Cytological Features of Fibrin-associated Diffuse Large B-cell Lymphoma Arising in Retroperitoneal Pseudocyst: The First Case Report.

BACKGROUND/AIM: Fibrin-associated diffuse large B-cell lymphoma (FA-DLBCL) is frequently associated with the Epstein-Barr virus (EBV) and manifests as non-mass-forming microscopic lesions within fibrin-rich lesions. Herein, we describe the cytological features of FA-DLBCL. CASE REPORT: A 72-year-old man presented with a large retroperitoneal cystic mass that was treated by cyst aspiration and laparoscopic excision. Individually dispersed large, atypical cells in a necrotic background contained scant cytoplasm and hyperchromatic nuclei with irregular nuclear contours, frequent karyorrhectic debris, and mitotic figures. A fibrinous exudate with necrotic material attached to the inner surface of the cystic mass contained large, atypical cells that were individually scattered or arranged in small clusters. These were positive for cluster of differentiation 20 and Epstein-Barr virus-encoded RNA in situ hybridization. CONCLUSION: We cytologically characterized FA-DLBCL as large, atypical cells that were individually scattered or arranged in small clusters in a necrotic background. To the best of our knowledge, we revealed the cytological features of FA-DLBCL.

Genipin crosslinking promotes biomechanical reinforcement and pro-regenerative macrophage polarization in bioartificial tubular substitutes.

Traumatic nerve injuries are nowadays a significant clinical challenge and new substitutes with adequate biological and mechanical properties are in need. In this context, fibrin-agarose hydrogels (FA) have shown the possibility to generate tubular scaffolds with promising results for nerve repair. However, to be clinically viable, these scaffolds need to possess enhanced mechanical properties. In this line, genipin (GP) crosslinking has demonstrated to improve biomechanical properties with good biological properties compared to other crosslinkers. In this study, we evaluated the impact of different GP concentrations (0.05, 0.1 and 0.2% (m/v)) and reaction times (6, 12, 24, 72 h) on bioartificial nerve substitutes (BNS) consisting of nanostructured FA scaffolds. First, crosslinked BNS were studied histologically, ultrastructurally and biomechanically and then, its biocompatibility and immunomodulatory effects were ex vivo assessed with a macrophage cell line. Results showed that GP was able to improve the biomechanical resistance of BNS, which were dependent on both the GP treatment time and concentration without altering the structure. Moreover, biocompatibility analyses on macrophages confirmed high cell viability and a minimal reduction of their metabolic activity by WST-1. In addition, GP-crosslinked BNS effectively directed macrophage polarization from a pro-inflammatory (M1) towards a pro-regenerative (M2) phenotype, which was in line with the cytokines release profile. In conclusion, this study considers time and dose-dependent effects of GP in FA substitutes which exhibited increased biomechanical properties while reducing immunogenicity and promoting pro-regenerative macrophage shift. These tubular substitutes could be useful for nerve application or even other tissue engineering applications such as urethra.

Hydroxytyrosol Alleviates Methotrexate-Induced Pulmonary Fibrosis in Rats: Involvement of TGF-ß1, Tissue Factor, and VEGF.

Methotrexate (MTX) is an indispensable drug used for the treatment of many autoimmune and cancerous diseases. However, its clinical use is associated with serious side effects, such as lung fibrosis. The main objective of this study is to test the hypothesis that hydroxytyrosol (HT) can mitigate MTX-induced lung fibrosis in rats while synergizing MTX anticancer effects. Pulmonary fibrosis was induced in the rats using MTX (14 mg/kg/week, per os (p.o.)). The rats were treated with or without HT (10, 20, and 40 mg/kg/d p.o.) or dexamethasone (DEX; 0.5 mg/kg/d, intraperitoneally (i.p.)) for two weeks concomitantly with MTX. Transforming growth factor beta 1 (TGF-ß1), interleukin-4 (IL-4), thromboxane A2 (TXA2), vascular endothelial growth factor (VEGF), 8-hydroxy-2-deoxy-guanosine (8-OHdG), tissue factor (TF) and fibrin were assessed using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and RT-PCR. Pulmonary fibrosis was manifested by an excessive extracellular matrix (ECM) deposition and a marked increase in TGF-ß1 and IL-4 in lung tissues. Furthermore, cotreatment with HT or dexamethasone (DEX) significantly attenuated MTX-induced ECM deposition, TGF-ß1, and IL-4 expression. Similarly, HT or DEX notably reduced hydroxyproline contents, TXA2, fibrin, and TF expression in lung tissues. Moreover, using HT or DEX downregulated the gene expression of TF. A significant decrease in lung contents of VEGF, IL-8, and 8-OHdG was also observed in HT + MTX- or DEX + MTX -treated animals in a dose-dependent manner. Collectively, the results of our study suggest that HT might represent a potential protective agent against MTX-induced pulmonary fibrosis.

Advanced platelet rich fibrin demonstrates improved osteogenic induction potential in human periodontal ligament cells, growth factor production and mechanical properties as compared to leukocyte and platelet fibrin and injectable platelet rich fibrin.

OBJECTIVES: This cross-sectional invitro research aimed to compare and contrast the macroscopic and microscopic, mechanical and biochemical features of leukocyte-rich platelet-rich fibrin, advanced platelet-rich fibrin, and injectable platelet-rich fibrin. MATERIALS AND METHODS: In all, 150 samples were taken from males aged 18 to 25 with good systemic health (n = 50 each for i-PRF, A-PRF, and L-PRF). The samples were assessed for clot length, clot width, membrane length and width. Microscopic parameters assessed were the distribution of cells and fibrin structure. Mechanical tests were performed for tensile strength using a universal testing machine and growth factor analysis was performed for platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)- ß on Days 1, 3 and 7 using commercially available ELISA kits. The osteogenic potential was analyzed in a culture of human periodontal ligament cells for 21 days using cell viability assay, alkaline phosphatase formation and alizarin red staining for mineralization. RESULTS: L-PRF demonstrates statistically superior clot length, width, weight, membrane length, width and weight in comparison to A-PRF (p < 0.05). L-PRF demonstrates a denser fibrin structure in comparison to A-PRF and i-PRF (p < 0.05). The cells in L-PRF are most commonly situated in the proximal of the clot where as they are distributed in the proximal and middle aspect for A-PRF(p < 0.05). A-PRF demonstrates the highest tensile strength followed by L-PRF (p < 0.05). When growth factor release was evaluated, A-PRF showed noticeably increased release of all growth factors, namely PDGF-BB, TGF-ß, and VEGF, in comparison to i-PRF and L-PRF (p < 0.05). On days 7 and 14, the cell viability of human periodontal ligament cells in co-culture with A-PRF was statistically substantially greater than that of L-PRF and i-PRF (p < 0.05). Alkaline phosphatase levels were statistically substantially higher in A-PRF, followed by i-PRF and L-PRF on days 14 and 21 (p < 0.05). After 21 days of culture, A-PRF treated cultures had much more Alizarin Red staining than L-PRF and i-PRF cultures did (p < 0.05). CONCLUSION: It was determined that although L-PRF exhibits greater size and weight in comparison to A-PRF and i-PRF, A-PRF has superior mechanical properties, increased growth factor releases of TGF-b, PDGF-BB, and VEGF as well as superior cell viability, alkaline phosphatase production, and mineralization on human periodontal ligament cells. CLINICAL RELEVANCE: Based on these findings, A-PRF can be recommended for improved delivery of growth factors and osteogenesis whereas L-PRF is better-suited for applications relying on the size of membrane.

Identification of Neural (N)-Cadherin as a Novel Endothelial Cell Receptor for Fibrin and Localization of the Complementary Binding Sites.

Based on the high structural homology between vascular endothelial (VE)-cadherin and neural (N)-cadherin, we hypothesized that fibrin, which is known to interact with VE-cadherin and promote angiogenesis through this interaction, may also interact with N-cadherin. To test this hypothesis, we prepared fibrin and its plasmin-produced and recombinant fragments covering practically all parts of the fibrin molecule. We also prepared the soluble extracellular portion of N-cadherin (sN-cadherin), which includes all five extracellular N-cadherin domains, and studied its interaction with fibrinogen, fibrin, and the aforementioned fibrin fragments using two independent methods, ELISA and SPR. The experiments confirmed our hypothesis, revealing that fibrin interacts with sN-cadherin with high affinity. Furthermore, the experiments localized the N-cadherin binding site within the fibrin ßN-domains. Notably, the recombinant dimeric (ß15-66)2 fragment, corresponding to these domains and mimicking their dimeric arrangement in fibrin, preserved the N-cadherin-binding properties of fibrin. To localize the fibrin binding site within N-cadherin, we performed ELISA and SPR experiments with (ß15-66)2 and recombinant N-cadherin fragments representing its individual extracellular domains and combinations thereof. The results obtained indicate that the interaction of fibrin with N-cadherin occurs through the third and fifth extracellular domains of the latter. This is in contrast to our previous study, which revealed that fibrin interacts only with the third extracellular domain of VE-cadherin. In conclusion, our study identified N-cadherin as a novel receptor for fibrin and localized complementary binding sites within both fibrin and N-cadherin. The pathophysiological role of this interaction remains to be established.

A New Bioactive Fibrin Formulation Provided Superior Cartilage Regeneration in a Caprine Model.

The effective and long-term treatment of cartilage defects is an unmet need among patients worldwide. In the past, several synthetic and natural biomaterials have been designed to support functional articular cartilage formation. However, they have mostly failed to enhance the terminal stage of chondrogenic differentiation, leading to scar tissue formation after the operation. Growth factors substantially regulate cartilage regeneration by acting on receptors to trigger intracellular signaling and cell recruitment for tissue regeneration. In this study, we investigated the effect of recombinant insulin-like growth factor 1 (rIGF-1), loaded in fibrin microbeads (FibIGF1), on cartilage regeneration. rIGF-1-loaded fibrin microbeads were injected into full-thickness cartilage defects in the knees of goats. The stability, integration, and quality of tissue repair were evaluated at 1 and 6 months by gross morphology, histology, and collagen type II staining. The in vivo results showed that compared to plain fibrin samples, particularly at 6 months, FibIGF1 improved the functional cartilage formation, confirmed through gross morphology, histology, and collagen type II immunostaining. FibIGF1 could be a promising candidate for cartilage repair in the clinic.

Diagnostic and prognostic role of synovial tissue analysis in juvenile idiopathic arthritis: a monocentric study.

OBJECTIVES: This study investigates the diagnostic role of synovial tissue analysis in children presenting with arthritis and assesses its prognostic significance to predict clinical outcome in juvenile idiopathic arthritis (JIA). METHODS: Synovial samples of paediatric patients undergoing synovial biopsy between 1995 and 2020 were analysed histologically and immunohistochemically. Relationships between histological/immunohistochemical parameters and clinical variables were assessed. RESULTS: Synovial biopsy was performed for diagnosis in 65 cases allowing to correctly classify 79% of patients.At histological analysis on 42 JIA samples, any difference in the number of synovial lining layers, subsynovial elementary lesions, fibrin deposit, Krenn Synovitis Score, inflammatory infiltrate score and pattern emerged between JIA subsets or on treatment exposure. Synovial tissue analysis predicted outcome: higher number of synovial layers predicted worse disease course (>4 flares during follow-up; 4.5 vs 3.0, p=0.035), even after adjusting for age at diagnosis and observation time (OR 2.2, p=0.007); subjects who had switched>2 biological disease-modifying antirheumatic drugs had higher prevalence of subsynovial elementary lesions (55.6% vs 10.3%, p=0.005) and fibrin deposits in synovial lining (60.0% vs 22.6%, p=0.049), even after adjustment for observation time and age at diagnosis (OR 8.1, p=0.047). At immunohistochemistry on 31 JIA samples, higher CD3 expression was described in polyarticular compared with oligoarticular subset (p=0.040). Patients with severe disease course had higher CD20+ rate (OR 7, p=0.023), regardless of JIA subset and treatment exposure. CONCLUSIONS: Synovial tissue analysis might support the clinicians in the diagnostic approach of paediatric patients presenting with arthritis and guide the clinical management in JIA.

[Effects of advanced platelet-rich fibrin on deep partial-thickness burn wounds in nude mice].

Objective: To explore the effects of advanced platelet-rich fibrin (A-PRF) on deep partial-thickness burn wounds in nude mice and its mechanism. Methods: The experimental study method was adopted. Forty healthy volunteers in Subei People's Hospital were recruited, including 32 females and 8 males, aged 60 to 72 years. Leukocyte platelet-rich fibrin (L-PRF) and A-PRF membranes were prepared after venous blood was extracted from them. The microstructure of two kinds of platelet-rich fibrin (PRF) membranes was observed by field emission scanning electron microscope. The number of samples was 3 in the following experiments. The L-PRF and A-PRF membranes were divided into L-PRF group and A-PRF group and cultured, and then the release concentrations of platelet-derived growth factor-AB (PDGF-AB) and vascular endothelial growth factor (VEGF) in culture supernatant were determined by enzyme-linked immunosorbent assay on culture day 1, 3, 7, and 14. Mice L929 fibroblasts (Fbs) were divided into L-PRF group and A-PRF group, and cultured with L-PRF or A-PRF conditioned medium, respectively. On culture day 1, 3, and 7, the cell proliferation activity was detected by thiazole blue method. The cell migration rate was detected and calculated at 24 h after scratching by scratch test. Thirty-six male BALB/c nude mice aged 6-8 weeks were selected to make a deep partial-thickness burn wound on one hind leg, and then divided into normal saline group, L-PRF group, and A-PRF group, according to the random number table, with 12 mice in each group. The wounds of nude mice in normal saline group were only washed by normal saline, while the wounds of nude mice in L-PRF group and A-PRF group were covered with the corresponding membranes in addition. The wounds of nude mice in the 3 groups were all bandaged and fixed with dressings. On treatment day 4, 7, and 14, the wound healing was observed and the wound healing rate was calculated. Masson staining was used to observe the new collagen in wound tissue, and immunohistochemical staining was used to detect the percentage of CD31 positive cells in the wound. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, analysis of variance for factorial design, one-way analysis of variance, and least significant difference test. Results: L-PRF membrane's dense network structure was composed of coarse fibrin bundles, with scattered white blood cells and platelets with complete morphology. A-PRF membrane's loose network structure was composed of fine fibrin bundles, with scattered small amount of deformed white blood cells and platelets. On culture day 1, the release concentration of PDGF-AB in PRF culture supernatant in A-PRF group was significantly higher than that in L-PRF group (t=5.73, P<0.05), while the release concentrations of VEGF in PRF culture supernatant in the two groups were similar (P>0.05). On culture day 3, 7, and 14, the release concentrations of PDGF-AB and VEGF in PRF culture supernatant in A-PRF group were significantly higher than those in L-PRF group (with t values of 6.93, 7.45, 5.49, 6.97, 8.97, and 13.64, respectively, P<0.05). On culture day 3, 7, and 14, the release concentrations of PDGF-AB and VEGF in PRF culture supernatant in the two groups were all significantly higher than those in the previous time points within the group (P<0.05). On culture day 1, 3, and 7, the proliferation activity of mice Fbs in A-PRF group was 0.293±0.034, 0.582±0.054, and 0.775±0.040, respectively, which were significantly stronger than 0.117±0.013, 0.390±0.036, and 0.581±0.037 in L-PRF group (with t values of 8.38, 5.14, and 6.16, respectively, P<0.05). At 24 h after scratching, the migration rate of mice Fbs in A-PRF group was (60.9±2.2)%, which was significantly higher than (39.1±2.3)% in L-PRF group (t=11.74, P<0.05). On treatment day 4, the wound exudates of nude mice in L-PRF group and A-PRF group were less with no obvious signs of infection, while the wounds of nude mice in normal saline group showed more exudation. On treatment day 7, the wounds of nude mice in L-PRF group and A-PRF group were dry and crusted, while there was still a small amount of exudate in the wounds of nude mice in normal saline group. On treatment day 14, the wounds of nude mice in A-PRF group tended to heal; a small portion of wounds remained in nude mice in L-PRF group; the wound of nude mice was still covered with eschar in normal saline group. On treatment day 4, 7, and 14, the wound healing rate and percentage of CD31 positive cells of nude mice in L-PRF group were all significantly higher than those in normal saline group (P<0.05); compared with those in normal saline group and L-PRF group, the wound healing rate of nude mice in A-PRF group was significantly increased (P<0.05), the newborn collagen was orderly and evenly distributed, with no excessive deposition, and the percentage of CD31 positive cells was significantly increased (P<0.05). Conclusions: The stable fibrin network structure of A-PRF can maintain the sustained release of growth factors, accelerate cell proliferation, and promote cell migration, so as to shorten the healing time and improve the healing quality of deep partial-thickness burn wounds in nude mice.

Bone marrow concentration in combination with hyaluronan and fibrin for the treatment of primary and non-primary in orthopaedic cases of osteochondral defects of the ankle.

This research examined the effects of varying amounts of fibrinogen (the fibrin precursor) and HA-MA on mechanical strength, BMSC proliferation, and chondrogenesis potential in vitro. In order to start a culture, fibrinogen, aprotinin, doxycycline, and HA-MA were well mixed in 200 L of AMEM containing 1% (w/v) photoinitiator. In order to produce a fibrin gel, thrombin was used at 1, 3,5, and 7 days after implantation, live/dead staining and a metabolic-activity test were used to examine the viability and proliferation of BMSCs inside fibrin/HA-MA. The cell lysis solution from the Real Time ready Cell Lysis Kit was added to each gel. Both primer and probe mixes, as well as DNA polymerase, deoxyribonucleotide triphosphates, an activator, and an enhancer, were mixed before the lysis process began to asses mRNA expression. The mechanical strength of fibrin hydrogels was shown to be proportional to the quantity of HA-MA utilised in the reinforcement. Quantitative polymerase chain reaction demonstrated a reduction in the expression of collagen type 1 alpha 1 mRNA in BMSCs after they were treated in a fibrin/HA-MA hydrogel. Using fibrin/HA-MA hydrogel as a delivery technique for bone marrow stem cells may induce BMSC differentiation into chondrocytes and perhaps aid in articular cartilage repair for OA therapy (BMSCs). We concluded that the utilisation of bone marrow concentration in conjunction with a combination of fibrin and Hyaluronan treatment is safe for patients suffering from OCD of the ankle and is well tolerated by these patients as both a primary treatment and a non-primary treatment option.

Antimicrobial Effects of Mouse Adipose-Derived Mesenchymal Stem Cells Encapsulated in Collagen-Fibrin Hydrogel Scaffolds on Bacteroides fragilis Wound Infection in vivo.

Background: Anaerobes are the causative agents of many wound infections. B. fragilis is the most prevalent endogenous anaerobic bacterium causes a wide range of diseases, including wound infections. This study aimed to assess the antibacterial effect of mouse adipocyte derived-mesenchymal stem cell (AD-MSCs) encapsulated in collagen-fibrin (CF) hydrogel scaffolds on B. fragilis wound infection in an animal model. Methods: Stem cells were extracted from mouse adipose tissue and confirmed by surface markers using flow cytometry analysis. The possibility of differentiation of stem cells into osteoblast and adipocyte cells was also assessed. The extracted stem cells were encapsulated in the CF scaffold. B. fragilis wound infection was induced in rats, and then following 24 h, collagen and fibrin-encapsulated mesenchymal stem cells (MSCs) were applied to dress the wound. One week later, a standard colony count test monitored the bacterial load in the infected rats. Results: MSCs were characterized as positive for CD44, CD90, and CD105 markers and negative for CD34, which were able to differentiate into osteoblast and adipocyte cells. AD-MSCs encapsulated with collagen and fibrin scaffolds showed ameliorating effects on B. fragilis wound infection. Additionally, AD-MSCs with a collagen scaffold (54 CFU/g) indicated a greater effect on wound infection than AD-MSCs with a fibrin scaffold (97 CFU/g). The combined CF scaffold demonstrated the highest reduction in colony count (the bacteria load down to 29 CFU/g) in the wound infection. Conclusion: Our findings reveal that the use of collagen and fibrin scaffold in combination with mouse AD-MSCs is a promising alternative treatment for B. fragilis.

Immunohistochemical evaluation of fibrin/fibrinogen, d-dimers, and intravascular thrombosis in brains of dogs with meningoencephalitis of unknown origin.

Granulomatous meningoencephalitis (GME) and necrotizing encephalitides (NE) are the most common immune-mediated inflammatory diseases of the central nervous system in dogs. Activation of the fibrinolytic system in multiple sclerosis, a similar immune-mediated disease affecting the central nervous system in humans, seems to be related to disease progression. The aim of this study was to identify fibrin/fibrinogen and D-dimer deposition, as well as presence of intravascular thrombosis (IVT) in brains of dogs with a diagnosis of GME or NE. Immunohistochemical studies using antibodies against fibrin/fibrinogen and D-dimers were performed. Statistical analyses were performed to determine whether there were differences in the presence and location of fibrin/fibrinogen, D-dimers deposits, and IVT between GME and NE. Samples from sixty-four dogs were included in the study: 32 with a diagnosis of GME and 32 with a diagnosis of NE. Fibrin/fibrinogen depositions were detected in all samples and d-dimers were detected in 43/64 samples. IVT was present in 29/64 samples, with a significantly higher score in samples from dogs with NE than in samples from dogs with GME (P = 0.001). These data support hemostatic system activation in both diseases, especially NE. This finding might be related to the origin of the necrotic lesions seen in NE, which could represent chronic ischemic lesions. Further studies are needed to investigate the association between vascular lesions and the histopathological differences between GME and NE and the hemostatic system as a potential therapeutic target.

Plasma fibrin membranes loaded with bone marrow mesenchymal stem cells and corneal epithelial cells promote corneal injury healing via attenuating inflammation and fibrosis after corneal burns.

The shortage of corneal donors has prompted the development of tissue-engineered corneal grafts as an alternative solution. Currently, amniotic membranes with good biocompatibility are widely used as scaffolds for loading stem cells in the treatment of corneal injury. However, this approach has its limitations. In this study, BMSCs were induced to differentiate into corneal epithelial cells via direct contact co-culture, and platelet-poor plasma was used to prepare fibrin gels, which were compressed to remove excess liquid and then lyophilized to obtain plasma fibrin membranes (PFMs). A tissue-engineered corneal implant with PFMs as a scaffold loaded with BMSCs and corneal epithelial cells was designed and obtained. Scanning electron microscopy showed that PFMs have a uniformly distributed microporous surface that facilitates cell attachment and nutrient transport. The rheological results showed that the freeze-dried and rehydrated PFMs were more rigid than fresh membranes, which makes it easier to use them for transplantation after cell loading. The experimental results of a rat alkali burn cornea injury model showed that PFMs effectively reduced the inflammatory reaction, inhibited fibrosis, and accelerated the healing of corneal wounds. It was also found that some of the BMSCs were successfully implanted into the corneal injury site in rats and differentiated into corneal epithelial cells. These results demonstrate the potential of tissue-engineered corneal implants using BMSCs and corneal epithelial cells and PFMs as scaffolds as a new treatment option for corneal injury.

Intersection of Coagulation and Fibrinolysis by the Glycosylphosphatidylinositol (GPI)-Anchored Serine Protease Testisin.

Hemostasis is a delicate balance between coagulation and fibrinolysis that regulates the formation and removal of fibrin, respectively. Positive and negative feedback loops and crosstalk between coagulation and fibrinolytic serine proteases maintain the hemostatic balance to prevent both excessive bleeding and thrombosis. Here, we identify a novel role for the glycosylphosphatidylinositol (GPI)-anchored serine protease testisin in the regulation of pericellular hemostasis. Using in vitro cell-based fibrin generation assays, we found that the expression of catalytically active testisin on the cell surface accelerates thrombin-dependent fibrin polymerization, and intriguingly, that it subsequently promotes accelerated fibrinolysis. We find that the testisin-dependent fibrin formation is inhibited by rivaroxaban, a specific inhibitor of the central prothrombin-activating serine protease factor Xa (FXa), demonstrating that cell-surface testisin acts upstream of factor X (FX) to promote fibrin formation at the cell surface. Unexpectedly, testisin was also found to accelerate fibrinolysis by stimulating the plasmin-dependent degradation of fibrin and enhancing plasmin-dependent cell invasion through polymerized fibrin. Testisin was not a direct activator of plasminogen, but it is able to induce zymogen cleavage and the activation of pro-urokinase plasminogen activator (pro-uPA), which converts plasminogen to plasmin. These data identify a new proteolytic component that can regulate pericellular hemostatic cascades at the cell surface, which has implications for angiogenesis, cancer biology, and male fertility.

Role of SHP2 (PTPN11) in glycoprotein VI-dependent thrombus formation: Improved platelet responsiveness by the allosteric drug SHP099 in Noonan syndrome patients.

INTRODUCTION: The protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. MATERIALS AND METHODS: Washed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. RESULTS: Pharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbß3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11-mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. CONCLUSION: Pharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients.

Macrophages-derived Factor XIII links coagulation to inflammation in COPD.

Background: The local, extravascular, activation of the coagulation system in response to injury is a key factor mediating the resulting inflammatory response. Coagulation Factor XIIIA (FXIIIA) found in alveolar macrophages (AM) and dendritic cells (DC), by influencing fibrin stability, might be an inflammatory modifier in COPD. Aims: To study the expression of FXIIIA in AM and Langerin+DC (DC-1) and their relation to the inflammatory response and disease progression in COPD. Methods: In 47 surgical lungs, 36 from smokers (22 COPD and 14 no-COPD) and 11 from non-smokers we quantified by immunohistochemistry FXIIIA expression in AM and DC-1 along with numbers of CD8+Tcells and CXCR3 expression in lung parenchyma and airways. Lung function was measured prior to surgery. Results: The percentage of AM expressing FXIII (%FXIII+AM) was higher in COPD than no-COPD and non-smokers. DC-1 expressed FXIIIA and their numbers were higher in COPD than no-COPD and non-smokers. DC-1 positively correlated with %FXIII+AM (r=0.43; p<0.018). CD8+Tcells, which were higher in COPD than in no-COPD, were correlated with DC-1 (p<0.01) and %FXIII+AM. CXCR3+ cells were increased in COPD and correlated with %FXIII+AM (p<0.05). Both %FXIII+AM (r=-0.6; p=0.001) and DC-1 (r=-0.7; p=0.001) correlated inversely with FEV1. Conclusion: FXIIIA, an important link between the extravascular coagulation cascade and inflammatory response, is significantly expressed in alveolar macrophages and dendritic cells of smokers with COPD, suggesting that it could play an important role in the adaptive inflammatory reaction characteristic of the disease.

Altered fibrinogen γ-chain cross-linking in mutant fibrinogen-γΔ5 mice drives acute liver injury.

BACKGROUND: Hepatic deposition of cross-linked fibrin(ogen) occurs alongside platelet accumulation as a hallmark of acetaminophen (APAP)-induced liver injury. OBJECTIVES: We sought to define the precise role of the fibrinogen γ-chain C-terminal integrin αIIbß3 binding domain in APAP-induced liver injury. METHODS: Mice expressing mutant fibrinogen incapable of engaging integrin αIIbß3 due to a C-terminal fibrinogen γ-chain truncation (mutant fibrinogen-γΔ5 [FibγΔ5] mice) and wild-type mice were challenged with APAP (300 mg/kg, intraperitoneally). RESULTS: We observed an altered pattern of fibrin(ogen) deposition in the livers of APAP-challenged FibγΔ5 mice. This led to the unexpected discovery that fibrinogen γ-chain cross-linking was altered in the livers of APAP-challenged FibγΔ5 mice compared with that in wild-type mice, including absence of γ-γ dimer and accumulation of larger molecular weight cross-linked γ-chain complexes. This finding was not unique to the injured liver because activation of coagulation did not produce γ-γ dimer in plasma from FibγΔ5 mice or purified FibγΔ5 fibrinogen. Sanger sequencing predicted that the fibrinogen-γΔ5 γ-polypeptide would terminate at lysine residue 406, but liquid chromatography tandem mass spectrometry analysis revealed that this critical lysine residue was absent in purified fibrinogen-γΔ5 protein. Interestingly, hepatic deposition of this uniquely aberrantly cross-linked fibrin(ogen) in FibγΔ5 mice was associated with exacerbated hepatic injury, an effect not recapitulated by pharmacologic inhibition of integrin αIIbß3. CONCLUSION: The results indicate that fibrinogen-γΔ5 lacks critical residues essential to form γ-γ dimer in response to thrombin and suggest that hepatic accumulation of abnormally cross-linked fibrin(ogen) can exacerbate hepatic injury.

Preservation of critical quality attributes of mesenchymal stromal cells in 3D bioprinted structures by using natural hydrogel scaffolds.

Three dimensional (3D) bioprinting is an emerging technology that enables complex spatial modeling of cell-based tissue engineering products, whose therapeutic potential in regenerative medicine is enormous. However, its success largely depends on the definition of a bioprintable zone, which is specific for each combination of cell-loaded hydrogels (or bioinks) and scaffolds, matching the mechanical and biological characteristics of the target tissue to be repaired. Therefore proper adjustment of the bioink formulation requires a compromise between: (i) the maintenance of cellular critical quality attributes (CQA) within a defined range of specifications to cell component, and (ii) the mechanical characteristics of the printed tissue to biofabricate. Herein, we investigated the advantages of using natural hydrogel-based bioinks to preserve the most relevant CQA in bone tissue regeneration applications, particularly focusing on cell viability and osteogenic potential of multipotent mesenchymal stromal cells (MSCs) displaying tripotency in vitro, and a phenotypic profile of 99.9% CD105+ /CD45,- 10.3% HLA-DR,+ 100.0% CD90,+ and 99.2% CD73+ /CD31- expression. Remarkably, hyaluronic acid, fibrin, and gelatin allowed for optimal recovery of viable cells, while preserving MSC's proliferation capacity and osteogenic potency in vitro. This was achieved by providing a 3D structure with a compression module below 8.8 ± 0.5 kPa, given that higher values resulted in cell loss by mechanical stress. Beyond the biocompatibility of naturally occurring polymers, our results highlight the enhanced protection on CQA exerted by bioinks of natural origin (preferably HA, gelatin, and fibrin) on MSC, bone marrow during the 3D bioprinting process, reducing shear stress and offering structural support for proliferation and osteogenic differentiation.

Antibodies against Noncatalytic B Subunit of Factor XIII Inhibit Activation of Factor XIII and Fibrin Crosslinking.

BACKGROUND: Coagulation factor XIII (FXIII) is a proenzyme of plasma transglutaminase. It comprises two catalytic A subunits (FXIII-A) and two carrier B subunits (FXIII-B). We previously reported that alloantibodies against FXIII-B could promote FXIII clearance in a patient with congenital FXIII-B deficiency who had received infusions of plasma-derived human FXIII (A2B2 heterotetramer). OBJECTIVES: We aimed to investigate whether anti-FXIII-B antibodies affect the catalytic function of FXIII. METHODS: FXIII activation and fibrin crosslinking were examined in the presence of patient plasma, isolated patient IgG, or rat anti-FXIII-B monoclonal antibodies. RESULTS: Alloantibody levels were increased by repeated infusions of plasma-derived A2B2 heterotetramer, which enhanced binding to the functionally important FXIII-B sushi domains. The patient plasma strongly inhibited cleavage of the FXIII-A activation peptide, amine incorporation, and fibrin crosslinking in normal plasma. Furthermore, anti-FXIII-B alloantibodies blocked the formation of the complex of FXIII-B with FXIII-A, and fibrinogen. Rat monoclonal antibodies against the 10th sushi domain of FXIII-B inhibited the incorporation of FXIII-B to fibrin, FXIII activation (i.e., cleavage of FXIII-A activation peptide), and ultimately fibrin crosslinking in normal plasma, independent of their effect on heterotetramer assembly with FXIII-A. Alloantibody binding to the A2B2 heterotetramer blocked the access of thrombin to the FXIII-A cleavage site, as indicated by the reaction of the alloantibodies to the A2B2 heterotetramer and FXIII-B, but not to FXIII-A. CONCLUSION: Anti-FXIII-B antibodies binding to the A2B2 heterotetramer and FXIII-B inhibited FXIII activation and its crosslinking function despite being directed against its noncatalytic subunit (FXIII-B).

Plasmin and plasminogen prevent sepsis severity by reducing neutrophil extracellular traps and systemic inflammation.

Sepsis is a lethal syndrome characterized by systemic inflammation and abnormal coagulation. Despite therapeutic advances, sepsis mortality remains substantially high. Herein, we investigated the role of the plasminogen/plasmin (Plg/Pla) system during sepsis. Plasma levels of Plg were significantly lower in mice subjected to severe compared with nonsevere sepsis, whereas systemic levels of IL-6, a marker of sepsis severity, were higher in severe sepsis. Plg levels correlated negatively with IL-6 in both septic mice and patients, whereas plasminogen activator inhibitor-1 levels correlated positively with IL-6. Plg deficiency render mice susceptible to nonsevere sepsis induced by cecal ligation and puncture (CLP), resulting in greater numbers of neutrophils and M1 macrophages, liver fibrin(ogen) deposition, lower efferocytosis, and increased IL-6 and neutrophil extracellular trap (NET) release associated with organ damage. Conversely, inflammatory features, fibrin(ogen), and organ damage were substantially reduced, and efferocytosis was increased by exogenous Pla given during CLP- and LPS-induced endotoxemia. Plg or Pla protected mice from sepsis-induced lethality and enhanced the protective effect of antibiotics. Mechanistically, Plg/Pla-afforded protection was associated with regulation of NET release, requiring Pla-protease activity and lysine binding sites. Plg/Pla are important host-protective players during sepsis, controlling local and systemic inflammation and collateral organ damage.

Magnetic microrheometry of tumor-relevant stiffness levels and probabilistic quantification of viscoelasticity differences inside 3D cell culture matrices.

The progression of breast cancer involves cancer-cell invasions of extracellular matrices. To investigate the progression, 3D cell cultures are widely used along with different types of matrices. Currently, the matrices are often characterized using parallel-plate rheometry for matrix viscoelasticity, or liquid-like viscous and stiffness-related elastic characteristics. The characterization reveals averaged information and sample-to-sample variation, yet, it neglects internal heterogeneity within matrices, experienced by cancer cells in 3D culture. Techniques using optical tweezers and magnetic microrheometry have measured heterogeneity in viscoelasticity in 3D culture. However, there is a lack of probabilistic heterogeneity quantification and cell-size-relevant, microscale-viscoelasticity measurements at breast-tumor tissue stiffness up to ≃10 kPa in Young's modulus. Here, we have advanced methods, for the purpose, which use a magnetic microrheometer that applies forces on magnetic spheres within matrices, and detects the spheres displacements. We present probabilistic heterogeneity quantification using microscale-viscoelasticity measurements in 3D culture matrices at breast-tumor-relevant stiffness levels. Bayesian multilevel modeling was employed to distinguish heterogeneity in viscoelasticity from the effects of experimental design and measurement errors. We report about the heterogeneity of breast-tumor-relevant agarose, GrowDex, GrowDex-collagen and fibrin matrices. The degree of heterogeneity differs for stiffness, and phase angle (i.e. ratio between viscous and elastic characteristics). Concerning stiffness, agarose and GrowDex show the lowest and highest heterogeneity, respectively. Concerning phase angle, fibrin and GrowDex-collagen present the lowest and the highest heterogeneity, respectively. While this heterogeneity information involves softer matrices, probed by ≃30 µm magnetic spheres, we employ larger ≃100 µm spheres to increase magnetic forces and acquire a sufficient displacement signal-to-noise ratio in stiffer matrices. Thus, we show pointwise microscale viscoelasticity measurements within agarose matrices up to Young's moduli of 10 kPa. These results establish methods that combine magnetic microrheometry and Bayesian multilevel modeling for enhanced heterogeneity analysis within 3D culture matrices.