Tumor-Associated Macrophages: Therapeutic Targets for Skin Cancer

Tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) are significant components of the microenvironment of solid tumors in the majority of cancers. TAMs sequentially develop from monocytes into functional macrophages. In each differentiation stage, TAMs obtain various immunosuppressive functions to maintain the tumor microenvironment (e.g., expression of immune checkpoint molecules, production of Treg-related chemokines and cytokines, production of arginase I). Although the main population of TAMs is immunosuppressive M2 macrophages, TAMs can be modulated into M1-type macrophages in each differential stage, leading to the suppression of tumor growth. Because the administration of certain drugs or stromal factors can stimulate TAMs to produce specific chemokines, leading to the recruitment of various tumor-infiltrating lymphocytes, TAMs can serve as targets for cancer immunotherapy. In this review, we discuss the differentiation, activation, and immunosuppressive function of TAMs, as well as their benefits in cancer immunotherapy

Efficient Assay for Total Antioxidant Capacity in Human Plasma Using a 96-Well Microplate

In the present study, we tried to establish an efficient assay for total antioxidant capacity (TAC) in human plasma using a 96-well microplate. TAC was assessed using lag time by antioxidants against the myoglobin-induced oxidation of 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) with hydrogen peroxide, and expressed as Trolox equivalent. The linearity of the calibration curve with Trolox was maintained with the Trolox concentration range from 2.5 µM to 25 µM (R2 = 0.997). The assay was applied to the measurement of TAC in healthy human plasma. Coefficient of variation in intraday assay was 2.4%. Difference was not observed in interday assay. Plasma TAC of men ((569 ± 41) µM Trolox equivalent; n = 6) was higher than that of women ((430 ± 28) µM Trolox equivalent; n = 4). After the vegetable juice was drunk for 1 week, the increase in plasma TAC was observed in almost all the volunteers. In summary, we developed the efficient assay for plasma TAC using a 96-well microplate

Preparation and Characterization of a Polyclonal Antibody against Brominated Protein

(Di)bromotyrosine is formed by the specific reaction of eosinophil peroxidase and can be used as an eosinophil activation marker. In the present study, an antibody for (di)bromotyrosine in proteins was prepared to investigate the pathogenesis of eosinophil-related diseases such as allergic responses. A rabbit polyclonal antibody was raised against brominated keyhole limpet hemocyanin. The specificity of the antiserum was investigated with an enzyme-linked immunosorbent assay (ELISA). The antiserum recognized brominated bovine serum albumin (BSA) and dibromotyrosine-conjugated BSA. The antiserum also reacted with chlorinated BSA and di-iodotyrosine-conjugated BSA. Moreover, the specificity of the antiserum was investigated using competitive ELISA. Dibromotyrosine and di-iodotyrosine inhibited the recognition of brominated BSA by the antiserum. However, the recognition of brominated BSA by the antiserum was not inhibited by bromotyrosine, chlorotyrosine, iodotyrosine, nitrotyrosine, aminotyrosine, phosphotyrosine, or tyrosine. These results suggested that the epitope of the antiserum is dihalogenated tyrosine. Immunohistochemically, the antiserum stained brominated rat eosinophils but not chlorinated or nitrated eosinophils. In conclusion, an antiserum for dihalogenated protein was prepared. It is expected that the antiserum will be useful for the analysis of the pathogenesis of allergic diseases such as asthma and atopic dermatitis

Biochemical characterization of reactive nitrogen species by eosinophil peroxidase in tyrosine nitration

It is well known that eosinophils are involved in tyrosine nitration. In this study, we evaluated tyrosine nitration by rat eosinophils isolated from peritoneal fl uid and constituent eosinophils in the stomach. Rat peritoneal eosinophils activated with 1 &#956;M phorbol myristate acetate (PMA) and 50 &#956;M NO2 &#65437; showed immunostaining for nitrotyrosine only in smaller cells, despite the fact that eosinophils are capable of producing superoxide (O2·&#65437;). Free tyrosine nitrating capacity after incubation with PMA and NO2 &#65437; was 4-fold higher in eosinophils than in neutrophils. Catalase and &#65400;- and &#65402; -tocopherol inhibited free tyrosine nitration by reactive nitrogen species from eosinophils but not that by peroxynitrite. Superoxide dismutase augmented free tyrosine nitration by activated eosinophils and peroxynitrite. The concentration of nitric oxide released from eosinophils was relatively low (0.32 &#956;M/106 cells/h) and did not contribute to the formation of nitrotyrosine. On the other hand, most constituent eosinophils constituent in the rat stomach stimulated by PMA and NO2 &#65437; showed tyrosine nitration capacity. These results suggest that intact cells other than apoptotic-like eosinophils eluted in the intraperitoneal cavity could not generate reactive species responsible for nitration by a peroxidase-dependent mechanism. In contrast, normal eosinophils in the stomach were capable of nitration, suggesting that the characteristics of eosinophils in gastric mucosa are diff erent from those eluted in the peritoneal cavity.</p

Formation of Superoxide Anion during Ferrous Ion-Induced Decomposition of Linoleic Acid Hydroperoxide under Aerobic Conditions

金沢大学医薬保健研究域医学系We studied the mechanism of formation of oxygen radicals during ferrous ion-induced decomposition of linoleic acid hydroperoxide using the spin trapping and chemiluminescence methods. The formation of the superoxide anion (O2.-) was verified in the present study. The hydroxyl radical is also generated through Fenton type decomposition of hydrogen peroxide produced on disproportionation of O2.-. A carbon-centered radical was detected using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) as a spin trap. Alkoxyl radical formation is essential for the conversion of linoleic acid hydroperoxide into the peroxyl radical by ferrous ion. It is likely that the alkoxyl radical [R1CH(O.)R2] is converted into the hydroxylcarbon radical [R1C.(OH)R2] in water, and that this carbon radical reacts with oxygen to give the α-hydroxyperoxyl radical [R1R2C(OH)OO.], which decomposes into the carbocation [R1C+(OH)R 2] and O2.-

Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD

金沢大学医薬保健研究域医学系3-Nitrotyrosine (NTyr) is considered as a biomarker of the generation of reactive nitrogen species (RNS). However, it is still difficult to determine its concentration in biological samples. To develop a reliable and high-throughput method, we optimized the conditions for high performance liquid chromatography and electrochemical detection (HPLC-ECD). The best separation of NTyr was achieved using a highly acidic mobile phase (pH 2.5). The concentration of protein-bound NTyr in plasma protein was 593.6 ± 53.8 fmol/mg in rats treated with lipopolysaccharide (LPS) and 114.4 ± 27.6 fmol/mg in control. After intravenous administration of in vitro-nitrated plasma protein, NTyr concentration decreased; the half-life was 63.4 ± 16.8 h. Consistently, protein-bound NTyr concentration in plasma after LPS treatment declined gradually, but was detectable for 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo. © 2007 The Japanese Biochemical Society

Exacerbation of daily cough and allergic symptoms in adult patients with chronic cough by Asian dust: A hospital-based study in Kanazawa

The health effects associated with Asian dust have attracted attention due to the rapid increase in the number of Asian dust events in East Asia in recent years. The aim of this study was to investigate the associations between Asian dust and daily cough, as well as allergic symptoms, in adult patients who suffer from chronic cough. We enrolled 86 adult patients from Kanazawa University Hospital, Japan, who were diagnosed with asthma, cough variant asthma, atopic cough or a combination of these conditions. From January to June 2011, subjects recorded their symptoms in a diary every day. Asian dust and non-Asian dust periods were defined according to the dust extinction coefficient, measured using the light detection and ranging (LIDAR). The daily levels of total suspended particulates, polycyclic aromatic hydrocarbons (PAHs) and coexisting factors related to allergies, such as the Japanese cedar pollen count, were measured. McNemar\u27s test showed that there were significantly more cough-positive patients during Asian dust periods than during the non-Asian dust period (p = 0.022). In addition, during Asian dust periods when the daily levels of Japanese cedar pollen, Japanese cypress pollen and PAHs were elevated, there were significantly more patients who experienced itchy eyes than during the non-Asian dust period (p < 0.05). On the other hand, there were no significant differences in the allergic symptoms, including sneezing or a runny nose and nasal congestion. This is the first report to show that Asian dust triggers cough and allergic symptoms in adult patients with chronic cough. © 2014 Elsevier Ltd. All rights reserved

Effects of Asian dust on daily cough occurrence in patients with chronic cough: A panel study

Asian dust, known as kosa in Japanese, is a major public health concern. In this panel study, we evaluated the effects of exposure to kosa on daily cough occurrence. The study subjects were 86 patients being treated for asthma, cough variant asthma, or atopic cough in Kanazawa University Hospital from January 2011 to June 2011. Daily mean concentrations of kosa and spherical particles were obtained from light detection and ranging (LIDAR) measurements, and were categorized from Grade 1 (0μg/m3) to 5 (over 100μg/m3). The association between kosa and cough was analyzed by logistic regression with a generalized estimating equation. Kosa effects on cough were seen for all Grades with potential time lag effect. Particularly at Lag 0 (the day of exposure), a dose-response relationship was observed: the odds ratios for Grades 2, 3, 4, and 5 above the referent (Grade 1) were 1.111 (95% confidence interval (CI): 0.995-1.239), 1.171 (95% CI: 1.006-1.363), 1.357 (95% CI: 1.029-1.788), and 1.414 (95% CI: 0.983-2.036), respectively. Among the patients without asthma, the association was higher: the odds ratios for Grades 2, 3, 4 and 5 were 1.223 (95% CI: 0.999-1.497), 1.309 (95% CI: 0.987-1.737), 1.738 (95% CI: 1.029-2.935) and 2.403 (95% CI: 1.158-4.985), respectively. These associations remained after adjusting for the concentration of spherical particles or particulate matter with an aerodynamic diameter of less than 2.5μm (PM2.5). Our findings demonstrate that kosa is an environmental factor which induces cough in a dose-response relationship. © 2014 Elsevier Ltd

PRMT5 Associates With the FOXP3 Homomer and When Disabled Enhances Targeted p185erbB2/neu Tumor Immunotherapy

Regulatory T cells (Tregs) are a subpopulation of T cells that are specialized in suppressing immune responses. Here we show that the arginine methyl transferase protein PRMT5 can complex with FOXP3 transcription factors in Tregs. Mice with conditional knock out (cKO) of PRMT5 expression in Tregs develop severe scurfy-like autoimmunity. In these PRMT5 cKO mice, the spleen has reduced numbers of Tregs, but normal numbers of Tregs are found in the peripheral lymph nodes. These peripheral Tregs that lack PRMT5, however, display a limited suppressive function. Mass spectrometric analysis showed that FOXP3 can be di-methylated at positions R27, R51, and R146. A point mutation of Arginine (R) 51 to Lysine (K) led to defective suppressive functions in human CD4 T cells. Pharmacological inhibition of PRMT5 by DS-437 also reduced human Treg functions and inhibited the methylation of FOXP3. In addition, DS-437 significantly enhanced the anti-tumor effects of anti-erbB2/neu monoclonal antibody targeted therapy in Balb/c mice bearing CT26Her2 tumors by inhibiting Treg function and induction of tumor immunity. Controlling PRMT5 activity is a promising strategy for cancer therapy in situations where host immunity against tumors is attenuated in a FOXP3 dependent manner