ICAM-1 Research Articles

Laminar flow substantially affects the morphology and functional phenotype of glomerular endothelial cells.

Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.

Assessment of ICAM-1 gene polymorphism in Acne Vulgaris

Background: Considering the recognized roles of ICAM-1 in inflammation and rising an evidence that inflammation is evident at all stages of AV. The current study aimed to investigate the incidence of ICAM-1 gene expression and polymorphism with special reference to amino acids at position 469 and 241 with acne vulgaris and its possible correlation with the lesion severity. Patient and methods: The present case control study included 60 participants of matched age, sex and of Egyptian origin where there were 45 acne patients assigned into three groups according to acne severity (mild, moderate and severe), 15 patients in each group, and there were 15 participants as control.All studied participants were tested for the genotypes of ICAM-1 gene polymorphism by PCR where four primers have been used to detect all possible polymorphism at codon 241 and 469 Results: There was statistically significant difference among four studied groups as regard ICAM (496) and ICAM (241) polymorphism where the highest percentage of GG were among severe acne group and none of controls had GG genotype. There was significant association between ICAM (496) polymorphism and patients’ BMI where the highest BMI Conclusion: ICAM-1 496 and 241 polymorphism might be associated with AV. Further understanding of functional significance of the ICAM-1 polymorphism will help delineate immunopathogenesis in certain subgroups of AV obese patients

Impact of radiation therapy on vascular endothelial adhesion receptor expression

The adjunction of PD-1/PD-L1 immunotherapy and radiation therapy (RT) has proven to be an effective approach to combat cancer. Clinical studies recently confirmed that patients show increased response to immune checkpoint inhibitors if also treated with RT. One effect of RT in this setting is the facilitation of recruitment of immune cells to tumors in which T cell infiltration is low or absent. Recruitment of immune cells is primarily dependent on endothelial activation: activated endothelial cells exhibit an increased expression of endothelial adhesion receptors that mediate recruitment from the blood. Despite evidence that RT can boost T cell recruitment to tumors an in-depth analysis of endothelial adhesion receptor expression and which fractionation doses are best suited in this context are unknown. We hypothesize that RT leads to activation of the endothelium. Using the endothelial mouse cell line bEnd5, we investigated the expression of the adhesion receptors ICAM-1, ICAM-2, VCAM-1, E-selectin, and P-selectin following RT ranging from 0.5 to 10 Gy at different times points (1.5h, 4h, and 24h) with or without prior stimulation with TNF-α by qPCR, flow cytometry, and immunofluorescence. We observed upregulation of E-selectin, P-selectin, and VCAM-1 as early as 1.5h after RT. Early upregulation of selectins was independent of dose; however, sustained upregulation of selectins and upregulation of VCAM-1 was dose-dependent. Following preceding stimulation with TNF-α led to an increased baseline expression of E-Selectin and VCAM-1. E-selectin, P-selectin, and VCAM-1 expression was further increased after RT, and to a much greater extent than without prior stimulation. Again, effects were more pronounced at early time points and showed a dose dependency for VCAM-1. In contrast, while ICAM-2 was not changed by RT or was even downregulated, the addition of RT following TNF-α treatment did increase ICAM-1 levels. This effect was not observed without prior TNF-α stimulation. In vivo experiments of KrasG12D;Tp53fl/fl NSCLC tumors revealed changes in P-selectin, E-selectin, and VCAM-1 that mimicked changes of in vitro experiments with prior TNF-α stimulation. Performing adoptive T cell transfer experiments, we found that both timing and dose of RT is important for successful recruitment of transferred cells to the tissue. Our study demonstrates that RT alters endothelial receptor display on endothelial cells in vitro and in vivo and is influenced significantly by the local cytokine milieu.

O17: HYALURONIC ACID DEPENDENT ADHESION IN COLORECTAL CANCER PERITONEAL METASTASIS

Abstract Introduction Peritoneal Metastasis (PM) in Colorectal Cancer (CRC) undoubtedly remains a challenge to treat and often portends a poor prognosis for patients. Hyaluronic acid (HA) is found throughout the body, including coating of the peritoneum. Interaction of HA with HA-dependent adhesion molecules can facilitate cell adhesion within the peritoneum. HA may play a role in spread of PM in CRC in association with known HA-receptor molecules CD44, RHAMM and ICAM-1. Method Expression of HA-dependent and HA-independent adhesion molecules were examined in CRC using tissue microarray datasets and matched to clinicopathological data. In-vitro peritoneal modelling assessed cellular adhesion when treated with a competitive HA-inhibitor (HAi) or excess exogenous HA. An in-vivo Xenograft peritoneal model, using CD1 nude mice injected with CRC cells, were treated with either HAi or excess exogenous HA and compared to a control (PPL: PE9445FC2). Result There is a significant increase in expression of HA-dependent adhesion molecules seen in CD44, RHAMM and ICAM-1 in CRC (p=<0.0001). Whilst Non-HA-dependent adhesion molecules demonstrated either significant downregulation or no expression difference. Cellular adhesion was decreased in three CRC cell lines when treated with HAi or excess HA. Treatment with HAi and HA in-vivo confirmed a significant reduction in PM, compared to controls (HAi p=0.0094, HA p=0.0009). Conclusion HA-dependent adhesion molecules and HA appear to play a role in CRC. Targeting HA-dependent interaction in CRC influences cellular adhesion and may have a potential therapeutic use in treatment of PM in CRC. Further in-vitro and in vivo modelling is needed. Take-home message HA receptor adhesion molecules CD44, RHAMM and ICAM-1 have all been independently implicated in adverse outcomes in colorectal cancer. Targeting HA receptor interaction appears to reduce peritoneal dissemination in colorectal cancer.

Hspa8 and ICAM-1 as damage-induced mediators of γδ T cell activation.

Tissue-resident γδ T cells form the first line of defense at barrier surfaces where they survey host tissue for signs of stress or damage. Following recognition of injury, γδ T cells play a crucial role in the wound-healing response through the production of growth factors and cytokines that promote proliferation in surrounding epithelial cells. To initiate this response, γδ T cells require interactions with a variety of epithelial-expressed costimulatory molecules in addition to primary signaling through their TCR. In the epidermis these signals include the coxsackie and adenovirus receptor (CAR), histocompatibility antigen 60c (H60c), and plexin B2, which interact with γδ T cell-expressed junctional adhesion molecule-like protein (JAML), NKG2D, and CD100, respectively. Here we identify heat shock protein family A member 8 (Hspa8) and ICAM-1 as two additional keratinocyte-expressed costimulatory molecules for epidermal resident γδ T cells (termed DETC). These molecules were rapidly up-regulated in the epidermis following wounding in both mouse and human tissue. Both Hspa8 and ICAM-1 had a costimulatory effect on DETC, inducing proliferation, CD25 up-regulation, and IL-2 production. We also provide evidence that DETC can be activated through the potential ICAM-1 and Hspa8 receptors LFA-1 and CD316. Finally, knockdown of Hspa8 in keratinocytes reduced their ability to activate DETC in culture and ICAM-1-/- mice exhibited impaired rates of healing in skin-organ culture suggesting a role for these proteins in the DETC-mediated damage response. Together with previous work on CAR, H60c, and plexin B2, these results add to a picture of a complex keratinocyte wound signature that is required for efficient DETC activation.

The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity

Current therapies for treating heterogeneous cancers such as head and neck squamous cell carcinoma (HNSCC) are non-selective and are administered independent of response biomarkers. Therapy resistance subsequently emerges, resulting in increased cellular proliferation that is associated with loss of differentiation. Whether a cancer cell differentiation potential can dictate therapy responsiveness is still currently unknown. A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity was employed in a HNSCC mouse model, primary patients’ data, and human cell lines to assess the potential of functional differentiation in predicting therapy response. Interestingly, a subset of HNSCC with effective GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-Myc, and STAT3 signaling. Furthermore, we identified the GRHL3-differentiation target gene Filaggrin (FLG) as a response biomarker and more importantly, stratified HNSCC subsets as treatment resistant based on their FLG mutational profile. The loss of FLG in sensitive HNSCC resulted in a dramatic resistance to targeted therapies while the GRHL3-FLG signature predicted a favorable patient prognosis. This study provides evidence for a functional GRHL3-FLG tumor-specific differentiation axis that regulates targeted therapy response in HNSCC and establishes a rationale for clinical investigation of differentiation-paired targeted therapy in heterogeneous cancers.

Effect of dual PPAR-α/γ agonist saroglitazar on diabetic retinopathy and oxygen-induced retinopathy

Diabetic retinopathy is a serious complication of diabetes, marked by retinal vascular damage, inflammation, and angiogenesis. This study's objective was to assess the potential benefits of saroglitazar, a peroxisome proliferator-activated receptor-alpha/gamma (PPAR-α/γ) agonist in diabetic retinopathy. Diabetic retinopathy was induced by streptozotocin in Sprague Dawley rats. The effect of saroglitazar was also assessed in the oxygen-induced retinopathy model in newborn rats and VEGF-induced angiogenesis in the chick chorioallantoic membrane (CAM) assay.Treatment of saroglitazar (1 and 4 mg/kg, oral) for 12 weeks significantly ameliorated retinal vascular leakage and leukostasis in the diabetic rats. Saroglitazar decreased oxidative stress, VEGF receptor signalling, NF-κBp65, and ICAM-1 in the retina of diabetic rats. The beneficial effects of saroglitazar (1 and 4 mg/kg, oral) were also observed on the neovascularization in oxygen-induced retinopathy in newborn rats. Saroglitazar also reduced VEGF-induced angiogenesis in CAM assay.This study reveals that saroglitazar has the potential to prevent the progression of retinopathy in diabetic patients.

IL-36 and IL-17A Cooperatively Induce a Psoriasis-Like Gene Expression Response in Human Keratinocytes

IL-36 and IL-17A Cooperatively Induce a Psoriasis-Like Gene Expression Response in Human Keratinocytes

Abstract P337: RAPID-LVO for Automated Detection of Intracranial Large Vessel Occlusion in Ct Angiography of the Brain

Background: Identification of large vessel occlusion (LVO) is paramount in the urgent evaluation of acute ischemic stroke (AIS). Emergent interpretation of large and high-complexity data sets, however, may impose strains upon imaging and clinical workflows, motivating development of fast and accurate computer-aided approaches to facilitate LVO detection in the emergency setting. This study investigates the performance of a fully automated LVO detection platform in a mixed cohort of stroke subjects with and without LVO on head and neck CT angiography (CTA). Methods: CTA from two cerebrovascular trials were enriched with cases from eleven global sites. Imaging and clinical variables were balanced between populations including in LVO positivity and across demographic and imaging environments to the extent achievable. Independent and fully blinded review for intracranial ICA or MCA M1 LVO was performed by two subspecialty neuroradiologists. A novel, user-independent imaging analysis application ( RAPID-LVO , iSchemaview inc) was used to predict LVO presence, location, and overall performance relative to reader consensus. Any discordance between readers was adjudicated by a blinded tertiary reader with subspecialty training. Sensitivity, specificity, and receiver-operating characteristics were determined by an independent statistician. Performance thresholds were set a priori, including a lower bound of the 95% CI of sensitivity and specificity of ≥0.8 at mean times-to-notification <3.5 minutes. Results: 217 CTA (median age 65.5, 53% male, 109 LVO(+)) were included. Lower confidence limits of sensitivity and specificity exceeded 90% (sensitivity 0.963, 95% CI 0.909-0.986; specificity 0.981, 95% CI 0.935-0.995), surpassing pre-specified performance benchmarks. Subgroup analyses revealed no decrement in performance relative to subject age or sex, vendor systems, or location of the examination within or outside the United States. The area under the receiver operating characteristics curve was 0.99 (95% CI: 0.971-0.999) and average time-to-notification was 3.18 minutes. Conclusion: RAPID-LVO offers fast, highly accurate, and fully user-independent large vessel occlusion detection across all tested clinical and imaging environments.

IgG acquisition against PfEMP1 PF11_0521 domain cassette DC13, DBL\u03b23_D4 domain, and peptides located within these constructs in children with cerebral malaria

The Plasmodium falciparum erythrocyte-membrane-protein-1 (PF3D7_1150400/PF11_0521) contains both domain cassette DC13 and DBLβ3 domain binding to EPCR and ICAM-1 receptors, respectively. This type of PfEMP1 proteins with dual binding specificity mediate specific interactions with brain micro-vessels endothelium leading to the development of cerebral malaria (CM). Using plasma collected from children at time of hospital admission and after 30 days, we study an acquisition of IgG response to PF3D7_1150400/PF11_0521 DC13 and DBLβ3_D4 recombinant constructs, and five peptides located within these constructs, specifically in DBLα1.7_D2 and DBLβ3_D4 domains. We found significant IgG responses against the entire DC13, PF11_0521_DBLβ3_D4 domain, and peptides. The responses varied against different peptides and depended on the clinical status of children. The response was stronger at day 30, and mostly did not differ between CM and uncomplicated malaria (UM) groups. Specifically, the DBLβ3 B3-34 peptide that contains essential residues involved in the interaction between PF11_0521 DBLβ3_D4 domain and ICAM-1 receptor demonstrated significant increase in reactivity to IgG1 and IgG3 antibodies at convalescence. Further, IgG reactivity in CM group at time of admission against functionally active (ICAM-1-binding) PF11_0521 DBLβ3_D4 domain was associated with protection against severe anemia. These results support development of vaccine based on the PF3D7_1150400/PF11_0521 structures to prevent CM.

Deficiency of the novel high mobility group protein HMGXB4 protects against systemic inflammation-induced endotoxemia in mice

Sepsis is a major cause of mortality in intensive care units, which results from a severely dysregulated inflammatory response that ultimately leads to organ failure. While antibiotics can help in the early stages, effective strategies to curtail inflammation remain limited. The high mobility group (HMG) proteins are chromosomal proteins with important roles in regulating gene transcription. While HMGB1 has been shown to play a role in sepsis, the role of other family members including HMGXB4 remains unknown. We found that expression of HMGXB4 is strongly induced in response to lipopolysaccharide (LPS)-elicited inflammation in murine peritoneal macrophages. Genetic deletion of Hmgxb4 protected against LPS-induced lung injury and lethality and cecal ligation and puncture (CLP)-induced lethality in mice, and attenuated LPS-induced proinflammatory gene expression in cultured macrophages. By integrating genome-wide transcriptome profiling and a publicly available ChIP-seq dataset, we identified HMGXB4 as a transcriptional activator that regulates the expression of the proinflammatory gene, Nos2 (inducible nitric oxide synthase 2) by binding to its promoter region, leading to NOS2 induction and excessive NO production and tissue damage. Similar to Hmgxb4 ablation in mice, administration of a pharmacological inhibitor of NOS2 robustly decreased LPS-induced pulmonary vascular permeability and lethality in mice. Additionally, we identified the cell adhesion molecule, ICAM1, as a target of HMGXB4 in endothelial cells that facilitates inflammation by promoting monocyte attachment. In summary, our study reveals a critical role of HMGXB4 in exacerbating endotoxemia via transcriptional induction of Nos2 and Icam1 gene expression and thus targeting HMGXB4 may be an effective therapeutic strategy for the treatment of sepsis.

TRPV4 channel activation induces the transition of venous and arterial endothelial cells toward a pro-inflammatory phenotype.

The Transient Receptor Potential Vanilloid 4 (TRPV4) of endothelial cells contributes to many important functions including the regulation of Ca2+ homeostasis, cell volume, endothelial barrier permeability, and smooth muscle tone. However, its role in the transition of endothelial cells toward a pro‐inflammatory phenotype has not been studied so far. Using both arterial and venous endothelial cells, we first show that the pharmacological activation of TRPV4 channels with GSK1016790A, a potent TRPV4 agonist, triggers robust and sustained Ca2+ increases, which are blocked by both TRPV4 antagonists HC067047 and RN9893. TRPV4 activation also triggers the actin cytoskeleton and adherens junction (VE‐Cadherin) rearrangement in both arterial and venous endothelial cells and leads to rapid decreases of trans‐endothelial electrical resistance. In addition to its effect on endothelial barrier integrity, TRPV4 activation selectively increases ICAM‐1 surface expression in arterial and venous endothelial cells, due to the stimulation of ICAM‐1 gene expression through the NF‐κB transcription factor. TRPV4 channel activation also induced apoptosis of venous and arterial endothelial cells, while TRPV4 blockade reduced apoptosis, even in the absence of TRPV4 activation. As altered barrier integrity, increased adhesion molecule expression and apoptosis are hallmarks of the pro‐inflammatory state of endothelial cells, our results indicate that TRPV4 channel activity can induce the transition of both venous and arterial endothelial cells toward a pro‐inflammatory phenotype.

Selection of a novel AAV2/TNFAIP3 vector for local suppression of islet xenograft inflammation.

Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non-human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof-of-concept experiments the potential of the cytoplasmic ubiquitin-editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene. We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF-stimulated NF-κB activation and NPI graft function. As adeno-associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation. Forcing the expression of A20 in NPI with Ad5 vector blocked NF-κB activation by inhibiting IκBα phosphorylation and degradation, and reduced the induction of pro-inflammatory genes Cxcl10 and Icam1. A20-expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14-day culture period maximized NPI transduction efficiency (>70% transduction rate), and suppressed NF-κB-dependent gene expression without adverse impact upon NPI maturation. We report a new protocol that allows for high-efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement.

Mapping the Multi-Organ miRNA-mRNA Regulatory Network in LPS-Mediated Endotoxemic Mice: Exploring the Shared Underlying Key Genes and Mechanisms.

BackgroundTo this day, the molecular mechanism of endotoxin-induced multi-organ failure has not been completely clarified. This study aimed to construct an miRNA-mRNA regulatory network and identify main pathways and key genes in multi-organ of LPS-mediated endotoxemic mice.MethodsPublic datasets from six mRNA and three miRNA microarray datasets were downloaded from the GEO website to screen final differentially expressed genes (FDEGs) and hub genes in the heart, lung, liver, and kidney of LPS-mediated endotoxemic mice. Functional and pathway enrichment analysis of FDEGs was used to identify the main pathways in multi-organ damage of LPS-treated mice. Finally, hub genes of each organ were intersected to obtain the key genes of multi-organ.ResultsFirstly, 158, 358, 299, and 91 FDEGs were identified in the heart, lung, liver, and kidney, respectively. The pathway enrichment analysis of the FDEGs then showed that the TNF signaling pathway, Toll-like receptor signaling pathway, and some viral-infection-related pathways (influenza A, measles, and herpes simplex) were the main pathways in multi-organ damage of LPS-mediated endotoxemic mice. Moreover, miRNA-mRNA or PPI regulatory networks were constructed based on FDEGs. According to these networks, 31, 34, 34, and 31 hub genes were identified in the heart, lung, liver, and kidney, respectively. Among them, nine key genes (Cd274, Cxcl1, Cxcl9, Icam1, Ifit2, Isg15, Stat1, Tlr2, and Usp18) were enriched in Toll-like receptor signaling pathway and chemokine signaling pathway. Finally, seven potential drugs were predicted based on these key genes.ConclusionThe shared underlying molecular pathways in endotoxin-induced multi-organ damage that have been identified include Toll-like receptor signaling pathway and TNF signaling pathway. Besides, nine key genes (Cd274, Cxcl1, Cxcl9, Icam1, Ifit2, Isg15, Stat1, Tlr2, and Usp18) and seven potential drugs were identified. Our data provide a new sight and potential target for future therapy in endotoxemia-induced multi-organ failure.

Transcriptomic Analyses Reveal the Protective Immune Regulation of Conjugated Linoleic Acids in Sheep Ruminal Epithelial Cells.

The ruminal epithelium is continuously challenged by antigens released by the lysis of dead microbial cells within the rumen. However, the innate immune system of the ruminal epithelium can almost always actively respond to these challenges. The cross talk between the ruminal microbiota and innate immune cells in the ruminal epithelium has been suggested to play an important role in sustaining the balance of immune tolerance and inflammatory response in the rumen. We hypothesized that conjugated linoleic acid (CLA), a functional microbial metabolite in the rumen, may contribute to the immune regulation in rumen epithelial cells (RECs); therefore, we first established an immortal REC line and then investigated the regulatory effects of CLA on the immune responses in these RECs. The results showed that long-term REC cultures were successfully established via SV40T-induced immortalization. Transcriptome analysis showed that a 100 μM CLA mixture consisting of 50:50 cis-9, trans-11:trans-10, cis-12 CLA significantly downregulated the expression of the inflammatory response-related genes TNF-α, IL-6, CX3CL1, IRF1, ICAM1 and EDN1, and upregulated the expression of the cell proliferation-related genes FGF7, FGF21, EREG, AREG and HBEGF and the lipid metabolism-related genes PLIN2, CPT1A, ANGPTL4, ABHD5 and SREBF1 in the RECs upon LPS stimulation. Correspondingly, the GO terms regulation of cell adhesion, response to stimulus and cytokine production and KEGG pathways TNF and HIF-1 signaling, ECM-receptor interaction and cell adhesion molecules were identified for the significantly downregulated genes, while the GO terms epithelial cell proliferation and regulation of epithelial cell migration and the KEGG pathways PPAR, ErbB and adipocytokine signaling were identified for the RECs with significantly upregulated CLA-pretreated genes upon LPS stimulation. These findings revealed that CLA conferred protective immunity onto the RECs by inhibiting proinflammatory processes, promoting cell proliferation and regulating lipid metabolism related to the immune response.

Epitopes prediction of PfEMP1-DBL2? recombinant protein from Indonesian Plasmodium falciparum Isolate for malaria vaccine development

The development of an effective vaccine against malaria is essential. Domain Duffy-binding-like (DBL)2?in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) protein is one of the vaccinecandidate proteins because of its binding capacity to ICAM-1 receptor. However, the high polymorphismneeds epitope identification before formulating a peptide-based vaccine. This study aimed to identify theconserved epitopes in PfEMP1-DBL2? by an immunoinformatic approach. The protein sequences wereanalyzed to predict the hydrophobicity attributes, T-cell and B-cell epitopes. T-cell epitopes were identifiedusing the NetCTL and Propred server and analyzed for population coverage rate using the IEDB analysistool. Moreover, the Bepipred 2.0 and Kolaskar Tangaonkar method combined with the K-means clusteringwere used to predict the B-cell epitopes. This study found that the hydrophobicity value of PfEMP1-DBL2?recombinant protein is 32.62, indicating that this protein is soluble and potentially fit into HLA alleles activesite. Two conserved antigenic CTL epitopes with near 90% population coverage rate in the malaria targetpopulation were identified. For Th cell epitopes, the NN-align algorithm showed no overlapping strongbinding epitope positions for three chosen Indonesian and African alleles. Three B-cell conserved epitopeswere identified at the position of 77-89, 236-254 and 360-377 amino acids with one cluster overlapping withICAM-1 determinant binding area. The predicted conserved epitopes within the protein in this study arevaluable in constructing a subunit peptide-based malaria vaccine candidate. A further experimental study isneeded to validate this approach as the next step in vaccine development.

Wound Healing After Alkali Burn Injury of the Cornea Involves Nox4-Type NADPH Oxidase.

PurposeCorneal injury that occurs after burning with alkali initiates wound-healing processes, including inflammation, neovascularization, and fibrosis. Excessive reactions to injury can reduce corneal transparency and thereby compromise vision. The NADPH oxidase (Nox) enzyme complex is known to be involved in cell signaling for wound-healing angiogenesis, but its role in corneal neovascularization has been little studied.MethodsThe center corneas of wild-type and Nox4 knockout (KO) mice were injured with 3 µL 1 M NaOH, while the contralateral corneas remained untouched. On day 7, mRNA expression levels of NADPH oxidase isoforms, the proangiogenic factors VEGF-A and TGFβ1, and proinflammatory genes ICAM-1 and VCAM-1 were determined. Corneal neovascularization and fibrosis were visualized using PECAM-1 antibody and picrosirius red staining, respectively, on the same day.ResultsExpressions of both Nox2 and Nox4 gene isoforms as well as the above genes were markedly increased in the injured corneas at 7 days. Injured corneas showed neovascularization and fibrosis as well as an increase in clinical opacity score. All responses stimulated by alkali burn were abrogated in Nox4 KO mice.ConclusionsNox4 could be a new target to treat pathologic corneal wound-healing responses and such targeting might prevent blindness caused by burn injuries.

Enhancing Bioavailability and Stability of Curcumin Using Solid Lipid Nanoparticles (CLEN): A Covenant for Its Effectiveness.

Curcumin, very rightly referred to as “a wonder drug” is proven to be efficacious in a variety of inflammatory disorders including cancers. Antiaging, anti-inflammatory, antioxidant, antitumor, chemosensitizing, P-gp efflux inhibiting, and antiproliferative activity are some of the striking features of curcumin, highlighting its importance in chemotherapy. Curcumin inhibits Bcl-2, Bcl-XL, VEGF, c-Myc, ICAM-1, EGFR, STAT3 phosphorylation, and cyclin D1 genes involved in the various stages of breast, prostate, and gastric cancer proliferation, angiogenesis, invasion, and metastasis. The full therapeutic potential of curcumin however remains under explored mainly due to poor absorption, rapid metabolism and systemic elimination culminating in its poor bioavailability. Furthermore, curcumin is insoluble, unstable at various pH and is also prone to undergo photodegradation. Nanotechnology can help improve the therapeutic potential of drug molecules with compromised biopharmaceutical profiles. Solid lipid nanoparticles (SLNs) are the latest offshoot of nanomedicine with proven advantages of high drug payload, longer shelf life, biocompatibility and biodegradability, and industrial amenability of the production process. We successfully developed CLEN (Curcumin encapsulated lipidic nanoconstructs) containing 15 mg curcumin per ml of the SLN dispersion with highest (till date, to our knowledge) increase in solubility of curcumin in an aqueous system by 1.4 × 106 times as compared to its intrinsic solubility of 11 ng/ml and high drug loading (15% w/v with respect to lipid matrix). Zero-order release kinetics observed for CLEN versus first order release for free curcumin establish controlled release nature of the developed CLEN. It showed 69.78 times higher oral bioavailability with respect to free curcumin; 9.00 times higher than a bioavailable marketed formulation (CurcuWIN®). The formulation showed 104, 13.3, and 10-times enhanced stability at pH 6.8, 1.2, and 7.4, respectively. All these factors ensure the efficacy of CLEN in treating cancer and other inflammatory diseases.

Assessment of Amphiphilic Poly-N-vinylpyrrolidone Nanoparticles' Biocompatibility with Endothelial Cells in Vitro and Delivery of an Anti-Inflammatory Drug.

Nanoparticles (NPs) produced from amphiphilic derivatives of poly-N-vinylpyrrolidone (Amph-PVP), composed of various molecular weight polymeric hydrophilic fragments linked into hydrophobic n-alkyl chains of varying lengths, were previously shown to exert excellent biocompatibility. Although routes of administration can be different, finally, most nanosystems enter the blood circulation or lymphatic vessels, and by this, they establish direct contact with endothelial cells. In this study, Amph-PVP NPs and fluorescently labeled Amph-PVP-based NPs, namely "PVP" NPs (Amph-PVP-NPs (6000 Da) unloaded) and "F"-NPs (Amph-PVP-NPs (6000 Da) loaded with fluorescent FITC), were synthesized to study Amph-PVP NPs interactions with HMEC-1 endothelial cells. PVP NPs were readily uptaken by HMEC-1 cells in a concentration-dependent manner, as demonstrated by immunofluorescence imaging. Upon uptake, the FITC dye was localized to the perinuclear region and cytoplasm of treated cells. The generation of lipopolysaccharide (LPS)-induced activated endothelium model revealed an increased uptake of PVPNPs, as shown by confocal microscopy. Both unloaded PVP NPs and F-NPs did not affect EC viability in the 0.01 to 0.066 mg/mL range. Furthermore, we focused on the potential immunological activation of HMEC-1 endothelial cells upon PVPNPs treatment by assessing the expression of their E-Selectin, ICAM-1, and VCAM-1 adhesion receptors. None of the adhesion molecules were affected by NP treatments of both activated by LPS and nonactivated HMEC-1 cells, at the utilized concentrations (p = NS). In this study, PVP (6000 Da) NPs were used to encapsulate indomethacin, a widely used anti-inflammatory drug. The synthesized drug carrier complex did not affect HMEC-1 cell growth and expression of E-selectin, ICAM-1, and VCAM-1 adhesion receptors. In summary, PVP-based NPs are safe for use on both basal and activated endothelium, which more accurately mimics pathological conditions. Amph-PVP NPs are a promising drug delivery system.

Dietary Methionine Supplementation Exacerbates Gastrointestinal Toxicity in a Mouse Model of Abdominal Irradiation

Identification of appropriate dietary strategies for prevention of weight and muscle loss in cancer patients is crucial for successful treatment and prolonged patient survival. High-protein oral nutritional supplements decrease mortality and improve indices of nutritional status in cancer patients; however, high-protein diets are often rich in methionine, and experimental evidence indicates that a methionine-supplemented diet (MSD) exacerbates gastrointestinal toxicity after total body irradiation. Here, we sought to investigate whether MSD can exacerbate gastrointestinal toxicity after local abdominal irradiation, an exposure regimen more relevant to clinical settings. Male CBA/CaJ mice fed either a methionine-adequate diet or MSD (6.5 mg methionine/kg diet vs 19.5 mg/kg) received localized abdominal X-irradiation (220 kV, 13 mA) using the Small Animal Radiation Research Platform, and tissues were harvested 4, 7, and 10 days after irradiation. MSD exacerbated gastrointestinal toxicity after local abdominal irradiation with 12.5 Gy. This was evident as impaired nutrient absorption was paralleled by reduced body weight recovery. Mechanistically, significant shifts in the gut ecology, evident as decreased microbiome diversity, and substantially increased bacterial species that belong to the genus Bacteroides triggered proinflammatory responses. The latter were evident as increases in circulating neutrophils with corresponding decreases in lymphocytes and associated molecular alterations, exhibited as increases in mRNA levels of proinflammatory genes Icam1, Casp1, Cd14, and Myd88. Altered expression of the tight junction-related proteins Cldn2, Cldn5, and Cldn6 indicated a possible increase in intestinal permeability and bacterial translocation to the liver. We report that dietary supplementation with methionine exacerbates gastrointestinal syndrome in locally irradiated mice. This study demonstrates the important roles registered dieticians should play in clinical oncology and furtherunderlines the necessity of preclinical and clinical investigations in the role of diet in the success of cancer therapy.