Yoshinari Myoken

Immunohistochemical study of overexpression of fibroblast growth factor-1 (FGF-1), FGF-2, and FGF receptor-1 in human malignant salivary gland tumours.

Fibroblast growth factor‐1 (FGF‐1) and FGF‐2 are broad spectrum mitogens. The expression of FGF‐1, FGF‐2, and their receptor, FGF receptor‐1 (FGFR‐1), was examined in malignant salivary gland tumours and normal salivary glands, using immunohistochemical methods. In seven cases of adenoid cystic carcinoma (ACC), both duct‐like cells and modified myoepithelial cells were apparently immunopositive for FGF‐1, FGF‐2, and FGFR‐1. In five cases of mucoepidermoid carcinoma (MC), all three types of tumour cells including epidermoid cells, mucous cells, and intermediate cells expressed immunoreactive FGF‐1, FGF‐2, and FGFR‐1. In these malignant salivary gland tumours, increased expression of FGFR‐1 correlated with the intensity of both FGF‐1 and FGF‐2 immunoreactivity. In contrast to malignant salivary gland tumours, eight cases of normal salivary gland showed negative immunostaining for FGF‐1, FGF‐2, and FGFR‐1 while four cases were weakly immunoreactive for FGF and its receptor. These results demonstrate that malignant salivary gland tumours overexpress FGF‐1, FGF‐2, and FGFR‐1 compared with normal salivary glands and suggest that these growth factors may play an important role in facilitating neoplastic progression in human salivary glands.

Invasive Aspergillus Stomatitis in Patients with Acute Leukemia: Report of 12 Cases

An 8-year retrospective analysis of invasive Aspergillus stomatitis in neutropenic patients with acute leukemia was performed to characterize the epidemiology and clinical features of the infection. Twelve cases of invasive Aspergillus stomatitis were identified with both clinicohistological and microbiological evidence, and the majority of cases were caused by Aspergillus flavus (10 [83%] of 12 patients). The infection was strongly suspected when a neutropenic patient developed persistent fever without a known source, symptoms of gingival pain and facial swelling, and a solitary ulcerating lesion of mucogingiva covered with a gray necrotic pseudomembrane. Aspergillus stomatitis was diagnosed a median 23 days after admission. In all 12 patients, the diagnosis was made during the period of neutropenia. Ten patients (83%) were treated with amphotericin B and surgery and survived with recovery of neutrophils. Two patients died, and disseminated aspergillosis was identified in 1 patient.

Pathologic features of invasive oral aspergillosis in patients with hematologic malignancies

PURPOSE Little is known about the characteristic macroscopic and microscopic changes that take place during the progression of oral invasive aspergillosis in immunocompromised patients. The aim of this study was to determine the relationship between the oral and histopathologic findings in these patients. Such a study would aid in understanding the early development of subsequent progression of the disease. PATIENTS AND METHODS Twelve patients with hematologic malignancies who developed invasive oral aspergillosis were studied. The condition was divided into three stages according to the oral findings at the time biopsy procedures were performed. Tissue sections from biopsy specimens were stained with hematoxylin and eosin for histopathologic study and the findings were evaluated in relation to the oral findings. Fungal cultures of biopsy specimens were also performed to confirm the causative organisms. RESULTS The diagnosis of oral aspergillosis was established in terms of both histologic and microbiologic evidence in all 12 patients. In the early stage (three patients), isolated areas of violaceous marginal gingiva consisted of degenerated epithelium and connective tissue infiltrated by fungal hyphae. In the advanced stage (four patients), the violaceous marginal gingiva had become transformed into gray necrotic lesions that extended to the attached gingiva. The necrotic lesions showed ulceration and were covered by a pseudomembrane containing fungal hyphae. At the base of the ulcers, connective tissue was occupied by proliferating fungal hyphae, with vascular invasion being observed. In the late stage (five patients), the ulcerated lesions had progressed, showing destruction of the alveolar bone and surrounding facial muscles, with infiltration of fungal hyphae unto the tissues. No inflammatory cellular reaction was observed until the hematologic status of the patients improved. CONCLUSION These findings indicate that invasive oral aspergillosis has three distinctive clinicopathological stages. Recognition of the different stages of invasive Aspergillus infections is helpful for correct diagnosis of the disease.

Expression of fibroblast growth factor-1 (FGF-1), FGF-2 and FGF receptor-1 in a human salivary-gland adenocarcinoma cell line : Evidence of autocrine growth

Fibroblast growth factor‐1 (FGF‐1) and FGF‐2 are heparin‐binding polypeptides which express potent mitogenic properties in neoplastic cells. In the present study, we have examined the contribution of endogenous FGF‐1 and FGF‐2 to the autocrine growth of HSY human salivary‐gland adenocarcinoma cells in vitro. Using specific monoclonal antibodies against FGF‐1 and FGF‐2, immunohistochemical analysis of HSY cells revealed strong expression of both FGF‐1 and FGF‐2 in the cytoplasm and nucleus. Consistent with these data, 2 molecular mass species of FGF‐1 (16 and 18 kDa) and 3 FGF‐2 (18, 24 and 27 kDa) were identified in HSY cells by Western‐blot analysis. Scatchard analysis of FGF binding sites on HSY cells indicated the presence of 23,000 [125I] FGF‐1 binding sites/cells with a dissociation constant (KD) of 178 pM and 13,000 [125I]FGF‐2 binding sites/cell with a KD of 102 pM. In addition, HSY cells were shown to express the mRNA for FGF receptor‐I (FGFR‐1) by reverse transcription‐polymerase chain reaction (RT‐PCR), confirming the existence of high‐affinity FGF binding sites. The influence of endogenous FGF‐1 and FGF‐2 on HSY cell growth was evaluated by suppressing the expression and activity of FGF by using anti‐sense oligonucleotides and neutralizing antibodies. The addition of 50 μM FGF‐1‐specific anti‐sense oligonucleotides to HSY cells resulted in a 61% inhibition of cell growth, while 50 μM FGF‐2‐specific anti‐sense oligonucleotides resulted in a 76% inhibition. These effects were dose‐dependent and specific, since sense oligonucleotides were ineffective in inhibiting HSY cell growth at the same concentration. Furthermore, HSY cell growth was suppressed in the presence of anti‐FGF‐1 or anti‐FGF‐2 neutralizing antibody, resulting in a 58% inhibition at 8 μmg/ml. Our observations suggest that FGF‐1 and FGF‐2 may act as autocrine regulators by interacting with FGF receptors on HSY cells.

Invasive oral aspergillosis in immunocompromised patients with leukemia

The clinicopathologic characteristics of invasive oral aspergillosis in 16 immunocompromised patients who developed this infection during antileukemic chemotherapy are described. The primary site of the infection was the marginal gingiva, there was severe spontaneous pain, and the patients developed spiking fever and granulocytopenia. Necrotic ulceration of the gingiva rapidly extended to the contiguous mucosa, muscle, and bone. Microscopically, the necrotic tissue contained thrombotic vascular infarcts and there were hyphae that showed frequent transverse septa and dichotomous branching. The invasive organisms were not responsive to amphotericin B in the absence of remission of the leukemia and restoration of the depressed host defenses. In 15 patients who showed improvement of hematologic status, oral aspergillosis was controlled by the combination of antifungal chemotherapy and debridement of necrotic tissues.

Effect of fibroblast growth factor-1 on the three-dimensional growth and morphogenesis of human salivary gland epithelial cells embedded in collagen gels

Dear Editor: The induction of epithelial tubular morphogenesis and glandular histogenesis occurs in vivo during the fetal development of the salivary glands (6). Several in vitro studies have shown that the culture in a three-dimensional collagen gel of epithelial cells from thyroid (9), mammary glands (10), or kidney (8) promotes their organization into tubelike structures. The epithelial structures can mimic the dichotomous branching morphogenesis of tubules and the preliminary steps of glandular development. In fact, the morphogenesis of epithelial tissues can be controlled by the cell-extracellular matrix interactions and by soluble factors released by autocrine and paracrine activation of the epithelial and adjacent mesenchymal cells (3). Fetal salivary gland development is known to correlate with the expression of fibroblast growth factor-1 (FGF-1) (2), which has numerous effects on cellular growth and differentiation in a wide variety of epithelial cells (1). In this report, we cultured human salivary gland epithelial (HSGE) cells within a collagen gel in a serum-free defined medium and found that the sustained growth and duct formation of these cells occurred in the presence of FGF-1. HSGE cells were isolated from labial salivary glands (7). The three-dimensional collagen gels were prepared by mixing 0.4 ml of type I collagen solution (Nitta Gelatin, Tokyo, Japan) and 0.05 ml of 10)< concentrated RD153 medium (RPMI1640:DMEM: MCDB 153; 1:1:2 vol/vol/vol) and 0.05 ml of reconstitution buffer (0.2 M HEPES, 0.08 M NaOH). Collagen (0.5 ml), containing 106 cells, was overlaid on a 0.5 ml base of gelled collagen in each well of 24 well tissue culture plates (Falcon, Mountain View, CA). Cultures were fed with serum-free RD153 medium containing insulin at 10 #g/ml, transferrin at 10 #g/ml, 10/.tM 2-mercaptoethanol, 10 ttM 2-aminoethanol, 10 nM selenite (Sigma Chemical Co., St. Louis, MO) with 20 ng/ml of FGF-1 (Upstate Biotechnology, Lake Placid, NY) and heparin (1000-fold excess by weight). The medium was changed every 3 d. The growth kinetics and morphogenesis was analyzed on Days O, 7, and 14. The number of cells in each well was estimated using a hemocytometer after digestion of collagen with 40 U/ml collagenase (type IA-S, Sigma) followed by a 0.25% trypsin digestion. At the same time, HSGE cells in a collagen gel were fixed with 10% formalin and processed for paraffin embedding, sectioning and stained with hematoxylin and eosin (H-E). In addition, HSGE cells in paraffin sections were immunohistochemically examined using avidin-biotin-peroxidase technique (4) with rabbit polyclonal antisecretory component antibody and antilactoferrin antibody (Dako A/S, Copenhagen, Denmark). As shown in Fig. 1, the growth of HSGE cells in collagen gels was significantly enhanced by FGF-1 in a concentration dependent manner in the presence of heparin. A sixfold increase in cell number could be accomplished at 20 ng/ml of FGF-1 in 14 d. Morphologically, the embedded HSGE cells extended duct-like structures into the collagen matrix overtime. On Day 0, isolated cells and small cell aggregates were seen dispersed within the collagen matrix (Fig. 2 A,B). On Day 7, ductal projections initiated in culture (Fig. 2 C), creating three-dimensional duct-like structures (Fig. 2 D), which became extensive with time (Fig. 2 E,F). When FGF-1 was omitted from medium, HSGE cells did not form duct-like structures; they remained either individual or they formed small clusters (data not shown). Immunohistochemical analysis revealed the presence of secretory component (Fig. 3 A) and lactoferrin (Fig. 3 B) in HSGE cells forming duct-like structures. These polypeptides have been reported to be present in salivary gland duct ceils (5). The results reported here provide evidence that human salivary gland epithelial cells were capable of reconstructing epithelial ductlike structures similar to developing salivary glands when cells were

Monoclonal antibodies against human oral squamous cell carcinoma reacting with keratin proteins

Two mouse monoclonal antibodies (MoAbs), B10 and 1H5, were generated by fusing mouse myeloma NS‐1 cells with spleen cells from a BALB/c mouse immunized with Ueda‐1 cells derived from human squamous cell carcinoma (SCC) of the floor of the mouth. Immunohistochemical analysis revealed that these MoAbs recognize the filamentous components of cytoplasm which were protein in nature. While the pattern of antigen distribution in various cell lines was not cell‐type specific, reactivity of these antibodies with tissue sections was informative. MoAb 1H5 was preferentially reactive with well‐differentiated squamous cell carcinoma, however, reaction with adenocarcinoma was observed infrequently. This antibody also preferentially reacted with the spinous layer of normal stratified squamous epithelium. MoAb B10, however, was reactive with nonepithelial tissues as well as with epithelial ones, and its level of binding bore no relationship to the grade of histologic malignancy. SDS‐PAGE and Western blotting analysis, using cytokeratin extracts of Ueda‐1 cells and human epidermis, demonstrated that MoAb B10 reacted with a wide range of keratin proteins of 65–67K, 58K, 56.5K, 56K, 52K, 50K, 48K, 45K, 40K, 38K, 36K, and 34K molecular weight (MW), while MoAb 1H5 reacted with keratin proteins of 65–67K, 58K, 56.5K, 56K, 52K, 48K, and 34K MW. These results suggest that MoAb 1H5 may recognize keratin subfamilies related to squamous differentiation, whereas MoAb B10 recognizes a wide range of keratin proteins, and may even react with other kinds of intermediate filament proteins (IFPs).

Release of fibroblast growth factor-1 by human squamous cell carcinoma correlates with autocrine cell growth

SummaryA squamous cell carcinoma cell line Nakata proliferated in serum-free culture and was not responsive to exogenous fibroblast growth factor-1 (FGF-1). Immunostaining revealed that Nakata cells expressed FGF-1 in their cytoplasms and nuclei. Two molecular mass species of FGF-1 (16 and 18 kDa) were identified in cell extracts by Western blot. These cells also expressed high-affinity FGF-1 binding sites (Kd=360 pM, 28 000 sites/cell). The results of cross-linking with [125I]FGF-1 demonstrated the presence of two bands with molecular masses of 160 and 140 kDa. The addition of FGF-1 specific antisense oligonucleotides at 25 µM to Nakata cells resulted in an 82% inhibition in cell growth and suppressed FGF-1 expression. This effect was dose-dependent and specific, because sense oligonucleotides were ineffective in inhibiting cell growth. In addition, Nakata cell growth was suppressed by an anti-FGF-1 neutralizing antibody, which resulted in a 52% inhibition at 8 µg/ml. These results demonstrate that Nakata cells produce FGF-1, and indicate that this growth factor acts in an autocrine manner by interacting with FGF-1 binding sites on Nakata cells.

Identification of Aspergillus Species in Oral Tissue Samples of Patients With Hematologic Malignancies by In Situ Hybridization: A Preliminary Report

PURPOSE A definitive diagnosis of invasive oral aspergillosis can be difficult because the culturing of tissue samples frequently fails to isolate Aspergillus species. In addition, the mycelial elements of Aspergillus species seen in tissue sections are histopathologically indistinguishable from those of non-Aspergillus species. We analyzed the usefulness of a DNA probe directed against the alkaline proteinase (ALP) gene of Aspergillus fumigatus for the identification of Aspergillus species by the in situ hybridization (ISH) technique in patients with oral mycosis. PATIENTS AND METHODS The ALP probe was tested on tissue specimens from 16 patients with hematologic malignancies who had invasive, orofacial fungal infections and a positive culture for one of the following organisms: Aspergillus species in 13 patients (A. flavus in 10, A. terreus in 2, and A. fumigatus in 1), and Exophiala dermatitis, Trichoderma longibrachiatum, and Candida albicans in 1 patient each. In situ hybridization with the ALP probe was performed using formalin-fixed, paraffin-embedded tissue samples. RESULTS The ALP probe showed a strong reaction with specimens from all 13 patients who had culture-proven aspergillosis specimens attributable to A. flavus, A. terreus, and A. fumigatus. On the other hand, the ALP probe showed no cross-reactivity with other fungi (Exophiala dermatitis, Trichoderma longibrachiatum, and Candida albicans). CONCLUSION These findings indicate that ISH using an ALP probe may increase the accuracy of diagnosing invasive oral aspergillosis in immunocompromised patients, and facilitate the provision of adequate antifungal treatment.

Acromegaly identified in a patient with a complaint of malocclusion

Acromegaly in a female patient was first diagnosed after a dental examination at which time the patient complained of the inability to incise properly. An elevated serum growth hormone level and enlarged sella turcica confirmed the diagnosis of acromegaly. Based on examination of her plaster study models, it was apparent the patient had been developing a change in her dentition for years.