In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.     

Toll-like receptor 3 signaling converts tumor-supporting myeloid cells to tumoricidal effectors

Smoldering inflammation often increases the risk of progression for malignant tumors and simultaneously matures myeloid dendritic cells (mDCs) for cell-mediated immunity. PolyI:C, a dsRNA analog, is reported to induce inflammation and potent antitumor immune responses via the Toll-like receptor 3/Toll-IL-1 receptor domain-containing adaptor molecule 1 (TICAM-1) and melanoma differentiation-associated protein 5/IFN-β promoter stimulator 1 (IPS-1) pathways in mDCs to drive activation of natural killer cells and cytotoxic T lymphocytes. Here, we found that i.p. or s.c. injection of polyI:C to Lewis lung carcinoma tumor-implant mice resulted in tumor regression by converting tumor-supporting macrophages (Mfs) to tumor suppressors. F4/80(+)/Gr1(-) Mfs infiltrating the tumor respond to polyI:C to rapidly produce inflammatory cytokines and thereafter accelerate M1 polarization. TNF-α was increased within 1 h in both tumor and serum upon polyI:C injection into tumor-bearing mice, followed by tumor hemorrhagic necrosis and growth suppression. These tumor responses were abolished in TNF-α(-/-) mice. Furthermore, F4/80(+) Mfs in tumors extracted from polyI:C-injected mice sustained Lewis lung carcinoma cytotoxic activity, and this activity was partly abrogated by anti-TNF-α Ab. Genes for supporting M1 polarization were subsequently up-regulated in the tumor-infiltrating Mfs. These responses were completely abrogated in TICAM-1(-/-) mice, and unaffected in myeloid differentiation factor 88(-/-) and IPS-1(-/-) mice. Thus, the TICAM-1 pathway is not only important to mature mDCs for cross-priming and natural killer cell activation in the induction of tumor immunity, but also critically engaged in tumor suppression by converting tumor-supporting Mfs to those with tumoricidal properties.     

Hepatocyte growth factor reduces cardiac fibrosis by inhibiting endothelial-mesenchymal transition

The purpose of this study was to investigate the effect of hepatocyte growth factor (HGF) on the pathogenesis of cardiac fibrosis induced by pressure overload in mice. Although cardiac fibrosis is attributed to excess pathological deposition of extracellular matrix components, the mechanism remains unclear. Recent reports revealed that α-smooth muscle actin-expressing myofibroblasts are primarily responsible for fibrosis. It is believed that myofibroblasts are differentiated from resident fibroblasts, whereas the transformation of vascular endothelial cells into myofibroblasts, known as endothelial-mesenchymal transition, has been suggested to be intimately associated with perivascular fibrosis. Thus, we hypothesized that HGF prevents cardiac fibrosis by blocking these pathways. We analyzed the pressure-overloaded HGF-transgenic mouse model made by transverse aortic constriction. Human coronary artery endothelial cells and human cardiac fibroblasts were examined in vitro after being treated with transforming growth factor-β1 or angiotensin II with or without HGF. The amount of cardiac fibrosis significantly decreased in pressure-overloaded HGF-transgenic mice compared with pressure-overloaded nontransgenic controls, particularly in the perivascular region. This was accompanied by a reduction in the expression levels of fibrosis-related genes and by significant preservation of echocardiographic measurements of cardiac function in the HGF-transgenic mice (P<0.05). The survival rate 2 months after transverse aortic constriction was higher by 45% (P<0.05). HGF inhibited the differentiation of human coronary artery endothelial cells into myofibroblasts induced by transforming growth factor-β1 and the phenotypic conversion of human cardiac fibroblasts into myofibroblasts. We conclude that HGF reduced cardiac fibrosis by inhibiting endothelial-mesenchymal transition and the transformation of fibroblasts into myofibroblasts.     

Conversion Therapy Using mFOLFOX6 With Panitumumab for Unresectable Liver Metastases From Multiple Colorectal Cancers With Familial Adenomatous Polyposis

A 39-year-old man received a diagnosis of unresectable multiple liver metastases from multiple colorectal cancers with familial adenomatous polyposis. After construction of an ileostomy, modified FOLFOX6 (mFOLFOX6) with panitumumab was administrated because rectal cancer and sigmoid colon cancer are KRAS wild type. The 13 courses of chemotherapy resulted in a marked reduction in the size of liver metastases and sigmoid colon cancer. Consequently, curative resection with total colectomy, ileal pouch anal anastomosis, and liver metastasis resection with radiofrequency ablation was performed. Progression of KRAS wild-type rectal cancer after chemotherapy suggested that each clone from rectal and sigmoid colon cancer might have a different sensitivity to epidermal growth factor receptor antibody. Immunohistochemical analysis revealed loss of PTEN expression in rectal cancer compared with liver metastases from sigmoid colon cancer, showing that the difference of mFOLFOX6 with panitumumab might be related to activation of the PI3K-AKT pathway.Key words: Panitumumab, mFOLFOX6, Colorectal cancer, Liver metastases, Familial adenomatous polyposisThe only available treatment associated with long-term survival in patients with liver metastases from colorectal cancer is complete liver tumor resection, with 5-year survival rates ranging from 25% to 57%.¹ However, only 40% to 50% of patients with colorectal metastasis to the liver are eligible for surgical resection.² Therefore, other liver metastasis patients undergo palliative chemotherapy to stabilize the disease and prolong their overall survival.During the past decade, the biggest advance made regarding unresectable liver metastases from colorectal cancer has been the ability of oncologists to convert inoperable liver disease to resectable disease using various molecular targeting drugs.^3,4 Several clinical studies have shown that the association of chemotherapy with bevacizumab (vascular endothelial growth factor monoclonal antibody), or cetuximab [epidermal growth factor receptor (EGFR) monoclonal antibody] is particularly promising in improving the resectability rate and, ultimately, survival.⁵Panitumumab is a fully human monoclonal antibody that binds specifically to the EGFR, and consequently, severe panitumumab-related infusion reactions are rare. Panitumumab, when added to FOLFOX4 (folinic acid, 5-fluorouracil, and oxaliplatin), increased response rate and improved progression-free survival in previously untreated metastatic colorectal cancer.⁶ Retrospective analyses of phase 3 trials of anti-EGFR antibodies, including cetuximab and panitumumab, found KRAS status to be an important predictive marker of efficacy, with only wild-type patients benefiting from treatment.⁷Here, we report a successful conversion therapy using modified FOLFOX6 (mFOLFOX6) plus panitumumab in a patient with familial adenomatous polyposis (FAP) who had unresectable multiple liver metastases from multiple colorectal cancers. To the best of our knowledge, we are the first researchers to demonstrate treatment of multiple target tumors derived from different clones with mFOLFOX6 and panitumumab, and to show differential panitumumab sensitivity for multiple primary tumors and liver metastases.     

Dynamic pathology for circulating free DNA in a dextran sodium sulfate colitis mouse model

Purpose

In sepsis, circulating free DNA (cf-DNA) is increased, and is a marker of severity and prognosis of septic patients. This study aimed to evaluate cf-DNA in a dextran sodium sulfate-induced colitis mouse model, and its clinical implications.

Methods

Dynamic pathology of the cecum wall in the DSS-induced colitis mouse model was analyzed using multiphoton microscopy (MPM). Plasma cf-DNA concentrations in colitis mouse were quantified using PicoGreen dsDNA Assay Kit. Plasma cf-DNA was also measured in 123 human ulcerative colitis (UC) patients [mean age: 35.9 years (3–75 years) with 20 pediatric patients] to assess its relationships with clinical severity and Matt’s grade.

Results

Real-time images of cf-DNA were detected in the colitis model. The amount of labeled cf-DNA in the circulation of the colitis mice group was significantly higher compared with that in the control group (P < 0.05). In human UC blood samples, plasma cf-DNA concentrations in UC patients were significantly positively correlated with the clinical severity of UC and Matt’s grade (P < 0.05, P < 0.05, respectively).

Conclusions

Using MPM, we observed and analyzed real-time images of cf-DNA in a colitis mouse model. Plasma cf-DNA is a potential non-invasive blood marker for reflecting clinical severity and mucosal damage in UC patients.     

Nonagonistic Dectin-1 ligand transforms CpG into a multitask nanoparticulate TLR9 agonist

CpG DNA, a ligand for Toll-like receptor 9 (TLR9), has been one of the most promising immunotherapeutic agents. Although there are several types of potent humanized CpG oligodeoxynucleotide (ODN), developing “all-in-one” CpG ODNs activating both B cells and plasmacytoid dendritic cells forming a stable nanoparticle without aggregation has not been successful. In this study, we generated a novel nanoparticulate K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand schizophyllan (SPG), K3-SPG. In sharp contrast to K3 alone, K3-SPG stimulates human peripheral blood mononuclear cells to produce a large amount of both type I and type II IFN, targeting the same endosome where IFN-inducing D CpG ODN resides without losing its K-type activity. K3-SPG thus became a potent adjuvant for induction of both humoral and cellular immune responses, particularly CTL induction, to coadministered protein antigens without conjugation. Such potent adjuvant activity of K3-SPG is attributed to its nature of being a nanoparticle rather than targeting Dectin-1 by SPG, accumulating and activating antigen-bearing macrophages and dendritic cells in the draining lymph node. K3-SPG acting as an influenza vaccine adjuvant was demonstrated in vivo in both murine and nonhuman primate models. Taken together, K3-SPG may be useful for immunotherapeutic applications that require type I and type II IFN as well as CTL induction.CpG oligodeoxynucleotide (CpG ODN) is a short (∼20 bases), single-stranded synthetic DNA fragment containing the immunostimulatory CpG motif, a potent agonist for Toll-like receptor 9 (TLR9), which activates dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (1, 2) and acts as an adjuvant toward both Th1-type humoral and cellular immune responses, including cytotoxic T-lymphocyte (CTL) responses (3, 4). Therefore, CpG ODN has been postulated as a possible immunotherapeutic agent against infectious diseases, cancer, asthma, and pollinosis (2, 5).There are at least four types of CpG ODN, each of which has a different backbone, sequence, and immunostimulatory properties (6). D-type (also called A) CpG ODNs typically comprise one palindromic CpG motif with a phosphodiester (PO) backbone and phosphorothioate (PS) poly(G) tail, and activates plasmacytoid DCs (pDCs) to produce a large amount of IFN-α but fails to induce pDC maturation and B-cell activation (7, 8). The three other types of ODN consist of a PS backbone. K-type (also called B) CpG ODN contains nonpalindromic multiple CpG motifs, and strongly activates B cells to produce IL-6 and pDCs to maturation but barely produces IFN-α (8, 9). Recently, C and P CpG ODNs have been developed; these contain one and two palindromic CpG sequences, respectively, both of which can activate B cells like K-type and pDC like D-type, although C CpG ODN induces weaker IFN-α production compared with P CpG ODN (10–12).D and P CpG ODNs have been shown to form higher-order structures, Hoogsteen base pairing to form parallel quadruplex structures called G tetrads, and Watson–Crick base pairing between cis- and trans-palindromic portions, respectively, that are required for robust IFN-α production by pDCs (12–14). Although such higher-order structures appear necessary for localization to early endosomes and signaling via TLR9, they suffer from product polymorphisms, aggregation, and precipitation, thereby hampering their clinical application (15). Therefore, only K and C CpG ODNs are generally available as immunotherapeutic agents and vaccine adjuvants for human use (16, 17). Although K CpG ODN enhances the immunogenicity of vaccines targeting infectious diseases and cancers in human clinical trials (6, 17), chemical or physical conjugation between antigen and K CpG ODN is necessary for optimal adjuvant effects. These results indicate that these four (K, D, P, and C) types of CpG ODN have advantages and disadvantages; however, the development of an “all-in-one” CpG ODN activating both B cells and pDCs that forms a stable nanoparticle without aggregation has yet to be accomplished. A better strategy, targeting CpG ODN toward antigen-presenting cells (APCs), is desired to improve immunostimulatory specificity and immunotherapeutic efficacy of CpG ODNs.Schizophyllan (SPG), a soluble β-glucan derived from Schizophyllum commune, is a drug that has been approved in Japan as an enhancer of radiotherapy in cervical carcinoma patients for the last three decades (18). It has been shown to form a complex with polydeoxyadenylic acid (dA) as a triple-helical structure (19). Although we previously demonstrated that mouse and humanized CpG ODN with PO poly(dA) at the 5′ end complexed with SPG enhanced cytokine production and acted as an influenza vaccine adjuvant (20, 21), it has been difficult to achieve high yields of the CpG–SPG complex toward its more efficient and cost-effective preclinical as well as clinical development. Recently, when the PS backbone of the dA sequence was linked to CpG ODN, the efficacy of complex formation was elevated by nearly 100% (22). However, a thorough investigation has yet to be conducted to identify the best humanized CpG sequence and optimization of factors to gain all-in-one activities of the four types of CpG ODN.To do this, we sought to optimize a humanized CpG–SPG complex as a vaccine adjuvant and immunostimulatory agent in humans (in vitro), mice (in vitro and in vivo), and nonhuman primates (in vivo). In this study, we identified a novel K CpG ODN (K3) and SPG complex, namely K3-SPG. It forms a higher-order nanoparticle that can be completely solubilized. We found that this all-in-one K3-SPG displayed a more potent activity than, and different characteristics from, any other type of CpG ODN and previous CpG–SPG complexes.