To purify CMVpp65495–503-specific CD8+ T cells, purified total CD8+ T cells from CMVpent+ subjects were stained with the biotin mAb cocktail for CD8+ T-cell isolation and subsequently with Streptavidin-PE and CMVpent-APC (Proimmune). CMVpent+ cells were sorted in a FACSAria to 95% purity. Human neonatal CD8+ T cells from UCBMC were labeled with anti-CD8 microbeads (Miltenyi) and purified using find more POSELD2 program (purity of CD3+CD8+≥90%). Purified

CD8+ T cells were cultured (5×105 cells/mL) with medium alone (RPMI-glutamax medium (Invitrogen) supplemented with 10% FCS (Sigma) and 1% penicillin/streptomycin (Invitrogen)) or medium containing IFN-α2b, IFN-α5, anti-CD3/CD28-Beads (Beads coated with anti-human CD3 and CD28 mAb)

(Invitrogen) alone or together with IFN-α (IFN-α2b or IFN-α5). The IFN-α dose was 500 IU/mL. Beads were used at a 1:10 Beads:cell ratio. Purified CMVpp65495–503-specific CD8+ T cells were left unstimulated or stimulated with anti-CD3/CD28-Beads alone or together selleck screening library with IFN-α, in IL-2-conditioned medium (50 IU/mL) (Peprotech). In some cases, previously to stimulation, CD8+ T cells were labeled with 1.25 μM of CFSE (Sigma-Aldrich). In some cases, freshly purified CD8+ T cells were directly co-cultured (4 h) (i) with control IgG- or anti-CD3 OKT3 mAb-loaded p815 target cells (E:T ratio=10:1) or with (ii) HLA-A2+ T2 cells (E:T=5:1) loaded with HLA-A2-restricted control peptide (Leukocyte Proteinase-3169–177) or CMV peptide (Proimmune), in the presence or absence of IFN-α. To facilitate

IFN-γ detection by intracellular staining, cells were cultured in the presence of Brefeldin A (10 μg/mL) (Sigma-Aldrich) for the last 6 h of culture or along the culture (in the case of 4 h short-term assay). For the detection of CD107a, cells were cultured in the presence of anti-human CD107a-PE mAb (H4A3) or mouse IgG1-PE (10 μg/mL) (BD Biosciences) and Monensine (1 μg/mL) (Sigma-Aldrich). Total Rucaparib in vitro RNA was extracted using the nucleic Acid Purification lysis solution and the semiautomated ABI Prism 6100 Nucleic Acid PrepStation system (Applied Biosystems). Total RNA was treated with DNase prior to RT with M-MLV reverse transcriptase in the presence of RNaseOUT (all from Invitrogen). Real-time RT-PCR was performed using the CFX96 Real-time system, the IQ SYBR Green Mix (BioRad) and specific primers for each gene (Supporting Information Table 3). Results were normalized to β-actin. The amount of each transcript was expressed by the formula: 2Δct [Δct=ct(β-actin)-ct(gene], with ct as the point at which fluorescence rises appreciably above background fluorescence. Cells stained with fluorochrome-labeled mAb and/or CFSE were acquired on a FACSCalibur (BD Biosciences) and analyzed using FlowJo (Tree Star). Fold expansion was calculated as the output/input ratio of the absolute numbers of cells determined using Trucount beads (BD Biosciences).

It caused a small outbreak in Jiangsu Province in 1998 and then, most recently, the largest outbreak in Sichuan Province in 2005 (9, 10). The ST7 S. suis strain has not been isolated outside of China. In the present study, our results indicate that four of the five MLVA types identified among the 1998 isolates of ST7 S. suis Vadimezan were also detected in Sichuan in 2005. This suggests the pathogens responsible for the two outbreaks in China in 1998 and 2005 are closely associated. The one in Sichuan may have been

transmitted from Jiangsu province (9). In regard to the S. suis outbreak surveillance and investigations, because all of the ST7 isolates showed identical PFGE restriction patterns, we could not determine the transmission route from the PFGE data. The MLVA method described here has a higher discriminative typing power than PFGE (9). Therefore the MLVA scheme may better enable identification of transmission routes. During the outbreak in 2005, our epidemiology team found that one patient had become ill after slaughtering a diseased pig. The patient died Crenolanib cost 18 hr after the onset of the illness (9, 26). No samples were left from the diseased pig that he had processed. However one strain (SC16) from a diseased pig in the same herd and strain SC22

isolated from the patient (9) were both typed as MLVA16. Our data supports the epidemiological observations and may confirm the transmission route. The MLVA analysis was consistent with the results of investigations suggesting that the S. suis in Jiangxi province was derived from the outbreak in Jiangsu province in 2005 (9, 10). One patient had become ill after processing pork in a cold storage house. A strain named JX1 was isolated from this patient. Three other strains, Jxs1, Jxs2 and Jxs3, were isolated from pork in the cold storage house (9). All of these old four strains were typed as MLVA31 and a retrospective investigation found that the pork had been transported from Jiangsu province. Interestingly, three strains in

1998 and one strain in 2005 (both from Jiangsu province) were also typed as MLVA31. These data suggest the ST7 S. suis in Jiangxi province could have been derived from Jiangsu province via the pork trade (Fig. 2) (9, 10). To our knowledge, this is the first report of applying the MLVA method to S. suis. The MLVA developed here shows many advantages. First, it has a greater power to distinguish serotype 2 strains than PFGE and this makes it more useful for epidemiological purposes. Second, the MLVA method is a high-throughput screening method which is comparatively inexpensive, easy to perform, rapid, and reliable (19, 27, 28). The method is well suited for inter-laboratory comparisons of S. suis outbreak investigations. Third, reference strains are not required to ascertain consistency between inter-laboratory results (24).

4).

Analysis of differences in microbiota composition between the pIgR KO and WT mice indicated that the abundance of some bacterial groups decreased significantly in pIgR KO (p < 0.05, q Selleck Imatinib = 0.1). This included Bifidobacterium, Dorea, Anaerovorax, Acholeplasma, and relatives of Escherichia coli while Helicobacter abundance increased in the pIgR KO group (p = 0.006, q = 0.1). Further analysis of differences in microbiota composition in the four groups combined showed that some bacterial groups were differentially abundant (Supporting Information Table 5). To examine how DSS-induced colitis in pIgR KO mice was affected by the commensal microbiota, we subjected mice to the microbial depletion protocol described above for 1 week prior to initiation of DSS treatment. Successful depletion was verified by culturing see more and quantification of bacteria in fecal pellets both before switching mice to DSS-containing water and at the end of the experiment. Interestingly, we found that depletion of the cultivable commensal microbiota completely cured both pIgR KO and WT mice of weight loss and mortality induced by 1.5% DSS for 1 week (Fig. 5A). Although some pIgR KO mice still showed modest signs of diarrhea or rectal bleeding in presence of the antibiotic treatment, there was a significant improvement

compared with mice receiving DSS only (Fig. 5B). Thus, the colitis observed in both pIgR KO and WT was dependent on the presence of an intact intestinal microbiota. Here, we have shown that pIgR KO mice, which fail to actively transport secretory antibodies to the lumen, have a disturbed relationship with their intestinal microbes. This is evidenced by an increased expression of

AMPs by the epithelium in pIgR KO mice compared with WT counterparts that was reversed when the intestinal microbes were suppressed by oral antibiotics. Furthermore, pIgR KO mice had an altered intestinal microbiota composition and showed increased susceptibility to DSS-induced Thiamet G colitis. For both pIgR KO and WT mice, susceptibility to DSS-induced colitis depended on intestinal microbes, because both genotypes were completely resistant when the microbiota was suppressed by gavage with a concoction containing broad-spectrum antibiotic. Gene expression profiling of isolated colonic ECs found that the genes most highly upregulated in the absence of secretory antibodies encode innate epithelial defense factors. This compensation probably partially masks the functional importance played by secretory antibodies in WT mice, but reveals an important redundancy between innate and adaptive mucosal immune functions. The several “layers” of mucosal immunity highlight the importance of keeping the mucosal barrier intact [9].

It was shown previously to recruit NK cells in an atherosclerotic plaque [11]. Therefore, it is likely that IL-15 plays an important role in the recruitment of activated leucocytes from the blood stream in the infracted myocardial region of persons who died early after Selleck Acalabrutinib an acute coronary event. This hypothesis is supported by the abundant IL-15 expression in the border-necrotic, viable myocardiocytes that surround lymphocytes infiltration in the form of necklace. Although the CD56+bright NK cell subset represents mostly

cytokine-producing, regulatory NK cells in a steady state condition, they are able to become highly cytotoxic under tissue-specific inflammatory Th1 cytokine stimulation, such as the combination of IL-15 with other cytokines [12]. This was confirmed in vitro even with decidual CD56+bright NK cells [27], whose cytotoxicity is normally strongly down-regulated in situ by local immune-endocrine interactions during the first trimester of pregnancy. However, there is no clear evidence for selleck kinase inhibitor the involvement of particular cytotoxic mediator(s) in the

apoptosis of myocardial tissue after infarction. Here, we show for the first time the presence of the pro-apoptotic molecule GNLY in the cytoplasm of CD3+ and CD56+ cells, which take part in lymphocyte infiltration in the centre of MI in the patients who died in the first week after coronary artery thrombosis. GNLY can be easily released from the cells upon pro-inflammatory stimulation [19], what is supported with significantly lower MFI for GNLY in peripheral blood

lymphocytes of MI patients when compared with healthy control. Phenylethanolamine N-methyltransferase In turn, the soluble mature form of GNLY could enhance secretion of Th1 chemokines from macrophages and exhibit chemotactic properties for monocytes, mature dendritic cells, NK cells, and CD4+ and CD8+ T cells with a CD45RO+ phenotype, but not naïve CD45RA+ cells, as was shown previously [19], thus contributing to the accumulation of immune effectors in the myocardium after infarction [2]. On the other side, GNLY could hasten resolution rather than worsen cardiac post-infarction inflammation because of the finding of GNLY+ cells within accumulations of apoptotic leucocytes 1 week after the acute coronary event. K562 killing represents a model for in vitro testing of NK cell-mediated self-aggression, because K562 cells do not express MHC class I protein forms, as is known for damaged tissue cells [30]. Significant spontaneous peripheral blood NK cell- and GNLY-mediated apoptosis of K562 cells, which occurs in the first week after the acute coronary event, disappeared on day 14, with a concomitant decrease in the percentage of GNLY+ cells and the GNLY+ CD56+ bright NK cell subset in the circulation.

For bioinformatics analyses, the JCVI annotation service (JCVI, Rockville, MD, USA) was used as well as the antiSmash 2.0 server,[36, 37] the NCBI BLAST® server and the PKS/NRPS tool[38] to predict and annotate putative biosynthetic gene clusters. Burkholderia Smoothened inhibitor gladioli was grown in potato dextrose broth for 4 days. To achieve a

large surface area to volume ratio 30 Fernbach flasks were filled with 500 ml PDB (Difco™, Becton, Dickinson and Company, Heidelberg, Germany) medium and sterilised. Flasks were inoculated with 2.5 ml bacteria suspension (24 h grown in PDB at 30 °C and 110 rpm orbital shaking) and incubated at 28 °C for 4 days. The cultures were extracted with ethyl acetate (1 : 1) and concentrated under reduced pressure. Burkholderia gladioli was cultivated under

various conditions to monitor secondary metabolite production. The following media were used: nutrient agar and nutrient broth, both according to DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) protocol, a previously described rich liquid medium for secondary metabolite production[39] and MGY liquid medium consisting of yeast extract (1.25 g l−1) and M9 salts (50×, part A: 350 g l−1 K2HPO4; 100 g l−1 KH2PO4; part B: 29.4 g l−1 tri-Na-citrate-dihydrate; 50 g l−1 (NH4)2SO4; 5 g l−1 MgSO4) and glycerol (10 g l−1). All cultures were incubated at 30 °C and liquid cultures were shaken at 110 rpm in baffled flasks. Bacterial–fungal cocultivation was performed on 94 mm petri dishes containing 20 ml PDA at 30 °C. see more The fungus was inoculated on a small spot on the agar plate by transferring a small inoculation loop of spore and hyphae material from a sporulating plate. At the same time, a small inoculation loop containing bacteria from a well growing agar plate was streaked out on the other half of the plate. PH indicator

containing PDA agar plates were supplemented with a 0.06 g ml−1 Litmus solution (1 : 30; Merck, Darmstadt, Germany) and incubated at 30 °C for 7 days. Analytical HPLC was performed on a Shimadzu Sclareol LC-10Avp series HPLC system consisting of an autosampler, high-pressure pumps, column oven and PDA. HPLC conditions: C18 column (Eurospher 100-5, 250 × 4.6 mm, Knauer GmbH Berlin, Germany) and gradient elution (MeCN/0.1% (v/v) TFA 0.5/99.5 in 30 min to MeCN/0.1% (v/v) TFA 100/0, MeCN 100% for 10 min), flow rate 1 ml min−1; injection volume: 20 μl. Compounds were quantified by integrating the peak area (UV max, Shimadzu Deutschland GmbH, Duisburg, Germany) using Shimadzu Class-VP software (version 6.14 SP1). LC-MS measurements were performed using an Exactive Orbitrap High Performance Benchtop LC-MS with an electrospray ion source and an Accela HPLC system (Thermo Fisher Scientific, Bremen, Germany). HPLC conditions: C18 column (Betasil C18 3 μm 150 × 2.1 mm, Thermo Fisher Scientific, Bremen, Germany) and gradient elution (MeCN/0.

Serological studies conducted in countries where malaria is endemic suggest that high titres of cytophilic IgG3 and IgG1 or weakly cytophilic IgG2 antibody subclasses are associated with protection against severe malaria [81]. Malaria parasites were shown to be killed in vitro by monocytes, and this was enhanced in the presence of various antibody subclasses [82], CDK activation which facilitated phagocytosis of parasites by binding to Fcγ receptors on the phagocytes through their Fc domain; the parasites were then killed by the respiratory burst generated by Fc receptor cross-linking. This antibody-dependent

cellular inhibition (ADCI) of parasites was positively associated with protection against malaria [83-85]. Antibody responses against three P. falciparum blood-stage antigens–MSP-1 [86], MSP2 [87] and AMA-1 [88]–were skewed towards the cytophilic isotypes

IgG1 and IgG3, responses associated with protection against malaria. How long protective antibody responses are retained after recovery from malaria is of great interest. The absence of a memory B-cell response (MBC), or the presence of a dysregulated B-cell response, has been attributed to the highly polymorphic find more and clonally variant nature of P. falciparum blood-stage antigens. However memory B cells apparently existed in vaccinated mice that acquired sterilizing immunity after rechallenge [89]. During self-resolving P. chabaudi infections, the expression of a memory B-cell phenotype was detectable for at least 60 days after primary Unoprostone infection, and after rechallenge, they rapidly formed germinal centres in the spleen and differentiated into plasma cells giving a more efficient and rapid antibody response than in the primary infection [90]. Studies with transgenic mice carrying a TCR specific for an epitope of MSP-1 of P. chabaudi showed that some MSP-1-specific B cells were found in the spleen up to 8 months after a primary infection, although not in high numbers [91]. While present in the spleens of immune mice, similar B cells

have been found more recently among peripheral blood mononuclear cells in human malaria infections [92-95]. The cellular basis of humoral immunity has been clarified by the introduction of the B-cell ELISPOT assay that has enabled the prevalence, specificity and life-span of malaria-specific memory B cells to be determined, both after natural infections with P. falciparum [93-95] and in studies in mice [91]. The number of individuals with malaria-specific memory B cells has been found to increase with age [93, 94, 96], indicating that protective immunity depends on the range of these cells as well as the antibody response [97]. Over 75 years ago, Taliaferro and Mulligan demonstrated that blood-stage malaria in mice was associated with activation and expansion of the mononuclear phagocyte system.

At 1 month, 13 out of 16 (81%) patients in the levamisole group as compared with six out of 18 (33%) patients in the placebo group developed protective anti-tetanus IgG levels

(relative risk = 2.44, 95% confidence interval (CI) = 1.21, 4.88). From 1 to 6 months post-vaccination, one more patient in the levamisole group and two more patients in the placebo group were excluded because of renal transplantation. LY2606368 nmr At 6 months, 11 out of 15 (73%) patients in the levamisole group as compared with four out of 16 (25%) patients in the placebo group still had protective anti-tetanus IgG levels (relative risk = 2.93, 95% CI = 1.19, 7.23). Supplementation of Td vaccination with levamisole may enhance seroconversion against tetanus in haemodialysis patients. Compared with healthy population, haemodialysis patients are more susceptible to infections like tetanus[1-3] and experience lower seroconversion rate following vaccination because of their impaired immune system.[3-5] One of the strategies to increase the rate of seroconversion in these patients is to boost the immune system with immunostimulatory agents.[6] Levamisole is an immunomodulatory drug that stimulates depressed T cell activity

and enhances the production of antibodies by B cells.[6, 7] This anti-helminthic drug has been reported to increase the seroconversion rate following hepatitis B virus (HBV) vaccination.[6, 7] However, the possible enhancing effects of this drug on the response rates https://www.selleckchem.com/products/LBH-589.html of other vaccines like tetanus have not been studied. Therefore, the aim of the current study was to evaluate the effect of levamisole supplementation on tetanus vaccine response rate in haemodialysis patients. This

randomized double-blind placebo-controlled trial was approved by the Institutional Review Board of Shiraz University of Medical Sciences and was done in accordance with Flavopiridol (Alvocidib) the Declaration of Helsinki and Good Clinical Practice guidelines. Eligible participants were 20- to 80-year-old patients on regular haemodialysis in Faghihi Hospital Dialysis Center for more than 3 months who had unprotective anti-tetanus immunoglobulin G (IgG) levels as defined by the World Health Organization (WHO) (<0.1 international unit (IU)/mL). Exclusion criteria were: tetanus diphtheria (Td) vaccination in past year, leukopenia (white blood cell count < 1500 cells/mcL), immunosuppressive drug exposure in past 2 months, and recent hospitalization or history of transfusion of blood products in the past 3 months. In accordance with previous studies evaluating the effect of levamisole on HBV vaccination response in haemodialysis patients,[8, 9] a sample size of 12 patients per group was calculated considering two-sided significance level of 5%, power of 80% and response rates of 75% and 25% in levamisole and placebo groups, respectively. Considering a drop-out rate of 40%, a sample size of 20 patients per group was finalized.

01 EU/μg pDNA by the Triton X-114 extraction. For polyI:C and imiquimod, polymyxin B, which binds to LPS, was added to cells at a final concentration of 5 μg/mL. ODNs, nucleotides and nucleosides were used as obtained without further purification or addition of polymyxin B. TLR9 KO mice were purchased from the Oriental Yeast Company (Tokyo, Japan). C57BL/6 WT mice selleck kinase inhibitor and Institute for Cancer Research (ICR)

mice were purchased from Japan SLC (Shizuoka, Japan) and maintained on a standard food and water diet under conventional housing conditions. All animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for animal experiments were approved by the Institutional Animal Experimentation Committee of the Graduate School of Pharmaceutical Sciences, Kyoto University. In the experiment of subcutaneous injection

of ODN into mouse footpad, 3 nmol of ODN1668 in 20 μL PBS were subcutaneously injected into the footpad of the right hind leg of male ICR mice with or without 10 nmol DNase I-treated or untreated ODN1720. Before and 24 h after injection of ODN, the thickness of footpad was measured using a micrometer caliper with a minimum scale of 10 μm (Mitutoyo, Kawasaki, Japan). Separately, the footpad was removed at 24 h after injection and submerged into

4% paraformaldehyde in PBS for 24 h at 4°C. The fixed footpad tissues ABT-263 in vitro were decalcified and embedded in paraffin and sectioned into 3-μm slices. The paraffin sections were stained with hematoxylin and eosin to evaluate the infiltration of blood cells. The number of mononuclear cells and neutrophils infiltrating into the injection site in 25 mm2 was counted. Splenic macrophages were collected as previously described 16 and cultured on 96-well culture plates at a density of 3×105 cells/well in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/mL), streptomycin (100 μg/mL), L-glutamine (292 μg/mL) and 2-mercaptoethanol (10−5 M). They were used for the cytokine release experiment soon after isolation. The murine macrophage-like cell line, RAW264.7 cells, was cultured Fludarabine on 96-well culture plates at a density of 5×104 cells/well in RPMI-1640 supplemented with 10% FBS, penicillin G (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (292 μg/mL). They were cultured for 24 h prior to use. The human leukemic plasmacytoid DC line, PMDC05 cells 17, was cultured on 96-well culture plates at a density of 4×105 cells/well in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS, penicillin G (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (292 μg/mL). They were plated before the cytokine release experiment. RAW264.

g. foreign carbohydrate surfaces (and the absence of cellular and humoral

inhibitors) Fulvestrant price leading to formation of the AP C3-convertase C3bBb, stabilized by properdin. The LP is activated mainly when mannose-binding lectin (MBL) or ficolins bind to restricted patterns of non-self carbohydrate structures on target surfaces. This recognition leads to the activation of the MBL/ficolin-associated serine proteases (MASPs), of which MASP-2 has been shown to activate C4 and C2 leading to the LP C3-convertase C4bC2a [6]. With a prevalence of 5–10% in the Caucasian population, MBL deficiency is the most common immunodeficiency [7]. Functional MBL deficiency is explained largely by three single point mutations in codons 52, 54 and 57 of exon 1 in the MBL2 gene. These variants are referred to as variants D, B and C, respectively (often pooled into one O allele, while the wild-type is referred to as A). They result in unstable MBL variant proteins characterized by a low avidity towards ligands and an inability to initiate the MBL pathway [8,9]. Promoter polymorphisms, including the variants upstream of the MBL-2 gene, H/L (at position −550), X/Y variant (at position −221) and the P/Q variant (at position +4), are correlated with lower promoter activity in the order HY > LY > LX, leading to decreased amounts of an otherwise fully functional MBL [10]. Numerous studies

have reported an association between MBL deficiency and increased susceptibility to different types of infection. In particular, these are infections caused by extracellular

bacteria causing acute respiratory tract infections during early childhood [11–13]. However, see more studies have indicated that diseases correlated with MBL deficiency may require one or more co-existing immune malfunctions. For example, a study on meningitis caused Resminostat by Neisseria meningitidis showed an increased probability of the disease when MBL deficiency was associated with properdin deficiency [14]. Another area where complement deficiencies may play an important pathogenic role is in various autoimmune diseases, where elimination of immune complexes is hampered. Thus, screening of patients suffering from frequent and/or opportunistic infections and suspected of an underlying immunodeficiency or screening of patients suffering from autoimmune diseases, especially type III diseases, often involves assessment and evaluation of functional complement activity. For autoimmune diseases, monitoring of complement function also allows for an assessment of actual disease activity. In clinical laboratories the most commonly used method to measure functional complement activity is haemolysis of erythrocytes due to complement activation, either via the classical complement pathway in which sheep erythrocytes coated with antibodies are used as targets (CH50), or via the alternative complement pathway where rabbit erythrocytes are used as targets (AP50) [15].

20–22 When compared with BM-MSCs, human buy LY2835219 adipose-derived mesenchymal stem cells (hASCs) are equally capable of differentiating into cells and tissues of mesodermal origin.22–26 Abundant numbers of hASCs can be easily derived from lipoaspirate, the waste product of liposuction surgery and rapidly expanded in vitro to generate a clinically effective dosage. Moreover, recent studies have reported that hASCs share some of the immunomodulatory properties that characterize the BM-MSCs.16,22–26 Some researchers

have reported that ASCs exert profound immunomodulatory properties and protective effects on acute graft-versus-host disease and experimental arthritis.16,24–26 Our results show that hASC administration has therapeutic effects. Notably, the suppression of EAHL by hASCs was associated with the induction of CD25+ CD4+ Foxp3+  regulatory T (Treg) Selleck Sirolimus cells and interleukin-10 (IL-10) that could suppress the in vivo-induced T helper type 1 (Th1) responses in an in vitro co-culture assay. Female BALB/c mice (Jackson Laboratory, Bar Harbor, ME) were used in this study, and auditory brain responses (ABRs) were

measured bilaterally, both pre-treatment and post-treatment, for all the mice to ensure their normal hearing function. Mice were maintained in the animal facility at the University of Tennessee Health Science Center, according to the institutional guidelines for animal care and use. These studies were approved by the Institutional Animal Care Thalidomide and Use Committee of the University of Tennessee. At 6 weeks of age, mice were immunized subcutaneously with 300 μg β-tubulin (recombinant full-length human β-tubulin; Abcam, Cambridge, MA) emulsified with an equal volume of complete Freund’s adjuvant (Difco Laboratories, Detroit, MI) containing 2 mg/ml H37Ra Mycobacterium tuberculosis (Difco). The mice were given boosters by subcutaneous injection with β-tubulin emulsified with incomplete Freund’s adjuvant (Difco) twice at 1-week intervals, 2 weeks after the initial immunization. The therapeutic treatment was begun after the onset of hearing

loss, 2 weeks after immunization. Mice with EAHL received 2 × 106 hASCs (RNL Life Science Inc., Korea) or PBS intraperitoneally, once a week for 6 consecutive weeks. During ABR measurements, mice were anaesthetized with avertin (500 mg/kg bodyweight). The far-field auditory brainstem-evoked response was conducted in a sound-attenuating booth and the ABRs were recorded subcutaneously between vertex (active), posterior bulla (reference), and lower back (ground). Click and tone burst stimuli of 8, 16 and 32 kHz were generated and delivered to both ears through a high-frequency transducer. A maximum sound pressure level was stimulated in tone bursts of 100 dB. The evoked potentials were amplified 5000 times and averaged from 600 evoked responses for the first 10-millisecond period following stimulation.