Evaluation of ABCB5 immunostained epithelial stem cells in oral squamous cell carcinoma, inflammatory gingival hyperplasia and normal mucosa.

Oral cancer is the most prevalent head and neck cancer. Although tumor markers have been investigated for detecting the progression and prognosis of oral cancer, no reliable marker has been identified. We investigated the expression of ATP binding cassette subfamily B member 5 (ABCB5) positive stem cells in oral squamous cell carcinoma (OSCC) and in inflammatory gingival hyperplasia. We used tissue samples from normal subjects, patients with inflammatory gingival hyperplasia, and patients with OSCC. Samples were investigated using anti-ABCB5 monoclonal antibody immunohistochemistry to detect epithelial stem cells. Staining density, intensity, and immunoreactive scores of ABCB5 were analyzed for the three study groups. We found ABCB5 immunostaining in all three study groups, but different distributions of ABCB5 expression in different layers of the epithelium. We found no significant difference in staining intensity between inflammatory hyperplasia and normal mucosa, but we found significantly stronger expression in OSCC compared to normal and inflammatory hyperplasia individually. Elevated expression of ABCB5 in OSCC suggests an increased subpopulation of tumor cells with an undifferentiated stem cell phenotype, which facilitates cancer initiation and progression.

8-Isoprostane: A salivary oxidative stress biomarker for oral submucous fibrosis and oral squamous cell carcinoma.

BACKGROUND: 8-isoprostane is one of the stable oxidative stress marker formed by the lipid peroxidation of arachidonic acid. It is present in detectable quantities in all biological fluids. Elevation of 8-Isoprostane has been reported in various neurological, cardiological disorders, and periodontal diseases. AIM: The present study was conducted to estimate and compare the level of 8-isoprostane in plasma and saliva in patients with oral squamous cell carcinoma (OSCC), oral submucous fibrosis (OSMF), and in controls. The study also aimed to find out if 8-isoprostane can be used as an effective oxidative stress marker in evaluating the disease progression in OSCC. MATERIALS AND METHODS: Plasma and salivary samples were taken from 10 cases each of clinically diagnosed OSMF, clinically and hisotpathologically diagnosed cases of OSCC and controls. The samples were subjected to 8-Isoprostane ELISA procedure and analyzed. Statistical analysis was performed using the SPSS software. RESULTS: The levels of 8-isoprostane in plasma showed an average increase from normal to OSMF to OSCC but was not statistically significant. The variations in the level of salivary 8-isoprostane were found to be statistically significant (P = 0.037) suggesting that there is a gradual increase in levels of isoprostane from controls to OSMF to OSCC. CONCLUSION: The results showed that the concentration of isoprostane in saliva showed a progressive and steady increase from control through OSMF to OSCC indicating that saliva could be used as an effective diagnostic tool in estimating tumor markers. Large scale studies correlating with other potentially malignant oral disorders are required to ascertain the role of 8-Isoprostane as an ideal tumor marker.

Antioxidants: Friend or foe?

Reactive oxygen species are the intermediates that are formed during the normal metabolic process which are effectively neutralized by the antioxidant system of the body. Any imbalance in this neutralization process causes oxidative stress which has been implicated as one of the cause in diseases such as Alzheimer's disease, cardiovascular disorders, cancer etc. Research has enabled the use of antioxidants as therapeutic agents in the treatment of various diseases. Literature also puts forth the negative effects of using antioxidants in the treatment of diseases. This review is a compilation of both the beneficial and detrimental effects of use of antioxidants in the treatment of diseases such as cancer, cardiovascular diseases, diabetes and oral diseases.

A Study of Diabetes Associated Oral Manifestations.

AIM: The aim of this work was to determine the frequency of oral changes in diabetic patients and to study the relationship between periodontal disease and diabetes mellitus. MATERIALS AND METHODS: The study sample consisted of 440 known diabetic patients between the age group of 20-80 years, of which 212 were males and 228 were females. One hundred and six patients were below 40 years, 138 patients between 41 and 50 years, 97 in 51-60 years, and 99 above 60 years of age. Data were statistically analyzed by Student's t-test. RESULTS: Nearly 57% of the patients showed a Russell's Periodontal Index score of 2-4.9, which suggested an established periodontal disease. Risk factors for the people above the age of 40 years to develop diabetes were 76%. CONCLUSION: The frequency of oral manifestations in diabetic patients was significantly high, hence showing a relationship of gingival and periodontal diseases with diabetes mellitus.

Traumatic ulcerative granuloma with stromal eosinophilia - Mystery of pathogenesis revisited.

Oral ulcers are a common symptom in clinical practice. Among various causative factors, different types of ulcers in oral cavity exist. Among this, traumatic ulcerative granuloma with stromal eosinophilia (TUGSE) appears to be quite neglected by the clinicians due to the limited knowledge and awareness. On reviewing with a detailed approach to titles and abstracts of articles eliminating duplicates, 40 relevant articles were considered. Randomized studies, review articles, case reports and abstracts were included while conference papers and posters were excluded. Of importance, TUGSE cases been reported only to a minimal extent in the literature. Lack of its awareness tends to lead clinicians to a misconception of cancer. Thus, this particular lesion needs to be differentiated from other malignant lesions to provide a proper mode of treatment. The present article reviews various aspects of the TUGSE with emphasis on the clinical manifestation, pathogenesis, histological, and immunohistochemical study. This study provides the clinician contemporaries, a humble expansion to their knowledge of the disease, based on the searched literature, enabling a more comprehensive management of this rare occurrence.

Pseudoepitheliomatous Hyperplasia: Relevance in Oral Pathology.

Pseudoepitheliomatous hyperplasia (PEH), a neglected entity by oral pathologist possesses utmost importance in the field of research. Of all the investigative challenges, PEH, a reactive epithelial proliferation is seen secondary to lesions with infectious, inflammatory, reactive, and degenerative origin. Small sized samples, incomplete excision, improper orientation, and dense inflammatory changes render diagnostic confront to the oral pathologist in exclusion of frankly invasive malignant lesions like squamous cell carcinoma from lesions exhibiting PEH. The diagnosis can occasionally be difficult as they mimic other lesions also, on clinic-pathological assessment. Thus, this article gives an insight regarding the various concepts of etiopathogenesis, histopathology, differential diagnosis, and malignant potential of PEH. A combined effort of a clinician and pathologist benefits every patient to rule out malignancy and render appropriate treatment as the only local conservative approach is essential to remove PEH associated lesions.

Abnormal translocation of tyrosinase and tyrosinase-related protein 1 in cutaneous melanocytes of Hermansky-Pudlak Syndrome and in melanoma cells transfected with anti-sense HPS1 cDNA.

Hermansky-Pudlak syndrome is an autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding disorder, and, in some patients, ceroid storage and progressive lung disease. Although Hermansky-Pudlak syndrome exhibits locus heterogeneity, most patients have mutations in the HPS1 gene. Melanocytes in the basal epithelial layer of skin from patients with different mutations in the HPS1 gene exhibited occasional large complexes containing dihydroxyphenylalanine-positive cisterna and 50 nm vesicles. To characterize the role of the HPS1 protein in cells, human HPS1 cDNA was transfected into pigmented SK-MEL-188 melanoma cells (M-188) in either the sense (S-188) or the antisense (A-188) orientation. Expression of the 79 kDa HPS1 protein (in M-188 and S-188 cells) or lack of expression (in A-188 cells) was confirmed by Western blotting using two HPS1-protein-specific polyclonal antibodies. Significant reduction in expression of HPS1 protein in A-188 cells resulted in a significant decrease in tyrosinase activity and melanin content compared with M-188 and S-188 cells using an intact cell assay for tyrosinase. In contrast, tyrosinase activities in cell lysates of M-188, S-188, and A-188 cells were not significantly different. Knockout of HPS1 protein expression in A-188 cells caused both tyrosinase and tyrosinase-related protein 1 to be localized to large granular complexes in the cell cytosol and dendrites. Electron microscope analysis of the A-188 cells revealed that absence of HPS1 protein resulted in the deposition of dihydroxyphenylalanine reaction products (i.e., tyrosinase) confined to large membrane-bound structures with limiting membranes. We conclude that lack of HPS1 protein expression results in mistranslocation of tyrosinase and tyrosinase-related protein 1 to large granular complexes rather than melanosomes, compromising melanin synthesis.

AP-3 mediates tyrosinase but not TRP-1 trafficking in human melanocytes.

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.

Molecular and functional characterization of sodium--hydrogen exchanger in skin as well as cultured keratinocytes and melanocytes.

The sodium--hydrogen (Na(+)/H(+)) exchanger is one of the few transporter proteins involved in the regulation and maintenance of intracellular pH and cell volume in most eukaryotic cell types. The current study investigates the expression of isoforms of the Na(+)/H(+) exchanger (NHE) in human skin and in cultured keratinocytes, melanocytes, and melanoma cells by reverse transcription-polymerase chain reaction (RT--PCR), immunohistochemical analysis and functional studies. Neonatal foreskins were used to isolate RNA from epidermis and dermis, and to initiate cultures of keratinocytes and melanocytes. RT--PCR on RNA isolated from epidermis, dermis, keratinocytes, melanocytes and melanoma cells using PCR primers specific for NHE-1 yielded a 463 bp PCR product. RT--PCR performed using primers specific for NHE isoforms 2, 3, 4 and 5 did not yield any products. Western blotting analysis (of keratinocyte and melanocyte cell cultures) and indirect immunohistochemistry on neonatal foreskin, keratinocytes, melanocytes and melanoma cells using a NHE-1-specific polyclonal antibody demonstrated NHE-1 expression at the protein level. Physiological regulation of intracellular pH using a pH-sensitive dye, BCECF, detected an amiloride-sensitive NHE activity in human keratinocyte, melanocyte and melanoma cell cultures. These results indicate that cultures of human keratinocytes and melanocytes established from human skin and melanoma cells express the NHE-1 isoform of the sodium--hydrogen exchanger.

Primitive neuroectodermal tumors of the biliary and gastrointestinal tracts: clinicopathologic and molecular diagnostic study of two cases.

Primitive neuroectodermal tumor (PNET) is a prototypic malignant small round cell tumor of childhood that is characterized in most cases by t(11;22) resulting in an EWS-FLI1 gene fusion. Once thought to be uncommon, PNET now accounts for almost 20% of malignant soft tissue tumors in children. Increased recognition of PNET is partly due to advances in immunohistochemistry and molecular diagnostics, which have led to the identification of the tumor in non-classical sites. We report the clinical, histologic, immunohistochemical, and molecular findings of two visceral PNETs of the digestive system--one involving the small intestine and the other involving the hepatic duct. Histologically, each tumor was composed of malignant small cells growing in sheets, nests, and lobules; the tumor cells of both cases showed characteristic immunoreactivity for vimentin and O13 (CD99). Reverse transcription-polymerase chain reaction (RT-PCR) analysis for t(11;22) using nested primers was performed with RNA extracted from paraffin-embedded, formalin-fixed tissue and demonstrated an EWS exon 7 to FLI1 exon 5 fusion in both cases, confirmed by Southern blot hybridization and DNA sequence analysis. These results illustrate the expanded clinicopathologic profile of PNET, and demonstrate that visceral PNETs, despite their unusual sites of presentation, maintain the characteristic immunohistochemical and genetic features of PNETs at more conventional sites.

Tyrp1 and oculocutaneous albinism type 3.

Tyrosinase-related protein 1 (Tyrp1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutations in the mouse Tyrp1 gene are associated with brown pelage, and in the human TYRP1 gene with oculocutaneous albinism type 3 (OCA3). In the murine system, Tyrp1 expresses significant dihydroxyindole carboxylic acid oxidase (i.e. DHICA oxidase) activity. However, in humans, TYRP1 is enigmatic in that despite extensive efforts focused on the study of its function, its actual role in the human melanocyte is still unclear. There is mounting evidence demonstrating that in addition to its role in eumelanin synthesis, Tyrp1 is involved in maintaining stability of tyrosinase protein and modulating its catalytic activity. Tyrp1 is also involved in maintenance of melanosome ultrastructure and affects melanocyte proliferation and melanocyte cell death. The current review is an attempt to consolidate our understanding of the role of Tyrp1 in the melanocyte.

'VIT1', a novel gene associated with vitiligo.

To define genes associated with the pigmentary disorder vitiligo, gene expression was compared in non-lesional melanocytes cultured from three vitiligo patients and from three control melanocyte cultures by differential display. A basic local alignment search tool search did not reveal homology of six differentially expressed cDNA fragments to previously identified expressed sequence tags; thus, one was used to screen a melanocyte cDNA library. The underlying VIT1 gene maps to chromosome 2p16. The 3' portion of the VIT1 message is complementary to the 3' end of hMSH6 mRNA, enabling the formation of RNA-RNA hybrids, which may interfere with G/T mismatch repair function. Moreover, the aligned cDNA sequence revealed an open reading frame identical to a hypothetical protein expressed in brain, with a similarity to Drosophila calmodulin, and containing a zinc-finger motif partially identical to N-recognin. Expression of ORF mRNA was confirmed for multiple skin cell types, suggesting its importance for skin physiology.

Mutant alleles at the brown locus encoding tyrosinase-related protein-1 (TRP-1) affect proliferation of mouse melanocytes in culture.

Tyrosinase related protein-1 (TRP-1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP-1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1b (brown) and Tyrp1b-cj (cordovan) compared with wild type Tyrp1B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP-1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP-1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.

PIG3V, an immortalized human vitiligo melanocyte cell line, expresses dilated endoplasmic reticulum.

Vitiligo is an enigmatic pigmentary disorder of the skin. Factors potentially involved in the progressive loss of melanocytes from the basal layer of the epidermis include genetically determined aberrancies of the vitiligo melanocyte. It follows that analysis of melanocytes cultured from vitiligo donors can contribute to a further understanding of the etiopathomechanism. A setback for vitiligo research has been the limited availability of vitiligo-derived melanocytes. To overcome this limitation, we have generated a vitiligo melanocyte cell line according to a protocol established previously for the immortalization of normal human melanocytes. Vitiligo melanocytes Ma9308P4 were transfected with HPV16 E6 and E7 genes using the retroviral construct LXSN16E6E7. Successful transformants were selected using geneticin and subsequently cloned to ensure genetic homogeneity. The resulting cell line PIG3V has undergone more than 100 cell population doublings since its establishment as a confluent primary culture, whereas untransfected melanocytes derived from adult skin senesce after a maximum of 50 population doublings. Cells immortalized by this transfection procedure retain lineage-specific characteristics and proliferate significantly faster than parental cells. In this study, the phenotype of PIG3V resembled melanocytes rather than melanoma cells in culture. Tyrosinase was processed properly and melanosomes remained pigmented. Importantly, ultrastructural characterization of PIG3V cells revealed dilated endoplasmic reticulum profiles characteristic of vitiligo melanocytes. An explanation for this dilation may be found in the retention of proteins with molecular weight of 37.5. 47.5, and 56.5 kDa, as determined by gel electrophoresis of microsomal proteins isolated from radiolabeled cells.

The cytotoxicity and apoptosis induced by 4-tertiary butylphenol in human melanocytes are independent of tyrosinase activity.

It has been known for several decades that cutaneous depigmentation, i.e., contact/occupational vitiligo, can be caused by some phenolic derivatives that have a similar structure to tyrosine. Among these phenolic depigmenting agents, 4-tertiary butylphenol is the most potent. The cutaneous depigmentation induced by phenolic derivatives results from the loss of functional melanocytes. Tyrosinase is a melanocyte specific copper-containing enzyme that catalyzes the conversion of the amino acid tyrosine, through a complex series of intermediates, to melanin. In this study we tested the hypothesis that the cytotoxicity induced by 4-tertiary butylphenol is mediated by tyrosinase and occurs via an apoptotic process. Melanocyte cultures derived from African-American and Caucasian donors exhibiting a 3-fold difference in tyrosinase activity and 14-fold difference in melanin content demonstrate comparable concentration-dependent sensitivity to 4-tertiary butylphenol. In addition, cultures of dermal fibroblasts and epidermal keratinocytes exhibited similar and reduced sensitivity, respectively, to 4-tertiary butylphenol compared with autologous melanocytes. Two melanoma cell lines, one melanotic and one amelanotic lacking the expression of both tyrosinase protein and activity, when transfected with the tyrosinase cDNA, exhibited no alteration in its sensitivity to 4-tertiary butylphenol. These data suggest that 4-tertiary butylphenol cytotoxicity is not mediated via tyrosinase. Melanocytes treated with 4-tertiary butylphenol, however, did exhibit plasma membrane blebbing, DNA fragmentation, and phosphatidylserine relocalization indicating that 4-tertiary butylphenol induced melanocyte destruction occurs by an apoptotic process.

Normalization of hyperglycaemia by oral vanadyl sulfate does not reverse diabetes-induced protection against cisplatin nephrotoxicity in streptozotocin-diabetic rats.

Streptozotocin- and galactose-induced diabetic rats are protected against nephrotoxic effects of cisplatin. While the mechanism remains to be defined, protection is associated with a decrease in the accumulation of platinum in renal cortical tissues of streptozotocin-diabetic versus non-diabetic rats. A physiological abnormality common to streptozotocin and galactosemic models of diabetes is hyperglycaemia, suggesting that elevated sugars are involved in mediating protection of diabetic kidney against cisplatin nephrotoxicity. The current study focused on the effect of normalization of hyperglycaemia by vanadyl sulfate trihydrate on the initiation of protection and accumulation of platinum in kidneys of streptozotocin-diabetic rats. Streptozotocin-diabetic rats were treated with 0.75 mg/ml of vanadyl sulfate trihydrate in drinking water to normalize streptozotocin-induced hyperglycaemia. Vanadyl sulfate treatment normalized plasma glucose and glycosylated haemoglobin levels in streptozotocin-diabetic rats to values observed for non-diabetic rats. Intraperitoneal administration of cisplatin (5 mg/kg body weight) increased blood urea nitrogen by a factor >2.5 over baseline in both untreated and vanadyl-treated non-diabetic groups. Cisplatin-induced increases in blood urea nitrogen were 1.6 times baseline in both untreated and vanadyl-treated streptozotocin-diabetic rats. Renal platinum accumulation was significantly lower in streptozotocin-diabetic versus non-diabetic rats regardless of vanadyl sulfate treatment. Renal vanadium levels in all groups of diabetic rats were not significantly different from each other. These results indicate that normalization of plasma glucose levels with vanadyl sulfate in streptozotocin-diabetic rats did not reverse protection of streptozotocin-diabetic kidney against cisplatin nephrotoxicity.

Cranial and extracranial fasciitis of childhood: a clinicopathologic and immunohistochemical study.

Fasciitis of various types constitutes a distinctive category of soft tissue lesions whose microscopic features are known for their potential to evoke a pathological diagnosis of one or another type of sarcoma. Nodular fasciitis is the archetype of this group of "fibrous tumors." Cranial fasciitis is considered a nonneoplastic lesion similar to nodular fasciitis, which is seen almost exclusively in infants and children and has unique clinicopathologic features. The current study documents our experience with fibrous-myofibroblastic proliferations in extracranial sites, mainly in the head and neck region of young children, whose histological features resemble those of cranial fasciitis. These lesions were composed of loosely arranged spindle to stellate cells in a myxoid background. One patient in the study had both cranial and extracranial involvement. Some histological overlap between nodular fasciitis and cranial-extracranial fasciitis was noted, but the latter lesion tended to be more uniform in appearance than nodular fasciitis with its fascicular, spindle cell features and variability in histological patterns within the same lesion, unlike the more consistently uniform myxoid appearance of the cranial and extracranial lesions. Immunohistochemically, the spindle cells of the extracranial lesions and the one case of cranial fasciitis co-expressed vimentin and smooth muscle actin. The extracranial lesions had a predilection for children in the first year of life with all cases occurring at or before 2 years of age, unlike nodular fasciitis, which is rarely seen in the first 4 to 5 years of life. The cranial and extracranial fasciitides should be differentiated from the fibromatoses that tend to locally recur, unlike the nonrecurring behavior of these lesions in common with the other types of fasciitis.

NHE-1 is the sodium-hydrogen exchanger isoform present in erythroid cells.

Erythrocyte sodium hydrogen exchanger (NHE) represents one of a limited number of sodium entry pathway in erythrocytes. At least five NHE isoforms have been identified, differing in tissue specificity, regulatory characteristics, and pharmacological sensitivities. Although physiological characteristics of erythrocyte NHE suggest that the widely expressed NHE-1 isoform may be present, evidence is not conclusive and does not exclude the existence of other isoforms. In this study, Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses were used to test for five NHE isoforms in erythroid cells. Blood from patients with sickle cell disease was depleted of white blood cells (WBC) by passage through leukocyte filters and cellulose column. RT-PCR performed on WBC depleted reticulocyte RNA using a NHE-1 primer set yielded product a of expected size, the sequence of which was identical to the published human NHE-1 sequence. Northern blot analysis of the reticulocyte RNA using a 1.6 kb probe revealed a message of approximately 5.0 kb in size. RT-PCR analysis of rat kidney RNA using primers specific for NHE isoforms -2, -3, -4 and rat brain RNA using primer specific for NHE-5 isoform yielded products of expected size, whereas WBC depleted RNA under identical conditions yielded no products. These results identify the erythroid isoform of the sodium hydrogen exchanger as NHE-1.