Temporal kinetics and cellular phenotype of TNF p55/p75 receptors in experimental allergic encephalomyelitis.

TNF-alpha and LT-alpha are thought to be involved in the immunopathology of CNS demyelinating diseases. Both cytokines induce cellular effects through 55-kDa type-1 receptors (R1) and 75-kDa type-2 receptors (R2). To date, no study has specifically identified the various cell populations that express TNF receptors (TNFR) in the inflammatory and demyelinating mouse model, EAE. Phenotyping the TNFR positive cells is important in determining when and where the ligands may be acting and playing a role in disease pathology. We observed an upregulation of TNF R1 and R2 mRNA in high endothelial venules (HEVs) in the lymph node and CNS before the onset of EAE (preclinical phase). This upregulation of TNFR expression in HEVs was followed by a rapid increase in leukocytes within the CNS after the onset of clinical disease. The temporal kinetics of these data suggest that HEVs become activated early, probably through the release of pro-inflammatory cytokines originating from circulating leukocytes. An increase in TNFR on HEVs would make these cells more susceptible to TNF-induced changes, such as increasing cellular adhesion molecules, thereby further facilitating the trafficking of leukocytes into the CNS parenchyma.

Demonstration of human papillomavirus DNA in a peripheral ameloblastoma by in situ hybridization.

The peripheral ameloblastoma (PA) is a rare, extraosseous, odontogenic lesion usually located on the mandibular or maxillary gingiva. It often appears as an asymptomatic, firm, pink-red nodule with occasional secondary involvement of the underlying bone. Although histologically identical to primary intraosseous ameloblastoma, the PA is notably less aggressive in biologic behavior and rarely recurs following conservative treatment. It has a proposed dual histogenesis, while its etiology remains obscure. Several benign oral mucosal lesions have been found to be associated with various types of human papillomavirus (HPV). The following study uses a commercially available HPV DNA in situ hybridization technique to confirm the presence of HPV in a previously reported case of PA. Results were positive for HPV types 16/18 but negative for HPV types 6/11. The implications of these results with regard to etiology are discussed.

CNTF regulation of astrogliosis and the activation of microglia in the developing rat central nervous system.

In response to physical or chemical brain injury, the mammalian central nervous system (CNS) often reacts by evoking astrogliosis. The most prominent feature describing this state is an upregulation of glial fibrillary acidic protein (GFAP). The agent(s) responsible for inducing astrogliosis remains unclear; however, recent observations have shown cytokines may play a pivotal role. During CNS trauma, macrophages and lymphocytes infiltrate the CNS where they are thought to synthesize and secrete cytokines; moreover, activated microglia and reactive astrocytes are known to be capable of cytokine production. We are the first to report that an intracerebral injection of the pleiotropic cytokine, ciliary neurotrophic factor (CNTF), increases astrogliosis and the appearance of activated microglia in the neonatal rat. This response to CNTF was comparable to the response observed in animals receiving a well known pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). Only a moderate increase was observed in the proliferative index of cytokine-injected animals; therefore, we conclude that GFAP is largely upregulated in a pre-existing GFAP negative cell population. Interestingly, coinjections of CNTF and TNF-alpha appeared to act synergistically. Coinjected animals displayed a wave of hypertrophied astrocytes reaching far into the contralateral hemisphere. No contralateral spreading of microglia was observed. This article clearly provides interesting information regarding the regulatory mechanisms that govern astrogliosis and discusses the probable relationship of reactive astrocytes to microglia.

CNTF induces GFAP in a S-100 alpha brain cell population: the pattern of CNTF-alpha R suggests an indirect mode of action.

In a recent report, we demonstrated that intracerebral injections of the pleiotropic cytokine, ciliary neurotrophic factor (CNTF), into developing postnatal rats evoked a severe inflammatory response as determined by the appearance of reactive astrocytes and activated microglia. Considering the likely involvement of CNTF in the injury response, we felt it was important to further understand the role of CNTF in the developing rat CNS. In this study, we examined the responsiveness of other cell populations to intracerebral injections of CNTF. We report that CNTF increases glial fibrillary acidic protein (GFAP), while having no appreciable effect on the levels of other intermediate filaments including vimentin and neurofilament. Moreover, CNTF did not affect the expression of the mature oligodendrocyte gene, myelin basic protein. These results suggest that CNTF is highly specific in its regulation of GFAP. In our previous study, we showed CNTF to increase GFAP in a cell population that already exists in the CNS parenchyma. To determine the origin of the CNTF-induced reactive astrocytes, therefore, we have utilized a technique of combined in situ hybridization and immunocytochemistry. To examine the possibility that CNTF acts on oligodendrocyte precursors to give rise to reactive astrocytes, the platelet-derived growth factor alpha receptor (PDGF-alpha R) was utilized as a riboprobe in conjunction with an antibody to GFAP. Examination of CNTF-induced GFAP+ astrocytes revealed no colocalization with PDGF-alpha R mRNA. In contrast, when we utilized an S100 alpha antibody recognizing a calcium binding protein in immature astrocytes, we found colocalization of S100 alpha and GFAP mRNA. These data suggest that CNTF induces an upregulation of GFAP in immature S100 alpha + astrocytes. Examination of the CNTF-alpha receptor mRNA revealed no change in expression following CNTF treatment. Unexpectedly, however, the CNTF-induced astrogliotic response appears to be indirect since the CNTF-alpha receptor was solely expressed by neurons in the cytokine-treated animals.

Molecular mechanism of the excellent emulsifying properties of phosvitin-galactomannan conjugate.

The emulsifying properties of native and N- and C-terminal-deleted phosvitin (protease digests) were compared after conjugation with galactomannan. The emulsifying properties of Maillard-type phosvitin-galactomannan conjugates were greatly improved, whereas those of the protease-digested phosvitin-galactomannan conjugates were not so dramatically improved. Phosvitin was highly glycosylated with galactomannan, whereas the protease-digested phosvitin conjugate consisting of a highly phosphorylated core peptide fragment was not. The results suggest that both N and C termini of the peptide moiety, digested by protease, were essential for the improvement of emulsifying properties of phosvitin-galactomannan conjugates. In addition, the role of N and C termini as anchors in oil droplets was supported from the comparative studies of native phosvitin, phosvitin-galactomannan conjugates, and protease-digested phosvitin-galactomannan conjugates.

RET protooncogene mutational analysis in multiple endocrine neoplasia syndrome type 2B: case report and review of the literature.

Multiple endocrine neoplasia, type 2B (MEN 2B), is a phenotypic variant of a group of autosomal-dominant neurocristopathies. MEN 2B is associated with medullary thyroid carcinoma and pheochromocytoma with oral, ocular, and alimentary submucosal ganglioneuromas and Marfanoid body features. Approximately 50% of cases are thought to be spontaneous mutations. The RET protooncogene (RET) is a 21-exon gene encoding a tyrosine kinase receptor. A codon 918 germ line mutation, which converts a highly conserved methionine to a threonine in the intracellular tyrosine kinase portion of this receptor of RET, has been identified in 95% of patients with MEN 2B. This mutation is easily detected by a direct deoxyribonucleic acid sequencing or restriction enzyme (Fok 1) analysis of amplified polymerase chain reaction products. The RET gene is normally expressed in the oral and gastrointestinal submucosal neural ganglia, and the codon 918 mutation is thought to cause neuromas by virtue of its transforming activity in these ganglia. Identifying clinical features of MEN 2B in an 11-year-old boy by an oral pathologist led to confirmation by mutational analysis. Before genetic testing was available, the patient, and at a later date his mother, underwent thyroidectomies based solely on biochemical testing. Results indicated the patient had the codon 918 mutation, whereas his phenotypically normal mother, father, and older brother had normal RET analyses. Studies in families have demonstrated that the mutant allele is derived from the father with possible acquisition during spermatogenesis. We believe the mother of our affected patient to be normal; the absence of phenotypic features of MEN 2B and a normal genotype suggest her calcitonin abnormalities and minimal evidence for C-cell hyperplasia were inconsequential. Molecular analysis for RET abnormalities will likely supplant biochemical methods of diagnosis in patients with MEN 2B.

Polymorphous low-grade adenocarcinoma: flow cytometric, p53, and PCNA analysis.

Polymorphous low-grade adenocarcinoma of minor salivary glands (terminal duct carcinoma, lobular carcinoma) was first defined more than a decade ago. A 17% recurrence rate and a 9% metastasis rate have been reported. Fifteen formalin-fixed, paraffin-embedded archival cases were analyzed. Ploidy and proliferative activity were evaluated with flow cytometric analysis. Demonstration of an abnormal p53 gene product and proliferative cell nuclear antigen analyses were also performed with routine immunohistochemical procedures. The purpose of this investigation was to evaluate these parameters and determine if a correlation existed. Flow cytometry was performed on 10 cases; 3 showed an aneuploid cell line (mean, S-phase diploid tumor cells 5.9%; S-phase aneuploid 26.7%). Products of a mutation of the p53 tumor suppressor gene have been noted to accumulate in salivary gland tumors, both benign and malignant. Qualitative assessment revealed p53 positive staining in 4 of 15 tumors; positive cells comprised 5% to 10% of the tumor. The percentage of tumor cells positive for proliferative cell nuclear antigen staining ranged from 0.5% to 70%. There was no correlation between proliferative activity as determined by proliferative cell nuclear antigen when compared with results of flow cytometric analysis except for one case that exhibited p53 staining, a 26% proliferative cell nuclear antigen fraction, and a distinct aneuploid cell line.

Oral mucosal melanomas: the WESTOP Banff workshop proceedings. Western Society of Teachers of Oral Pathology.

A workshop to discuss primary oral melanomas was convened at the annual Western Society of Teachers of Oral Pathology meeting in Bannf, Alberta, Canada. Fifty oral melanomas, identified from the files of the participants, were reviewed in order to better understand the clinical features, histologic spectrum, and natural history of these perplexing lesions. Results confirmed that oral melanomas occur in adults almost three times more frequently in men than women and have a decided predilection for the palate and gingiva. Some lesions exhibit a clinically detectable and prolonged in situ growth phase, whereas others seem to lack this property and exhibit only or predominantly invasive characteristics. Recurrences, metastases, and death from tumor were characteristic of the follow-up of a limited number of patients. Until definitive prospective data are collected that elucidate natural history, oral mucosal melanomas should be tracked separately from cutaneous lesions. All oral pigmented lesions that are not clinically diagnostic should be biopsied. Lesions with equivocal histopathologic features might be referred to as "atypical melanocytic proliferation" and should be excised. Recognition of lesions in an early in situ phase and aggressive treatment should have a favorable effect on prognosis. To enhance future or prospective study of these rare neoplasms, guidelines for reporting oral melanomas are suggested.

Comparative pharmacokinetics of submucosal vs. intravenous flumazenil (Romazicon) in an animal model.

PURPOSE: This study was performed to determine the bioavailability and local tissue toxicological safety of flumazenil (Romazicon) when administered by oral submucosal (SM) as opposed to intravenous (i.v.) injection. METHODS: Six dogs each received SM flumazenil (0.2 mg), and their serum was collected at predetermined time intervals (0-2 h) and frozen (-70 degrees C). Seven days later, the dogs received an identical dose of i.v. flumazenil, and serum samples were again collected, as above. Comparative quantitation of flumazenil levels (i.v. vs. SM) was made using a sensitive HPLC assay (UV detection). Direct/local drug toxicity was visually scored by unbiased raters of color photographs (test and control mucosa) taken at 1, 2, and 7 days following SM flumazenil injection. An oral pathologist examined slides processed from control and treatment tissues (hematoxylin and eosin staining) taken (punch biopsy) 1 week following SM injection to compare with direct clinical scores. RESULTS: Serum flumazenil levels reached a plateau (8.5 +/- 1.5 ng/mL, mean +/- SD) within 4 min of SM drug injection and declined thereafter to -2 ng/mL by 2 h. Bioavailability of SM flumazenil was 101 +/- 14%, based upon measuring the area under the serum concentration-time curves over 1.5 h (AUC 0-1.5 h, SM vs. i.v. drug). Thus, serum drug levels following SM drug administration were broadly comparable to those obtained during the elimination phase of corresponding i.v. drug delivery. Regarding drug tissue toxicity, no evidence of direct drug toxicity was observed by unbiased raters of color photographs (test and control mucosa) taken at 1, 2, and 7 days following SM flumazenil injection. Following pathologic review, no difference was seen in the degree of inflammation between treatment and control tissue. CONCLUSION: At the quantity and concentration used, SM drug flumazenil administration appears to be both a safe and a viable alternative to bolus i.v. drug delivery and worthy of further investigation.

The dos and don'ts of an oral mucosal biopsy performed by the general dentist.

The recognition of a pathologic entity is only the first step in the chain of events which must be performed for accurate diagnoses and appropriate patient management. Other steps include selection of the type of biopsy to be performed; proper biopsy technique, instrumentation and specimen handling; appropriate fixation; detailed documentation; and postoperative patient instructions. If the clinician performs each step carefully and correctly, the patient is ensured of receiving an accurate diagnosis in the shortest possible period of time.