Proliferative activity as detected by immunostaining with Ki-67 and proliferating cell nuclear antigen in benign and malignant epithelial lesions of the human parotid gland.

OBJECTIVE: A retrospective immunohistochemical study of parotid gland lesions was designed to evaluate the diagnostic and prognostic value of the proliferating cell nuclear antigen (PCNA) and Ki-67 with monoclonal antibodies PC 10 and MIB-1, respectively. STUDY DESIGN: Tissue samples comprised normal parotid gland (N, n = 10), chronic sialadenitis (CS, n = 8), Warthin's tumor (W, n = 10), benign pleomorphic adenoma (BPA, n = 8), mucoepidermoid carcinoma (MEC, n = 13), carcinoma in pleomorphic adenoma (CPA, n = 8) and adenoid cystic carcinoma (ACC, n = 12). The morphometric parameters for PCNA and MIB-1 comprised the PI and MI labelling indices (the numerical percentage of positive nuclei), NAP and NAM (the numerical density of positive nuclei), and NPI and NMI (volume corrected index). RESULTS: The values of MIB-1 parameters increased progressively in benign lesions in comparison with the N group and in malignant neoplasms in comparison with nonneoplastic groups and benign lesions. Values for all parameters in BPA were significantly lower than those in malignant groups. Spearman rank correlation analysis showed a highly positive correlation between the morphometric parameters and severity of the lesions. The mean values of MI and NMI were significantly higher in patients who died of the malignant tumors than in those who survived. The same quantitative parameters for PCNA did not differ significantly from those obtained for MIB-1 and showed similar trends. CONCLUSION: PCNA and MIB-1 indices are reliable markers for discriminating between benign and malignant tumors of the parotid gland, and the parameters PI, MI, NPI and NMI may have prognostic applications.

Quantitative assessment of normal and potentially premalignant epithelium at different levels of human colorectal crypts.

The present study uses morphometric techniques to assess whether altered differentiation patterns exist in PPM which might reflect its premalignant status. Samples were obtained from resected malignant lesions of large bowels of 10 Chinese patients. Normal (N) samples were biopsied from the margins of each resected large bowel. Potentially premalignant (PPM) mucosae were obtained from within 2 cm of the margins of the malignant lesions. Tissues were processed for histological examination and using strict criteria, colorectal crypts were divided into basal (B), intermediate (I) and surface (S) segments. Interactive digitisation of sections from each group was used to generate the following morphometric parameters in each segment: nuclear profile circularity indices (NSF and NCI); nuclear numerical density (NA and NV); the degree of deviation of the major nuclear axis in relation to the epithelial-connective junction (AGDMAX); cell height (CH); the distance between nuclear apex to cell apex (DNACA); the distance between cell base to nuclear apex (DCBNA); stratification index (SI)--the ratio of DCBNA and CH; and the volume density of mucous vacuoles in the reference epithelium (VVMV,EP). In comparisons of different segments within groups, the nuclei at the S segment of N and PPM crypts were more irregular and less circular in shape than nuclei from other segments. There was a shift of nuclear profile shape (NSF and NCI) from circular to ellipsiodal between B and S segments. In comparisons of similar segments between groups, no significant nuclear shape changes were detected in nuclei of PPM crypts when compared with nuclei in similar segments of N crypts and the pattern of nuclear shape alterations resembled those of normal crypts. In comparisons of different segments within groups of N and PPM crypts, AGDMAX, DNACA, DCBNA, CH and SI parameters demonstrated that epithelial cells at the I segments have more centrally positioned nuclei with the tallest epithelial height when compared with epithelial cells in other segments of both crypts. In B segments, nuclear NA and NV were almost double those of other segments in both N and PPM crypts, with marked reductions in these parameters between B and I segments. VVMV,EP was significantly highest in the I segment and significantly lowest in the S segment of both groups. Both N and PPM crypts showed similar trends in VVMV,EP within the crypt segments but when comparing similar segments between both crypts, a significant difference was detected only between S segments. The alterations of nuclear shape and packing densities, orientation and mucous content in N crypts were similarly expressed in PPM crypts and distinct differences in numerical density (NV) and stratification index existed in crypts between these two groups when comparing similar segments. All values in PPM were consistently lower when compared with N crypts. These preliminary observations may represent a subtly altered state of cellular differentiation in PPM which may be a reflection of early preneoplastic transformation.

[Relationship between the mRNA transcriptions of bFGF and KGF in the carcinogenesis of oral mucosa].

The aim of the study was to determine the expressions and effects of KGF and bFGF of FGF superfamily in oral mucosa. The mRNA transcriptional levels of bFGF and KGF in dysplasia (DYS) and squamous cell carcinoma (SCC) of buccal mucosa were observed and evaluated by in situ hybridization method. The results showed that bFGF and KGF mRNA were expressed by the mesenchymal cells in both DYS and SCC groups, but not in any of the epithelial cells. The positive reactions were located in fibroblasts, endothelial cells and some of the chronic inflammatory cells. There is an increasing mRNA transcriptional level in SCC than in DYS. The results indicated that bFGF and KGF may not only enhance the neovascularization and formation of stroma by autocrine and paracrine action, but also involved in the growth of neoplastic cells.

Expression of BMP-2 and TGF-beta 1 mRNA during healing of the rabbit mandible.

To identify the cell types which produce BMP and TGF-beta during fracture healing and to elucidate the interactions between BMP and TGF-beta in regulating cell proliferation and differentiation at various stages, an experimental model of fracture healing in the rabbit mandible was established and the expression of BMP-2 and TGF-beta 1 mRNA was studied at different healing stages by in situ hybridization. The results showed that undifferentiated mesenchymal cells, differentiating osteoblasts and chondroblasts, had higher levels of BMP-2 mRNA at the stage of intramembranous bone formation and early chondrogenesis, while the level of TGF-beta 1 mRNA was higher in chondrocytes and active differentiated osteoblasts during chondrogenesis and endochondral ossification, respectively. We conclude that BMP-2 expression was correlated with the differentiation of mesenchymal cells into osteoblasts and chondrocytes. TGF-beta 1 mRNA expression was closely associated with the active synthetic stage of osteoblasts and chondrocytes. These observations suggest that both BMP and TGF-beta are involved in the regulation of fracture healing. BMP may play an important role in bone induction and early chondrogenesis, while TGF-beta regulates the proliferation and active synthetic ability of chondrocytes and osteoblasts.

p53 oncoprotein accumulation in adenoid cystic carcinoma of parotid and palatine salivary glands.

Previous studies have suggested that alterations of the p53 gene are the most common genetic abnormality in human cancer. The aims of the present study were to evaluate p53 protein (p53P) immunostaining in adenoid cystic carcinoma (ACC) of the salivary gland and to correlate the expression with patient survival. A total of 27 cases of ACC in the parotid gland (n = 12) and the minor palatine glands (n = 15) were studied, with ten cases each of normal parotid and palatine glands as non-neoplastic controls. Staining was performed with mouse monoclonal antibody DO-7 against p53 (Dako, USA) using the ABC method. Stained nuclei irrespective of intensity or frequency were considered as positive. The frequency of positive nuclei was evaluated as the p53P index (p53PI), the percentage of the total nuclei in the reference epithelium. Clinical survival data were available for patients for periods up to 156 months. Our data showed that no normal tissues showed immunoreactivity with p53P in their nuclei. Thirteen of 15 (87%) cases of palatal and two of 12 (17%) cases of parotid neoplasms stained with p53P and the p53PI ranged from 0.01 to 10%. The number of p53P positive tumors was significantly higher in palatal than in parotid neoplasms, suggesting that palatal ACCs may be more aggressive in comparison with parotid ACCs. Our data also showed that the number of p53P positive tumors was significantly increased in patients who died of tumors than in patients with no evidence of disease at the end of the follow-up period between 60 to 156 months. These results suggest that p53P may be involved in the development of salivary gland ACCs and that p53P analysis may be a useful indicator of poor prognosis.

The assessment of proliferating cell nuclear antigen (PCNA) immunostaining in human benign and malignant epithelial lesions of the parotid gland.

Immunoreactivity of proliferating cell nuclear antigen (PCNA) was assessed in formalin-fixed, paraffin-embedded sections from human normal parotid gland (N; n = 12), chronic sialadenitis (CS; n = 8), Warthin's tumour (W; n = 10), benign pleomorphic adenoma (BPA; n = 11), mucoepidermoid carcinoma (MEC; n = 14), carcinoma in pleomorphic adenoma (CPA; n = 10) and adenoid cystic carcinoma (ACC; n = 12) of the parotid gland, using the monoclonal antibody PC 10. The morphometric parameters measured comprised PCNA labelling induces (PI = the numerical percentage of PCNA positive nuclei) and volume densities of PCNA positive nuclei(VV, PEP = the relative volume of positive nuclei per unit volume of reference epithelium). All parameters were expressed in relation to total positive, as well as to strongly- and weakly-positive nuclei. In general, the values of PCNA parameters increased progressively in benign lesions in comparison with the N group, and in malignant neoplasms in comparison with non-neoplastic groups and benign lesions. The strongly-positive parameters showed more statistically significant differences than weakly-positive ones, suggesting that weakly-stained nuclei may include some non-cycling cells and, therefore, that weakly-positive parameters may not be reliable proliferation markers. Values for all parameters in CPA were significantly higher than those in BPA, suggesting that these parameters may be used as diagnostic discriminators. Spearman rank correlation analysis showed a highly positive correlation between the morphometric parameters and the severity of the lesions. Furthermore, the mean values of PISP were significantly higher in patients who died of the malignant tumours than in those patients who survived. Our results indicate that PCNA indices might be useful markers for discriminating between benign (BPA) and malignant tumours of the parotid gland and that the parameter PISP may have prognostic applications.

An evaluation of the role of nuclear cytoplasmic ratios and nuclear volume densities as diagnostic indicators in metaplastic, dysplastic and neoplastic lesions of the human cheek.

The increase in nuclear cytoplasmic (N/C) ratio is one of the features of cellular atypia which is used in the histopathological assessment of premalignant lesions of the oral mucosa. Since this feature is readily quantifiable using morphometry, we have analysed both N/C and nuclear volume densities in basal and spinous cells from human cheek lesions with and without malignant potential in order to ascertain the validity of this parameter as a predictor. Using a strictly standardised sampling procedure, measurements of cellular and nuclear areas of basal and spinous cells from normal and pathological human cheek mucosa were made on haematoxylin and eosin-stained sections using a VIDAS image analyser. Cases examined comprised fibrous hyperplasia (FH), traumatic inflammation (IF), benign hyperkeratosis (HK), lichen planus (LI), leukoplakia with dysplasia (DYS), squamous cell papilloma (PP), dysplastic epithelium from the edges adjacent to invasive carcinoma (CE) and islands from invasive squamous cell carcinoma (CI). In basal cells, N/C ratios and nuclear volume densities were lower than values obtained for the normal controls. In spinous cells, these parameters were elevated in the potentially premalignant lesions (DYS, CE) as well as in CI but values were similarly elevated in FH, IF, HK and PP, lesions which appear to have no malignant potential. The N/C ratio is of no value as a predictor of malignant potential in basal or spinous cells from cheek lesions. The putative increase in N/C which has been previously described qualitatively is probably due to increased nuclear hyperchromatism, which may provide an illusory increase in relative nuclear size at the expense of the cytoplasm.

A quantitative study of silver-stained NORs in different segments of the normal human colorectal crypt.

Quantification of silver-stained nucleolar organiser regions (AgNORs) in paraffin sections may provide clues about the proliferation and differentiation in normal and neoplastic tissues. The aim of the present investigation was to determine whether AgNOR quantification could provide useful data about proliferation in the different segments of the normal human colorectal crypt. Samples of histologically 'normal' large intestine (n = 8) were obtained from colorectal cancer resections at a distance of > 5 cm from the tumour margins and were routinely processed for paraffin embedding using strictly standardised procedures. Sections were cut and stained with a one-stage silver colloid impregnation technique. The longitudinally sectioned crypts were divided into proliferative (P), intermediate (I) and surface (S) segments using strict criteria. Clearly defined AgNORs, which appeared as black dots within the nuclear profile, were quantified from each segment for volume density (Vv) and number per unit area (NA) estimates using traditional point-counting techniques. A 1-way analysis of variance followed by Scheffe's test indicated significant progressive reductions of AgNOR Vv and NA from P to S segments. Our data suggest that both volume and frequency of AgNORs may be related to cellular proliferation since both parameters are highest in the P segment. The further exploitation of stereological tools in conjunction with AgNOR staining may be valuable in assessing normal differentiation and proliferation patterns and in predicting the biological behaviour of neoplastic tissues in which increased proliferation is a feature.

The relationship between vascularity and cell proliferation in human normal and pathological lesions of the oral cheek epithelium.

The present study investigates relationships between neovascularisation and PCNA cell proliferation markers in different pathological lesions of the oral cheek mucosa. All specimens were fixed in 10% formalin and routinely processed for histology. Six normal (N) samples were taken from resection margins of benign lesions. The pathological lesions consisted of chronic inflammation (n = 10), lichen planus (n = 7), fibrous hyperplasia (n = 11), dysplasia (n = 5), squamous cell carcinoma (n = 22) and epithelium adjacent to carcinoma (n = 6). Adjacent 5 microns sections were stained with monoclonal antibodies against vimentin (clone no. V9) for identification of stromal blood vessels and against proliferating nuclear antigen (PCNA/PC10) using ABC immunoperoxidase techniques. Point counting was used to obtain the primary morphometric data using a Zeiss VIDAS image analyser. No attempt was made to classify the different types of blood vessels. The morphometric blood vessel parameters estimated were volume density, number per unit area, length per unit volume and mean transverse sectional area. PCNA indices were determined by estimating the percentage frequency of PCNA positive nuclei in basal and spinous strata. Generally, there were significant increases in all PCNA indices and blood vessel parameters between the N group and the different pathological lesions. A highly positive correlation was detected between all PCNA indices and blood vessel parameters. These data suggest that increased vascularity and angiogenesis occur in support of actively proliferating and transforming oral epithelial cells in order to permit growth. PCNA indices and blood vessel parameters may have a potential application as diagnostic and prognostic indicators.

Preliminary assessment of the epithelial nuclear-cytoplasmic ratio and nuclear volume density in human palatal lesions.

We have analysed both the nuclear-cytoplasmic (N/C) ratio and nuclear volume densities (VVN) in defined strata from human hard palate lesions with and without malignant potential to determine the prognostic reliability and/or validity of this parameter. Measurements of cellular and nuclear areas of basal and spinous cells from normal (N) and pathological palatal epithelium were made on histological sections using an image analyser. The lesions comprised fibrous hyperplasia (FH), traumatic inflammation (INF), benign hyperkeratosis (HK), squamous cell papilloma (PP), dysplastic epithelium adjacent to invasive carcinoma (CE) and islands of invasive squamous cell carcinoma (CI). In basal cells, no significant differences were detected in comparisons of N/C and VVN between all pathological groups and the N control group. The mean value for CE was lower than that obtained for N. In spinous cells, the only statistically significant comparison was between IF and FH for both N/C and VVN. Both parameters were lower in CE than in N. Of all groups analysed except CI, the CE group is the only one likely to possess an increased malignant potential. The N/C ratio therefore seems to be of no value as a predictor of malignancy in palatal epithelial lesions.

Blood vessel morphometry in human colorectal lesions.

Neovascularisation in tumours of different cell origins has been well documented qualitatively. In this report, we have assessed vascular architecture in different pathological lesions of the colorectum by quantifying blood vessel parameters in order to detect subtle morphological changes using objective methods. Colorectal tissue samples were obtained from resected large bowels containing malignant tumours. Biopsies were taken from defined sites in the resected specimen and were classified as normal (N), potentially premalignant mucosa (PPM), adenomatous polyp (P) and adenocarcinoma (ADCA). All tissues were fixed in modified Karnovsky's fixative for 4 hrs and postfixed in 1% OsO4 for 1 hr. Samples were processed for EM under standardized procedures and embedded in Epon. 0.5 microns semithin sections from five patients per group were stained with toluidine blue. A multistage systematic sampling procedure was adopted. The inner outlines of all blood vessels in the lamina propria (LP) were digitised using a Zeiss VIDAS Image Analyzer at a final magnification of x1,050. The area of the reference (LP) was also measured. No attempt was made to distinguish between the different types of vessel. The morphometric blood vessels parameters quantified were volume density (Vv), numerical density (NA), length density (LV) and mean transverse sectional area (A). Statistically significant differences in Vv and A were detected between all groups except between N and PPM and between P and ADCA. No significant differences in NA and LV were present in any group comparisons. The mean values of all parameters were the highest in ADCA.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of high-performance liquid chromatography to determination of modifier activity in alpha-lactalbumin and other proteins.

High-performance liquid chromatography has been applied to determination of modifier activity in alpha-lactalbumin (alpha-LA). An amino-bonded column separates uridine diphosphate (UDP) (product), UDPgalactose (substrate), and uridine monophosphate (UMP). From an aliquot of the same sample, a column for carbohydrate analysis separates lactose (the other product) and galactose-1,2-cyclic phosphate (Gal-c-P). Nucleotide peaks are detected by measurement of A262 and those of carbohydrate by 3H counting, the isotope originating from UDP-galactose-3H. A pH of 6.3 was taken as optimal for production of UDP since, at this level, the unwanted side reaction is minimized, by which UMP and Gal-c-P are formed. Thus, the conservation of substrate so effected may have contributed to an enhanced production of UDP. The reaction by which UDP and lactose are produced was linear for 120 min, as followed by UDP formation, but it continued to at least 300 min. Production of lactose was equivalent to that of UDP, when alpha-LA was the modifying protein. From a survey of seven other proteins, only lysozyme and ovalbumin showed ability to produce UDP. However, failure of the last two proteins to produce lactose indicates absence of modifier activity and demonstrates the need for monitoring both products.

A quantitative investigation of immunocytochemically stained blood vessels in normal, benign, premalignant and malignant human oral cheek epithelium.

The present study was designed to determine whether increased vascularity occurs during malignant transformation of human oral cheek epithelium. Nine normal (N) samples were taken from the resection margins of benign lesions; the pathological lesions were classified as chronic inflammation (CI; n = 11), fibrous hyperplasia (FH; n = 12), lichen planus (LIP; n = 8), dysplasia (DYS; n = 5), squamous cell carcinoma (SCC; n = 25; well differentiated [SCCWD]; n = 10; moderately to poorly differentiated [SCCMPD]; n = 15) and epithelium adjacent to carcinomas (EAC; n = 6). Sections were stained with monoclonal antibody (mAb) against vimentin using an ABC immunoperoxidase technique. All blood vessels present within a depth of 0.9 mm of lamina propria were quantified irrespective of their morphology. The blood vessel parameters quantified were volume density (VVBV, CT), number per unit area (NABV, CT), length per unit volume (LVBV, CT) and mean transverse sectional area (ABV). VVBV, CT increased significantly between normal and all pathological groups. Amongst the pathological groups, statistical differences were detected between CI and SCC, CI and EAC, FH and SCCWD, FH and EAC, LIP and SCC, LIP and EAC, DYS and SCCWD and DYS and EAC. The EAC group had the highest VVBV, CT and the values of NABV, CT and LVBV, CT were significantly higher in all the pathological groups when compared with the normal group. No significant differences were detected between any of the pathological group. The parameter ABV increased significantly between normal and DYS, FH, SCC, EAC, FH and EAC, FH and SCC, CI and EAC, CI and SCC, LIP and EAC and LIP and SCC. Spearman rank correlations detected a positive correlation between the severity of oral lesions and all of the blood vessel parameters. We conclude that a mAb against vimentin improved the identification of smaller blood vessels and the blood vessel data suggest that angiogenesis occurs in premalignant and malignant lesions of human oral cheek epithelium. Angiogenesis seems to play an essential role in sustaining the actively growing and transforming cells.

An immunocytochemical study of bone morphogenetic protein in experimental fracture healing of the rabbit mandible.

A monoclonal antibody raised against bone morphogenetic protein (BMP-McAb) has been used to demonstrate the presence of bone morphogenetic protein (BMP) in experimental fracture healing. Rabbit mandibles were fractured using standardized methods and left to heal for 3, 7, 14, 21 and 24 d, respectively. The avidin-biotin complex (ABC) method demonstrated an accumulation of positively stained primitive mesenchymal cells at the fracture site in the hematoma stage of bone repair. These cells appeared to undergo differentiation into positively-stained chondroblasts and osteoblasts during the phase of callus formation. Undifferentiated mesenchymal cells showed a high positive reactivity in the early post-fracture stages but a much lower reactivity during the remodelling phase. The results of our study suggest that bone inductive processes are accompanied by the presence of BMP in osteoprogenitor cells during fracture healing of the mandible and that BMP may play a significant role in osteogenesis during bone healing.

Quantitative cellular and nuclear volumetric alterations in epithelium from lichen planus lesions of human buccal mucosa.

Patients with oral lichen planus lesions may represent a relatively high risk population for subsequent development of oral cancer. Little is known of the relative effects of chronic inflammation and the process of malignant transformation itself on the histological structure of transforming epithelia. We have assessed cellular and nuclear volumes in defined basal and spinous cells from normal buccal mucosa epithelium, from epithelium associated with a non-specific chronic inflammatory infiltrate and from lichen planus lesions. Normal (N) tissues were obtained from the margins of non-neoplastic buccal mucosa lesions. Inflammatory (INF) lesions were from areas of the buccal mucosa diagnosed clinically as traumatic irritation without ulceration, and lichen planus (LI) lesions were biopsied from areas exhibiting Wickham's striae. Basal and spinous epithelial cells from normal and pathological human buccal mucosa were measured on haematoxylin and eosin-stained sections imaged through a video camera using a Zeiss VIDAS analyser and from these measurements, nuclear (VN) and cellular (VCELL) volumes were determined. VN and VCELL derived for both basal and spinous strata were similar in N and INF groups but were almost doubled in the LI group. Comparisons between LI and all other groups were significantly elevated. The effects of the inflammatory infiltrate on the oral epithelium in lichen planus and in non-specific inflammation thus differ significantly. VN and VCELL may serve as potential discriminators between benign lesions and premalignant lichen planus.

Tissue reactions to titanium implants containing bovine bone morphogenetic protein: a scanning electron microscopic investigation.

Cylindric titanium implants treated with bovine bone morphogenetic protein (bBMP) (Group I) and with bovine serum albumin (BSA) (Group II) were implanted in the edentulous mandibles of 15 adult dogs. They were examined 1, 2, 4, 8, and 12 weeks after insertion of the implants by scanning electron microscopy (SEM). In all specimens from Group I, active bone formation had occurred between the implant and host bone, with a well-adapted fit between the implant and the newly formed bone 4 weeks after implantation. The surrounding bone was of normal structure by week 8. However, 3 months after implantation, fibrous tissue was consistently seen surrounding parts of the implants in Group II. The results of this study suggest that the implants treated with bBMP are capable of forming new bone of normal structure at a faster rate than BSA controls and that this new bone is closely adapted to the implant surface.

Early histologic response to titanium implants complexed with bovine bone morphogenetic protein.

This study established a method for combining bovine bone morphogenetic protein (bBMP) with titanium and evaluated the early bone formation induced by the bBMP/titanium complex in edentulous dogs. Results showed newly formed bone within the interface bone 2 weeks after implantation and nearly complete osseointegration 4 weeks after implantation. Bone formed with the apical opening of the implant within 1 month. This study indicates that osseointegration can be enhanced by bBMP-bone induction. The apical opening of the implant may provide a site in which osteogenesis can occur with protection from implant stress before and after loading.

Bovine bone morphogenetic protein-induced dentinogenesis.

Differentiation of odontoblasts is important for dentin formation in tooth germs and mature teeth. Although previous reports have indicated that there may be a kind of inductive agent that could induce mesenchymal cells in dental pulps to differentiate into odontoblasts, and secrete dentin matrix, the primary inductive factor of odontoblasts has not been found. Bone morphogenetic protein (BMP), which induces the formation of cartilage and bone when implanted in muscle tissue, is found in dentin matrix. The relationship between the differentiation of odontoblasts and BMP was observed by means of immunohistochemical staining with monoclonal antibody (MAb) against BMP in dental pulp tissue and cell culture; [3H]thymidine incorporation; and measurement of alkaline phosphatase activity. The conclusions are: (1) BMP exists in odontoblasts, ameloblasts, and dentin matrix (the positive reaction in ameloblasts appeared earlier and remained stronger); (2) BMP promotes incorporation of [3H]thymidine and increases the activity of alkaline phosphatase in cultured dental pulp cells; (3) BMP-induced dental pulp cells in dental pulp tissue cultures differentiate from mesenchymal to odontoblast-like cells; and (4) BMP induces formation of osteodentin and tubular dentin when used as a dental capping agent of dogs' teeth. Bone morphogenetic protein plays an important role in differentiation of odontoblasts and might be one of the inductive agents of odontoblasts. Further investigations of BMP as a biologic dental capping agent are warranted.

A histological morphometric study of nuclear size in benign and malignant neoplasms of the human cheek.

This report describes the application of simple morphometric methods to generate quantitative data on nuclear size from tissue sections of normal, benign and malignant oral epithelium of the cheek. Measurements of nuclear areas of basal and spinous cells from cheek mucosa were made on haematoxylin and eosin-stained sections using a Zeiss VIDAS image analyser. The lesions examined comprised benign squamous cell papillomas and islands of cells from invasive squamous cell carcinomas. Normal control epithelium was obtained from the biopsy margins of non-neoplastic lesions. The nuclear areas (AN) were obtained by direct measurement whereas the nuclear diameters (DN) were determined automatically. In both basal and spinous strata, values for both these nuclear parameters were lowest in normal tissue and increased progressively through benign papillomas, with the highest values being found invariably in carcinomas. Statistically significant differences were detected between both normal and carcinoma and between papilloma and carcinoma. The morphometric parameters AN and DN are of value in distinguishing benign from malignant lesions of the human cheek.

Further studies on the lysozyme-like activity of alpha-lactalbumin: development of alternative methods of assay.

Earlier, a sensitive turbidimetric method was reported (H.A. McKenzie and F.H. White, Jr. Biochem. Int. 14, 347-356, 1987), with which evidence was found for weak lysozyme-like activity in alpha-lactalbumin (alpha-LA) against Micrococcus luteus. Alternative methods have been developed for the further study of trace cell-lytic activity, and the results are compared with those of the turbidimetric technique. These methods involve (1) determination of weight loss from a suspension of bacterial cells after exposure to the protein under investigation, and (2) viability studies on the exposed cells. In addition, exposed and control cells were subjected to microscopic examination. Results from all studies were consistent with lysis of cells by alpha-LA as well as by lysozyme. Activities of alpha-LA from the three methods of assay, expressed as ratios to those of lysozyme, were 2.2-5.2 x 10(-5) (mean = 3.6 x 10(-5). The methods were assessed with respect to sources of error characteristic of each and to protein dose requirements for a specified level of cell killing. The turbidimetric approach remains useful for measuring cell-lytic activities as described here. However, caution is urged in its general use.