Immune complexome analysis of serum samples from non-small-cell lung cancer patients identifies predictive biomarkers for nivolumab therapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have achieved important outcomes in cancer treatment. However, current clinical biomarker tests are not suitable for some patients because they require tumor tissues and have poor predictive value for treatment responses. Therefore, the identification of biomarkers that enable screening tests in all patients is necessary. METHODS: We performed an immune complexome analysis of non-small cell lung cancer patients treated with nivolumab to comprehensively identify and compare antigens incorporated into immune complexes (IC-antigens) in serum samples from the responders (n = 15) and non-responders (n = 20). Additionally, combinations of IC-antigens characteristic to the responder group were evaluated by logistic regression analysis and receiver operating characteristics curves to examine their predictiveness for ICI treatment responses. RESULTS: The combination of predictive biomarkers detected before treatment was profilin-1, purine nucleoside phosphorylase, alpha-enolase, and nucleoside diphosphate kinase A [p = 0.0043, odds ratio = 2.26, 95% confidence interval (CI) = 1.19-4.28, area under the curve = 0.76]. The combination of predictive biomarkers detected after treatment was peptidyl-prolyl cis-trans isomerase A, ubiquitin-like modifier-activating enzyme 1, complement component C8 beta chain, and apolipoprotein L1 (p = 0.0039, odds ratio = 2.56, 95% CI = 1.25-5.23, area under the curve = 0.77). CONCLUSION: Combinations of serum IC-antigens may predict the therapeutic effect of nivolumab in non-small cell lung cancer patients.

Novel siRNA delivery system using a ternary polymer complex with strong silencing effect and no cytotoxicity.

We developed a novel small interfering RNA (siRNA) delivery system using a ternary complex with polyethyleneimine (PEI) and γ-polyglutamic acid (γ-PGA), which showed silencing effect and no cytotoxicity. The binary complexes of siRNA with PEI were approximately 73-102 nm in particle size and 45-52 mV in ζ-potential. The silencing effect of siRNA/PEI complexes increased with an increase of PEI, and siRNA/PEI complexes with a charge ratio greater than 16 showed significant luciferase knockdown in a mouse colon carcinoma cell line regularly expressing luciferase (Colon26/Luc cells). However, strong cytotoxicity and blood agglutination were observed in the siRNA/Lipofectamine complex and siRNA/PEI16 complex. Recharging cationic complexes with an anionic compound was reported to be a promising method for overcoming these toxicities. We therefore prepared ternary complexes of siRNA with PEI (charge ratio 16) by the addition of γ-PGA to reduce cytotoxicity and deliver siRNA. As expected, the cytotoxicity of the ternary complexes decreased with an increase of γ-PGA content, which decreased the ζ-potential of the complexes. A strong silencing effect comparable to siRNA/Lipofectamine complex was discovered in ternary complexes including γ-PGA with an anionic surface charge. The high incorporation of ternary complexes into Colon26/Luc cells was confirmed with fluorescence microcopy. Having achieved knockdown of an exogenously transfected gene, the ability of the complex to mediate knockdown of an endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was assessed in B16-F10 cells. The ternary complex (siRNA/PEI16/γ-PGA12 complex) exhibited a significant GAPDH knockdown effect. Thus, we developed a useful siRNA delivery system.

Secure and effective gene vector of polyamidoamine dendrimer pharmaceutically modified with anionic polymer.

The purpose of this study was to develop a new type of gene vector, polyamidoamine (PAMAM) dendriplex pharmaceutically modified, based on electrostatic interactions, by various anionic polymers. The γ-polyglutamic acid (γ-PGA)/PAMAM dendriplex and the α-PGA/PAMAM dendriplex formed a stable complex, although α-polyaspartic acid and heparin released pDNA from the complex. The addition of anionic polymer decreased the ζ-potential, although it did not greatly affect the size of the complex. As a result of an in vitro gene expression study of mouse melanoma cells, we found that the γ-PGA/PAMAM dendriplex showed high gene expression comparable to the PAMAM dendriplex, although the α-PGA/PAMAM dendriplex showed lower gene expression. Tail vein injection of the γ-PGA/PAMAM dendriplex into mice also led to high gene expression in the spleen and lung. The γ-PGA/PAMAM dendriplex showed no cytotoxicity and no agglutination, although severe cytotoxicity and agglutination were observed in the PAMAM dendriplex. Thus, we discovered that complexes of pDNA, PAMAM dendrimers, and γ-PGA showed higher gene expression in vitro and in vivo, and markedly lower toxicity. This complex is valuable and is expected to be a safe and effective gene vector.

Carbazochrome sodium sulfonate (AC-17) reverses endothelial barrier dysfunction through inhibition of phosphatidylinositol hydrolysis in cultured porcine endothelial cells.

The effect of carbazochrome sodium sulfonate (AC-17), a hemostatic drug with capillary stabilising action, on the endothelial barrier dysfunction induced by a variety of vasoactive substances or agents that increase the vascular permeability was investigated in the monolayers of cultured porcine aortic endothelial cells (PAECs). The endothelial barrier function was determined by the transendothelial transport of albumin-conjugated Evans blue. AC-17 (0.1-1 M) reversed the barrier dysfunction induced by tryptase, thrombin and bradykinin without affecting the endothelial permeability enhanced by Ca(2+) ionophores such as ionomycin and A23187 or phorbol 12-myristate 13-acetate. Immunofluorescence analysis showed that AC-17 reversed the tryptase-induced formation of actin stress fibres and disruption of VE-cadherin in PAECs. On the other hand, AC-17 (0.1-10 M) reduced concentration-dependently the enhancement of [(3)H]inositol triphosphate formation from [(3)H]myo-inositol induced by bradykinin and thrombin.Therefore, it is suggested that AC-17 reduces the vascular hyperpermeability induced by a variety of vasoactive agents through inhibition of agonist-induced phosphoinositide hydrolysis.

Involvement of proteinase-activated receptor-2 in mast cell tryptase-induced barrier dysfunction in bovine aortic endothelial cells.

We report here a direct modulation by mast cell tryptase of endothelial barrier function through activation of proteinase-activated receptor-2 (PAR-2). In cultured bovine aortic endothelial cells (BAECs), tryptase, trypsin and PAR-2 activating peptide impaired the barrier function as determined by the permeability of protein-conjugated Evans blue. The tryptase-induced barrier dysfunction was completely blocked by U73122, and partially reversed by xestospongin C, calphostin C or Y27632. The intracellular Ca(2+) was elevated by tryptase. It was notable that ioxaglate, a contrast material that degranulates mast cells, markedly increased the permeability when applied to BAECs in combination with mast cells, an action that was blocked by nafamostat, a potent tryptase inhibitor. Immunofluorescence analysis showed that actin stress fibre formation and disruption of VE-cadherin were observed after exposure to tryptase or ioxaglate in combination with mast cells. Therefore, it is suggested that mast cell tryptase impairs endothelial barrier function through activation of endothelial PAR-2 in a manner dependent on the phospholipase C activity.

A potent tryptase inhibitor nafamostat mesilate dramatically suppressed pulmonary dysfunction induced in rats by a radiographic contrast medium.

(1) Intravenous injection of ioxaglate (4 g iodine kg(-1)), an iodinated radiographic contrast medium, caused a marked protein extravasation, pulmonary oedema and a decrease in the arterial partial oxygen pressure in rats. (2) All of these reactions to ioxaglate were reversed by the pretreatment with gabexate mesilate (10 and 50 mg kg(-1), 5 min prior to injection) or nafamostat mesilate (3 and 10 mg kg(-1)), in which the inhibition was complete after injection of nafamostat mesilate (10 mg kg(-1)). (3) Both gabexate mesilate and nafamostat mesilate inhibited the activity of purified human lung tryptase, although the latter compound was far more potent than the former. (4) Ioxaglate enhanced the nafamostat-sensitive protease activity in the extracellular fluid of rat peritoneal mast cell suspensions. (5) Tryptase enhanced the permeability of protein through the monolayer of cultured human pulmonary arterial endothelial cells. Ioxaglate, when applied in combination with rat peritoneal mast cells, also produced the endothelial barrier dysfunction. These effects of tryptase and ioxaglate were reversed by nafamostat mesilate. (6) Consistent with these findings, immunofluorescence morphological analysis revealed that tryptase or ioxaglate in combination with mast cells increased actin stress fibre formation while decreasing VE-cadherin immunoreactivity. Both of these actions of tryptase and ioxaglate were reversed by nafamostat mesilate. (7) These findings suggest that tryptase liberated from mast cells plays a crucial role in the ioxaglate-induced pulmonary dysfunction. In this respect, nafamostat mesilate may become a useful agent for the cure or prevention of severe adverse reactions to radiographic contrast media.

Carbazochrome attenuates pulmonary dysfunction induced by a radiographic contrast medium in rats.

The effects of carbazochrome sodium sulfonate (AC-17), a capillary stabilizer, on pulmonary edema and dysfunction induced by ioxaglate, an ionic radiographic contrast medium, were evaluated in rats. The pulmonary edema was evaluated by the extravasation of intravenously injected Evans blue into lung tissues, while pulmonary dysfunction was determined by monitoring blood gasses including pO(2). Ioxaglate (4 g I/kg, i.v.) caused a marked increase in vascular permeability and a decrease in arterial pO(2). AC-17 reversed the ioxaglate-induced vascular hyperpermeability in a dose-dependent manner. In addition, AC-17 (10 mg/kg) significantly inhibited the decrease in arterial pO(2). In isolated rat pulmonary mast cells, ioxaglate markedly enhanced the histamine release, which was not affected by AC-17. On the other hand, AC-17 did significantly blocked the hyperpermeability induced in cultured bovine endothelial cells by tryptase, thrombin and proteinase-activated receptor-2 agonist peptide (SLIGKV-NH(2)). These findings suggest that AC-17 blocks radiographic contrast medium-induced pulmonary dysfunction by maintaining the endothelial barrier function. Thus, the compound is potentially useful for the prophylaxis of contrast media-induced acute pulmonary adverse events during angiography.