An immunohistochemical study of keratin expression in ameloblastoma from a Kenyan population.

OBJECTIVES: Ameloblastomas appear to exhibit biological heterogeneity and, except in the case of malignancy, histological appearances that do not always allow their behaviour to be predicted. The aim of this study was to assess keratin expression in African ameloblastomas and to correlate this with their clinical and histological features. MATERIALS AND METHODS: Expression of simple keratins 7, 8, 18 and 19; cornification keratins 1 and 10; basal and differentiation keratins 5 and 14 and hyperproliferation-related keratins 6 and 16 in 14-39 cases of ameloblastoma was assessed by immunohistochemical methods. RESULTS: There was patchy expression of keratin 7 in the suprabasal and stellate reticulum-like cells in some cases. All cases showed similar weak expression for keratins 8 and 18 in suprabasal and stellate reticulum-like cells but none showed keratin 1 or 10 expression. There was intense expression of keratins 5, 14 and 19 by all tumour cells suggesting that they may retain basal cell characteristics with a potential for proliferation. No consistent relationship was seen between histological types and keratin expression pattern. However, keratins 6 and 16, expressed by suprabasal and stellate reticulum-like cells, showed a marked variation within and between cases, with the highest levels of expression in squamous strands. CONCLUSIONS: We propose that squamous strands may represent the sites of most active growth within individual tumours and expression of keratins 6, 16 and 19 may be predictors of rapid growth. There is a need for further investigation of this in longitudinal clinical studies.

Protease-activated receptor-2 involvement in hypotension in normal and endotoxemic rats in vivo.

BACKGROUND: The protease-activated receptor-2 (PAR-2) is expressed by vascular endothelial cells and upregulated by lipopolysaccharide (LPS) in vitro. PAR-2 is activated by a tethered ligand created after proteolytic cleavage by trypsin or experimentally by a synthetic agonist peptide (PAR-2AP) corresponding to the new amino terminus of the tethered ligand. METHODS AND RESULTS: Intravenous administration of PAR-2AP (0.1, 0.3, and 1 mg/kg) to rats caused a dose-dependent hypotension. A scrambled peptide was without effect. A specific trypsin inhibitor, biotin-SGKR-chloromethylketone, inhibited trypsin-induced hypotension but not that stimulated by PAR-2AP. In animals treated with LPS 20 hours earlier, we found an increased sensitivity to trypsin and PAR-2AP in the hypotensive response. In particular, PAR-2AP caused hypotension at a low concentration of 30 ng/kg. Moreover, PAR-2 was immunolocalized to endothelial and smooth muscle cells in aorta and jugular vein in LPS-treated rats, and increased levels of PAR-2 mRNA were shown by reverse transcription-polymerase chain reaction analysis. CONCLUSIONS: Our findings suggest that PAR-2 is important in the regulation of blood pressure in vivo. A functional upregulation of PAR-2 by LPS was demonstrated by the activity of concentrations of PAR-2AP that were inactive in normal animals. We conclude that PAR-2 may play an important role in the hypotension associated with endotoxic shock and may represent a new therapeutic target.

Immunolocalization of protease-activated receptor-2 in skin: receptor activation stimulates interleukin-8 secretion by keratinocytes in vitro.

The protease-activated receptor-2 (PAR-2) is a seven transmembrane domain receptor related to the thrombin receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions.

Protease-activated receptors and their role in IL-6 and NF-IL-6 expression in human gingival fibroblasts.

The serine protease thrombin is formed at sites of coagulation and inflammation and has been shown to have important proinflammatory cellular effects relevant to the pathogenesis of periodontal disease. Thrombin acts via specific cell surface receptors termed protease-activated receptor-1 (PAR-1) and PAR-3, which have a distinctive method of activation. Proteolytic cleavage of the extracellular domain by thrombin reveals a hidden amino terminus which then acts as a "tethered ligand". A short synthetic peptide (SFLLRN) can also mimic the tethered ligand and activate PAR-1 but not PAR-3. Also, a trypsin-sensitive receptor termed PAR-2 has been described which is activated by the PAR-1 activating peptide SFLLRN. Here we show conclusively by flow cytometric and Northern blot analysis that human gingival fibroblasts (HGF) express PAR-1 but not PAR-2. In functional studies we also show that thrombin and SFLLRN stimulated increased expression of mRNA encoding nuclear transcription factor NF-IL-6 and IL-6 in vitro. At optimal concentrations, thrombin (10(-7) M) induced 7.6 +/- 0.01 ng/ml immunoactive IL-6 and PAR-1 activating peptide (5 x 10(-5) M) induced 2.2 +/- 0.2 ng/ml (mean +/- standard error of mean). A proteolytically inactive recombinant thrombin (serine 195 to alanine) was without activity. These data show that HGF express PAR-1 and suggest that PAR-1 activation stimulates increased NF-IL-6 and IL-6 gene expression and IL-6 secretion by HGF in vitro. Whether HGF express PAR-3 is unknown, but the fact that SFLLRN was not a complete replacement for thrombin raises the possibility that HGF may express additional thrombin receptors. These findings add weight to the importance of the cytokine-like role played by thrombin and raise the possibility that protease-activated receptors may play a role in the pathogenesis of inflammatory periodontal disease.

Multiparameter flow cytometric analysis of polymorphonuclear leucocytes in whole blood from patients with adult rapidly progressive periodontitis reveals low expression of the adhesion molecule L-selectin (Cd62L).

Numerous studies of polymorphonuclear leucocyte (PMN) function in patients with adult periodontitis, including rapidly progressive periodontitis, have yielded conflicting findings, perhaps because most of the assays were performed on PMNs that had been separated from whole blood by a variety of procedures. To avoid the problems associated with in vitro analysis of isolated cells, PMN function and antigen expression in live whole unmanipulated blood of eight patients with rapidly progressive periodontitis were compared with those of age-, race-, and sex-matched controls. Using multiparameter flow cytometry, a) L-selectin (CD62L) expression, b) cell size, and c) respiratory burst activity were measured in PMNs in whole blood immediately ex vivo and during incubation with Porphyromonas gingivalis and Staphylococcus aureus. By comparison with PMNs from the control group, PMNs from the patient group expressed significantly lower levels of CD62L and had an increased size before stimulation. PMNs from both groups produced respiratory bursts similar to those of the two bacteria, but in both groups the responses to S. aureus were significantly greater than those to P. gingivalis. The significantly reduced expression of the adhesion molecule CD62L on PMNs in the patient group may lead to reduced tethering of neutrophils at sites of inflammation and may partly explain the susceptibility of these individuals to recurrent and severe periodontal infections.

A CD4+ proliferation of large granular lymphocytes expresses the protease activated receptor-1.

The platelet-type thrombin receptor was the first member to be identified in a family of protease activated receptors (PARs) and has been designated PAR-1. We recently reported that the large granular lymphocytes (LGLs) in patients with proliferations of CD8+ cells co-expressed PAR-1 and the expression of PAR-1 correlated with the expression of CD57. Here we show, by three-colour immunofluorescence, that the LGLs from a patient with a rare CD4+ CD57+ monoclonal expansion also expressed PAR-1. Northern blot analysis confirmed the presence of high levels of mRNA for PAR-1 in these LGLs.

Ki-67 antigen in ameloblastomas: correlation with clinical and histological parameters in 54 cases from Kenya.

The aim of this study was to assess the cell proliferation in ameloblastomas and to correlate this with clinical features and histology. Immunohistochemistry with Ki-67 monoclonal antibody was performed on fresh tissue from 54 ameloblastomas. A labelling index (LI) was calculated by expressing the percentage of Ki-67 positive cells. There was no significant correlation between LI and clinical features: age, sex or tumour size. Follicular ameloblastomas had significantly higher LI (5.0 +/- 0.5; mean +/- SEM) than plexiform tumours (3.2 +/- 0.6; P < 0.05). Plexiform ameloblastomas from the anterior mandible had a significantly lower LI (1.8 +/- 0.5) than those from the posterior (3.9 +/- 0.8; P < 0.05). LI was higher in squamous arcades (6.4 +/- 3.1%) than in epithelial cords and cysts (1.4 +/- 1.3%; P < 0.001). These results suggest that LI correlates most closely with the histological pattern of the epithelium of ameloblastoma, both within and between different tumours.

Proteinase-activated receptor-2: expression by human neutrophils.

Neutrophils were shown to express the proteinase-activated receptor-2 (PAR-2), a seven transmembrane domain receptor, which is activated by cleavage by trypsin. Granulocytes from 14 donors stained positively for PAR-2 with affinity-purified rabbit antibodies raised against a peptide corresponding to the trypsin cleavage site of human PAR-2. Neutrophil activation in response to a receptor activating peptide (RAP) varied between donors. RAP (Ser-Leu-Ile-Gly-Lys-Val-NH2) alone induced an increase in the forward and side light scatter after 5-10 minutes and a small increase in the expression of the activation molecule CD11b. The increased expression of CD11b induced by RAP was markedly enhanced by priming the neutrophils with a low concentration (1 nM) of formyl-Leu-Met-Phe. Trypsin and RAP also induced an increase in intracellular calcium, but there were large variations in the magnitude of responses between donors also in this assay. The effects of RAP in the different assays were specific; acetylated RAP was completely without activity.

Hemangiopericytoma: a report of two cases arising on the lip.

Hemangiopericytoma is a rare tumour of pericytes; represents 1% of all vasoformative tumours and 15-25% of those which occur in the head and neck. We present two cases of hemangiopericytoma occurring on the lower lip and report the use of cryosurgery to treat the most recent case.

Analysis of the platelet-type thrombin receptor in 20 cases of large granular lymphocyte proliferations.

The platelet-type thrombin receptor was expressed by large granular lymphocytes (LGLs) in a variety of proliferative diseases. Twenty patients with LGL proliferative disease were examined, including five T cell clones and a variety of polyclonal proliferations, some secondary to rheumatoid arthritis and Felty's syndrome; 17/20 showed high number of CD3+, CD8+, and CD57+ lymphocytes and 9/20 also had high numbers of CD16+ or CD 56+ positive lymphocytes. The thrombin receptor was present on more than 20% of the LGLs in 13/20 patients. The clonal T cell expansions showed the highest receptor expression with greater than 75% cells positive. Regression analysis of all 20 cases showed striking and highly statistically significant positive Spearman rank correlation between the proportion of thrombin receptor and CD57-positive LGLs (r = 0.56, P = 0.009). A negative correlation with CD56 was also found (r = -0.46, P= 0.043). Dual antibody flow cytometry showed the receptor was more often co-expressed with CD57 (64%) than with CD16 (19%) or CD56 (11%). The expression of the platelet-type thrombin receptor by LGLs of this phenotype raises the possibility of a functional role for thrombin in the pathogenesis of LGL proliferative diseases.

Cytokine networks in destructive periodontal disease.

BACKGROUND: Cytokines are important regulatory proteins, produced by activated cells, which act by binding high affinity cell surface receptors. They are involved in almost all aspects of cell biology and form interacting networks, with cascades of sequential cell activation. They often show overlapping activities (redundancy) or the same cytokine may have a variety of different effects (pleiotropy). In excess, certain cytokines are damaging and proinflammatory. Tumour necrosis factor alpha (TNF alpha) and interleukin-I (IL-I) are markedly proinflammatory, inducing bone resorption, collagenase and prostaglandin E2 production. OBJECTIVE: This paper focuses on the role of TNF alpha and IL-I in the cytokine networks of destructive chronic periodontitis; specifically their regulation by T cell cytokines, receptor antagonists and inhibitory soluble forms of the IL-I and TNF receptors. CONCLUSION: A hypothesis is proposed that destructive periodontal disease may be due to disregulation of these inhibitors, rather than an overproduction of IL-I and TNF alpha per se.

The response to thrombin of human neutrophils: evidence for two novel receptors.

Human alpha-thrombin was a chemoattractant for human neutrophils yielding a maximal response of similar magnitude to that observed with formyl-Met-Leu-Phe. The observed chemotaxis was not due to stimulation of the proteolytically activated thrombin receptor since: (1) this receptor was not detected by flow cytometry; (2) the inactive thrombin mutant Ser195-->Ala elicited a chemotactic response indistinguishable from that caused by wild-type thrombin; (3) antibodies to the cleavage site of the proteolytically activated receptor did not affect thrombin-induced chemotaxis; (4) a thrombin receptor activating peptide (TRAP) failed to stimulate chemotaxis. These data indicate the existence of a thrombin receptor for neutrophil chemotaxis which is not activated by proteolysis. In addition, although wild-type and ser195-->Ala thrombin did not cause an increase in intracellular Ca2+, a Ca2+ response to TRAP was observed with neutrophils from some donors. The TRAP-induced increase in Ca2+ was reproducible, dose dependent and specific. The use of alanine-substituted peptides demonstrated that the Ca2+ response was due to TRAP stimulation of a receptor other than the proteolytically activated thrombin receptor. Thus, it is necessary to re-evaluate the assumption made in previous studies that responses to TRAP are mediated by the proteolytically activated thrombin receptor.

Malignant ameloblastoma revisited.

A case of a malignant ameloblastoma in a 49-year old Sri Lankan woman with widespread pulmonary metastases is presented, the diagnosis confirmed by needle biopsy. The current histological classification of odontogenic carcinomas and the management of metastatic pulmonary deposits are discussed.

Thrombin receptor expression and function in large granular lymphocyte proliferative disorders.

It has recently been shown that peripheral blood NK-cells and a fraction of T-cells which co-express CD16 and either CD56 or CD57 express the platelet type thrombin receptor. Large granular lymphocytes exhibit a T- or NK-cell phenotype, and therefore these results raise the possibility that thrombin and its receptor may be involved in the biology of large granular lymphocytes in health and disease. It is difficult, however, to perform functional studies using normal blood as a source of large granular lymphocytes, because the small fraction of large granular lymphocytes cannot be separated from other lymphocytes in numbers sufficient for most in vitro experiments. Therefore patients with large granular lymphocyte proliferative disorders have been screened in order to identify a population of cells enriched in large granular lymphocytes that express the thrombin receptor. Expression of the receptor was analysed in polyclonal and clonal large granular lymphocyte proliferative disorders. Using flow cytometry, it was found that the proportion of thrombin receptor positive large granular lymphocytes varied from 3% to 86%. Northern analysis indicated a high level of expression of mRNA in a clonal expansion of large granular lymphocytes that stained positively for the receptor by flow cytometry. Thrombin was found to act as a chemotactic stimulus for large granular lymphocytes from a polyclonal expansion with high numbers of thrombin receptor positive cells. At an optimal concentration of 10(-9) M the chemotactic response to thrombin was roughly equivalent to that obtained with the potent chemoattractant 1-oleoyl 2-acetyl glycerol. These findings suggest that thrombin may play a role in the recruitment of large granular lymphocytes in sites of inflammation.

Interleukin-11 inhibits bone formation in vitro.

The effects of interleukin-11(IL-11) on the differentiation of osteoblast precursors was tested using a bone nodule forming assay in rat calvaria cell cultures. IL-11 caused a dose dependent inhibition of nodule formation, with 500 U/ml IL-11 resulting in complete inhibition of nodule formation. IL-11 also caused a reduction in alkaline phosphatase expression in these cultures. These effects are similar to, but more potent than, the actions of IL-6 on these cells. These results indicate that IL-11 is an osteotropic cytokine and suggest that IL-11 may be an important inhibitor of bone formation in health and disease.

Platelet activation by Protease I of Porphyromonas gingivalis W83.

Porphyromonas gingivalis produces a trypsin-like enzyme, Protease I, which is thought to be an important virulence determinant of the organism in adult periodontal disease. Protease I is transiently inhibited by physiological inhibitors of human thrombin. The aim of the present work was to establish whether Protease I was able to mimic thrombin by activation of the thrombin receptor on human platelets. Protease I caused true platelet activation at concentrations comparable to thrombin as measured by aggregometry, morphology and fluorescence flow cytometric analysis of CD63 expression. The effect was blocked by protease inhibitors but not by anti-thrombin receptor antibodies which, by contrast, blocked platelet activation by thrombin. We conclude that the activation of platelets by P. gingivalis Protease I involves proteolysis, but not scission of the thrombin cleavage site of the thrombin receptor.

Peripheral blood lymphocytes express the platelet-type thrombin receptor.

Northern blot analysis of human mononuclear cells indicated that the platelet thrombin receptor may be expressed by lymphocytes. In order to investigate this, we prepared affinity purified rabbit antibodies against the thrombin receptor which bound platelets and blocked thrombin activation. Using flow cytometry on peripheral blood cells, we found that the vast majority of NK cells (CD16/CD56 positive) and a fraction of CD3/CD4 positive T cells expressed the thrombin receptor. B cells, neutrophils and monocytes were negative. These data suggest that potentially thrombin may play a direct role in regulating NK and T cell function.

Interleukin-6 inhibits bone formation in vitro.

Interleukin-6 (IL-6) is a pluripotent cytokine which is made by osteoblasts, but its role in bone metabolism is uncertain. The aim of this study was to test the effect of IL-6 on bone formation in vitro using a nodule-forming assay. Osteoblast-enriched calvaria cells were isolated from 2-day-old Sprague-Dawley rats and cultured in the presence of 10(-8) M dexamethasone. After 2 days, calvaria cells were treated with recombinant human IL-6 for 72 h, washed and maintained for a further 18 days before fixation. IL-6 caused a dose-dependent inhibition of bone nodule formation, with a maximum reduction of 53% with 5000 U/ml IL-6. IL-6 also inhibited alkaline phosphatase activity in a dose-dependent manner (e.g. control: 114 +/- 9.2; IL-6: 68 +/- 10.6 nmol p-nitrophenol (pNP)/mg/min). IL-6 did not affect cell numbers during early cell growth up to 6 days but caused a small but significant reduction in cell number at confluence (8 days). These results demonstrate that IL-6 inhibits bone nodule formation by rat calvaria cells in vitro and suggest that IL-6 may inhibit osteoblast differentiation.

Platelet-derived interleukin 1 induces human endothelial adhesion molecule expression and cytokine production.

Interleukin 1 (IL-1) plays a central role in the regulation of the body's response to infectious and inflammatory stimuli. Recent evidence has shown that human platelets express a cell associated form of this proinflammatory cytokine very rapidly following activation. Since one of the earliest events in inflammation is frequently the rapid adhesion of platelets to injured endothelium, it was of interest to determine whether platelets express IL-1 in a functionally relevant form that can alter the phenotype of human endothelial cells in vitro. Thrombin activated platelets induced significant expression of the adhesion molecule intercellular adhesion molecule 1, as well as secretion of the IL-1 inducible cytokines IL-6 and granulocyte macrophage colony stimulating factor by cultured human umbilical cord and saphenous vein endothelial cells. This was inhibited by prior treatment of the platelets with antibody specific for IL-1. These results suggest that platelet delivered IL-1 might initiate and regulate some of the earliest phases of the inflammatory response. An additional observation of interest was differential induction of endothelial leucocyte adhesion molecule 1 by activated platelets on saphenous vein but not umbilical vein but not umbilical vein endothelial cells, which suggests functional heterogeneity of the endothelial cells.