Molecular Mechanisms of Malignant Transformation of Oral Submucous Fibrosis by Different Betel Quid Constituents-Does Fibroblast Senescence Play a Role?

Betel quid (BQ) is a package of mixed constituents that is chewed by more than 600 million people worldwide, particularly in Asia. The formulation of BQ depends on a variety of factors but typically includes areca nut, betel leaf, and slaked lime and may or may not contain tobacco. BQ chewing is strongly associated with the development of potentially malignant and malignant diseases of the mouth such as oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC), respectively. We have shown recently that the constituents of BQ vary geographically and that the capacity to induce disease reflects the distinct chemical composition of the BQ. In this review, we examined the diverse chemical constituents of BQ and their putative role in oral carcinogenesis. Four major areca alkaloids-arecoline, arecaidine, guvacoline and guvacine-together with the polyphenols, were identified as being potentially involved in oral carcinogenesis. Further, we propose that fibroblast senescence, which is induced by certain BQ components, may be a key driver of tumour progression in OSMF and OSCC. Our study emphasizes that the characterization of the detrimental or protective effects of specific BQ ingredients may facilitate the development of targeted BQ formulations to prevent and/or treat potentially malignant oral disorders and oral cancer in BQ users.

Efficacy of Oil Pulling in the Management of Oral Submucous Fibrosis: A Preliminary Study

ABSTRACT Objective: To evaluate the effectiveness of oil pulling in the management of oral submucous fibrosis (OSMF). Material and Methods: A total of 62 patients clinically and histopathologically diagnosed as OSMF were incorporated in the present study. The subjects are randomly divided equally into two groups, Group A (oil pulling group) and Group B (placebo group). Subjects in Group A were asked to perform OP with sesame oil on an empty stomach in the morning for 3 months and Group B was given placebo capsules for 3 months. Assessment of various clinical parameters was done regularly, and data were analyzed using the Chi-square test. Results: Eighty-two percent had a habit of betel nut chewing, while 18% of the patients had tobacco chewing habits, which were among the main causative factors for OSMF. Clinical improvements in mouth opening, tongue protrusion, difficulty in speech and deglutition, and burning sensation were significant in the Group A. None of the patients reported any discomfort or side effects. The symptoms were not severe in nature and resolved in few days without stopping the therapy. Conclusion: Oil pulling can bring about significant clinical improvements in the symptoms like a mouth opening and tongue protrusion.

Tanshinone Suppresses Arecoline-Induced Epithelial-Mesenchymal Transition in Oral Submucous Fibrosis by Epigenetically Reactivating the p53 Pathway.

Oral submucous fibrosis (OSF) induced by chewing of the areca nut has been considered to be a precancerous lesion with a high probability of developing oral squamous cell carcinoma. Tanshinone (TSN) is the main component extracted from Salvia miltiorrhiza, a traditional Chinese medicine, which was found to have diverse pharmacological effects, such as anti-inflammatory and antitumor. In the current study, we aimed to identify the inhibitory effects and the underlying mechanism of TSN on OSF progress. We found that treatment with TSN inhibited the arecoline-mediated proliferation of primary human oral mucosal fibroblasts and reversed the promotive effects of arecoline on the EMT process. By RNA deep sequencing, we screened two possible targets for TSN: LSD1 and p53. We confirmed that p53 is much lower in OSF than in normal mucous tissues. In addition, p53 and its downstream molecules were decreased by arecoline treatment in oral mucosal fibroblasts, which was reversed by treatment with TSN in a dose-dependent manner. Our results also revealed that arecoline stimulation resulted in hypermethylation of the promoter of TP53 and subsequent downregulation of p53 levels, which was reversed by TSN. Furthermore, we identified that LSD1 could epigenetically activate TP53 by recruiting H3K27me1 and H3K4m2 to its promoter. Our findings provide new insights into the mechanism by which TSN influences arecoline-induced OSF and rationale for the development of clinical intervention strategies for OSF and even oral squamous cell carcinoma.

Characterization of a Novel Dermal Fibrosis Model Induced by Areca Nut Extract that Mimics Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is an oral potentially malignant disorder and areca quid chewing is the main etiological factor. However, the molecular mechanism underlying OSF remains unclear, partly due to the lack of an appropriate animal model. The present study aimed to establish and characterize an animal model of areca nut extract (ANE)-induced skin fibrosis that mimics OSF. Mice were divided into 4 groups: the control group; the bleomycin group; and the ANE10 and ANE20 groups, which received 10mg/ml and 20mg/ml subcutaneous (SC) injection of ANE, respectively. Skin fibrosis was evaluated by histological analyses. Additionally, the expression levels of the fibrotic marker genes were determined by immunohistochemical staining and immunoblotting. ANE administration significantly increased dermal thickness and collagen deposition compared with the control group. Moreover, ANE induced the expression of the fibrotic marker genes alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) in the skin lesions. The SC injection of ANE successfully induced skin fibrosis, exhibiting characteristics similar to those of OSF. This model may facilitate future studies of the mechanism underlying OSF.

Research on curcumin: A meta-analysis of potentially malignant disorders.

INTRODUCTION: Turmeric has been described in ayurveda, and is referred by different names in different cultures, the active principle called curcumin or diferuloylmethane, has been shown to exhibit numerous activities. Extensive research over the last half century has revealed several important functions of curcumin. It binds to a variety of proteins and inhibits the activity of various kinases. By modulating the activation of various transcription factors, curcumin regulates the expression of inflammatory enzymes, cytokines, adhesion molecules, and cell survival proteins. Various preclinical, clinical, and animal studies suggest that curcumin has potential as an antiproliferative, anti-invasive, and antiangiogenic, as a mediator of chemoresistance, chemopreventive, and as a therapeutic agent. Thus, curcumin a spice once relegated to the kitchen shelf has moved into the clinic and may prove to be "Curecumin." Methodology and Objectives: The focus of this publication is to provide research on curcumin with scientific publications on curcumin indexed in PubMed, Google J-Gate including systematic reviews, randomized controlled trials (RCT's), observational studies, or case series reports for various potentially malignant disorders (PMD's) with special attention to studies on oral submucous fibrosis. This research will be valuable in terms of identifying opportunities to provide recommendations for future research, in terms of the populations to research, the types of interventions needed, the types of outcomes to be measured, the study designs needed, to initiate a pathway for a low-cost research plan for future clinical trials in this field with an emphasis on conducting studies in regions of the world where PMD's are prevalent. CONCLUSION: There is a lacunae for scientific review of curcumin for PMDs specially on OSMF. Appropriate therapeutic interventions are needed for the initial, intermediate, and advanced stages of the disease. High-quality RCTs should be initiated.

Epithelial atrophy in oral submucous fibrosis is mediated by copper (II) and arecoline of areca nut.

Exposure of oral cavity to areca nut is associated with several pathological conditions including oral submucous fibrosis (OSF). Histopathologically OSF is characterized by epithelial atrophy, chronic inflammation, juxtaepithelial hyalinization, leading to fibrosis of submucosal tissue and affects 0.5% of the population in the Indian subcontinent. As the molecular mechanisms leading to atrophied epithelium and fibrosis are poorly understood, we studied areca nut actions on human keratinocyte and gingival fibroblast cells. Areca nut water extract (ANW) was cytotoxic to epithelial cells and had a pro-proliferative effect on fibroblasts. This opposite effect of ANW on epithelial and fibroblast cells was intriguing but reflects the OSF histopathology such as epithelial atrophy and proliferation of fibroblasts. We demonstrate that the pro-proliferative effects of ANW on fibroblasts are dependent on insulin-like growth factor signalling while the cytotoxic effects on keratinocytes are dependent on the generation of reactive oxygen species. Treatment of keratinocytes with arecoline which is a component of ANW along with copper resulted in enhanced cytotoxicity which becomes comparable to IC(50) of ANW. Furthermore, studies using cyclic voltammetry, mass spectrometry and plasmid cleavage assay suggested that the presence of arecoline increases oxidation reduction potential of copper leading to enhanced cleavage of DNA which could generate an apoptotic response. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling assay and Ki-67 index of OSF tissue sections suggested epithelial apoptosis, which could be responsible for the atrophy of OSF epithelium.

Efficacy of oxitard capsules in the treatment of oral submucous fibrosis.

AIM: Oral submucous fibrosis (OSMF) is a high-risk premalignant condition predominantly seen in the Indian subcontinent. The aim of the present study was to evaluate the efficacy of oxitard capsules in the management of OSMF. MATERIALS AND METHODS: Total of 120 subjects with clinico-pathologically diagnosed OSMF were included in the study and divided equally in 2 groups; Group A (oxitard group) and Group B (placebo group). Group A was administered 2 oxitard capsules twice daily and Group B was given placebo tablets twice daily, for 3 months. Evaluation for different clinical parameters was done at regular intervals and data was analyzed using the Student's paired t test and Chi-square test. P < 0.001 was considered to be statistically significant. RESULTS: Clinical improvements in mouth-opening and tongue protrusion were significant in the Group A (P < 0.001). Subjective symptoms of burning sensation (P = 0.0001), pain associated with the lesion (P = 0.000), difficulty in swallowing (P = 0.0003) and speech (P = 0.0005) also significantly improved in the Group A. There was a mild to moderate decrease in the size of the lesion. CONCLUSION: Though there is no definitive treatment for the condition; however, oxitard capsules can bring about significant clinical improvements in the symptoms like mouth-opening, tongue protrusion, burning sensation, difficulty in swallowing and speech and pain associated with the lesion, thereby improving the quality of life of the affected individuals.

Panax notoginseng saponins inhibit areca nut extract-induced oral submucous fibrosis in vitro.

BACKGROUND: Oral submucous fibrosis (OSF) is a premalignant and fibrosing disease, which is closely associated with the habit of chewing areca nut. Panax notoginseng Buck F. H. Chen is an often used antifibrotic and antitumor agent. To treat areca nut-induced OSF, we have developed a chewable tablet, in which one of the major medicines is total Panax notoginseng saponins (PNS). In this study, we have investigated the antifibrotic effect and mechanism of PNS on areca nut-induced OSF in vitro. METHODS: Through human procollagen gene promoter luciferase reporter plasmid, hydroxyproline assay, gelatin zymography, qRT-PCR, ELISA, and Western blot, the influences of PNS on areca nut extract (ANE)-induced cell growth, collagen accumulation, procollagen gene transcription, MMP-2/-9 activity, MMP-1/-13 and TIMP-1/-2 expression, cytokine secretion, and the activation of PI3K/AKT, ERK/JNK/p38 MAPK, and TGFß/Smads pathways were detected. RESULTS: Panax notoginseng saponins could inhibit the ANE-induced abnormal growth and collagen accumulation of oral mucosal fibroblasts in a concentration-dependent manner. PNS (25 µg/ml) could significantly inhibit the ANE-induced expression of Col1A1 and Col3A1, augment the ANE-induced decrease of MMP-2/-9 activity, inhibit the ANE-induced increase of TIMP-1/-2 expression, and decrease the ANE-induced transcription and release of CTGF, TGFß1, IL-6, and TNFα. PNS (25 µg/ml) also significantly inhibited the ANE-induced activation of AKT and ERK/JNK/p38 MAPK pathways in oral mucosal fibroblasts and the ANE-induced activation of TGFß/smad pathway in HaCaT cells. CONCLUSION: Panax notoginseng saponins possess excellent anti-OSF activity, and its mechanism may be related to its ability to inhibit the ANE-induced activation of PI3K/AKT, ERK/JNK/p38 MAPK, and TGFß/smad pathways.

Areca nut-induced buccal mucosa fibroblast contraction and its signaling: a potential role in oral submucous fibrosis--a precancer condition.

Betel quid (BQ) chewing is an oral habit that increases the risk of oral cancer and oral submucous fibrosis (OSF), a precancerous condition showing epithelial atrophy and tissue fibrosis. Persistent fibroblast contraction may induce the fibrotic contracture of tissue. In this study, we found that areca nut extract (ANE) (200-1200 µg/ml) stimulated buccal mucosa fibroblast (OMF)-populated collagen gel contraction. Arecoline but not arecaidine-two areca alkaloids, slightly induced the OMF contraction. Exogenous addition of carboxylesterase (2U/ml) prevented the arecoline- but not ANE-induced OMF contraction. OMF expressed inositol triphosphate (IP3) receptors. ANE-induced OMF (800 µg/ml) contraction was inhibited by U73122 [phospholipase C (PLC) inhibitor] and 2-aminoethoxydiphenyl borate (IP3 receptor antagonist), respectively. Ethylene glycol tetraacetic acid and verapamil, two calcium mobilization modulators, also suppressed the ANE-induced OMF contraction. ANE induced calcium/calmodulin kinase II and myosin light chain (MLC) phosphorylation in OMF. Moreover, W7 (a Ca(2+)/calmodulin inhibitor), HA1077 (Rho kinase inhibitor), ML-7 (MLC kinase inhibitor) and cytochalasin B (actin filament polymerization inhibitor) inhibited the ANE-induced OMF contraction. Although ANE elevated reactive oxygen species (ROS) level in OMF, catalase, superoxide dismutase and N-acetyl-L-cysteine showed no obvious effect on ANE-elicited OMF contraction. These results indicate that BQ chewing may affect the wound healing and fibrotic processes in OSF via inducing OMF contraction by ANE and areca alkaloids. AN components-induced OMF contraction was related to PLC/IP3/Ca(2+)/calmodulin and Rho signaling pathway as well as actin filament polymerization, but not solely due to ROS production.

Oral submucous fibrosis in a 11-year-old Bangladeshi girl living in the United Kingdom.

A case of oral submucous fibrosis occurring in a 11-year-old Bangladeshi girl is presented. This paper reviews the aetiology, clinical presentation and treatment modalities of oral submucous fibrosis. This case highlights the link between oral submucous fibrosis and the regular use of areca-nut (paan) and the newer transcultural oral tobacco products. This association has been reported among children resident in the Indian sub-continent but is unrecorded for United Kingdom residents. The case report underlines the danger that children face with products which are clearly targeted at them by the tobacco industry.

Raised tissue copper levels in oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a well-recognised-potentially malignant condition of the oral cavity associated with areca nut chewing. Areca nut has been shown to have a high copper content compared to other commonly eaten nuts, and chewing areca nut for 5-30 min significantly increases soluble copper in whole mouth fluids. Our aims were to determine if tissue and serum concentrations of copper were raised in patients with OSF as a result of chewing areca nut. A panel of buccal mucosal biopsies from patients with OSF from Nagpur, India, was used to measure the tissue concentrations of copper by mass absorption spectrometry (MAS). By MAS, the mean tissue copper level was 5.5+/-2.9 microg/g in the OSF specimens (n=11) compared with 4+/-1.9 microg/g in the non-areca chewing controls (n=7) (P=0.2). Energy dispersive x-ray microanalysis (EDX) was used to identify the presence and distribution of the metal element. EDX showed distinct peaks corresponding to copper (Kalpha 8.04 keV; Kbeta, 8.91 keV) in the epithelium (21/23) and in the connective tissue (17/23) of the OSF specimens compared to spectra obtained from control oral biopsies from non-areca chewing subjects (n=7). These findings were confirmed by secondary ion mass spectrometry (SIMS) analysis in a small number of samples. Serum copper (17.23+/-1.80 pmol/l), caeruloplasmin (0.32+/-0.04 g/l) levels and urinary copper (0.52+/-0.26 micromol/l) in OSF patients (n=14) were within the laboratory reference ranges. The finding of copper in OSF tissue supports the hypothesis of copper as an initiating factor in OSF, playing a role in stimulating fibrogenesis by the upregulation of lysyl oxidase activity.

Clinical and functional staging of oral submucous fibrosis.

OBJECTIVE: To investigate the association of location of bands in oral submucous fibrosis and extent of mouth-opening. DESIGN: Cross-sectional study. SETTING: University hospital, Pakistan. SUBJECTS: 325 patients who presented with oral submucous fibrosis in Karachi between January 1992 and October 1994, of whom 288 had data sufficient for analysis. MAIN MEASURES: Location of fibrous bands in the mouth and interincisal distance (mm). RESULTS: All subjects with labial bands had bands in the fauces, and all but one who had labial bands also had buccal bands. All those with buccal bands also had bands in the fauces. Of those with buccal bands, 42% did not have labial bands. The proportion of patients with bands in all three sites increased from functional stage A (interincisal opening < or = 10 mm) to stage C (interincisal opening > or = 20 mm) with a simultaneous reduction in the proportion of people with bands in one or two sites (X2 = 105, df = 4, P < 0.0001). CONCLUSIONS: Bands are common at the back of the mouth in mild cases of oral submucous fibrosis and, as the disease increases in severity, are more likely to be found anteriorly as well.

Oral mucosal lesions associated with betel quid, areca nut and tobacco chewing habits: consensus from a workshop held in Kuala Lumpur, Malaysia, November 25-27, 1996.

A variety of betel/areca nut/tobacco habits have been reviewed and categorized because of their possible causal association with oral cancer and various oral precancerous lesions and conditions, and on account of their widespread occurrence in different parts of the world. At a recent workshop in Kuala Lumpur it was recommended that "quid" be defined as "a substance, or mixture of substances, placed in the mouth or chewed and remaining in contact with the mucosa, usually containing one or both of the two basic ingredients, tobacco and/or areca nut, in raw or any manufactured or processed form." Clear delineations on contents of the quid (areca nut quid, tobacco quid, and tobacco and areca nut quid) are recommended as absolute criteria with finer subdivisions to be added if necessary. The betel quid refers to any quid wrapped in betel leaf and is therefore a specific variety of quid. The workshop proposed that quid-related lesions should be categorized conceptually into two categories: first, those that are diffusely outlined and second, those localized at the site where a quid is regularly placed. Additional or expanded criteria and guidelines were proposed to define, describe or identify lesions such as chewer's mucosa, areca nut chewer's lesion, oral submucous fibrosis and other quid-related lesions. A new clinical entity, betel-quid lichenoid lesion, was also proposed to describe an oral lichen planus-like lesion associated with the betel quid habit.

[Research on inhibition of areca nut extract on human buccal keratinocytes].

OBJECTIVE: To culture human oral mucosa epithelial cells in vitro and study the mechanism of changes in the epithelia of oral submucous fibrosis (OSF). METHODS: The keratinocytes were obtained from normal buccal mucosa (NM-KC) and OSF buccal mucosa (OSF-KC) with dispase trypsine and cultured in vitro. Then the cell proliferation of keratinocytes incubated with or without areca nut extract in the presence of 10% fetal calf serum for 48 hours at 37 degrees C in 5% CO2 and air were monitored by use of thiazolyl blue (MTT) assay. RESULTS: Primary culture grew in small islands that coalesced at confluency. Ultrastructurally the cells contained a large of tonofilament and microvilli. Immunohistochemistry demonstrated uniform staining of the cells with antibodies to keratins. There was no difference in cell proliferation between OSF-KC and NM-KC, and areca nut extract inhibited keratinocyte proliferation in a concentration-dependent manner. CONCLUSION: The results suggested that utilizing dispase II can establish fibroblast-free culture for growing of human oral mucosa keratinocytes and areca nut extract is a cytotoxic agent to human buccal keratinocytes.

Oesophageal subepithelial fibrosis: an extension of oral submucosal fibrosis.

Fifty-five patients with oral submucosal fibrosis and an equal number of patients with no evidence of the disease were studied. All patients underwent upper gastrointestinal endoscopy and any abnormality was noted. Multiple oesophageal biopsies were obtained from the upper end of the oesophagus and from any endoscopically observed abnormality. The histological changes in the two groups were assessed blindly by an experienced histopathologist. Histological abnormalities were noted in the oesophageal mucosa in 2% of controls and 66% of patients with oral submucosal fibrosis (p < 0.0001). In the control group, acanthosis was seen in one patient, while in the patient group atrophy of the squamous epithelium was evident in 52%, hyperkeratosis in 52%, parakeratosis in 30%, dyskeratosis in 14%, acanthosis in 14%, and papillomatosis and mild dysplasia in 2% patients. Subepithelial collagenization was seen in 32 (64%) patients. The oesophageal abnormalities were seen more frequently in patients who had consumed Pan masala, Gutka, betel nut, tobacco or a combination of some or all of these, with or without betel leaf, for > or = 5 years compared to those consuming them for a shorter period of time (91% vs 46%, p < 0.001). It is concluded that oral submucosal fibrosis is not a disease confined to the oral cavity; the oesophagus may also be involved in about two-thirds of patients.

Increased severity of oral submucous fibrosis in young Pakistani men.

OBJECTIVE: To investigate the influence of age and sex on the incidence of severe oral submucous fibrosis (mouth opening 15 mm or less). DESIGN: Case (severe disease) control (mild disease) study. SETTING: University hospital, Pakistan. SUBJECTS: 147 patients with severe (mouth opening 15 mm or less) and 83 with mild disease (mouth opening 20 mm or more). MAIN MEASURES: Influence of sex and age on severity of oral submucous fibrosis. RESULTS: Men were twice as likely as women to have severe disease (odds ratio (OR) 2.0, 95% confidence interval (CI) of OR 1.15 to 3.47, P=0.02). Men in the 20-39 year age group were 4.8 times as likely as women to have severe disease (OR 5.8, 95% CI 2.15 to 15.5, P=0.00004). Men aged 40 years or more were 40% less likely to have severe disease than women (OR 0.6, 95% CI 0.19 to 1.88, P=0.39). CONCLUSIONS: Smoking increases the carcinogenic potential of submucous fibrosis. As men in this population smoke more than women, men with severe oral submucous fibrosis may develop oral cancer and die younger. Alternatively, young men may consume more areca nut than women.

[Experimental study on aqueous areca nut extracts inducing oral submucous fibrosis in rats. I. Observation of histomorphology].

The purpose of this study was to establish the animal model of oral submucous fibrosis (OSF) in buccal mucosa of SD rats by submucosa injecting and surface painting aqueous areca nut extracts (AANE). The animals of all groups were killed randomly in the 12th, 16th, 22nd and 28th week. The buccal mucosae were collected, and were detected by light microscope and transmission electron microscope. The results were obtained: at different stages, the OSF-like lesions appeared in different degrees among some animals of the model groups, including atrophic epithelium with loss or partial loss of rate pegs, inflammation, accumulation and disorder of collagen fibre in juxta-epithelial region, along with concomitant muscle degeneration. The lesions didn't reverse obviously themselves after the authors stopped using AANE. Histomorphologic changes didn't appear in any animal of the control groups. The results indicated that the typical and stable OSF animal model were induced rapidly by submucosa injecting and surface painting AANE.

[Experimental study on aqueous areca nut extracts inducing oral submucous fibrosis in rats. II. Effection of mast cells on collagen metabolism].

The effection of mast cells (MC) on collagen metabolism of oral submucous fibrosis (OSF) induced by aqueous areca nut extracts (AANE) in rats was studied by transmission electron microscope, histochemical and biochemical analyses. The results revealed that there was a close relationship between MC and fibroblats (FB) in the pathogensis of OSF; MC which appeared in the buccal mucosa in the model groups increased significantly and became more active in the function. A great deal of collagen compacted in the buccal mucosa in the model groups. The contents of tissue hydroxyproline (Hyp) in the model groups were obviously higher than those in the control groups. Moreover, the longer the time of irritation continued, the higher the contents were. The number of MC had a higher positive correlation with the contents of tissue Hyp at the same time. Total serum Hyp had no significant difference among all the groups and any stages. The results indicated that some aqueous components of areca nuts might disturb collagen metabolism by accumulation and activation of MC.

Oral submucous fibrosis. The South African experience.

A review of research related to oral submucous fibrosis (OSF) among South Africans of Indian descent shows a certain uniqueness compared to other countries. In South Africa the betel habit is more common among women, only 60% of chewers prefer the betel quid while the rest like the nut by itself, the majority of chewers prefer the baked (black) nut variety and a minority add tobacco to their chew. This pattern reflects in the distribution of OSF and the practice of the habit by OSF subjects. Compared to chewers without OSF, OSF subjects are younger and have shorter histories of chewing. Yet the profile of systemic diseases were similar among subjects with and without OSF. The habit as practised in South Africa also determines the pattern of oral squamous carcinomas. They are more common in women, with buccal mucosa cancers being the most frequent. The latter are commonly found in subjects not using any tobacco, indicating the carcinogenicity of the areca nut. It was also shown that oral cancer can develop in chewers without an intermediate precancerous OSF phase. A follow-up of OSF cases after cessation of the habit revealed that once present the disease is permanent. An analysis of cultured OSF fibroblasts demonstrated a permanent shift to larger cells theoretically capable of producing larger amounts of collagen. Thus the agents in the nut could be the initiators of the disease and its permanent character the result of a phenotypic alteration in cells from changes in gene expression.