Expression and significance of miR-223 in rats with pulmonary fibrosis.

OBJECTIVE: To explore the expression and significance of miR-223 in mice with pulmonary fibrosis. MATERIALS AND METHODS: The rats were separated into a control group (n=15), a sham operation group (n=15), and a model group (n=45) (which was then divided into a 3-day group, a 7-day group, and a 14-day group, with 15 rats in each group). The rat model of pulmonary fibrosis was established. The rats in the model group were injected with bleomycin solution, while those in the control group and sham operation group were given the same operation and injected with the same amount of normal saline. After observing the pulmonary function indexes of the rats on the 3rd, 7th and 14th days after modeling, the rats were sacrificed by cervical dislocation, the pulmonary inflammation and fibrosis of the rats were observed, and the HYP (hydroxyproline) content and miR-223 expression level were determined. Pearson correlation analysis was employed to analyze the correlation between miR-223 and HYP. RESULTS: The pulmonary inflammation score of the model group was significantly higher than that of the sham group and the control group, and the pulmonary inflammation of the model group significantly increased with the increase of time (p<0.05). The pulmonary fibrosis score in the model group was markedly higher than that in the rest two groups, and the pulmonary fibrosis in the model group elevated significantly with the passage of time (p<0.05). The relevant pulmonary function indexes of the model group rats were significantly lower than those of the other two groups, and the pulmonary function of the model group rats gradually decreased with time (p<0.05). As to the HYP, it presented notably higher content in the model group than in the remaining two groups, and its content in the model group rats increased significantly with time (p<0.05). The expression of miR-223 decreased with the increase of fibrosis (p<0.05), and the expression level of miR-223 was negatively correlated with the HYP content (p<0.05). CONCLUSIONS: MiR-572 targeted CDH1 to promote cell metastasis in WT by suppressing EMT.

Effects of survivin antagonists on growth of established tumors and B7-1 immunogene therapy.

BACKGROUND: Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is detectable in most types of cancer, and its presence is associated with a poor prognosis. We determined the effects of gene-based therapies that inhibit survivin function in a mouse tumor model. METHODS: Using five to six mice per treatment group, we injected tumors derived from mouse EL-4 thymic lymphoma cells with plasmids encoding antisense survivin, a dominant-negative mutant survivin, and the T-cell costimulator B7-1. Expression of endogenous survivin and the proteins encoded by the injected plasmids were examined by immunohistochemical staining of tumor sections and by western blot and flow cytometry analyses of isolated tumor cells. Tumor growth, the generation of antitumor cytotoxic T-lymphocyte (CTL) activity, apoptosis, and the contribution of leukocyte subsets to antitumor activity were measured. All statistical tests were two-sided. RESULTS: Large (1.0-cm diameter) tumors had approximately 10-fold more survivin than small (0.2-cm diameter) tumors. At 28 days after injection, antisense and dominant-negative mutant survivin plasmids statistically significantly inhibited the growth of both small (P =.006 and P =.0018, respectively) and large (P<.001 for both plasmids) EL-4 tumors compared with tumors injected with empty plasmid. The growth of large tumors was further inhibited by intratumoral injection with antisense survivin and B7-1 (P =.004); thus, inhibition of survivin expression renders large tumors susceptible to B7-1-mediated immunotherapy. Mice whose tumors were completely eradicated by injection of B7-1 remained tumor free for 26 days after re-injection with EL-4 cells (when the experiment ended). Compared with tumors injected with empty plasmid, tumors injected with survivin-based plasmids had increased apoptosis, and animals bearing such tumors generated more antitumor CTLs. CONCLUSION: Intratumoral injection of plasmids that block survivin expression and stimulate the generation of tumor-specific CTLs may be beneficial for the treatment of large lymphomas.

T20/DP178, an ectodomain peptide of human immunodeficiency virus type 1 gp41, is an activator of human phagocyte N-formyl peptide receptor.

Human immunodeficiency virus type 1 (HIV-1) envelope protein gp41 mediates viral fusion with human host cells. The peptide segment T20/DP178, located in the C-terminus of the ectodomain of gp41, interacts with the N-terminal leucine zipper-like domain on gp41 to establish the fusogenic conformation of the virus. Synthetic T20/DP178 peptide is highly efficacious in inhibiting HIV-1 infection in vitro by disrupting the transformation of fusogenic status of viral gp41; thus, it has been proposed for clinical trial. We report that synthetic T20/DP178 is a chemoattractant and activator of human peripheral blood phagocytes but not of T lymphocytes. We further demonstrate that T20/DP178 specifically activates a seven-transmembrane, G-protein-coupled phagocyte receptor for N-formylated chemotactic peptides, formyl peptide receptor (FPR). Moreover, synthetic T20/DP178 analogs lacking N-terminal amino acids acted as FPR antagonists. Our results suggest that gp41 peptides regulate phagocyte function via FPR and identify a novel mechanism by which HIV-1 may modulate innate immunity.

Development of infant botulism in a 3-year-old female with neuroblastoma following autologous bone marrow transplantation: potential use of human botulism immune globulin.

Infant botulism is a rare disease caused by the release of toxin produced in the intestinal tract by Clostridium botulinum. The disease primarily affects infants under 1 year of age. We report a 3-year-old child with stage IV neuroblastoma who developed symptoms of progressive motor weakness, bulbar palsy and respiratory failure 42 days after autologous BMT. The diagnosis of infant botulism was established by identifying botulism toxin in the stool. Human botulism immune globulin (HBIG) was administered. Following the diagnosis, the patient made significant recovery over the next 7 weeks and was successfully extubated from mechanical ventilation. However, her neuroblastoma eventually recurred and she subsequently died of progressive disease. Although the etiology of the development of infant botulism in this case following autologous BMT still remains unclear, alteration of the intestinal microbial environment from gut sterilization and laminar airflow room isolation or, alternatively, immune suppression during pre- and post-autologous BMT and activation of endogenous spores may have contributed to the development of this disease. The use of HBIG in children with botulism over 1 year of age may be beneficial.

Molecular analysis of a myxoid chondrosarcoma with rearrangements of chromosomes 10 and 22.

Myxoid chondrosarcoma is a rare tumor of adulthood. An associated nonrandom reciprocal translocation between chromosome 9 and 22 was previously reported in this tumor. We performed cytogenetic and molecular genetic analysis of a myxoid chondrosarcoma derived from the sphenoid bone. Using restriction fragment length polymorphism (RFLP) analysis, we demonstrated rearrangement and a possible allele loss close to the chromosome 22 breakpoint. In addition, structural rearrangement in the chromosome 10q21.1 region and an allele loss in the chromosome 10q21-q23 region were also detected. In tumor DNA an additional hybridization fragment was detected by pAS-1 probe, which recognizes multiple pseudogenes of argininosuccinate synthetase dispersed in various chromosomes. We were unable to detect chromosomal abnormalities with two additional chromosome 9q probes. This study suggests that multiple gene rearrangements occurred in the myxoid chondrosarcoma and the significance of this is discussed.

Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells.

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.

Expression of normal and mutant ras proteins in human acute leukemia.

The expression of normal and mutant ras genes in human acute leukemias was assessed by the direct analysis of p21ras polypeptides, using immunoprecipitation with monoclonal antibodies. High-resolution two-dimensional gel electrophoresis permits the identification of a wide array of activated ras alleles encoding proteins with single amino acid substitutions at any of several positions. The products of three ras genes, H-ras, N-ras, and K-ras, were detected in each of 33 specimens of fresh leukemic cells. The normal K-ras and N-ras polypeptides were substantially more abundant than H-ras p21 in all samples. In over three-fourths of the cases the total amount of p21ras exceeded that seen in control hematopoietic cell lines. The level of ras expression did not correlate simply with clinical parameters, although the two samples with the most abundant p21ras were obtained from patients with relapsed T-cell acute lymphocytic leukemia (ALL). Abnormal p21ras, consistent with oncogenic activation, was found in eight patients. Six of 11 samples from acute myelocytic leukemia (AML) patients displayed a mutant N-ras p21, while only one of 20 ALL specimens had abnormal N-ras, and one had a mutant H-ras. In every case the mutant protein comprised a minority of total p21ras. In two T-cell ALL cell lines both normal and activated N-ras gene products were expressed at equal levels. By contrast, in five fresh AML samples the abnormal N-ras protein was several-fold less abundant than the normal N-ras p21. This finding implies that only a proportion of leukemic cells in an individual patient may carry the mutant ras oncogene.