TLR9 mediated regulatory B10 cell amplification following sub-total body irradiation: Implications in attenuating EAE.

Autoimmunity and inflammation are controlled in part by regulatory B (Breg) cells, including the recently identified IL-10-competent B10 cell subset that represents 1%-3% of mouse spleen B cells. In this study, the influence of irradiation on Breg/B10 cell generation and IL-10 production mediated by TLR9 signaling pathways was investigated. Spleen and peritoneal cavity Breg/B10 cell frequencies were significantly expanded three weeks after sub-total body irradiation (sub-TBI, 5Gy or 10Gy) in adult male wild type (WT) C57BL/6(B6) mice but not in TLR9-/- mice. TLR9 agonist ODN1826 stimulation in vitro for 5h induced more B10 cells to express cytoplasmic IL-10 in sub-TBI WT mice than in TLR9-/- mice. Prolonged ODN1826 stimulation (48h) induced additional spleen CD19hiCD5+CD1dhi B cells to express IL-10. TLR9-dependent signaling molecules, MyD88, TRAF6 and IRF8 are involved in sub-TBI induced Breg/B10 cells development and expansion. Furthermore, using a mouse model for multiple sclerosis, we show here that sub-TBI induced Breg/B10 cells dramatically inhibit disease onset and severity when transferred into mice with established experimental autoimmune encephalomyelitis (EAE). Adoptively transferred sub-TBI induced Breg cells significantly suppress inflammatory T cell responses of TH17 and TH1 types in EAE mice. In conclusion, sub-TBI can drive Breg/B10 cell development and expansion, which could be used as a novel tool for suppressing undesirable immunity. The ex vivo expansion and reinfusion of autologous Breg/B10 cells may provide a novel and effective in vivo treatment for severe autoimmune diseases that are resistant to current therapies.

Triptolide Mitigates Radiation-Induced Pulmonary Fibrosis.

Triptolide (TPL) may mitigate radiation-induced late pulmonary side effects through its inhibition of global pro-inflammatory cytokines. In this study, we evaluated the effect of TPL in C57BL/6 mice, the animals were exposed to radiation with vehicle (15 Gy), radiation with TPL (0.25 mg/kg i.v., twice weekly for 1, 2 and 3 months), radiation and celecoxib (CLX) (30 mg/kg) and sham irradiation. Cultured supernatant of irradiated RAW 264.7 and MLE-15 cells and lung lysate in different groups were enzyme-linked immunosorbent assays at 33 h. Respiratory rate, pulmonary compliance and pulmonary density were measured at 5 months in all groups. The groups exposed to radiation with vehicle and radiation with TPL exhibited significant differences in respiratory rate and pulmonary compliance (480 ± 75/min vs. 378 ± 76/min; 0.6 ± 0.1 ml/cm H2O/p kg vs. 0.9 ± 0.2 ml/cm H2O/p kg). Seventeen cytokines were significantly reduced in the lung lysate of the radiation exposure with TPL group at 5 months compared to that of the radiation with vehicle group, including profibrotic cytokines implicated in pulmonary fibrosis, such as IL-1ß, TGF- ß1 and IL-13. The radiation exposure with TPL mice exhibited a 41% reduction of pulmonary density and a 25% reduction of hydroxyproline in the lung, compared to that of radiation with vehicle mice. The trichrome-stained area of fibrotic foci and pathological scaling in sections of the mice treated with radiation and TPL mice were significantly less than those of the radiation with vehicle-treated group. In addition, the radiation with TPL-treated mice exhibited a trend of improved survival rate compared to that of the radiation with vehicle-treated mice at 5 months (83% vs. 53%). Three radiation-induced profibrotic cytokines in the radiation with vehicle-treated group were significantly reduced by TPL treatment, and this partly contributed to the trend of improved survival rate and pulmonary density and function and the decreased severity of pulmonary fibrosis at 5 months. Our findings indicate that TPL could be a potential new agent to mitigate radiation-induced pulmonary fibrosis.

PicoGreen assay of circular DNA for radiation biodosimetry.

We developed a simple, rapid and quantitative assay using the fluorescent probe PicoGreen to measure the concentration of ionizing radiation-induced double-stranded DNA (dsDNA) in mouse plasma, and we correlated this concentration with the radiation dose. With 70 µl of blood obtained by fingerstick, this 30 min assay reduces protein interference without extending sample processing time. Plasma from nonirradiated mice (BALB/c and NIH Swiss) was pooled, diluted and spiked with dsDNA to establish sensitivity and reproducibility of the assay to quantify plasma dsDNA. The assay was then used to directly quantify dsDNA in plasma at 0-48 h after mice received 0-10 Gy total-body irradiation (TBI). There are three optimal conditions for this assay: 1:10 dilution of plasma in water; 1:200 dilution of PicoGreen reagent in water; and calibration of radiation-induced dsDNA concentration through a standard addition method using serial spiking of samples with genomic dsDNA. Using the internal standard calibration curve of the spiked samples method, the signal developed within 5 min, exhibiting a linear signal (r(2) = 0.997). The radiation-induced elevation of plasma DNA in mice started at 1-3 h, peaked at 9 h and gradually returned to baseline at 24 h after TBI (6 Gy). DNA levels in plasma collected from mice 9 h after 0-10 Gy TBI correlated strongly with dose (r(2) = 0.991 and 0.947 for BALB/c and NIH Swiss, respectively). Using the PicoGreen assay, we observed a radiation dose-dependent response in extracellular plasma DNA 9 h after irradiation with an assay time ≤ 30 min.

[Effect of α-melanocyte stimulating hormone and its novel analogue on the production of tissue factor pathway inhibitor in mice with endotoxemia].

OBJECTIVE: To evaluate the effect of α-melanocyte stimulating hormone (α-MSH) and its novel analogue STY39 on the production of tissue factor pathway inhibitor (TFPI) in mice with endotoxemia. METHODS: Female BALB/c mice were randomly divided into eight groups with 9 mice in each group. Endotoxemia was reproduced by intraperitoneal injection of lipopolysaccharide (LPS, 25 µg/kg) and D-galactosamine (D-Gal, 100 mg/kg). The animals of the control group were given phosphate buffered solution (PBS) instead. In the experimental groups, the mice were injected intraperitoneally with 2.5 mg/kg α-MSH or STY39 at 1, 2 or 3 hours following LPS injection. The orbital blood was collected at different time points, and tissues of lung, liver, and kidney were collected 8 hours after the administration of LPS. The plasma TFPI levels were determined by enzyme linked immunosorbent assay (ELISA), and the expression of TFPI mRNA in different tissues was determined with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The plasma TFPI levels began to increase (11.84±1.55 µg/L) in the endotoxemia mice 4 hours after LPS challenge and reached the peak (23.49 ± 1.12 µg/L) at 8 hours. α-MSH or STY39 treatment at 1, 2 or 3 hours after LPS challenge could significantly increase the TFPI content, with the best drug effect at 1 hour after LPS challenge (the blood was collected 8 hours after LPS challenge, α-MSH group: 58.79±2.67 µg/L vs. 28.49±1.69 µg/L, STY39 group: 71.08±2.13 µg/L vs. 28.49±1.69 µg/L, both P<0.01), and the effect of STY39 was better than that of α-MSH (P<0.01). A small amount of TFPI mRNA expression was observed in each tissue of the healthy mice. After LPS challenge, TFPI mRNA expression was increased in all the tissues, especially in the lung, liver and kidney. α-MSH or STY39 treatment at 1 hour after LPS challenge could significantly up-regulate the expression of TFPI mRNA in the lung and liver (A value, α-MSH in lung: 51.10±2.89 vs. 32.43±2.51, STY39 in lung: 72.11±3.48 vs. 32.43±2.51; α-MSH in liver: 43.21±2.12 vs. 29.29±2.06, STY39 in liver: 66.82±1.76 vs. 29.29±2.06, both P<0.01). The treatment with STY39 at 1 hour after LPS challenge could significantly up-regulate the expression of TFPI mRNA in the kidney (A value: 45.21±1.80 vs. 30.44±2.23, P<0.01), but the treatment with α-MSH had no obvious effect (A value: 24.61±1.98 vs. 30.44±2.23, P>0.05). The enhancing effect of early administration of STY39 on TFPI mRNA expression in the lung, liver and kidney tissues of endotoxemia mice was more powerful than that of α-MSH (all P<0.01). CONCLUSIONS: The early administration of α-MSH or STY39 may up-regulate TFPI production in the mice with endotoxemia, and the effect of STY39 is superior to α-MSH.

Mitochondrial DNA and functional investigations into the radiosensitivity of four mouse strains.

We investigated whether genetic radiosensitivity-related changes in mtDNA/nDNA ratios are significant to mitochondrial function and if a material effect on mtDNA content and function exists. BALB/c (radiosensitive), C57BL/6 (radioresistant), and F1 hybrid mouse strains were exposed to total body irradiation. Hepatic genomic DNA was extracted, and mitochondria were isolated. Mitochondrial oxygen consumption, ROS, and calcium-induced mitochondrial swelling were measured. Radiation influenced strain-specific survival in vivo. F1 hybrid survival was influenced by maternal input. Changes in mitochondrial content corresponded to survival in vivo among the 4 strains. Calcium-induced mitochondrial swelling was strain dependent. Isolated mitochondria from BALB/c mice were significantly more sensitive to calcium overload than mitochondria from C57BL/6 mice. Maternal input partially influenced the recovery effect of radiation on calcium-induced mitochondrial swelling in F1 hybrids; the hybrid with a radiosensitive maternal lineage exhibited a lower rate of recovery. Hybrids had a survival rate that was biased toward maternal input. mtDNA content and mitochondrial permeability transition pores (MPTP) measured in these strains before irradiation reflected a dominant input from the parent. After irradiation, the MPTP opened sooner in radiosensitive and hybrid strains, likely triggering intrinsic apoptotic pathways. These findings have important implications for translation into predictors of radiation sensitivity/resistance.

SVα-MSH, a novel α-melanocyte stimulating hormone analog, ameliorates autoimmune encephalomyelitis through inhibiting autoreactive CD4(+) T cells activation.

Alpha-melanocyte stimulating hormone (α-MSH) plays a crucial role in the regulation of immune and inflammatory reactions. Here we report that SVα-MSH, a novel α-MSH analog, could ameliorate the clinical severity of experimental autoimmune encephalomyelitis (EAE) in a preventive and therapeutic manner. SVα-MSH treatment induced the production of regulatory T (Treg) cells and reduced the Th17 cells in the CNS of EAE mice. SVα-MSH-treated PLP peptide 139-151-specific T cells showed a down-regulation of T cell activation markers CD69 and CD134. SVα-MSH did not induce apoptosis but blocked the G1/S phase transition, reduced the expression of cyclin E, Cdk2 and the activity of NFAT and AP-1 transcription factors. Thus, SVα-MSH acts as a novel immunotherapeutic approach in the treatment of autoimmune attack on the CNS.

Alteration of the inflammatory molecule network after irradiation of soft tissue.

Inflammatory molecules (IMs) play an important role in ionizing radiation (IR)-induced soft tissue damage. The alteration of IMs as a function of time was studied with a protein array containing 62 IMs in mouse cutaneous soft tissues exposed to 30 Gy. The results showed that: (1) 2 days after irradiation, the levels of TGF-ß1, MIP-1γ, IL-1α, and sTNF RI increased, while IGFBP-3, CXCL16, and IL-1ß decreased in IR skin as compared to control skin; (2) 21 days after IR, TGF-ß1, and MIP-1 γ, IL-1α remained high, while CXCL16 and IL-1ß remained low; (3) 3 months after IR, the cytokine pattern exhibited reversals. The levels of MIP-1γ decreased, while VCAM-1, IGFBP-3, and TGF-ß1 production increased. The data indicated that: (a) IMs change as a function of time after soft tissue irradiation; (b) changing IM levels may reflect the altered balance of the cytokine network, leading to imbalance or homeostasis; and (c) an antibody-based protein array can be used to assess multiple IMs simultaneously, making it useful for bulk screening for changes in tissue cytokine levels.

The chemokine, macrophage inflammatory protein-2γ, reduces the expression of glutamate transporter-1 on astrocytes and increases neuronal sensitivity to glutamate excitotoxicity.

BACKGROUND: Changes in glutamatergic neurotransmission via decreased glutamate transporter (GLT) activity or expression contributes to multiple neurological disorders. Chemokines and their receptors are involved in neurological diseases but the role of chemokines in the expression of glutamate transporters is unclear. METHODS: Primary astrocytes were prepared from neonatal (<24 hours old) SJL/J mouse brains and incubated with 5 µg/ml lipopolysaccharide (LPS) or 50 ng/ml tumor necrosis factor α (TNF-α) for 24 hours. Soluble macrophage inflammatory protein-2γ (MIP-2γ) in culture supernatants was determined using a sandwich ELISA. The MIP-2γ effect on the expression of GLT-1 was measured by quantitative RT-PCR, flow cytometric analysis or western blot assay. Detergent-resistant membranes from astrocytes were isolated on the basis of their ability to float in density gradients. Raft-containing fractions were tracked by the enrichment of caveolin-1 and the dendritic lipid raft marker, flotillin-1. Cell viability was determined by measuring either the leakage of lactate dehydrogenase or the reduction of 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide by viable cells and confirmed by visual inspection. RESULTS: The production of the chemokine MIP-2γ by mouse cortical astrocytes increased significantly after stimulation with LPS or TNF-α in vitro. Astrocytes over-expressing MIP-2γ down-regulated the expression of GLT-1 at the mRNA and protein level and caused redistribution of GLT-1 out of the lipid rafts that mediate glutamate uptake. We used pharmacological inhibitors to identify the downstream signaling pathways underlying MIP-2γ activity. We also found complementary results by knocking down MIP-2γ activity in astrocytes with MIP-2γ small interfering RNA (siRNA). MIP-2γ overexpression in astrocytes enhanced the neuronal toxicity of glutamate by decreasing GLT-1 activity, but MIP-2γ itself was not toxic to neurons. CONCLUSIONS: These results suggest that MIP-2γ mediates the pathogenesis of central nervous system disorders associated with neutrophil infiltration in the brain and decreased GLT-1 activity.

Fibroblast growth factor-peptide improves barrier function and proliferation in human keratinocytes after radiation.

PURPOSE: Epidermal keratinocytes, which can be severely damaged after ionizing radiation (IR), are rapid turnover cells that function as a barrier, protecting the host from pathogenic invasion and fluid loss. We tested fibroblast growth factor-peptide (FGF-P), a small peptide derived from the receptor-binding domain of FGF-2, as a potential mitigator of radiation effects via proliferation and the barrier function of keratinocytes. METHODS AND MATERIALS: Keratinocytes isolated from neonatal foreskin were grown on transwells. After being exposed to 0, 5, or 10 Gy IR, the cells were treated with a vehicle or FGF-P. The permeability of IR cells was assessed by using transepithelial electrical resistance (TEER) and a paracellular tracer flux of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) with Ussing chambers. The cell proliferation was measured with yellow tetrazolium salt (MTT) and tritiated thymidine ([3H]-TdR) assays. The phosphorylation of extracellular signal-regulated kinases (ERK) was measured in an enzyme-linked immunosorbent (ELISA)-like assay, and the proteins related to tight junctions (TJ) and adherens junctions (AJ) were examined with Western blotting. We used a mouse model to assess the ability of FGF-P to promote the healing of skin ß burns created with a strontium applicator. RESULTS: We found (1) FGF-P reduced the permeability of irradiated keratinocytes, as evidenced by increased TEER and decreased diffusion of FITC-BSA, both associated with the regulation of different proteins and levels of TJ and AJ; and (2) FGF-P enhanced the proliferation of irradiated keratinocytes, as evidenced by increased MTT activity and [3H]-TdR incorporation, which was associated with activation of the ERK pathway; and (3) FGF-P promoted the healing of skin ß burns. CONCLUSIONS: FGF-P enhances the barrier function, including up-regulation of TJ proteins, increases proliferation of human keratinocytes, and accelerates the healing of skin ß burns. FGF-P is a promising mitigator that improves the proliferation and barrier function of keratinocytes after IR.

Antioxidant properties of select radiation mitigators based on semicarbazone and pyrazole derivatives of curcumin.

Fifty-eight semicarbazone and pyrazole derivatives of curcumin have been developed as potential mitigation agents to treat acute radiation syndrome (ARS). Pyridyl (D12, D13), furyl (D56), and phenyl (D68) derivatives of curcumin semi-carbazones were found to provide the highest dose modifying factors (DMF) with respect to survival in sub-TBI (bone marrow sparing) exposures in mouse models. To investigate the basis for the mitigating effects of these agents on ARS, we examined their oxidation potentials and radical scavenging properties in comparison to other semicarbazone and pyrazole curcumin derivatives with less effective DMFs. Comparisons between D12, D13, D56, and D68 and other semicarbazone and pyrazole derivatives of curcumin did not show a sufficient difference in reducing properties and hydrogen atom donating properties for these properties to be the basis of the dose modifying activities of these compounds. Therefore, their DMFs likely reflect structure-activity relationship(s),wherein interaction with key receptors or alteration of enzyme expression result in modifications of cellular or tissue responses to radiation, rather than on the derivatives' ability to modify radiation-induced flux of free radicals through direct interaction with these radicals.

Antioxidant properties of quercetin.

UNLABELLED: Quercetin, a plant-derived aglycone form of flavonoid glycosides, has been used as a nutritional supplement and may be beneficial against a variety of diseases, including cancer. We examined the antioxidant properties of quercetin. The reduction potential of quercetin was measured at various pH values using voltammetric methods, and its total antioxidant capacity (TAC) was measured using the phosphomolybdenum method. The effect of quercetin on production of reactive oxygen species (ROS) and nitric oxide (NO) in LPS-stimulated human THP-1 acute monocytic leukemia cells was determined by flow cytometry using CM-H2DCFDA dye. The results were compared with curcumin, a natural product exhibiting a similar range of reported health benefits. RESULTS: 1) Quercetin has a higher reduction potential compared with curcumin at three different pH settings and is comparable to Trolox at pH 7-9.5; 2) its TAC is 3.5 fold higher than curcumin; 3) it reduced LPS-induced ROS to near normal levels; 4) it reduced LPS-induced NO production. These data provide a physico-chemical basis for comparing antioxidants, with potential benefits individually or in combination.

STY39, a novel alpha-melanocyte-stimulating hormone analogue, attenuates bleomycin-induced pulmonary inflammation and fibrosis in mice.

Various anti-inflammatory agents have been used to treat acute or chronic lung injury-induced pulmonary fibrosis (PF). However, the efficacy of the available treatments is disappointing, and new therapies are urgently needed. In the current study, we investigated the effect of a novel α-melanocyte-stimulating hormone analog, STY39, on bleomycin (BLM)-induced pulmonary inflammation and fibrosis in mice. C57BL/6 mice received an intratracheal injection of BLM before being treated with STY39 (0.625, 1.25, or 2.5 mg/kg, i.p.) once a day for 14 consecutive days. Various parameters, reflecting the inflammatory reaction, metabolism of extracellular matrix, myofibroblast proliferation, and degree of fibrosis in the lung, were evaluated. We found that STY39 significantly improved the survival of mice with lethal BLM-induced lung injury, limited body weight loss and the increase in the lung index, reduced the mRNA expression of types I and III procollagen and the production of hydroxyproline in the lung, diminished myofibroblast proliferation, and ultimately reduced BLM-induced lung damage. Further investigation revealed that, in a dose-dependent manner, STY39 treatment inhibited leukocyte migration into the lung; reduced the production of TNF-α, IL-6, macrophage inflammatory protein 2, and transforming growth factor ß1 in the lung; and altered the ratio of matrix metalloproteinase 1 to tissue inhibitors of metalloproteinase 1. These findings suggest that STY39 attenuates BLM-induced experimental PF by limiting the inflammatory reaction through the inhibition of proinflammatory and profibrosis cytokines and by accelerating the metabolism of extracellular matrix. Therefore, STY39 may be an effective therapy for preventing PF.

α-MSH inhibits TNF-α-induced maturation of human dendritic cells in vitro through the up-regulation of ANXA1.

α-Melanocyte-stimulating hormone (α-MSH), an anti-inflammatory and immunomodulatory neuropeptide, has been shown to be effective in the experimental treatment of autoimmune diseases and allograft rejection. However, its regulatory mechanism is still unclear. Mature dendritic cells (DCs) are pivotal initiators of immune response and inflammation. We hypothesized that the regulatory role of α-MSH in DC maturation would contribute to the effects of α-MSH in immune-response-mediated disease models. It was found that α-MSH inhibited tumor necrosis factor-alpha (TNF-α)-induced maturation of human peripheral-monocyte-derived DCs (MoDCs), both phenotypically and functionally. This occurred through the down-regulation of the expression of co-stimulatory molecules CD83 and CD86, the production of IL-12, the promotion of IL-10 secretion, and the MoDC phagocytic activity, suggesting that the inhibition of DC maturation by α-MSH could contribute to the anti-inflammatory effect of this neuropeptide. Furthermore, increased expression of annexin A1 (ANXA1) was found to be responsible for the α-MSH inhibiting effect on TNF-α-induced MoDC maturation, which could be abolished by the treatment of MoDCs with specific, small interfering RNAs targeting ANXA1 (ANXA1-siRNA), suggesting that α-MSH-induced ANXA1 mediates the inhibition. Therefore, α-MSH inhibits TNF-α-induced maturation of human DCs through α-MSH-up-regulated ANXA1, suggesting that inhibition of the maturation of DCs by α-MSH could mediate the anti-inflammatory effect of the neuropeptide. Furthermore, ANXA1 could be identified as a new therapeutic drug target based on the role of DCs in immune-mediated inflammatory diseases.

Mitigation effect of an FGF-2 peptide on acute gastrointestinal syndrome after high-dose ionizing radiation.

PURPOSE: Acute gastrointestinal syndrome (AGS) resulting from ionizing radiation causes death within 7 days. Currently, no satisfactory agent exists for mitigation of AGS. A peptide derived from the receptor binding domain of fibroblast growth factor 2 (FGF-P) was synthesized and its mitigation effect on AGS was examined. METHODS AND MATERIALS: A subtotal body irradiation (sub-TBI) model was created to induce gastrointestinal (GI) death while avoiding bone marrow death. After 10.5 to 16 Gy sub-TBI, mice received an intramuscular injection of FGF-P (10 mg/kg/day) or saline (0.2 ml/day) for 5 days; survival (frequency and duration) was measured. Crypt cells and their proliferation were assessed by hematoxylin, eosin, and BrdU staining. In addition, GI hemoccult score, stool formation, and plasma levels of endotoxin, insulin, amylase, interleukin (IL)-6, keratinocyte-derived chemokine (KC) monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor (TNF)-alpha were evaluated. RESULTS: Treatment with FGF-P rescued a significant fraction of four strains of mice (33-50%) exposed to a lethal dose of sub-TBI. Use of FGF-P improved crypt survival and repopulation and partially preserved or restored GI function. Furthermore, whereas sub-TBI increased plasma endotoxin levels and several pro-inflammation cytokines (IL-6, KC, MCP-1, and TNF-alpha), FGF-P reduced these adverse responses. CONCLUSIONS: The study data support pursuing FGF-P as a mitigator for AGS.

[Detection of binding activity and biologic effect of a novel alpha-melanocyte-stimulating hormone analogue].

Binding activity and biologic effect of a novel alpha-melanocyte-stimulating hormone analogue were tested on cells transiently expressing the human melanocortin-1 (MC1), MC3, MC4, and MC5 receptors. The human MC1 and MC5 receptor genes were cloned into the expression vector pcDNA3. 1/ myc-his(-) B. The vectors were transferred to HEK-293 cells by the calcium phosphate method. Stable receptor populations were generated using G418 selection (900 microg x mL(-1)) for subsequent bioassay analysis. K(i) values of the novel alpha-MSH analogue for MC1, MC3, MC4, and MC5 receptors were obtained in competition with [125I]-NDP-MSH for binding studies. The cyclic AMP level was tested by using [3H]-cyclic AMP kit. It is showed that K(i) values of the novel alpha-MSH analogue for MC1, MC3, MC4, and MC5 receptors were (0.159 +/- 0.040), (35.430 +/- 6.743), (19.293 +/- 2.780) and (2.230 +/- 0.670) nmol L(-1), respectively. Its EC50 values for MC1, MC3, MC4, and MC5 receptors were (0.45 +/- 0.07), (7.80 +/- 0.65), (2.55 +/- 0.23) and (0.33 +/- 0.09) nmol L(-1), respectively. In these tests, the novel alpha-MSH analogue is a MC1R and MC5R selective agonist.

[Protein profiling of human dendritic cells infected with mycobacterium tuberculosis].

The response of dendritic cells (DCs) plays an essential role in the initiation of immune responses following Mycobacterium tuberculosis (MTB) challenge. Two-dimensional electrophoresis (2-DE) was employed to compare the global protein patterns between human DCs infected and that uninfected with MTB H37Rv ATCC 27294 strains, and 45 protein spots were found to express differentially. Four protein spots which remarkably changed in DCs infected with MTB H37Rv ATCC 27294 strains were measured by matrix assisted laser desorption/ionization tandem time-of-flight (TOF/TOF) mass spectrometry. The data obtained from peptide mass fingerprinting were used in protein database search. Four protein spots in gel were identified as Human Arsenite-stimulated ATPase (hASNA-I), Annexin IV, gamma-actin and Heat shock protein27 (HSP27). These data provide insight into the changed global protein patterns of the DCs after infection and may prove useful for further study in the interaction between MTB and host.

[Effect of alpha-melanocyte stimulating hormone on the apoptosis of the vascular endothelial cell of the lung in two-hit acute respiratory distress syndrome in rat].

OBJECTIVE: To investigate the effect of alpha-melanocyte stimulating hormone (alpha-MSH) on the apoptosis of the vascular endothelial cells of the lung in acute respiratory distress syndrome (ARDS) reproduced with acute hemorrhagic shock followed by intratracheal lipopolysaccharide (LPS, two-hit model) in rat. METHODS: Ten male Sprague Dawley rats, weighing (33.7+/-2.5) g, were randomly divided into two groups (A and B) with 5 in each group. All rats were anesthesized and ventilated mechanically with fractional concentration of inspired oxygen(FiO(2)) of 0.5, breath rate 100 times/min, tidal volume(V(T)) 12 ml/kg and inspiratory/expiratory ratio (I/E) 1:15. The blood was withdrawn to induce hemorrhagic shock via the carotid artery until blood pressure reached (45+/-5) mm Hg (1 mm Hg=0.133 kPa), which was maintained for 1 hour, and the shed blood and Ringer's lactate in volume equal to the shed blood were reinfused in 2 hours for resuscitation. Afterwards, LPS was given via the tracheal (200 microg/kg, in 500 microl normal saline) to establish the ARDS model. Group A was ARDS control group, group B was alpha-MSH administration group. alpha-MSH was intravenously administrated simultaneously, 3 hours and 6 hours after LPS given, the dosage was 17 mg/kg at each time point. The rats were sacrificed at 9 hours after LPS challenge, and the lung tissue was examined with microscope and electron microscope to observe the pathological changes and apoptosis of the vascular endothelial cells. RESULTS: In ARDS control group, remarkable infiltration of inflammatory cells was found in the alveoli, and the apoptosis of the vascular endothelial cells had developed to late stage. In alpha-MSH treatment group, few inflammatory cells were found in the alveoli, and the apoptosis of the endothelial cells was still in an early stage. CONCLUSION: alpha-MSH could inhibit the apoptosis of the vascular endothelial cells of the lung in the two-hit ARDS in rats. Therefore, it might have a protective effect on the lung after hemorrhagic shock and endotoxin challenge.