Quantitative Evaluation of Interleukin-4 by Immunowall Devices Made of Gelatin Methacryloyl Hydrogel

Immunoassays, which use antigen–antibody reactions, are the primary techniques used to selectively quantify specific disease markers in blood. Conventional immunoassays, such as the microplate-based enzyme-linked immunosorbent assay (ELISA) and paper-based immunochromatography, are widely used, but they have advantages and disadvantages in terms of sensitivity and operating time. Therefore, in recent years, microfluidic-chip-based immunoassay devices with high sensitivity, rapidity and simplicity, which are compatible with whole blood assays and multiplex assays, have been actively investigated. In this study, we developed a microfluidic device using gelatin methacryloyl (GelMA) hydrogel to form a wall-like structure in a microfluidic channel and perform immunoassays inside the wall-like structure, which can be used for rapid and highly sensitive multiplex assays with extremely small sample amounts of ~1 μL. The characteristics of GelMA hydrogel, such as swelling rate, optical absorption and fluorescence spectra, and morphology, were carefully studied to adapt the iImmunowall device and immunoassay. Using this device, a quantitative analysis of interleukin-4 (IL-4), a biomarker of chronic inflammatory diseases, was performed and a limit of detection (LOD) of 0.98 ng/mL was achieved with only 1 μL sample and 25 min incubation time. The superior optical transparency over a wide range of wavelengths and lack of autofluorescence will help to expand the application field of the iImmunowall device, such as to a simultaneous multiple assay in a single microfluidic channel, and provide a fast and cost-effective immunoassay method

The Association of Tumor Immune Microenvironment of the Primary Lesion with Time to Metastasis in Patients with Renal Cell Carcinoma: A Retrospective Analysis

Biological or immunological differences in primary lesions between synchronous and metachronous metastatic renal cell carcinoma (mRCC) have been reported. However, the association between the tumor immune microenvironment (TIME) of primary lesions and time to metastasis remains unknown. We investigated the differences in the TIME of primary lesions based on time intervals to metastasis, mainly between the synchronous group (SG; metastasis within 3 months) and metachronous group (MG; metastasis after 3 months), and its association with clinicopathological parameters in patients with mRCC. Overall, 568 patients treated first-line with vascular endothelial growth factor receptor inhibitors comprised the analysis population (SG: N = 307 [54.0%]; MG: N = 261 [46.0%]). SG had a higher proportion of patients with poor prognostic pathological feature tumors: WHO/ISUP grade 4, necrosis, lymphovascular invasion, infiltrative growth pattern, and sarcomatoid differentiation. Regarding the TIME, more immunogenic features were seen in SG than MG, with a higher PD-L1 positivity and a lower proportion of the desert phenotype. This is the first study to examine the differences in the TIME of primary lesions in patients with mRCC based on the time intervals to metastasis. The TIME of primary lesions could affect the time to metastasis

The Association of Tumor Immune Microenvironment of the Primary Lesion with Time to Metastasis in Patients with Renal Cell Carcinoma : A Retrospective Analysis

The association between the tumor immune microenvironment (TIME) of primary lesions and time to metastasis remains unknown. The aim of our retrospective study was to investigate the differences in the TIME of primary lesions based on time intervals to metastasis, mainly between the synchronous group (SG; metastasis within 3 months) and metachronous group (MG; metastasis after 3 months), and its association with clinicopathological parameters in patients with metastatic renal cell carcinoma (mRCC). SG showed more immunogenic feature of TIME (PD-L1 positivity, CD8+ TIL infiltration) and poor prognostic pathological features (WHO/ISUP grade 4, necrosis, lymphovascular invasion, infiltrative growth pattern, and sarcomatoid differentiation). In addition, we observed that the time to metastasis differed by TIME characteristics (PD-L1 status, immunophenotype), which were associated with the WHO/ISUP grade. The TIME of primary lesions could affect the time to metastasis.Biological or immunological differences in primary lesions between synchronous and metachronous metastatic renal cell carcinoma (mRCC) have been reported. However, the association between the tumor immune microenvironment (TIME) of primary lesions and time to metastasis remains unknown. We investigated the differences in the TIME of primary lesions based on time intervals to metastasis, mainly between the synchronous group (SG; metastasis within 3 months) and metachronous group (MG; metastasis after 3 months), and its association with clinicopathological parameters in patients with mRCC. Overall, 568 patients treated first-line with vascular endothelial growth factor receptor inhibitors comprised the analysis population (SG: N = 307 [54.0%]; MG: N = 261 [46.0%]). SG had a higher proportion of patients with poor prognostic pathological feature tumors: WHO/ISUP grade 4, necrosis, lymphovascular invasion, infiltrative growth pattern, and sarcomatoid differentiation. Regarding the TIME, more immunogenic features were seen in SG than MG, with a higher PD-L1 positivity and a lower proportion of the desert phenotype. This is the first study to examine the differences in the TIME of primary lesions in patients with mRCC based on the time intervals to metastasis. The TIME of primary lesions could affect the time to metastasis

Quantitative Evaluation of Interleukin-4 by Immunowall Devices Made of Gelatin Methacryloyl Hydrogel

Immunoassays, which use antigen–antibody reactions, are the primary techniques used to selectively quantify specific disease markers in blood. Conventional immunoassays, such as the microplate-based enzyme-linked immunosorbent assay (ELISA) and paper-based immunochromatography, are widely used, but they have advantages and disadvantages in terms of sensitivity and operating time. Therefore, in recent years, microfluidic-chip-based immunoassay devices with high sensitivity, rapidity and simplicity, which are compatible with whole blood assays and multiplex assays, have been actively investigated. In this study, we developed a microfluidic device using gelatin methacryloyl (GelMA) hydrogel to form a wall-like structure in a microfluidic channel and perform immunoassays inside the wall-like structure, which can be used for rapid and highly sensitive multiplex assays with extremely small sample amounts of ~1 L. The characteristics of GelMA hydrogel, such as swelling rate, optical absorption and fluorescence spectra, and morphology, were carefully studied to adapt the iImmunowall device and immunoassay. Using this device, a quantitative analysis of interleukin-4 (IL-4), a biomarker of chronic inflammatory diseases, was performed and a limit of detection (LOD) of 0.98 ng/mL was achieved with only 1 L sample and 25 min incubation time. The superior optical transparency over a wide range of wavelengths and lack of autofluorescence will help to expand the application field of the iImmunowall device, such as to a simultaneous multiple assay in a single microfluidic channel, and provide a fast and cost-effective immunoassay method

Resequencing and Association Analysis of <i>PTPRA</i>, a Possible Susceptibility Gene for Schizophrenia and Autism Spectrum Disorders

<div><p>Background</p><p>The <i>PTPRA</i> gene, which encodes the protein RPTP-α, is critical to neurodevelopment. Previous linkage studies, genome-wide association studies, controlled expression analyses and animal models support an association with both schizophrenia and autism spectrum disorders, both of which share a substantial portion of genetic risks.</p><p>Methods</p><p>We sequenced the protein-encoding areas of the <i>PTPRA</i> gene for single nucleotide polymorphisms or small insertions/deletions (InDel) in 382 schizophrenia patients. To validate their association with the disorders, rare (minor allele frequency <1%), missense mutations as well as one InDel in the 3′UTR region were then genotyped in another independent sample set comprising 944 schizophrenia patients, 336 autism spectrum disorders patients, and 912 healthy controls.</p><p>Results</p><p>Eight rare mutations, including 3 novel variants, were identified during the mutation-screening phase. In the following association analysis, L59P, one of the two missense mutations, was only observed among patients of schizophrenia. Additionally, a novel duplication in the 3′UTR region, 174620_174623dupTGAT, was predicted to be located within a Musashi Binding Element.</p><p>Major Conclusions</p><p>No evidence was seen for the association of rare, missense mutations in the <i>PTPRA</i> gene with schizophrenia or autism spectrum disorders; however, we did find some rare variants with possibly damaging effects that may increase the susceptibility of carriers to the disorders.</p></div

In silico functional effect prediction for rs61742029 and L59P.

<p>In silico functional effect prediction for rs61742029 and L59P.</p

Targeted sequencing areas of the <i>PTPRA</i> Gene.

<p>Targeted sequencing areas of the <i>PTPRA</i> Gene.</p

Evolutionary conservation information for rs61742029 and L59P

<p>*Marks the position of the amino acid change due to mutation.</p><p>Evolutionary conservation information for rs61742029 and L59P</p

Structure of the <i>PTPRA</i> gene, RPTP-α, and position of discovered rare missense mutations.

<p>Structure of the <i>PTPRA</i> gene, RPTP-α, and position of discovered rare missense mutations.</p

Profiles of participants in the resequencing and association sample sets.

<p>Note: Some samples in the association study group were not identified by sex.</p><p>Profiles of participants in the resequencing and association sample sets.</p