[Effect of severe thoracic trauma combined with endotoxin challenge on phagocytosis function of alveolar macrophages and pulmonary interstitial macrophages].

OBJECTIVE: To observe the difference of phagocytosis between alveolar macrophages and pulmonary interstitial macrophages, and to investigate their responses to severe thoracic trauma with or without lipopolysaccharide (LPS) challenge. METHODS: A rat model of severe thoracic trauma was reproduced by thoracic impact machine. The alveolar macrophages and interstitial macrophages were isolated before injury and at 2, 4, 8, 16, 24 hours after injury respectively. The dynamic changes of these macrophage phagocytosis were tested by malachite green colorimetry. RESULTS: Macrophage phagocytosis function was increased during the early stage after trauma (2 and 4 hours) and then decreased. The phagocytosis function of alveolar macrophages was stronger than that of interstitial macrophages in all time points before and after trauma. After challenge with LPS, no further significant effect on the alveolar macrophages was found, while LPS challenge could stimulate the phagocytosis of interstitial macrophages. CONCLUSION: Alveolar macrophages and pulmonary interstitial macrophages are functional heterogeneous, and their response to trauma and combined with endotoxin challenge are different. The results indicate that the two subgroups of macrophages play different roles in immune function disorder after trauma.

[Changes in human leukocyte antigen-DR expression on monocytes and its value of prediction on infection complication in trauma patients].

OBJECTIVE: To observe the changes of human leukocyte antigen DR (HLA-DR) expression in monocytes of trauma patients and its value of prediction on infection complications. METHODS: Fifty-four trauma patients were divided into three groups according to severity of injury: severe trauma group injury severity score (ISS) >or=25, moderate trauma group (16or=16) were divided into three groups according to infection or not: no infection group, localized infection group and systemic infection group. Blood samples were collected immediately after admission and serially at 8:30 to 9:00 a.m. on days 1, 2, 4, 6, 8, 14 after admission, and monocyte HLA-DR expression was determined with monoclonal staining and flow cytometry. RESULTS: The HLA-DR expression in monocytes was reduced in the trauma patients. The lowest levels of HLA-DR were recorded on day 2 after trauma. Subsequently HLA-DR expression in monocytes increased gradually. During the whole observation, the HLA-DR expression was significantly decreased in both severe trauma group and moderate trauma group versus control group, but no significant differences were found between severe trauma group and moderate trauma group, mild trauma group and control group, Immediately after trauma, HLA-DR expression in monocytes was significantly lower in the localized infection group than that in the patients without infection, and lasted until day 4 after trauma. The mean fluorescence intensity of HLA-DR expression in monocytes on the day 2 to 14 after trauma, the percentage of HLA-DR monocytes on the day 1 to 14 after trauma were significantly lower in the systemic infection group than those in the localized infection group. The level of HLA-DR expression in monocyte in the 2 died trauma patients was lowered till died. CONCLUSION: In severe trauma patients, the HLA-DR expression in monocytes is significantly decreased, and decreased levels of HLA-DR expression in monocytes might be the early indicators of an immune deviation associated with the development of infection complications and prognosis.

Identification of synthetic peptides that inhibit lipopolysaccharide (LPS) binding to myeloid differentiation protein-2 (MD-2).

Many studies have suggested that the synergic effect of myeloid differential protein-2 (MD-2) on bacterial lipopolysaccharide (LPS) stimulation of toll-like receptor 4 (TLR4) may be a critical step during the LPS-TLR4 response signaling pathway. We performed a bioinformatic analysis on the MD-2 protein and identified the amino acid sequence NH2-FSKGKYKCV-COOH (K128-132) as a possible key sequence involved in the binding between MD-2 and LPS. We then screened a random phage display peptide library using this sequence as bait in order to identify antagonistic peptides. After 3 rounds of selection, 3 positive clones were identified. All 3 peptides were shown to inhibit, in a dose-dependent manner the production of tumor necrosis factor-α and interleukin-6 in human U937 and THP-1 cell lines as well as human peripheral blood monocytes stimulated by LPS. Only 2 of the 3 peptides were able to bind MD-2 directly as shown by sulfo-SBED biotin label transfer experiments. BALB/C mice were used to estimate the protection of these peptides from LPS challenge, and 2 of the 3 peptides (Lys-Thr-Val-Pro-Asp-Asn-His and Ile-Gly-Lys-Phe-Leu-Tyr-Arg) reduced mortality of the challenged mice from 100% to 53.8%. This study has demonstrated that interfering with the binding between MD-2 and LPS might be a potential therapeutic strategy for treating LPS-induced sepsis, and in doing so has identified 2 potential peptide candidates.

[Cloning and identification of NF-kappaB p50 Rel homology domain and detection of its autonomous reporter gene activity].

AIM: To clone NF-kappaB p50 Rel homology domain (RHD) gene and construct the "bait" vector in yeast two-hybrid system, and detect the yeast cell toxicity and autonomous reporter gene activity of target gene. METHODS: Total RNA was extracted from human peripheral blood mononuclear cells and NF-kappaB p50 RHD gene was amplified by RT-PCR, and cloned into pGBKT7. The recombinant plasmid was transformed into yeast AH109. The growth condition of the transformants was observed in the selected medium SD/-Trp. The reporter gene activity of target gene in the yeast cells was verified by filter blotting. RESULTS: Using restriction enzyme digestion analysis and PCR, the length of inserted gene was confirmed correct. Sequencing result indicated that the sequence of the inserted gene and its open reading frame were completely correct, and then the recombinant plasmid was named pGBKT7-p50. p50 RHD gene had neither autonomous reporter gene activity nor yeast cytotoxicity. CONCLUSION: As a bait plasmid, pGBKT7-p50 could be used in yeast two-hybrid system to screen and capture the polypeptides which interact with p50 RHD.