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We measured section thickness (ST) after slicing using a film thickness meter and investigated the relationship between ST and the percent area of positive staining using computer-assisted image analysis., Methods: Sections were prepared from a paraffin-only block and formalin-fixed paraffin-embedded (FFPE) blocks containing fish sausage and human liver specimens. The ST was compared between the sections prepared with cooling using an ice pack (IP) or a continuous cooling device (CCD) paired with a sliding microtome set at an ST of 4 µm. The sections were stained with eosin or aniline blue, and the association between the percent area of positive staining and ST was determined using computer-aided analysis of images captured with a whole slide scanner., Results: The average STs of the paraffin-only block sections measured by four practitioners were 5.01-5.41 and 4.09-4.33 µm in samples prepared using an IP and a CCD, respectively. Therefore, subsequent analyses included sections prepared using the CCD. The ST of the tissue surface was significantly thinner than that of the paraffin surrounding the tissue section. Furthermore, the percent areas of positive staining for eosin and aniline blue were significantly correlated with ST in both the fish sausage and liver sections. The analysis of the ST and percent area of positive staining in 60 sections of the same block, which were categorized into quantiles based on ST, revealed a significant difference in the percent area of positive staining between the thicker and thinner sections., Discussion: Specimen sectioning should be performed with a CCD, ST should be measured before the staining of pathologic specimens prepared for quantitative analysis, and histologic examination should be performed using specimens with uniform ST., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

To date, no report has compared section thickness (ST) between the sliding microtome (SM) and rotary microtome (RM). We used the ice pack (IP) condition, in which the paraffin block was not cooled during slicing, and a continuous cooling device (CCD) for continuous cooling during slicing. The ST was greater for the SM than the RM in the IP condition, but it was identical between the devices under the CCD condition. Thus, we used the CCD condition for subsequent studies. In formalin-fixed, paraffin-embedded (FFPE) fish sausage blocks, the ST of the tissue surface (T-surface) was significantly concaved compared to that of the paraffin surface (P-surface) for both microtomes. On the contrary, in FFPE human kidney blocks, ST did not differ between the T-surface and P-surface. Furthermore, the eosin-positive area of PAM-stained specimens was affected by ST, and the color tone of the thickest sample differed from that of the median or thinnest sample. Our data indicated that we should use CCD conditions to ensure that ST is uniform regardless of the type of microtome. In addition, for quantitative analysis, we should utilize ST measure equipment and specimens with a constant ST., Competing Interests: Declaration of Competing Interest The author(s) declare they have no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Emerin and lamins not only influence nuclear morphology but are also involved in differentiation. We herein examined 82 resected cases of invasive lung adenocarcinoma using computer-assisted image analysis of nuclear morphology on Feulgen-stained and immunohistochemical sections of lamin A, B1, B2, and emerin (four proteins) to calculate the rank sum of the cell positivity rates for these four proteins. The rank sum of four proteins showed weak negative correlations with the nuclear area and perimeter and a weak positive correlation with the nuclear shape factor. Interestingly, the top three cases with the highest rank sum were papillary adenocarcinoma, and the bottom three cases were acinar adenocarcinomas containing cribriform patterns. We compared the rank sum for grading (differentiation: G1, G2, and G3) and predominant histological subtypes and found that the rank sum of G3 was lower than that of G1 and G2. Furthermore, the rank sum was lower for acinar adenocarcinoma with >20 % cribriform pattern (acinar+cribri) and solid adenocarcinoma than for lepidic and papillary adenocarcinoma. Individual examination of the four proteins revealed that emerin expression was lower in G3 than in G1, and lamin B2 expression was lower in G3 than in G1 and G2. Compared with lepidic adenocarcinoma, acinar+cribri showed significantly lower expression of all four proteins among histological subtypes. These data indicated that the expression of lamin A, B1, B2, and emerin was markedly decreased in poorly differentiated adenocarcinoma (i.e., G3), especially in acinar+cribri. Our data suggested that changes in these four proteins can not only affect nuclear morphology but also histological structure in lung adenocarcinoma., Competing Interests: Declaration of Competing Interest On behalf of all authors of our manuscript, I would like to declare that none of us has a conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The detection of a single-enzyme catalytic reaction by surfaced-enhanced Raman scattering (SERS) is presented by utilizing DNA origami-based plasmonic antennas. A single horseradish peroxidase (HRP) was accommodated on a DNA origami nanofork plasmonic antenna (DONA) containing gold nanoparticles, enabling the tracing of single-molecule SERS signals during the peroxide reduction reaction. This allows monitoring of the structure of a single enzymatic catalytic center and products under suitable liquid conditions. Herein, we demonstrate the chemical changes of HRP and the appearance of tetramethylbenzidine (TMB), which works as a hydrogen donor before and after the catalytic reaction. The results show that the iron in HRP adopts Fe 4+ and low spin states with the introduction of H 2 O 2 , indicating compound-I formation. Density functional theory (DFT) calculations were performed for later catalytic steps to rationalize the experimental Raman/SERS spectra. The presented data provide several possibilities for tracking single biomolecules in situ during a chemical reaction and further developing plasmon-enhanced biocatalysis.

Cytochrome C, an evolutionarily conserved protein, plays pivotal roles in cellular respiration and apoptosis. Understanding its molecular intricacies is essential for both academic inquiry and potential biomedical applications. This study introduces an advanced single-molecule surface-enhanced Raman scattering (SM-SERS) system based on DNA origami nanoantennas (DONAs), optimized to provide unparalleled insights into protein structure and interactions. Our system effectively detects shifts in the Amide III band, thereby elucidating protein dynamics and conformational changes. Additionally, the system permits concurrent observations of oxidation processes and Amide bands, offering an integrated view of protein structural and chemical modifications. Notably, our approach diverges from traditional SM-SERS techniques by de-emphasizing resonance conditions for SERS excitation, aiming to mitigate challenges like peak oversaturation. Our findings underscore the capability of our DONAs to illuminate single-molecule behaviors, even within aggregate systems, providing clarity on molecular interactions and behaviors.

Background: A distinctive pathological feature of idiopathic pulmonary fibrosis (IPF) is the aberrant accumulation of extracellular matrix components in the alveoli in abnormal remodeling and reconstruction following scarring of the alveolar structure. The current antifibrotic agents used for IPF therapy frequently result in systemic side effects because these agents are distributed, through the blood, to many different tissues after oral administration. In contrast to oral administration, the intrapulmonary administration of aerosolized drugs is believed to be an efficient method for their direct delivery to the focus sites in the lungs. However, how fibrotic lesions alter the distribution of aerosolized drugs following intrapulmonary administration remains largely unknown. In this study, we evaluate the intrapulmonary distribution characteristics of aerosolized model compounds in mice with bleomycin-induced pulmonary fibrosis through imaging the organs and alveoli. Methods: Aerosolized model compounds were administered to mice with bleomycin-induced pulmonary fibrosis using a Liquid MicroSprayer ® . The intrapulmonary distribution characteristics of aerosolized model compounds were evaluated through several imaging techniques, including noninvasive lung imaging using X-ray computed tomography, ex vivo imaging using zoom fluorescence microscopy, frozen tissue section observation, and three-dimensional imaging with tissue-clearing treatment using confocal laser microscopy. Results: In fibrotic lungs, the aerosolized model compounds were heterogeneously distributed. In observations of frozen tissue sections, model compounds were observed only in the fibrotic foci near airless spaces called honeycombs. In three-dimensional imaging of cleared tissue from fibrotic lungs, the area of the model compound in the alveolar space was smaller than in healthy lungs. Conclusion: The intrapulmonary deposition of extracellular matrix associated with pulmonary fibrosis limits the intrapulmonary distribution of aerosolized drugs. The development of delivery systems for antifibrotic agents to improve the distribution characteristics in fibrotic foci is necessary for effective IPF therapy.

A versatile generation of plasmonic nanoparticle dimers for surface-enhanced Raman scattering (SERS) is presented by combining a DNA origami nanofork and spherical and nonspherical Au or Ag nanoparticles. Combining different nanoparticle species with a DNA origami nanofork to form DNA origami nanoantennas (DONAs), the plasmonic nanoparticle dimers can be optimized for a specific excitation wavelength in SERS. The preparation of such nanoparticle dimers is robust enough to enable the characterization of SERS intensities and SERS enhancement factors of dye-modified DONAs on a single dimer level by measuring in total several thousands of dimers from five different dimer designs, each functionalized with three different Raman reporter molecules and measured at four different excitation wavelengths. Based on these data, SERS enhancement factor (EF) distributions have been determined for each dimer design and excitation wavelengths. The structures and measurement conditions with the highest EFs are suitable for single-molecule SERS (SM-SERS), which is realized by placing single dye molecules into hot spots. We demonstrate that the probability of placing single molecules in a strongly enhancing hot spot for SM-SERS can be increased by using anisotropic nanoparticles with several sharp edges, such as nanoflowers. Combining a Ag nanoparticle with a Au particle in one dimer structure allows for a broadband excitation covering almost the whole visible range. The most versatile plasmonic dimer structure for SERS combines a spherical Ag nanoparticle with a Au nanoflower. Employing the discontinuous Galerkin time domain method, we numerically investigate the bare, symmetric dimers with respect to spectral and near-field properties, showing that, indeed, the nanoflowers induce multiple hot spots located at the edges which surpass the intensity of the spherical dimers, indicating the possibility for SM-SERS. The presented DONA structures and SERS data provide a robust basis for applying such designs as versatile SERS tags and as substrates for SM-SERS measurements.

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 10 11 -fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

Optical fibers equipped with plasmonic flow sensors at their tips are fabricated and investigated as photothermomechanical nanopumps for the active transport of target analytes to the sensor surface. The nanopumps are prepared using a bottom-up strategy: i.e., by sequentially stacking a monolayer of a thermoresponsive polymer and a plasmonic nanohole array on an optical fiber tip. The temperature-dependent collapse and swelling of the polymer is used to create a flow-through pumping mechanism. The heat required for pumping is generated by exploiting the photothermal effect in the plasmonic nanohole array upon irradiation with laser light (405 nm). Simultaneous detection of analytes by the plasmonic sensor is achieved by monitoring changes in its optical response at longer wavelengths (∼500-800 nm). Active mass transport by pumping through the holes of the plasmonic nanohole array is visualized by particle imaging velocimetry. Finally, the performance of the photothermomechanical nanopumps is investigated for two types of analytes, namely nanoscale objects (gold nanoparticles) and molecules (11-mercaptoundecanoic acid). In the presence of the pumping mechanism, a 4-fold increase in sensitivity was observed compared to the purely photothermal effect, demonstrating the potential of the presented photothermomechanical nanopumps for sensing applications.

The study of biologically relevant molecules and their interaction with external stimuli on a single molecular scale is of high importance due to the availability of distributed rather than averaged information. Surface enhanced Raman scattering (SERS) provides direct chemical information, but is rather challenging on the single molecule (SM) level, where it is often assumed to require a direct contact of analyte molecules with the metal surface. Here, we detect and investigate the molecular states of single hemin by SM-SERS. A DNA aptamer based G-quadruplex mediated recognition of hemin directs its placement in the SERS hot-spot of a DNA Origami Nanofork Antenna (DONA). The configuration of the DONA structure allows the molecule to be trapped at the plasmonic hot-spot preferentially in no-contact configuration with the metal surface. Owing to high field enhancement at the plasmonic hot spot, the detection of a single folded G-quadruplex becomes possible. For the first time, we present a systematic study by SM-SERS where most hemin molecule adopt a high spin and oxidation state (III) that showed state crossover to low spin upon strong-field-ligand binding. The present study therefore, provides a platform for studying biologically relevant molecules and their properties at SM sensitivity along with demonstrating a conceptual advancement towards successful monitoring of single molecular chemical interaction using DNA aptamers.

DNA origami technology allows for the precise nanoscale assembly of chemical entities that give rise to sophisticated functional materials. We have created a versatile DNA origami nanofork antenna (DONA) by assembling Au or Ag nanoparticle dimers with different gap sizes down to 1.17 nm, enabling signal enhancements in surface-enhanced Raman scattering (SERS) of up to 10 11 . This allows for single-molecule SERS measurements, which can even be performed with larger gap sizes to accommodate differently sized molecules, at various excitation wavelengths. A general scheme is presented to place single analyte molecules into the SERS hot spots using the DNA origami structure exploiting covalent and noncovalent coupling schemes. By using Au and Ag dimers, single-molecule SERS measurements of three dyes and cytochrome c and horseradish peroxidase proteins are demonstrated even under nonresonant excitation conditions, thus providing long photostability during time-series measurement and enabling optical monitoring of single molecules.

Combination therapy of active vitamin D 3 with some bisphosphonates (BPs) has been reported to be clinically beneficial. However, combination therapy of eldecalcitol (ELD) with BP has to date not been validated as to whether it is beneficial in the clinical setting. Preclinical studies suggested that simultaneous treatment with ELD and some BPs is more effective than monotherapy. However, the relative potency of various BPs, when used in combination with ELD, is completely unknown. In this study, we examined and compared the effects of risedronate (RIS), alendronate (ALN), and minodronate (MIN) alone or in combination with ELD on bone mass, microarchitecture, strength, and material properties in ovariectomized Sprague-Dawley rats aged 13 weeks. RIS, ALN, MIN, and ELD were administered five times weekly for 16 weeks. Micro-computed tomography analysis, compression test, and Fourier transform infrared (FTIR) imaging analysis were performed 16 weeks after treatment initiation. Trabecular and cortical bone mineral density (BMD) in the fourth lumbar vertebra (L4) significantly increased in the RIS + ELD, ALN + ELD, and MIN + ELD groups compared with the vehicle group. Moreover, the bone microarchitecture of L4 in all the BP + ELD groups also significantly improved. On mechanical testing of L4, the maximum load was significantly increased in the RIS + ELD and ALN + ELD groups. FTIR analysis revealed that the mineral-to-collagen ratio of trabecular bone in L3 of all the BP + ELD groups was significantly increased compared with the vehicle group. By contrast, the carbonate-to-phosphate ratio, a parameter of mineral immaturity, was significantly decreased in the RIS + ELD and ALN + ELD groups. BP + ELD improved the BMD and structural properties of the bone to a similar extent. RIS + ELD and ALN + ELD also improved bone strength. Furthermore, treatment with BP + ELD improved the bone material. These results suggest that the combination therapy of BP and ELD is beneficial and warrants further clinical trials., Competing Interests: The authors declare that they have no conflict of interest in this study. HK has received research funding from 10.13039/100014421EA pharma (formerly known as Ajinomoto Pharmaceutical). DI has received honoraria and research funding from 10.13039/501100004948Astellas Pharma, 10.13039/100002429Amgen, 10.13039/100014421EA Pharma, 10.13039/100010795Chugai Pharmaceutical, 10.13039/501100002336Daiichi Sankyo, Taisyo Pharmaceutical, and 10.13039/501100010486Teijin Pharma., (© 2021 The Author(s).)

Background: Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive respiratory disease characterized by the destruction of the alveolar structure. In pulmonary fibrosis, aerosolized drugs are easily transferred to the systemic circulation via leakage through the injured alveolar epithelium. Therefore, pulmonary drug delivery systems for sustained distribution in fibrotic lungs are needed., Objective: We evaluated the intrapulmonary pharmacokinetics of aerosolized liposomes as pulmonary drug delivery systems in mice with bleomycin-induced pulmonary fibrosis., Methods: The aerosolized liposomal formulations and solutions of model compounds, including indocyanine green and 6-carboxyfluorescein (6-CF), were intrapulmonary administered to mice with bleomycin-induced pulmonary fibrosis. In vivo imaging for indocyanine green and 6-CF measurements in lung tissues and plasma were performed. Additionally, in vitro permeation experiments using NCI-H441 cell monolayers as a model of alveolar epithelial cells were performed., Results: The fluorescence signals of indocyanine green following the administration of liposomal formulations were observed longer in the lungs than those in solution-treated mice. Compared with the solution, the 6-CF concentrations in lung tissues after the administration of liposomal formulations were determined higher, whereas those in the plasma were lower. 6-CF permeability was significantly increased by transforming growth factor-β1 in NCI-H441 cell monolayers treated with the solution but unchanged in the presence of the liposomal formulation., Conclusion: The aerosolized liposomal formulation can prevent enhanced drug transfer from fibrotic lungs into the systemic circulation via the injured alveolar epithelium. This system may be useful for the sustained distribution of anti-fibrotic agents in fibrotic lungs and the optimization of IPF therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

This study evaluated the delivery efficiency and antitumor effects of the intrapulmonary administration of antitumor small interfering ribonucleic acid (siRNA)-containing nanoparticles to mice with metastatic lung tumor. Fluorescence-labeled, siRNA-containing nanoparticles were administered using Liquid MicroSprayer ® to mice with metastatic lung tumors induced by the murine melanoma cell line B16F10. Fluorescent signals in the whole lung and in the tumor region following the intrapulmonary administration of siRNA-containing nanoparticles were stronger than those following intravenous administration. The intrapulmonary administration of nanoparticles containing a mixture of siRNA against MDM2, c-Myc, and vascular endothelial growth factor (VEGF) significantly improved survival and prolonged the survival of mice with metastatic lung tumor. In addition, after the intrapulmonary or intravenous administration of the mixture, the activity levels of interleukin-6 and -12, markers of systemic toxicity, were similar to those of nontreatment. These results indicate that the antitumor siRNA-containing nanoparticles were delivered efficiently and specifically to tumor cells, effectively silencing the oncogenes in the lung metastasis without any significant systemic toxicity.

Background signals from in situ-formed amorphous carbon, despite not being fully understood, are known to be a common issue in few-molecule surface-enhanced Raman scattering (SERS). Here, discrete gold and silver nanoparticle aggregates assembled by DNA origami were used to study the conditions for the formation of amorphous carbon during SERS measurements. Gold and silver dimers were exposed to laser light of varied power densities and wavelengths. Amorphous carbon prevalently formed on silver aggregates and at high power densities. Time-resolved measurements enabled us to follow the formation of amorphous carbon. Silver nanolenses consisting of three differently-sized silver nanoparticles were used to follow the generation of amorphous carbon at the single-nanostructure level. This allowed observation of the many sharp peaks that constitute the broad amorphous carbon signal found in ensemble measurements. In conclusion, we highlight strategies to prevent amorphous carbon formation, especially for DNA-assembled SERS substrates.

This study demonstrates the bottom-up synthesis of silver nanolenses. A robust coating protocol enabled the functionalization of differently sized silver nanoparticles with DNA single strands of orthogonal sequence. Coated particles 10 nm, 20 nm, and 60 nm in diameter were self-assembled by DNA origami scaffolds to form silver nanolenses. Single molecules of the protein streptavidin were selectively placed in the gap of highest electric field enhancement. Streptavidin labelled with alkyne groups served as model analyte in surface-enhanced Raman scattering (SERS) experiments. By correlated Raman mapping and atomic force microscopy, SERS signals of the alkyne labels of a single streptavidin molecule, from a single silver nanolens, were detected. The discrete, self-similar aggregates of solid silver nanoparticles are promising for plasmonic applications., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Patient-reported outcomes are increasingly commonly used as a method of evaluating treatments. This cross-sectional study aimed to evaluate implant treatment from the perspective of patient-reported outcomes., Materials and Methods: Subjects were 804 patients who visited the Department of Oral Implantology at Osaka Dental University. The participants were categorized into a pre-implant group and a post-implant group. They were further categorized into five subgroups based on the number of occlusal supports provided by the remaining teeth according to the Eichner classification. The participants answered a basic questionnaire and the General Oral Health Assessment Index (GOHAI) questionnaire, an oral health-related quality of life (QOL) scale. GOHAI scores were compared according to the number of occlusal supports within each group and between the two groups., Results: The results revealed a significant difference in terms of the number of occlusal supports within the pre-implant group; GOHAI scores decreased as the number of occlusal supports decreased ( P <.001). However, no significant difference was observed in GOHAI scores in terms of the number of occlusal supports in the post-implant group ( P >.05). GOHAI scores significantly improved in both pre- and post-implant groups in all occlusal support subgroups ( P <.001)., Conclusion: GOHAI scores decrease as occlusal support is lost. However, implant treatment performed in areas of loss improves the GOHAI score when occlusal support is restored.

Extensive isolation work on the 1-BuOH-soluble fraction of a MeOH extract of the aerial parts of Dianthus japonicus afforded three further triterpene glycosyl estsers, termed dianthosaponins G-I, an anthranilic acid amide glucoside and a C-glycosyl flavonoid along with one known triterpene saponin. Their structures were elucidated from spectroscopic evidence. The cytotoxicity of the isolated compounds toward A549 cells was evaluated.

Pirfenidone is the first and only clinically used anti-fibrotic drug for the treatment of idiopathic pulmonary fibrosis (IPF). It was reported previously that pirfenidone metabolites (5-hydroxypirfenidone and 5-carboxypirfenidone) also have anti-fibrotic effects. The present study evaluated the distribution of pirfenidone and its metabolites in the lung, liver and kidney tissues in rats. The time course for the different concentrations of pirfenidone, 5-hydroxypirfenidone and 5-carboxypirfenidone in the lung tissue following oral administration (30 mg/kg) to rats was lower than that in plasma, and the area under the drug concentration-time curve (AUC) ratios of lung/plasma for pirfenidone, 5-hydroxypirfenidone and 5-carboxypirfenidone were 0.52, 0.40 and 0.61, respectively. In in vitro transport experiments, the basolateral-to-apical transport of pirfenidone and its metabolites through the model of lung epithelial cell (Calu-3) monolayers was not significantly different from their apical-to-basolateral transport. In binding experiments, the binding rate of these drugs to the lung tissue was lower than that to the plasma protein. These findings suggest that the low distribution of pirfenidone and its metabolites in the lungs was based on their low affinities with lung tissue and not the transport characteristics of lung epithelial cells. On the other hand, the AUC ratios of liver/plasma for pirfenidone and 5-carboxypirfenidone were 2.3 and 6.5 and the AUC ratios of kidney/plasma were 1.5 and 20, respectively. The binding rates to the liver and kidney tissues were higher than those to the plasma protein. These results suggest that high concentrations of these drugs were found in the liver and kidney tissues., (Copyright © 2014 John Wiley & Sons, Ltd.)

In the present in vitro study, we assessed the delivery of pirfenidone incorporated into liposomes modified with truncated basic fibroblast growth factor (tbFGF) to lung fibroblasts and investigated the anti-fibrotic effect of the drug. The tbFGF peptide, KRTGQYKLC, was used to modify the surface of liposomes (tbFGF-liposomes). We used the thin-layer evaporation method, followed by sonication, to prepare tbFGF-liposomes containing pirfenidone. The cellular accumulation of tbFGF-liposomes was 1.7-fold greater than that of non-modified liposomes in WI-38 cells used as a model of lung fibroblasts. Confocal laser scanning microscopy showed that tbFGF-liposomes were widely localized in WI-38 cells. The inhibitory effects of pirfenidone incorporated into tbFGF-liposomes on transforming growth factor-β1 (TGF-β1)-induced collagen synthesis in WI-38 cells were evaluated by measuring the level of intracellular hydroxyproline, a major component of the protein collagen. Pirfenidone incorporated into tbFGF-liposomes at concentrations of 10, 30, and 100 µM significantly decreased the TGF-β1-induced hydroxyproline content in WI-38 cells. The anti-fibrotic effect of pirfenidone incorporated into tbFGF-liposomes was enhanced compared with that of pirfenidone solution. These results indicate that tbFGF-liposomes are a useful drug delivery system of anti-fibrotic drugs to lung fibroblasts for the treatment of idiopathic pulmonary fibrosis.

Pirfenidone (PFD) is the first and only clinically used antifibrotic drug for the treatment of idiopathic pulmonary fibrosis (IPF). This study evaluated the antifibrotic effects of two metabolites of PFD, 5-hydroxypirfenidone (PFD-OH) and 5-carboxypirfenidone (PFD-COOH), on WI-38 cells in an in vitro lung fibroblast model. The inhibitory effects of PFD-OH and PFD-COOH on transforming growth factor-β1 (TGF-β1)-induced collagen synthesis in WI-38 cells were evaluated by measuring intracellular hydroxyproline, a major component of the protein collagen. PFD-OH and PFD-COOH at 300 and 1000 µM concentrations significantly decreased the TGF-β1-induced hydroxyproline content in WI-38 cells. These results indicate that PFD-OH and PFD-COOH have antifibrotic activities, which inhibit collagen synthesis in fibroblasts. This study suggests that the concentrations of PFD and its metabolites should be considered in clinical therapy for IPF.

Background: Airway eosinophilia is one of the hallmarks of asthma. Eotaxin may play an important role in eosinophil recruitment., Objectives: To examine the relationship between eotaxin levels in the sputum and eosinophilic inflammation., Methods: The sputum was obtained from 11 non-smokers, 14 smokers and 13 asthmatic patients using a sputum induction method. Eotaxin and interleukin (IL)-5 levels in the sputum were determined by ELISA and immunocytochemical analysis., Results: Asthmatic patients had eosinophilia and smokers showed neutrophilia in their sputum. The eotaxin level in the sputum was significantly higher in smokers (median 412.5, range 91.1-872.2 pg/ml) and asthmatic patients (351.0, 185.0-928.0 pg/ml) compared with non-smokers (123.2, 0-369.0 pg/ml; both p < 0.05). IL-5 was detected in the sputum of 1 non-smoker, none of the smokers and 4 asthmatic patients. The percentage of eotaxin-positive cells was higher in smokers and asthmatic patients than in non-smokers, but the percentage of IL-5-positive cells was significantly higher only in asthmatic patients (p < 0.05)., Conclusions: These findings suggest that the elevated eotaxin level in the sputum does not always accompany the increase in eosinophils, and cooperation with another cytokine such as IL-5 may be required for the recruitment of eosinophils., (Copyright 2003 S. Karger AG, Basel)

Background: Transforming growth factor-beta1 (TGF- beta1) is a multifunctional factor and is known to affect tumor growth in malignant tumors. The effects of TGF-beta1 on angiogenesis, stromal formation, and immune function suggest its possible involvement in tumor progression. The authors examined whether TGF-beta1 levels may be correlated with angiogenesis, clinicopathologic factors, and survival in patients with surgically resected lung carcinoma., Methods: TGF-beta1 protein was extracted from 53 nonsmall cell lung carcinoma tissue samples (19 squamous cell carcinomas, 33 adenocarcinomas, and 1 adenosquamous cell carcinoma), and its level was measured by enzyme-linked immunosorbent assay. To assess tumor angiogenesis, microvessel density (MVD) was determined by CD31 immunostaining., Results: The protein level of TGF-beta1 was 289 picograms per milligram of protein (pg/mg protein), ranging from 94 pg/mg protein to 584 pg/mg protein. The TGF-beta1 protein level was significantly higher in patients with lymph node metastasis compared with patients who were without lymph node metastasis (P = 0.02), and the TGF-beta1 protein level was significantly higher in patients with Stage III disease (TNM classification) compared with patients who had Stage I and II disease (P = 0.03). There was no significant correlation between the TGF-beta1 protein level and any of the other clinicopathologic factors that were considered. A significant positive correlation between TGF-beta1 protein level and MVD was noted (P < 0.01). Furthermore, in patients with adenocarcinoma, a significant correlation between TGF-beta1 protein level and prognosis was detected by multivariate analysis (P = 0.028)., Conclusions: TGF-beta1 seems to affect tumor angiogenesis and to play an important role in tumor progression in patients with nonsmall cell lung carcinoma. Furthermore, the TGF-beta1 protein level may be an independent predictor of survival in patients with adenocarcinoma of the lung., (Copyright 2001 American Cancer Society.)