STIMULATING EFFECT OF HIGH DOSE HEPARIN ON MIGRATION ACTIVITY AND MSC STEMNESS PRESERVATION IN THE PRESENCE OF BONE-SUBSTITUTING MATERIALS

Synthetic materials used in regenerative medicine, upon implantation, induce the development of an inflammatory reaction necessary for the effective regeneration of damaged bone tissue. Implant contact with tissues is accompanied by the deposition of blood proteins and interstitial fluid on its surface, contributing to the activation of the complement system, components of innate immunity, initiating coagulation hemostasis, leading to the formation of a fibrin clot. An extracellular matrix based on fibrin, collagen and elastin forms on the implant’s surface, which provides the basis for the formation of tissue structure through the adhesion of stem cells to the forming bone callus before the formation of bone regenerate. To prevent the development of postoperative pathological conditions caused by hypercoagulable syndrome, therapeutic strategies are used to use anticoagulants (heparin, warfarin). However, their use limits the normal formation of a fibrin clot in vivo. This can slow down the migration of mesenchymal stem cells (MSC) and disrupt the formation of callus, inhibiting the processes of osseointegration of the implant and bone healing. The study’s goal was to study the effect of heparin in a gradient of low and high concentrations on the migration activity and stem capacity of human MSCs under in vitro cultivation conditions. According to the results of flow cytometry, it was revealed that high concentrations of heparin (130, 260 IU/ml) in a 2D cultivation model contribute to an increase in the number of cells expressing surface markers CD73 and CD90, which indicates that MSCs retain high clonogenic potential. A 3D model of in vitro cultivation with the addition of heparin and osteosubstituting implants bearing a CF coating with a roughness index of Ra = 2.6-4.9 μm contributed to preserving the “stemness” character of MSCs through the expression of surface markers CD73 and CD90. According to the results obtained using the xCELLigence system, heparin at a later time (from 20-40 hours) increases the invasion of MSCs through micropores that simulate the state of the blood vessel walls. However, in the presence of HAP nanoparticles that mimic the remodeling processes of the mineral bone matrix and/or resorption of bone cement, the effect of heparin was less pronounced. The results can be used in the field of regenerative medicine associated with the introduction of MSCs. The data can serve as a prerequisite for developing new therapeutic strategies for surgical patients with a high risk of postoperative thrombosis after osteosynthesis

Mesenchymal stem cells: a brief review of classis concepts and new factors of osteogenic differentiation

Molecular genetic mechanisms, signaling pathways, cultural conditions, factors, and markers of osteogenic differentiation of mesenchymal stem cells (MSC) are actively studied despite numerous works in this area of cellular technologies. This is largely due to the accumulating contradictions in seemingly classical knowledge, as well as permanent updating of the results in the field. In this regard, we focused on the main classical concepts and some new factors and mechanisms that have a noticeable regulatory effect on the differentiation potential of postnatal MSCs. The present review considers the significance of MSC sources for their differentiation capacity, as well as the role of the cellular microenvironment. The issues of classification, terminology, and functional activity of MSCs from various sources are discussed. The paracrine potential of MSCs in tissue regeneration has been considered; sufficient importance of inflammation in osteogenesis is noted, in particular, the presence of inflammatory cytokines and chemokines in the lesion focus, produced not only by microenvironmental cells but also by blood cells, including mononuclear leukocytes, migrating to the affected site. An important role in this review is given to biomechanical signals and to influence of conformational changes in cell cytoskeleton (cell shape) upon MSC differentiation, since the morphological features of cells and the structure of cytoskeleton are modulated by interactions of the cell surface with environmental factors, including hydrostatic pressure, fluid flow, compression/stretching loads. The data are presented concerning elasticity of extracellular matrix being a determining factor of cell differentiation. We conclude that one should switch from point studies of individual gene effects to multiple measurements of the gene-regulatory profile and biomolecules responsible for multiple, still poorly studied osteogenic factors of endogenous and exogenous origin. Among cornerstones in future (epi)genetic studies will be to decide if osteomodulatory effects are realized through specific signaling pathways and/or via cross-signaling with known genes controlling osteogenic differentiation of MSCs

Analysis of ground reaction force and electromyographic activity of the gastrocnemius muscle during double support

O documento em anexo encontra-se na versão post-print (versão corrigida pelo editor).Purpose: Mechanisms associated with energy expenditure during gait have been extensively researched and studied. According to the double-inverted pendulum model energy expenditure is higher during double support, as lower limbs need to work to redirect the centre of mass velocity. This study looks into how the ground reaction force (GRF) of one limb affects the muscle activity required by the medial gastrocnemius (MG) of the contralateral limb during step-to-step transition. Methods: Thirty-five subjects were monitored as to the MG electromyographic activity (EMGa) of one limb and the GRF of the contralateral limb during double support. Results: After determination of the Pearson correlation coefficient (r), a moderate correlation was observed between the MG EMGa of the dominant leg and the vertical (Fz) and anteroposterior (Fy) components of GRF of the non-dominant leg (r=0.797, p<0.0001; r=-0.807, p<0.0001) and a weak and moderate correlation was observed between the MG EMGa of the non-dominant leg and the Fz and Fy of the dominant leg, respectively (r=0.442, p=0.018; r=-0.684 p<0.0001). Conclusions: The results obtained suggest that during double support, GRF is associated with the EMGa of the contralateral MG and that there is an increased dependence between the GRF of the non-dominant leg and the EMGa of the dominant MG

Simian virus 40 vectors for pulmonary gene therapy

<p>Abstract</p> <p>Background</p> <p>Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS). Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40) vectors for pulmonary gene therapy.</p> <p>Methods</p> <p>Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP). SV40 vectors carrying the luciferase reporter gene (SV/<it>luc) </it>were administered intratracheally immediately after sepsis induction. Sham operated (SO) as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by <it>in vivo </it>light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C). Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector.</p> <p>Results</p> <p>Luc expression measured by <it>in vivo </it>light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response.</p> <p>Conclusion</p> <p>In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of <it>in vivo </it>transduction of alveolar type II cells and may thus become a future therapeutic tool.</p

Graft-versus-host disease in recipients of male unrelated donor compared with parous female sibling donor transplants

Optimal donor selection is critical for successful allogeneic hematopoietic cell transplantation (HCT). Donor sex and parity are well-established risk factors for graft-versus-host disease (GVHD), with male donors typically associated with lower rates of GVHD. Well-matched unrelated donors (URDs) have also been associated with increased risks of GVHD as compared with matched sibling donors. These observations raise the question of whether male URDs would lead to more (or less) favorable transplant outcomes as compared with parous female sibling donors. We used the Center for International Blood and Marrow Transplant Research registry to complete a retrospective cohort study in adults with acute myeloid leukemia, acute lymphoblastic leukemia, or myelodysplastic syndrome, who underwent T-cell replete HCT from these 2 donor types (parous female sibling or male URD) between 2000 and 2012. Primary outcomes included grade 2 to 4 acute GVHD (aGVHD), chronic GVHD (cGVHD), and overall survival. Secondary outcomes included disease-free survival, transplant-related mortality, and relapse. In 2813 recipients, patients receiving male URD transplants (n = 1921) had 1.6 times higher risk of grade 2 to 4 aGVHD (P \u3c .0001). For cGVHD, recipient sex was a significant factor, so donor/recipient pairs were evaluated. Female recipients of male URD grafts had a higher risk of cGVHD than those receiving parous female sibling grafts (relative risk [RR] = 1.43, P \u3c .0001), whereas male recipients had similar rates of cGVHD regardless of donor type (RR = 1.09, P = .23). Donor type did not significantly affect any other end point. We conclude that when available, parous female siblings are preferred over male URDs

Hematopoietic Cell Transplantation Outcomes in Monosomal Karyotype Myeloid Malignancies

The presence of monosomal karyotype (MK+) in acute myeloid leukemia (AML) is associated with dismal outcomes. We evaluated the impact of MK+ in AML (MK+AML, N=240) and in myelodysplastic syndrome (MK+MDS, N=221) on hematopoietic cell transplantation (HCT) outcomes compared to other cytogenetically defined groups (AML, N=3,360; MDS, N=1,373) as reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) from 1998 to 2011. MK+AML was associated with higher disease relapse (hazard ratio [HR] 1.98, p<0.01), similar transplant related mortality (TRM, HR 1.01, p=0.9) and worse survival (HR 1.67, p<0.01) compared to other cytogenetically defined AML. Among patients with MDS, MK+MDS was associated with higher disease relapse (HR 2.39, p<0.01), higher TRM (HR 1.80, p<0.01) and worse survival (HR 2.02, p<0.01). Subset analyses comparing chromosome 7 abnormalities (del7/7q) with or without MK+ demonstrated higher mortality for MK+ disease in for both AML (HR 1.72, p<0.01) and MDS (HR1.79, p<0.01). The strong negative impact of MK+ in myeloid malignancies was observed in all age groups and using either myeloablative or reduced intensity conditioning regimens. Alternative approaches to mitigate disease relapse in this population are needed

DNA-Free Recombinant SV40 Capsids Protect Mice from Acute Renal Failure by Inducing Stress Response, Survival Pathway and Apoptotic Arrest

Viruses induce signaling and host defense during infection. Employing these natural trigger mechanisms to combat organ or tissue failure is hampered by harmful effects of most viruses. Here we demonstrate that SV40 empty capsids (Virus Like Particles-VLPs), with no DNA, induce host Hsp/c70 and Akt-1 survival pathways, key players in cellular survival mechanisms. We postulated that this signaling might protect against organ damage in vivo. Acute kidney injury (AKI) was chosen as target. AKI is critical, prevalent disorder in humans, caused by nephrotoxic agents, sepsis or ischemia, via apoptosis/necrosis of renal tubular cells, with high morbidity and mortality. Systemic administration of VLPs activated Akt-1 and upregulated Hsp/c70 in vivo. Experiments in mercury-induced AKI mouse model demonstrated that apoptosis, oxidative stress and toxic renal failure were significantly attenuated by pretreatment with capsids prior to the mercury insult. Survival rate increased from 12% to >60%, with wide dose response. This study demonstrates that SV40 VLPs, devoid of DNA, may potentially be used as prophylactic agent for AKI. We anticipate that these finding may be projected to a wide range of organ failure, using empty capsids of SV40 as well as other viruses

Mechanisms of viral entry: sneaking in the front door

Recent developments in methods to study virus internalisation are providing clearer insights into mechanisms used by viruses to enter host cells. The use of dominant negative constructs, specific inhibitory drugs and RNAi to selectively prevent entry through particular pathways has provided evidence for the clathrin-mediated entry of hepatitis C virus (HCV) as well as the caveolar entry of Simian Virus 40. Moreover, the ability to image and track fluorescent-labelled virus particles in real-time has begun to challenge the classical plasma membrane entry mechanisms described for poliovirus and human immunodeficiency virus. This review will cover both well-documented entry mechanisms as well as more recent discoveries in the entry pathways of enveloped and non-enveloped viruses. This will include viruses which enter the cytosol directly at the plasma membrane and those which enter via endocytosis and traversal of internal membrane barrier(s). Recent developments in imaging and inhibition of entry pathways have provided insights into the ill-defined entry mechanism of HCV, bringing it to the forefront of viral entry research. Finally, as high-affinity receptors often define viral internalisation pathways, and tropism in vivo, host membrane proteins to which viral particles specifically bind will be discussed throughout

Secondary solid cancer screening following hematopoietic cell transplantation

Hematopoietic stem cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers, particularly beyond 5 years after HCT and without reaching a plateau overtime. A working group was established through the Center for International Blood and Marrow Transplant Research and the European Group for Blood and Marrow Transplantation with the goal to facilitate implementation of cancer screening appropriate to HCT recipients. The working group reviewed guidelines and methods for cancer screening applicable to the general population and reviewed the incidence and risk factors for secondary cancers after HCT. A consensus approach was used to establish recommendations for individual secondary cancers. The most common sites include oral cavity, skin, breast and thyroid. Risks of cancers are increased after HCT compared with the general population in skin, thyroid, oral cavity, esophagus, liver, nervous system, bone and connective tissues. Myeloablative TBI, young age at HCT, chronic GVHD and prolonged immunosuppressive treatment beyond 24 months were well-documented risk factors for many types of secondary cancers. All HCT recipients should be advised of the risks of secondary cancers annually and encouraged to undergo recommended screening based on their predisposition. Here we propose guidelines to help clinicians in providing screening and preventive care for secondary cancers among HCT recipients