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Jens Dannull1, Zhen Su1, David Rizzieri2, Benjamin K. Yang1, Doris Coleman1, Donna Yancey1, Aijing Zhang1, Philipp Dahm1, Nelson Chao2, Eli Gilboa1 and Johannes Vieweg1
1Genitourinary Cancer Immunotherapy Program, Division of Urology, Department of Surgery, and 2Division of Cellular Therapy, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Address correspondence to: Johannes Vieweg, Duke University Medical Center, MSRB, Suite 455, DUMC Box 2626, Durham, North Carolina 27710, USA. Phone: (919) 684-9949; Fax: (919) 681-7414; E-mail: email@example.com.
First published November 1, 2005Submitted: June 13,
2005; Accepted: October 4,
In this study, we investigated whether elimination of CD4+/CD25+ Tregs using the recombinant IL-2 diphtheria toxin conjugate DAB389IL-2 (also known as denileukin diftitox and ONTAK) is capable of enhancing the immunostimulatory efficacy of tumor RNA-transfected DC vaccines. We show that DAB389IL-2 is capable of selectively eliminating CD25-expressing Tregs from the PBMCs of cancer patients without inducing toxicity on other cellular subsets with intermediate or low expression of CD25. DAB389IL-2–mediated Treg depletion resulted in enhanced stimulation of proliferative and cytotoxic T cell responses in vitro but only when DAB389IL-2 was omitted during T cell priming. DAB389IL-2 significantly reduced the number of Tregs present in the peripheral blood of metastatic renal cell carcinoma (RCC) patients and abrogated Treg-mediated immunosuppressive activity in vivo. Moreover, DAB389IL-2–mediated elimination of Tregs followed by vaccination with RNA-transfected DCs significantly improved the stimulation of tumor-specific T cell responses in RCC patients when compared with vaccination alone. Our findings may have implications in the design of immune-based strategies that may incorporate the Treg depletion strategy to achieve potent antitumor immunity with therapeutic impact.
We have previously shown that vaccination with RNA-transfected DCs is an effective strategy to stimulate potent T cell responses in patients with metastatic cancers (1, 2). In this study, we investigated whether the immunostimulatory properties of RNA-transfected DC vaccines can be further enhanced by disrupting regulatory pathways that suppress the activation and function of tumor-specific T effector cells in the cancer patient. Recent research has shown that CD4+ T cells constitutively expressing the IL-2 receptor α-chain (CD25) act in a regulatory capacity by suppressing the activation and function of other T cells (3). Their physiological role is to protect the host against the development of autoimmunity by regulating immune responses against antigens expressed by normal tissues (4, 5). Since tumor antigens are largely self antigens, Tregs may also prevent the tumor-bearing host from mounting an effective antitumor immune response. Previous studies have shown that elevated numbers of CD4+CD25+ Tregs can be found in advanced cancer patients (6) and that high Treg frequencies are associated with reduced survival (7). The important role of CD4+CD25+ Tregs in controlling tumor growth was further highlighted by the demonstration that depletion of Tregs using anti-CD25 antibodies can evoke effective antitumor immunity in mice (8, 9). Moreover, anti-CD25 therapy enhanced the therapeutic efficacy of GM-CSF–secreting B16 tumor cells and prolonged survival of tumor-bearing animals (10). Cumulatively, these experimental data suggest that the efficacy of cancer vaccination could be enhanced by treatment with agents that lead to the preferential depletion of CD4+CD25+ Tregs, such as compounds that target cells expressing the IL-2 receptor CD25 subunit.
In this study, we used the recombinant IL-2 diphtheria toxin conjugate DAB389IL-2 (also known as denileukin diftitox and ONTAK) to eliminate CD25-expressing Tregs in metastatic renal cell carcinoma (RCC) patients. DAB389IL-2 contains the catalytical and membrane translocation domain of diphtheria toxin (11). The binding domain for the diphtheria toxin receptor, however, is deleted and replaced by the human IL-2 gene, which allows for targeting of CD25-expressing cells. The cytotoxic action of DAB389IL-2 occurs as a result of binding to the high-affinity IL-2 receptor, subsequent internalization, and enzymatic inhibition of protein synthesis, ultimately leading to cell death.
Here we show that DAB389IL-2 is capable of selectively eliminating Tregs from human PBMCs in a dose-dependent manner without apparent bystander toxicity to other PBMCs or CD4+ T cells with intermediate- or low-level expression of CD25. Treg depletion resulted in enhanced stimulation of proliferative and cytotoxic T cell responses in vitro but only when DAB389IL-2 was used prior to and omitted during the T cell priming phase. Depletion of Tregs in RCC patients followed by vaccination with tumor RNA-transfected DCs led to improved stimulation of tumor-specific T cells when compared with vaccination alone. To our knowledge, we provide the first clinical evidence that in vivo elimination of Tregs is capable of enhancing the magnitude of vaccine-mediated, tumor-specific T cell responses in humans.
Phenotypic and functional characterization of Tregs. The definition and enumeration of human Tregs is complicated by the fact that CD25 is a marker of T cell activation (12, 13). Human CD4+ T cells expressing CD25 represent a heterogeneous cell population containing not only regulatory but also effector/memory T cells (14). Analysis of PBMCs from healthy donors and RCC patients revealed the presence of CD4+ T cell populations that express increasing levels of CD25 (14). As shown in Figure 1A, 1 major subset of CD4+ T cells, isolated from the PBMCs of an RCC patient, lacked CD25 expression while a second population was characterized by intermediate levels of CD25, and a third, albeit small portion, exhibited high CD25 cell surface expression levels. To further characterize these 3 subsets, CD4+/CD25neg, CD4+/CD25int, and CD4+/CD25high T cells were isolated from the PBMCs of RCC patients by FACS and functionally analyzed in vitro (Figure 1B). CD4+/CD25neg cells expressed cell surface markers characteristic of naive/resting T cells and demonstrated reduced proliferative activity following exposure to tetanus toxoid, renal tumor RNA, benign renal epithelium (RE) RNA, and PBMC RNA-loaded DCs. In contrast, CD4+/CD25int cells produced a strong proliferative response against tetanus toxoid, and a significant, albeit weaker, response, against RCC RNA-encoded antigens. No proliferative response against RE RNA- or PBMC RNA-transfected DCs (latter not shown) was observed. CD4+/CD25high Tregs exhibited profound immunosuppressive activity in vitro, as evidenced by inhibition of allogeneic DC-stimulated mixed-lymphocyte reaction (MLR) cultures. The addition of increasing numbers of CD4+/CD25high cells (1:5 responder cells; 1:1 responder cells) to MLRs led to a dose-dependent inhibition of responder T cell proliferation while CD4+/CD25high T cells did not proliferate significantly upon stimulation with DCs (DC+Treg). Tregs demonstrated strong cell surface expression of glucocorticoid-induced TNF receptor family gene (GITR) as well as intracellular CTL-associated antigen-4 (CTLA-4) and forkhead box P3 transcriptional regulator (FoxP3) (Figure 1C). Stimulation of CD4+/CD25high T cells using anti-CD3/CD28 antibodies resulted in enhanced expression of GITR, CTLA-4, and FoxP3 while CD4+ T cells with negative or intermediate levels of CD25 exhibited significantly lower levels of these markers after nonspecific stimulation.
Characterization of CD4+ T cell subsets. (A) PBMCs from an RCC patient were stained with anti-CD4/CD25 mAbs and analyzed by FACS. (B) CD4+CD25neg, CD4+CD25int (R1), and CD4+CD25high (R2) T cells were sorted. For functional analysis, CD4+CD25neg (left panel) and CD4+CD25int (middle panel) T cells were stimulated with tetanus toxoid–loaded DCs (tetanus), DCs transfected with autologous RCC RNA (RCC), or with DCs transfected with autologous RE RNA (RE) at the indicated stimulator to responder ratios. After 48 hours, cells were pulsed with 3H-thymidine, and incorporation was determined using a liquid scintillation counter. CD4+CD25high cells (right panel) were functionally validated by MLR. Mixture ratios of 1 CD4+CD25high cell per T cell (Treg 1:1) or 1 CD4+CD25high cell per 5 T cells (Treg 1:5) were added to the reaction, and inhibition of cell proliferation was analyzed. As a negative control, proliferation of CD4+CD25high cells was determined in the presence of allogeneic DCs only (DC+Treg). Results are presented as means with SD calculated from triplicate wells. (C) FACS-based detection of GITR, CTLA-4, and FoxP3 by CD4+CD25neg/int and CD4+CD25high T cells subsets with or without stimulation using anti-CD3/CD28 mAb. Gray histograms represent isotypic controls. (D) Left panel: analysis of FoxP3 transcripts was performed by real-time PCR on indicated T cell populations. FoxP3 mRNA copy numbers were normalized to 1 × 107 copies of β-actin mRNA. A representative result from 3 subjects is shown. Right panel: FoxP3 mRNA was amplified from CD4+ T cells isolated from RCC patients (n = 5) and healthy donors (n = 5). Differences in FoxP3 mRNA expression among groups were significant (P = 0.009).
Since CD25 is not an optimal marker to enumerate Tregs in human subjects, as it is upregulated upon T cell activation, the number of FoxP3 transcripts was determined in various T cell populations using quantitative real-time PCR. FoxP3 is the most specific Treg marker currently available while other molecules expressed by Tregs (i.e., CD45RB, CD38, and CD62L) previously failed to demonstrate specificity for detecting Tregs with immunosuppressive activity (15, 16). To determine the specificity of FoxP3 for CD4+/CD25+ Tregs, CD3+, CD8+, CD4+, CD4+/CD25–, and CD4+/CD25+ T cells were isolated from the PBMCs of healthy volunteers and from RCC patients by magnetic bead separation, and copy numbers of FoxP3 mRNA were analyzed. Consistent with other reports (7, 17), high expression of FoxP3 protein (Figure 1C) and message (Figure 1D) were observed in CD4+/CD25+ T cells while CD4+/CD25– and all other T cell subsets exhibited only low or background levels of FoxP3 mRNA (Figure 1D, left panel). When corrected for cell population frequencies, CD4+/CD25+ T cells contributed 64% of FoxP3 transcripts in the PBMC pool of healthy volunteers (84% in RCC patients) while CD4+/CD25– cells accounted for 25% (14% in RCC patients). Less than 10% of FoxP3 transcripts were expressed by CD8+ T cells and CD3– cells (B cells, monocytes/macrophages, and NK cells; data not shown). When assayed by FACS, CD4+/CD25high/FoxP3+ Treg frequencies detected in the peripheral blood of metastatic RCC patients after tumor nephrectomy (n = 10) ranged from 2.5% to 4.6% and were significantly elevated (2.1 ± 1.2-fold increase) when compared with healthy volunteer controls (n = 10). Accordingly, there was a statistically significant increase in the number of FoxP3 transcripts that could be amplified from isolated CD4+ T cells of RCC patients when compared with those of healthy volunteers (P = 0.009) (Figure 1D, right panel).
In summary, we show that CD4+/CD25high T cells isolated from the PBMCs of RCC patients exhibited suppressive activity while CD4+ cells with negative or intermediate CD25 levels represented either naive/resting or memory/effector T cells. Therefore, in clinical settings, it will be important to identify suitable reagents that allow selective elimination of CD25high Tregs while sparing other cells expressing low or intermediate levels of CD25. Consistent with other reports (7, 18), higher Treg frequencies were measured in the peripheral blood of metastatic RCC patients when compared with healthy donor controls.
Selective elimination of Tregs in vitro. Human malignant cells overexpressing CD25 can be inactivated or eliminated using the recombinant IL-2 diphtheria toxin conjugate, denileukin difititox (DAB389IL-2) (19). In order to determine whether DAB389IL-2 could serve as a suitable reagent to achieve Treg depletion under clinically relevant conditions, we analyzed Treg susceptibility to DAB389IL-2 in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide salt (MTT) assays. In these experiments, we chose conditions that resembled the pharmacokinetics of a single intravenous dose of DAB389IL-2 (18 μg/kg) corresponding to 5 nM peak plasma concentrations. In previous clinical trials, this dose level demonstrated optimal clinical efficacy and acceptable toxicity profiles (20). Given a plasma half-life of approximately 60 minutes and a dissociation constant of 1 pM for DAB389IL-2 and the high-affinity IL-2 receptor, DAB389IL-2 plasma levels were projected to reach suboptimal concentrations after 6 hours. Therefore, in the experiments shown in Figure 2A, the viability of isolated CD4+/CD25high T cells was analyzed after a 6-hour exposure to increasing concentrations of DAB389IL-2 (range 0.05–5.0 nM) in vitro over 48 hours. For CD4+/CD25high Tregs, a significant reduction in cell viability was observed 24 hours after exposure to DAB389IL-2. Efficient killing of CD4+/CD25high cells was noted at 0.5 nM concentrations after 48 hours while complete depletion was achieved at a 5 nM concentration. In contrast, exposure of CD4+/CD25neg and CD4+/CD25int cells to DAB389IL-2 did not result in significant cell death except when these cells were exposed to DAB389IL-2 concentrations higher than 10 nM (latter not shown). In another set of experiments, DAB389IL-2 used at a 5 nM concentration resulted in specific killing of Tregs but not of other bystander cells in vitro. As shown in Figure 2A, there were no significant differences in PBMC viability over time when DAB389IL-2 (5 nM) was added to the culture. In contrast, when PBMCs and Tregs were mixed (1:1 ratio), cell viability was reduced by more than 60% after 48 hours, suggesting selective, diphtheria toxin–mediated Treg killing without bystander toxicity. In order to corroborate these findings, 7-amino-actinomycin D (7-AAD) staining was performed on CD4+ T cell populations with increasing densities of CD25 (Figure 2B). Consistent with the experiments shown in Figure 2A, optimal killing of CD4+/CD25high Tregs was achieved using 5-nM DAB389IL-2 concentrations while DAB389IL-2 exposure to other CD4+ T cells with lower or no CD25 expression revealed only background levels of 7-AAD staining.
Enhancement of T cell responses after Treg depletion. (A and B) CD4+CD25high cells were isolated by FACS sorting and incubated for 6 hours in the presence of increasing concentrations of DAB389IL-2 (left panel). In order to determine DAB389IL-2–mediated toxicity, PBMCs and PBMCs admixed with CD4+/CD25high cells at a 1:1 ratio were incubated with or without DAB389IL-2 (5 nM) for 6 hours. In all experiments, cell viability was determined through MTT assays (A) or 7-AAD staining (B). Representative results from 3 evaluable subjects are presented. (C) Treg-depleted PBMCs (PBMC+DAB) or nondepleted PBMCs (PBMC–DAB) from an RCC patient were analyzed in allogeneic MLRs using DCs at a responder to stimulator ratio of 20:1. Cell proliferation was significantly inhibited when isolated CD4+/CD25high cells were added to PBMCs at a 1:1 PBMC/CD4+CD25high cell ratio (DC+Treg). This inhibition was reversible when the added CD4+/CD25high cells were pretreated with DAB389IL-2 (5 nM) for 48 hours (DC+Treg+DAB). Exposure of PBMCs to DAB389IL-2 during the T cell–priming phase (day 2) led to complete inhibition of T cell proliferation (DC+DAB). (D) DCs transfected with mRNA encoding hTERT or MART-1 were used to stimulate CTL from Treg-depleted (filled symbols) or nondepleted (open symbols) human PBMCs.In addition, DCs loaded with MART-1–derived peptide 26-35 ELAGIGILTV (MART-1 pep) were used as stimulators. Following 2 stimulation cycles, CTLs were analyzed for their capacity to lyse their cognate (squares) or control targets (circles). As control targets, DCs loaded with GFP mRNA (mock transfected) or irrelevant peptide were used.
Next, the impact of DAB389IL-2 on freshly activated lymphocytes was analyzed after stimulation with allogeneic DCs in MLR cultures. DAB389IL-2–mediated Treg depletion prior to initiation of MLR culture resulted in a 2-fold increase in proliferation of responder cells (PBMC±DAB). Conversely, the addition of isolated Tregs (DC+Treg) resulted in an approximately 80% reduction in T cell proliferation (1:1 Treg/responder ratio) (Figure 2C). Preincubation of Tregs with 5-nM DAB389IL-2 (DC+Treg+DAB) significantly abrogated their inhibitory effect when added to MLR; however, responder cells did not proliferate as vigorously as in the absence of Tregs, suggesting potential contact inhibition by Tregs, as reported previously (21). Importantly, the addition of DAB389IL-2 two days after initiation of MLR (DC+DAB) completely abrogated the proliferation of responder cells, suggesting that DAB389IL-2 eliminated not only Tregs but also freshly activated naive T cells that acquired CD25 expression.
These experiments suggest that DAB389IL-2 is a suitable reagent for selectively eliminating Tregs in vitro without affecting other lymphocytes, including naive and memory T cells with negative or intermediate expression levels of CD25, respectively. Our data further indicate that in a vaccination setting, DAB389IL-2 should only be applied prior to immunization but not during the vaccination phase, since activated effector T cells appear susceptible to DAB389IL-2–mediated toxicity.
Enhancement of T cell responses after Treg depletion in vitro. To provide evidence that DAB389IL-2–mediated Treg depletion is capable of augmenting antigen-specific T cell responses in vitro, CTLs were stimulated from PBMCs that were pretreated with or without DAB389IL-2 (5 nM) (Figure 2D). PBMCs were stimulated twice with autologous DCs transfected with human telomerase reverse transcriptase (hTERT) and melanocyte antigen recognized by T cells 1 (MART-1) mRNA. In addition, DCs pulsed with an HLA-A0201–restricted MART-1 peptide were used as stimulators. RNA-transfected DCs were not only used as stimulators but also served as specific or control targets, as described previously (2, 22–24). The ability of the stimulated, antigen-specific CTLs to recognize their cognate but not control target cells was analyzed in standard cytotoxicity assays. As shown in Figure 2D, CTLs stimulated from Treg-depleted PBMCs exhibited significantly higher lytic activity against antigens encoded by hTERT or MART-1 mRNA than CTLs stimulated from nondepleted PBMCs.In contrast, only modest, statistically insignificant improvement of antigen-specific killing was observed when MART-1 peptide–pulsed DCs were used as stimulators.
These data suggest that depletion of Tregs from human PBMCs enhances the stimulation of antigen-specific CTLs in vitro. It appears that among other factors, the impact of this strategy is influenced by the strength of the antigenic signal (25), as evidenced by the fact that T cell responses against mRNA-encoded self antigens was significantly enhanced. In contrast, the Treg-depletion strategy was less effective in improving CTL responses when DCs presenting high densities of peptide-MHC complexes (peptide pulsing) were used for stimulation.
Depletion of Tregs in vivo. Having shown that Treg elimination is capable of enhancing T cell responses in vitro, a clinical study was initiated to test the Treg-depletion concept in a human vaccination setting. A total of 11 patients, including 10 with metastatic RCC and 1 with disseminated ovarian carcinoma (OVA), were treated in a study approved by the Duke University Institutional Review Board and the FDA. Seven subjects received a single intravenous dose of DAB389IL-2 (18 μg/kg) 4 days prior to vaccination with tumor RNA-transfected DCs while a second cohort of 4 subjects was treated with the vaccine alone (Table 1). Outside the study, PBMCs were obtained from 1 additional RCC subject who received a single dose of DAB389IL-2 (18 μg/kg) but no vaccine. Consistent with reports of others, DAB389IL-2–related toxicities included grade I constitutional symptoms such as low-grade fever and malaise (n = 4), grade I elevation of serum rheumatoid factor (n = 1), and transient grade II serum alanine aminotransferase (ALT) elevations (n = 1) (11). RNA-transfected DC injections were well tolerated without any major clinical toxicities or serologic/immunologic evidence of autoimmunity (2, 26).
Patient characteristics and treatment assignments
In order to quantify the presence of CD4+/CD25neg, CD4+/CD25int, and CD4+/CD25high T cells in PBMC samples collected prior to and after DAB389IL-2 treatment, flow cytometry was performed on patient-derived PBMC samples (Figure 3A). Gates were set on CD4+/CD25high T cells that constitutively expressed FoxP3 protein, as shown in Figure 1C. In all 7 subjects analyzed, DAB389IL-2 administration resulted in significant reduction (range 26% to 76%) of CD4+/CD25high Tregs 4 days following intravenous infusion. Additional evidence that Tregs were depleted and that CD25neg/int T cell subsets were unaffected was provided by the observation that the number of total CD25pos cells measured in each patient after DAB389IL-2 administration decreased correspondingly with the number of depleted CD4+/CD25high Tregs (data not shown). Moreover, as shown in 4 RCC patients from whom sufficient cells were available for analysis (02-RCC-DAB, 03-RCC-DAB, 05-RCC-DAB, and 06-RCC-DAB), DAB389IL-2 treatment resulted in a significant reduction of FoxP3 transcripts after DAB389IL-2 treatment, as determined by quantitative real-time PCR (Figure 3B). In these subjects, FoxP3 mRNA copy numbers (normalized to β-actin transcripts) were reduced by approximately 30% to 80% within purified blood CD4+ cells 4 days after DAB389IL-2 administration. Finally, CD4+/CD25+ T cells isolated prior to but not 4 days after DAB389IL-2 treatment consistently inhibited anti-CD3/CD28-mediated activation of CD4+/CD25– indicator T cells in all RCC subjects analyzed (Figure 3C), suggesting abrogation of Treg-mediated immunosuppressive activity in vivo. Notably, DAB389IL-2–mediated Treg elimination was transient, since approximately 75% of Tregs were restored within 2 months in the patients' peripheral T cell pool (Figure 4A).
Depletion of Tregs in study subjects. (A) CD4+ T cells isolated from all DAB389IL-2–treated study patients were analyzed by flow cytometry for expression of CD25 prior to and 4 days after intravenous administration; percentages of CD4+/CD25high T cells are shown. (B) Reduction of FoxP3 mRNA copy numbers before and after DAB389IL-2 treatment was determined by CD4+ T cells derived from 4 study subjects, as described in the legend to Figure 1D. The average FoxP3 mRNA copy number averaged from 5 healthy volunteers was used as control (C). (C) Functional analysis of Tregs isolated from study subjects prior to and after DAB386IL-2 administration. CD4+/CD25+ and CD4+/CD25– T cell subsets were isolated from PBMC samples by magnetic bead separation, and Treg-mediated inhibition of activated CD4+/CD25– indicator T cells was measured according to a protocol described previously (28).
Specificity of Treg depletion. (A) Calculated CD4+/CD25high Treg frequencies in 2 study subjects (01-RCC-DAB; 03-RCC-DAB) prior to, 4 days after, and 2 weeks after final vaccination (study week 8; d 56). (B) IFN-γ ELISPOT were performed on sorted CD4+CD25neg, CD4+CD25int, and CD4+CD25high T cell subsets using tetanus toxoid and CMV lysate–pulsed DCs as stimulators. (C–E) In separate experiments, IFN-γ ELISPOT and antigen-specific proliferation analyses were performed on T cells isolated prior to vaccination and 4 and 28 days after DAB389IL-2 treatment (results from patient RCC-01-DAB). For ELISPOT assays, 1 × 105 purified CD4+ T cells (C) or 1 × 105 purified CD8+ T cells (D) were stimulated with 1 × 104 DCs that were transfected with fluM1 mRNA, autologous PBMC RNA, CMV lysate (20 μg/ml), or tetanus toxoid (0.5 μg/ml). After 18 hours, visible spots were enumerated using an automated ELISPOT reader. The same stimulators and RCC RNA-transfected DCs were used in proliferation assays (E). For proliferation assays, isolated CD3+ T cells were used as responders. Assays were performed at a stimulator/responder ratio of 1:10. After 4 days, cells were pulsed with 1 μCi of 3H-thymidine, and incorporated radioactivity was determined after 16 hours by liquid scintillation counting.
In 5 of 6 evaluable DAB389IL-2–treated subjects, minor reductions (averaging 10%) in absolute neutrophil counts were observed while 1 subject exhibited a decrease of 20%. Accordingly, among these 5 with only minor absolute neutrophil count reductions, there were no significant changes in the relative number of CD3+, CD4+, and CD8+ T cells, B cells, monocytes/macrophages (CD14), and NK cells after treatment. In order to address the concern that DAB389IL-2–mediated Treg depletion may induce toxicity within the memory T cell pool (CD4+/CD25int), a series of experiments was performed analyzing CD4+/CD25int T cell function prior to and 4 days after DAB389IL-2 administration. First, IFN-γ ELISPOT analyses were performed on sorted CD4+/CD25neg, CD4+/CD25int, and CD4+/CD25high T cell subsets using tetanus toxoid or CMV lysate–pulsed DCs as stimulators. As shown in Figure 4B, only CD4+/CD25int memory T cells were capable of stimulating T cell responses against tetanus or CMV antigens while naive (CD4+/CD25neg) and CD4+/CD25high T cells failed to stimulate T cell responses of a significant magnitude. In a second set of experiments, the frequency of IFN-γ secreting T cells was analyzed using CD4+ (Figure 4C) and CD8+ responder T cells isolated from human PBMCs (Figure 4D) prior to and 4 days after DAB389IL-2 administration (DAB) and 2 weeks after 3 vaccination cycles. Over the entire treatment cycle, no differences were observed in the absolute number of T cells with specificity against PBMC RNA-transfected DCs, influenza virus matrix protein 1 (fluM1) mRNA-transfected DCs, CMV lysate–loaded DCs, or tetanus toxoid–loaded DCs. Furthermore, antigen-specific proliferation assays (Figure 4E) revealed strong reactivities against renal tumor antigens and unchanged reactivities against the prototype recall antigens fluM1/tetanus toxoid.
Cumulatively, the experiments shown in Figures 3 and 4 demonstrate that administration of a single dose of DAB389IL-2 resulted in significantly reduced numbers of Tregs in the peripheral blood of RCC patients (Figure 3, A and B) and in significant abrogation of Treg-mediated suppressive activity (Figure 3C). Our data further suggest that DAB389IL-2–mediated toxicities against other hematopoietic cells expressing CD25 are unlikely and that lymphopenia-induced T cell proliferation (27) may not represent a significant issue in a vaccination setting.
In vivo stimulation of tumor-specific T cell responses. In order to determine whether DAB389IL-2–mediated Treg depletion is capable of augmenting vaccine-induced CD8+ and CD4+ T cell responses in cancer patients, IFN-γ ELISPOT analyses were performed to determine the frequencies of vaccine-induced, tumor-specific T cells from PBMC samples collected before and 2 weeks after the third vaccination. CD8+ and CD4+ T cells were isolated from pre- and post-vaccination PBMCs and cultured overnight with tumor RNA-transfected DC targets. As controls, autologous PBMC RNA and/or autologous benign RE-derived RNA-transfected DCs were used for short-term antigenic stimulation. As shown in Figure 5, A and B, only background levels of RE- or PBMC-specific T cells were observed prior to or after vaccination. Also, tumor-specific T cell responses did not significantly increase in independently evaluated subjects after treatment with DAB389IL-2 alone over a period of 28 days (data not shown). In contrast, all but 1 (08-RCC) patient immunized with tumor RNA-transfected DCs exhibited significant increases in tumor-specific CD8+ and CD4+ T cell frequencies after vaccination (Figure 5B and Table 1). A 2.7-fold median increase (range 0 to 5.1) of tumor-specific CD8+ T cells and a 2.0-fold median increase in tumor-specific CD4+ T cells (range 1.0 to 4.5) was observed in the subjects receiving vaccination alone (Table 1). Although there was significant patient-to-patient variability in the magnitude of T cell responses measured in each patient, vaccination after Treg depletion stimulated significantly higher numbers of tumor-specific CD8+ T cells in RCC patients receiving DAB389IL-2 plus RCC RNA-transfected DCs, when compared with RCC patients receiving vaccination alone (P = 0.011). Moreover, there was a trend toward improved CD4+ T cell responses (P = 0.055) in RCC patients treated with combined therapy (Figure 5B). A 7.9-fold median increase in the number of tumor-specific CD8+ T cells (range 5.4 to 16.2) and a 7.2-fold median increase in CD4+ T cells (range 2.2 to 31.0) was detected in the 6 RCC patients receiving DAB389IL-2 plus vaccination with RCC RNA-transfected DCs. The absolute CD8+ T cell frequencies achieved with combined therapy were remarkably high with up to 0.90% of CD8+ T cells exhibiting tumor specificity after 3 vaccinations. In order to evaluate the temporal evolution of the T cell response stimulated by vaccination with or without DAB389IL-2 therapy, longitudinal monitoring of vaccine-induced CD8+ T cell responses was performed using IFN-γ ELISPOT in 2 subjects from whom sufficient numbers of cells were available for analysis (11-RCC and 05-RCC-DAB). DAB389IL-2–mediated Treg depletion followed by vaccination resulted in a significantly enhanced and also prolonged CD8+ T cell response when compared with the subject receiving vaccination alone. As demonstrated recently (26), the vaccine-induced and tumor-specific CTL response surged over the entire treatment course and peaked approximately 2 weeks after the third and final dose (Figure 5C).
In vivo induction of tumor-specific T cell responses. (A) CD8+ and CD4+ T cells were isolated from prevaccination (white bars) and postvaccination (black bars) PBMCs of patients treated with DAB389IL-2 and RCC RNA-transfected DCs. For vaccination, 3 doses of 1 × 107 cells injected intradermally every other week were administered. Isolated CD8+ and CD4+ T cells were stimulated for 18 hours with tumor RNA-transfected DCs, RE, or PBMC RNA-transfected DCs (controls). Visible spots were enumerated, and antigen-specific T cell frequencies were expressed as the number of spots forming cells per 1 × 105 T cells. (B) Left panels: stimulation of tumor-specific CD8+ in 4 subjects treated with tumor RNA-transfected DCs alone. Right panels: summary of CD8+ and CD4+ T cell responses from 4 subjects receiving immunization alone (–DAB) or from 7 patients treated with combined therapy (+DAB). Bars indicate the median value of all subjects analyzed. Filled triangles represent T cell frequencies of individual patients. (C) Temporal evolution of tumor-specific CD8+ T cells after vaccination. IFN-γ ELISPOT analyses on sorted CD8+ T cells were performed as described in A. Frequencies of tumor-specific T cells prior to, during, and after immunization are presented for 2 patients who received 3 vaccinations with tumor mRNA-transfected DCs alone (11-RCC) or were treated with DAB389IL-2 (01-RCC-DAB) followed by vaccination.
In additional experiments, the cytokine secretion profiles by vaccine-induced and tumor-specific CD4+ T cells were analyzed after combined DAB389IL-2 and active immunotherapy (Figure 6, A and B). PBMCs were collected from 3 study subjects at baseline and 2 weeks after the final vaccination, and CD4+ T cells were isolated by magnetic bead sorting. CD4+ T cells were restimulated for 18 hours with renal tumor RNA-transfected DCs and analyzed for IFN-γ and IL-4 secretion using ELISPOT analysis. As control targets, GFP mRNA-, RE RNA-transfected DCs, and staphylococcal enterotoxin B (SEB) were used. As shown in Figure 6A, vaccination after Treg depletion resulted in significant stimulation of IFN-γ but not IL-4–secreting renal tumor–specific CD4+ T cells while no stimulation against GFP or RE-expressing targets was noted. In addition, human Th1/Th2 flow cytometry–based bead arrays (Figure 6B) confirmed secretion of the Th-1 type cytokines IL-2 (increase from 680 to 1270 pg/ml), IFN-γ (160 to 270 pg/ml), and TNF-α (34 to 59 pg/ml) but not Th-2 type cytokines (IL-10 and IL-4) by vaccine-induced CD4+ T cells 18 hours after stimulation with RCC RNA-transfected DCs. This effect was not seen when RE RNA-transfected DCs were used as stimulators. These data further suggest that the vaccine-induced tumor-specific T cell response is directed against tumor-associated antigens but not gene products expressed by normal RE.
In vivo induction and cytokine profile of RCC-specific CD4+ T cell responses. (A) CD4+ T cells were isolated from pre- (white bars) and post-vaccination (black bars) PBMC samples of 3 study subjects (representative data from patient RCC-01-DAB are shown) who received DAB389IL-2 (18 μg/kg) followed by vaccination with RCC RNA-transfected DCs (2 cycles of 1 × 107 cells per treatment). Cells were stimulated for 18 hours with autologous PBMC RNA-, RE RNA-, or RCC RNA-transfected DCs. IFN-γ (left panel) or IL-4–expressing T cells (right panel) were enumerated using an automated ELISPOT reader, and antigen-specific T cell frequencies were expressed as the number of spot-forming cells per 1 × 105 CD4+ T cells. Staphylococcal enterotoxin B (SEB) at a concentration of 10 μg/ml was used as a positive control in the IL-4 ELISPOT assays (right panel). (B) The cytokine expression profile of CD4+ T cells prior to (gray) and after (white) vaccination was measured after overnight (18 hours) stimulation with RCC (DC+RCC) or RE RNA-transfected DCs (DC+RE) using human Th1/Th2 cytometric bead arrays. Culture supernatants were used to determine expression of the Th-1 cytokines IFN-γ, TNF-α, and IL-2 as well as the Th-2 type cytokines IL-4 and IL-10.
In conclusion, we show that Treg depletion using the diphtheria fusion protein DAB389IL-2 is capable of enhancing a vaccine-induced T cell response in patients with advanced RCC. Although only a limited number of patients were studied in this clinical trial, an up to 16-fold increase in tumor-specific CTL frequencies could be measured in subjects receiving combined treatment when compared with individuals receiving vaccination alone. The vaccine-induced T cell frequencies achieved without Treg depletion were similar to those observed in a prior study in which immature tumor RNA-transfected DCs were used for vaccination (1).
The objective of this study was to enhance the immunostimulatory efficacy of RNA-transfected DC vaccines by selectively eliminating CD4+/CD25high Tregs in metastatic RCC patients. For Treg depletion, we used the recombinant fusion protein denileukin diftitox (DAB389IL-2) (20) in a human vaccination setting. We show that human CD4+CD25high Tregs can be eliminated using a single dose of DAB389IL-2 without apparent bystander toxicity and without having an impact on the function of other cells expressing CD25. However, DAB389IL-2 also abrogated DC-mediated activation of T cells in vitro, suggesting that the applicability of this reagent should be restricted to a prevaccination setting (Figure 2C). These preclinical results provided important information regarding the design of our clinical study, in which DAB389IL-2 was administered to RCC patients 4 days prior to DC-based vaccination. This time interval was chosen since, unlike antibodies, DAB389IL-2 is characterized by a short duration of action, with a half-life of approximately 60 minutes, thereby minimizing the possibility of interfering with the ongoing vaccine-induced T cell response.
In this study, DAB389IL-2 profoundly reduced the number of Tregs present in the peripheral blood of RCC patients, reduced levels of peripheral blood–derived FoxP3 transcripts, and abrogated Treg-mediated immunosuppressive activity in vivo. Moreover, significantly higher frequencies of tumor-specific CD8+ T cells could be measured in patients treated with combined DAB389IL-2 and vaccination when compared with subjects receiving the vaccine alone. Also, there was a trend toward an improved CD4+ T cell response after combined therapy. Cumulatively, these data provide several independent lines of evidence that Tregs were depleted in the peripheral blood of RCC patients by using a single dose of the fusion protein DAB389IL-2.
The T cell frequencies achieved after Treg depletion and 3 vaccination cycles were remarkably high with up to 0.90% of all CD8+ T cells demonstrating tumor specificity. No clear correlation between the efficacy of Treg depletion and the magnitude of the vaccine-induced T cell response was observed. Also, serum diphtheria titers did not appear to have an impact on vaccine efficacy (11). The present study further suggests that the degree of Treg depletion achieved using a single dose of 18 μg/kg may be quite variable and that Treg depletion was transient, with most cells returning after 2 months. However, it should be pointed out that the exact enumeration of Tregs in a vaccination setting is complicated by the fact that CD4+ T cells with negative or intermediate expression levels of CD25 may upregulate expression of CD25 in response to antigenic stimulation, thereby biasing results towards increased detection of Tregs. Our preclinical studies also suggest that the Treg strategy may be geared toward the improvement of T cell responses against relatively weak self antigens such as hTERT or MART-1 antigens but not against immunodominant peptide-derived antigens (Figure 2D). Accordingly, other studies have recently shown that Tregs effectively suppress the physiologic activation of autoreactive T cells associated with low strength of the antigenic signal while T cells activated with high antigenic signal strength were refractory to this mechanism of suppression (25). Although in this study, the concept of Treg elimination has been employed in context with RNA-transfected DC-based vaccination, this strategy could potentially be applied to many immune-based approaches of active and passive immunotherapy as well as to classical adjuvants. The information gained from this study will serve as a baseline for further clinical investigation to better define the full potential of this strategy in ultimately achieving antitumor immunity with clinical impact. For such studies it will be critical to collect precise information on Treg depletion and vaccine-induced T cell response and, ultimately, address the clinical efficacy of such strategy in cancer patients.
Clinical trial design and patient eligibility. Treatment of patients was performed following written informed consent as part of a protocol approved by an Institutional Review Board and the FDA. Patients with histologically confirmed metastatic RCC were eligible for this study. One patient with disseminated OVA was included and treated on a compassionate basis. All patients were required to have adequate hepatic, renal, and neurological function, a life expectancy of more than 6 months, and a Karnofsky performance status of greater than or equal to 70%. Patients had to have recovered from all toxicities related to any prior therapy and not received any chemotherapy, radiation therapy, or immunotherapy for at least 6 weeks prior to study entry. Excluded from the study were patients with CNS metastases, with a history of autoimmune disease, and with serious intercurrent chronic or acute illnesses. Patients on immunosuppressive agents were also excluded. Eligible subjects were randomized with equal probability to receive either a single dose of DAB389IL-2 (18 μg/kg) followed by vaccination with tumor RNA-transfected DCs or vaccination alone. All subjects received 3 intradermal injections of tumor RNA-transfected DCs. The injections were given intradermally at biweekly intervals and consisted of 1 × 107 cells suspended in 200 μl 0.9% sodium chloride at each vaccination. Following treatment, subjects were evaluated for clinical toxicity and immunological and clinical responses. Due to regulatory restrictions and, in some subjects, limited access to tumor tissue, no tumor biopsies were performed.
DAB389IL-2 and vaccine preparation. DAB389IL-2 (ONTAK; Ligand Pharmaceuticals) was provided as a frozen, sterile solution formulated in citrate buffer in 2 ml single-use vials at a concentration of 150 μg/ml. After thawing, DAB389IL-2 was diluted with sterile normal saline to a final concentration of 15 μg/ml and delivered by intravenous infusion over a 30-minute period. Patients were permitted to receive acetaminophen (600 mg) and antihistamines 30 to 60 minutes prior to infusion. For DC culture, a concentrated leukocyte fraction was harvested by leukapheresis. PBMCs were isolated from the leukapheresis product by density gradient centrifugation (Histopaque; Sigma-Aldrich). The semiadherent cell fraction was used for DC culture in serum-free X-VIVO 15 medium (Cambrex Corp.) supplemented with recombinant human IL-4 (500 U/ml) (R&D Systems) and recombinant human GM-CSF (rhGM-CSF) (800 U/ml) (Immunex Corp.). After 7 days, immature DCs were harvested and transfected with total RNA extracted from tumor tissues histologically classified as clear cell carcinoma. Control RNA used for immunological monitoring studies was isolated from autologous benign renal tissues (RE) or from PBMCs. Transfection of immature DCs was carried out by electroporation. DCs were washed in PBS and resuspended at a concentration of 4 × 107 cells/ml in ViaSpan (Barr Laboratories). Cells were then coincubated for 5 minutes with 5 μg RNA per 1 × 106 cells and electroporated in 0.4 cm cuvettes via exponential decay delivery at 300 V and 150 μF (Gene Pulser II; Bio-Rad). After electroporation, cells were resuspended in X-VIVO 15 medium and matured for 20 hours in the presence of 10 ng/ml TNF-α, 10 ng/ml IL-1β, 150 ng/ml IL-6 (R&D Systems), and 1 μg/ml prostaglandin E2 (PGE2) (Cayman Chemical Co.). Prior to administration, cells were characterized to ensure that they met the typical phenotype of fully mature DCs: Linneg, HLA class I and IIhigh, CD86high, and CD83high.
Evaluation of immune status. IFN-γ and IL-4 ELISPOT analyses were performed using PBMCs obtained prior to, during, and after vaccination. PBMCs were cultured overnight in complete RPMI 1640 medium. CD4+ and CD8+ T cells were isolated from PBMCs by negative depletion (Miltenyi Biotec). After blocking, 1 × 105 T cells and 1 × 104 RNA-transfected DCs were added to each well of 96-well nitrocellulose plates (Multiscreen-IP; Millipore) precoated with 2 μg/ml IFN-γ capture antibody (Pierce Biotechonology Inc.) or with IL-4 capture antibody (BD Biosciences — Pharmingen). Plates were incubated for 20 hours at 37°C, and biotinylated IFN-γ detection antibody (Pierce Biotechonology Inc.) or biotinylated IL-4 antibody (BD Biosciences — Pharmingen) was added to each well. Cells were then incubated for an additional 2 hours at room temperature, then with streptavidin-alkaline phosphatase (1 μg/ml; Sigma-Aldrich); plates were developed with substrate (KPL). After washing, spots were counted using an automated ELISPOT reader (Zeiss). CTL assays were performed by coculturing RNA-transfected DCs with autologous PBMCs. Cells were restimulated once, and IL-2 (20 units/ml) was added after 5 days and every other day thereafter. After 12 days of culture, effector cells were harvested. Target cells were labeled with 100 μCi of Na2[51CrO4] (PerkinElmer) in 200 μl of complete RPMI 1640 for 1 hour at 37°C in 5% CO2, and 51Cr-labeled target cells were incubated in complete RPMI 1640 medium with effector cells for 5 hours at 37°C. Then 50 μl of supernatant was harvested, and release of 51Cr was measured with a scintillation counter. For proliferation assays, purified CD3+ T cells were seeded into round-bottomed microplates in the presence of mRNA-transfected DCs. T cells alone were used as the background control. After 4 days, 1 μCi of [methyl-3H] thymidine (PerkinElmer) was added to each well for an additional 16 hours. Incorporation of thymidine was determined using a liquid scintillation counter. Cytotoxicity of DAB389IL-2 was determined in MTT assays. After 6 hours incubation with varying concentrations of DAB389IL-2, cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After 48 hours of incubation, 20 μl MTT from a 5 mg/ml stock was added. After 4 hours, the formazan crystals were solubilized by adding 100 μl isopropanol/0.1 M hydrochloric acid. The absorbance of the formazan product was measured on an ELISA plate reader at 570 nm. Cytokine secretion by vaccine-induced CD4+ T cells was measured using the human Th-1/Th-2 cytokine kit (Cytokine Bead Array; BD Biosciences — Pharmingen) according to the manufacturer's instructions. Isolated CD4+ T cells were restimulated overnight with RNA-transfected DCs at a ratio of 10:1.
FACS analysis. Four-color FACS analyses were performed using the following antibodies: anti-CD4 FITC, anti-CD45RO, anti-CD45RA (CALTAG Laboratories), anti-CD25 PE (BD Biosciences — Pharmingen), and anti-GITR (R&D Systems) as well as isotypic controls (CALTAG Laboratories). Sorting of CD4+/CD25neg, CD4+/CD25int and CD4+/CD25high T cells was performed using a BD FACSAria cell sorter after antibody labeling. For intracellular detection of FoxP3, cells were permeabilized with 30 μg/ml digitonin for 45 minutes at 4°C. Subsequently, cells were stained with anti-FoxP3 antibody (Abcam), and R-phycoerythrin anti-goat IgG in the presence of 10 μg/ml digitonin for 30 minutes at 4°C. Following staining, cells were fixed and analyzed by FACS. For intracellular CTLA-4 detection, T cells were permeabilized, fixed, and stained with biotinylated anti-CD152 (BD Biosciences — Pharmingen) followed by APC-strepavidin (BD Biosciences — Pharmingen). A total of 1 × 106 cells were suspended in staining buffer (PBS with 1% FCS, 2 mM EDTA, and 0.1% sodium azide) and incubated for 20 minutes at 4°C with the antibody.
Treg functional evaluation. The suppressive activity of Tregs isolated from PBMCs of study subjects prior to and 4 days after DAB386IL-2 administration was analyzed, as described previously (28). CD4+/CD25+ T cells were isolated from the PBMCs of study subjects using magnetic bead separation techniques. Cells were washed with PBS, resuspended in complete RPMI 1640 medium, and placed into 96-well round bottom plates precoated with anti-CD3/CD28 antibodies (0.4 μg/well) (CALTAG Laboratories). CD4+/CD25– cells were plated at 2.0 × 104/well alone or in combination with CD4+/CD25+ cells in triplicate wells at a ratio of 1:2 (CD4+/CD25–:CD4+CD25+). On day 5, 1 μCi of 3H thymidine was added for the final 16 hours of the cultures. Cells were then harvested on glass fiber filters and assessed for uptake of radiolabeled thymidine.
Detection of FoxP3 transcripts. Details of real-time PCR–based quantification of β-actin transcripts were previously provided by Heiser et al. (2). FoxP3 mRNA transcripts were quantified using the Hs00203958_ml TaqMan gene expression assay (Applied Biosystems) according to the protocol provided by the manufacturer. A plasmid containing the full-length FoxP3 insert was used to generate standard curves.
Statistics. T cell analysis before and after treatment was performed by IFN-γ ELISPOT on all patients who completed immunotherapy. Increases of antigen-specific CD4+ and CD8+ T cells after vaccination were compared using the Wilcoxon matched-pairs signed rank test, analyzing the null hypothesis that the rates of change in T cell response were equivalent prior to and after therapy. A 2-sided P value of less than 0.05 was considered statistically significant.
This work was supported in part by NIH grants R21-CA098446-02 (National Cancer Institute) and M01-RR-30 (National Center for Research Resources, General Clinical Research Centers Program). We would like to thank Ligand Pharmaceuticals for provision of the fusion protein for the clinical study.
Nonstandard abbreviations used: 7-AAD, 7-amino-actinomycin D; CTLA-4, CTL-associated antigen-4; DAB389IL-2, recombinant IL-2 diphtheria toxin conjugate; fluM1, influenza virus matrix protein 1; FoxP3, forkhead box P3 transcriptional regulator; GITR, glucocorticoid-induced TNF receptor family gene; hTERT, human telomerase reverse transcriptase; MART-1, melanocyte antigen recognized by T cells 1; MLR, mixed-lymphocyte reaction; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide salt; OVA, ovarian carcinoma; RCC, renal cell carcinoma; RE, renal epithelium.
Conflict of interest: The authors have declared that no conflict of interest exists.
Copyright © 2015
American Society for Clinical Investigation


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Part Time Jobs in Placentia, California ( CA )
If you're looking for part time jobs, student jobs or jobs for teens in Placentia, California, you've come to the right place! Chances are that GrooveJob.com has the right job for you and wants to be your only source for finding part time jobs in Placentia, California.
We've got plenty of part time and summer jobs in Placentia, California. Check out the list of employers below who are accepting applications for part time jobs in your town. Remember to check back with Groovejob.com on a regular basis. We add new part time job listings to our database every week, so you'll find new opportunities every time you check back with us.
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Part time nanny needed for 14 months old toddler
…I'm looking for someone who's caring, reliable, energetic, and would love to spend time with my toddler. The right candidate Should...
Anaheim, CA
Consultative Sales Associate Trainee - Brea, CA ( Part Time )
…The Consultative Sales Associate is responsible for enhancing the experience of customers and driving profitable sales by providing...
Brea, CA
Cook - Part - Time
…written and verbal skills for effective communication, be competent in organizational, time management skills and demonstrate good...
Fullerton, CA
Seeking Part - Time Weekly Tutoring Help For 2 Boys Elementary School
…Ideal situation would be meeting at a library after school at 2:30 pm on Tuesdays and Thursdays, duration should be about 1-2 hours at...
Yorba Linda, CA
UberX Driver Partner ( Part Time Independent Contractor)
…drive and earn when you have time , whether you're a student, working somewhere part time , or looking for that perfect entry-level...
Santa Ana, CA
Inside Sales Rep ($15 - $17 hourly )
Job Snapshot Hiring Company: Kimco Staffing Employee Type: Full- time / Part - time Location: Santa Ana, CA Job Type: Skilled Industrial...
Santa Ana, CA
Looking for Part Time Work? Earn up to $20/Hr in fares Driving Your Ow
…Driving with Uber is for independent contractors only. Uber is looking for partners who know the local area and can get our community of...
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Seeking Part - Time Babysitter In
…A car is not provided - please have your own. We need a babysitter to take care of my children in Anaheim. A car is not provided -...
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Hourly Shift Coordinator
…As a leader, you are expected to help guide, motivate, and mentor others in the restaurant, while also developing your management skills...
Orange, CA
Mail / Package Clerk ( part time or full time )
DHL is seeking seeking part time and full time Mail / Package Clerk positions to support our distribution and logistics operations....
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Congratulations to DramaQueen for being our first Quad Loser!!!!
Congratulations to DramaQueen for being our first Quad Loser!!!!
Delta Mu Chi Alpha ΔΜΧΑ
Wow that is impressive. Congrats DQ!
Holy Loserdom!! Now that's what I call a loser! Congrats DQ!!
Founding member of the B.A. I LOVE US!!
FratSor Sister-Delta Mu Chi Alpha ΔΜΧΑ"If all my friends were to jump off a bridge, I wouldn't jump with them, I'd be at the bottom to catch them"
I bring the magic!!!
Originally Posted by Reverend DMother
Somebody take me out side and shoot me so I will shut up already!
WOW!!!!!!! All Hail DQ!!!!
I give myself very good advice, but I very seldom follow it.That explains the trouble that I'm always in...
Unreal. We are so proud of our little loserette!!!
MiceChat 101: Be NICE! If you don't play well with others, you are in the wrong sandbox.
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You may want to spread the 40 DMCA free shots over the course of your next 1000 posts. Don't want you getting sick or anything.
Anything listed on the park maps as an attraction is fair game for inclusion in the Rally.
I cannot confirm nor deny the inclusion of any attraction in the Rally.
May the Gumballs Be with You...Always.
ROFL. Congrats.... loser
Unusually and exceedingly peculiar and altogether quite impossible to describe...
now thats impressive.
4000 huh?
Way to go DQ
You clicked Submit 4000+ times!!! Most impressive!!!
Does this mean that DQ is the most submissive person on the board?Originally Posted by stinkerbell
Sorry, that is wrong isn't it?
Carry on.
Ooo, heaven is a place on earth.
Submissive? Only sometimes
Congrats DQ. There is something now for me to aspire to!
Fratsor Sister - Delta Mu Chi Alpha
Who in the hell is Dramaqueen? I don't think I've ever seen her on the boards.
I find it hardIt's hard to findOh well, whatever, nevermind


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I, I'm just not seeing the (incredibly obvious) metaphor.
I, I'm just not seeing the (incredibly obvious) metaphor.
You see guys, the Risky Business dance scene only works if you take your pants off first!
Reminds me of the last time I was put under for dental surgery... wait I mean best wedding ever!!
Hmmm, ill-fitting suit, juvenile ass-jokes... In what state do they marry 14-year olds?
Out of all our 200+ wedding photos, this one is everyone's favorite. For obvious reasons, but c'mon, the bride isn't even in it!
Well obviously you're just about to summon her from the dark portal or something, right?
Pantless groomsmen will always have a special place here on Wedinator.
The guy second from the left just lost a bet and now has to pounce on the groom.


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Kia Soul Questions Any type
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How Can I See An Old Listing?
http://www.cargurus.com/Cars/m... viewSavedListings.action This is a 2012 green kia soul listed by delaware cadillac a little over a week ago.
Where Do I Put The Ac Gas On A Kia Soul
All Sensor Lights Staying On While I Drive, What Does This Mean?
My traction, e-brake, low tire pressure lights are all staying as I drive. Not sure what this means, this isn't the first time its happened.
Something Fell Into The Defrost Vent Of My Kia Soul.
Now the piece that closes for floor air versus window air doesn't close. How can I get it out without paying tons of money.
Do You Have To Replace Rotors When You Replace Brake Pads?
I was told by my mechanic than rotors HAVE to be replaced when replacing brake pads. I did not think this was true but he said the car would shake if I did not.
What Happened To My "welcome Home Sound?"
I somehow no longer have a "doorbell" audible sound when I arrive home. It was a feature I loved & no one at dealership has a clue ... it is the whole shebang package I have!
I Have A 2012 Kia Soul Automatic And Its Having A Hard Time Starting And Sw...
My Kia Soul Tempest When I Put Fuel In The Mpg Re Sets To Zero
I am trying to find out if anyone else with an 11 plate soul tempest has this problem of the mpg resets to zero whenever I put fuel in so on a long journey I am unable to get the true mpg
Why Can't I Get My Blue Tooth To Work Again?
I had my blue tooth paired and working well with my cell phone but turned off the blue tooth a couple times when I was in the passenger seat. Now I can't get it to work again. I have done everythi...
Kia Soul Interior Leak
I have a 2010 KIA soul, and lately I have been having a problem with every time it rains the passenger floor is very very wet. The dealership the last time I took it told me that there was no leak....
Does The Rear (hatchback) Window Open On A Kia Soul?
Why The Ac On My Kia Soul Won't Blow Cold Air Unless The Car Is Moving?
How Many Gallons Of Gas Does A 2014 Kia Soul Hold?
Only The Driverside Window Works On My 06 Kia Soul
just the driver side window works the rest do not function at all ,why is that??
Common Maintenance Problems, Or Major Work To Kia Soul?
I just purchase a used 2011 Kia Soul +, manual trans. Has anyone had to do any major maintenance to their souls? Like engine work, transmission,, etc.. Any common problems I should be aware of? So f...
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Upper Colfax News
|A message from Mayor Michael B. Hancock
Dear Neighbor –
Today, a new unauthorized camping law that prohibits camping on public and private property without written consent goes into effect.
This new ordinance provides the City with an additional tool to continue Denver's compassionate work of connecting vulnerable populations with vital services, while also maintaining the integrity of our vibrant City.
In Denver, everyone matters, and we must take care of those who live, work and play here. This... Read More »
|Notice of Street Closures for InnovAge Moonlight Classic 2012
Hello Colfax Business Improvement District!
We are excited to announce the return of the InnovAge Moonlight Classic presented by Kaiser Permanente on July 14, 2012. In its 20th year, the ride will once again feature a 10-mile course that is partially or fully closed to automobile traffic.
It is our hope you will support this event by sharing the street closure information with your neighborhood or district. Please see the attached letter with more information including... Read More »
|2012 Film & Fork Independent Film + Independent Restaurants Make Film & Fork...
Film & Fork on Colfax is a great opportunity to enjoy a meal Sunday through Thursday on Upper Colfax. With a minimum purchase of $25, guests can use their dated meal receipt to get a 'buy one-get one free' movie pass to the Denver FilmCenter//Colfax. Best of all, guests can redeem their free movie pass at any regular movie, during Film & Fork's summer promotion: June 17th - August 31st.
Meal & a movie, Colfax-style!
Films at the Denver... Read More »
|Don't miss this amazing line up of summer events on Colfax, sponsored by the...
CHUN People's Fair, June 2 - June 3, 2012
For more information, go to chundenver.org
The Fax Partnership's Feast on the Fax, Tuesday, June 12, 2012
For more information, go to thefaxdenver.com
Denver Film Center //Colfax's Raise the Roof Block Parties, June 14, July 12, August 9, 2012
For more information, go to denverfilm.org.
GLBT's Denver PrideFest 2012, June 16 - June 17, 2012
For more details, go to... Read More »
|Colfax Real Estate Tour a Success
On May 9, the Fillmore Auditorium and Cheeky Monk hosted over 100 real estate professionals for a half-day event promoting development projects and opportunities on Colfax Avenue. The event, organized by the Urban Land Institute, CBID, and three other Colfax business districts, demonstrated that Denver's 13-mile-long Main Street is flourishing. CBID statistics tell the same story: Upper Colfax Business Demographics:
55,000 population within 1 mile radius... Read More »
|Showing 121 - 125 of 281


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Best gearless car in india
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New Cars
Maruti Omni
Maruti Omni is a MUV car which is powered by a 796 CC engine. The car has a Manual type of transmission/gearbox. The car runs on LPG and sports a mileage of 10.9 Km/L. The dimensions of Maruti Omni are 3370X1410X1640 mm (length X width X height). Maruti Omni is priced at Rs. 2,49,546 (ex-showroom, New Delhi). ... read more
Used Cars
Chevrolet Optra 1.6 (2005)
It is like devil on road ...... It is very powerfull .....1600 cc engine Best car in low price ... read more
|Registration no: Hr 06 h 0460
|Colour: Gold
|City: Gurgaon
|Asking Price: 1,60,000
Fiat Siena 1.6 ELX (2002)
This Car was purchased New In July 2002 in Delhi. It has 1600 cc DOHC Petrol engine It has been driven by the owner sparingly, mostly in South Delhi. In city it gives an average of 11/12 Km/ lt with AC It is extremely well maintained car in Delhi, milage covered 40,500Km. It has best of the fittings.The car is mostly imported parts from Itly and Brazils etc and assembled in India. It is considered among very safe cars to drive. I expect, it can run another 2,00,000 approx. with regular servicing ... read more
|Registration no: Dl 3C V 8438
|Colour: Tan
|City: New Delhi
|Asking Price: 1,20,000
Upcoming Cars
Fiat Panda
Fiat needs to launch new models in India. Only fresh launches will add to Fiat India's bid to revive its image in India. The best way is to launch a n ... read more
Hyundai New Elantra
Hyundai Elantra is one of the best cars in the D segment in India. Hyundai has been working on the latest iteration of the Elantra and we expect it to ... read more
User Reviews
Skoda Octavia 2001-2013 Reviews
i like it very much. it is most best car of seller in india. read more
Tata Indica eV2 Reviews
Good car wiith good looks which i really like. It has good features but comfort is the best in class. It has excellent space for carrying my luggage. I like to drive more as my family prefers me to drive as my husband likes to relax on long drive. I love driving this car and my son also loves it verread more
India Yamaha Motor is planning to launch gearless scooters in India soon. "We have completed the feasibility study and plan to launch new gearless sco Read more detail
Introduction A limousine car price in India varies a lot. To start with, a limousine (also referred to as limo in short) is usually a luxury and Read more detail
Expert Reviews
New WagonR: The blue-eyed boy
What would you do if you manufacture the 2nd best selling car in India, six years on the trot? If the car in question is the darling of the In read more
Volkswagen Polo Diesel: A great value for money
This is a car thoroughly enjoying its success in India. With a past history of uncountable awards like the ECOTY to the best advertisements in read more
Mercedes-Benz India First Initiatives, Turbo: 62
Mercedes-Benz is one of the best luxury car brands in the country known for their high performance innovative machinery that is adored by people world ...view video
Mercedes GL Class India Review, GAADI.COM
The Mercedes GL is probably the best real SUV under a crore in India. What makes it so special? We find out.. ...view video
Forum Discussions
Hatch back Cars
How many people will Say that Skoda Fabia Is the Best hatchback Car on INDIA Roads
which one is best gearless scooter
hi binny here, i am planning to buy new gearless two wheeler and also good milage and power. Please advise me which one is best scooter from the following: honda activa honda aviator suzuki access
New Car Dealers
Porsche Centre Bengaluru ( Porsche)
Friendly Automotives (India) Private Limited
Mobile: +91 8884 000 911
Metro Ford ( Ford)
No 1 , Palace Cross Road, Bangalore, Karnataka, India
Phone: 080 - 40100800
Phone: 080 - 40100800
Used Car Dealers
Nemuri Motors
5-4-200/5, Behind Budha Bhavanam Opp Super Car Decors, M G Road, M G RoadHyderabad , Andhra Pradesh , India .Hyderabad, M G Road
Phone: 040-65212360
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Jemi Autos
Chennai , Tamil Nadu, IndiaChennai
Phone: 044-45530059
Mobile: 0-9443122758


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700 Club Interactive – July 19, 2010
Topic: Getting a Job in a Great Recession
1837 results for '700clubi'. Showing results 1261 - 1278
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I'm curious if this could have a positive effect on a beer that's already been brewed? I have about 4 gallons of a Belgian Quad in a keg, and I get a distinct chlorinated-tasting solvent taste from it.
Could I just pop open the keg and stir a very, very amount of potassium metabisulfite into the Quad? What effect would this have on an already brewed beer?
Probably not but try it in the glass first. Bisulfite appears to offer promise as it has the potential to reduce nasty things like acetaldehyde and diacetyl in finished beer to less bothersome compounds but it also forms adducts some of which seem to taste almost as bad as the things you are trying to deal with.
How long do the Campden tablets last??? I have a bottle in the fridge for 2 years or soo and though I don't mind getting more.... if I don't have to, why bother with the trip to the LHBS?
I notice on my bottle (LD carlson) there is no "born on" or "use by" date
The fact that they smell of sulfur dioxide says there is some decomposition taking place but as so little is needed to treat most water I don't think you need to worry with tabs that are 2 yrs old.
But I can't really answer your question. Ten years?
Since I have switched to AG I have been getting a strong plastic smell and taste to my beer. I have switched tubing, gone over my mash tun and made sure it's clean and I can attest that it is. I have, on 2 occasions now, used RO water to build my own brewing water. Low and behold no more plastic taste/smell! I've tried using a campden tablet in my tap water without success, however, I was adding it to the wort just as I was bringing it to a boil. Should I be adding it to my mash/sparge water instead?
I don't mind building my own water it's just that I typically brew on a whim and picking up RO water would require pre-planning (which is my wife's department).
Thanks for your time.
Correct. I add my potassium metabisulphite to my Hot Liquor Tank as the water is heating up in order to treate all my brewing liquor (both strike and sparge water).
The original post states what to do if your water has chloromine, but I'm not sure I understand what to do if like me, your water is treated with chlorine. Do you follow the same steps?
I've been using RO water with added Calcium Chloride up till now, but it takes forever to get 7 gallons of RO water from my system, so I thought I would try out my tap water on my next brew.


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Tag: diana mini
Analog Sounds with Simeon Smithwritten by hannah_brown on 2015-03-31 in #people #lifestyle
Simeon Smith is a musician who recorded the sounds of our film cameras in action and made these samples available as a free download. We couldn't resist interviewing him about this project and taking a look at some of his photos. Meet the man behind the cams here.7 Share Tweet
Wide-Angle Trio: So Simple, So Differentwritten by Alexander Krolikowski on 2015-02-25 in #gear #tipster
Lomographers know that once you start collecting cameras, it's difficult to stop yourself. It has a very logical explanation: every camera produces unique images that are impossible to get using another camera. In this article, I decided to compare three cameras with wide-angle lenses.1 4 Share Tweet
10 Reasons Why Diana Cameras are Perfect for the Holidayswritten by chooolss on 2014-12-09 in #lifestyle
Looking for the perfect analogue companion during the holidays? Well, look no further as we list down 10 reasons why the Diana F+, Mini, and Baby are perfect for capturing those bright and festive scenes, happy reunions, and exciting trips on film!6 Share Tweet
Scarlet Snaps Taken with the Diana Miniwritten by cheeo on 2014-11-10 in #lifestyle
Fire it up with the queen of retro cool's little sister, the Diana Mini!5 Share Tweet
Creative Diptychs Taken with the Diana Miniwritten by cheeo on 2014-10-10 in #lifestyle
Trying to save on film? Whip out the Diana Mini and put it on half frame mode to make the most out of your roll!3 Share Tweet
September Events @ LomographySGwritten by lomographysg on 2014-09-05 in #events
Aside from the DIANA MINI 5th BIRTHDAY PROMO which is ongoing until September 30, Lomography Singapore has prepared a series of exciting and worthwhile events this month.Share Tweet
The Diana Mini Turns 5 - Get Free Lomography Film With a Diana Mini Purchase!written by shhquiet on 2014-08-19 in #news
Happy birthday, Diana Mini! The sweet and petite 35mm princess of Lomography is celebrating its fifth birthday by way of a special treat. This week, buy a Diana Mini camera and get a Lomography Color Negative 100 35mm (3-Pack) for free! Simply type the code HIGH5MINI at checkout.1 Share Tweet
Take a Look at the Five Stylish Years of the Diana Miniwritten by shhquiet on 2014-08-18 in #news
The Diana Mini is turning five years old this month! Through the years we have seen this sweet and petite 35mm camera transform from a classic analogue beauty to a blinged-out snapshooter and everything else in between. Remember the Love Letters edition? How about the Premier Cru? To refresh your memory here's a gallery of all the Diana Mini styles we've released in the past five years. Which among these limited edition Clones is your favorite?1 Share Tweet
How to Use the Diana Miniwritten by chooolss on 2014-08-17 in #lifestyle
Taking your Diana Mini out for a spin for the first time? We got you covered!Share Tweet
Fun Half-Frame Portraits Taken with the Diana Miniwritten by chooolss on 2014-05-06 in #lifestyle
Have people taking a second look with your quirky, diptych-type portraits courtesy of the Diana Mini!3 1 Share Tweet
Simple Color Gels or Filters Holder for the Diana Mini's Flashwritten by grungyjodie on 2014-04-25 in #gear #tipster
I've always been looking for a really simple solution to hold my color gels of my Diana Mini's flash WITH the camera and make them easy to grab when I want to use them. I also wanted something to keep them from getting damaged. Let me show you how I found a simple way to make it.15 Share Tweet
More Photos of Kylie Minogue with the Diana Mini and Flash Gold Edition!written by plasticpopsicle on 2014-04-24 in #news
Remember the Stylist magazine issue featuring international pop icon Kylie Minogue with a Diana Mini Gold Edition on the cover? We're thrilled to share with everyone some more stunning snaps from the issue's shoot! Read on and take a look!3 Share Tweet
May Workshops at Lomography Chicago!written by lomographychicago on 2014-04-20 in #news
The weather is finally warning up here in Chicago so let's take advantage of these beautiful days by taking pictures with some of our favorite Lomography cameras! We've got some really cool workshops lined up this month so grab a friend and come hang with us!Share Tweet
A Salute to the Masters: Homes for Italians (A Tribute to Dan Graham)written by sirio174 on 2014-03-31 in #lifestyle
In 1966, American artist Dan Graham published an article about typical one-family homes in ordinary American suburbs built after World War II. He used a cheap Kodak Instamatic camera, with a deliberately amateur approach. In this article, I wrote a tribute to him with a series of photos taken in the suburbs of my city, Como, using my pretty Diana Mini camera. Read more after the jump!4 Share Tweet
Spotted: Kylie Minogue with Diana Mini and Flash Gold Edition on Stylistwritten by plasticpopsicle on 2014-03-21 in #news
International pop icon Kylie Minogue graced the cover story of a UK lifestyle magazine's issue this week, along with the equally glowing Diana Mini and Flash Gold Edition!2 13 Share Tweet
Diana Mini Superstar: The name says it allwritten by dopa on 2014-03-09 in #reviews
She may be small, but she can easily beat the big cameras out there! Read on to find out what makes this version of the Diana Mini a "Superstar!"1 1 Share Tweet
A New and Recycled Look for My Diana Miniwritten by teng1208 on 2014-02-25 in #gear #tipster
Attention Diana Mini owners, I would like to share my first experience on how I changed the look of my beloved camera. It's fun playing with you camera and be their private designer! Be proud and excited with your very own design!2 7 Share Tweet
LomoAmigo: Takeshi Sugawritten by hannah_brown on 2014-02-10 in #people #lomoamigos
Takeshi Suga is a Japanese music photographer who has recently moved to London. He has a passion for film photography and especially loves Lomography. Takeshi is exhibiting his photographs at our East London store and has agreed to be our Lom Amigo for the month. We gave him a La Sardina and sent him out into the city. Read on to find out what he got up to.1 2 Share Tweet
The Lomography Rainbow Rumblewritten by soundfoodaround on 2014-02-07 in #competitions
We wanted to do something to raise awareness of basic human rights. The Sochi 2014 Winter Olympics starts today as gay rights advocates across the globe rally to challenge the Russian government's anti-gay law. This rumble is meant to symbolize our support and inspire the Lomographic community to do and standup for what they believe in.5 37 Share Tweet
Beautiful Multiple Exposure Snaps Shot with the Diana F+!written by chooolss on 2014-02-06 in #news
Get a pair of Diana F+ cameras for the price of one when you avail of Lomography's special Valentine offer!2 Share Tweet
Awesome Day and Night Shots Taken with the Diana Miniwritten by chooolss on 2014-01-23 in #lifestyle
Beneath the Diana Mini's small exterior lies a myriad of features that makes it one powerful camera – don't let its size fool you!2 2 Share Tweet
Joyful Snapshots of New Year Celebrations Captured with the Diana Miniwritten by chooolss on 2014-01-02 in #lifestyle
Wherever you are in the globe right now, we hope you'll have an awesome year ahead!2 2 Share Tweet
Photo of the Day by mczoumwritten by jillytanrad on 2013-12-30 in #news
It's another one of those multiple exposure shots that deserve recognition as our Photo of the Day.2 3 Share Tweet
Decorate Your Diana Mini with Fabricwritten by philip0723 on 2013-12-27 in #gear #tipster
See all those Diana Minis decorated with colorful fabrics on the Online Shop but you already own one? Here's how you can decorate your own Diana Mini with your favorite fabric or pattern!1 15 Share Tweet
Double Exposure Portraits Shot with the Diana Miniwritten by chooolss on 2013-11-25 in #lifestyle
The next time you shoot a few portraits, remember to give them an extra oomph by utilizing your camera's multiple exposure setting!5 2 Share Tweet
Poster Art by Tara McPhersonwritten by jillytanrad on 2013-11-19 in #lifestyle
New York-based artist Tara McPherson has been creating art in different mediums for over a decade. If you're a fan, you've probably seen them all---paintings, posters, toys, prints, illustrations for magazines, and all sorts of merchandise clad in her designs. You might have had the opportunity to go to one of her exhibits and ask for her autograph, while gushing with adoration and marveling at her talent.3 Share Tweet
Keep Your Cool With These Chilled Blue Photographs Taken Using the Diana Mini!written by cheeo on 2013-11-18 in #lifestyle
It's starting to get chilly this time of year and we all feel it in the cool breeze. To add to that chill factor, we bring you these bluer than blue photographs from the community!6 Share Tweet
Creative Half-Frame Shots Taken with the Diana Miniwritten by chooolss on 2013-11-12 in #lifestyle
The adage "good things come in twos" rings true when it comes to shooting artsy half-frames with the Diana Mini.1 5 Share Tweet
Introducing the Diana Mini Double Rainbow by Tara McPhersonwritten by shhquiet on 2013-11-12 in #news
Combining the sweetness of the Diana Mini with the ethereal charm of Tara McPherson's artwork comes the new Diana Mini Double Rainbow.5 Share Tweet
View Lomography's 35mm Gallery of Art Courtesy of the Diana Mini!written by cheeo on 2013-11-11 in #lifestyle
Art is relative. Art is evocative. Art is art. Well, we really can't argue over these musings on the subject, but there's one thing we know, we love art! See more of our artsy-themed photo series from the community after the jump.4 Share Tweet


text: Web exclusive! "Shelby" has a secret, namely that she's a great bag for toting everything from the day's essentials to a small notebook computer, with enough room left over for a few groceries. Price: Price pending Padded carrying handles can be used as shoulder straps Padded core back panel and bottom protect contents Open inner pocket fits most 15" notebook computers Large, deluxe multi-pocket organizer with key fob Zippered accessory pocket on front panel Open cell phone pocket on back panel Large, zippered main compartment Side water bottle pocket 16" x 14" x 5" 600 denier Duralite Imported More


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Miele 4842 -- Good idea at $799?
I am in the market for a new washer and dryer and stopped into our local appliance store today.
I am looking for a washer that will get my clothes clean and perform well in a second floor laundry room -- minimal vibration is important.
The salesperson showed me the Miele 4842, which they have available (new, in the box) for $799. She told me it is superior to anything else I could buy, in terms of (gently) getting clothes clean and in build quality and longevity. She also said that it will work well on the second floor because it is heavy (heavier than other brands) and "doesn't move."
I have several questions for you laundry experts:
*i know this washer would have been manufactured years ago; does it matter that it's been sitting in a warehouse?
*Does this model have a good track record? I read a lot of posts here in which people were happy (or not) with the washer when they got it, but am wondering if people are still pleased (or not).
*Will this be a better performer than other brands? I was thinking of an Electrolux Wave- or IQ-Touch when I went into the store.
*Any idea if it will be good in a second-floor laundry room?
In short, I'm wondering if I should consider going with the Miele or with the Electrolux (or something else). Any advice?
That model has been discontinued but Miele will do repairs on it.
Make sure you do have a certified Miele repair/service center near you first instead of after purchase.
The price is a good for a Miele since the machines went for around $2000 so the shop is really trying to get rid of that machine.
Quick review from Good Housekeeping:
Although the machine has a large drum (for Miele standards (4.00 cu. ft.) it's not as huge as say an Electrolux.
Cycle times are way longer on the Miele.
You'll have to see what others have to say, it's a tough decision.
Any machine you put on a second floor may need the floor strengthened to keep silent. See the thread here about a month ago or less on that topic. I put 3/4 inch pliywood under my Samsung washer and tiled over that. to make the floor heavier. I don't know anything about Miele washers other than that the brand has ardent supporters in these forums.
Wow, I paid twice that in USD for ours, brand new, four years ago.
Our Miele 4842 doesn't move around for a couple of reasons. First, it sits right next to an outside wall in the laundry room, so the "trampoline effect" is minimized. Second, it is bolted via brackets to the Miele dryer we bough that the same time. Our Miele machines weigh a ton, figuratively speaking. Possibly more than any other brand? I will get some vibration from time to time, depending on what is spinning in there.
Our laundry room is upstairs over a garage, but more like a main floor because the washer sits next to an outer wall, and concrete block is holding the floor joists steady.
Where on the second floor is crucial.
I have a friend whose laundry room is in right the middle of the main floor, over a garage/basement, and her Whirlpool Duet will walk across the room with certain loads. THAT is the trampoline effect of being in the middle of the house.
Thank you for your replies. It is a tough decision for me!
The Miele would involve some work (for DH). There are no pedestals available, so I would want a platform built to raise it to a good height. In addition, the fact that the washer only has a right-side hinge means we will have to reroute hoses and cords to accommodate it. The salesperson says this is not a big deal, but my husband is reluctant to change things (even though the store will take care of it as part of installation).
I just keep thinking the price is great for a well-reviewed machine that *should* last a long time. Even if it were only to last for ten years, it is a good buy at $799.
On the other hand, it might just be easier to purchase the Electolux with its matching pedestals and call it good.
The store only has a handful of these Miele machines left. If I keep spinning in circles on making the decision, there won't be one available. ðÂÂÂ
If the door hinge means you would have to place the washer where the dryer currently sits, essentially reverse current locations, that will be no problem. We had to do it in our laundry room. The installers used new hoses (old ones were 16 or 17 years old & time to be replaced) and they are plenty long enough. We;'ve had no problems.
I would be a bit careful. We've been buying Miele appliances for over 20 years now. Currently have 2 washing machines, dryer, 2 dishwashers, convection cooktop and oven split between a home in Europe and one in the USA. In Europe, where high quality appliances are far more readily available, we're down to an old Miele washing machine that never gives up. Everything else has been replaced with another brand (not distributed in the USA).
Over the years their quality, along with their prices, has gone down and their maintenance costs have increased. Their once vaunted reputation has been severely bruised in this household with several calls to repair the washing machine, culminating in a $900 drum replacement right after the warranty ran out because the bearing they installed could not handle the weight of their drum. The latest dishwasher is their top-of-the line model. Cheaper than our old lower than top-of-the-line Miele, noisier, more difficult to fill and not as robustly made.
Other than ovens and vacuum cleaners, we've about had it with the brand and it now competes with any of the other better brands. They tend to be like BMW's, you see your service person a lot the first year or 2. After the warranty runs out, you sell it.
My mom bought a Miele washer and dryer years ago and they were really good quality machines. Both the washing machine and dryer are almost silent. However, my dad was more hesitant because of the same reason others have mentioned - he heard repair costs are more because you have to go with their technician, and ordering parts isn't like running down to home depot to grab what you need. However, my mom talked him into it and it turned out they've never needed a repair. A couple of years ago they bought a new set for their home and moved the old set to their business. They went with Miele again, and my mom loves them. The new set is also very quiet, and I haven't ever noticed any bouncing or movement, especially not compared to my own w/d, which are pretty good quality but still make noise and rattle the laundry baskets on the spin cycle. They also haven't ever needed to repair their dishwasher, although that's only a couple of years old.
Yes, I can see your hesitance because it's a risk, but I have to tell you, after seeing the performance of my parents, if I were in your position it would be hard for me to pass up. I don't have any experience with electrolux to compare, I just know I would love to have a Miele. In fact, after visiting my parents and using their Miele vacuum I joked with my husband that when we have new carpet later this year, it would only be protecting our investment to buy one . . .
I have an almost-6-year-old W4840, the model that preceded the W4842. Got a 5 year warranty under an offer that was available at the time, and had the door seal replaced (once) and adjusted (once) under warranty. I really don't know what I'd replace this machine with, if it were to die. Are there any washers of comparable capacity with "true" hot, warm, cold wash temperatures on the market today? Last time I looked into it, even sanitary cycles didn't run so hot anymore, rather they relied on chemicals for part of the sanitization process. Not that I feel a need to sanitize my laundry, but at least a sanitary cycle is or was a way to get a really hot wash to get stuff like dishtowels clean. The Miele goes one better, because it gives you evenly spaced temperature settings at 10degC intervals between 30degC and 70degC, because you may want a wash warmer than lukewarm, but not 70degC which will damage some items.
Thank you all so much for your responses. I purchased the Miele washer last week and am excited to get it delivered and up and running!
Now I'm working on choosing a dryer. My sense of order makes me want the matching Miele dryer, but there are none to be had. I hope I am so pleased with my washer that I don't notice they're not a matched set. :)
We have that washer and dryer set. We are WAY happier with the washer than with the dryer, mostly because of the limited settings on the dryer and no way to set a timer to run. I really miss having a timed setting.
But we LOVE the washer. We've had if for I think 2 1/2 years now and no problems yet.
We have the matching dryer and, honestly, any decent brand dryer should do the trick. Check the consumer reports ratings for starters. I like our matching dryer, and have been quite happy with it. It doesn't bake things like our last (circa 1995 purchase) dryer.


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Vilas is a populated place located in Watauga County at latitude 36.246 and longitude -81.768.
The elevation is 2,746 feet. Vilas appears on the Valle Crucis U.S. Geological Survey Map. Watauga County is in the Eastern Time Zone (UTC -5 hours).
Watauga County: Cities & Towns | ZIP Codes | Schools | Features (airports, churches, hospitals, etc.)
BIG Map |
Driving Directions |
Data Source - USGS GNIS FID: 1023082
BOUNDARY MAPS: Township of Brushy Fork | Watauga County
Vilas, NC
(< 0.1 mile)
Sugar Grove, NC (1.4 miles NW)
Sherwood, NC (1.9 miles NNW)
Lovill, NC (1.9 miles ESE)
Amantha, NC (2.4 miles NNW)
Valle Crucis, NC
(2.7 miles SSW)
Silverstone, NC (3.1 miles NNE)
Oak Grove, NC (3.6 miles ESE)
Rominger, NC (4.1 miles WSW)
Sweetwater, NC (4.3 miles NW)
Hodges Gap, NC
(4.5 miles SE)
Hounds Ear, NC (4.9 miles SSE)
Dougherty Heights, NC (5.3 miles ESE)
Valley Creek, NC (7.2 miles SSW)
Mill Ridge, NC (7.4 miles S)
New River Heights, NC
(7.4 miles ESE)
Blairmont, NC (7.5 miles ESE)
Moretz, NC (8.3 miles E)
Ski Mountain, NC (8.7 miles SE)
Sorrento Skies, NC (8.8 miles ESE)


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Weather & Clock Widget Android
10,000,000 - 50,000,000 downloads
- Useful
- Highly complete
- Many similar apps
+ By Devexpert.NET
"The Weather Predictor"
Reviewed by Anna Grace / Jul 09, 2014
What to wear? Find out if it's going to be a sunny day
Weather & Clock Widget Android is a weather application including a helpful widget that helps you figure out what's the weather like in a more direct way.
Discover the temperature, humidity, pressure and wind properties in order to decide what to wear, where to go and make plans for the weekend.
Devexpert.NET is the developer of Weather & Clock Widget Android, an excellent weather app with a complete forecast and an interesting interface.
Weather & Clock Widget for Android comes with different widget sizes, 2x1, 4x1, 4x2 and 4x3 for normal screens, and 5x1, 5x2 and 5x3 for large screens and tablets.
Weather & Clock Widget for Android supports a wide range of Android phones; it supports Android Version 2.3 and higher.
- Provides current weather condition and five days weather forecast
- 10-Day and Hourly Weather with precipitation
- Sunrise and Sunset time
- Atmospheric pressure with different units
- Temperature in status bar
- Share weather and location information with friends.
- Home screen widgets, 5x3, 5x2, 5x1 for large screen only and 4x3, 4x2, 4x1 and 2x1 for all screens.
- Searches for all cities in the world by country and city or zip code
- Ability to set your internet source to Wi-Fi only
- Ability to disable internet access from operators while you are in roaming
- Ability to set your preferred temperature unit, Celsius or Fahrenheit
- Automatic update intervals are: 1, 3, 6, 9, 12 hours and Manual update.
- Auto location detection
- Up to 10 different locations
- Weather map with ability to add any city to your city list
- Notification alerts (with option to disable it)
- Moon phase
- UV Index and Dew Point
- Available in: English, Dansk, Deutsch, Ελληνικά, Български, Suomi, Norsk Bokmål, Nederlands, Slovenčina, Slovenščina, Svenska, Română, العربية, كوردى, ئۇيغۇرچە, Français, Hrvatski, Türkçe, Čeština, Español, Català, Italiano, Português, Magyar, Македонски, Pусский, Українська, Қазақ тілі, Polski, Português(Brasil), Српски, Lietuvių, հայերեն, Indonesia, Việt, 日本語, 中文 (简体), 中文 (繁體), 한국어.
For any questions or issues, please visit FAQ page at: http://www.devexpert.net/weather
* Android is a trademark of Google Inc.
FAQ page at: http://www.devexpert.net/weather
- Optimized update frequency
- We apologize for update problem, we have removed faulty service providers and we are working hard to incorporate more providers, we appreciate your patience and support.
Some lists where this application appears: Reviewed by Anna Grace, Weather apps.
This doesn't work anymore since yesterday's update. Please fix!
Gr8 app, the only one i use for weather. After downloading update 22.214.171.124 the app doesn't work anymore, can't get the weather updated.
Will not sinc
Used to love it...not working anymore at all...deleted it and found another one.
Wouldn't update
Doesnt work anymore. Move along.