Figure 2
Figure 2. rTMD1 reduces LPS-induced inflammatory response and lethality, attenuates LPS-induced pulmonary accumulation of PMNs and renal injury, and enhances LPS clearance in vivo. (A,B) rTMD1 was intravenously administered before intraperitoneal injection of LPS (20 mg/kg). Sera were collected 6 hours after administration of LPS for assay of (A) TNF-α and (B) NO production. Values are the mean plus or minus SD (n = 10). *P < .05, **P < .01, ***P < .001 compared with the LPS-treated group; ###P < .001 compared with the PBS-treated group. (C) Representative microscopic images of hematoxylin-and-eosin–stained sections of lung are shown. (D) The number of infiltrated PMNs in each alveolus was observed by light microscope (original magnification ×630). The number of PMNs was counted from 4 randomly fields per slide on each experimental mouse and normalized to fields of alveolus numbers on each slide. Values are the mean plus or minus SD (n = 10). ***P < .001 compared with the LPS-treated group; ###P < .001 compared with the PBS-treated group. These graphs represent the results from 3 independent experiments. (E-G) rTMD1 (5 mg/kg and 25 mg/kg) was intravenously administered before LPS (20 mg/kg) was intraperitoneally injected into mice. After 12 hours, mice were killed and kidney tissues were removed. Kidney sections were observed by histologic hematoxylin-and-eosin staining. Representative microscopic images are shown. (E) rTMD1 suppression of renal injury. Arrowheads indicate the site of the glomerulus (scale bars represent 50 μm). rTMD1 decreased the levels of renal injury markers BUN (F) and creatinine (G) in mice sera. For each experimental group, n = 4; ###P < .001 compared with the LPS-untreated control mice; **P < .01, ***P < .001 compared with the LPS-treated control mice. These graphs represent the results from 3 independent experiments. (H) Mice received LPS (40 mg/kg) and rTMD1 (4 intravenous doses of 2 mg/kg at 0, 6, 12, and 24 hours after LPS injection). Survival was determined. For each experimental group, n = 20. ***P < .001 compared with the LPS-treated group. (I) Clearance of rTMD1 in circulation. The half-life of rTMD1 in the circulation was determined by intravenous injection of rTMD1 (10 mg/kg) and the levels of rTMD1 in serum samples were measured by a sandwich ELISA using anti–c-Myc and TM-H300 as capture and detection antibodies, respectively. Values are the mean plus or minus SD (n = 5). (J) Clearance of LPS in circulation without or with rTMD1 treatment. LPS (20 mg/kg) was intraperitoneally administered to male FVB mice without or with rTMD1 (10 mg/kg; intravenously), and serum samples were collected at various time intervals and the amount of LPS was determined by the Limulus amebocyte lysate test. Values are the mean plus or minus SD. For each time interval group, n = 5; *P < .05 and ***P < .001 compared with the LPS-treated mice.

rTMD1 reduces LPS-induced inflammatory response and lethality, attenuates LPS-induced pulmonary accumulation of PMNs and renal injury, and enhances LPS clearance in vivo. (A,B) rTMD1 was intravenously administered before intraperitoneal injection of LPS (20 mg/kg). Sera were collected 6 hours after administration of LPS for assay of (A) TNF-α and (B) NO production. Values are the mean plus or minus SD (n = 10). *P < .05, **P < .01, ***P < .001 compared with the LPS-treated group; ###P < .001 compared with the PBS-treated group. (C) Representative microscopic images of hematoxylin-and-eosin–stained sections of lung are shown. (D) The number of infiltrated PMNs in each alveolus was observed by light microscope (original magnification ×630). The number of PMNs was counted from 4 randomly fields per slide on each experimental mouse and normalized to fields of alveolus numbers on each slide. Values are the mean plus or minus SD (n = 10). ***P < .001 compared with the LPS-treated group; ###P < .001 compared with the PBS-treated group. These graphs represent the results from 3 independent experiments. (E-G) rTMD1 (5 mg/kg and 25 mg/kg) was intravenously administered before LPS (20 mg/kg) was intraperitoneally injected into mice. After 12 hours, mice were killed and kidney tissues were removed. Kidney sections were observed by histologic hematoxylin-and-eosin staining. Representative microscopic images are shown. (E) rTMD1 suppression of renal injury. Arrowheads indicate the site of the glomerulus (scale bars represent 50 μm). rTMD1 decreased the levels of renal injury markers BUN (F) and creatinine (G) in mice sera. For each experimental group, n = 4; ###P < .001 compared with the LPS-untreated control mice; **P < .01, ***P < .001 compared with the LPS-treated control mice. These graphs represent the results from 3 independent experiments. (H) Mice received LPS (40 mg/kg) and rTMD1 (4 intravenous doses of 2 mg/kg at 0, 6, 12, and 24 hours after LPS injection). Survival was determined. For each experimental group, n = 20. ***P < .001 compared with the LPS-treated group. (I) Clearance of rTMD1 in circulation. The half-life of rTMD1 in the circulation was determined by intravenous injection of rTMD1 (10 mg/kg) and the levels of rTMD1 in serum samples were measured by a sandwich ELISA using anti–c-Myc and TM-H300 as capture and detection antibodies, respectively. Values are the mean plus or minus SD (n = 5). (J) Clearance of LPS in circulation without or with rTMD1 treatment. LPS (20 mg/kg) was intraperitoneally administered to male FVB mice without or with rTMD1 (10 mg/kg; intravenously), and serum samples were collected at various time intervals and the amount of LPS was determined by the Limulus amebocyte lysate test. Values are the mean plus or minus SD. For each time interval group, n = 5; *P < .05 and ***P < .001 compared with the LPS-treated mice.

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