Transcription factor DEC1 is required for maximal experimentally induced periodontal inflammation.

BACKGROUND AND OBJECTIVES: Disruption of transcriptional regulation is a confounding factor associated with a wide range of human inflammatory diseases. To investigate mechanistic links between transcription factor DEC1 and pathways underlying inflammation, wild-type and DEC1 knockout (KO) C57BL/6 mice were treated with Porphyromonas gingivalis (or carboxymethyl cellulose as a control) to induce periodontal inflammation. It provoked an inflammatory response within the oral environment, which showed robust variation in alveolar bone resorption and expression of inflammatory cytokines. MATERIAL AND METHODS: Male DEC1KO mice and their wild-type littermates were used for the experimental periodontitis model. Measurement of alveolar bone resorption, micro-computed tomography, isolation of gingival mononuclear cells (GMCs), flow cytometry and immunohistochemical analysis were used in this study. Human gingival fibroblast cells (HGF-1) were used for DEC1 over-expression and short interference RNA (siRNA) studies and quantitative real-time polymerase chain reaction and western blot analysis were performed. RESULTS: Micro-computed tomography analysis demonstrated that P. gingivalis caused a decrease in bone area of wild-type mice compared with DEC1KO mice. Expression of inflammatory and immune markers in GMCs was significantly decreased in DEC1KO mice after treatment with P. gingivalis. Conversely, interleukin (IL)-4 and IL-10 mRNAs were significantly increased in GMCs isolated from DEC1KO mice. The results show that treatment of DEC1KO mice with P. gingivalis decreased the numbers of CD11b+ F4/80+ and CD4+ RANKL+ T cells. Moreover, expression of CD4, F4/80, RANKL and cathepsin K in inflammatory cell infiltrates was significantly reduced in DEC1KO mice treated with P. gingivalis compared with controls. Furthermore, over-expression of DEC1 in HGF-1 cells increased the expression of IL-1ß and tumor necrosis factor-α mRNAs and their expression levels reached a maximum in response to treatment with lipopolysaccharide. Inhibition of DEC1 by short interference RNA interference suppressed the P. gingivalis-derived lipopolysaccharide-induced expression of IL-1ß, tumor necrosis factor-α and toll-like receptor4. CONCLUSION: These results suggest that transcription factor DEC1 can modulate P. gingivalis-induced periodontitis in the oral mucosa.

Quantitative assessment of provability of radiation-related cancers considering unavoidable existence of unadjusted risk factors.

The attribution of stochastic effects to exposure to ionizing radiation has been qualitatively discussed by introducing two distinct concepts of provability and probability. This study aims to develop a method of quantitatively assessing the provability of radiation-related cancers. To this end, the 'minimum provable dose' (MPD) was developed and applied to actual cancer mortality in Japan. The background lifetime risk of cancer mortality was calculated for the esophagus, stomach, colon, liver, lungs, skin, breasts, ovaries, bladder, and bone marrow as well as the age-specific risk coefficients reproducing those given in the 2007 Recommendations of the International Commission on Radiological Protection (ICRP). Comparing the relative ratio of MPDs, which was defined herein as the 'provability index' (PI), we quantitatively ranked radiation-related cancers for different tissues and organs predicated on provability for ages of 10, 30, 50, and 0-85+ years at exposure. We discuss the radiological protection of male emergency workers focusing on cancers highly prioritized according to the ranking (i.e. colon, bone marrow, and bladder). The present study proposed the system to quantitatively evaluate the level of radiological protection taking into account the variations of the background cancer risk on the provability of radiation-related cancers.

Potential Effects of Climate Change on Ecological Interaction Outcomes Between Two Disease-Vector Mosquitoes: A Mesocosm Experimental Study.

The objective of this study was to experimentally assess the effects of different climate change scenarios on the outcomes of interactions between Aedes aegypti (L.) and Culex quinquefasciatus (Say) (Diptera: Culicidae) larvae. The experimental design maintained a constant density of specimens while the proportion of the species in different experimental climate change scenarios varied. Our results indicate that survival of the two species was not affected, but larval development and pupation times decreased under elevated atmospheric CO(2) concentration and high air temperature. In climate change scenarios with both species together, the survival of Ae. aegypti increased and its larval development time decreased with increasing density of Cx. quinquefasciatus. This may be attributed to the effects of intraspecific competition being more significant than interspecific competition in Ae. aegypti. Our study also reveals that climatic changes may affect the patterns of interactions between Cx. quinquefasciatus and Ae. aegypti. Alterations in climatic conditions changed the response of context-dependent competition, indicating the importance of studies on how ecological interactions will be affected by projected future climatic change.

Performance of a rapid human metapneumovirus antigen test during an outbreak in a long-term care facility.

Using a newly developed rapid test, an outbreak of human metapneumovirus (HMPV) infection in a long-term care facility was detected within only 2 days after the onset of symptoms in a putative index case. The outbreak was almost under control within 8 days mainly by zoning patients, with the exception of two cases of HMPV that were diagnosed 16 and 17 days after the onset of the outbreak. According to an immunological diagnosis as well as the rapid test, it was eventually proven that 18 patients had HMPV infections. We suspected that even asymptomatic residents, who had not been completely separated from the facility population, were a source of infection. That suggested that all asymptomatic residents should be tested and that the separation of the infected patients should be absolute, if an outbreak of HMPV infection is suspected in such a facility.

Influence of calcium channel blockers in patients with gastrointestinal disease in Japanese community pharmacies.

WHAT IS KNOWN AND OBJECTIVE: Calcium channel blockers (CCBs), which have been widely used for the treatment of hypertension and angina pectoris, decrease lower oesophageal sphincter pressure and, as a result, can exacerbate gastrointestinal disease. In a previous study, increased risk of exacerbation of gastrointestinal disease among elderly patients following treatment with CCBs was identified. The prevalence of gastrointestinal diseases has increased in elderly patients, and it is possible that treatment with CCBs may have undesirably influenced this increase. The change in risk of gastrointestinal disease can be estimated by analysing changes in the prescription of antisecretory drugs as an outcome of exacerbation of gastrointestinal disease caused by CCBs. METHODS: It was hypothesized that patients who were prescribed CCBs would also change their use of antisecretory drugs. From September 2005 to August 2009, a dynamic retrospective cohort study was performed at five community pharmacies in Nagasaki, Japan, to assess alteration of antisecretory drug therapy following treatment with CCBs. Correlations with alterations of antisecretory drug therapy were determined by the Cox proportional hazards model. RESULTS AND DISCUSSION: The proposed study included 260 patients who were prescribed CCBs and 155 controls. During the study period, 53 patients were prescribed CCBs and 13 controls altered their antisecretory drug therapy; the hazard ratio was 2·22 (95% CI 1·25-4·26). WHAT IS NEW AND CONCLUSION: Calcium channel blocker treatment of patients with gastrointestinal disease was associated with alteration in frequency of prescription and an increase in dosage of antisecretory drugs. For clinical management of hypertension, alternative antihypertensive drugs may be considered for patients with gastrointestinal diseases. Further studies are required to determine the influence of CCB therapy on gastroesophageal diseases, suggested by the increase in use of antisecretory drugs.

How Habitat Filtering Can Affect Taxonomic and Functional Composition of Aquatic Insect Communities in Small Amazonian Streams.

Responses in taxonomic and functional composition of communities were analysed in small Amazonian streams at the small and large scale (habitat patches, river segment scale, and catchment scale). We hypothesised that similar responses in community structure to local environmental factors were a correlation between taxonomic and functional composition. To evaluate the response of taxonomic composition to environmental variables, redundancy analysis (RDA) and RLQ analysis were performed to investigate the response of community abundance (L) as a function of the environment (R) and traits (Q). The fourth-corner analysis was applied to summarize specific interactions between environmental variables and traits. Then, community taxonomic composition was associated with models at multiple scales of habitat (i.e. riparian/channel, substrates, and water variables). Likewise, the fourth-corner tests and RLQ axes showed associations between trait composition and environmental variables related to variables, such as riparian cover and channel morphology followed by variation in substrate size and composition. Unexpectedly, these results did not show specific associations between unique environmental variables and traits. At last, results showed that local conditions of stream habitat regulated community structure and functional composition of aquatic insects. Thus, these findings indicate that the local environmental filtering appears to be strongly associated with selected species traits adapted to occur in a range of habitat conditions. Despite the low number of analysed streams, these results provide important information for understanding the simultaneous variation in functional trait composition and community composition of aquatic insect assemblages.

No evidence for DNA and early cytogenetic damage in bystander cells after heavy-ion microirradiation at two facilities.

The occurrence of bystander effects has challenged the evaluation of risk for heavy ions, mainly in the context of space exploration and the increasing application of carbon ions in radiotherapy. In the present study, we addressed whether heavy-ion-induced DNA and cytogenetic damage is detectable in bystander cells. The formation of gamma-H2AX foci, sister chromatid exchanges and micronuclei were used as markers of damage to DNA. Normal human fibroblasts were exposed to low fluences of carbon and uranium ions, and alternatively single cells were targeted with heavy ions using the GSI microbeam. We did not observe a significant increase in the bystander formation of gamma-H2AX foci, sister chromatid exchanges or micronuclei. In addition, we performed for the first time parallel experiments at two microbeam facilities (GSI, JAEA) using the same cell line, culture conditions and irradiation protocols. No significant enhancement of the micronucleus frequencies in bystander cells was detected after targeted carbon-ion irradiation, confirming the results. Details regarding the history, culture conditions or support of the cells might be affecting the detection of bystander effects. On the other hand, the potential X-ray- and heavy-ion-induced bystander effects investigated herein clearly do not exceed the experimental error and thus are either lacking or are less pronounced than the effects reported in the literature for similar end points after alpha-particle and X-ray exposure.

Cervical sympathectomy causes alveolar bone loss in an experimental rat model.

BACKGROUND AND OBJECTIVE: Periodontal disease, a pathological destructive inflammatory condition, is characterized by alveolar bone loss. Recent studies have suggested a correlation between the sympathetic nervous system and bone remodeling. To confirm the importance of the sympathetic nervous system in bone resorption, we investigated the effects of superior cervical ganglionectomy and oral challenge with Porphyromonas gingivalis on alveolar bone loss in rats. MATERIAL AND METHODS: Rats were divided into three groups: group A underwent a sham operation as the control group; group B underwent superior cervical ganglionectomy; and group C underwent a sham operation and oral challenge with P. gingivalis. Horizontal alveolar bone loss was evaluated by measuring the distance between the cemento-enamel junction and the alveolar bone crest. Cytokine gene expression in the gingival tissues was assessed using reverse transcription-polymerase chain reaction analyses. The furcation areas of the mandibular molars were examined histologically. RESULTS: Both superior cervical ganglionectomy and oral challenge with P. gingivalis resulted in accelerated alveolar bone loss. Gingival tissues in the superior cervical ganglionectomy group showed increased expression of the cytokines interleukin-1 alpha, tumor necrosis factor-alpha and interleukin-6. The density of neuropeptide Y-immunoreactive fibers was decreased following superior cervical ganglionectomy. Osteoclasts were observed in the superior cervical ganglionectomy and P. gingivalis-challenged groups. CONCLUSION: Both superior cervical ganglionectomy and oral challenge with P. gingivalis induced alveolar bone loss. These results provide new information on the occurrence of alveolar bone loss, in that both oral challenge with P. gingivalis and superior cervical ganglionectomy are important accelerating factors for alveolar bone loss. Thus, we suggest that the sympathetic nervous system is linked with the prevention of alveolar bone loss.

Nosocomial outbreak of epidemic keratoconjunctivitis accompanying environmental contamination with adenoviruses.

An outbreak of acute keratoconjunctivitis involving 27 patients occurred in the Department of Ophthalmology, Kurume University Hospital. Adenoviral DNA was detected in four inpatients, one outpatient and one healthcare worker. Sequence-based typing of adenoviral DNA indicated serotype 3 from one inpatient, the rest being serotype 37. At a later stage of the outbreak adenoviral DNA types 37 and/or 3 were also detected from almost all environmental instruments and commonly used eye drops, despite thorough disinfection of the environment and enforcement of various infection control measures. The detection rate of adenoviral DNA in environmental swabs was 81%. A further second disinfection of the environment reduced the detection rate of adenoviral DNA to 38%. The outbreak ceased after closing the ophthalmology ward and outpatient consulting room, accompanied by enhanced cleaning of environmental instruments and the introduction of disposable eye drops for individual patients.

Increased risk of exacerbating gastrointestinal disease among elderly patients following treatment with calcium channel blockers.

BACKGROUND AND OBJECTIVE: Calcium channel blockers (CCBs) have been widely used for the treatment of hypertension and angina pectoris. It is presumed that CCBs decrease the lower esophageal sphincter pressure and as a result, the risk of gastrointestinal disease may be increased. Since the prevalence of gastrointestinal diseases has increased in elderly patients, it is possible that treatment with CCBs may have contributed to this increase. Therefore, we considered that the risk of exacerbating gastrointestinal disease among elderly patients by CCBs can be estimated by using the prescription ratio of antisecretory drugs as an outcome. METHOD: We hypothesized that patients who are prescribed CCBs would increase the use of antisecretory drugs involving H(2)-receptor antagonists and proton pump inhibitors (PPIs). From January 2001 to December 2005, a dynamic retrospective cohort study was performed at three community pharmacies in Nagasaki city, Japan, to assess the use of antisecretory drugs following treatment with CCBs among elderly patients. The correlation of initiation of antisecretory drugs treatment to maintenance therapy with PPIs was determined by the Cox proportional hazards model. RESULTS: The proposed study includes 303 patients prescribed CCBs and 258 controls. During the study period, 138 patients prescribed CCBs and 66 controls were initiated by giving antisecretory drugs; the hazard ratio was 1.40 (95% confidence interval 1.21-1.63). Eighty two patients taking CCBs and 32 controls were initiated by the maintenance therapy with PPIs; the hazard ratio was 1.48 (95% confidence interval 1.21-1.83). CONCLUSION: Patients who simultaneously initiated the use of antisecretory drugs with CCBs could not be found. Therefore, antisecretory drugs have not been used to prevent the gastrointestinal diseases caused by CCBs. The results obtained in this study suggest that the risk of gastrointestinal disease could be increased by long-term treatment with CCBs for elderly patients.

Characterization and isolation of thyroid microsomal antigen.

We investigated the structure of the 107-kD thyroid protein recognized as microsomal antigen. Solubilized microsomes were incubated with affinity gels consisting of IgG, from thyroiditis patients or controls, linked to Reacti-gel. Eluates were analyzed by SDS polyacrylamide electrophoresis and Western blot. 107- and 101-kD proteins were augmented in eluates from gels containing patient IgG and had microsomal antigenicity. In a Western blot of microsomes run under unreduced conditions, poorly defined large proteins were identified by antibody. When eluted electrophoretically and reanalyzed in reducing conditions, they demonstrated the 107-kD antigen. The 107-kD protein identified in reducing conditions was extracted and reanalyzed under nonreducing conditions. Large molecular mass proteins were then observed. On two-dimensional electrophoresis, a 107-kD antigen was isolated with isoelectric point of 7.0. The microsomal antigen may be complexes or multimers of a 107-kD peptide with isoelectric point of 7.0.

Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies.

MAb directed to the thyroid microsomal antigen have been developed. All bound to 101- and 107-kD bands in Western blot analysis using thyroid microsomal fraction as antigen. The MAb also bound to microsomal proteins immunoprecipitated by serum from patients having a high titer of anti-microsomal antibody but no antibodies to thyroglobulin or thyrotropin-stimulating hormone receptor. The pattern of binding was related to the amount of reducing agent. The 101- and 107-kD bands were increased by addition of dithiothreitol whereas, in its absence, numerous bands of higher molecular weight were present, suggesting a multimeric protein structure. Despite the inability to immunoprecipitate thyroid peroxidase (TPO) enzymatic activity, the MAb bound intensively in Western blot to denatured purified hog TPO and to denatured immunopurified human TPO. Trypsin digestion of the 101-107-kD antigen produced a doublet of 84-88 kD that was still immunoreactive with MAb. One of five polyclonal sera tested (with a microsomal antibody titer greater than 1/20,480 measured by the tanned red cell hemagglutination technique) also recognized the 84-88 kD trypsin fragments. Addition of V8 protease led to a disappearance of the 107-kD protein, but not the 101-kD protein, proving that this antigen is formed by two different polypeptides. The MAb bound strongly to thyroid epithelium, whereas binding to papillary carcinoma was absent or low and moderate for follicular and Hurthle cell carcinoma. This study indicates that the thyroid microsomal antigen and TPO are identical and are constituted of two different polypeptides. On SDS-PAGE the antigen appears as two contiguous bands which share common epitopes but are not identical, as proven by their size and difference in susceptibility to proteolytic digestion. The immunoreactivity of the molecule is highly dependent on a trypsin-sensitive site, which appears important in the recognition of the antigen by polyclonal sera and may have biological importance. The expression of microsomal antigenicity is variable among various thyroid carcinomas.

Thermoelectric power calculation by the Boltzmann equation: Na(x)CoO(2).

A full-potential augmented-plane-wave (FLAPW) band-structure calculation in the local density approximation (LDA) was carried out for hexagonal Na(x)CoO(2) (x = 0.45, 0.55, 0.66 and 0.75). The Seebeck tensor was estimated by the Boltzmann theory, assuming that the relaxation time is constant on the Fermi surface. The Seebeck tensor is extremely anisotropic; the c-axis Seebeck coefficient varies dramatically with the Na content. The calculation reproduces the experiment semiquantitatively.

Residue level of polycyclic aromatic hydrocarbons in Japanese paddy soils from 1959 to 2002.

The residue level of 21 polycyclic aromatic hydrocarbons (PAHs) and the temporal changes in this level were investigated in paddy soils collected from particular experimental sites in Japan from 1959 to 2002. The average total PAH concentration in all the samples was 496 microg kg(-1), and it ranged from 52.9 to 2180 microg kg(-1). The residue level of the PAHs was the highest during the 1960s, rapidly decreased during the 1970s, and remained almost constant thereafter. Relatively high PAH concentrations were observed in soils from areas that experienced heavy snowfall and that had relatively low air temperature. The predominant PAHs were phenanthrene, fluoranthene, naphthalene, and pyrene, and their concentration overall and in relation to that of the total PAHs decreased each year since the 1960s. Similarities in the PAH profiles among the locations were determined using the concentration correlation matrix and cluster analysis, and ratios of the levels of specific PAH pairs were also calculated to determine their origin. The collected data suggested that the origins of soil PAHs changed chronologically from the burning of agricultural wastes such as stubble before the mid-1970s to the combustion of fossil fuel and its secondary products after the mid-1970s.

Effects of an oil spill and discharge of domestic sewage on the insect fauna of Cururu stream, Manaus, AM, Brazil.

Assemblages of aquatic and edaphic insects in three streams in Central Amazonia were used to evaluate the impact on one of the streams (Cururu) caused by an oil spill that occurred in August 1999. The Cururu stream had already been impacted by domestic sewage. These three streams were evaluated during four different periods of the regional hydrological cycle (falling-, low-, rising- and high-water), from September 2000 to May 2001. An Eckman dredge, an aquatic entomological net and a manual auger were used to collect insects, which were used to estimate the taxonomic richness (at the family level) and insect density in each stream. Anoxic and eutrophic conditions were determined in each stream based on the mean concentration of dissolved oxygen, total nitrogen and phosphorus. Richness and abundance of insect fauna were higher in the Cristalino stream (not impacted) than in the Bom Jardim stream (impacted by domestic sewage) and in the Cururu stream, with the exception of samples collected along the stream banks (littoral fauna). The mean dissolved oxygen was lower in the Cururu stream than in the other two, while the mean concentrations of total nitrogen and phosphorus were higher. These elements showed intermediate values in the Bom Jardim stream, while the opposite pattern was observed in the Cristalino stream, with higher concentrations of dissolved oxygen and lower concentrations of total nitrogen and phosphorus. The concentrations of these variables reflect the modifications resulting from anthropogenic eutrophication caused by the discharge of domestic sewage into Bom Jardim and Cururu and by the oil spill in Cururu, which negatively affected the richness and abundance of insects in these streams.

Dietary restriction permits normal parturition and lactation but suppresses mouse mammary tumor virus proviral transcription even after mammary involution.

Dietary restriction of C3H/Ou mice prevents development of spontaneous mammary adenocarcinoma by suppressing mammary expression of the mouse mammary tumor virus (MMTV) via a mechanism which may involve prolactin. In the present study, dietary restriction of 40% was imposed for 16 weeks on nulliparous C3H/Ou mice, interrupted by ad libitum consumption at mating and continued only during pregnancy and lactation, with 40% energy restriction reimposed at the end of lactation. The results show that mammary MMTV mRNA expression levels of chronic energy intake restricted (CEIR) mice and ad libitum fed mice are similar and elevated during early lactation, when all mice of both groups are being fed ad libitum energy levels. In spite of this, and in marked contrast, when CEIR dams are returned to 40% dietary restriction following the weaning of litters, mammary MMTV transcription is suppressed to levels 4-5-fold less than those measured in mammary glands from ad libitum fed controls. Within the 38 weeks of study, 73% of ad libitum fed uniparous mice at risk and 11% of CEIR uniparous mice at risk developed mammary tumors, yet mice of both dietary groups delivered and weaned healthy litters with comparable efficiency. When dietary restriction is maintained in CEIR mice during pregnancy and lactation, efficiency of conception and litter size are reduced, and MMTV transcription is suppressed even during lactation. Mean serum prolactin levels were not significantly different among dietary groups. These findings show that the level of MMTV transcription is rigorously influenced by dietary energy level, and that 40% dietary restriction of C3H/Ou mice not only suppresses mammary MMTV transcription and prevents mammary tumor development in uniparous mice, but also permits normal conception, gestation, lactation, and the production of healthy litters as long as the nutritional demands of gestation and lactation are met.

A new species of Enderleina Jewett (Plecoptera, Perlidae) from Amazonas State, Brazil.

Enderleina Jewett, 1960 is one of the least-known Perlidae genera in northern South America, including six species. During an aquatic insect survey of the northern portion of Amazonas State, Brazil, we collected a male Enderleina with a distinct pronotum pattern and genitalic morphology. The objectives of this study are to describe a new species of Enderleina based on male morphology. The new species can be distinguished from all other Enderleina species in the male by the presence of a paired thin dark band on the mesal region of pronotum and by the structure of the penial armature.

The p53-Mdm2 association in epithelial cells in idiopathic pulmonary fibrosis and non-specific interstitial pneumonia.

BACKGROUND: Wild-type p53 is increased during cellular responses to various stresses. Mdm2, which is induced by p53, regulates p53 protein concentrations through the ubiquitin-proteasome pathway. AIM: To investigate whether the Mdm2 mediated ubiquitination of p53 is associated with epithelial cell apoptosis in idiopathic pulmonary fibrosis (IPF). METHODS: Immunohistochemistry and western blot analysis were carried out on lung samples obtained by lung biopsy from patients with IPF and non-specific interstitial pneumonia (NSIP). RESULTS: The expression of p53, phosphorylated p53, Mdm2, p21, and Bax was upregulated in epithelial cells from patients with IPF and NSIP compared with normal lung parenchyma. Except for p21, there was a significant increase in the expression of these factors in IPF compared with NSIP. In addition, the number of apoptotic cells and the number of p53 and Bax positive cells was increased compared with controls. p53 conjugated with Mdm2 was decreased in IPF compared with NSIP and controls. Ubiquitinated p53 was increased in both IPF and NSIP compared with controls. CONCLUSIONS: Signalling molecules associated with p53 mediated apoptosis may participate in epithelial cell apoptosis, and the attenuation of p53-Mdm2 conjugation and of p53 degradation may be involved in the epithelial cell apoptosis seen in IPF. Augmented epithelial apoptosis in IPF may lead to the poor prognosis compared with NSIP.

Expression of fas (CD95/APO-1) antigen induced by radiation therapy for diffuse B-cell lymphoma: immunohistochemical study.

Most malignant lymphomas show relatively high degrees of radiosensitivity, in which apoptosis has been shown to play an important role. Recently, the Fas (CD95/APO-1)/Fas ligand system has been identified as a key regulator of apoptosis in some types of lymphoma cell lines. In this study, we aimed to determine whether Fas antigen expression is induced by radiotherapy for malignant lymphoma and to clarify its possible correlation with the therapeutic effect of radiation therapy. Fifty-six patients with tumors of the tongue, oropharynx, and maxillary sinus were examined; four were confirmed as malignant lymphoma, and the rest were identified as squamous cell carcinoma. After obtaining the patients' informed consent, biopsies were performed before treatment and at doses of 4, 10, and 20 Gy of radiotherapy, and specimens were preserved in liquid nitrogen until further examination. Serial sectioning of 6 micrometer was performed using a cryostat, and samples were immunohistochemically stained using the streptoavidin-biotin peroxidase method and a monoclonal antibody against Fas. Two of the four patients with malignant lymphoma showed Fas antigen expression on their tumor tissue at 4 and 10 Gy of radiotherapy. These tumors showed high radiosensitivity and disappeared at a dose of 20 Gy of radiotherapy. In samples from these two patients, DNA ladder formation was identified at 10 Gy. In 52 squamous cell carcinomas, staining for the Fas antigen showed negative or only slightly positive results. However, in one of the cases of squamous cell carcinoma, lymphocytes infiltrating into cancer tissue showed Fas antigen expression at 4 Gy of irradiation, and these lymphocytes disappeared on the tumor tissue at 10 Gy. Therefore, the high radiosensitivity of malignant lymphoma among our samples could be explained by the overexpression of Fas antigen induced by small doses of radiation therapy, and Fas ligand could be produced by infiltrating lymphocytes or may be expressed simultaneously on the lymphoma cells.

Immunohistochemical study of c-fos-positive lymphocytes infiltrated into human squamous cell carcinomas of the head and neck during radiation therapy and its clinical significance.

C-fos has been reported to be one of the immediate early genes in signal transduction systems after many kinds of stresses, including ionizing radiation. Changes in c-fos expression induced by radiation therapy in tumor tissues have not yet been reported. In this study, we have attempted to determine whether c-fos expression is induced by radiotherapy in human squamous cell carcinomas of the head and neck and to establish a possible correlation between c-fos expression and the therapeutic effects of radiation therapy. Twenty-seven patients with tumors of the oral cavity, oropharynx, and maxillary sinus were examined, all of which were confirmed as squamous cell carcinomas. After obtaining the patients' informed consent, biopsies were performed before treatment and at doses of 4, 10, and 20 Gy of radiotherapy, and the specimens were preserved in liquid nitrogen for further examination. Serial sectioning of 6 micrometer was performed using a cryostat, and samples were immunohistochemically stained using the streptoavidin-biotin peroxidase method and a monoclonal antibody against c-fos. Three of the 27 patients with squamous cell carcinoma showed slight expression of c-fos in their tumor cells before and/or at 4 or 10 Gy of radiotherapy. The tumors showed high radiosensitivity. Concerning tumor-infiltrating lymphocytes, the rate of moderate or remarkable grades of c-fos-positive lymphocytes before radiotherapy and at radiation doses of 4, 10, and 20 Gy was 8.0, 29.2, 4.8, and 0%, respectively. The relationship between the immunohistochemical findings and the antitumor effect at a radiation dose of 20 Gy was examined on the corresponding H&E-stained sections. In patients whose infiltration of c-fos-positive lymphocytes into tumor tissues were moderate or remarkable at 4 Gy of radiotherapy, the tumors responded significantly well to radiation therapy (P < 0.025, chi2 test), and the patients took a significantly favorable clinical course (P < 0.05, chi2 test). In a sample from one of the patients, c-fos-positive lymphocytes were identified as CD4 positive and CD8 negative. Therefore, the high radiosensitivity of squamous cell carcinomas in our samples could be explained by an overexpression of c-fos in the tumor-infiltrating lymphocytes induced by small doses of radiation therapy, and these activated lymphocytes exerted a cytotoxic effect against the cancer cells.