Cell Repopulation Research Articles

The CCL2/CCR2 axis is critical to recruiting macrophages into acellular nerve allograft bridging a nerve gap to promote angiogenesis and regeneration

Acellular nerve allografts (ANAs) are increasingly used to repair nerve gaps following injuries. However, these nerve scaffolds have yet to surpass the regenerative capabilities of cellular nerve autografts; improved understanding of their regenerative mechanisms could improve design. Due to their acellular nature, both angiogenesis and diverse cell recruitment is necessary to repopulate these scaffolds to promote functional regeneration. We determined the contribution of angiogenesis to initial cellular repopulation of ANAs used to repair nerve gaps, as well as the signaling that drives a significant portion of this angiogenesis. Wild-type (WT) mice with nerve gaps repaired using ANAs that were treated with an inhibitor of VEGF receptor signaling severely impaired angiogenesis within ANAs, as well as hampered cell repopulation and axon extension into ANAs. Similarly, systemic depletion of hematogenous-derived macrophages, but not neutrophils, in these mice models severely impeded angiogenesis and subsequent nerve regeneration across ANAs suggesting hematogenous-derived macrophages were major contributors to angiogenesis within ANAs. This finding was reinforced using CCR2 knockout (KO) models. As macrophages represented the majority of CCR2 expressing cells, a CCR2 deficiency impaired angiogenesis and subsequent nerve regeneration across ANAs. Furthermore, an essential role for CCL2 during nerve regeneration across ANAs was identified, as nerves repaired using ANAs had reduced angiogenesis and subsequent nerve regeneration in CCL2 KO vs WT mice. Our data demonstrate the CCL2/CCR2 axis is important for macrophage recruitment, which promotes angiogenesis, cell repopulation, and subsequent nerve regeneration and recovery across ANAs used to repair nerve gaps.

Smad2/4 Signaling Pathway Is Critical for Epidermal Langerhans Cell Repopulation Under Inflammatory Condition but Not Required for Their Homeostasis at Steady State.

Epidermal Langerhans cells (LCs) are skin-resident dendritic cells that are essential for the induction of skin immunity and tolerance. Transforming growth factor-β 1 (TGFβ1) is a crucial factor for LC maintenance and function. However, the underlying TGFβ1 signaling pathways remain unclear. Our previous research has shown that the TGFβ1/Smad3 signaling pathway does not impact LC homeostasis and maturation. In this study, we generated mice with conditional deletions of either individual Smad2, Smad4, or both Smad2 and Smad4 in the LC lineage or myeloid lineage, to further explore the impact of TGFβ1/Smad signaling pathways on LCs. We found that interruption of Smad2 or Smad4 individually or simultaneously in the LC lineage did not significantly impact the maintenance, maturation, antigen uptake, and migration of LCs in vivo or in vitro during steady state. However, the interruption of both Smad2 and Smad4 pathways in the myeloid lineage led to a dramatic inhibition of bone marrow-derived LCs in the inflammatory state. Overall, our data suggest that canonical TGFβ1/Smad2/4 signaling pathways are dispensable for epidermal LC homeostasis and maturation at steady state, but are critical for the long-term LC repopulation directly originating from the bone marrow in the inflammatory state.

Fundamentals of Extracellular Matrix Biomaterial Assimilation: Effect of Suture Type on Attachment Strength and Cell Repopulation.

Background:The clinical results with extracellular matrix biomaterials are confounded by expectations of material response based on years of experience with permanent or degradable synthetic polymers. However, the remodeling or assimilation of extracellular matrix biomaterials is dictated by cell-mediated processes rather than fibrous encapsulation or hydrolytic degradation. Previously, we found that tissue adherence and revascularization were dictated by proximity with de-epithelialized host tissue. We now investigate the effects of polymer and fixation type on attachment strength and rate of cell repopulation in an intra-abdominal implant model.Methods:An intra-abdominal implant model in rats was used to probe assimilation properties at 4 weeks and 12 weeks with permanent and degradable suture types as well a combination of suture and biologic attachment (mesothelial abrasion). The mechanical strength of the attachment was measured by peel testing and the repopulation by automated cell counting of histologic sections.Results:The intensity of the biologic response was greater with degradable polymers than permanent polypropylene. Tissue attachment strength ranged from 2 to 15 N but changed in elasticity with time. The magnitude and distribution of cell repopulation was highly variable by suture type but ultimately did not affect the long-term strength of the soft tissue attachment.Conclusions:The tissue approximating polymer sutures were stretchy and of similar strength regardless of degradation rate or polymer type. The strongest attachment, most rapid repopulation of the deep matrix regions, and most uniform distribution of cells were found with the addition of biologic attachment.

Analysis of Immune Cell Repopulation After Anti-thymocyte Globulin Administration for Steroid-Resistant T-cell–mediated Rejection

BackgroundAnti-thymocyte globulin (ATG) is a treatment option for steroid-resistant T-cell–mediated rejection after kidney transplantation. However, the extent to which immune-cell subsets can repopulate the peripheral blood is unknown. MethodsSix patients with steroid-resistant T-cell–mediated rejection were recruited and underwent analysis of their immune cells for 1 year after ATG administration. Multicolor flow cytometric analysis was used to evaluate the proportions of T cells, B cells, natural killer cells, and monocytes. ResultsT-cell repopulation from 24% to 75% occurred in the treatment group. The major repopulated cells were effector memory CD8+ T cells followed by effector memory CD4+ T cells. The population of effector memory CD8+ T cells with low expression of interleukin-7 receptor α increased over time. The population of regulatory T cells (eg, CD8+CD28-CD56+ T cells and CD4+CD25bright T cells) increased after ATG administration. However, the populations of other immune-cell subsets, including B cells, natural killer cells, and monocytes, were not significantly altered by ATG. ConclusionsOur findings on immune cell repopulation after ATG administration will enable future studies aiming to unravel the steroid-resistance mechanism underlying T-cell–mediated rejection.

Complex Stability and an Irrevertible Transition Reverted by Peptide and Fibroblasts in a Dynamic Model of Innate Immunity.

We here apply a control analysis and various types of stability analysis to an in silico model of innate immunity that addresses the management of inflammation by a therapeutic peptide. Motivation is the observation, both in silico and in experiments, that this therapy is not robust. Our modeling results demonstrate how (1) the biological phenomena of acute and chronic modes of inflammation may reflect an inherently complex bistability with an irrevertible flip between the two modes, (2) the chronic mode of the model has stable, sometimes unique, steady states, while its acute-mode steady states are stable but not unique, (3) as witnessed by TNF levels, acute inflammation is controlled by multiple processes, whereas its chronic-mode inflammation is only controlled by TNF synthesis and washout, (4) only when the antigen load is close to the acute mode's flipping point, many processes impact very strongly on cells and cytokines, (5) there is no antigen exposure level below which reduction of the antigen load alone initiates a flip back to the acute mode, and (6) adding healthy fibroblasts makes the transition from acute to chronic inflammation revertible, although (7) there is a window of antigen load where such a therapy cannot be effective. This suggests that triple therapies may be essential to overcome chronic inflammation. These may comprise (1) anti-immunoglobulin light chain peptides, (2) a temporarily reduced antigen load, and (3a) fibroblast repopulation or (3b) stem cell strategies.

Quantification of the foreign body reaction by means of a miniaturized imaging window for intravital nonlinear microscopy

Brand new biomaterials, intended to be used on humans, must undergo in vivo quantification standardized, expensive and unethical procedures mainly based on histopathological analysis, from dissections, as defined by the ISO 10993 normative set. The aim is to prove the biomaterials biocompatibility. There exist no methods based on intravital microscopy able to satisfy the normative quantification requirements both reducing the number of employed animals and related costs. We developed a miniaturized imaging window, the Microatlas, which allows subcutaneous repeated observations in vivo of the foreign body reactions, for example to the implantation of a biomaterial. Confocal and twophoton microscopy inspections at Microatlas implantation sites demonstrated growth of the recipient tissue inside the microgrids both with micro vascularization formation and collagen generation. In conclusion, the Microatlas guided in vivo a quantifiable localized reaction inside its microscaffold, both in terms of cell repopulation, collagen and capillary formation as a probable foreign body reaction.

Sequential adaptation of perfusion and transport conditions significantly improves vascular construct recellularization and biomechanics.

Recellularization of ex vivo-derived scaffolds remains a significant hurdle primarily due to the scaffolds subcellular pore size that restricts initial cell seeding to the scaffolds periphery and inhibits migration over time. With the aim to improve cell migration, repopulation, and graft mechanics, the effects of a four-step culture approach were assessed. Using an ex vivo-derived vein as a model scaffold, human smooth muscle cells were first seeded onto its ablumen (Step 1: 3 hr) and an aggressive 0-100% nutrient gradient (lumenal flow under hypotensive pressure) was created to initiate cell migration across the scaffold (Step 2: Day 0 to 19). The effects of a prolonged aggressive nutrient gradient created by this single lumenal flow was then compared with a dual flow (lumenal and ablumenal) in Step 3 (Day 20 to 30). Analyses showed that a single lumenal flow maintained for 30 days resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source), with improved distribution. In Step 4 (Day 31 to 45), the transition from hypotensive pressure (12/8 mmHg) to normotensive (arterial-like) pressure (120/80 mmHg) was assessed. It demonstrated that recellularized scaffolds exposed to arterial pressures have increased glycosaminoglycan deposition, physiological modulus, and Young's modulus. By using this stepwise conditioning, the challenging recellularization of a vein-based scaffold and its positive remodeling toward arterial biomechanics were obtained.

PDK4-Deficiency Reprograms Intrahepatic Glucose and Lipid Metabolism to Facilitate Liver Regeneration in Mice.

Liver regeneration requires intrahepatic and extrahepatic metabolic reprogramming to meet the high hepatic bioenergy demand for liver cell repopulation. This study aims to elucidate how pyruvate dehydrogenase kinase 4 (PDK4), a critical regulator of glucose and lipid metabolism, coordinates metabolic regulation with efficient liver growth. We found that hepatic Pdk4 expression was elevated after two‐thirds partial hepatectomy (PHx). In Pdk4 −/− PHx mice, the liver/body weight ratio was more rapidly restored, accompanied by more aggressive hepatic DNA replication; however, Pdk4 −/− mice developed more severe hypoglycemia. In Pdk4 −/− PHx livers, the pro‐regenerative insulin signaling was potentiated, as demonstrated by early peaking of the phosphorylation of insulin receptor, more remarkable induction of the insulin receptor substrate proteins, IRS1 and IRS2, and more striking activation of Akt. The hepatic up‐regulation of CD36 contributed to the enhanced transient regeneration‐associated steatosis in Pdk4 −/− PHx mice. Notably, CD36 overexpression in mice promoted the recovery of liver/body weight ratio and elevated intrahepatic adenosine triphosphate after PHx. CD36 expression was transcriptionally suppressed by FOXO1 (forkhead box protein O1), which was stabilized and translocated to the nucleus following AMPK (adenosine monophosphate–activated protein kinase) activation. PHx remarkably induced AMPK activation, which became incompetent to respond in Pdk4 −/− livers. Moreover, we defined that PDK4‐regulated AMPK activation directly depended on intracellular adenosine monophosphate in vitro and in regenerative livers. Conclusion: PDK4 inhibition reprograms glucose and lipid metabolism to promote liver regeneration by enhancing hepatic insulin/Akt signaling and activating an AMPK/FOXO1/CD36 regulatory axis of lipid. These findings may lead to potential therapeutic strategies to prevent hepatic insufficiency and liver failure.

Handheld instrument for wound-conformal delivery of skin precursor sheets improves healing in full-thickness burns

The current standard of care for patients with severe large-area burns consists of autologous skin grafting or acellular dermal substitutes. While emerging options to accelerate wound healing involve treatment with allogeneic or autologous cells, delivering cells to clinically relevant wound topologies, orientations, and sizes remains a challenge. Here, we report the one-step in situ formation of cell-containing biomaterial sheets using a handheld instrument that accommodates the topography of the wound. In an approach that maintained cell viability and proliferation, we demonstrated conformal delivery to surfaces that were inclined up to 45° with respect to the horizontal. In porcine pre-clinical models of full-thickness burn, we delivered mesenchymal stem/stromal cell-containing fibrin sheets directly to the wound bed, improving re-epithelialization, dermal cell repopulation, and neovascularization, indicating that this device could be introduced in a clinical setting improving dermal and epidermal regeneration.

A novel hydrogel-based treatment for complete transection spinal cord injury repair is driven by microglia/macrophages repopulation

Microglia/macrophage mediated-inflammation, a main contributor to the microenvironment after spinal cord injury (SCI), persists for a long period of time and affects SCI repair. However, the effects of microglia/macrophage mediated-inflammation on neurogenic differentiation of endogenous neural stem/progenitor cells (NSPCs) are not well understood. In this study, to attenuate activated microglia/macrophage mediated-inflammation in the spinal cord of complete transection SCI mice, a combination of photo-crosslinked hydrogel transplantation and CSF1R inhibitor (PLX3397) treatment was used to replace the prolonged, activated microglia/macrophages via cell depletion and repopulation. This combined treatment in SCI mice produced a significant reduction in CD68-positive reactive microglia/macrophages and mRNA levels of pro-inflammatory factors, and a substantial increase in the number of Tuj1-positive neurons in the lesion area compared with single treatment methods. Moreover, most of the newborn Tuj1-positive neurons were confirmed to be generated from endogenous NSPCs using a genetic fate mapping mouse line (Nestin-CreERT2; LSL-tdTomato) that can label and trace NSPC marker-nestin expressing cells and their progenies. Collectively, our findings show that the combined treatment method for inhibiting microglia/macrophage mediated-inflammation promotes endogenous NSPC neurogenesis and improves functional recovery, which provides a promising therapeutic strategy for complete transection SCI.

Design, Implementation, and in Vivo Validation of a Novel Proton FLASH Radiation Therapy System

Recent studies suggest that ultrahigh-dose-rate, "FLASH," electron radiation therapy (RT) decreases normal tissue damage while maintaining tumor response compared with conventional dose rate RT. Here, we describe a novel RT apparatus that delivers FLASH proton RT (PRT) using double scattered protons with computed tomography guidance and provide the first report of proton FLASH RT-mediated normal tissue radioprotection. Absolute dose was measured at multiple depths in solid water and validated against an absolute integral charge measurement using a Faraday cup. Real-time dose rate was obtained using a NaI detector to measure prompt gamma rays. The effect of FLASH versus standard dose rate PRT on tumors and normal tissues was measured using pancreatic flank tumors (MH641905) derived from the KPC autochthonous PanCa model in syngeneic C57BL/6J mice with analysis of fibrosis and stem cell repopulation in small intestine after abdominal irradiation. The double scattering and collimation apparatus was dosimetrically validated with dose rates of 78 ± 9 Gy per second and 0.9 ± 0.08 Gy per second for the FLASH and standard PRT. Whole abdominal FLASH PRT at 15 Gy significantly reduced the loss of proliferating cells in intestinal crypts compared with standard PRT. Studies with local intestinal irradiation at 18 Gy revealed a reduction to near baseline levels of intestinal fibrosis for FLASH-PRT compared with standard PRT. Despite this difference, FLASH-PRT did not demonstrate tumor radioprotection in MH641905 pancreatic cancer flank tumors after 12 or 18 Gy irradiation. We have designed and dosimetrically validated a FLASH-PRT system with accurate control of beam flux on a millisecond time scale and online monitoring of the integral and dose delivery time structure. Using this system, we found that FLASH-PRT decreases acute cell loss and late fibrosis after whole-abdomen and focal intestinal RT, whereas tumor growth inhibition is preserved between the 2 modalities.

The Effects of Continual Intermittent Parathyroid Hormone Treatment on Human Periodontal Ligament Fibroblasts in an In Vitro Wound Repopulation Model.

Periodontal ligament fibroblasts (PDLFs) play a vital role in periodontal regeneration. Parathyroid hormone (PTH) is important in catabolic regulation on osteoclasts; it also has anabolic effects on hard tissue formation. Using an in vitro wound repopulation model, this study investigated the effect of continual intermittent administration of PTH on PDLFs wound repopulation. Methods and Materials: PDLFs were grown in 12-well plates and divided into 0 (control), 5, 10, 20, 40, and 80nM of PTH treatments. A 3-mm wound was created on confluent and synchronized cells. Six PTH treatments were initiated using serum-free medium with supplements. Cell repopulation was measured at four time points: 5, 10, 15, and 20days. A 5% increase wound repopulation showed an enhancement on day 10 for all treatment groups as compared to control groups. On days 15 and 20, treatment groups showed a decrease in proliferation and migration compared to controls with significant decreases at concentrations of 40 and 80nM. Continual intermittent treatment with PTH has the potential to enhance proliferation and migration of PDLFs for wound repopulation at early time points. A dose-dependent correlation was seen with a positive trend on day 10 while a significant decrease on day 20.

Post-rituximab immunoglobulin M (IgM) hypogammaglobulinemia.

Rituximab is a B cell depleting monoclonal antibody that targets the B cell-specific cell surface antigen CD20 and is currently used to treat several autoimmune diseases. The elimination of mature CD20-positive B lymphocytes committed to differentiate into autoantibody-producing plasma cells is considered to be the major effect of rituximab, that makes it a beneficial biological agent in treating autoimmune diseases. Hypogammaglobulinemia has been reported after rituximab therapy in patients with lymphoma and rheumatoid arthritis. Similar data are scarce for other autoimmune diseases. Low immunoglobulin G (IgG) or hypogammaglobulinemia has attracted the most attention because of its significant role in protective immunity. However, the incidence and clinical implications of low immunoglobulin M (IgM) or hypogammaglobulinemia have not been studied in detail. This review will focus on the frequency and the clinical concerns of low IgM levels that result as a consequence of the administration of rituximab. The etiopathogenic mechanisms underlying post-rituximab IgM hypogammaglobulinemia and its implications are presented. The long-term consequences, if any, are not known or documented. Multiple factors may be involved in whether IgG or IgM decreases secondary to rituximab therapy. It is possible that the autoimmune disease itself may be one of the important factors. The dose, frequency and number of infusions appear to be important variables. Post-rituximab therapy immunoglobulin levels return to normal. During this process. IgM levels take a longer time to return to normal levels when compared to IgG or other immunoglobulins. IgM deficiency persists after B cell repopulation to normal levels has occurred. Laboratory animals and humans deficient in IgM can have multiple infections. Specific pharmacologic agents or biologic therapy that address and resolve IgM deficiency are currently unavailable. If the clinical situation so warrants, then prophylactic antibiotics may be indicated and perhaps helpful. Research in this iatrogenic phenomenon will provide a better understanding of not only the biology of IgM, but also the factor(s) that control its production and regulation, besides its influence if any, on rituximab therapy.

A long-term follow-up of rituximab treatment in 20 Chinese patients with neuromyelitis optica spectrum disorders

BackgroundRituximab has been recommended as the first-line therapy for neuromyelitis optica spectrum disorders (NMOSD); however, its regimen is yet to be optimised. We aimed to present our experience of treating adult Chinese patients having NMOSD with low-dose rituximab, and to further investigate its efficacy and safety in a long-term follow-up. MethodsTwenty adult Chinese patients with NMOSD, who had received rituximab injections, were retrospectively analysed, and followed up over a long time in our hospital. Efficacy was evaluated mainly by the change in expanded disability status scale and annual relapse ratio before and after rituximab therapy. Safety was reflected by the incidence and severity of side effects. ResultsRituximab regimens, including doses and intervals, varied widely across patients. The median dose was 500 mg, ranging from 100 mg to 1000 mg. The median interval was 6.1 months, ranging from 2 months to 18 months. After rituximab therapy, the median EDSS was stable, whereas median ARR decreased by 90%. Of the 20 patients, 9 suffered from 11 relapses, with relapse-free rate of 55%. Six relapsing cases occurred in less than 6 months from the preceding rituximab injection. Seven cases occurred without CD19+ B cell repopulation. There were 4 cases of infection, including lung and urinary infection; severity ranged from mild to severe across the patients. ConclusionsWe confirmed the long-term efficacy and safety of low-dose rituximab in adult Chinese patients with NMOSD. We recommend monitoring of B cell repopulation to instruct individualised regimens for patients with NMOSD in clinical practice.

Efficacy and safety of rituximab in patients with refractory neuromyelitis optica spectrum disorders: A prospective observation in Iranian cases.

Background:Rituximab has been used successfully in the recent years for treatment of neuromyelitis optica spectrum disorders (NMOSD). However, a uniform treatment protocol for maintenance therapy and the best interval for evaluation and retreatment have not been postulated. We evaluated the efficacy and safety of rituximab treatment as second line therapy, in Iranian patients with refractory NMOSD, based on annualized relapse rate (ARR) and expanded disability status scale (EDSS).Methods:In this prospective before-after study, a total of 18 patients were treated with a loading dose of rituximab (375 mg/m2 weekly in 4 consecutive weeks). Flow cytometric determination of CD19+ B cell in peripheral blood sample was carried every 6 weeks and patients were re-treated based on B cell repopulation with a single dose of 375 mg/m2. Wilcoxon signed rank test was used to evaluate the ARR and EDSS before and after treatment. A p-value of <0.05 was considered statistically significant.Results:Of the 18 patients, 10 (55.5%) were relapse-free during the period of follow up. The EDSS scores were reduced in nine (50%) patients and stable in the remaining nine (50%). The mean EDSS score before and after treatment were 4.1±0.4 and 3.7±0.3, respectively, which was statistically significant. There was also a statistically significant reduction in median ARR after treatment (1.48 (range 0.47-5) vs. 0 (range 0-2)). Rituximab administration did not have significant adverse effect in 94% of patients.Conclusion:Repeated treatment with Rituximab is an effective and well-tolerated treatment in refractory NMOSD.

Novel morphological multi-scale evaluation system for quality assessment of decellularized liver scaffolds

Decellularized scaffolds can serve as an excellent three-dimensional environment for cell repopulation. They maintain tissue-specific microarchitecture of extracellular matrix proteins with important spatial cues for cell adhesion, migration, growth, and differentiation. However, criteria for quality assessment of the three-dimensional structure of decellularized scaffolds are rather fragmented, usually study-specific, and mostly semi-quantitative. Thus, we aimed to develop a robust structural assessment system for decellularized porcine liver scaffolds. Five scaffolds of different quality were used to establish the new evaluation system. We combined conventional semi-quantitative scoring criteria with a quantitative scaffold evaluation based on automated image analysis. For the quantitation, we developed a specific open source software tool (ScaffAn) applying algorithms designed for texture analysis, segmentation, and skeletonization. ScaffAn calculates selected parameters characterizing structural features of porcine liver scaffolds such as the sinusoidal network. After evaluating individual scaffolds, the total scores predicted scaffold interaction with cells in terms of cell adhesion. Higher scores corresponded to higher numbers of cells attached to the scaffolds. Moreover, our analysis revealed that the conventional system could not identify fine differences between good quality scaffolds while the additional use of ScaffAn allowed discrimination. This led us to the conclusion that only using the combined score resulted in the best discrimination between different quality scaffolds. Overall, our newly defined evaluation system has the potential to select the liver scaffolds most suitable for recellularization, and can represent a step toward better success in liver tissue engineering.

Mathematical modeling of radiotherapy cancer treatment using Caputo fractional derivative

BackgroundThis paper presents a mathematical model that simulates a radiotherapy cancer treatment process. The model takes into consideration two important radiobiological factors, which are repair and repopulation of cells. The model was used to simulate the fractionated treatment process of six patients. The results gave the population changes in the cells and the final volumes of the normal and cancer cells. MethodThe model was formulated by integrating the Caputo fractional derivative with the previous cancer treatment model. Thereafter, the linear-quadratic with the repopulation model was coupled into the model to account for the cells’ population decay due to radiation. The treatment process was then simulated with numerical variables, numerical parameters, and radiation parameters. The numerical parameters which included the proliferation coefficients of the cells, competition coefficients of the cells, and the perturbation constant of the normal cells were obtained from previous literature. The radiation and numerical parameters were obtained from reported clinical data of six patients treated with radiotherapy. The patients had tumor volumes of 24.1cm3, 17.4cm3, 28.4cm3, 18.8cm3, 30.6cm3, and 12.6cm3 with fractionated doses of 2 Gy for the first two patients and 1.8 Gy for the other four. The initial tumor volumes were used to obtain initial populations of cells after which the treatment process was simulated in MATLAB. Subsequently, a global sensitivity analysis was done to corroborate the model with clinical data. Finally, 96 radiation protocols were simulated by using the biologically effective dose formula. These protocols were used to obtain a regression equation connecting the value of the Caputo fractional derivative with the fractionated dose. ResultsThe final tumor volumes, from the results of the simulations, were 3.58cm3, 8.61cm3, 5.68cm3, 4.36cm3, 5.75cm3, and 6.12cm3, while those of the normal cells were 23.87cm3, 17.29cm3, 28.17cm3, 18.68cm3, 30.33cm3, and 12.55cm3. The sensitivity analysis showed that the most sensitive model factors were the value of the Caputo fractional derivative and the proliferation coefficient of the cancer cells. Lastly, the obtained regression equation accounted for 99.14% of the prediction. ConclusionThe model can simulate a cancer treatment process and predict the results of other radiation protocols.

Cirrhotic Human Liver Extracellular Matrix 3D Scaffolds Promote Smad-Dependent TGF-β1 Epithelial Mesenchymal Transition.

An altered liver microenvironment characterized by a dysregulated extracellular matrix (ECM) supports the development and progression of hepatocellular carcinoma (HCC). The development of experimental platforms able to reproduce these physio-pathological conditions is essential in order to identify and validate new therapeutic targets for HCC. The aim of this work was to validate a new in vitro model based on engineering three-dimensional (3D) healthy and cirrhotic human liver scaffolds with HCC cells recreating the micro-environmental features favoring HCC. Healthy and cirrhotic human livers ECM scaffolds were developed using a high shear stress oscillation-decellularization procedure. The scaffolds bio-physical/bio-chemical properties were analyzed by qualitative and quantitative approaches. Cirrhotic 3D scaffolds were characterized by biomechanical properties and microarchitecture typical of the native cirrhotic tissue. Proteomic analysis was employed on decellularized 3D scaffolds and showed specific enriched proteins in cirrhotic ECM in comparison to healthy ECM proteins. Cell repopulation of cirrhotic scaffolds highlighted a unique up-regulation in genes related to epithelial to mesenchymal transition (EMT) and TGFβ signaling. This was also supported by the presence and release of higher concentration of endogenous TGFβ1 in cirrhotic scaffolds in comparison to healthy scaffolds. Fibronectin secretion was significantly upregulated in cells grown in cirrhotic scaffolds in comparison to cells engrafted in healthy scaffolds. TGFβ1 induced the phosphorylation of canonical proteins Smad2/3, which was ECM scaffold-dependent. Important, TGFβ1-induced phosphorylation of Smad2/3 was significantly reduced and ECM scaffold-independent when pre/simultaneously treated with the TGFβ-R1 kinase inhibitor Galunisertib. In conclusion, the inherent features of cirrhotic human liver ECM micro-environment were dissected and characterized for the first time as key pro-carcinogenic components in HCC development.

Belimumab after B cell depletion therapy in patients with systemic lupus erythematosus (BEAT Lupus) protocol: a prospective multicentre, double-blind, randomised, placebo-controlled, 52-week phase II clinical trial

IntroductionFew treatment options exist for patients with systemic lupus erythematosus (SLE) who fail conventional therapy. Although widely used to treat lupus, the efficacy of B cell depletion therapy using rituximab...

Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells.

The periosteum is critical for bone maintenance and healing. However, the invivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible forlifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration invivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.