Masaru Ohyama

Molecular epidemiology of Epstein‐Barr virus (EBV) in EBV‐related malignancies

The prevalent strain of Epstein‐Barr virus (EBV) in EBV‐related malignancies and in healthy adults in Southern Japan was examined by means of polymerase chain reaction (PCR) and/or restriction fragment length polymorphism (RFLP) analysis. In EBV‐related gastric cancers, 51/73 cases were subtype A, 4 were subtype B and the EBNA‐2 region was not amplified in 18 cases. Sixty‐three were wild‐type F, and only one was variant “f”. Sixty‐one cases had type C and 2 type D. EBNA‐2 subtype A was found in 10/12 EBV‐related T/NK‐cell lymphomas, and 11 samples harbored the wild‐type F. Neither subtype B nor the “f” variant was detected. Type C EBV was found in 8 cases and type D in 3 specimens. Two Japanese nasopharyngeal carcinomas (NPC) harbored subtype A with wild‐type F and type C. Throat washings from healthy adults harbored wild‐type F virus in 60/153 cases, and 25 of these samples were EBNA‐2 subtype A. Type C viruses were detected in 92% of cases and type D in 7.4%. Therefore, the prevalent strain in EBV‐related malignancies in Southern Japan was the same as in the healthy population in this geographical region. Int. J. Cancer 72:72–76, 1997.

Type V collagen selectively inhibits human endothelial cell proliferation.

Type V collagen from human placenta remarkably inhibited human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner when coated on the culture dishes. Other types of collagen (I, III, IV) and fibronectin enhanced HUVEC proliferation under the same conditions. The inhibitory activity of type V collagen was seen not only when it was coated on the dishes, but also when it was directly added into cell culture. The attachment effect of type V collagen did not differ from that of type I collagen. The inhibitory activity is a phenomenon selective for endothelial cells, since type V collagen did not affect the proliferation of human umbilical vein smooth muscle cells, aortic smooth muscle cells, or nasal mucosa fibroblasts.

Human papillomavirus DNA sequences and p53 over‐expression in laryngeal squamous cell carcinomas in Northeast China

One‐hundred‐two patients with laryngeal squamous cell carcinomas in Northeast China were examined for human papillomavirus (HPV) DNA by the polymerase chain reaction (PCR) coupled with Southern blot hybridization, and for p53 over‐expression by immunohistochemical staining. HPV DNAs were found in 60 cases (58.8%). HPV‐16, ‐18, ‐6, ‐11, and ‐33 DNAs were detected in 30 cases, 22 cases, 25 cases, two cases, and one case, respectively. In addition, coinfection either with HPV‐6 and ‐16 or with HPV‐6 and ‐18 was detected in 20 cases (33.3% of HPV DNA‐positive cases). p53 over‐expression was observed in 60 patients (58.8%). p53 was over‐expressed significanty in the poorly‐differentiated SCC and in patients with metastasis to lymph nodes (P < 0.05, respectively). Both HPV DNA and p53‐expression were positive in 35 patients, and negative in 17 patients. Either HPV DNA or p53‐expression were positive in 50 patients (25 cases each). Although p53 was detected in 35 (58.3%) of HPV‐positive patients, there was no significant correlation between HPV infection and p53 over‐expression in laryngeal squamous cell carcinomas of Northeast China. J. Med. Virol. 54:186–191, 1998.

Establishment of human mucosal microvascular endothelial cells from inferior turbinate in culture

Human microvascular endothelial cells were isolated and cultured from the mucosa of inferior turbinates. Using dish-coated collagen and a medium composed of a 1:1 mixture of sarcoma 180-conditioned medium and Dulbeccos modified Eagles medium (containing 10% fetal bovine serum and 75 micrograms/mL endothelial cell growth factors prepared from bovine pituitary glands), these cells grew rapidly to confluence and survived serial passages until the 16th population doubling level. The cells were identified as endothelial cells by their morphology, immunostaining of factor VIII antigen, and cytochemical staining with Ulex europeus agglutinin. Furthermore, Weibel-Palade bodies and numerous pinocytotic vesicles were confirmed by electron microscopy. Proliferation experiments demonstrated the need for either endothelial cell growth factor or tumor-conditioned medium. An exogenous matrix was also required for these cells in tissue culture. A tubule-like morphology appeared in the original monolayer of human microvascular endothelial cells after 1 month in the same plate, indicating that these cells have the ability to form tubules in the presence of sarcoma 180-conditioned medium.

Ciliogenesis and Mucus Synthesis in Cultured Human Respiratory Epithelial Cells

The mechanisms for the regulation of ciliogenesis and for the synthesis of mucus are not well understood. We sought to develop a culture system for differentiating ciliated and secretory types of human respiratory epithelial (HRE) cells. Dissociated HRE cells obtained from nasal polyps and maxillary sinus mucosa were cultured on type I collagen gel. Cells grown to confluence on collagen gel lost their cilia and exhibited a flat, squamouslike appearance. After reaching confluence, the cultured cells with a collagen gel substrate were removed from plastic dishes and floated in the culture medium. After 7 days in the floating culture, some cells exhibited several centrioles or basal bodies, while others showed secretory granules. The secretory phenotype predominated after 7 days. After 14 days in the floating culture, nearly all cells were ciliated. The results demonstrate that the differentiation of HRE cells can be induced by floating cultured cells with a collagen gel substrate in a defined culture medium.

Ciliary Beat of Cultured Human Respiratory Cells Studied with Differential Interference Microscope and High Speed Video System

The ciliary beating of upper respiratory tract cells cultured on cover glasses was studied by using differential interference microscope equipped with high speed video. By culturing the cells on collagen coated cover glasses, objectives with higher magnifications could be used. With this system we could evaluate not only ciliary beat frequency, but also amplitude, wave form, orientation and synchrony of ciliary beating. Also some structural anomalies such as compound cilia and tide cilia bundles could be recognized. Ciliary beat frequency measured from 1,026 ciliated cells was 20.6 +/- 4.7 Hz (mean +/- SD). The orientation of ciliary beat directions was random and the mean standard deviation for measured angles was 73.0 degrees +/- 28.9 degrees (mean SD +/- SD). When the ciliary beat frequency was 20 Hz, the time used for effective phase was 0.022 +/- 0.002 s (mean +/- SD), and 0.028 +/- 0.004 s (mean +/- SD) for the recovery phase of beat. This system is advantageous for studying ciliary function because all parts can be studied simultaneously with higher magnification, and the effects of chemical physical mediators can be studied without disturbing effects of the autonomic nervous system or secretory cells. Also, the same cells could be observed before and after challenge with test medication and thus evaluated more accurately.

Degeneration of human respiratory cell ciliary beat in monolayer cell cultures

SummaryThe degeneration of ciliary beat of human respiratory cells was studied in monolayer cell cultures by using a differential interference microscope equipped with a high speed video system. This method for studying ciliary beat in cell cultures on collagen-coated cover glasses is quite advantageous, because it allows for detailed study of all parts of ciliary function and not just ciliary beat frequency (CBF). In the present study both CBF and ciliary beat amplitude (CBA) were found to decrease continuously from the 1st day after plating but the wave form of ciliary beat did not change. Cultures with high cell density provided better preservation of normal ciliary beat for a longer period. In contrast, ciliary beat degenerated quickly in cultures with low cell density. CBF and CBA in cell cultures less than 5 days after plating were always high, supporting use of these cultures for studies of normal ciliary motility.

Effect of Exogenous ATP on Ciliary Beat of Human Ciliated Cells Studied with Differential Interference Microscope Equipped with High Speed Video

To study the detailed effects of exogenous adenosine triphosphate (ATP) on ciliary function, we used the differential interference microscope equipped with high speed video and evaluated ciliated cells from the human sinus mucosa in monolayer culture. With this system it was possible to evaluate all parts of ciliary motility with a minimum of interference from the mucous membrane, secretory cells and the autonomic nervous system. The best direct ciliostimulative effect of exogenous ATP on ciliary beat frequency (CBF) and ciliary beat amplitude (CBA) was observed at concentrations of ATP ranging from 10(-5) M to 10(-3) M. Exogenous ATP appeared to normalize the slightly damaged ciliary motility in groups with an initial CBF of 14 Hz or higher. When the CBF was less than 14 Hz, ATP produced an increase in CBF greater than 40%, an increase in CBA greater than 30%, but these did not reach the normal level in 5 min. The biggest increases: 59.9% in CBF and 40.7% in CBA were seen in the group with an initial CBF less than 9 Hz. In the cells with a low initial CBF and unsynchronized motility exogenous ATP increased the synchrony of ciliary movement together with an increase in CBF and CBA.

Effects of free radicals on ciliary movement in the human nasal epithelial cells

OBJECTIVE There have been few reports on the effects of free oxygen radicals on ciliary mobility of nasal respiratory epithelial cells. The aim of this study was to determine the effects of free radicals and antioxidants on human nasal epithelial cells (HNECs) using video-computerized analysis. METHODS Human nasal epithelial cells were obtained from the nasal cavity of normal volunteers. Ciliary beat frequency (CBF) was calculated as the mean value of ten randomly selected cells. The proportion of the area with normal CBF (above 8 Hz) was calculated from 10 randomly selected sites per specimen. Free radicals were produced by xanthine-xanthine oxidase enzymatic system. The generation of free radicals was confirmed by chemoilluminometer. CBF and the proportion of the area with normal CBF were measured at every 5 min for 30 min after the addition of enzyme. For the evaluation of the antioxidant effects on free radical-mediated ciliary slowing in HNECs, cells were incubated in superoxide dismutase solution (300 unit/ml) for 30 min and 3-aminobenzamide (5 mM). RESULTS Superoxide produced by 0.4 mM xanthine and 400 miliunit/ml xanthine oxidase decreased CBF (7.71 +/- 1.91 Hz). A total of 2 min later, ciliary slowing was evident (3.87 +/- 1.10 Hz). Regarding the changes in proportion of epithelial area that showed normal CBF experimental group showed a significant decrease in percentage of epithelial area with normal CBF over time. Superoxide dismutase prevented ciliary slowing (8.76 +/- 0.99 Hz). Moreover, 3-aminobenzamide, an inhibitor of the DNA repair enzyme poly-ADP ribose polymerase, prevented inhibition of CBF (8.32 +/- 0.61 Hz). CONCLUSIONS These results suggest that oxygen-mediated damage to DNA may be the mechanism of the deterioration effects of oxygen radicals on the ciliated respiratory nasal epithelium.

Retention fluids of chronic sinusitis induce neutrophil adherence to microvascular endothelial cells

The adherence of circulating leukocytes to the vascular endothelium is a critical step in the emigration of leukocytes through blood vessel walls to inflammatory lesions. The influence of nasal secretions on the adherence of neutrophils to the vascular endothelium was investigated using monolayers of human mucosal microvascular endothelial cells derived from the inferior turbinate. Preincubation of vascular endothelial cells with retention fluids from the maxillary sinus of the patients with chronic sinusitis showed increased neutrophil adherence. Recombinant IL-1 beta was also tested and found to induce adherence of neutrophils to human mucosal microvascular endothelial cells. However, no adhesive effect was observed with the nasal secretions of nasal allergy. An enzyme-linked immunosorbent assay detected considerable amounts of IL-1 beta in the chronic sinusitis retention fluids, while the amounts of IL-1 alpha and TNF-alpha were very low. The increased adhesion of the neutrophils by the retention fluids of chronic sinusitis was also neutralized by the incubation with anti-IL-1 beta antibody in a dose dependent manner. These findings suggest that IL-1 beta in the paranasal secretion of chronic sinusitis induces the adherence of neutrophils to vascular endothelium and subsequent infiltration of neutrophils in the paranasal sinuses, thus contributing to the persistence of chronic sinusitis.