Nickel oxide nanoparticles can recruit eosinophils in the lungs of rats by the direct release of intracellular eotaxin.

BACKGROUND: Instillation of highly soluble nanoparticles (NPs) into the lungs of rodents can cause acute eosinophilia without any previous sensitizations by the role of dissolved ions. However, whether gradually dissolving NPs can cause the same type of eosinophilia remains to be elucidated. We selected nickel oxide (NiO) as a gradually dissolving NP and evaluated the time course pulmonary inflammation pattern as well as its mechanisms. METHODS: NiO NPs were intratracheally instilled into female Wistar rats at various concentrations (50, 100, and 200 cm(2)/rat) and the lung inflammation was evaluated at various time-points (1, 2, 3, and 4 days). As positive controls, NiCl2 and the ovalbumin-induced allergic airway inflammation model was applied. NiCl2 was instilled at 171.1 µg Ni/rat, which is equivalent nickel concentration of 200 cm(2)/rat of NiO NPs. Cytological analysis and biochemical analysis including lactate dehydrogenase (LDH), total protein, and pro-inflammatory cytokines were measured in bronchoalveolar lavage fluid (BALF). The levels of total immunoglobulin E (IgE) and anaphylatoxins (C3a and C5a) were measured in BALF and serum. The levels of eotaxin were measured in the alveolar macrophages and normal lung tissue before and after addition of cell lysis buffer to evaluate whether the direct lysis of cells can release intracellular eotaxin. RESULTS: NiO NPs produced acute neutrophilic inflammation throughout the study. However, eosinophils were recruited at 3 and 4 days post-instillation of NiO NPs and the magnitude and pattern of inflammation was similar with NiCl2 at 24 h post-instillation. The eosinophil recruitment by NiO NPs was not related with either the levels of total IgE or anaphylatoxins. The lysis of alveolar macrophages and normal lung tissue showed high levels of intracellular eotaxin and the levels of LDH showed positive correlation with the levels of eotaxin. CONCLUSIONS: Instillation of NiO NPs produced neutrophilia at 1 and 2 days after instillation, while the mixed type of neutrophilic and eosinophilic inflammation was produced at 3 and 4 days post-instillation, which was consistent with NiCl2. The mechanism of the eosinophilia involves the direct release of intracellular eotaxin due to the rupture of cells by the accumulated solubilized nickel ions in the phagolysosome.

NF-kappaB/STAT3/PI3K signaling crosstalk in iMyc E mu B lymphoma.

BACKGROUND: Myc is a well known driver of lymphomagenesis, and Myc-activating chromosomal translocation is the recognized hallmark of Burkitt lymphoma, an aggressive form of non-Hodgkin's lymphoma. We developed a model that mimics this translocation event by inserting a mouse Myc cDNA gene into the immunoglobulin heavy chain locus, just upstream of the intronic Emu enhancer. These mice, designated iMyc E mu, readily develop B-cell lymphoma. To study the mechanism of Myc-induced lymphoma, we analyzed signaling pathways in lymphoblastic B-cell lymphomas (LBLs) from iMyc E mu mice, and an LBL-derived cell line, iMyc E mu-1. RESULTS: Nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) were constitutively activated in iMyc E mu mice, not only in LBLs but also in the splenic B-lymphocytes of young animals months before tumors developed. Moreover, inhibition of either transcription factor in iMyc E mu-1 cells suppressed growth and caused apoptosis, and the abrogation of NF-kappaB activity reduced DNA binding by both STAT3 and Myc, as well as Myc expression. Inhibition of STAT3 signaling eliminated the activity of both NF-kappaB and Myc, and resulted in a corresponding decrease in the level of Myc. Thus, in iMyc E mu-1 cells NF-kappaB and STAT3 are co-dependent and can both regulate Myc. Consistent with this, NF-kappaB and phosphorylated STAT3 were physically associated with one another. In addition, LBLs and iMyc E mu-1 cells also showed constitutive AKT phosphorylation. Blocking AKT activation by inhibiting PI3K reduced iMyc E mu-1 cell proliferation and caused apoptosis, via downregulation of NF-kappaB and STAT3 activity and a reduction of Myc levels. Co-treatment with NF-kappaB, STAT3 or/and PI3K inhibitors led to additive inhibition of iMyc E mu-1 cell proliferation, suggesting that these signaling pathways converge. CONCLUSIONS: Our findings support the notion that constitutive activation of NF-kappaB and STAT3 depends on upstream signaling through PI3K, and that this activation is important for cell survival and proliferation, as well as for maintaining the level of Myc. Together, these data implicate crosstalk among NF-kappaB, STAT3 and PI3K in the development of iMyc E mu B-cell lymphomas.

Genetic and phenotypic characterization of the novel mouse substrain C57BL/6N Korl with increased body weight.

In inbred mouse lines, there is generally little genetic difference between individuals. This small genetic variability facilitates carrying out research on minute changes of various traits and the gene pool. Also, characterizing the diversity and detecting selective genetic and phenotypic signatures are crucial to understanding the genomic basis of a population and to identify specific patterns of evolutionary change. In this study, we investigated the underlying genetic profiles of a newly developed mouse strain, C57BL/6NKorl (Korl), established through sibling mating over 30 generations. To analyse the distinctive genomic features of Korl mice, we used whole-genome sequencing from six samples, which were compared to those of other C57BL/6N-based mouse strains. Korl strain-specific polymorphisms were identified and signatures of a selective sweep were detected. In particular, the candidate genes related to the increased body weight of the Korl strain were identified. Establishment of the genetic profile of Korl mice can provide insight into the inbreeding-induced changes to the gene pool, and help to establish this strain as a useful model for practical and targeted research purposes.

CDDO-Imidazolide inhibits growth and survival of c-Myc-induced mouse B cell and plasma cell neoplasms.

BACKGROUND: Gene-targeted iMycEmu mice that carry a His6-tagged mouse Myc(c-myc)cDNA, MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Emu, are prone to B cell and plasma cell neoplasms, such as lymphoblastic B-cell lymphoma (LBL) and plasmacytoma (PCT). Cell lines derived from Myc-induced neoplasms of this sort may provide a good model system for the design and testing of new approaches to prevent and treat MYC-driven B cell and plasma cell neoplasms in human beings. To test this hypothesis, we used the LBL-derived cell line, iMycEmu-1, and the newly established PCT-derived cell line, iMycEmu-2, to evaluate the growth inhibitory and death inducing potency of the cancer drug candidate, CDDO-imidazolide (CDDO-Im). METHODS: Morphological features and surface marker expression of iMycEmu-2 cells were evaluated using cytological methods and FACS, respectively. mRNA expression levels of the inserted MycHis and normal Myc genes were determined by allele-specific RT-PCR and qPCR. Myc protein was detected by immunoblotting. Cell cycle progression and apoptosis were analyzed by FACS. The expression of 384 "pathway" genes was assessed with the help of Superarray cDNA macroarrays and verified, in part, by RT-PCR. RESULTS: Sub-micromolar concentrations of CDDO-Im caused growth arrest and apoptosis in iMycEmu-1 and iMycEmu-2 cells. CDDO-Im-dependent growth inhibition and apoptosis were associated in both cell lines with the up-regulation of 30 genes involved in apoptosis, cell cycling, NFkappaB signaling, and stress and toxicity responses. Strongly induced (> or = 10 fold) were genes encoding caspase 14, heme oxygenase 1 (Hmox1), flavin-containing monooxygenase 4 (Fmo4), and three members of the cytochrome P450 subfamily 2 of mixed-function oxygenases (Cyp2a4, Cyp2b9, Cyp2c29). CDDO-Im-dependent gene induction coincided with a decrease in Myc protein. CONCLUSION: Growth arrest and killing of neoplastic mouse B cells and plasma cells by CDDO-Im, a closely related derivative of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid, appears to be caused, in part, by drug-induced stress responses and reduction of Myc.

Evaluation of Haemagglutinin Content by RP-HPLC to Generate Pandemic Influenza Vaccine.

The potency of influenza vaccine is determined based on its hemagglutinin (HA) content. In general, single radial immunodiffusion (SRID) assay has been utilized as the standard method to measure HA content. However, preparation of reagents for SRID such as antigen and antibody takes approximately 2~3 months, which causes delays in the development of influenza vaccine. Therefore, quantification of HA content by other alternative methods is required. In this study, we measured HA contents of H1N1 antigen and H1N1 influenza vaccine by reverse phase-high performance liquid chromatography (RP-HPLC) methods. The presence of HA1 and HA2 was investigated by silver staining and Western blot assay. In addition, accuracy and repeatability of HA measurement by RP-HPLC were evaluated. Comparison of HA concentration by SRID and RP-HPLC revealed a precise correlation between the two methods. Our results suggest that RP-HPLC assay can replace SRID in the event of a pandemic flu outbreak for rapid vaccine development.

Molecular and cytological features of the mouse B-cell lymphoma line iMycEmu-1.

BACKGROUND: Myc-induced lymphoblastic B-cell lymphoma (LBL) in iMycEmu mice may provide a model system for the study of the mechanism by which human MYC facilitates the initiation and progression of B cell and plasma cell neoplasms in human beings. We have recently shown that gene-targeted iMycEmu mice that carry a His6-tagged mouse Myc cDNA, MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Emu, are prone to B cell and plasma cell tumors. The predominant tumor (approximately 50%) that arose in the iMycEmu mice on the mixed genetic background of segregating C57BL/6 and 129/SvJ alleles was LBL. The purpose of this study was to establish and characterize a cell line, designated iMycEmu-1, for the in-depth evaluation of LBL in vitro. METHODS: The morphological features and the surface marker expression profile of the iMycEmu-1 cells were evaluated using cytological methods and FACS, respectively. The cytogenetic make-up of the iMycEmu-1 cells was assessed by spectral karyotyping (SKY). The expression of the inserted MycHis gene was determined using RT-PCR and qPCR. Clonotypic immunoglobulin gene arrangements were detected by Southern blotting. The global gene expression program of the iMycEmu-1 cells and the expression of 768 "pathway" genes were determined with the help of the Mouse Lymphochip(c) and Superarray(c) cDNA micro- and macroarrays, respectively. Array results were verified, in part, by RT-PCR and qPCR. RESULTS: Consistent with their derivation from LBL, the iMycEmu-1 cells were found to be neoplastic IgMhighIgDlow lymphoblasts that expressed typical B-cell surface markers including CD40, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). The iMycEmu-1 cells harbored a reciprocal T(9;11) and three non-reciprocal chromosomal translocations, over-expressed MycHis at the expense of normal Myc, and exhibited gene expression changes on Mouse Lymphochip microarrays that were consistent with MycHis-driven B-cell neoplasia. Upon comparison to normal B cells using eight different Superarray cDNA macroarrays, the iMycEmu-1 cells showed the highest number of changes on the NFkappaB array. CONCLUSION: The iMycEmu-1 cells may provide a uniquely useful model system to study the growth and survival requirements of Myc-driven mouse LBL in vitro.

Immunomodulatory effect of caffeic acid phenethyl ester in Balb/c mice.

Caffeic acid phenethyl ester (CAPE), an the active component of propolis, is known to have anticarcinogenic, antiviral and various biological activities; however, the effect of CAPE on the immunomodulatory activity in vivo remains unknown. We have investigated the effect of CAPE on the immune system in female Balb/c mice. CAPE (0, 5, 10, 20 mg/kg) was given to mice orally for 14 days. Immunomodulatory activity was evaluated by assessment of body and organ weight, lymphocyte blastogenesis, plaque-forming cell (PFC) assay, lymphocyte subpopulation by flow cytometry and cytokine production. Even though the change of body weight was not observed in CAPE-administered group, thymus weight and/or cellularity of thymus and spleen are decreased at the all dose groups of CAPE (5, 10, 20 mg/kg). On the other hand, CAPE had no effect on B lymphocyte proliferation induced by lipopolysaccharide (LPS) but increased T lymphocyte blastogenesis induced by concanavalin A (Con A) at the dose of 20 mg/kg. In the case of lymphocyte subpopulation, the population of T and B cells was not changed but CD4(+) T cell subsets are significantly increased in exposure to CAPE. The antibody responses to T lymphocyte dependent antigen, sheep red blood cell and keyhole limpet hemocyanin (KLH) were increased more than 10 mg/kg in CAPE-treated group. Likewise, the cytokine, IL-2, IL-4 and IFN-gamma were significantly increased at the dose of 20 mg/kg CAPE group. These results suggest that CAPE could have immunomodulatory effects in vivo.

Evaluation of cell proliferation in ear and lymph node using BrdU immunohistochemistry for mouse ear swelling test.

The mouse ear swelling test (MEST) was developed as an alternative to guinea pig models for measuring the contact sensitization potential. However, the MEST relies on the quantitative measurement of ear swelling by micrometer as the means of determining the endpoint. The purpose of this study was to investigate the possibility of using cell proliferation in the ear and lymph node by bromodeoxyuridine (BrdU) immunohistochemistry as a reliable marker for MEST. Female Balb/c mice were treated by the topical application of various sensitizers, 2,4-dinitrochlorobenzene (DNCB), toluene diisocyanate (TDI) and α-hexylcinnamaldehyde (HCA) and an irritant, sodium lauryl sulfate (SLS) following the protocol of MEST. The proliferation of cells in the ear and auricular lymph node was analyzed by BrdU incorporations into cells. There were significant increases in the cell proliferations of the ear and auricular lymph node in mice treated with DNCB and TDI compared to the vehicle control. All allergens and the irritant were correctly identified by the MEST using BrdU immunohistochemistry of lymph node responses. The standard MEST assay showed positive results in the case of the strong sensitizers, DNCB and TDI. However, HCA and SLS were not correctly identified in the ear swelling assay. These results suggest that the measurement of cell proliferation in the auricular lymph node using BrdU immunohistochemistry could provide a reliable marker for MEST.