Zinc is an essential trace element required for enzymatic activity and for maintaining the conformation of many transcription factors; thus, zinc homeostasis is tightly regulated. Although zinc affects several signaling molecules and may act as a neurotransmitter, it remains unknown whether zinc acts as an intracellular second messenger capable of transducing extracellular stimuli into intracellular signaling events. In this study, we report that the cross-linking of the high affinity immunoglobin E receptor (Fcɛ receptor I [FcɛRI]) induced a release of free zinc from the perinuclear area, including the endoplasmic reticulum in mast cells, a phenomenon we call the zinc wave. The zinc wave was dependent on calcium influx and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase activation. The results suggest that the zinc wave is involved in intracellular signaling events, at least in part by modulating the duration and strength of FcɛRI-mediated signaling. Collectively, our findings indicate that zinc is a novel intracellular second messenger.

Zinc (Zn) is an essential nutrient, and its deficiency causes growth retardation, immunodeficiency, and neuronal degeneration. However, the precise roles and molecular mechanism(s) of Zn function in immune response have not been clarified. Mast cells (MCs) are granulated cells that play a pivotal role in allergic reactions and inflammation. The granules of MCs contain various chemical mediators and inflammatory cytokines that are released upon FcεRI cross-linking. In this study, we report that Zn is essential for MC activation both in vitro and in vivo. We showed that a Zn chelator, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine, inhibited in vivo allergic reactions such as PCA and PSA. Consistent with this, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine significantly inhibited the FcεRI-induced degranulation and cytokine production. We found that Zn was required for FcεRI-induced translocation of granules to the plasma membrane, a process that we have shown to be important for MC degranulation. In addition, we showed that Zn was essential for plasma membrane translocation of protein kinase C and subsequent nuclear translocation of NF-κB, leading to cytokine production, such as IL-6 and TNF-α. These results revealed that Zn was involved in multiple steps of FcεRI-induced MC activation and required for degranulation and cytokine production.

Zinc (Zn) is an essential nutrient and its deficiency causes immunodeficiency. However, it remains unknown how Zn homeostasis is regulated in mast cells and if Zn transporters are involved in allergic reactions. We show that Znt5/Slc30a5 is required for contact hypersensitivity and mast cell–mediated delayed-type allergic response but not for immediate passive cutaneous anaphylaxis. In mast cells from Znt5−/− mice, Fcϵ receptor I (FcϵRI)–induced cytokine production was diminished, but degranulation was intact. Znt5 was involved in FcϵRI-induced translocation of protein kinase C (PKC) to the plasma membrane and the nuclear translocation of nuclear factor κB. In addition, the Zn finger–like motif of PKC was required for its plasma membrane translocation and binding to diacylglycerol. Thus, Znt5 is selectively required for the mast cell–mediated delayed-type allergic response, and it is a novel player in mast cell activation.

The quantitation of hepatitis C virus (HCV) viremia RNA is measured by molecular techniques such as comcan be helpful in the diagnosis, therapy, and monitoring petitive reverse-transcription polymerase chain reacof patients with chronic hepatitis C. A sensitive and tion and branched DNA (bDNA) signal amplification. 5,6 quantitative fluorescence enzyme immunoassay (FEIA) More recently, a sensitive and quantitative fluoreshas recently been developed for assaying HCV core pro-cence enzyme immunoassay (FEIA) has been developed tein in serum. To assess the utility of measurements of to measure serum HCV core protein, which may also serum HCV core protein during the course of treatment correlate with levels of HCV viremia. 8 We have evaluof chronic hepatitis C, we studied 27 patients who were ated the clinical usefulness of serum HCV core protein treated with a single schedule of interferon alfa (IFN-a) measurements in patients with chronic hepatitis C be- This study was conducted at Shinshu University 27 patients (93%). The initial serum concentration of Hospital and affiliated hospitals between July 1993 and Sep-HCV core protein was significantly (P õ .01) higher in tember 1994. Informed consent was obtained from all pathe nonresponders versus the responders. Two weeks tients. Twenty-seven Japanese patients with chronic hepatiafter initiating IFN-a therapy, HCV core protein was not tis type C who were treated with IFN-a therapy (19 men detectable in any of the 11 responders, but was detected and 8 women aged 32 to 65 years; mean, 50.6 years) were in 8 of 16 nonresponders (P õ .01). All responders, but evaluated. All had antibodies to HCV and HCV RNA in senone of the nonresponders, remained negative for core rum, and were negative for hepatitis B virus surface antigen protein after IFN-a therapy. The measurement of HCV and antibodies to human immunodeficiency virus. Chronic core protein by FEIA may be useful for predicting the hepatitis was diagnosed on the basis of persistence of serum response to IFN-a and for monitoring its therapeutic alanine transaminase (ALT) elevations, determined in serum efficacy. (HEPATOLOGY 1996;23:1330-1333.) tests performed monthly for at least 6 months, and by liver biopsy taken within the previous 6 months. Seven patients had mild, 6 had moderate, and 14 had severe chronic hepatiInfection by hepatitis C virus (HCV) can lead to tis. 9 chronic hepatitis and cirrhosis, and may also be inRecombinant IFN-a 2a (Roche, Tokyo, Japan, and Takeda, volved in the pathogenesis of hepatocellular carci-Osaka, Japan) was administered in a dose of 9 million units intramuscularly daily for 2 weeks, followed by three times a noma. 1,2 Treatment with interferon alfa (IFN-a) is efweek for 22 weeks. Patients were seen 2 and 4 weeks after fective in some patients with chronic hepatitis C, the initiation of therapy, and then every 4 weeks for at least resulting in a rapid decrease in aminotransferase activ-24 weeks after its completion. Serum samples were collected ities into the normal range and a clea...

Recent studies have shown that zinc ion (Zn) can behave as an intracellular signaling molecule. We previously demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI) rapidly release intracellular Zn from the endoplasmic reticulum (ER), and we named this phenomenon the “Zn wave”. However, the molecules responsible for releasing Zn and the roles of the Zn wave were elusive. Here we identified the pore-forming α1 subunit of the Cav1.3 (α1D) L-type calcium channel (LTCC) as the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, α1D was mainly localized to the ER rather than the plasma membrane in mast cells, and the Zn wave was impaired by α1D knockdown. We further found that the LTCC-mediated Zn wave positively controlled cytokine gene induction by enhancing the DNA-binding activity of NF- κB. Consistent with this finding, LTCC antagonists inhibited the cytokine-mediated delayed-type allergic reaction in mice without affecting the immediate-type allergic reaction. These findings indicated that the LTCC α1D subunit located on the ER membrane has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling and the delayed-type allergic reaction.

Mast cells are major players in allergic responses. IgE-dependent activation through FcεR leads to degranulation and cytokine production, both of which require Gab2. To clarify how the signals diverge at Gab2, we established Gab2 knock-in mice that express Gab2 mutated at either the PI3K or SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) binding sites. Examination of these mutants showed that both binding sites were required for the degranulation and anaphylaxis response but not for cytokine production or contact hypersensitivity. Furthermore, the PI3K, but not the SHP2, binding site was important for granule translocation during degranulation. We also identified a small GTPase, ADP-ribosylation factor (ARF)1, as the downstream target of PI3K that regulates granule translocation. FcεRI stimulation induced ARF1 activation, and this response was dependent on Fyn and the PI3K binding site of Gab2. ARF1 activity was required for FcεRI-mediated granule translocation. These data indicated that Fyn/Gab2/PI3K/ARF1-mediated signaling is specifically involved in granule translocation and the anaphylaxis response.

Zinc (Zn) is an essential nutrient and its deficiency causes immunodeficiency and skin disorders. Various cells including mast cells release Zn-containing granules when activated; however, the biological role of the released Zn is currently unclear. Here we report our findings that Zn transporter ZnT2 is required for the release of Zn from mast cells. In addition, we found that Zn and mast cells induce IL-6 production from inflammatory cells such as skin fibroblasts and promote wound healing, a process that involves inflammation. Zn induces the production of a variety of pro-inflammatory cytokines including IL-6 through signaling pathways mediated by the Zn receptor GPR39. Consistent with these findings, wound healing was impaired in mice lacking IL-6 or GPR39. Thus, our results show that Zn and mast cells play a critical role in wound healing through activation of the GPR39/IL-6 signaling axis.

The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. The manufacturing process of CAR-T cells should be optimized to prevent early T cell exhaustion during expansion. Activation of T cells by monoclonal antibodies is a critical step for T cell expansion, which may sometimes induce excess stimulation and exhaustion of T cells. Given that piggyBac transposon (PB)-based gene transfer could circumvent the conventional pre-activation of T cells, we established a manufacturing method of PB-mediated HER2-specific CAR-T cells (PB-HER2-CAR-T cells) that maintains their memory phenotype without early T cell exhaustion. Through stimulation of CAR-transduced T cells with autologous peripheral blood mononuclear cell-derived feeder cells expressing both truncated HER2, CD80, and 4-1BBL proteins, we could effectively propagate memory-rich, PD-1-negative PB-HER2-CAR-T cells. PB-HER2-CAR-T cells demonstrated sustained antitumor efficacy in vitro and debulked the HER2-positive tumors in vivo . Mice treated with PB-HER2-CAR-T cells rejected the second tumor establishment owing to the in vivo expansion of PB-HER2-CAR-T cells. Our simple and effective manufacturing process using PB system and genetically modified donor-derived feeder cells is a promising strategy for the use of PB-CAR-T cell therapy.