Evolution toward beta common chain receptor usage links the matrix proteins of HIV-1 and its ancestors to human erythropoietin.

The HIV-1 matrix protein p17 (p17) is a pleiotropic molecule impacting on different cell types. Its interaction with many cellular proteins underlines the importance of the viral protein as a major determinant of human specific adaptation. We previously showed the proangiogenic capability of p17. Here, by integrating functional analysis and receptor binding, we identify a functional epitope that displays molecular mimicry with human erythropoietin (EPO) and promotes angiogenesis through common beta chain receptor (ßCR) activation. The functional EPO-like epitope was found to be present in the matrix protein of HIV-1 ancestors SIV originated in chimpanzees (SIVcpz) and gorillas (SIVgor) but not in that of HIV-2 and its ancestor SIVsmm from sooty mangabeys. According to biological data, evolution of the EPO-like epitope showed a clear differentiation between HIV-1/SIVcpz-gor and HIV-2/SIVsmm branches, thus highlighting this epitope on p17 as a divergent signature discriminating HIV-1 and HIV-2 ancestors. P17 is known to enhance HIV-1 replication. Similarly to other ßCR ligands, p17 is capable of attracting and activating HIV-1 target cells and promoting a proinflammatory microenvironment. Thus, it is tempting to speculate that acquisition of an epitope on the matrix proteins of HIV-1 ancestors capable of triggering ßCR may have represented a critical step to enhance viral aggressiveness and early human-to-human SIVcpz/gor dissemination. The hypothesis that the p17/ßCR interaction and ßCR abnormal stimulation may also play a role in sustaining chronic activation and inflammation, thus marking the difference between HIV-1 and HIV-2 in term of pathogenicity, needs further investigation.

mCRP-Induced Focal Adhesion Kinase-Dependent Monocyte Aggregation and M1 Polarization, Which Was Partially Blocked by the C10M Inhibitor.

Monomeric C-reactive protein (mCRP) has recently been implicated in the abnormal vascular activation associated with development of atherosclerosis, but it may act more specifically through mechanisms perpetuating damaged vessel inflammation and subsequent aggregation and internalization of resident macrophages. Whilst the direct effects of mCRP on endothelial cells have been characterized, the interaction with blood monocytes has, to our knowledge, not been fully defined. Here we showed that mCRP caused a strong aggregation of both U937 cell line and primary peripheral blood monocytes (PBMs) obtained from healthy donors. Moreover, this increase in clustering was dependent on focal adhesion kinase (FAK) activation (blocked by a specific inhibitor), as was the concomitant adhesive attachment to the plate, which was suggestive of macrophage differentiation. Confocal microscopy confirmed the increased expression and nuclear localization of p-FAK, and cell surface marker expression associated with M1 macrophage polarization (CD11b, CD14, and CD80, as well as iNOS) in the presence of mCRP. Inclusion of a specific CRP dissociation/mCRP inhibitor (C10M) effectively inhibited PBMs clustering, as well as abrogating p-FAK expression, and partially reduced the expression of markers associated with M1 macrophage differentiation. mCRP also increased the secretion of pro-inflammatory cytokines Interleukin-8 (IL-8) and Interleukin-1ß (IL-1ß), without notably affecting MAP kinase signaling pathways; inclusion of C10M did not perturb or modify these effects. In conclusion, mCRP modulates PBMs through a mechanism that involves FAK and results in cell clustering and adhesion concomitant with changes consistent with M1 phenotypical polarization. C10M has potential therapeutic utility in blocking the primary interaction of mCRP with the cells-for example, by protecting against monocyte accumulation and residence at damaged vessels that may be predisposed to plaque development and atherosclerosis.

Micro-Fragmented Adipose Tissue as a Natural Scaffold for Targeted Drug Delivery in Brain Cancer.

Major limitations in the effective treatment of neurological cancer include systemic cytotoxicity of chemotherapy, inaccessibility, and inoperability. The capability to successfully target a drug to the tumor site(s) without incurring serious side effects-especially in the case of aggressive tumors, such as glioblastoma and neuroblastoma-would represent a significant breakthrough in therapy. Orthotopic systems, capable of storing and releasing proteins over a prolonged period at the site of a tumor, that utilize nanoparticles, liposomes, and hydrogels have been proposed. One candidate for drug delivery is Micro-Fragmented Adipose Tissue (MFAT). Easily obtained from the patient by abdominal subcutaneous liposuction (autologous), and with a high content of Mesenchymal Stem Cells (MSCs), mechanically derived nanofat is a natural tissue graft with a structural scaffold organization. It has a well-preserved stromal vascular fraction and a prolonged capacity to secrete anti-tumorigenic concentrations of pre-absorbed chemotherapeutics within extracellular vesicles. This review discusses current evidence supporting the potential of drug-modified MFAT for the treatment of neurological cancer with respect to recent preclinical and in vitro studies. Possible limitations and future perspectives are considered.

Astrocyte Involvement in Blood-Brain Barrier Function: A Critical Update Highlighting Novel, Complex, Neurovascular Interactions.

Neurological disorders have been linked to a defective blood-brain barrier (BBB), with dysfunctions triggered by stage-specific disease mechanisms, some of these being generated through interactions in the neurovascular unit (NVU). Advanced knowledge of molecular and signaling mechanisms in the NVU and the emergence of improved experimental models allow BBB permeability prediction and the development of new brain-targeted therapies. As NVU constituents, astrocytes are the most numerous glial cells, characterized by a heterogeneity that occurs as a result of developmental and context-based gene expression profiles and the differential expression of non-coding ribonucleic acids (RNAs). Due to their heterogeneity and dynamic responses to different signals, astrocytes may have a beneficial or detrimental role in the BBB's barrier function, with deep effects on the pathophysiology of (and on the progression of) central nervous system diseases. The implication of astrocytic-derived extracellular vesicles in pathological mechanisms, due to their ability to pass the BBB, must also be considered. The molecular mechanisms of astrocytes' interaction with endothelial cells at the BBB level are considered promising therapeutic targets in different neurological conditions. Nevertheless, a personalized and well-founded approach must be addressed, due to the temporal and spatial heterogeneity of reactive astrogliosis states during disease.

GPR43 regulates sodium butyrate-induced angiogenesis and matrix remodeling.

Butyrate is a short-chain fatty acid (SCFA) derived from microbiota and is involved in a range of cell processes in a concentration-dependent manner. Low concentrations of sodium butyrate (NaBu) were shown to be proangiogenic. However, the mechanisms associated with these effects are not yet fully known. Here, we investigated the contribution of the SCFA receptor GPR43 in the proangiogenic effects of local treatment with NaBu and its effects on matrix remodeling using the sponge-induced fibrovascular tissue model in mice lacking the Gpr43 gene (Gpr43-KO) and the wild-type (WT) mice. We demonstrated that NaBu (0.2 mM intraimplant) treatment enhanced the neovascularization process, blood flow, and VEGF levels in a GPR43-dependent manner in the implants. Moreover, NaBu was able to modulate matrix remodeling aspects of the granulation tissue such as proteoglycan production, collagen deposition, and α-smooth muscle actin (α-SMA) expression in vivo, besides increasing transforming growth factor (TGF)-ß1 levels in the fibrovascular tissue, in a GPR43-dependent manner. Interestingly, NaBu directly stimulated L929 murine fibroblast migration and TGF-ß1 and collagen production in vitro. GPR43 was found to be expressed in human dermal fibroblasts, myofibroblasts, and endothelial cells. Overall, our findings evidence that the metabolite-sensing receptor GPR43 contributes to the effects of low dose of NaBu in inducing angiogenesis and matrix remodeling during granulation tissue formation. These data provide important insights for the proposition of new therapeutic approaches based on NaBu, beyond the highly explored intestinal, anti-inflammatory, and anticancer purposes, as a local treatment to improve tissue repair, particularly, by modulating granulation tissue components.NEW & NOTEWORTHY Our data show the contribution of the metabolite-sensing receptor GPR43 in the effects of low dose of sodium butyrate (NaBu) on stimulating angiogenesis and extracellular matrix remodeling in a model of granulation tissue formation in mice. We also show that human dermal fibroblasts, myofibroblasts, and endothelial cells express the receptor GPR43. These data provide important insights for the use of NaBu in local therapeutic approaches applicable to tissue repair in sites other than the intestine.

Changes in muscle-tendon unit length-force characteristics following experimentally induced photothrombotic stroke cannot be explained by changes in muscle belly structure.

PURPOSE: The aim of this study was to assess the effects of experimentally induced photothrombotic stroke on structural and mechanical properties of rat m. flexor carpi ulnaris. METHODS: Two groups of Young-adult male Sprague-Dawley rats were measured: stroke (n = 9) and control (n = 7). Photothrombotic stroke was induced in the forelimb region of the primary sensorimotor cortex. Four weeks later, muscle-tendon unit and muscle belly length-force characteristics of the m. flexor carpi ulnaris, mechanical interaction with the neighbouring m. palmaris longus, the number of sarcomeres in series within muscle fibres, and the physiological cross-sectional area were measured. RESULTS: Stroke resulted in higher force and stiffness of the m. flexor carpi ulnaris at optimum muscle-tendon unit length, but only for the passive conditions. Stroke did not alter the length-force characteristics of m. flexor carpi ulnaris muscle belly, morphological characteristics, and the extent of mechanical interaction with m. palmaris longus muscle. CONCLUSION: The higher passive force and passive stiffness at the muscle-tendon unit level in the absence of changes in structural and mechanical characteristics of the muscle belly indicates that the experimentally induced stroke resulted in an increased stiffness of the tendon.

Characterisation of Novel Angiogenic and Potent Anti-Inflammatory Effects of Micro-Fragmented Adipose Tissue.

Adipose tissue and more specifically micro-fragmented adipose tissue (MFAT) obtained from liposuction has recently been shown to possess interesting medicinal properties whereby its application supports pain reduction and may enhance tissue regeneration particularly in osteoarthritis. Here we have characterised samples of MFAT produced using the Lipogems® International Spa system from eight volunteer individuals in order to understand the critical biological mechanisms through which they act. A variation was found in the MFAT cluster size between individual samples and this translated into a similar variation in the ability of purified mesenchymal stem cells (MSCs) to form colony-forming units. Almost all of the isolated cells were CD105/CD90/CD45+ indicating stemness. An analysis of the secretions of cytokines from MFAT samples in a culture using targeted arrays and an enzyme-linked immunosorbent assay (ELISA) showed a long-term specific and significant expression of proteins associated with anti-inflammation (e.g., interleukin-1 receptor alpha (Il-1Rα) antagonist), pro-regeneration (e.g., hepatocyte growth factor), anti-scarring and pro-angiogenesis (e.g., transforming growth factor beta 1 and 2 (TGFß1/2) and anti-bacterial (e.g., chemokine C-X-C motif ligand-9 (CXCL-9). Angiogenesis and angiogenic signalling were notably increased in primary bovine aortic endothelial cells (BAEC) to a different extent in each individual sample of the conditioned medium whilst a direct capacity of the conditioned medium to block inflammation induced by lipopolysaccharides was shown. This work characterises the biological mechanisms through which a strong, long-lasting, and potentially beneficial effect can be observed regarding pain reduction, protection and regeneration in osteoarthritic joints treated with MFAT.

Sex Comparison of Knee Extensor Size, Strength, and Fatigue Adaptation to Sprint Interval Training.

ABSTRACT: Bagley, L, Al-Shanti, N, Bradburn, S, Baig, O, Slevin, M, and McPhee, JS. Sex comparison of knee extensor size, strength, and fatigue adaptation to sprint interval training. J Strength Cond Res 35(1): 64-71, 2021-Regular sprint interval training (SIT) improves whole-body aerobic capacity and muscle oxidative potential, but very little is known about knee extensor anabolic or fatigue resistance adaptations, or whether effects are similar for men and women. The purpose of this study was to compare sex-related differences in knee extensor size, torque-velocity relationship, and fatigability adaptations to 12-week SIT. Sixteen men and 15 women (mean [SEM] age: 41 [±2.5] years) completed measurements of total body composition assessed by dual energy X-ray absorptiometry, quadriceps muscle cross-sectional area (CSAQ) assessed by magnetic resonance imaging, the knee extensor torque-velocity relationship (covering 0-240°·s-1) and fatigue resistance, which was measured as the decline in torque from the first to the last of 60 repeated concentric knee extensions performed at 180°·s-1. Sprint interval training consisted of 4 × 20-second sprints on a cycle ergometer set at an initial power output of 175% of power at V̇o2max, 3 times per week for 12 weeks. Quadriceps muscle cross-sectional area increased by 5% (p = 0.023) and fatigue resistance improved 4.8% (p = 0.048), with no sex differences in these adaptations (sex comparisons: p = 0.140 and p = 0.282, respectively). Knee extensor isometric and concentric torque was unaffected by SIT in both men and women (p > 0.05 for all velocities). Twelve-week SIT, totaling 4 minutes of very intense cycling per week, significantly increased fatigue resistance and CSAQ similarly in men and women, but did not significantly increase torque in men or women. These results suggest that SIT is a time-effective training modality for men and women to increase leg muscle size and fatigue resistance.

Prevalence and management of diabetic neuropathy in secondary care in Qatar.

AIMS: Diabetic neuropathy (DN) is a "Cinderella" complication, particularly in the Middle East. A high prevalence of undiagnosed DN and those at risk of diabetic foot ulceration (DFU) is a major concern. We have determined the prevalence of DN and its risk factors, DFU, and those at risk of DFU in patients with type 2 diabetes mellitus (T2DM) in secondary care in Qatar. MATERIALS AND METHODS: Adults with T2DM were randomly selected from the two National Diabetes Centers in Qatar. DN was defined by the presence of neuropathic symptoms and a vibration perception threshold (VPT) ≥ 15 V. Participants with a VPT ≥ 25 V were categorized as high risk for DFU. Painful DN was defined by a DN4 score ≥4. Logistic regression analysis was used to identify predictors of DN. RESULTS: In 1082 adults with T2DM (age 54 ± 11 years, duration of diabetes 10.0 ± 7.7 years, 60.6% males), the prevalence of DN was 23.0% (95% CI, 20.5%-25.5%) of whom 33.7% (95% CI, 27.9%-39.6%) were at high risk of DFU, and 6.3% had DFU; 82.0% of the patients with DN were previously undiagnosed. The prevalence of DN increased with age and duration of diabetes and was associated with poor glycaemic control (HbA1c ≥ 9%) AOR = 2.1 (95% CI, 1.3-3.2), hyperlipidaemia AOR = 2.7 (95% CI, 1.5-5.0), and hypertension AOR = 2.0 (95% CI, 1.2-3.4). CONCLUSIONS: Despite DN affecting 23% of adults with T2DM, 82% had not been previously diagnosed with one-third at high risk for DFU. This argues for annual screening and identification of patients with DN. Furthermore, we identify hyperglycaemia, hyperlipidaemia, and hypertension as predictors of DN.

Role of Autophagy in Von Willebrand Factor Secretion by Endothelial Cells and in the In Vivo Thrombin-Antithrombin Complex Formation Promoted by the HIV-1 Matrix Protein p17.

Although the advent of combined antiretroviral therapy has substantially improved the survival of HIV-1-infected individuals, non-AIDS-related diseases are becoming increasingly prevalent in HIV-1-infected patients. Persistent abnormalities in coagulation appear to contribute to excess risk for a broad spectrum of non-AIDS defining complications. Alterations in coagulation biology in the context of HIV infection seem to be largely a consequence of a chronically inflammatory microenvironment leading to endothelial cell (EC) dysfunction. A possible direct role of HIV-1 proteins in sustaining EC dysfunction has been postulated but not yet investigated. The HIV-1 matrix protein p17 (p17) is secreted from HIV-1-infected cells and is known to sustain inflammatory processes by activating ECs. The aim of this study was to investigate the possibility that p17-driven stimulation of human ECs is associated with increased production of critical coagulation factors. Here we show the involvement of autophagy in the p17-induced accumulation and secretion of von Willebrand factor (vWF) by ECs. In vivo experiments confirmed the capability of p17 to exert a potent pro-coagulant activity soon after its intravenous administration.

p17 from HIV induces brain endothelial cell angiogenesis through EGFR-1-mediated cell signalling activation.

HIV-associated neurocognitive disorder in HIV patients substantially reduces their quality of life. We previously showed that the HIV matrix protein, p17 could stimulate lymph-angiogenesis in vitro potentially contributing to lymphoma tumour growth and in addition is associated with vascular activation in neuro-degenerating brain tissue; here, therefore, we have investigated the detailed molecular mechanisms of this action. We performed in vitro cell culture, angiogenesis experiments, phospho-protein microarrays and Western blotting to identify cellular signalling induced by p17 within human brain endothelial cells (HbMEC), and inhibitor studies to block p17-induced vascular growth. We also characterised the effects of hippocampal CA1 injection of p17 on epidermal growth factor receptor-1 (EGFR1) expression linked to our murine model of dementia. p17 strongly induced angiogenesis of HbMEC (migration, tube formation and spheroid growth). p17 concomitantly increased phosphorylation of EGFR1 as well as down-stream intermediates ERK1/2, FAK, PLC-γ and PKC-ß whilst an inhibitor peptide of EGFR, blocked cell signalling and angiogenesis. Finally, Mice that showed reduced cognitive function and behavioural deficiencies after p17 injection, demonstrated that p17 localised in cortical microvessels and also neurones many of which stained positive for p-EGFR1 by histology/IHC. This work provides strong support that p17 may be involved in initiating and/or perpetuating vascular tissue pathophysiology associated with comorbidity in HIV patients.

Clinical Course and Outcomes of Small Supratentorial Intracerebral Hematomas.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) volume, particularly if ≥30 mL, is a major determinant of poor outcome. We used a multinational ICH data registry to study the characteristics, course, and outcomes of supratentorial hematomas with volumes <30 mL. METHODS: Basic characteristics, clinical and radiological course, and 30-day outcomes of these patients were recorded. Outcomes were categorized as early neurological deterioration (END), hematoma expansion, Glasgow Outcome Scale (GOS), and in-hospital death. Poor outcome was defined as composite of in-hospital death and severe disability (GOS ≤ 3). Comparison was conducted based on hemorrhage location. Logistic regression using dichotomized outcome scales was applied to determine predictors of poor outcome. RESULTS: Among 375 cases of supratentorial ICH with volumes <30 mL, expansion and END rates were 19.2% and 7.5%, respectively. Hemorrhage growth was independently associated with END (odds ratio: 28.7, 95% confidence interval [CI]: 8.51-96.5; P < .0001). Expansion rates did not differ according to ICH location. Overall, 13.9% (exact binomial 95% CI: 10.5-17.8) died in the hospital and 29.1% (CI: 24.5-34.0) had severe disability at 30 days; there was a cumulative poor outcome rate of 42.9% (CI: 37.9-48.1). Age, admission Glasgow Coma Scale, intraventricular extension, and END were independently associated with poor outcome. There was no difference in poor outcome rates between lobar and deep locations (40.2% versus 43.8%, P = .56). CONCLUSION: Patients with supratentorial ICH <30 mL have high rates of poor outcome at 30 days, regardless of location. Nearly 1 in 5 hematomas <30 mL expands, leading to END or death.

HIV-1 matrix protein p17 promotes lymphangiogenesis and activates the endothelin-1/endothelin B receptor axis.

OBJECTIVE: AIDS-related lymphomas are high grade and aggressively metastatic with poor prognosis. Lymphangiogenesis is essential in supporting proliferation and survival of lymphoma, as well as tumor dissemination. Data suggest that aberrant lymphangiogenesis relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. HIV-1 matrix protein p17 was found to accumulate and persist in lymph nodes of patients even under highly active antiretroviral therapy. Because p17 was recently found to exert a potent proangiogenic activity by interacting with chemokine (C-X-C motif) receptors 1 and 2, we tested the prolymphangiogenic activity of the viral protein. APPROACH AND RESULTS: Human primary lymph node-derived lymphatic endothelial cells were used to perform capillary-like structure formation, wound healing, spheroids, and Western blot assays after stimulation with or without p17. Here, we show that p17 promotes lymphangiogenesis by binding to chemokine (C-X-C motif) receptor-1 and chemokine (C-X-C motif) receptor-2 expressed on lymph node-derived lymphatic endothelial cells and activating the Akt/extracellular signal-regulated kinase signaling pathway. In particular, it was found to induce capillary-like structure formation, sprout formation from spheroids, and increase lymph node-derived lymphatic endothelial cells motility. The p17 lymphangiogenic activity was, in part, sustained by activation of the endothelin-1/endothelin receptor B axis. A Matrigel plug assay showed that p17 was able to promote the outgrowth of lymphatic vessels in vivo, demonstrating that p17 directly regulates lymphatic vessel formation. CONCLUSIONS: Our results suggest that p17 may generate a prolymphangiogenic microenvironment and plays a role in predisposing the lymph node to lymphoma growth and metastasis. This finding offers new opportunities to identify treatment strategies in combating AIDS-related lymphomas.

HIV-1 matrix protein p17 promotes angiogenesis via chemokine receptors CXCR1 and CXCR2.

Vascular diseases supported by aberrant angiogenesis have increased incidence in HIV-1-infected patients. Several data suggest that endothelium dysfunction relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. The HIV-1 matrix protein p17 is known to deregulate the biological activity of different immune cells. Recently, p17 was found to mimic IL-8 chemokine activity by binding to the IL-8 receptor CXCR1. Here we show that p17 binds with high affinity to CXCR2, a CXCR1-related receptor, and promotes the formation of capillary-like structures on human endothelial cells (ECs) by interacting with both CXCR1 and CXCR2 expressed on the EC surface. ERK signaling via Akt was defined as the pathway responsible for p17-induced tube formation. Ex vivo and in vivo experimental models confirmed the provasculogenic activity of p17, which was comparable to that induced by VEGF-A. The hypothesis of a major role for p17 in HIV-1-induced aberrant angiogenesis is enforced by the finding that p17 is detected, as a single protein, in blood vessels of HIV-1-patients and in particular in the nucleus of ECs. Localization of p17 in the nucleus of ECs was evidenced also in in vitro experiments, suggesting the internalization of exogenous p17 in ECs by mechanisms of receptor-mediated endocytosis. Recognizing p17 interaction with CXCR1 and CXCR2 as the key event in sustaining EC aberrant angiogenesis could help us to identify new treatment strategies in combating AIDS-related vascular diseases.

C-reactive protein predicts hematoma growth in intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Early hematoma growth (EHG) occurs in about one third of patients with spontaneous intracerebral hemorrhage. The main aim of this study was to investigate the potential of plasma C-reactive protein (CRP) for predicting EHG after acute spontaneous intracerebral hemorrhage. METHODS: Plasma CRP was measured within 6 hours of onset (median, 120 minutes) in 399 patients with primary or vitamin K antagonist-associated spontaneous intracerebral hemorrhage and without recent infection. Computed tomography brain scans were performed at baseline and repeated within 24 hours (median, 22 hours). The primary outcome was EHG, defined as absolute growth>12.5 cm3 or relative growth>33%. Secondary outcomes included early neurological worsening (ENW) using the Glasgow Coma Scale and 30-day mortality. Multivariable regression analyses were used to evaluate associations of CRP concentration and outcomes. Kaplan-Meier analysis was used for survival. RESULTS: EHG occurred in 25.8%, ENW in 19.3%, and mortality was 31.8% at 30 days. Thirty-day mortality was significantly higher in patients with ENW (hazard ratio, 3.21; 95% confidence interval, 2.00-5.17; P<0.0001) and in patients with EHG (hazard ratio, 2.13; 95% confidence interval, 1.42-3.18; P<0.0001, log-rank test). Median CRP was 12 mg/L (interquartile range, 10-17) in the EHG group and 7 mg/L (interquartile range, 4-12.1) in those without EHG (P<0.0001). In multivariable analyses, plasma CRP>10 mg/L independently predicted EHG (odds ratio, 4.71; 95% confidence interval, 2.75-8.06; P<0.0001) and ENW (odds ratio, 2.70; 95% confidence interval, 1.50-4.84; P=0.0009). CONCLUSIONS: CRP>10 mg/L is independently predictive of EHG and ENW, both of which are associated with increased mortality. Inflammation may be important in contributing to EHG and warrants further investigation.

Monomeric C-reactive protein and Notch-3 co-operatively increase angiogenesis through PI3K signalling pathway.

C-reactive protein (CRP) is the most acute-phase reactant serum protein of inflammation and a strong predictor of cardiovascular disease. Its expression is associated with atherosclerotic plaque instability and the formation of immature micro-vessels. We have previously shown that CRP upregulates endothelial-derived Notch-3, a key receptor involved in vascular development, remodelling and maturation. In this study, we investigated the links between the bioactive monomeric CRP (mCRP) and Notch-3 signalling in angiogenesis. We used in vitro (cell counting, wound-healing and tubulogenesis assays) and in vivo (chorioallantoic membrane) angiogenic assays and Western blotting to study the angiogenic signalling pathways induced by mCRP and Notch-3 activator chimera protein (Notch-3/Fc). Our results showed an additive effect on angiogenesis of mCRP stimulatory effect combined with Notch-3/Fc promoting bovine aortic endothelial cell (BAEC) proliferation, migration, tube formation in Matrigel(TM) with up-regulation of phospho-Akt expression. The pharmacological blockade of PI3K/Akt survival pathway by LY294002 fully inhibited in vitro and in vivo angiogenesis induced by mCRP/Notch-3/Fc combination while blocking Notch signalling by gamma-secretase inhibitor (DAPT) partially inhibited mCRP/Notch-3/Fc-induced angiogenesis. Using a BAEC vascular smooth muscle cell co-culture sprouting angiogenesis assay and transmission electron microscopy, we showed that activation of both mCRP and Notch-3 signalling induced the formation of thicker sprouts which were shown later by Western blotting to be associated with an up-regulation of N-cadherin expression and a down-regulation of VE-cadherin expression. Thus, mCRP combined with Notch-3 activator promote angiogenesis through the PI3K/Akt pathway and their therapeutic combination has potential to promote and stabilize vessel formation whilst reducing the risk of haemorrhage from unstable plaques.

C-reactive Protein: An Inflammatory Biomarker and a Predictor of Neurodegenerative Disease in Patients With Inflammatory Bowel Disease?

Inflammatory bowel disease (IBD) refers to two chronic conditions of the digestive tract: ulcerative colitis (UC) and Crohn's disease (CD), representing a progressive inflammatory process that mainly occurs in the gut, with frequent extra-intestinal manifestations. Even if remission is periodically obtained for some patients, the histological activity and digestive symptoms may continue, maintaining a persistent systemic inflammation that could induce further extra-intestinal complications and contribute to the development of neurodegenerative disease. C-reactive protein (CRP) is an acute-phase reactant that is widely accepted as a dominant serum biomarker in IBD. CRP consequently activates the complement cascade, supports the release of pro-inflammatory cytokines, and the clearance of microbial pathogens. All these processes facilitate further processes, including atherosclerosis and hypercoagulability, alteration of the intestinal microbiota, and the increased permeability of the intestinal barrier for neurotoxic substances produced by gut microorganisms, due to the presence of a high level of lipopolysaccharides. For IBD, the connection between intestinal inflammation and central nervous system inflammation could be explained through the activity of the vagus nerve, a carrier of cytokines, CRP, and toxic materials to the brain, potentially inducing vascular lesions and damage of the glial vascular unit, with further risk for degeneration within the central nervous system. CRP is a key marker for IBD pathogenesis and is able to dissociate into its monomeric form, mCRP, on contact with activated cell and tissue components via the systemic circulation. We hypothesize that the chronic inflammatory process within IBD could initiate neuroinflammation and neurodegeneration, and therefore, further investigation of the significance of chronically raised plasma of CRP and mCRP in patients with IBD is warranted, as it may represent a critical predictive factor associated with a later neurodegenerative risk. Any future initiative aimed at pharmacologic modulation of CRP (e.g., blocking CRP-mCRP dissociation), could represent a new therapeutic approach protecting against intestinal inflammation and concomitantly reducing the risk of neuroinflammation, neurodegeneration, and cognitive decline.

Monomeric C-Reactive Protein Potential Utilization in the Histological Assessment of Inflammatory Bowel Disease (IBD) Patients.

Introduction Inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn's disease (CD), represent chronic progressive inflammatory gastrointestinal disorders, without a single reference standard for their diagnosis. The histological assessment gained an important role in accurately measuring disease activity, and mucosal healing (MH) was recently proposed to be an ideal treatment goal for patients with IBD because of its favorable prognosis, with a lower risk of recurrence or surgical treatment. This paper aims to add to the histological classical findings for IBD patients the identification of the monomeric form of the C-reactive protein (mCRP) as a supplementary marker that could be stained at the level of tissue samples and could be correlated with the pathogenic mechanism. Methods Two groups of 10 patients were each selected for the study, for both UC and CD, together with a control group. All samples collected through digestive endoscopy were analyzed by using H&E-stained slides, followed by immunohistochemical examination with antibodies to mCRP (M8C10), and markers of inflammatory activity through CD3, CD45(leukocyte common antigen (LCA)), CD138/syndecan-1 and CD68. Results For the CD study group, all histological elements identified with H&E and afterward stained with CD138, CD68, CD3, and CD45/LCA were correlated with the standards imposed by the European Crohn's and Colitis Organization (ECCO). For the group of patients with UC, histological images obtained with H&E and IHC stainings also confirmed the recommendation of ECCO. The main cells considered in the literature as histological markers for IBD are neutrophils, lymphocytes, and plasmocytes, stained in our study with CD45/LCA, CD3, and CD138. For all 20 cases of IBD (UC and CD), the staining with anti-Ab8C10 antibodies for mCRP was positive, while negative results were noticed within the control group. An mCRP protein visualized with anti-Ab8C10 antibodies presented an intracytoplasmatic localization in the neutrophils, plasma cells, lymphocytes, and macrophages from the lamina propria and glandular epithelium, without expression in endothelial cells. Conclusions Our study represents one of the first papers that identifies the localization of mCRP molecules within the intestinal mucosa of patients with IBD (both UC and CD) by using immunohistochemistry (IHC) staining. This finding opens a new perspective for considering mCRP as a marker correlated with histological disease activity and/or definition of histological remission in IBD.

A Gender-Bias-Mitigated, Data-Driven Precision Medicine System to Assist in the Selection of Biological Treatments of Grade 3 and 4 Knee Osteoarthritis: Development and Preliminary Validation of precisionKNEE.

Objective To identify key variables predictive of patient responses to microfragmented adipose tissue (MFAT) treatment in knee osteoarthritis (KOA) and evaluate its potential to delay or mitigate the need for total knee replacement (TKR). Methods We utilised a dataset comprising 329 patients treated with MFAT for KOA, incorporating variables such as gender, age, BMI, arthritic aetiology, radiological grade, and Oxford Knee Scores (OKS) pre- and post-treatment. We employed random forest regressors for model training and testing, with gender bias mitigation and outlier detection to enhance prediction accuracy. Model performance was assessed through root mean squared error (RMSE) and mean absolute error (MAE), with further validation in a TKR-suitable patient subset. Results The model achieved a test RMSE of 6.72 and an MAE of 5.38, reflecting moderate predictive accuracy across the patient cohort. Stratification by gender revealed no statistically significant differences between actual and predicted OKS improvements (p-values: males = 0.93, females = 0.92). For the subset of patients suitable for TKR, the model presented an increased RMSE of 9.77 and MAE of 7.81, indicating reduced accuracy in this group. The decision tree analysis identified pre-operative OKS, radiological grade, and gender as significant predictors of post-treatment outcomes, with pre-operative OKS being the most critical determinant. Patients with lower pre-operative OKS showed varying responses based on radiological severity and gender, suggesting a nuanced interaction between these factors in determining treatment efficacy. Conclusion This study highlights the potential of MFAT as a non-surgical alternative for KOA treatment, emphasising the importance of personalised patient assessments. While promising, the predictive model warrants further refinement and validation with a larger, more diverse dataset to improve its utility in clinical decision-making for KOA management.

A Quantum-Enhanced Precision Medicine Application to Support Data-Driven Clinical Decisions for the Personalized Treatment of Advanced Knee Osteoarthritis: The Development and Preliminary Validation of precisionKNEE_QNN.

Background Quantum computing and quantum machine learning (QML) are promising experimental technologies that can improve precision medicine applications by reducing the computational complexity of algorithms driven by big, unstructured, real-world data. The clinical problem of knee osteoarthritis is that, although some novel therapies are safe and effective, the response is variable, and defining the characteristics of an individual who will respond remains a challenge. In this study, we tested a quantum neural network (QNN) application to support precision data-driven clinical decisions to select personalized treatments for advanced knee osteoarthritis. Methodology After obtaining patients' consent and Research Ethics Committee approval, we collected the clinicodemographic data before and after the treatment from 170 patients eligible for knee arthroplasty (Kellgren-Lawrence grade ≥3, Oxford Knee Score (OKS) ≤27, age ≥64 years, and idiopathic aetiology of arthritis) treated over a two-year period with a single injection of microfragmented fat. Gender classes were balanced (76 males and 94 females) to mitigate gender bias. A patient with an improvement ≥7 OKS was considered a responder. We trained our QNN classifier on a randomly selected training subset of 113 patients to classify responders from non-responders (73 responders and 40 non-responders) in pain and function at one year. Outliers were hidden from the training dataset but not from the validation set. Results We tested our QNN classifier on a randomly selected test subset of 57 patients (34 responders, 23 non-responders) including outliers. The no information rate was 0.59. Our application correctly classified 28 responders out of 34 and 6 non-responders out of 23 (sensitivity = 0.82, specificity = 0.26, F1 Statistic = 0.71). The positive and negative likelihood ratios were 1.11 and 0.68, respectively. The diagnostic odds ratio was 2. Conclusions Preliminary results on a small validation dataset showed that QML applied to data-driven clinical decisions for the personalized treatment of advanced knee osteoarthritis is a promising technology to reduce computational complexity and improve prognostic performance. Our results need further research validation with larger, real-world unstructured datasets, as well as clinical validation with an artificial intelligence clinical trial to test model efficacy, safety, clinical significance, and relevance at a public health level.