The paracrine effect of mesenchymal human stem cells restored hearing in ß-tubulin induced autoimmune sensorineural hearing loss.

The aim of this study was to examine the activities of hASCs (Human Adipose tissue Derived Stem Cells) on experimental autoimmune hearing loss (EAHL) and how human stem cells regenerated mouse cochlea cells. We have restored hearing in 19 years old white female with autoimmune hearing loss with autologous adipose tissue derived stem cells and we wish to understand the mechanism of restoration of hearing in animal model. BALB/c mice underwent to develop EAHL; mice with EAHL were given hASCs intraperitoneally once a week for 6 consecutive weeks. ABR were examined over time. The helper type 1 autoreactive responses and T-reg cells were examined. H&E staining or immunostaining with APC conjugated anti-HLA-ABC antibody were conducted. The organ of Corti, stria vascularis, spira ligament and spiral ganglion in stem cell group are normal. In control group, without receiving stem cells, the organ of Corti is replaced by a single layer of cells, atrophy of stria vascularis. Systemic infusion of hASCs significantly improved hearing function and protected hair cells in established EAHL. The hASCs decreased the proliferation of antigen specific Th1/Th17 cells and induced the production of anti-inflammatory cytokine interleukin10 in splenocytes. They also induced the generation of antigen specific CD4(+)CD25(+)Foxp3(+)T-reg cells. The experiment showed the restoration is due to the paracrine activities of human stem cells, since there are newly regenerated mice spiral ganglion cells, not human mesenchymal stem cells derived tissue given by intraperitoneally.

Prevention and treatment of DNA vaccine encoding cockroach allergen Bla g 1 in a mouse model of allergic airway inflammation.

BACKGROUND: One-fourth of the US population is sensitized to the German cockroach. Primary German cockroach allergen Bla g 1 is detected in 63% of homes and 52% of childcare facilities in the United States. No effective treatment or vaccination strategies are yet available. OBJECTIVES: We evaluated the prophylactic and therapeutic efficacy of a plasmid DNA-mediated vaccination using the Bla g 1 gene in a mouse model of allergic inflammatory airway disease. METHODS: A plasmid DNA vector coding for the Bla g 1 allergen controlled by cytomegalovirus promoter was constructed. To estimate the protective efficacy, BALB/c mice were given three injections of plasmid DNA-Bla g 1 prior to sensitization with two priming doses of recombinant Bla g 1 (rBla g 1) antigens, followed by nebulized rBla g 1 challenge. In the therapeutic approach, sensitization was followed by administering Bla g 1 DNA vaccine. RESULTS: Bla g 1 vaccination significantly reduced allergen-induced airway inflammation, even after mice were presensitized and a Th2-dominant response was established. The Bla g 1 vaccination significantly reduced total inflammatory cell infiltrate, eosinophilia, secretion of Th2 cytokines IL-4 and IL-5 in bronchoalveolar lavage fluid, allergen-induced inflammatory infiltrates in the lungs, and Bla g 1-specific IgE in serum upon challenge with rBla g 1. Importantly, Bla g 1 DNA vaccination was able to induce IL-10-secreting regulatory T cells that could suppress the allergen-specific Th2 cells. CONCLUSION: DNA vaccination showed protective and therapeutic efficacy against a clinically relevant allergen Bla g 1.

Experimental autoimmune hearing loss is exacerbated in IL-10-deficient mice and reversed by IL-10 gene transfer.

Interleukin-10 (IL-10) has an important role in the homeostatic regulation of autoreactive T-cell repertoire. We hypothesized that endogenous IL-10 would regulate the severity of ß-tubulin-induced experimental autoimmune hearing loss (EAHL) and that exogenous IL-10 would abrogate it. BALB/c wild-type (WT) and homozygous IL-10-deficient mice (IL-10(-/-)) underwent ß-tubulin immunization to develop EAHL; some IL-10 mice with EAHL were administered IL-10 DNA at the peak of EAHL. Auditory brainstem responses were examined over time. EAHL developed progressively in both WT and IL-10(-/-) mice. However, the severity of hearing loss in the IL-10(-/-) mice was significantly greater than that in WT animals. Moreover, disease severity was associated with a significantly enhanced interferon-γ level and loss of hair cells in IL-10(-/-) mice. IL-10 administered to EAHL IL-10(-/-) mice promoted IL-10 expression. Consequently, hearing significantly improved by protecting hair cells in established EAHL. Importantly, IL-10 treatment suppressed proliferation of antigen-specific T-helper type 1 (Th1) cells, and the suppression can be attributed to inducing IL-10-secreting regulatory T cells that suppressed autoreactive T cells. We demonstrated that the lack of IL-10 exacerbated hearing loss, and the exogenous administration of IL-10 improved hearing. Mechanistically, our results indicate that IL-10 is capable of controlling autoimmune reaction severity by suppressing Th1-type proinflammatory responses and inducing IL-10-secreting regulatory T cells.

Comparison of skin tests to aeroallergens in Ankara and Seoul.

BACKGROUND: The environment contains many allergenic proteins, and skin test reactivity to aeroallergens may be different among people living in different regions. OBJECTIVE: To compare skin test results of Turkish and Korean patients with respiratory allergies. METHODS: The charts of 304 (160 male, 144 female) patients from Ankara, Turkey, and 208 (111 male, 97 female) patients' charts from Seoul, Korea, who had undergone skin prick tests were reviewed. Skin tests were classified as positive when the allergen-induced wheal size was the same size or larger than that caused by histamine. RESULTS: Grass pollens were found to be major allergens more often in Ankara than in Seoul (74.34% vs. 15.87%, p < 0.001). Skin test reactivities in Ankara were significantly lower (p < 0.001) than in Seoul to weed (6.91% vs. 37.50%) and tree pollens (4.61% vs. 39.42%). Allergic reactions to indoor allergens were significantly higher (p < 0.001) in Seoul than in Ankara: house dust mites (HDM) (83.17% vs. 32.90%), cockroaches (45.67% vs. 1.97%), and cats (17.79% vs. 1.65%). CONCLUSION: Due to the different aeroallergen environment, the positive skin test results were different in both cities: grass pollens were the most common allergens in Ankara, while patients from Seoul reacted more commonly to indoor allergens, especially to HDMs and cockroaches.

Intravenous infusion of recombinant tissue plasminogen activator for the treatment of patients with sudden and/or chronic hearing loss.

Seventeen patients with sudden hearing loss and 10 patients with chronic hearing loss were treated with intravenous infusion of tissue plasminogen activator. For sudden hearing loss, the recombinant tissue plasminogen activator was used, and 3 mg (in 3 mL of diluent) was diluted into 250 mL of physiological saline solution and given intravenously every 12 hours. Sixteen patients of the sudden hearing loss group and all 10 patients of the chronic hearing loss group showed an improvement after this treatment. No patients had side effects from the treatment. The results indicate that this would be an excellent mode of therapy for patients with hearing loss.

Restoration of hearing loss with tissue plasminogen activator. Case report.

A 59-year-old man with a 2-year history of sudden onset of hearing loss in the left ear was treated with tissue plasminogen activator (tPA) for myocardial infarction. The patient had 50 dB of hearing recovery after the full dose (100 mg of tPA followed by 3 mg/d of tPA for 2 weeks).

Association of HLA-DR and type II collagen autoimmunity with Meniere's disease.

To investigate HLA-associated genetic susceptibility to Meniere's disease in relation to type II collagen (CII) autoimmunity status, HLA-DRB1 genotyping and ELISA measurement of anti-CII antibody were performed in 41 Korean patients with Meniere's disease. In the anti-CII positive subgroup (20%) of patients, the frequency of HLA-DRB1*0405 was significantly increased (uncorrected) compared with both controls (63% vs 16%) and anti-CII negative patients (63% vs 12%). In the anti-CII negative subgroup, HLA-DRB1*1201 was significantly increased (uncorrected) (27% vs 10%) and DRB1*13 was decreased (6% vs 24%) compared with controls; these alleles appeared to confer susceptibility and resistance to the development of the disease. Association of HLA-DRB1*0405 with anti-CII positive Meniere's disease in this study suggests that it shares a specific HLA-DR sequence, QRRAA, as a genetic susceptibility factor with the anti-CII positive rheumatoid arthritis. In conclusion, whilst type II collagen autoimmunity may have a partial role in Meniere's disease, different HLA-DR alleles may also be associated with either susceptibility or resistance to the development of the disease in relation to anti-CII antibody status.

Molecular mechanism of autoimmune hearing loss.

Molecules considered as autoantigens in autoimmune sensorineural hearing loss and Meniere's diseases are type II collagens, type IX collagens, 30 kD proteins of inner ear membranes, laminin, 68 kD proteins of inner ear, PO protein, Raf I protein and beta-tubulin are reviewed in relation to molecular mechanisms of autoimmune injury of inner ear resulting in hearing loss.

[Allergic rhinitis: evaluation of prick test positivity for German and American cockroaches (Blatella Germanica, Periplaneta Americana)]. / Rinite allergica: valutazione della positività al test cutaneo per lo scarafaggio (blatella germanica e periplaneta americana).

Allergies are a widespread phenomenon and one that is in continuous expansion, especially in large cities. A heretofore underestimated allergen, at least in Italy, is the cockroach. Between April and June 2000, we administered a prick test (including the cockroach antigen in the allergen kit) to 163 patients. The prick test was executed utilizing histamine as the positive control and a normal diluent as the negative control; both indoor allergens (including dermatophagoids and dog and cat epithelium) and outdoor allergens (including trees, grasses, pollens and spores) were employed. The results obtained were evaluated by comparing the reaction provoked by these allergens to that of the histamine. About 20% of the patients who reacted to the other indoor allergens also tested positive to the cockroach antigen. Also on the basis of experiences previously carried out in other countries (United States, Korea, Japan, Turkey), the cockroach must be borne in mind as a possible significant cause of allergic reactions in Italy, too.

Induction of endolymphatic hydrops by directly infused monoclonal antibody against type II collagen CB11 peptide.

Autoimmune ear disease induced by type II collagen has been investigated by Yoo et al. In the present study, we investigated the effects of direct chronic infusion of type II collagen-specific monoclonal antibodies into guinea pig cochlea. Type II collagen fragment CB11 peptide-specific monoclonal antibodies (anti-CB11 Mabs) were infused directly into the scala tympani of guinea pigs with an Alzet mini-osmotic pump (anti-CB11 Mab group). As a control, normal mouse serum was infused by the same method (control group). To evaluate the auditory function, we recorded brain stem auditory-evoked potentials (BAEPs). In the anti-CB11 Mab group, 80% of the animals showed an increased hearing threshold of more than 25 dB at 7 days after infusion. The hearing threshold shift observed in the guinea pigs of the control group was minimal (15 dB or less). To detect the structural changes, we performed histopathologic studies using hematoxylin and eosin staining. Inflammatory cell migration was detected mainly in the scala tympani of the guinea pigs of both groups. In the anti-CB11 Mab group, endolymphatic hydrops was also observed. The results of this experiment suggest that type II collagen autoimmunity is responsible for the production of hearing loss and endolymphatic hydrops.

The effect of vaccination with DNA encoding murine T-cell epitopes on the Der p 1 and 2 induced immunoglobulin E synthesis.

BACKGROUND: Immunization with naked plasmid DNA leads to strong and persistent cell-mediated and humoral immune response to plasmid encoded antigen. Vaccination of DNA encoded whole allergen has been tried, but little information is currently available on the efficacy of DNA encoding T-cell epitopes in allergic disease. The purpose of this study was to determine whether the vaccination of naked plasmid DNA encoding only T-cell epitopes suppresses the allergic reaction as effectively as naked DNA encoding whole segments of allergen. METHODS: We immunized mice with a mixed naked plasmid DNA encoding the five classes of murine T-cell epitopes on Der p 1 and Der p 2 three times at weekly intervals via an intramuscular injection of BALB/c mice. Control mice were injected with the pcDNA 3.1 blank vector. After 3 weeks, the mice were actively sensitized twice and allowed to inhale the Der p extracts intranasally six times at weekly intervals. RESULTS: The vaccinated mice showed a significant attenuated induction of Der p-specific immunoglobulin E synthesis compared to controls. In terms of the Der p-specific IgG2a antibody response, the vaccinated mice showed more prominent responses than the control mice group. In addition, analysis of the cytokine profile after Der p stimulation of the lymph-node cells revealed that the level of the mRNA expression of the interferon-gamma gene was higher in the vaccinated mice than in the controls. Histologic studies showed a much reduced infiltration of inflammatory cells in lung tissue of the gene-vaccinated mice in comparison with the controls. CONCLUSIONS: These results suggest that vaccination with DNA encoding T-cell epitopes effectively inhibits allergen-induced IgE synthesis and reduces cell infiltration in lung tissue. Thus, gene therapy using T-cell epitope-encoding DNA presents an ideal way of combating allergic disease in the future.

Oral administration of collagen conjugated with cholera toxin induces tolerance to type II collagen and suppresses chondritis in an animal model of autoimmune ear disease.

B10.RIII (H-2r) mice were orally administered cyanogen bromide peptide 11 (CB11) or cholera toxin B (CTB)-conjugated CB11 to induce tolerance in collagen-induced autoimmune ear disease. Oral administration of a high dosage of CB11 provided partial protection from chondritis. However, administration of a tiny amount of CTB-CB11 conjugate effectively suppressed chondritis. Oral administration of CTB-CB11 conjugate did not alter the stimulation of T cells in vitro or the fine specificities of B cells. The oral administration of CTB-CB11 caused a higher level of type II collagen-specific IgG and its subclass. Interestingly, increases of TH1 cytokine (interferon-gamma) in Peyer's patches and of TH1/TH2 cytokines (interleukin-2 and interleukin-4) in lymph nodes were detected in mice that had been fed CTB-CB11. An increase of CD8+ T cells in the Peyer's patches with a decrease of CD8+ T cells in lymph nodes was seen in mice that had been fed CTB-CB11. These results suggest that protection from chondritis by oral administration of minute amounts of CTB-CB11 conjugate can be achieved by a mechanism distinct from that of conventional oral tolerance induction.

Presence of autoantibodies in the sera of Meniere's disease.

We examined the sera of patients with Meniere's disease for the presence of antibodies against 8 inner ear antigens by enzyme-linked immunosorbent assay (ELISA). One hundred eight patients with Meniere's disease and 28 control subjects were studied. The antibodies against chicken type II collagen, bovine type II collagen, the cyanogen bromide cleaved peptide 11 (CB11) of each, type IX and XI collagens, C-Raf, and tubulin were measured by ELISA. The sensitivity of each antigen was between 37% and 60% individually, and was 91% when all 8 inner ear antigens were combined. These results showed that 91% of Meniere's disease sera have antibody activities to 1 or more of these inner ear antigens. The results suggest that performing ELISA for these 8 inner ear antigens was useful as a diagnostic tool for Meniere's disease. Further study is required for elucidating the role of these antigens in the pathogenesis of Meniere's disease, which might eventually result in better therapy.

Proto-oncogene Raf-1 as an autoantigen in Meniere's disease.

The 28 kd protein extracted from the guinea pig inner ear membranous fraction, which reacted with sera from patients with Meniere's disease, has been subjected to microsequencing. Nineteen amino acids were obtained (IVQQFGFQRRASDDGKLTQ). A protein data bank search showed that this sequence corresponded to residues 41 to 60 of human Raf-1 protein. Sera from 16 of 27 patients with Meniere's disease showed reactivity to the recombinant purified glutathione-S-transferase-Raf-1 protein. These results support the hypothesis that a subgroup of patients who suffer from Meniere's disease, as well as some other kinds of autoimmune inner ear diseases, have an autoantibody against Raf-1 protein.

Murine model of autoimmune hearing loss induced by myelin protein P0.

Myelin protein P0 has been identified as an autoantigen in inner ear diseases. In order to study autoimmune hearing loss, we performed brain stem auditory-evoked potential (BAEP) studies on P0-sensitized mice. Two P0-sensitized mice showed hunched posture, poor coat, loss of body weight, and abnormal walking with a waddling gait. About 25% of the P0-sensitized mice developed hearing loss. In the BAEP study, peak latencies of waves I, III, and V and the interpeak latency I-III were prolonged in the P0-sensitized hearing loss group of mice. Hearing thresholds were elevated in this group of mice in comparison with the control mice. Inflammatory cell infiltration was observed in the cochlear nerve region, and a reduced number of spiral ganglion cells was also detected. These results suggest that P0-sensitized mice are a useful model for studying autoimmune inflammation of the peripheral portion of the auditory system.

Successful prevention of retrocochlear hearing loss in murine experimental allergic encephalomyelitis with T cell receptor Vbeta8-specific antibody.

Experimental allergic encephalomyelitis is an animal model of a T cell-mediated autoimmune disease -- for example, multiple sclerosis. We demonstrated that mice with experimental allergic encephalomyelitis developed retrocochlear hearing loss, and that the lesion of the auditory pathway might be related to T cell receptor Vbeta8-expressing T cells. To investigate whether anti-Vbeta8 antibody could prevent hearing loss, we carried out brain stem auditory evoked potential testing, histologic examinations, and flow cytometry in antibody-treated and control myelin basic protein-immunized B10.PL mice. The antibody was administered just before immunization of myelin basic protein. The disease incidence and severity were significantly reduced in the mice injected with the antibody. The results of brain stem auditory evoked potential testing, histologic examinations, and flow cytometry indicated that the depletion of Vbeta8-expressing T cells brings the prevention of hearing loss, as well as prevention of other neurologic deficits. The development of T cell receptor-specific antibody therapy might help treat retrocochlear hearing loss in multiple sclerosis.

Clonal expansion of T-cell receptor beta gene segment in the retrocochlear lesions of EAE mice.

It has been reported that the T cell receptor V beta 8.2 (TcrbV8.2) gene segment is predominantly expressed in encephalomyelitic T cells responding to myelin basic protein (MBP) in experimental allergic encephalomyelitis (EAE) mice. We have demonstrated retrocochlear hearing loss in EAE mice in previous studies. Administration of a monoclonal antibody specific to the T cell receptor V beta 8 (TcrbV8) subfamily prevented both this type of hearing loss and the central nerve disease. In this study, we examined the role of the TcrbV8.2 gene segment in the retrocochlear lesions of EAE mice. A clonal expression of T cell receptor beta chain gene segment (TcrbV8.2-TcrbD2-TcrbJ2.7) was identified in the retrocochlear lesions. The TcrbV8.2 gene segment appears to recombine only with TcrbJ2.1 (32.1%) and TcrbJ2.7 (67.9%) gene segments. The TcrbJ2.7 gene segment has also been previously identified as the dominant TcrbJ gene in the lymph nodes of EAE mice. Only TcrbD2, with a length of 4 amino acids, was observed recombining with these TcrbV8.2 sequences. G and C nucleotides are predominantly expressed at the N regions between the V-D and D-J junctions. This dominant TcrbV gene segment (TcrbV8.2-TcrbD2-TcrbJ2.7) observed in the retrocochlear lesions has been identified in the MBP-specific T cells from the lymph nodes of EAE mice. These results suggest that a small subset of antigen-specific T cells migrate to, and expand at, the retrocochlear lesions, which leads to hearing loss.

House dust mite-induced sensitivity in mice.

Sensitivity induced by house dust mite (HDM) extract in mice was investigated in this study. Sensitized B10.RIII mice (H-2r background) had T-cell proliferative responses to HDM extract in vitro and an HDM-specific IgE response. When mice were immunized by injection and intranasal inhalation with HDM extract, a histologic study showed eosinophils and mononuclear cell infiltration in the lung tissue and bronchial wall. Tcr alphabeta-positive cells were also found in the cell infiltration area of the lung lesions. In the control mice that were immunized by injection or intranasal inhalation (but not both), we did not observe cell accumulation in the lung tissue or in the bronchial wall. Epitope studies suggest that T cells recognize multiple epitopes. Molecular analysis of these HDM-specific T-cell hybridoma clones suggest that T-cell receptor use is restricted to members of the V alpha 8 and Vbeta 6 subfamilies.

TCRV and TCRJ gene usage in MBP responding T cells from (B10.PL x PL/J)F1 mice is biased towards that of B10.PL mice.

We analyzed myelin basic protein (MBP) specific T cell hybridoma clones from (B10.PL x PL/J)F1 mice. MBP-reacting T cell hybridomas from F1 mice preferentially expressed B10.PL TcraV2.3 (53%) and B10.PL TcraV4.2 (13%) with minor expression of TcraV4.4 (13%) gene segments. A dominant expression of TcrbV8.2 (73%) accompanying with TcrbV8.1 (20%) and TcrbV13 (7%) gene segments have been identified in these MBP-reacting T cell hybridomas from F1 mice. There was less restrictive but non-random usage of the TcraJ and TcrbJ gene segments. Overall, the MBP-reacting T cell hybridomas from (B10.PL x PL/J)F1 mice were dominated by the MBP-reacting T cell pattern seen in B10.PL mice.

Molecular basis of type II collagen autoimmune disease: observations of arthritis, auricular chondritis and tympanitis in mice.

A type II collagen autoimmune response results in arthritis, auricular chondritis and tympanosclerosis in humans and animals. The purpose of this study is to further define the molecular and pathogenic events involved in these lesions in rodents. Type II collagen fragment CB11-specific monoclonal antibodies induced lesions in joints, ear lobes and tympanic membranes. In immunized mice, the thickness of tympanic membranes increased to two- to fourfold normal size. Electron micrography showed that the arrangement of collagen fibers is irregular in both radial and auricular layers, containing fibroblasts, a homogeneous material resembling low-density cholesterol crystals and cell infiltration. The mice with auricular chondritis had lymphocytes expressing V beta-8 T cell receptor (TCR) in arthritic joints and lymphocytes expressing V beta-6 TCR in ear lobe lesions. A monoclonal antibody specific to the TCR V beta-8 subfamily suppressed the onset of arthritis. Sequence analyses of the V beta structure of TCR involved in the lesions confirm the immunohistologic study.