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CD4+ T cells not only direct immune responses against infectious micro-organisms but are also involved in the pathogenesis of inflammatory diseases. In the last two to three decades, various researchers have identified and characterized several functional CD4+ T-cell subsets, including T-helper 1 (Th1), Th2, Th9 and Th17 cells and regulatory T (Treg) cells. In this mini-review, we introduce the concept of pathogenic Th cells that induce inflammatory diseases with a model of disease induction by a population of pathogenic Th cells: the ‘pathogenic Th population disease-induction model’. We will focus on Th2 cells that induce allergic airway inflammation—pathogenic Th2 cells (Tpath2 cells)—and discuss the nature of Tpath2 cells that shape the pathology of chronic inflammatory diseases. Various Tpath2-cell subsets have been identified and their unique features are summarized in mouse and human systems. Second, we will discuss how Th cells migrate and are maintained in chronic inflammatory lesions. We propose a model known as the ‘CD69–Myl9 system’. CD69 is a cell surface molecule expressed on activated T cells and interaction with its ligand myosin light chain 9 (Myl9) is required for the induction of inflammatory diseases. Myl9 molecules in the small vessels of inflamed lungs may play a crucial role in the migration of activated T cells into inflammatory lesions. Emerging evidence may provide new insight into the pathogenesis of chronic inflammatory diseases and contribute to the development of new therapeutic strategies for intractable inflammatory disorders.

Invariant natural killer T (iNKT) cells are innate‐like CD1d‐restricted T cells that express the invariant T cell receptor (TCR) composed of Vα24 and Vβ11 in humans. iNKT cells specifically recognize glycolipid antigens such as α‐galactosylceramide (αGalCer) presented by CD1d. iNKT cells show direct cytotoxicity toward CD1d‐positive tumor cells, especially when CD1d presents glycolipid antigens. However, iNKT cell recognition of CD1d‐negative tumor cells is unknown, and direct cytotoxicity of iNKT cells toward CD1d‐negative tumor cells remains controversial. Here, we demonstrate that activated iNKT cells recognize leukemia cells in a CD1d‐independent manner, however still in a TCR‐mediated way. iNKT cells degranulated and released Th1 cytokines toward CD1d‐negative leukemia cells (K562, HL‐60, REH) as well as αGalCer‐loaded CD1d‐positive Jurkat cells. The CD1d‐independent cytotoxicity was enhanced by natural killer cell‐activating receptors such as NKG2D, 2B4, DNAM‐1, LFA‐1 and CD2, but iNKT cells did not depend on these receptors for the recognition of CD1d‐negative leukemia cells. In contrast, TCR was essential for CD1d‐independent recognition and cytotoxicity. iNKT cells degranulated toward patient‐derived leukemia cells independently of CD1d expression. iNKT cells targeted myeloid malignancies more than acute lymphoblastic leukemia. These findings reveal a novel anti–tumor mechanism of iNKT cells in targeting CD1d‐negative tumor cells and indicate the potential of iNKT cells for clinical application to treat leukemia independently of CD1d., Activated human iNKT cells directly recognize leukemia cells, especially myeloid malignancies, in a CD1d‐independent manner. The CD1d‐independent cytotoxicity of iNKT cells depends on Vα24Vβ11‐T cell receptor and is enhanced by NK cell‐activating receptors.

CD69 is an activation marker on leukocytes. Early studies showed that the CD69+ cells were detected in the lung of patients with asthmatic and eosinophilic pneumonia, suggesting that CD69 might play crucial roles in the pathogenesis of such inflammatory diseases, rather than simply being an activation marker. Intensive studies using mouse models have since clarified that CD69 is a functional molecule regulating the immune responses. We discovered that Myosin light chain 9, 12a, 12b (Myl9/12) are ligands for CD69 and that platelet-derived Myl9 forms a net-like structure (Myl9 nets) that is strongly detected inside blood vessels in inflamed lung. CD69-expressing activated T cells attached to the Myl9 nets can thereby migrate into the inflamed tissues through a system known as the CD69-Myl9 system. In this review, we summarize the discovery of the CD69-Myl9 system and discuss how this system is important in inflammatory immune responses. In addition, we discuss our recent finding that CD69 controls the exhaustion status of tumor-infiltrating T cells and that the blockade of the CD69 function enhances anti-tumor immunity. Finally, we discuss the possibility of CD69 as a new therapeutic target for patients with intractable inflammatory disorders and tumors.

The numbers of patients with inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD), have been increasing over time, worldwide; however, the pathogenesis of IBD is multifactorial and has not been fully understood. Myosin light chain 9 and 12a and 12b (Myl9/12) are known as ligands of the CD69 molecule. They create “Myl9 nets” that are often detected in inflamed site, which play a crucial role in regulating the recruitment and retention of CD69-expressing effector cells in inflamed tissues. We demonstrated the strong expression of Myl9/12 in the inflamed gut of IBD patients and mice with DSS-induced colitis. The administration of anti-Myl9/12 Ab to mice with DSS-induced colitis ameliorated the inflammation and prolonged their survival. The plasma Myl9 levels in the patients with active UC and CD were significantly higher than those in patients with disease remission, and may depict the disease severity of IBD patients, especially those with UC. Thus, our results indicate that Myl9/12 are involved in the pathogenesis of IBD, and are likely to be a new therapeutic target for patients suffering from IBD.

While IFNγ is a well-known cytokine that actively promotes the type I immune response, it is also known to suppress the type II response by inhibiting the differentiation and proliferation of Th2 cells. However, the mechanism by which IFNγ suppresses Th2 cell proliferation is still not fully understood. We found that IFNγ decreases the expression of growth factor independent-1 transcriptional repressor (GFI1) in Th2 cells, resulting in the inhibition of Th2 cell proliferation. The deletion of theGfi1gene in Th2 cells results in the failure of their proliferation, accompanied by an impaired cell cycle progression. In contrast, the enforced expression of GFI1 restores the defective Th2 cell proliferation, even in the presence of IFNγ. These results demonstrate that GFI1 is a key molecule in the IFNγ-mediated inhibition of Th2 cell proliferation.

Memory helper T (Th) cells are crucial for secondary immune responses against infectious microorganisms but also drive the pathogenesis of chronic inflammatory diseases. Therefore, it is of fundamental importance to understand how memory T cells are generated. However, the molecular mechanisms governing memory Th cell generation remain incompletely understood. Here, we identified CD30 as a molecule heterogeneously expressed on effector Th1 and Th17 cells, and CD30(hi)effector Th1 and Th17 cells preferentially generated memory Th1 and Th17 cells. We found that CD30 mediated signal induced Transglutaminase-2 (TG2) expression, and that the TG2 expression in effector Th cells is essential for memory Th cell generation. In fact,Cd30-deficiency resulted in the impaired generation of memory Th1 and Th17 cells, which can be rescued by overexpression of TG2. Furthermore, transglutaminase-2(Tgm2)-deficient CD4 T cells failed to become memory Th cells. As a result, T cells fromTgm2-deficient mice displayed impaired antigen-specific antibody production and attenuated Th17-mediated allergic responses. Our data indicate that CD30-induced TG2 expression in effector Th cells is essential for the generation of memory Th1 and Th17 cells, and that CD30 can be a marker for precursors of memory Th1 and Th17 cells. Refereed/Peer-reviewed

Multiple sclerosis (MS) is a disabling demyelinating autoimmune disorder of the central nervous system (CNS) which is driven by IL-23- and IL-1β-induced autoreactive Th17 cells that traffic to the CNS and secrete proinflammatory cytokines. Th17 pathogenicity in MS has been correlated with the dysregulation of microRNA (miRNA) expression, and specific miRNAs have been shown to promote the pathogenic Th17 phenotype. In the present study, we demonstrate, using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), that let-7 miRNAs confer protection against EAE by negatively regulating the proliferation, differentiation and chemokine-mediated migration of pathogenic Th17 cells to the CNS. Specifically, we found that let-7 miRNAs may directly target the cytokine receptors Il1r1 and Il23r, as well as the chemokine receptors Ccr2 and Ccr5. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for disease treatment.

Maintaining the diversity and constant numbers of naïve T cells throughout the organism's lifetime is necessary for efficient immune responses. Naïve T cell homeostasis, which consists of prolonged survival, occasional proliferation and enforcement of quiescence, is tightly regulated by multiple signaling pathways which are in turn controlled by various transcription factors. However, full understanding of the molecular mechanisms underlying the maintenance of the peripheral T cell pool has not been achieved. In the present study, we demonstrate that T cell-specific deficiency in let-7 miRNAs results in peripheral T cell lymphopenia resembling that of Dicer1 knockout mice. Deletion of let-7 leads to profound T cell apoptosis while overexpression prevents it. We further show that in the absence of let-7, T cells cannot sustain optimal levels of the pro-survival factor Bcl2 in spite of the intact IL-7 signaling, and re-expression of Bcl2 in let-7 deficient T cells completely rescues the survival defect. Thus, we have uncovered a novel let-7-dependent mechanism of post-transcriptional regulation of naïve T cell survival in vivo.

Iodinated contrast media (ICM) are nonmutagenic agents administered for X-ray imaging of soft tissues. ICM can reach μg/L levels in surface waters because they are administered in high doses, excreted largely unmetabolized, and poorly removed by wastewater treatment. Iodinated disinfection byproducts (I-DBPs) are highly genotoxic and have been reported in disinfected waters containing ICM. We assessed the mutagenicity in Salmonella of extracts of chlorinated source water containing one of four ICM (iopamidol, iopromide, iohexol, and diatrizoate). We quantified 21 regulated and nonregulated DBPs and 11 target I-DBPs and conducted a nontarget, comprehensive broad-screen identification of I-DBPs. We detected one new iodomethane (trichloroiodomethane), three new iodoacids (dichloroiodoacetic acid, chlorodiiodoacetic acid, bromochloroiodoacetic acid), and two new nitrogenous I-DBPs (iodoacetonitrile and chloroiodoacetonitrile). Their formation depended on the presence of iopamidol as the iodine source; identities were confirmed with authentic standards when available. This is the first identification in simulated drinking water of chloroiodoacetonitrile and iodoacetonitrile, the latter of which is highly cytotoxic and genotoxic in mammalian cells. Iopamidol (5 μM) altered the concentrations and relative distribution of several DBP classes, increasing total haloacetonitriles by >10-fold. Chlorination of ICM-containing source water increased I-DBP concentrations but not mutagenicity, indicating that such I-DBPs were either not mutagenic or at concentrations too low to affect mutagenicity.

The introduction of immune checkpoint inhibitors in cancer treatment highlights the negative regulation of anti-tumor immunity, such as effector T-cell exhaustion in the tumor microenvironment. However, the mechanisms underlying the induction and prevention of T-cell exhaustion remain largely unknown. We found that CD69, a type II glycoprotein known to regulate inflammation through T-cell migration and retention in tissues, plays an important role in inducing the exhaustion of tumor-infiltrating T cells. Cd69-/- mice showed reduced tumor growth and metastasis in a 4T1-luc2 murine breast cancer model, in which increased numbers of tumor-infiltrating lymphocytes, relatively little T-cell exhaustion, and enhanced IFNγ production were observed. Anti-CD69 monoclonal antibody treatment attenuated the T-cell exhaustion and tumor progression in tumor-bearing mice. These findings highlight a novel role of CD69 in controlling the tumor immune escape mediated by T-cell exhaustion and indicate that CD69 is a novel target for cancer immunotherapy.

While CD69 may regulate thymocyte egress by inhibiting S1P

The introduction of drinking water disinfection greatly reduced waterborne diseases. However, the reaction between disinfectants and natural organic matter in the source water leads to an unintended consequence, the formation of drinking water disinfection byproducts (DBPs). The haloace-taldehydes (HALs) are the third largest group by weight of identified DBPs in drinking water. The primary objective of this study was to analyze the occurrence and comparative toxicity of the emerging HAL DBPs. A new HAL DBP, iodoacetaldehyde (IAL) was identified. This study provided the first systematic, quantitative comparison of HAL toxicity in Chinese hamster ovary cells. The rank order of HAL cytotoxicity is tribromoacetaldehyde (TBAL) ≈ chloroacetaldehyde (CAL) > dibromoacetaldehyde (DBAL) ≈ bromochloroacetaldehyde (BCAL) ≈ dibromochloroacetaldehyde (DBCAL) > IAL > bromoacetaldehyde (BAL) ≈ bromodichloroacetaldehyde (BDCAL) > dichloroacetaldehyde (DCAL) > trichloroacetaldehyde (TCAL). The HALs were highly cytotoxic compared to other DBP chemical classes. The rank order of HAL genotoxicity is DBAL > CAL ≈ DBCAL > TBAL ≈ BAL > BDCAL > BCAL ≈ DCAL > IAL. TCAL was not genotoxic. Because of their toxicity and abundance, further research is needed to investigate their mode of action to protect the public health and the environment.

Lethal-7 (let-7) microRNAs (miRNAs) are the most abundant miRNAs in the genome, but their role in developing thymocytes is unclear. We found that let-7 miRNAs targeted Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and thereby the effector functions of natural killer T cells (NKT cells). Dynamic upregulation of let-7 miRNAs during the development of NKT thymocytes downregulated PLZF expression and directed their terminal differentiation into interferon-γ (IFN-γ)-producing NKT1 cells. Without upregulation of let-7 miRNAs, NKT thymocytes maintained high PLZF expression and terminally differentiated into interleukin 4 (IL-4)-producing NKT2 cells or IL-17-producing NKT17 cells. Upregulation of let-7 miRNAs in developing NKT thymocytes was signaled by IL-15, vitamin D and retinoic acid. Such targeting of a lineage-specific transcription factor by miRNA represents a previously unknown level of developmental regulation in the thymus.

Disinfectants inactivate pathogens in source water; however, they also react with organic matter and bromide/iodide to form disinfection byproducts (DBPs). Although only a few DBP classes have been systematically analyzed for toxicity, iodinated and brominated DBPs tend to be the most toxic. The objectives of this research were (1) to determine if monochloramine (NH2Cl) disinfection generated drinking water with less toxicity than water disinfected with free chlorine (HOCl) and (2) to determine the impact of added bromide and iodide in conjunction with HOCl or NH2Cl disinfection on mammalian cell cytotoxicity and genomic DNA damage induction. Water disinfected with chlorine was less cytotoxic but more genotoxic than water disinfected with chloramine. For both disinfectants, the addition of Br(-) and I(-) increased cytotoxicity and genotoxicity with a greater response observed with NH2Cl disinfection. Both cytotoxicity and genotoxicity were highly correlated with TOBr and TOI. However, toxicity was weakly and inversely correlated with TOCl. Thus, the forcing agents for cytotoxicity and genotoxicity were the generation of brominated and iodinated DBPs rather than the formation of chlorinated DBPs. Disinfection practices need careful consideration especially when using source waters containing elevated bromide and iodide.

Summary CD69 has been known as an early activation marker of lymphocytes; whereas, recent studies demonstrate that CD69 also has critical functions in immune responses. Early studies using human samples revealed the involvement of CD69 in various inflammatory diseases including asthma. Moreover, murine disease models using Cd69−/− mice and/or anti-CD69 antibody (Ab) treatment have revealed crucial roles for CD69 in inflammatory responses. However, it had not been clear how the CD69 molecule contributes to the pathogenesis of inflammatory diseases. We recently elucidated a novel mechanism, in which the interaction between CD69 and its ligands, myosin light chain 9, 12a and 12b (Myl9/12) play a critical role in the recruitment of activated T cells into the inflammatory lung. In this review, we first summarize CD69 function based on its structure and then introduce the evidence for the involvement of CD69 in human diseases and murine disease models. Then, we will describe how we discovered CD69 ligands, Myl9 and Myl12, and how the CD69-Myl9 system regulates airway inflammation. Finally, we will discuss possible therapeutic usages of the blocking Ab to the CD69-Myl9 system.

Background: Natural killer T (NKT) cells express a T-cell receptor that recognizes endogenous and environmental glycolipid antigens. Several subsets of NKT cells have been identified, including IFN-γ–producing NKT1 cells, IL-4–producing NKT2 cells, and IL-17–producing NKT17 cells. However, little is known about the factors that regulate their differentiation and respective functions within the immune system. Objective: We sought to determine whether the polycomb repressive complex 2 protein enhancer of zeste homolog 2 (Ezh2) restrains pathogenicity of NKT cells in the context of asthma-like lung disease. Methods: Numbers of invariant natural killer T (iNKT) 1, iNKT2, and iNKT17 cells and tissue distribution, cytokine production, lymphoid tissue localization, and transcriptional profiles of iNKT cells from wild-type and Ezh2 knockout (KO) iNKT mice were determined. The contribution of NKT cells to development of spontaneous and house dust mite–induced airways pathology, including airways hyperreactivity (AHR) to methacholine, was also assessed in wild-type, Ezh2 KO, and Ezh2 KO mice lacking NKT cells. Results: Ezh2 restrains development of pathogenic NKT cells, which induce spontaneous asthma-like disease in mice. Deletion of Ezh2 increased production of IL-4 and IL-13 and induced spontaneous AHR, lung inflammation, mucus production, and IgE. Increased IL-4 and IL-13 levels, AHR, lung inflammation, and IgE levels were all dependent on iNKT cells. In house dust mite–exposed animals Ezh2 KO resulted in enhanced AHR that was also dependent on iNKT cells. Conclusion: Ezh2 is a central regulator of iNKT pathogenicity and suppresses the ability of iNKT cells to induce asthma-like pathology. Refereed/Peer-reviewed

Combined chlorine is increasingly being used as an alternative disinfectant to free chlorine to maintain a residual in drinking water distribution systems mainly because it would reduce the formation of regulated disinfection byproducts (DBPs) trihalomethanes and haloacetic acids. However, the use of combined chlorine could promote the formation of currently unregulated nitrogenous DBPs (N-DBPs) such as haloacetonitriles and haloacetamides that are found to be more cyto- and genotoxic than regulated DBPs. Monochloramine quickly reacts with chloroacetaldehyde, a DBP formed during primary disinfection with free chlorine, forming and reaching pseudoequilibrium (equilibrium constant K1 = 1.87 × 10(3) M(-1)) with the carbinolamine 2-chloro-1-(chloroamino)ethanol. 2-Chloro-1-(chloroamino)ethanol undergoes slow dehydration to form the imine 1-chloro-2-(chloroimino)ethane that decomposes at a faster rate to chloroacetonitrile. 2-Chloro-1-(chloroamino)ethanol is also oxidized by monochloramine to produce the previously unreported DBP N,2-dichloroacetamide. The carbinolamine dehydration step was found to be acid/base catalyzed (k2(0) = 3.30 × 10(-6) s(-1), k2(H) = 2.43 M(-1) s(-1), k2(OH) = 3.90 M(-1) s(-1)). In contrast, N,2-dichloroacetamide formation was observed to be only base catalyzed (k3(OH) = 3.03 × 10(4) M(-2) s(-1)). N,2-dichloroacetamide cytotoxicity (LC50 = 2.56 × 10(-4) M) was found to be slightly lower compared to that reported for chloroacetamide but higher than those of di- and trichloroacetamide.

Summary Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αβ T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αβTCR that are restricted by major histocompatibility complex (MHC)-encoded determinants. Here, we report that Lck proteins that were coreceptor associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αβTCR depending on whether Lck is coreceptor associated or coreceptor free. We conclude that the intracellular state of Lck determines the specificity of thymic selection and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αβTCR repertoire.

SummaryImmune tolerance requires regulatory T (Treg) cells to prevent autoimmune disease, with the transcription factor Foxp3 functioning as the critical regulator of Treg cell development and function. We report here that Foxp3 was lethal to developing Treg cells in the thymus because it induced a unique proapoptotic protein signature (Puma++p-Bim++p-JNK++DUSP6−) and repressed expression of prosurvival Bcl-2 molecules. However, Foxp3 lethality was prevented by common gamma chain (γc)-dependent cytokine signals that were present in the thymus in limiting amounts sufficient to support only ∼1 million Treg cells. Consequently, most newly arising Treg cells in the thymus were deprived of this signal and underwent Foxp3-induced death, with Foxp3+CD25− Treg precursor cells being the most susceptible. Thus, we identify Foxp3 as a proapoptotic protein that requires developing Treg cells to compete with one another for limiting amounts of γc-dependent survival signals in the thymus.

Major histocompatibility complex class I (MHC I) positive selection of CD8(+) T cells in the thymus requires that T cell antigen receptor (TCR) signaling end in time for cytokines to induce Runx3d, the CD8-lineage transcription factor. We examined the time required for these events and found that the overall duration of positive selection was similar for all CD8(+) thymocytes in mice, despite markedly different TCR signaling times. Notably, prolonged TCR signaling times were counter-balanced by accelerated Runx3d induction by cytokines and accelerated differentiation into CD8(+) T cells. Consequently, lineage errors did not occur except when MHC I–TCR signaling was so prolonged that the CD4-lineage-specifying transcription factor ThPOK was expressed, preventing Runx3d induction. Thus, our results identify a compensatory signaling mechanism that prevents lineage-fate errors by dynamically modulating Runx3d induction rates during MHC I positive selection.

Zusammenfassung Uber die monoaminergischen Systeme des Gehirns in Beziehung zur Ejakulation III. Weitere Untersuchung unter Vetwendung eines peripheren Decarboxylase-Inhibitors In den vorausgegangenen Arbeiten war berichtet worden, das die Ejakulation erleichtert wird durch das dopaminergische System des Gehirns, teilweise uber den vorderen Hypothalamus und das die Ejakulation durch das serotonergische System gehemmt wird. Auf dem Wege, diese Resultate praeziser zu klaren wurden jetzt Untersuchungen durchgefuhrt, bei welchen Hunden ein peripherer Decarboxylase-Inhibitor (Ro 4–4602) in Kombination mit Tetrabenazin und L-dopa verabfolgt wurde. Es kann gezeigt werden, das zusatzlich zu dem dopaminergischen System des Gehirns der Anstieg des Serotoninwertes im limbischen System und der Abfall des Serotininspiegels im Stammhirn die Ejakulation erleichtert.

We conducted a field survey to estimate the relationship between embryo production and the body condition score (BCS) on a 5-point scale, as well as blood concentrations of insulin and glucose, in superovulated Holstein yearling heifers housed in a free-stall barn. They were provided total mixed rations to meet the nutrient requirements. The daily ration was divided between 2 feeding times, utilizing stanchions to separate heifers to avoid social status preventing inferior heifers from having enough feed. The recovered fluid after uterine flushing from heifers (n = 88, 13 mo old) was examined microscopically for the morphological grade and the development stage. The number of heifers in which BCS was 2.75, 3.00, 3.25, and 3.50 was 6, 35, 40, and 7, respectively. The 3.50 BCS heifers produced fewer excellent grade embryos than 3.00 or 3.25 BCS heifers significantly. The 3.50 BCS heifers produced significantly more morula than 2.75, 3.00, or 3.25 BCS heifers. In contrast, 2.75 BCS heifers produced more blastocysts than 3.25 or 3.50 BCS heifers. The 3.50 BCS heifers were hyperinsulinemic. Our results suggested no significant effect of BCS around 3.00 on embryo production, whereas 3.50 BCS heifers may have poorer embryo production.

Elevated plasma A-type natriuretic peptide (ANP) levels in sepsis cause fluid transfer into extravascular spaces. We investigated the changes in ANP concentrations and natriuretic peptide receptor (NPR) expression induced by thiorphan, a neutral endopeptidase (NEP) inhibitor, in a rat model of sepsis. Fifteen male rats were divided into three groups: a control group (n = 5), a lipopolysaccharide (LPS) group (n = 5), and an LPS-thiorphan group (n = 5). We measured ANP concentrations in the plasma and lung, and NPR mRNA expression in the lung 4 h after administering LPS, and compared the values with those in the control group. Plasma and lung ANP levels in the LPS group were significantly higher than those in the control group (P < 0.05), but were significantly decreased by thiorphan administration (P < 0.05). NPR-A mRNA levels did not differ significantly among the groups. NPR-C mRNA levels in the LPS-thiorphan group were significantly higher than those in the other groups (P < 0.05). Elevated ANP levels were decreased by thiorphan administration, which increased NPR-C mRNA levels in the lung. Thus, thiorphan might be effective for reducing elevated ANP levels in sepsis.

Nitriles and amides are two classes of nitrogenous disinfection byproducts (DBPs) associated with chloramination that are more cytotoxic and genotoxic than regulated DBPs. Monochloramine reacts with acetaldehyde, a common ozone and free chlorine disinfection byproduct, to form 1-(chloroamino)ethanol. Equilibrium (K1) and forward and reverse rate (k1,k-1) constants for the reaction between initial reactants and 1-(chloroamino)ethanol were determined between 2 and 30 °C. Activation energies for k1 and k-1 were 3.04 and 45.2 kJ·mol(-1), respectively, and enthalpy change for K1 was -42.1 kJ·mol(-1). In parallel reactions, 1-(chloroamino)ethanol (1) slowly dehydrated (k2) to (chloroimino)ethane that further decomposed to acetonitrile and (2) was oxidized (k3) by monochloramine to produce N-chloroacetamide. Both reactions were acid/base catalyzed, and rate constants were characterized at 10, 18, and 25 °C. Modeling for drinking water distribution system conditions showed that N-chloroacetamide and acetonitrile concentrations were 5-9 times higher at pH 9.0 compared to 7.8. Furthermore, acetonitrile concentration was found to form 7-10 times higher than N-chloroacetamide under typical monochloramine and acetaldehyde concentrations. N-chloroacetamide cytotoxicity (LC50 = 1.78 × 10(-3) M) was comparable to dichloroacetamide and trichloroacetamide, but less potent than N,2-dichloroacetamide and chloroacetamide. While N-chloroacetamide was not found to be genotoxic, N,2-dichloroacetamide genotoxic potency (5.19 × 10(-3) M) was on the same order of magnitude as chloroacetamide and trichloroacetamide.

Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the inter-leukin- 5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methyla-tion- mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3. Refereed/Peer-reviewed

The incidence, distribution, and macro- and microscopic structures of left ventricular false tendons (LVFTs) in the cat heart were studied using 25 normal and 57 diseased hearts. The fibrous bands were observed in the left ventricle of all 82 cat hearts examined and most commonly extended between the papillary muscles and the ventricular septum. Histologically, the LVFTs were composed of central Purkinje fibres and surrounding dense collagenous fibres covered by endothelium. There was no appreciable difference in the incidence, distribution or morphology of LVFTs between the normal and the diseased hearts, indicating that LVFTs are a common anatomic variant in the cat heart.

Background Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice raised in nonair-controlled conventional circumstances spontaneously develop atopic dermatitis-like skin lesions; however, the underlying mechanisms remain unclear. Objective We wanted to identify the critical intracellular signaling molecules in T cells that induce atopic dermatitis-like skin legions in NC/Nga mice. Methods We examined the levels of signal transduction and cytokine production in T cells, particularly those in atopic dermatitis-like lesions induced by the topical injection of mite antigens in NC/Nga mice under specific pathogen-free conditions. Results In NC/Nga mice maintained under specific pathogen-free conditions, the capability of T H 1/T H 2 and T cytotoxic 1/T cytotoxic 2 (Tc1/Tc2) cell differentiation increased significantly. T-cell antigen receptor–mediated activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase cascade and nuclear factor-κB (NF-κB) signaling were enhanced in NC/Nga T cells. The expression of T H 2 cytokines (IL-4, IL-13, and IL-5) and that of GATA-binding protein 3 (GATA3), avian musculoaponeurotic fibrosarcoma (c-Maf), NF-κB, and activator protein 1 (AP1) selectively increased in draining lymph node T cells of NC/Nga mice. Moreover, the cell transfer of inhibitory NF-κB mutant-infected T H 2 cells reduced ear thickness in the mite antigen-induced skin lesion of NC/Nga mice. Conclusion Hyperresponsive T H 2 cells with an enhanced activity of NF-κB and AP1 play a crucial role in the pathogenesis of atopic dermatitis-like skin lesions in NC/Nga mice. Clinical implications These results indicate potential therapeutic usefulness of developing selective inhibitors for NF-κB in the treatment of human atopic dermatitis.

Background/Aim: Liver regeneration is a finely tuned process that is closely regulated by multiple cell cycle steps. Although the portal blood flow affects liver regeneration, the molecular mechanism by which the blood flow regulates gene expression and liver function is largely unknown. The aim of this study was to investigate the molecular effect of portal blood flow on hepatocyte proliferation and gene regulation during liver regeneration. Materials and Methods: We developed a simple surgical rat model to investigate the relation between portal blood flow and liver regeneration by partially ligating the portal trunk with 8-0 Proline sutures under microscopy to reduce the blood flow by 40%. We investigated recovery of liver volume, DNA synthesis, and gene expression associated with cell cycle regulators, comparing partially hepatectomized (PH) rats without (PH group; n = 30) and with partial portal ligation (PHPL group; n = 30) for 7 days after the operation. Results: The hepatic tissue blood flow and the recovery ratio between liver weight and body weight in the PHPL group were significantly lower than in the PH group after hepatectomy. The peak 5-bromo-2′-deoxyuridine labeling index in the PHPL group was delayed and weak compared with the PH group. The expression of CT-1 and cyclin D, E, and B mRNAs indicated that the liver regeneration in the PHPL group was delayed and weak. In addition, there was reciprocal expression of C/EBPα and C/EBPβ mRNAs, an observation supported by their nuclear protein levels. Furthermore, the cytochrome P-450 protein level in the PHPL group was higher than that in the PH group 1 day after hepatectomy. Conclusion: The portal blood flow regulates the activity of liver regeneration and the gene expression associated with cell cycle regulators, while the functions are maintained.

Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.

Both CD4 and CD8 T cells play crucial roles in immune responses in transplantation. Immunosuppressive drugs, such as FK506 and cyclosporin A, block the priming of alloreactive CD4 T(h) cells and the subsequent induction of allospecific CD8 cytotoxic effector T cells and inhibit allograft rejection. However, the desire to minimize chronic complications that may arise from the use of immunosuppressive agents drives the search for additional strategies for immunosuppression of allograft rejection. In this study, CD4 or CD8 T cells into which the IL-10 gene is introduced using an adenovirus vector containing human IL-10 (hIL-10) cDNA (Ad-hIL-10) and into mouse T cells transgenic for the Coxsackie virus and adenovirus receptor form a model system to study the effect of administration of IL-10-secreting T cells on the survival of the allogenic skin grafts. Ad-hIL-10-infected CD4 and CD8 T cells secreted a large amount of hIL-10 for 3-4 days in culture in vitro. Ad-hIL-10-infected CD4 T cells administered in vivo could be detected in the spleen for 7 days post-transfer. Significantly prolonged survival of grafts was observed in animals that received either Ad-hIL-10-infected activated CD4 T cells or T(h)2-skewed CD4 T cells as compared with controls. Furthermore, substantial enhancement of the effect was observed in B6.C-H2(bm1)/ByJ transplants. Thus, a direct manipulation of T cells through the introduction of the immunosuppressive cytokine gene IL-10 may be a novel strategy for the control of allograft rejection.

Natural killer type-1 (NK1)/NK2 terminology was proposed as an analogy to T helper type-1 (Th1)/Th2 subsets of CD4 T cells. In addition to well recognized IFN-gamma-producing NK1 cells, recent accumulating evidence suggests the presence and the regulatory function of NK2 cells, which produce type-2 cytokines, such as IL-5 and IL-13. Most studies on the production of cytokines by NK1/NK2 subsets have been performed in the human system. Recently, a mouse in vitro NK1/NK2 cell differentiation system was established. A linear 2-0-1 model has been proposed for the differentiation of NK1/NK2 cell subsets, but the branching NK1/NK2 cell differentiation model, comparable to Thl/Th2 cell differentiation, remains a likely explanation. In this review, we summarize the evidence indicating the unique features of NK1/NK2 cells and discuss the above two NK cell differentiation models. In addition, we point to interesting similarities in the expression of transcription factors that regulate cytokine production in NK cells and CD8 T cells.

Schnurri (Shn) is a large zinc finger protein implicated in cell growth, signal transduction, and lymphocyte development. Vertebrates possess at least three Shn orthologues (Shn-1, Shn-2, and Shn-3), which appear to act within the bone morphogenetic protein, transforming growth factor beta, and activin signaling pathways. However, the physiological functions of the Shn proteins remain largely unknown. In Shn-2-deficient mice, mature peripheral T cells exhibited normal anti-T cell receptor-induced proliferation, although there was dramatic enhancement in the differentiation into T helper type (Th)2 cells and a marginal effect on Th1 cell differentiation. Shn-2-deficient developing Th2 cells showed constitutive activation of nuclear factor kappaB (NF-kappaB) and enhanced GATA3 induction. Shn-2 was able to compete with p50 NF-kappaB for binding to a consensus NF-kappaB motif and inhibit NF-kappaB-driven promoter activity. Thus, Shn-2 plays a crucial role in the control of Th2 cell differentiation by regulating NF-kappaB function.

During development in the thymus, mature CD4+ or CD8+ cells are derived from immature CD4+CD8+ cells through a series of selection events. One of the hallmarks of this maturation process is the expression of CD69, which first appears on thymocytes as they begin positive selection. We have used blockade and overexpression of CD69 to determine the role of CD69 in thymocyte development. Blockade of CD69 led to a reduction in single-positive cells and a concomitant increase in double-positive cells in the thymus. Overexpression of a CD69 transgene in the thymus resulted in a dramatic increase in both CD8SP and CD4SP cells. Coexpression with a TCR transgene demonstrated that both positive and negative selection were enhanced by the increased levels of CD69 on thymocytes. Finally, mice overexpressing CD69 displayed a sharp reduction in the number of T cells in the spleen and lymph node. Taken as a whole, these data suggest the involvement of CD69 in the process of selection and maturation during the trafficking of thymocytes to the medulla.

Lineage fate in the thymus is determined by mutually exclusive expression of the transcription factors ThPOK and Runx3, with ThPOK imposing the CD4(+) lineage fate and Runx3 promoting the CD8(+) lineage fate. While it is known that cytokine signals induce thymocytes to express Runx3, it is not known how ThPOK prevents thymocytes from expressing Runx3 and adopting the CD8(+) lineage fate, nor is it understood why ThPOK itself imposes the CD4(+) lineage fate on thymocytes. We now report that genes encoding members of the SOCS (suppressor of cytokine signaling) family are critical targets of ThPOK and that their induction by ThPOK represses Runx3 expression and promotes the CD4(+) lineage fate. Thus, induction of SOCS-encoding genes is the main mechanism by which ThPOK imposes the CD4(+) lineage fate in the thymus.

Polycomb group ( PcG ) gene products regulate homeobox gene expression in Drosophila and vertebrates and also cell cycle progression of immature lymphocytes. In a gene-disrupted mouse for polycomb group gene mel-18 , mature peripheral T cells exhibited normal anti-TCR-induced proliferation; however, the production of Th2 cytokines (IL-4, IL-5, and IL-13) was significantly reduced, whereas production of IFNγ was modestly enhanced. Th2 cell differentiation was impaired, and the defect was associated with decreased levels in demethylation of the IL-4 gene. Significantly, reduced GATA3 induction was demonstrated. In vivo antigen-induced IgG1 production and Nippostrongylus brasiliensis -induced eosinophilia were significantly affected, reflecting the deficit in Th2 cell differentiation. Thus, the PcG gene products play a critical role in the control of Th2 cell differentiation and Th2-dependent immune responses.

To study graft-versus-host reaction (GVHR) in small-bowel transplantation and its underlying mechanisms and to find methods for circumventing GVHR, we used an unidirectional GVHR model in which F1 Lewis (LEW) × Wistar King A (WKA) hybrid rats received small-bowel transplants from either LEW or WKA parent rats. The survival time of F1 hybrid rats that received full-length small-bowel transplantation from LEW and WKA was 16.3 ± 2.1 days and 18.2 ± 3.4 days, respectively. When one-quarter of LEW small bowel was transplanted to an F1 hybrid recipient, the survival time was significantly longer at 44.0 ± 23.4 days compared with rats that had received full-length LEW small-bowel transplantation. The survival time of F1 hybrid rats which received an injection of high-dose (5 × 108 cells) LEW or WKA spleen cells was 11.9 ± 4.0 days and 13.1 ± 3.6 days, respectively. However, when an injection containing a low dose (1 × 108 cells) of LEW spleen cells was used, survival was > 100 days, showing significance compared with the survival of rats receiving the higher dose LEW spleen-cell injection. Both small-bowel transplantation and spleen-cell injection were compared for the effective period of recipient resistance to donor cell or small-bowel transplantation as second challenge. When the F1 rats given a quarter LEW small-bowel transplant as first challenge were treated with a high-dose of spleen cells 30 days after transplantation, they survived for > 30 days without GVHR. F1 rats that were treated with a low-dose LEW spleen-cell injection, followed 30 days later by full LEW small-bowel transplantation, had a survival time of > 100 days. These results indicate that segmental small-bowel transplantation and spleen-cell injection as first challenge may facilitate the prevention of GVHR, resulting in resistance to subsequent immunological challenge

A nonapeptide, H-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2, corresponding to amino acids 6 to 14 of bombesin and its four analogues were synthesized by a solid-phase method and were tested for their comparative effect on reducing activity on food intake using male Wistar rats. The synthetic bombesin-fragment6-14 showed reducing effect on food intake using male Wistar rats. Of the synthetic analogues, [Phe(4F)13]bombesin-fragment6-14 exhibited the most potent effect. The reducing effect on food intake of [Sar11]bombesin-fragment6-14 was lower than that of our synthetic bombesin-fragment6-14, but the other two analogues, [betaAla11]bombesin-fragment6-14 and [1-Nal8]bombesin-fragment6-14 showed no reducing effect on food intake in rats.

Clonal anergy is one of the mechanisms that may account for self tolerance induced in T cells in the periphery. In this study we used the well-documented system of in vivo administration of a superantigen, staphylococcal enterotoxin B (SEB), to induce a state of hyporesponsiveness (anergy) in murine peripheral T cells to decipher the intracellular biochemical basis for this process. The TCR-induced Ca response of in vitro activated T cells was found to be impaired with significant defects in the phosphorylation of phospholipase C-gamma 1. Experiments with calcium ionophore and newly established transgenic mouse lines that express an active form of calcineurin suggested that in vivo SEB-induced anergy is established and/or maintained by a selective impairment in the TCR-induced activation of the Ca/calcineurin pathway.

The central role of type-2 helper T (Th2) cells in the development of allergic responses and immune responses against helminthic parasites is well documented. The differentiation of Th2 cells from naive T cells requires both the recognition of antigen by T cell antigen receptors (TCR) and the activation of downstream signal-transduction molecules of the interleukin 4 receptor (IL-4R) pathway, including Jak1, Jak3, and STAT6. Little is known, however, about how these two distinct pathways cooperate with each other to induce Th2 cells. Here, we use a T cell-specific H-Ras-dominant-negative transgenic mouse to show that TCR-mediated activation of the Ras/mitogen-activated protein kinase pathway alters IL-4R function and is required for Th2 cell differentiation. The enhancement of IL-4R signaling seems to be a consequence of both direct “crosstalk” with the TCR signaling pathway and increased protein expression of downstream signaling molecules of the IL-4R pathway. Therefore, successful Th2 differentiation depends on the effectiveness of the TCR-mediated activation of the Ras/mitogen-activated protein kinase pathway in modifying the IL-4R-mediated signaling pathway.

Murine Valpha14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Valpha14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-gamma upon in vivo stimulation with a specific ligand, alpha-galactosylceramide (alpha-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Valpha14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Valpha14 NKT-deficient mice that Valpha14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with alpha-GalCer, Valpha14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Valpha14 NKT- or IFN-gamma-deficient mice. Consistent with these results, we also found that ligand-activated Valpha14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Valpha14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, our studies have revealed that IL-4 produced by Valpha14 NKT cells has only a minor effect on Th2 cell differentiation.

A novel immunosuppressant, FTY720, was generated by chemical modification of ISP-I, an immunosuppressive compound purified from culture filtrates of Isaria sinclairii. FTY720 directly induces apoptotic cell death in lymphocytes, which is believed to be the mechanism by which this drug exerts its immunosuppressive effect. We examined the effect of FTY720 treatment on antigen-induced apoptotic cell death in peripheral T cells and thymocytes. A superantigen, staphylococcus enterotoxin B (SEB), induces T-cell antigen receptor (TCR) Vbeta-specific apoptotic cell death in mature T cells in vivo. In this well-documented experimental system, FTY720 administration significantly enhanced the efficiency of superantigen-induced T-cell deletion. We also determined that apoptotic cell death with DNA fragmentation induced in T-hybridoma cells after stimulation in vitro with anti-TCR antibodies was enhanced in the presence of non-cytolytic doses of FTY720. In sharp contrast, negative selection of T cells in the thymus, another example of antigen-induced apoptosis, was found to be inhibited by FTY720 treatment. A rescue effect was observed on clonal deletion in the H-Y-specific TCRalpha beta transgenic male thymus. In a chicken egg albumin (OVA)-specific TCRalphabeta transgenic system, OVA-induced apoptotic cell death of CD4+CD8+ thymocytes was also inhibited by FTY720 injection. Thus, FTY720 increased the susceptibility of mature T cells to TCR-mediated apoptosis but decreased that of immature thymocytes. The results in this report suggest that the potent immunosuppressive effect of FTY720 is, in part, a result of the augmentation of effects on antigen-induced apoptosis in mature T cells, and that two distinct apoptotic cell death pathways are operating in mature and immature T cells.

The number of γδ T cells in the peritoneal cavity was increased after an intraperitoneal (i.p.) infection with Escherichia coli in lipopolysaccharide (LPS)-responsive C3H/HeN mice but not in LPS-hyporesponsive C3H/HeJ mice. The γδ T cells preferentially expressed invariant Vγ6 and Vδ1 chains and proliferated to produce a large amount of gamma interferon in the presence of LPS. Mice depleted of γδ T cells by T-cell receptor δ gene mutation showed impaired resistance against E. coli as assessed by bacterial growth. Macrophages from C3H/HeN mice infected with E. coli expressed higher levels of interleukin-15 (IL-15) mRNA than those from the infected C3H/HeJ mice. Administration of anti-IL-15 monoclonal antibody inhibited, albeit partially, the appearance of γδ T cells in C3H/HeN mice after E. coli infection and diminished the host defense against the infection. These results suggest that LPS-stimulated γδ T cells play an important role in the host defense against E. coli infection and that IL-15 may be partly involved in the protection via an increase in the γδ T cells.

The lymphocyte-specific protein tyrosine kinase p56(lck) (Lck) is well documented with regard to its role in regulating T cell activation and thymocyte development through delivery of signals via the mature alphabeta TCR as well as the pre-TCR. Little is known, however, about the role of Lck in Th cell subset differentiation in the periphery. Here, we assess the requirement for tyrosine kinase activation of Lck in Th1 and Th2 cell differentiation by using a dominant-negative Lck (DLGKR) transgenic (Tg) mice under the control of a lck distal promoter that directs high expression in mature T cells, in which splenic CD4 T cells developed normally. This Tg mouse provides a good experimental model system to investigate the roles of Lck in mature T cell function in vivo. We show that the catalytically inactive Lck protein at about twice-normal concentrations inhibits Th2 subset differentiation in vivo and in vitro, whilst leaving the maturation of the other T cell subset, Th1, intact. These data indicate a requirement for Lck activity in Th2 cell differentiation, and a differential dependence for Lck activity between Th2 and Th1 cell differentiation.

The effect of flavoxate hydrochloride (flavoxate), an anti-pollakiurea agent, on cyclic AMP (cAMP) formation was investigated in the rat brain and a possible involvement of brain G proteins in the action of flavoxate on the bladder function was subsequently examined. Flavoxate (10(-8)-10(-5) M) inhibited cAMP formation in a concentration-dependent manner, an action which was completely abolished by pretreating the membranes with pertussis toxin (PTX). The inhibitory effect of flavoxate was also completely antagonized by combined treatment with any two antagonists for adenosine A1 (8-cyclopentyl-1,3-dipropylxanthine), dopamine D2 (sulpiride) or adrenergic alpha 2 (yohimbine) receptors, although each antagonist alone did not significantly block the flavoxate-induced inhibition of cAMP formation. Radioligand binding studies indicated that flavoxate at micro- or submicromolar concentrations has affinity for Gi-coupled receptors such as A1, D2 and alpha 2 receptors. Therefore, flavoxate may inhibit cAMP formation by the stimulation of A1, D2 and alpha 2 receptors. To clarify the involvement of brain Gi proteins in the flavoxate-induced inhibition of the micturition reflex, the effect of pretreatment with PTX (i.c.v.) on the flavoxate-induced inhibition of isovolumetric rhythmic bladder contractions was examined in rats. Flavoxate (3 mg/kg, i.v.) completely abolished rhythmic bladder contractions in vehicle-pretreated rats, but not in PTX-pretreated rats. These findings suggest that signal transduction via Gi-coupled receptors is involved, at least in part, in the inhibition of the micturition reflex by flavoxate in rats. These results also provide the first evidence suggesting a negative role of brain PTX-sensitive G proteins in the micturition reflex.

Receptor binding studies have shown that the combination of some new quinolone antibacterial agents with 4-biphenylacetic acid (BPAA), a metabolite of fenbufen, inhibits GABAA receptors. In order to elucidate further the mechanism of these drug interactions, the effect of quinolone antibacterial agents on muscimol-stimulated 36Cl- uptake in rat cerebral cortical synaptoneurosomes was investigated in the absence or presence of BPAA. In the absence of BPAA, quinolones such as norfloxacin (NFLX) and enoxacin attenuated muscimol-stimulated 36Cl- uptake at 10 microM and above. In combination with 10 microM BPAA, the inhibitory effect of these drugs was potentiated and there was a parallel shift of the inhibition curves to the left for these drugs. BPAA alone (1 and 10 microM) did not affect basal or muscimol-stimulated 36Cl- uptake. Hybrid molecules of NFLX and BPAA were synthesized and their inhibitory potency was also investigated. Inhibition curves of muscimol-stimulated 36Cl- uptake revealed that a hybrid with a -CONH(CH2)3- chain between NFLX and BPAA (flexible structure) (1 nM-20 microM) inhibited muscimol-stimulated 36Cl- uptake more potently than did the combination of NFLX (10 nm-100 microM) and 10 microM BPAA. In contrast, another hybrid linked by -CONH-(stretched structure) exhibited a weak inhibitory effect at 10 microM. These results suggest that quinolones in combination with BPAA bind to GABAA receptors, thus inhibiting Cl- channel activity, and that the inhibitory potency of quinolones may be enhanced by an intermolecular interaction with BPAA.

The maintenance of naive CD8(+) T cells is necessary for lifelong immunocompetence but for unknown reasons requires signaling via both interleukin 7 (IL-7) and the T cell antigen receptor (TCR). We now report that naive CD8(+) T cells required IL-7 signaling to be intermittent, not continuous, because prolonged IL-7 signaling induced naive CD8(+) T cells to proliferate, produce interferon-γ (IFN-γ) and undergo IFN-γ-triggered cell death. Homeostatic engagement of the TCR interrupted IL-7 signaling and thereby supported the survival and quiescence of CD8(+) T cells. However, CD8(+) T cells with insufficient TCR affinity for self ligands received prolonged IL-7 signaling and died during homeostasis. In this study we identified regulation of the duration of IL-7 signaling by homeostatic engagement of the TCR as the basis for in vivo CD8(+) T cell homeostasis.

We have succeeded in transplanting human osteogenic sarcoma into nude mice. Morphologically, the transplanted tumor is chondrosarcoma and manifests calcification, but not ossification. This tumor is thought to be an excellent model for studying the process of morbid endochondral calcification. In this study, we have used in situ hybridization to examine expression of collagen type I, II, and III mRNAs in this tumor. In situ hybridization was carried out using biotinylated DNA probes. Hybridized probes were detected using a streptavidin-biotin-alkaline phosphatease reagent. The results showed that collagen type I and II mRNAs were produced by cells of the transplanted tumor. Collagen type I mRNA was chiefly localized in the marginal region of the tumor. Collagen type II mRNA, which was predominantly found in the premineralized region of the transplanted tumor, gradually decreased toward the mineralized region. Collagen type III mRNA was not expressed in the transplanted tumor. These results suggest that the character of progenitor chondrogenic cells might be transferred to the transplanted tumor, and that the tumor cells may change the expression of collagen genes with the differentiation or maturation.

Two distinct cDNAs encoding bradykinin receptors (BKRs) were cloned from NG108-15 neuroblastoma-glioma hybrid cells. One was identical with rat uterus B2 BKR, whereas the other one (mBKR) had 91% amino acid homology to the rat B2 BKR and 82% homology to human B2 BKR. Southern blot analysis and genomic DNA cloning revealed that mBKR is derived from the mouse genome. The mBKR, expressed in Xenopus oocytes and COS-7 cells, produced functional BKRs that exhibited the properties of smooth muscle type B2 BKR. These results suggest that both the rat and mouse B2 BKRs of the smooth muscle type are expressed in NG108-15 cells.

The combination of some new quinolone antibacterials with 4-biphenylacetic acid (BPAA) functionally inhibits the gamma-amino-butyric acid (GABA) receptors and thereby induces clonic convulsions. We examined the effects of ofloxacin and its optical isomers on this quinolone-induced neurotoxicity. Norfloxacin at 10(-5) M alone or at 10(-7) M in combination with BPAA (10(-4) M) inhibited [3H]muscimol binding to rat brain synaptic membranes. Ofloxacin and its optical isomers did not affect muscimol binding by themselves. While they slightly reduced muscimol binding at 10(-4) M in combination with BPAA, the inhibitory activity of the l-isomer levofloxacin (DR-3355) on muscimol binding was slightly, but significantly, weaker than that of the d-isomer DR-3354 and ofloxacin. Intracisternal injection of norfloxacin (5 micrograms), ofloxacin, levofloxacin or DR-3354 (50 micrograms each) induced clonic convulsions in mice. The incidence of these convulsions was enhanced by the combination with BPAA (50 micrograms). The epileptogenic activity of levofloxacin was also weaker than that of DR-3354 or ofloxacin when quinolones were given alone or in combination with BPAA. These results suggest that epileptogenic activity of quinolones is closely related to the inhibitory potency in GABA receptor binding and that levofloxacin may have lower neurotoxicity than ofloxacin and DR-3354.

We evaluated the biocompatibility of tetracalcium phosphate (4CP) cement, made of 4CP powder and 40 wt% copolymer of polyacrylic acid/itaconic acid and 10 wt% citric acid solution. Light and electron microscopic characteristics were studied 3, 10 and 30 d after implantation. Neither inflammation nor foreign-body giant cell reaction was observed in the tissue adjacent to the implanted material. After 30 d, this material was surrounded with newly formed bone. Ultrastructural examination showed that osteogenesis occurred directly on the surface of the material. These findings suggest that this 4CP cement is biocompatible and possesses osteoconductive properties.