Rasheedunnisa Begum

Vitiligo: interplay between oxidative stress and immune system

Vitiligo is a multifactorial polygenic disorder with a complex pathogenesis, linked with both genetic and non‐genetic factors. The precise modus operandi for vitiligo pathogenesis has remained elusive. Theories regarding loss of melanocytes are based on autoimmune, cytotoxic, oxidant–antioxidant and neural mechanisms. Reactive oxygen species (ROS) in excess have been documented in active vitiligo skin. Numerous proteins in addition to tyrosinase are affected. It is possible that oxidative stress is one among the main principal causes of vitiligo. However, there also exists ample evidence for altered immunological processes in vitiligo, particularly in chronic and progressive conditions. Both innate and adaptive arms of the immune system appear to be involved as a primary event or as a secondary promotive consequence. There is speculation on the interplay, if any, between ROS and the immune system in the pathogenesis of vitiligo. The article focuses on the scientific evidences linking oxidative stress and immune system to vitiligo pathogenesis giving credence to a convergent terminal pathway of oxidative stress–autoimmunity‐mediated melanocyte loss.

Decreased regulatory T-cells and CD4+/CD8+ ratio correlate with disease onset and progression in patients with generalized vitiligo

The aim of present study was to evaluate CD4+/CD8+ ratio and CD4+CD25hiFoxP3+ Tregs in GV patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4+/CD8+ ratio and Tregs in 82 patients and 50 controls. CD8+ T‐cell counts were significantly higher in GV patients as compared with controls (p = 0.003). Active GV patients showed higher CD8+ T‐cell counts compared with stable GV patients (p = 0.001). The CD4+/CD8+ ratio decreased significantly in patients as compared with controls (p = 0.001). Moreover, the ratio in active GV patients significantly lowered as compared with stable GV patients (p = 0.002). Significant decrease in Treg cell percentage and counts in GV patients was observed compared with controls (p = 0.009, p = 0.008) with significant reduction in FoxP3 expression (p = 0.024). Treg cell percentage and counts were significantly decreased in active GV patients compared with stable GV patients (p = 0.007, p = 0.002). Our results suggest that an imbalance of CD4+/CD8+ ratio and natural Tregs in frequency and function might be involved in the T‐cell mediated pathogenesis of GV and its progression.

Increased Tumor Necrosis Factor (TNF)-α and Its Promoter Polymorphisms Correlate with Disease Progression and Higher Susceptibility towards Vitiligo

Abstract Tumor Necrosis Factor (TNF)-α, is a paracrine inhibitor of melanocytes, which plays a critical role in the pathogenesis of several autoimmune diseases including vitiligo, as abnormal immune responses have frequently been observed in vitiligo patients. Moreover, vitiligo patients show higher lesion levels of TNF-α. Genetic polymorphisms in the promoter region of TNF-α are involved in the regulation of its expression. The present study explores TNF-α promoter polymorphisms and correlates them with TNF-α transcript and protein levels in vitiligo patients and controls of Gujarat along with its effect on disease onset and progression. PCR-RFLP technique was used for genotyping of these polymorphisms in 977 vitiligo patients and 990 controls. TNF-α transcript and protein levels were measured by Real time PCR and ELISA respectively. The genotype and allele frequencies for the investigated polymorphisms were significantly associated with vitiligo patients. The study revealed significant increase in TNF-α transcript and protein levels in vitiligo patients compared to controls. In particular, haplotypes: AATCC, AACCT, AGTCT, GATCT, GATCC and AGCCT were found to increase the TNF-α levels in vitiligo patients. Analysis of TNF-α levels based on the gender and disease progression suggests that female patients and patients with active vitiligo had higher levels of TNF-α. Also, the TNF-α levels were high in patients with generalized vitiligo as compared to localized vitiligo. Age of onset analysis of the disease suggests that the haplotypes: AACAT, AACCT, AATCC and AATCT had a profound effect in the early onset of the disease. Moreover, the analysis suggests that female patients had an early onset of vitiligo. Overall, our results suggest that TNF-α promoter polymorphisms may be genetic risk factors for susceptibility and progression of the disease. The up-regulation of TNF-α transcript and protein levels in individuals with susceptible haplotypes advocates the crucial role of TNF-α in autoimmune pathogenesis of vitiligo.

A Review on Salivary Genomics and Proteomics Biomarkers in Oral Cancer

Oral cancer has emerged as an alarming public health problem with increasing incidence and mortality rates all over the world. Therefore, the implementation of newer screening and early detection approaches are of utmost importance which could reduce the morbidity and mortality associated with this disease. Sensitive and specific biomarkers for oral cancer are likely to be most effective for screening, diagnosis, staging and follow-up for this dreaded malignancy. Unlike other deep cancers, oral cancer is located in oral cavity. Hence, the direct contact between saliva and oral cancer lesion makes the measurement of tumor markers in saliva an attractive alternative to serum and tissue testing. The DNA, RNA and protein molecules derived from the living cancer cells can be conveniently obtained from saliva. Thus, salivary biomarkers, a non-invasive alternative to serum and tissue-based biomarkers may be an effective modality for early diagnosis, prognostication and monitoring post therapy status. In the current post-genomic era, various technologies provide opportunities for high-throughput approaches to genomics and proteomics; which have been used to evaluate altered expressions of gene and protein targets in saliva of oral cancer patients. The emerging field of salivary biomarkers has great potentials to prove its clinical significance to combat oral cancer. Hence, we have reviewed importance of several salivary genomics and proteomics biomarkers for oral cancer.

HLA Alleles and Amino-Acid Signatures of the Peptide-Binding Pockets of HLA Molecules in Vitiligo

Vitiligo is a depigmenting disorder of the skin that is characterized by the loss of functional melanocytes from the lesional sites. Although the exact etiology is not understood, autoimmunity is thought to be a crucial deterministic factor. A recurring theme of several autoimmune disorders is the aberrant presentation of self-antigens to the immune system, which triggers downstream perturbations. Here we examine the role of alleles of HLA class I and class II loci to delineate vitiligo manifestation in two distinct populations. Our studies have identified three specific alleles, HLA-A*33:01, HLA-B*44:03, and HLA-DRB1*07:01, to be significantly increased in vitiligo patients as compared with controls in both the initial study on North Indians (N=1,404) and the replication study in Gujarat (N=355) cases, establishing their positive association with vitiligo. Both generalized and localized vitiligo have the same predisposing major histocompatibility complex alleles, i.e., B*44:03 and DRB1*07:01, in both the populations studied, beside the differences in the frequencies of other alleles, suggesting that localized vitiligo too may be an autoimmune disorder. Significant differences in the amino-acid signatures of the peptide-binding pockets of HLA-A and HLA-B α-chain and HLA-DR β-chain were observed between vitiligo patients and unaffected controls.

Antioxidants prevent UV-induced apoptosis by inhibiting mitochondrial cytochrome c release and caspase activation in Spodoptera frugiperda (Sf9) cells.

Oxidative stress has been shown to be associated with apoptosis (programmed cell death) in a number of cell systems. We earlier reported in vitro cultured Spodoptera frugiperda (Sf9) cells as a model system to study oxidative stress induced apoptosis (J Biosciences 24 (1999) 13) and the inhibition of UV‐induced apoptosis by the baculovirus antiapoptotic p35 protein that acts as a sink to sequester reactive oxygen species (Proc Natl Acad Sci USA 96 (1999) 4838). We now show that UV‐induced apoptosis in Sf9 cells, is preceded by the release of mitochondrial cytochrome c into the cytosol and consequent activation of Sf‐caspase‐1. The inhibitory effect of different antioxidants including scavengers of oxygen radicals such as butylated hydroxyanisole (BHA), alpha tocopherol acetate, benzoate and reduced‐glutathione (GSH) on ultra violet B (UVB)‐induced apoptosis in cultured Sf9 cells was assessed. Both, cytochrome c release as well as Sf‐caspase‐1 activation was inhibited by pre‐treatment with antioxidants such as BHA and alpha tocopherol acetate, suggesting that these antioxidants inhibit apoptosis by acting quite upstream in the apoptosis cascade at the mitochondrial level, as well as downstream at the caspase level.

Interleukin-4 genetic variants correlate with its transcript and protein levels in patients with vitiligo

Background  Vitiligo is an acquired pigmentary disorder resulting from loss of melanocytes. Interleukin (IL)‐4 has been shown to stimulate B‐cell proliferation, to regulate immunoglobulin class switching (IgG1 and IgE) and to promote T‐cell development. Polymorphisms in the IL4 gene are known to increase its expression, thereby implicating its role in vitiligo susceptibility.

Vitiligo: Clinical profiles in Vadodara, Gujarat

Purpose: Vitiligo is an acquired depigmentary condition involving a progressive loss of melanocytes from the epidermis and hair follicles We have earlier reported impairment of systemic antioxidant status of Baroda vitiligo patients ( Pigment Cell Res 2004; 17; 289-94) and we now show analysis of the clinical profiles of these patients. Procedure: The study comprised of 424 vitiligo patients. Clinical and demographic details of all the patients were obtained from the vitiligo clinical proformas. Lipid peroxidation levels (LPO) in erythrocytes of vitiligo patients and healthy controls were estimated. Result: Out of four hundred and twenty four outpatients, males constituted 38.44% and females were 61.56%. Mean age of the patients was 25.59 years. The sites of onset were the lower limb, face, trunk, upper limb, genital, hand, labia and scalp in the descending order of frequency. Koebners phenomenon was observed in 12.74%, diabetes mellitus in 1.18%, leukotrichia in 9.2% and premature graying of hair in 23.11% patients. A family history of vitiligo was present in 21.93% of the patients. Significant increase ( P <0.002) in the LPO levels of the vitiliginous patients was observed compared to the controls. Conclusion: Vitiligo vulgaris was the most common form of the disease which constituted 52.36% of the patients followed by focal vitiligo (28.54%), segmental vitiligo (6.84), acrofacial (7.55%), mucosal (2.83%) and universal vitiligo (1.89%). Systemic oxidative stress may have a pathophysiological role in precipitating all clinical types of vitiligo in Vadodara vitiliginous patients.

The ACE gene I/ D polymorphism is not associated with generalized vitiligo susceptibility in Gujarat population.

Dear Sir, Vitiligo is a hypomelanotic disease characterized by circumscribed depigmented macules and affects 0.5–2% of the world population (Passeron and Ortonne, 2005). However, in India Gujarat has the highest prevalence i.e., approximately 8.8% (Valia and Dutta, 1996). Vitiligo is hypothesized to be of autoimmune origin and genetic factors may also involve in precipitating this disease. Our earlier study suggests that 13.7% of Gujarat vitiliginous patients have at least one firstdegree relative affected (Shajil et al., 2006). Angiotensin-converting enzyme (ACE), a key enzyme in the renin–angiotensin system, plays an important role in the physiology of the vasculature, blood pressure and inflammation. ACE has also been found to be associated with several autoimmune disorders (Scholzen et al., 2003; Vuk-Pavlovic and Rohrbach, 1988). The ACE gene is located on chromosome 17q23.3 and an insertion ⁄ deletion (I ⁄ D) polymorphism of a 287-base pair repetitive sequence in intron 16 of the ACE gene (NCBI: AF118569; repeat region 14094–14381) is reported to be associated with the development of vitiligo (Jin et al., 2004). The I ⁄ D polymorphism of the ACE gene accounts for the variability of serum ACE activity, D ⁄ D genotype having the highest and I ⁄ I genotype having the lowest ACE activity (Rigat et al., 1990). The aim of this study was to investigate the distribution of ACE I ⁄ D genotypes in generalized vitiligo patients and in healthy controls of Gujarat population to find the relationship between ACE I ⁄ D polymorphism and vitiligo. A total of 125 generalized vitiligo patients and 156 ethnically matched healthy controls of Gujarat population were examined for ACE I ⁄ D polymorphism. The patients selected for this study had no other associated diseases. The DNA was prepared from blood samples after written informed consent was obtained. Oligonucleotide primers used for ACE intron 16 PCR amplification were 5¢-CTGGAGACCACTCCCATCCTTTCT 3¢-(forward primer) and 5¢-GATGTGGCCATCACATTCGTCAGAT 3¢ (reverse primer). PCR amplification was performed in 25 ll reaction system containing 50 ng of genomic DNA and 20 pmol of each primer under the following conditions: 94 C for 10 min, 94 C for 30 s, 58 C for 1 min, 72 C for 1 min and final extension at 72 C for 10 min. Amplified PCR products were analyzed on 2% agarose gel electrophoresis with 100 bp DNA ladder to identify the three genotype patterns: I ⁄ I (a 480 bp fragment), D ⁄ D (a 193 bp fragment) and I ⁄ D (two fragments, i.e., 480 and 193 bp). The distribution of the ACE I ⁄ D genotypes for patients and control subjects were compared by using the chi-square test with 2 · 2 and 3 · 2 contingency tables and P-values less than 0.05 were considered statistically significant. The statistical power of the current study was determined by using the G * Power software (Faul et al., 2007). The observed allele and genotype frequencies for the ACE I ⁄ D polymorphism in controls and patient population are shown in Table 1. No significant difference in the genotype frequencies of I ⁄ I, I ⁄ D and D ⁄ D genotypes was observed between vitiligo patients and control subjects (P = 0.459) (Table 1), suggesting that there is no association of the ACE I ⁄ D polymorphism with vitiligo. The observed ACE allele frequencies for I and D alleles were 0.56 and 0.44 in controls; and 0.61 and 0.39 in vitiligo patients, respectively (Table 1). The allele frequencies of ACE I ⁄ D polymorphism did not differ significantly between the controls and patient population (P = 0.252) (Table 1). The observed genotype frequencies of the vitiligo patients did not show significant difference as predicted by the Hardy–Weinberg equation (P < 0.964). Also the control population did not deviate from Hardy– Weinberg equilibrium (P < 0.905). This study has 86% statistical power for the effect size 0.2 to detect

Biochemical basis of the high resistance to oxidative stress in Dictyostelium discoideum

Aerobic organisms experience oxidative stress due to generation of reactive oxygen species during normal aerobic metabolism. In addition, several chemicals also generate reactive oxygen species which induce oxidative stress. Thus oxidative stress constitutes a major threat to organisms living in aerobic environments. Programmed cell death or apoptosis is a physiological mechanism of cell death, that probably evolved with multicellularity, and is indispensable for normal growth and development.Dictyostelium discoideum, an eukaryotic developmental model, shows both unicellular and multicellular forms in its life cycle and exhibits apparent caspase-independent programmed cell death, and also shows high resistance to oxidative stress. An attempt has been made to investigate the biochemical basis for high resistance ofD. discoideum cell death induced by different oxidants. Dose-dependent induction of cell death by exogenous addition of hydrogen peroxide (H2O2),in situ generation of H2O2 by hydroxylamine, and nitric oxide (NO) generation by sodium nitroprusside treatment inD. discoideum were studied. The AD50 doses (concentration of the oxidants cusing 50% of the cells to die) after 24 h of treatment were found to be 0.45 mM, 4 mM and 1 mM, respectively. Studies on enzymatic antioxidant status ofD. discoideum when subjected to oxidative stress, NO and nutrient stress reveal that superoxide dismutase and catalase were unchanged; a significant induction of glutathione peroxidase was observed. Interestingly, oxidative stress-induced lipid membrane peroxidative damage could not be detected. The results shed light on the biochemical basis for the observed high resistance to oxidative stress inD. discoideum.