T cell-mediated hypersensitivity to quinolones: mechanisms and cross-reactivity

BACKGROUND: Quinolones are widely used, broad spectrum antibiotics that can induce immediate- and delayed-type hypersensitivity reactions, presumably either IgE or T cell mediated, in about 2-3% of treated patients. OBJECTIVE: To better understand how T cells interact with quinolones, we analysed six patients with delayed hypersensitivity reactions to ciprofloxacin (CPFX), norfloxacin (NRFX) or moxifloxacin (MXFX). METHODS: We confirmed the involvement of T cells in vivo by patch test and in vitro by means of the lymphocyte proliferation test (LTT). The nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones were investigated through the generation and analysis (flow cytometry and proliferation assays) of quinolone-specific T cell clones (TCC). RESULTS: The LTT confirmed the involvement of T cells because peripheral blood mononuclear cells (PBMC) mounted an enhanced in vitro proliferative response to CPFX and/or NRFX or MXFX in all patients. Patch tests were positive after 24 and 48 h in three out of the six patients. From two patients, CPFX- and MXFX-specific CD4(+)/CD8(+) T cell receptor (TCR) alphabeta(+) TCC were generated to investigate the nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones. The use of eight different quinolones as antigens (Ag) revealed three patterns of cross-reactivity: clones exclusively reacting with the eliciting drug, clones with a limited cross-reactivity and clones showing a broad cross-reactivity. The TCC recognized quinolones directly without need of processing and without covalent association with the major histocompatability complex (MHC)-peptide complex, as glutaraldehyde-fixed Ag-presenting cells (APC) could present the drug and washing quinolone-pulsed APC removed the drug, abrogating the reactivity of quinolone-specific TCC. CONCLUSION: Our data show that T cells are involved in delayed immune reactions to quinolones and that cross-reactivity among the different quinolones is frequent.

Expression of zebra fish aromatase cyp19a and cyp19b genes in response to the ligands of estrogen receptor and aryl hydrocarbon receptor

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17beta-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERalpha, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligands.

Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.

Copper and human health: biochemistry, genetics, and strategies for modeling dose-response relationships

Copper (Cu) and its alloys are used extensively in domestic and industrial applications. Cu is also an essential element in mammalian nutrition. Since both copper deficiency and copper excess produce adverse health effects, the dose-response curve is U-shaped, although the precise form has not yet been well characterized. Many animal and human studies were conducted on copper to provide a rich database from which data suitable for modeling the dose-response relationship for copper may be extracted. Possible dose-response modeling strategies are considered in this review, including those based on the benchmark dose and categorical regression. The usefulness of biologically based dose-response modeling techniques in understanding copper toxicity was difficult to assess at this time since the mechanisms underlying copper-induced toxicity have yet to be fully elucidated. A dose-response modeling strategy for copper toxicity was proposed associated with both deficiency and excess. This modeling strategy was applied to multiple studies of copper-induced toxicity, standardized with respect to severity of adverse health outcomes and selected on the basis of criteria reflecting the quality and relevance of individual studies. The use of a comprehensive database on copper-induced toxicity is essential for dose-response modeling since there is insufficient information in any single study to adequately characterize copper dose-response relationships. The dose-response modeling strategy envisioned here is designed to determine whether the existing toxicity data for copper excess or deficiency may be effectively utilized in defining the limits of the homeostatic range in humans and other species. By considering alternative techniques for determining a point of departure and low-dose extrapolation (including categorical regression, the benchmark dose, and identification of observed no-effect levels) this strategy will identify which techniques are most suitable for this purpose. This analysis also serves to identify areas in which additional data are needed to better define the characteristics of dose-response relationships for copper-induced toxicity in relation to excess or deficiency.

Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint

Upon the incidence of DNA stress, the ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signaling kinases activate a transient cell cycle arrest that allows cells to repair DNA before proceeding into mitosis. Although the ATM-ATR pathway is highly conserved over species, the mechanisms by which plant cells stop their cell cycle in response to the loss of genome integrity are unclear. We demonstrate that the cell cycle regulatory WEE1 kinase gene of Arabidopsis thaliana is transcriptionally activated upon the cessation of DNA replication or DNA damage in an ATR- or ATM-dependent manner, respectively. In accordance with a role for WEE1 in DNA stress signaling, WEE1-deficient plants showed no obvious cell division or endoreduplication phenotype when grown under nonstress conditions but were hypersensitive to agents that impair DNA replication. Induced WEE1 expression inhibited plant growth by arresting dividing cells in the G2-phase of the cell cycle. We conclude that the plant WEE1 gene is not rate-limiting for cycle progression under normal growth conditions but is a critical target of the ATR-ATM signaling cascades that inhibit the cell cycle upon activation of the DNA integrity checkpoints, coupling mitosis to DNA repair in cells that suffer DNA damage.

Systemic response of the GH/IGF-I axis in timely versus delayed fracture healing

The GH-IGF axis has profound effects on the local and systemic regulation of bone metabolism and may be important for quality of fracture healing. To test the hypothesis that deficiency of the GH/IGF axis may play a role in the pathogenesis of fracture non-union we investigated whether alterations of serum concentrations of the GH-IGF axis could be related to failed fracture healing compared to timely fracture healing in trauma patients. Serum probes were prospectively collected from 186 patients with surgical treatment of long bone fractures up to 6 months after surgery. Samples from 14 patients with atrophic type of non-union have been compared to 14 matched patients with normal bone healing. Postoperative time courses of serum concentrations have been analyzed using commercially available chemiluminescence sandwich assays (GH), fully automated assay systems (IGF-I, IGFBP-3) or sandwich immunometric assays (ALS). Comparison between both collectives revealed significantly lower serum concentrations of GH dependent ALS during early (1st week after surgery) and of both IGFBP-3 and ALS during late stages of fracture healing (6 and 8 weeks after surgery) in non-union patients, coinciding clinically with failed fracture healing. Tendentially lower serum levels of IGF-I in the non-union group over the entire investigation period were statistically not significant. We have been able to show time courses of serum concentrations of the GH/IGF-I axis during normal and failed fracture healing in humans. An impairment of the GH/IGF-I axis might be involved in the biochemical mechanisms determining delayed or failed fracture healing.

Cardiac c-kit+AT2+ cell population is increased in response to ischemic injury and supports cardiomyocyte performance

The expression pattern of angiotensin AT2 receptors with predominance during fetal life and upregulation under pathological conditions during tissue injury/repair process suggests that AT2 receptors may exert an important action in injury/repair adaptive mechanisms. Less is known about AT2 receptors in acute ischemia-induced cardiac injury. We aimed here to elucidate the role of AT2 receptors after acute myocardial infarction. Double immunofluorescence staining showed that cardiac AT2 receptors were mainly detected in clusters of small c-kit+ cells accumulating in peri-infarct zone and c-kit+AT2+ cells increased in response to acute cardiac injury. Further, we isolated cardiac c-kit+AT2+ cell population by modified magnetic activated cell sorting and fluorescence activated cell sorting. These cardiac c-kit+AT2+ cells, represented approximately 0.19% of total cardiac cells in infarcted heart, were characterized by upregulated transcription factors implicated in cardiogenic differentiation (Gata-4, Notch-2, Nkx-2.5) and genes required for self-renewal (Tbx-3, c-Myc, Akt). When adult cardiomyocytes and cardiac c-kit+AT2+ cells isolated from infarcted rat hearts were cocultured, AT2 receptor stimulation in vitro inhibited apoptosis of these cocultured cardiomyocytes. Moreover, in vivo AT2 receptor stimulation led to an increased c-kit+AT2+ cell population in the infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute myocardial infarction. These data suggest that cardiac c-kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports performance of cardiomyocytes, thus contributing to cardioprotection via cardiac c-kit+AT2+ cell population.

Altered response control and anterior cingulate function in attention-deficit/hyperactivity disorder boys

To investigate mechanisms and structures underlying prefrontal response control and inhibition in boys suffering from attention-deficit/hyperactivity disorder (ADHD).

The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response - Biological and Clinical Aspects

Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

Early gene expression analysis in 9L orthotopic tumor-bearing rats identifies immune modulation in molecular response to synchrotron microbeam radiation therapy

Synchrotron Microbeam Radiation Therapy (MRT) relies on the spatial fractionation of the synchrotron photon beam into parallel micro-beams applying several hundred of grays in their paths. Several works have reported the therapeutic interest of the radiotherapy modality at preclinical level, but biological mechanisms responsible for the described efficacy are not fully understood to date. The aim of this study was to identify the early transcriptomic responses of normal brain and glioma tissue in rats after MRT irradiation (400Gy). The transcriptomic analysis of similarly irradiated normal brain and tumor tissues was performed 6 hours after irradiation of 9 L orthotopically tumor-bearing rats. Pangenomic analysis revealed 1012 overexpressed and 497 repressed genes in the irradiated contralateral normal tissue and 344 induced and 210 repressed genes in tumor tissue. These genes were grouped in a total of 135 canonical pathways. More than half were common to both tissues with a predominance for immunity or inflammation (64 and 67% of genes for normal and tumor tissues, respectively). Several pathways involving HMGB1, toll-like receptors, C-type lectins and CD36 may serve as a link between biochemical changes triggered by irradiation and inflammation and immunological challenge. Most immune cell populations were involved: macrophages, dendritic cells, natural killer, T and B lymphocytes. Among them, our results highlighted the involvement of Th17 cell population, recently described in tumor. The immune response was regulated by a large network of mediators comprising growth factors, cytokines, lymphokines. In conclusion, early response to MRT is mainly based on inflammation and immunity which appear therefore as major contributors to MRT efficacy.

Acquired thrombotic thrombocytopenic purpura. Development of an autoimmune response

The von Willebrand factor (VWF)-cleaving metalloprotease, ADAMTS13 (adisintegrin and metalloprotease with thrombospondin type 1 motifs-13) is the only known target of the dysregulated immune response in acquired TTP. Autoantibodies to ADAMTS13 either neutralize its activity or accelerate its clearance, thereby causing a severe deficiency of ADAMTS13 in plasma. As a consequence, size regulation of VWF is impaired and the persistence of ultra-large VWF (ULVWF) multimers facilitates microvascular platelet aggregation causing microangiopathic haemolytic anaemia and ischaemic organ damage. Autoimmune TTP although a rare disease with an annual incidence of 1.72 cases has a mortality rate of 20% even with adequate therapy. We describe the mechanisms involved in ADAMTS13 autoimmunity with a focus on the role of B- and T-cells in the pathogenesis of this disorder. We discuss the potential translation of recent experimental findings into future therapeutic concepts for the treatment of acquired TTP.

European summer temperature response to annually dated volcanic eruptions over the past nine centuries

The drop in temperature following large volcanic eruptions has been identified as an important component of natural climate variability. However, due to the limited number of large eruptions that occurred during the period of instrumental observations, the precise amplitude of post-volcanic cooling is not well constrained. Here we present new evidence on summer temperature cooling over Europe in years following volcanic eruptions. We compile and analyze an updated network of tree-ring maximum latewood density chronologies, spanning the past nine centuries, and compare cooling signatures in this network with exceptionally long instrumental station records and state-of-the-art general circulation models. Results indicate post-volcanic June–August cooling is strongest in Northern Europe 2 years after an eruption (−0.52 ± 0.05 °C), whereas in Central Europe the temperature response is smaller and occurs 1 year after an eruption (−0.18 ± 0.07 °C). We validate these estimates by comparison with the shorter instrumental network and evaluate the statistical significance of post-volcanic summer temperature cooling in the context of natural climate variability over the past nine centuries. Finding no significant post-volcanic temperature cooling lasting longer than 2 years, our results question the ability of large eruptions to initiate long-term temperature changes through feedback mechanisms in the climate system. We discuss the implications of these findings with respect to the response seen in general circulation models and emphasize the importance of considering well-documented, annually dated eruptions when assessing the significance of volcanic forcing on continental-scale temperature variations.

Grazing response patterns indicate isolation of semi-natural European grasslands

Identifying drivers of species diversity is a major challenge in understanding and predicting the dynamics of species-rich semi-natural grasslands. In particular in temperate grasslands changes in land use and its consequences, i.e. increasing fragmentation, the on-going loss of habitat and the declining importance of regional processes such as seed dispersal by livestock, are considered key drivers of the diversity loss witnessed within the last decades. It is a largely unresolved question to what degree current temperate grassland communities already reflect a decline of regional processes such as longer distance seed dispersal. Answering this question is challenging since it requires both a mechanistic approach to community dynamics and a sufficient data basis that allows identifying general patterns. Here, we present results of a local individual- and trait-based community model that was initialized with plant functional types (PFTs) derived from an extensive empirical data set of species-rich grasslands within the `Biodiversity Exploratories' in Germany. Driving model processes included above- and belowground competition, dynamic resource allocation to shoots and roots, clonal growth, grazing, and local seed dispersal. To test for the impact of regional processes we also simulated seed input from a regional species pool. Model output, with and without regional seed input, was compared with empirical community response patterns along a grazing gradient. Simulated response patterns of changes in PFT richness, Shannon diversity, and biomass production matched observed grazing response patterns surprisingly well if only local processes were considered. Already low levels of additional regional seed input led to stronger deviations from empirical community pattern. While these findings cannot rule out that regional processes other than those considered in the modeling study potentially play a role in shaping the local grassland communities, our comparison indicates that European grasslands are largely isolated, i.e. local mechanisms explain observed community patterns to a large extent.

Induced carbon reallocation and compensatory growth as root herbivore tolerance mechanisms

Upon attack by leaf herbivores, many plants reallocate photoassimilates below ground. However, little is known about how plants respond when the roots themselves come under attack. We investigated induced resource allocation in maize plants that are infested by the larvae Western corn rootworm Diabrotica virgifera virgifera. Using radioactive 11CO2, we demonstrate that root-attacked maize plants allocate more new 11C carbon from source leaves to stems, but not to roots. Reduced meristematic activity and reduced invertase activity in attacked maize root systems are identified as possible drivers of this shoot reallocation response. The increased allocation of photoassimilates to stems is shown to be associated with a marked thickening of these tissues and increased growth of stem-borne crown roots. A strong quantitative correlation between stem thickness and root regrowth across different watering levels suggests that retaining photoassimilates in the shoots may help root-attacked plants to compensate for the loss of belowground tissues. Taken together, our results indicate that induced tolerance may be an important strategy of plants to withstand belowground attack. Furthermore, root herbivore-induced carbon reallocation needs to be taken into account when studying plant-mediated interactions between herbivores.