Optimization of large-scale production process for human prothrombin complex concentrates / 中国输血杂志

【Objective】 To determine the optimal process conditions for efficiently extracting human prothrombin complex concentrates from human plasma. 【Methods】 Using human plasma as the materials and the yield of prothrombin complex concentrates as the evaluation standard, the preparation process parameters were studied and optimized through design of exporement(DOE), orthogonal experiments, and single factor experiments. 【Results】 The optimal process conditions were as follows: DEAE Sephadex A50 gel was selected, which balanced to pH 7.6, and then the amount of 1.7-2.5 g/L of plasma weight is added into the cryoprecipitate supernatant for adsorption for 40 minutes; Washing solution (0.15-0.175 mol/L sodium chloride) with 3 times the volume of gel was washed 3 times, and eluent (0.5-2.0 mol/L sodium chloride) was washed 3 to 5 times; Add stabilizer (heparin 35 IU, sodium chloride 0.1 mol/L) for ultrafiltration dialysis. 【Conclusion】 By using the optimized process mentioned above, the yield(measured by human coagulation factor IX)can reach 620 000 to 630 000 IU/ton of plasma, which is suitable for large-scale production.

Effect of Feiliuping Ointment on Angiogenesis of Bone Marrow-derived Endothelial Progenitor Cells in Tumor Cells Supernatant via PI3K/AKT Pathway / 中国药学杂志

OBJECTIVE: To discuss the effect of feiliuping ointment on proliferation, angiogenesis and PI3K/AKT pathway of rat bone marrow-derived endothelial progenitor cells (EPCs) in A549 lung tumor cells supernatant. METHODS: Bone marrow mononuclear cells were isolated from Sprague-Dawley rats by density gradient centrifugation methods and cultured in EGM-2 MV medium. And fluorescent immunocytochemistry was used to detect CD133 and vascular endothelial growth factor receptor 2 (VEGFR2) expression. EPCs were treated with 0.9% sodium chloride solution (blank control) or serum of rats treated with high dose and low dose Feiliuping ointment for 48 h to explore the concentration-effect, or with serum of rats treated with high dose feiliuping ointment for 0, 12, 24, 48 h to explore the time-effect. MTT assay and Matrigel method were used to detect the proliferation and angiogenesis of EPCs. The NF-κB, p-Akt, PI3K p85 proteins of EPCs were detected by Western blot. RESULTS: After induced culture for 7 d, the isolated bone marrow mononuclear cells exhibited round or short spindle-shaped appearance, were Dil-Ac-LDL and FITC-UEA-1 positive, and expressed CD133+VEGFR2+. After being treated with high dose feiliuping ointment serum for 48 h, OD490 nm value was lower than that in the control group or low dose feiliuping ointment group (P<0.05). The inhibition effect of rat serum after Feiliuping ointment administration on EPCs was dose-dependent and time-dependent. Matrigel tube formation assays showed that the counts of tube formation of EPCs in high dose Feiliuping ointment group was lower than that in control group or low dose of feiliuping ointment group (P<0.05). And the levels of NF-κB, p-Akt, PI3K p85 proteins in high dose of feiliuping ointment group were lower than those in control group(P<0.01). CONCLUSION:S Treatment with administration can Feiliuping ointment serum would inhibit the proliferation and tube formation of bone marrow-derived EPCs in A549 supernatant via the down-regulation of PI3K/AKT signaling pathway.

Cell culture supernatant of olfactory ensheathing cells promotes nerve regeneration after peripheral nerve injury in rats / 中国组织工程研究

BACKGROUND: Previous studies have found that the culture supernatant of the olfactory ensheathing cells is capable of promoting axonal regeneration and functional recovery after spinal cord injury, but there is a lack of research in the field of peripheral nerve. OBJECTIVE: To investigate whether olfactory ensheathing cell culture supernatant is beneficial for nerve repair after peripheral nerve injury. METHODS: Olfactory ensheathing cells were isolated and purified, to prepare the supernatant. The olfactory ensheathing cell culture supernatant was applied to the dorsal root ganglion tissue block in vitro to observe the axon growth of the dorsal root ganglion. The olfactory ensheathing cell culture supernatant was applied to a rat sciatic nerve defect model in vivo to examine its effect on axonal regeneration and myelinization of the injured nerve. RESULTS AND CONCLUSION: The purity of olfactory ensheathing cells was (94.4±3.1)%. Compared with the blank control and low dose olfactory ensheathing cell culture groups, the average length of five longest axons in dorsal root ganglion tissue mass in the high dose olfactory ensheathing cells culture group was significantly increased (P < 0.05). Immunofluorescence showed that the regenerated nerve penetrated through the defect area and the regenerated nerve was arranged orderly in the olfactory ensheathing cell culture and the autologous nerve groups, which was significantly superior to that in the blank control group. Transmission electron microscope observed that the number of regenerated nerve axons and the thickness of myelin sheath in the olfactory ensheathing cell culture group were significantly higher than those in the blank control group (P < 0.05). These results indicate that the supernatant of the olfactory ensheathing cells can promote axonal regeneration after peripheral nerve injury and the myelination of the regenerated axons, which provides a new olfactory ensheathing cells-based acellular therapy for peripheral nerve injury.

Antisickling Activity Evaluation Of Fractions Obtained From Whole Extracts Of Newbouldia Laevis P. Beauv (BIGNONIACEAE)

Objective: This study aims to evaluate the fractions from whole extracts of roots and stem barks of Newbouldia laevis and their effect on sickling. Methods: Hydroethanolic extracts of stem barks and roots of Newbouldia laevis were fractionated by the technics of cold precipitation in ethanol. The fractions obtained after phytochemical screening were subjected to Emmel test to evaluate their anti-sickling activity. Active fractions were tested for DPPH and AAPH assay (AAPH induced membrane lipoperoxidation and evaluation of reduction of hemolysis). Results: Two fractions were obtained from each whole extract: supernatant and pellet fractions. Supernatants fractions obtained from whole roots barks extract and stem barks extract at a concentration of 30 mg/ml reduced sickling up respectively to 7% and 10% against 86% for the control. Pellets fractions obtained from the both extracts induced coagulation of SS blood at 30 mg/ml against 86% for the control. Conclusion: Supernatants fractions of hydroethanolic whole extract of roots and stem backs of Newbouldia laevis promise as the potential source of active molecules against sickle cell disease.

Effect of limbal stem cell culture medium on vascular endothelial cell proliferation / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effect of cultured limbal stem cells on proliferating of vascular endothelial cells and validate the theory of limbal stem cells.<p>METHODS: The limbal stem cells and epithelium cells of bulbar conjunctiva were cultured and determined by immunohistochemistry staining with AE-5. The supernatant was collected and added into the cultured human umbilical vein endothelium cells(ECV-304). The negative control was set up. After 24h, MTT was done to detect endothelium cell proliferation. The statistical analysis was done by analysis of Variance.<p>RESULTS: Negative stain of AE-5 can be seen in limbal stem cells and positive stain can be seen in conjunctiva cells. The ratio of MTT added by limbal stem cell supernatant, conjunctiva epithelium cell and negative control were 2.097±0.079, 1.981±0.034 and 1.990±0.044, respectively. There were significant difference among the three groups(<i>F</i>=9.169, <i>P</i>=0.000). The proliferate activity of vascular endothelial cells added by supernatant of limbal stem cells was higher than the other two groups(<i>P</i>=0.005 and <i>P</i>=0.007, respectively).<p>CONCLUSION:The supernatant of limbal stem cells can improve the proliferation of vascular endothelium cell. This may be the unique characteristics of limbal stem cells. This study provides more evidences for the theory of limbal stem cells by determining the functional methods.

Comparison of cobas EGFR Mutation Test v2 and PANAMutyper-R-EGFR for Detection and Semi-Quantification of Epidermal Growth Factor Receptor Mutations in Plasma and Pleural Effusion Supernatant

BACKGROUND: Plasma epidermal growth factor receptor (EGFR) mutation tests are less invasive than tissue EGFR mutation tests. We determined which of two kits is more efficient: cobas EGFR Mutation test v2 (cobasv2; Roche Molecular Systems, Pleasanton, CA, USA) or PANAMutyper-R-EGFR (Mutyper; Panagene, Daejeon, Korea). We also evaluated whether pleural effusion supernatant (PE-SUP) samples are assayable, similar to plasma samples, using these two kits. METHODS: We analyzed 156 plasma and PE-SUP samples (31 paired samples) from 116 individuals. We compared the kits in terms of accuracy, assessed genotype concordance (weighted κ with 95% confidence intervals), and calculated Spearman's rho between semi-quantitatively measured EGFR-mutant levels (SQIs) measured by each kit. We also compared sensitivity using 47 EGFR-mutant harboring samples divided into more-dilute and less-dilute samples (dilution ratio: ≥ or <1:1,000). RESULTS: cobasv2 tended to have higher accuracy than Mutyper (73% vs 69%, P=0.53), and PE-SUP samples had significantly higher accuracy than plasma samples (97% vs 55–71%) for both kits. Genotype concordance was 98% (κ=0.92, 0.88–0.96). SQIs showed strong positive correlations (P<0.0001). In less-dilute samples, accuracy and sensitivity did not differ significantly between kits. In more-dilute samples, cobasv2 tended to have higher sensitivity than Mutyper (43% vs 20%, P=0.07). CONCLUSIONS: The kits have similar performance in terms of EGFR mutation detection and semi-quantification in plasma and PE-SUP samples. cobasv2 tends to outperform Mutyper in detecting less-abundant EGFR-mutants. PE-SUP samples are assayable using either kit.

Tumor cells supernatant promotes the healing of diabetic foot ulcers in mice through increasing the expression of VEGF-A, α-SMA and Vimentin / 中华内分泌外科杂志

Objective To investigate the effect of tumor cells supernatant on treatment of diabetic foot ulcer in mice and on the expression of VEGF-A,α-SMA and Vimentin.Methods A total of 45 male BALB/c mice were randomly divided into three groups:normal control group (group A),tumor cell supernatant treated group (group B),and diabetic control group (group C).Mouse models of type 2 diabetic foot ulcers were established in group B and group C.After the first day of modeling,group B were treated with tumor cells supernatant and the other two groups were injected with equal volume of medium.At the 1st,3rd and 7th day following model established,mouse ulcer area was observed in each group.The ulcer infection rate and mortality of mice were compared between each group.The ulcer tissue of each group was HE-stained and the expression of VEGF-A,α-SMA and Vimentin in each group was detected by immunohistochemistry (IHC).ELISA assay was used to detect the relative protein levels and stability in tumor cells supernatant.Results The healing degree in group A (66.7％) and group B(80.0％) was better than that in group C(33.3％) and the infection rate (group A=0,group B=7.1％) and mortality (group A=0,group B=6.7％) were significantly lower than those of group C (40.0％,33.3％),and the difference was statistically significant(P＜0.05).Compared with group C,HE staining showed that the healing time of group A and B was shorter than group C,and the epidermal coverage was more obvious.The expression levels of VEGF-A,α-SMA and Vimentin detected by IHC in group A and B were significantly higher than those in group C.ELISA results showed high-level and stable TGF-β expression in the tumor cells supernatant.Conclusion The tumor cells supernatant can effectively promote the healing of diabetic foot ulcers in mice and TGF-β,VEGF-A,α-SMA and Vimentin play a very important role in ulcers healing process.

Efecto bioconservante del sobrenadante de Lactobacillus plantarum y Lactobacillus lactis en lomo de cerdo (Longisimus dorsi)* / Bioconservant effect of the supernatant of Lactobacillus plantarum and Lactobacillus lactis on pork loin (Longisimus dorsi) / Efeito bioconservante do sobrenadante de Latobacillus plantarum e Latobacillus lactis em lombo de porco (Longisimus dorsi)

Resumen La bioconservación incrementa la vida útil de los alimentos y mejora su calidad. Se determinó el efecto del sobrenadante de Lactobacillus plantarum y Lactobacillus lactis en lomo de cerdo. Se utilizó el sobrenadante (500 µl) en 50 g de lomo, y como testigos, ácido láctico (2 %) y suero fisiológico (sin aditivo). Se evaluó cada tratamiento a 4 y 20 °C por 15 días. Durante los días 0, 7 y 15, se realizaron mediciones microbiológicas, físicoquímicas y sensoriales; las dos primeras se evaluaron con un diseño de bloques con factorial 4 x 2. El bloque fueron los días de medición, y los factores, aditivo y temperatura. La evaluación sensorial se hizo por análisis de varianza. No hubo interacción entre factores (p > 0,05). Los coliformes totales, Clostridium sulfito reductor y pH no mostraron diferencia en el factor aditivo (p > 0,05), pero coliformes fecales, acidez y capacidad de retención de agua (CRA), sí (p < 0,05). La refrigeración (4 °C) mostró mejores resultados. Se concluye que el sobrenadante de L. plantarum conserva el lomo de cerdo; además, mantiene las características organolépticas y evita el crecimiento microbiano. L. lactis mostró resultados similares al ácido láctico y por encima del tratamiento sin aditivo, aunque los valores de esta cepa fueron inferiores a los encontrados en L. plantarum.

Abstract Bioconservation extends the useful life of food and improves its quality. This paper aimed to determine the effect of the supernatant of Lactobacillus plantarum and Lactobacillus lactis on pork loin. The supernatant (500 µl) was used in 50 g of loin, and as controls, lactic acid (2%) and physiological saline (without additive). Each treatment was evaluated at 4 and 20 °C for 15 days. On days 0, 7 and 15, microbiological, physicalchemical and sensorial measurements were performed; the first two were evaluated with a 4x2 factorial block design. The block consisted of measurement days, and the factors were additive and temperature. Sensory evaluation was done by analysis of variance. There was no interaction between factors (p > 0.05). Total coliforms, sulfite-reducing clostridium, and pH showed no difference in the additive factor (p > 0.05), but fecal coliforms, acidity and water retention capacity did (p < 0.05). Refrigeration (4 °C) had better results. It is concluded that the supernatant of L. plantarum preserves pork loin; in addition, it maintains organoleptic characteristics and avoids microbial growth. L. lactis showed similar results to lactic acid and was better than the treatment without additive, although the values of this strain were inferior to those found in L. plantarum.

Resumo A bioconservação aumenta a vida útil dos alimentos e melhora a sua qualidade. Determinou-se o efeito do sobrenadante de Latobacillus plantarum e Latobacillus lactis em lombo de porco. Utilizou-se o sobrenadante (500 µl) em 50 g de lombo, ácido láctico (2 %) e soro fisiológico (sem aditivo). Avaliou-se cada tratamento a 4 e 20 °C por 15 dias. Durante os dias 0, 7 e 15, se realizaram medições microbiológicas, físico-químicas e sensoriais; as duas primeiras se avaliaram com um desenho de blocos com fatorial 4 x 2. O bloco foram os dias de medição, e os fatores, aditivo e temperatura. A avaliação sensorial foi realizada por análise de variação. Não houve interação entre fatores (p > 0,05). Coliformes totais, clostridium sulfito redutor e pH não mostraram diferencia no fator aditivo (p > 0,05), pero coliformes fecais, acidez e capacidade de retenção de água sim (p < 0,05). A refrigeração (4 °C) mostrou melhores resultados. Conclui-se que o sobrenadante de L. plantarum conserva o lombo de porco; além do mais, mantém as características organolépticas e evita o crescimento microbiano. L. lactis mostrou resultados similares ao ácido láctico e por cima do tratamento sem aditivo, mesmo que os valores desta cepa tenham sido inferiores aos encontrados em L. plantarum.

Effect of Acinetobacter baumannii secretions on Pseudomonas aeruginosa cell growth and biofilm formation / 临床检验杂志

Objective To investigate the effects of Acinetobacter baumannii culture supernatants on planktonic cell growth and biofilm formation of Pseudomonas aeruginosa.Methods The standard isolates (ATCC 19606,ATCC 1195) and clinical isolates (AB23,AB39,AB53) of Acinetobacter baumannii were collected and the 6,12,16,24 and 48 hour-cultured supernatants were extracted.The effects of the culture supernatants on the biofilm formation of Pseudomonas aeruginosa PAO1 were detected on the 96-well plate combined with crystal violet staining.Two-fold concentration of LB medium was prepared to eliminate the effects of nutrition consumption of Acinetobacter baumannii during culture on Pseudomonas aeruginosa growth.The active ingredients in the supernatant of Acinetobacter baumannii culture medium were investigated by using the concentrated tube containing protein with relative molecular mass 3 000.Results The most suitable period for Acinetobacter baumannii culture supernatant extraction was between 12 to 24 hours,so the 16 hourcultured supernatant was chosen for next experiments.The 50％ culture supernatant of Acinetobacter baumannii ATCC 1195 and ATCC 19606 significantly inhibited the planktonic cell growth of Pseudomonas aeruginosa PAO1,in which the absorbance at 630 nm reduced from(0.688 ± 0.014) and(0.692 ± 0.014) to (0.431 ± 0.023) and (0.428 ± 0.020) respectively (t =16.780,P ＜ 0.05;t =18.500,P ＜ 0.05).The 50％ culture supernatant of Acinetobacter baumannii ATCC 1195 and ATCC 19606 also significantly inhibited the biofilm formation of Pseudomonas aeruginosa PAO1 with decreased absorbance at 570 nm from (2.071 ± 0.068) and (1.986 ±0.023) to (1.639 ± 0.042) and (1.525 ± 0.202) respectively (t =9.358,P ＜ 0.05;t =3.924,P ＜ 0.05).The biofilm inhibitory effect of the protein with relative molecular mass less than 3 000 was obviously observed by reducing amount of biofilm formation from (1.177 ± 0.040) to(1.056 ± 0.030) (t =4.192,P ＜ 0.05),while there was no inhibitory effect of the proteins with relative molecular mass more than 3 000 in the composition.Conclusion Acinetobacter baumannii culture supernatant could effectively inhibit the planktonic cell growth and biofilm formation of Pseudomonas aeruginosa and the relative molecular mass of active ingredients in the culture supernatant may be less than 3 000.

Improved protein-A chromatography for monoclonal antibody purification / 生物工程学报

Therapeutic monoclonal antibodies become the major product class within the biopharmaceutical market. Protein A as the first capture step is still dominant in current platforms for purification of monoclonal antibodies. In this study, we developed a new antibody harvest process that incorporates acidic treatment of cell harvest, demonstrating high process yield, improved clearance of host cell associated contaminants, like non-histone host cell protein, histone, DNA and heteroaggregates. Host protein contamination was reduced about 10-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Turbidity increase of eluted IgG upon pH neutralization was nearly eliminated. Residual levels of impurities in the protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. The mechanism of host cell associated contaminants removal during acidic treatment was also explored. After a polishing step by Capto adhere, host cell protein was reduced to less than 5 ppm, DNA less than 1 ppb, histone to undetectable level, heteroaggregates less than 0.01% with total IgG recovery around 87%. This efficient process can be easily integrated into current IgG purification platforms, and may overcome downstream processing challenges.

A simple and rapid method for the quantitation of secreted hepatitis B virions in cell culture models.

Cell culture models for hepatitis B virus (HBV) remain the mainstay for screening and testing the efficacy of anti‑hepatitis B virus agents. Gradient‑based ultracentrifugation followed by Southern Blotting is used for hepatitis B virion estimation in cell culture; this method has several limitations. We report the development of an assay using a commercially available HBsAg‑ELISA plate for immunocapture followed by real‑time PCR for quantification of hepatitis B virions in cell cultures. This assay is rapid, highly sensitive (50 copies/reaction) and highly specific for virion‑associated DNA. In addition, the assay requires only 20 μL of supernatant, allowing scaling down of transfections.

Antioxidant and Radical Scavenging Properties of β-Carotene on Cisplatin Induced Cardiotoxicity.

Objective: To investigate the protective role of β-Carotene against cisplatin induced cardiotoxicity in male albino rats. Methods: Various biochemical parameters such as Creatine kinase-MB, Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Triglycerides (TG) and Total cholesterol (TC) are being assessed. Also the levels of the in vivo antioxidants such as Reduced glutathione (GSH), Catalase (CAT), and Malondialdehyde (MDA) in the post mitochondrial supernatant of heart were measured. In addition, the histopathological studies were performed to study the protective activity of β-carotene. Results: Cisplatin administration has shown the elevated levels of the cardiac markers and diminished the endogenous antioxidant levels when compared with the normal rats. β-carotene treatment showed the inhibitory effect on the free radicals showing decreased levels of the cardiac markers like CK-MB, LDH, AST, ALT and ALP. The β-carotene treated rats showed significant (p<0.001) decrease in lipid peroxidation in both prophylactic and curative groups when compared to the cisplatin group. Also showed a significant (p<0.05, p<0.001) increase in the levels of GSH in prophylactic and curative group respectively when compared with the cisplatin group. Both prophylactic and curative groups have shown a significant (p<0.001) increase in the levels of CAT. Further, the histopathological studies confirm the protective effect of β-carotene. Conclusion: These findings justify the biological and traditional uses of β-carotene as confirmed by its promising radical scavenging activity against cisplatin induced cardiotoxicity.

Effects of bone marrow mesenchymal stem cells supernatant on hippocampal GFAP expression and learning and memory in rats with neonatal hypoxic ischemic brain damage / 中华行为医学与脑科学杂志

Objective To analyze the expression of glial fibrillary acidic protein(GFAP) in hippocampal of rats with hypoxic-ischemic brain damage(HIBD) after injection of bone marrow stromal cells(BMSCs) supernatant into the lateral ventricle of the neonate rats,assess the ability of learning and memory,and explore the mechanism.Methods 7-day-old SD rats were randomly divided into sham operation group,model group,cells and supernatant group,with 20 rats in each group.The HIBD model was established via the ligation of left carotid arteries followed by 2-hour hypoxia.One week later,rats in control group were injected with 0.01 mol/L PBS 2 μl via the left ventricle.Meanwhile,the cell and the supernatant groups were injected with BMSCs and supernatant harvested from BMSC culture,respectively,via the same route.In the sham operation group,the left carotid arteries were separated but not ligated,and no hypoxia treatment was imposed on this group.They also received 0.01 mol/L PBS injection 2 μl per weak post surgery.8 weeks later,Morris water maze test was performed to assess the learning and memory,and the expression of GFAP in the dentate gyrus of rats was measured by immunohistochemistry.Results Morris water maze showed that the searching time of the probe trail(T1:(15.40±2.80) s) and reversal probe trail (T2:(16.45± 1.16) s) of the model group was shorter than that of the sham operation group (T1:(19.96±2.57) s,T2:(2:25.32±2.54)s,P＜0.05),while the searching time of the cell group(T1:(17.54± 1.80)s,T2:(18.99± 1.47) s) and supernatant group (T1:(17.40±2.37) s,T2:(17.96± 1.09) s) was prolonged compared with that of the model group (P＜0.05).No significant difference between the cell group and the supernatant group (P＞0.05).The integral optical density (IOD) value of GFAP positive cells was higher in the model group than that in the sham operation group(15.26±1.49 vs 12.82±2.56,P＜0.05),while the IOD of cell group(18.13±1.97) and that of the supernatant group(17.38± 1.64) were higher than that of the model group (P＜0.05).However,there was no significant difference between the cell group and the supernatant group (9＞0.05).Conclusion BMSC supernatant injected through lateral ventricle can improve the HIBD rats' abilities of learning and memory.The mechanism might be that MSCs secrete some cytokines to promote central nervous system repair.

Isolation of bacteriocin - producing lactic acid bacteria from 'Ugba' and 'Okpiye', two locally fermented nigerian food condiments

In this work, 100 samples each of 'ugba' and 'okpiye' were evaluated for the presence of bacteriocin producing lactic acid bacteria. Thirty strains showing antibacterial activity against at least one of the indicator organisms were selected from a total of 752 colonies isolated from the condiments. Out of the 30, only five strains retained activity after the pH of the broth supernatant was adjusted to 6.5. When evaluated by the agar-well diffusion assay, the spectra of inhibitory activity showed that Staphylococcus aureus was the most sensitive indicator organism tested, while Listeria monocytogenes was the most resistant. One strain (UG 2) was active against Escherichia coli. The assays using the cell-free supernatant of the cultures showed that the bacteriocins were completely inactivated by the proteolyses as well as by the chloroform treatment. In ethanol, the activity of the compounds was only partially modified. When incubated in a water bath at 80°C for 30 min, no significant activity loss was recorded. The antimicrobial activity of the bacteriocins produced by the lactic acid bacteria has potential for use in biopreservation of condiments against the spoilage and food - borne pathogens.

In Vitro Evaluation of Antimicrobial Activity of Lactic Acid Bacteria against Clostridium difficile

Clostridium difficile infection (CDI) has become a significant threat to public health. Although broad-spectrum antibiotic therapy is the primary treatment option for CDI, its use has evident limitations. Probiotics have been proved to be effective in the treatment of CDI and are a promising therapeutic option for CDI. In this study, 4 strains of lactic acid bacteria (LAB), namely, Lactobacillus rhamnosus (LR5), Lactococ-cuslactis (SL3), Bifidobacterium breve (BR3), and Bifidobacterium lactis (BL3) were evaluated for their anti-C. difficile activity. Co-culture incubation of C. difficile (106 and 1010 CFU/ml) with each strain of LAB indicated that SL3 possessed the highest antimicrobial activity over a 24-hr period. The cell-free supernatants of the 4 LAB strains exhibited MIC50 values between 0.424 mg/ml (SL3) and 1.318 (BR3) mg/ml. These results may provide a basis for alternative therapies for the treatment of C. difficile-associated gut disorders.

Anti-tumor effects of supernatant from B16 melanoma cells mixedly cultured with lymphocytes and macrophages in vitro / 肿瘤研究与临床

Objective To study the anti-tumor effects of mixed cultured B16 melanoma cells supernatant.Methods The supernatant from purely cultured B16 melanoma cells or mixedly cultured B16 melanoma cells with lymphocytes and macrophages at indicated time points were collected,respectively.The chemotaxis of the two different cell supernatants was determined by Boyden room method.The activation effects towards lymphocytes of the two different supernatants were determined by CCK-8 method.Results When the cells were mixed cultured for 0.5,1,2,4,8 and 12 h,the numbers of lymphocytes to travel from the upper well to the bottom well were (1.00±0.82) × 104,(7.00±1.63) × 104,(9.50±0.58) × 104,(11.25±2.36) ×104,(17.25±1.71) × 104 and (21.50±1.29) × 104,respectively.When the cells were purely cultured for 0.5,1,2,4,8 and 12 h,the numbers of lymphocytes to travel from the upper well to the bottom well were (0.00±0.00) ×104,(0.25±0.50) × 104,(1.75±0.96) × 104,(5.25±0.96) × 104,(5.75±1.26) × 104 and (10.75±3.20) × 104,respectively.The mixed cultured group showed higher chemotaxis effects towards lymphocytes in comparison with the purely cuhured one at the same points except for 0.5 h (P ＜ 0.05).The mixed cultured group showed higher activation effects towards lymphocytes in comparison with the purely cultured at the same points except for 0.5 and 1 h (P ＜ 0.05).Each group showed higher chemotaxis and activation effects towards lymphocytes when they were cultured for 12 h than the other time points (P ＜0.05).Conclusion The supernatant from mixed cultured cells shows much higher chemotaxis and activation effects towards lymphocytes to kill tumor cells.

Morphine Attenuates Endothelial Cell Adhesion Molecules Induced by the Supernatant of LPS-Stimulated Colon Cancer Cells

A large reservoir of bacterial lipopolysaccharide (LPS) is available in the colon and this could promote colon cancer metastasis by enhancing tumor cell adhesion, intravasation, and extravasation. Furthermore, adhesion molecules like ICAM-1, VCAM-1, and E-selectin play important roles in the adhesion of tumor cells to endothelium. This study was designed to determine whether morphine can attenuate the expressions of adhesion molecules up-regulated by the supernatant of LPS-stimulated HCT 116 colon cancer cells (LPS-Sup). In this study, we divided to three groups by cell-growth medium of human umbilical vascular endothelial cells (HUVECs): the control group was incubated in growth factor-free endothelial medium, the Sup group was incubated in the supernatant of HCT 116 cells (Sup), and the LPS-Sup group was incubated in LPS-Sup. To observe effect of morphine to the adhesion molecules expressions in the LPS-Sup group, we co-treated morphine with LPS or added it to LPS-Sup. Adhesion molecule expressions on HUVECs in all three groups were measured during incubation period. Consquentially, ICAM-1, VCAM-1, and E-selectin expressions on HUVECs were significantly lower when morphine was co-treated with LPS than not co-treated. Thus, we suggest that morphine affects the expressions of adhesion molecules primarily by attenuating LPS stimuli on tumor cells.

Spent culture supernatant of Lactobacillus acidophilus influences contraction of isolated intestinal smooth muscle of rabbits / 第二军医大学学报

Objective: To observe the influence of spent culture supernatant (SCS) of Lactobacillus acidophilus strain LA14 on the contraction of isolated intestinal smooth muscle and to discuss the related mechanism. Methods: The ileum samples of rabbits were prepared and the contraction frequency and amplitude of intestinal smooth muscle were observed as the normal control. Then the SCS, bacterium suspension, and SCS with bacterium suspension were added by an accumulative dose to the culture media (0.3 ml per times, at an interval of 6 min), respectively. Four minutes after each administration, the contractive curves were recorded for 2 min. The influences of various groups of Lactobacillus acidophilus on the contraction of isolated intestinal smooth muscle were observed. The effect of SCS on M cholinoceptor was observed by adding in order pilocarpine, atropine or SCS, and pilocarpine. Results: After continuous administration of SCS or SCS with bacterium suspension (0.6-1.5 ml), the contraction frequency of the intestinal smooth muscle was significantly lowered compared with before administration (P0.05). Within the range of 0.3-1.5 ml, the SCS, bacterium suspension, and SCS with bacterium suspension resulted in no significant difference in reducing the contraction amplitude, except for SCS with bacterium suspension at 1.5 ml(P<0.05). SCS or atropine significantly inhibited pilocarpine-induced increase of contraction amplitude(P<0.05 or P<0.01). SCS also reduced the contraction frequency of the intestinal smooth muscle(P<0.01). Conclusion: SCS of Lactobacillus acidophilus may inhibit the peristalsis of the intestinal smooth muscle of rabbits by blocking M cholinoceptor.

Protein Profile Changes in Platelet Concentrates According to Storage and Leukoreduction- Analysis Using Proteomics Technology / 대한진단검사의학회지

BACKGROUND: Knowing how the protein profile of platelet products changes with storage or leukoreduction may give us greater insight into cell physiology and the cause of transfusion reactions other than cytokines and chemokines. METHODS: We filtered four packs of platelet concentrates (PC) within 24 hr of blood collection and after 120 hrs of storage. Four aliquots of each supernatant in PC were obtained: pre-storage+prefiltration, pre-storage+post-filtration, post-storage+pre-filtration and post-storage+post-filtration. Routine chemistry tests and a two-dimensional electrophoresis (2-DE) were performed. The stained images were analyzed and the significant spots were identified using a peptide mass finger printing (PMF) with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis after trypsin digestion. RESULTS: The protein spots increased with storage and decreased after filtration (P<0.05, prestorage+post-filtration). The spot density of various proteins, including macrophage inflammatory protein-2 alpha, megakaryocyte colony stimulating factor and interleukin-22 changed with storage and leukoreduction. CONCLUSIONS: The database of identified protein spots and their changes produced in this study is a useful basic tool for future studies on the mechanism of transfusion reactions. Further studies should validate the significance of each protein spot.

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5'-nucleotidase subclass I and purine-specific 5'-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5'-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.