Production of specific antibodies against SARS-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses 229E and OC43

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43

NaCl plus chitosan as a dietary salt to prevent the development of hypertension in spontaneously hypertensive rats

The effect of NaCl plus 3% chitosan on the systolic blood pressure of spontaneously hypertensive rats (SHR) were evaluated and compared with NaCl plus KCl (NaCl, 49.36% + KCl 49.36%) and chitosan or NaCl treatment alone. In SHR, administration of NaCl plus chitosan (44 mM Na/day) for two months significantly decreased the systolic blood pressure greater than of NaCl plus KCl and NaCl alone. NaCl plus chitosan resulted, though not statistically significant, in decreased urinary Na+ excretion and decreased blood urea nitrogen levels. Urinary creatinine of NaCl plus chitosan was slightly decreased compared to 3 treated groups. Serum electrolytes levels, however, remained unchanged. The combination of NaCl and chitosan may be superior to the conventional use of NaCl plus KCl or NaCl alone in the prevention of hypertension. Even though these supplementary diets have demonstrated potential anti-hypertensive effects in the experimental animal model, further research is needed before any recommendations can be made

In vivo alternative testing with zebrafish in ecotoxicology

Although rodents have previously been used in ecotoxicological studies, they are expensive, time-consuming, and are limited by strict legal restrictions. The present study used a zebrafish (Danio rerio) model and generated data that was useful for extrapolating toxicant effects in this system to that of humans. Here we treated embryos of the naive-type as well as a transiently transfected zebrafish liver cell line carrying a plasmid (phAhRE-EGFP), for comparing toxicity levels with the well-known aryl hydrocarbon receptor (AhR)-binding toxicants: 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,7,8-tetrachlorodibenzo-p-dioxin, and 3-methylcholanthrene. These toxicants induced a concentration-dependent increase in morphological disruption, indicating toxicity at early life-stages. The transient transgenic zebrafish liver cell line was sensitive enough to these toxicants to express the CYP1A1 regulated enhanced green fluorescent protein. The findings of this study demonstrated that the zebrafish in vivo model might allow for extremely rapid and reproducible toxicological profiling of early life-stage embryo development. We have also shown that the transient transgenic zebrafish liver cell line can be used for research on AhR mechanism studies

E prostanoid receptor 4 expressing macrophages promote the regeneration of the intestinal epithelial barrier upon inflammation

status: Published onlin

Cyclooxygenase-2 inhibition blocks M2 macrophage differentiation and suppresses metastasis in murine breast cancer model.

Tumor cells are often associated with abundant macrophages that resemble the alternatively activated M2 subset. Tumor-associated macrophages (TAMs) inhibit anti-tumor immune responses and promote metastasis. Cyclooxygenase-2 (COX-2) inhibition is known to prevent breast cancer metastasis. This study hypothesized that COX-2 inhibition affects TAM characteristics potentially relevant to tumor cell metastasis. We found that the specific COX-2 inhibitor, etodolac, inhibited human M2 macrophage differentiation, as determined by decreased CD14 and CD163 expressions and increased TNFα production. Several key metastasis-related mediators, such as vascular endothelial growth factor-A, vascular endothelial growth factor-C, and matrix metalloproteinase-9, were inhibited in the presence of etodolac as compared to untreated M2 macrophages. Murine bone marrow derived M2 macrophages also showed enhanced surface MHCII IA/IE and CD80, CD86 expressions together with enhanced TNFα expressions with etodolac treatment during differentiation. Using a BALB/c breast cancer model, we found that etodolac significantly reduced lung metastasis, possibly due to macrophages expressing increased IA/IE and TNFα, but decreased M2 macrophage-related genes expressions (Ym1, TGFβ). In conclusion, COX-2 inhibition caused loss of the M2 macrophage characteristics of TAMs and may assist prevention of breast cancer metastasis

Macrophages in intestinal inflammation and resolution: a potential therapeutic target in IBD

Macrophages are the gatekeepers of intestinal immune homeostasis as they discriminate between innocuous antigens and potential pathogens to maintain oral tolerance. However, in individuals with a genetic and environmental predisposition, regulation of intestinal immunity is impaired, leading to chronic relapsing immune activation and pathologies of the gastrointestinal tract, such as IBD. As evidence suggests a causal link between defects in the resolution of intestinal inflammation and altered monocyte-macrophage differentiation in patients with IBD, macrophages have been considered as a novel potential target to develop new treatment approaches. This Review discusses the molecular and cellular mechanisms involved in the differentiation and function of intestinal macrophages in homeostasis and inflammation, and their role in resolving the inflammatory process. Understanding the molecular pathways involved in the specification of intestinal macrophages might lead to a new class of targets that promote remission in patients with IBD.N

Etodolac induced more immune activated macrophages in breast cancer.

<p><b><i>Panel A</i></b><b>:</b> Dot plots from primary breast tumor single cell isolates. Inset gate, CD11b F4/80 double positive macrophages as backgating using FACS. <b><i>Panel B</i></b><b>:</b> Merged histograms for IA/IE expression on macrophage surfaces by FACS. Representative for five animals per each group. <b><i>Panel C</i></b><b>:</b> Merged histograms for intracellular TNFα (top left) and IL-10 (top right), all gated on F4/80⁺CD11b⁺ macrophages from naïve peritoneal fluid cells and primary breast tumors. Bottom graphs represents quantitative MFI. n = 3, **, <i>p</i><0.01 by student <i>t</i>-test. <b><i>Panel D</i></b><b>:</b> Merged histograms for intracellular TNFα (top left) and IL-10 (top right), all gated on F4/80⁺CD11b⁺ macrophages from naïve and tumor implanted splenocytes. Bottom graphs represents quantitative MFI. n = 3, *, <i>p</i><0.05 by student <i>t</i>-test.</p

Etodolac induced more immune activated murine macrophages.

<p><b><i>Panel A</i></b><b>:</b> BALB/c bone marrow cells were differentiated with 10% L929 culture supernatants during 7 days and CD11b F4/80 double positive macrophage populations were backgated for further examinations by FACS. Etodolac 20 µM or 100 µM was applied from the start of culture and until examination. <b><i>Panel B</i></b><b>:</b> Merged histograms represents macrophage surface IA/IE, CD80 and CD86 expressions. Representatives for five independent experiments. <b><i>Panel C</i></b><b>:</b> MFI for intracellular TNFα and IL-10 measured by FACS. Golgitransporter inhibitor was applied during 5 hrs with or without 100 ng/ml LPS stimulation. Error bars, SEM, *, <i>p</i><0.05 by Student's <i>t</i>-test.</p

Etodolac inhibits lung metastasis in a murine syngeneic breast cancer model.

<p><b><b><i>Panel A</i></b><b>:</b></b> Primary tumor volumes were measured at the indicated time point. Feeding with 500 ppm etodolac-containing food started 7 days before tumor cell injection (10⁵ cells) into the right inguinal mammary fat pad of 7-week-old female BALB/c mice and continued until sacrifice. n = 6. <b><i>Panel B</i></b><b>:</b> Metastatic lung nodules on lung surfaces were manually counted and measured in size after resecting lungs from sacrificed mice. Means of a total of three separate counts. <i>p</i><0.05, Mann-whitney U test, n = 6. <b><i>Panel C</i></b><b>:</b> Lung metastasis images obtained by fluorescence microscopy after 4T1_GFP cell implantation (Left panel). Right graph represents quantitatively analyzed fluorescence density measured using Image J software (<i>p</i> = 0.0377, Student's unpaired <i>t</i>-test, <i>n</i> = 3). <b><i>Panel D</i></b><b>:</b> Lung histology by HE staining. Scale bars, left bottom.</p

Etodolac inhibited <i>YmI</i> and <i>TGFβ</i> of TAMs in breast cancer.

<p><i>YmI </i><b><i>(Panel A)</i></b> and <i>TGFβ</i>, <i>VEGFA</i> and <i>VEGFC </i><b><i>(Panel B)</i></b> gene expression levels were quantified by real-time PCR. Macrophages were obtained from primary breast tumors using CD11b-positive magnetic bead selection after single cell digestion followed by RNA extraction. Macrophages were quantified using the relative C_T method after endogenous <i>GAPDH</i> normalization. *, <i>p</i><0.05, unpaired Student's <i>t</i> test.</p