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We report here that islet-specific expression of TNF-α can play a dual role in autoimmune diabetes, depending on its precise timing in relation to the ongoing autoimmune process. In a transgenic model (rat insulin promoter-lymphocytic choriomeningitis virus) of virally induced diabetes, TNF-α enhanced disease incidence when induced through an islet-specific tetracycline-dependent promoter system early during pathogenesis. Blockade of TNF-α during this phase prevented diabetes completely, suggesting its pathogenetic importance early in disease development. In contrast, TNF-α expression abrogated the autoimmune process when induced late, which was associated with a reduction of autoreactive CD8 lymphocytes in islets and their lytic activities. Thus, the fine-tuned kinetics of an autoreactive process undergo distinct stages that respond in a differential way to the presence of TNF-α. This observation has importance for understanding the complex role of inflammatory cytokines in autoimmunity.
Inflammatory cytokines such as TNF-α are thought to play an important role in the pathogenesis of autoimmune type 1 diabetes. TNF-α is directly implicated in the destruction of β-cells from in vitro studies on isolated islets (1) and has profound inflammatory effects in vivo by acting directly on APCs and autoreactive T lymphocytes (2, 3, 4, 5). Studies in animal models for type 1 diabetes have predominantly revealed functions that accelerate the autoimmune process. For example, constitutive TNF-α expression in islets in rat insulin promoter (RIP)3-TNF-α-transgenic C57BL/6 mice leads to profound intraislet infiltration but, surprisingly, not to overt autoimmune diabetes (6). Only when the costimulatory molecule B7.1 is coexpressed with TNF-α does diabetes develop (7). It has recently become clear by using an islet-specific tetracycline (Tet)-regulated TNF-α in vivo model that the initial duration of TNF-α expression in this TNF-α-B7.1 diabetes model is crucial for determining the fate of autoaggressive CD8 lymphocytes (8). Only if TNF-α is "on" for >21 days will CD8 lymphocytes enter the islets and destroy them. It is quite likely that enhanced presentation of islet Ags plays an important role in this situation. Evidence for the profound effect of TNF-α expression on islet Ag presentation to autoaggressive CD8 lymphocytes early during diabetes pathogenesis has been generated in RIP-TNF-α-transgenic nonobese diabetic (NOD) mice. Such animals exhibit markedly accelerated spontaneous diabetes, and presentation of β cell Ags to islet-infiltrating CD4 as well as to CD8 lymphocytes is enhanced. Interestingly, only CD8 lymphocytes show a clear pathogenetic potential in this model from adoptive transfer studies, whereas CD4 lymphocytes might even have regulatory function (4, 9, 10, 11). Similar to these transgenic models in which expression of TNF-α was specifically targeted to the islets, early systemic TNF-α administration enhanced diabetes in NOD mice (2, 5). However, another study reported that systemic TNF-α administration late during diabetes development could abrogate the disease process, probably by affecting expansion, migration, and function of autoreactive lymphocytes (12). This finding was mirrored by another in vivo study in NOD mice in which TNF-α was expressed later during pathogenesis and not in the neonatal period, and diabetes was prevented. In this model, massive insulitis was observed, but these lymphocytes did not have sufficient autodestructive capability to induce diabetes, and, in addition, TNF-α expression down-modulated the capacity of autoreactive T lymphocytes in these NOD mice to destroy islets in adoptive transfer experiments (3). Thus, TNF-α appears to play a dual role in the regulation and propagation of the diabetogenic autoimmune process, and its precise function appears to depend on the timing of expression. Based on these previous studies, our intention was to precisely dissect the time factor of islet-specific TNF-α expression in relation to the ongoing autoimmune process. For this endeavor, we chose to develop a double-transgenic mouse model in which islet TNF-α expression could be repressed and derepressed by Tet administration, and a viral self Ag was constitutively expressed under the RIP as a target autoantigen. This RIP-lymphocytic choriomeningitis virus (LCMV) × Tet-TNF-α model (see next paragraph) had the advantages that autoreactive lymphocytes could be traced precisely, the time point of initiation of autoimmunity could be chosen experimentally, and TNF-α could be switched on for any period of time during the pathogenesis of diabetes.
The RIP-LCMV-transgenic mouse models for virally induced autoimmune diabetes were first developed by the laboratories of Zinkernagel et al. (13) and Oldstone et al. (14) in 1990. RIP-LCMV mice express the gp of LCMV under control of the RIP selectively in pancreatic β cells as a target/marker autoantigen. Interestingly, these mice are not tolerant but are rather unresponsive to the viral (self) protein, because infection with LCMV leads to autoimmune diabetes in 80–100% of such transgenic animals. Further analysis of the Oldstone model revealed a three-stage pathogenetic process. First (the initiation stage), systemic infection with LCMV leads to up-regulation of MHC class I molecules on islet cells (15), activation of APCs, and production of type 1 IFNs as early as 2 days after infection in most major organs, including the pancreas (but not the islets) (15, 16). At this time, no lymphocytes are found in the islets or pancreas. However, these events are pathogenetically crucial, because adoptive transfer of LCMV-specific lymphocytes into uninfected RIP-LCMV mice leads to insulitis but not diabetes (14, 16), unless the presence of activated professional APCs is mimicked by the expression of B7.1 on islets (17). Second (the effector stage), 7 days after infection, the first LCMV-specific autoreactive CD4 and CD8 lymphocytes enter the pancreas and islets. In contrast to the Zinkernagel model, diabetes occurs in our RIP-LCMV mice (18) later (day 10–14 after infection) and is not only dependent on perforin-dependent direct lysis of β cells through autoreactive cytotoxic T lymphocytes (19), but is also dependent on IFN-γ producing autoreactive CD8 lymphocytes (15, 18, 20). Likely, β cells are destroyed by perforin as well as by inflammatory cytokines involving IFN-γ and TNF-α. This killing of β cells by inflammatory cytokines requires prolonged insulitis characterized by local production of inflammatory cytokines, a process that we view as the distinct third stage in our RIP-LCMV model. From earlier studies, it is known that constitutive expression of TNF-α enhances diabetes in RIP-LCMV-gp mice (21) and that lack of the TNFR hampers disease development. However, no clear knowledge exists about which role TNF-α plays during each of the three phases (initiation, infiltration of autoreactive lymphocytes, and inflammation and β cell destruction) leading to RIP-LCMV diabetes. Based on the earlier studies mentioned previously, we expected to find differential effects, which formed the working hypothesis for our present report.
Using double-transgenic RIP-LCMV-gp × Tet-TNF-α mice, we were able to precisely define the period during which TNF-α production was switched on specifically in the islets. This transgenically mediated TNF-α expression was superimposed on the endogenously occurring TNF-α secretion during LCMV infection of RIP-LCMV-gp mice that peaks between days 6 to 8 after infection in the pancreas (15). We found that when transgenically regulated TNF-α expression coincides with the peak of inflammatory TNF-α production that occurs relatively early during pathogenesis, disease is enhanced, similar to previously reported findings in RIP-LCMV (21) as well as NOD (8) mice. In contrast, late induction of transgenic TNF-α abrogated diabetes and even led to disease reversion in up to 50% of already overtly diabetic RIP-LCMV mice. This was associated with a sudden and marked decrease in autoreactive CD8 lymphocytes. Thus, autoaggressive CD8 lymphocytes benefit from the presence of TNF-α early during pathogenesis (8) but are adversely affected by TNF-α at the latest stages of the prediabetic phase, in which TNF-α might be of therapeutic value.
Materials and Methods
Mice breeding scheme and origin
Generation of the H-2b RIP-LCMV-gp-transgenic mice used for this study has been described previously (14, 18). Tet-TNF-α mice (8) expressing TNF-α under the control of a Tet-regulated gene transcription system (22) were kindly provided by R. A. Flavell (Yale University School of Medicine (New Haven, CT). Briefly, the expression of the murine TNF-α gene is controlled by the Tet operator sequences (TetO-TNF-α). In addition, the gene encoding the Tet-responsive transactivator (tTA) was placed under the control of the RIP II (RIP-tTA) to ensure β cell-specific TNF-α expression. Thus, expression of transgenic TNF-α is either blocked or induced in the presence or absence of the Tet derivative doxycycline (Dox), respectively (8). Three different preparations of Dox were used: 1) Dox hyclate (300 μg/ml in drinking water), obtained from Mutual Pharmaceuticals (Philadelphia, PA); 2) Dox-HCl (300 μg/ml in drinking water), obtained from Sigma (St. Louis, MO); and 3) Dox Diet (200 μg/g in food pellets), obtained from Bioserv (Frenchtown, NJ). We initially tested the reliability of different Dox preparations in blocking TNF-α expression in RIP-gp-TNF-α mice by determining pancreatic TNF-α expression by RNase protection analysis (RPA). The amount of protected TNF-α mRNA was normalized against protected L32 mRNA, which was used as an internal standard. Mice that exhibited a >2-fold increase in TNF-α mRNA compared with the mean concentrations detected in uninfected RIP-gp mice were scored as "TNF expressers." Dox preparations using Dox hyclate added to the drinking water resulted in a considerable amount of "leakiness," which was reflected in a high (50%) frequency of TNF expressers in the presence of Dox. In contrast, Dox preparations using Dox-HCl in drinking water or Dox food pellets resulted in a frequency of TNF expressers that was as low as for uninfected single-transgenic RIP-gp mice. As a consequence, Dox food pellets were used for all our following experiments. H-2b (C57BL/6) Tet-TNF-α mice were intercrossed with the H-2b (C57BL/6) RIP-LCMV-gp line, resulting in double-transgenic Tet-TNF-α-RIP-LCMV-gp mice on the H-2b (C57BL/6) background, which were designated as RIP-gp-TNF-α mice. RIP-gp-TNF-α mice were constantly bred in presence of Dox food pellets.
Mice genotyping
The presence of transgenic RIP-gp, TetO-TNF-α, and RIP-tTA sequences was determined by performing three independent standard PCRs with genomic DNA obtained from mouse tails. The primer pairs used were as follows: RIP-gp sense, 5′-TGG ACA GGC TCA GAT GGC AAG-3′ and RIP-gp antisense, 5′-CTC AAA GCA GCC TTG TTG TAG TC-3′; TetO-TNF-α sense, 5′-TGA CCT CCA TAG AAG ACA CC-3′ and TetO-TNF-α antisense, 5′-TGT GAG GGT CTG GGC CAT AGA ACT ACT GAT-3′; RIP-tTA sense, 5′-ATT TGA GGG ACG CTG TGG GCT CTT-3′ and RIP-tTA antisense, 5′-ACT TCA ATG GCT AAG GCG TC-3′. In the Tet-TNF-α-transgenic line we used, both components of the tTA system, the RIP-tTA and TetO-TNF-α transgenes, always cosegregated to the offspring.
Virus used was LCMV strain Armstrong (Arm) clone 53b (20). LCMV was plaque purified three times on Vero cells and stocks prepared by a single passage on BHK-21 cells. Mice were infected with a single i.p. dose of 105 PFU LCMV-Arm unless indicated otherwise.
Viral titers
LCMV viral titers of organ homogenates were determined by infection of Vero cells as described elsewhere (18). Briefly, organs' homogenates were diluted serially and cultured with Vero cells for 5 days. Viral titers were determined from the number of counted plaques.
Blood glucose (BG) measurements
Blood samples were obtained from the retro-orbital plexus, and plasma glucose concentration was determined using Accu-Check III (Roche, Indianapolis, IN). Mice with blood glucose values >300 mg/dl were considered diabetic (17). Mice that were scored diabetic after 2–5 wk but returned to nondiabetic BG concentrations of <200 mg/dl were considered as "revertants."
Islet cell enrichment
Islets were isolated as previously described (15). Briefly, the pancreas was removed, cut into little pieces, and digested with collagenase P (Roche). Islets were purified on HISTOPAQUE-1077 density gradients (Sigma). The obtained islet-enriched fraction and the remaining portion of the pancreas containing mainly acinar cells were immediately homogenized in Tri-Reagent (Molecular Research Center, Cincinnati, OH) for subsequent isolation of total RNA.
The RPA
Total RNA was isolated either from whole pancreas homogenates or from islet- or acinar cell-enriched fractions using Tri-Reagent. RNA was extracted with chloroform followed by isopropanol precipitation and washing with ethanol. A total of 20 μg of total pancreatic RNA was used for hybridization with a [32P]UTP-labeled multitemplate set containing specific probes for TNF-α, IFN-γ, and lymphotoxin-β (LTβ) provided by a commercial kit (Riboquant, mCK-3b; BD PharMingen, La Jolla, CA). The RPA was conducted according to the manufacturer's guidelines. The resulting analytical acrylamide gel was scanned using a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and the intensity of bands corresponding to protected mRNAs was quantified using the ImageQuant image analysis software (Molecular Dynamics) using L32 as a reference gene.
Organs were harvested at wk 6 after LCMV infection unless indicated otherwise, immersed in Tissue-Tek OCT (Bayer, Elkhart, IN), and quick-frozen on dry ice. Using cryomicrotome and sialin-coated Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), 6- to 10-μm tissue sections were cut. Sections were then fixed with 90% ethanol at −20°C, and, after washing in PBS, an avidin-biotin blocking step was included (Vector Laboratories, Burlingame, CA). Primary and biotinylated secondary Abs (Vector Laboratories) were reacted with the sections for 30 min each, and color reaction was obtained by sequential incubation with avidin-peroxidase conjugate (Vector Laboratories) and diaminobenzidine-hydrogen peroxide. Primary Abs used were rat anti-mouse CD8a (Ly2), rat anti-mouse CD8b (Ly3), rat anti-mouse ICAM-1 (CD54) (BD PharMingen), and rat anti-mouse MHC class I (Bachem, King of Prussia, PA).
Staining for apoptosis
Quick-frozen 6- to 10-μm tissue sections (pancreas and pancreatic draining lymph node (PDLN)) were probed for the presence of apoptotic cells with a basic TUNEL assay using the ApopTag peroxidase in situ apoptosis detection kit (Intergen, Purchase, NY) according to the manufacturer's guidelines. Briefly, the TUNEL assay detects DNA stand breaks that are characteristic for DNA fragmentation as occurs during apoptosis. Tissue sections were fixed with 1% paraformaldehyde and ethanol-acetic acid (2:1), washed, and incubated with TdT to label free 3′OH DNA termini with digoxigenin-dNTP. Incorporated digoxigenin-dNTP was detected by sequential incubation with peroxidase-conjugated anti-digoxigenin mAb and diaminobenzidine-hydrogen peroxide.
Cytotoxicity assays
LCMV-specific CTL activity in the spleen was analyzed in a 5-h in vitro 51Cr release assay (23, 24). All samples were run in triplicates. Primary CTL activity was tested by harvesting spleens at day 7 after i.p. infection with 105 PFU LCMV-Arm. Splenocytes were coincubated with MHC-matched (MC57 H-2b) and mismatched (BALB/c17 H-2d) target cells that have been loaded with 51Cr. Target cells were either LCMV infected, uninfected but coated with the immunodominant LCMV MHC class I peptide GP33 (KAVYNFATC), or uninfected and uncoated. For determination of secondary CTL activity, spleens were harvested 6 wk after LCMV infection, and splenocytes were cultured for 8 days on LCMV-infected irradiated peritoneal exudate cells before testing CTL activity in a 51Cr release assay.
Flow Cytometry
Spleen and PDLNs were harvested at 6 wk after LCMV infection. Single-cell suspensions were stimulated with 100 U/ml IL-2 and 2 μg/ml brefeldin A (Sigma) for 5–16 h at 37°C. Cells were stained for cell surface markers using mAbs against CD8 and CD4, permeabilized and fixed with paraformaldehyde-saponin, and stained for intracellular cytokines using FITC-conjugated anti-mouse TNF-α mAb and PE-conjugated anti-mouse IFN-γ mAb (BD PharMingen). Staining for LCMV (gp33)-specific CD8 lymphocytes was performed with PE-conjugated H-2Db-gp33 tetramers (Tetramer Core Facility, Emory University, Atlanta, GA). Cells were acquired and analyzed on a FACSort or FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences).
Expression of inducible TNF-α in double-transgenic RIP-gp-TNF-α mice is blocked in the presence of Dox and is specifically expressed in islets after removal of Dox
Generation of Tet-TNF-α-transgenic mice expressing inducible TNF-α specifically in the islets of Langerhans under the control of a Tet-dependent gene transcription system (tTA-system) has been reported previously (8). We crossed these Tet-TNF-α mice with syngeneic RIP-LCMV-gp-transgenic H-2b (C57BL/6) mice. The resulting RIP-gp-TNF-α mice were genotyped for the presence of transgenic LCMV-gp, as well as the TetO-TNF-α and RIP-tTA sequences using a standard PCR screening protocol.
All breeding of RIP-gp-TNF-α mice was conducted in the presence of Dox to repress expression of transgenic TNF-α. We initially tested the reliability of different Dox preparations in blocking TNF-α expression in RIP-gp-TNF-α mice by determining pancreatic TNF-α expression by RPA (see Materials and Methods for details). Mice that exhibited a >2-fold increase in TNF-α mRNA compared with the mean concentrations detected in uninfected RIP-gp mice were scored as TNF expressers. RIP-gp-TNF-α mice expressed elevated levels of TNF-α in presence of Dox at a frequency of ∼15%. The elevated TNF-α mRNA in these spontaneous TNF expressers probably results from other sources than the Tet-TNF-α transgene, because it is similar to the frequency found in non-Tet-TNF-α-transgenic RIP-gp mice (Fig. 1⇓).
Time and magnitude of transgenic TNF-α expression was analyzed by RPA. The frequency of TNF expressers increased from ∼15% (Dox on) to >50% after 4 days. Fourteen days after Dox removal, a maximum frequency of >80% TNF expressers was reached that did not further increase when Dox was removed for a longer time period (Fig. 1⇑). The mean pancreatic TNF-α mRNA expression level was increased by a factor of 4 after 14 days of Dox withdrawal (data not shown). It is important to note that even after 2 mo of Dox-free diet, not all of the RIP-gp-TNF-α-transgenic mice were exhibiting elevated pancreatic TNF-α levels (Fig. 1⇑). These mice were genotyped again, and all were transgenic for the Tet-TNF-α system. Thus, there appears to be a rate of ∼15% nonresponders that are incapable of up-regulating TNF-α.
The specificity of TNF-α induction for the islets of Langerhans was tested by RPA on purified islets using a multiprobe template set including gene probes for several other cytokines. TNF-α, IFN-γ, and LTβ mRNA expression was determined in uninfected RIP-gp-TNF-α mice that were bred in the presence or absence (2 wk) of Dox. Total RNA was isolated either from islet cell- or acinar cell-enriched fractions that were obtained by limited collagenase P digestion of pancreas pools of three mice per group. Cytokine mRNA was detected specifically in islet-enriched fractions (Fig. 2⇓A). Quantification of the obtained RPA data revealed that only TNF-α mRNA, but not other cytokines, was up-regulated in this islet cell-enriched fraction (Fig. 2⇓B). In contrast, IFN-γ and LTβ mRNA concentrations even decreased after Dox removal (Fig. 2⇓B). In addition, islet cell enrichment resulted in a 5- to 6-fold higher TNF-α mRNA concentration than found in the crude pancreas homogenate of TNF-α expressers after Dox removal (data not shown). These data clearly demonstrate the specific expression of TNF-α in the islets of Langerhans of RIP-gp-TNF-α-transgenic mice after Dox removal and confirm previous results from Tet-TNF-α-transgenic mice (8).
Expression of TNF-α late but not early during pathogenesis decreases the incidence of type 1 diabetes
RIP-gp-TNF-α double-transgenic mice were infected with 105 PFU LCMV-Arm, and TNF-α expression was induced by removal of Dox at several times after infection. BG was determined in weekly intervals for at least 12 wk, and values >300 mg/dl were considered diabetic. Mice that scored diabetic after 2–5 wk but returned to nondiabetic BG levels of <200 mg/dl were considered to be revertants. Control mice that were kept on a Dox diet had an ∼70% incidence of diabetes 14 days after LCMV infection, comparable to findings reported by us previously using single-transgenic RIP-LCMV-gp mice and indicating that the presence of Dox did not profoundly influence diabetes development in RIP-LCMV-gp mice (Fig. 3⇓A). Interestingly, expression of TNF-α had a differential effect on diabetes incidence depending on the time of its induction. When Dox was removed at the time of LCMV infection (day 0), the incidence increased to ∼90%. In contrast, when Dox was removed late (day 10 after infection) or very late (day 14 after infection), the diabetes incidence decreased to ∼30% (Fig. 3⇓A). In parallel to this decrease in overall diabetes incidence, the frequency of revertant mice increased from 0% (day 0) up to 45 or 35% (days 10 and 14, respectively) (Fig. 3⇓A). These data show that expression of TNF-α late during pathogenesis, while islet infiltration and destruction is already ongoing, can have a beneficial effect in reverting diabetic to nondiabetic mice. Importantly, the revertants remained nondiabetic for the rest of their lives (observation time of >6 mo), and the late pulse of TNF-α can thus be considered therapeutic.
Systemic blockade of TNF-α starting early after LCMV infection abrogates autoimmune diabetes
Virally induced inflammatory TNF-α expression in the pancreas of RIP-gp mice is highest at day 7 after LCMV infection and returns back to preinfection levels 21 days after infection (15). Therefore, when expressed early (i.e., at the same time as the LCMV infection), transgenic TNF-α is superimposed onto inflammatory TNF-α expression in the pancreas of RIP-gp-TNF-α mice. Based on the enhancement of diabetes seen when transgenic TNF-α was turned on early, we first sought to assess the pathogenetic importance of this early TNF-α expression in our RIP-gp mice. Groups of four RIP-gp mice were infected with LCMV, and one group of animals was treated with a rTNFR55-IgG1 fusion protein known to neutralize TNF-α. TNFR55-IgG1 (kindly provided by Dr. W. Lesslauer, Hoffmann-LaRoche, Basel, Switzerland) was constructed using human TNF-α sequences (25, 26) but was found to effectively block mouse TNF-α activity (27). As expected, diabetes occurred between days 10 and 28 after infection in all untreated control RIP-gp mice (Fig. 3⇑B). In contrast, mice that received three i.v. injections of 100 μg TNFR55-IgG1 at days 4, 7, and 10 after LCMV infection never developed diabetes (Fig. 3⇑B). This experiment demonstrates the importance of early TNF-α activity for initiation of virally induced type 1 diabetes in the RIP-gp mouse model. It also agrees with the fact that we observed an increase in diabetes incidence when we induced expression of transgenic TNF-α in RIP-gp-TNF-α mice at an early time (i.e., day 4 after infection).
Infiltration of islets by CD8 lymphocytes is reduced but not absent in revertant RIP-gp-TNF-α mice
Revertant mice can be identified at wk 4–6 after LCMV infection and 2–4 wk after transgenically (Dox withdrawal) induced TNF-α expression. At that time, we examined pancreas sections of either revertant or diabetic RIP-gp-TNF-α mice that received Dox-free diets starting at day 10 after infection. Fig. 4⇓A shows sequential pancreas sections of representative revertant and diabetic RIP-gp-TNF-α mice stained for CD8, MHC I, and ICAM-1 to show the representative degree of islet infiltration found in such animals. Infiltration of various types of lymphocytes such as CD8 and CD4 lymphocytes, B cells, dendritic cells, and macrophages was much more pronounced in islets of diabetic RIP-gp-TNF-α mice, as shown for CD8 lymphocytes in particular (Fig. 4⇓A). However, it is important to note that some infiltrating CD8 lymphocytes are still present in islets of revertant RIP-gp-TNF-α mice, even though most of these mice had nondiabetic BG values for 1–2 wk before the pancreas was removed (Fig. 4⇓A). There were no obvious differences in MHC class I expression when comparing diabetic and revertant mice (Fig. 4⇓A). The degree of ongoing overall inflammation, as reflected by ICAM-1 expression, was much stronger in diabetic animals, possibly due to a higher rate of cellular infiltration (Fig. 4⇓A). In this context, it may be important to add that induction of TNF-α in β cells itself leads to enhanced expression of ICAM-1 in and around islets, as displayed in a pancreas section of an uninfected RIP-gp-TNF-α mouse 2 mo after removal of Dox food (Fig. 4⇓B). In contrast, only minor expression of ICAM-1 could be detected in pancreas sections of an uninfected mouse that was kept on Dox throughout the experiment (Fig. 4⇓B). Furthermore, LCMV infection alone can lead to enhanced presence of ICAM-1 expression in and around the islets, likely as a direct result of the primary anti-LCMV-driven inflammatory process (Fig. 4⇓B).
RIP-gp-TNF-α double-transgenic mice clear LCMV and generate a primary LCMV-specific immune response similar to single-transgenic RIP-gp mice
We tested whether the presence of the additional transgene (tTA) that is under the control of the same promoter as the viral gp gene (RIP) affected the antiviral immune response following LCMV infection, which could have influenced overall incidence of diabetes. Therefore, a series of experiments was conducted. First, viral clearance of LCMV infection was tested. RIP-gp, RIP-gp-TNF-α, and wild-type C57BL/6 mice were infected with 105 PFU LCMV-Arm. Mice were euthanized after 5 or 13 days, and the pancreata, livers, kidneys, and spleens were removed and examined for viral presence in a plaque assay. At day 5, viral organ titers were comparable in all groups. After 13 days, no virus was detectable in any organs from all groups of mice described previously for i.p. LCMV infection (data not shown). Thus, as expected, control of LCMV infection is not affected by expression of the two transgenes in β cells.
In the second experiment, LCMV-specific anti-self CTL activities were analyzed at day 7 after LCMV infection. Three groups of mice were used for these studies: RIP-gp mice, RIP-gp-TNF-α mice receiving a Dox diet, and RIP-gp-TNF-α mice receiving a Dox-free diet for 2 mo. Splenocytes harvested ex vivo were directly incubated with uninfected H-2b target cells, with cells coated with the immunodominant LCMV CTL peptide gp33, or with LCMV-infected target cells, and cytotoxic activity was measured in a standard 51Cr release assay. Similar to splenocytes isolated from infected RIP-gp mice, cells from RIP-gp-TNF-α mice specifically lysed LCMV-infected and gp33-coated but not uninfected C57BL/6 H-2b fibroblasts (Fig. 5⇓). In contrast, no significant killing was observed when LCMV-infected or gp33 peptide-coated BALB/c (H-2d) fibroblasts were used as target cells (data not shown). In addition, removal of Dox 2 mo before LCMV infection had no influence CTL activity at 7 days after LCMV infection (Fig. 5⇓). Thus, the presence or absence of Dox as well as islet-specific TNF-α early after LCMV infection did not influence the magnitude of the anti-LCMV response.
Frequency and effector functions of experienced LCMV-specific CD8 lymphocytes is reduced in revertant RIP-gp-TNF-α mice
We defined the cause for the reduction of diabetes in mice with late-induced TNF-α expression (Dox removal at day 10). Spleens of such mice were harvested at wk 6 after infection to assess LCMV-specific CTL activity. They were cultured for 8 days on LCMV-infected irradiated peritoneal exudate cells before testing CTL activity in a 51Cr release assay. MHC class I-matched (H-2b) and mismatched (H-2d) fibroblasts that were either infected with LCMV or coated with the immunodominant gp33 peptide were used as target cells. No significant lysis of MHC class I-mismatched target cells could be detected (data not shown). Splenocytes from diabetic double-transgenic RIP-gp-TNF-α mice and single-transgenic RIP-gp mice exhibited similar CTL activity against LCMV-infected or gp33-coated target cells. In contrast, killing of both LCMV-infected and gp33-coated target cells was significantly lower when splenocytes obtained from revertant RIP-gp-TNF-α mice were tested (Fig. 6⇓A). Thus, autoreactive CTL activity is reduced after induction of TNF-α and correlates with the reduction of insulitis (Fig. 4⇑) and prevention of autoimmune disease.
To further compare CD8 lymphocytes found in diabetic vs revertant mice, RIP-gp-TNF-α mice were infected with LCMV and received a Dox-free diet at day 10 after infection. Similar to the previous experiment, mice were scored into diabetic and revertant gourps at wk 6 after infection. Spleens and PDLNs were probed for LCMV-specific production of IFN-γ and TNF-α by flow cytometry after stimulation in vitro for 5 h with LCMV-gp33 peptide. Control staining of cells obtained from spleens or PDLNs of LCMV-infected RIP-gp mice revealed that CD8 lymphocytes harvested at day 7 after LCMV infection produce predominantly IFN-γ. In contrast, experienced CD8 lymphocytes harvested at day 60 after infection produced IFN-γ as well as TNF-α (data not shown). Thus, the parallel production of both IFN-γ and TNF-α can be used as an indication for the presence of more experienced CD8 lymphocytes, whereas a predominant IFN-γ production characterizes inexperienced CD8 lymphocytes found directly after their initial phase of activation (28, 29, 30). In splenocytes of either revertant or diabetic RIP-gp-TNF-α mice, we found CD8 lymphocytes of the more experienced phenotype producing both IFN-γ and TNF-α (Fig. 6⇑C), and the mean ratio of experienced (IFN-γ- and TNF-α-producing) to inexperienced activated (IFN-γ-producing) CD8 lymphocytes in all analyzed mice was not significantly different (Fig. 6⇑D). However, in PDLN cell suspensions, we detected a clear difference between revertant and diabetic RIP-gp-TNF-α mice, and CD8 lymphocytes from revertant mice exhibited a more inexperienced phenotype reflecting their recent activation, whereas CD8 lymphocytes from diabetic mice were of the more experienced phenotype (Fig. 6⇑C). On average, the ratio of experienced to inexperienced CD8 lymphocytes in all analyzed mice was >5-fold lower in revertant mice (Fig. 6⇑D). In addition, CD8 lymphocytes from diabetic RIP-gp-TNF-α mice had a higher density of surface CD8 molecules compared with revertant RIP-gp-TNF-α mice, as manifested by an ∼1.5-fold higher mean fluorescence intensity (data not shown). Up-regulation of surface CD8 expression was previously demonstrated to be characteristic for experienced CD8 lymphocytes by others (30).
Furthermore, we analyzed the frequency of LCMV-specific CD8 cells in revertant vs diabetic mice using H-2Db-gp33 tetramers. Cells were harvested from the spleens and the PDLNs of five revertant or diabetic mice at wk 6 after infection and were stained for CD8 and H-2Db-gp33 reactive TCR. Revertant mice had a reduced frequency of gp33-specific CD8 lymphocytes (Fig. 6⇑E) in the PDLN. In contrast, no significant difference could be detected in the spleen (Fig. 6⇑E). The mean frequency of gp33-specific CD8 lymphocytes in the spleen was ∼4–5% in both diabetic and revertant mice (Fig. 6⇑F). However, revertant mice had a mean gp33-specific CD8 lymphocyte frequency of only ∼2.5% in the PDLN, a value that is considerably lower than the ∼9% found in diabetic mice (Fig. 6⇑F).
Possible explanations for these findings include lack of generation, migration away from the PDLN, and deletion of experienced LCMV-specific CD8 lymphocytes in revertant, but not in diabetic, RIP-gp-TNF-α mice.
Apoptosis of self (gp)-reactive CD8 lymphocytes is the probable mechanism for abrogation of diabetes after induction of TNF-α
Apoptosis of self (gp)-reactive CD8 lymphocytes could be an explanation for the abrogation of diabetes in RIP-gp-TNF-α mice, because TNF-α was demonstrated to directly induce apoptosis in CD8 lymphocytes by binding to either of its receptors, TNFR55 (TNFR1, CD120a) (31) or TNFR75 (TNFR2, CD120b) (32). TNFR at the cell surface is the starting point of a pathway that may ultimately lead to apoptosis via activation of the caspase cascade (33). Because we observed a systemic reduction in autoreactive CTL combined with local lack of experienced CTL in the PDLN, we determined the number of apoptotic cells in the pancreata and PDLNs of revertant and protected mice. Indeed, TUNEL analysis revealed an increased number of apoptotic cells in pancreata of revertant RIP-gp-TNF-α mice that were protected from diabetes compared with diabetic controls (Fig. 7⇓A). Islets of histological sections of three mice per group were examined for the number or apoptotic cells per islet. The overall frequency of islets containing apoptotic cells, as well as the average number of apoptotic cells per islet, was significantly increased in revertant compared with diabetic RIP-gp-TNF-α mice (Fig. 7⇓B).
This is, in our opinion, the first situation in which completely opposite effects of islet-specific TNF-α on the ongoing diabetogenic autoimmune process have been demonstrated in the same in vivo model. When expressed late, TNF-α can abrogate diabetes, whereas early TNF-α production is required for and seems to enhance disease development. The use of a transgenic mouse model in which we could experimentally control both the initiation of the autoimmune process (LCMV infection) and the time of expression of an important inflammatory factor (Tet-TNF-α system) allowed us to precisely define the late prediabetic period during which TNF-α is capable of abrogating the ongoing autoimmune process. The paradigm that too much of an inflammatory cytokine can collapse an autoimmune reaction at a certain point is going to be important. Indeed, similar effects are suspected for other cytokines (i.e., IFN-γ). IFNs usually enhance diabetes (34) and break tolerance to self Ags (35). However, IFN-γ-mediated intraductal islet regeneration can also occur and counterbalance this detrimental effect by dampening diabetes development (36). Further, IFN-γ can lessen the effect of viral infections (37) that are otherwise capable of exacerbating disease (38). It would reach too far to attribute a therapeutic effect to these cytokines at this point; however, their blockade is time sensitive, and precise knowledge of the distinct phases of an individual autoimmune process might be required.
In the RIP-gp mouse model, for fast-onset type 1 diabetes, the pathogenesis is precipitated predominantly by the action of self (gp)-reactive CD8 lymphocytes with lytic as well as cytokine-secreting activity (15, 18). Possible mechanisms for how islet-specific TNF-α expression might reverse the ongoing autoaggressive action of CD8 lymphocytes include 1) induction of regulatory cell populations, 2) migration of CD8 lymphocytes away from the islets, and 3) induction of apoptosis resulting in depletion of autoaggressive T lymphocytes. We found that the overall infiltration of islets by CD8 (and CD4) lymphocytes is reduced in revertant RIP-gp-TNF-α mice in which TNF-α was switched on late during pathogenesis compared with nonprotected diabetic animals. At wk 6 after infection, the number of experienced LCMV-specific CD8 lymphocytes able to express both IFN-γ and TNF-α was much lower in revertant RIP-gp-TNF-α mice (Fig. 6⇑, C and D), suggesting a disappearance of those cells since their generation at 1–2 wk after infection (Fig. 6⇑, E and F). This is further supported by the finding that revertant RIP-gp-TNF-α mice had decreased LCMV-specific CTL activity (Fig. 6⇑, A and B). It is striking that numbers of autoaggressive lymphocytes were only reduced significantly in PDLNs (Fig. 6⇑F) and not spleens but that their systemic lytic activities (Fig. 6⇑B) were found to be reduced. A similar segregation between lytic effector function and numbers of CTL present has been observed previously in persistently LCMV-infected mice (39) and is an interesting phenomenon validating further exploration.
No evidence for production of regulatory cytokines was detected in RPAs comparing protected with diabetic mice (identical levels of TGF-β, IL-4, and IL-10; data not shown). Furthermore, entry of lymphocytes into the pancreas was not inhibited because insulitis occurred and ICAM-1 expression was enhanced by TNF-α, which should have facilitated lymphocyte entry. Thus, the data obtained from our RIP-gp-TNF-α model can be used to suggest the following hypothesis of protective as well as detrimental TNF-α effects in autoimmune diabetes. By infection with LCMV, the autoimmune process (diabetes) is initiated as reflected in early (day 2) activation of APCs and up-regulation of MHC class I in islets (16). Next, self (gp)-reactive CD8 lymphocytes remain in the pancreas after the elimination of the virus itself and start destroying (day 7) the insulin-producing β cells (expressing gp at the cell surface). This stage coincides with the peak of endogenous TNF-α expression, and, if transgenic TNF-α is induced in addition, disease kinetics are enhanced. During the following 2 wk (8–21 days after infection), islet infiltration slowly increases, and β cells die as a consequence of lytic CTL killing as well as cytokine-mediated effects (15). If transgenic TNF-α is expressed at this late time during pathogenesis, the autoimmune process collapses, and 60% of the mice are either protected or revert to normoglycemic BG values. Judging from our data, it is most likely that this increase in TNF-α concentration locally in the islets results in the deletion of experienced, activated self (gp)-reactive CD8 lymphocytes (Fig. 6⇑, C and D) by induction of apoptosis, leaving behind a reduced population of self (gp)-reactive CD8 lymphocytes (Fig. 6⇑, E and F) that are inexperienced and have a lower amount of lytic CTL activity (Fig. 6⇑, A and B). Indeed, the frequency of apoptotic cells was higher in islets from revertant mice than in diabetic mice. Possible mechanisms, such as TNF-α-induced apoptosis, that cause abrogation of disease are likely to precede the actual read out for revertant mice as manifested in nondiabetic BG values. Therefore, the nature of the experiments conducted might have made it impossible for us to analyze the actual peak of the mechanisms involved.
In conclusion, autoimmune processes as involved in type 1 diabetes, and other diseases are at least initially driven by an inflammatory event. Mediators that are traditionally termed proinflammatory, such as TNF-α, often play a major role during this initial phase of inflammation and promote rather than suppress the ongoing destruction (2, 4). Here, the precise duration of TNF-α expression is an important factor determining the progress of an ongoing autoimmune process to diabetes (8). However, later during pathogenesis, too much of such inflammatory factors is not beneficial to the propagation of the autoimmune process. This could occur in vivo, for example, by a second infection by the same or another unrelated virus capable of inducing inflammatory factors. Indeed, we have recently observed such a scenario in our RIP-LCMV model, in which secondary viral infection can abrogate ongoing autoimmunity by enhancing apoptosis of autoreactive lymphocytes (M. G. von Herrath and U. Christen, unpublished observations). Furthermore, TNF-α was demonstrated in various other occasions to confer immunoregulatory activities by down-regulation of type 1 cytokines (40), suppression of T cell proliferation and cytokine production (5), or preventing the development of self-reactive T lymphocytes (3). Thus, one has to postulate that inflammatory cytokines exist in a fine-tuned time-sensitive equilibrium, because we showed here that TNF-α has opposite effects in the same animal depending on the time of expression. Future studies on the role of cytokines in autoimmunity and other diseases should attempt to include the element of timing as a central factor that determines effector function and outcome.
We thank D. Frye for assistance with the manuscript preparation.
↵1 This work was supported by National Institutes of Health Grants R01AI4451 and R29 DK51091 (to M.G.v.H.). U.C. is supported by Juvenile Diabetes Fellowship Award 3-2000-510. This is Publication Number 13619-NP from the Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA.
↵2 Address correspondence and reprint requests to Dr. Matthias G. von Herrath, Department of Neuropharmacology and Immunology, IMM6, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: matthias{at}scripps.edu
↵3 Abbreviations used in this paper: RIP, rat insulin promotor; Tet, tetracycline; NOD, nonobese diabetic; LCMV, lymphocytic choriomeningitis virus; TetO, Tet operator; tTA, Tet-responsive transactivator; Dox, doxycycline; RPA, RNase protection analysis; RIP-tTA, tTA under the control of RIP II; Arm, Armstrong strain; BG, blood glucose; PDLN, pancreatic draining lymph node; LTβ, lymphotoxin-β.
- Received January 29, 2001.
- Accepted April 2, 2001.
- Copyright © 2001 by The American Association of Immunologists


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I am an actor with film and stage experience, and would love to offer my vocal skills to your production! As an avid reader of sci-fi and fanfic, I have a very soft spot in my heart for fan-made films and parodies, as well as space epics. A bartender by day, I can appreciate a good story, terrible dad jokes, and creating a world separate from this one to entertain others. Let's create amazing stories together!
My vocal range is a mezzo-alto, going from B2 up to F5. I am fluent in English, decent with both Chinese and Spanish, and am beginning Italian. I can also do accents as well as vocally pass as both male and female.
I am normally available for conference during the evening (US Eastern Time) but can adjust to fit your schedule, if given enough notice ahead of time!

subset: commoncrawl


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Operation X is an special stage available in Ace Combat X: Skies of Deception. It is unlocked by completing all of the game's missions. The game's equivalent to "The Gauntlet" from Ace Combat Zero: The Belkan War, Operation X has players engage AI-controlled versions of the game's fictional aircraft, such as the XFA-24A Apalis and the YR-302 Fregata.
A non-canon mission, the player's mission in Operation X is to fight through formations of all of Ace Combat X's original aircraft, except for the Fenrir. As you progress through the mission, enemies will begin spawning all around your position, often overwhelming you in higher difficulties. Once the X-02 wave is destroyed, you will be engaged by a squadron of four ADF-01 FALKEN aircraft, the main targets of the mission, two of which are armed with TLS laser pods. Destroying the FALKEN formation will finish the mission.
In Normal mode and higher, once a single ADF-01 remains, two unidentified aircraft will engage the battle from both sides of the map, the aircraft in question being the aces ADF-01 "Z.O.E." and XFA-27 "Scarface" from Ace Combat 2, their respective aircraft bearing their original paintjobs (simple red and white with red trim, respectively.) Killing both pilots will unlock their respective aircraft's Special paint scheme.
Mission overview
- 2x XFA-24A Apalis
- 2x YR-302 Fregata
- 2x XR-45 Cariburn
- 2x YR-99 Forneus
- 2x XFA-27
- 2x X-02 Wyvern
- 4x ADF-01A/F FALKEN (TGT)
- 1x XFA-27 "Scarface 1"
- 1x ADF-01A/F Falken "Z.O.E."
- First playthrough: ADF-01; all Leasath 1 paint schemes for all original aircraft except FALKEN and Fenrir if S-ranked.
- Operation X marks the first occasion where an enemy-controlled ADF-01 is able to use the TLS. Unlike the player, however, the TLS-wielding FALKENs will often hit with quick swipes that deal low to medium damage, instead of outright killing the player.
- All the enemy aircraft are the ones that can be tuned in the Hanger for the player.


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Christmas is coming. Santa was delivering presents to cute children. But these gifts were very naughty and scattered in different places. Can you help Santa to pick up the presents? Be careful about the ice pitons! They are very dangerous. Make sure to avoid them. Good luck!
Instructions: tap or click arrow key to play
Category: Mobile Games
Plays: 1016
Tags: Avoid, Boy, Christmas, Collecting, Highscore, Ice, Kids


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Watch Will Ferrell choke Mike O'Brien in new 7 Minutes in Heaven clip
A lot of emotions can flare to the surface when trapped in a tight closet. The latest episode of Mike O'Brien's 7 Minutes in Heaven webseries, for example, stars Will Ferrell and covers a wide spectrum of feelings. The interview starts in a nostalgic mood, as O'Brien asks the California-born Ferrell, "Is there any truth to the saying that if you grow up in Irvine, Calif., you basically grow up in a Dell Taco restaurant, you ride your bike around in flannel shirts, you drink cans of beer, you lose your virginity in a mall, and you respect the band Soundgarden?"
The next emotion is straight-up pain, thanks to a new game called Meme This Choke. Ferrell chokes O'Brien for several minutes so that the next time someone wants to create a meme about their displeasure at someone, they have a handy photo at the ready. Mission accomplished.
In the grand tradition of 7 Minutes in Heaven, the situation gets more awkward as it progresses. When O'Brien asks about Ferrell's parents' divorce, the actor responds, "What do you think, a–hole?"
Eventually they kiss and make up, thanks to O'Brien cheerfully bringing up Anchorman.
Watch the clip below.
7 Minutes in Heaven


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Listen To The NAG BUZZ Podcast HERE!
This week we hear from guests Claudia Bracco and Mike Philp, local podcast addicionados and lifestyle hackers extraordinaire. We cover the coming week's events, including the Belgian Beer Festival and long-awaited next installment in The Sexy Parties legend, The Pink Sexy at Club Mago on April 28th.
Podcast Sponsored by:
Natural English School, The Sexy Parties, and NAGMAG
In addition to being from the land of roos and barbies, host Dan Speight has something in common with guests Claudia and Mike. And it's not his favorite sex position. In fact, believe it or not, the conversation this week didn't even get that far below the belt. So when you think about it you can't completely rule that one out. But these three have developed a shared love for The Joe Rogan Experience Podcast and all the inspirational, hilarious, motivational, entertaining, fascinating, mind-expanding and lifestyle-improving topics that emerge from the whirlpool of cultural wisdom that is the man, Joe Rogan. We've been profoundly affected since tuning into Rogan's podcast, and continue to glean priceless pearls from the many hours of free entertainment and education made available every week. This is a highly recommended resource for anyone interested in bettering themselves, getting fit, eating healthy, and finding no-nonsense alternatives to the run-of-the-mill media content you tend to be bombarded with when you turn on a television or pick up a newspaper. Plus, a healthy measure of tongue-in-cheek bullshit to keep you laughing.
Below is a list of some of our favorite past JRE guests and related links.
Stanley Krippner – Professor of Psychology at Saybrook University in San Francisco. humanistic psychologist, having written extensively on
dreams, altered states of consciousness, hypnosis, shamanism, dissociation, and parapsychological subjects
Justin Wren – pygmies congo. fight for the the forgotten raised 53grand.
Dave Asprey -mycotoxins
Neil Degrasse Tyson – quantum physics, stephen hawking doco http://www.youtube.com/watch?v=1zblTCsThDE
Dr Amit Goswami – quantum physics
Tim Ferriss – author of popular books, The Four Hour Work Week, The Four Hour Body, The Four Hour Chef
Cara Santa Maria – American neuroscience enthusiast and science educator. She was the senior science correspondent and host of the "Talk Nerdy to Me" series for The Huffington Post until April 2013
Les Stroud – survivorman http://en.wikipedia.org/wiki/Survivorman
Peter Duesberg – with theory that HIV does not cause AIDS.
Phillip Coppens – ancient aliens founder who died of cancer since being on the podcast.
Duncan Trussel – comedian writer actor
Recent Duncan Trussel explanation on the philosophy of how your reality is created: here, which is taken from the philosophy of Indian-born Jiddu Krishnamurti
Famous Duncan Trussel rant: Killing the vampires in Your Life
Kat Von D – tattoo artist
Immortal Technique – rapper
Aubrey Marcus – Supplement company CEO (use the code name sp8 for a 10% discount!)
Michael Ruppert – ex-cop turned investigative journalist and writer.
Freeway Rick Ross – ex-drug dealer who unknowingly sold cocaine for the CIA
Everlast – musician
Ronda Rousey – MMA fighter, UFC World Champion
Daniele Bolelli – Italian writer, martial artist, and university professor. He is the author of On the Warrior's Path, one of the modern classics on the philosophy of martial arts
Graham Hancock – writer, journalist, psychedelic adventurer
Bert Kreischer – comedian
Joey Diaz – comedian and ex-criminal
David Choe and Eddie Huang
Shane Smith – from popular online documentary website Vice.com
Recommended podcast management app for smartphone: Downcast
The Obvious Circus with Daniel Ostrander: This Week's Events (with cameo appearance from NAGMAG media magnate Carter Witt)
The Pink Sexy, April 28th at Mago, 7pm-1am


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- Published: 26 March 2019
Join State Senators Julie Morrison and Ann Gillespie and State Representatives Mark Walker and Jonathan Carroll for a Town Hall discussion on Cannabis Legalization.
The event is being held on Saturday, March 30 from 10:00a until 11:30a at the Mt. Prospect Public Library (10 S. Emerson Street, Mt. Prospect).
Contact Senator Morrison's office in Deerfield at (847) 945-5200.
Click on the flyer for more information


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We specialize in working with qualitative researchers to transform their media files into actionable insights.
Try it for free . NO CREDIT CARD REQUIRED.
4 Steps to take before transcribing the data in your qualitative research
by Diana Carrillo / Landmark Associates Inc. / Transcription / 23/09/19
One of the biggest logistical challenges in qualitative research is the transcription of recorded video or audio data into a written document that can be coded, organized and analyzed. While on the surface, data transcription may seem like a pretty straight forward process, there are actually several considerations you need to have in mind before you settle on a particular source or method of transcription. These considerations multiply in importance when you are dealing with many hours of recorded data and if you are working in multilingual settings or with international teams.
The very first thing you need to do is to be clear about the type of transcription you will need for your research. There are basically three main types of transcription to choose from:
• Verbatim
• Intelligent
• Edited
If you are working on a tight budget and can't assign someone in-house to take on the transcription, then you can choose an automated transcription platform which relies on speech recognition technology to convert audio to text. While this is definitely the quickest and easiest option, you may experience problems with the resulting transcript's accuracy since automated systems are currently limited particularly where the audio quality is poor, or there are heavy accents, industry jargon, and numerous people speaking simultaneously.
We specialize in working with qualitative researchers to transform their media files into actionable insights.
Try it for free . NO CREDIT CARD REQUIRED.


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I usually don't upgrade my iPhone to the latest OS right away, as you know there is going to be 9.0.1 and 9.0.2 and so on in the upcoming weeks. But since the Discover Apple Pay promo required it, I upgraded immediately this time.
One of the new features of iOS 9 is something called WiFi Assist. Previously, your iPhone would try to cling onto that WiFi signal as long as it could before finally giving up and switching to cellular data. Sometimes it would interrupt your Netflix or Spotify streaming. If you found that annoying, the good news is that WiFi Assist will now seamlessly transfer you earlier onto cellular 3G or 4G data.
However, I liked the old way. If I have weak WiFi and my ESPN3 video won't stream, I don't want it to seamlessly switch to my cell data. I want it to cut off and let me know. Video streaming at high-definition can use up 1-2 gigabytes per hour. That's $15 to $30 an hour at my overage rates. Some folks argue an early handoff won't cause a "massive" overage, but I want the choice of either moving to improve the WiFi signal or turning it off. (With WiFi Assist on, your only indicator will be a greyed-out WiFi icon, if it is even visible)
WiFi Assist is turned on by default in iOS 9. You might like this feature, or you might not. If you also manage your cell data somewhat carefully, you may decide to turn off this feature off. Here's how:
- Open Settings.
- Tap on Cellular.
- Scroll down to the bottom.
- Turn Wi-Fi Assist off.
More: Ting Blog, Houston Chronicle


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Samarpan Dhyan Aur Aarti New
Inspired By H.H.Shivkrupanand Swamiji
The MP3 begins with a track containing Guru-Mantra in H.H. Swamiji's voice. The period of meditation is ended by prayer in H.H. Swamiji's voice. After meditation there is a track of Aarti which is sung by H.H. Guruma.
Available in : Hindi, & German
Samarpan Dhyan Aur Aarti New
- Brand: Babaswami Printing & Multimedia Pvt. Ltd.
- Product Code: Meditation 3
- Availability: In Stock