We compared the effect of the specific type IV phosphodiesterase inhibitor rolipram on intracellular cAMP concentration, nitric oxide (NO) and tumour necrosis factor-α (TNF) formation in the murine ...macrophage cell line RAW 264.7. We found a dose-dependent increase of nitrite accumulation in LPS-stimulated macrophages from 17.5 to 25.1 μM nitrite with rolipram, whereas TNF synthesis was suppressed to less than 30% of control. This was accompanied by an increase from 7.4 to a maximum of 10.5 nM cAMP in RAW cells incubated with rolipram. These results were confirmed with the stable cAMP analogue (
S)-
p-adenosine 3′,5′-cyclic phosphorothioate (
S)-
p-cAMPS. These findings demonstrate that elevation of cAMP in RAW 264.7 cells by rolipram decreases TNF synthesis and increases NO formation.
Human diseases frequently occur in discrete, genetically susceptible groups rather than being distributed randomly in the population. As discussed at a recent symposium
*
*
The symposium ‘MHC-based ...Therapeutics in Immunological Disorders’ was part of The American Association of Immunologists Meeting and was held in San Francisco, CA, USA, on 21 April 1998.
one set of well-known disease-linked genes, the major histocompatibility complex, are leading the way into the clinic.
Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell ...of origin. EV cargo in liquid biopsies is of widespread interest owing to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. To facilitate robust EV repertoire studies, we developed multiplex analysis post-acquisition analysis (MPAPASS) open-source software for stitched multiplex analysis, EV database-compatible reporting, and visualization of EV repertoires.
Display omitted
•High-parameter EV multiplex analysis is introduced•MPAPASS enables multiplex data stitching and multidimensional data visualization•EV multiplex experiment design, performance, analysis, and reporting are presented
Assessment of EV repertoire profiles is a priority for many research groups, but methods for performing and analyzing EV markers with multimarker multiplex bead sets are not well established. MPAPASS enables stitched multiplex EV analyses, and our results illustrate key considerations for designing, performing, and analyzing EV multiplex studies.
Welsh et al. demonstrate the utility of EV multiplex analysis with MPAPASS software for EV repertoire studies. MPAPASS enables the standard reporting and ergonomic stitching of multiplex datasets for extracellular vesicle repertoire identification.
Preclinical studies with murine tumor models have demonstrated that tumor cell vaccines engineered to secrete certain cytokines in a paracrine fashion elicit systemic immune responses capable of ...eliminating small amounts of established tumor. In particular, tumors that express the cytokine GM-CSF produce potent systemic antitumor immune responses against poorly immunogenic murine tumors. These results have encouraged the development of paracrine-cytokine secreting tumor vaccines for gene therapy of human cancer. GM-CSF recruits professional antigen-presenting cells, which in turn activate effector T cells. These findings suggest that allogeneic as well as autologous tumor cells can be used as the tumor source for developing cancer vaccines. A major obstacle to creating genetically modified human allogeneic tumor vaccines is the absence of stable cell lines required for efficient gene transfer, because most human tumors isolated from primary surgical specimens fail to proliferate in long-term culture. We have developed a method for the routine generation of in vitro cell lines from primary tumors of the pancreas. This method overcomes the common problem of stromal and fibroblast overgrowth that can inhibit the in vitro expansion of many histologic types of tumors. In addition, we have analyzed 12 of these cell lines for cytokeritin and mutated K-ras expression to demonstrate that they derive from the original epithelial tumor tissue. The lines can be genetically modified to stably express the cytokine GM-CSF. These methods should be helpful to investigators attempting to establish cell lines from other histologic tumor types for the development of allogeneic genetically modified tumor vaccines.
Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% ...antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-gamma secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.
NF-...B is a key transcriptional regulator of inflammatory responses, but also controls expression of prosurvival genes, whose products protect tissues from damage and may thus act indirectly in an ...antiinflammatory fashion. The variable importance of these two distinct NF-...B-controlled responses impacts the potential utility of NF-...B inhibition as a treatment strategy for intractable inflammatory conditions, such as inflammatory bowel disease. Here, we show in murine models that inhibition of IKKβ-dependent NF-...B activation exacerbates acute inflammation, but allenuates chronic inflammatory disease in the intestinal tract. Acute ulcerating inflammation is aggravated because of diminished NF-...B-mediated protection against epithelial cell apoptosis and delayed mucosal regeneration secondary to reduced NF-...B-dependent recruitment of inflammatory cells that secrete cytoprotective factors. In contrast, in IL-b-deficient mice, which serve as a model of chronic T cell-dependent colitis, ablation of IKKβ in the intestinal epithelium has no impact, yet IKKβ deficiency in myeloid cells attenuates inflammation and prolongs survival. These results highlight the striking context and tissue dependence of the proinflammatory and antiapoptotic functions of NF-...B. Our findings caution against the therapeutic use of IKKβ/NF-...B inhibitors in acute inflammatory settings dominated by cell loss and ulceration. (ProQuest: ... denotes formulae/symbols omitted.)
NF-κB is a key transcriptional regulator of inflammatory responses, but also controls expression of prosurvival genes, whose products protect tissues from damage and may thus act indirectly in an ...antiinflammatory fashion. The variable importance of these two distinct NF-κB-controlled responses impacts the potential utility of NF-κB inhibition as a treatment strategy for intractable inflammatory conditions, such as inflammatory bowel disease. Here, we show in murine models that inhibition of IKKβ-dependent NF-κB activation exacerbates acute inflammation, but attenuates chronic inflammatory disease in the intestinal tract. Acute ulcerating inflammation is aggravated because of diminished NF-κB-mediated protection against epithelial cell apoptosis and delayed mucosal regeneration secondary to reduced NF-κB-dependent recruitment of inflammatory cells that secrete cytoprotective factors. In contrast, in IL-10-deficient mice, which serve as a model of chronic T cell-dependent colitis, ablation of IKKβ in the intestinal epithelium has no impact, yet IKKβ deficiency in myeloid cells attenuates inflammation and prolongs survival. These results highlight the striking context and tissue dependence of the proinflammatory and antiapoptotic functions of NF-κB. Our findings caution against the therapeutic use of IKKβ/NF-κB inhibitors in acute inflammatory settings dominated by cell loss and ulceration.
NF-kappaB is a key transcriptional regulator of inflammatory responses, but also controls expression of prosurvival genes, whose products protect tissues from damage and may thus act indirectly in an ...antiinflammatory fashion. The variable importance of these two distinct NF-kappaB-controlled responses impacts the potential utility of NF-kappaB inhibition as a treatment strategy for intractable inflammatory conditions, such as inflammatory bowel disease. Here, we show in murine models that inhibition of IKKbeta-dependent NF-kappaB activation exacerbates acute inflammation, but attenuates chronic inflammatory disease in the intestinal tract. Acute ulcerating inflammation is aggravated because of diminished NF-kappaB-mediated protection against epithelial cell apoptosis and delayed mucosal regeneration secondary to reduced NF-kappaB-dependent recruitment of inflammatory cells that secrete cytoprotective factors. In contrast, in IL-10-deficient mice, which serve as a model of chronic T cell-dependent colitis, ablation of IKKbeta in the intestinal epithelium has no impact, yet IKKbeta deficiency in myeloid cells attenuates inflammation and prolongs survival. These results highlight the striking context and tissue dependence of the proinflammatory and antiapoptotic functions of NF-kappaB. Our findings caution against the therapeutic use of IKKbeta/NF-kappaB inhibitors in acute inflammatory settings dominated by cell loss and ulceration.
PDF
