Paired box 6 gene delivery preserves beta cells and improves islet transplantation efficacy.

Loss of pancreatic beta cells is the central feature of all forms of diabetes. Current therapies fail to halt the declined beta cell mass. Thus, strategies to preserve beta cells are imperatively needed. In this study, we identified paired box 6 (PAX6) as a critical regulator of beta cell survival. Under diabetic conditions, the human beta cell line EndoC-ßH1, db/db mouse and human islets displayed dampened insulin and incretin signalings and reduced beta cell survival, which were alleviated by PAX6 overexpression. Adeno-associated virus (AAV)-mediated PAX6 overexpression in beta cells of streptozotocin-induced diabetic mice and db/db mice led to a sustained maintenance of glucose homeostasis. AAV-PAX6 transduction in human islets reduced islet graft loss and improved glycemic control after transplantation into immunodeficient diabetic mice. Our study highlights a previously unappreciated role for PAX6 in beta cell survival and raises the possibility that ex vivo PAX6 gene transfer into islets prior to transplantation might enhance islet graft function and transplantation outcome.

Emerging Preclinical Applications of Humanized Mouse Models in the Discovery and Validation of Novel Immunotherapeutics and Their Mechanisms of Action for Improved Cancer Treatment.

Cancer therapeutics have undergone immense research over the past decade. While chemotherapies remain the mainstay treatments for many cancers, the advent of new molecular techniques has opened doors for more targeted modalities towards cancer cells. Although immune checkpoint inhibitors (ICIs) have demonstrated therapeutic efficacy in treating cancer, adverse side effects related to excessive inflammation are often reported. There is a lack of clinically relevant animal models to probe the human immune response towards ICI-based interventions. Humanized mouse models have emerged as valuable tools for pre-clinical research to evaluate the efficacy and safety of immunotherapy. This review focuses on the establishment of humanized mouse models, highlighting the challenges and recent advances in these models for targeted drug discovery and the validation of therapeutic strategies in cancer treatment. Furthermore, the potential of these models in the process of uncovering novel disease mechanisms is discussed.

Successful targeting of PD-1/PD-L1 with chimeric antigen receptor-natural killer cells and nivolumab in a humanized mouse cancer model.

In recent decades, chimeric antigen receptor (CAR)-engineered immune effector cells have demonstrated promising antileukemic activity. Nevertheless, their efficacy remains unsatisfactory on solid cancers, plausibly due to the influence of tumor microenvironments (TME). In a novel mouse cancer model with a humanized immune system, tumor-infiltrating immunosuppressive leukocytes and exhausted programmed death protein-1 (PD-1)high T cells were found, which better mimic patient TME, allowing the screening and assessment of immune therapeutics. Particularly, membrane-bound programmed death ligand 1 (PD-L1) level was elevated on a tumor cell surface, which serves as an attractive target for natural killer (NK) cell-mediated therapy. Hematopoietic stem cell-derived CAR-NK (CAR pNK) cells targeting the PD-L1 showed enhanced in vitro and in vivo anti-solid tumor function. The CAR pNK cells and nivolumab resulted in a synergistic anti-solid tumor response. Together, our study highlights a robust platform to develop and evaluate the antitumor efficacy and safety of previously unexplored therapeutic regimens.

Paired box 6 programs essential exocytotic genes in the regulation of glucose-stimulated insulin secretion and glucose homeostasis.

The paired box 6 (PAX6) transcription factor is crucial for normal pancreatic islet development and function. Heterozygous mutations of PAX6 are associated with impaired insulin secretion and early-onset diabetes mellitus in humans. However, the molecular mechanism of PAX6 in controlling insulin secretion in human beta cells and its pathophysiological role in type 2 diabetes (T2D) remain ambiguous. We investigated the molecular pathway of PAX6 in the regulation of insulin secretion and the potential therapeutic value of PAX6 in T2D by using human pancreatic beta cell line EndoC-ßH1, the db/db mouse model, and primary human pancreatic islets. Through loss- and gain-of-function approaches, we uncovered a mechanism by which PAX6 modulates glucose-stimulated insulin secretion (GSIS) through a cAMP response element-binding protein (CREB)/Munc18-1/2 pathway. Moreover, under diabetic conditions, beta cells and pancreatic islets displayed dampened PAX6/CREB/Munc18-1/2 pathway activity and impaired GSIS, which were reversed by PAX6 replenishment. Adeno-associated virus-mediated PAX6 overexpression in db/db mouse pancreatic beta cells led to a sustained amelioration of glycemic perturbation in vivo but did not affect insulin resistance. Our study highlights the pathophysiological role of PAX6 in T2D-associated beta cell dysfunction in humans and suggests the potential of PAX6 gene transfer in preserving and restoring beta cell function.

Analysis and Validation of Human Targets and Treatments Using a Hepatocellular Carcinoma-Immune Humanized Mouse Model.

BACKGROUND AND AIMS: Recent development of multiple treatments for human hepatocellular carcinoma (HCC) has allowed for the selection of combination therapy to enhance the effectiveness of monotherapy. Optimal selection of therapies is based on both HCC and its microenvironment. Therefore, it is critical to develop and validate preclinical animal models for testing clinical therapeutic solutions. APPROACH AND RESULTS: We established cell line-based or patient-derived xenograft-based humanized-immune-system mouse models with subcutaneous and orthotopic HCC. Mice were injected with human-specific antibodies (Abs) to deplete human immune cells. We analyzed the transcription profiles of HCC cells and human immune cells by using real-time PCR and RNA sequencing. The protein level of HCC tumor cells/tissues or human immune cells was determined by using flow cytometry, western blotting, and immunohistochemistry. The HCC tumor size was measured after single, dual-combination, and triple-combination treatment using N-(1',2-Dihydroxy-1,2'-binaphthalen-4'-yl)-4-methoxybenzenesulfonamide (C188-9), bevacizumab, and pembrolizumab. In this study, human immune cells in the tumor microenvironment were strongly selected and modulated by HCC, which promoted the activation of the IL-6/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in tumor cells and led to augmented HCC proliferation and angiogenesis by releasing angiogenic cytokines in humanized-immune-system mice with HCC. In particular, intratumor human cluster of differentiation-positive (hCD14+ ) cells could produce IL-33 through damage-associated molecular pattern/Toll-like receptor 4/activator protein 1, which up-regulated IL-6 in other intratumor immune cells and activated the JAK2/STAT3 pathway in HCC. Specific knockdown of the CD14 gene in human monocytes could impair IL-33 production induced by cell lysates. Subsequently, we evaluated the in vivo anti-HCC effect of C188-9, bevacizumab, and pembrolizumab. The results showed that the anti-HCC effect of triple-combination therapy was superior to that of single or dual treatments. CONCLUSIONS: Humanized-immune-system HCC mouse models are suitable for identifying targets from cancer and immune components and for testing combinational therapies.

Establishment and Characterization of Humanized Mouse NPC-PDX Model for Testing Immunotherapy.

Immune checkpoint blockade (ICB) monotherapy shows early promise for the treatment of nasopharyngeal carcinoma (NPC) in patients. Nevertheless, limited representative NPC models hamper preclinical studies to evaluate the efficacy of novel ICB and combination regimens. In the present study, we engrafted NPC biopsies in non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain-null (NSG) mice and established humanized mouse NPC-patient-derived xenograft (NPC-PDX) model successfully. Epstein-Barr virus was detected in the NPC in both NSG and humanized mice as revealed by Epstein-Barr virus-encoded small RNA (EBER) in situ hybridization (ISH) and immunohistochemical (IHC) staining. In the NPC-bearing humanized mice, the percentage of tumor-infiltrating CD8+ cytotoxic T cells was lowered, and the T cells expressed higher levels of various inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) than those in blood. The mice were then treated with nivolumab and ipilimumab, and the anti-tumor efficacy of combination immunotherapy was examined. In line with paired clinical data, the NPC-PDX did not respond to the treatment in terms of tumor burden, whilst an immunomodulatory response was elicited in the humanized mice. From our results, human proinflammatory cytokines, such as interferon-gamma (IFN-γ) and interleukin-6 (IL-6) were significantly upregulated in plasma. After treatment, there was a decrease in CD4/CD8 ratio in the NPC-PDX, which also simulated the modulation of intratumoral CD4/CD8 profile from the corresponding donor. In addition, tumor-infiltrating T cells were re-activated and secreted more IFN-γ towards ex vivo stimulation, suggesting that other factors, including soluble mediators and metabolic milieu in tumor microenvironment may counteract the effect of ICB treatment and contribute to the tumor progression in the mice. Taken together, we have established and characterized a novel humanized mouse NPC-PDX model, which plausibly serves as a robust platform to test for the efficacy of immunotherapy and may predict clinical outcomes in NPC patients.

Cancer Immunotherapies and Humanized Mouse Drug Testing Platforms.

Cancer immunotherapy is a type of treatment that restores and stimulates human immune system to inhibit cancer growth or eradicate cancer. It serves as one of the latest systemic therapies, which has been approved to treat different types of cancer in patients. Nevertheless, the clinical response rate is unsatisfactory and the response observed is mostly a partial response in patients. Despite the continuous improvement and identification of novel cancer immunotherapy, there is a pressing need to establish a robust platform to evaluate the efficacy and safety of pre-clinical drugs, simulate the interaction between patients' tumor and immune system, and predict patients' responses to the treatment. In this review, we summarize the pros and cons of existing immuno-oncology assay platforms, especially the humanized mouse models for the screening of cancer immunotherapy drugs. In addition, various emerging trends and progress of utilizing humanized mouse models as the screening tool are discussed. Of note, humanized mouse models can also be used for further development of personalized precision medicines to treat cancer. Collectively, these highlight the significance of humanized mouse models as the important platform for the screening of next generation cancer immunotherapy in vivo.

Enterovirus A71 Infection Activates Human Immune Responses and Induces Pathological Changes in Humanized Mice.

Since the discovery of enterovirus A71 (EV-A71) half a century ago, it has been recognized as the cause of large-scale outbreaks of hand-foot-and-mouth disease worldwide, particularly in the Asia-Pacific region, causing great concern for public health and economic burdens. Detailed mechanisms on the modulation of immune responses after EV-A71 infection have not been fully known, and the lack of appropriate models hinders the development of promising vaccines and drugs. In the present study, NOD-scid IL2Rγ-/- (NSG) mice with a human immune system (humanized mice) at the age of 4 weeks were found to be susceptible to a human isolate of EV-A71 infection. After infection, humanized mice displayed limb weakness, which is similar to the clinical features found in some of the EV-A71-infected patients. Histopathological examination indicated the presence of vacuolation, gliosis, or meningomyelitis in brain stem and spinal cord, which were accompanied by high viral loads detected in these organs. The numbers of activated human CD4+ and CD8+ T cells were upregulated after EV-A71 infection, and EV-A71-specific human T cell responses were found. Furthermore, the secretion of several proinflammatory cytokines, such as human gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-17A, was elevated in the EV-A71-infected humanized mice. Taken together, our results suggested that the humanized mouse model permits insights into the human immune responses and the pathogenesis of EV-A71 infection, which may provide a platform for the evaluation of anti-EV-A71 drug candidates in the future.IMPORTANCE Despite causing self-limited hand-food-and-mouth disease in younger children, EV-A71 is consistently associated with severe forms of neurological complications and pulmonary edema. Nevertheless, only limited vaccines and drugs have been developed over the years, which is possibly due to a lack of models that can more accurately recapitulate human specificity, since human is the only natural host for wild-type EV-A71 infection. Our humanized mouse model not only mimics histological symptoms in patients but also allows us to investigate the function of the human immune system during infection. It was found that human T cell responses were activated, accompanied by an increase in the production of proinflammatory cytokines in EV-A71-infected humanized mice, which might contribute to the exacerbation of disease pathogenesis. Collectively, this model allows us to delineate the modulation of human immune responses during EV-A71 infection and may provide a platform to evaluate anti-EV-A71 drug candidates in the future.

A thirty-year quest for a role of R-Ras in cancer: from an oncogene to a multitasking GTPase.

Since the identification of R-Ras, which is the first Ras-related GTPase isolated based on sequence similarity to the classical RAS oncogene, more than 160 members of the Ras superfamily of GTPases have been identified and classified into the Ras, Rho, Rap, Rab, Ran, Arf, Rheb, RGK, Rad, Rit, and Miro subfamilies. R-Ras belongs to the Ras subfamily of small G-proteins, which are frequently implicated in cell growth and differentiation. Although the roles of R-Ras in cellular transformation and integrin-mediated cell adhesion have been extensively studied, the physiological function of this enigmatic G-protein was only revealed when a mouse strain deficient in R-Ras was generated. In parallel, a plethora of research findings also linked R-Ras with processes including tumor angiogenesis, axon guidance, and immune cell trafficking. Several upstream factors that modulate R-Ras GTP-binding were identified including Notch, semaphorin, and chemokine C-C motif ligand 21. A review of our evolving understanding of the role of R-Ras in oncogenesis is timely, as this year marks the 30th anniversary of the publication describing the cloning of R-Ras.

Anti-allergic effect of the naturally-occurring conjugated linolenic acid isomer, jacaric acid, on the activated human mast cell line-1.

The present study aimed to investigate the immunomodulatory effect of jacaric acid, a naturally-occurring conjugated linolenic acid isomer that can be found in jacaranda seed oil, on the activated human mast cell line-1 (HMC-1). Our previous studies have demonstrated that jacaric acid only exerted minimal, if any, cytotoxicity on normal murine cells. In the present study, jacaric acid at concentrations ≤100 µM did not exhibit direct cytotoxicity on human peripheral blood mononuclear cells after 72 h of incubation, as determined by the MTT reduction assay. By contrast, jacaric acid could alleviate the calcium ionophore A23187 and phorbol 12-myristate 13-acetate-triggered allergic response in the HMC-1 cells at concentrations that were non-cytotoxic to the HMC-1 cells. Following pre-treatment with jacaric acid, the secretion of two inflammatory mediators, ß-N-acetylglucosaminidase and tryptase, as well as the T helper 2 cytokines [interleukin (IL)-4 and IL-13] was significantly reduced in HMC-1 cells. The alleviation of allergic response was accompanied by downregulation of the matrix metalloproteinase-2 and -9 proteins and upregulation of the tissue inhibitor of metalloproteinase-1 protein. Collectively, the results indicated that the naturally-occurring jacaric acid exhibits a suppressive effect on the allergic response in activated human mast cells in vitro, and this could not be attributed to the direct cytotoxicity of jacaric acid on the treated cells.

The Immunomodulatory Activity of Jacaric Acid, a Conjugated Linolenic Acid Isomer, on Murine Peritoneal Macrophages.

This study aims at demonstrating the immunomodulatory property of jacaric acid, a conjugated linolenic acid (CLNA) isomer that is present in jacaranda seed oil, on murine peritoneal macrophages. Our results showed that jacaric acid exhibited no significant cytotoxicity on the thioglycollate-elicited murine peritoneal macrophages as revealed by the neutral red uptake assay, but markedly increased their cytostatic activity on the T-cell lymphoma MBL-2 cells as measured by the fluorometric CyQuant® NF Cell Proliferation Assay Kit. Flow cytometric analysis indicated that jacaric acid could enhance the endocytic activity of macrophages and elevated their intracellular production of superoxide anion. Moreover, jacaric acid-treated macrophages showed an increase in the production of nitric oxide which was accompanied by an increase in the expression level of inducible nitric oxide synthase protein. In addition, the secretion of several pro-inflammatory cytokines, including interferon-γ, interleukin-1ß and tumor necrosis factor-α, was up-regulated. Collectively, our results indicated that the naturally-occurring CLNA isomer, jacaric acid, could exhibit immunomodulating activity on the murine peritoneal macrophages in vitro, suggesting that this CLNA isomer may act as an immunopotentiator which can be exploited for the treatment of some immunological disorders with minimal toxicity and fewer side effects.

Jacaric acid inhibits the growth of murine macrophage-like leukemia PU5-1.8 cells by inducing cell cycle arrest and apoptosis.

BACKGROUND: Conjugated linolenic acids (CLN) refer to the positional and geometric isomers of octadecatrienoic acids with three conjugated double bonds (C18:3). Previous researches have demonstrated that CLN can inhibit the growth of a wide variety of cancer cells, whereas the modulatory effect of CLN on various myeloid leukemia cells remains unclear. This study aims at demonstrating the in vitro anti-tumor effect and action mechanisms of jacaric acid, a CLN isomer which is present in jacaranda seed oil, on the murine macrophage-like leukemia PU5-1.8 cells. METHODS AND RESULTS: It was found that jacaric acid inhibited the proliferation of PU5-1.8 cells in a time- and concentration-dependent manner, as determined by the MTT reduction assay and by using CyQUANT(®) NF Cell Proliferation Assay Kit, while it exerted minimal cytotoxicity on normal murine cells. Besides, the reactive oxygen species production in jacaric acid-treated PU5-1.8 cells was elevated in a concentration-dependent mannar. Flow cytometric analysis revealed the induction of G0/G1 cell cycle arrest, accompanied by a decrease in CDK2 and cyclin E proteins. Jacaric acid also triggered apoptosis as reflected by induction of DNA fragmentation, phosphatidylserine externalization, mitochondrial membrane depolarization, up-regulation of pro-apoptotic Bax protein and down-regulation of anti-apoptotic Bcl-2 and Bcl-xL proteins. CONCLUSIONS: Our results demonstrated the growth-inhibitory effect of jacaric acid on PU5-1.8 cells through inducing cell cycle arrest and apoptosis, while exhibiting minimal cytotoxicity to normal murine cells. Therefore, jacaric acid is a potential candidate for the treatment of some forms of myeloid leukemia with minimal toxicity and fewer side effects.

Omega-3 Polyunsaturated Fatty Acids Trigger Cell Cycle Arrest and Induce Apoptosis in Human Neuroblastoma LA-N-1 Cells.

Omega-3 (n-3) fatty acids are dietary long-chain fatty acids with an array of health benefits. Previous research has demonstrated the growth-inhibitory effect of n-3 fatty acids on different cancer cell lines in vitro, yet their anti-tumor effects and underlying action mechanisms on human neuroblastoma LA-N-1 cells have not yet been reported. In this study, we showed that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exhibited time- and concentration-dependent anti-proliferative effect on the human neuroblastoma LA-N-1 cells, but had minimal cytotoxicity on the normal or non-tumorigenic cells, as measured by MTT reduction assay. Mechanistic studies indicated that DHA and EPA triggered G0/G1 cell cycle arrest in LA-N-1 cells, as detected by flow cytometry, which was accompanied by a decrease in the expression of CDK2 and cyclin E proteins. Moreover, DHA and EPA could also induce apoptosis in LA-N-1 cells as revealed by an increase in DNA fragmentation, phosphatidylserine externalization and mitochondrial membrane depolarization. Up-regulation of Bax, activated caspase-3 and caspase-9 proteins, and down-regulation of Bcl-XL protein, might account for the occurrence of apoptotic events. Collectively, our results suggest that the growth-inhibitory effect of DHA and EPA on LA-N-1 cells might be mediated, at least in part, via triggering of cell cycle arrest and apoptosis. Therefore, DHA and EPA are potential anti-cancer agents which might be used for the adjuvant therapy or combination therapy with the conventional anti-cancer drugs for the treatment of some forms of human neuroblastoma with minimal toxicity.

Apoptosis- and differentiation-inducing activities of jacaric acid, a conjugated linolenic acid isomer, on human eosinophilic leukemia EoL-1 cells.

Conjugated linolenic acids (CLNAs) are a group of naturally occurring positional and geometrical isomers of the C18 polyunsaturated essential fatty acid, linolenic acid (LNA), with three conjugated double bonds (C18:3). Although previous research has demonstrated the growth-inhibitory effects of CLNA on a wide variety of cancer cell lines in vitro, their action mechanisms and therapeutic potential on human myeloid leukemia cells remain poorly understood. In the present study, we found that jacaric acid (8Z,10E,12Z-octadecatrienoic acid), a CLNA isomer which is present in jacaranda seed oil, inhibited the in vitro growth of human eosinophilic leukemia EoL-1 cells in a time- and concentration-dependent manner. Mechanistic studies showed that jacaric acid triggered cell cycle arrest of EoL-1 cells at the G0/G1 phase and induced apoptosis of the EoL-1 cells, as measured by the Cell Death Detection ELISAPLUS kit, Annexin V assay and JC-1 dye staining. Notably, the jacaric acid-treated EoL-1 cells also underwent differentiation as revealed by morphological and phenotypic analysis. Collectively, our results demonstrated the capability of jacaric acid to inhibit the growth of EoL-1 cells in vitro through triggering cell cycle arrest and by inducing apoptosis and differentiation of the leukemia cells. Therefore, jacaric acid might be developed as a potential candidate for the treatment of certain forms of myeloid leukemia with minimal toxicity and few side effects.