Eggshell membrane modulates gut microbiota to prevent murine pre-cachexia through suppression of T helper cell differentiation.

BACKGROUND: Cachexia is a life-threatening condition observed in several pathologies, such as cancer or chronic diseases. Interleukin 10 (Il10) gene transfer is known to improve cachexia by downregulating Il6. Here, we used an IL10-knockout mouse model to simulate cachexia and investigate the effects of eggshell membrane (ESM), a resistant protein, on general pre-cachexia symptoms, which is particularly important for the development of cachexia therapeutics. METHODS: Five-week-old male C57BL6/J mice were fed an AIN-93G powdered diet (WT), and 5-week-old male B6.129P2-Il10 < tm1Cgn>/J (IL10-/- ) mice were fed either the AIN-93G diet (KO) or an 8% ESM-containing diet (KOE) for 28 weeks. The tissue weight and levels of anaemia-, blood glucose-, lipid metabolism-, and muscular and colonic inflammation-related biochemical markers were measured. Transcriptomic analysis on liver and colon mucus and proteomic analysis on skeletal muscle were performed. Ingenuity Pathway Analysis was used to identify molecular pathways and networks. Caecal short-chain fatty acids (SCFAs) were identified using HPLC, and caecal bacteria DNA were subjected to metagenomic analysis. Flow cytometry analysis was performed to measure the CD4+ IL17+ T cells in mesenteric lymph nodes. RESULTS: The body weight, weight of gastrocnemius muscle and fat tissues, colon weight/length ratio, plasma HDL and NEFA, muscular PECAM-1 levels (P < 0.01), plasma glucose and colonic mucosal myeloperoxidase activity (P < 0.05) and T helper (Th) 17 cell abundance (P = 0.071) were improved in KOE mice over KO mice. Proteomic analysis indicated the protective role of ESM in muscle weakness and maintenance of muscle formation (>1.5-fold). Transcriptomic analysis revealed that ESM supplementation suppressed the LPS/IL1-mediated inhibition of RXR function pathway in the liver and downregulated the colonic mucosal expression of chemokines and Th cell differentiation-related markers (P < 0.01) by suppressing the upstream BATF pathway. Analysis of the intestinal microenvironment revealed that ESM supplementation ameliorated the microbial alpha diversity and the abundance of microbiota associated with the degree of inflammation (P < 0.05) and increased the level of total organic acids, particularly of SCFAs such as butyrate (2.3-fold), which could inhibit Th1 and Th17 production. CONCLUSIONS: ESM supplementation ameliorated the chief symptoms of cachexia, including anorexia, lean fat tissue mass, skeletal muscle wasting and reduced physical function. ESM also improved colon and skeletal muscle inflammation, lipid metabolism and microbial dysbiosis. These results along with the suppressed differentiation of Th cells could be associated with the beneficial intestinal microenvironment and, subsequently, attenuation of pre-cachexia. Our findings provide insights into the potential of ESM in complementary interventions for pre-cachexia prevention.

Occlusional Modifications Reversibly Alter Aquaporin 5 Expression and Localization in Rat Salivary Glands.

BACKGROUND: Aquaporin 5 (AQP5) is a water channel-forming protein that plays a key role in saliva secretion. A decrease in masticatory function associated with the molar extraction adversely affects the submandibular salivary gland (SMG) in rats, inducing hypertrophic changes in the acinar cells and the expression of AQP5 in acinar cells or intercalated duct of the SMG. However, changes in AQP5 expression and localization in the SMG in association with occlusal modification have not been fully characterized. METHODS: We examined the influence of the decline and recovery of masticatory function on expression and localization of AQP5 in the rat SMG by inserting and removing an incisor bite plate (IBP). Thirty 5-week-old male Wistar rats were randomly divided into IBP (n = 12), recovery (REC) (n = 6), and control (CON) (n = 12) groups. Each rat in both the IBP and REC groups was fitted with the IBP on its maxillary incisors. Rats without the IBPs served as controls. All rats were fed powder diet and water ad libitum. Rats in the IBP and CON groups were sacrificed after 14 (n = 6) and 28 (n = 6) days after the IBP attachment. In the REC group, the IBP was detached on the 14th day and sacrificed on 28th day after the IBP attachment. AQP5 mRNA expression was quantified by reverse transcription-polymerase chain reaction. Changes in the localization of AQP5 were tracked by immunohistochemical staining. RESULTS: Attachment of IBP resulted in a decrease in the expression of AQP5 in the IBP group. Changes in the localization of AQP5 were observed between 14 and 28 days in the IBP group. In contrast, changes in the expression and localization of AQP5 were not observed in the REC group. CONCLUSION: Findings suggested that a loss of molar occlusion, due to the IBP attachment, altered AQP5 expression and localization in the rat SMG. However, removal of the bite plate allowed the recovery of both AQP5 expression and its normal localization in the SMGs.

Alterations in the gustatory papillae after anterior bite plate insertion in growing rats.

OBJECTIVES: To determine whether the modification of dental occlusion, without molar extraction, affected the gustatory papillae located in the tongue of growing rats. MATERIALS AND METHODS: Five-week-old male Wistar rats were randomly divided into an anterior bite plate (ABP) group and a control group. Under general anesthesia, ABPs were placed on the occlusal surfaces of the maxillary incisors, while metal caps covered the mandibular incisal edges of the rats in the ABP group. The control group rats underwent a sham operation. The rats in both groups were euthanized 14 days after the procedure. The circumvallate papillae and taste buds were analyzed by immunohistochemical methods, and the fungiform papillae were observed and counted after immersion of the tongue in 1% methylene blue. RESULTS: Two weeks after ABP insertion and mandibular incisal cap placement, the gustatory papillae exhibited morphological and structural changes. The rats in the ABP group had exhibited significantly fewer fungiform papillae, and narrower circumvallate papillae, with greater trench depths, larger trench profile areas, smaller taste bud profile areas, lower ratios of the taste bud profile area to the trench profile area, and more taste buds than those in the control group. CONCLUSIONS: Our findings support the association between occlusal and taste functions and provide a basis for further studies on the gustatory function. In conclusion, loss of molar occlusion, resulting from the ABP and metal cap insertion, altered the peripheral gustatory receptors in the growing rats.

Analysis of microvascular density in early gastric carcinoma using magnifying endoscopy with narrow-band imaging.

BACKGROUND AND STUDY AIMS: Intramucosal vascular density differs between differentiated and undifferentiated type gastric carcinomas. This study aimed to evaluate the microvascular density characteristics of these two types of carcinoma using magnifying endoscopy with narrow-band imaging (ME-NBI). PATIENTS AND METHODS: In total, 42 differentiated and 10 undifferentiated types were evaluated. The microvessels observed using ME-NBI were extracted from stored still images and the microvascular density in the two carcinoma types was analyzed. Histological vascular density in resected specimens was also evaluated using CD34 immunostaining. RESULTS: There were significant differences between the microvascular density in the differentiated and undifferentiated types of carcinoma (10.02 ±â€Š4.72 % vs 4.02 ±â€Š0.40 %; P < 0.001) using ME-NBI. Vascular density assessed histologically also differed significantly between differentiated and undifferentiated types in both the whole mucosal (5.81 ±â€Š3.17 % vs 3.25 ±â€Š1.21 %) and the superficial mucosal layers (0 - 100 µm) (6.38 ±â€Š3.73 % vs 3.66 ±â€Š1.46 %). However, the vascular density in the surrounding non-carcinomatous mucosa assessed using ME-NBI and histologically, was significantly lower in the differentiated than in the undifferentiated types (P < 0.001). There was good agreement between ME-NBI and histologically assessed microvascular density in both the whole (r = 0.740; P < 0.001) and superficial mucosal layers (r = 0.764; P < 0.001). White opaque substance (WOS) was seen in eight patients who had the differentiated type carcinoma. In almost all cases with WOS, the appearance of the carcinoma was discolored. CONCLUSIONS: There was a close relationship between ME-NBI assessed microvascular density and histologically assessed vascular density in the mucosal layer. Microvascular density differed significantly between the differentiated and undifferentiated types of carcinoma assessed using ME-NBI.

Biochemical features of a catalytic antibody light chain, 22F6, prepared from human lymphocytes.

Human antibody light chains belonging to subgroup II of germ line genes were amplified by a seminested PCR technique using B-lymphocytes taken from a human adult infected with influenza virus. Each gene of the human light chains was transferred into the Escherichia coli system. The recovered light chain was highly purified using a two-step purification system. Light chain 22F6 showed interesting catalytic features. The light chain cleaved a peptide bond of synthetic peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates, such as QAR-MCA and EAR-MCA, indicating amidase activity. It also hydrolyzed a phosphodiester bond of both DNA and RNA. From the analysis of amino acid sequences and molecular modeling, the 22F6 light chain possesses two kinds of active sites as amidase and nuclease in close distances. The 22F6 catalytic light chain could suppress the infection of influenza virus type A (H1N1) of Madin-Darby canine kidney cells in an in vitro assay. In addition, the catalytic light chain clearly inhibited the infection of the influenza virus of BALB/c mice via nasal administration in an in vivo assay. In the experiment, the titer in the serum of the mice coinfected with the 22F6 light chain and H1N1 virus became considerably lowered compared with that of 22F6-non-coinfected mice. Note that the catalytic light chain was prepared from human peripheral lymphocyte and plays an important role in preventing infection by influenza virus. Considering the fact that the human light chain did not show any acute toxicity for mice, our procedure developed in this study must be unique and noteworthy for developing new drugs.

Role of chlorogenic acid quinone and interaction of chlorogenic acid quinone and catechins in the enzymatic browning of apple.

Chlorogenic acid (CQA) is one of the major polyphenols in apple and a good substrate for the polyphenol oxidase (PPO) in apple. Apple contains catechins as well as CQA, and the role of CQA quinone and its interaction with catechins in the enzymatic browning of apple were examined. Browning was repressed and 2-cysteinyl-CQA was formed when cysteine was added to apple juice. CQA quinone was essential for browning to occur. Although catechins and CQA were oxidized by PPO, some catechins seemed to be non-enzymatically oxidized by CQA quinone.