T-PCR analysis of FcγR expression on pulmonary DC Purified lung

T-PCR analysis of FcγR expression on pulmonary DC. Purified lung DC were taken up in TriZol® Reagent (Invitrogen, Karlsruhe, Germany), total RNA was isolated from frozen samples with a chlorophorm-propanol-ethanol extraction procedure and cDNA synthesis was carried out via reverse PF-02341066 manufacturer transcriptase (Qiagen, Hilden, Germany). Quantitative real-time RT-PCR analysis was performed with an iCycler® (Biorad, Munich, Germany) and QuantiTect SYBR® Green PCR kit (Qiagen) in order to determine the levels of FcγRI-IV mRNA, normalized to tubulin and using published FcγRI-III primers 33, 34. For detection of FcγRIV transcripts,

the following FcγRIV-specific primers were used:

sense, 5′-CAGAGGGCTCATTGGACA-3′; antisense, 5′-GTGATTTGATGCCACGGT-3′. The PCR condition was 95°C, 15 min one cycle, followed learn more by 94°C, 15 s, 52.5°C, 30 s and 72°C, 30 s for 40 cycles for all primer pairs. DC were isolated from mouse spleen or lungs as previously described 35–37. In brief, the organs were cut into small fragments, digested with collagenase and DNase I (Sigma) and enriched by gradient centrifugation using Nycodenz reagents (Axis-Shield, Oslo, Norway) with a density of 1.073 for lung DC and 1.077 for splenic DC. DC were then enriched by negative depletion using magnetic separation and an antibody cocktail containing anti-Gr1, anti-B220, anti-erythrocytes, anti-CD19 and anti-CD3. To prevent

DC maturation during the isolation protocol, the procedure was carried out on ice, with the exception of the initial 20 min digestion with collagenase/DNase, which was performed at room temperature. This protocol excluded B220+ “plasmacytoid DC” from the DC preparation 38. DC were labeled with CD11c (HL3, FITC or PE), CD4 (GK1.5, FITC or PE), and CD8 (53-6.7, APC) monoclonal antibodies (all BD Biosciences, Heidelberg, Germany). Lung DC were stained for CD11c and MHC class II (2G9), CD11b (M1-70), CD103 (M290) (all BD PharMingen, Germany), CD16 (275005, IgG2a, Alexa 647), CD32 (K9 361, IgG2b, Alexa 647), CD64 (290322, IgG2a, plus goat-anti-rat APC, Invitrogen) (all R&D Systems, Germany) or isotype control antibodies. Analytical and RAS p21 protein activator 1 preparative fluorescent-activated cell sorting was done on a FACSAria (BD Biosciences, San Jose, CA, USA), or a Mo-Flo (Cytomation, Fort Collins, CO, USA) instrument and sorts were usually 95–98% pure. Gating strategy for analysis and sort of lung DC and lung macrophages (CD11c+MHC class IIlow) is shown in Fig. 2B. For spleen-derived DC, dead cells were excluded by DAPI or PI-staining, and CD11c+ cells were gated and analyzed for CD4 and CD8 expression. BMDC were generated by flushing out the BM from tibia and fibula of B6 mice.

In this region, elongated tumor cells were observed radiating tow

In this region, elongated tumor cells were observed radiating toward a central vessel to form characteristic papillary structures. Immunohistochemically,

three cases showed strong reactivity for GFAP, and one exhibited Fludarabine in vitro weak reactivity. All cases were focally positive for epithelial membrane antigen, CD34 and D2-40, but negative for neurofilament protein (NFP). Several ultrastructural investigations have supported the ependymal origin of chordoid glioma. In some cases of immunoreactivity for NFP, some authors have supposed that chordoid glioma originates from a multipotential stem cell with glial and neuronal cell differentiation. With regard to the present four cases with immunoreactivity for D2-40 (an ependymal marker) and CD34 (undifferentiated neural precursors) and based on previously published data, we considered that the majority of chordoid gliomas had an ependymal origin, and that a small minority might have originated from a multipotential stem cell having ependymal and neuronal cell differentiation. “
“This chapter contains sections titled: Introduction Anatomy and Function of the Olfactory Mucosa and Olfactory Tract Preparation of the Olfactory Mucosa for Neuropathology Examination Special Procedures for Neuropathology Evaluation of the Olfactory

Mucosa Neuropathology of the Olfactory Mucosa and Olfactory Tract: Basic Principles Comparative Neuropathology Selumetinib of the Olfactory Mucosa and Olfactory Brain Toxicological Neuropathology of the Vomeronasal Organ References “
“CNS involvement by systemic Hodgkin lymphoma (HL) is quite rare, but the disease limited to the CNS is an exceptionally rare entity. The incidence of CNS-HL has been estimated at 0.2–0.5% of cases, but a more recent study has modified that figure to less than 0.02%. Like the conventional form, the diagnosis of primary CNS-HL rests upon distinct morphological and immunohistochemical characteristics, including diagnostic Reed-Sternberg cells, in addition to staging studies demonstrating a lack of disease elsewhere. The paucity Sodium butyrate of cases

in the literature precludes reliable clinical and demographic data, as well as a consensus on treatment and prognosis. We present two cases of primary cerebellar HL, one with 10-year follow-up, and a relevant review of the literature. “
“Aceruloplasminemia is characterized by progressive neurodegeneration with brain iron accumulation due to the complete lack of ceruloplasmin ferroxidase activity caused by mutations in the ceruloplasmin gene. Redox-active iron accumulation was found to be more prominent in the astrocytes than in the neurons. The most characteristic findings were abnormal or deformed astrocytes and globular structures of astrocytes. The lack of ceruloplasmin may primarily damage astrocytes in the aceruloplasminemic brains as a result of lipid peroxidation due to massive iron deposition.

9 and 7 0 pg mL−1, respectively Secretions of IFN-γ and IL-10 in

9 and 7.0 pg mL−1, respectively. Secretions of IFN-γ and IL-10 in response to a given antigen were considered positive when absolute concentrations were ≥100 and ≥29 pg mL−1, respectively, and E/C was ≥2 (Brock et al., 2004; Moura et al., 2004; Al-Attiyah & Mustafa, 2008). A positive response for both cytokines was considered strong

at ≥60%, moderate at 40% to <60% and weak at <40% (Mustafa, 2009a, b). The ratios of IFN-γ : IL-10 were calculated to determine Th1 vs. anti-inflammatory biases in response to Con A, complex mycobacterial antigens and peptides of RD1 and RD15. The ratios of ≥2 were considered to be Th1, <0.5 to be anti-inflammatory and 0.5 to <2 to be neither Th1 nor anti-inflammatory. Moreover, Th1 responses were considered strong, moderate and weak with IFN-γ : IL-10 ratios of >20, 5–20 and 2 to <5, respectively. The antigen-induced cell proliferation and IFN-γ secretion results CH5424802 with Con A, complex

mycobacterial antigens and peptide pools were statistically analyzed for significant differences between TB patients and healthy subjects using the nonparametric Mann–Whitney U-test for two independent samples. P-values of <0.05 were considered significant. In lymphocyte proliferation assays, Con A and the complex mycobacterial antigens were strong stimulators of PBMC from TB patients and healthy subjects, as indicated by high percentages of positive responders (83–100%) (Fig. 1a and https://www.selleckchem.com/products/ulixertinib-bvd-523-vrt752271.html b). Furthermore, the proliferation of PBMC

from TB patients was strong in response to RD1 peptide pool (70% positive responders) and weak in response to peptide pools of RD15 and all of its ORFs (<40% positive responders) (Fig. 1c). In healthy subjects, the RD1 peptide pool induced moderate responses (47% positive responders), whereas the peptide pool of RD15 and 1502 induced strong responses (70% and 63% positive responders, respectively), and RD1501, RD1504 and RD1505 induce moderate responses (40%, 43% and 43% positive responders, respectively) (Fig. 1d). Peptide pools of other ORFs of RD15 induced weak proliferation of PBMC (<40% positive responders) (Fig. 1d). Statistical analysis of the results showed that positive responses induced by RD15 and RD1502 were significantly higher (P<0.05) in healthy MycoClean Mycoplasma Removal Kit subjects than in TB patients (Fig. 1c and d). To further determine the secretion of Th1 and anti-inflammatory cytokines and their ratios in response to complex mycobacterial antigens and peptides of RD1 and RD15, we studied secretion of Th1 cytokine IFN-γ and the anti-inflammatory cytokine IL-10 with PBMC from 20 TB patients and 12 healthy subjects using FlowCytomix assays. The results showed that PBMC from both TB patients and healthy subjects secreted high concentrations of IFN-γ (median values=6727–10 986 pg mL−1) with strong responses to complex mycobacterial antigens (positive responders =92–100%) (Fig. 2a and b).

In the presence of polarizing cytokines, this APC-independent act

In the presence of polarizing cytokines, this APC-independent activation regimen generated effector T cells producing equivalent amounts selleckchem of IFN-γ and IL-17, irrespective of the naive T-cell donor age (Fig. 2B). When T-cell activation was titrated to include lower doses of anti-CD3 in the absence of polarizing cytokines, 2-week-old T cells produced even higher amounts of IFN-γ and slightly elevated levels of IL-17 (Supporting Information Fig. 1). These findings highlight that T cells are generally capable of differentiating into encephalitogenic Th1 and Th17 cells at the age of 2 weeks, suggesting that an immaturity of peripheral T cells is unlikely to explain EAE resistance

in 2-week-old mice. Activation and proinflammatory differentiation of CD4+ T cells depends on recognition of Ag provided by Ag-presenting cells, such as DCs, monocytes, and B cells [13]. Accordingly, we next investigated whether the insufficiency of young mice to generate encephalitogenic T cells may relate to an age-dependent alteration Fostamatinib molecular weight within the APC compartment. Similar to the investigations on T cells, we first

determined that the overall frequency of DCs, monocytes, and B cells in 2-week-old mice was comparable with that in adult mice (Fig. 2C–E and Table 1). Recent findings suggest that subclasses of DCs and myeloid cells may differ in their capacity to activate T cells, with subtypes rather suppressing than promoting proinflammatory T-cell differentiation. In this regard, further phenotyping of DCs revealed that at an age of 2 weeks, mice contained a higher frequency of CD11cintPDCA+Siglec-H+ plasmacytoid DCs, which can promote development of Treg cells and inhibit CNS autoimmune disease [14]. In contrast, the frequency of CD11b+ myeloid DCs with a strong

capacity to generate Th1 and Th17 cell responses, but also to reactivate encephalitogenic T cells in the inflamed CNS [15] was reduced (Fig. 2C and Table 1). Along the same lines, the frequency of CD115+Gr-1+ myeloid-derived suppressor cells, which can impair expansion and homeostasis of proinflammatory T cells [16] and development of EAE [17] was elevated in 2-week-old mice (Fig. 2D and Table 1). Taken together, within the compartment of APCs of myeloid origin young mice contained a markedly higher Sinomenine percentage of phenotypes with the potential to suppress autoimmune T-cell responses. Proinflammatory differentiation of CD4+ T cells requires two signals [18]. The first signal is Ag recognition in the context of MHC II via their T-cell receptor, the second mandatory interaction consists of ligation of co-stimulatory molecules. In order to investigate whether APC from 2-week-old mice may differ in quantity or quality of these signals, myeloid CD11b+ APCs as well as B cells from 2- or 8-week-old mice were evaluated for surface expression of MHC II and the co-stimulatory molecules CD40, CD80, and CD86.

In addition, the immune cross reaction between rCp23 and rCp15–23

In addition, the immune cross reaction between rCp23 and rCp15–23 was observed. To examine the generation of the specific cellular immune responses to rCp15–23 fusion protein, rCp23 protein and crude extract of C. parvum, single spleen cell suspensions from different protein immunized or control (adjuvant-immunized) mice collected 14 days after the final immunization were prepared and used for T cell characterization analysis. The antigen-specific lymphocytes were examined by direct staining with antibodies for surface expression of cluster of differentiation CD4+ and CD8+. The results showed that the number of

CD4+ and CD8+ T cells was increased in all three immunized groups compared with adjuvant control group (P < 0·01), whereas the number of CD4+ T cells was much more than that of CD8+ T cells. In addition, the stimulation of cells from rCp15–23 fusion

protein immunized mice generated higher CD4+, see more CD8+ T cells and the ratio of CD4+/CD8+ than either Crizotinib molecular weight crude extract or rCp23 protein groups (P < 0·01) (Figure 5). ELISA was used to detect the concentrations of cytokines in the supernatants of in vitro activated lymphocytes at day 14 after the third doses of vaccine. In the spleen cells, significantly higher concentrations of IFN-γ or IL-12 were found in all antigen immunized groups, whereas no IFN-γ or IL-12 was detected in the adjuvant control group. The IFN-γ and IL-12 levels were found to be significantly higher in rCp15–23 fusion protein immunized mice compared with the

crude extract immunized mice (P < 0·05) (Figure 6). No significant difference was observed in crude extract immunized mice compared with adjuvant control group mice. Very low level of IL-4 was found in our study in all the groups and no difference was found between different groups. To examine differences in protection of C. parvum Pregnenolone infection after different protein immunization, faecal oocyst shedding was detected. The faecal oocyst shedding was noted between days 3 and 7 post-infection in both the crude extract protein immunized group and adjuvant control group, in the rCp23 protein immunized group between days 4 and 8 post-infection, in the rCp15–23 fusion protein immunized group between days 5 and 9 post-infection. The manifestations of C. parvum infection, i.e. oocyst shedding was not noted or was minimal on days 10 and thereafter. The prepatent period of oocysts shedding was longer after immunization with both rCp23 protein and rCp15–23 fusion protein. However, the increase in the prepatent period in mice immunized with rCp15–23 fusion protein was obvious compared with those in mice immunized with either crude extract or rCp23 protein (Figure 7). In addition, the oocyst shedding number was reduced in C. parvum challenged mice following immunization. In rCP15–23 recombinant protein immunized group, the oocyst shedding number was reduced 31·4% compared with the adjuvant control group (P < 0·05).

In the human, the ascending uterine arteries give rise to approxi

In the human, the ascending uterine arteries give rise to approximately eight arcuate arteries that are embedded in the myometrium and form anastomoses with those emanating from the contralateral ascending uterine arteries [16]. These vessels then branch centripetally into radial arteries that penetrate the middle third of the myometrium and give rise to ~200 spiral

arteries [16]. The vascular pattern differs somewhat in the guinea pig or the rat. In these species, the arcuate (syn. mesometrial) arteries are located within the planar mesometrium and are therefore external to the uterus. Radial arteries emanate from the arcuates and are also external to the uterus. During pregnancy, these vessels may further ramify into those that supply a placenta Proteases inhibitor (pre-placental or spiral arteries) vs. myometrium (pre-myometrial or basal arteries). Although both types of radial arteries remodel BMS-354825 chemical structure during pregnancy, they may (rabbits [12]) or may not (rats [25]) do so to a different extent, depending upon species. Such interspecies variation in vascular anatomy presents an opportunity to dissect the potential contributions of placenta-specific vs. whole uterine (or horn-specific in the case of species with bicornuate uterus) influences on pregnancy vascular remodeling and its consequences. The time course of the proliferative responses

in the arcuate and radial arteries differs from that seen in the larger (main) uterine arteries, also with some variation occurring among species. In the guinea pig, DNA synthesis continues to rise until term in the radial artery, which is longer than seen in the main uterine artery [31]. Just the reverse occurs in the rat, as DNA synthesis peaks at mid-pregnancy in the radial artery

but later in pregnancy in the upstream main uterine artery (measured on day 20 of a 22 day gestation [13]). As discussed below, endothelial NO appears to be a key modulator of circumferential remodeling and can be stimulated by a variety of factors such as shear stress, estrogen, and VEGF [81, 55, 9, Rebamipide 28]. The literature on uterine veins is quite limited relative to that on arteries. Significant increases in venous diameter and length occur during pregnancy as well and comprise an important means for accommodating the ~40% rise in blood volume. The venous responses are associated with changes in connective tissue elements such as elastin and collagen; these, in turn, lead to altered biomechanical properties such as increased compliance [60]. In summary, uterine vascular remodeling in the upstream vessels begins earlier and is at least in part independent from downstream, placentation-related changes. In many respects, the changes in the uterine artery are anticipatory, enabling the maternal circulation to accommodate the exponential rise in fetal demand occurring near the end of gestation.

It is reported that different Fcγ receptors on neutrophils posses

It is reported that different Fcγ receptors on neutrophils possess different phagocytosis capabilities, and CD32 (FcγRIIA) is the most MAPK Inhibitor Library ic50 efficient receptor among them (Rivas-Fuentes et al., 2010). The affinity of human CD32 increases during neutrophil activation leading to CD32-dependent ligand binding and signaling (Nagarajan et al., 2000). It has been documented that BCG has the capacity to increase the expression of CD32 (Suttmann et al., 2003). Similarly, in this study,

expression of CD32 was increased in BCG- and H37Rv-infected neutrophils indicating activation followed by functional upregulation of neutrophils. Another important FCγ receptor CD64 (FcγRI) that induces high respiratory burst (Hoffmeyer et al., 1997) was also upregulated in H37Rv-infected neutrophils, which further indicates a physiological response to infection (Allen et al., 2002). Neutrophils recognize pathogens via TLRs and activate various pathways

that contribute to the repertoire of defense mechanisms utilized by the immune system. Among TLRs, TLR2 is important in MTB infection and has been extensively studied. Another receptor TLR4, although important in innate immunity, Roxadustat datasheet has no direct role in protective immunity in mycobacterial infections (Reiling et al., 2002). However, it mediates the signals responsible for the production of MTB-induced IL-17A response, which strongly relies on the endogenous IL-1 pathway (van de Veerdonk Mirabegron et al., 2010). In another study, it was demonstrated that after Mtb infection neither TLR2,

-4 and -9, nor MyD88 is required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, -4 and -9, is critical for triggering macrophage effector mechanisms central to antimycobacterial defense (Hölscher et al., 2008). In this study, an increased TLR4 expression was observed in H37Rv-stimulated neutrophils, which reflects the fact that TLR4 mediated activation of neutrophils occur during MTB infections; however, the activation does not necessarily lead to protective immune response. Neutrophils are traditionally known to express limited number of chemokine receptors; however, under inflammatory conditions, they undergo phenotypic changes, enabling them to expand their chemokine receptor expression pattern and respond to chemokines that are functionally inactive under resting conditions. The chemokine receptor CXCR3 that is normally inactive on neutrophils gets expressed when induced with TLR ligands (Hartl et al., 2008). Here, the increased expression of CXCR3 on H37Rv-infected neutrophils indicates that H37Rv has the capacity to induce the expression of CXCR3, whereas BCG and Mw are not effective enough to stimulate its expression. Neutrophils undergo spontaneous apoptosis that make them susceptible to engulfment by monocytes/macrophages.

In addition, residue MOG113–127 was found to be a B-cell epitope,

In addition, residue MOG113–127 was found to be a B-cell epitope, suggesting that this may be a useful adjunct for the Linsitinib induction of EAE as well as for immunological studies

in C57BL/6 mice, which are increasingly being used to study immune function through the use of transgenic and gene knockout technology. Multiple sclerosis (MS) is an immune-mediated, demyelinating and neurodegenerative disease of the central nervous system (CNS).[1] These aspects of MS can be modelled using experimental autoimmune encephalomyelitis (EAE) in animals.[2] EAE can be induced following immunization with a variety of myelin proteins,[2] notably with CNS-specific antigens such as proteolipid protein and myelin oligodendrocyte glycoprotein (MOG).[2, 3] Whereas proteolipid protein, an extremely hydrophobic protein, is the major myelin protein in CNS myelin, MOG is a minor CNS myelin protein present as a transmembrane protein expressed exclusively on the surface of oligodendrocytes and myelin. Despite comprising only 2·5% of the myelin proteins,[4] MOG is a powerful encephalitogen inducing EAE in a range of species including mice, rats and monkeys.[2-5] The full-length protein contains 218 amino acids that form a single extracellular region containing an immunoglobulin-like domain (residues 1–125), anchored

by a hydrophobic transmembrane domain (residues 126–146), an intracytoplasmic domain (residues 147–181), a second hydrophobic transmembrane domain (residues IWR-1 research buy 182–202) and another extracellular domain (residues 203–218). Many immunological studies in EAE and MS make use of recombinant proteins

representing the extracellular immunoglobulin-like domain of MOG, which is expressed on the surface of oligodendrocyte and myelin and is therefore readily available for recognition by autoreactive antibody responses.[2, 3, 6] However, the use of recombinant protein and peptides fails to address the possible pathogenic role of the full-length myelin-derived protein, expression of conformational epitopes, peptide targets within the transmembrane and intracytoplasmic Sclareol domains as well as post-translational modifications.[7, 8] More recently, several of these aspects have been addressed with the use of myelin from wild-type (WT) and MOG-deficient (MOG−/−) mice.[9] Immunization with myelin from these animals demonstrates that immune responses to MOG in myelin can be crucial for chronic demyelinating EAE in mice and common marmosets.[4, 5] Having established that MOG-specific peptides can induce EAE in rodents,[3, 10] an important finding arising from the early studies on the encephalitogenic potential of MOG was the identification of an epitope of human MOG35–55 (hMOG35–55) that induced EAE in C57BL/6 mice.

KIR genotyping with PCR–SSP method   The KIR genotyping was perfo

KIR genotyping with PCR–SSP method.  The KIR genotyping was performed using polymerase chain reaction with sequence-specific primers (PCR–SSP) in all samples collected from recruited subjects for the following 18 KIR Selleckchem NU7441 genes: 2DL1-5, 3DL1-3, 2DS1-5, 3DS1, 2DP1, 3DP1, KIR1D and 3DP1v. We used newly extracted DNA from frozen peripheral blood mononuclear cells in this study to avoid false-negative results

of KIR genes in the PCR–SSP typing because most KIR-specific amplicons are longer than 1000 bp. DNA was extracted with an EZ Bead System-32 DNA workstation (Texas BioGene Inc., Richardson, TX, USA) according to the manufacturer’s instruction. The human growth hormone gene was served as a positive control for PCR. The primers were as follows: 5′CTTCCCAACCATTCCCTTA3′ and 5′CGGATTTCTGTTGTGTTT C3′. The PCR without DNA template was served as a negative control. The PCR sequence-specific polymorphism primers used for the detection of KIR genes were described previously BAY 57-1293 [4, 19] (Table 1). All primers were synthesized and validated by BOYA. Bio Co., Ltd., Shanghai, China. In detail, 20–50 ng DNA was amplified in 10 μl volume containing

0.2 mm dNTP, 0.5 U Taq DNA polymerase (Promega Corporation, Madison, WI, USA), 0.4 μm primers (except for KIR2DS1, 0.8 μm) and 1× PCR buffer. PCR amplification was carried out in a 9700 thermal cycler (PerkinElmer, Waltham, MA, USA) under the following conditions: initial denaturing at 94 °C for 4 min, followed by 30 cycles of 94 °C for 30 s, 65 °C for 30 s, 72 °C for 90 s, plus a final extension at 72 °C for 10 min. Some annealing temperatures changed as follows: KIR2DS2 (63 °C), KIR2DS3 (63 °C), KIR2DS4 (61 °C) and KIR2DS5 (63 °C). The amplification products were analysed in 1–2% agarose gel with

fluorescence dye and visualized by a Gene Genius Bio Imaging System (Syngene Low-density-lipoprotein receptor kinase Ltd., Cambridge, UK). Genotype and haplotype analyses.  Each genotype was given the putative haplotype combination according to the model described by Hsu et al. [4]. In assigning genes to specific haplotypes, the following assumptions were made: (1) all haplotypes contained KIR3DL3, 2DL4 and 3DL2; (2) haplotypes contained either 2DL2 or 2DL3, but not both; and (3) haplotypes contained either 3DP1 or 3DP1 variant (3DP1v), but not both [4]. In the assessment of the KIR haplotypes, haplotype B was defined by the presence of one or more of the following genes: KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1 [20]. Conversely, haplotype A was defined by the absence of all these genes. Genotypes for the Cen and Tel parts of the KIR locus were defined according to the description of Cooley et al. [5].

The more severely inflamed thyroids (4–5+ severity scores) also h

The more severely inflamed thyroids (4–5+ severity scores) also had microabscess formation, necrosis, and focal fibrosis, and inflammation generally extended beyond the thyroid to involve adjacent muscle and connective tissue.1–5,19 G-EAT lesions in IFN-γ−/− Selleckchem Dasatinib mice with 4–5+ severity scores differed from those

in WT mice in that they generally had less fibrosis and minimal necrosis and lesions generally did not extend outside the thyroid. G-EAT lesions in IFN-γ−/− mice had very few neutrophils, but many eosinophils.6–8 Neutrophils were detected in frozen thyroid sections using a rat anti-neutrophil monoclonal antibody (mAb) (RB6-8C5; American Type Culture Collection, Rockville, MD).6,20 Frozen thyroid sections were fixed in acetone for 10 min at 4°. Goat anti-rat antibody (1 : 500; Caltag Laboratories, Burlingame, CA) was used as the secondary antibody, with 3-diaminobenzidine tetrahydrochloride (DAB; Sigma) as the chromogen. Slides were counterstained with haematoxylin. Rat IgG was used as a negative control and staining was always negative. The same method was used for IL-5 staining except that the primary antibody was rabbit anti-IL-5 polyclonal Ab

(Santa Cruz Biotechnology, Staurosporine chemical structure Santa Cruz, CA) and anti-rabbit IgG (Santa Cruz) was used as the secondary antibody. NovaRED (Vector Laboratories, Burlingame, CA) was used as the chromogen. Serum T4 levels were determined using a T4 enzyme immunoassay kit (Biotecx Labs, Houston, TX) according to the manufacturer’s instructions. Results are expressed

as μg T4/dl of serum. Using this assay, T4 values for normal mouse serum ranged from 4 to 10 μg/dl; values < 3 μg/dl are considered low.20 RNA was isolated from individual thyroid lobes of recipient mice using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed as previously acetylcholine described.20–22 Levels of IL-10, IL-17, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 11 (CCL11) were quantified by real-time PCR using the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Amplification was performed in a total volume of 25 μl for 40 cycles, and product was detected using SYBR Green (ABgene, Rochester, NY). Samples were run in triplicate and relative expression levels were determined by normalizing expression of each target to hypoxanthine-guanine phosphoribosyl transferase. Expression levels of normalized samples are shown as relative expression units. Real-time PCR primers for IL-10 and IL-17 were previously described.22,23 Primers for CXCL1 were: sense, 5′-TGCACCCAAACCGAAGTCAT-3′; antisense, 5′-TTGTCAGAAGCCAGCGTTGAC-3′; and for CCL11, they were: sense, 5′-CTGCTTGATTCCTTCTCTTTCCTAA-3′; antisense, 5′-GGAACTACATGAAGCCAAGTCCTT-3′. Experiments were repeated at least three times.