Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates

There is no licensed vaccine for the prevention of shigellosis. Our approach to the development of a Shigella vaccines is based on inducing serum IgG antibodies to the O-specific polysaccharide (O-SP) domain of their lipopolysaccharides (LPS). We have shown that low molecular mass O-SP-core (O-SPC) fragments isolated from Shigella sonnei LPS conjugated to proteins induced significantly higher antibody levels in mice than the full length O-SP conjugates. This finding is now extended to the O-SPC of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1. The structures of O-SPC, containing core plus 1-4 O-SP repeat units (RUs), were analyzed by NMR and mass spectroscopy. The first RUs attached to the cores of S. flexneri 2a and 6 LPS were different from the following RUs in their O-acetylation and/or glucosylation. Conjugates of core plus more than 1 RU were necessary to induce LPS antibodies in mice. The resulting antibody levels were comparable to those induced by the full length O-SP conjugates. In S. dysenteriae type 1, the first RU was identical to the following RUs, with the exception that the GlcNAc was bound to the core in the β-configuration, while in all other RUs the GlcNAc was present in the α-configuration. In spite of this difference, conjugates of S. dysenteriae type 1 core with 1, 2, or 3 RUs induced LPS antibodies in mice with levels statistically higher than those of the full size O-SP conjugates. O-SPC conjugates are easy to prepare, characterize, and standardize, and their clinical evaluation is planned.     

Parasitoid Mark-Release-Recapture Techniques-- II. Development and Application of a Protein Marking Technique for Eretmocerus spp., Parasitoids of Bemisia argentifolii

In this study, we validate and apply techniques for marking and capturing small parasitoids of the silverleaf whitefly, Bemisia argentifolii Bellows & Perring [ = B. tabaci (Gennadius), strain B] for mark-release-recapture (MRR) studies. The marker is the purified protein, rabbit immunoglobulin G (IgG), which was applied externally by topical spray or internally by feeding. Marked parasitoids were then assayed using a sandwich enzyme-linked immunosorbent assay (ELISA) for the presence of the protein marker using an antibody specific to rabbit IgG. Virtually all of the externally marked Eretmocerus sp. (Ethiopia, M96076) (98.0%) contained enough rabbit IgG to be easily distinguished from unmarked parasitoids, regardless of the amount of protein applied or the post-marking interval. A field MRR study was then conducted to examine the dispersal characteristics of E. emiratus Zolnerowich & Rose. Parasitoids marked externally and internally with protein were released on three separate trial dates into the center of a cotton field bordered by cantaloupe and okra. Overall, a total of 1388, 637, and 397 marked and unmarked wasps were captured in suction traps during each trial, respectively with the majority of parasitoids captured between 0600 and 0800 h. Furthermore, even though we released an equal proportion of males to females, our traps consistently contained more males. Our results suggest that there are gender-specific differences in the dispersal behavior of E. emiratus . Almost 40% of the captured parasitoids collected during the three release trials were positively identified for the presence of the protein marker. The distribution of the marked parasitoids revealed two distinct patterns. First, almost all of the marked parasitoids recaptured in the cotton plot were in suction traps at or adjacent to the     

Short Communication: The effect of CYP1A1 induction on the formation of benzo a pyrene adducts in liver and lung DNA and plasma albumin in rats exposed to benzo a pyrene:adduct quantitation by immunoassay and an HPLC method

Induction of cytochrome P450 enzymes by exposure to polycyclic aromatic hydrocarbons (PAH) can result in both decreased or increased PAH adduct levels. The lung is a main target site for PAH-carcinogenesis. By HPLC determination of B[ a]P-r-7, t-8-dihydrodiol, t-9, 10-epoxide (BPDE-I)-DNA adducts in rat, the level of the ultimate carcinogenic B[a]P-metabolite was higher in lungs than in liver. However, measured by immunoassay, the total benzo[a]pyrene (B[a]P)-DNA adduct levels were higher in liver than in lungs. Induction of CYP1A1 in vivo in rat by repeated i.p. doses of methylcholanthrene (MC) prior to a single dose of B[a]P resulted in a 2.4 times increase in CYP1A1 activity in liver tissue and 1.5 times higher levelsof total B[a]P-DNA adducts in lung and liver compared with controls which only received B[a]P. Increased levels of BPDE-I-DNA adducts were significantly correlated to increased CYP1A1 activity in induced lung tissue but not in liver. The times to reach maximum adduct levels were similar for both controls and MC-induced rats in both lung and liver,and plasma albumin. The BPDE-I-albumin adducts reached a maximum level around 1 day after B[a]P exposure and could not be used as a reliable marker of the short term PAH exposure in this study.     

SDF-1 在乳腺癌患者血清中表达水平及临床意义

目的:分析SDF-1在乳腺癌患者外周血中的表达水平,探讨其临床意义。方法:Elisa法检测乳腺癌患者术前及术后血清SDF-1水平,分析其与乳腺癌临床病理特征的关系。结果:乳腺癌患者术前(69例)血清SDF-1水平明显高于正常对照组(20例)(6406.7±1302.5 pg/m L vs 5217.4±1225.7 pg/m L),有明显统计学差异(P<0.01),发生远处转移的乳腺癌患者(11例)血清SDF-1水平明显高于未发生转移者(58例)(7656.4±784.1 pg/m L vs 6169.7±1364.6 pg/m L),差异具有明显统计学意义(P<0.01)。ER及Her-2表达阳性乳腺癌的患者SDF-1水平较ER及Her-2表达阴性者低,差异亦有统计学意义(P<0.05)。结论:SDF-1可能是预测乳腺癌发生及远处转移的重要标志物。     

Bayesian analysis of serial dilution assays

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.     

Production by non-O1 Vibrio cholerae of hemolysin related to thermostable direct hemolysin of Vibrio parahaemolyticus

Abstract The production by non-O1 Vibrio cholerae of a hemolysin immunologically related to the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus was demonstrated by enzyme-linked immunosorbent assay and a double gel diffusion test. Although results by the double gel diffusion test suggested the immunological identities of TDH and the TDH-related hemolysin of non-O1 V. cholerae , conventional polyacrylamide gel disc electrophoresis and immunoelectrophoresis suggested some differences between the two, at least with respect to charge. The TDH-related hemolysin of non-O1 V. cholerae was also shown to differ from the hemolysin of non-O1 V. cholerae reported previously.     

A Species-specific Monoclonal Antibody System for Detecting the Remains of Field Slugs, Deroceras reticulatum (Muller) (Mollusca: Pulmonata), in Carabid Beetles (Coleoptera: Carabidae)

The field slug, Deroceras reticulatum (Muller), is probably the most damaging and widespread species of slug, causing severe economic losses in a broad range of crops in temperate zones throughout the world. Investigations into the role of predation in the population dynamics of this species required a biochemical system which was capable of identifying the remains of this slug in the crop contents of predators, and distinguishing them from those of other molluscs. A monoclonal antibody was developed (IgM isotype). This was capable of separating, in an enzyme-linked immunosorbent assay, D. reticulatum from the related D. caruanae (Pollonera) and all molluscs and other invertebrates tested, with the unexpected exceptions of New Zealand flatworms, Artioposthia triangulata (Deny) and the millipede Polymicrodon polydesmoides (Leach). Characterization of the antibody and assay demonstrated that the system could clearly detect as little as 11.6 ng of D. reticulatum protein in 200 mu l of buffer. Slug remains could be identified as such in the crops of the carabid predator Pterostichus melanarius Illiger for 38.1 h, while the antibody-antigen reaction declined to half of that measured immediately following consumption, after 12.9 h. A practical and highly sensitive system was therefore developed, using the first species-specific monoclonal antibody available for the investigation of predation on slugs.     

Large-size fattening calves’ lots fed with automatic milk feeders may have an increased risk for Mycoplasma bovis infection spread and for antibiotic use

Bovine respiratory disease is the leading user of antibiotics (AB) in calf production. Mycoplasma (M.) bovis could lead to greater use of AB as it is a persistent and AB resistant causative agent for respiratory diseases. Two cross-sectional studies were set up to assess the effects of lot size and feeding system on M. bovis infection and the effects of M. bovis seroconversion, lot size and feeding system on AB use in calves’ feedlots. Twenty-six lots in 22 fattening farms were monitored for 41–81 days, from all-in entry of calves until three consecutive weeks without using any collective antibiotics. M. bovis spread was estimated by measuring seroconversion at entry and at the end of study period in 10–15 calves randomly sampled in each lot. All AB treatments used in the meanwhile were recorded. The lots were selected according to feeding system, i.e. individual bucket (n = 7) vs. automated milk feeder (AMF, n = 19), and lot size (30–519 calves), less than 50 calves (n = 9) vs. more than 50 calves (n = 17). Statistical analysis was performed using multivariable generalised linear models with fattening farms as random effect. M. bovis spread increased with lot size (odds ratio (OR) 2.9[1.4; 5.8] per two-fold increase in lot size). This proportion of seroconverted calves was lower in bucket-fed lots compared to lots fed with the AMF using a shared nipple (OR = 0.03[0.003; 0.41]). The main risk factor for AB use was the lot size, with an increase of 1.5[0.94; 1.98] treatments per two-fold increase in lot size. For same size lots, the use of bucket can decrease AB consumption by up to 1.03[−2.18; 0.14] treatments per calf compared to AMF. Analysis of the association between seroconversion to M. bovis and AB use was inconclusive. We found that bucket feeding in small-size lots, i.e. up to a maximum of 50 calves in the same space, limits seroconversion to M. bovis and enables lower use of AB in veal calf production.     

Development of a monoclonal antibody for ELISA of CYP1A in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes

Induction of cytochrome P4501A CYP1A in cultured cells can be used to determine the induction potencies of xenobiotics or complex environmental samples. This report describes the development of an enzyme linked immunosorbent assay ELISA for measurement of CYP1A expression in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes. Juvenile rainbow trout were injected with naphthoflavone BNF 25 mg kg-1 body weight to induce the synthesis of CYP1A. The CYP1A isoenzyme was purified, characterized by immunological cross reactivity and N terminal sequencing and used to prepare a monoclonal antibody in Balb C mice. The specificity of the antibody for CYP1A was proved by Western blotting of samples from control and BNF injected fish. Two ELISA methods, a direct and a competitive one, were evaluated, with both methods being of comparable sensitivity. Rainbow trout hepatocytes, maintained as monolayers in serum free, chemically defined medium, were exposed to naphthoflavone, and the induction response was measured both by 7 ethoxyresorufin O deethylase EROD activity and the direct ELISA method. Comparison between EROD activity and immunodetectable CYP1A protein can provide information on the catalytic efficiency of CYP1A.