Profile of p53 expression in bladder and oral tumours. Effects of in vitro manipulations of p53 on the behaviour of established human tumour cell lines.

In this investigation the profile of p53 and epidermal growth factor receptor (EGFR) expression in tumour tissue biopsies of transitional cell carcinoma of bladder (TCC) and of oral-pharyngeal carcinoma (OP) were compared using an immunocytochemical staining method. In addition, various techniques including sodium dodecyl sulphate-polyacrylamide gel elecrophoresis (SDS-PAGE), colorimetric assay and gene transfection were used to investigate the influence of p53 on the behaviour of human tumour cell lines in vitro. The results showed that: (a) p53 was detectable in more than 45% of cases in both tumour types, although the profile and intensity of expression differed. (b) Concomitant strong expression of EGFR and p53 for TCC and OP was 21% and 38% (P>0.05%), respectively. (c) Treatment of tumour cells by either gamma radiation or by cisplatin resulted in the induction of p53 independent of the origin of the tumour. (d) Susceptibility of two cell lines, one with and one without constitutive expression of p53 showed that the expressing cells were more sensitive to gamma radiation (the percentage inhibition at 250 cGy was 57% versus -15%, P<0.01), and also cisplatin (the percentage inhibition at 1 microgram/ml was 71.0+/-6.0 versus 2.6+/-7.0, P<0.001). (e) Transfection of wild-type TP53 gene into a bladder tumour cell line resulted in a rapid cell apoptosis (by as much as 90%) whereas cells receiving mutated TP53 survived. A similar frequency of TP53 mutation in TCCs and OPs was observed. In addition, the pattern of p53 expression within the squamous type of TCC was similar to that in OPs. If the data from the in vitro studies could be translated into an in vivo setting, one could envisage a situation where the introduction of wild-type TP53 gene by gene transfection into tumour cells (independent of their TP53 gene mutational status), would prove to be beneficial. If the cellular TP53 gene is mutated, then an introduction of the normal TP53 gene would induce cells to undergo apoptosis. Alternatively, if TP53 is wild-type, then the increased levels of p53 expression would enable the cells to become more susceptible to DNA damaging treatments such as cisplatin or gamma radiation.

Profile of epidermal growth factor receptor (EGFr) expression in human malignancies: effects of exposure to EGF and its biological influence on established human tumour cell lines.

The aim of this study was to compare the profile of EGFr expression in transitional cell carcinoma of the bladder (TCC) and in oral squamous cell carcinoma (OSCC). In addition, to study the influence of EGF stimulation on the expression of major histocompatibility complex class I antigens, placental alkaline phosphatase (PLAP) as well as changes in tumour cell sensitivity to cisplatin using immunocytochemical staining, a colorimetric assay and SDS-gel electrophoresis. The results showed that: a) strong EGFr expression could be seen in 22/88 (27%) cases of TCCs. In oral tumours the values for non-invasive ameloblastoma and invasive OSCC were 4/25 (16%) and 30/41 (73%) respectively. b) EGFr expression in tumour cell lines paralleled that of tumour biopsies. The number of lines expressing high and low EGFr expression amongst TCCs were 4 and 4 and in OSCCs were 3 and 1 respectively. c) Exposure of tumour cell lines to EGF led to: i) an increase in EGFr expression (stimulatory indices SI, ranged from 1.06 to 2.58) for TCCs but a decrease in the case of OSCCs (SI ranged from 0.01 to 0.85). The corresponding SI values for class I antigens were 0.95-1.16 and 0.10-0.84. ii) A significant reduction in expression of PLAP by OSCC cell lines. iii) An increased susceptibility of OSCC cell lines to cisplatin by as much as 14% (p<0.001). These data demonstrated the overexpression of EGFr in a significant proportion of TCCs. As for oral tumours it depended on whether they were of an invasive or non-invasive type. In the invasive cases the majority overexpressed EGFr. The exposure of OSCC but not TCC tumour cells to EGF resulted in down regulation of EGFr and class I antigens. The expression of PLAP was also significantly reduced. Exposure of OSCC cells to EGF resulted in their increased susceptibility to cisplatin. The data supports the notion that the mitogenic activation of some tumour cells by EGF resulted in a reduction of their immune visibility, differentiation status and an increase in chemosensitivity.

Concordance between tumour cell activation by epidermal growth factor and alteration of cell sensitivity to cisplatin and lymphokine-activated killer cell activity.

In this study the impact of epidermal growth factor (EGF) on chemosensitivity and susceptibility to lymphokine-activated killer (LAK) cytolysis on six cell lines (one super expressing EGFr i.e. HN5, three high expressing i.e. Hep2, KB and MCF-7 and two low expressing i.e. Fen and HN15) was investigated using the tetrazolium salt reduction assay (MTT) as measured by optical density (OD). Hep2, KB, MCF-7 and Fen lines showed a dose-related inhibition to cisplatin (from 19% to 80%). Treatment of EGFr positive cell lines, Hep2, KB and MCF-7 but not EGFr negative Fen by EGF prior to exposure to cisplatin inhibited the cells by between 10-15% (p<0.05). Exposure of HN5 line to EGF (0.1 ng/ml) prior to LAK assay, led to a decrease in tumour killing (13%, p<0.05). However, at 0.01 ng/ml the pre-treatment enhanced tumour sensitivity. These data indicated that pre-exposure of tumour cells to EGF altered their response to cisplatin and LAK killing and this depended on the degree of EGFr expression. These data may prove helpful for pre-selection of patients for an appropriate therapy.

Profile of placental alkaline phosphatase expression in human malignancies: effect of tumour cell activation on alkaline phosphatase expression.

Cellular alkaline phosphatases (ALP) are increasingly recognised as important markers for monitoring tumour cell behaviour in human malignancies. Colorimetric, flow-cytometric, and immunocytochemical assays were employed to assess the influence of activation on expression of cellular ALP in human tumour cell lines. The results showed the following: (1) Testis tumour biopsies (16/16) unlike bladder (0/14) and head and neck (0/16) tumours showed positive staining for ALP, particularly the placental type, i.e. PLAP, although this was not always present on all the cells of non-seminoma biopsies. (2) The intensity of ALP expression differed widely in tumour cell lines. Based on biochemical analysis, the profile of ALP fell into two categories: (a) low expressing (MW 70 kD, placental type ALP) like Hep2 and KB lines, and (b) those expressing both low and high molecular (MW 95 kD) bands like testis lines Tera II and Ep2102. In all cases treatment of tumour cell lysates with heat prior to biochemical analysis showed the disappearance of the higher and sharpening of the lower molecular weight ALP band. (3) Exposure of tumour cells to epidermal growth factor (EGF) expressing EGF receptor led to a decreased ALP expression by as much as 54% as assessed by biochemical or flow-cytometric techniques. These data demonstrated that testis tumour tissues and cell lines expressed ALP which were different from others. The data also showed that exposure of tumour cell lines expressing EGFr to EGF resulted in suppression of ALP expression. These observations are consistent with the notion that EGFr and PLAP expression may be taken as a marker of proliferation and differentiation in human malignancies, respectively.

Stomatitis glandularis--two confirmed cases of a rare condition.

Stomatitis glandularis is an unusual chronic inflammatory condition of the minor salivary glands, mainly affecting the lower lip. Two cases are reported, one of which was progressive and affected the glands of both the cheeks as well as the lips. After confirmation of the clinical diagnosis by histopathological appearances, treatment was by excision of the suppurating areas as staged procedures. Chronic irritation from dentures may have contributed to the condition.

Gender influences tumour growth in a model of human oral squamous cell carcinoma.

The pattern of tumour growth was investigated using our unique syngeneic animal model of human oral squamous cell carcinoma of the head and neck (SCCHN). Gender-conditioned tumour fragments of near equal size were transplanted into recipient animals and the pattern of tumour growth was assessed by monitoring tumour size in vivo and/or the weight of tumour al termination of the experiment. Our results confirmed the epithelial nature of tumour cells on fresh tumour tissue sections using morphological and immunocytochemical examination. The mean weights of tumour per animal (n=10) after transplant from female (F) to F recipients, and across and between same gender showed differences if the animals were sexually mature. The results indicated an endocrine sensitive (sex hormone) component of tumour growth. In separate experiments, the recipient animals were immunised with a mechanically dissociated single cell suspension from fresh tumour after irradiation (500 cGys). The test,group (n=5) and control group (n=5, saline injected) animals received tumour fragments by grafting and following a Eur ther three weeks post immunisation, the tumours were removed and weighed. The test and control groups were different with a p-value of 0.001, suggesting an immune participation in the growth of this tumour. The results, taken together, thus suggest the participation of both the endocrine and the immune systems on the pattern of tumour growth in this animal model. The possible clinical relevance of these results is discussed.

The possible relevance of the expression of MHC antigens and of EGF receptor in aggressive oral tumours.

In this study the intensity of expression of major histocompatibility complex (MHC) class I and II antigens, adhesion molecule i.e. ICAM-1, epidermal growth factor receptor i.e. EGFr, T cell marker and cytokeratin were compared in oral squamous cell carcinoma (OSCC) and in the benign ameloblastoma of the jaws. The results showed that: a) There was strong expression of both monomorphic and of polymorphic class I MHC antigens (90% of cases) in both basal and suprabasal cells of controls from normal mucose. b) Whereas up to 4% of OSCCs and 27% of ameloblastomas showed complete loss of monomorphic class I antigens, the frequency of polymorphic class I abnormalities was even more marked in both tumour types. c) Strong expression of class II MHC antigens and of ECFr was observed in the basal cells of most normal controls. d) Both class II (50% of cases) and ICAM-1 (30% of cases) showed strong expression in OSCC but not in ameloblastoma. The statistical values between OSCC and normal basal cells for class II and ICAM-1 were not significant whilst the corresponding values for OSCC compared with ameloblastoma were p<0.001 and p<0.001. In the case of OSCC, there were a large number of infiltrating T cells expressing activation marker i.e. class II antigen. e) Strong expression of EGFr was seen in more than 90% of the OSCC cases compared with only 16% of ameloblastomas (0.01

0.001). Our working hypothesis to explain these abnormalities is that although both tumour types (more so in the case of ameloblastoma) have in place an escape mechanism from the immune system, the overexpression of EGFr in OSCC may in part be responsible for the more aggressive behaviour of the malignancy compared with the locally invasive but benign ameloblastoma.

Local anergy rather than systemic anti-tumour immunity to explain tumour growth in an animal model of oral squamous cell carcinoma.

A chemically induced syngeneic hamster tumour model of human oral squamous cell carcinoma (OSCC) was used to investigate the possible influence of locally transplanted growing rumours on the immune system of the recipient. Cell activation and cell cytotoxicity assays were performed in vitro using the colorimetric MTT assay to measure any possible changes. The fast growing nature of the tumour model if grafted locally as a fragment was confirmed but not if injected as a single cell suspension (SCS). Stimulation (Concanavalin A) of spleen cells from normal and from tumour bearing animals showed that there was a minor though statistically significant decrease in the mitogenic response of the latter. Thus, the respective stimulatory indices (SI) were 4.06+/-1.61 and 2.06+/-0.87 (0.02

0.01). No significant difference was observed when spleen cells were stimulated with interleukin-2 (IL-2), although there was a similar trend. Pre-immunisation of animals with irradiated autologous SCS three weeks prior to grafting, resulted in a significant decrease in the tumour growth rate of subsequently grafted tumour. Thus, the mean If: SD (weight of takes in mg) for the successful takes of untreated (n=10) and treated (n=9) groups were 52.0+/-52.2 and 25.7+/-19.4 (0.02

0.05) respectively. The number of cases with no tumour takes were 2 of 10 (20%) and 6 of 9 (66%) respectively. In a separate experiment groups of 5 animals were immunized with an increasing number of cells as irradiated SCS, the results of which demonstrated an inverse correlation between the rate of tumour growth and the number of injected tumour cells. The addition of irradiated SCS to IL-2 activated normal spleen cells (LAK cells) in vitro led to a dose-related decrease in the efficiency of cytotoxicity of latter when tested against an xenogeneic super-sensitive surrogate tumour target cell line (Fen cells). Thus, the percent killing by IL-2-activated normal spleen cells was 56.4%. The corresponding mean values for IL-2 activated normal spleen cells in the presence of tumour SCS at 25/1 and 50/1 ratios were 35.9% (p<0.05) and 11.9% (p<0.001) respectively. Ln an attempt to establish the presence of T suppressor cells, spleen cells from tumour bearing animals were injected concomitantly with SCS into 5 recipients. After four weeks no tumour growth had occurred. In conclusion we demonstrated that the presence of injected or grafted tumour had only a minor effect on systemic immune function but induced a strong local anergic effect. This local anergic effect was demonstrable as blocking of LAK activity and thus perhaps allowed suppression of the functional activities of incoming immunocompetent cells.

Evidence for safety margins of lignocaine local anaesthetics for peri-oral use.

The evidence for the safety margins of doses of lignocaine local anaesthetics for standard injections for oral and dental purposes has been reviewed. Trials of peri-oral dosages leading to toxicity in humans have not been reported. The overwhelming evidence from trials of the normal dose range used clinically points to a restriction of total doses much lower than the 25 ml (500 mg) as published for 2% lignocaine with 1:80,000 adrenaline for a healthy adult. The recommendations are: 1. In mixtures of two pharmacologically active drugs (dual formulations) for peri-oral injections, to base the safety limit of local anaesthetic on the circulating level of lignocaine, rather than the amount of adrenaline contained in the injected volume. 2. Thus the suggested usual upper limit for dosage for a healthy adult patient is four and a half 2 ml (or 2.2-ml) cartridges of lignocaine with adrenaline (180-198 mg lignocaine or 2.57-2.82 mg/kg) body weight, if carefully given. 3. For some medically compromised patients, minimal doses only of lignocaine and adrenaline (about one cartridge) should be used and especial caution is necessary in patients likely to react adversely to the exogenous adrenaline of the dual formulation. 4. For both children and adults, the dosages should broadly be related to body size and note taken of the total dose which accrues from topical use of other formulations of lignocaine, such as pastes or creams or sprays. The doses injected should be the minimum that allow the treatment to proceed. If necessary the doses are better given at a series of appointments rather than as a large volume on one single occasion.

Maxillofacial injuries in multiply injured patients.

Experience in team management of multiply injured patients with maxillofacial injuries is reported. During 1992, out of 169 patients transferred to the Royal London Hospital, UK by the Helicopter Emergency medical Service 38 (22.4%) had injuries to the maxillofacial region, 17 of whom were scored on the Abbreviated Injury Scale (AIS) as having sustained facial AIS > 2. The median Injury Severity Score (ISS) was 22, while the ISS was 17.7 for survivors and 34.5 for those who died (chi 2 = 7.3, 0.05 < P > 0.02). Facial AIS (median 4) and facial AIS contribution to ISS were found to be poor indicators of severity of injury. Revised Trauma Score (RTS) and percentage probability of survival (Ps%) were found to be useful discriminators of severity of overall injuries. RTS compared between survivors and those who died was 0.05 < P > 0.02 (chi 2), while Ps% was 0.01 < P > 0.001 (chi 2). It was concluded that the severity of maxillofacial injuries, and hence their contribution to total injury assessments, tended to be underscored. We propose that refined facial injury assessment methods be tested.

Bilateral mandibular nerve damage following root canal therapy.

A case of bilateral inferior dental nerve paraesthesia following accidental introduction of root canal filling material into the canal is described. The significance of early recognition of the complication and prompt surgical intervention is highlighted.

Maxillofacial injuries in the multiply injured.

A survey of patients with maxillofacial injuries (MFI) was carried out in a triaged cohort of multiply injured patients (n = 802) evacuated from accident scenes by the helicopter emergency medical service (HEMS). Despite intubation at accident scenes, some patients required further airway protection on arrival at hospital. One hundred and ninety-six patients (24.5%) had MFI and 90 (11.2%) were classified as severe with ISS of facial region > 2 or more severe (ISS up to 16, median 4). The latter group were commonly found to be associated with other severe injuries (median ISS of 36) leading to death in 41 patients (5.1% of the total). Out of the 196 multiply injured patients with MFI, 57 (29%) had diagnostic peritoneal lavage for suspected haemorrhage, 27 (14%) subsequently underwent laparotomy. Six (3%) had thoracotomies and 29 (15%) had chest drains inserted. Seventeen (9%) had emergency craniotomies and 14 (7%) required intracranial pressure monitoring. Orthopaedic injuries were most commonly associated with MFI and 91 patients (46%) underwent surgical interventions. The purpose of the present study was primarily to establish a database for MFI patients with multiple injuries. The longer term objective being to gain evidence for early definitive management of these complicated cases rather than the more traditional expectant policies. In our view delayed management of MFI can rarely correct all the consequent facial deformities.

Informed consent and the practice of good dentistry.

The importance of obtaining informed consent in dentistry is increasingly recognised for moral and legal arguments which are explored. Morally, patients have the right to self-determination and respect for it underpins the relationship of trust deemed so important for clinical success. Legally, this right is reinforced through the risk of dentists being sued for battery or negligence if they do no adequately respect it. An explanation is offered as to why it can be difficult for patients in the UK to win such litigation. The practical implications of the doctrine of informed consent are sometimes unclear in clinical work--the reasons for this are assessed and illustrated. Standards of good practice in obtaining informed consent are suggested. Readers are invited to evaluate the degree to which a hypothetical case conforms to these standards and to compare their conclusions with those of the authors.

Comparative efficacy of oral doses of clindamycin and erythromycin in the prevention of bacteraemia.

Two antibiotics, clindamycin and erythromycin, were compared in a double-blind trial to test their efficacy in the prevention of post-dental extraction bacteraemia with streptococci in a group of 40 healthy patients. Tolerance to the oral doses was tested by questionnaire. Levels of drug in the serum were estimated using a microbiological assay. An in-vitro blood culture system was used as an analogy of the persistence of a bacteraemia in the presence of high levels of antibiotic. Isolates of streptococci were identified to species level. Minimum inhibitory concentrations of clindamycin and of erythromycin for each isolate were estimated. Results showed satisfactory levels of antibiotics in the blood for activity against oral streptococci. Clindamycin was slightly more effective than erythromycin in the prevention of post-extraction streptococcal bacteraemia but that efficacy was only 45%. Clindamycin as a single oral dose of 600 mg was well tolerated by patients compared with erythromycin 1.5 g.

Are intraligamentary injections intravascular?

A pressure type syringe was used to give intraligamentary injections (IL) to upper teeth of two formulations commonly used in general practice, lignocaine and prilocaine. Assay of plasma levels of drug was carried out by high performance liquid chromatography. Results of assays after intraligamentary injections were then compared with results of assays after intravenous injections of plain drug in the same subjects. Both formulations of local anaesthetic were found as peak levels in the circulation, presumably after intraosseous spread, by 2 minutes following the intraligamentary injections. For lignocaine the peak amount was nearly 7% of the intravenous dose and for prilocaine the peak amount was 25% of the intravenous dose, at 2 minutes after injection. It was concluded that IL injections for healthy adults were unlikely to cause systemic unwanted effects when given in small doses.

Early management of multiply injured patients with maxillofacial injuries transferred to hospital by helicopter.

It has been suggested that early intensive medical treatment will improve the outcome for trauma patients with severe injuries. A Helicopter Emergency Medical Service (HEMS) based in an urban area was inaugurated by The Royal London Hospital. Specially trained medical and paramedical personnel flew with the helicopter to accident scenes. On return to the hospital, multi-disciplinary teams, including maxillofacial, were called to manage the patients. Operations from its own helipad commenced from the end of August 1990. A retrospective study of 192 patients transferred by HEMS to the Royal London Hospital during 16 months was undertaken. 18% (34) patients had facial injuries recognised at primary and secondary surveys according to Advanced Trauma Life Support (ATLS) protocols. The problems of early management are described and routine techniques for the control of haemorrhage from the maxillofacial area were developed.

Neurilemmoma of the head and neck.

The neurilemmoma is an uncommon tumour of the oral cavity. In this present series of soft tissue neurilemmomas the majority occurred within or around the mouth. Initially these were provisionally diagnosed on clinical appearances and behaviour as commoner benign lesions, including the fibro-epithelial polyp, mucocele and papilloma. On sampling they were found to have the histological features of a neurilemmoma. This suggests that the solitary neurilemmoma occurs more frequently than previously reported and should be included in the differential diagnosis of these more frequent benign lesions occurring in and around the mouth.