T cells engineered to express CD19-specific chimeric antigen receptors (CARs) have shown breakthrough clinical successes in patients with B-cell lymphoid malignancies. However, similar therapeutic efficacy of CAR T cells in solid tumors is yet to be achieved. In this study we systematically evaluated a series of CAR constructs targeting glypican-3 (GPC3), which is selectively expressed on several solid tumors. We compared GPC3-specific CARs that encoded CD3ζ (Gz) alone or with costimulatory domains derived from CD28 (G28z), 4-1BB (GBBz), or CD28 and 4-1BB (G28BBz). All GPC3-CARs rendered T cells highly cytotoxic to GPC3-positive hepatocellular carcinoma, hepatoblastoma, and malignant rhabdoid tumor cell lines in vitro. GBBz induced the preferential production of Th1 cytokines (interferon γ/granulocyte macrophage colony-stimulating factor) while G28z preferentially induced Th2 cytokines (interleukin-4/interleukin-10). Inclusion of 4-1BB in G28BBz could only partially ameliorate the Th2-polarizing effect of CD28. 4-1BB induced superior expansion of CAR T cells in vitro and in vivo. T cells expressing GPC3-CARs incorporating CD28, 4-1BB, or both induced sustained tumor regressions in two xenogeneic tumor models. Thus, GBBz CAR endows T cells with superior proliferative potential, potent antitumor activity, and a Th1-biased cytokine profile, justifying further clinical development of GBBz CAR for immunotherapy of GPC3-positive solid tumors.

Background: Dysfunction in survival and differentiation of osteoblasts commonly occurs in patients with osteoporosis. Cannabinoid receptor type 2 (CNR2) is a major receptor of endocannabinoid system that is crucial for bone mass homeostasis. Our group prior demonstrated that activation of CNR2 signaling promoted osteogenic differentiation of bone marrow derived mesenchymal stem cells in vitro. Autophagy is reported to participate in osteoblastic differentiation. Whether autophagy is regulated by CNR2-mediated cannabinoid signaling is unknown, and how the autophagy-CNR2 interaction affects osteoblastic differentiation requires further elucidation. Methods: hFOB 1.19 osteoblasts were treated with CNR2 agonists HU308 (5, 10, 25, 50 or 100 nM) and JWH133 (1, 2, 5, 10 or 20 μM) in presence or absence of autophagy inhibitor 3-Methyladenine (3-MA). The differentiation of hFOB 1.19 cells was determined via evaluating their alkaline phosphatase (ALP) activity and mineralization ability (Alizarin red staining). Alterations in autophagy-related molecules and osteogenic markers were analyzed via real-time PCR and/or immunoblotting assays. Results: hFOB 1.19 cells spontaneously differentiated towards mature osteoblasts under 39°C, during which CNR2 expression increased, and autophagy was activated. The strongest autophagy flux was observed at 192 h post differentiation─LC3I to LC3II conversion was enhanced and Beclin 1 expression was upregulated considerably, while p62 expression was downregulated. Treatment of HU308 and JWH133 promoted autophagy in a dose-dependent manner, and suppressed mTOR signaling pathway in hFOB 1.19 cells. In CNR2-silenced cells, HU308's and JWH133's effects on autophagy were weakened. HU308 and JWH133 enhanced the ALP activity and mineralization, and upregulated the expression of osteogenic markers, osteopontin and osteocalcin, in hFOB 1.19 cells. Intriguingly, such pro-osteogenic effects induced by CNR2 activation were markedly mitigated by 3-MA. In addition to provoking autophagy, CNR2 agonists also reduced nuclear Nrf2 accumulation and increased Keap1 expression. Further, reexpression of p62 inhibited CNR2 agonists-induced Nrf2 degradation. Conclusions: Osteogenic differentiation induced by CNR2 signaling activation involves autophagy induction and p62mediated Nrf2 deactivation.

Background/Aim: In literature, few studies have examined the diagnostic or prognostic potential of matrix metalloproteinases (MMP) in pterygium, whose formation and progression are closely related to imbalance in the extracellular microenvironment. In this study, we investigated the contribution of MMP7 promoter (A-181G and C-153T) polymorphic genotypes to pterygium risk. Materials and Methods: A total of 134 cases and 268 controls were collected and their MMP7 genotypes at A-181G and C-153T were examined by polymerase chain reaction-restriction fragment length polymorphism methodology. Results: The AA, AG and GG genotypes at MMP7 promoter A-181G were nonsignificantly differentially distributed between the two groups at 85.8, 11.2 and 3.0%, respectively, in pterygium cases and 88.4, 9.7 and 1.9% in controls, respectively (p for trend=0.6822). There was no polymorphic genotype for MMP7 C-153T among our Taiwanese cohort. Conclusion: A-181G and C-153T genotypes at MMP7 do not have a direct role in determining Taiwanese susceptibility to pterygium. Pterygium is a disease presenting an abnormal wing-shaped outgrowth of fibrovascular conjunctival tissues invading the clear cornea. The etiology of pterygium is still largely unclear. The incidence of pterygium is epidemiologically due to several factors, such as overexposure to sunshine, UV light, heat, dust, and other particles in the atmosphere (1-3). In the past two decades, mounting evidence has supported the concept that variations in our genome play a critical role in the determination of the etiology and development of pterygium (4-11). Many theories for pterygium have been proposed, such as imbalances in immunological mechanisms, growth factors, cytokines, apoptosis, angiogenesis (12-15), and most interestingly but complicatedly, an imbalance in the extracellular microenvironment and the involvement of matrix metalloproteinases (MMPs) (16-19). MMPs are a group of zinc-binding endopeptidases responsible for regulating the components of the extracellular matrix microenvironment (20). The activation of MMPs generally takes place in the extracellular space, and interacts with various other proteases, teaming up to regulate the behaviors of cancer cells including viability, cell differentiation, programmed cell death, angiogenesis, immune surveillance, invasiveness and migration capacity (21, 22). The smallest MMP member, MMP7 (matrilysin), is responsible for the metabolism of a broad spectrum of substrates including fibronectin, vitronectin, elastin, collagen IV, aggrecan, and proteoglycans (23). In addition, MMP7 is involved in inflammatory responses via its capacity to promote cell-surface processing of cytokines such as tumor necrosis factor a (24). MMP7 activation was first proposed to be involved in the pathogenesis of pterygium as early as 2001 by Girolamo and colleagues (25). In their analysis of cultured pterygial and conjunctival tissues from eight pterygium cases at Wales hospital in Sydney, basal and activated MMP7 levels were 1.4-and 2.7-fold higher, resp...

Our results indicate that the MMP7 A-181G genotype interacts with age and gender and may serve as an early and predictive biomarker for childhood ALL.

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