A clinical study of the local injection of a freshly manufactured 35 kDa hyaluronan fragment for treating chronic wounds.

This study manufactured a 35 kDa hyaluronan fragment (HA35) by enzymatically degrading high-molecular-weight HA using hyaluronidase PH20 derived from bovine testis. The research then examined the therapeutic efficacy of locally administered, tissue-permeable HA35 in alleviating chronic wounds and their associated neuropathic pain. For 20 patients with nonhealing wounds and associated pain lasting over three months, 100 mg of HA35 was injected daily into the healthy skin surrounding the chronic wound for 10 days. Self-assessments before and after treatment indicated that HA35 significantly enhanced wound healing. This was evidenced by the formation of fresh granulation tissue on the wounds (p < 0.0001); reduced darkness, redness, dryness, and damage in the skin surrounding the wounds (p < 0.0001), and a decrease in wound size (p < 0.001). Remarkably, HA35 injections alleviated pain associated with chronic wounds within 24 hours (p < 0.0001). It can be concluded that the low-molecular-weight hyaluronan fragment HA35 potentially enhances the immune response and angiogenesis during wound healing.

Characterization, Bioactivity, and Biodistribution of 35 kDa Hyaluronan Fragment.

It has been reported that hyaluronic acid (HA) with a 35 kDa molecular weight (HA35) acts biologically to protect tissue from injury, but its biological properties are not yet fully characterized. This study aimed to evaluate the cellular effects and biodistribution of HA35 compared to HA with a 1600 kDa molecular weight (HA1600). We assessed the effects of HA35 and HA1600 on cell migration, NO and ROS generation, and gene expression in cultured macrophages, microglia, and lymphocytes. HA35 was separately radiolabeled with 99mTc and 125I and administered to C57BL/6J mice for in vivo biodistribution imaging. In vitro studies indicated that HA35 and HA1600 similarly enhanced cell migration through HA receptor binding mechanisms, reduced the generation of NO and ROS, and upregulated gene expression profiles related to cell signaling pathways in immune cells. HA35 showed a more pronounced effect in regulating a broader range of genes in macrophages and microglia than HA1600. Upon intradermal or intravenous administration, radiolabeled HA35 rapidly accumulated in the liver, spleen, and lymph nodes. In conclusion, HA35 not only exhibits effects on cellular bioactivity comparable to those of HA1600 but also exerts biological effects on a broader range of immune cell gene expression. The findings herein offer valuable insights for further research into the therapeutic potential of HA35 in inflammation-mediated tissue injury.

Protective Properties of Intestinal Alkaline Phosphatase Supplementation on the Intestinal Barrier: Interactions and Effects.

The intestinal barrier is critical for maintaining intestinal homeostasis, and its dysfunction is associated with various diseases. Recent findings have revealed the multifunctional role of intestinal alkaline phosphatase (IAP) in diverse biological processes, including gut health maintenance and function. This review summarizes the protective effects of IAP on intestinal barrier integrity, encompassing the physical, chemical, microbial, and immune barriers. We discuss the results and insights from in vitro, animal model, and clinical studies as well as the available evidence regarding the impact of diet on IAP activity and expression. IAP can also be used as an indicator to assess intestinal-barrier-related diseases. Further research into the mechanisms of action and long-term health effects of IAP in maintaining overall intestinal health is essential for its future use as a dietary supplement or functional component in medical foods.

Anti-Inflammatory Effects of the 35kDa Hyaluronic Acid Fragment (B-HA/HA35).

Background: Hyaluronic acid (HA) and HA fragments interact with a variety of human body receptors and are involved in the regulation of various physiological functions and leukocyte trafficking in the body. Accordingly, the development of an injectable HA fragment with good tissue permeability, the identification of its indications, and molecular mechanisms are of great significance for its clinical application. The previous studies showed that the clinical effects of injectable 35kDa B-HA result from B-HA binding to multiple receptors in different cells, tissues, and organs. This study lays the foundation for further studies on the comprehensive clinical effects of injectable B-HA. Methods: We elaborated on the production process, bioactivity assay, efficacy analyses, and safety evaluation of an injectable novel HA fragment with an average molecular weight of 35 kDa (35 kDa B-HA), produced by recombinant human hyaluronidase PH20 digestion. Results: The results showed that 35 kDa B-HA induced human erythrocyte aggregation (rouleaux formation) and accelerated erythrocyte sedimentation rates through the CD44 receptor. B-HA application and injection treatment significantly promoted the removal of mononuclear cells from the site of inflammation and into the lymphatic circulation. At a low concentration, 35 kDa B-HA inhibited production of reactive oxygen species and tumor necrosis factor by neutrophils; at a higher concentration, 35 kDa B-HA promoted the migration of monocytes. Furthermore, 35 kDa B-HA significantly inhibited the migration of neutrophils with or without lipopolysaccharide treatment, suggesting that in local tissues, higher concentrations of 35 kDa B-HA have antiinflammatory effects. After 99mTc radiolabeled 35 kDa B-HA was intravenously injected into mice, it quickly entered into the spleen, liver, lungs, kidneys and other organs through the blood circulation. Conclusion: This study demonstrated that the HA fragment B-HA has good tissue permeability and antiinflammatory effects, laying a theoretical foundation for further clinical studies.

Anti-Inflammatory and Antioxidant Effects of Liposoluble C60 at the Cellular, Molecular, and Whole-Animal Levels.

Introduction: Liposoluble carbon-60 (C60) has potential applications in many fields, including cosmetics, medical devices, and medicine, but its specific mechanism of action remains unclear. This study explored whether liposoluble C60 could be delivered to human organs, tissues, and cells through blood, extracellular fluid, and cell culture fluid and whether it exerts anti-inflammatory and antioxidant effects at the molecular, cellular, and whole-animal levels. Methods: At the cellular level, we mixed C60 dissolved in grape seed oil with cell culture medium containing 10% serum and investigated its effects on tumor necrosis factor-α (TNF-α) release, migration, phagocytosis, respiratory burst, and apoptosis in freshly isolated human neutrophils. At the molecular level, we mixed a trace amount of C60 dissolved in grape seed oil with aqueous and ethanolic solutions and studied its antioxidant effect. At the animal level, we investigated the inhibitory effect of C60 on the serum inflammatory marker C-reactive protein (CRP) in beagle dogs after oral administration of C60 dissolved in grape seed oil. Results: The results showed that the trace amount of C60 dissolved in grape seed oil significantly inhibited TNF-α release, cell migration, phagocytosis, and respiratory burst in freshly isolated human neutrophils. In addition, the trace amount of C60 dissolved in grape seed oil had a significant scavenging effect on superoxide free radicals and 1,1-diphenyl-2-trinitrophenylhydrazine free radicals. Oral administration of C60 dissolved in grape seed oil markedly reduced the level of the serum inflammatory marker CRP in beagle dogs. Conclusion: In summary, a trace amount of hydrophobic C60 in hydrophilic media effectively produced anti-inflammatory and antioxidant effects in cells and animals. C60 dissolved in grape seed oil is a novel anti-inflammatory and antioxidant drug candidate.

Modification of fermented whey protein concentrates: Impact of sequential ultrasound and TGase cross-linking.

This study aimed to examine the impact of fermentation process on whey protein and improve the general properties of fermented whey protein concentrate (FWPC) recovered by a combined ultrafiltration-diafiltration (UF-DF) operation. Impacts of sequential ultrasound (US) pretreatment and transglutaminase (TGase) crosslinking on structural, functional, and physicochemical properties of FWPCs were investigated. Partially denatured and hydrolyzed fermented whey protein could replace heat denaturation prior to the TGase addition to a whey protein system. Sequential treatment increased the molecular weight of FWPCs as exhibited by both SEM and SDS-PAGE, which demonstrates that modification can lead to the polymers and oligomers production. The zeta potential value increased significantly after US treatment and enzyme catalysis, and all the modified FWPCs were strongly negatively charged. Compared with the secondary structure of untreated FWPCs, the percentage of α-helix and random coil in modified FWPCs significantly increased, while the percentage of ß-sheet and ß-turns reduced. Solubility, free sulfhydryl groups, and surface hydrophobicity of all FWPCs were significantly improved compared to non-fermented WPC (P < 0.05). Sequential treatment induced a substantial impact on the emulsifying activity and stability of modified samples in comparison with untreated FWPCs. Scanning electron microscope pictures confirmed the positive effects of sequential treatments on texture and void size reduction. Therefore, the application of recovering modified FWPCs is fully recommended as a commercially viable approach for enhanced protein production at the industrial scale.

A replication-deficient H9N2 influenza virus carrying H5 hemagglutinin conferred protection against H9N2 and H5N1 influenza viruses in mice.

H5N1 and H9N2 influenza viruses have been reported to cause human infections and are believed to have pandemic potential. The vaccine is an effective tool to prevent influenza virus infection. However, inactivated influenza vaccines sometimes result in low antigenicity as result leads to generating of incomplete immune protection in the form of low cellular and humoral immunity. While the low temperature adapted, traditional live attenuated influenza vaccine (LAIV) is associated with the potential risk to revert to a virulent phenotype, there appears an essential need for an alternative potent methodology to design and develop influenza vaccines with substantial safety and efficacy which may confer solid protection against H9N2 or H5N1 influenza virus infections. In the present study, a replication-deficient recombinant influenza virus, WM01ma-HA(H5), expressing hemagglutinin (HA) of both H9N2 and H5N1 subtypes was developed. The chimeric gene segment expressing HA(H5), was designed using the sequence of an open reading frame (ORF) of HA adopted from A/wild duck/Hunan/021/2005(H5N1)(HN021ma) which was flanked by the NA packaging signals of mouse-adapted strain A/Mink/Shandong/WM01/2014(H9N2)(WM01ma). Due to the absence of ORF of structural protein NA, the replication of this engineered H9N2 influenza viruses WM01ma-HA(H5) was hampered in vitro and in vivo but was well competent in MDCK cells stably expressing the NA protein of WM01ma. Intranasal vaccination of mice with WM01ma-HA(H5) stimulated robust immune response without any clinical signs and conferred complete protection from infection by H5N1 or H9N2 subtype influenza viruses.

Intestinal alkaline phosphatase (IAP, IAP Enhancer) attenuates intestinal inflammation and alleviates insulin resistance.

In this study, we investigated the effects of intestinal alkaline phosphatase (IAP) in controlled intestinal inflammation and alleviated associated insulin resistance (IR). We also explored the possible underlying molecular mechanisms, showed the preventive effect of IAP on IR in vivo, and verified the dephosphorylation of IAP for the inhibition of intestinal inflammation in vitro. Furthermore, we examined the preventive role of IAP in IR induced by a high-fat diet in mice. We found that an IAP + IAP enhancer significantly ameliorated blood glucose, insulin, low-density lipoprotein, gut barrier function, inflammatory markers, and lipopolysaccharide (LPS) in serum. IAP could dephosphorylate LPS and nucleoside triphosphate in a pH-dependent manner in vitro. Firstly, LPS is inactivated by IAP and IAP reduces LPS-induced inflammation. Secondly, adenosine, a dephosphorylated product of adenosine triphosphate, elicited anti-inflammatory effects by binding to the A2A receptor, which inhibits NF-κB, TNF, and PI3K-Akt signalling pathways. Hence, IAP can be used as a natural anti-inflammatory agent to reduce intestinal inflammation-induced IR.

Carbon 60 Dissolved in Grapeseed Oil Inhibits Dextran Sodium Sulfate-Induced Experimental Colitis.

Introduction: Carbon 60 (C60) and its derivatives have various biological applications. In our laboratory, we have demonstrated that C60 dissolved in grape seed oil (C60-Oil) has antioxidant and anti-inflammatory properties; however, the effectiveness of this formulation to treat diseases of the intestinal tract and specifically ulcerative colitis has not been studied. In this study, we intend to explore the effects of C60-Oil against experimental ulcerative colitis induced by Dextran Sulfate Sodium (DSS) in rats and a human colorectal cell line, HT-29. Methods: The rats were randomly distributed into three groups: a negative control group with no induced damage and two other groups were treated with DSS to induce UC for seven days: one as untreated control and the other group treated with C60-Oil 3 mg/kg/day. We quantified the clinical manifestations of the disease, body weight, colon weight, microscopic damage score, and colonic content of IL-6, TNF-alpha, IL-1B, and IL-10. As part of the cell studies, HT-29 cells were pretreated with C60-Oil at different concentrations (0.1, 1, 5, 10, 50, 30 µg/mL) and then stimulated with DSS (10 µg/mL). We measured the levels of IL-8 and NO secreted in the medium and the intracellular levels of ROS. Results: Oral treatment with C60-Oil significantly prevented the change in body weight, reduced most of the clinical signs of the disease, colon weight, microscopic damage score, and considerably improved the profile of cytokines analyzed. The pretreatment of HT-29 cells also protected the cells from the action of DSS as it reduced the levels of IL-8, NO, and ROS. Conclusion: According to our results, we can suggest C60-Oil, as a formulation with pharmacological potential for treating ulcerative colitis.

Dual Functional Eudragit® S100/L30D-55 and PLGA Colon-Targeted Nanoparticles of Iridoid Glycoside for Improved Treatment of Induced Ulcerative Colitis.

AIM: Iridoid glycosides (IG) as the major active fraction of Syringa oblata Lindl. has a proven anti-inflammatory effect for ulcerative colitis (UC). However, its current commercial formulations are hampered by low bioavailability and unable to reach inflamed colon. To overcome the limitation, dual functional IG-loaded nanoparticles (DFNPs) were prepared to increase the residence time of IG in colon. The protective mechanism of DFNPs on DSS-induced colonic injury was evaluated in rats. MATERIALS AND METHODS: We prepared DFNPs using the oil-in-water emulsion method. PLGA was selected as sustained-release polymer, and ES100 and EL30D-55 as pH-responsive polymers. The morphology and size distribution of NPs were measured by SEM and DLS technique. To evaluate colon targeting of DFNPs, DiR, was encapsulated as a fluorescent probe into NPs. Fluorescent distribution of NPs were investigated. The therapeutic potential and in vivo transportation of NPs in gastrointestinal tract were evaluated in a colitis model. RESULTS: SEM images and zeta data indicated the successful preparation of DFNPs. This formulation exhibited high loading capacity. Drug release results suggested DFNPs released less than 20% at the first 6 h in simulated gastric fluid (pH1.2) and simulated small intestine fluid (pH6.8). A high amount of 84.7% sustained release from NPs in simulated colonic fluid (pH7.4) was beyond 24 h. DiR-loaded NPs demonstrated a much higher colon accumulation, suggesting effective targeting due to functionalization with pH and time-dependent polymers. DFNPs could significantly ameliorate the colonic damage by reducing DAI, macroscopic score, histological damage and cell apoptosis. Our results also proved that the potent anti-inflammatory effect of DFNPs is contributed by decrease of NADPH, gene expression of COX-2 and MMP-9 and the production of TNF-α, IL-17, IL-23 and PGE2. CONCLUSION: We confirm that DFNPs exert protective effects through inhibiting the inflammatory response, which could be developed as a potential colon-targeted system.

Bioactive hyaluronic acid fragments inhibit lipopolysaccharide-induced inflammatory responses via the Toll-like receptor 4 signaling pathway.

The high- and the low-molecular weight hyaluronic acids (HMW-HA and LMW-HA, respectively) showed different biological activities in inflammation. However, the role of LMW-HA in inflammatory response is controversial. In this study, we aimed to investigate the effect of bioactive hyaluronan (B-HA) on lipopolysaccharide (LPS)-induced inflammatory responses in human macrophages and mice. B-HA was produced from HA treated with glycosylated recombinant human hyaluronidase PH20. Human THP-1 cells were induced to differentiate into macrophages. THP-1-derived macrophages were treated with B-HA, LPS, or B-HA + LPS. The mRNA expression and the production of inflammatory cytokines were determined using quantitative real-time PCR and enzyme-linked immunosorbent assay. The phosphorylation levels of proteins in the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and IRF-3 signaling pathways were measured using Western blot. The in vivo efficacy of B-HA was assessed in a mouse model of LPS-induced inflammation. Results showed that B-HA inhibited the expression of TNF-α, IL-6, IL-1, and IFN-ß, and enhanced the expression of the antiinflammatory cytokine IL-10 in LPS-induced inflammatory responses in THP-1-derived macrophages and in vivo. B-HA significantly suppressed the phosphorylation of the TLR4 signaling pathway proteins p65, IKKα/ß, IκBα, JNK1/2, ERK1/2, p38, and IRF-3. In conclusion, our results demonstrated that the B-HA attenuated the LPS-stimulated inflammatory response by inhibiting the activation of the TLR4 signaling pathway. B-HA could be a potential anti-inflammatory drug in the treatment of inflammatory disease.

Inflammation imaging of atherosclerosis in Apo-E-deficient mice using a (99m)Tc-labeled dual-domain cytokine ligand.

UNLABELLED: Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) play a critical role in initiating and accelerating atherosclerosis. This study evaluated the imaging properties of (99m)Tc-TNFR2-Fc-IL-1RA ((99m)Tc-TFI), a dual-domain cytokine radioligand that targets TNF-α and IL-1ß pathways, in assessing atherosclerosis development in apolipoprotein-E-deficient (ApoE(-)(/)(-)) mice. METHODS: The feasibility and specificity of detecting atherosclerosis with (99m)Tc-TFI SPECT imaging were investigated in ApoE(-)(/)(-) and ApoE(+)(/)(+) mice. Fifty-four ApoE(-)(/)(-) mice were fed either an atherogenic diet (AGD) or a normal diet (ND) beginning at 5 weeks of age. Eighteen Apo-E wild-type (ApoE(+)(/)(+)) mice were fed an ND. Two groups of ApoE(-)(/)(-) mice (n=12 each group) on AGD and ND were imaged three times with (99m)Tc-TFI and a high-resolution SPECT system at 20-25, 30-40, and 48-52 weeks to study the evolution of atherosclerotic plaque. RESULTS: Focal radioactive accumulations in the aortic arch region were observed in the ApoE(-)(/)(-) mice (n=12) on AGD but not in the ApoE(+)(/)(+) mice on ND (n=10). Apo-E(-)(/)(-) mice on ND (n=11) exhibited lower radioactive uptake than ApoE(-)(/)(-) mice on AGD (P<0.05). Co-injection of an excess of cold ligand with (99m)Tc-TFI resulted in significant reduction of (99m)Tc-TFI uptake in the ApoE(-)(/)(-) mice on AGD. Longitudinal studies showed that (99m)Tc-TFI uptake in the aortas of ApoE(-)(/)(-) mice progressively increased from 20 to 48 weeks. Real-time PCR assays demonstrated that atherosclerotic aortas expressed significantly higher IL-1ß and TNF-α than the aortas from wild-type controls. CONCLUSIONS: Atherosclerotic plaques were detected by (99m)Tc-TFI imaging in ApoE(-)(/)(-) mice. (99m)Tc-TFI is promising for specific detection of inflammatory response in atherosclerotic plaques.

Cross talk between vascular smooth muscle cells and monocytes through interleukin-1ß/interleukin-18 signaling promotes vein graft thickening.

OBJECTIVE: Interleukin (IL)-1ß and IL-18 are key proinflammatory cytokines that play important roles in the pathophysiology of vein graft remodeling. However, the mechanism of IL-1ß/IL-18 production and its role in the development of graft remodeling remain unclear. APPROACH AND RESULTS: IL-1ß/IL-18 were rapidly expressed in venous interposition grafts. Vascular smooth muscle cell (VSMC) death and monocytic inflammasome activation occurred in grafted veins. Necrotic VSMCs induced the expression of IL-1ß, IL-18, and other inflammasome-associated proteins in monocytes, which was partially inhibited by their antagonist, recombinant IL-1ra-Fc-IL-18bp. Activated monocytes stimulated proliferation of VSMCs by activating cell growth-related signaling molecules (AKT, STAT3, ERK1/2, and mTOR [AKT/protein kinase B, signal transducer and activator of transcription 3, extracellular signal-regulated kinase 1/2, mammalian target of rapamycin]) and increasing production of platelet-derived growth factor-bb; these effects were suppressed by IL-1ra-Fc-IL-18bp. Activated monocytes also promoted migration of VSMCs, which was independent of IL-1ß/IL-18 signaling. Importantly, administration of IL-1ra-Fc-IL-18bp inhibited activation of cell growth-related signaling molecules, VSMC proliferation, and vein graft thickening in vivo. CONCLUSIONS: Our work identified an interaction among necrotic VSMCs, monocytes, and viable VSMCs through IL-1ß/IL-18 signaling, which might be exploited as a therapeutic target in vein graft remodeling.

5TNF-α and IL-1ß neutralization ameliorates angiotensin II-induced cardiac damage in male mice.

Inflammation is a key event in hypertensive organ damage, and TNF-α and IL-1ß are elevated in hypertension. In this study, we evaluated the effects of TNF-α and IL-1ß elevation on hypertensive cardiac damage by treatment with a bifunctional inflammatory inhibitor, TNF receptor 2-fragment crystalization-IL-1 receptor antagonist (TFI), which can neutralize these 2 cytokines simultaneously. A mouse hypertension model of angiotensin II (Ang II) infusion (1500 ng/kg·min for 7 d) was induced in wild-type mice. TNF-α and IL-1ß were inhibited by TFI administration (5 mg/kg, every other day), the effects of inhibition on cardiac damage were examined, and its mechanism on inflammatory infiltration was further studied in vivo and in vitro. Ang II infusion induced cardiac injury, including increased macrophage infiltration, expression of inflammatory cytokines (IL-12, IL-6, etc), and cardiac fibrosis, such as elevated α-smooth muscle actin, collagen I, and TGF-ß expression. Importantly, the Ang II-induced cardiac injury was suppressed by TFI treatment. Moreover, TFI reduced the expression of adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) and monocyte chemotactic protein-1 expression in Ang II-treated hearts. Additionally, blockade of TNF-α and IL-1ß by TFI reduced monocyte adherence to endothelia cell and macrophage migration. This study demonstrates that blocking TNF-α and IL-1ß by TFI prevents cardiac damage in response to Ang II, and targeting these 2 cytokines simultaneously might be a novel tool to treat hypertensive heart injury.

SPECT imaging of inflammatory response in ischemic-reperfused rat hearts using a 99mTc-labeled dual-domain cytokine ligand.

UNLABELLED: Soluble tumor necrosis factor (TNF) receptor-2 (TNFR2) and interleukin-1 receptor antagonist (IL-1ra) were fused to the Fc portion of IgG1 using recombinant DNA technology. The resulting dual-domain cytokine ligand, TNFR2-Fc-IL-1ra, specifically binds to TNF and to the type I IL-1 receptor (IL-1RI). This study was designed to characterize the kinetic profile of (99m)Tc-labeled TNFR2-Fc-IL-1ra (TFI) for imaging inflammatory response in an ischemic-reperfused (IR) rat heart model. METHODS: The IR model was created by ligating the left coronary artery for 45 min, followed by 2-h reperfusion. Cardiac SPECT images of TFI in the IR model (n = 6) were dynamically acquired for 3 h. Correlative data of myocardial TFI distribution versus microsphere-determined tissue blood flow were acquired in 3 extra IR hearts. Inflammation targeting affinity of TFI was compared with 2 individual cytokine radioligands, (99m)Tc-IL-1ra-Fc (IF) and (99m)Tc-TNFR2-Fc (TF) (n = 6 each group). Myocardial cytokine expression was evaluated by immunochemical assay. RESULTS: Increased TFI uptake was found in the ischemic area and correlated with the severity of ischemia. At 3 h after injection, the ratio of hot-spot accumulation in the ischemic area to a remote viable zone was 5.39 ± 1.11 for TFI, which was greater than that for IF (3.28 ± 0.81) and TF (3.29 ± 0.75) (P < 0.05). The in vivo uptake profiles of TFI, TF, and IF were consistent with ex vivo radioactive measurements and correlated with upregulated IL-1 and TNF expression. CONCLUSION: The dual-domain TFI is promising for noninvasive detection of inflammatory reactions in IR myocardium because of its more potent affinity to the inflammatory sites compared with TF and IF.

A novel bifunctional protein TNFR2-Fc-IL-1ra (TFI): expression, purification and its neutralization activity of inflammatory factors.

Tumor necrosis factor receptor (TNF) and internleukin-1 (IL-1) are the most potent proinflammatory cytokines involving in autoimmune and inflammatory human diseases. Many anti-inflammatory agents have been exploited for anti-inflammation treatments by targeting cytokines including TNF and IL-1. Theoretically, simultaneously neutralizing or blocking two important inflammatory mediators may achieve a synergistic therapeutic effect. We have developed a recombinant fusion protein, TNFR2-Fc-IL-1ra (TFI), which consists of a TNF-neutralizing domain that specifically binds to TNF-α, an IL-1 receptor antagonist domain, and a dimerization Fc portion of human IgG1, for bifunctional inflammatory inhibitor. Recombinant DNA expressing the sequence of this fusion protein was expressed in CHO-S cells. The protein product was purified using a two-step purification protocol and the identity of the protein was confirmed by western blot analysis. The purified recombinant protein had a purity of about 98 % as determined by HPLC, and a molecular mass of 164.6 kDa as determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The results of cell binding inhibition indicate that TFI was able to strongly neutralize TNF activity and antagonize IL-1r activity, suggesting that TFI may be used as a bifunctional ligand with enhanced anti-inflammatory effect. The result obtained in this study may provide a platform for extending bifunctional anti-inflammatory drug development.

Characterization of 99mTc-labeled cytokine ligands for inflammation imaging via TNF and IL-1 pathways.

INTRODUCTION: TNFR2-Fc and IL-1ra-Fc are recombinant cytokine ligands that target TNF and IL-1. TNFR2-Fc-IL-1ra, a dual-domain agent that incorporates both ligands, allows bifunctional binding of IL-1 receptors and TNF. This study was designed to characterize (99m)Tc-labeled forms of these ligands, (99m)Tc-IL-1ra-Fc (IF), (99m)Tc-TNFR2-Fc (TF), and (99m)Tc-TNFR2-Fc-IL-1ra (TFI), for inflammation imaging. METHODS: The cytokine ligands were labeled with (99m)Tc by a direct approach via 2-iminothiolane (2-IT) reduction at various 2-IT/protein molar ratios. In vivo inflammation targeting studies were carried out in a mouse ear edema model created by topical application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the right ear of ICR mice. RESULTS: Radiolabeling yields increased with increasing amounts of 2-IT. When the 2-IT/protein ratio reached 1000, the radiolabeling yield was greater than 90% without significant colloid production. TPA-treated ears showed high radioligand uptake, which was clearly detected by SPECT and autoradiographic imaging. The activities (%ID/g) in the inflamed and control ears at 3h after injection were 2.76 ± 0.20 vs. 0.69 ± 0.12 for IF, 5.86 ± 0.40 vs. 2.86 ± 0.61 for TF, and 7.61 ± 0.86 vs. 1.99 ± 0.31 for TFI (P<0.05 vs. controls). TFI showed significantly higher uptake in the inflamed ears compared to TF and IF (P<0.05). Blocking study results indicated specificity of radioligand binding with decreased radioactive uptake in the inflamed ears. Western blotting and ELISA analysis further confirmed a high expression of IL-1ß and TNF-α in the inflamed ears. CONCLUSIONS: (99m)Tc-labeled cytokine ligands are a promising approach for detecting and understanding the inflammatory process. TFI may be more useful than the single-domain ligands for noninvasive detection of inflammatory sites.

A (99m)Tc-labeled dual-domain cytokine ligand for imaging of inflammation.

INTRODUCTION: Interleukin (IL)-1 and IL-18 are potent proinflammatory cytokines in inflammation-related diseases. Their actions are regulated by IL-1 receptor antagonist (IL-1ra) and IL-18 binding protein (IL-18bp). This study was designed to (99m)Tc-radiolabel an IL-1ra and IL-18bp dual-domain cytokine ligand, IL-18bp-Fc-IL-1ra, for specific inflammation targeting. METHODS: The (99m)Tc-IL-18bp-Fc-IL-1ra was obtained by direct labeling via 2-iminothiolane reduction. Competitive binding of (99m)Tc-labeled and unlabeled IL-18bp-Fc-IL-1ra to rat polymorphonuclear leukocytes was assessed in vitro. A mouse ear edema model was used to evaluate specific targeting properties of (99m)Tc-IL-18bp-Fc-IL1ra in vivo. The correlation between (99m)Tc-IL-18bp-Fc-IL-1ra uptake and (111)In-labeled polymorphonuclear neutrophil infiltration was studied using ischemic-reperfused rat hearts. RESULTS: Direct (99m)Tc-labeling yielded a stable dual-domain cytokine radioligand with radiochemical purity greater than 95% after gel filtration. Competitive binding studies showed specific targeting of (99m)Tc-IL-18bp-Fc-IL-1ra to inflammatory cells. The (99m)Tc-IL-18bp-Fc-IL-1ra uptake was 1.80±0.17 % injected dose per gram (%ID/g) in the inflamed ear without blocking, whereas uptake in the presence of IL-18bp-Fc-IL-1ra was 1.09±0.08 %ID/g (P<.05). The amounts of IL-1ß and IL-18 were significantly increased in the inflamed ears compared to the vehicle controls. A significant correlation of (99m)Tc-IL-18bp-Fc-IL-1ra with (111)In-labeled neutrophil distribution was observed in the ischemic-reperfused hearts (P<.001). CONCLUSION: Targeting proinflammatory cytokines with (99m)Tc-IL-18bp-Fc-IL-1ra may provide a suitable approach for specific detection of inflammatory sites.

PET imaging of acute and chronic inflammation in living mice.

PURPOSE: In this study, we evaluated the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced acute and chronic inflammation in living mice by PET imaging of TNF-alpha and integrin alpha(v)beta(3) expression. METHODS: TPA was topically applied to the right ear of BALB/c mice every other day to create the inflammation model. (64)Cu-DOTA-etanercept and (64)Cu-DOTA-E{E[c(RGDyK)](2)}(2) were used for PET imaging of TNF-alpha and integrin alpha(v)beta(3) expression in both acute and chronic inflammation. Hematoxylin and eosin staining, ex vivo autoradiography, direct tissue sampling, and immunofluorescence staining were also performed to confirm the non-invasive PET imaging results. RESULTS: The ear thickness increased significantly and the TNF-alpha level more than tripled after a single TPA challenge. MicroPET imaging using (64)Cu-DOTA-etanercept revealed high activity accumulation in the inflamed ear, reaching 11.1 +/- 1.3, 13.0 +/- 2.0, 10.9 +/- 1.4, 10.2 +/- 2.2%ID/g at 1, 4, 16, and 24 h post injection, respectively (n = 3). Repeated TPA challenges caused TPA-specific chronic inflammation and reduced (64)Cu-DOTA-etanercept uptake due to lowered TNF-alpha expression. (64)Cu-DOTA-E{E[c(RGDyK)](2)}(2) uptake in the chronically inflamed ears (after four and eight TPA challenges) was significantly higher than in the control ears and those after one TPA challenge. Immunofluorescence staining revealed increased integrin beta(3) expression, consistent with the non-invasive PET imaging results using (64)Cu-DOTA-E{E[c(RGDyK)](2)}(2) as an integrin alpha(v)beta(3)-specific radiotracer. Biodistribution and autoradiography studies further confirmed the quantification capability of microPET imaging. CONCLUSION: Successful PET imaging of TNF-alpha expression in acute inflammation and integrin alpha(v)beta(3) expression in chronic inflammation provides the rationale for multiple target evaluation over time to fully understand the inflammation processes.