Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

Long-lasting reactivity and high frequency of drug-specific T cells after severe systemic drug hypersensitivity reactions

BACKGROUND: Drug-reactive T cells are involved in most drug-induced hypersensitivity reactions. The frequency of such cells in peripheral blood of patients with drug allergy after remission is unclear. OBJECTIVE: We determined the frequency of drug-reactive T cells in the peripheral blood of patients 4 months to 12 years after severe delayed-type drug hypersensitivity reactions, and whether the frequency of these cell differs from the frequency of tetanus toxoid-reactive T cells. METHODS: We analyzed 5 patients with delayed-type drug hypersensitivity reactions, applying 2 methods: quantification of cytokine-secreting T cells by enzyme-linked immunospot (ELISpot), and fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity distribution analysis of drug-reactive T cells. RESULTS: Frequencies found were between 0.02% and 0.4% of CD4(+) T cells reacting to the respective drugs measured by CFSE analysis, and between 0.01% and 0.08% of T cells as determined by ELISpot. Reactivity was seen neither to drugs to which the patients were not sensitized nor in healthy individuals after stimulation with any of the drugs used. CONCLUSION: About 1:250 to 1:10,000 of T cells of patients with drug allergy are reactive to the relevant drugs. This frequency of drug-reactive T cells is higher than the frequency of T cells able to recognize recall antigens like tetanus toxoid in the same subjects. A substantial frequency could be observed as long as 12 years later in 1 patient even after strict drug avoidance. Patients with severe delayed drug hypersensitivity reactions are therefore potentially prone to react again to the incriminated drug even years after strict drug avoidance.

Cytotoxic effects of camptothecin and cisplatin combined with tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in a model of primary culture of non-small cell lung cancer

The cytokine tumor-necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been shown to preferentially induce apoptosis in cancer cells. A previous study of our group demonstrated that non-small cell lung cancer cell lines can be sensitized to Apo2L/TRAIL-induced apoptosis by chemotherapeutic agents. The aim of the present study was the evaluation of these results in a model of primary culture of non-small cell lung cancer.

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

INTRODUCTION: Recent findings suggest that articular cartilage contains mesenchymal progenitor cells. The aim of this study was to examine the distribution of stem cell markers (Notch-1, Stro-1 and VCAM-1) and of molecules that modulate progenitor differentiation (Notch-1 and Sox9) in normal adult human articular cartilage and in osteoarthritis (OA) cartilage. METHODS: Expression of the markers was analyzed by immunohistochemistry (IHC) and flow cytometry. Hoechst 33342 dye was used to identify and sort the cartilage side population (SP). Multilineage differentiation assays including chondrogenesis, osteogenesis and adipogenesis were performed on SP and non-SP (NSP) cells. RESULTS: A surprisingly high number (>45%) of cells were positive for Notch-1, Stro-1 and VCAM-1 throughout normal cartilage. Expression of these markers was higher in the superficial zone (SZ) of normal cartilage as compared to the middle zone (MZ) and deep zone (DZ). Non-fibrillated OA cartilage SZ showed reduced Notch-1 and Sox9 staining frequency, while Notch-1, Stro-1 and VCAM-1 positive cells were increased in the MZ. Most cells in OA clusters were positive for each molecule tested. The frequency of SP cells in cartilage was 0.14 +/- 0.05% and no difference was found between normal and OA. SP cells displayed chondrogenic and osteogenic but not adipogenic differentiation potential. CONCLUSIONS: These results show a surprisingly high number of cells that express putative progenitor cell markers in human cartilage. In contrast, the percentage of SP cells is much lower and within the range of expected stem cell frequency. Thus, markers such as Notch-1, Stro-1 or VCAM-1 may not be useful to identify progenitors in cartilage. Instead, their increased expression in OA cartilage implicates involvement in the abnormal cell activation and differentiation process characteristic of OA.

The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes

Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.

Allopurinol hypersensitivity is primarily mediated by dose-dependent oxypurinol-specific T cell response

BACKGROUND Allopurinol is a main cause of severe cutaneous adverse reactions (SCAR). How allopurinol induces hypersensitivity remains unknown. Pre-disposing factors are the presence of the HLA-B*58:01 allele, renal failure and possibly the dose taken. OBJECTIVE Using an in vitro model, we sought to decipher the relationship among allopurinol metabolism, HLA-B*58:01 phenotype and drug concentrations in stimulating drug-specific T cells. METHODS Lymphocyte transformation test (LTT) results of patients who had developed allopurinol hypersensitivity were analysed. We generated allopurinol or oxypurinol-specific T cell lines (ALP/OXP-TCLs) from allopurinol naïve HLA-B*58:01(+) and HLA-B*58:01(-) individuals using various drug concentrations. Their reactivity patterns were analysed by flow cytometry and (51) Cr release assay. RESULTS Allopurinol allergic patients are primarily sensitized to oxypurinol in a dose-dependent manner. TCL induction data show that both the presence of HLA-B*58:01 allele and high concentration of drug are important for the generation of drug-specific T cells. The predominance of oxypurinol-specific lymphocyte response in allopurinol allergic patients can be explained by the rapid conversion of allopurinol to oxypurinol in vivo rather than to its intrinsic immunogenicity. OXP-TCLs do not recognize allopurinol and vice versa. Finally, functional avidity of ALP/OXP-TCL is dependent on both the induction dose and HLA-B*58:01 status. CONCLUSIONS AND CLINICAL RELEVANCE This study establishes the important synergistic role of drug concentration and HLA-B*58:01 allele in the allopurinol or oxypurinol-specific T cell responses. Despite the prevailing dogma that Type B adverse drug reactions are dose independent, allopurinol hypersensitivity is primarily driven by oxypurinol-specific T cell response in a dose-dependent manner, particular in the presence of HLA-B*58:01 allele.

Holocene flood frequency across the Central Alps – solar forcing and evidence for variations in North Atlantic atmospheric circulation

The frequency of large-scale heavy precipitation events in the European Alps is expected to undergo substantial changes with current climate change. Hence, knowledge about the past natural variability of floods caused by heavy precipitation constitutes important input for climate projections. We present a comprehensive Holocene (10,000 years) reconstruction of the flood frequency in the Central European Alps combining 15 lacustrine sediment records. These records provide an extensive catalog of flood deposits, which were generated by flood-induced underflows delivering terrestrial material to the lake floors. The multi-archive approach allows suppressing local weather patterns, such as thunderstorms, from the obtained climate signal. We reconstructed mainly late spring to fall events since ice cover and precipitation in form of snow in winter at high-altitude study sites do inhibit the generation of flood layers. We found that flood frequency was higher during cool periods, coinciding with lows in solar activity. In addition, flood occurrence shows periodicities that are also observed in reconstructions of solar activity from C-14 and Be-10 records (2500-3000, 900-1200, as well as of about 710, 500, 350, 208 (Suess cycle), 150, 104 and 87 (Gleissberg cycle) years). As atmospheric mechanism, we propose an expansion/shrinking of the Hadley cell with increasing/decreasing air temperature, causing dry/wet conditions in Central Europe during phases of high/low solar activity. Furthermore, differences between the flood patterns from the Northern Alps and the Southern Alps indicate changes in North Atlantic circulation. Enhanced flood occurrence in the South compared to the North suggests a pronounced southward position of the Westerlies and/or blocking over the northern North Atlantic, hence resembling a negative NAO state (most distinct from 4.2 to 2.4 kyr BP and during the Little Ice Age). South-Alpine flood activity therefore provides a qualitative record of variations in a paleo-NAO pattern during the Holocene. Additionally, increased South Alpine flood activity contrasts to low precipitation in tropical Central America (Cariaco Basin) on the Holocene and centennial time scale. This observation is consistent with a Holocene southward migration of the Atlantic circulation system, and hence of the ITCZ, driven by decreasing summer insolation in the Northern hemisphere, as well as with shorter-term fluctuations probably driven by solar activity. (C) 2013 Elsevier Ltd. All rights reserved.

Impact of solar versus volcanic activity variations on tropospheric temperatures and precipitation during the Dalton Minimum

The aim of this work is to elucidate the impact of changes in solar irradiance and energetic particles versus volcanic eruptions on tropospheric global climate during the Dalton Minimum (DM, AD 1780–1840). Separate variations in the (i) solar irradiance in the UV-C with wavelengths λ < 250 nm, (ii) irradiance at wavelengths λ > 250 nm, (iii) in energetic particle spectrum, and (iv) volcanic aerosol forcing were analyzed separately, and (v) in combination, by means of small ensemble calculations using a coupled atmosphere–ocean chemistry–climate model. Global and hemispheric mean surface temperatures show a significant dependence on solar irradiance at λ > 250 nm. Also, powerful volcanic eruptions in 1809, 1815, 1831 and 1835 significantly decreased global mean temperature by up to 0.5 K for 2–3 years after the eruption. However, while the volcanic effect is clearly discernible in the Southern Hemispheric mean temperature, it is less significant in the Northern Hemisphere, partly because the two largest volcanic eruptions occurred in the SH tropics and during seasons when the aerosols were mainly transported southward, partly because of the higher northern internal variability. In the simulation including all forcings, temperatures are in reasonable agreement with the tree ring-based temperature anomalies of the Northern Hemisphere. Interestingly, the model suggests that solar irradiance changes at λ < 250 nm and in energetic particle spectra have only an insignificant impact on the climate during the Dalton Minimum. This downscales the importance of top–down processes (stemming from changes at λ < 250 nm) relative to bottom–up processes (from λ > 250 nm). Reduction of irradiance at λ > 250 nm leads to a significant (up to 2%) decrease in the ocean heat content (OHC) between 0 and 300 m in depth, whereas the changes in irradiance at λ < 250 nm or in energetic particles have virtually no effect. Also, volcanic aerosol yields a very strong response, reducing the OHC of the upper ocean by up to 1.5%. In the simulation with all forcings, the OHC of the uppermost levels recovers after 8–15 years after volcanic eruption, while the solar signal and the different volcanic eruptions dominate the OHC changes in the deeper ocean and prevent its recovery during the DM. Finally, the simulations suggest that the volcanic eruptions during the DM had a significant impact on the precipitation patterns caused by a widening of the Hadley cell and a shift in the intertropical convergence zone.

The ceramide kinase inhibitor NVP-231 inhibits breast and lung cancer cell proliferation by inducing M phase arrest and subsequent cell death

Background and Purpose Ceramide kinase (CerK) catalyzes the generation of ceramide-1-phosphate which may regulate various cellular functions, including inflammatory reactions and cell growth. Here, we studied the effect of a recently developed CerK inhibitor, NVP-231, on cancer cell proliferation and viability and investigated the role of cell cycle regulators implicated in these responses. Experimental Approach The breast and lung cancer cell lines MCF-7 and NCI-H358 were treated with increasing concentrations of NVP-231 and DNA synthesis, colony formation and cell death were determined. Flow cytometry was performed to analyse cell cycle distribution of cells and Western blot analysis was used to detect changes in cell cycle regulator expression and activation. Key Results In both cell lines, NVP-231 concentration-dependently reduced cell viability, DNA synthesis and colony formation. Moreover it induced apoptosis, as measured by increased DNA fragmentation and caspase-3 and caspase-9 cleavage. Cell cycle analysis revealed that NVP-231 decreased the number of cells in S phase and induced M phase arrest with an increased mitotic index, as determined by increased histone H3 phosphorylation. The effect on the cell cycle was even more pronounced when NVP-231 treatment was combined with staurosporine. Finally, overexpression of CerK protected, whereas down-regulation of CerK with siRNA sensitized, cells for staurosporine-induced apoptosis. Conclusions and Implications Our data demonstrate for the first time a crucial role for CerK in the M phase control in cancer cells and suggest its targeted inhibition, using drugs such as NVP-231, in combination with conventional pro-apoptotic chemotherapy.