Homology analysis between clinically isolated extraintestinal and enteral Klebsiella pneumoniae among neonates.

BACKGROUND: Klebsiella pneumoniae is a leading cause of hospital-associated (HA) infections. It has been reported that gastrointestinal colonization (GI) is likely to be a common and significant reservoir for the transmission and infections of K. pneumoniae in both adults and neonates. However, the homologous relationship between clinically isolated extraintestinal and enteral K. pneumoniae in neonates hasn't been characterized yet. RESULTS: Forty-three isolates from 21 neonatal patients were collected in this study. The proportion of carbapenem resistance was 62.8%. There were 12 patients (12/21, 57.4%) whose antibiotic resistance phenotypes, genotypes, and ST types (STs) were concordant. Six sequence types were detected using MLST, with ST37 and ST54 being the dominant types. The results of MLST were consist with the results of PFGE. CONCLUSIONS: These data showed that there might be a close homologous relationship between extraintestinal K. pneumoniae (EXKP) and enteral K. pneumoniae (EKP) in neonates, indicating that the K. pneumoniae from the GI tract is possibly to be a significant reservoir for causing extraintestinal infections.

Tolerability, pharmacokinetics and pharmacodynamics of CMAB001, an anti-CD11a antibody, in Chinese healthy volunteers and psoriatic patients.

AIM: To evaluate the pharmacokinetics (PK), pharmacodynamics (PD) and primary tolerability of an anti-CD11a monoclonal antibody (CMAB001) in Chinese healthy volunteers and psoriatic patients. METHODS: Two open-label studies were conducted. One was a parallel-group, single-center, dose-escalation test, including 24 healthy adult volunteers from 18 to 45 years in age. All subjects randomly received a single subcutaneous injection dose of 0.5, 1.0 or 2.0 mg/kg. The other was a multiple-dose study: 10 adult psoriatic patients were administered weekly subcutaneous injections of 1.0 mg/kg for 7 weeks. RESULTS: CMAB001 was well tolerated in the single- and multiple-dose studies. Slow absorption was observed in both studies. In the single-dose study, the concentration of CMAB001 reached its highest level 2 d later after the injection, and the C(max) increased in an approximate dose-proportionate manner, while the area under curve (AUC) showed much greater than dose-proportionate increase. In the multiple-dose study, the steady-state serum concentration level was attained following the 4th injection. CONCLUSION: CMAB001 exhibited a nonlinear pharmacokinetic profile over the dose range from 0.5 to 2.0 mg/kg, and was well tolerated in healthy volunteers and psoriatic patients.

Treatment of psoriasis with recombinant human LFA3-antibody fusion protein: a multi-center, randomized, double-blind trial in a Chinese population.

To evaluate clinical efficacy and safety of injectable recombinant human LFA3-antibody fusion protein (rhLFA3-IgFP), a multi-center, randomized, double-blind, double-dummy, parallel-controlled clinical trial was performed in 212 cases of moderate to severe psoriasis. Intramuscular injection of rhLFA3-IgFP (15 mg/week) and oral administration of blank dummy methotrexate at the dose of 7.5 mg/week was performed in the patients in the experimental group, and control patients were orally administered with methotrexate at the dose of 7.5 mg/week and intramuscularly injected with the blank dummy rhLFA3-IgFP (15 mg/week). PASI was determined prior to and at 2, 4, 6, 8, 12, 16, 20 weeks after the treatment. The efficacy evaluation was carried out on 192 patients, and no significant differences were found in PASI50, PASI75 & PASI90 between the two groups after twelve weeks' treatment (p>0.05). After discontinuation, PASI scores continued to decrease drastically in the experiment group, whereas they increased in the control group. At 8 weeks after discontinuation, PASI scores were decreased by 62.32% (p<0.05) and 52.67% (p<0.05) in the experimental and control groups, respectively. No serious adverse reactions were observed. In conclusion, the results of our investigation demonstrated that rhLFA3-IgFP was an effective therapy for chronic plaque psoriasis with lasting action and low incidence of adverse reactions.

[Bone mass change and its possible mechanisms in murine knockout apolipoprotein E(ApoE-/-)].

OBJECTIVE: To study bone mass changes and its mechanisms in murine knockout apolipoprotein E (ApoE-/-). METHODS: Both 28-week-old male ApoE-/- mice (n = 6) and wild-type (WT) mice (n = 10) were euthanized. The trabecular and cortical bone microarchitecture were assessed by micro-CT (microCT) in right distal femur. The total body BMD (bone mineral density) of left femur was determined by dual-energy X-ray absorptiometry (DXA). The serum concentrations of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) were analyzed using enzyme-linked immunosorbent assay (ELISA). The left tibias were dissected and fixed in 4% paraformaldehyde after decalcified in 0.15M EDTA buffer for 30 days. And the expressions of OPG and RANKL were detected by immunohistochemical staining. RESULTS: Compared with WT mice, the ApoE-/- mice showed an increased volumetric BMD (294 +/- 38) mg/mm(3), tissue BMD (627 +/- 23) mg/mm(3), BMC (bone mineral content) (0.57 +/- 0.08) mg, bone volume fraction (20.38 +/- 4.74)%, trabecular number (6.67 +/- 0.78) mm(-1), trabecular thickness (0.03 +/- 0.01) mm with an decreased bone surface fraction (69 +/- 18) mm(-1), trabecular separation (0.12 +/- 0.01) and structure mode index (1.73 +/- 0.24). The total body BMD was higher in ApoE-/- mice than WT mice [(0.063 +/- 0.004) g/cm(2) vs (0.056 +/- 0.004) g/cm(2)]. The serum level of OPG was significantly higher in ApoE-/- mice than WT mice [(4.98 +/- 0.97) ng/ml vs (3.54 +/- 0.65) ng/ml]. The results of immunohistochemical staining showed that the OPG expression was at a higher level in bone cell of ApoE-/- mice and RANKL showed no obvious change in either sera or bone cells. CONCLUSION: A decreased bone resorption causes an increased bone mass in ApoE-/- mice and leads to an imbalanced ratio of OPG/ RANKL.

Pharmacokinetic and pharmacodynamic study of LFA3Ig fusion protein in healthy volunteers and patients with psoriasis.

AIM: To evaluate the pharmacokinetics (PK) and pharmacodynamics of the LFA3Ig fusion protein (LFA3IgFP) in healthy volunteers and patients with chronic plaque psoriasis. METHODS: The clinical trials included 2 phase I open studies. Study 1 was an open-label dose escalation study in 24 healthy volunteers, and study 2 was a single-group, open-label study in 12 patients with chronic plaque psoriasis. The serum drug concentrations were measured, and the concentration-time data were analyzed by compartmental analysis using the Practical Pharmacokinetic Program. RESULTS: In study 1, after intramuscular (im) administration at a dosage of 5, 15, and 25 mg, the concentration-time curves of LFA3IgFP fitted well to a 1 compartment open model. Areas under the concentration-time curves increased linearly with dose. Clearance rates (Cls/ F) and elimination half-lives (T1/2ke) had no significant difference between different dose groups. A transient, slight decline of CD(4+) and CD(8+) T-cell subsets was observed after administration. In study 2, after im administration at a dosage of 15 mg weekly for 8 weeks, the concentration-time curve was best fitted to a 1 compartment open model, with a T(1/2ke ) of 307.9+/-32.7 h. The steady state was attained after the fifth administration. CONCLUSION: The PK behaviors of LFA3IgFP in healthy volunteers and patients with chronic plaque psoriasis complied with linear kinetics within the examined dose range. A significant accumulation was observed after repeated administration at a dose of 15 mg weekly for 8 weeks.

Phage displaying peptides mimic schistosoma antigenic epitopes selected by rat natural antibodies and protective immunity induced by their immunization in mice.

AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice. METHODS: Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced. Sixty female mice were immunized thrice with positive phage clones (0, 2nd), 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. RESULTS: NRS could specifically react to the molecules of 75,000, 47,000, 34,500 and 23,000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P=0.000) worm reduction and 67.6% (P=0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P=0.001), 14.5% (P=0.074) worm reduction and 61.2% (P=0.000), 35.7% (P=0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6,400 as detected by ELISA. CONCLUSION: Specific peptides mimic antigenic molecules can be obtained by biopanning the phage random peptide library and a partially protective immunity against schistosome infection can be stimulated by these phage epitopes in mice.

[Studies on the mimic short peptide-based vaccine: immunoprotection in mice against Schistosoma japonicum infection].

OBJECTIVE: To evaluate the immunoprotective effect of the mimic short peptide-based vaccine of Schistosoma japonicum (S.j) in mice. METHODS: Phage random peptide library of 12 amino acids was immunoscreened with purified IgG from sera of rats. Three rounds of biopanning were carried out. The positive clones were randomly selected, detected and sequenced. Kunming mice were immunized with positive clones three times (0, 2, 4 weeks). Each mouse was infected with 40 cercariae after 2 weeks of the 3rd immunization. After 42 days of infection, the mice were killed. Adult worms and the liver eggs were counted. RESULTS: The specific phages binding to IgG were enriched after 3 rounds of biopanning. Two short peptides were obtained. Compared with the control groups, the mixture of two mimic peptides induced 34.9% (P < 0.05) worm reduction and 67.6% (P < 0.001) total liver egg reduction in mice. Two different mimic peptides induced 31.0% (P < 0.05), 14.5% (P > 0.05) worm reduction and 61.2% (P < 0.001), 35.7% (P < 0.05) total liver egg reduction respectively. The specific antibody could be induced by the mimic peptides and detected by ELISA in immunized mice, and the antibody titer reached more than 1:6,400. CONCLUSION: The 2 different mimic peptides obtained by immunoscreening phage random peptide library show partial immunoprotection against S. j infection.

Adiponectin exerts its negative effect on bone metabolism via OPG/RANKL pathway: an in vivo study.

To explore the effects of adiponectin on the bone metabolism in vivo. Bone mineral density (BMD), bone microstructure, serum adiponectin levels, and biochemical markers of the bone turnover were measured in 12-week-old male Adipo-/- and WT mice. In addition, the osteoclast formation, osteoprotegerin (OPG), and the receptor activator of nuclear factor-κB ligand (RANKL) expression were examined. The serum adiponectin levels were normal in the WT mice while undetectable in the Adipo-/- mice. Compared with the WT mice, the Adipo-/- mice had higher BMD, more trabecular bone, greater bone volume fraction, and trabecular thickness in the left femur. On the contrary, fewer osteoclasts were observed in the Adipo-/- mice when compared with the WT mice. Meanwhile, the Adipo-/- mice had a significantly decreased serum carboxyl-terminal telopeptide of type 1 collagen (CTX)/osteocalcin (OC) ratio. Interestingly, both the adiponectin and RANKL would cause a significant increase of CTX/OC ratio in the co-culture of the CD14+ peripheral blood mononuclear cells and the osteoblasts from Adipo-/- mice. Further, immunohistochemistry assays in tibias and both the RT-PCR and immunoblot analyses in the cultured osteoblasts showed the Adipo-/- mice expressed lower levels of RANKL but higher levels of OPG. Adiponectin had a negative effect on the bone metabolism, and this negative effect might be mediated, at least in part, by the OPG/RANKL pathway.

Evidence-based novel changes in prevalence and symptom characteristics of spleen deficiency syndrome in persons of varied health status and different ages: a cross-sectional observational study.

Deficiency of the organs is a vital pathophysiologic characteristic in the elderly. A core TCM aging theory is known as aging caused by spleen deficiency syndrome (SDS) that can be found in ancient and modern literature. The key objectives of this study were to establish a full-scale trial to evaluate the prevalence, symptom severity, frequency, and distribution of SDS in different age groups as related to health status (healthy, subhealthy, and chronic disease) to elucidate the role of spleen deficiency in the aging process and deterioration of health status. This cross-sectional observational study was conducted in 4 hospitals in China. 1390 participants aged 20-79 were interviewed by investigators who completed questionnaires recording prevalence, severity, and frequency of symptoms as well as other relevant information. The results revealed that prevalence and symptom characteristics of SDS showed regularities with increasing age and deteriorating health status. It supports the TCM concept that spleen deficiency is an important mechanism of aging, subhealth, and chronic diseases. Early recognition of the warning signs and symptoms of SDS may lead to intervention and even prevention strategies for subhealth and chronic diseases as well as promotion of healthy aging.

Effects of 17ß-estradiol on adiponectin regulation of the expression of osteoprotegerin and receptor activator of nuclear factor-κB ligand.

Adiponectin may exert a negative effect on bone metabolism by regulating osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) expression. However, the action of adiponectin on bone may be influenced by estrogen in women. The present study was undertaken to investigate the effects of 17ß-estradiol (E2) on adiponectin-regulated OPG and RANKL expression in human osteoblast. Human osteoblasts were treated with α-MEM containing 10µg/ml adiponectin alone or together with 10(-10) to 10(-8)M E2 for 12-48h. Cells were also treated with α-MEM containing 10µg/ml adiponectin together with 10(-8)M E2 plus p38 agonist-anisomycin or estrogen receptor (ER) antagonist ICI182780 for 48h. The effects of E2 were also investigated by knockdown of ERs or overexpression of p38 MAPK in osteoblasts. Further, we examined the effects of E2 on adiponectin-dependent osteoclastogenesis by the co-culture systems of osteoblast and CD14+ peripheral blood monocytes (PBMCs). Real-time quantitative PCR (RT-PCR) and ELISA were used to detect OPG/RANKL mRNA and their corresponding protein expression, Western Blot was used to analyze the phosphorylated p38 (p-p38) levels. The results showed that E2 blocked adiponectin-induced p38 phosphorylation, decreased adiponectin-regulated OPG/RANKL mRNA and protein expression in a dose- and time-dependent manner. ICI182780 or knockdown of ERs abolished the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. Furthermore, anisomycin or overexpression of p38 also reserved the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. E2 inhibited adiponectin-dependent osteoclastogenesis in the co-culture systems of osteoblast and CD14+ PBMCs, whereas anisomycin, ICI182780, knockdown of ERs and overexpression of p38 significantly reversed this response. In conclusions, our findings demonstrated, through blocking the activation of adiponectin-induced p38 MAPK, E2 suppressed the adiponectin-regulated OPG/RANKL expression and then inhibited osteoclastogenesis, which suggested that estrogen would suppress the effect of adiponectin on bone metabolism.

[Effect of peptide seals specific to CD59 on the expression of apoptosis-related genes in HeLa cells].

AIM: To study the effect of peptide seals specific to CD59 on the expression of apoptosis-related survivin, caspase-3 and bax in HeLa cells and investigate the mechanism of peptide seals specific to CD59 in inducing apoptosis of HeLa cells. METHODS: The peptide seals specific to CD59 were put into HeLa cells and HeLa cells with CD59-transfected gene, respectively. After 24 hours of interaction, the inhibitory ratio of cell proliferation was measured by MTT and the expression of survivin, caspase-3 and bax was detected by immunohistochemistry. RESULTS: The inhibitory ratio of HeLa cells with CD59-transfected gene + peptide seal group was higher than that of HeLa cells + peptide seal group. There was a significant difference in depressing the proliferation between two groups (P < 0.01). Compared with HeLa cells + peptide seal group, the survivin expression of HeLa cells with CD59-transfected gene + peptide seal group was decreased (P < 0.05) while caspase-3 expression was increased (P < 0.05). There was no significant difference in the expression of bax in four groups. CONCLUSION: The peptide seal specific to CD59 can down-regulate the expression of survivin, activate caspase-3 and enhance the apoptosis of HeLa cells.

[Knocking down human CD59 gene expression decreased protection to complement-mediated cytolysis].

AIM: To construct recombinant vectors expressing siRNA that target CD59 gene and a stable-inhibit cell line A2780 in order to analyze the role of CD59 in the protection to complement-mediated cytolysis. METHODS: The 60 bp encoded targeting CD59 gene shRNA sequence was cloned into pSUPER vector with DNA recombinant technique. The ovary cancer cell A2780 was transfected with this recombinant plasmid using liposome and the stable strains was selected by using G418-medium, CD59 mRNA and protein level was detected by RT-PCR and Western blot, and its function was analysed by dye release assay. RESULTS: The pSUPER-siRNA expressing vector was successfully constructed. And a stable cell line A2780 was selected and was detected the expression of GFP. The siRNA vector effectively inhibited the CD59 gene expression from mRNA and protein level. Dye release assay suggested that CD59's protection to complement-mediated cytolysis decreased. CONCLUSION: The siRNA vector targeting CD59 gene could consistently inhibit CD59 expression. Furthermore, it decreased CD59's protection against complement. These results may pave the way for studying the role of CD59 in the immune escape of tumors cells as well as in tumor therapy.