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Abstract Background In order to elucidate tumoral progression and drug resistance, cultured cell lines are valuable tools applied on tumor related assays provided they are well established and characterized. Our laboratory settled the NG97 cell line derived from a human astrocytoma grade III, which started to develop and express important phenotypical characteristics of an astrocytoma grade IV after injection in the flank of nude mice. Astrocytomas are extremely aggressive malignancies of the Central Nervous System (CNS) and account for 46% of all primary malignant brain tumors. Progression to worse prognosis occurs in 85% of the cases possibly due to changes in cell tumor microenvironment and through biological pathways that are still unclear. Methods This work focused on characterizing the NG97 cell line specifically after being recovered from the xenotransplant, who maintained their undifferentiated characteristics along the following 60th passages in vitro. These cells were subcultivated to evaluate the possible contribution of these undifferentiated characteristics to the malignant progression phenotype. These characteristics were the expression of molecules involved in the processes of migration, dedifferentiation and chromosomal instability. Results Results showed that NG97(ht) had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP) and vimentin. In addition, β1 integrins were present in intermediate levels while α5 integrins had a high expression profile as well as fibronectin and laminin. Cytogenetic analysis of NG97(ht) revealed several chromosomal abnormalities, 89% of the cells showed to be hyperdiploid and the modal number was assigned to be 63. Several acrocentric chromosomes were visualized and at least 30 figures were attributed to be murine. These findings suggest a possible fusion between the original NG97 cells with stromal murine cells in the xenotransplant. Conclusion In this study the NG97(ht) cells were characterized to embryonic recovery patterns of intermediate filaments, adhesion molecules expression, chromosomal imbalances and murine chromosomes. In the latter case, these presumably chromosomes were originated as fusions between murine stroma cells and NG97 cell lineage in the xenotransplant. Our results emphasize important queries about astrocytomas tumor progression.

The fibrotic tumor microenvironment is a pivotal therapeutic target. Nintedanib, a clinically approved multikinase antifibrotic inhibitor, is effective against lung adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). Previous studies have implicated the secretome of tumor-associated fibroblasts (TAFs) in the selective effects of nintedanib in ADC, but the driving factor(s) remained unidentified. Here we examined the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a tumor-promoting cytokine overproduced in ADC-TAFs. To this aim, we combined genetic approaches with in vitro and in vivo preclinical models based on patient-derived TAFs. Nintedanib reduced TIMP-1 production more efficiently in ADC-TAFs than SCC-TAFs through a SMAD3-dependent mechanism. Cell culture experiments indicated that silencing TIMP1 in ADC-TAFs abolished the therapeutic effects of nintedanib on cancer cell growth and invasion, which were otherwise enhanced by the TAF secretome. Consistently, co-injecting ADC cells with TIMP1-knockdown ADC-TAFs into immunocompromised mice elicited a less effective reduction of tumor growth and invasion under nintedanib treatment compared to tumors bearing unmodified fibroblasts. Our results unveil a key mechanism underlying the selective mode of action of nintedanib in ADC based on the excessive production of TIMP-1 in ADC-TAFs. We further pinpoint reduced SMAD3 expression and consequent limited TIMP-1 production in SCC-TAFs as key for the resistance of SCC to nintedanib. These observations strongly support the emerging role of TIMP-1 as a critical regulator of therapy response in solid tumors., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: The TGF-β1 transcription factor SMAD3 is epigenetically repressed in tumour-associated fibroblasts (TAFs) from lung squamous cell carcinoma (SCC) but not adenocarcinoma (ADC) patients, which elicits a compensatory increase in SMAD2 that renders SCC-TAFs less fibrotic. Here we examined the effects of altered SMAD2/3 in fibroblast migration and its impact on the desmoplastic stroma formation in lung cancer., Methods: We used a microfluidic device to examine descriptors of early protrusions and subsequent migration in 3D collagen gels upon knocking down SMAD2 or SMAD3 by shRNA in control fibroblasts and TAFs., Results: High SMAD3 conditions as in shSMAD2 fibroblasts and ADC-TAFs exhibited a migratory advantage in terms of protrusions (fewer and longer) and migration (faster and more directional) selectively without TGF-β1 along with Erk1/2 hyperactivation. This enhanced migration was abrogated by TGF-β1 as well as low glucose medium and the MEK inhibitor Trametinib. In contrast, high SMAD2 fibroblasts were poorly responsive to TGF-β1, high glucose and Trametinib, exhibiting impaired migration in all conditions., Conclusions: The basal migration advantage of high SMAD3 fibroblasts provides a straightforward mechanism underlying the larger accumulation of TAFs previously reported in ADC compared to SCC. Moreover, our results encourage using MEK inhibitors in ADC-TAFs but not SCC-TAFs., (© 2022. The Author(s).)

Background: Since the first isolation of rubella virus (RuV) in 1962, comprehensive data regarding the quantitative evaluation of RuV shedding remain unavailable. In this study, we evaluated the shedding of viral RNA and infectious virus in patients with acute RuV infection., Study Design: We analyzed 767 specimens, including serum/plasma, peripheral blood mononuclear cells (PBMCs), throat swabs, and urine, obtained from 251 patients with rubella. The viral RNA load and the presence of infectious RuV were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and virus isolation., Results: Virus excretion peaked 0-2 days after rash onset and decreased over time. The median viral RNA load dropped to an undetectable level on day 3 after rash onset in serum/plasma, day 2 in PBMCs, days 10-13 in throat swabs, and days 6-7 in urine. Infectious virus could be isolated for up to day 2 after rash onset in serum/plasma, day 1 in PBMCs, days 8-9 in throat swabs, and days 4-5 in urine. The minimum viral RNA load that allowed virus isolation was 961 copies/mL in serum/plasma, 784 copies/mL in PBMCs, 650 copies/mL in throat swabs, and 304 copies/mL in urine. A higher viral RNA load indicated a higher likelihood of the presence of infectious virus., Conclusion: These findings would contribute to improve algorithms for rubella surveillance and diagnosis. In addition, this study indicates that the results of RT-qPCR enable efficient rubella control by estimating candidate patients excreting infectious virus, which could help prevent viral transmission at an early stage and eliminate rubella ultimately., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Japan is one of the countries conducting longitudinal serosurveillance of vaccine-preventable diseases. We conducted surveillance of the local measles-specific antibody titer, calculated the effective reproduction number (R e ), and compared data of four terms: term 1, 2003-2006 (before the introduction of the second shot of measles-containing vaccine); term 2, 2007-2010 (early term toward measles elimination); term 3, 2011-2014 (later term toward measles elimination); and term 4, 2015-2020 (after elimination of measles in Japan). Approximately 250 sera from volunteers aged 0 to ≥ 40 years were collected and examined for measles-specific IgG using the gelatin particle agglutination (PA) method annually from 2003 to 2020. Seroprevalence and the geometric mean of the PA antibody titer were examined by term. R e was calculated using the age-dependent proportion immune and contact matrix for each term. Of the 4,716 sera, 886 in term 1, 1,217 in term 2, 1,069 in term 3, and 1,544 in term 4 were collected. The seroprevalence gradually increased from term 1 (88.3% CI 86.0-90.3) to term 4 (95.7% CI 94.6-96.7), and the seroprevalence of term 1 was significantly lower than those of other terms (Fisher's exact test, p < 0.001), with PA titer ≥ 16 as positive. By contrast, PA antibody titers significantly decreased from term 1 (median 1,024) to term 4 (median 256) (Mann-Whitney U test, p < 0.001). With the protection level (PA titer ≥ 128 and ≥ 256) as positive, R e gradually increased from term 1 (1.8 and 2.3) to term 4 (2.5 and 4.8, respectively). Waning levels of measles antibodies potentially increase the measles susceptibility in Osaka, Japan. This trend might imply a limitation of vaccine-induced immunity in the absence of a natural booster for wild strains after measles elimination. This study provides a cue for maintaining continuous measles elimination status in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important regulator of extracellular matrix turnover that has been traditionally regarded as a potential tumor suppressor owing to its inhibitory effects of matrix metalloproteinases. Intriguingly, this interpretation has been challenged by the consistent observation that increased expression of TIMP-1 is associated with poor prognosis in virtually all cancer types including lung cancer, supporting a tumor-promoting function. However, how TIMP-1 is dysregulated within the tumor microenvironment and how it drives tumor progression in lung cancer is poorly understood. We analyzed the expression of TIMP-1 and its cell surface receptor CD63 in two major lung cancer subtypes: lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), and defined the tumor-promoting effects of their interaction. We found that TIMP-1 is aberrantly overexpressed in tumor-associated fibroblasts (TAFs) in ADC compared to SCC. Mechanistically, TIMP-1 overexpression was mediated by the selective hyperactivity of the pro-fibrotic TGF-β1/SMAD3 pathway in ADC-TAFs. Likewise, CD63 was upregulated in ADC compared to SCC cells. Genetic analyses revealed that TIMP-1 secreted by TGF-β1-activated ADC-TAFs is both necessary and sufficient to enhance growth and invasion of ADC cancer cells in culture, and that tumor cell expression of CD63 was required for these effects. Consistently, in vivo analyses revealed that ADC cells co-injected with fibroblasts with reduced SMAD3 or TIMP-1 expression into immunocompromised mice attenuated tumor aggressiveness compared to tumors bearing parental fibroblasts. We also found that high TIMP1 and CD63 mRNA levels combined define a stronger prognostic biomarker than TIMP1 alone. Our results identify an excessive stromal TIMP-1 within the tumor microenvironment selectively in lung ADC, and implicate it in a novel tumor-promoting TAF-carcinoma crosstalk, thereby pointing to TIMP-1/CD63 interaction as a novel therapeutic target in lung cancer., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Lung cancer is the leading cause of cancer-related death worldwide. The desmoplastic stroma of lung cancer and other solid tumors is rich in tumor-associated fibroblasts (TAFs) exhibiting an activated/myofibroblast-like phenotype. There is growing awareness that TAFs support key steps of tumor progression and are epigenetically reprogrammed compared to healthy fibroblasts. Although the mechanisms underlying such epigenetic reprogramming are incompletely understood, there is increasing evidence that they involve interactions with either cancer cells, pro-fibrotic cytokines such as TGF-β, the stiffening of the surrounding extracellular matrix, smoking cigarette particles and other environmental cues. These aberrant interactions elicit a global DNA hypomethylation and a selective transcriptional repression through hypermethylation of the TGF-β transcription factor SMAD3 in lung TAFs. Likewise, similar DNA methylation changes have been reported in TAFs from other cancer types, as well as histone core modifications and altered microRNA expression. In this review we summarize the evidence of the epigenetic reprogramming of TAFs, how this reprogramming contributes to the acquisition and maintenance of a tumor-promoting phenotype, and how it provides novel venues for therapeutic intervention, with a special focus on lung TAFs.

Severe fever with thrombocytopenia syndrome (SFTS) is a novel tick-borne infectious disease, therefore, the information on the whole genome of the SFTS virus (SFTSV) is still limited. This study demonstrates a nearly whole genome of the SFTSV identified in Osaka in 2017 and 2018 by next-generation sequencing (NGS). The evolutionary lineage of two genotypes, C5 and J1, was identified in Osaka. The first case in Osaka belongs to suspect reassortment (L:C5, M:C5, S:C4), the other is genotype J1 (L: J1, M: J1, S: J1) according to the classification by a Japanese group. C5 was identified in China, indicating that C5 identified in this study may be transmitted by birds between China and Japan. This study revealed that different SFTSV genotypes were distributed in two local areas, suggesting the separate or focal transmission patterns in Osaka.

The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in non-small cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs in vitro and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs was associated with increased promoter methylation of the profibrotic TGFβ transcription factor SMAD3 compared with ADC-TAFs, which elicited a compensatory increase in TGFβ1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFβ1/SMAD3 activation, whereas genetic downregulation of SMAD3 in ADC-TAFs and control fibroblasts increased TGFβ1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib in vitro and in vivo . Our results also support that smoking and/or the anatomic location of SCC in the proximal airways, which are more exposed to cigarette smoke particles, may prime SCC-TAFs to stronger SMAD3 epigenetic repression, because cigarette smoke condensate selectively increased SMAD3 promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential SMAD3 promoter methylation in TAFs and provide new mechanistic insights on the selective poor response of SCC-TAFs to nintedanib. Moreover, our findings support that patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFβ1/SMAD3 activation. SIGNIFICANCE: This study implicates the selective epigenetic repression of SMAD3 in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFβ1/SMAD3., (©2019 American Association for Cancer Research.)

An epidemiological survey focused on farmers engaged in vinyl-house culture of strawberry was carried out among the inhabitants of a farming village. For avoiding residential and habitual factors, all residents (851 in number) over 12 years of age in the area were inquired about symptoms, personal and family histories. There were no marked difference in age and sex among three groups, i.e. farmers with and without vinyl-house culture and people with no relation to farming in this area. Incidence of one of the inquired symptoms were 54% and 45% in farmers with and without strawberry culture in vinyl-house respectively, and 34% in the non-farming people. This difference became more larger up to 83% in the strawberry farmers of allergic constitution. Symptoms found more in the farmers than the other were headache, dizziness, conjunctivitis, rhinorrea, sneeze, palpitation and neuralgia, and the last four symptoms were more marked among farmers in charge of strawberry culture. Incidence of the allergic symptom consisting of conjunctivitis, rhinorrea and sneeze was more marked among the persons of allergic constitution . In conclusion, health effects of strawberry culture in vinyl-house in farmers can be look upon from three aspects; overwork, a particular stance during the work and allergic responses to the dust in the vinyl-house.

Sulbactam/cefoperazone (SBT/CPZ), a new antibacterial drug, was administered to 14 cases with respiratory infections for a duration of 5 approximately 13 days at a daily dose of 4 g. Diagnoses of these patients were 7 respiratory tract infections, and 7 bronchopneumonias. The underlying diseases were chronic pulmonary emphysema in 6 cases, bronchial asthma in 2 cases, and one each of bronchiectasis, diffuse panbronchiolitis and lung cancer with bronchoesophageal fistula. All patients had underlying respiratory diseases and/or were more than 70 years old. The rate of clinical efficacy was 78.6%. The incidence of penicillinase production by isolated bacteria was 18.2% and that of cephalosporinase was 63.6%. SBT/CPZ was expected to be more effective than CPZ alone in 3 cases judging from the susceptibility of the bacterial strains concerned, to antibiotics. No side effects were observed. We conclude that SBT/CPZ is useful in the treatment of respiratory infections of patients of advanced age and/or with underlying respiratory diseases.

Prevalence studies of Japanese cedar pollinosis in densely planted areas were performed using pollen counts, patient studies, and population surveys with questionnaires. The incidence of pollinosis was increased and appeared to be related to dispersed pollen, which resulted in high immunologic responses. Onset of symptoms was influenced by hereditary factors as well as pollution from automobile exhausts.

Decreased response of beta-adrenergic receptor has been considered to be one of the causes of increased responsiveness of the bronchi in asthma. Since beta-adrenergic receptor has two subtypes, beta 1 and beta 2, and the bronchodilating effect of beta stimulants is mediated by beta 2-receptor, responsiveness of the bronchi is expected to correlate to the cyclic AMP response of lymphocytes to a beta 2-stimulant. Responsiveness of the bronchi was expressed as respiratory threshold to acetylcholine (RT-Ach), which was the minimal concentration of acetylcholine solution to cause an initial decrease of FEV1 of more than 20% of the baseline value. Beta 1- and beta 2-responses were expressed as the increments of cyclic AMP content of 10(6) lymphocytes incubated with norepinephrine (beta 1-stimulant) and salbutamol (beta 2-stimulant). RT-Ach showed a significant correlation with the beta 2-cyclic AMP response of lymphocytes, but not with the beta 1-response among patients with asthma. Sixteen symptomatic patients on continuous beta-stimulants showed lower RT-Ach value and diminished beta 2-receptor activity of lymphocytes compared with 14 patients in remission. These results suggest that selective beta 2-adrenergic blockade may be one of the causes of bronchial hypersensitivity in asthma, though it should be noted that in this study beta-adrenergic responses were examined in lymphocytes and were compared with the responsiveness of the bronchi. Possible beta-receptor subsensitivity induced by administration of beta-stimulants is discussed.

Respiratory infections of 10 subjects with underlying respiratory diseases were treated with cefmetazole (CMZ) and its clinical effects were studied. Five subjects of them were respiratory tract infection, 3 subjects were pneumonia and 2 subjects were pneumonia followed by empyema. The underlying diseases were chronic pulmonary emphysema in 4 subjects, diffuse panbronchiolitis in 3, chronic bronchitis in 2 and bronchial asthma in 1. The doses of CMZ were 4 to 8 grams per day and the durations of administration ranged 3 to 39 days. The clinical effects were judged from the changes of fever, cough, amount of sputum, dyspnea, rale, chest X-ray, white blood cell counts, erythrocyte sedimentation rates, sputum culture and PaO2. The clinical effects of 6 subjects were evaluated as good, those of 3 were fair and that of 1 was poor. In 3 subjects H. influenzae in the sputum was eliminated and in 1 subject both H. aphrophilus and alpha-Streptococcus found in the pleural effusion were eliminated. In 1 subject Klebsiella in the sputum was eliminated and replaced by Enterobacter. No side effects were observed. We conclude that CMZ is considerably useful in the treatment of respiratory infections of the patients with underlying respiratory diseases.

Fibrin clots formed from normal plasma dissolve readily in 1% monochloroacetic acid at 37 C. However, if the clots are washed thoroughly before the acid is added, they are no longer soluble. The agent present in the serum which causes dissolution of the fibrin clot was isolated and identified as pepsinogen. Because of the low pH of monochloroacetic acid the pepsinogen is activated and the clots are digested, simulating the dispersion of a fibrin clot which occurs in the absence of fibrin-stabilizing factor (factor XIII). Because of its higher pH, urea will not activate pepsinogen and is therefore a better agent to screen factor XIII deficiencies.

A variety of systemic reactions associated with exercise are recognized. This article describes three cases of anaphylaxis induced by exercise within 2 hours after the ingestion of causative foods. Their symptoms were urticaria, angioedema, bronchial asthma, fall in blood pressure and syncope. Cases #1 and #3 showed exercise-induced anaphylaxis after ingestion of wheat. However, case #1 reacted only to toasted wheat. In case #2 the most likely responsible food was also wheat, although we could not definitely confirm this. Two subjects exhibited an elevation in plasma histamine level when symptoms developed. In addition, we observed inhibition of these symptoms by pretreatment with anti-allergic drugs. These observations suggest that type I allergic reaction is involved in the development of these symptoms. However, why exercise combined with ingestion of food is needed for this type of anaphylaxis still remains unknown.

Our previous study indicated that the IgE antibody responses in mice immunized with intraperitoneal injection of the antigens mixed with diesel-exhaust particulates (DEP) were higher than those in the animals immunized with the antigens alone. We examined the adjuvant activity of DEP inoculated by the intranasal route, i.e., the natural entrance of DEP. In 3-week interval immunization, the IgE antibody responses in mice immunized with intranasal inoculation of ovalbumin (OA) mixed with DEP were higher than responses in the animals immunized with OA alone. DEP had an adjuvant activity for anti-OA IgE antibody production, even in a small dose such as 1 micrograms administered with a 3-week interval. Also in 1-week interval immunization, the enhancing effect of DEP on anti-OA IgE antibody production was demonstrated when mice were immunized with intranasal inoculation of OA and DEP. The possibility cannot be excluded that DEP, which are kept buoyant in the environmental atmosphere of urban districts, may exert an adjuvant activity for IgE antibody production after being inhaled into the human body and have some relation to the mechanism of the outbreak of allergic rhinitis caused by pollens in Japan.

Clinical mass surveys were carried out on the residents to whom questionnaires on symptoms with reference to strawberry culture in the vinyl-house had been delivered. Two hundred and eleven persons attended at the first survey in September, 1976 (off the season of strawberry culture) and the 58 attended at the second survey in March, 1977 (within the season). The participants of the first survey consisted of three groups, i.e. farmers with and without strawberry culture and people without farming. The participants of the second survey consisted only of farmers with strawberry culture. Results are summarized as follows. 1) There were no difference of incidence of various clinical findings detected by an ophthalmologist and otolaryngologists among the three groups. 2) There were also no difference of incidence of abnormal findings in urine tests, liver functions, respiratory test, blood examinations and blood pressure test among the three groups. However, the comparison of persons in charge of strawberry culture and attended at both tof the first and the second surveys, such changes were detected as increases in the value of GOT (within normal limits), of stab neutrophiles, lymphocytes and of monocytes within the season of strawberry culture. 3) Increases of positive rates in skintest by extracts of house dust and Candida sp. were observed in the farmers with and without strawberry culture. Positive rates of skintest by strawberry pollen and honey bee extracts among the farmers with strawberry culture were markedly higher than the allergic patients in Dokkyo Hospital having no relation to this culture. This positive rate was markedly elevated among the persons who complained the symptom of rhinorrea within the season.