Production of cytokines by peritoneal macrophages and splenocytes after exposures of mice to low doses of X-rays

We have shown previously that irradiations of mice with 0.1 or 0.2 Gy of X-rays stimulate anti-tumour cytotoxic activities of peritoneal macrophages and splenocytes enriched for NK lymphocytes and suppress the development of pulmonary tumour colonies. The up-regulated cytotoxicities were related to the production of nitric oxide by macrophages, and perforin and Fas ligand by the splenocytes, but specific blockade of these pathways did not totally suppress the effector cell-mediated cytolysis of the tumour target. Hence, other factors such as cytotoxic/cytostatic cytokines might have been produced by the effector cells. To test this possibility peritoneal macrophages and splenocytes were isolated from BALB/c mice which had been either once or tentimes whole body-irradiated with the total doses of 0.1 and 0.2 Gy of X-rays and assayed for the levels of IL-1beta, IL-2, IL-12, IFN-gamma and TNF-alpha in the incubation medium using the respective ELISA kits. The results demonstrate that both single and multiple exposures to the two low doses of X-rays significantly stimulate secretion of IL-1beta, TNF-alpha and IL-12 by macrophages and IL-2 and IFN-gamma by splenocytes, but the kinetics and magnitude of the induced changes in the production of these cytokines differ between the two irradiation protocols.     

Adaptation to P element transposon invasion in Drosophila melanogaster

Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.     

Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model

High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 μm in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases.     

Neuron‐specific translational control shift ensures proteostatic resilience during ER stress

Proteostasis is essential for cellular survival and particularly important for highly specialised post‐mitotic cells such as neurons. Transient reduction in protein synthesis by protein kinase R‐like endoplasmic reticulum (ER) kinase (PERK)‐mediated phosphorylation of eukaryotic translation initiation factor 2α (p‐eIF2α) is a major proteostatic survival response during ER stress. Paradoxically, neurons are remarkably tolerant to PERK dysfunction, which suggests the existence of cell type‐specific mechanisms that secure proteostatic stress resilience. Here, we demonstrate that PERK‐deficient neurons, unlike other cell types, fully retain the capacity to control translation during ER stress. We observe rescaling of the ATF4 response, while the reduction in protein synthesis is fully retained. We identify two molecular pathways that jointly drive translational control in PERK‐deficient neurons. Haem‐regulated inhibitor (HRI) mediates p‐eIF2α and the ATF4 response and is complemented by the tRNA cleaving RNase angiogenin (ANG) to reduce protein synthesis. Overall, our study elucidates an intricate back‐up mechanism to ascertain translational control during ER stress in neurons that provides a mechanistic explanation for the thus far unresolved observation of neuronal resilience to proteostatic stress.     

The role of mast cells and macrophages in amiodarone induced pulmonary fibrosis and the possible attenuating role of atorvastatin

Amiodarone (AM) is an effective anti-arrhythmic drug. We investigated the role of mast cells and macrophages on AM induced pulmonary fibrosis and the action of atorvastatin on this fibrosis. Rats were allocated into four groups; negative control (1), positive control (2), 30 mg/kg body weight/day AM (3) and AM + 10 mg/kg/day atorvastatin (4). Lungs were harvested and prepared for histology and immunohistochemistry. Hematoxylin and eosin stained sections of group 3 exhibited disorganized lung architecture. We found cellular debris in the lumen of both intrapulmonary bronchi and bronchioles with partial disruption of the thickened epithelial lining and mononuclear cellular infiltration into the lamina propria. We also observed thickening of the epithelial lining and the smooth muscle layer. Congested, dilated and thickened blood capillaries and thickened inter-alveolar septa were observed with mononuclear cellular infiltrates in the lung of group 3. Most alveoli were collapsed, but some dilated ones were detected. In some alveoli, type ?? pneumocytes were increased, while type I cells were decreased. We observed significant increases in the amount of collagen in the thickened inter-alveolar septa, around bronchioles and around blood capillaries in sections from group 3. We found a significant increase in mast cells and alveolar macrophages in group 3 compared to group 1. Mast cells and macrophages appear to play important roles in AM induced pulmonary fibrosis. Atorvastatin appears to attenuate this condition.     

DNA mismatch repair-induced double-strand breaks

Escherichia coli dam mutants are sensitized to the cytotoxic action of base analogs, cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while their mismatch repair (MMR)-deficient derivatives are tolerant to these agents. We showed previously, using pulse field gel electrophoresis (PFGE), that MMR-mediated double-strand breaks (DSBs) are produced by cisplatin in dam recB(Ts) cells at the non-permissive temperature. We demonstrate here that the majority of these DSBs require DNA replication for their formation, consistent with a model in which replication forks collapse at nicks or gaps formed during MMR. DSBs were also detected in dam recB(Ts) ada ogt cells exposed to MNNG in a dose- and MMR-dependent manner. In contrast to cisplatin, the formation of these DSBs was not affected by DNA replication and it is proposed that two separate mechanisms result in DSB formation. Replication-independent DSBs arise from overlapping base excision and MMR repair tracts on complementary strands and constitute the majority of detectable DSBs in dam recB(Ts) ada ogt cells exposed to MNNG. Replication-dependent DSBs result from replication fork collapse at O(6)-methylguanine (O(6)-meG) base pairs undergoing MMR futile cycling and are more likely to contribute to cytotoxicity. This model is consistent with the observation that fast-growing dam recB(Ts) ada ogt cells, which have more chromosome replication origins, are more sensitive to the cytotoxic effect of MNNG than the same cells growing slowly.     

Mechanisms of oral tolerance revisited

Oral tolerance induction is thought to depend on special antigen presenting cells in the gut. A new report in the previous issue of Arthritis Research & Therapy supports this idea by demonstrating that indoleamine 2,3-dioxygenase-expressing dendritic cells in Peyer's patches from orally tolerized mice suppress T-cell responses via the generation of CD4+CD25+ regulatory T cells. This finding provides novel input into the mechanisms of oral tolerance that could further facilitate its use for the treatment of autoimmunity and chronic inflammatory reactions.     

Single low doses of X rays inhibit the development of experimental tumor metastases and trigger the activities of NK cells in mice

There is evidence indicating that low-level exposures to low- LET radiation may inhibit the development of tumors, but the mechanism of this effect is virtually unknown. In the present study, BALB/c mice were irradiated with single doses of 0.1 or 0.2 Gy X rays and injected intravenously 2 h later with syngeneic L1 sarcoma cells. Compared to the values obtained for sham-irradiated control mice, the numbers of pulmonary tumor colonies were significantly reduced in the animals exposed to either 0.1 or 0.2 Gy X rays. Concurrently, a significant stimulation of NK cell-mediated cytotoxic activity was detected in splenocyte suspensions obtained from irradiated mice compared to sham-exposed mice. Intraperitoneal injection of the NK-suppressive anti-asialo GM1 antibody totally abrogated the tumor inhibitory effect of the exposures to 0.1 and 0.2 Gy X rays. These results indicate that single irradiations of mice with either 0.1 or 0.2 Gy X rays suppress the development of experimental tumor metastases primarily due to the stimulation of the cytolytic function of NK cells by radiation.