Anti-Inflammatory Activity of Ferula assafoetida Oleo-Gum-Resin (Asafoetida) against TNF-α-Stimulated Human Umbilical Vein Endothelial Cells (HUVECs).

Inflammation is the body's biological reaction to endogenous and exogenous stimuli. Recent studies have demonstrated several anti-inflammatory properties of Ferula species. In this paper, we decided to study the anti-inflammatory effect of ethanolic extract of Ferula assafoetida oleo-gum-resin (asafoetida) against TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in a flat-bottom plate and then treated with ethanolic extract of asafoetida (EEA, 0-500 µg/ml) and TNF-α (0-100 ng/ml) for 24 h. We used the MTT test to assess cell survival. In addition, the LC-MS analysis was performed to determine the active substances. HUVECs were pretreated with EEA and then induced by TNF-α. Intracellular reactive oxygen species (ROS) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs were evaluated with DCFH-DA and CFSE fluorescent probes, respectively. Gene expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin and surface expression of ICAM-1 protein were measured using real-time PCR and flow cytometry methods, respectively. While TNF-α significantly increased intracellular ROS formation and PBMC adhesion to TNF-α-induced HUVECs, the pretreatment of HUVECs with EEA (125 and 250 µg/ml) significantly reduced the parameters. In addition, EEA pretreatment decreased TNF-α-induced mRNA expression of VCAM-1 and surface protein expression of ICAM-1 in the target cells. Taken together, the results indicated that EEA prevented ROS generation, triggered by TNF-α, and inhibited the expression of VCAM-1 and ICAM-1, leading to reduced PBMC adhesion. These findings suggest that EEA can probably have anti-inflammatory properties.

Enforced sialyl-Lewis-X (sLeX) display in E-selectin ligands by exofucosylation is dispensable for CD19-CAR T-cell activity and bone marrow homing.

CD19-directed chimeric antigen receptors (CAR) T cells induce impressive rates of complete response in advanced B-cell malignancies, specially in B-cell acute lymphoblastic leukemia (B-ALL). However, CAR T-cell-treated patients eventually progress due to poor CAR T-cell persistence and/or disease relapse. The bone marrow (BM) is the primary location for acute leukemia. The rapid/efficient colonization of the BM by systemically infused CD19-CAR T cells might enhance CAR T-cell activity and persistence, thus, offering clinical benefits. Circulating cells traffic to BM upon binding of tetrasaccharide sialyl-Lewis X (sLeX)-decorated E-selectin ligands (sialofucosylated) to the E-selectin receptor expressed in the vascular endothelium. sLeX-installation in E-selectin ligands is achieved through an ex vivo fucosylation reaction. Here, we sought to characterize the basal and cell-autonomous display of sLeX in CAR T-cells activated using different cytokines, and to assess whether exofucosylation of E-selectin ligands improves CD19-CAR T-cell activity and BM homing. We report that cell-autonomous sialofucosylation (sLeX display) steadily increases in culture- and in vivo-expanded CAR T cells, and that, the cytokines used during T-cell activation influence both the degree of such endogenous sialofucosylation and the CD19-CAR T-cell efficacy and persistence in vivo. However, glycoengineered enforced sialofucosylation of E-selectin ligands was dispensable for CD19-CAR T-cell activity and BM homing in multiple xenograft models regardless the cytokines employed for T-cell expansion, thus, representing a dispensable strategy for CD19-CAR T-cell therapy.

Interferon Gamma Mediates Hematopoietic Stem Cell Activation and Niche Relocalization through BST2.

During chronic infection, the inflammatory cytokine interferon gamma (IFNγ) damages hematopoietic stem cells (HSCs) by disrupting quiescence and promoting excessive terminal differentiation. However, the mechanism by which IFNγ hinders HSC quiescence remains undefined. Using intravital 3-dimensional microscopy, we find that IFNγ disrupts the normally close interaction between HSCs and CXCL12-abundant reticular (CAR) cells in the HSC niche. IFNγ stimulation increases expression of the cell surface protein BST2, which we find is required for IFNγ-dependent HSC relocalization and activation. IFNγ stimulation of HSCs increases their E-selectin binding by BST2 and homing to the bone marrow, which depends on E-selectin binding. Upon chronic infection, HSCs from mice lacking BST2 are more quiescent and more resistant to depletion than HSCs from wild-type mice. Overall, this study defines a critical mechanism by which IFNγ promotes niche relocalization and activation in response to inflammatory stimulation and identifies BST2 as a key regulator of HSC quiescence. VIDEO ABSTRACT.

Dietary γ-Glutamyl Valine Ameliorates TNF-α-Induced Vascular Inflammation via Endothelial Calcium-Sensing Receptors.

γ-Glutamyl valine (γ-EV), commonly found in edible beans, was shown to reduce gastrointestinal inflammation via activation of calcium-sensing receptors (CaSRs). The present study aimed to evaluate the efficacy of γ-EV in modulating the tumor necrosis factor-α-induced inflammatory responses in endothelial cells (ECs) via CaSR-mediated pathways. Human aortic ECs (HAoECs) were pretreated (2 h) with γ-EV (0.01, 0.1, and 1 mM). 1 mM pretreatment of γ-EV significantly reduced the upregulation of inflammatory adhesion molecules, VCAM-1 and E-selectin, by 44.56 and 57.41%, respectively. The production of cytokines IL-8 and IL-6 was significantly reduced by 40 and 51%, respectively, with 1 mM pretreatment of γ-EV. Similarly, there was a significant reduction in chemokine MCP-1 from a positive control of 9.70 ± 0.52 to 6.6 ± 0.43 ng/mL, after γ-EV treatment. The anti-inflammatory effect of γ-EV was attenuated by the treatment of the CaSR-specific inhibitor, NPS-2143, suggesting the involvement of CaSR-mediated pathways. Further studies identified the critical role of key modulators, such as ß-arrestin2 and cyclic adenosine monophosphate response element-binding protein, in mediating the CaSR-dependent anti-inflammatory effect of γ-EV. Finally, the transport efficiency of γ-EV was evaluated through a monolayer of intestinal epithelial cells (Caco-2), and the apparent permeability (Papp) of the peptide was found to be 1.56 × 10-6 cm/s.

E-selectin targeted immunoliposomes for rapamycin delivery to activated endothelial cells.

Activated endothelial cells play a pivotal role in the pathology of inflammatory disorders and thus present a target for therapeutic intervention by drugs that intervene in inflammatory signaling cascades, such as rapamycin (mammalian target of rapamycin (mTOR) inhibitor). In this study we developed anti-E-selectin immunoliposomes for targeted delivery to E-selectin over-expressing tumor necrosis factor-α (TNF-α) activated endothelial cells. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3.;hosphocholine (DPPC), Cholesterol, and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-maleimide (DSPE-PEG-Mal) were loaded with rapamycin via lipid film hydration, after which they were further functionalized by coupling N-succinimidyl-S-acetylthioacetate (SATA)-modified mouse anti human E-selectin antibodies to the distal ends of the maleimidyl (Mal)-PEG groups. In cell binding assays, these immunoliposomes bound specifically to TNF-α activated endothelial cells. Upon internalization, rapamycin loaded immunoliposomes inhibited proliferation and migration of endothelial cells, as well as expression of inflammatory mediators. Our findings demonstrate that rapamycin-loaded immunoliposomes can specifically inhibit inflammatory responses in inflamed endothelial cells.

The First Step in Adoptive Cell Immunotherapeutics: Assuring Cell Delivery via Glycoengineering.

Despite decades of intensive attention directed to creation of genetically altered cells (e.g., as in development of chimeric antigen receptor (CAR) T-cells) and/or to achieve requisite in vitro accumulation of desired immunologic effectors (e.g., elaboration of virus-specific T cells, expansion of NK cells, differentiation of dendritic cells, isolation, and propagation of Tregs, etc.), there has been essentially no interest in the most fundamental of all hurdles: assuring tissue-specific delivery of administered therapeutic cells to sites where they are needed. With regards to use of CAR T-cells, the absence of information on the efficacy of cell delivery is striking, especially in light of the clear association between administered cell dose and adverse events, and the obvious fact that pertinent cell acquisition/expansion costs would be dramatically curtailed with more efficient delivery of the administered cell bolus. Herein, based on information garnered from studies of human leukocytes and adult stem cells, the logic underlying the use of cell surface glycoengineering to enforce E-selectin ligand expression will be conveyed in the context of how this approach offers strategies to enhance delivery of CAR T-cells to marrow and to tumor beds. This application of glycoscience principles and techniques with intention to optimize cell therapeutics is a prime example of the emerging field of "translational glycobiology."

Lunasin abrogates monocytes to endothelial cells.

The adherence of monocytes to endothelial cells plays a causal role in the early development of atherosclerosis and is driven by several inflammatory stimuli, which includes oxidized low-density lipoprotein (ox-LDL). Lunasin, a natural peptide identified in soybean seeds, soy-derived food products, other grains and herbal plants, has been found to exert numerous biological activities, including anti-inflammatory and antioxidant properties. However, little is known regarding the mechanism of action of lunasin in ox-LDL-induced endothelial inflammation. The results of the present study indicate that lunasin significantly ameliorated ox-LDL-induced adhesion of THP-1 monocytes to the surface of human umbilical vein endothelial cells (HUVECs). Lunasin also suppressed expression of the adhesion molecules VCAM-1 and E-selectin, but not ICAM-1. Notably, the inhibitory mechanism of lunasin is associated with its stimulatory effects on expression of the KLF2 transcriptional factor. In addition, lunasin treatment could reverse the effects of ox-LDL on the expression of eNOS and PAI-1, the direct target genes of KLF2. Mechanistically, it was proven that the MEK5/ERK5 pathway mediates the effects of lunasin on KLF2 expression. Taken together, the results of this study suggest that dietary or supplementary intake of lunasin may have a prophylactic or therapeutic capacity in cardiovascular diseases such as atherosclerosis.

PBCA-based polymeric microbubbles for molecular imaging and drug delivery.

Microbubbles (MB) are routinely used as contrast agents for ultrasound (US) imaging. We describe different types of targeted and drug-loaded poly(n-butyl cyanoacrylate) (PBCA) MB, and demonstrate their suitability for multiple biomedical applications, including molecular US imaging and US-mediated drug delivery. Molecular imaging of angiogenic tumor blood vessels and inflamed atherosclerotic endothelium is performed by modifying the surface of PBCA MB with peptides and antibodies recognizing E-selectin and VCAM-1. Stable and inertial cavitation of PBCA MB enables sonoporation and permeabilization of blood vessels in tumors and in the brain, which can be employed for direct and indirect drug delivery. Direct drug delivery is based on US-induced release of (model) drug molecules from the MB shell. Indirect drug delivery refers to US- and MB-mediated enhancement of extravasation and penetration of co-administered drugs and drug delivery systems. These findings are in line with recently reported pioneering proof-of-principle studies showing the usefulness of (phospholipid) MB for molecular US imaging and sonoporation-enhanced drug delivery in patients. They aim to exemplify the potential and the broad applicability of combining MB with US to improve disease diagnosis and therapy.

Upregulation of endothelial cell adhesion molecules characterizes veins close to granulomatous infiltrates in the renal cortex of cats with feline infectious peritonitis and is indirectly triggered by feline infectious peritonitis virus-infected monocytes in vitro.

One of the most characteristic pathological changes in cats that have succumbed to feline infectious peritonitis (FIP) is a multifocal granulomatous phlebitis. Although it is now well established that leukocyte extravasation elicits the inflammation typically associated with FIP lesions, relatively few studies have aimed at elucidating this key pathogenic event. The upregulation of adhesion molecules on the endothelium is a prerequisite for stable leukocyte-endothelial cell (EC) adhesion that necessarily precedes leukocyte diapedesis. Therefore, the present work focused on the expression of the EC adhesion molecules and possible triggers of EC activation during the development of FIP. Immunofluorescence analysis revealed that the endothelial expression of P-selectin, E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) was elevated in veins close to granulomatous infiltrates in the renal cortex of FIP patients compared to non-infiltrated regions and specimens from healthy cats. Next, we showed that feline venous ECs become activated when exposed to supernatant from feline infectious peritonitis virus (FIPV)-infected monocytes, as indicated by increased adhesion molecule expression. Active viral replication seemed to be required to induce the EC-stimulating activity in monocytes. Finally, adhesion assays revealed an increased adhesion of naive monocytes to ECs treated with supernatant from FIPV-infected monocytes. Taken together, our results strongly indicate that FIPV activates ECs to increase monocyte adhesion by an indirect route, in which proinflammatory factors released from virus-infected monocytes act as key intermediates.

Fulfilling Koch's postulates in glycoscience: HCELL, GPS and translational glycobiology.

Glycoscience-based research that is performed expressly to address medical necessity and improve patient outcomes is called "translational glycobiology". In the 19th century, Robert Koch proposed a set of postulates to rigorously establish causality in microbial pathogenesis, and these postulates can be reshaped to guide knowledge into how naturally-expressed glycoconjugates direct molecular processes critical to human well-being. Studies in the 1990s indicated that E-selectin, an endothelial lectin that binds sialofucosylated carbohydrate determinants, is constitutively expressed on marrow microvessels, and investigations in my laboratory indicated that human hematopoietic stem cells (HSCs) uniquely express high levels of a specialized glycoform of CD44 called "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a highly potent E-selectin ligand. To assess the role of HCELL in directing HSC migration to marrow, a method called "glycosyltransferase-programmed stereosubstitution" (GPS) was developed to custom-modify CD44 glycans to enforce HCELL expression on viable cell surfaces. Human mesenchymal stem cells (MSCs) are devoid of E-selectin ligands, but GPS-based glycoengineering of CD44 on MSCs licenses homing of these cells to marrow in vivo, providing direct evidence that HCELL serves as a "bone marrow homing receptor". This review will discuss the molecular basis of cell migration in historical context, will describe the discovery of HCELL and its function as the bone marrow homing receptor, and will inform on how glycoengineering of CD44 serves as a model for adapting Koch's postulates to elucidate the key roles that glycoconjugates play in human biology and for realizing the immense impact of translational glycobiology in clinical medicine.

Tricin, flavonoid from Njavara reduces inflammatory responses in hPBMCs by modulating the p38MAPK and PI3K/Akt pathways and prevents inflammation associated endothelial dysfunction in HUVECs.

Previous studies revealed the potent anti-inflammatory activity of tricin, the active component of Njavara rice bran. Here, we report the involvement of specific signaling pathways in the protective effect of tricin against LPS induced inflammation in hPBMCs and the role of tricin in modulating endothelial dysfunction in LPS induced HUVECs. Pretreatment with tricin (15µM) significantly inhibited the release of TNF-α and was comparable to the specific pathway blockers like ERK inhibitor (PD98059), JNK inhibitor (SP600125) and p38 inhibitor (SB203580), whereas an increased release of TNF-α was observed in PI3K/Akt inhibitor (LY294002) treated cells. Tricin alone and combination treatment of tricin and SB203580 showed more significant inhibition of activation of COX-2 and TNF-α than that of SB203580 alone treated group. Combination treatment of tricin and LY294002 showed increased activation of COX-2 and TNF-α, proved that PI3K activation is essential for the anti-inflammatory effect of tricin. Studies conducted on HUVECs revealed the protective effect of tricin against endothelial dysfunction associated with LPS induced inflammation by inhibiting the activation of proinflammatory mediators like TNF-α, IFN-γ, MCP 1 by modulating NF-κB and MAPK signaling pathways. ELISA and flow cytometric analysis again confirmed the protection of tricin against endothelial damage, especially from the decreased activation of cell adhesion molecules like ICAM-1, VCAM-1 and E-Selectin upon tricin treatment. This work establishes the mechanism behind the potent anti-inflammatory activity of the flavonoid tricin.

Identification and Expression Analysis of Zebrafish (Danio rerio) E-Selectin during Embryonic Development.

In this study, we cloned the full-length cDNA of E-selectin of zebrafish (Danio rerio), analyzed its expression pattern and preliminarily explored its biological function. Zebrafish E-selectin cDNA is 3146 bp and encodes a putative 871 amino acid protein. All structural domains involved in E-selectin function are conserved in the putative protein. Whole-mount in situ hybridization of zebrafish at 24 and 48 h post-fertilization (hpf) revealed E-selectin expression mainly in vascular/endothelial progenitor cells in the posterior trunk and blood cells in the intermediate cell mass and posterior cardinal vein regions. Real-time quantitative RT-PCR analysis detected E-selectin expression at 0.2, 24 and 48 hpf and significantly decreased from 48 to 72 hpf. The expression of E-selectin, tumor necrosis factor-α and interleukin-1ß was significantly upregulated at 22 to 72 h after induction with bacterial lipopolysaccharide. Thus, the structure of E-selectin protein is highly conserved among species, and E-selectin may be involved in embryonic development and essential for hematopoiesis and angiogenesis during embryonic development in zebrafish. Furthermore, we provide the first evidence of inflammatory mediators inducing E-selectin expression in non-mammalian vertebrates, which suggests that zebrafish E-selectin may be involved in inflammation and probably has similar biological function to mammalian E-selectin.

Cross-Talk between Shp1 and PIPKIγ Controls Leukocyte Recruitment.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLß2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly). Despite the clinical importance, cell-intrinsic molecular mechanisms of negative regulation of integrin adhesiveness and neutrophil recruitment are poorly understood. Mice deficient in the tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase 1 (Shp1) show increased leukocyte adhesion, but the interpretation of these data is limited by the severe global phenotype of these mice. In this study, we used mice with global and myeloid-restricted deletion of Shp1 to study neutrophil arrest, adhesion, crawling, and transendothelial migration in vitro and in vivo. Shp1 deficiency results in increased neutrophil adhesion in vivo; however, neutrophil crawling, transmigration, and chemotaxis were reduced in these mice. Mechanistically, Shp1 binds and controls PIPKIγ activity and, thereby, modulates phosphatidylinositol (4,5)-bisphosphate levels and adhesion. Thus, Shp1 is involved in the deactivation of integrins and regulation of neutrophil recruitment into inflamed tissue.

A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

Oral administration of non-absorbable delayed release 6-mercaptopurine is locally active in the gut, exerts a systemic immune effect and alleviates Crohn's disease with low rate of side effects: results of double blind Phase II clinical trial.

Therapy for Crohn's disease (CD) with thiopurines is limited by systemic side effects. A novel formulation of fixed-dose, delayed-release 6-mercaptopurine (DR-6MP) was developed, with local effect on the gut immune system and minimal absorption. The aim of this study was to evaluate the safety and efficacy of DR-6MP in patients with moderately severe CD compared to systemically delivered 6-mercaptopurine (Purinethol). Seventy CD patients were enrolled into a 12-week, double-blind controlled trial. The primary end-point was the percentage of subjects with clinical remission [Crohn's Disease Activity Index (CDAI) < 150] or clinical response (100-point CDAI reduction). Twenty-six (56·5%) and 13 (54·2%) subjects from the DR-6MP and Purinethol cohorts, respectively, completed the study. DR-6MP had similar efficacy to Purinethol following 12 weeks of treatment. However, the time to maximal clinical response was 8 weeks for DR-6MP versus 12 weeks for Purinethol. A higher proportion of patients on DR-6MP showed clinical remission at week 8. A greater improvement in Inflammatory Bowel Disease Questionnaire (IBDQ) score was noted in the DR-6MP group. DR-6MP led to a decrease of CD62(+) expression on T cells, implying a reduction of lymphocyte adhesion to site of inflammation. DR-6MP was safer than Purinethol, with significantly fewer adverse events (AEs). There was no evidence of drug-induced leucopenia in the DR-6MP group; the proportion of subjects who developed hepatotoxicity was lower for the DR-6MP. Non-absorbable DR-6MP is safe and biologically active in the gut. It is clinically effective, exerting a systemic immune response with low systemic bioavailability and a low incidence of side effects.

Characterization of Adsorbents for Cytokine Removal from Blood in an In Vitro Model.

INTRODUCTION: Cytokines are basic targets that have to be removed effectively in order to improve the patient's health status in treating severe inflammation, sepsis, and septic shock. Although there are different adsorbents commercially available, the success of their clinical use is limited. Here, we tested different adsorbents for their effective removal of cytokines from plasma and the resulting effect on endothelial cell activation. METHODS: The three polystyrene divinylbenzene (PS-DVB) based adsorbents Amberchrom CG161c and CG300m and a clinically approved haemoperfusion adsorbent (HAC) were studied with regard to cytokine removal in human blood. To induce cytokine release from leucocytes, human blood cells were stimulated with 1 ng/ml LPS for 4 hours. Plasma was separated and adsorption experiments in a dynamic model were performed. The effect of cytokine removal on endothelial cell activation was evaluated using a HUVEC-based cell culture model. The beneficial outcome was assessed by measuring ICAM-1, E-selectin, and secreted cytokines IL-8 and IL-6. Additionally the threshold concentration for HUVEC activation by TNF-α and IL-1ß was determined using this cell culture model. RESULTS: CG161c showed promising results in removing the investigated cytokines. Due to its pore size the adsorbent efficiently removed the key factor TNF-α, outperforming the commercially available adsorbents. The CG161c treatment reduced cytokine secretion and expression of cell adhesion molecules by HUVEC which underlines the importance of effective removal of TNF-α in inflammatory diseases. CONCLUSION: These results confirm the hypothesis that cytokine removal from the blood should approach physiological levels in order to reduce endothelial cell activation.

Hibernation induces immune changes in the lung of 13-lined ground squirrels (Ictidomys tridecemlineatus).

During hibernation, significant changes occur in the systemic and intestinal immune populations. We found that the lungs of hibernating 13-lined ground squirrels (Ictidomys tridecemlineatus) also undergo shifts in immune phenotype. Within the population of mononuclear cells, the percentage of T cells increases and the percentage of CD11b/c(+) cells decreases in hibernators. E-selectin, which promotes endothelial attachment, increases during arousal from torpor. Levels of the anti-inflammatory cytokine interleukin (IL)-10 in the lung are lower during hibernation while levels of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α remain constant. Expression of suppressor of cytokine signaling (SOCS) proteins is also decreased in torpid hibernators. Our data point to a unique immune phenotype in the lung of hibernating ground squirrels in which certain immunosuppressive proteins are downregulated while some potentially inflammatory proteins are maintained or amplified. This indicates that the lung houses an immune population that can potentially respond to antigenic challenge during hibernation.

Effect of P/E-selectin blockage on antisperm antibody development and histopathological alterations in experimental orchitis.

AIM: This study aimed to evaluate the effect of P/E-selectin blockage on antisperm antibody (ASA) development and histopathological alterations in experimental orchitis. MATERIALS AND METHODS: Thirty-six Wistar albino-type male rats weighing 100-150 g were included in the study. Rats were allocated into six groups (n = 6) including control (CG), sham (SG), orchitis (OG), antimicrobial treatment (AG), P/E-selectin blockage (PESG), and both antimicrobial and P/E-selectin treatment (TG) groups. In CG, serum samples were taken from the tail vein prior to the procedure and followed by extraction of both testes. In SG, 1 ml of saline solution was injected in testicular parenchyma. OG was obtained by injecting 0.1 ml 106 cfu/ml Escherichia coli (0:6 strain) and 1 ml saline solution into the right testes. AG received ciprofloxacin (50 mg/kg/day) twice a day through gastrogavage 24 hours after generating orchitis. In PESG, P/E-selectin antibody (100 µg) was administered intravenously via the tail vein 24 hours after the induction of orchitis. Finally, both ciprofloxacin and P/E-selectin antibody were administered in TG 24 hours after the induction of orchitis for 14 days. At the end of treatment, 1 ml of serum sample was obtained to evaluate the ASA, P-selectin and E-selectin levels. In order to evaluate spermatogenesis (Johnsen score) and testicular injury (Cosentino score), both testes were extracted at the end of the 14th day. RESULTS: In orchitis-induced groups (OG, ATG, PSEG, TG), ASA levels were significantly increased at the 14th day when compared to SG (p < 0.05). In TG, ASA levels were decreased when compared to AG. However, similar alteration in ASA levels was not detected in PSEG (p > 0.05). In OG and AG, P-selectin levels were decreased at the 14th day when compared to levels observed on 0 day (p < 0.05). E-selectin levels on 0 day showed that each group had higher levels of E-selectin when compared to CG (p > 0.05). There was no significant difference regarding E-selectin when compared to CG (p > 0.05). No significant differences regarding E-selectin levels were detected on the 0th and 14th days between AG and CG (p > 0.05). When the Cosentino and Johnsen scores were compared among groups, TG and PSEG has decreased scores of Cosentino than OG on the right testicle (p < 0.05). In contrast, an increased Johnsen score was detected in TG and PSEG when compared to OG (p < 0/05). No significant difference was detected for both Cosentino and Johnsen scores on the left testicle (p > 0.05). There was no difference with regard to the right and left testicular injury in TG. In P/E-blocked groups, decreased histopathological alterations were observed in the contralateral testis. CONCLUSION: P/E-selectin blockage may reduce ASA production after orchitis when combined with antimicrobial treatment. P/E-selectin blockage not only has a protective effect on blood-testis barrier but also decreases the histopathological alterations in both the affected and contralateral testis. Histopathological parameters of spermatogenesis may also be prevented by P/E-selectin blockage in experimental orchitis.

Anti-VCAM-1 and anti-E-selectin SAINT-O-Somes for selective delivery of siRNA into inflammation-activated primary endothelial cells.

Activated endothelial cells play a pivotal role in the pathology of inflammatory diseases and present a rational target for therapeutic intervention by endothelial specific delivery of short interfering RNAs (siRNA). This study demonstrates the potential of the recently developed new generation of liposomes based on cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) for functional and selective delivery of siRNA into inflamed primary endothelial cells. To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1) or respectively E-selectin and tested in TNF-α activated primary endothelial cells from venous and aortic vascular beds. Both targeted SAINT-O-Somes carrying siRNA against the endothelial gene VE-cadherin specifically downregulated its target mRNA and protein without exerting cellular toxicity. SAINT-O-Somes formulated with siRNA formed small particles (106 nm) with a 71% siRNA encapsulation efficiency. SAINT-O-Somes were stable in the presence of serum at 37 °C, protected siRNA from degradation by serum RNases, and after i.v. injection displayed pharmacokinetic comparable to conventional long circulating liposomes. These anti-VCAM-1 and anti-E-selectin SAINT-O-Somes are thus a novel drug delivery system that can achieve specific and effective delivery of siRNA into inflamed primary endothelial cells and have physicochemical features that comply with in vivo application demands.