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Cytotoxic T lymphocytes (CTLs) play a crucial role in cancer rejection. However, CTLs encounter dysfunction and exhaustion in the immunosuppressive tumor microenvironment (TME). Although the reactive oxygen species (ROS)-rich TME attenuates CTL function, the underlying molecular mechanism remains poorly understood. The nuclear factor erythroid 2-related 2 (Nrf2) is the ROS-responsible factor implicated in increasing susceptibility to cancer progression. Therefore, we examined how Nrf2 is involved in anti-tumor responses of CD8 + T and chimeric antigen receptor (CAR) T cells in the ROS-rich TME. Here, we demonstrated that tumor growth in Nrf2 -/- mice was significantly controlled and was reversed by T cell depletion and further confirmed that Nrf2 deficiency in T cells promotes anti-tumor responses using an adoptive transfer model of antigen-specific CD8 + T cells. Nrf2-deficient CTLs are resistant to ROS, and their effector functions are sustained in the TME. Furthermore, Nrf2 knockdown in human CAR-T cells enhanced the survival and function of intratumoral CAR-T cells in a solid tumor xenograft model and effectively controlled tumor growth. ROS-sensing Nrf2 inhibits the anti-tumor T cell responses, indicating that Nrf2 may be a potential target for T cell immunotherapy strategies against solid tumors., Competing Interests: Declaration of interests C.H. received funding from NeoImmuneTech, Inc. D.C., S.-K.I., and B.H.L. are currently employed by NeoImmuneTech, Inc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable remission in patients with B-cell malignancies. However, its efficacy in treating solid tumors remains limited. Here, we investigated a combination therapy approach using an engineered long-acting interleukin (IL)-7 (rhIL-7-hyFc or NT-I7) and CAR-T cells targeting three antigens, glypican-2 (GPC2), glypican-3 (GPC3), and mesothelin (MSLN), against multiple solid tumor types including liver cancer, neuroblastoma, ovarian cancer, and pancreatic cancer in mice., Methods: CAR-T cells targeting GPC2, GPC3, and MSLN were used in combination with NT-I7 to assess the anticancer activity. Xenograft tumor models, including the liver cancer orthotopic model, were established using NOD scid gamma mice engrafted with cell lines derived from hepatocellular carcinoma, neuroblastoma, ovarian cancer, and pancreatic cancer. The mice were monitored by bioluminescence in vivo tumor imaging and tumor volume measurement using a caliper. Immunophenotyping of CAR-T cells on NT-I7 stimulation was evaluated for memory markers, exhaust markers, and T-cell signaling molecules by flow cytometry and western blotting., Results: Compared with the IL-2 combination, preclinical evaluation of NT-I7 exhibited regression of solid tumors via enhanced occupancy of CD4 + CAR-T, improved T-cell expansion, reduced exhaustion markers (programmed cell death protein 1 or PD-1 and lymphocyte-activation gene 3 or LAG-3) expression, and increased generation of stem cell-like memory CAR-T cells. The STAT5 pathway was demonstrated to be downstream of NT-I7 signaling, mediated by increased expression of the IL-7 receptor expression in CAR-T cells. Furthermore, CAR-T cells improved efficacy against tumors with low antigen density when combined with NT-I7 in mice, presenting an avenue for patients with heterogeneous antigenic profiles., Conclusion: This study provides a rationale for NT-I7 plus CAR-T cell combination therapy for solid tumors in humans., Competing Interests: Competing interests: MH and DL are inventors on the related patent application no. 63/196556 assigned to the NIH, “Combination immunotherapy for the treatment of glypican-3 (GPC3)-expressing tumors”. MH and DL have patents related to antibody and cell-based immunotherapies and may receive blind royalties from the NIH. MH received research funds from NeoImmuneTech via Cooperative Research and Development Agreement (CRADA) assigned to the NIH. AW, SF-M, and BHL are employees of NeoImmuneTech. The authors declare no other conflicts of interest., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

T-cell malignancies are associated with frequent relapse and high morbidity, which is partly due to the lack of effective or targeted treatment options. To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogeneic "universal" CD2-targeting CAR-T cell (UCART2), in which the CD2 antigen is deleted to prevent fratricide, and the T-cell receptor is removed to prevent GvHD. UCART2 demonstrated efficacy against T-ALL and CTCL and prolonged the survival of tumor-engrafted NSG mice in vivo. To evaluate the impact of CD2 on CAR-T function, we generated CD19 CAR-T cells (UCART19) with or without CD2 deletion, single-cell secretome analysis revealed that CD2 deletion in UCART19 reduced frequencies of the effector cytokines (Granzyme-B and IFN-γ). We also observed that UCART19ΔCD2 had reduced anti-tumor efficacy compared to UCART19 in a CD19+NALM6 xenograft model. Of note is that the reduced efficacy resulting from CD2 deletion was reversed when combined with rhIL-7-hyFc, a long-acting recombinant human interleukin-7. Treatment with rhIL-7-hyFc prolonged UCART2 persistence and increased survival in both the tumor re-challenge model and primary patient T-ALL model in vivo. Together, these data suggest that allogeneic fratricide-resistant UCART2, in combination with rhIL-7-hyFc, could be a suitable approach for treating T-cell malignancies., (© 2023. The Author(s).)

Multiple myeloma (MM), a malignancy of mature plasma cells, remains incurable. B-cell maturation antigen (BCMA) is the lead protein target for chimeric antigen receptor (CAR) therapy because of its high expression in most MM, with limited expression in other cell types, resulting in favorable on-target, off tumor toxicity. The response rate to autologous BCMA CAR-T therapy is high; however, it is not curative and is associated with risks of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome. Outcomes in patients treated with BCMA CAR-T cells (CAR-Ts) may improve with allogeneic CAR T-cell therapy, which offer higher cell fitness and reduced time to treatment. However, to prevent the risk of graft-versus-host disease (GVHD), allogenic BCMA CAR-Ts require genetic deletion of the T-cell receptor (TCR), which has potential for unexpected functional or phenotype changes. Invariant natural killer T cells (iNKTs) have an invariant TCR that does not cause GVHD and, as a result, can be used in an allogeneic setting without the need for TCR gene editing. We demonstrate significant anti-myeloma activity of BCMA CAR-iNKTs in a xenograft mouse model of myeloma. We found that a long-acting interleukin-7 (IL-7), rhIL-7-hyFc, significantly prolonged survival and reduced tumor burden in BCMA CAR-iNKT-treated mice in both primary and re-challenge settings. Furthermore, in CRS in vitro assays, CAR-iNKTs induced less IL-6 than CAR-Ts, suggesting a reduced likelihood of CAR-iNKT therapy to induce CRS in patients. These data suggest that BCMA CAR-iNKTs are potentially a safer, effective alternative to BCMA CAR-Ts and that BCMA CAR-iNKT efficacy is further potentiated with rhIL-7-hyFc., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Interleukin-7 (IL-7) is an essential cytokine for T-cell homeostatic proliferation and maintenance. Clinical studies have shown the potential benefits of IL-7 therapy in various diseases associated with lymphopenia. However, the kinetics of the T-cell response to a single administration of IL-7 in humans have not been fully elucidated. Here, we investigated the effects of Fc-fused long-acting recombinant human IL-7 (hIL-7-hyFc, efineptakin alfa) on lymphocytes in healthy adults after a single subcutaneous or intramuscular administration. Administration of hIL-7-hyFc increased the CD8+ and CD4+ T-cell numbers up to 2.5-fold, with corresponding upregulation of Ki-67 and Bcl-2 expression, peaking at day 3 or 7. Regulatory T cells (Tregs) did not expand. Among CD8+ and CD4+ T cells, all T-cell subsets (TN, TEM, TCM, TEMRA, and TSCM) increased for 56 days. The T-cell receptor repertoire diversity of naive CD8+ and CD4+ T cells was increased by hIL-7-hyFc, whereas the memory T-cell subsets did not differ between day 56 and day 0. Transcriptomic analysis revealed that hIL-7-hyFc induced robust T-cell expansion without changes in gene expression profiles associated with T-cell functions or genes related to T-cell exhaustion, senescence, and anergy. The effector functions of antigen-specific CD8+ T cells were preserved after hIL-7-hyFc administration. Our results suggest that hIL-7-hyFc administration induced a sustained increase in the numbers of CD8+ and CD4+ T cells, but not Tregs, without qualitative changes. These results support the potential of hIL-7-hyFc as a treatment for patients with compromised T-cell immunity or as a vaccine adjuvant., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Immunotherapies for the treatment of solid tumors continue to develop in preclinical and clinical research settings. Unfortunately, for many patients the tumor fails to respond or becomes resistant to therapies such as checkpoint inhibitors (CPIs) targeting programmed cell death protein-1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T lymphocyte antigen-4 (CTLA-4). In many cancers, failed response to CPIs can be attributed to poor T cell infiltration, dominant immunosuppression, and exhausted immune responses. In gastrointestinal (GI) cancers T cell infiltration can be dismal, with several reports finding that CD8 + T cells compose less than 2% of all cells within the tumor. Organized aggregates of lymphocytes, antigen-presenting cells, and vessels, together termed tertiary lymphoid structures (TLSs), are hypothesized to be a major source of T cells within solid tumors. The intratumoral formation of these organized immune centers appears to rely on intricate cytokine and chemokine signaling to heterogeneous cell populations such as B and T cells, innate lymphoid cells, fibroblasts, and dendritic cells. In GI cancers, the presence and density of TLSs provide prognostic value for predicting outcome and survival. Further, TLS presence and density associates with favorable responses to CPIs in many cancers. This review highlights the prognostic value of TLSs in GI cancers, the role of the homeostatic cytokine interleukin-7 (IL-7) in TLS formation, and the induction of TLSs in solid tumors by novel therapeutics., Competing Interests: Conflict of Interest: None.

Chimeric antigen receptor (CAR) T cell therapy is routinely used to treat patients with refractory hematologic malignancies. However, a significant proportion of patients experience suboptimal CAR T cell cytotoxicity and persistence that can permit tumor cell escape and disease relapse. Here we show that a prototype pro-lymphoid growth factor is able to enhance CAR T cell efficacy. We demonstrate that a long-acting form of recombinant human interleukin-7 (IL-7) fused with hybrid Fc (rhIL-7-hyFc) promotes proliferation, persistence and cytotoxicity of human CAR T cells in xenogeneic mouse models, and murine CAR T cells in syngeneic mouse models, resulting in long-term tumor-free survival. Thus, rhIL-7-hyFc represents a tunable clinic-ready adjuvant for improving suboptimal CAR T cell activity., (© 2022. The Author(s).)

Purpose: Patients with glioblastoma (GBM) are treated with radiotherapy (RT) and temozolomide (TMZ). These treatments may cause prolonged systemic lymphopenia, which itself is associated with poor outcomes. NT-I7 is a long-acting IL7 that expands CD4 and CD8 T-cell numbers in humans and mice. We tested whether NT-I7 prevents systemic lymphopenia and improves survival in mouse models of GBM., Experimental Design: C57BL/6 mice bearing intracranial tumors (GL261 or CT2A) were treated with RT (1.8 Gy/day × 5 days), TMZ (33 mg/kg/day × 5 days), and/or NT-I7 (10 mg/kg on the final day of RT). We followed the mice for survival while serially analyzing levels of circulating T lymphocytes. We assessed regulatory T cells (Treg) and cytotoxic T lymphocytes in the tumor microenvironment, cervical lymph nodes, spleen, and thymus, and hematopoietic stem and progenitor cells in the bone marrow., Results: GBM tumor-bearing mice treated with RT+NT-I7 increased T lymphocytes in the lymph nodes, thymus, and spleen, enhanced IFNγ production, and decreased Tregs in the tumor which was associated with a significant increase in survival. NT-I7 also enhanced central memory and effector memory CD8 T cells in lymphoid organs and tumor. Depleting CD8 T cells abrogated the effects of NT-I7. Furthermore, NT-I7 treatment decreased progenitor cells in the bone marrow., Conclusions: In orthotopic glioma-bearing mice, NT-I7 mitigates RT-related lymphopenia, increases cytotoxic CD8 T lymphocytes systemically and in the tumor, and improves survival. A phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957)., (©2022 American Association for Cancer Research.)

The approval of immunotherapies such as checkpoint inhibitors (CPIs), adoptive cell therapies and cancer vaccines has revolutionized the way cancer treatment is approached. While immunotherapies have improved clinical outcome in a variety of tumor types, some cancers have proven harder to combat using single agents, underscoring the need for multi-targeted immunotherapy approaches. Efficacy of CPIs and cancer vaccines requires patients to have a competent immune system with adequate cell numbers while the efficacy of adoptive cellular therapy is limited by the expansion and persistence of cells after infusion. A promising strategy to overcome these challenges is combination treatment with common gamma-chain cytokines. Gamma-chain cytokines play a critical role in the survival, proliferation, differentiation and function of multiple immune cell types, including CD8 T-cells and NK cells, which are at the center of the anti-tumor response. While the short half-life of recombinant cytokines initially limited their application in the clinic, advancements in protein engineering have led to the development of several next-generation drug candidates with dramatically increased half-life and bioactivity. When combining these cytokines with other immunotherapies, strong evidence of synergy has been observed in preclinical and clinical cancer settings. This promising data has led to the initiation of 70 ongoing clinical trials including IL-2, IL-7, IL-15 and IL-21. This review summarizes the recent advancements of common gamma-chain cytokines and their potential as a cancer immunotherapy., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest., (Copyright © 2022. The Korean Association of Immunologists.)

Pollen apertures, the characteristic gaps in pollen wall exine, have emerged as a model for studying the formation of distinct plasma membrane domains. In each species, aperture number, position, and morphology are typically fixed; across species they vary widely. During pollen development, certain plasma membrane domains attract specific proteins and lipids and become protected from exine deposition, developing into apertures. However, how these aperture domains are selected is unknown. Here, we demonstrate that patterns of aperture domains in Arabidopsis are controlled by the members of the ancient ELMOD protein family, which, although important in animals, has not been studied in plants. We show that two members of this family, MACARON (MCR) and ELMOD_A, act upstream of the previously discovered aperture proteins and that their expression levels influence the number of aperture domains that form on the surface of developing pollen grains. We also show that a third ELMOD family member, ELMOD_E, can interfere with MCR and ELMOD_A activities, changing aperture morphology and producing new aperture patterns. Our findings reveal key players controlling early steps in aperture domain formation, identify residues important for their function, and open new avenues for investigating how diversity of aperture patterns in nature is achieved., Competing Interests: YZ, PA, SR, BL, AH, AD No competing interests declared, (© 2021, Zhou et al.)

Purpose: Patients with triple-negative breast cancer (TNBC) who do not achieve pathological complete response (pCR) following neoadjuvant chemotherapy have a high risk of recurrence and death. Molecular characterization may identify patients unlikely to achieve pCR. This neoadjuvant trial was conducted to determine the pCR rate with docetaxel and carboplatin and to identify molecular alterations and/or immune gene signatures predicting pCR., Experimental Design: Patients with clinical stages II/III TNBC received 6 cycles of docetaxel and carboplatin. The primary objective was to determine if neoadjuvant docetaxel and carboplatin would increase the pCR rate in TNBC compared to historical expectations. We performed whole-exome sequencing (WES) and immune profiling on pre-treatment tumor samples to identify alterations that may predict pCR. Thirteen matching on-treatment samples were also analyzed to assess changes in molecular profiles., Results: Fifty-eight of 127 (45.7%) patients achieved pCR. There was a non-significant trend toward higher mutation burden for patients with residual cancer burden (RCB) 0/I versus RCB II/III (median 80 versus 68 variants, p 0.88). TP53 was the most frequently mutated gene, observed in 85.7% of tumors. EGFR, RB1, RAD51AP2, SDK2, L1CAM, KPRP, PCDHA1, CACNA1S, CFAP58, COL22A1, and COL4A5 mutations were observed almost exclusively in pre-treatment samples from patients who achieved pCR. Seven mutations in PCDHA1 were observed in pre-treatment samples from patients who did not achieve pCR. Several immune gene signatures including IDO1, PD-L1, interferon gamma signaling, CTLA4, cytotoxicity, tumor inflammation signature, inflammatory chemokines, cytotoxic cells, lymphoid, PD-L2, exhausted CD8, Tregs, and immunoproteasome were upregulated in pre-treatment samples from patients who achieved pCR., Conclusion: Neoadjuvant docetaxel and carboplatin resulted in a pCR of 45.7%. WES and immune profiling differentiated patients with and without pCR., Trial Registration: Clinical trial information: NCT02124902, Registered 24 April 2014 & NCT02547987, Registered 10 September 2015., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Pollen apertures are an interesting model for the formation of specialized plasma-membrane domains. The plant-specific protein INP1 serves as a key aperture factor in such distantly related species as Arabidopsis, rice and maize. Although INP1 orthologues probably play similar roles throughout flowering plants, they show substantial sequence divergence and often cannot substitute for each other, suggesting that INP1 might require species-specific partners. Here, we present a new aperture factor, INP2, which satisfies the criteria for being a species-specific partner for INP1. Both INP proteins display similar structural features, including the plant-specific DOG1 domain, similar patterns of expression and mutant phenotypes, as well as signs of co-evolution. These proteins interact with each other in a species-specific manner and can restore apertures in a heterologous system when both are expressed but not when expressed individually. Our findings suggest that the INP proteins form a species-specific functional module that underlies formation of pollen apertures., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF ( p < 0.01) or GF ( p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM ( p < 0.01), IgG ( p < 0.05), IL-2 ( p < 0.01), IL-4 ( p < 0.01), IL-6 ( p < 0.01), and IFN-γ ( p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

A low lymphocyte count puts immune-compromised patients at risk of mortality. hIL-7-hyFc is a homodimeric interleukin-7 (IL-7), a potent T-cell amplifier, fused to the hybridizing IgD/IgG4 immunoglobulin domain. We performed a randomized, double-blind, placebo-controlled, dose-escalation, phase I study to assess the pharmacokinetic, pharmacodynamic, safety, tolerability, and immunogenicity profiles of hIL-7-hyFc administered s.c. and i.m. to healthy volunteers. Thirty subjects randomly received hIL-7-hyFc or its matching placebo in an 8:2 ratio at 20, 60 μg/kg s.c., or 60 μg/kg i.m. The hIL-7-hyFc was slowly absorbed and its terminal half-life was 63.26 hours after i.m. administration. The hIL-7-hyFc increased absolute lymphocyte count, mostly in T-cells, which peaked 3 weeks after administration and then lasted for several additional weeks. The hIL-7-hyFc was well-tolerated after a single s.c. and i.m. administration. Injection site reaction was the most common treatment-emergent adverse event, which resolved spontaneously without treatment. The hIL-7-hyFc can be developed into a beneficial treatment option for patients with compromised T-cell immunity. This trial was registered at www.clinicaltrials.gov as #NCT02860715., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society of Clinical Pharmacology and Therapeutics.)

Objectives: Emerging oncotherapeutic strategies require the induction of an immunostimulatory tumor microenvironment (TME) containing numerous tumor-reactive CD8 + T cells. Interleukin-7 (IL-7), a T-cell homeostatic cytokine, induces an antitumor response; however, the detailed mechanisms underlying the contributions of the IL-7 to TME remain unclear. Here, we aimed to investigate the mechanism underlying the induction of antitumor response by hybrid Fc-fused long-acting recombinant human IL-7 (rhIL-7-hyFc) through regulation of both adaptive and innate immune cells in the TME., Methods: We evaluated rhIL-7-hyFc-mediated antitumor responses in murine syngeneic tumor models. We analysed the cellular and molecular features of tumor-infiltrating lymphocytes (TILs) and changes in the TME after rhIL-7-hyFc treatment. Furthermore, we evaluated the antitumor efficacy of rhIL-7-hyFc combined with chemotherapy and checkpoint inhibitors (CPIs)., Results: Systemic delivery of rhIL-7-hyFc induced significant therapeutic benefits by expanding CD8 + T cells with enhanced tumor tropism. In tumors, rhIL-7-hyFc increased both tumor-reactive and bystander CD8 + TILs, all of which displayed enhanced effector functions but less exhausted phenotypes. Moreover, rhIL-7-hyFc suppressed the generation of immunosuppressive myeloid cells in the bone marrow of tumor-bearing mice, resulting in the immunostimulatory TME. Combination therapy with chemotherapy and CPIs, rhIL-7-hyFc elicited a strong antitumor response and even under a T lymphopenic condition by restoring CD8 + T cells. When combined with chemotherapy and CPIs, rhIL-7-hyFc administration enhanced antitumor response under intact andlymphopenic conditions by restoring CD8 + T cells., Conclusion: Taken together, these data demonstrate that rhIL-7-hyFc induces antitumor responses by generating T-cell-inflamed TME and provide a preclinical proof of concept of immunotherapy with rhIL-7-hyFc to enhance therapeutic responses in the clinic., Competing Interests: DC and BHL are employees of the Research Institute of NeoImmuneTech, Inc., which supports rhIL‐7‐hyFc (the registered international nonproprietary name) and its formulation buffer reagents presented in this article. The remaining authors declare no conflict of interests., (© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Novel engineered T cells containing chimeric antigen receptors (CAR-T cells) that combine the benefits of antigen recognition and T cell response have been developed, and their effect in the anti-tumor immunotherapy of patients with relapsed/refractory leukemia has been dramatic. Thus, CAR-T cell immunotherapy is rapidly emerging as a new therapy. However, it has limitations that prevent consistency in therapeutic effects in solid tumors, which accounts for over 90% of all cancer patients. Here, we review the literature regarding various obstacles to CAR-T cell immunotherapy for solid tumors, including those that cause CAR-T cell dysfunction in the immunosuppressive tumor microenvironment, such as reactive oxygen species, pH, O 2 , immunosuppressive cells, cytokines, and metabolites, as well as those that impair cell trafficking into the tumor microenvironment. Next-generation CAR-T cell therapy is currently undergoing clinical trials to overcome these challenges. Therefore, novel approaches to address the challenges faced by CAR-T cell immunotherapy in solid tumors are also discussed here.

Certain regions on the surfaces of developing pollen grains exhibit very limited deposition of pollen wall exine. These regions give rise to pollen apertures, which are highly diverse in their patterns and specific for individual species. Arabidopsis thaliana pollen develops three equidistant longitudinal apertures. The precision of aperture formation suggests that, to create them, pollen employs robust mechanisms that generate distinct cellular domains. To identify players involved in this mechanism, we screened natural Arabidopsis accessions and discovered one accession, Martuba, whose apertures form abnormally due to the disruption of the protein kinase D6PKL3. During pollen development, D6PKL3 accumulates at the three plasma membrane domains underlying future aperture sites. Both D6PKL3 localization and aperture formation require kinase activity. Proper D6PKL3 localization is also dependent on a polybasic motif for phosphoinositide interactions, and we identified two phosphoinositides that are specifically enriched at the future aperture sites. The other known aperture factor, INAPERTURATE POLLEN1, fails to aggregate at the aperture sites in d6pkl3 mutants, changes its localization when D6PKL3 is mislocalized, and, in turn, affects D6PKL3 localization. The discovery of aperture factors provides important insights into the mechanisms cells utilize to generate distinct membrane domains, develop cell polarity, and pattern their surfaces., (© 2018 American Society of Plant Biologists. All rights reserved.)

We investigated the biological roles of the Arabidopsis ( Arabidopsis thaliana ) GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) transcriptional complex in the development of gynoecia and anthers. There are nine GRFs and three GIFs in Arabidopsis, and seven GRFs are posttranscriptionally silenced by microRNA396 (miR396). We found that overexpression of MIR396 in the gif1 gif2 double mutant background ( gif1 gif2 35S : MIR396 ) resulted in neither ovary nor pollen. Histological and molecular marker-based analyses revealed that the mutant gynoecial primordia failed to develop carpel margin meristems and mature flowers lacked the ovary, consisting only of the stigma, style, and replum-like tissues. The mutant anther primordia were not able to form the pluripotent archesporial cells that produce pollen mother cells and microsporangia. Multiple combinations of GRF mutations also displayed the same phenotypes, indicating that the GRF-GIF duo is required for the formation of those meristematic and pluripotent cells. Most GRF proteins are localized and abundant in those cells. We also found that the weak gynoecial defects of pinoid-3 ( pid-3 ) mutants were remarkably exacerbated by gif1 gif2 double mutations and 35S : MIR396 , so that none of the gynoecia produced by gif1 gif2 pid-3 and 35S : MIR396 pid-3 developed ovaries at all. Moreover, gif1 gif2 double mutations and 35S : MIR396 also acted synergistically with 1- N -naphthylphthalamic acid in forming aberrant gynoecia. The results altogether suggest that the GRF-GIF duo regulates the meristematic and pluripotent competence of carpel margin meristems and the archesporial cell lineage and that this regulation is implemented in association with auxin action, ultimately conferring reproductive competence on Arabidopsis., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures-openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores-the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them.

Background: Psoriasis is one of the most common chronic inflammatory diseases of the skin. Recently, IL-17-producing T cells have been shown to play a critical role in psoriatic inflammation. Programmed cell death 1 (PD-1) is a coinhibitory receptor expressed on T cells in various chronic inflammatory diseases; however, the expression and function of PD-1 during psoriatic inflammation have not previously been characterized., Objective: We examined PD-1 expression on IL-17A-producing T cells from imiquimod-treated mice and patients with psoriasis. Additionally, we investigated the therapeutic effect of recombinant programmed cell death ligand 1 (PD-L1) protein on imiquimod-induced psoriatic inflammation., Methods: PD-1 expression on IL-17A-producing γδ T cells from imiquimod-treated mice was examined by means of multicolor flow cytometric analysis. In the psoriatic skin of patients, PD-1 and IL-17A expression was analyzed by using immunofluorescence. The therapeutic effect of PD-L1-Fc fusion protein (PD-L1-Fc) was assessed in imiquimod-treated mice ex vivo and in vivo., Results: During imiquimod-induced psoriatic inflammation, PD-1 is overexpressed on CD27(-)Vγ1(-) γδ T cells. Furthermore, PD-1 expression on IL-17A(+) T cells was confirmed in psoriatic skin tissues from patients and imiquimod-treated mice. In the CD27(-)Vγ1(-) γδ T-cell population, Vγ4(-) γδ T cells with Vγ6 mRNA expression showed a high level of PD-1 expression. Furthermore, these PD-1(hi)Vγ4(-) (Vγ6(+)) γδ T cells were specialized for anti-CD3-induced IL-17A production, which was inhibited by PD-L1-Fc treatment. In imiquimod-treated mice PD-L1-Fc reduced psoriatic inflammation when given alone and enhanced the therapeutic effect of anti-p40 when given in combination., Conclusion: PD-1 is overexpressed in IL-17A-producing T cells in both imiquimod-treated mice and patients with psoriasis. Moreover, recombinant PD-L1-Fc alleviates psoriatic inflammation in imiquimod-treated mice., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The cell proliferation process of aerial lateral organs, such as leaves and flowers, is coordinated by complex genetic networks that, in general, converge on the cell cycle. The Arabidopsis thaliana NGATHA (AtNGA) family comprises four members that belong to the B3-type transcription factor superfamily, and has been suggested to be involved in growth and development of aerial lateral organs, although its role in the cell proliferation and expansion processes remains to be resolved in more detail. In order to clarify the role of AtNGAs in lateral organ growth, we took a systematic approach using both the loss- and gain-of-functional mutants of all four members. Our results showed that overexpressors of AtNGA1 to AtNGA4 developed small, narrow lateral organs, whereas the nga1 nga2 nga3 nga4 quadruple mutant produced large, wide lateral organs. We found that cell numbers of the lateral organs were significantly affected: a decrease in overexpressors and, inversely, an increase in the quadruple mutant. Kinematic analyses on leaf growth revealed that, compared with the wild type, the overexpressors displayed a lower activity of cell proliferation and yet the mutant a higher activity. Changes in expression of cell cycle-regulating genes were well in accordance with the cell proliferation activities, establishing that the AtNGA transcription factors act as bona fide negative regulators of the cell proliferation of aerial lateral organs.

The Arabidopsis thaliana GROWTH-REGULATING FACTOR (GRF) gene family comprises 9 members and encodes a class of transcription factors. We previously demonstrated that GRF genes played an essential role in formation of the boundary region between cotyledons, since their loss-of-function mutants developed fused cotyledons. Our present study shows that the grf mutants display fused floral organs as well. Such fusion phenotypes of embryonic and post-embryonic floral organs are highly reminiscent of the cup-shaped cotyledon (cuc) mutants. In order to test a genetic interaction between GRFs and CUCs, we constructed cuc1 grf1/2/3, cuc2 grf1/2/3, and cuc3 grf1/2/3 quadruple mutants, and found that the mutants showed dramatic increases in cotyledon fusion as well as floral organ fusion. The results suggest that the signaling pathway of GRFs may be genetically associated with that of CUCs in the organ separation process.

Reproductive success of angiosperms relies on the precise development of the gynoecium and the anther, because their primary function is to bear and to nurture the embryo sac/female gametophyte and pollen, in which the egg and sperm cells, respectively, are generated. It has been known that the GRF-INTERACTING FACTOR (GIF) transcription co-activator family of Arabidopsis thaliana (Arabidopsis) consists of three members and acts as a positive regulator of cell proliferation. Here, we demonstrate that GIF proteins also play an essential role in development of reproductive organs and generation of the gamete cells. The gif1 gif2 gif3 triple mutant, but not the single or double mutants, failed to establish normal carpel margin meristem (CMM) and its derivative tissues, such as the ovule and the septum, resulting in a split gynoecium and no observable embryo sac. The gif triple mutant also displayed severe structural and functional defects in the anther, producing neither microsporangium nor pollen grains. Therefore, we propose that the GIF family of Arabidopsis is a novel and essential component required for the cell specification maintenance during reproductive organ development and, ultimately, for the reproductive competence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Developmental biologists have been fascinated with the long-standing mystery of how multicellular organisms, such as plants and animals, sense and control their organ size. In plants, leaves are a suitable experimental system for elucidation of the mystery, because they, like animal organs, inherently exhibit a determinate growth pattern, meaning that they possess genetic information for the control of their final size. The cell proliferation and expansion processes are prerequisites for growth, so that the genetic controls should converge on the 2 cellular processes and decide their rate or duration during leaf growth. Plant scientists have found dozens of genes involved in the control of the cellular processes, including the Arabidopsis thaliana GRF-INTERACTING FACTOR (GIF) family. The GIF family consists of 3 members, GIF1 to GIF3, and encodes a class of transcription co-activators. Although the GIF family genes have been shown to play an essential role in the control of cell proliferation of the leaf organ, understanding of the spatio-temporal behaviors of GIF expression, in both aspects of their promoters and proteins, has been limited to GIF1 (also known as ANGUSTIFOLIA3, AN3). Here, we define kinematic growth properties of wild-type and gif leaf organs and present spatio-temporal expression patterns of all GIF genes, thus providing comprehensive insights into biological roles and expression behaviors of the whole GIF family members during leaf growth.

Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS.

Introduction: Sjögren's syndrome (SjS) is a systemic autoimmune disease characterized by decreased salivary and lacrimal gland secretions, resulting in severe dry mouth and dry eyes. Recent studies have suggested that TH17 cells and its signature cytokine IL-17 are involved in the underlying pathogenic mechanisms leading to destructive inflammation and autoimmunity. In the present study, we examined whether IL-27, a natural inhibitor of TH17 activity, could down-regulate or reverse SjS in C57BL/6.NOD-Aec1Aec2 mice, a model of primary-SjS., Methods: Recombinant serotype 2 adeno-associated viral (AAV2) vectors expressing either IL-27 (rAAV2-IL27) or LacZ (rAAV2-LacZ) were injected into 6 or 14 week-old C57BL/6.NOD-Aec1Aec2 mice. Changes in IL-27, IL-17, and IL-10 cytokine levels in peripheral blood were determined by ELISAs, while flow cytometry analyses were used to quantify cytokine-positive splenocytes. Histological assessment of salivary glands, anti-nuclear autoantibody (ANA) staining, and stimulated saliva flow rates were used to profile SjS disease severity., Results: Mice systemically treated with intravenous rAAV2-IL27 injections at either 6 or 14 weeks of age exhibited long-term elevated levels of serum IL-27 with concomitantly reduced levels of IL-17 compared with sera from mice injected with rAAV2-LacZ or saline out to 20 weeks post-inoculation. Most importantly, disease profiles revealed that rAAV2-IL27 treatment had little effect on lymphocytic focus (LF) scores, but resulted in structural changes in LF, lower titers of ANAs with changes in staining patterns, and a less severe clinical disease as determined by saliva flow rates., Conclusions: These data support the concept that IL-27, when provided exogenously, can induce a suppressive effect on SjS development and thus may be an effective therapeutic agent for regulating TH17 pro-inflammatory activity in autoimmune diseases where the TH17 system has been shown to play an important role in their pathogenesis.

The mouse model is the one of the most frequently used and well-established animal models, and is currently used in many research areas. To date, various mouse models have been utilized to elucidate underlying causes of multifactorial autoimmune conditions, including pathological immune components and specific signaling pathways. This review summarizes the more recent mouse models for Sjögren's syndrome, a systemic autoimmune disease characterized by lymphocytic infiltration in the exocrine glands, such as the salivary and lacrimal glands, and loss of secretory function, resulting in dry mouth and dry eyes in patients. Although every Sjögren's syndrome mouse model resembles the major symptoms or phenotypes of Sjögren's syndrome conditions in humans, the characteristics of each model are variable. Moreover, to date, there is no single mouse model that can completely replicate the human conditions. However, unique features of each mouse model provide insights into the roles of potential etiological and immunological factors in the development and progression of Sjögren's syndrome. Here, we will overview the Sjögren's syndrome mouse models. Lessons from these mouse models will aid us to understand underlying immune dysregulation in autoimmune diseases in general, and will guide us to direct future research towards appropriate diagnostic and therapeutic strategies.

Sjögren's syndrome (SjS) is a complex chronic autoimmune disease of unknown etiology which primarily targets the exocrine glands, resulting in eventual loss of secretory function. The disease can present as either primary SjS or secondary SjS, the latter of which occurs concomitantly with another autoimmune disease such as rheumatoid arthritis, systemic lupus erythematosus, scleroderma, or primary biliary cirrhosis. Current advancements in therapeutic prevention and treatment for SjS are impeded by lack of understanding in the pathophysiological and clinical progression of the disease. Development of appropriate mouse models for both primary and secondary SjS is needed in order to advance knowledge of this disease. This paper details important features, advantages, and pitfalls of current animal models of SjS, including spontaneous, transgenic, knockout, immunization, and transplantation chimera mouse models, and emphasizes the need for a better model in representing the human SjS phenotype.

Sjögren's syndrome (SS) involves a chronic, progressive inflammation primarily of the salivary and lacrimal glands leading to decreased levels of saliva and tears that eventually result in dry mouth and dry eye diseases. T(H)17 cell populations secreting IL17A have been shown to have an important function in an increasing number of autoimmune diseases, including SS. In this study, we investigated the function of IL17A on SS development and onset. Adenovirus-5 vectors expressing either IL17R:fragment of crystallization (Fc) fusion protein or LacZ were injected through retrograde cannulation into the salivary glands of SS-susceptible (SS(S)) C57BL/6.NOD-Aec1Aec2 mice between 6 and 8 weeks of age (a pre-disease stage) or 15 and 17 weeks of age (a diseased stage). The mice were subsequently characterized for their SS phenotypes. Mice cannulated with the Ad5-IL17R:Fc viral vector at either 7 or 16 weeks of age exhibited a rapid temporal, yet persistent, decrease in the levels of serum IL17 as well as the overall numbers of CD4+IL17+T cells present in their spleens. Disease profiling indicated that these mice showed decreased lymphocytic infiltrations of their salivary glands, normalization of their antinuclear antibodies repertoire, and increased saliva secretion. In contrast, mice cannulated with the control Ad5-LacZ viral vector did not exhibit similar changes and progressed to the overt disease stage. The capacity of the Ad5-IL17R:Fc-blocking factor to reduce SS pathology in SS(S) mice strongly suggests that IL17 is an important inflammatory cytokine in salivary gland dysfunction. Thus, therapeutic approach targeting IL17 may be effective in preventing glandular dysfunction.

Introduction: Sjögren's syndrome (SS) involves a chronic, progressive inflammation primarily of the salivary and lacrimal glands leading to decreased levels of saliva and tears resulting in dry mouth and dry eye diseases. Seminal findings regarding TH17 cell populations that secrete predominantly interleukin (IL)-17A have been shown to play an important role in an increasing number of autoimmune diseases, including SS. In the present study, we investigated the function of IL-17A on the development and onset of SS., Methods: Adenovirus serotype 5 (Ad5) vectors expressing either IL-17A or LacZ were infused via retrograde cannulation into the salivary glands of C57BL/6J mice between 6 and 8 weeks of age or between 15 and 17 weeks of age. The mice were characterized for SS phenotypes., Results: Disease profiling indicated that SS-non-susceptible C57BL/6J mice whose salivary glands received the Ad5-IL17A vector developed a SS-like disease profile, including the appearance of lymphocytic foci, increased cytokine levels, changes in antinuclear antibody profiles, and temporal loss of saliva flow., Conclusions: Induction of SS pathology by IL-17A in SS-non-susceptible mice strongly suggests that IL-17A is an important inflammatory cytokine in salivary gland dysfunction. Thus, localized anti-IL17 therapy may be effective in preventing glandular dysfunction.

In an effort to elucidate biological functions of transcription factors of Brassica rapa L. (ssp. pekinensis), an NGATHA homolog, BrNGA1, that belongs to the B3-type transcription factor superfamily was identified and expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Arabidopsis plants overexpressing BrNGA1, named BrNGA1ox, displayed markedly reduced organ growth compared with the wild type: lateral organs, such as leaves, flowers and cotyledons, were small and distinctively narrow, and their root growth was also severely retarded. Reduced sizes of BrNGA1ox organs were mainly due to reduction in cell numbers. Kinematic analysis of leaf growth revealed that both the rate and duration of cell proliferation declined during organogenesis, which was consistent with the reduced expression of cyclin genes. Reduction in organ growth was strongly correlated with the small size of meristematic cell pools in the shoot and root meristems. Taken together, these data indicate that BrNGA1 acts as a negative regulator of cell proliferation and may do so, in part, by regulating the size of the meristematic cell pool.

Previously, the GRF-INTERACTING FACTOR1 (GIF1)/ANGUSTIFOLIA3 (AN3) transcription coactivator gene, a member of a small gene family comprising three genes, was characterized as a positive regulator of cell proliferation in lateral organs, such as leaves and flowers, of Arabidopsis (Arabidopsis thaliana). As yet, it remains unclear how GIF1/AN3 affects the cell proliferation process. In this study, we demonstrate that the other members of the GIF gene family, GIF2 and GIF3, are also required for cell proliferation and lateral organ growth, as gif1, gif2, and gif3 mutations cause a synergistic reduction in cell numbers, leading to small lateral organs. Furthermore, GIF1, GIF2, and GIF3 overexpression complemented a cell proliferation defect of the gif1 mutant and significantly increased lateral organ growth of wild-type plants as well, indicating that members of the GIF gene family are functionally redundant. Kinematic analysis on leaf growth revealed that the gif triple mutant as well as other strong gif mutants developed leaf primordia with fewer cells, which was due to the low rate of cell proliferation, eventually resulting in earlier exit from the proliferative phase of organ growth. The low proliferative activity of primordial leaves was accompanied by decreased expression of cell cycle-regulating genes, indicating that GIF genes may act upstream of cell cycle regulators. Analysis of gif double and triple mutants clarified a previously undescribed role of the GIF gene family: gif mutants had small vegetative shoot apical meristems, which was correlated with the development of small leaf primordia. gif triple mutants also displayed defective structures of floral organs. Taken together, our results suggest that the GIF gene family plays important roles in the control of cell proliferation via cell cycle regulation and in other developmental properties that are associated with shoot apical meristem function.

Purpose: As a membrane protein at the insertion site of the slit diaphragm (SD) complex in podocyte foot processes, podocin has been reported to act as a scaffolding protein required to maintain or regulate the structural integrity of the SD. In order to identify proteins that associate or interact with podocin, we screened a mouse kidney complementary DNA (cDNA) library using a yeast 2-hybrid system., Materials and Methods: 1) The full-length cDNA of podocin from the mouse kidney was amplified by Polymerase Chain Reaction (PCR), 2) The PCR product was cloned into a pGBKT7 vector, pGBKT7-podocin, 3) After the pGBKT7-podocin was transformed into AH109, the AH109/pGBKT7-podocin product was obtained, 4) The mouse kidney cDNA library was transformed into the AH109/pGBKT7-podocin and screened by selection steps, 5) Next, twelve clones were cultured and isolated, 6) The yeast-purified plasmids were transformed into Escherichia coli (E. coli) by heat shock, and 7) To identify the activation domain (AD)/library inserts, we digested them with Him III, and the fragments were then sequenced., Results: 12 positive clones that interacted with podocin were obtained by screening a mouse kidney cDNA library using pGBKT7-podocin. Among them, only 4 clones were found to function at the podocyte where podocin is present., Conclusion: Additional studies are needed to clarify the role and interaction with podocin and candidates.

Sjögren's syndrome (SjS) is chronic autoimmune disease manifested by the loss of saliva and/or tear secretion by salivary and/or lacrimal glands, respectively. The pathogenesis of the disease remains elusive, perhaps due to the multiple triggers of the disease. However, substantial advances have been made in attempting to resolve the complexity of SjS using both animal models and human subjects. The primary objectives of this review are to provide a better understanding of the disease processes with major emphasis on the use of mouse models, how genetic predisposition plays a role in the natural history of the disease, as well as a presentation of new findings pertaining to the role of T(H)1, T(H)2, and T(H)17 cells in the pathogenesis of SjS.

Introduction: Recently, we reported the development of the C57BL/6.NOD-Aec1Aec2 mouse that carries two genetic intervals derived from the non-obese diabetic (NOD) mouse capable of conferring Sjögren's syndrome (SjS)-like disease in SjS-non-susceptible C57BL/6 mice. In an attempt to define the molecular bases underlying the onset of stomatitis sicca (xerostomia) in this C57BL/6.NOD-Aec1Aec2 mouse model, we have carried out a study using genomic microarray technology., Methods: By means of oligonucleotide microarrays, gene expression profiles of salivary glands at 4, 8, 12, 16, and 20 weeks of age were generated for C57BL/6.NOD-Aec1Aec2 male mice. Using Linear Models for Microarray Analysis and B-statistics software, 480 genes were identified as being differentially expressed (P < 0.01 and Q < 0.0001) during the development of SjS-like disease in the salivary glands., Results: The 480 genes could be arranged into four clusters, with each cluster defining a unique pattern of temporal expression, while the individual genes within each cluster could be grouped according to related biological functions. By means of pair-wise analysis, temporal changes in transcript expressions provided profiles indicating that many additional genes are differentially expressed at specific time points during the development of disease. Multiple genes reportedly showing an association with autoimmunity and/or SjS, in either humans or mouse models, were found to exhibit differential expressions, both quantitatively and temporally. Selecting various families of genes associated with specific functions (for example, antibody production, complement, and chemokines), we noted that only a limited number of family members showed differential expressions and these correlated with specific phases of disease., Conclusions: Taking advantage of known functions of these genes, investigators can construct interactive gene pathways, leading to modeling of possible underlying events inducing salivary gland dysfunction. Thus, these different approaches to analyzing microarray data permit the identification of multiple sets of genes of interest whose expressions and expression profiles may correlate with molecular mechanisms, signaling pathways, and/or immunological processes involved in the development and onset of SjS.

Introduction: Sjögren syndrome (SjS) is a systemic autoimmune disease in which an immunological attack primarily against the salivary and lacrimal glands results in the loss of acinar cell tissue and function, leading to stomatitis sicca and keratoconjunctivitis sicca. In recent years, two genetic regions, one on chromosome 1 (designated autoimmune exocrinopathy 2 or Aec2) and the second on chromosome 3 (designated autoimmune exocrinopathy 1 or Aec1) derived from nonobese diabetic (NOD) mice, have been shown to be necessary and sufficient to replicate SjS-like disease in nonsusceptible C57BL/6 mice., Methods: Starting with the SjS-susceptible C57BL/6-derived mouse, referred to as C57BL/6.NOD-Aec1Aec2, we generated a large set of recombinant inbred (RI) lines containing portions of Aec2 as a means of identifying more precisely the genetic elements of chromosome 1 responsible for disease development., Results: Disease profiling of these RI lines has revealed that the SjS susceptibility genes of Aec2 lie within a region located at approximately 79 +/- 5 cM distal to the centromere, as defined by microsatellite markers. This chromosomal region contains several sets of genes known to correlate with various immunopathological features of SjS as well as disease susceptibility genes for both type 1 diabetes and systemic lupus erythematosus in mice. One gene in particular, tumor necrosis factor (ligand) superfamily member 4 (or Ox40 ligand), encoding a product whose biological functions correlate with both physiological homeostasis and immune regulations, could be a potential candidate SjS susceptibility gene., Conclusions: These new RI lines represent the first step not only in fine mapping SjS susceptibility loci but also in identifying potential candidate SjS susceptibility genes. Identification of possible candidate genes permits construction of models describing underlying molecular pathogenic mechanisms in this model of SjS and establishes a basis for construction of specific gene knockout mice.

Background: Most commonly resulting from intimal hyperplasia at the venous anastomosis, stenosis leading to thrombosis is a major cause of failure of polytetrafluoroethylene (PTFE) dialysis grafts. We recently reported that coating haemodialysis grafts with paclitaxel could reduce neointimal hyperplasia. This study tested whether paclitaxel-coating could prolong graft survival in a porcine model., Methods: PTFE grafts were double-coated with paclitaxel. Bilateral grafts were created between the carotid arteries and the external jugular veins, and we evaluated graft survival by weekly measurements of blood flow for 12 weeks., Results: We successfully implanted four pairs of paclitaxel-coated grafts and four pairs of control grafts in eight Landrace pigs. One control pig had to be euthanized at 4 weeks after graft placement. The grafts in the other three controls and four paclitaxel pigs survived until harvesting of the grafts. All paclitaxel-coated grafts remained patent for 12 weeks without decrease of blood flow. Median blood flow was 702 ml/min at three weeks and 818 ml/min at 12 weeks after placement. In contrast, the four control grafts lost luminal patency at 5, 6, 6 and 8 weeks, respectively. In Kaplan-Meier analysis, paclitaxel-coated grafts showed better survival than uncoated grafts (P = 0.011)., Conclusions: Double-coating with paclitaxel improved graft survival. Coated PTFE grafts may be effective for the prevention of graft failure in patients on haemodialysis.

The major cause of hemodialysis vascular access dysfunction (HVAD) is the occurrence of stenosis followed by thrombosis at venous anastomosis sites due to the aggressive development of venous neointimal hyperplasia. Local delivery of antiproliferative drugs may be effective in inhibiting hyperplasia without causing systemic side effects. We have previously demonstrated that paclitaxel-coated expanded poly(tetrafluoroethylene) (ePTFE) grafts, by a dipping method, could prevent neointimal hyperplasia and stenosis of arteriovenous (AV) hemodialysis grafts, especially at the graft-venous anastomoses; however, large quntities of initial burst release have remained a problem. To achieve controlled drug release, paclitaxel (Ptx)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Ptx-PLGA-NPs) were prepared by the emulsion-solvent evaporation method and then transferred to the luminal surface and inner part of ePTFE vascular grafts through our micro tube pumping and spin penetration techniques. Scanning electron microscope (SEM) images of various stages of Ptx-PLGA-NPs unequivocally showed that micro tube pumping followed by spin penetration effectively transferred Ptx-PLGA-NPs to the inner part, as well as the luminal surface, of an ePTFE graft. In addition, the in vitro release profiles of paclitaxel demonstrated that this new system achieved controlled drug delivery with a reduced initial burst release. These results suggest that loading of Ptx-PLGA-NPs to the luminal surface and the inner part of an ePTFE graft is a promising strategy to ultimately inhibit the development of venous neointimal hyperplasia.

Background: The main pathology of haemodialysis graft stenosis is venous neointimal hyperplasia at graft-venous anastomoses. Neointimal hyperplasia is also observed in cases of coronary artery in-stent restenosis. Paclitaxel is a chemotherapeutic agent used to treat cancer, and has been proven to inhibit neointimal hyperplasia of coronary artery in-stent restenosis. In this study, we examined whether a paclitaxel-coated haemodialysis graft could inhibit neointimal hyperplasia and prevent stenosis., Methods: We dip-coated paclitaxel on expanded polytetrafluoroethylene (ePTFE) grafts at a dose density of 0.59 microg/mm(2). In vitro release tests showed an initial paclitaxel burst followed by a long-term slow release. Using ePTFE grafts with (coated group, n = 8) or without a paclitaxel coating (control group, n = 11), we constructed arteriovenous (AV) grafts connecting the common carotid artery and the external jugular vein in Landrace pigs., Results: After excluding seven pigs for technical failure, cross-sections of graft-venous anastomoses obtained 6 weeks after placing the AV grafts were analysed. Percentage luminal stenosis, ratios of intima to media in whole cross-sections, areas of intima in the peri-junctional areas (within 2 mm above and 2 mm below the graft-venous junction), and the mean thickness of intima within venous sides of cross-sections, were 60.5% (range, 41.5-60.7), 13.0 (range, 8.6-20.4), 23.7 mm(2) (range, 10.8-32.1) and 2.1 mm (range, 1.1-3.0), respectively, in the control group, whereas corresponding median values in the coated group were 10.4% (range, 1.0-17.8), 1.0 (range, 0.7-5.1), 1.6 mm(2) (range, 0.2-8.0) and 0.3 mm (range, 0.1-2.2). All parameters were significantly different between the two groups (P<0.05 by Mann-Whitney test)., Conclusion: Paclitaxel-coated ePTFE grafts could prevent neointimal hyperplasia and the stenosis of AV haemodialysis grafts.

Pulmonary delivery provides the most promising non-parenteral route of insulin administration. Insulin was used as a model protein to demonstrate the feasibility of using protein crystals for the pulmonary delivery of a sustained-release protein drug formulation. Insulin microcrystals with a mean diameter of 3 microm were prepared using a seed zone method. The yield of crystallization was very high (95.8 +/- 0.97%), and the microcrystals were recovered with high efficiency (>98%) by centrifugation. Morphological examination using scanning electron microphotography showed the microcrystals to be of a homogeneous rhombohedral shape, with some rhombus forms, without aggregates. After the administration of 32 U/kg of the microcrystal suspension to STZ-induced diabetic SD rats by intratracheal instillation, the blood glucose levels were reduced and hypoglycemia was prolonged over 13 h, as compared to the insulin solution. The percent minimum reductions of the blood glucose concentration (% MRBG) produced by the microcrystal suspension and insulin solution reached 36.5 and 37.2%, respectively, of the initial level, and the percent total reductions in blood glucose (% TRBG(13 h)) were 34.4 and 25.0%, respectively. In the case of inhalation using a sieve-type ultrasonic nebulizer, the % MRBG produced by the microcrystal suspension and insulin solution were 21.7 and 26.3%, respectively, of the initial level, and the % TRBG(13 h) were 66.7 and 58.4%, respectively. However, the hypoglycemic effects of the microcrystal suspension were prolonged over 7 h, which compares favorably with the insulin solution (P<0.5 by unpaired t-test). These results could be attributed to the sustained-release of insulin from the microcrystals, which were deposited widely throughout the entire lung.