Treg-independent manner . Furthermore, Foxp3 2 Tr1 cells that produce immunosuppressive IL-10 have been strongly implicated in modulating allergic responses ,. Concomitantly, IL-10 plays a non-redundant role in maintaining tolerance at mucosal surfaces including the lungs . Altogether these studies Figure 5. No influence of genetic susceptibility on the effect of Treg depletion duringallergen challenge. (A) DEREG-C57BL/6 mice were sensitized to OVA with Alum on day 0, day 14 and day 21. One week post last sensitization DT was administered to mice i.p. Mice were challenged with 30 mg OVA via intra-nasal route for two consecutive days and analyzed 24 hr post last challenge. (B) Total cellular infiltration in BAL was determined by live cell counting using trypan blue exclusion dye. (C) Differential cell count was performed on Diff-Quik stained cytospots made from BAL cells. 200 cells were counted from random fields under light microscope in a single blinded manner. Data shown is a representative of three individual experiments using 3–6 mice in each group. Each dot represents data from individual mouse. Horizontal bar indicate mean value. Mann Whitney test was used to determine statistical significance. n.s. = non-significant.
A previous study showed a critical role of Fc receptor gamma chain in the sensitization phase ofallergicairwayinflammation . In the present study, absenceof FccRIIb increased levels ofallergen specific IgE after sensitization. This indicated that FccRIIb can specifically attenuate IgE humoral responses, suggesting its specific regulatory role in allergic lung inflammation. IgE production by the differentiating B cell requires class switch recombination (CSR) to Ce that is CD40 and IL-4 depen- dent[67,68]. FccRIIb deficient splenocytes made similar amount of IL-4 as wild type splenocytes in allergen recall assay. Thus, T cell-secreted IL-4 might not be involved in the FccRIIb-mediated suppression of Ce class switch. One possibility is that FccRIIb suppresses CD40L expression on Tcells thus reducing the stimulus for IgE class switch. Another mechanism might involve regulation of IgE production by DCs. CSR in B cells is regulated by the expression of BAFF (BLyS) and APRIL on DCs[69–71]. One report showed inhibition of B cell IgE production by DCs via direct cell-cell interaction as well as by soluble factors including TGF-b and IFN-c. It is possible that FccRIIb expression affects the ability of DCs to regulate IgE production by B cells. Yet another possibility is that the enhanced IgE response in FccRIIb deficient mice is independent of the Th2 T cell response.
In the absenceof adjuvant, stimulation of TLR4 on airway structural cells has been shown to be necessary for priming of innate immune responses and for the development ofairway disease in response to inhaled house dust mite allergen (HDM).  Indeed HDM and LPS airway sensitization and challenge reduced Th2 responses and enhanced Th1 inflammatory respons- es, increasing both TNFa and IL-17 secretion.  Recently, Tan et al., have also reported that TLR4 expression on stromal cells promotes Th2-biased allergic sensitization to OVA via the airways and the development of subsequent airway disease.  The levels of contaminating LPS in our study are comparable to the low- moderate level referred to by Tan et al. Here, we show that systemic OVA sensitization in conjunction with an exogenous adjuvant (alum) leads to AHR that is largely independent of the contaminating endotoxin and TLR4 signaling. However, our data reveal that although i.p. OVA sensitization with alum can elicit subsequent allergicairway disease independently of TLR4, as has been previously reported, this doesnot mean that the contami- nating LPS in OVA and associated TLR4 signaling are devoid of any influence upon the ensuing allergen challenge-associated inflammatory response in an alum-dependent OVA-asthma model. Indeed, observations regarding the phenomenon of LPS tolerance by both Peters et al  and more recently by Chapman et al,  elegantly demonstrate the significant effect of repeated exposure to LPS and the respective LPS levels have on the cytokine levels and allergic lung response. LPS tolerance also results in long lasting effects on murine macrophage gene expression and phenotypes.  Therefore, our study provides a degree of refinement in our understanding of the mechanisms underlying such experimental asthma models. Specifically, in agreement with Tan et al., despite the sensitization with alum adjuvant, we find that the associated Th2 inflammatory response remains partially dependent on the contaminating endotoxin present with OVA allergen challenge. Significantly, the inflam- matory cytokine, Tregulatory cell, and neutrophil responses Figure 5. LPS-rich OVA induces a cytokine response in human PBMCs similar to LPS. PBMCs were stimulated for 20 hrs with saline, LPS- free OVA, LPS-rich OVA and LPS, the supernatants were the analysed by MSD electrochemical ELISA for the levels of secreted cytokines, (a) IL-12p40, (b) TNFa (c) IFNc, (d) IL-1b, (e) IL-2, (f) IL4, (g) IL-5 and (h) IL-10. Values are shown as Mean 6 SEM. (n = 6). Statistical significance was determined using ANOVA.
of Treg cells isolated from peripheral blood and or bone marrow could have impact in MM outcome. In Giannopou- los et al.  study, patients were divided into two cohorts according to median Treg frequency. Those with a high per- centage of Treg lived significantly less (median overall sur- vival [OS] of 21 months) as compared with those with lower Treg frequency (median OS not reached at median follow-up of 32 months). The difference in survival was also observed when only non-transplanted patients were analyzed. On the other hand, patients submitted to autologous transplant pre- sented no difference in OS when both groups of Treg fre- quencies were compared . Raja et al.  evaluated Treg cells in both peripheral blood and bone marrow in a larger number of cases of MM using flow cytometry. They found a significantly elevated frequency of Treg cells in a newly diagnosed and relapsed MM patients compared with healthy controls. Functional studies showed that Treg cells from both MM and controls were similar in their inhibitory function. Using cutoffs very close to Giannopoulos et al. group , they showed that MM patients with high percentage of Treg cells had inferior time to progression and this variable had independent prognostic impact by multivariate analysis . Unfortunately, our study could not confirm the above results using two Treg-related gene expressions.
cohort consists of 200 households across two study sites, the Nagongera sub-county in Tor- oro district and the Walukuba sub-county in Jinja district. Description of the study and results have been published in  In all households, one adult caregiver and all eligible children aged 6 months to 11 years were enrolled into the study. Samples used here were taken from cross-sectional bleeds of study participants taken between August 2013 and March 2014. Household-level mosquito exposure was calculated based on mosquito counts obtained from CDC light traps placed monthly within the household of each individual trial participant in the 2012 .
The authors of the published article have identified concerns about the reliability of a subset of the reported data, specifically the data underlying Figures 1 and 2. Potential discrepancies have been discovered between the machine-generated raw data and the data for publication pro- vided by the murine pulmonary function laboratory, and the authors have become aware of potential issues in methodology that could make the data unreliable. Figures 3 and 4 are not affected.
expression, we first characterized the iRBC-induced cytokine profile. As shown in Figure 9A, the pro-inflammatory cytokines IL-6 and IFNc and the anti-inflammatory cytokine IL-10 were induced as early as 24 h after the addition of iRBCs and remained at high levels throughout the duration of the co-culture. IL-2 levels in the supernatant peaked at day 2–3 of co-culture and then declined to barely detectable levels on day 5 and 6 (Figure 9A). None of these cytokines were detectable in the absenceof iRBCs. High levels of total TGFb1 (latent plus bioactive) were measured in the supernatant of untreated PBMCs on day 6 of culture, ranging from 0.6–13.5 ng/ml (23 donors; Med- ian = 3.48 ng/ml; data not shown). Interestingly, day 6 TGFb1 levels were significantly decreased when PBMCs were co-cultured with iRBCs (12 donors, P ,0.01 for 2:1 ratio and P,0.05 for 1:5 ratio, one-way ANOVA with Neuman-Keuls’ post-test; Figure 9B), but unchanged when PBMCs were co-cultured with uninfected RBCs (15 donors, data not shown). This decrease in total TGFb1 was already evident and significant from 24 hours of co-culture onwards (data not shown) and dependent on the amount of iRBCs added to the co-culture (Figure 9B; P,0.0001, one-way ANOVA with linear trend post-test). A decrease in total TGFb1 would be consistent with either increased conversion into bioactive TGFb and immediate consumption, or decreased production and secretion. To test the latter, TGFb1 mRNA levels for 2 donors were monitored over a time course of 6 days, showing no decrease in TGFb1 mRNA in iRBC co-cultured PBMCs compared to control cultures, but instead a 2–3 fold increase on day 5–6 of co- culture (data not shown), suggesting a possible counter-regulatory mechanism to enhanced TGFb consumption and therefore
sensitized offspring were anaesthetized with chloral hydrate (300 mg/kg, i.p.) and the trachea was exposed through a midline ventral incision of approximately 0.5 cm length in the neck. With the aid of a 26.5 gauge needle, 0.4 ml of 0.25% solution of OVA was injected into the airways. Immediately after this procedure, the animals were sutured and allowed to recover from the anaesthesia. Bronchoal- veolar lavage was performed 48 h after OVA chal- lenge. Briefly, the animals were again anaesthetized with chloral hydrate (300 mg/kg, i.p.) and exsangui- nated by cutting the abdominal aorta. The trachea was exposed and cannulated with a polyethylene tube (1 mm diameter) connected to a syringe. The lungs were washed by flushing with phosphate- buffered saline (PBS) solution containing heparin (20 IU/ml) and 0.03% serum albumin. The PBS buffer was instilled through the tracheal cannula as one 10-ml aliquot followed by three 5-ml aliquots. The fluid recovered after each aliquot instillation was combined and centrifuged (1000 g for 10 min at 20°C). The cell supernatant was discarded and the cell pellet was resuspended in 2 ml of PBS buffer. The total cell numbers were counted in T¨ urk’s solution while differential counts were carried out on air-dried smears stained with May–Gr¨ unwald – Giemsa. A minimum of 400 cells was counted and classified as neutrophils, eosinophils and mono- nuclear cells based on normal morphological cri- teria. The sensitization procedure here employed is efficient, since challenge of the trachea strips with OVA (10 mg) causes a significant contraction of the tissues obtained from OVA-sensitized rats, whereas strips obtained from non-sensitized animals do not show any contractile effect. 7
Cultures were also supplemented with varying amounts (0–2 ng/ mL) of recombinant human TGF-b1 (PeproTech, Rocky Hill, NJ) and 10 mg/mL of SA-4-1BBL or an equimolar concentration of SA protein as control. SA-4-1BBL and SA control proteins were produced using S2 cells with undetectable endotoxin levels . Cells were stained with Abs to CD4-APC (Clone RM4–5) or CD4- PE (Clone GK1.5), CD25-PE or CD25 PE-Cy7 (Clone PC61), and intracellular FoxP3-PE according to the manufacturer’s protocol (eBioscience). Isotype Abs with matched fluorochromes were used as controls. All samples were collected using FACSCalibur or BD LSR II (BD Biosciences) and analyzed using FlowJo (Tree Star) or FACSDiva software.
On the surface, we could simply accept that CD8+ T cell control of HIV infection acts via non-cytolytic mechanisms such as the production of cytokines and/or chemo- kines, and make that our new credo. Such a shift in beliefs would be supported by the demonstration that the ability of CD8+ Tcells to produce multiple cytokines is associated with good viral control in HIV, suggesting an important role for cytokines [9,10]. However, it is difficult to reconcile the results of the Klatt and Wong papers with other recent in vivo and in vitro results (see Table 1). First amongst these are studies of CD8+ T cell depletion in acute SIV infection [3,11]. These studies dem- onstrate that in the absenceof CD8+ Tcells, the virus level rises to its ‘‘normal’’ peak in acute infection, but then doesnot decline from the peak for a prolonged period. The simplest interpretation of this result is that, in the absenceof CD8+ Tcells, the peak number of infected cellsdoesnot decline, but remains constant because these cells are not dying. However, two other observations make this unlikely: First, the increased loss of CD4+ Tcells in CD8- depleted animals indicates that many cells are dying. Second, the Klatt and Wong results show that infected cells die at the same rate independent of the presence of CD8+ Tcells in chronic infection, so why would things be so different in primary
Allergicairway disease was induced using an adapted house dust mite model . Briefly, mice were sensitized to 10 μg of filtered house dust mite extract (HDM, GREER Labs) or saline through intraperitoneal (i.p) injection on days 0 and 7 followed by sensitization on days 14 and 15 with 5 μg HDM or saline administered intratracheally using a microsprayer (Penn Century). On day 17, mice were anesthetized by an i.p. injection of pentobarbital sodium (60 mg/kg). Fol- lowing tracheostomy, pancuronium bromide (0.25 mg/kg) was administered, and mice were ventilated on a small animal ventilator (flexiVent FV-FXM1; SCIREQ). Airway resistance was measured through forced oscillation techniques (flexiVent FV-FXM1; SCIREQ) over increasing doses of methacholine. Following procedure, a cardiac stick was performed to euthanize the mouse and collect blood, then whole lung lavage (WLL) was collected. Lung tissue was perfused with phosphate buffered saline then snap frozen in liquid nitrogen. Cytokines in the lung lavage
syndrome and die at 3–4 wks after birth [10,11]. We attempted to prolong their life by injecting galectin-1, which has been reported to suppress other autoimmune diseases , starting at day 7 of birth. P3U1-muTGF-b cells were injected i.p. every other day 5 times beginning at day 8 after birth, and spleen cells were taken at day 22 after birth and fused with P3U1 myeloma cells. The hybridoma cells were grown in hypoxanthine-aminopterin-thymi- dine (HAT)-supplemented methylcellulose medium. Approximate- ly 2,800 clones were picked from the plates, and transferred to hypoxanthine-thymidine (HT)-supplemented DMEM in 96-well plates. The culture supernatants were screened by surface staining of P3U1-muTGF-b cells by FACS. Thirty-six positive clones were selected and recovered (TW7 series) (Figure S2). Of the 36 clones, 32 clones were IgG and 4 clones were IgM. To check their specificity, we tested the ability of the antibodies to immunopre- cipitate Flag-tagged mouse LAP (Flag-mLAP) produced by retrovirally Flag-mLAP-transduced P3U1 cells. Of the 32 IgG clones, 26 clones, including TW7-16B4 and TW7-20B9, immu- noprecipitated Flag-mLAP (Figure S3, underlined) and thus were true anti-mouse LAP mAbs. Several clones, including TW7- 28G11, did not immunoprecipitate Flag-mLAP (Figure S3). TW7- 28G11, however, stained human latent TGF-b-coated beads, but not human LAP- or human active TGF-b-coated beads (Figure S4A). TW7-28G11 immunoprecipiated Flag-mLAP only when active TGF-b was exogenously added to Flag-mLAP solution (Figure S4B), and immunoprecipiated pro-TGF-b and latent TGF-b from the culture supernatant of P3U1-muTGF-b cells (Figure S5A). These results indicate that TW7-28G11 is a conformation specific anti-mouse/human latent TGF-b/pro- TGF-b mAb which recognize LAP and TGF-b in combination. The specificity of some clones, including TW7-16B4, TW7-20B9 and TW7-28G11, were further confirmed by testing their ability to detect mouse pro-TGF-b and/or LAP by Western blot (Figure S5B), and by their ability to immunoprecipiate pro-TGF-b and latent TGF-b from culture supernatant of P3U1-muTGF-b cells (Figure S5A).
Diminished thymic function has been associated with inflammatory diseases [13,14,17] due to compensatory proliferative mechanisms in the periphery and increased Th1 and Th17 responses in relation to a dysfunctional Treg activity. Additionally, lower output of thymic- dependent naturally occurring Tregs may result in lower suppression of inflammatory responses in the periphery. Our study revealed hypermethylation of the FOXP3 promotor region—potentially resulting in reduced immunosuppressive Treg function—in female but not in male patients with panic disorders, corroborates the idea of a prematurely aged immune sys- tem in this particular patient subgroup. Although a strong sex bias of autoimmunity, with most autoimmune diseases predominantly affecting females, is well known [51,52], the underlying mechanisms are not well understood. The absenceof a second (inactivated) X chromosome in males, sex hormones, and sex-specific differences in gene regulation due to internal and exter- nal (i.e. environmental) factors, all can influence the susceptibility to disease. Particularly, hor- mone factors may explain the higher differences regarding methylation status of CpG regions within the FOXP3 promoter [53,54] and lower TRECs in females .
homologue deleted on chromosome ten (PTEN) and Src homology region 2 domain-containing inositol 59-phosphatase-1 (SHIP-1). SHIP-1 dephosphorylates PI(3,4,5)P3 to generate PI(3,4)P2 [7,8]. SHIP-1 is believed to be a negative regulator in a variety of cytokine, immunoreceptor, and growth factor signaling pathways in different cell types, including Tcells, B cells, mast cells, basophils, and neutrophils [8,9,10,11,12,13,14]. SHIP-1 deficiency as in gene-targeted deletion resulted in spontaneous inflammatory cell infiltration in the lung of some mice [11,12], which has been recently identified by our group as a Th2-like allergic inflammatory phenotype that may be related to enhanced mast cell response . Adoptively transferred SHIP-1 deficient mast cells were shown to enhance allergic and anaphylactic responses in vivo . In Tcells, SHIP-1 was reported to regulate cytokine activation in a way favoring Th2 response but limiting Th1 cytotoxicity . However, a regulatory role of SHIP-1 in adaptive immune response to allergen stimulation in the airway has not been established.
covering with a thin internal catheter and a balloon at the end and measured 170cm in length and 8mm in diameter. It was introduced into the esophagus through the right nostril up to the mediastinum, approximately 135-145cm from the nostril, and only then was the balloon tip inflated with 10mL of air pushed through a syringe. According to the technique described by KLEIN & DEEGEN (1984), accurate placement of the probe is confirmed by recording a sequence of 10 regular respiratory cycles. Differences in pressure measured by the balloon were transmitted pneumatically to the ventigraph through the probe and registered graphically on the recording paper provided with the equipment by the manufacturer. The recording paper speed was 2mm/s for 5min. Interpleural pressure differences ≤4cm H 2 O were considered as physiological, 5-8cm
(Magicmark, Invitrogen) on a NuPAGE 4–12% bis-tris gel (Invitro- gen). The proteins were electroblotted onto a PVDF membrane (Amersham Life Science). Unspeciﬁc binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 8C. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji). Membranes were incubated in stripping buffer and re-blocked for 1 h. The membranes were re-probed using anti-GATA3 (HG3–31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnol- ogy), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Tech- nology),
Ten micron thick cryosections of quadriceps, tibialis anterior, and triceps were stained with hematoxylin and eosin-phloxine as described . Four images from different areas of the muscle section were collected on a Zeiss Axiovert 25 microscope using ImagePro Software. These images were imported into Scion Image to determine the proportion of centrally nucleated fibers (800–1000 fibers counted/muscle) at 1, 3, and 12 month old mice (n = 4 for each genotype at each timepoint). Fiber diameter distribution was determined in 12 month old triceps sections as described  from at least 700 fibers/genotype. Membrane permeability was deter- mined by quantifying serum creatine kinase levels on Vitros CK DT slides (Ortho-Clinical Diagnostics catalogue number DT1975580) using a Kodak Ektachem DT60 analyzer.
Consistent with previous observations, HIF-1a protein levels in the nasal mucosa were substantially elevated in our mouse model of OVA-induced AR, suggesting that HIF-1a was activated. The increased HIF-1a levels were significantly reduced following administration of 2ME2. In addition, the increased VEGF levels in nasal mucosa following OVA challenge were also significantly reduced by administration of 2ME2 in vivo. Meanwhile, the titer of OVA-specific IgE was significantly lower following 2ME2 treatment. This decrease in IgE may have been caused by inhibitory effects on B-cell activation and reduced release of Th2 cytokines, which correlated well with the changes in IL-4 and IL-5 levels in NLF. IL-4 promotes Th2 cell differentiation and induces B cells to switch to IgE production . IL-5 increases eosinophilic inflammation and airway infiltration . As major Th2 cytokines, both of them are key mediators in allergicinflammation. In addition, eosinophils play a central role in the pathogenesis ofallergicinflammation . Our present findings show that 2ME2 prevented infiltration of OVA-induced inflammatory cells into the upper airways as shown by a significant drop in eosinophil counts in the nasal mucosa. Therefore, because of the reduction in nasal mucosa eosinophilia, IL-4 and IL-5 levels in NLF, and OVA- specific IgE levels in sera, 2ME2 treatment likely inhibits the nasal allergic responses. Because there was also a significant decrease in sneezing and nasal rubbing counts, this inhibition of nasal allergies may also contribute to the clinical alleviation of AR. Taken together, these findings suggest that HIF-1a inhibition by 2ME2 suppresses VEGF expression and greatly reduces nasal allergicinflammation.
Total RNA was purified from approximately 100 mg each of lung tissue by liquid phase separation using ISOGEN (NIPPON GENE) in combination with High-Salt Precipitation Solution (NIPPON GENE). The RNA concentrations were measured on a NanoDrop 1000 spectrophotometer (NanoDrop Technologies). The integrity of RNA was verified by denaturing agarose gel electrophoresis. The complementary DNA (cDNA) was synthe- sized from 1 mg each of total RNA in a 20 ml reaction volume using PrimeScript 1st strand cDNA Synthesis Kit (Takara 6110) with the Oligo dT primer according to the manufacturer’s instructions. An aliquot of cDNA was used as a template for quantitative PCR using SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara RR420) with the ROX dye passive reference on StepOnePlus Real-Time PCR Systems (Applied Biosystems; the Central Research Laboratory, Okayama University Medical School) operated in the relative gene expression mode using the ROX dye as a passive reference. The primers used are listed in Table 1. The primers were chosen from either PrimerBank  or qPrimerDepot , such that each amplicon spans at least one intron. The relative expression levels were calculated by the DDC T
for 4 d[14, 19]. In some experiments, the following were included in the coculture conditions: (a) Anti–human TGF-β1 (Peprotech) or rat IgG1 isotype control antibody (eBioscience) was added to cultures at a final concentration of 1 μg/ml. (b) Recombinant human TGF-β1 (Pepro- tech) at varying concentrations; and (c) RA receptor inhibitors LE540 (Wako Chemicals, Japan) and LE135 (Tocris Bioscience, UK), each at 1μM. The differentiated Treg cells in the 4 d co-cultures were identified by flow cytometry using FITC-conjugated anti-CD4 and APC-con- jugated anti-Foxp3 in a Treg cell identification kit (eBioscience). At the same time, the ability of the PB eosinophils to induce a Treg response during co-cultivation was also determined as above.