Tyrosinase as an autoantigen in patients with vitiligo

Vitiligo is considered an autoimmune disorder due to the generation and presence of autoantibodies directed against melanocyte antigens in the patients' sera. In the present study we point towards a newly defined autoantigen in vitiligo, the enzyme tyrosinase, which participates in the process of melanogenesis. Anti-tyrosinase antibodies were detected in the sera of seven patients with diffuse and 11 patients with localized vitiligo. Employing solid-phase ELISA to mushroom tyrosinase, we found that patients with diffuse vitiligo had significantly higher titres of IgG anti-tyrosinase autoantibodies than patients with localized disease or healthy subjects. These anti-tyrosinase autoantibodies have relatively high functional affinity to tyrosinase and can be recovered from vitiligo patients’ sera by affinity purification. The anti-tyrosinase antibodies do not cross-react with other enzymes recognized as autoantigens in different autoimmune disorders and the autoantibodies do not block the enzymatic activity of tyrosinase, indicating that they are not reacting with the catalytic site of the enzyme. These data point to tyrosinase as an autoantigen in vitiligo and suggest that anti-tyrosinase titres can serve as a marker for disease activity.     

The clinical significance of antityrosinase antibodies in melanoma and related hypopigmentary lesions

Summary Antityrosinase antibody is a newly detected antibody in the sera of patients with melanoma or vitiligo. The serum level of the antibody is measured by enzyme-linked immunosorbent assay (ELISA). The autoantigen is tyrosinase itself, the enzyme that participates in pigment (melanin) formation by both melanocytes and melanoma cells Antityrosinase IgG antibodies were found to be present in high titers in sera of patients with vitiligo in comparison to patients with melanoma or healthy volunteers. The level of antityrosinase antibodies in patients with metastatic melanoma was significantly higher than the level in healthy subjects, but insignificantly higher than the level in patients with no evidence of disease. Patients with melanoma and MAH (melanoma-associated hypopigmentation; vitiligo-like) had the same level of antityrosinase antibodies as the controls or the patients with metastatic melanoma. This observation reflected the possible absorption of antityrosinase antibodies by melanoma antigens, and pointed to the participation of the antibodies in the destruction of normal melanocytes in patients with melanoma, as part of the immune reaction towards this disease. The most interesting observation was the high level of antityrosinase antibodies in patients with vitiligo in comparison with the low level in patients with melanoma, patients with MAH, and patients with NED. Although the cutaneous manifestations of vitiligo and MAH are similar and result from destruction of melanocytes by specific antibodies, the two situations are immunologically different. The serum level of free antityrosinase antibodies could not serve as marker for the state of the disease or disease progression or relapse, as no significant difference could be detected between the levels in patients without evidence of disease to those with metastatic melanoma; nor could the levels of antityrosinase antibodies differentiate between the different sites of the primary lesion. However, we have shown that antityrosinase antibodies could be used for monitoring the response to active specific immunotherapy by injection of anti-idiotypic antibodies mimicking the HMW-MAA. In the future, antityrosinase antibodies may be incorporated into immunotherapy for malignant melanoma.     

Immunoreactivity to food antigens in patients with chronic urticaria

The goal of study was better understanding of complex immune mechanisms that can help to evaluate patients with chronic urticaria (CU), especially those with unknown etiology. The study involved 55 patients with CU. Control group consisted of up to 90 healthy persons. The presence and intensity of serum IgG, IgA, IgM and IgE antibodies to common food antigens: cow’s milk proteins (CMP), gliadin and phytohemagglutinin were determined by ELISA. Determination of subpopulations of immunocompetent cells was performed by flow cytometry. Significantly enhanced IgE, but also IgA immunity to CMP was found in patients with CU in comparison to healthy controls: (p?<?0.000004) and (p?<?0.002), respectively. Notably, in 40 out of 55 CU patients, the increased levels of some type of immunoglobulin reactivity to CMP were found. Regarding gliadin, only the levels of serum IgE anti-gliadin antibodies were significantly enhanced in patients with CU (p?<?0.04). Significantly enhanced percentage of CD89+ cells accompanied with significantly lower percentage of lymphocytes and significantly higher mean fluorescence intensity of CD26 expression on lymphocytes were found in patients with CU in comparison to healthy controls (p?<?0.04), (p?<?0.02) and (p?<?0.003), respectively. Results of this study may help in better understanding the complex immune disturbances in patients with CU.     

Stimulation of Melanogenesis by Nordihydroguaiaretic Acid in Human Melanoma Cells

Nordihydroguaiaretic acid (NDGA), a lignan found in vegetables, fruits and legumin, has been shown to possess antineoplastic, antiviral and antioxidant characteristics. In this study, we examined the effect of NDGA on melanogenesis in human melanoma cells (HMVII). In vitro, NDGA does not alter mushroom tyrosinase activity. However, in NDGA-treated HMVII cells, cellular tyrosinase activity increased in both a time- and dose-dependent manner. The concomitant increases in melanin content in NDGA-treated cells indicated an elevation of melanin synthesis by tyrosinase activation. In addition, after a 7-day incubation, melanin content in 20 μM NDGA-treated cells increased 5.02 fold. Tyrosinase protein also increased by treatment with NDGA. Nevertheless, tyrosinase mRNA was not altered in NDGA-treated cells. Our results suggest that NDGA can increase tyrosinase activity and de novo synthesis of melanin in human melanoma cells. We found that NDGA is a novel potent stimulator of melanogenesis in human melanoma cells.     

Peripheral T-cell subset imbalance in patients with vitiligo and in their apparently healthy first-degree relatives

Twenty-two patients with vitiligo had lesions stable for at least 1 year and 23 apparently healthy first-degree relatives were studied for T-cell subpopulations using monoclonal antibody technique. High levels of CD4+ lymphocytes and high CD4/CD8 ratios were found in both patients and their relatives, as compared with normal age-matched and sex-matched controls. This immunologic abnormality, together with the presence of autoantibodies previously reported in the apparently normal relatives, seems to be the hallmark of the disease. Further support of this interpretation comes from the observation that two out of the 23 apparently healthy relatives went on to develop vitiligo in a 2-year follow-up.     

反义核酸抑制B16黑色素瘤细胞tyr基因的表达

目的：用反义核酸技术抑制小鼠B16黑色素瘤细胞酪氨酸酶基因（tyr）的表达。 方法： 构建tyr反义重组体pcDNA3.1(-)-tyr，用脂质体法导入B16细胞，用酪氨酸酶活性及黑色素含量测定，多巴染色及透射电镜等检测由反义重组体pcDNA3.1(-)-tyr转录产生的tyr反义核酸对B16细胞tyr表达的抑制情况。 结果： 成功构建了反义重组体pcDNA3.1(-)-tyr，转染了反义重组体的B16细胞其酪氨酸酶活性为0.0498±0.0036，显著低于对未转染组B16细胞的0.0916±0.0132(P<0.01);转染空质粒pcDNA3.1(-)及真核表达重组体pcDNA3.1(+)-tyr组B16细胞的氨酸酶活性分别为0.1015±0.0166和0.0948±0.0096,两者同对照组相比均无显著差异(P>0.05)。多巴染色及电镜观察结果表明转染了反义重组体的B16细胞其黑色素颗粒的含量明显低于对照组。 结论： 反义核酸可以显著抑制B16黑色素瘤细胞tyr的表达。     

Evaluation of the effect and mechanism of action of local phenytoin in treatment of vitiligo

There are many theories explaining vitiligo such as genetic, autoimmune, neural, free radicals, biochemical, intrinsic defect, melanocytorrhagy, and convergent theories. Phenytoin is a widely used anticonvulsant, which is used in cutaneous medicine for treatment of ulcers and epidermolysis bullosa. The aim of this study is to assess the effectiveness of topical phenytoin gel in the treatment of vitiligo patients and explaining the underlying mechanism using immunohistochemistry for evaluation of HMB45, CD4, and CD8. Only 9 patients out of 28 experienced response to phenytoin in the form of dull, white color change and light brown color. Post-phenytoin treatment biopsies showed decreased density of inflammation, increased melanin and increased HMB45 positive cells together with an increased number of CD4 positive lymphocytes and decreased number of CD8 positive lymphocytes. These observations did not reach significant level (P > 0.05). A high percentage of CD4 positive lymphocytes was significantly associated with a long duration of vitiligo (p = 0.03) and segmental vitiligo type (p = 0.02). The current study applied phenytoin as 2% concentrated gel for 3 months, which is a relatively short duration without observed side effects throughout the period. These results indicate that topical phenytoin of low concentrations may have beneficial effects through immunomodulatory activity by affecting CD4 and CD8 counts and subsequently the ratio between them. Further studies are recommended to combine phenytoin with other antivitiligo agents as local corticosteroids or phototherapy to clarify if it could potentiate their effects.     

IgA autoreactivity: a feature common to inflammatory bowel and connective tissue diseases

The immunopathogenic mechanisms in inflammatory bowel disease (IBD) are not yet fully established. The aim of this study was to determine the profile and magnitude of IgA and IgG autoantibodies in IBD patients. The autoantigen profile defined by IgA and IgG antibodies from 24 IBD (14 Crohn's disease CD], 10 ulcerative colitis UC]), three coeliac, 12 connective tissue disease (CTD) patients and 10 healthy individuals was studied in human cellular extracts by Western blotting. The magnitude of the IgA and IgG1-4 subclass responses was measured by ELISA. IBD patients could not be distinguished from healthy individuals on the basis of IgG autoantibodies to Western blotted proteins. IgG subclass analysis indicated no clear bias towards Th1 or Th2 immune responses in IBD or CTD. In accordance with previous work, we found that IgA autoreactivity was strongest in coeliac disease patients. Unexpectedly, IBD as well as CTD patients exhibited strong IgA autoantibody reactivities to components of similar molecular weights (16-80 kD) in intestinal and non-intestinal epithelial cell lines. Our data indicate immunopathogenic similarities between IBD and CTD.     

Branched-chain amino acids complex inhibits melanogenesis in B16F0 melanoma cells

Present study was investigated the effect of each or complex of three branched-chain amino acids (BCAAs; isoleucine, leucine, and valine) on melanin production in B16F0 melanoma cells treated with various concentrations (1-16?mM) for 72?h. Among the 20 amino acids, lysine and glycine showed the highest activities of DPPH radical scavenging and mushroom tyrosinase inhibition, respectively. Each and combination of BCAAs reduced melanogenesis in a concentration-dependent manner without any morphological changes and cell viability in melanoma cells. Present study was also investigated the inhibitory effects of each or complex of BCAAs at each 10?mM concentration on the 100 μM IBMX-mediated stimulation of melanogenesis in melanoma cells for 72?h and found that IBMX treatment was stimulated to enhance melanin synthesis and that the complex of BCAAs was the most effectively inhibited in the melanin amounts of cellular and extracellular and the whitening the cell pellet. When the inhibitory effect of BCAAs on tyrosinase was examined by intracellular tyrosinase assay, both isoleucine and valine exhibit slightly inhibition, but leucine and combination of BCAAs did not inhibit the cell-derived tyrosinase activity. Present study demonstrated that complex of BCAAs inhibited melanin production without changes intercellular tyrosinase activity. Thus, the complex of BCAAs may be used in development of safe potentially depigmenting agents.     

龙胆草对人黑素细胞增殖及黑素合成的影响

目的探讨龙胆草对人黑素瘤细胞增殖及黑素合成的影响。方法采用MTT法测定龙胆草对人A375黑素瘤细胞增殖的影响,NaOH裂解法测定黑素合成,并与8-MOP的结果进行比较。结果0．1mg／mL、0．2mg／mL和0．5mg／mL龙胆草对人A375黑素瘤细胞增殖无影响,0．1mg／mL、0．2mg／mL和0．5mg／mL龙胆草对人A375黑素瘤细胞酪氨酸酶活性及黑素生成均有较强的剂量相关性激活作用,0．2mg／mL及0．5mg／mL龙胆草与对照组相比差异有统计学意义（P〈0．01）。结论龙胆草对培养的人黑素瘤细胞酪氨酸酶活性及黑素生成有较强的剂量相关性激活作用。     

Branched-chain amino acids complex inhibits melanogenesis in B16F0 melanoma cells

Present study was investigated the effect of each or complex of three branched-chain amino acids (BCAAs; isoleucine, leucine, and valine) on melanin production in B16F0 melanoma cells treated with various concentrations (1-16?mM) for 72?h. Among the 20 amino acids, lysine and glycine showed the highest activities of DPPH radical scavenging and mushroom tyrosinase inhibition, respectively. Each and combination of BCAAs reduced melanogenesis in a concentration-dependent manner without any morphological changes and cell viability in melanoma cells. Present study was also investigated the inhibitory effects of each or complex of BCAAs at each 10?mM concentration on the 100 μM IBMX-mediated stimulation of melanogenesis in melanoma cells for 72?h and found that IBMX treatment was stimulated to enhance melanin synthesis and that the complex of BCAAs was the most effectively inhibited in the melanin amounts of cellular and extracellular and the whitening the cell pellet. When the inhibitory effect of BCAAs on tyrosinase was examined by intracellular tyrosinase assay, both isoleucine and valine exhibit slightly inhibition, but leucine and combination of BCAAs did not inhibit the cell-derived tyrosinase activity. Present study demonstrated that complex of BCAAs inhibited melanin production without changes intercellular tyrosinase activity. Thus, the complex of BCAAs may be used in development of safe potentially depigmenting agents.     

Function-blocking autoantibodies to the melanin-concentrating hormone receptor in vitiligo patients

Vitiligo is a common depigmenting skin disorder resulting from the loss of melanocytes in the cutaneous epidermis. Although the cause of the disease remains obscure, autoimmune mechanisms are thought to be involved. Recently, melanin-concentrating hormone receptor (MCHR)-binding autoantibodies have been identified in vitiligo patients. In the present study, we aimed to determine if MCHR autoantibodies could also affect receptor function either by direct activation or by blocking its response to melanin-concentrating hormone. The results indicated that 10/18 (56%) vitiligo patient IgG samples inhibited the function of MCHR expressed in a Chinese hamster ovary cell line. In contrast, neither control (n=20) nor SLE patient (n=10) IgG samples blocked receptor function. Compared with healthy controls, MCHR function-blocking autoantibodies were found at a significantly increased frequency in the vitiligo patient group (P=0.0004). No MCHR-activating autoantibodies were detected in any of the vitiligo patient, SLE patient or control IgG samples that were analysed. In addition, vitiligo patient IgGs were tested for MCHR autoantibodies that could mediate antibody-dependent cell-mediated cytotoxicity via the receptor. However, this could only be demonstrated in two vitiligo patient sera. Overall, this work has provided additional evidence that MCHR is a B-cell autoantigen in vitiligo and has demonstrated the existence of MCHR function-blocking autoantibodies further to the receptor-binding autoantibodies previously reported.     

Immunohistochemical Study of Melanocyte–Melanocyte Stem Cell lineage in Vitiligo; A Clue to Interfollicular Melanocyte Stem Cell Reservoir

ABSTRACT

There has been a long lasting controversy over whether melanocytes (MCs) in vitiligo are actually lost or still present but functionally inactive. We aimed to evaluate the MC cell lineage in follicular and interfollicular vitiliginous epidermis through immunohistochemical localization of Human Melanoma Black-45 (HMB-45) and Tyrosinase Related Protein 2 (TRP2) and to correlate it with clinicopathologic parameters. Using immunohistochemical techniques, skin biopsies from 50 vitiligo patients and 20 age- and gender-matched healthy subjects were examined. Differentiated active MCs were detected in 44% of interfollicular epidermis (IFE) and 46.7% of follicular epidermis (FE) in lesional skin. Melanocyte precursors/stem cells were detected in 54% of IFE and 63.3% of FE in lesional skin. Melanocyte precursors/stem cells of IFE were significantly associated with residual melanin pigment (p?=?0.007) and with absence of angiogenesis (p?=?0.05). HMB-45 percentage of expression in IFE was positively correlated with MC precursors/stem cells percentage in FE (r?=?+0.65, p?<?0.001) and IFE (r?=?+0.33, p?=?0.01). Melanocyte precursors/stem cells positivity (p?<?0.001) was progressively decreasing with advanced histopathologic grading. There was no significant association between interfollicular or follicular expression of HMB-45, TRP2 or MC precursors/stem cells and the clinical type of vitiligo or its duration. In conclusion, functioning MCs may exist in vitiligo. The presence of MC precursors/stem cells in IFE may provide an additional reservoir needed for repigmentation.     

Photoperiod and testosterone modulate growth and melanogenesis of s91 murine melanoma

In vivo and in vitro assays were performed with S91 murine melanoma cells aiming to investigate the effects of testosterone and photoperiod on tumor growth and melanogenesis (tyrosinase activity). In vivo assays were performed by inducing melanoma tumors in castrated mice receiving increasing concentrations of testosterone and submitted to varying photoperiod regimens. The results demonstrated that the increase of melanin content was higher in animals submitted to the longest days, thus demonstrating the importance of photoperiod length in melanin synthesis. Increase in tumor growth and protein content was observed in testosterone-treated animals submitted to 12L:12D; in testosterone-treated animals submitted to 4L:20D and 20L:4D tumor growth was significantly smaller. In S91 cultured cells, testosterone increased cell proliferation and reduced tyrosinase activity in a dose-dependent manner. Radioactive binding assays demonstrated that the hormone was acting through low affinity testosterone receptors, since the presence of aromatase inhibitor did not affect the binding assay in a statistically significant way, and all the in vitro experiments were performed in the presence of the inhibitor. Our in vivo data added to the in vitro results corroborate the hypothesis that S91 melanoma cells directly respond to testosterone and that this effect is modulated by light.     

Serum DPPIV activity and CD26 expression on lymphocytes in patients with benign or malignant breast tumors

The aim of this work was to determine serum DPPIV activity as well as the percentage of CD26+ white blood cells and of CD26+ lymphocytes and the mean fluorescence intensity (MFI) of CD26 expression on lymphocytes in groups of patients with benign or malignant breast tumors and in healthy control people. Serum DPPIV activity was determined by colorimetric test, while CD26+ cells were counted using flow cytometer. Results of this study show that there is no statistically significant difference in serum DPPIV activity between examined groups of patients and healthy controls. However, two times higher frequency of patients with breast cancers had the enhanced DPPIV enzymatic activity in comparison to controls. Significant decrease in the percentage of CD26+ total white blood cells was found in the group of breast cancer patients and in patients with benign breast tumors compared to that found for healthy people. Although there was decrease in the percentage of lymphocytes in patients with breast tumors it was not statistically significant. The MFI of CD26 expression on these cells was significantly lower for cancer patients in comparison to healthy controls.In conclusion, this work showed the enhanced frequency of breast cancer patients with higher serum DPPIV activity. Decreased percentage of CD26+ white blood cells and decreased CD26 expression on lymphocytes are also characteristics of this group of patients.Determination of the clinical outcome of analyzed patients, 1 and 2 years after the surgical resection of the tumor, would clarify potential prognostic values of examined parameters for breast cancer.     

Celiac disease in North Italian patients with autoimmune thyroid diseases

Background: Patients with autoimmune thyroid diseases (AITDs) are prone to develop other autoimmune manifestations and to display autoimmune polyendocrine syndromes.

An increased prevalence of celiac disease (CD) was demonstrated in adult European and Italian patients with AITDs; conversely, an increased prevalence of AITDs was demonstrated in patients with CD. An IgA deficiency is the most frequent immunodeficiency in humans and, in general, high frequency of this disorder was demonstrated in those with autoimmune diseases.

Aim: To define the prevalence of both CD and IgA deficiency in North Italian patients with AITDs.

Methods: 276 Italian patients with AITD were enrolled (mean age 42.6 years range 12–89, 186 of whom had chronic thyroiditis and 90 had Graves' disease). The tissue transglutaminase autoantibodies of the IgA class (IgA-tTGAbs) were evaluated using an ELISA method in these patients. Furthermore, the serological levels of the IgA were determined.

Results: Five of the patients (1.8%) were affected by previously diagnosed CD and were on a gluten-free diet. Ten out of the remaining 271 patients (3.6%) were found to be positive for celiac-related autoantibodies. All of these patients agreed to undergo endoscopy and duodenal biopsies and silent CD was found in 5 of them but 5 had not histopathological signs of CD.

CD (clinical, silent or latent) was present in 15/276 (5.4%) of the North Italian patients with AITD; this prevalence is significantly higher with respect to the general population (p < 0.00001).

The genetic pattern of the 10 patients with both AITDs and CD was characterized by the presence of DQ2 in 8 patients and DQ8 in 2. An IgA deficiency was present in 2/276 of the patients (0.72%).

Conclusions: CD is significantly increased in patients with thyroid autoimmune disorders for this reason it is important to screen for CD in patients with AITDs.     

Hypogalactosylation of serum IgG in patients with coeliac disease

Coeliac disease (CD) is described as an autoimmune enteropathy associated with the presence of IgG and IgA antigliadin and antitransglutaminase autoantibodies. While of diagnostic significance, the role of these autoantibodies in the immunopathogenesis of CD is elucidated. An inappropriate T cell immune response to gluten is also involved in the pathogenesis of CD, as evidenced by autoantibody switching. The N-glycans released from serum IgG of CD patients and three groups of healthy controls, of differing age ranges, were analysed by NH2-high performance liquid chromatography (HPLC). The fucosylated biantennary N- glycans were the most abundant neutral oligosaccharides; in particular, the agalacto form (G0F) showed a mean value of 42% (s.d. +/- 7.4), 30% (s.d. +/- 5.9), 26% (s.d. +/- 4.2) and 35% (s.d. +/- 6.8) for CD patients, healthy children, healthy adults under 40 and healthy adults over 40 years old, respectively. The ratio of asialo agalacto fucosylated biantenna to asialo monogalacto fucosylated biantenna (G0F)/(G1F) for CD patients showed a significant increase compared to healthy children (P < 0.0002), healthy adults under 40 (P < 0.0002) and healthy adults over 40 years old (P < 0.01). Hypogalactosylation was more pronounced for CD patients than for the patients with other autoimmune diseases such as rheumatoid arthritis or psoriatic arthritis.     

Isotype and IgG Subclass Distribution of Autoantibody Response to Alpha-enolase Protein in Adult Patients with Severe Asthma

Purpose

A possible involvement of autoimmune mechanism in the pathogenesis of bronchial asthma has been proposed. Recently, alpha-enolase protein was identified as a major autoantigen recognized by circulating IgG autoantibodies in patients with severe asthma. To evaluate a possible pathogenetic significance of these autoantibodies in severe asthma, isotype (IgG, IgA, IgM, and IgE) and IgG subclass (IgG1, IgG2, IgG3, and IgG4) distributions of autoantibodies to recombinant human alpha-enolase protein were analyzed.

Patients and Methods

We examined serum samples from 10 patients with severe asthma and 7 patients with mild-to-moderate asthma, and 5 healthy controls by immunoblot analysis. Severe asthma was defined as patients having at least 1 severe asthmatic exacerbation requiring an emergency department visit or admission in the last year despite continuous typical therapies.

Results

IgG1 was the predominant IgG subclass antibody response to alpha-enolase protein in patients with severe asthma. IgG1 autoantibody to alpha-enolase protein was detected in 7 of 10 patients with severe asthma (70%), 1 of 7 patients with mild-to-moderate asthma (14.3%), and none of 5 healthy controls (0%) (chi-square test; p < 0.05). IgA, IgM, and IgE autoantibodies to alpha-enolase protein could not be detected in patients with severe asthma.

Conclusion

IgG1 subclass was the predominant type of autoantibody response to alpha-enolase protein in patients with severe asthma, suggests a possibility of IgG1 autoantibody-mediated complement activation in the pathogenesis of severe asthma.