SECRETORY MONOCLONAL IGA ANTIBODY TO HUMAN SPERM PRODUCED BY GASTROINTESTINAL IMMUNIZATION INHIBITS HUMAN SPERM ACTIVITY AND MOUSE INVITRO FERTILIZATION

BALB/c mice were immunized intragastrically with human sperm. Cells from the Peyer's patches and spleens of the immunized mice were for the preparation of hybridomas secreting antisperm monoclonal IgA (mcIgA). The specific ratio of IgA-secreting cells in Peyer's patches was much higher than that in spleen. The binding site on human sperm of 9 of 19 mcIgA was in the post-acrosomal region using an immunofluorescent assay. Two of eight selected mcIgA caused strong human sperm agglutination and three of them produced significant inhibition of mouse in vitro fertilization. No mcIgA tested caused obvious human sperm immobilization or inhibited mouse in vivo fertilization. In vitro assembly of selected mcIgA in ascites with mouse secretory component (SC) caused no significant changes in effects on sperm function and in vitro fertilization. By use of Western blotting, dimer or higher polymers were demonstrated in all selected mcIgAs and corresponding protein antigens in 6 of 8 selected mcIgAs. These results suggest that human sperm function may be inhibited and fertilization rate reduced by specific secretory IgA to human sperm and that secretory immunity to protein antigens of human sperm could be induced by intragastrointestinal immunization.

Short-term in vitro responses of human peripheral blood monocytes to ferritic stainless steel fiber networks.

Beneficial effects on bone-implant bonding may accrue from ferromagnetic fiber networks on implants which can deform in vivo inducing controlled levels of mechanical strain directly in growing bone. This approach requires ferromagnetic fibers that can be implanted in vivo without stimulating undue inflammatory cell responses or cytotoxicity. This study examines the short-term in vitro responses, including attachment, viability, and inflammatory stimulation, of human peripheral blood monocytes to 444 ferritic stainless steel fiber networks. Two types of 444 networks, differing in fiber cross section and thus surface area, were considered alongside austenitic stainless steel fiber networks, made of 316L, a widely established implant material. Similar high percent seeding efficiencies were measured by CyQuant® on all fiber networks after 48 h of cell culture. Extensive cell attachment was confirmed by fluorescence and scanning electron microscopy, which showed round monocytes attached at various depths into the fiber networks. Medium concentrations of lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-α) were determined as indicators of viability and inflammatory responses, respectively. Percent LDH concentrations were similar for both 444 fiber networks at all time points, whereas significantly lower than those of 316L control networks at 24 h. All networks elicited low-level secretions of TNF-α, which were significantly lower than that of the positive control wells containing zymosan. Collectively, the results indicate that 444 networks produce comparable responses to medical implant grade 316L networks and are able to support human peripheral blood monocytes in short-term in vitro cultures without inducing significant inflammatory or cytotoxic effects.

Apoptosis induction on human hepatoma cells Hep G2 of decabrominated diphenyl ether (PBDE-209)

Polybrominated diphenyl ethers (PBDEs) are an important class of halogenated organic brominated flame retardants. Because of their presence in abiotic and biotic environments widely and their structural similarity to polychlorinated biphenyls (PCBs), concern has been raised on their possible adverse health effects to humans. This study was designed to determine the anti-proliferative, apoptotic properties of decabrominated diphenyl ether (PBDE-209), using a human hepatoma Hep G2 line as a model system. Hep G2 cells were cultured in the presence of PBDE-209 at various concentrations (1.0-100.0 mu mol/L) for 72 h and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that PBDE-209 inhibited the cells viability in time and concentration-dependent characteristics at concentrations (10.0-100.0 mu mol/L). We found that anti-proliferative effect of PBDE-209 was associated with apoptosis on Hep G2 cells by determinations of morphological changes, cell cycle and apoptosis. Mechanism study showed that PBDE-209 could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. The mechanism for its hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation. In addition, activity of lactate dehydrogenase (LDH) release increased when the cells incubated with PBDE-209 at various concentrations and times. These results suggested that PBDE-209 had the toxicity activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed in vitro to carbon ions and argon ions at the HIRFL

Human hepatoma (SMMC-7721) and normal liver (L02) cells were irradiated with c-rays, 12C6+ and 36Ar18+ ion beams at the Heavy Ion Research Facility in Lanzhou (HIRFL). By using the Calyculin-A induced premature chromosome condensation technique, chromatid-type breaks and isochromatid-type breaks were scored separately. Tumor cells irradiated with heavy ions produced a majority of isochromatid break, while chromatid breaks were dominant when cells were exposed to c-rays. The relative biological effectiveness (RBE) for irradiation-induced chromatid breaks were 3.6 for L02 and 3.5 for SMMC-7721 cell lines at the LET peak of 96 keVlm 1 12C6+ ions, and 2.9 for both of the two cell lines of 512 keVlm 1 36Ar18+ ions. It suggested that the RBE of isochromatid-type breaks was pretty high when high-LET radiations were induced. Thus we concluded that the high production of isochromatid-type breaks, induced by the densely ionizing track structure, could be regarded as a signature of high-LET radiation exposure.

Comparison of clonogenic assay with premature chromosome condensation assay in prediction of human cell radiosensitivity

Aim: To determine whether the number of non-rejoining G2-chromatid breaks can predict the radiosensitivity of human cell lines. Methods: Cell lines of human ovary carcinoma cells (HO8910), human hepatoma cells (HepG2) and liver cells (L02) were irradiated with a range of doses and assessed both of cell survival and non-rejoining G2-chromatid breaks at 24 h after irradiation. Cell survival was documented by a colony assay. Non-rejoining G2-chromatid breaks were measured by counting the number of non-rejoining G2 chromatid breaks at 24 h after irradiation, detected by the prematurely chromosome condensed (PCC) technique. Results: A linear-quadratic survival curve was observed in three cell lines, and HepG2 was the most sensitive to gamma-radiation. A dose-dependent linear increase was observed in radiation-induced non-rejoining G2-PCC breaks measured at 24 h after irradiation in all cell lines, and HepG2 was the most susceptible to induction of non-rejoining G2-PCC breaks. A close correlation was found between the clonogenic radiosensitivity and the radiation-induced non-rejoining G2-PCC breaks (r=0.923). Furthermore, survival-aberration correlations for two or more than two doses lever were also significant. Conclusion: The number of non-rejoining G2 PCC breaks holds considerable promise for predicting the radiosensitivity of normal and tumor cells when two or more than two doses lever is tested.

Radiobiological response of human hepatoma and normal liver cells exposed to carbon ions generated by Heavy Ion Research Facility in Lanzhou

Human hepatoma and normal liver cells were irradiated with C-12(6+), ion beams (LET= 96.05 keV/mu m) and gamma-rays at Heavy Ion Research Facility in Lanzhou (HIRFL). The chromatid breaks and break types were detected using the premature chromosome condensation technique. Our experimental results showed that chromatid breaks seem to have a good relation with C-12(6+) absorbed dose and C-12(6+) are more effective to induce chromatid breaks as compared to they-rays. For C-12(6+) ion irradiation the major break was isochromatid break, while chromatid breaks were dominant for gamma-ray irradiation. We also observed that the Relative Biology Effectiveness (RBE) of C-12(6+) ion is about 2.5 times higher than that of gamma-rays.

Inhibiting Survivin Expression Increases The Radiosensitivity of Human Hepatoma HepG2 Cells to High-LET Radiation

The influence of survivin expression on the radiosensitivity of tumor cells to high linear energy transfer (LET) radiation is investigated. Survivin-specific short-interfering RNA (siRNA) oligonucleotides were synthesized based on the survivin sequence provided by GenBank. Human hepatoma HepG2 cells were transfected with survivin-specific siRNA to inhibit its expressions. It was found that the transfection with surviving-specific siRNA increased the levels of G2/M arrest and the apoptotic rates induced by radiation in HepG2 cells. After exposure to high-LET carbon ions, a reduced clonogenic survival effect was observed in the cells treated with siRNA. These results show that survivin plays a key role in mediating the radioresistance of cells to high-LET radiation.

Early effects of low dose C-12(6+) ion or X-ray irradiation on human peripheral blood lymphocytes

The aim of this study was to estimate the acute effects of low dose C-12(6+) ions or X-ray radiation on human immune function. The human peripheral blood lymphocytes (HPBL) of seven healthy donors were exposed to 0.05 Gy C-12(6+) ions or X-ray radiation and cell responses were measured at 24 h after exposure. The cytotoxic activities of HPBL were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); the percentages of T and NK cells subsets were detected by flow cytometry; mRNA expression of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were examined by real time quantitative RT-PCR (qRT-PCR); and these cytokines protein levels in supematant of cultured cells were assayed by enzyme-linked immunosorbent assays (ELISA). The results showed that the cytotoxic activity of HPBL, mRNA expression of IL-2, IFN-gamma and TNF-alpha in HPBL and their protein levels in supernatant were significantly increased at 24 h after exposure to 0.05 Gy C-12(6+) ions radiation and the effects were stronger than observed for X-ray exposure. However, there was no significant change in the percentage of T and NK cells subsets of HPBL. These results suggested that 0.05 Gy high linear energy transfer (LET) C-12(6+) radiation was a more effective approach to host immune enhancement than that of low LET X-ray. We conclude that cytokines production might be used as sensitive indicators of acute response to LDL (C) 2009 COSPAR. Published by Elsevier Ltd. All rights reserved.

A novel anti-tumor protein extracted from Meretrix meretrix Linnaeus induces cell death by increasing cell permeability and inhibiting tubulin polymerization

Discovery and development of new pharmaceuticals from marine organisms are attracting increasing interest. Several agents derived from marine organisms are under preclinical and clinical evaluation as potential anticancer drugs. We extracted and purified a novel anti-tumor protein from the coelomic fluid of Meretrix meretrix Linnaeus by ammonium sulphate fractionation, ion exchange and hydrophobic interaction chromatography. The molecular weight of the highly purified protein, designated MML, was 40 kDa as determined by SDS-PAGE analysis. MML exhibited significant cytotoxicity to several cancer cell types, including human hepatoma BEL-7402, human breast cancer MCF-7 and human colon cancer HCT116 cells. However, no inhibitory effect was found when treating murine normal fibroblasts NIH3T3 and benign human breast MCF-10A cells with MML. The cell death induced by MML was characterized by cell morphological changes. The induction of apoptosis of BEL-7402 cells by MML was weak by DNA ladder assay. The possible mechanisms of its anti-tumor effect might be the changes in cell membrane permeability and inhibition of tubulin polymerization. MML may be developed as a novel, highly selective and effective anti-cancer drug.

cDNA cloning and mRNA expression of the translationally controlled tumor protein (TCTP) gene from Japanese sea perch (Lateolabrax japonicus)

A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.

Proteomic detection of changes in protein expression induced by cordycepin in human hepatocellular carcinoma BEL-7402 cells

The nucleoside analogue cordycepin (3'-deoxyodenosine, 3'-dA), one of the components of cordyceps militaris, has been shown to inhibit the growth of various tumor cells. However, the probable mechanism is still obscure. In this study, the inhibition of cell growth and changes in protein expression induced by cordycepin were investigated in BEL-7402 cells. Using the MTT assay and flow cytometry, we found that cordycepin inhibits cell viability and induces apoptosis in BEL 7402 cells. Additionally. the proteins were separated using two-dimensional polyacrylamide gel electrophoresis, and eight proteins were found to be significantly, affected by cordycepin compared to untreated control; among them, two were downregulated and six were upregulated. Of the eight proteins, six were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after in-gel trypsin digestion. These proteins are involved in various aspects of cellular metabolism. It is suggested that the effect of cordycepin on the growth of tumor cells is significantly related to the metabolism-associated protein expression induced by cordycepin. Copyright 2008 Prous Science, S.A.U. or its licensors. All rights reserved.

Emodin-induced apoptosis in human breast cancer BCap-37 cells through the mitochondrial signaling pathway

Emodin, a natural anthraquinone compound isolated from the rhizome of rhubarb, is reported to suppress the growth of tumor in many clinical situations. In this study, we focused on the effect of emodin in human breast cancer BCap-37 cells and further understand the underlying molecular mechanism in treating breast cancer. Using MTT assay and flow cytometry, we demonstrated the critical role of emodin in the suppression of the proliferation of BCap-37 cells based on a concentration- and time-dependent manner. The increase of apoptotic rate was also observed after incubation of BCap-37 cells on emodin at 20 mu M and 50 mu M for 48 h. The cells exhibited typical apoptotic features including cellular morphological change, chromatin condensation and membrane blebbing. The results of the study further showed that Bcl-2 level decreased, while Bax and cytosolic cytochrome c levels in sample cells increased after the emodin treatment by using Western blot. The decline in the Bcl-2/Bax ratio and the increase of cytosolic cytochrome c concentration were consistent with the increase of the apoptotic ratio. The results strongly suggest that the disruption of the mitochondrial signaling pathway was involved in emodin-induced apoptosis in BCap-37 cells.

Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.

Expression of C-reactive protein in human lung epithelial cells and up-regulation by cytokines and carbon particles.

C-reactive protein (CRP) is the prototypic human acute-phase protein and is found at increased levels in the blood during episodes of inflammation. CRP was generally thought to be produced only by hepatocytes; however, several studies have shown extrahepatic synthesis of CRP. A previous study showed that PM10 and ultrafine carbon black (ufCB) were able to induce CRP expression in A549 cells. This study aims to examine the factors that lead to the production of CRP in A549 cells. A549 human lung epithelial cells were treated with cytokines (interleukin 6, tumor necrosis factor , interferon , or interleukin 1) or carbon particles (CB and ufCB) for 18 h. It was found that CRP could be expressed within the cells and that CRP was secreted from the cells particularly with tumor necrosis factor , CB and ufCB treatments. It was also found that this expression of CRP with CB and ufCB treatments was dependent on nuclear factor kappa B (NFB). The expression of CRP in A549 cells may indicate an important role for CRP expression and secretion from lung epithelial cells in response to inflammatory stimuli.

Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from Mycobacterium tuberculosis.

A number of different interferon-gamma ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-gamma spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.