characterized. Wistar rats were subjected to blunt SCI and were divided into four groups. Two experimental groups (EG): EG1 received one ADSC infusion after SCI, and EG2 received two infusions, the first one after SCI and the second infusion seven days after the injury. Control groups (CG1 and CG2) received infusion with a culture medium. In vivo, cell tracking was performed 48 h and seven days after ADSC infusion. The animals were followed up for 40 days after SCI, and immunohistochemical quantification of myelin, neurons, and astrocytes was performed. Cellular tracking showed cell migration towards the injury site. ADSC infusion significantly reduced neuronal loss, although it did not prevent the myelin loss or enhance the area occupied by astrocytes compared to the control group. The results were similar when comparing one or two cell infusions. The injection of ADSC distal to the injured area was shown to be a safe and effective method for cellular administration in spinal cord injury.

Biocompatibility of ABS and PLA Polymers with Dental Pulp Stem Cells Enhance Their Potential Biomedical Applications.

Polylactic Acid (PLA) and Acrylonitrile-Butadiene-Styrene (ABS) are commonly used polymers in 3D printing for biomedical applications. Dental Pulp Stem Cells (DPSCs) are an accessible and proliferative source of stem cells with significant differentiation potential. Limited knowledge exists regarding the biocompatibility and genetic safety of ABS and PLA when in contact with DPSCs. This study aimed to investigate the impact of PLA and ABS on the adhesion, proliferation, osteogenic differentiation, genetic stability, proteomics, and immunophenotypic profile of DPSCs. A total of three groups, 1- DPSC-control, 2- DPSC+ABS, and 3- DPSC+PLA, were used in in vitro experiments to evaluate cell morphology, proliferation, differentiation capabilities, genetic stability, proteomics (secretome), and immunophenotypic profiles regarding the interaction between DPSCs and polymers. Both ABS and PLA supported the adhesion and proliferation of DPSCs without exhibiting significant cytotoxic effects and maintaining the capacity for osteogenic differentiation. Genetic stability, proteomics, and immunophenotypic profiles were unaltered in DPSCs post-contact with these polymers, highlighting their biosafety. Our findings suggest that ABS and PLA are biocompatible with DPSCs and demonstrate potential in dental or orthopedic applications; the choice of the polymer will depend on the properties required in treatment. These promising results stimulate further studies to explore the potential therapeutic applications in vivo using prototyped polymers in personalized medicine.

Recovery of motricity and micturition after transplantation of mesenchymal stem cells in rats subjected to spinal cord injury.

The objective was to evaluate the effect of human adipose-derived stem cell (hADSC) infusion on impaired hindlimb function and urinary continence after spinal cord contusion in rats. hADSCs were transplanted into the injured spinal cords of rats 7 and 14 days after injury in two groups (B and C). Group C also received methylprednisolone sodium succinate (MPSS) after 3 h of injury. The control group (group A) did not receive corticoids or stem cells. Voiding and motor performance evaluations were performed daily for 90 days post-transplantation. Cells were labeled with PKH26 or PKH67 for in vitro monitoring. For in vivo screening, the cells were evaluated for bioluminescence. The levels of some cytokines were quantified in different times. Euthanasia was performed 90 days post-transplant. ß-tubulin III expression was evaluated in the spinal cord of the animals from all groups. As a result, we observed a recovery of 66.6 % and 61.9 % in urinary continence of animals from groups B and C, respectively. Partial recovery of motor was observed in 23.8 % and 19 % of the animals from groups B and C, respectively. Cells remained viable at the site up to 90 days after transplantation. No significant difference was observed in levels of cytokines and thickness of urinary bladders between groups. A smaller percentage of tissue injury and higher concentrations of neuropils were observed in the spinal cords of the animals from groups B and C than control group. Thus, hADSCs transplantation with or without MPSS, contributed to the improvement in voiding and motor performance of Wistar rats submitted to compressive spinal cord injury.

Management of Airway Remodeling in a Mouse Model of Allergic Airways Inflammation Using Extracellular Vesicles from Human Bone Marrow-Derived Mesenchymal Stromal Cells

Abstract: Asthma is a chronic respiratory disease affecting 300 million people worldwide. It results in several structural changes in the airways, which are minimally accessible in clinical practice. Cell therapy using mesenchymal stromal cells (MSCs) is a promising strategy for treating asthma due to the paracrine activity of MSCs, which influences tissue regeneration and modulates the immune response. Studies using extracellular vesicles (EV) released by MSCs have demonstrated their regenerative properties in animal models. The aim of this study was to evaluate the potential of EVs isolated from human bone marrow MSCs (hBM-MSCs) to control lung tissue remodeling in ovalbumin-induced allergic asthma in Balb/c mice. We isolated hBM-MSCs from a single donor, expanded and characterized them, and then isolated EVs. Asthma was induced in 43 male Balb/c mice, divided into four groups: control, asthmatic (AS), asthmatic plus systemic EVs (EV-S), and asthmatic plus intratracheal EVs (EV-IT). Upon completion of asthma induction, animals were treated with EVs either locally (EV-IT) or intravenously (EV-S). Seven days after, we performed bronchoalveolar lavage (BAL) and the total nuclear cells were counted. The animals were euthanized, and the lungs were collected for histopathological analysis of the airways. The EV-S group showed improvement in only the total BAL cell count compared with the AS group, while the EV-IT group showed significant improvement in almost all evaluated criteria. Therefore, we demonstrate that the local application of EVs derived from hBM-MSCs may be a potential treatment in controlling asthma.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.