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Serial Dilutions (serial + dilution)
Selected AbstractsBayesian Analysis of Serial Dilution AssaysBIOMETRICS, Issue 2 2004Andrew Gelman Summary. In a serial dilution assay, the concentration of a compound is estimated by combining measurements of several different dilutions of an unknown sample. The relation between concentration and measurement is nonlinear and heteroscedastic, and so it is not appropriate to weight these measurements equally. In the standard existing approach for analysis of these data, a large proportion of the measurements are discarded as being above or below detection limits. We present a Bayesian method for jointly estimating the calibration curve and the unknown concentrations using all the data. Compared to the existing method, our estimates have much lower standard errors and give estimates even when all the measurements are outside the "detection limits." We evaluate our method empirically using laboratory data on cockroach allergens measured in house dust samples. Our estimates are much more accurate than those obtained using the usual approach. In addition, we develop a method for determining the "effective weight" attached to each measurement, based on a local linearization of the estimated model. The effective weight can give insight into the information conveyed by each data point and suggests potential improvements in design of serial dilution experiments. [source] HOST GROWTH CONDITIONS INFLUENCE EXPERIMENTAL EVOLUTION OF LIFE HISTORY AND VIRULENCE OF A PARASITE WITH VERTICAL AND HORIZONTAL TRANSMISSIONEVOLUTION, Issue 7 2010Hélène Magalon In parasites with mixed modes of transmission, ecological conditions may determine the relative importance of vertical and horizontal transmission for parasite fitness. This may lead to differential selection pressure on the efficiency of the two modes of transmission and on parasite virulence. In populations with high birth rates, increased opportunities for vertical transmission may select for higher vertical transmissibility and possibly lower virulence. We tested this idea in experimental populations of the protozoan Paramecium caudatum and its bacterial parasite Holospora undulata. Serial dilution produced constant host population growth and frequent vertical transmission. Consistent with predictions, evolved parasites from this "high-growth" treatment had higher fidelity of vertical transmission and lower virulence than parasites from host populations constantly kept near their carrying capacity ("low-growth treatment"). High-growth parasites also produced fewer, but more infectious horizontal transmission stages, suggesting the compensation of trade-offs between vertical and horizontal transmission components in this treatment. These results illustrate how environmentally driven changes in host demography can promote evolutionary divergence of parasite life history and transmission strategies. [source] Dose,response and time course relationships for vitellogenin induction in male western fence lizards (Sceloporus occidentalis) exposed to ethinylestradiolENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2002Sandra M. Brasfield Abstract The long-term goal of this research is to develop and validate an in vivo reptile model for endocrine-mediated toxicity using fence lizards (Sceloporus spp.). One of the best defined estrogenic responses in oviparous vertebrates is induction of the yolk precursor protein, vitellogenin (Vtg). In this study, dose,response and time course relationships for Vtg induction were determined in male western fence lizards (Sceloporus occidentalis) given intraperitoneal injections of 17,-ethinylestradiol (EE2). Plasma Vtg was quantified directly with an antibody-capture enzyme-linked immunosorbent assay (ELISA) and indirectly using plasma alkalinelabile phosphate (ALP) in order to compare these two methods. Both ELISA and ALP predicted similar median effective dose (ED50 [dose causing a 50% maximal response]) values for plasma Vtg induction (0.167 mg/kg for ELISA and 0.095 mg/kg for ALP). In addition, both ELISA and ALP detected significant Vtg induction at a dose of 0.0003 mg/kg of EE2, which was the lowest dose used in our study. A decrease in body weight at the highest dose (10 mg/kg) and an increase in hepatosomatic index at the four highest doses were observed. Serial dilutions of plasma from an EE2 -exposed male revealed a high correlation between plasma Vtg and ALP determinations in this species. In conclusion, our data show that plasma ALP may be a suitable alternative for measuring plasma Vtg compared with developing a Vtg ELISA in fence lizards exposed to estrogenic compounds. [source] Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growthEQUINE VETERINARY JOURNAL, Issue 7 2003J. S. Weese Summary Reasons for performing study: Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. Objectives: To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. Methods: Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests. Serial dilutions of smectite were tested for the ability to inhibit growth of C. perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C. perfringens in culture broth. Results: Clostridium difficile toxins A and B, and C. perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16. Partial binding of C. difficile toxins occurred at dilutions up to 1:256 while partial binding of C. perfringens enterotoxin occurred up to a dilution of 1:128. Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32. No inhibition of growth of C. difficile or C. perfringens was present at any dilution, and there was no effect on the action of metronidazole. Conclusions: Di-tri-octahedral smectite possesses the ability to bind C. difficile toxins A and B, C. perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole. Potential relevance: In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colifis and endotoxaemia in horses. [source] Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio LJOURNAL OF FISH DISEASES, Issue 4 2008R B Shivappa Abstract Spring viraemia of carp virus (SVCV) is a rhabdovirus associated with systemic illness and mortality in cyprinids. Several diagnostic tests are available for detection of SVCV. However, most of these tests are time consuming and are not well adapted for field-based diagnostics. In this study, a diagnostic tool for SVCV detection based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed. Based on the nucleotide sequence of the glycoprotein (G) gene of SVCV North Carolina (NC) isolate, four sets (each set containing two outer and two inner) of primers were designed. Temperature and time conditions were optimized to 65 °C and 60 min, respectively, for LAMP and RT-LAMP using one primer set. In vitro specificity was evaluated using four different strains of fish rhabdoviruses and RT-LAMP was found to be specific to SVCV. Serial dilutions of SVCV NC isolate was used to evaluate the in vitro sensitivity of RT-LAMP. Sensitivity of the assays was similar to RT-PCR and detected SVCV even at the lowest dilution of 101 TCID50 mL,1. The ability of RT-LAMP to detect SVCV from infected carp was also tested and the assay detected SVCV from all infected fish. The isothermal temperature requirements, high specificity and sensitivity, and short incubation time of the RT-LAMP assay make it an excellent choice as a field diagnostic test for SVCV. [source] Isolation and Characterization of Lactobacillus Species Having Potential for Use as Probiotic Cultures for DogsJOURNAL OF FOOD SCIENCE, Issue 3 2007S. McCoy ABSTRACT:, The need to control pathogenic microorganisms in the intestinal tract of dogs is a growing concern. There is interest in using probiotics such as species of Lactobacillus to help control canine intestinal infections. For successful use as a probiotic, the bacterial species should be of canine intestinal origin since these species exhibit host specificity. Serial dilutions of freshly voided dog feces were plated on Lactobacillus selection (LBS) agar to isolate the cultures. Isolates were identified based on Gram stain, catalase test, and fermentation patterns using API 50 CH kits. All potential isolates were compared for bile resistance based on relative ability to grow in broth containing 0.3% Oxgall, the ability to inhibit Salmonella Typhimurium in associative broth cultures, and the production of reuterin. Of the lactobacilli isolated, Lactobacillus reuteri was the dominant species. However, some cultures of L. acidophilus also were isolated. We found variations among the isolates of L. reuteri and L. acidophilus with respect to bile tolerance. In general, isolates of L. reuteri appeared to be more bile resistant than were isolates of L. acidophilus. There were also variations in the ability to inhibit growth of S. Typhimurium. Some isolates of L. reuteri produced reuterin while others did not. [source] In vitro study of the inactivation by dentine of some endodontic medicaments and their basesAUSTRALIAN DENTAL JOURNAL, Issue 3 2010B Athanassiadis Abstract Background:, The aim of this study was to investigate the antimicrobial effect of endodontic medicaments and their bases in the presence of dentine powder. Methods:, The medicaments tested were Ledermix paste, Pulpdent paste, a 50:50 combination of the Pulpdent:Ledermix and their bases. The test organism was Enterococcus faecalis ATCC 29212. The presence or absence of dentine was examined as well as the effect of autoclaving dentine. Serial dilutions of samples at 1 hour, 1 day and 3 days were used for colony counting. The effects of dentine powder on pH for saturated Ca(OH)2 solution and Pulpdent paste at 1 hour and 24 hours were also measured. Results:, Pulpdent and the 50:50 combination of Pulpdent:Ledermix completely inhibited the growth of E. faecalis from 1 hour onwards, and these results were not affected by the presence/absence of dentine powder, pre-incubation period, timing of autoclaving, or exposure time. Saturated solutions of Ca(OH)2 are prone to inactivation by dentine powder unlike Pulpdent paste. Ledermix paste took 3 days to exert a significant effect on the growth of E. faecalis. Conclusions:, In this laboratory study, both Pulpdent and the 50:50 mixture of Pulpdent with Ledermix were effective medicaments against E. faecalis in the presence of dentine powder. [source] Guidelines on use of anti-IFN- , antibody measurements in multiple sclerosis: report of an EFNS Task Force on IFN- , antibodies in multiple sclerosisEUROPEAN JOURNAL OF NEUROLOGY, Issue 11 2005P. S. Sørensen Therapy-induced binding and neutralizing antibodies is a major problem in interferon (IFN)- , treatment of multiple sclerosis. The objective of this study was to provide guidelines outlining the methods and clinical use of the measurements of binding and neutralizing antibodies. Systematic search of the Medline database for available publications on binding and neutralizing antibodies was undertaken. Appropriate publications were reviewed by one or more of the task force members. Grading of evidence and recommendations was based on consensus by all task force members. Measurements of binding antibodies are recommended for IFN- , antibody screening before performing a neutralizing antibody (NAB) assay (Level A recommendation). Measurement of NABs should be performed in specialized laboratories with a validated cytopathic effect assay or MxA production assay using serial dilution of the test sera. The NAB titre should be calculated using the Kawade formula (Level A recommendation). Tests for the presence of NABs should be performed in all patients at 12 and 24 months of therapy (Level A recommendation). In patients who remain NAB-negative during this period measurements of NABs can be discontinued (Level B recommendation). In patient with NABs, measurements should be repeated, and therapy with IFN- , should be discontinued in patients with high titres of NABs sustained at repeated measurements with 3- to 6-month intervals (Level A recommendation). [source] Dual-association of gnotobiotic Il-10,/, mice with 2 nonpathogenic commensal bacteria induces aggressive pancolitisINFLAMMATORY BOWEL DISEASES, Issue 12 2007Sandra C. Kim MD Abstract Background: Monoassociating gnotobiotic IL-10-deficient (,/,) mice with either nonpathogenic Enterococcus faecalis or a nonpathogenic Escherichia coli strain induces T-cell-mediated colitis with different kinetics and anatomical location (E. faecalis: late onset, distal colonic; E. coli: early onset, cecal). Hypothesis: E. faecalis and E. coli act in an additive manner to induce more aggressive colitis than disease induced by each bacterial species independently. Methods: Germ-free (GF) inbred 129S6/SvEv IL-10,/, and wildtype (WT) mice inoculated with nonpathogenic E. faecalis and/or E. coli were killed 3,7 weeks later. Colonic segments were scored histologically for inflammation (0 to 4) or incubated in media overnight to measure spontaneous IL-12/IL-23p40 secretion. Bacterial species were quantified by serial dilution and plated on culture media. Mesenteric lymph node (MLN) CD4+ cells were stimulated with antigen-presenting cells pulsed with bacterial lysate (E. faecalis, E. coli, Bacteroides vulgatus) or KLH (unrelated antigen control). IFN-, and IL-17 levels were measured in the supernatants. Results: Dual-associated IL-10,/, (but not WT) mice developed mild-to-moderate pancolitis by 3 weeks that progressed to severe distal colonic-predominant pancolitis with reactive atypia and duodenal inflammation by 7 weeks. NF-,B was activated in the duodenum and colon in dual-associated IL-10,/, × NF-,BEGFP mice. The aggressiveness of intestinal inflammation and the degree of antigen-specific CD4+ cell activation were greater in dual- versus monoassociated IL-10,/, mice. Conclusion: Two commensal bacteria that individually induce phenotypically distinct colitis in gnotobiotic IL-10,/, mice act additively to induce aggressive pancolitis and duodenal inflammation. (Inflamm Bowel Dis 2007) [source] Efficacy of various concentrations of NaOCl and instrumentation techniques in reducing Enterococcus faecalis within root canals and dentinal tubulesINTERNATIONAL ENDODONTIC JOURNAL, Issue 1 2006V. B. Berber Abstract Aim, To evaluate the efficacy of 0.5%, 2.5% and 5.25% sodium hypochlorite (NaOCl) as intracanal irrigants associated with hand and rotary instrumentation techniques against Enterococcus faecalis within root canals and dentinal tubules. Methodology, A total of 180 extracted human premolar teeth were infected for 21 days with E. faecalis. The specimens were divided into 12 groups, as follows: group 1: 5.25% NaOCl + Hybrid technique (Valdrighi et al. 1998); group 2: 5.25% NaOCl + nickel,titanium (NiTi) rotary technique 4 mm shorter than the apex (by FOP-UNICAMP); group 3: 5, 25% NaOCl + NiTi rotary technique (Hero 642); group 4: 2.5% NaOCl +Hybrid technique; group 5: 2.5% NaOCl + NiTi rotary technique 4 mm shorter than the apex; group 6: 2.5% NaOCl + NiTi rotary technique (Hero 642); group 7: 0.5% NaOCl + Hybrid technique; group 8: 0.5% NaOCl + NiTi rotary technique 4 mm shorter than the apex; group 9: 0.5% NaOCl + NiTi rotary technique (Hero 642); group 10: sterile saline solution + Hybrid technique; group 11: sterile saline solution + NiTi rotary technique 4 mm shorter than the apex; group 12: sterile saline solution + NiTi rotary technique (Hero 642). Canals were sampled before and after preparation. After serial dilution, samples were plated onto brain heart infusion (BHI) agar, and the colony forming units (CFU) that were grown were counted. The teeth were sectioned into three thirds and dentine chips were removed from the canals with conical burs. The samples obtained with each bur were immediately collected into test tubes containing BHI broth, and were incubated at 37 °C and plated onto BHI agar. The CFU were counted and analysed. Results, At all depths and thirds of the root canals and for all techniques used, 5.25% NaOCl was shown to be the most effective irrigant solution tested when dentinal tubules were analysed, followed by 2.5% NaOCl. No differences among concentrations in cleaning the canals were found. Conclusions, Especially at higher concentrations, NaOCl, was able to disinfect the dentinal tubules, independent of the canal preparation technique used. [source] Antibacterial activity of novel insoluble bead-shaped polymer-supported multiquaternary ammonium saltsJOURNAL OF APPLIED POLYMER SCIENCE, Issue 6 2010E. Murugan Abstract This study describes the effect of antibacterial activity of newly reported five different novel insoluble bead-shaped polymer-supported multiquaternary ammonium salts (PM quats) viz., bis-quat, tris-quat (2 Nos.), tetrakis-quat, hexakis-quat containing two, three, four, and six quaternary ammonium groups, respectively. The presence of number of quaternary ammonium groups in each salt was established already through Fourier-transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, and chloride ion analyzes. The antibacterial activities of these five different PM quats against three different bacteria viz., Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa were investigated by serial dilution and spread plate method and compared the same with a monoquat containing single quaternary ammonium group. The extent of antibacterial activity has been measured in terms of colony forming units (CFU) at different time intervals. The observed results show that all the PM quats exhibited excellent-antibacterial activity against each bacterium. On the basis of the CFU values, the antibacterial activity was found to increase from bis-quat to hexakis-quat, which reveals that the activity of PM quats increases with increase in the number of quaternary ammonium groups. The mechanism of interaction of quats with bacterial cytoplasmic membrane has been explained as an adsorption-like phenomenon. The reusability of highly active hexakis-quat against Staphylococcus aureus was studied and the activity was found to reduce after first cycle. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source] Estimating numbers of infectious units from serial dilution assaysJOURNAL OF THE ROYAL STATISTICAL SOCIETY: SERIES C (APPLIED STATISTICS), Issue 1 2006Nigel Stallard Summary., The paper concerns the design and analysis of serial dilution assays to estimate the infectivity of a sample of tissue when it is assumed that the sample contains a finite number of indivisible infectious units such that a subsample will be infectious if it contains one or more of these units. The aim of the study is to estimate the number of infectious units in the original sample. The standard approach to the analysis of data from such a study is based on the assumption of independence of aliquots both at the same dilution level and at different dilution levels, so that the numbers of infectious units in the aliquots follow independent Poisson distributions. An alternative approach is based on calculation of the expected value of the total number of samples tested that are not infectious. We derive the likelihood for the data on the basis of the discrete number of infectious units, enabling calculation of the maximum likelihood estimate and likelihood-based confidence intervals. We use the exact probabilities that are obtained to compare the maximum likelihood estimate with those given by the other methods in terms of bias and standard error and to compare the coverage of the confidence intervals. We show that the methods have very similar properties and conclude that for practical use the method that is based on the Poisson assumption is to be recommended, since it can be implemented by using standard statistical software. Finally we consider the design of serial dilution assays, concluding that it is important that neither the dilution factor nor the number of samples that remain untested should be too large. [source] Microbial status in seawater and coastal sediments during pre- and post-tsunami periods in the Bay of Bengal, IndiaMARINE ECOLOGY, Issue 3 2006Subramani Ramesh Abstract Tsunami, the natural disaster, which occurred on December 26, 2004 in the Indian Ocean, caused severe damage to mankind in the coastal areas. Total loss of life and economic loss because of this disaster have been estimated by various agencies but its effect on microbial density has not been probed. With our previous results on microbial populations in four locations of the Chennai coast of the Bay of Bengal, India in the pre-tsunami period, the change in microbial populations was studied after the tsunami at regular intervals in the same locations. Coastal sediment and seawater samples were collected from four different locations after 5,10 h and thereafter at intervals of every 7 days up to 28 days post-tsunami. Bacteria, fungi and actinomycetes were isolated from the marine samples by serial dilution on respective media. Before the tsunami, the bacterial population was higher in seawater samples than the sediments, whereas fungi and actinomycetes were recorded only in the sediments. The microbial population remarkably increased 5,10 h post-tsunami in all the marine samples irrespective of the location. However, it slowly declined in the subsequent days and became similar to that of the population recorded before the tsunami. The population of gram-positive bacteria increased whereas the gram-negative bacterial population decreased after the tsunami. Further, populations of pathogenic bacteria such as coliform and vibrios did not increase after the tsunami. It has been observed that the increase in populations of bacteria and actinomycetes even after 28 days of tsunami may be due to the introduction of foreign microorganisms that developed the ability to survive in the extreme environment by exhibiting special characteristics such as pigmentation and production of exopolysaccharides. [source] Microbiological examination of infected dental root canalsMOLECULAR ORAL MICROBIOLOGY, Issue 2 2004B. P. F. A. Gomes Objectives:, The aim of this study was to investigate the root canal microbiota of primary and secondary root-infected canals and the association of constituent species with specific endodontic signs and symptoms. Methods:, Microbial samples were taken from 60 root canals, 41 with necrotic pulp tissues (primary infection) and 19 with failed endodontic treatment (secondary infection). Strict anaerobic techniques were used for serial dilution, plating, incubation and identification. Results:, A total of 224 cultivable isolates were recovered belonging to 56 different bacterial species. Individual root canals yielded a maximum of 10 bacterial species. Of the bacterial isolates, 70% were either strict anaerobes or microphilic. The anaerobes most frequently isolated were: Peptostreptococcus micros (35%), Fusobacterium necrophorum (23.3%), Fusobacterium nucleatum (11.7%), Prevotella intermedia/nigrescens (16.7%), Porphyromonas gingivalis (6.7%) and Porphyromonas endodontalis (5%). The root canal microflora of untreated teeth with apical periodontitis was found to be mixed, comprising gram-negative and gram-positive and mostly anaerobic microorganisms and usually containing more than 3 species per canal. On the other hand, facultative anaerobic and gram-positive bacteria predominated in canals with failed endodontic treatment, which harbored 1,2 species per canal. Suggested relationships were found between anaerobes, especially gram-negatives, and the presence or history of pain, tenderness to percussion and swelling (P < 0.05). In particular, associations were found between: a) pain (n = 29) and P. micros (P < 0.01), P. intermedia/nigrescens and Eubacterium spp. (both P < 0.05); b) history of pain (n = 31) and P. micros (P < 0.01) Porphyromonas and Fusobacterium spp. (P < 0.05); c) tenderness to percussion (n = 29) and Porphyromonas spp. (P < 0.01), Peptostreptococcus and Fusobacterium spp. (P < 0.001); d) swelling (n = 20) and Peptostreptococcus spp. (P < 0.01), Porphyromonas and Enterococcus spp. (P < 0.05); e) wet canals (n = 33) and Porphyromonas and Fusobacterium spp. (P < 0.05); f) purulent exudate (n = 20) and Porphyromonas, Peptostreptococcus and Fusobacterium spp. (P < 0.05); previous endodontic treatment and Enterococcus faecalis, Streptococcus spp., P. micros, F. necrophorum (P < 0.05). Conclusions:, Our findings indicate potential complex interactions of species resulting in characteristic clinical pictures which cannot be achieved by individual species alone. They also indicate that the microbiota of primary infected canals with apical periodontitis differs in number and in species from the secondary infected canals by using the culture technique. [source] Bayesian Analysis of Serial Dilution AssaysBIOMETRICS, Issue 2 2004Andrew Gelman Summary. In a serial dilution assay, the concentration of a compound is estimated by combining measurements of several different dilutions of an unknown sample. The relation between concentration and measurement is nonlinear and heteroscedastic, and so it is not appropriate to weight these measurements equally. In the standard existing approach for analysis of these data, a large proportion of the measurements are discarded as being above or below detection limits. We present a Bayesian method for jointly estimating the calibration curve and the unknown concentrations using all the data. Compared to the existing method, our estimates have much lower standard errors and give estimates even when all the measurements are outside the "detection limits." We evaluate our method empirically using laboratory data on cockroach allergens measured in house dust samples. Our estimates are much more accurate than those obtained using the usual approach. In addition, we develop a method for determining the "effective weight" attached to each measurement, based on a local linearization of the estimated model. The effective weight can give insight into the information conveyed by each data point and suggests potential improvements in design of serial dilution experiments. [source] Cross-reactivity between nickel and palladium demonstrated by systemic administration of nickelCONTACT DERMATITIS, Issue 1 2005M. Hindsén Concomitant patch test reactions to nickel and palladium have frequently been reported in patients undergoing investigation because of suspected allergic contact dermatitis. Theoretically, these reactions can be explained by multiple, concomitant, simultaneous sensitization as well as cross-sensitization. We studied whether concomitant reactions to nickel and palladium could represent cross-sensitization in females hypersensitive to combinations of nickel, palladium and cobalt. Females were patch tested with serial dilutions of nickel sulfate, cobalt chloride and palladium chloride on the upper back. 1 month later, when the patch test reactions were gone, the patients were randomized into 2 groups that were challenged orally with either nickel or placebo. 1 day later, the areas of previous positive patch test reactions were read in a blind way looking for flare-up reactions. Nickel provocation but not placebo yielded flare-up reactions on sites previously tested with nickel (P = 0.012) and palladium (P = 0.006), but were also observed on sites previously tested with cobalt, even though this was not statistically significant. Flare-up reactions of previous patch test reactions to nickel and palladium after oral challenge with nickel speak in favour of a cross-reactivity mechanism. [source] Flow cytometry antibody screening using pooled red cells,CYTOMETRY, Issue 2 2010Dong Il Won Abstract Background: For red cell alloantibody screening, the column agglutination technique (CAT) is used extensively, and flow cytometry (FC) screening has recently been demonstrated to be accurate, rapid, and cost effective. We attempted to determine whether the high sensitivity of FC allows pooling of screening red cells, which is generally not an acceptable technique in CAT. Methods: For FC screening, a commercial two-cell screening panel was utilized for the preparation of individual cells (CSi), as well as pooled cells diluted 1 in 2 (CSp), and 1 in 3 (CS1/3). Another panel was pooled from 120 randomly selected group O donors (RSp). Results: Comparing the endpoint titrations of serial dilutions, CS1/3 was found to be one dilution, on the average, less sensitive than CSi. In 33 CAT-positive patient samples, the sensitivities of CSi and CSp did not differ significantly without polyethylene glycol (PEG) (30/33, 26/33, respectively, P = 0.125), although they did differ significantly with PEG (32/33, 25/33, respectively, P = 0.016). The percentages of reactive cells among the total cells from RSp were roughly proportional to the relevant antigen frequencies of the local donors. Conclusions: A trend toward reduced sensitivity was observed using pooled red cells, even via FC. Pooled cells from randomly selected group O donors may be employed as another method by which the characteristics of known antibodies might be assessed. © 2009 Clinical Cytometry Society [source] Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growthEQUINE VETERINARY JOURNAL, Issue 7 2003J. S. Weese Summary Reasons for performing study: Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. Objectives: To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. Methods: Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests. Serial dilutions of smectite were tested for the ability to inhibit growth of C. perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C. perfringens in culture broth. Results: Clostridium difficile toxins A and B, and C. perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16. Partial binding of C. difficile toxins occurred at dilutions up to 1:256 while partial binding of C. perfringens enterotoxin occurred up to a dilution of 1:128. Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32. No inhibition of growth of C. difficile or C. perfringens was present at any dilution, and there was no effect on the action of metronidazole. Conclusions: Di-tri-octahedral smectite possesses the ability to bind C. difficile toxins A and B, C. perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole. Potential relevance: In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colifis and endotoxaemia in horses. [source] High sensitivity of chemiluminescent methodology for detection of clonal CDR3 sequences in patients with acute lymphoblastic leukemiaHEMATOLOGICAL ONCOLOGY, Issue 2 2004E. Leal Abstract Detection of minimal residual disease (MRD) in patients with B-cell acute lymphoblastic leukemia (B-ALL) has been achieved using several radioactive labelling methodologies; however, limited information exists about the use of chemiluminescent labelling. Although many malignant disorders are related to cytogenetic alterations, there is not a consistent chromosomal translocation that could serve as a tumour marker for the monitoring of MRD. ALL are derived from B-lymphocytes in 80% of cases. In the early stages of their maturation, the immunoglobulin heavy chain genes (IgH) undergo rearrangements among their V, D, and J segments, giving rise to the Complementary Determining Regions (CDR). Among these, CDR3 is considered unique for each lymphocyte and used as a tumour-specific marker in B-ALL patients. In this study, the CDR3 was labelled with digoxigenin and used as a patient-specific probe to test its sensitivity for further detection of MRD. Fourteen pretreatment samples of bone marrow (BM) or peripheral blood (PB) from B-ALL patients were included. Tumour-specific probes were designed from each clonal product by elimination of the consensus sequences. Ten digoxigenin-labelled probes were hybridized with a mixture of their respective clonal DNA and the polyclonal product from a normal healthy donor, in serial dilutions from 1:1 up to 1:107. A sensitivity range of 1:103,1:106 was obtained, with an average of 1:105. Crossed tests performed in four patients, showed right probe specificity in all cases. We propose that the design of allele-specific probes with chemiluminescent labelling, represents a reliable, sure and sensitive alternative methodology for MRD detection in patients with B-cell lymphoproliferative disorders. Copyright © 2004 John Wiley & Sons, Ltd. [source] Active MMP-2 effectively identifies the presence of colorectal cancerINTERNATIONAL JOURNAL OF CANCER, Issue 12 2009Mary Jo Murnane Abstract Fully active MMP-2 is expressed at such low levels in human tissues that studies often fail to confirm its value as a cancer marker despite strong associations with malignancy. Our study utilized careful extraction, accurate activity measurements, standardization to purified controls and a new statistical metric to determine whether active MMP-2 is an effective indicator of colorectal cancer compared to pro-MMP-2 or pro-MMP-9. MMP-2 and MMP-9 activities were analyzed in matched normal and cancer samples from 269 patients by gelatin zymography, computer-assisted image analysis, serial dilutions of strong samples and standardization to controls. An index of effect size was designed for comparative evaluation of active MMP-2, pro-MMP-2 and pro-MMP-9 activities. For each gelatinase, mean activity and protein levels/mg soluble protein in normal mucosa and colorectal cancer were calculated for the first time with respect to commercial standards. Active MMP-2 activity, detected in 99% of colorectal cancers, was higher in 95% of cancers (on average 10-fold) than in normal mucosa. Levels of pro-MMP-2 and pro-MMP-9, but not active MMP-9, activities were also significantly higher in cancers versus normal. However, active MMP-2 activity provided the most effective test for the presence of cancer (p < 0.0.0001) with an effect size statistically significantly larger than for either pro-MMP-2 or pro-MMP-9. Receiver operating characteristic (ROC) curves demonstrated that a cut-off for active MMP-2 of >44 SDU activity/mg soluble protein (>180 pg/mg), which is three times mean normal levels, would permit detection of colorectal cancer with an estimated sensitivity of 84% and estimated specificity of 93%. © 2009 UICC [source] Rapid and sensitive detection of mumps virus RNA directly from clinical samples by real-time PCRJOURNAL OF MEDICAL VIROLOGY, Issue 3 2005Kazue Uchida Abstract A rapid, sensitive, and specific assay to detect mumps virus RNA directly from clinical specimens using a real-time PCR assay was developed. The assay was capable of detecting five copies of standard plasmid containing cDNA from the mumps virus F gene. No cross-reactions were observed with other members of Paramyxoviridae, or with viruses or bacteria known to be meningitis pathogens. Seventy-three clinical samples consisting of throat swabs collected from patients with parotitis, and cerebrospinal fluid (CSF) collected from patients with aseptic meningitis, were examined with a real-time PCR assay developed by the authors, reverse-transcription nested-PCR (RT-n-PCR), and virus isolation using cell culture. Like the RT-n-PCR assay, the real-time PCR assay could detect mumps virus RNA in approximately 70% of both throat swabs and CSF samples, while, by tissue culture, mumps virus was isolated from only approximately 20% of CSF and 50% of throat swab samples. In addition, the real-time PCR assay could be developed easily into a quantitative assay for clinical specimens containing more than 1,800 copies of mumps virus RNA/ml by using serial dilutions of the standard plasmid. The results suggest that the real-time PCR assay is useful for identification of mumps virus infections, not only in typical cases, but also in suspected cases, which show only symptoms of meningitis or encephalitis. J. Med. Virol. 75:470,474, 2005. © 2005 Wiley-Liss, Inc. [source] Adverse effects of Sudanese toombak vs.JOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 2 2010Swedish snuff on human oral cells J Oral Pathol Med (2010) 39 128,140 Background:, The high incidence of oral cancer in Sudan has been associated with the use of toombak, the local type of smokeless tobacco. However, its specific effects on human oral cells are not known. We aimed to investigate the effects of toombak on primary normal human oral keratinocytes, fibroblasts, and a dysplastic oral keratinocytic cell line, and to compare them with the effects induced by Swedish snuff. Method:, Aqueous extracts were prepared from moist toombak and Swedish snuff and added in serial dilutions on in vitro monolayer cultured cells. Cell viability, morphology and growth, DNA double-strand breaks (,H2AX staining), expression of phosphatidylserine (Annexin V staining), and cell cycle were assessed after various exposure time periods. Results:, Significant decrease in cell number, occurrence of DNA double-strain breaks, morphological and biochemical signs of programmed cell death were detected in all oral cell types exposed to clinically relevant dilutions of toombak extract, although to a lesser extent in normal oral fibroblasts and dysplastic keratinocytes. G2/M-block was also detected in normal oral keratinocytes and fibroblasts exposed to clinically relevant dilutions of toombak extract. Swedish snuff extract had less adverse effects on oral cells, mainly at non-clinically relevant dilutions. Conclusion:, This study indicates a potential for toombak, higher than for Swedish snuff, to damage human oral epithelium. Dysplastic oral keratinocytes were less sensitive than their normal counterparts, suggesting that they might have acquired a partially resistant phenotype to toombak -induced cytotoxic effects while still being prone to DNA damage that could lead to further malignant progression. [source] Electronic nose analysis of volatile compounds from poultry meat samples, fresh and after refrigerated storage,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 3 2002Dorothy D, H Boothe Abstract Electronic nose technology has previously been applied to the assessment of the quality of red meats, pork and fish, but not poultry products. In the present study the ability of the electronic nose to assess the microbiological quality of raw poultry meat as a function of storage time and temperature was investigated. Four types of chicken pieces (boneless breast with and without skin, wings and thighs) were stored for up to 2 days at 13,°C (the maximum allowable temperature in poultry processing environments) or for up to 5 days at 4,°C (refrigeration temperature for raw poultry products prior to shipping or further processing). Saline rinses of meat samples were serially diluted in tryptic soy broth to 10,10. The rinses and their associated serial dilutions were analysed on an electronic nose with 12 metal oxide sensors in order to determine the specificity and sensitivity respectively of the assay. Principal component analysis (PCA) maps of the data confirmed that the electronic nose could differentiate volatile compounds associated with individual types of meat samples properly stored at 4,°C from those maintained at processing temperature, 13,°C, for a comparable time, even as early as day 1 of storage. Differences in headspace gases from any type of meat sample stored at one temperature could also be determined with increased storage time. However, data from samples stored at 4,°C clustered more tightly in PCA maps than those associated with samples maintained at 13,°C, indicating a greater diversity in volatile compounds at the higher temperature. We have shown herein that the electronic nose can detect changes in the volatile compounds associated with chicken meat based on product storage time and temperature; the technology can assess length of sample storage as well as deviation from refrigeration temperature. Published in 2002 for SCI by John Wiley & Sons, Ltd. [source] ANTICOAGULANT EFFECTS OF LOW MOLECULAR WEIGHT HEPARIN IN HEALTHY CATSJOURNAL OF VETERINARY EMERGENCY AND CRITICAL CARE, Issue S1 2004AJ Alwood Objectives: 1) Validate a chromogenic assay to measure Factor Xa inhibitory activity (anti-Xa activity) in normal feline plasma and following administration of low molecular weight heparins and unfractionated heparin. 2) Compare the effects of two commercially available low molecular weight heparins (LMWH), unfractionated heparin (UFH), and placebo on TEG, anti-Xa activity, PT/aPTT, PCV/TS and platelet count in healthy cats. Methods: Our study consisted of two phases: 1) the evaluation of a commercially available chromogenic anti-Xa assay (Rotachrom Heparin, Diagnostic Stago) for use in cats, and 2) the evaluation of hemostatic effects of LMWH in healthy cats. Phase 1: The anti-Xa assay was validated for use in cats using feline plasma and serial dilutions of the plasma spiked with UFH, enoxaparin, and dalteparin. Phase 2: Five healthy cats were included in a randomized Latin Squares model crossover-design to compare the effects of UFH and LMWH in cats. The cats then received one of the following subcutaneously: 1) 250 IU/kg UFH QID, 2) 100 IU/kg dalteparin BID, 3) 1 mg/kg enoxaparin BID, 4) 0.25 mL/kg 0.9% saline (placebo) QID. A minimum of a two-week washout period separated each treatment period. Each drug was administered for 5 days. Blood samples were obtained to measure anti-Xa, TEG, PT/aPTT, platelet count, and PCV/TS on Days 1, 3, 5, and 6 of each treatment cycle. Samples were collected at time 0 on each sample day for all parameters and on select days at hours 4, 8, and 12 for anti-Xa and TEG. Results: Preliminary results using the validated anti-Xa assay (from the first part of this study) demonstrate that LMWH treatment results in peak anti-Xa activity at the 4-hour sampling time that returned toward baseline by 8 hours (in 5/6 cats treated with LMWH thus far). Similar anticoagulant effects were noted in the TEG parameters of cats receiving LMWH (i.e., peak effects were noted at 4 hours). Analysis of current data by linear regression identifies a relationship between anti-Xa measurements and TEG parameters for cats treated with all heparin therapies (p<0.001). A similar relationship exists between anti-Xa and aPTT. Conclusions: Preliminary results suggest an anticoagulant effect of LMWH in cats that may not be uniform across individuals. Anti-Xa activity or TEG may provide useful tools for monitoring LMWH. [source] Ferutinin stability in human plasma and interaction with human serum albuminBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 2 2008H. Greige-Gerges Abstract Ferutinin is a potent phytoestrogen extracted from plants of the genus Ferula. The biological activity of this sesquiterpene is associated with the esterification of p -hydroxybenzoic acid with the daucane alcohol, jaeschkeanadiol. A HPLC method was developed to investigate the stability of ferutinin in acidic and basic solutions (pH 1.5 and 9.0, respectively), in buffer (pH 7.4) as well as in serial dilutions of albumin and in human plasma. The degradation of ferutinin was relatively slow at physiological pH 7.4 compared with low or high pH. Ferutinin was fully stable in human plasma as well as in albumin solution and the stability increased with albumin concentration. The binding of ferutinin to albumin was investigated by fluorescence spectroscopy. Ferutinin decreased the fluorescence of HSA and that of the only tryptophan residue located in domain IIA. As a result of the interaction between ferutinin and albumin, the binding of bilirubin decreased. The stability of ferutinin in plasma is attributable to ferutinin,albumin binding. Copyright © 2007 John Wiley & Sons, Ltd. [source] Aerobic Heterotrophic Bacterial and Fungal Communities in the Topsoil of Omo Biosphere Reserve in Southwestern Nigeria,BIOTROPICA, Issue 2 2000A. I. Okoh ABSTRACT As a part of the surveillance effort to monitor the ecological status of Omo Biosphere Reserve in the southwestern region of Nigeria, the aerobic heterotrophic bacterial and fungal communities of the topsoil were investigated in March 1995 and April 1996, before the onset of the rainy season. Four distinct wood-tree plantations, a core strict nature reserve (SNR) area, and a buffer zone were sampled. The topsoil samples (7.5 cm depth), including the litter, were taken with an auger (8 cm diameter) and transported to the laboratory in polyethylene bags. One-gram dry weight equivalent of sample was suspended in 10 ml sterile water, and serial dilutions from it were used for the estimation of bacterial and fungal densities. The bacterial and fungal densities ranged in the order of 106 and 103 cfu/g, respectively. Out of the 18 bacterial and 16 fungal species that were obtained, 13 and 12, respectively, were isolated from the core SNR. About 46 to 69 percent of the bacteria and 50 to 83 percent of the fungi species found in the SNR were absent in different combinations in the plantations and the buffer zone; these variations were significant among the sites monitored. The bacterial and fungal species compositions were significantly different between the SNR and each of the other sites. Proportional distributions within the sites were significant only for the bacterial communities. It would appear that plantation and human activities have caused significant changes in the distribution and species richness of the heterotrophic bacterial and fungal communities relative to the undisturbed SNR area of the Omo Biosphere Reserve. [source] The topical glucocorticoids beclomethasone dipropionate and fluticasone propionate inhibit human T-cell allergen-induced production of IL-5, IL-3 and GM-CSF mRNA and proteinCLINICAL & EXPERIMENTAL ALLERGY, Issue 1 2001N. Powell T-cell production of eosinophil-active cytokines (IL-5, IL-3, GM-CSF) is thought to be fundamental to asthma pathogenesis. Inhaled aeroallergens may be one important stimulus for T-cell cytokine production in asthma. To compare the potency and efficacy of the topical anti-asthma glucocorticoids beclomethasone dipropionate (BDP) and fluticasone propionate (FP) in inhibiting allergen-driven peripheral blood T-cell proliferation and production of IL-3, IL-5 and GM-CSF mRNA and protein. Peripheral blood mononuclear cells from six atopic asthmatics sensitized to house dust mite (HDM) were cultured in the presence of HDM and serial dilutions of BDP or FP in vitro. Cellular proliferation (7 days) and culture supernatant cytokine concentrations (6 days) were measured by uptake of tritiated thymidine and ELISA, respectively. Cytokine mRNA expression (24 h) was measured in three subjects using a quantitative PCR technique. Both BDP and FP inhibited allergen-induced T-cell proliferation, expression of IL-3, IL-5 and GM-CSF mRNA, and secretion of the corresponding proteins in a concentration-dependent fashion. FP was considerably more potent, but not more efficacious, in exerting these actions. Both BDP and FP have the potential markedly to inhibit allergen-induced T-cell production of asthma-relevant cytokines. This activity is effected at the level of T-cell proliferation and cytokine gene transcription. These properties may be key features of the anti-asthma activity of these drugs. The greater potency of FP in vitro may be responsible for its greater clinical potency. [source] | ||||||||||||||||||||||