Impact of NaOH based perfusion-decellularization protocol on mechanical resistance of structural bone allografts.

INTRODUCTION: To mitigate the post-operative complication rates associated with massive bone allografts, tissue engineering techniques have been employed to decellularize entire bones through perfusion with a sequence of solvents. Mechanical assessment was performed in order to compare conventional massive bone allografts and perfusion/decellularized massive bone allografts. MATERIAL AND METHODS: Ten porcine femurs were included. Five were decellularized by perfusion. The remaining 5 were left untreated as the "control" group. Biomechanical testing was conducted on each bone, encompassing five different assessments: screw pull-out, 3-points bending, torsion, compression and Vickers indentation. RESULTS: Under the experimental conditions of this study, all five destructive tested variables (maximum force until screw pull-out, maximum elongation until screw pull-out, energy to pull out the screw, fracture resistance in flexion and maximum constrain of compression) were statistically significantly superior in the control group. All seven nondestructive variables (Young's modulus in flexion, Young's modulus in shear stress, Young's modulus in compression, Elastic conventional limit in compression, lengthening to rupture in compression, resilience in compression and Vickers Hardness) showed no significant difference. DISCUSSION: Descriptive statistical results suggest a tendency for the biomechanical characteristics of decellularized bone to decrease compared with the control group. However, statistical inferences demonstrated a slight significant superiority of the control group with destructive mechanical stresses. Nondestructive mechanical tests (within the elastic phase of Young's modulus) were not significantly different.

Collagen molecular organization preservation in human fascia lata and periosteum after tissue engineering.

Large bone defect regeneration remains a major challenge for orthopedic surgeons. Tissue engineering approaches are therefore emerging in order to overcome this limitation. However, these processes can alter some of essential native tissue properties such as intermolecular crosslinks of collagen triple helices, which are known for their essential role in tissue structure and function. We assessed the persistence of extracellular matrix (ECM) properties in human fascia lata (HFL) and periosteum (HP) after tissue engineering processes such as decellularization and sterilization. Harvested from cadaveric donors (N = 3), samples from each HFL and HP were decellularized following five different chemical protocols with and without detergents (D1-D4 and D5, respectively). D1 to D4 consisted of different combinations of Triton, Sodium dodecyl sulfate and Deoxyribonuclease, while D5 is routinely used in the institutional tissue bank. Decellularized HFL tissues were further gamma-irradiated (minimum 25 kGy) in order to study the impact of sterilization on the ECM. Polarized light microscopy (PLM) was used to estimate the thickness and density of collagen fibers. Tissue hydration and content of hydroxyproline, enzymatic crosslinks, and non-enzymatic crosslinks (pentosidine) were semi-quantified with Raman spectroscopy. ELISA was also used to analyze the maintenance of the decorin (DCN), an important small leucine rich proteoglycan for fibrillogenesis. Among the decellularization protocols, detergent-free treatments tended to further disorganize HFL samples, as more thin fibers (+53.7%) and less thick ones (-32.6%) were recorded, as well as less collagen enzymatic crosslinks (-25.2%, p = 0.19) and a significant decrease of DCN (p = 0.036). GAG content was significantly reduced in both tissue types after all decellularization protocols. On the other hand, HP samples were more sensitive to the D1 detergent-based treatments, with more disrupted collagen organization and greater, though not significant loss of enzymatic crosslinks (-37.4%, p = 0.137). Irradiation of D5 HFL samples, led to a further and significant loss in the content of enzymatic crosslinks (-29.4%, p = 0.037) than what was observed with the decellularization process. Overall, the results suggest that the decellularization processes did not significantly alter the matrix. However, the addition of a gamma-irradiation is deleterious to the collagen structural integrity of the tissue.

Decellularization of Massive Bone Allografts By Perfusion: A New Protocol for Tissue Engineering.

In terms of large bone defect reconstructions, massive bone allografts may sometimes be the only solution. However, they are still burdened with a high postoperative complication rate. Our hypothesis is that the immunogenicity of residual cells in the graft is involved in this issue. Decellularization by perfusion might therefore be the answer to process and create more biologically effective massive bone allografts. Seventy-two porcine bones were used to characterize the efficiency of our sodium hydroxide-based decellularization protocol. A sequence of solvent perfusion through each nutrient artery was set up to ensure the complete decellularization of whole long bones. Qualitative (histology and immunohistochemistry [IHC]) and quantitative (fluoroscopic absorbance and enzyme-linked immunosorbent assay) evaluations were performed to assess the decellularization and the preservation of the extracellular matrix in the bone grafts. Cytotoxicity and compatibility were also tested. Comparatively to nontreated bones, our experiments showed a very high decellularization quality, demonstrating that perfusion is mandatory to achieve an entire decellularization. Moreover, results showed a good preservation of the bone composition and microarchitecture, Haversian systems and vascular network included. This protocol reduces the human leukocyte antigen antigenic load of the graft by >50%. The majority of measured growth factors is still present in the same amount in the decellularized bones compared to the nontreated bones. Histology and IHC show that the bones were cell compatible, noncytotoxic, and capable of inducing osteoblastic differentiation of mesenchymal stem cells. Our decellularization/perfusion protocol allowed to create decellularized long bone graft models, thanks to their inner vascular network, ready for in vivo implantation or to be further used as seeding matrices.

Biochemical and cellular insights into the Baz2B protein, a non-catalytic subunit of the chromatin remodeling complex.

Baz2B is a regulatory subunit of the ATP-dependent chromatin remodeling complexes BRF1 and BRF5, which control access to DNA during DNA-templated processes. Baz2B has been implicated in several diseases and also in unhealthy ageing, however limited information is available on the domains and cellular roles of Baz2B. To gain more insight into the Baz2B function, we biochemically characterized the TAM (Tip5/ARBP/MBD) domain with the auxiliary AT-hook motifs and the bromodomain (BRD). We observed alterations in histone code recognition in bromodomains carrying cancer-associated point mutations, suggesting their potential involvement in disease. Furthermore, the depletion of Baz2B in the Hap1 cell line resulted in altered cell morphology, reduced colony formation and perturbed transcriptional profiles. Despite that, super-resolution microscopy images revealed no changes in the overall chromatin structure in the absence of Baz2B. These findings provide insights into the biological function of Baz2B.

Diagnostic Superiority of Dual-Time Point [18F]FDG PET/CT to Differentiate Malignant from Benign Soft Tissue Tumors.

[18F]FDG PET/CT is used in the workup of indeterminate soft tissue tumors (STTs) but lacks accuracy in the detection of malignant STTs. The aim of this study is to evaluate whether dual-time point [18F]FDG PET/CT imaging (DTPI) can be useful in this indication. In this prospective study, [18F]FDG PET/CT imaging was performed 1 h (t1) and 3 h (t2) after injection. Tumor uptake (SUVmax) was calculated at each time point to define a retention index (RI) corresponding to the variation between t1 and t2 (%). Sixty-eight patients were included, representing 20 benign and 48 malignant tumors (including 40 sarcomas). The RI was significantly higher in malignant STTs than in benign STTs (median: +21.8% vs. -2%, p < 0.001). An RI of >14.3% predicted STT malignancy with a specificity (Sp) of 90% and a sensitivity (Se) of 69%. An SUVmaxt1 of >4.5 was less accurate with an Sp of 80% and an Se of 60%. In a subgroup of tumors with at least mild [18F]FDG uptake (SUVmax ≥ 3; n = 46), the RI significantly outperformed the diagnostic accuracy of SUVmax (AUC: 0.88 vs. 0.68, p = 0.01). DTPI identifies malignant STT tumors with high specificity and outperforms the diagnostic accuracy of standard PET/CT.

Corrigendum to "Anti- E. coli immunoglobulin yolk (IgY): Reduction of pathogen receptors and inflammation factors could be caused by decrease in E. coli load" [Heliyon 9(3) (February 21, 2023) e13876]>.

[This corrects the article DOI: 10.1016/j.heliyon.2023.e13876.].

Scalable biomimetic sensing system with membrane receptor dual-monolayer probe and graphene transistor arrays.

Affinity-based biosensing can enable point-of-care diagnostics and continuous health monitoring, which commonly follows bottom-up approaches and is inherently constrained by bioprobes' intrinsic properties, batch-to-batch consistency, and stability in biofluids. We present a biomimetic top-down platform to circumvent such difficulties by combining a "dual-monolayer" biorecognition construct with graphene-based field-effect-transistor arrays. The construct adopts redesigned water-soluble membrane receptors as specific sensing units, positioned by two-dimensional crystalline S-layer proteins as dense antifouling linkers guiding their orientations. Hundreds of transistors provide statistical significance from transduced signals. System feasibility was demonstrated with rSbpA-ZZ/CXCR4QTY-Fc combination. Nature-like specific interactions were achieved toward CXCL12 ligand and HIV coat glycoprotein in physiologically relevant concentrations, without notable sensitivity loss in 100% human serum. The construct is regeneratable by acidic buffer, allowing device reuse and functional tuning. The modular and generalizable architecture behaves similarly to natural systems but gives electrical outputs, which enables fabrication of multiplex sensors with tailored receptor panels for designated diagnostic purposes.

Understanding the role of imidazolium-based ionic liquids in the electrochemical CO2 reduction reaction.

The development of efficient CO2 capture and utilization technologies driven by renewable energy sources is mandatory to reduce the impact of climate change. Herein, seven imidazolium-based ionic liquids (ILs) with different anions and cations were tested as catholytes for the CO2 electrocatalytic reduction to CO over Ag electrode. Relevant activity and stability, but different selectivities for CO2 reduction or the side H2 evolution were observed. Density functional theory results show that depending on the IL anions the CO2 is captured or converted. Acetate anions (being strong Lewis bases) enhance CO2 capture and H2 evolution, while fluorinated anions (being weaker Lewis bases) favour the CO2 electroreduction. Differently from the hydrolytically unstable 1-butyl-3-methylimidazolium tetrafluoroborate, 1-Butyl-3-Methylimidazolium Triflate was the most promising IL, showing the highest Faradaic efficiency to CO (>95%), and up to 8 h of stable operation at high current rates (-20 mA & -60 mA), which opens the way for a prospective process scale-up.

Anti-E. coli Immunoglobulin Yolk (IgY): Reduction of pathogen receptors and inflammation factors could be caused by decrease in E. coli load.

Graft versus host disease (GVHD) remains the major cause of morbidity and mortality after allogeneic stem cell transplantation, especially for intestinal GVHD, as steroid resistant GVHD results in high mortality. For this reason, new treatments of GVHD are needed. One approach is the reduction of pathogenic bacteria using anti-E. coli Immunoglobulin Yolk (IgY). In a haploidentical murine model, B6D2F1 mice conditioned with total body irradiation (TBI), received bone marrow cells (BM) and splenocytes (SC) from either syngeneic (Syn = B6D2F1) or allogeneic (Allo = C57BL/6) donors. Following this, animals received from day -2 until day +28 chow contained IgY or control chow. Thereafter the incidence and severity of aGVHD, the cytokines, chemokines, IDO1 and different pathogen-recognition receptors (PRR) were analyzed and compared to control animals (received chow without IgY). We found that animals receiving chow with IgY antibody showed reduced GVHD severity compared to control animals. On day28 after alloBMT, IDO, NOD2, TLR2, TLR4 and the inflammatory chemokine CCL3, were reduced in the colon and correlated with a significant decrease in E. coli bacteria. In summary chow containing chicken antibodies (IgY) improved GVHD via decrease in bacterial load of E coli conducting to reduction of pathogen receptors (NOD2, TLR2 and 4), IDO, chemokines and cytokines.

A New Osteogenic Membrane to Enhance Bone Healing: At the Crossroads between the Periosteum, the Induced Membrane, and the Diamond Concept.

The lack of viability of massive bone allografts for critical-size bone defect treatment remains a challenge in orthopedic surgery. The literature has reviewed the advantages of a multi-combined treatment with the synergy of an osteoconductive extracellular matrix (ECM), osteogenic stem cells, and growth factors (GFs). Questions are still open about the need for ECM components, the influence of the decellularization process on the latter, the related potential loss of function, and the necessity of using pre-differentiated cells. In order to fill in this gap, a bone allograft surrounded by an osteogenic membrane made of a decellularized collagen matrix from human fascia lata and seeded with periosteal mesenchymal stem cells (PMSCs) was analyzed in terms of de-/recellularization, osteogenic properties, PMSC self-differentiation, and angiogenic potential. While the decellularization processes altered the ECM content differently, the main GF content was decreased in soft tissues but relatively increased in hard bone tissues. The spontaneous osteogenic differentiation was necessarily obtained through contact with a mineralized bone matrix. Trying to deepen the knowledge on the complex matrix-cell interplay could further propel these tissue engineering concepts and lead us to provide the biological elements that allow bone integration in vivo.

Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluation.

INTRODUCTION: Large bone defects are challenging for surgeons. Available reimplanted bone substitutes can't properly restore optimal function along and long term osteointegration of the bone graft. Bone substitute based on the perfusion-decellularization technique seem to be interesting in order to overcome these limitations. We present here an evaluation of the biomechanics of the bones thus obtained. MATERIAL AND METHODS: Two decellularization protocols were chosen for this study. One using Sodium Dodecyl Sulfate (SDS) (D1) and one using NaOH and H2O2 (D2). The decellularization was performed on porcine forearms. We then carried out compression, three-point bending, indentation and screw pull-out tests on each sample. Once these tests were completed, we compared the results obtained between the different decellularization protocols and with samples left native. RESULTS: The difference in the means was similar between the tests performed on bones decellularized with the SDS protocol and native bones for pull-out test: +1.4% (CI95% [-10.5%- 12.4%]) of mean differences when comparing Native vs D1, compression -14.9% (CI95% [-42.7%- 12.5%]), 3-point bending -5.7% (CI95% [-22.5%- 11.1%]) and indentation -10.8% (CI95% [-19.5%- 4.6%]). Bones decellularized with the NaOH protocol showed different results from those obtained with the SDS protocol or native bones during the pull-out screw +40.7% (CI95% [24.3%- 57%]) for Native vs D2 protocol and 3-point bending tests +39.2% (CI95% [13.7%- 64.6%]) for Native vs D2 protocol. The other tests, compression and indentation, gave similar results for all our samples. CONCLUSION: Vascularized decellularized grafts seem to be an interesting means for bone reconstruction. Our study shows that the decellularization method affects the mechanical results of our specimens. Some methods seem to limit these alterations and could be used in the future for bone decellularization.

MIA/CD-RAP Regulates MMP13 and Is a Potential New Disease-Modifying Target for Osteoarthritis Therapy.

Melanoma inhibitory activity/cartilage-derived retinoicacid-sensitive protein (MIA/CD-RAP) is a protein expressed and secreted by chondrocytes and cartilaginous tissues. MIA/CD-RAP-deficient mice develop milder osteoarthritis than wildtype mice. In this study, we investigated MIA/CD-RAP downstream targets to explain this reduced disease development. As a possible mediator, we could detect matrix metalloproteinase 13 (MMP13), and the influence of MIA/CD-RAP on MMP13 regulation was analyzed in vitro using SW1353 chondrosarcoma cells and primary chondrocytes. The femoral head cartilage of WT and MIA/CD-RAP -/- mice were cultured ex vivo to further investigate MMP13 activity. Finally, osteoarthritis was surgically induced via DMM in C57BL/6 mice, and the animals were treated with an MIA/CD-RAP inhibitory peptide by subcutaneously implanted pellets. MMP13 was regulated by MIA/CD-RAP in SW1353 cells, and MIA/CD-RAP -/- murine chondrocytes showed less expression of MMP13. Further, IL-1ß-treated MIA/CD-RAP -/- chondrocytes displayed less MMP13 expression and activity. Additionally, MIA/CD-RAP-deficient ex vivo cultured cartilage explants showed less MMP13 activity as well as reduced cartilage degradation. The mice treated with the MIA/CD-RAP inhibitory peptide showed less osteoarthritis development. Our findings revealed MIA/CD-RAP as a new regulator of MMP13 and highlighted its role as a potential new target for osteoarthritis therapy.

Periosteum and fascia lata: Are they so different?

Introduction: The human fascia lata (HFL) is used widely in reconstructive surgery in indications other than fracture repair. The goal of this study was to compare microscopic, molecular, and mechanical properties of HFL and periosteum (HP) from a bone tissue engineering perspective. Material and Methods: Cadaveric HP and HFL (N = 4 each) microscopic morphology was characterized using histology and immunohistochemistry (IHC), and the extracellular matrix (ECM) ultrastructure assessed by means of scanning electron microscopy (SEM). DNA, collagen, elastin, glycosaminoglycans, major histocompatibility complex Type 1, and bone morphogenetic protein (BMP) contents were quantified. HP (N = 6) and HFL (N = 11) were submitted to stretch tests. Results: Histology and IHC highlighted similarities (Type I collagen fibers and two-layer organization) but also differences (fiber thickness and compaction and cell type) between both tissues, as confirmed using SEM. The collagen content was statistically higher in HFL than HP (735 vs. 160.2 µg/mg dry weight, respectively, p < 0.0001). On the contrary, DNA content was lower in HFL than HP (404.75 vs. 1,102.2 µg/mg dry weight, respectively, p = 0.0032), as was the immunogenic potential (p = 0.0033). BMP-2 and BMP-7 contents did not differ between both tissues (p = 0.132 and p = 0.699, respectively). HFL supported a significantly higher tension stress than HP. Conclusion: HP and HFL display morphological differences, despite their similar molecular ECM components. The stronger stretching resistance of HFL can specifically be explained by its higher collagen content. However, HFL contains many fewer cells and is less immunogenic than HP, as latter is rich in periosteal stem cells. In conclusion, HFL is likely suitable to replace HP architecture to confer a guide for bone consolidation, with an absence of osteogenicity. This study could pave the way to a bio-engineered periosteum built from HFL.

Mid and long-term overall survival after carcinologic resections of thyroid cancer bone metastases.

Background: Bone metastases in thyroid cancer impair the patient's quality of life and prognosis. Interestingly, wide margins resection as the surgical treatment of bone metastases might improve the overall survival (OS). Nonetheless, data are lacking regarding the potential benefits of this strategy. Methods: In order to assess the OS of patients with thyroid cancer after a bone metastases carcinologic resection, a retrospective multicentric study was performed, evaluating the 1, 5, 10 and 15 years-OS along with the potential prognosis associated factors. Results: 40 patients have been included in this multicentric study, with a mean follow-up after surgery of 46.6 ± 58 months. We observed 25 (62.5%) unimestastatic patients and 15 multimetastatic patients (37.5%). The median overall survival after resection was 48 ± 57.3 months. OS at 1, 5, 10, and 15 years was respectively 76.2%, 63.6%, 63.6%, and 31.8%. Survival for patients with a single bone metastasis at 15 year was 82.3%, compared with 0.0% (Log Rank, p = 0.022) for multi-metastatic bone patients. Conclusions: This study advocates for an increased long term 10-year OS in patients with thyroid cancer, after resection of a single bone metastasis, suggesting the benefits of this strategy in this population.

Valence, sensations and appraisals co-occurring with feeling moved: evidence on kama muta theory from intra-individually cross-correlated time series.

Emotional experiences typically labelled "being moved" or "feeling touched" may belong to one universal emotion. This emotion, which has been labelled "kama muta", is hypothesised to have a positive valence, be elicited by sudden intensifications of social closeness, and be accompanied by warmth, goosebumps and tears. Initial evidence on correlations among the kama muta components has been collected with self-reports after or during the emotion. Continuous measures during the emotion seem particularly informative, but previous work allows only restricted inferences on intra-individual processes because time series were cross-correlated across samples. In the current studies, we instead use a within-subject design to replicate and extend prior work. We compute intra-individual cross-correlations between continuous self-reports on feeling moved and (1) positive and negative affect; (2) goosebumps and subjective warmth and (3) appraisals of closeness and morality. Results confirm the predictions of kama muta theory that feeling moved by intensified communal sharing cross-correlates with appraised closeness, positive affect, warmth and (less so) goosebumps, but not with negative affect. Contrary to predictions, appraised morality cross-correlated with feeling moved as much as appraised closeness did. We conclude that strong inferences on emotional processes are possible using continuous measures, replace earlier findings, and are largely in line with theorising.

Quality of resection margin with patient specific instrument for bone tumor resection.

Background: Patient Specific Instruments (PSI) is currently a proven technique for bone tumor resection. In a previous publication, we analyzed the quality of margin resection of pelvic sarcoma resections with the use of PSI (by pathologic evaluation of the margins). In this new study, we compare preoperative resection planning and actual resection margins by MRI analysis of the resection specimens. Methods: Between 2011 and 2020, 31 patients underwent bone tumor resection with the use of PSI. Preoperatively, the margins were planned with a software and PSI were made according to these margins. Postoperatively, the surgical resection specimens were analyzed with MRI. Resection margins were measured with the same software used in the preoperative planning. Results: All margins were safe (free of tumor). The differences between preoperative planned margins and the obtained ones were within the range -5 to +5 mm. The correlation between planned margin and the obtained one was excellent (R2 = 0.841; p < 0.0001). Conclusions: This study demonstrates the accuracy of PSI. In our series, all resection margins were safe. A minimal 5 mm-margin has to be planned but a larger sample is needed to give recommendations.

Investigation of a Fluorine-Free Phosphonium-Based Ionic Liquid Electrolyte and Its Compatibility with Lithium Metal.

A novel fluorine-free ionic liquid electrolyte comprising lithium dicyanamide (LiDCA) and trimethyl(isobutyl)phosphonium tricyanomethanide (P111i4TCM) in a 1:9 molar ratio is studied as an electrolyte for lithium metal batteries. At room temperature, it demonstrates high ionic conductivity and viscosity of about 4.5 mS cm-1 and 64.9 mPa s, respectively, as well as a 4 V electrochemical stability window (ESW). Li stripping/plating tests prove the excellent electrolyte compatibility with Li metal, evidenced by the remarkable cycling stability over 800 cycles. The evolution of the Li-electrolyte interface upon cycling was investigated via electrochemical impedance spectroscopy, displaying a relatively low impedance increase after the initial formation cycles. Finally, the solid electrolyte interphase (SEI) formed on Li metal appeared to have a bilayer structure mostly consisting of DCA and TCM reduction products. Additionally, decomposition products of the phosphonium cation were also detected, despite prior studies reporting its stability against Li metal.

Moved by Social Justice: The Role of Kama Muta in Collective Action Toward Racial Equality.

Participation in collective action is known to be driven by two appraisals of a social situation: Beliefs that the situation is unfair (injustice appraisal) and beliefs that a group can change the situation (collective efficacy appraisal). Anger has been repeatedly found to mediate the relationship between injustice appraisals and collective action. Recent work suggests that the emotion of being moved mediates the relationship between efficacy appraisals and collective action. Building on this prior work, the present research applies kama muta theory to further investigate the relationship between efficacy appraisals and collective action. Kama muta is a positive emotion that is evoked by a sudden intensification of communal sharing, and largely overlaps with the English concept being moved. We investigated its relationship with collective action in both advantaged and disadvantaged racial groups in the context of the Black Lives Matter Movement (BLM) in Spring of 2020. In one pilot study (N = 78) and one main study (N = 215), we confirmed that anger toward the system of racial inequalities mediated between injustice and collective action intentions, and that kama muta toward the movement mediated between collective efficacy and collective action intentions. Both mediations were found for both Black and White participants. We also observed additional unpredicted paths from anger to kama muta and from efficacy to anger. Together, this provides evidence for the pivotal role of emotions in collective action intentions, but also points out that appraisals need to be better understood.

GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.

Hepatocellular carcinoma is the most frequent primary liver cancer. Macroautophagy/autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two hepatocellular carcinoma in vivo models. We showed that due to its lysosomotropic properties, GNS561 could reach and specifically inhibited its enzyme target, PPT1 (palmitoyl-protein thioesterase 1), resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of MTOR (mechanistic target of rapamycin kinase), lysosomal membrane permeabilization, caspase activation and cell death. Accordingly, GNS561, for which a global phase 1b clinical trial in liver cancers was just successfully achieved, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment.Abbreviations: ANXA5:annexin A5; ATCC: American type culture collection; BafA1: bafilomycin A1; BSA: bovine serum albumin; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CCND1: cyclin D1; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; CQ: chloroquine; iCCA: intrahepatic cholangiocarcinoma; DEN: diethylnitrosamine; DMEM: Dulbelcco's modified Eagle medium; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HDSF: hexadecylsulfonylfluoride; IC50: mean half-maximal inhibitory concentration; LAMP: lysosomal associated membrane protein; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3; LMP: lysosomal membrane permeabilization; MALDI: matrix assisted laser desorption ionization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MRI: magnetic resonance imaging; NH4Cl: ammonium chloride; NtBuHA: N-tert-butylhydroxylamine; PARP: poly(ADP-ribose) polymerase; PBS: phosphate-buffered saline; PPT1: palmitoyl-protein thioesterase 1; SD: standard deviation; SEM: standard error mean; vs, versus; Zn2+: zinc ion; Z-Phe: Z-Phe-Tyt(tBu)-diazomethylketone; Z-VAD-FMK: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone.

Compound A Increases Cell Infiltration in Target Organs of Acute Graft-versus-Host Disease (aGVHD) in a Mouse Model.

Systemic steroids are used to treat acute graft-versus-host disease (aGVHD) caused by allogenic bone marrow transplantation (allo-BMT); however, their prolonged use results in complications. Hence, new agents for treating aGVHD are required. Recently, a new compound A (CpdA), with anti-inflammatory activity and reduced side effects compared to steroids, has been identified. Here, we aimed to determine whether CpdA can improve the outcome of aGVHD when administered after transplantation in a mouse model (C57BL/6 in B6D2F1). After conditioning with 9Gy total body irradiation, mice were infused with bone marrow (BM) cells and splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. The animals were subsequently treated (3 days/week) with 7.5 mg/kg CpdA from day +15 to day +28; the controls received 0.9% NaCl. Thereafter, the incidence and severity of aGVHD in aGVHD target organs were analyzed. Survival and clinical scores did not differ significantly; however, CpdA-treated animals showed high cell infiltration in the target organs. In bulk mixed lymphocyte reactions, CpdA treatment reduced the cell proliferation and expression of inflammatory cytokines and chemokines compared to controls, whereas levels of TNF, IL-23, chemokines, and chemokine receptors increased. CpdA significantly reduced proliferation in vitro but increased T cell infiltration in target organs.