Persistent elevation of lysophosphatidylcholine promotes radiation brain necrosis with microglial recruitment by P2RX4 activation

Brain radiation necrosis (RN) or neurocognitive disorder is a severe adverse effect that may occur after radiation therapy for malignant brain tumors or head and neck cancers. RN accompanies inflammation which causes edema or micro-bleeding, and no fundamental treatment has been developed. In inflammation, lysophospholipids (LPLs) are produced by phospholipase A2 and function as bioactive lipids involved in sterile inflammation in atherosclerosis or brain disorders. To elucidate its underlying mechanisms, we investigated the possible associations between lysophospholipids (LPLs) and RN development in terms of microglial activation with the purinergic receptor P2X purinoceptor 4 (P2RX4). We previously developed a mouse model of RN and in this study, measured phospholipids and LPLs in the brains of RN model by liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses. We immune-stained microglia and the P2RX4 in the brains of RN model with time-course. We treated RN model mice with ivermectin, an allosteric modulator of P2RX4 and investigate the effect on microglial activation with P2RX4 and LPLs’ production, and resulting effects on overall survival and working memory. We revealed that LPLs (lysophosphatidylcholine (LPC), lysophosphatidyl acid, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylglycerol) remained at high levels during the progression of RN with microglial accumulation, though phospholipids elevations were limited. Both microglial accumulation and activation of the P2RX4 were attenuated by ivermectin. Moreover, the elevation of all LPLs except LPC was also attenuated by ivermectin. However, there was limited prolongation of survival time and improvement of working memory disorders. Our findings suggest that uncontrollable increased LPC, even with ivermectin treatment, promoted the development of RN and working memory disorders. Therefore, LPC suppression will be essential for controlling RN and neurocognitive disorder after radiation therapy

Dietary Intake of Green Nut Oil or DHA Ameliorates DHA Distribution in the Brain of a Mouse Model of Dementia Accompanied by Memory Recovery

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, has significant health benefits. Previous studies reported decreased levels of DHA and DHA-containing phosphatidylcholines in the brain of animals suffering from Alzheimer’s disease, the most common type of dementia; furthermore, DHA supplementation has been found to improve brain DHA levels and memory efficiency in dementia. Oil extracted from the seeds of Plukenetia volubilis (green nut oil; GNO) is also expected to have DHA like effects as it contains approximately 50% α-linolenic acid, a precursor of DHA. Despite this, changes in the spatial distribution of DHA in the brain of animals with dementia following GNO or DHA supplementation remain unexplored. In this study, desorption electrospray ionization imaging mass spectrometry (DESI-IMS) was applied to observe the effects of GNO or DHA supplementation upon the distribution of DHA in the brain of male senescence-accelerated mouse-prone 8 (SAMP8) mice, a mouse model of dementia. DESI-IMS revealed that brain DHA distribution increased 1.85-fold and 3.67-fold in GNO-fed and DHA-fed SAMP8 mice, respectively, compared to corn oil-fed SAMP8 mice. Memory efficiency in SAMP8 mice was also improved by GNO or DHA supplementation. In summary, this study suggests the possibility of GNO or DHA supplementation for the prevention of dementia

Spatial distribution of the Shannon entropy for mass spectrometry imaging.

Mass spectrometry imaging (MSI) allows us to visualize the spatial distribution of molecular components in a sample. A large amount of mass spectrometry data comprehensively provides molecular distributions. In this study, we focus on the information in the obtained data and use the Shannon entropy as a quantity to analyze MSI data. By calculating the Shannon entropy at each pixel on a sample, the spatial distribution of the Shannon entropy is obtained from MSI data. We found that low-entropy pixels in entropy heat maps for kidneys of mice had different structures between two ages (3 months and 31 months). Such changes cannot be visualized by conventional imaging techniques. We further propose a method to find informative molecules. As a demonstration of the proposed scheme, we identified two molecules by setting a region of interest which contained low-entropy pixels and by exploring changes of peaks in the region

Spatial distribution of the Shannon entropy for mass spectrometry imaging

Mass spectrometry imaging (MSI) allows us to visualize the spatial distribution of molecular components in a sample. A large amount of mass spectrometry data comprehensively provides molecular distributions. In this study, we focus on the information in the obtained data and use the Shannon entropy as a quantity to analyze MSI data. By calculating the Shannon entropy at each pixel on a sample, the spatial distribution of the Shannon entropy is obtained from MSI data. We found that low-entropy pixels in entropy heat maps for kidneys of mice had different structures between two ages (3 months and 31 months). Such changes cannot be visualized by conventional imaging techniques. We further propose a method to find informative molecules. As a demonstration of the proposed scheme, we identified two molecules by setting a region of interest which contained low-entropy pixels and by exploring changes of peaks in the region

Establishment of HLA-DR4 Transgenic Mice for the Identification of CD4⁺ T Cell Epitopes of Tumor-Associated Antigens

<div><p>Reports have shown that activation of tumor-specific CD4⁺ helper T (Th) cells is crucial for effective anti-tumor immunity and identification of Th-cell epitopes is critical for peptide vaccine-based cancer immunotherapy. Although computer algorithms are available to predict peptides with high binding affinity to a specific HLA class II molecule, the ability of those peptides to induce Th-cell responses must be evaluated. We have established HLA-DR4 (<i>HLA-DRA*01:01/HLA-DRB1*04:05</i>) transgenic mice (Tgm), since this HLA-DR allele is most frequent (13.6%) in Japanese population, to evaluate HLA-DR4-restricted Th-cell responses to tumor-associated antigen (TAA)-derived peptides predicted to bind to HLA-DR4. To avoid weak binding between mouse CD4 and HLA-DR4, Tgm were designed to express chimeric HLA-DR4/I-E^d, where I-E^d α1 and β1 domains were replaced with those from HLA-DR4. Th cells isolated from Tgm immunized with adjuvant and HLA-DR4-binding cytomegalovirus-derived peptide proliferated when stimulated with peptide-pulsed HLA-DR4-transduced mouse L cells, indicating chimeric HLA-DR4/I-E^d has equivalent antigen presenting capacity to HLA-DR4. Immunization with CDCA1_55-78 peptide, a computer algorithm-predicted HLA-DR4-binding peptide derived from TAA CDCA1, successfully induced Th-cell responses in Tgm, while immunization of HLA-DR4-binding Wilms' tumor 1 antigen-derived peptide with identical amino acid sequence to mouse ortholog failed. This was overcome by using peptide-pulsed syngeneic bone marrow-derived dendritic cells (BM-DC) followed by immunization with peptide/CFA booster. BM-DC-based immunization of KIF20A_494-517 peptide from another TAA KIF20A, with an almost identical HLA-binding core amino acid sequence to mouse ortholog, successfully induced Th-cell responses in Tgm. Notably, both CDCA1_55-78 and KIF20A_494-517 peptides induced human Th-cell responses in PBMCs from HLA-DR4-positive donors. Finally, an HLA-DR4 binding DEPDC1_191-213 peptide from a new TAA DEPDC1 overexpressed in bladder cancer induced strong Th-cell responses both in Tgm and in PBMCs from an HLA-DR4-positive donor. Thus, the HLA-DR4 Tgm combined with computer algorithm was useful for preliminary screening of candidate peptides for vaccination.</p> </div

Chimeric HLA-DR4/I-E^d molecules induced the differentiation of mouse CD4⁺ cells.

<p>(Upper panels) PBMCs (gated on lymphocytes) from ^{<i>MHC-II</i>} knock-out mice (DR^-/-MHC-II^-/-), Tgm (DR^+/-MHC-II^+/+) and Tgm that lack ^{<i>MHC-II</i>} (DR^+/-MHC-II^-/-) were stained with anti-HLA-DR and anti-mouse CD4 mAbs. (Lower panels) PBMC (gated on lymphocytes) from each mouse was stained with anti-B220 mAb and anti-mouse MHC-II mAb.</p

Selection of transgene-positive founder mice (F₀).

<p>Twenty five F₀ mice were arbitrarily numbered #1~#25. PBMCs were analyzed for HLA-DR4/I-E^d expression by flow-cytometric analyses (gated on lymphocytes) using anti-HLA-DRα and β mAbs (A) and genomic PCR analyses (B). A typical dot blot of PBMCs from other mice negative for transgenes is shown (null (#9) in A) and results from representative 10 mice from 25 F₀ mice are shown in B.</p