Cent Serum Research Articles

Cytotoxic and antiproliferative potential of methanolic extracts of Asparagus racemosus in MDAMB231 cells

The present study was carried out to evaluate the cytotoxic and antiproliferative activity of methanolic extract of Asparagus racemosus in MDAB-231 cells. The qualitative phytochemical analysis of the extract revealed the presence of steroids, alkaloids, flavonoids, glycosides, saponins and diterpenes. MDAMB-231 cells were maintained in RPMI media containing 10 per cent serum and 1 per cent antibiotic and antimycotic solution. The cells were harvested and seeded to 96 well plate at 5×103 cells/mL, incubated for 24 hours at 37˚C with 5 per cent CO2. The cells were treated with 160, 80, 40, 20 and 10µg/mL of the extract for 24 hours and viability was accessed using MTT Assay. Also cells were plated in 6 well plates at concentrations 3×105 cells/mL and exposed to 80, 40, 20 and 10µg/mL of the extract of Asparagus racemosus for acridine orange ethidium bromide (AO/EB) staining. There was a dose dependent decrease in viability of cells exposed to methanolic extracts of Asparagus racemosus with a viability of 15.55+0.193 per cent for cells treated with 160µg/mL. The IC50 was found to be 91.36+ 0.87 µg/mL. The AO/EB staining revealed that most of the cells were dead in extract treated with 80µg/mL where more than 90 per cent of cells were live when treated with 10µg/mL of the extract.

Killing Activity of Micafungin Against Candida albicans, C. dubliniensis and Candida africana in the Presence of Human Serum.

We compared killing activity of micafungin in time-kill experiments in RPMI-1640 with and without 50% serum against Candida albicans, Candida dubliniensis and Candida africana reference strains and clinical isolates. Killing rates (k values) were determined for each strain and concentration. In RPMI-1640 MIC ranges were 0.015-0.03, 0.015-0.03 and 0.015mg/L against C. albicans, C. dubliniensis and C. africana, respectively. In 50% serum MIC values for the three species increased 16- to 64-fold. In RPMI-1640 micafungin was fungicidal against two of three C. albicans isolates at 16 and 32mg/L within 14.54h and fungistatic against all C. africana and C. dubliniensis. Fifty per cent serum significantly decreased the growth rate of C. africana, but not of the other two species; weak in vivo replication ability of C. africana was confirmed in murine model. In 50% serum micafungin at 0.25 and 1mg/L did not inhibit any of the three species (k values were always negative). Micafungin killing rate in 50% serum at 4, 16 and 32mg/L was significantly decreased for C. albicans, but increased for C. dubliniensis compared to RPMI-1640. Killing activity of micafungin against C. africana was comparable or higher in 50% serum than in RPMI-1640. Although micafungin is a highly protein-bound drug, it was equally effective against the species of the C. albicans complex in 50% serum at therapeutic trough concentration (4mg/L). Both in vitro and in vivo data confirmed the low virulence of C. africana compared to the two sibling species.

Effect of supplementing broiler chicken diets with green tea extract on the growth performance, lipid profile, antioxidant status and immune response

A trial was conducted to explore the effect of feeding graded concentrations of green tea extract (GTE; 125, 250, 500, 1000 and 2000 mg/kg) on the growth performance, serum lipid profile, liver glutathione-reduced, thigh muscle malondialdehyde and humoral immune response against Newcastle disease virus vaccines of broiler chickens from hatching to 42 d of age. The results of broilers fed on GTE were contrasted to those fed on butylated hydroxytoluene (BHT; 125 mg/kg) or unsupplemented controls.The obtained results revealed no significant difference among treatments in the measured growth performance parameters (body weight, average daily gain, average daily feed intake and feed conversion ratio), per cent liveability and serum lipid profile (total lipid, total cholesterol, high-density, low-density and very low-density lipoprotein cholesterols).Dietary intake of GTE resulted in a significant increase in the liver glutathione-reduced level compared to the control. The glutathione-reduced level of broilers fed on the BHT diet was not significantly different from that of broilers fed on GTE or control diets. Feeding GTE or BHT resulted in a significant decrease in the malondialdehyde level of meat tissue.The specific antibody titre against Newcastle disease virus vaccines was significantly increased at 28 and 35 d of age in broilers fed on diets supplemented with GTE but not with BHT.In conclusion, GTE was found to possess antioxidant and immunostimulant characteristics for broilers when supplemented to their diets, and its optimum inclusion level ranged from 125 to 500 mg/kg.

Modification of the slide agglutination test for the detection of sperm-agglutinins.

We have modified the slide agglutination test (SAT) in order to make it available for the routine detection and titration of sperm agglutinins. Serum and seminal plasma samples of 156 subfertile patients were tested by the modified slide agglutination test (MSAT) in two trials. Fifteen per cent of serum and 7.5% of siminal plasma samples gave positive results and MSAT showed excellent reproducibility: a highly significant statistical correlation between the positive titres in the two determinations was observed. One hundred and ten samples were tested simultaneously by the MSAT and the tray agglutination test (TAT). The two tests showed a similar sensitivity with a significant statistical correlation between the positive titres. Using the MSAT the reading of results was easier and more precise. Therefore we propose the MSAT for the routine detection and titration of sperm-agglutinins.

The influence of swing phase load on the electrochemical response, friction, and ion release of metal-on-metal hip prostheses in a friction simulator

Second-generation metal-on-metal (MoM) hip replacements have been considered as an alternative to commonly used polyethylene-on-metal joint prostheses due to polyethylene wear debris-induced osteolysis. However, the role of corrosion is still not fully understood. Bench tests have been carried out to characterize the relationship and interactions between electrochemical reactions ( corrosion), mechanical damage ( wear), and biological environments. An investigation of the corrosion response of MoM hip replacements has been conducted in a friction simulator in an initial attempt to reveal the tribocorrosion behaviour of clinically used metallic hip implants. These are the first reported results where electrochemical measurements have been made in-situ in a joint simulator. A ProSim friction simulator was used with an integrated electrochemical cell in this study of biotribocorrosion. Peak loads of 2 kN with three different swing phase loads, 25, 100, and 300 N were used to determine the effect of different swing loads on the corrosion processes (through electrochemical measurements and the release of metal ions). The three lubricants, NaCl solution, 25 per cent serum, and bovine synovial fluid, were employed as lubricants in this study. All femoral heads of the tested hip implants were 36 mm in diameter and made from a CoCrMo alloy. The open circuit potential (OCP) was monitored while implants were subjected to dynamic loading cycles. The link between swing phase load, lubricant, and ion release is discussed in relation to corrosion processes occurring at the tribological interface.

Two Distinct Phenotypes of Rat Vascular Smooth Muscle Cells: Growth Rate and Production of Tumor Necrosis Factor-α

The monoclonal theory of atherosclerosis postulates that a subpopulation of vascular smooth muscle cells (VSMCs) is selectively expanded in response to pathologic stimuli and accumulates in vascular intima. The purpose of this research was to clone VSMC, determine growth rates of the clones and their ability to release the mitogenic cytokine tumor necrosis factor-alpha (TNF-alpha). With approval of the institutional animal care and use committee, VSMCs were isolated and cultured from the thoracic aortas of three rats. To confirm that the cells in primary culture were of smooth muscle origin, they were immunostained with anti-alpha-smooth muscle-actin antibodies. Single cell-derived individual colonies with uniform appearance were surrounded by cloning rings, released with trypsin, and expanded. Growth rates of the clones were assessed by the mitochondrial dependent reduction of methyltetrazolium (MTT) to formazan after 24-hour stimulation with 10 per cent serum. Additionally, cloned cells were stimulated with 0.1, 1, 10, and 20 microg/mL lipopolysaccharide (LPS) for 24 hours, and TNF-alpha was determined in the culture medium. Data were analyzed by ANOVA. Two clones were isolated that could be divided into categories based on distinctly different morphology: 1) spindle-shaped (SP) or 2) epithelioid-shaped (EP) VSMCs. The SP clone had a growth rate that was 25 per cent higher than the EP clone (P < 0.05). Also, the SP clone had significantly higher release of TNF-alpha in response to LPS. For instance, TNF-alpha released in response to 0.1 microg/mL of LPS in the SP clone was 157 +/- 45 pg/mL versus 21 +/- 8.5 pg/mL in the EP clone (P < 0.05). Primary cultures of rat VSMCs are heterogeneous and consist of at least two morphologically distinct cell types. These clones are different in growth rate and cytokine production. It is possible that selective expansion of one of these clones contributes to formation of stenotic vascular lesions.

Transforming Growth Factor-β1 Regulates Hepatocyte Growth Factor-induced Trophoblast Motility and Invasion

During placental development extravillous trophoblasts invade the uterine wall in a tightly regulated manner dependent on both pro- and anti-invasive molecules. We have shown using the extravillous trophoblast cell line, SGHPL-4, that both cellular invasion and motility are stimulated by hepatocyte growth factor (HGF). It has previously been demonstrated that transforming growth factor=β1 (TGF-β1), produced by the decidua, inhibits extravillous trophoblast proliferation and invasion. It was the aim of this study to determine whether TGF-β1 could modulate HGF-induced motility and invasion and, if so, examine the mechanism involved. TGF-β1 significantly inhibited the growth of SGHPL-4 cells stimulated with 10 per cent serum. HGF-stimulated trophoblast cell invasion and motility were significantly inhibited by TGF-β1. Neither HGF nor TGF-β1 had an effect on SGHPL-4 cell growth under the conditions used for the invasion and motility experiments (0.5 per cent serum). Previous studies suggest that both HGF-stimulated trophoblast invasion and motility may be regulated by the production of nitric oxide. TGF-β1 was found to significantly decrease HGF-induced iNOS expression therefore suggesting a novel mechanism by which TGF-β1 could regulate motility and invasion.

Transforming growth factor-beta1 regulates hepatocyte growth factor-induced trophoblast motility and invasion.

During placental development extravillous trophoblasts invade the uterine wall in a tightly regulated manner dependent on both pro- and anti-invasive molecules. We have shown using the extravillous trophoblast cell line, SGHPL-4, that both cellular invasion and motility are stimulated by hepatocyte growth factor (HGF). It has previously been demonstrated that transforming growth factor=beta1 (TGF-beta1), produced by the decidua, inhibits extravillous trophoblast proliferation and invasion. It was the aim of this study to determine whether TGF-beta1 could modulate HGF-induced motility and invasion and, if so, examine the mechanism involved. TGF-beta1 significantly inhibited the growth of SGHPL-4 cells stimulated with 10 per cent serum. HGF-stimulated trophoblast cell invasion and motility were significantly inhibited by TGF-beta1. Neither HGF nor TGF-beta1 had an effect on SGHPL-4 cell growth under the conditions used for the invasion and motility experiments (0.5 per cent serum). Previous studies suggest that both HGF-stimulated trophoblast invasion and motility may be regulated by the production of nitric oxide. TGF-beta1 was found to significantly decrease HGF-induced iNOS expression therefore suggesting a novel mechanism by which TGF-beta1 could regulate motility and invasion.

Vasoactive and proliferative effects of parathyroid hormone and parathyroid hormone-related peptide on human vascular smooth muscle.

Hyperparathyroidism is associated with an increased incidence of hypertension and cardiovascular disease. Experiments in rats suggest that this results from desensitization of the normally inhibitory effects of parathyroid hormone-related peptide (PTHrP) on vascular smooth muscle cells (VSMCs) by previous exposure to parathyroid hormone (PTH). Isometric tension in vitro was measured in segments of vessels obtained at neck surgery from six normotensive patients with hyperparathyroidism and 12 eucalcaemic controls undergoing thyroid surgery. Vessels were precontracted with noradrenaline to allow the vasodilatory effects of PTH and PTHrP to be observed. Human VSMCs were cultured in 0.5, 5 and 15 per cent serum with or without PTH (10(-11) to 10(-7) nmol l-(1)) and proliferation was assessed by cell counts after 14 days. Vasodilatation of arterial segments produced by both PTH 20 nmol l(-1) and PTHrP 100 nmol l(-1) did not differ significantly between control and hyperparathyroidism groups (P > 0.08, Mann-Whitney U test). Vasodilatation by PTHrP was not reduced by preincubation of vessels in vitro with PTH 50 nmol l(-1) for 1 h (P > 0.36, Wilcoxon test). No effects of PTH on serum-induced proliferation of human VSMCs were observed. These data imply that homologous desensitization to PTHrP is not implicated in the pathogenesis of vascular disease in patients with hyperparathyroidism.

Characterization of a new in vitro burn wound model

Previous studies of the healing process in burn wounds have largely been dependent on different animal models. These models are not only ethically questionable but are also combined with several systemic variables that are difficult to control and standardize. The aim of this study was to develop a standardized and repeatable in vitro model of the burn wound in human skin. Burn wounds with a standardized area and depth were created in human skin from routine breast reduction operations and incubated in vitro. The re-epithelialization of the wounds was followed throughout the time of incubation (14 days) by fixing and staining wounds every second day. After 14 days of incubation, the viability of the cells in the epidermis and dermis was confirmed by isolation and culture in vitro. The wounds incubated in 10 per cent fetal calf serum were shown to heal after 7 days, whereas wounds incubated in 2 per cent serum did not show any sign of re-epithelization. However, both epidermal and dermal cells from wounds incubated in 2 per cent serum were shown to be viable after 2 weeks of incubation. These findings indicate that the in vitro model can be of great value for studies of the different phases of the healing process in burn wounds, as well as stimulatory effects on the wound healing process by different pharmacological agents and treatments.

Porphyromonas gingivalis: Presence of a Species-specific Antigen which is Discriminatory in Chronic Inflammatory Adult Periodontal Disease

Serum from adults with and without evidence of past periodontitis was assayed for the presence of antibodies to outer membrane components of Porphyromonas gingivalis . A protein of 47 000 Da in Sarcosyl-insoluble outer membrane preparations was shown to be a discriminatory antigen insofar as serum from 29 of 31 (94 per cent) patients with a history ofchronic adult periodontitis contained immunoglobulin G antibodies against this protein compared to four of 24 (17 per cent) serum samples from individuals without disease. The binding of antibodies to this antigen was shown to be F(ab’) 2 mediated. Type and reference strains of other oral and intestinal anaerobic bacteria were studied to see if the immunoreactive protein of 47 000 Da was exclusive to this species. None of the other microorganisms tested possessed this antigen, indicating that it is specific to P. gingivalis . Keywords: Porphyromonas gingivalis ; Bacteroides gingivalis ; Periodontal disease.

Bactericidal effects of human neutrophils and sera on selected endodotic pathogenic bacteria in an anaerobic environment

Phagocytosis by normal human neutrophils and the bactericidal activity of normal human pooled serum were measured under anaerobic conditions on six endodontic pathogenic bacterial species from the genera Actinomyces, Bacteroides, Fusobacterium, Peptostreptococcus and Streptococcus. The results revealed that Actinomyces viscosus was not killed either by human neutrophils or by 20 per cent serum; Peptostreptococcus anaerobius was not killed by either 20 per cent or 95 per cent serum; and Bacteroides intermedius was susceptible to both 20 per cent and 95 per cent serum, but was not killed by neutrophils after exposure for 20 minutes. The other species showed intermediate susceptibility patterns. Such results suggest that oral pathogens vary with regard to the bactericidal effects of these two host defense mechanisms, and that this variation may at least in part explain the relative quantities of microbes recovered in certain oral infections.

Stimulation of tumor cell growth in vitro by a monoclonal antibody to a tumor specific protein (TSP-180) present on the cell surface of 3LL cells.

Proliferation capacity and MHC class I antigen expression of two Lewis lung carcinoma (3LL) metastatic variants (C87, BC215) grown under defined experimental conditions (serum-free defined medium or 10 per cent serum) have been studied following exposure to MoAb 135-13C which recognizes on these cells a tumor surface protein of 180,000 daltons (TSP-180). The results of this study indicate that the high metastatic clone (C87) binds higher amounts of MoAb to TSP-180 and Db antigens than does the low metastatic one (BC215), while both clones express very low amounts of Kb antigens. 3LL clones grown in 10 per cent serum or adapted in serum-free, defined medium show the same metastatic phenotype and MHC class I antigen expression, but when grown in defined medium exhibit increased capacity to bind MoAb 135-13C. However, the relative binding rate of 3LL clones grown in 10 per cent serum or in defined medium is unchanged: the high metastatic clone always showing higher capacity to bind MoAb to TSP-180. Furthermore, comparison of EGF binding sites on the cell surface of 3LL clones, grown in different culture conditions, demonstrates that the C87 clone binds higher amounts of labelled EGF and that this amount increases in serum-free defined medium, exactly as reported for TSP-180. In addition, competition experiments demonstrated that MoAb 135-13C does not compete for EGF binding sites on 3LL cell surface. Studies on cell proliferation following exposure to MoAb 135-13C, revealed that the low metastatic clone (BC215) is more actively stimulated than the high metastatic one. Moreover, similar data were obtained after exposure of 3LL clones to physiological amounts of different growth factors (i.e. EGF, MSA, insulin). Analysis of MHC class I antigen expression following exposure to MoAb 135-13C indicated that MoAb 135-13C induces on the cell surface of the C87 clone a transient low modulation of Db antigens. These results suggest that 3LL cells endowed with lower metastatic potential are more dependent on the microenvironmental conditions than the high metastasizing ones, and that MoAb 135-13C binding to 3LL cell surface stimulates proliferation as reported for several known growth factors.

Attachment, spreading and growth in vitro of highly malignant and low malignant murine fibrosarcoma cells.

Highly malignant cell lines and low-malignant cell lines isolated from three different methylcholanthrene-induced murine fibrosarcomas were examined for their ability to attach to plastic dishes and collagen-coated dishes under serum-free conditions and in the presence of serum. Most of the cells from the three highly malignant lines attached and spread under all conditions. By 72 h, there was a significant increase in the number of cells indicating that at least some of the cells had undergone division (even in the absence of serum). In contrast, fewer of the cells from the three low-malignant lines attached and spread on the plastic or collagen substrates in the absence of serum or in the presence of 0.1 per cent serum. However, when 15 micrograms laminin per dish was added along with the low-malignant cells, they then attached and spread on the plastic and collagen-coated dishes. Previous studies have indicated that the highly malignant lines express cell surface antigens that cross-react with laminin while the low-malignant cell lines do not. We speculate that the differences between the high- and low-malignant cells in the expression of cell surface laminin-like antigens contribute to the dissimilarities in attachment and spreading capacity. These differences may also contribute to the dissimilarity between these cells in malignant potential.

Effect of bovine horn cancer serum on E-rosetting capacity of peripheral blood lymphocytes from normal unaffected animals

The effect of serum from horn cancer affected bullocks and cows on E-rosetting capacity of peripheral blood lymphocytes (PBL) from unaffected control animals was examined. The E-rosette counts were made using 2-aminoethyl isothiouronium bromide (AET) treated sheep red blood cells. A significant decrease in the percentage of EAET rosette forming cells was noticed when PBL were incubated with 50 per cent serum from animals affected with horn cancer. However, no such effect was noticed when PBL were treated with 50 per cent serum from unaffected control animals. A linear relationship was observed between percentage of EAET rosette forming cells of animals affected with horn cancer and E-rosette inhibitory activity of the corresponding serum on PBL from control animals.

A Survey about Blood Bank Policies and Procedures

A questionnaire about blood bank policies and procedures was prepared and sent out to 170 hospitals across the nation, 50 per cent responded. There is wide variation in both technical and nontechnical policies and procedures. Serum is separated from the original tube within 8 hours by 68 per cent of the hospitals and in 32 per cent serum is never separated from the original tube. Auto-control is included by 66 per cent of hospitals in at least one phase of the antibody testing, 34 per cent do not include auto-control in any phase. Du testing of the recipient is done in 85 per cent of the hospitals and in 15 per cent Rho type is determined by the immediate spin reactions alone. Slide type is the only method used to determine ABO group of cells of the recipients in 10 per cent of the hospitals, 18 per cent use serum-cell suspension, 10 per cent use the applicator stick method and only 62 per cent use washed saline-cell suspension routinely. Compatiblity testing between a recipient and a donor vary from a one-tube major crossmatch to a three-tube major crossmatch and minor crossmatch.

In vitro stimulation of ovine lymphocytes by various mitogens

A technique for the separation and in vitro culture of ovine lymphocytes is described and applied to the study of their blastogenic responses to various mitogens. Lymphocytes were separated on a Ficoll-Triosil gradient, resuspended in RPMI 1640 medium supplemented with 10 per cent serum to a concentration of 1 x 10(6) cells/ml and cultured in 200 microliter volumes in microculture plates in the presence of mitogens for varying lengths of time. A total culture period of 66 h was found to be satisfactory with 1 muCi of [3H]-thymidine being added to each well 18 h before termination of culture. Optimal blastogenic stimulation of the lymphocytes occurred with phytohaemagglutinin at 2-5 microliter/ml, pokeweed mitogen at 10 microliter/ml, concanavalin A at 6-25 microgram/ml and lipopolysaccharide of Escherichia coli (LPS) at 6-25 microgram/ml. Proliferation due to stimulation by LPS appeared to be of a lower order than that achieved with the other mitogens tested.

Inactivation of African horse-sickness virus by betapropiolactone and by pH

The inactivation of several types of African horse sickness virus (AHSV) by pH and by betapropiolactone (BPL) was studied. At 19 degrees - 22 degrees C the virus was stable between pH 6.0 and 10.4, whether suspended in mouse brain or in serumfree buffer. Below pH 5.6 and above pH 10.9, more than 99 per cent of infectivity was inactivated within 15 minutes. The addition of 50 per cent serum did not influence pH stability. Disinfection in the presence of citric acid and caustic soda is briefly discussed. Inactivation by BPL was complete within 30 minutes at 37 degrees C, yet incomplete after 15 hours at 4 degrees C. Types 3 and 9 virus grown in suckling mouse brain and types 1, 3 and 9 produced in pig kidney cells were equally susceptible to 0.1 per cent BPL, more than 99.9 per cent being inactivated. The effectiveness of BPL was reduced at least 10-fold by the addition of 50 per cent serum. No infective virus was detected following incubation of either tissue culture virus with 0.2 per cent BPL or of mouse brain virus with 0.3 per cent BPL. Virus suspensions exposed to 0.3 per cent BPL required buffering with Tris of at least 0.05 molar strength in order to maintain the pH within an acceptable range. Inactivated antigens prepared with 0.4 per cent or lower concentrations of BPL were immunogenic in guinea pigs.

Effect of heparin, histamine and serotonin on the density-dependent inhibition of replication in two fibroblastic cell lines.

The effects of heparin, histamine and serotonin—constituents of mast cells—on the density-dependent inhibition of replication was studied in two fibroblastic cell lines. Normal human foetal lung fibroblast-like cells (in subculture generations 10 through 14) and the established 3T3 murine cell line were used. In most experiments, the cultures were more than 14 days old and fed medium supplemented by 30 per cent serum. The human cells formed multilayered, tissue-like sheets whereas the 3T3 cells formed confluent monolayers.

Eosinophilia and elevated IgE concentration in a serous pleural effusion following trauma.

Pleural effusions with eosinophilia have been reported after trauma, but the fluid is usually sanguineous. This patient presented with a fractured rib, a lung hematoma, and a subsequent pleural effusion that was serous in nature and had 46 per cent eosinophils. In addition, there was a 10 per cent serum eosinophilia. Measurements of pleural fluid and serum IgE were also elevated, and the IgE value and eosinophil count returned to normal with resolution of the hematoma and pleural effusion. A trauma-related serous pleural fluid eosinophilia with peripheral eosinophilia has not been described previously.