Chemerin-ChemR23 signaling in tooth development.

Our long-term goal is to identify and characterize molecular mechanisms regulating tooth development, including those mediating the critical dental epithelial-dental mesenchymal (DE-DM) cell interactions required for normal tooth development. The goal of this study was to investigate Chemerin (Rarres2)/ChemR23(Cmklr1) signaling in DE-DM cell interactions in normal tooth development. Here we present, for the first time, tissue-specific expression patterns of Chemerin and ChemR23 in mouse tooth development. We show that Chemerin is expressed in cultured DE progenitor cells, while ChemR23 is expressed in cultured DM cells. Moreover, we demonstrate that ribosomal protein S6 (rS6) and Akt, downstream targets of Chemerin/ChemR23 signaling, are phosphorylated in response to Chemerin/ChemR23 signaling in vitro and are expressed in mouse tooth development. Together, these results suggest roles for Chemerin/ChemR23-mediated DE-DM cell signaling during tooth morphogenesis.

RSV infection modulates IL-15 production and MICA levels in respiratory epithelial cells.

The cytokine interleukin (IL)-15, major histocompatibility complex (MHC) class I molecules and MHC class I chain-related proteins (MIC) A and B are involved in cellular immune responses to virus infections but their role in respiratory syncytial virus (RSV) infection has not been studied. We aimed to determine how RSV infection modulates IL-15 production, MHC class I and MICA expression in respiratory epithelial cells, the molecular pathways implicated in virus-induced IL-15 production and how interferon (IFN)-γ alters RSV-induced IL-15 production and MHC class I and MICA expression. We infected respiratory epithelial cell lines (A549 and BEAS-2B cells) and primary bronchial epithelial cells with RSV and measured production of IL-15, expression of MHC I and MICA and the role of the transcription factor nuclear factor (NF)-κB. We report here that RSV increases IL-15 in respiratory epithelial cells via virus replication and NF-κB-dependent mechanisms. Furthermore, RSV infection of epithelial cells upregulated cell surface expression of MICA and levels of soluble MICA. IFN-γ upregulated RSV induction of soluble IL-15 but inhibited induction of MICA. Upregulation of IL-15, MHC I and MICA are likely to be important mechanisms in activating immune responses to RSV by epithelial cells.

IL-15 plays a major role in the persistence of Tax-specific CD8 cells in HAM/TSP patients.

IL-15 is a critical cytokine for the maintenance of memory-phenotype CD8 cells in mice. Here, we investigated the role of IL-15 in the neurological disease termed human T cell lymphotropic virus I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The high number of viral-specific CD8 cells in these patients is associated with inflammatory responses in the central nervous system. Because IL-15 is overexpressed in these patients, we asked whether IL-15 contributes to the persistence of human T cell lymphotropic virus I viral-specific CD8 cells. Using ex vivo cultures of HAM/TSP peripheral blood mononuclear cells, we demonstrated that in the majority of patients examined here blocking IL-15 action resulted in a decrease in the number of viral-specific CD8 cells. This decrease was caused by both inhibition of proliferation and induction of apoptosis in these cells. The data indicate that IL-15 plays a major role in the maintenance of viral-specific CD8 cells in HAM/TSP.

Onset of natural killer cell lymphomas in transgenic mice carrying a truncated HMGI-C gene by the chronic stimulation of the IL-2 and IL-15 pathway.

Rearrangements of the high mobility group protein I-C (HMGI-C) gene, consisting in the loss of the carboxyl-terminal tail, have been frequently detected in benign human tumors of mesenchymal origin. We have previously demonstrated that transgenic (TG) mice carrying a truncated HMGI-C construct (HMGI-C/T) exhibit a giant phenotype together with a predominantly abdominal/pelvic lipomatosis. Here, we report that HMGI-C/T TG mice develop natural killer (NK)-T/NK cell lymphomas starting from 12 months of age. We found an increased expression of IL-2 and IL-15 proteins and their receptors in these lymphomas, and we demonstrate that HMGI-C/T protein positively regulates their expression in vitro. Therefore, the HMGI-C/T-mediated chronic stimulation of the IL-2/IL-15 pathway could be responsible for the onset of NK-T/NK cell lymphomas in HMGI-C/T TG mice.

Human T cell lymphotropic virus type I Tax activates IL-15R alpha gene expression through an NF-kappa B site.

IL-15 mRNA levels are increased in diseases caused by human T cell lymphotropic virus type I (HTLV-I). In this study, we demonstrated that IL-15Ralpha, the IL-15-specific binding receptor, mRNA and protein levels were also elevated in HTLV-I-infected cells. We showed that transient HTLV-I Tax expression lead to increased IL-15Ralpha mRNA levels. In addition, by using a reporter construct that bears the human IL-15Ralpha promoter, we demonstrated that Tax expression increased promoter activity by at least 4-fold. Furthermore, using promoter deletion constructs and gel shift analysis, we defined a functional NF-kappaB-binding motif in the human IL-15Ralpha promoter, suggesting that Tax activation of IL-15Ralpha is due, in part, to the induction of NF-kappaB. These data indicate that IL-15Ralpha is transcriptionally regulated by the HTLV-I Tax protein through the action of NF-kappaB. These findings suggest a role for IL-15Ralpha in aberrant T cell proliferation observed in HTLV-I-associated diseases.

How does interleukin 15 contribute to the pathogenesis of HTLV type 1-associated myelopathy/tropical spastic paraparesis?

HTLV-1 is the etiological agent of a neurological disease named HAM/TSP that has clinical characteristics similar to those of multiple sclerosis (MS). The PBMC obtained from HAM/TSP patients undergo spontaneous proliferation in the absence of addition of any exogenous cytokines in an ex vivo culture. This spontaneous proliferation has been thought to be due to the proliferation of T cells. It was demonstrated that this proliferation is, in part, due to the production of IL-2 and its receptor (IL-2Ralpha) by HTLV-1-infected T cells. In this review, we demonstrate that IL-15 production also contributes to the spontaneous proliferation of T cells obtained from HAM/TSP PBMC. We provide data indicating that IL-15 expression is elevated in HAM/TSP PBMC when compared to that of the normal donors. Furthermore, we demonstrate that IL-15 overexpression by HTLV-1 is due to Tax trans-activation of its promoter and induction of NF-kappaB transcription factors. On the basis of these studies, we propose a model in which HTLV-1 infection of T cells results in the production of both IL-2 and IL-15 cytokines, growth factors that support the proliferation of T cells.

Overexpression of p21(waf1) in human T-cell lymphotropic virus type 1-infected cells and its association with cyclin A/cdk2.

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5'-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G(1), S, or G(2)/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G(1) phase prior to gamma-irradiation, arrested in G(1) and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G(1)/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry.

Viral activation of interleukin-15 (IL-15): characterization of a virus-inducible element in the IL-15 promoter region.

We identified an interferon regulatory factor motif (IRF-E) upstream of an NF-kappaB binding site in the interleukin-15 (IL-15) promoter. Since these two motifs are part of the virus-inducible enhancer region of the beta interferon promoter, we speculated that there might be similar responses of these two genes to stimuli such as viruses. To test this hypothesis, L929 cells were infected with Newcastle disease virus (NDV), which led to the induction of IL-15 mRNA and protein expression. Using IL-15 promoter-reporter deletion constructs, a virus-inducible region, encompassing IRF-E, NF-kappaB, and a 13-nucleotide sequence flanked by these two motifs, was mapped to the -295-to--243 position relative to the transcription initiation site. Using cotransfection studies, it was demonstrated that all three motifs were essential to achieve the maximum promoter activity induced by IRF-1 and NF-kappaB expression plasmids. The presence of a virus-inducible region in the IL-15 promoter suggests a role for IL-15 as a component of host antiviral defense mechanisms.

Transcriptional up-regulation of the cyclin D2 gene and acquisition of new cyclin-dependent kinase partners in human T-cell leukemia virus type 1-infected cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis. Tax(1) is a 40-kDa phosphoprotein, predominantly localized in the nucleus of the host cell, which functions to transactivate both viral and cellular promoters. It seems likely that HTLV-1, through expression of the viral regulatory protein Tax(1), provides some initial alteration in cell metabolism predisposing the development of ATL. Here, we demonstrate that HTLV-1 infection in T-cell lines and patient samples causes overexpression of an early G(1) cyclin, cyclin D2. The transcriptional up-regulation of the cyclin D2 gene is due to activation of Tax on the cyclin D2 gene. More important, we find that overexpression of cyclin D2 is accompanied by acquisition of new partners such as cyclin-dependent kinase 2 (cdk2), cdk4, and cdk6 in infected cells. This is in contrast to uninfected T cells, where cyclin D2 associates only with cdk6. Functional effects of these cyclin-cdk complexes in infected cells are shown by hyperphosphorylation of Rb and histone H1, indicators of active progression into S phase as well as changes in cellular chromatin and transcription machinery. These studies link HTLV-1 infection with changes of cellular cyclin gene expression, hence providing clues to development of T-cell leukemia.

Involvement of IL-15 in the pathogenesis of human T lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis: implications for therapy with a monoclonal antibody directed to the IL-2/15R beta receptor.

Human T lymphotropic virus type I (HTLV-I) is the causative agent of an inflammatory neurological disease termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). An ongoing lymphocyte activation exists in patients with HAM/TSP, which was demonstrated by the spontaneous proliferation of their PBMC ex vivo. It was shown that spontaneous proliferation present in HAM/TSP is due, in part, to an IL-2/IL-2R autocrine loop. However, addition of Abs against IL-2 or IL-2R alpha only partially inhibited the spontaneous proliferation. Since IL-15 is a cytokine with similar functional characteristics to those of IL-2, we reasoned that IL-15 might be an additional growth factor that contributes to the spontaneous proliferation observed in HAM/TSP. In this study, we demonstrated that IL-15 mRNA expression was elevated in PBMC obtained from HAM/TSP patients when compared with those of the normal donors. Furthermore, we showed that the addition of blocking Abs against IL-15 or its receptor inhibited the spontaneous proliferation of HAM/TSP PBMC. Addition of Abs directed toward both IL-15 and IL-2, or their receptors, inhibited the proliferation almost completely. These data suggest the existence of two autocrine loops involving IL-15/IL-15R and IL-2/IL-2R, both contributing to the spontaneous proliferation of HAM/TSP PBMC.

The effectiveness of cost-effectiveness analysis in containing costs.

OBJECTIVE: Although cost-effectiveness analyses (CEAs) have been advocated as a tool to critically appraise the value of health expenditures, it has been widely hoped that they might also help contain health care costs. To determine how often they discourage additional expenditures, we reviewed the conclusions of recently published CEAs. DATA SOURCES: A search of the Abridged Index Medicus (a subset of MEDLINE designed to afford rapid access to the literature of "immediate interest" to the practicing physician) between 1990 and 1996. STUDY SELECTION: We only included articles that reported an explicit cost-effectiveness (CE) ratio (a cost for some given health effect) in the abstract. DATA ABSTRACTION: From each abstract, we collected the value for the incremental CE ratio and the measure of health effect (life-years, quality-adjusted life-years [QALYs], other). We then categorized the authors' conclusion into one of three categories: supports strategy requiring additional expenditure, no firm conclusion, and supports low-cost alternative. Finally, we obtained the article and collected information on funding source. DATA SYNTHESIS: Among the 109 eligible articles, the authors' conclusion supported strategies requiring additional expenditure in 58 (53%) and supported the low-cost alternative in 28 (26%). We then focused on the 65 articles reporting either life-years or QALYs. Cost-effectiveness ratios ranged from $400 to $166,000 (per life-year or QALY) in the 39 articles (60%) in which authors supported additional expenditure, and ranged from $61,500 to $11,600,000 in the 13 articles (20%) in which authors supported the low-cost alternative. Despite identifying similar CE ratios, authors arrived at different conclusions in the overlapping range ($61,500 to $166,000). Of the 10 articles acknowledging industry funding, 9 supported a strategy requiring additional expenditure (p = .01 as compared with those without such funding). CONCLUSIONS: Authors of CEAs are more likely to support strategies requiring additional expenditure than the low-cost alternative. There is no obvious consensus about how small the CE ratio should be to warrant additional expenditure. Finally, concerns about funding source seem to be warranted.

The 5' untranslated region, signal peptide, and the coding sequence of the carboxyl terminus of IL-15 participate in its multifaceted translational control.

We previously reported that the AUG-burdened 5' untranslated region (UTR) of IL-15 mRNA impedes its translation. Here we demonstrate that the nucleotide or protein sequences of the IL-15 signal peptide and carboxyl terminus also contribute to the poor translation of IL-15 transcripts. In particular, the exchange of the IL-15 signal peptide coding sequence with that of IL-2 increased cellular and secreted levels of IL-15 protein 15- to 20-fold in COS cells, while IL-2 transcripts with the IL-15 signal peptide generated 30- to 50-fold less IL-2 protein than wild-type IL-2. Furthermore, the addition of an artificial epitope tag to the 3' coding sequence of IL-15 increased its protein production 5- to 10-fold. Combining these two IL-15 message modifications, in addition to removing the 5' UTR, increased IL-15 synthesis 250-fold compared with a wild-type construct with an intact 5' UTR. These data suggest that IL-15 mRNA, unlike IL-2 mRNA, may exist in translationally inactive pools. By storing translationally quiescent IL-15 mRNA, cells might respond to intracellular infections or other stimuli by rapidly transforming IL-15 message into one that can be efficiently translated.

Human T cell lymphotropic virus type I Tax protein trans-activates interleukin 15 gene transcription through an NF-kappaB site.

Interleukin 15 (IL-15) mRNA is expressed in a wide variety of tissue types. However, with the exception of some T cell lines, IL-15 transcript expression has not been described in T cells. Herein we demonstrate that IL-15 mRNA can be detected in freshly isolated normal T cells and T cell lines. Furthermore, its expression is 3- to 4-fold higher in human T cell lymphotropic virus type I (HTLV-I)-infected T cells. By using reporter constructs bearing the 5' regulatory region of the IL-15 gene, we observed a positive correlation between HTLV-I Tax protein expression and IL-15 promoter activity in HTLV-I-infected T cells. Additionally, by using a Jurkat T cell transfectant that expresses Tax under an inducible promoter, we demonstrated that the expression of IL-15 mRNA increased 3-fold as Tax was expressed, suggesting that the Tax protein activates IL-15 transcription. An NF-kappaB consensus sequence is located at the -75 and -65 region of the IL-15 5' regulatory region. Mutations in the NF-kappaB motif or deletion of this sequence abrogated the promoter activity in both HTLV-I-positive and Jurkat Tax-transfectant cells. These data represent evidence for trans-activation of the IL-15 gene by the HTLV-I Tax protein through an NF-kappaB motif and suggest a potential role for IL-15 in HTLV-I-associated diseases such as adult T cell leukemia and HTLV-I-associated myopathy/tropical spastic paraparesis.

Requirement for IRF-1 in the microenvironment supporting development of natural killer cells.

Natural killer (NK) cells are critical for both innate and adaptive immunity. The development of NK cells requires interactions between their progenitors and the bone-marrow microenvironment; however, little is known about the molecular nature of such interactions. Mice that do not express the transcription factor interferon-regulatory factor-1 (IRF-1; such mice are IRF-1(-/-) mice) have been shown to exhibit a severe NK-cell deficiency. Here we demonstrate that the lack of IRF-1 affects the radiation-resistant cells that constitute the microenvironment required for NK-cell development, but not the NK-cell progenitors themselves. We also show that IRF-1(-/-) bone-marrow cells can generate functional NK cells when cultured with the cytokine interleukin-15 and that the interleukin-15 gene is transcriptionally regulated by IRF-1. These results reveal, for the first time, a molecular mechanism by which the bone-marrow microenvironment supports NK-cell development.

Generation of secretable and nonsecretable interleukin 15 isoforms through alternate usage of signal peptides.

Two isoforms of human interleukin 15 (IL-15) exist. One isoform has a shorter putative signal peptide (21 amino acids) and its transcript shows a tissue distribution pattern that is distinct from that of the alternative IL-15 isoform with a 48-aa signal peptide. The 21-aa signal isoform is preferentially expressed in tissues such as testis and thymus. Experiments using different combinations of signal peptides and mature proteins (IL-2, IL-15, and green fluorescent protein) showed that the short signal peptide regulates the fate of the mature protein by controlling the intracellular trafficking to nonendoplasmic reticulum sites, whereas the long signal peptide both regulates the rate of protein translation and functions as a secretory signal peptide. As a consequence, the IL-15 associated with the short signal peptide is not secreted, but rather is stored intracellularly, appearing in the nucleus and cytoplasmic components. Such production of an intracellular lymphokine is not typical of other soluble interleukin systems, suggesting a biological function for IL-15 as an intracellular molecule.

Reverse iontophoresis: noninvasive glucose monitoring in vivo in humans.

PURPOSE: To demonstrate that "reverse iontophoresis" can be used to noninvasively obtain information about systemic glucose levels in vivo in humans. METHODS: The passage of current across the skin in vivo drives ions into the tissue, from the electrode chambers positioned on the skin surface, and simultaneously pulls ions from the body in the opposite direction. Because of the net negative charge on the skin, under normal conditions, the membrane is permselective to cations, and a potential gradient also results, therefore, in electroosmotic convection of solvent in the direction of counterion flow (i.e., from anode to cathode). Thus, it is also possible to enhance the transport of polar, yet uncharged, species using iontophoresis. In an earlier study, the in vitro extraction of glucose, by "reverse iontophoresis" was established, and extension of the approach to an in vivo model was indicated. The idea has therefore been further explored in vivo in humans. RESULTS: Using small, simple, prototypical electrode chambers, attached to the ventral forearm surface, direct current iontophoresis at 0.25 mA/cm2 for periods of up to 1 hour, and a sensitive analytical procedure to measure the quantities of glucose extracted, it has been shown that iontophoretic sampling of glucose is feasible. However, the shorter periods (15 minutes or less) of extraction considered yield results which are "contaminated" (it is believed) by glucose that is a product of lipid metabolism within the skin. While this material is expected to complicate the initial calibration of the approach, the problem is effectively resolved within one hour, by which time the glucose arriving in the electrode chambers on the skin surface is expected to directly reflect the subcutaneous tissue concentration. CONCLUSIONS: Based upon these initial observations, further investigation can now be directed towards optimization of electroosmotic flow and sampling time, improved reproducibility and the development of a practical assay methodology.