Cryptopatches (CPs) are part of the murine intestinal immune compartment. Cells isolated from CPs of the small intestine that were c-kit positive (c-kit+) but lineage markers negative (Lin-) gave rise to T cell receptor (TCR) alphabeta and TCR gammadelta intestinal intraepithelial T cells after in vivo transfer or tissue engraftment into severe combined immunodeficient mice. In contrast, cells from Peyer's patches and mesenteric lymph nodes, which belong in the same intestinal immune compartment but lack c-kit+Lin- cells, failed to do so. These findings and results of electron microscopic analysis provide evidence of a local intestinal T cell precursor that develops in the CPs.

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Acidic extracellular pH is a common feature of tumor tissues. We have reported that culturing cells at acidic pH (5.4-6.5) induced matrix metalloproteinase-9 expression through phospholipase D, extracellular signal regulated kinase 1 ⁄ 2 and p38 mitogen-activated protein kinases and nuclear factor-jB. Here, we show that acidic extracellular pH signaling involves both pathways of phospholipase D triggered by Ca 2+ influx and acidic sphingomyelinase in mouse B16 melanoma cells. We found that BAPTA-AM [1,2-bis(2-aminophenoxy)-ethane-N,N,N¢,N¢-tetraacetic acid tetrakis (acetoxymethyl) ester], a chelator of intracellular free calcium, and the voltage dependent Ca 2+ channel blockers, mibefradil (for T-type) and nimodipine (for L-type), dose-dependently inhibited acidic extracellular pH-induced matrix metalloproteinase-9 expression. Intracellular free calcium concentration ( 2+ channel blocker and acidic sphingomyelinase inhibitor, attenuated matrix metalloproteinase-9 induction but did not affect calcium influx. We found that acidic sphingomyelinase activity was induced by acidic extracellular pH and that the specific acidic sphingomyelinase inhibitors (perhexiline and desipramine) and siRNA targeting aSMase ⁄ smpd1 could inhibit acidic extracellular pH-induced matrix metalloproteinase-9 expression. BAPTA-AM reduced acidic extracellular pH-induced phospholipase D but not acidic sphingomyelinase acitivity. The acidic Abbreviations aSMase, acidic sphingomyelinase; BAPTA-AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N¢,N¢-tetraacetic acid tetrakis (acetoxymethyl) ester; CM, conditioned medium; [Ca 2+ ] i ,

Lympho-hemopoietic progenitors residing in murine gut cryptopatches (CP) have been shown to generate intestinal intraepithelial T cells (IEL). To investigate the role of CP in progenitor maturation, we analyzed IEL in male mice with a truncated mutation of common cytokine receptor γ-chain (CRγ−/Y) in which CP were undetectable. IEL-expressing TCR-γδ (γδ-IEL) were absent, and a drastically reduced number of Thy-1highCD4+ and Thy-1highCD8αβ+ αβ-IEL were present in CRγ−/Y mice, whereas these αβ-IEL disappeared from athymic CRγ−/Y littermate mice. Athymic CRγ−/Y mice possessed a small TCR- and αEβ7 integrin-negative IEL population, characterized by the disappearance of the extrathymic CD8αα+ subset, that expressed pre-Tα, RAG-2, and TCR-Cβ but not CD3ε transcripts. These TCR− IEL from athymic CRγ−/Y mice did not undergo Dβ-Jβ and Vδ-Jδ joinings, despite normal rearrangements at the TCR-β and -δ loci in thymocytes from euthymic CRγ−/Y mice. In contrast, athymic severe combined immunodeficient mice in which CP developed normally possessed two major TCR−αEβ7+ CD8αα+ and CD8− IEL populations that expressed pre-Tα, RAG-2, TCR-Cβ, and CD3ε transcripts. These findings underscore the role of gut CP in the early extrathymic maturation of CD8αα+ IEL, including cell-surface expression of αEβ7 integrin, CD3ε gene transcription, and TCR gene rearrangements.

Clinical efficacy of gefitinib (ZD1839, Iressa), which is an inhibitor specific for epidermal growth factor (EGF) receptor tyrosine kinase, has been shown in non-small-cell lung carcinoma patients with EGF receptor mutations, so these mutations are useful marker(s) to find a responder for the drug. Recent studies have shown that the EGF receptor gene mutation is rare in squamous cell carcinoma in the esophageal and head and neck regions. We previously reported that the expression of the chemokine BRAK/CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells was downregulated by EGF treatment, and that forced expression of BRAK in tumor cells decreased the tumorigenicity of the cells in xenografts. Thus, we investigated the relationship between restoration of BRAK expression by gefitinib and the efficacy of the drug for tumor suppression. We found that EGF down-regulated BRAK expression through the MEK-extracellular signal regulated kinase pathway and that this down-regulated expression was restored by gefitinib in vitro. Oral administration of gefitinib significantly (P < 0.001) reduced tumor growth of xenografts of three HNSCC cell lines (HSC-2, HSC-3, and HSC-4), in female athymic nude mice, accompanied by an increase in BRAK expression specifically in tumor tissue. This tumor-suppressing effect of the drug was not observed in the case of BRAK non-expressing cells. Furthermore introduction of BRAK shRNA vector reduced both the expression levels of BRAK in HSC-3 cells and the antitumor efficacy of gefitinib in vivo. Our data showing an inverse relationship between BRAK expression levels in tumor cells and the tumor growth rate indicate that the gefitinib-induced increase in BRAK expression is beneficial for tumor suppression in

In the temporomandibular joint (TMJ), it has been hypothesized that mechanical stresses lead to the oxidative stress of articular tissues. It has also been postulated that cells pertinent to arthritis-including endothelial cells and synovial cells-when stimulated by mechanical stresses and/or pro-inflammatory cytokines, promote oxidative damage. To determine the involvement of reactive oxygen species (ROS) in the diseased joint, we studied the generation of ROS in synovial fluid (SF) from interleukin-1alpha (IL-1alpha)-induced TMJ arthritis by electron spin resonance (ESR) spectroscopy, using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The TMJ arthritis was experimentally induced in rats by the injection of human recombinant IL-1alpha into the TMJ; control rats were treated with normal saline solution. We found that the detected radicals in the collected SF were identified as a 1:2:2:1 quartet, characteristic of the hydroxyl radical-DMPO spin adduct. The ESR signal intensity of the hydroxyl radical-DMPO spin adduct in the SF from IL-1-treated rats was significantly higher than that from the control rats (P < 0.01). The results of ESR study also showed that hydroxyl radical (HO*) was increased in a time-dependent fashion in the presence of superoxide anion radical (O2*-) scavenger superoxide dismutase (SOD); the formation of DMPO-HO* was strongly inhibited by the iron chelater deferoxamine. We could measure higher levels of free iron (Fe2- and Fe3-) in the SF from TMJ arthritis than in that from controls (P < 0.05). Analysis of the data obtained from the present study suggests that the HO* radical detected in SF from IL-1-induced TMJ arthritis is generated via a modified Haber-Weiss reaction (biological Fenton reaction) in which O2*- can subsequently result in the production of H2O2 through dismutation reaction by SOD. Thus, HO* may be generated from the reaction of resultant H2O2 with free iron ions. The results presented here provide the first evidence of involvement of ROS in IL-1-induced TMJ arthritis.