Assessing 12(S)-lipoxygenase inhibitory activity using colorectal cancer cells overexpressing the enzyme.

12(S)-Lipoxygenase (LOX) is regarded as a pro-tumorigenic enzyme and as a potential target for therapy and prevention of cancer so that the search for specific 12(S)-LOX inhibitors is part of drug development strategies. To facilitate the identification of specific 12(S)-LOX inhibitors we have created an assay cell line by introducing a12(S)-LOX expression vector into SW480 colorectal cancer cells. When arachidonic acid was supplied in the medium both transiently and stably overexpressing cells produced 12(S)-hydroxytetraenic acid (HETE) originating from the transfected gene at 4-5-fold the amount obtained from control transfectants. 12(S)-HETE production was 1913.7+/-17.2pg/ml and reached a steady state level 24h after addition of arachidonic acid. To demonstrate the models suitability of 12(S)-LOX overexpressing SW480 cells they were used to measure the inhibitory activity of the plant phenols baicalein, kaempferol, quercetin, nordihydroguaretic acid and resveratrol which are known for their chemopreventive as well as LOX-inhibitory activity in different tumour models. All 5 compounds inhibited 12(S)-HETE production at concentrations below those necessary for growth inhibition.     

A reevaluation of the status of cholesterol in erythrocytes infected by Plasmodium knowlesi and P. falciparum

The cholesterol synthesis of rhesus monkey erythrocytes parasitized by Plasmodium knowlesi and human erythrocytes infected by P. falciparum, as measured by incorporation of [1-¹⁴C]acetate and ³H₂O, was almost undetectable, concordant with very low levels of measurable 3-hydroxy-3-methyl glutaryl-CoA reductase activity. In addition, both types of infected cells exchanged cholesterol with the plasma at the same rate as uninfected cells. The data do not exclude the possibility of cholesterol transfer from uninfected to infected cells.     

Specific and non-specific components in the triggering of proliferative and cytotoxic responses of T lymphocytes with different cell density

We hve analyzed the functional behavior of lymphocyte subsets separated on the basis of cell density. Low and high density subpopulations were cultured in FCS, alone or with allogeneic irradiated PBL, and then examined for proliferation and cytotoxic activity against autologous (responder) and allogeneic (stimulator) PHA-induced blasts, K562 and Daudi. In the high density subset proliferation and generation of anti-K562 and anti-Daudi effects were induced by FCS and to higher extent by allospecific stimulation. Exposure to alloantigens induced allospecific cytotoxicity. Autologous PHA blasts were not affected. The results with the low density subset differed. Independently of the type of stimulus imposed, the low density fraction showed little if any proliferation, but its cytotoxic activity was stronger against all targets tested. In some of the experiments, anti-alloblast cytotoxicity was generated in the control cultures. Thus, polyclonal activation induced by FCS triggered in this fraction allospecific cytotoxicity. In this subset, the effect against allogeneic PHA blasts comprised a specific and a non-specific component because autologous PHA blasts were also lysed. Limiting dilution analysis involving allostimulation showed higher frequency of cytotoxic precursors in the low density subset. Split minicultures were tested for lysis of auto- and allogeneic blasts. Alloreactive cultures that did not lyse the autologous target were more frequent in the cultures initiated with the high density cells. There was no conclusive evidence for the existence of autoreactive cultures that did not lyse the allogeneic blasts.     

A simple technique for evaluation of methods of cell separation

A simple method is described for labelling cells with fluorescein and using them in artificial mixtures to assess cell separation procedures. The method facilitates the examination of the variables in a separation procedure. It is thus possible to tailor a separation procedure (for example panning with monoclonal antibody) to suit the specific requirements of the experiment.     

An enzyme-linked immunosorbent assay (ELISA) for detection of Fc alpha receptors on isotype-specific T cells

A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) has been developed using T cell hybridomas as coating antigen, for detection of Fc receptors for IgA (Fc alpha R). T-T hybridomas were generated from fusions of Fc alpha R+ T cell clones from mouse Peyer's patches with the Fc alpha R- R1.1 T lymphoma cell line. The 2 T-T hybridomas (designated Th HA) used here express Fc alpha R as determined by a rosette method and by ELISA. Th HA cells were cultured under conditions for maximum Fc alpha R expression, were added to individual wells of 96-well EIA plates, and were fixed in situ with glutaraldehyde. Plates were incubated with purified mouse monoclonal IgA, IgM or IgG1 and were developed with beta-galactosidase-coupled goat IgG antibodies specific for mouse heavy chains. Using the ELISA, both Th HA cell lines were shown to express significant levels of Fc alpha R, lower but detectable Fc mu R, and no discernible Fc gamma 1R. Interestingly, the rosette assay only allowed detection of receptors for IgA. When splenic lymphocytes were used, good Fc mu R and less Fc alpha R expression occurred on these cells as determined by ELISA and rosetting; however, no Fc gamma 1R cells were detected by either method. Thus, the ELISA is sensitive and reproducible, and allows an objective measurement of FcR expressed on T cells.     

Regulatory T cell abundance and activation status before and after priming with HIVIS-DNA and boosting with MVA-HIV/rgp140/GLA-AF may impact the magnitude of the vaccine-induced immune responses

Background

Little is known about regulatory CD4 T cells (Tregs) in the context of HIV vaccines. Tregs can be differentiated into resting (FoxP3⁺CD45RA⁺ – rTregs), activated (FoxP3^HighCD45RA^? – aTregs) and memory (FoxP3^LowCD45RA^? – mTregs). Tregs, as CD4 T cells, are also frequent targets for HIV infection. We studied how the abundance and phenotypes of Tregs in terms of activation status and expression of HIV-1 binding molecules would have changed during vaccination in healthy volunteers participating in a phase IIa HIV vaccine clinical trial. Subjects were primed three times with HIVIS-DNA and boosted twice with MVA-CMDR-HIV alone (n?=?12) or MVA-CMDR combined with protein CN54rgp140 (n?=?13). The proportions of β7 integrin in all CD4 T cells and in the Tregs subset decreased moderately after the final vaccination (p?=?0.001 and p?=?0.033, respectively) and the rTregs proportion within the total Tregs were also decreased after the final vaccination (p?=?0.038). All these proportions returned to normal values within the three months after the final vaccination. The magnitude of HIV-Envelope-specific IFNγ?+?T cells after vaccination (r?=?0.66; p?=?0.021) correlated directly with the proportion of Tregs, and correlated inversely correlated with ratios of Th17/Tregs (r?=??0.75; p?=?0.0057) and Th17/mTregs (r?=??0.78; p?=?0.0065). Higher titers of IgG gp140 antibodies were observed in subjects with higher mTregs proportions (r?=?0.52; p?=?0.022). Interestingly, pre-vaccination levels of mTregs correlated with vaccine-induced Env-binding antibodies (r?=?0.57; p?=?0.01) and presence of neutralizing antibodies (r?=?0.61; p?=?0.01), while the pre-vaccination Th17/mTregs ratio correlated inversely with the magnitude of cellular IFN-γ ELISpot responses (r?=??0.9; p?=?0.002). Taken together, these results suggest that pre- and post-vaccination Tregs, their activation status, the Th17/Tregs ratio and other host factors affecting Treg abundance, have an impact on the magnitude of HIV vaccine-induced immune responses. Moreover, the DNA-HIVIS/MVA-HIV regimen, alone or in combination with CN54rgp140 induced moderate and temporary alterations of the Tregs activation status. We also show a decrease in expression of the HIV-1 ligand β7 integrin on Tregs and all CD4 T cells.     

New immunomodulatory role of neuropeptide Y (NPY) in Salmo salar leucocytes

Neuropeptide Y (NPY) plays different roles in mammals such as: regulate food intake, memory retention, cardiovascular functions, and anxiety. It has also been shown in the modulation of chemotaxis, T lymphocyte differentiation, and leukocyte migration. In fish, NPY expression and functions have been studied but its immunomodulatory role remains undescribed. This study confirmed the expression and synthesis of NPY in S. salar under inflammation, and validated a commercial antibody for NPY detection in teleost. Additionally, immunomodulatory effects of NPY were assayed in vitro and in vivo. Phagocytosis and superoxide anion production in leukocytes and SHK cells were induced under stimulation with a synthetic peptide. IL-8 mRNA was selectively and strongly induced in the spleen, head kidney, and isolated cells, after in vivo challenge with NPY. All together suggest that NPY is expressed in immune tissues and modulates the immune response in teleost fish.     

Human papillomavirus 16 L1-E7 chimeric virus like particles show prophylactic and therapeutic efficacy in murine model of cervical cancer

Cervical cancer is found to be associated with human papillomavirus (HPV) infection, with HPV16 being the most prevalent. An effective vaccine against HPV can thus, be instrumental in controlling cervical cancer. An ideal HPV vaccine should aim to generate both humoral immune response to prevent new infection as well as cell-mediated immunity to eliminate established infection. In this study, we have generated a potential preventive and therapeutic candidate vaccine against HPV16. We expressed and purified recombinant HPV16 L1(ΔN26)-E7(ΔC38) protein in E. coli which was assembled into chimeric virus like particles (CVLPs) in vitro. These CVLPs were able to induce neutralizing antibodies and trigger cell-mediated immune response, in murine model of cervical cancer, exhibiting antitumor efficacy. Hence, this study has aimed to provide a vaccine candidate possessing both, prophylactic and therapeutic efficacy against HPV16 associated cervical cancer.     

Chemotypical variations in Withania somnifera lead to differentially modulated immune response in BALB/c mice

Withania somnifera (Ashwagandha) is a plant with known ethnomedicinal properties and its use in Ayurvedic medicine in India is well documented. The present investigation reports on immunomodulatory efficacy of aqueous-ethanol extracts of roots of three selected Withania somnifera chemotypes designated as NMITLI 101R, NMITLI 118R and NMITLI 128R. Each chemotype was administered 10-100 mg/kg orally to BALB/c mice once daily for 14 days. The immunomodulatory consequences were recorded by determining the humoral immune response with the help of hemagglutination, plaque forming cell assay and cellular response by measuring delayed type hypersensitivity reaction. Additionally, other immune parameters such as proliferation of T and B cells, intracellular and secreted Th1 and Th2 cytokines along with modulation in ROS production by peritoneal macrophages were monitored after feeding with lower doses (3-30 mg/kg/day) of these three chemotypes individually. NMITLI 101R incited both humoral and cellular immune response in terms of higher number of antibody producing cells and enhanced foot pad swelling at the 10 mg dose as also dose dependent B and T cell proliferations. Levels of intracellular and secreted cytokines post-NMITLI 101R treatment illustrated generation of mixed Th1/Th2 response that remained more polarized towards Th1. This chemotype also generated maximum reactive oxygen species. NMITLI 118R provoked comparatively reduced immune response in all humoral and cellular parameters at lower doses but induced highly polarized Th1 cytokine response. In contrast, NMITLI 128R led to enhanced antibody production with minimal cellular response demonstrating marginally Th2 dominance at a lower dose. Taken together, it may therefore be concluded that there were distinct modulation in the immune response exhibited by the three chemotypes of Withania somnifera and NMITLI 101R appeared to possess a better immunostimulatory activity than the other chemotypes at lower doses.     

IP-10-induced recruitment of CXCR3 host T cells is required for small bowel allograft rejection

BACKGROUND & AIMS: Chemokines mediate cell trafficking in inflammatory states such as allograft rejection. However, their role in small-bowel allograft rejection has not been defined. The aim of this study was to examine the roles of type 1 helper T-cell chemokines in small-bowel allograft rejection. METHODS: Mucosal histology, chemokine messenger RNA (real-time polymerase chain reaction), and cell isolates were examined in small-bowel allografts and isografts. Interferon-gamma-inducible protein-10/CXC chemokine receptor (CXCR) 3 interactions were specifically evaluated by using allografts from interferon-gamma-inducible protein-10(-/-) donors and adoptive transfer of CXCR3(-/-) T cells into recombination activating gene (RAG)-1(-/-) recipients of small-bowel allografts. RESULTS: Type 1 helper T-cell cytokine (interferon-gamma) and chemokine (interferon-gamma-inducible protein-10, monokine induced by interferon-gamma, macrophage-inflammatory protein-1 alpha, and regulated on activation, normal T cells expressed and secreted) messenger RNA up-regulation was detected (real-time polymerase chain reaction) by postoperative day 3 in small-bowel allografts. Interferon-gamma-inducible protein-10(+/+) small-bowel allograft rejection was associated with a dramatic (>7-fold) increase in CXCR3(+) host T cells in the graft lamina propria. With interferon-gamma-inducible protein-10(-/-) small-bowel allografts, CXCR3(+) host T-cell infiltration of the graft lamina propria was markedly decreased and rejection was significantly delayed. Whereas adoptive transfer of wild-type B6 (CXCR3(+/+)) T cells into B6 (RAG-1(-/-)) recipients induced rapid rejection of CB6F1 small-bowel allografts, rejection was significantly delayed (29.2 +/- 8.7 days vs. 16.5 +/- 3.1 days; P < 0.01) in B6 (RAG-1(-/-)) mice reconstituted with T cells from B6 (CXCR3(-/-)) mice. CONCLUSIONS: Recruitment of CXCR3(+) host T cells by donor derived interferon-gamma-inducible protein-10 may precipitate small-bowel allograft rejection. These data highlight the importance of type 1 helper T cell-related chemokines in promoting cell-mediated rejection responses in small-bowel allografts and suggest that interferon-gamma-inducible protein-10 is an attractive therapeutic target for humanized monoclonal antibody strategies.