Development of biotinylated and magnetic bead-immobilized enzymes for efficient glyco-engineering and isolation of antibodies.

The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.

A common glycan structure on immunoglobulin G for enhancement of effector functions.

Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.

The Pathogenicity of Shewanella algae and Ability to Tolerate a Wide Range of Temperatures and Salinities.

Shewanella algae is a rod-shaped Gram-negative marine bacterium frequently found in nonhuman sources such as aquatic ecosystems and has been shown to be the pathogenic agent in various clinical cases due to the ingestion of raw seafood. The results of this study showed that S. algae was present in approximately one in four samples, including water and shellfish samples. Positive reactions (API systems) in S. algae strains were seen for gelatinase (gelatin); however, negative reactions were found for indole production (tryptophan). S. algae is adapted to a wide range of temperatures (4°C, 25°C, 37°C, and 42°C) and salinity. Temperature is a key parameter in the pathogenicity of S. algae as it appears to induce hemolysis at 25°C and 37°C. S. algae exhibits pathogenic characteristics at widely varying temperatures, which suggests that it may have the ability to adapt to climate change.

Litchi seed extract inhibits epidermal growth factor receptor signaling and growth of Two Non-small cell lung carcinoma cells.

BACKGROUND: Litchi seeds possess rich amounts of phenolics and have been shown to inhibit proliferation of several types of cancer cells. However, the suppression of EGFR signaling in non-small cell lung cancer (NSCLC) by litchi seed extract (LCSE) has not been fully understood. METHODS: In this study, the effects of LCSE on EGFR signaling, cell proliferation, the cell cycle and apoptosis in A549 adenocarcinoma cells and NCI- H661 large-cell carcinoma cells were examined. RESULTS: The results demonstrated that LCSE potently reduced the number of cancer cells and induced growth inhibition, cell-cycle arrest in the G1 or G2/M phase, and apoptotic death in the cellular experiment. Only low cytotoxicity effect was noted in normal lung MRC-5 cells. LCSE also suppressed cyclins and Bcl-2 and elevated Kip1/p27, Bax and caspase 8, 9 and 3 activities, which are closely associated with the downregulation of EGFR and its downstream Akt and Erk-1/-2 signaling. CONCLUSION: The results implied that LCSE suppressed EGFR signaling and inhibited NSCLC cell growth. This study provided in vitro evidence that LCSE could serve as a potential agent for the adjuvant treatment of NSCLC.

Different cellular responses of dexmedetomidine at infected site and peripheral blood of emdotoxemic BALB/c mice.

Various sedative agents, including dexmedetomidine (dex), induce immunosuppression, and enhance infection progression. However, there was no information on how anesthetic affects local and systemic cellular immune function. We conducted this study to examine the impact of dex on the differentiation and function of immune cells at site of inflammation and in peripheral blood during endotoxemia of mice. In BALB/c mice with and without endotoxemia, we evaluated the influence of two dosages of 5 and 50 mcg/kg/h intravenous dex on immune cells: including number of T cells (CD3), B cells (CD19), natural killer cells (CD8a), monocytes (CD11b), and macrophages (Mac-3) in peripheral blood, the activities of macrophages in peripheral blood and in peritoneal lavage, and proliferation of B and T cells and of natural killer cells activity in the spleen. Endotoxemia increased the number of CD3 T cells, CD 19 B cells and macrophages in the peripheral blood, augmented macrophage activity in the peritoneum, and increased T cell proliferation and natural killer cell activity in the spleen. Further administration of 5 mcg/kg/h dex attenuated systemic increase in number of T cells, B cells, and macrophages during endotoxemia and 50 mcg/kg/h dex significantly attenuated the increase in activity of macrophages in the peripheral blood during endotoxemia. In the peritoneum, however, 5 mcg/kg/h dex preserved and 50 mcg/kg/h dexmedetomidine enhanced the activity of macrophages during endotoxemia. Increased in proliferation of T cells in spleen during endotoxemia was attenuated by both doses of dex. Last, 50 mcg/kg/h dex enhanced natural killer cells activity during endotoxemia. While preserving the effects of endotoxemia on macrophage's activity in the infection site and natural killer cell's activity in the spleen, dex decreased systemic fulminant immune reaction in endotoxemia, by attenuating the augmented response in the number of T cells, B cells and macrophages, activity of macrophages in the peripheral blood, and proliferation of T cells in spleen during endotoxemia.

Tocilizumab Exerts Anti-Tumor Effects on Colorectal Carcinoma Cell Xenografts Corresponding to Expression Levels of Interleukin-6 Receptor.

The use of tocilizumab against the interleukin-6 receptor (IL-6R) has been demonstrated as inhibiting the progression of diverse cancers in vitro and in vivo. Nonetheless, evidence regarding the anti-tumor effects of tocilizumab on human colorectal carcinoma (CRC) corresponding to IL-6R expression levels remains scarce. To investigate the influence of IL-6R expression, SW480 and HT-29 cells inoculated subcutaneously into NU/NU mice were used as human CRC xenograft models with anti-IL-6R antibody (tocilizumab) therapy. The IL-6R expression levels, histology of CRC growth/invasiveness, and tumor growth-related signaling pathway were estimated by H&E and immunohistochemical staining. SW480 tumor cells with higher IL-6R expression levels showed better responsiveness in tocilizumab therapy than in the treated HT-29 group. Likewise, therapeutic effects of tocilizumab on the proliferative ability with mitotic index and Ki-67 expressions, invasiveness with MMP-9 proteinase expressions, and ERK 1/2 and STAT3 signaling transduction in the SW480 treatment group were superior to the HT-29 treatment group. In light of our results, IL-6R is the key indicator for the efficacy of tocilizumab treatment in CRC xenografts. From the perspective of precision medicine, tumor response to anti-IL-6R antibody therapy could be predicted on the basis of IL-6R expression levels. In this manner, tocilizumab may serve as a targeted and promising anti-CRC therapy.

Reevaluation of Hemoparasites in the Black Spiny-Tailed Iguana (Ctenosaura similis) with the First Pathological and Molecular Characterizations of Lankesterella desseri n. sp. and Redescription of Hepatozoon gamezi.

Hemoprotozoa are microorganisms that parasitize the blood and possess intricate life cycles. Despite the complexity of their nature, little is known about the biology of hemoprotozoa in reptilian hosts. In this study, we conducted disease surveillance on blood samples collected from six black spiny-tailed iguanas (Ctenosaura similis) exhibiting clinical signs. We found two different types of hemoparasites in the blood films and further confirmed they belong to the genera Lakesterella and Hepatozoon through molecular methods. In the tissue section from a dead iguana infected only with Lakesterella sp., parasites were also found in melanomacrophages of the liver and kidney. Since Lakesterella sp. infection has not been reported in C. similis, we propose this hemococcidian as a new species, Lankesterella desseri n. sp. The Hepatozoon parasites discovered in this study were classified as Hepatozoon gamezi based on their morphological characteristics, particularly the notable deformation of all infected erythrocytes, and this classification was further corroborated through molecular biological and phylogenetic analyses. This is the first hemoprotozoa investigation in C. similis with pathological and molecular characterization of these pathogens. We suggest that more studies are needed to understand the epidemiology, transmission, and impact of these parasites on their hosts and ecosystems.

Induction of apoptosis and cell cycle arrest in human colorectal carcinoma by Litchi seed extract.

The Litchi (Litchi chinensis) fruit products possess rich amounts of flavanoids and proanthocyanidins. Its pericarp has been shown to inhibit breast and liver cancer cell growth. However, the anticolorectal cancer effect of Litchi seed extract has not yet been reported. In this study, the effects of polyphenol-rich Litchi seed ethanol extract (LCSP) on the proliferation, cell cycle, and apoptosis of two colorectal cancer cell lines Colo320DM and SW480 were examined. The results demonstrated that LCSP significantly induced apoptotic cell death in a dose-dependent manner and arrested cell cycle in G2/M in colorectal carcinoma cells. LCSP also suppressed cyclins and elevated the Bax : Bcl-2 ratio and caspase 3 activity. This study provides in vitro evidence that LCSP serves as a potential chemopreventive agent for colorectal cancer.

Anti-interleukin-6 receptor antibody inhibits the progression in human colon carcinoma cells.

BACKGROUND: Interleukin-6 (IL-6) promotes proliferation and invasion in colorectal carcinoma, and serum IL-6 levels are correlated with survival in patients with colorectal carcinoma. In this study, we attempted to clarify the signal pathway downstream of IL-6 and the role of the IL-6 receptor complex in terms of the biological effects of clonogenic growth and invasiveness in colorectal carcinoma cells. MATERIALS AND METHODS: IL-6-stimulated SW480 cells were treated with IL-6 receptor neutralization antibody, mitogen-activated protein kinase (MAPK) inhibitor and phosphatidylinositol 3-kinase inhibitor, and clonogenic growth and invasiveness were assessed. IL-6 and IL-6 receptor-expressing LoVo cells were also tested the IL-6 receptor antibody effect. The downstream molecules of the IL-6-mediated pathway were also evaluated. RESULTS: IL-6 effectively enhanced the clonogenicity and invasiveness of SW480; however, these abilities were reversed by treatment with anti-IL-6 receptor antibody, and MAPK and PI3K inhibitors exhibited partial ability to reduce these effects. Similar effects were also found in anti-IL-6 receptor antibody-treated LoVo cells in addition of modulating STAT3 pathway. Anti-IL-6 receptor antibody also inhibited matrix metalloproteinase-2 (MMP-2) and 9 (MMP-9) expressions in IL-6-stimulated SW480. CONCLUSIONS: IL-6 and the IL-6R complex could induce clonogenic growth and invasiveness by mediating signals in the Ras/MAPK and PI3K/AKt pathways, and the malignant phenotypes might be associated with the production of MMP-2 and MMP-9 after IL-6 stimulation in SW480 cancer cells.

Total sleep deprivation augments balloon angioplasty-induced neointimal hyperplasia in rats.

Sleep deprivation has been shown to be associated with an increase in inflammation that is also involved in the development of neointimal hyperplasia (or restenosis). The purpose of this study was to investigate whether total sleep deprivation (TSD) would worsen neointimal formation by balloon injury. Sixteen rats were randomly allocated into the following four groups: group 1, balloon angioplasty alone; group 2, TSD prior to angioplasty; group 3, angioplasty before TSD; and group 4, TSD before and after angioplasty. Total sleep deprivation was induced by the disc-over-water method, and balloon angioplasty was performed in the carotid artery. Histopathological analysis and assay of cytokines were applied to evaluate the effects of TSD in this study. Total sleep deprivation significantly increased the ratio of postinjury neointima-to-media area in groups 2, 3 and 4 (all P < 0.01) compared with group 1. Additionally, in all groups with TSD administration the serum level of interleukin 10 was also markedly decreased on day 3 after angioplasty injury (P < 0.05 or P < 0.01). Our findings suggest that perioperative TSD can significantly augment neointimal hyperplasia of the carotid artery in rats, which may be partly caused by a TSD-induced effect in suppressing the serum level of the anti-inflammatory cytokine, interleukin 10.

Antibody to Interleukin-6 Receptor Inhibits In Vivo Growth of Human Colorectal Carcinoma Cell Xenografts.

BACKGROUND: Interleukin-6 receptor antibody (IL6R) inhibits colony formation and invasion by colorectal carcinoma (CRC) in vitro. We examined the effect of IL6R antibody on tumor growth of CRC xenografts in vivo. MATERIALS AND METHODS: SW480 cells inoculated subcutaneously into NU/NU mice were treated with anti-IL6R and tumor histology and growth-related signaling were subsequently estimated by hematoxylin and eosin and immunohistochemical staining. RESULTS: Tumor growth was inhibited by anti-IL6R treatment at dosages of both 0.1 and 1.0 mg/kg. Tumor cells had invaded into surrounding tissues in untreated mice, while there was no invasion of tumors in the IL6R antibody-treated mice. The expression of Ki-67, signal transducer and activator of transcription protein 3 (STAT3) and phosphor-extracellular signal-regulated kinase 1 and 2 (ERK1/2) were suppressed in anti-IL6R-treated tumors. CONCLUSION: IL6R antibody inhibited tumor growth and invasiveness in vivo by suppressing the expression of Ki-67, STAT3 and phosphor-ERK1/2. The results imply that the anti-IL6R may be a promising targeted drug for CRC.

Correction: Development of glycosynthases with broad glycan specificity for the efficient glyco-remodeling of antibodies.

Correction for 'Development of glycosynthases with broad glycan specificity for the efficient glyco-remodeling of antibodies' by Sachin S. Shivatare et al., Chem. Commun., 2018, 54, 6161-6164.

Development of glycosynthases with broad glycan specificity for the efficient glyco-remodeling of antibodies.

The first systematic investigation of the effect of high mannose, hybrid, and bi- and tri-antennary complex type glycans on the effector functions of antibodies was achieved by the discovery of novel Endo-S2 mutants generated by site-directed mutagenesis as glycosynthases with broad substrate specificity.

WISP-1 promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-300 in human oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC), which accounts for nearly 90% of head and neck cancers, is characterized by a poor prognosis and a low survival rate. Vascular endothelial growth factor-C (VEGF-C) has been implicated in lymphangiogenesis and is correlated with cancer metastasis. WNT1-inducible signaling pathway protein-1 (WISP)-1/CCN4 is an extracellular matrix-related protein that belongs to the CCN family and stimulates many biological functions. Our previous studies showed that WISP-1 plays an important role in OSCC migration and angiogenesis. However, the effect of WISP-1 on VEGF-C regulation and lymphangiogenesis in OSCC is poorly understood. Here, we showed a correlation between WISP-1 and VEGF-C in tissue specimens from patients with OSCC. To examine the lymphangiogenic effect of WISP-1, we used human lymphatic endothelial cells (LECs) to mimic lymphatic vessel formation. The results showed that conditioned media from WISP-1-treated OSCC cells promoted tube formation and cell migration in LECs. We also found that WISP-1-induced VEGF-C is mediated via the integrin αvß3/integrin-linked kinase (ILK)/Akt signaling pathway. In addition, the expression of microRNA-300 (miR-300) was inhibited by WISP-1 via the integrin αvß3/ILK/Akt cascade. Collectively, these results reveal the detailed mechanism by which WISP-1 promotes lymphangiogenesis via upregulation of VEGF-C expression in OSCC. Therefore, WISP-1 could serve as therapeutic target to prevent metastasis and lymphangiogenesis in OSCC.

Alteration of extracellular collagen matrix in the myocardium of canines infected with Dirofilaria immitis.

The heart consists of cardiocytes and the interstitial extracellular matrix (ECM), which is made up mainly of collagens. The ECM has been suggested to be important in maintaining the structure and function of the heart. This investigation attempted to elucidate the changes in the ECM collagens in the hearts of canines with dirofilariasis. The ECM collagen fibrils of the heart are grouped into endomysial struts, epimysial weaves, and perimysial coils. In the present study, we used the modified silver impregnation technique to stain paraffin-embedded sections to demonstrate three types of ECM. The results revealed that the ECM content of the heart was significantly reduced in heartworm-infected dogs, and became fragmented and dissociated. In addition, the amounts of collagen in the septum (Sep), RVs and LVs in canines with dirofilariasis (Sep=11.55+/-0.65, RV=12.07+/-0.59, LV=11.72+/-0.62 microg/mg, n=24) were significantly lower (p<0.01) than that in the normal canines (Sep=15.09+/-0.72, RV=15.16+/-0.83, LV=14.91+/-0.89 microg/mg, n=8). These results indicated that heartworm infection induced the remodeling of the extracellular matrix, thus markedly altering the architecture and function of the heart.

An abortion storm in cattle associated with neosporosis in Taiwan.

An abortion storm associated with acute neosporosis involving 18 cattle was observed in a dairy farm in Taiwan. Aborted fetus age ranged from 3 to 8 months. Of the 38 cattle in that farm examined during the abortion storm, 52.6% (20/38), 13.2% (5/38) and 10.5% (4/38) contained both IgG and IgM, only IgG and only IgM antibodies to Neospora caninum, respectively. No antibody to N. caninum was detected prior to the abortion storm. Follow-up study conducted a year later showed that 23 out of 28 cattle had sero-converted. Since some cattle were positive to either only IgG or IgM, we suggest that both IgG and IgM should be tested for diagnosing neosporosis. Neosporosis surveillance of naive cattle herd is recommended.

Investigation of gastrointestinal parasites of dairy cattle around Taiwan.

BACKGROUND: Parasitic nematodes are one of the most important causes of production losses in most cattle-producing countries of the world. The aim of the present study is to make a through estimate of helminth and protozoan infection prevalence in dairy cattle around Taiwan. METHODS: Coprological techniques, including direct fecal smear, simple flotation, and simple sedimentation, were used to detect gastrointestinal helminths and protozoan in dairy cattle. A total of 1259 rectal fecal samples were collected from Holstein dairy cattle at 94 farms in 13 counties in Taiwan. RESULTS: The overall prevalence of gastrointestinal parasitic infection was 86.9%. The infection rates of protozoa, nematodes, trematodes, and cestodes were 81.3%, 7.9%, 1.6%, and 0.6%, respectively. Among all parasites, Buxtonella sulcata (61.7%) was the most predominant one, followed with Cryptosporidium spp. (32.6%) and Eimeria spp. (11.8%). There were significant differences in the prevalence of protozoa and nematodes between different age groups and distributional area groups. CONCLUSION: The present study demonstrated that gastrointestinal parasitic infections occur frequently in dairy cattle around Taiwan, especially protozoan infections. The results indicated that a superior management system and regular anthelmintic treatment should be used for the control of parasitic infections in dairy cattle farms.

Grape-seed procyanidins inhibit the in vitro growth and invasion of pancreatic carcinoma cells.

OBJECTIVES: Grape-seed procyanidins (GSPs) can inhibit cell proliferation and invasiveness in various human cancers. However, the effect of GSP on pancreatic carcinoma cells has not been investigated. METHODS: Pancreatic carcinoma cell lines MIA PaCa-2 and BxPC-3 treated with GSP were assessed for viability by trypan blue exclusion, for cell cycle distribution by flow cytometry, for increased apoptosis by annexin V labeling, for their adhesion and invasion potential by evaluating their ability to penetrate through a matrix gel-coated Boyden chamber, and for changes in the levels of proteins involved in cellular events by immunoblotting. RESULTS: Grape-seed procyanidin inhibited MIA PaCa-2 and BxPC-3 proliferation in a dose-dependent manner and induced G1-phase arrest of the cell cycle in BxPC-3 or mitochondria-mediated apoptosis in MIA PaCa-2. Grape-seed procyanidin also inhibited the adhesion and invasion potential of both cell lines in a dose-dependent manner, which are associated with the suppression of metalloproteases matrix metalloproteinase 9 or 2 (MMP-9 or -2) expression. CONCLUSIONS: Grape-seed procyanidin inhibited the proliferation of pancreatic carcinoma cells by cell cycle blockage or apoptotic induction. The invasiveness was also suppressed by GSP through down-regulation of MMP-2 or MMP-9 in pancreatic carcinoma cells. Grape-seed procyanidin is a potential chemotherapeutic or preventive agent for pancreatic carcinoma.

Effects of sleep deprivation on serum testosterone concentrations in the rat.

Sleep deprivation (SD) leads to decreases in circulating levels of testosterone with unknown mechanisms. We tested the hypothesis that decreased testosterone levels associated with SD may be caused by serotonin-mediated inhibition of its production. Male rats were subjected to SD for 24 or 48 h using the dish-over-water-method with a Rechtschaffen apparatus. Serum testosterone, corticosterone and serotonin (5-HT) concentrations were assessed thereafter, as were testicular StAR and 5-HT 2 receptor levels. SD, regardless of duration led to significant decreases in serum testosterone levels and testicular steroid acute regulatory protein (StAR) protein expression, while 5-HT levels were significantly elevated (all P<0.05). Corticosterone concentrations were significantly increased in 48 h SD rats (P<0.05). In primary Leydig cell cultures, 5-HT decreased chorionic gonadotropin-induced testosterone secretion and StAR expression, which appeared to be dependent on 5-HT 2 receptor activation but independent of cyclic AMP signaling. These findings suggest that decreased serum testosterone levels in SD rats may be the result of 5-HT-related inhibition of testosterone production and decreased testicular expression of StAR protein.

Parotid tumors: a 10-year experience.

PURPOSE: The aim of the present study was to analyze the clinical presentation, histopathology, and complications of parotid tumors, as well as the management of malignant parotid tumors. METHODS: We retrospectively reviewed the medical records of 271 patients who underwent parotidectomy from August 1996 to July 2006. Data including age, sex, clinical signs and symptoms, histologic findings, complications, malignant tumor stage, and prognosis were collected from medical charts. RESULTS: Of the 271 patients who underwent parotidectomy, 229 (85%) had benign tumors, 33 (12%) had malignant tumors, and 9 had chronic inflammatory disease (3%). The most common benign tumor was pleomorphic adenoma (51%), and the most common malignant tumor was mucoepidermoid carcinoma (3%). The 5-year overall survival rate was 42%, and the disease-specific survival rate for malignant tumor was 72%. Only disease stage was the statistically significant prognostic factor of malignancy. The most common complication of parotidectomy was transient facial palsy (18%). CONCLUSIONS: Standardized superficial and total parotidectomy are safe procedures for treating parotid tumors. Management of malignant tumors depends on tumor stage and histologic grade. Advanced tumor stage is a predictor of poor outcome.