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Objective We aimed to clarify the clinical significance of serum levels of MMPs in interstitial lung disease (ILD) complicated with PM/DM (PM/DM-ILD). Methods We retrospectively analysed serum levels of seven subsets of MMPs in 52 PM/DM-ILD patients diagnosed at Kyoto University Hospital or Tenri Hospital from January 2005 to December 2014. The patients were sub-grouped based on the presence of anti-amimoacyl-tRNA synthetase antibody (anti-ARS antibody), anti-melanoma differentiation-associated protein 5 antibody (anti-MDA5 antibody) or lack of the antibodies (ARS-ILD, MDA5-ILD and other-ILD groups, respectively) and independently analysed. Eighteen PM/DM patients without ILD and 55 healthy control were also analysed. Associations between serum levels of MMPs and clinical findings including mortality were analysed. Results Among the MMPs analysed, MMP-7 serum levels in the ARS-ILD group were significantly higher compared with those in any of the other groups of PM/DM patients or in healthy controls. On the other hand, in the MDA5-ILD group, serum MMP-7 levels >5.08 ng/ml were associated with worse overall survival both in univariate (P = 0.017; odds ratio 18.0; 95% CI 1.69, 192.00) and multivariate (P = 0.027; odds ratio 14.60; 95% CI 1.11, 192.00) analyses. Immunohistochemical analysis suggested that MMP-7 was expressed in type II alveolar epithelial cells adjacent to the fibrotic lesions. Conclusion Serum MMP-7 levels were higher in anti-ARS antibody-positive PM/DM-ILD patients, while higher serum MMP-7 levels among anti-MDA5 antibody-positive PM/DM-ILD patients were associated with a worse prognosis. Fibrotic processes may be associated with the elevation of serum MMP-7 levels. [ABSTRACT FROM AUTHOR]

The application of matrix-assisted laser desorption/ionization (MALDI)- based mass spectrometry (MS) to the proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue presents significant technical challenges. In situ enzymatic digestion is frequently used to unlock formalin-fixed tissues for analysis, but the results are often unsatisfactory. Here, we report a new, simplified in situ pretreatment method for preparing tissue sections for MS that involves heating with vapor containing acetonitrile in a small airtight pressurized space. The utility of the novel method is shown using FFPE tissue of human colon carcinoma. The number and intensity of MALDI peaks obtained from analysis of pretreated tissue was significantly higher than control tissue not subjected to pretreatment. A prominent peak (m/z 850) apparently specific to cancerous tissue was identified as a fragment of histone H2A in FFPE tissue pretreated using our method. This highly sensitive treatment may enable MALDI-MS analysis of archived pathological FFPE samples, thus leading to the identification of new biomarkers. [ABSTRACT FROM AUTHOR]

Lineages of structurally related alleles at minisatellite MS32 in human populations show considerable differentiation at the continental level. However, the regional specificity of these lineages remains unknown. We now describe the comparison of allele structures in Thai, Han Chinese, and Japanese populations with lineages previously established for North Europeans and Africans. The great majority of alignable Asian alleles showed their closest structural relative in Asia, with few instances of preferential alignment of Asian with European alleles and only one isolated incident showing a best match with an African allele. Further, there was a strong tendency, most marked for Japanese, for Asian alleles to align preferentially with other alleles from the same population, indicating strong regional specificity of allele lineages. This rapidly evolving minisatellite can therefore serve as a lineage marker for exploring recent events in human population history and dissecting population structure at the fine-scale level, as well as being an extremely informative DNA marker for personal identification. [ABSTRACT FROM AUTHOR]

Recent advancements in proteomics technologies using formalin-fixed paraffin-embedded (FFPE) samples have significantly advanced biomarker discovery. Yet, the effects of varying sample preparation protocols on proteomic analyses remain poorly understood. We analyzed mouse liver FFPE samples that varied in fixatives, fixation duration, and storage temperature using LC/MS. We found that variations in fixation duration significantly affected the abundance of specific proteins, showing that HNRNPA2/B1 demonstrated a significant decrease in abundance in samples fixed for long periods, whereas STT3B exhibited a significant increase in abundance in samples fixed for long durations. These findings were supported by immunohistochemical analysis across liver, spleen, and lung tissues, demonstrating a significant decrease in the nuclear staining of HNRNPA2/B1 in long-duration acid formalin(AF)-fixed FFPE samples, and an increase in cytoplasmic staining of STT3B in long-duration neutral buffered formalin-fixed liver and lung tissues and granular staining in all long-duration AF-fixed FFPE tissue types. Similar trends were observed in the long-duration fixed HeLa cells. These results demonstrate that fixation duration critically affects the proteomic integrity of FFPE samples, emphasizing the urgent need for standardized fixation protocols to ensure consistent and reliable proteomic data. The quality of FFPE samples is primarily influenced by the fixation and storage conditions. However, previous studies have mainly focused on their impact on nucleic acids and the extent to which different fixation conditions affect changes in proteins has not been evaluated. In addition, to our knowledge, proteomic research focusing on differences in formalin fixation conditions has not yet been conducted. Here, we analyzed FFPE samples with different formalin fixation and storage conditions using LC/MS and evaluated the impact of different fixation conditions on protein variations. Our study unequivocally established formalin fixation duration as a critical determinant of protein variation in FFPE specimens and successfully identified HNRNPA2/B1 and STT3B as potential biomarkers for predicting formalin fixation duration for the first time. The study findings open new avenues for quality assessment in biomedical research and diagnostics. [Display omitted] • HNRNPA2/B1 and STT3B serve as promising immunohistochemistry biomarkers to predict the duration of formalin fixation in FFPE samples. [ABSTRACT FROM AUTHOR]

Background: Programmed death-1 (PD-1) is a marker for human neoplastic T cells. Here, we evaluated whether or not PD-1 was also a marker for human mastocytosis, and explored the role of PD-1 in human mastocytosis cells. Methods: Immunohistochemical analysis was used to evaluate the expression of PD-1 in clinical samples of human cutaneous mastocytosis. The expression of PD-1 in human mastocytosis cell lines was checked by RT-PCR, western blotting and flow cytometry. We stimulated human mastocytosis cell lines (LAD2 and HMC1.2) with recombinant ligand for PD-, PD-L1 (rPD-L1), and tested the proliferative activity and the status of signal molecules by Cell Counting Kit-8 and ELISA, respectively. Results: Ten of 30 human cutaneous mastocytosis cases (33.3%) expressed PD-1 protein. We also found that a human mastocytosis line LAD2 cells expressed PD-1 protein on their surfaces. The administration with rPD-L1 suppressed the stem cell factor-dependent growth of the LAD2 cells. And, rPD-L1 activated SHP-1 and SHP-2 simultaneously, and decreased the phosphorylation of AKT, in LAD2 cells. In contrast, we could not detect the expression of PD-, and the significant effect of rPD-L1 on the mutated KIT-driven growth of HMC1.2 cells. Conclusions: PD-1 could be a marker for human cutaneous mastocytosis and regulate the growth of human PD-1-positive mastocytosis cells. [ABSTRACT FROM AUTHOR]