An overview of the complexities and subtleties of immunohistochemistry.

Immunohistochemistry has the potential to be a powerful research tool. However, immunohistochemical studies are frequently undertaken without regard to the complexities and subtleties of these useful techniques. This review aims to address the problems and limitations that are often encountered, and the procedures that should be considered in both the planning and interpretation of immunohistochemical studies. Particular reference is made to the generation of functionally different protein isoforms from a single gene by alternative splicing and post-translational modifications, primary antibody selection, the effects of tissue manipulation such as fixation and antigen retrieval, the need for appropriate controls and interpretation of staining patterns.

Human tenascin-C: identification of a novel type III repeat in oral cancer and of novel splice variants in normal, malignant and reactive oral mucosae.

Tenascin-C is a mosaic, linear glycoprotein that is up-regulated during many normal and pathological processes involving either cell migration or tissue morphogenesis, such as invasion of malignant cells and wound healing. Human tenascin-C contains 8 consecutive type III fibronectin (TNCfn) domains that are involved in alternative splicing and potentially generate a large number of isoforms that code for tenascin-C proteins with subtly different functions. Human tenascin-C splice variants were investigated by RT-PCR in a range of normal and pathological oral mucosal tissues. A novel, 9th human TNCfn domain involved in alternative splicing was identified. It shares 70% nucleic acid and 55% protein sequence homology with chicken TNCfn-ad2. As in avians, this novel repeat was located between TNCfn-B and TNCfn-ad1 and accordingly was designated human TNCfn-ad2. Human TNCfn-ad2 was detected in only 2 of 10 oral cancers. However, TNCfn-ad2 was absent from 40 normal, reactive, pre-malignant and other oral mucosal specimens investigated. Previous studies have described 8 splice variant transcripts for human tenascin-C. By systematic investigation we identified further novel splice variants for human tenascin-C. Furthermore, our results indicate that many potential splice variants probably do not exist in the tissues investigated. Thus, we have demonstrated that human tenascin-C transcripts generate a complex but selected repertoire of different alternative splice products.

RT-PCR investigation of fibronectin mRNA isoforms in malignant, normal and reactive oral mucosa.

This study aimed to establish patterns of cellular fibronectin mRNA splice variants in normal oral mucosa, oral squamous cell carcinoma, oral leukoplakias with and without atypia, and focal reactive overgrowths of oral mucosa. Particular emphasis was placed on evaluation of either the EDA or EDB domains as markers of malignancy. Total RNA was extracted from normal oral mucosa, oral squamous cell carcinoma, oral leukoplakias with and without atypia, reactive epulides, fibroepithelial polyps and denture-related hyperplasia. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify different fibronectin transcripts at three splice sites (EDA, EDB and IIICS). All the tissues investigated produced EDA+, EDA-, EDB+ and EDB- splice variants, and this study did not support RT-PCR-based detection of either EDA or EDB domains as markers of malignancy in oral tissues. Variations in IIICS splice patterns were observed, although these were not specific to any lesion group. In particular, there were differences in either the inclusion or omission of the domain coding for the CS-5 binding site for alpha 4 beta 1 integrin, whereas the CS-1 binding site for alpha 4 beta 1 integrin was typically present when additional domains were included at the IIICS splice site. In conclusion, complex patterns of fibronectin splice variant transcripts exist in normal and pathological oral mucosa. This may reflect the multiple biological functions identified for fibronectin proteins, although the significance of different specific fibronectin splice variants has yet to be fully elucidated.

Histochemical and immunohistochemical localisation of elastic system fibres in focal reactive overgrowths of oral mucosa.

Eight specimens each of the following groups were investigated: gingival pyogenic granuloma, fibrous epulis, calcifying fibrous epulis, peripheral giant cell granuloma, giant cell fibroma (four gingival, four non-gingival), denture-irritation hyperplasia and fibroepithelial polyp. These lesions have diverse histopathological appearances but the composition of their connective tissue is poorly defined. The elastic system consists of a complex mixture of glycoproteins that in normal oral mucosa form three differentially distributed fibre types; oxytalan, elaunin and elastic. The elastic system was investigated by Verhoeff's haematoxylin stain, aldehyde fuchsin staining and an anti-elastin monoclonal antibody. Elastin was identified in all fibroepithelial polyps and denture-irritation hyperplasias, but in none of the other lesions. In particular, this identified a distinct difference in the extracellular matrix between the giant cell fibroma and fibroepithelial polyp. Many of the epulides included only oxytalan fibres, but the presence of oxytalan fibres did not follow any pattern within either a single lesion group, or between different lesions. However, the presence of oxytalan fibres in the absence of elastin does not necessarily support a periodontal ligament origin for reactive epulides.

PCNA and Ki-67 immunoreactivity in multinucleated cells of giant cell fibroma and peripheral giant cell granuloma.

Immunohistochemical investigation of PCNA and Ki-67, two diverse nuclear proteins essential to the cell cycle, was undertaken in archival, formalin-fixed and paraffin-embedded specimens of giant cell fibroma (GCF) and peripheral giant cell granuloma (PGCG++). GCF multinucleated cell nuclei were mostly PCNA+, although there was variability in staining intensity. This indicates heterogeneity in nuclear PCNA metabolism of GCF multinucleated cells, and it is possible that the most intensely stained nuclei have passed through the cell cycle more recently compared to the less immunoreactive nuclei. However, the absence of Ki-67 immunoreactivity in GCF multinucleated cells, and absence of mitoses in GCF multinucleated cells, suggests that cell cycling in the absence of cytokinesis is not involved in GCF multinucleated cell formation. Alternatively, GCF multinucleated cells possibly form by fusion of mononuclear cells previously identified as fibroblasts, although this theory cannot be confirmed by the data presented in this study, and the histogenesis of GCF multinucleated cells remains unclear. In contrast, absence of either PCNA or Ki-67 immunoreactivity in PGCG multinucleated cells is consistent with an osteoclast lineage and formation from differentiated mononuclear cells.

Yeast artificial chromosome cloning and chromosomal localization of the abundant odontogenic keratocyst protein elafin.

An imbalance in human leucocyte elastase (HLE) activity is widely recognized to play an important pathological role in a number of human diseases. An earlier report has described greater transcription of elafin, an endogenous inhibitor of HLE, in epithelia of odontogenic keratocysts of the jaw than in normal oral mucosa. The elafin gene was now localized to chromosome 20q11.2-13.1 using a combination of somatic cell-hybrid panel screening and fluorescence in situ hybridization using a biotinylated DNA probe prepared from isolated yeast artificial chromosomes. No other positive fluorescent signals were observed. This eliminates the elafin gene as a candidate gene for naevoid basal-cell carcinoma syndrome, as the gene for this syndrome localizes to chromosome 9q23.1-31. The elafin yeast artificial chromosome DNA is to be subcloned to identify polymorphic microsatellite markers that will establish whether this gene is frequently amplified in oral neoplastic tissue.

Investigation of chromosome 9q22.3-q31 DNA marker loss in odontogenic keratocysts.

Multiple basal cell carcinomas and odontogenic keratocysts of the jaws are a feature of the inherited naevoid basal cell carcinoma syndrome (NBCCS), although both occur more commonly as single, sporadic cases. The NBCCS gene has been mapped to chromosome 9q22.3-q31 and loss of heterozygosity for DNA markers from this region has been observed in familial and sporadic basal cell carcinomas. Based on these observations, we undertook a pilot study to determine if a similar pattern of chromosome loss occurs in odontogenic keratocysts. DNA extracted from microdissected odontogenic keratocyst epithelium was examined for loss of heterozygosity for six polymorphic DNA markers mapping to human chromosome 9q22.3-q31. Allelotype loss was detected in epithelium from three, single, sporadic odontogenic keratocysts. These results implicate homozygous inactivation of the NBCCS gene in the initiation and progression of the odontogenic keratocyst.

Immunolocalisation of tenascin-C in focal reactive overgrowths of oral mucosa.

Focal reactive overgrowths of oral mucosa were investigated in the following groups: gingival pyogenic granuloma, fibrous epulis, calcifying fibrous epulis, peripheral giant cell granuloma, giant cell fibroma, fibroepithelial polyp and denture-related fibrous hyperplasia (n = 8 for each group). We hypothesised that immunoreactivity to tenascin-C, a functional protein associated with connective tissue organisation and cell migration, would be differentially distributed in individual lesions and between lesion groups. Staining patterns for giant cell fibromas and fibroepithelial polyps were similar to those reported for normal mucosa. By contrast, additional staining was observed in the other lesion groups, although immunoreactivity was variable and not specific to each lesion group. Strong immunoreactivity was observed around blood vessels lined with plump endothelial cells and in regions where keratinocytes were migrating over ulcerated surfaces. Interlacing collagenous fascicles could be either strongly or weakly immunoreactive, with either fibrillar or diffuse staining. Localised staining was observed around, but not within, areas of calcification.

The polymorphous odontogenic cyst.

Over the years there have been sporadic reports of unusual cystic lesions of the jaws, not readily classified under conventional headings but which have been variously diagnosed as median-mandibular, glandular, sialo-odontogenic or botryoid odontogenic cyst. We present five cases which do not fit into other categories of odontogenic cyst, two of which have recurred within a few years of conservative treatment. This paper aims to alert clinicians to the propensity for regrowth of these cysts, proposes the term polymorphous odontogenic cyst for these lesions, to encompass their varied histological appearances and discusses their distinction from other cyst types with mucous and papillary formations in epithelium.

Patterns of immunoreactivity to an anti-fibronectin polyclonal antibody in formalin-fixed, paraffin-embedded oral tissues are dependent on methods of antigen retrieval.

The influence of antigen retrieval (AR) technique on immunoreactivity in formalin-fixed, paraffin-embedded tissues is recognized. In focal reactive overgrowths of oral mucosa, we noted that patterns of immunoreactivity to a fibronectin polyclonal antibody were dependent on methods of AR. To establish these patterns we investigated eight pyogenic granulomas and eight fibroepithelial polyps. In the absence of AR no immunoreactivity was observed. After alpha-chymotrypsin AR a band of intense immunoreactivity was associated with vascular endothelial cells, with either minimal or no staining of connective tissue. After microwave AR immunoreactivity was observed in connective tissue, especially in the subepithelial region, but there was no specific vascular staining. After autoclave AR immunoreactivity was observed in connective tissue and also in epithelial nuclei. To determine if these results represented either changes induced by tissue fixation and processing or exposure of epitopes normally masked in vivo, we investigated frozen sections from two fibroepithelial polyps and one pyogenic granuloma. In frozen sections immunoreactivity was observed around vascular endothelial cells and within connective tissue, especially in the subepithelial region, but not in epithelia. This study provides indirect evidence that alpha-chymotrypsin and heat-mediated AR protocols expose different masked epitopes, and reinforces the need to undertake appropriate pilot studies for immunohistochemical investigation of archival tissue.

Peripheral giant cell granuloma: a clinical study of 77 cases from 62 patients, and literature review.

OBJECTIVES: The aim of this study was to identify the principle clinical features of the peripheral giant cell granuloma (PGCG), and to recognise clinical features of PGCG that are poorly defined. DESIGN: We reviewed retrospectively 77 cases of PGCG from 62 patients, from our files with respect to incidence, sex, patient age, race, clinical symptoms and signs, radiographic features and recurrence following excision. RESULTS AND CONCLUSIONS: Our results were largely in agreement with previous reports, although there is wide variation in the results published between series. In addition, some clinical features of PGCG are poorly defined. Little is known about the relative incidences of PGCG and central giant cell granuloma. An association between PGCG and tooth loss may exist, but is poorly defined, and not all PGCG that involve edentulous areas follow recent tooth loss. Information about PGCG recurrence after excision is limited, and does not necessarily follow incomplete excision. Despite the large number of reported cases of PGCG, clarification of some clinical features is required, and may help formulation and interpretation of future laboratory-based research into this poorly understood lesion.

Expression of human cytokeratin 14 in normal, premalignant and malignant oral tissue following isolation by plaque differential hybridisation.

Differences in gene transcription between RNA samples extracted from oral normal and squamous cell carcinoma (SCC) tissue were examined using the technique of cDNA library differential plaque screening. A differentially expressed transcript was selected on the basis of it being under-expressed in the cancer tissue and was identified, using DNA sequencing, as cytokeratin 14. The level of cytokeratin 14 transcription in RNA samples extracted from a range of oral SCC and normal tissue, as well as "white patch" lesions, was then investigated. Cytokeratin 14 appeared to be significantly under-expressed in oral cancer specimens studied compared to normal and white-patch tissue (P < 0.01). The trend for higher levels of cytokeratin 14 transcription in the dysplastic "white patch" samples compared to that observed for the malignant tissue (P < 0.05) suggests that the decrease in cytokeratin 14 transcription is a late event in the carcinogenic pathway.

Differential expression of protease inhibitor and small proline-rich protein genes between normal human oral tissue and odontogenic keratocysts.

The technique of differential hybridization was used to compare gene transcription between normal oral mucosa and odontogenic keratocyst lining. Protease inhibitors, elafin and stefin-B as well as beta-actin and two epithelial-specific small proline-rich (spr) proteins, which we have named SPRC and SPRK and which are distinct from salivary proline-rich proteins, were differentially expressed. Increased abundance of alpha I(I) collagen and elafin transcripts was demonstrated in the keratocyst, with decreased abundance of stefin B, SPRC and cytokeratins 4 and 13 transcripts compared to normal palatal mucosa. The deduced protein sequences of SPRC and SPRK were described and compared, and the relative abundance of their respective cDNAs in palatal and keratocyst libraries determined. Identification of factors controlling transcription of these genes could advance our understanding of the development of odontogenic keratocysts.

Rapid detection of human herpes simplex virus type 1 in saliva.

Herpes simplex virus type 1 (HSV1) can be rapidly identified in saliva from patients with acute herpetic gingivostomatitis, by in vitro amplification using the polymerase chain reaction and specific primers. Amplification of DNA results in a product of 110 bp length corresponding to the region 1381-1490 bp of the HSV1 thymidine kinase gene. The specificity of the reaction was demonstrated in three ways: (i) the presence of a Sma 1 restriction enzyme site in the amplified product sequence; (ii) Southern blot using a biotinylated HSV1-specific oligonucleotide probe and (iii) direct sequencing of amplified product. At high titres of virus (> 5 x 10(5) virions/ml saliva), saliva may be added directly to the amplification assay for detection purposes. However, at lower titres of HSV1 viral DNA must be purified from saliva before in vitro amplification. HSV was identified in the saliva from symptomatic patients with acute herpetic gingivostomatitis and was absent in saliva collected from controls.

Resorption of the crown of an unerupted permanent molar.

This case report describes an unusual phenomenon where resorption occurred in the crown of an unerupted permanent molar. It was an incidental radiological finding. After eruption the tooth was extracted. Histological examination revealed resorption of enamel and dentine, and partial replacement by calcific material. The possible aetiology of the condition is discussed.

Identification and partial characterisation of a serum enzyme which hydrolyses epidermal inhibitory pentapeptide.

We have identified an enzyme present in mammalian, avian, and reptilian serum which cleaves epidermal inhibitory pentapeptide (pGlu-Glu-Asp-Ser-GlyOH) to form a relatively stable tripeptide (pGlu-Glu-Asp). The enzyme has an inhibitor profile unlike any readily identifiable serum enzyme, and is stable at 4 degrees C for up to 3 months. As most experiments using the pentapeptide are carried out in the presence of serum this finding may have important implications on future research into the biological function of the pentapeptide.

Unusual presentation of a monomorphic adenoma.

This case is presented as an example of the diagnostic difficulty that can be associated with radiolucencies around the apices of teeth. The discovery of a monomorphic adenoma within bone was unexpected because of its very rare occurrence.

Atypical fibroxanthoma of oral mucosa: a variant of malignant fibrous histiocytoma.

Atypical fibroxanthoma, a more recently recognised variant of malignant fibrous histiocytoma, is described arising at the site of an earlier squamous cell carcinoma, in a patient treated with radiotherapy 21 years previously. Distinction between atypical fibroxanthoma and a poorly-differentiated squamous cell carcinoma proved to be difficult by ordinary light microscopy and it was only after immunohistochemical staining that the second lesion was confirmed as atypical fibroxanthoma. The natural history, differential diagnosis and histological pitfalls of this poorly characterised mesenchymal lesion are discussed.

Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm-dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium.

We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine [( 3H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5.8 +/- 0.3 h), a high S phase efflux and an initially low influx of cells from G1 into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 +/- 0.1 h), an initially low efflux and a moderate to high influx. Two time-points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15-16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300-1400 h, efflux rate falls from high (19-20 cells %/h) to low (4-8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two 'control' points on the circadian LI cycle that were previously unrecognized.