Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination.

The immune response to Mycoplasma pneumoniae infection plays a key role in clinical symptoms. Previous investigations focused on the pro-inflammatory effects of leukocytes and the pivotal role of epithelial cell metabolic status in finely modulating the inflammatory response have been neglected. Herein, we examined how glycolysis in airway epithelial cells is affected by M. pneumoniae infection in an in vitro model. Additionally, we investigated the contribution of ATP to pulmonary inflammation. Metabolic analysis revealed a marked metabolic shift in bronchial epithelial cells during M. pneumoniae infection, characterized by increased glucose uptake, enhanced aerobic glycolysis, and augmented ATP synthesis. Notably, these metabolic alterations are orchestrated by adaptor proteins, MyD88 and TRAM. The resulting synthesized ATP is released into the extracellular milieu via vesicular exocytosis and pannexin protein channels, leading to a substantial increase in extracellular ATP levels. The conditioned medium supernatant from M. pneumoniae-infected epithelial cells enhances the secretion of both interleukin (IL)-1ß and IL-18 by peripheral blood mononuclear cells, partially mediated by the P2X7 purine receptor (P2X7R). In vivo experiments confirm that addition of a conditioned medium exacerbates pulmonary inflammation, which can be attenuated by pre-treatment with a P2X7R inhibitor. Collectively, these findings highlight the significance of airway epithelial aerobic glycolysis in enhancing the pulmonary inflammatory response and aiding pathogen clearance.

Sphingosine 1-phosphate protects against radiation-induced ovarian injury in female rats-impact on mitochondrial-related genes.

The toxic effects of ionizing radiation on the gonads have been widely recognized. Sphingosine 1-phosphate (S1P) has a protective effect on ovarian injury, and although it is known that mitochondria are involved in this process, the specific mechanism is not fully understood. The present study analysed the changes in the serum AMH and ovarian histology in Sprague-Dawley female rats exposed to X-ray radiation only or co-administered with S1P. The mRNA expression profile of ovarian tissue was further analysed via next-generation sequencing and bioinformatics approaches to screen out candidate mitochondria-related genes. Finally, differentially expressed target genes were verified by real-time PCR. The results showed that ionizing radiation could reduce the serum AMH level, destroy ovarian structure and decrease the number of follicles in rats, while S1P administration significantly attenuated the impairment of ovarian function. Gene ontology (GO) and KEGG pathway analysis revealed that a variety of genes related to mitochondrial function were differentially expressed, and the protective effect of S1P on mitochondria was more obvious in the acute phase 24 h after radiation. The differentially expressed mitochondrial function-related genes associated with the protective effect of S1P were UQCRH, MICU2 and GPX4, which were subsequently verified by RT-PCR. Therefore, ionizing radiation has a significant effect on ovarian function, and S1P has a protective effect on radiation-induced ovarian injury, in which mitochondria may play an important role. This study sheds new light on the mechanism of radiation-induced ovarian injury and helps develop a novel potential strategy to control it.

The mpn668 gene of Mycoplasma pneumoniae encodes a novel organic hydroperoxide resistance protein.

Mycoplasma pneumoniae (M. pneumoniae), as an obligate parasite, has evolved a protective strategy for coping with oxidative challenges caused by M. pneumoniae itself as well as the host immune system. However, to date, few antioxidant enzymes have been identified in mycoplasmas. In this report, we identified a protein encoded by the mpn668 gene from M. pneumoniae with a putative function as an organic hydroperoxide reductase (Ohr). The results indicated that the recombinant 140 amino acid protein, designated rMPN668, displayed hydroperoxidase activity towards both organic (tert-butyl hydroperoxide) and inorganic (hydrogen peroxide) hydroperoxides in the presence of a reducing agent such as dithiothreitol. Moreover, the expression of mpn668 in M. pneumoniae is upregulated in response to oxidative stress. Additionally, homology modeling of MPN668 and a molecular dynamics simulation suggest that both Cys55 and Cys119 form part of the active site of the protein. Mutants in which Cys55 or Cys119 were replaced with a serine lack antioxidant activity, indicating that MPN668 is a Cys-based peroxidase, consistent with it representing a new member of the Ohr family.

Cyclophilin A is the potential receptor of the Mycoplasma genitalium adhesion protein.

The Mycoplasma genitalium adhesion protein (MgPa), the most important outer membrane protein of M. genitalium, plays a vital role in the adhesion to and invasion of host cells by M. genitalium. Identification of MgPa receptors will help elucidate the pathogenic mechanism of M. genitalium. However, the receptor protein of MgPa has not been reported to date. In this study, an MgPa-binding protein with a molecular weight of approximately 17 kDa was screened from SV-HUC-1 cell membrane proteins by a modified virus overlay protein binding assay (VOPBA). Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the protein components of the 17-kDa protein. The results demonstrated that the MgPa-binding protein was most likely Cyclophilin A (CyPA). The binding activity and distribution of CyPA in SV-HUC-1 cells were detected using indirect ELISA, western blotting, far-western blotting and indirect immunofluorescence. We found that recombinant MgPa (rMgPa) could bind with CyPA from SV-HUC-1 cell membrane proteins and to recombinant CyPA, which indicated that CyPA was predominant component of the 17-kDa protein band and can interact with rMgPa. In addition, an indirect immunofluorescence assay showed that CyPA was partially distributed on the membrane surfaces of SV-HUC-1 cells and could partially inhibit the adhesion of rMgPa and M. genitalium to SV-HUC-1 cells. Co-localization assays further indicated that rMgPa and M. genitalium can interact with CyPA. These results suggested that the CyPA located on SV-HUC-1 cell membranes may be the potential receptor of MgPa, which could provide an experimental basis for elucidating the function of MgPa and the possible pathogenic mechanism of M. genitalium.

Three polypeptides screened from phage display random peptide library may be the receptor polypeptide of Mycoplasma genitalium adhesion protein.

Mycoplasma genitalium adhesion protein (MgPa) is a major adhesin of M. genitalium, a human pathogen associated with a series of genitourinary tract diseases. MgPa plays a very important role in M. genitalium adhering to the host cells. However, the exact receptor peptides or proteins of MgPa are still poorly understood so far. Three polypeptides (V-H-W-D-F-R-Q-W-W-Q-P-S), (D-W-S-S-W-V -Y-R-D-P-Q-T) and (H-Y-I-D-F-R-W) were previously screened from a phage display random peptide library using recombinant MgPa (rMgPa) as a target molecule. In this study, three polypeptides were artificially synthesized and investigated as to whether they are potential receptors of MgPa. We found that rMgPa specifically bound to three synthesized polypeptides as determined via an indirect enzyme-linked immunosorbent assay (ELISA). Moreover, three polypeptides were further identified by indirect immunofluorescence microscopy (IFM). We confirmed that rMgPa and M. genitalium can adhere to SV-HUC-1 cells in vitro and that anti-rMgPa antibody and three synthesized polypeptides can partially inhibit the adherence of rMgPa and M. genitalium to SV-HUC-1 cells. In summary, these three polypeptides may be the essential receptor peptides of MgPa, and may aid in enhancing the understanding of biological function of MgPa and the possible pathogenic mechanism of M. genitalium.

Protective immunity induced by recombinant protein CPSIT_p8 of Chlamydia psittaci.

Chlamydia psittaci is a zoonotic pathogen with a broad host range that can lead to severe respiratory and systemic disease in humans. Currently, an effective commercial vaccine against C. psittaci infection is not available. The chlamydial plasmid is an important virulence factor and encodes plasmid proteins that play important roles in chlamydial infection and the corresponding immune response. In this study, we assessed the efficacy of vaccination with plasmid proteins at preventing C. psittaci lung infection in a murine model. BALB/c mice were immunized intraperitoneally, three times at 2-week intervals, with purified recombinant CPSIT_p8 protein and then infected with C. psittaci. Immunization significantly decreased chlamydial load in the lungs of infected mice, resulted in a lower level of IFN-γ, and reduced the extent of inflammation. In vivo or in vitro neutralization of C. psittaci with sera collected from immunized mice did not reduce the amount of viable C. psittaci in the lungs of mice, indicating that CPSIT_p8-specific antibodies do not have neutralizing capacity. Furthermore, confocal fluorescence microscopy using a mouse anti-CPSIT_p8 antibody revealed that CPSIT_p8 was localized inside the inclusion of C. psittaci 6BC-infected cells. Our results demonstrate that CPSIT_p8 protein induces significant protective immunity against challenge with C. psittaci in mice and represents a promising new vaccine candidate for the prevention of C. psittaci infection.

[Advances in the mechanism of bacterial escape neutrophil killing].

The innate immune system is the forefront defense against bacterial invasion in human body. Neutrophils, as major immune cells, can capture and phagocytose bacteria, which are indispensable for maintaining the health of the body. Pathogens such as Streptococcus, Neisseria meningitidis, Klebsiella, Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, Staphylococcus aureus, and Mycobacterium tuberculosis have evolved multiple mechanisms to circumvent killing by neutrophils outside and inside the cells, even taking cells as their survival and breeding sites. These mechanisms include interfering with neutral granulocyte collection and phagocytosis, degrading neutrophil extracellular traps, inhibiting the production of reactive oxygen and antimicrobial peptides, and manipulating the life-span and death of cells.

X-ray Irradiation Improves Neurological Function Recovery of Injured Spinal Cord by Inhibiting Inflammation and Glial Scar Formation.

Spinal cord injury (SCI) is a common clinical disease that can cause permanent disruption of nerve function. Inflammation and glial scar formation influence the recovery of injured spinal cord. X-ray irradiation can reduce inflammation, inhibit cell proliferation and increase cell apoptosis. However, the regulatory effects of X-ray irradiation on inflammation and glial scars and the underlying molecular mechanisms are still unclear. This study was aimed to investigate the therapeutic effect and molecular mechanism of X-ray irradiation on spinal cord injury. Behavioural experiments showed that X-ray irradiation can promote the recovery of nerve function after SCI. X-ray irradiation inhibited the inflammatory response by reducing the expression of inflammatory factors (TNF-α and IL-1ß) at the lesion site, thereby reducing neuronal apoptosis. X-ray irradiation inhibited the formation of the glial scar (GFAP and vimentin) in the lesion. P38MAPK and Akt signalling pathways were involved in these processes. Furthermore, the 10 Gy dose had the most significant effects among the 2 Gy, 10 Gy and 20 Gy doses. In summary, X-ray irradiation may exert an active therapeutic effect on SCI by inhibiting inflammation and glial scar formation, which may be related to the inhibition of p38MAPK and Akt signalling pathways.

Mycoplasma pneumoniae downregulates RECK to promote matrix metalloproteinase-9 secretion by bronchial epithelial cells.

Airway epithelial cells function as both a physical barrier against harmful substances and pathogenic microorganisms and as an important participant in the innate immune system. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in modulating inflammatory responses during respiratory infections. However, the signalling cascade that induces MMP-9 secretion from epithelial cells infected with Mycoplasma pneumoniae remains poorly understood. In this study, we investigated the mechanism of MMP-9 secretion in airway epithelial cells infected with M. pneumoniae. Our data clearly showed that M. pneumoniae induced the secretion of MMP-9 from bronchial epithelial cells and upregulated its enzymatic activity in a time- and dose-dependent manner. Using specific inhibitors and chromatin co-precipitation experiments, we confirmed that the expression of MMP-9 is reliant on the activation of the Toll-like receptor 2 (TLR2) and TLR6-dependent mitogen-activated protein kinase/nuclear factor- κB/activator protein-1 (MAPK/NF-κB/AP-1) pathways. Additionally, epigenetic modifications such as histone acetylation and the nuclear transcription factor Sp1 also regulate MMP-9 expression. M. pneumoniae infection also decreased the expression of the tumour suppressor reversion-inducing cysteine-rich protein with Kazal motifs (RECK) by inducing Sp1 phosphorylation. Overexpression of RECK significantly impaired the M. pneumoniae-triggered increase in MMP-9 enzymatic activity, although the level of MMP-9 protein remained constant. The study demonstrated that M. pneumoniae-triggered MMP-9 expression is modulated by TLR2 and 6, the MAPK/NF-κB/AP-1 signalling cascade, and histone acetylation, and M. pneumoniae downregulated the expression of RECK, thereby increasing MMP-9 activity to modulate the inflammatory response, which could play a role in airway remodelling.

Induction of autophagy promotes differentiation of glioma-initiating cells and their radiosensitivity.

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by increased proliferation and resistance to chemotherapy and radiotherapy. Recently, the identification of tumor-initiating cells with stem-like properties in diverse human cancers including GBM represents an important conceptual advance in cancer biology with therapeutic implications. However, the factors determining the differential development and radiosensitization of glioma-initiating cells (GICs) remain poorly defined. Here, we report that rapamycin induced differentiation of GICs and increased their sensitivity to radiation by activating autophagy. Transient in vitro exposure to rapamycin and radiation abolished the capacity of transplanted GICs to establish intracerebral GBMs. Most importantly, in vivo combination of rapamycin and radiation effectively blocked the tumor growth and associated mortality that occurs in mice after intracerebral grafting of human GICs. We demonstrate that rapamycin activated their autophagy and triggers the differentiation cascade in GICs isolated from human GBMs. This was followed by a reduction in proliferation, cell viability, clonogenic ability and increased expression of neural differentiation markers after radiation. Our results suggest that autophagy plays an essential role in the regulation of self-renewal, differentiation, tumorigenic potential and radiosensitization of GICs, suggesting autophagy could be a promising therapeutic target in a subset of GBMs. We propose that autophagy defect in GICs contributes to radioresistance of GICs by desensitizing GICs to normal differentiation cues. Activating autophagy may abrogate the resistance of GICs to radiation and could lead to the development of novel therapeutic approaches for the treatment of GBMs.

Identification of histone H2B as a potential receptor for Mycoplasma genitalium protein of adhesion.

Mycoplasma genitalium, the smallest prokaryotic microorganism capable of independent replication, is increasingly recognized as a sexually transmitted pathogen. M. genitalium protein of adhesion (MgPa) plays a pivotal role in the process of M. genitalium adhesion to host cells. We previously identified cyclophilin A as a cellular receptor of MgPa using the virus overlay protein binding assay (VOPBA) together with liquid chromatography-mass spectrometry (LC-MS). In the current study, we have evaluated H2B as an alternative cellular receptor for MgPa since H2B was assigned the second higher score as a potential binding partner of MgPa in the VOPBA and LC-MS screen. It was found that recombinant MgPa specifically bind to H2B both in the SV-HUC-1 cell membrane and in form of a recombinant protein. H2B was detected throughout the SV-HUC-1 cells, including the cytoplasmic membrane, cytosol and nucleus. Importantly, H2B partially inhibited the adhesion of M. genitalium to SV-HUC-1 cells. Finally, H2B was both co-precipitated with recombinant MgPa and co-localized with M. genitalium and recombinant MgPa in SV-HUC-1 cells. The above observations suggest that H2B may act as a potential cellular receptor of MgPa for mediating M. genitalium adhesion to host cells.

T-B cell epitope peptides induce protective immunity against Mycoplasma pneumoniae respiratory tract infection in BALB/c mice.

Mycoplasma pneumoniae is the most common pathogen of community-acquired pneumonia in humans. Due to its high rates of antibiotic resistance, vaccination has become the best method to control the dissemination of M. pneumoniae. The recombinant carboxyl terminus of the P1 (P1C) protein is an immunodominant antigen, but it has negative effects such as poor stability and lower purity. In the current study, T-B epitopes of the P1C protein were predicted according to bioinformatics analysis and assessed for efficacy in peptide vaccination. BALB/c mice were subcutaneously inoculated with the T-B epitope peptides four times and then infected with M. pneumoniae through the respiratory tract. The results showed that the T-B epitope peptides of the P1C protein (P1C103-117, P1C155-169, P1C224-238 and P1C244-258) induced strong antigen-specific serum antibody responses and cellular immune responses with high levels of serum IgG, IgA antibodies and Th1-biased (IFN-γ and IL-2) cytokines. Immunization with T-B epitope peptides significantly reduced the M. pneumoniae burden and the degree of inflammation in the challenged mice. Furthermore, the levels of IFN-γ and TNF-α in the supernatants of lung homogenates were observably reduced compared to those in the PBS group. Overall, our findings demonstrate that T-B epitopes (P1C103-117, P1C155-169, P1C224-238 and P1C244-258) play significant roles in the P1C protein and can be used to induce powerful humoral and cellular immune responses to provide significant protection against M. pneumoniae pulmonary infection, which provides new insight into the design of potential multiepitope vaccines to prevent host infection by M. pneumoniae.

Exogenous Hydrogen Sulfide Regulates Mycoplasma pneumoniae Lipid-Associated Membrane Proteins to Induce Expression of Heme Oxygenase-1 and Proinflammatory Cytokines.

This study was designed to investigate the effect of exogenous hydrogen sulfide (H2S) on the secretion of Heme oxygenase (HO-1) and proinflammatory cytokines in human mononuclear cell line THP-1 stimulated by lipid-associated membrane proteins (LAMPs) prepared from Mycoplasma pneumoniae (M. pneumoniae) and explore its regulatory mechanism. Cultured cells were stimulated with M. pneumoniae LAMPs after pretreatment with H2S to analyze the production of proinflammatory cytokines and HO-1 by enzyme-linked immunosorbent assay (ELISA) and Western blot. The results showed that THP-1 cells, which were stimulated by LAMPs after pretreatment with H2S, had decreased production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by inhibiting the mitogen-activated protein kinases (MAPKs)/nuclear factor-kappa B (NF-κB) signaling pathway and increased expression of HO-1 by activating the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. Our results indicate that H2S may play an important role in attenuating inflammation induced by M. pneumoniae LAMPs due to its ability to decrease the production of IL-6 and IL-8 and increase the expression of the HO-1. These findings support further studies for possible clinical applications.

MicroRNA­200a suppresses migration and invasion and enhances the radiosensitivity of NSCLC cells by inhibiting the HGF/c­Met signaling pathway.

Hepatocyte growth factor (HGF), an activator of the c­Met signaling pathway, is involved in tumor invasiveness, metastasis and radiotherapy resistance. In the present study, a novel HGF regulatory pathway in lung cancer involving micro-RNAs (miRNAs/miR) is described. Immunohistochemical staining and western blot analyses demonstrated that HGF was upregulated and associated with miR­200a downregulation in non­small cell lung cancer (NSCLC) samples compared with normal lung tissues. The association between HGF and miR­200a was associated with the degree of tumor malignancy and cell migration and invasion. miR­200a negatively regulated HGF expression by targeting the 3'­untranslated region of the HGF mRNA. miR­200a overexpression induced HGF downregulation, decreased NSCLC cell migration and invasion, promoted apoptosis, and decreased cell survival in A549 and H1299 cells in response to ionizing radiation. The present results revealed a previously uncharacterized role of miRNA­200a in regulating tumor malignancy and radiosensitivity by suppressing HGF expression, a key factor in the HGF/c­Met pathway.

NFAT3/c4-mediated excitotoxicity in hippocampal apoptosis during radiation-induced brain injury.

Whole brain irradiation (WBI) has become an indispensible tool in the treatment of head and neck cancer, and it has greatly improved patient survival rate and total survival time. In addition, prophylactic cranial irradiation (PCI) has dramatically decreased the incidence of brain metastatic carcinoma. However, WBI may induce temporary functional deficits or even progressive, irreversible cognitive dysfunction that compromises the quality of life for survivors. Unfortunately, the exact molecular mechanisms for cognitive damage remain elusive, and no treatment or preventative measures are available for use in the clinic. In the present study, the nuclear factor of activated T cells isoform 4 (NFAT3/c4) was found to play a vital role in excitotoxic hippocampus cell apoptosis induced by radiation. Sprague-Dawley (SD) rats received 20 Gy WBI, after which we detected NFAT3/c4-mediated excitotoxicity. We found that radiation caused hippocampus excitotoxicity, resulting from overactivation of the N-methyl-D-aspartate receptor (NMDAR) and always accompanied by subsequent elevation of the intracellular calcium level and activation of calcineurin (CaN). P-NFAT3/c4 was the principal downstream target of CaN, including regulation of its nuclear translocation as well as transcriptional activities. Radiation recruited NMDAR/NFAT3/c4 activation and subsequent Bax induction in hippocampus cells. Once treated with the NFAT3/c4 inhibitor 11R-VIVIT peptide pre-irradiation, hippocampal proliferation and neuron survival (dentate gyrus cells in particular) were protected from radiation-induced injury, resulting in inhibition of the apoptosis marker Bax. Our principal aim was to illuminate the role of NFAT3/c4-mediated excitotoxicity in hippocampal apoptosis during radiation-induced brain injury. This study is the first time that radiation-induced activation of NFAT3/c4 has been recorded, and our results suggest that NFAT3/c4 may be a novel target for prevention and treatment of radiation-induced brain injury.

Long-term outcome of changes in cognitive function of young rats after various/different doses of whole brain irradiation.

OBJECTIVE: To characterize the early delayed and late-delayed cognitive dysfunction induced by various doses of whole brain irradiation in young rats. METHODS: One-month-old Sprague-Dawley male rats were divided randomly into the 0 (control), 0 (anesthesia control), 2, 10, 20, and 30-Gy groups. Each group was then subdivided into 4 groups according to the experimental intervals: 1, 2, 3, and 6 months after radiation. Rats were irradiated using a 4-MeV electron beam, which was generated by a linear accelerator. Sequential behavioral tests, including open field, novel location and novel object recognition and Morris water maze were performed after radiation. Changes in gross neurological symptoms, body weight, topical skin response, and histopathology were observed. RESULTS: In the open field test, there were no radiation-induced alterations found. In the novel location and novel object recognition tests, rats of the 20-Gy group spent less time exploring the novel object and novel location 3 months after irradiation. During the place navigation test, the spatial working memory of the 30 and 20-Gy irradiated rats were impaired from 1 to 2 months after irradiation, respectively. In the spatial probe test, the 20 and 30-Gy irradiated rats spent less time in the critical region compared to control rats at 3 and 6 months post-irradiation. Morphological changes, including edema, vascular dilation, focal necrosis, demyelination, and adjacent reactive gliosis were observed in the 30-Gy irradiation group. CONCLUSION: More than 20 Gy of whole brain irradiation dose can cause significant cognitive dysfunction in young rats.

Macrophage-activating lipopeptide-2 requires Mal and PI3K for efficient induction of heme oxygenase-1.

AIMS: This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes. METHODS: Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay. RESULTS: MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, LY294002. CONCLUSIONS: These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.

[Mycoplasma genitalium lipid-associated membrane proteins induce endocervical epithelial cells to express ß-defensin-2].

OBJECTIVE: To observe the expression of human ß-defensin-2 (hBD-2) induced by Mycoplasma genitalium-derived lipid-associated membrane proteins (LAMPs) and its potential mechanism. METHODS: Human endocervical epithelial End1/E6E7 cells were cultured in vitro and stimulated by different concentrations of LAMPs for 48 hours, and the expressions of hBD-2 mRNA and protein were detected by real-time RT-PCR and Western blotting, respectively. Toll-like receptor 2 (TLR2) and TLR6 neutralizing antibodies for End1/E6E7 cell cultivation, and dominant negative plasmids for cell transfection were used to analyze the roles of TLR2, TLR6 and MyD88 in mediating hBD-2 expression. Nuclear translocation of the nuclear factor κB (NF-κB) p65 subunit, and its DNA-binding activity were detected by Western blotting and electrophoretic mobility shift assay (EMSA), respectively. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, was used to investigate the effect of NF-κB on hBD-2 expression. RESULTS: Mycoplasma genitalium-derived LAMPs induced the expressions of hBD-2 mRNA and protein in End1/E6E7 cells. The expressions could be abrogated by TLR2 and TLR6 neutralizing antibodies, or their dominant negative plasmids. In addition, dominant negative plasmids of MyD88 significantly decreased LAMPs-induced hBD-2 expression. Western blotting showed that p65 was translocated to the nucleus, and the DNA binding activity of NF-κB was enhanced after LAMPs treatment. Furthermore, PDTC treatment decreased LAMPs- induced hBD-2 expression. CONCLUSION: Mycoplasma genitalium-derived LAMPs can induce End1/E6E7 cells to express hBD-2, which may be involved in the TLR2, TLR6/Myd88/NF-κB pathways.

Effects of expression level of DNA repair-related genes involved in the NHEJ pathway on radiation-induced cognitive impairment.

Cranial radiation therapy can induce cognitive decline. Impairments of hippocampal neurogenesis are thought to be a paramountly important mechanism underlying radiation-induced cognitive dysfunction. In the mature nervous system, DNA double-strand breaks (DSBs) are mainly repaired by non-homologous end-joining (NHEJ) pathways. It has been demonstrated that NHEJ deficiencies are associated with impaired neurogenesis. In our study, rats were randomly divided into five groups to be irradiated by single doses of 0 (control), 0 (anesthesia control), 2, 10, and 20 Gy, respectively. The cognitive function of the irradiated rats was measured by open field, Morris water maze and passive avoidance tests. Real-time PCR was also used to detect the expression level of DNA DSB repair-related genes involved in the NHEJ pathway, such as XRCC4, XRCC5and XRCC6, in the hippocampus. The influence of different radiation doses on cognitive function in rats was investigated. From the results of the behavior tests, we found that rats receiving 20 Gy irradiation revealed poorer learning and memory, while no significant loss of learning and memory existed in rats receiving irradiation from 0-10 Gy. The real-time PCR and Western blot results showed no significant difference in the expression level of DNA repair-related genes between the 10 and 20 Gy groups, which may help to explain the behavioral results, i.e. DNA damage caused by 0-10 Gy exposure was appropriately repaired, however, damage induced by 20 Gy exceeded the body's maximum DSB repair ability. Ionizing radiation-induced cognitive impairments depend on the radiation dose, and more directly on the body's own ability to repair DNA DSBs via the NHEJ pathway.

Immunogenicity of Cpn0425 and its localization in cells infected with Chlamydophila pneumoniae.

The present study aimed to determine the intracellular localization of Cpn0425 in Chlamydophila pneumoniae-infected cells. The recombinant plasmid pGEX-6p/Cpn0425 was transformed into E.coli Bl21 cells to express the fusion protein. Following purification with glutathione S-transferase (GST) resin chromatography, the Cpn0425 fusion protein was used to induce immunity in mice to develop monoclonal and polyclonal antibodies, which were subsequently used to localize the endogenous Cpn0425 protein by indirect immunofluorescence assay (IFA). ELISA was used to determine the immunogenicity of the Cpn0425 plasmid protein by recognizing the pool sera of patients infected with Chlamydia trachomatis and the pool sera of mice immunized with the Cpn0425 fusion protein. The Cpn0425 gene was expressed as the GST-Cpn0425 fusion protein in E. coli and its antibody was prepared by immunizing mice with the fusion protein. An anti-GST-Cpn0425 antibody was used to localize the protein in cells infected with Chlamydophila pneumoniae AR-39 using an IFA. The anti-GST-CT058 antibody detected an inclusion signal in the IFA. Cpn0425 protein strongly reacted with antiserum. Although Cpn0425 protein is not a secreted protein, it has good immunogenicity. Therefore, this protein may be useful for developing vaccines against Chlamydophila pneumoniae infection.