Shape and volume of craniofacial cavities in intentional skull deformations.

Intentional cranial deformations (ICD) have been observed worldwide but are especially prevalent in preColombian cultures. The purpose of this study was to assess the consequences of ICD on three cranial cavities (intracranial cavity, orbits, and maxillary sinuses) and on cranial vault thickness, in order to screen for morphological changes due to the external constraints exerted by the deformation device. We acquired CT-scans for 39 deformed and 19 control skulls. We studied the thickness of the skull vault using qualitative and quantitative methods. We computed the volumes of the orbits, of the maxillary sinuses, and of the intracranial cavity using haptic-aided semi-automatic segmentation. We finally defined 3D distances and angles within orbits and maxillary sinuses based on 27 anatomical landmarks and measured these features on the 58 skulls. Our results show specific bone thickness patterns in some types of ICD, with localized thinning in regions subjected to increased pressure and thickening in other regions. Our findings confirm that volumes of the cranial cavities are not affected by ICDs but that the shapes of the orbits and of the maxillary sinuses are modified in circumferential deformations. We conclude that ICDs can modify the shape of the cranial cavities and the thickness of their walls but conserve their volumes. These results provide new insights into the morphological effects associated with ICDs and call for similar investigations in subjects with deformational plagiocephalies and craniosynostoses.

Inhibition of herpes simplex virus replication by tobacco extracts.

Herpes simplex virus type 1 (HSV-1) has been associated with the genesis of leukoplakias, epithelial atypia, and oral cancer. Tobacco habits, such as snuff dipping, are also definitely correlated with this type of lesion. The normal cytolytic HSV-1 infection can, after in vitro inactivation, transform cells. Extracts of snuff were prepared and assayed for their ability to inhibit HSV-1 replication. Plaque formation assays of HSV-1 in the presence of snuff extract showed that a reduced number of plaques was formed. Different batches of one brand of snuff were tested for inhibition of herpes simplex virus (HSV) production. More than 99% inhibition of 24-hr HSV production was obtained with undiluted batches. The 1:5 dilutions of snuff had an inhibitory effect of 85% and 1:25 dilutions, 39%. In agreement, the attachment of the virus to the host cell and penetration of the virus to the cell nuclei were found to be inhibited as was the synthesis of viral DNA. Nicotine had an inhibitory effect, while aromatic additions to snuff were found to have no major inhibitory effect on HSV replication. Snuff extracts were prepared from different brands of snuff reported to contain high and low quantities of tobacco-specific N-nitrosamines. Brands with reported high levels of tobacco-specific N-nitrosamines had significantly greater ability to inhibit HSV replication. In conclusion, this study has shown that extracts of snuff have inhibitory effects on the production of cytolytic HSV-1 infections. A chronic snuff dipper keeps tobacco in the mouth for the major part of the day. Thus, virus shed in the oral cavity in connection with a reactivated latent HSV-1 infection has great possibilities of being affected by snuff or derivatives of snuff. It is suggested that an interaction between tobacco products and HSV-1 might be involved in the development of dysplastic lesions in the oral cavity.

Snuff-induced carcinogenesis: effect of snuff in rats initiated with 4-nitroquinoline N-oxide.

A canal in the lower lip to function as a reservoir for snuff was surgically created in 150 male Sprague-Dawley rats. The animals were randomized into five groups of 30 each: Group I received snuff twice a day, 5 days a wk; Group II was painted with propylene glycol (solvent control) on the hard palate 3 times a wk during 4 wk; Group III underwent painting on the hard palate with 4-nitroquinoline N-oxide (4-NQO) dissolved in propylene glycol, 3 times a wk for 4 wk; Group IV received 4-NQO as in Group III followed by snuff application as in Group I; and Group V received a cotton pellet dipped in saline twice a day, 5 days a wk. Treatment continued for up to 108 wk. There was no significant difference in mean survival time between the groups. Squamous cell tumors of the lip, oral and nasal cavities, esophagus, and forestomach were seen only in Groups I, III, and IV. Nine tumors of these organs were found in Group I (six carcinomas and three papillomas), nine in Group III (seven carcinomas and two papillomas), and ten in Group IV (eight carcinomas and two papillomas). The difference between each of these groups and the control groups (II and V) with regard to tumor incidence is statistically significant (P less than 0.05). In Group I, four oral cavity or lip carcinomas were found in 29 rats, a significant difference in relation to control rats (P less than 0.05). In addition, hyperplastic lesions of the lip, palate, and forestomach were significantly more common in Groups I and IV compared with Groups II, III, and V. The study has shown that snuff and 4-NQO by themselves have the potential to induce malignant tumors. Initiation with 4-NQO followed by snuff did not significantly enhance tumor formation.

Hemodynamic effects of the use of oral snuff.

The hemodynamic effects during rest and exercise of oral snuff were investigated in an open, placebo-controlled study of nine habitual users of oral snuff. Blood pressure, heart rate, and central hemodynamics were measured noninvasively. Plasma concentrations of nicotine, cotinine, norepinephrine, and epinephrine, as well as neuropeptide Y-like immunoreactivity were measured before and after snuff intake during rest and exercise. Snuff intake induced a significant increase in heart rate and blood pressure and a decrease in stroke volume during rest. Hemodynamic changes were unrelated to nicotine or cotinine concentrations. Resting levels of norepinephrine and neuropeptide Y-like immunoreactivity were similar with or without snuff, whereas epinephrine was slightly increased 30 minutes after snuff intake. The exercise-induced increase in norepinephrine and neuropeptide Y-like immunoreactivity did not differ between the days subjects received snuff and the days they received placebo. In contrast, maximum work load was associated with a slight increase in circulating epinephrine after snuff intake. The findings suggest that snuff intake is associated with significant hemodynamic effects during rest but not during exercise. These effects could not be readily explained by activation of the sympathetic nervous system.

High-throughput DNA extraction from old paraffin-embedded biopsies.

We have developed a rapid and reliable microporous spin-column-based method for high-throughput extraction of DNA from old paraffin-embedded biopsies. The method was developed specifically for PCRs and molecular genetic studies. De-waxing and DNA extraction from tissues is performed in only one vessel, minimizing pipetting steps and eliminating supernatant/pellet procedures. All steps are performed at 60 degrees, 55 degrees and 95 degrees C minimize deleterious DNase activity. The amount of the extracted DNA was measured spectrophotometrically, and the quality of the extracted DNA and the PCR products was controlled on ethidium bromide-stained 3% agarose gel. The presented method for DNA extraction is less laborious and more convenient than previously published methods. It provides means by which extensive, paraffin-embedded material can be subjected to molecular genetic studies.

A randomized, double-blind, placebo-controlled clinical evaluation of a nicotine sublingual tablet in smoking cessation.

AIMS: Evaluation of the clinical efficacy and safety of a nicotine 2-mg sublingual tablet in smoking cessation. DESIGN: A randomized, double-blind, placebo-controlled study of smokers using the 2-mg tablet for 3-6 months with follow-up to 12 months. Dosing was established according to baseline nicotine dependence, scored on the Fagerström Tolerance Questionnaire (FTQ): FTQ > or = 7, two tablets/hour (maximum 40/day); FTQ < 7, one tablet/hour (maximum 20/day). SETTING: Smoking cessation programme in a department of oral and maxillofacial surgery. PARTICIPANTS: A total of 247 adult smokers, smoking > or = 10 cigarettes/day for > or = 3 years, of whom 123 received active and 124 placebo treatment. The study was powered to detect difference at 6 months. MEASUREMENTS: Efficacy and safety were evaluated at 6 weeks and 3, 6 and 12 months. Self-reported abstinence was verified by exhaled CO < 10 p.p.m. FINDINGS: Success rates for complete abstinence (no slips after 2 weeks) for active vs. placebo were 50% vs. 29% at 6 weeks, 42% vs. 23% at 3 months, 33% vs. 18% at 6 months and 23% vs. 15% at 12 months (p < 0.001, 0.001, 0.005 and p = 0.14), respectively. Craving during the first 8 days was significantly reduced among highly dependent smokers on active treatment compared to placebo. Baseline mucosal lesions among abstinent subjects were reduced during the treatment period and at the non-treatment follow-up. Adverse events were mild and tolerable, the most common being irritation and soreness in the mouth and throat. CONCLUSION: The nicotine sublingual tablet increased the smoking cessation rate compared to placebo, reduced craving in highly dependent smokers and was well tolerated.

The long-term effect of nicotine on the oral mucosa.

AIMS: Although nicotine replacement therapy (NRT) has been used to aid smoking cessation for the last 20 years, little information exists on the effect of nicotine products on the oral mucosa, particularly with regard to the direct effect at the site of application. This study aimed to assess the oral safety of a new sublingual tablet containing 2 mg nicotine with regard to lesions at the site of application. DESIGN: Prospective follow-up to 12 months of smokers using the 2-mg sublingual tablet over a period of 3-6 months. SETTING: A smoking cessation programme. PARTICIPANTS: Thirty smokers. MEASUREMENTS: Oral mucosa was inspected and photographed at each visit. At 6 months, subjects were asked for consent to take a biopsy from the site of application. FINDINGS: Spontaneous smoking cessation outcome at 12 months was 27% allowing for lapses. At baseline 21 mucosal lesions were diagnosed in 15 subjects. After 6 months eight lesions were observed in six subjects. The predominant diagnosis at all visits was melanin pigmentation. Eight subjects had lesions in the floor of the mouth during the 6-month medication period, all of which appeared in the first 1-6 weeks of treatment. By the 6-month visit all such lesions had resolved. The local symptoms were all mild and tolerable. CONCLUSION: The sublingual tablet appears to be a safe form of administration of nicotine with mild and transient effects on the floor of the mouth.

The Swedish snus and the Sudanese toombak: are they different?

In Sweden, snuff (locally known as snus), was introduced since the year 1637. Presently, Sweden has the highest per capita consumption and sale figures of snuff in the world, and the habit is becoming increasingly popular. Snus is manufactured into a dry form used in the nasal cavity and a moist form used in the oral cavity. Snus manufactured for oral use is a moist ground tobacco of Dark Kentucky or Virginia species mixed with an aqueous solution of water and other blending ingredients. This form of snuff is found in two types: (1) loose and (2) portion-bag-packed. These are the most widely used. The loose moist form (1-2 g a quid) is the most popular type consumed by 73% of the males, followed by the portion-bag-packed form (0.5-1 g a quid), consumed by 13% of the males, while 14% of the males are mixed users. The majority of snus users place the quid in the vestibular area of the upper lip, and the prevalence among persons 15 years of age or older in 15.9% among males and 0.2% among females. The pH of snus has declined from a previous range of 8-9 to a range of 7.8-8.5, moisture content ranges 35-60% and nicotine content is in the order of 5-11 mg/g dry wt tobacco-specific N-nitrosamines (TSNAs) in micrograms (N'-nitrosonornicotine: NNN 5-9; 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone: NNK 1-2; N'-nitrosoanatabine: NAT 2-5). In the Sudan, snuff, locally known as toombak, was introduced approximately 400 years ago. It is always processed into a loose moist form, and its use is widespread in the country. Tobacco used for manufacture of toombak is of the species Nicotiana rustica, and the fermented ground powder is mixed with an aqueous solution of sodium bicarbonate. The resultant product is moist, with a strong aroma, highly addictive and its use is widespread particularly among males. Its pH range is 8-11, moisture content ranges 6-60% and nicotine content is from 8 to 102 mg/g dry wt, and TSNAs contents in micrograms (NNN 420-1 550; NNK 620-7 870; NAT 20-290). Snus and toombak dippers develop a clinically and histologically characteristic lesion at the site of dipping. Probably due to control of the TSNAs in snus, this type of snuff is associated with a lower risk of cancer of the oral cavity (relative risk: RR 5-6-fold), whereas the risk for cancer of the oral cavity among toombak users was high (RR 7.3-73.0-fold). In conclusion, the two snuff products significantly differ in many aspects. Most notable differences are tobacco species, fermentation and ageing, nicotine and TSNAs content, pH, expression of the p53 tumour suppressor gene, and keratin types 13, 14, and 19. It was, therefore, the object of the present study to highlight the oral health hazards of toombak, and to compare it with snus regarding the aforementioned differences.

Human papilloma viruses in oral lesions.

Oral mucosa biopsies from 53 patients with different oral diseases, and from 12 healthy control patients, were examined by the PCR-technique using the L1 consensus primers and type-specific primers for HPV 6/11, 16 and 18. Three out of 24 (12.5%) oral cancer biopsies were HPV positive, 1 for HPV 18, 1 for HPV 16 and 6/11 and 1 for none of the specific primers. Six out of 22 (27.3%) lichen planus were HPV positive, 5 for HPV 18 and 1 for none of the specific primers. Seven leukoplakias were included in the study and 2 (29.6%) were positive for both HPV 16 and 6/11. None of the 12 control patients was HPV positive. No statistical difference between the use of tobacco and alcohol and HPV prevalence was found. An association between HPV infection and oral lesions was demonstrated in the studied population but the pathogenic influence of HPV infection remains unclear.

Effects of long-term administration of cancer-promoting substances on oral subepithelial mast cells in the rat.

The role of oral subepithelial mast cells in the defence against tumours is a matter of controversy. The effect of established and suggested carcinogens, such as the carcinogen 4-nitroquinoline-N-oxide (4-NQO) and Herpes simplex virus type 1 (HSV-1), in combination with oral snuff on lower lip subepithelial mast cells (MC) was studied in rats. The rats were exposed to prolonged use of oral snuff. The test substances were administered in a surgically created canal in the lower lip of the rats. There were 15 rats in each test group and 10 rats in the control group. The amount of countable subepithelial mast cells decreased significantly when the rat oral mucosa was exposed to the oral carcinogen 4-NQO but the effect of oral snuff and HSV-1 infection was weak. Our findings suggest that mast cells play a role in immunological cell defence against chemical carcinogens. Further studies are needed to clarify the mechanisms.

Non-random chromosome rearrangements in herpes simplex virus type 1 transformed diploid CHEF cells.

The effects of Herpes simplex virus type 1 (HSV-1) on diploid, non-tumourigenic Chinese hamster embryo fibroblasts (CHEF/18-1D-3) were studied. Six independent lines transformed by HSV-1 alone or by HSV-1 in combination with acyclovir or aqueous tobacco extract were isolated. In contrast to uninfected CHEF/18-1D-3 cells, all transformants grew in soft agar and were tumourigenic in nude mice. Neither infectious virus nor viral antigens could be detected in any of the lines. Cytogenetic analysis revealed clonal chromosome abnormalities in all lines including trisomy for the long arm of chromosome 3 in five lines. In three of these the extra 3q was translocated onto 6p. All lines showed loss of the corresponding 3p arm. The remaining line had a hypodiploid stemline with loss of one chromosome 7. This line also showed a pronounced chromosomal instability with a multitude of mainly sporadic rearrangements. These results show that HSV-1 induced transformation and tumourigenesis in CHEF cells is associated with the induction of chromosome rearrangements, in particular trisomy for 3q.

Expression of keratin 13, 14 and 19 in oral hyperplastic and dysplastic lesions from Sudanese and Swedish snuff-dippers: association with human papillomavirus infection.

Changes in the expression of keratins (Ks), indicating disturbed tissue differentiation, is one possible marker of malignant potential in stratified squamous epithelia. The presence of human papillomaviruses (HPVs) in the epithelium of the uterine cervix is increasingly regarded as a marker of risk for cervical cancer: However, a similar role in oral cancer and precancer remains controversial. To address these questions, potentially malignant oral mucosal lesions from Sudanese (9 hyperplasias/40 dysplasias) and Swedish (15 hyperplasias) snuff-dippers were examined by immunohistochemistry for expression of K types 13, 14 and 19 using monoclonal antibodies directed against each. HPV infection was searched for by in situ hybridization (ISH) using the cocktail HPV OmniProbe and the ViraType probe. For the Sudanese lesions, moderate to intense expression of both K13 (basal, basal/intermediate, basal/intermediate/superficial and intermediate/superficial cell layers) and K14 (basal, basal/intermediate cell layers) was found in 49/49 (100%). For the Swedish lesions, weak to moderate expression of K13 (basal, basal/intermediate cell layers) was found in 12/15 (80%) and 10/15 (67%), respectively. In the Sudanese lesions, expression of K13 showed a distinct pattern through the oral mucosa and its verrucous projections, with an increase towards the superficial cell layers of dysplastic, but not hyperplastic epithelium. K19 was expressed in the basal cell layer in 16/49 (33%) of the Sudanese lesions, while all the Swedish lesions were negative. HPV was found in only 2 Sudanese cases, both of which harboured both type 6 and type 11: both these cases demonstrated mild epithelial dysplasia, The present study shows that a) there is a high prevalence of expression of both K13 and K14 in oral lesions from Sudanese toombak dippers indicating dysregulation of keratinocyte maturation b) one-third of the Sudanese oral lesions expressed K19, regarded as a basal keratin representing epithelial dedifferentiation, which may prove to be a valuable risk marker in follow-up studies c) HPV genome is found infrequently in oral lesions from Sudanese toombak-dippers, suggesting that these viruses may not play a prominent role in the early stages of carcinogenesis in these subjects. These markers were less often expressed in the Swedish lesions, consistent with their much lower rate of malignant transformation.

Effect of repeated oral administration of tobacco snuff on natural killer-cell activity in the rat.

The possible immune effect of oral snuff was investigated. In male adult rats, oral snuff given for 15 weeks significantly decreased NK-cell activity in peripheral blood against murine NK cell-sensitive target cells (YAC-1 lymphoma).

Inhibition of herpes simplex virus replication and protein synthesis by non-smoked tobacco, tobacco alkaloids and nitrosamines.

Inhibitory effects of snuff extract and the tobacco chemicals nicotine, anabasine, diethyl-N-nitrosamine (DEN), and the tobacco-specific nitrosamines (TSNA), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on herpes simplex virus type 1 (HSV-1) replication in vitro and on HSV-1 protein synthesis in infected cells were analysed. Snuff extract and nicotine caused a significant reduction of HSV-1 attachment to cell membranes whereas anabasine, DEN, NNN and NNK did not affect adsorption of HSV-1. Virus production assays in the presence of snuff added after virus adsorption resulted in a significantly reduced production of virus at low multiplicities of infection (MOI), but at high MOI the inhibitory effect of snuff extract was less pronounced. DEN, NNN and NNK only affected virus production at toxic concentrations. Nicotine and anabasine reduced virus production in non-toxic doses but not at the concentrations present in snuff extract. In HSV-infected cells exposed to snuff extract, the immediate early (alpha-) infected cell proteins (ICPs) 4 and 27 (as well as the early (beta-) ICPs 6 and 8) were markedly increased, whereas the late (gamma-) ICPs 5, 11 and 29 were reduced. Nicotine had a less pronounced stimulating effect on the production of alpha-proteins but no detectable effect on production of beta- or gamma-proteins. Anabasine, DEN, NNN and NNK did not affect HSV protein synthesis at non-toxic concentrations. Synthesis of thymidine kinase and DNA polymerase was significantly reduced by snuff extract. Also nicotine and anabasine affected thymidine kinase and DNA polymerase but only at toxic concentrations. The production of the cellular protein actin, which almost disappears a few hours after HSV-1 infection, remained at a significant level in HSV-infected cells exposed to snuff. Thus snuff extract blocks the replicative cycle of HSV at an early stage, which results in an increased production of alpha-proteins in the infected cells and in prolonged maintenance of cellular functions. This may be of importance for HSV-induced transformation and the development of HSV-associated tumours.

Hydroxyapatite granule/carrier composites promote new bone formation in cortical defects.

BACKGROUND: A great deal of interest has been focused on finding substitutes for autogenous bone grafts. Among the most interesting materials are different calcium phosphate compositions (e.g., hydroxyapatite [HA]), due to their biocompatible properties in hard and soft tissue. PURPOSE: The bone response to porous ceramic HA granules in combination with two lipid and one polysaccharide carrier was evaluated in an experimental bone defect model in rabbits. MATERIALS AND METHODS: Circular defects (Ø 4 mm) were made in both tibias of 32 rabbits. The 64 defects were divided into four groups. Group A was augmented with a composite of HA granules and a phospholipid-diacetyl-glycerol carrier, group B with HA granules and a phospholipid carrier, group C received HA granules and a sodium hyaluronan carrier, and group D served as control. The animals were killed after 6 weeks and ground sections were evaluated using light microscopic morphometry. X-ray microfluorescence (XRF) was applied in order to evaluate the suitability of this method to examine bone-biomaterial interfaces. Calcium distribution was studied using x-ray fluorescence line scans at selected interface regions of two sections in group B. RESULTS: The HA/phospholipid composites were easier to shape and handle than the HA/hyaluronan composite. Group A had 36% newly formed bone area within the defect. Groups B and C showed significantly more newly formed bone within the defect (47% and 49%, respectively) compared to the control group (31%). The XRF analysis revealed that the amount of calcium in the newly formed bone was similar to that observed for the HA granules and slightly lower when compared to the mature, lamellar bone. CONCLUSIONS: Synchrotron radiation may be a new, suitable technique to study the interface between bone and biomaterials with regard to mineral content. The results suggest that HA granule/lipid and HA granule/hyaluronan composites have interesting properties as bone-substitute materials.

Autogenous free tooth transplantation in man by a 2-stage operation technique. A longitudinal intra-individual radiographic assessment.

Insufficient nutrition to the root surface of autotransplanted mature human teeth after transplantation could be an explanation for the high rates of complications such as root resorption and loss of the tooth transplant. Insufficient nutrition is suggested as being due to the distance between the recipient site tissue and the root surface, and the presence of a blood-clot between the tissues. Improved nutrition could be achieved by transplanting into contact with the vascularised connective tissue of a healing wound. To test this hypothesis, a series of human teeth with fully developed roots were autotransplanted to recipient sites prepared and left to heal for 14 days before transplantation. This study is a follow-up of 57 transplants with a minimum observation time of 1 year. Periodic radiographic examinations of the teeth were performed up to 5 years after transplantation. The results presented, showed a lower incidence of root resorption and extractions than previously reported for teeth with fully developed roots.

Epstein-Barr virus and human papillomavirus in snuff-induced lesions of the oral mucosa.

Epstein-Barr virus (EBV) and human papillomavirus (HPV) have been associated with benign as well as malignant oral lesions. We examined 43 patients with snuff-induced lesions and 22 control patients with clinically healthy oral mucosa for the presence of HPV and EBV. Polymerase chain reaction was performed on fresh frozen oral biopsies with degenerate consensus primers for HPV and nested primers for EBV. None of the 43 snuff-induced lesions or the 22 control biopsies were HPV-positive. Seven of the 43 (16.3%) snuff-induced lesions and one of the 22 (4.5%) controls were positive for EBV. The snuff-induced lesions were classified according to clinical severity, grade 1 lesions being the least severe and grade 4 the most severe. Eleven percent of grade 2 lesions, 15.8% of grade 3 lesions and 20% of grade 4 lesions were EBV-positive. Neither EBV nor HPV seem to be associated with snuff-induced lesions.

Measures of dental beliefs and attitudes: their relationships with measures of fear.

Emergency dental and fearful dental patients were questioned in order to investigate the relationship between dental fear and different dental beliefs. The instruments used were the dental anxiety scale and the dental beliefs survey. The study also evaluated a Swedish version of the beliefs survey. The mean anxiety scale scores were 9.4 for emergency and 16.8 for fearful patients. The average beliefs survey item values ranged from 1.6 to 2.6 and 1.7 to 3.8 among the two groups respectively. There were clinical meaningful and statistically significant correlations between the anxiety scale and the beliefs survey. Each of the four dimensions of the beliefs survey (communication, control, belittlement and trust), also correlated with the anxiety scale of which the most clearly defined was belittlement.

Fixture stability and nerve function after transposition and lateralization of the inferior alveolar nerve and fixture installation.

Twenty-four posterior mandibular segments in 18 patients were operated on placing implants after mobilisation of the neurovascular bundle. Two different surgical techniques, transposition and lateralization was used. The overall survival rate of fixtures was 92.1%. Performing lateralization resulted in 100% success, while transposition resulted in 80% stable fixtures in the involved segments. The mean time to full restoration of nerve function was 3.8 weeks after lateralization and 5.7 weeks after tranposing the nerve. Three patients exhibited persisting slight hyposthesia, but all, subjectively negligible disturbances in nerve function.

Assessment of p53 and Ki-67 expression in snuff-induced lesions.

An immunohistochemical study of snuff-induced lesions with a monoclonal antibody (DO-7) specific to p53 mutant and wildtype antioncogene product demonstrated nuclear overexpression of the mutant protein in 45.9 nuclear profiles/mm2 epithelium (SEM 10.8; n = 15) compared with only 0.18 positively stained nuclear profiles/mm2 in the control group (SEM 0.18; n = 4). Furthermore, the biopsy material was also stained with the antibody Ki-67, which has been shown to be excellent for the estimation of the growth fraction in both normal and malignant human tissues. Ki-67 stained positive in 566.1 nuclear profiles/mm2 epithelium (SEM 85.0; n = 15) in the snuff-group compared with 20.2 nuclear profiles/mm2 (SEM 4.9; n = 4) in the control group. To the best of our knowledge, this is the first study showing overexpression of p53 protein and Ki-67 in snuff-induced lesions. The results may indicate that the p53 gene is involved in the initial events leading to subsequent malignant transformation of oral mucosa exposed to snuff. Furthermore, mutations of the p53 gene have been associated with increased cellular proliferation with greater risk of perpetuation of mutations and malignant transformation.