OBJECTIVETo investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells.

METHODSRecombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining.

RESULTSThe tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry.

CONCLUSIONImmunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.

ADP Ribose Transferases ; genetics ; Animals ; Apoptosis ; Bacterial Toxins ; genetics ; Bone Neoplasms ; metabolism ; pathology ; Caspase 6 ; genetics ; metabolism ; Cell Line, Tumor ; Exotoxins ; genetics ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; Random Allocation ; Receptor, ErbB-2 ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden ; Virulence Factors ; genetics

OBJECTIVETo explore the genetic basis of using three species of Asarum as Herba Asari to determine the taxonomic positions of Asarum heterotropoides and A. siebodii; and to apply DNA molecular analysis as a tool for identification of Herba Asari.

METHODPCR, purification, sequence analysis were prerformed.

RESULTMS sequences of the three Asarum species were obtained. 3 botanical sources of Herba Asari are closely clustered together on the topology tree; one inner branch is composed of A. heterotropoides and A. sieboldii, whereas another branch contains A. sieboldii. Their ITS sequences are different.

CONCLUSIONThree plant species of Herba Asari are closely related, and there are genetic reasons that they are used as the sources of the same medicine. The classification placement of A. sieboldii is not certain. The differences of ITS sequences of the botanical sources of Herba Asari can be used as a means of identification.

Asarum ; classification ; genetics ; Base Sequence ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Molecular Sequence Data ; Phylogeny ; Plants, Medicinal ; genetics ; Species Specificity

OBJECTIVEIn order to explore the role of toll-like receptors 2 (TLR2) in initiating inflammatory response, the expression of TLR2 of the liver and IL-18, TNF-alpha and IFN-gamma of plasma in fulminant hepatic failure was analysed.

METHODSD-galactosamine (D-Gal, 900 mg/kg) and lipopolysaccharide (LPS, 10 microg/kg) were administered intraperitoneally into the BALB/C mice. To evaluate the hepatic injury, serum transaminase (ALT and AST) and plasma IL-18, TNF-alpha and IFN-gamma were determined and the mortality was observed at various time points following the intraperitoneal injection. The level of TLR2 mRNA was measured by semiquantitative RT-PCR. The protein expression of TLR2 in the liver was detected by immunohistochemistry. The data was analyzed by SAS software.

RESULTSAfter 4 hours of intraperitoneal injection of D-Gal/LPS, the serum transaminase and plasma IL-18, TNF-alpha and IFN-gamma levels were elevated. The treated mice began to die at 7 hours. The mortality reached up to 80% at 10 h. TLR2 mRNA was expressed at a low level in liver tissues of normal mice, while it was significantly increased and maintained at a higher level following intraperitoneal injection with D-Gal/LPS. The expression of TLR2 protein was similar to that of the TLR2 mRNA, and the expression of TLR2 mRNA was positively correlated with the concentration of plasma IL-18, TNF-alpha and IFN-gamma (r=0.36, P=0.02; r = 0.48, P 0.003; r = 0.72, P<0.001) at different time points.

CONCLUSIONSOur results showed that TLR2 was involved in initiating and inducing the expression of proinflammation cytokines in this model of fulminant hepatic failure. The results suggest that adjusting the expression of TLR2 might be a new strategy in preventing the development of infectious diseases

Animals ; Galactosamine ; Interferon-gamma ; blood ; Interleukin-18 ; blood ; Lipopolysaccharides ; Liver ; metabolism ; Liver Failure, Acute ; chemically induced ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; biosynthesis ; genetics ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; metabolism

After treating with chemotherapy or immunosuppressant, malignant diseases of hematopoietic system such as leukemia, malignant lymphoma and aplastic anemia usually induced severe infection such as sepsis. Sepsis which is hard to be diagnosed causes high death rate. This study was purposed to establish an experimental sepsis mouse model so as to provide a basis for pathogenesis and intervention study. A classic caecal ligation and puncture (CLP) was used to establish experimental sepsis model. ELISA was used to detect levels of C5a, IL-6, TNFalpha, and IFN-gamma. Flow Cytometry was applied to measure apoptosis of lymphocytes in thymus and mesentery. The pathologic changes of thymus and spleen were confirmed by HE staining. The results showed that almost 70%-80% mice died at 72 hours after CLP. Only approximate 20% animal survived during finite time, mice in CLP group had significant weight lose. Meanwhile large release of different inflammatory mediators which are related with sepsis (C5a, IL-6, TNF-alpha, and IFN-gamma) was observed after CLP. Apoptosis of lymphocytes in thymus and mesentery lymphonodus was enhanced markedly after CLP. Significantly pathologic injury was also observed in thymus and spleen. It is concluded that a mouse model of experimental sepsis was successfully established by caecal ligation and puncture which can well mimic the clinical symptom of sepsis. The experimental sepsis mouse model provides an excellent tool for exploring the pathogenesis and intervention ways for sepsis accompanied with complicated malignant hematological diseases in vivo.
Animals ; Apoptosis ; Cecum ; injuries ; Complement C5a ; metabolism ; Disease Models, Animal ; Interferon-gamma ; metabolism ; Interleukin-6 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Sepsis ; metabolism ; pathology ; Spleen ; pathology ; Thymus Gland ; pathology ; Tumor Necrosis Factor-alpha ; metabolism

Objective: This study aimed to assess the technical feasibility, efficacy, and safety of the safe triangular working zone (STWZ) approach applied in percutaneous vertebroplasty (PV) for spinal metastases involving the posterior part of the vertebral body. Materials and Methods: We prospectively enrolled 87 patients who underwent PV for spinal metastasis involving the posterior part of the vertebral body, with or without the STWZ approach, from January 2019 to April 2022. Forty-nine patients (27 females and 22 males; mean age ± standard deviation [SD], 57.2 ± 11.6 years; age range, 31–76 years) were included in group A (with STWZ approach), accounting for 54 vertebrae. Thirty-eight patients (18 females and 20 males; 59.1 ± 10.9 years; 29–81 years) were included in group B (without STWZ approach), accounting for 57 vertebrae. Patient demographics, procedure-related variables, and pain relief as assessed using the visual analog scale (VAS) were collected at different time points. Tumor recurrence in the vertebrae after PV was analyzed using Kaplan–Meier curves. Results: The STWZ approach was successful from T1 to L5 without severe complications. Cement filling was satisfactory in 47/54 (87.0%) and 25/57 (43.9%) vertebrae in groups A and B, respectively (v< 0.001). Cement leakage was not significantly different between groups A and B (p= 1.000). Mean VAS score ± SD before and 1 week and 1, 3, 6, 9, and 12 months after PV were 7.6 ± 1.8, 4.2 ± 2.0, 2.7 ± 1.9, 1.9 ± 1.5, 1.7 ± 1.4, 1.7 ± 1.1, and 1.6 ± 1.3, respectively, in group A and 7.2 ± 1.7, 4.0 ± 1.3, 3.4 ± 1.6, 2.4 ± 1.2, 1.8 ± 1.0, 1.4 ± 0.5, and 1.7 ± 0.9, respectively, in group B. Kaplan–Meier analysis showed a lower tumor recurrence rate in group A than in group B (p = 0.001). Conclusion The STWZ approach may represent a new, safe, alternative/auxiliary approach to target the posterior part of the vertebral body in the PV for spinal metastases.

Objective To study the impact of carbon monoxide(CO)on expression levels of inducible nitric oxide synthase(iNOS)mRNA in guinea pigs with allergic rhinitis(AR).Methods Twenty four guinea pigs were divided randomly into four study groups with 6 guinea pigs in each.The guinea pigs in the first group were treated with saline only(Group 1,the healthy controls).The remaing guinea pigs were sensitized by ovalbumin and thus establishing the AR models.After sensitization,the animals in the second group remained untreated(Group 2,AR control group).The third group was treated with Hemin as the induction group,and the fourth group was treated with Zinc protoporphyrin(ZnPP)as the suppression group.The plasma concentration of carboxyhemoglobin(COHb)was measured,which represents the concentration of CO.The expression levels of Heme oxygenase-1(HO-1)and NOS mRNAs in nasal mucosa were determined by fluorescent quantitative RT-PCR.Results AR models were established successfully in all study guinea pigs.The concentrations of COHb(x-±s)in plasma of the second group(2.27% ±1.13%)were significantly(q=4.10,P＜0.01)higher than those of healthy controls(1.08% ± 0.24%).The plasma concentration of COHb in the third group(3.17% ±0.68%)were also significantly higher(q=3.12,P＜0.05)than those in the second group.The expression levels of HO-1 and iNOS in nasal mucosa of the second group[(7.80 ± 1.60)×10~(-3) and(5.81 ±0.05)×10~(-3),respectively]were also significantly(q equals 5.52 and 7.21,respectively,P＜0.01)higher than those of controls[(1.96 ±0.71)×10~(-3) and(0.97 ±]0.05)×10~(-3),respectively].The expression levels of HO-1 and iNOS in the nasal mucosa of the third group[(11.89 ± 4.78)×10~(-3) and(7.42 ± 0.70)×10~(-3),respectively]were significantly(q equals 3.86 and 2.22,P＜0.05)higher than those of the second group.The expression levels of HO-1 and iNOS in nasal mucosa of the fourth group[(3.82 ±0.98)×10~(-3) and(2.34 ±0.04)×10~(-3),respectively]were significantly(q equals 3.76 and 5.18,P＜0.05)lower than those in the second group.Conclusions Endogenous carbon monoxide influenced the expression levels of iNOS in nasal mocusa in guinea pigs with AR.

PURPOSE: Although the polymorphisms of erythrocyte complement receptor type 1 (CR1) in patients with malaria have been extensively studied, a question of whether the polymorphisms of CR1 are associated with severe malaria remains controversial. Furthermore, no study has examined the association of CR1 polymorphisms with malaria in Chinese population. Therefore, we investigated the relationship of CR1 gene polymorphism and malaria in Chinese population. MATERIALS AND METHODS: We analyzed polymorphisms of CR1 gene rs2274567 G/A, rs4844600 G/A, and rs2296160 C/T in 509 patients with malaria and 503 controls, using the Taqman genotyping assay and PCR-direct sequencing. RESULTS: There were no significant differences in the genotype, allele and haplotype frequencies of CR1 gene rs2274567 G/A, rs4844600 G/A, and rs2296160 C/T polymorphisms between patients with malaria and controls. Furthermore, there was no association of polymorphisms in the CR1 gene with the severity of malaria in Chinese population. CONCLUSION: These findings suggest that CR1 gene rs2274567 G/A, rs4844600 G/A, and rs2296160 C/T polymorphisms may not be involved in susceptibility to malaria in Chinese population.
Adult ; Alleles ; Asian Continental Ancestry Group ; Case-Control Studies ; China ; Erythrocytes/parasitology ; Female ; Genetic Predisposition to Disease ; Genotype ; *Haplotypes ; Humans ; Malaria/ethnology/*genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide/*genetics ; Promoter Regions, Genetic/*genetics ; Receptors, Complement/blood/*genetics ; Taq Polymerase

OBJECTIVETo identify the clinical characteristics of and to explore the prognostic factors influencing mortality in children with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH).

METHODA retrospective study was conducted on 62 pediatric patients with EBV-HLH who were admitted to our hospital between 2003 and 2008. All their medical records were reviewed and analyzed. For each patient, demographic, clinical and laboratory data, genetic findings and outcome information were collected. The patients were divided into two groups: deceased or survived based on the follow-up results. Comparative analysis of the data was done by using independent-samples t test and Logistic multiple and univariate regression.

RESULT(1) Among the 62 EBV-HLH patients, 36 were male and 26 were female. The age of onset ranged from 2 months to 14 years and most of the patients were between 1 and 3 years of age. EBV-HLH occurred mainly in the setting of reactivation (61.3%). (2) All patients exhibited persistent or intermittent fever and cytopenia >/= 2 cell lines. Most of the patients presented with hepatomegaly (83.9%), splenomegaly (72.6%) and lymphadenopathy (69.4%). The main laboratory features showed an elevation of serum ferritin and aminotransferase levels. A reduction in serum albumin was observed and exhibited coagulopathy with hypofibrinogenemia and hypertriglyceridemia in most of the patients. Forty-eight of patients had hemophagocytosis in bone marrow at diagnosis of EBV-HLH. The serum EBV DNA level in 14 of 31 patients with EBV-HLH was in the range of 5.12 x 10(2) - 7.69 x 10(7) copies/ml with a mean value of 10(3.9) copies/ml. (3) Three heterozygous mutations in coding region were found, which resulted in amino acid change (C102F, S108N and T450M) in 3 patients. One patient had compound heterozygous mutations (S108N and T450M) in the PRF1 gene as the background defect and documented familial HLH type 2 (FHL2). (4) During the observational period, 35 of 57 patients (61.4%) died 3 months to 3 years after the onset, while 21 of whom died despite aggressive polychemotherapy, 15 of whom died within 2 months after hospitalization. The deceased patients were more likely to have lower albumin level and more prolonged activated partial thromboplastin time than the survived patients (P < 0.05 for all comparisons). Multivariate Logistic regression analysis revealed that duration of illness >/= 1 month, non-chemotherapy, albumin level < / = 25 microg/L and internal organs hemorrhage were related with the prognosis significantly (P < 0.05 for all comparisons).

CONCLUSIONThis study revealed that EBV-HLH infection in pediatric patients had severe clinical courses and prognosis was poor and the majority of cases underwent EBV reactivation. The early diagnosis, prompt and proper chemotherapy can improve the survival rate. The duration of illness >/= 1 month, non-chemotherapy, decreases in albumin and internal organs hemorrhage were the risk factors related to mortality in children with EBV-HLH.

Adolescent ; Child ; Child, Preschool ; Epstein-Barr Virus Infections ; complications ; physiopathology ; Female ; Herpesvirus 4, Human ; Humans ; Lymphohistiocytosis, Hemophagocytic ; complications ; diagnosis ; virology ; Male ; Prognosis ; Retrospective Studies ; Risk Factors

OBJECTIVETo investigate the effect of phosphatidylinositol 3-kinase inhibitor LY294002 combined with NF-κB P65 nuclear translocation inhibitor SN50 on the tumor cell growth and apoptosis using a nude mouse model of gastric cancer.

METHODSHuman gastric cancer cell strain SGC7901 was transplanted subcutaneously to nude mice to establish tumor models. Model mice were randomly divided into the control group, the LY294002 treatment group, the SN50 treatment group, and the LY294002+SN50 treatment group, with 5 in each group. After being treated for 10 days, the inhibition rate of tumor growth was ascertained by measuring the size of tumor. Immunohistochemical method was used to detect the expression levels of Bcl-2, P53 and Bax proteins and transmission electron microscopy to investigate the apoptosis of tumor cells.

RESULTSOn the 10th day after treatment, the inhibition rate of gastric cancer cellular growth in the LY294002+SN50 group was (49.2±2.5)%, which was significantly higher than that in the LY294002 group(29.4±1.5)% and SN50 group (19.7±1.6)%(P<0.05). In comparison with the other two groups, LY294002+SN50 group exhibited more severe apoptosis, with expression of Bcl-2 decreased and that of P53 and Bax increased more significantly(P<0.05).

CONCLUSIONLY294002 combined with SN50 inhibits the growth of SGC7901 transplanted tumor and aggravates the apoptosis of gastric cancer cells in nude mice model.

Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromones ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Morpholines ; pharmacology ; NF-kappa B ; antagonists & inhibitors ; Peptides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Tumor Suppressor Protein p53 ; metabolism ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

BACKGROUNDRecent studies have reported germline mutations in the perforin gene (PRF1) in some types of hemophagocytic lymphohistiocytosis (HLH). However, the prevalence of PRF1 mutations in HLH in Chinese pediatric patients has not been extensively studied. The aim of this study was to investigate the prevalence of mutations and sequence variations in the PRF1 gene in Chinese pediatric patients with HLH.

METHODSPolymerase chain reaction (PCR) was performed with five pairs of primers for the coding exons and the flanking intron sequences of PRF1. Sequencing of PCR products was subsequently applied in 30 pediatric patients with HLH and in 50 controls.

RESULTSThree heterozygous mutations in a coding region were found, which resulted in amino acid changes (C102F, S108N and T450M) in three patients. These mutations were not detected in control subjects. One patient had compound heterozygous mutations (S108N and T450M) in PRF1 as the background defect, and documented familial HLH type 2 (FHL2). One synonymous sequence variant (Q540Q) was observed in one patient but not in the controls. Two SNPs (A274A, H300H) in the coding region were detected in HLH patients and controls, but without differences in the heterozygosity rate between the two groups (P > 0.05 for all comparisons).

CONCLUSIONSWe have identified three patients with three heterozygous missense mutations in PRF1; two of those three mutations (C102F and S108N) have so far been found only from Chinese patients. These findings are useful in evaluating the prevalence of PRF1 mutations in Chinese pediatric patients with HLH, and to correlate their genotype with phenotype. Some patients without familial history probably have primary HLH, which should be suspected even beyond the usual age range.

Adolescent ; Amino Acid Sequence ; Child ; Child, Preschool ; Epstein-Barr Virus Infections ; genetics ; Female ; Humans ; Infant ; Lymphohistiocytosis, Hemophagocytic ; genetics ; Male ; Molecular Sequence Data ; Mutation ; Perforin ; Polymerase Chain Reaction ; Pore Forming Cytotoxic Proteins ; genetics