Transcriptional regulation of the IL-7Rα gene by dexamethasone and IL-7 in primary human CD8 T cells.

Interleukin-7 is essential for the development and maintenance of T cells, and the expression of the IL-7 receptor is tightly regulated at every stage of the T cell's lifespan. In mature CD8 T cells, IL-7 plays important roles in cell survival, peripheral homeostasis, and cytolytic function. The IL-7 receptor alpha-chain (CD127) is expressed at high levels on naïve and memory cells, but it is rapidly downregulated upon IL-7 stimulation. In this study, we illustrate the dynamicity of the CD127 promoter and show that it possesses positive as well as negative regulatory sites involved in upregulating and downregulating CD127 expression, respectively. We cloned the CD127 gene promoter and identified key cis-regulatory elements required for CD127 expression in mature resting primary CD8 T cells. The core promoter necessary for efficient basal transcription is contained within the first 262 bp upstream of the TATA box. Additional positive regulatory elements are located between -1200 and -2406 bp, conferring a further 2- to 4-fold enhancement in gene expression. While transcription of the CD127 gene is increased directly through a glucocorticoid response element located between -2255 and -2269 bp upstream of the TATA box, we identified a suppressive region that lies upstream of 1760 bp from the TATA box, which is likely involved in the IL-7-mediated suppression of CD127 transcription. Finally, we illustrated IL-7 does not bias alternative splicing of CD127 transcripts in primary human CD8 T cells.

IL-7 downregulates IL-7Rα expression in human CD8 T cells by two independent mechanisms.

Interleukin (IL)-7 is an essential nonredundant cytokine, and throughout the lifespan of a T-cell signaling via the IL-7 receptor influences cell survival, proliferation and differentiation. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. We have previously shown that IL-7 downregulates expression of CD127 at the cell surface and now elucidate the kinetics of that suppression and demonstrate that IL-7 downregulates CD127 transcripts and surface protein in primary human CD8 T cells by two separate pathways. We show that IL-7 induces the initial reduction in cell-surface CD127 protein independent of transcriptional suppression, which is delayed by 40-60 min. Although IL-7-mediated downregulation of CD127 transcripts is dependent on Janus kinase (JAK)/STAT5, the early downregulation of surface CD127 protein is independent of JAK activity. The data further illustrate that low levels of IL-7 induce smaller and transient decreases in CD127 transcripts and surface protein, whereas higher concentrations induce more profound and sustained suppression. Such flexibility in receptor expression likely allows for fine-tuned immune responses in human CD8 T cells in different microenvironments and in response to different immunological challenges.

Adult porcine (Sus scrofa) derived inner ear cells: Characteristics in in-vitro cultures.

There is a need for an animal model that closely parallels human cochlea gestational development. This study aims to document porcine inner ear anatomy, and in vitro porcine derived inner ear cell culture characteristics. Twenty-four temporal bone were harvested from 12 adult pigs (Sus scrofa). Six were formalin fixed and their maximal diameters were measured. The cochlea duct length was determined by the insertion length of a Nucleus 22 cochlear implant in two bones. Four formalin fixed bones were sectioned for histology. Cochlear and vestibular tissues were harvested from non-fixed bones, cultured and characterized at different passages (P). Gene and protein expression of multipotent stem/progenitor (Nestin and Sox2), inner ear hair (Myosin VIIa, Prestin) and supporting (Cytokeratin 18 and Vimentin) cell markers were determined. The porcine cochlea was a 3.5 turn spiral. There was a separate vestibular compartment. The cochlear mean maximal diameter and height was 7.99 and 3.77 mm, respectively. Sphere forming cells were identified on phase-contrast microscopy. The relative mRNA expression levels of KRT18, MYO7A and SLC26A5 were significantly positively correlated in cochlear cultures; and MYO7A and SLC26A5; SOX2 and KRT18; NES and SLC26A5 genes were positively correlated in vestibular cultures (p = .037, Spearman correlation [τ] = .900). Inner ear sensory and stem cell characteristics persist in passaged porcine inner ear cells. Further work is required to establish the usefulness of porcine inner ear cell cultures to the study of human inner ear disorders.

Soluble HIV Tat protein removes the IL-7 receptor alpha-chain from the surface of resting CD8 T cells and targets it for degradation.

IL-7 signaling is essential to CD8 T cell development, activation, and homeostasis. We have previously shown decreased expression of the IL-7R alpha-chain (CD127) on CD8 T cells in HIV(+) patients and that this downregulation is mediated at least in part by the HIV Tat protein. We show in this study that CD127 has a prolonged t(1/2) in resting CD8 T cells and continuously recycles on and off the cell membrane. We also demonstrate soluble Tat protein significantly decreases the t(1/2) of CD127. Soluble Tat is taken up from the medium and accumulates in CD8 T cells with a peak of 6 h. Once inside the cell, Tat exits the endosomes during their normal acidification and enters the cytosol. Tat then translocates to the inner leaflet of the cell membrane, where it binds directly to the cytoplasmic tail of CD127, inducing receptor aggregation and internalization through a process dependent on microtubules. Tat appears to then target CD127 for degradation via the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is thus able to reduce IL-7 signaling and impair CD8 T cell proliferation and function.

Systematic review of physical and chemical compatibility of commonly used medications administered by continuous infusion in intensive care units.

OBJECTIVE: To quantify the physical and chemical stability data published for commonly used continuously infused medications in the intensive care unit and to evaluate the quality of the studies providing these data. DATA SOURCES AND STUDY SELECTION: We conducted a systematic electronic literature search of MEDLINE, EMBASE, and International Pharmaceutical Abstracts as well as the references of electronic drug compatibility textbooks for all English and French language research publications evaluating the physical compatibility or chemical stability of the 820 possible two-drug combinations of 41 commonly used drugs in an adult intensive care unit. DATA EXTRACTION AND SYNTHESIS: A total of 93 studies comprised of 86 (92%) studies evaluating physical compatibility and 35 (38%) studies evaluating chemical compatibility of at least one drug combination of interest were included. Physical and/or chemical compatibility data exist for only 441 of the possible 820 two-drug combinations (54%), whereas chemical compatibility data exist for only 75 (9%) of the possible combinations. Of the 441 combinations for which compatibility data are available, 67 (15%) represent incompatible combinations and 39 (9%) had conflicting data in which both compatible and incompatible data were identified. CONCLUSIONS: Physical compatibility studies that provide the basis for y-site compatibility are lacking for commonly used medications in intensive care unit patients and may contribute to unsafe medication practices. Furthermore, the heterogeneity in the methodology of these studies likely contributes to the common finding of conflicting data for specific combinations of drugs. Future studies should apply similar methodologic and reporting principles to be able to reproduce and compare outcomes both clinically and in the laboratory.

IL-7 and the HIV Tat protein act synergistically to down-regulate CD127 expression on CD8 T cells.

IL-7 signaling is essential for optimal CD8 T cell function, homeostasis and establishment of memory. We have previously shown decreased expression of the IL-7 receptor alpha-chain (CD127) on CD8 T cells from HIV-infected patients with active viral replication. We have also shown that soluble HIV Tat protein specifically down-regulates CD127 on the surface of CD8 T cells and impairs cell proliferation and cytolytic potential following stimulation with IL-7 in vitro. We now show that soluble HIV Tat protein and IL-7 at near physiologic concentrations act synergistically to suppress CD127 expression. While soluble HIV Tat protein and IL-7 both independently reduce CD127 expression on the surface of CD8 T cells, Tat concentrations of 10 microg ml(-1) and IL-7 concentrations of 500 pg ml(-1) are required in vitro to have an appreciable effect. However, where 0.5 microg ml(-1) of Tat has no effect on CD127 expression and 200 pg ml(-1) of IL-7 decreases CD127 by only 14%, these two together at these same concentrations induce a 35% reduction in CD127 expression after 24 h. Inhibition of Janus kinase (JAK) completely blocks IL-7's ability to down-regulate CD127 on the surface of CD8 T cells and also abolishes synergy with Tat. Interestingly, while Tat acts synergistically with IL-7 to reduce CD127 expression, it antagonizes IL-7-induced cell proliferation and Ki-67 expression and has no effect on IL-7-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation or expression of the anti-apoptotic gene Bcl-2. Thus, by affecting different IL-7 signal transduction pathways, HIV Tat protein is able to impair both CD8 T cell activation and proliferation without inducing apoptosis.

Computerized Dynamic Posturography does not detect measured CVEMP and OVEMP abnormalities.

BACKGROUND: Computerized Dynamic Posturography (CDP)was developed by the American space program to assess imbalance in astronauts, and eventually evolved into a clinical diagnostic tool. However it is not a specific measure of vestibular function. Vestibular Evoked Myogenic Potential testing (VEMPs) is a new clinical tool which is sensitive and specific for measuring otolithic pathology, especially in the atypical vestibular patient. RESEARCH QUESTION: As posturography measures ability to maintain balance, and VEMP testing measures the structures responsible for this, we wondered if CDP results would correlate with VEMP abnormalities in the clinical setting. METHODS: We analysed 180 patients sequentially referred to our unit for vestibular complaints. All patients had a full battery of vestibular assessments. We correlated VEMP results with CDP results to look for abnormality patterns and correlations. An occasional patient's only abnormality was on CDP RESULTS: There was a high rate of VEMP abnormalities seen, which correlates with the fact that our referral base consists of patients with chronic vestibular complaints. The rate of VEMP abnormalities was the same in patients with normal CDP and those with abnormal CDP. SIGNIFICANCE: Our results do not suggest that CDP is unnecessary, but we feel that they emphasize the idea that these tests are measuring two different aspects of balance control. In some patients, all assessments are abnormal, but in some patients only one assessment is abnormal, suggesting that these modalities measure different things and are all important in the diagnostic armamentarium. Hopefully in the near future, the use of virtual reality will reduce the cost of CDP to the point where it can be made widely accessible to patients and clinicians.

The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: a critical review of the literature.

CD8+ tumor-infiltrating lymphocytes (TIL) are associated with survival in a variety of cancers. A second subpopulation of TIL, defined by forkhead box protein P3 (FoxP3) expression, has been reported to inhibit tumor immunity, resulting in decreased patient survival. On the basis of this premise, several groups are attempting to deplete FoxP3+ T cells to enhance tumor immunity. However, recent studies have challenged this paradigm by showing that FoxP3+ T cells exhibit heterogeneous phenotypes and, in some cohorts, are associated with favorable prognosis. These discrepant results could arise from differences in study methodologies or the biologic properties of specific cancer types. Here, we conduct the first systematic review of the prognostic significance of FoxP3+ T cells across nonlymphoid cancers (58 studies from 16 cancers). We assessed antibody specificity, cell-scoring strategy, multivariate modeling, use of single compared with multiple markers, and tumor site. Two factors proved important. First, when FoxP3 was combined with one additional marker, double-positive T cells were generally associated with poor prognosis. Second, tumor site had a major influence. FoxP3+ T cells were associated with poor prognosis in hepatocellular cancer and generally good prognosis in colorectal cancer, whereas other cancer types were inconsistent or understudied. We conclude that FoxP3+ T cells have heterogeneous properties that can be discerned by the use of additional markers. Furthermore, the net biologic effects of FoxP3+ T cells seem to depend on the tumor site, perhaps reflecting microenvironmental differences. Thus, depletion of FoxP3+ T cells might enhance tumor immunity in some patient groups but be detrimental in others.

Interleukin-7 receptor expression on CD8 T-cells is downregulated by the HIV Tat protein.

We have previously shown decreased expression of the interleukin (IL)-7 receptor alpha-chain (CD127) on CD8 T-cells in HIV-infected patients and an apparent recovery of this receptor in those receiving antiretroviral therapy with sustained viral suppression. Here, we demonstrate that the HIV Tat protein specifically downregulates cell surface expression of CD127 on human CD8 T-cells in a dose- and time-dependent manner. The effects of Tat on CD127 expression could be blocked with anti-Tat monoclonal antibodies or by preincubating Tat with heparin. Tat had no effect on the expression of other cell surface proteins examined, including CD132, or on cell viability over 72 hours. Further, CD127 expression was not altered by other HIV proteins, including gp160 or Nef. Preincubation of purified CD8 T-cells with Tat protein inhibited CD8 T-cell proliferation and perforin synthesis after stimulation with IL-7. Because IL-7 signaling is essential for optimal CD8 T-cell proliferation and function, the downregulation of CD127 and apparent inhibition of cytotoxic activity by Tat may play an important role in HIV-induced immune dysregulation and impaired cell-mediated immunity.