Transcriptional profiling of the L. monocytogenes PrfA regulon identifies six novel putative PrfA-regulated genes.

The transcriptional activator Positive Regulatory Factor A (PrfA) regulates expression of genes essential for virulence in Listeria monocytogenes. To define the PrfA regulon, the 10403S wildtype (WT) strain, a constitutively active prfA* mutant, and an isogenic ∆prfA mutant were grown under PrfA-inducing conditions in a medium containing glucose-1-phosphate and pre-treated with 0.2% activated charcoal. RNA-seq-generated transcript levels were compared as follows: (i) prfA* and WT; (ii) WT and ∆prfA and (iii) prfA* and ∆prfA. Significantly higher transcript levels in the induced WT or constitutively active PrfA* were identified for 18 genes and 2 ncRNAs in at least one of the three comparisons. These genes included: (i) 10/12 of the genes previously identified as directly PrfA-regulated; (ii) 2 genes previously identified as PrfA-regulated, albeit likely indirectly; and (iii) 6 genes newly identified as PrfA-regulated, including one (LMRG_0 2046) with a σA-dependent promoter and PrfA box located within an upstream open reading frame. LMRG_0 2046, which encodes a putative cyanate permease, is reported to be downregulated by a σB-dependent anti-sense RNA. This newly identified overlap between the σB and PrfA regulons highlights the complexity of regulatory networks important for fine-tuning bacterial gene expression in response to the rapidly changing environmental conditions associated with infection.

Nevertheless, She Resisted - Role of the Environment on Listeria monocytogenes Sensitivity to Nisin Treatment in a Laboratory Cheese Model.

The growth of Listeria monocytogenes on refrigerated, ready-to-eat food products is a major health and economic concern. The natural antimicrobial nisin targets the bacterial cell wall and can be used to inhibit L. monocytogenes growth on cheese. Cell wall composition and structure, and therefore the efficacy of cell wall acting control strategies, can be severely affected by environmental and stress conditions. The goal of this study was to determine the effect of a range of pH and temperatures on the efficacy of nisin against several strains of L. monocytogenes in a lab-scale, cheese model. Cheese was made with or without the addition of nisin at different pH and then inoculated with L. monocytogenes; L. monocytogenes numbers were quantified after 1, 7, and 14 days of incubation at 6, 14, or 22°C. While our data show that nisin treatment is able to reduce L. monocytogenes numbers, at least initially, growth of this pathogen can occur even in the presence of nisin, especially when cheese is stored at higher temperatures. Several environmental factors were found to affect nisin efficacy against L. monocytogenes. For example, nisin is more effective when cheese is stored at lower temperatures. Nisin is also more effective when cheese is made at higher pH (6 and 6.5), compared to cheese made at pH 5.5, and this effect is at least partially due to the activity of cell envelope modification genes dltA and mprF. Serotype was also found to affect nisin efficacy against L. monocytogenes; serotype 4b strains showed lower susceptibility to nisin treatment compared to serotype 1/2 strains. Overall, our results highlight the importance of considering environmental conditions specific to a food matrix when developing and applying nisin-based intervention strategies against L. monocytogenes.

Environmental conditions and serotype affect Listeria monocytogenes susceptibility to phage treatment in a laboratory cheese model.

Listeria monocytogenes can survive and grow in a variety of environments, including refrigeration, making it difficult to control and highlighting the importance of optimizing control strategies against this pathogen. Listeria phages are attractive biocontrol agents because phages bind to specific wall teichoic acids (WTA) on the bacterial cell wall, inhibiting pathogens without disrupting the normal microbiota or structure of the food. Common stresses found on dairy products can affect cell wall composition and structure and subsequently affect the efficiency of control strategies that target the cell wall. The goal of this study was to determine the effect of a range of pH and temperatures on the effectiveness of a commercial phage cocktail treatment against several strains of L. monocytogenes in a cheese matrix. We developed a laboratory-scale cheese model that was made at different pH, treated with phage, and then inoculated with L. monocytogenes. Cheeses were incubated at 6, 14, or 22°C for 14 d, and bacterial counts were determined on d 1, 7, and 14. Our data show that phage treatment has a limited ability to reduce L. monocytogenes counts at each temperature tested; however, it was more effective on specific strains of L. monocytogenes when cheese was stored at higher temperatures. More specifically, the average counts of L. monocytogenes on phage-treated cheese stored at 22°C were significantly lower than those on phage-treated cheese stored at 6 or 14°C. Similarly, phage treatment was significantly more effective at inhibiting L. monocytogenes on cheese made at higher pH (6 and 6.5) compared with counts on cheese made at pH 5.5, where L. monocytogenes did not grow. Furthermore, serotype was found to affect the susceptibility of L. monocytogenes to phage treatment; serotype 1/2 strains showed significantly higher susceptibility to phage treatment than serotype 4b strains. Overall, our results suggest the importance of considering the efficacy of phage under conditions (i.e., temperature and pH) specific to a given food matrix when applying interventions against this important foodborne pathogen.

Serum immunoglobulins and lymphocyte subset distributions in children and adults living in communities assessed for lead and cadmium exposure.

This study assessed the impact of environmental cadmium and lead exposure on the immune system of more than 2000 children and adults. Serum immunoglobulins [immunoglobulins (Ig) A, G, and M] and peripheral blood lymphocyte phenotypes (T cells, B cells, NK cells, and CD4/CD8 subsets) were measured in a total of 2041 children and adults who lived either in sites with elevated soil levels of cadmium and lead (n = 1561) or in comparison communities (n = 480). The blood lead and urine cadmium levels of participants were somewhat higher than national averages. Mean blood lead levels were 7 microg/dl for participants aged 6-35 mo; 6 microg/dl for participants aged 36-71 mo, 4 microg/dl for participants aged 6-15 yr; and 4.3 microg/dl for participants aged 16-75 yr. Multivariate analysis indicated no marked differences in any of the immune marker distributions attributed to lead for adults or children over 3 yr of age. However, in children under age 3, increased blood lead levels, principally those over 15 microg/dl, were associated with increases in IgA, IgG, IgM, and circulating B lymphocytes. Among adults, urine cadmium levels over 1.5 microg/g were associated with higher levels of IgA and circulating B lymphocytes. No evidence of immunosuppression was noted. The findings of potential immunologic effects at lead levels > 15 microg/dl in young children and at urine cadmium levels > 1.5 microg/g in adults are interesting, but too few participants had these high levels to delineate a threshold. Therefore, we find these results intriguing, but requiring confirmation in populations with higher exposure levels.

Fluorescence intensity calibration for immunophenotyping by flow cytometry.

Fluorescence intensity (FI) is the basis for classifying phenotypes by fluorescence-label flow cytometry. FI is customarily recorded as an arbitrary relative value, but with proper calibration it can be expressed in stoichiometric units called molecules of equivalent soluble fluorochrome (MESF) that reflect the concentrations of the fluorescent conjugates and the receptors they stain. Forthcoming availability of authoritative standards and consensus methods will alleviate many of the difficulties encountered in making valid MESF measurements. FI calibration establishes the true values for the critical parameters of the fluorescence measurement, a useful feature for quality control. It further allows the establishment of a comparable window of analysis across different times and laboratories, and it permits numeric assessment of antibody-binding capacity (ABC) values in selected cell populations. The relation between ABC values and receptor expression is complicated by several factors, but careful assessment of the binding chemistry can establish the actual number of receptors on cells stained by fluorescent conjugates.

Glutamic acid decarboxylase and IA-2 autoantibodies in type 1 diabetes: comparing sample substrates for autoantibody determinations.

Large-scale programs designed to assess risk for type 1 diabetes through serologic assessment of autoantibodies to recombinant beta-cell autoantigens are hampered by several limitations, including the methods for sample collection and assay performance, as well as the volume required for autoantibody determinations. The present study was designed to develop a low sample-volume, primary screening method for autoantibody detection of high specificity and sensitivity, and to determine the feasibility of dried blood spots collected on filter paper in serving as vehicles for such determinations. Autoantibodies to glutamic acid decarboxylase (GAD) and ICA512bdc (IA-2), both individually and in combination, were determined in persons with type 1 diabetes, healthy controls, or individuals with other autoimmune disorders. Autoantibody results for serum, plasma, and dried blood spots were compared. GAD, IA-2, and combined GAD/IA-2 autoantibodies were concordant in their measurement from minimal volumes of serum, plasma, and whole blood extracted from dried filter paper. The autoantibody levels from the dried blood spots were, however, lower than corresponding serum samples, and, as currently designed, failed to detect low-titer autoantibodies. Despite this limitation, screening for diabetes risk can be performed using small volumes of whole blood, serum, or plasma collected onto filter paper. These methodological improvements should simplify matters, reduce costs, and increase the efficacy of screening programs for type 1 diabetes. Further development of better substrates/methods for blood-specimen collection seems necessary to exploit the full potential of this and other autoantibody measurement strategies for screening large populations.

Current activities at the Centers for Disease Control and Prevention's National Diabetes Laboratory.

In 1997, the Centers for Disease Control and Prevention established the National Diabetes Laboratory in order to help prevent and treat type 1 diabetes. This state-of-the-art laboratory collaborates with research scientists and key national and international organizations throughout the world to identify and study risk factors for type 1 diabetes by developing measurements for glycosylated proteins, developing and evaluating technology for measuring genetic risk factors for the disease, and working to standardize autoantibody measurements. Developing improved technologies for diagnosing and managing diabetes and developing reference materials for properly calibrating and standardizing blood glucose meters are also critical aspects of the laboratory's work. In addition, the laboratory provides quality storage for valuable collections of biologics and other materials and facilitates sharing of specimens, associated epidemiologic data, and test results. Working with our partners in diabetes research, we are improving the diagnosis, treatment, and prevention of type 1 diabetes.

Terminology and nomenclature for standardization in quantitative fluorescence cytometry.

Terminology in any field is a complex mix of established conventions, accepted usages, disputed terms, and occasional misnomers. The terminology that has evolved for quantitative fluorescence cytometry (QFCM) is especially multifarious, in part because QFCM encompasses a range from subjective visual assessments to objective photon counts. Thus, while descriptive terms such as "dim" and "bright" are still quite useful, quantitative terms such as "binding capacity" should be used with collective understanding of their exact meanings. This article reviews current usage and proposes definitions that, with refinement from suppliers and users of QFCM technology, can provide the required clarity.

Titration of a CD45-FITC conjugate to determine the linearity and dynamic range of fluorescence intensity measurements on lymphocytes.

To produce biologic calibrators for relative fluorescence intensity (RFI) measurements, we stained leukocytes with serial dilutions of CD45-FITC conjugate and processed them using our regular whole blood lysis procedure. Cells were stained with conjugate concentrations ranging from twice recommended to a million-fold lower. At the highest concentrations of conjugate, the RFI reached a plateau near the top of the third decade, indicating saturation of CD45 binding sites. As the concentration decreased, the RFI declined in a highly linear relationship between the dilution factor and the histogram channel number. For channel numbers corresponding to the lowest percentiles of the RFI distribution, linearity persisted down to the first half decade. The slope of this relationship revealed a true dynamic range of 4.5 decades, which was comparable to the value obtained with microbead standards calibrated in molecules of equivalent soluble fluorochrome (MESF). Our results suggest that the lower limit of linearity for fluorescence intensity from fluorescein isothiocyanate (FITC)-stained lymphocytes is below 500 MESF and that cellular autofluorescence is the major limiting factor in detecting and quantifying FITC-specific staining. This procedure provides an adroit way of characterizing the linearity and dynamic range of measurements for quantitative fluorescence cytometry using exactly the same matrix, stains, and preparation methods as those used for cellular analytes.

An evaluation of the use of dried blood spots from newborn screening for monitoring the prevalence of cocaine use among childbearing women.

A collaborative March of Dimes study was designed to examine the utility of dried blood spot (DBS) materials routinely collected from newborns as a source for monitoring cocaine exposure and to assess the prevalence of cocaine use among childbearing women in Georgia. We used a modified urinary radioimmunoassay (RIA) to anonymously detect the cocaine metabolite benzoylecgonine (BE) in DBSs. Extensive efforts were undertaken to assure absolute nonlinkage of BE data to any individual. The positive results found by RIA were confirmed by a mass spectrometry (MS) method specifically developed to detect BE in DBSs. BE was measured in 23,141 DBSs collected during 2 months of routine newborn screening in Georgia. A good correlation was observed for RIA results versus MS results (r2 = 0.97). The estimated minimal statewide BE prevalence was 4.8 per 1000 childbearing women. We demonstrated that immunoassay testing for cocaine without confirmatory testing can yield falsely elevated prevalence rates. When proper confirmatory testing is done, DBSs are a valuable source for population-based monitoring of substance abuse among childbearing women.

Isotope dilution--mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I.

An enzymatic hydrolysis isotope dilution-mass spectrometric method was developed for reference quantification of specific proteins. The analytical procedure involved measuring a reproducibly hydrolyzed peptide (serving as the primary standard) unique to a specific protein. This new mass spectrometric method was evaluated by assessing the concentration of apolipoprotein (apo) A-I in the European Community Bureau of Reference (BCR) lyophilized Certified Reference Material (CRM 393). We used the method to make 96 measurements (4 replicate analyses of 4 enzymatic digests of 6 vials of BCR-CRM 393), which gave an average total protein mass of 1.048 mg (+/- 1.0% at 99% confidence limits). The total overall analytical CV was 3.95%. The results of this evaluation of our model approach to determine the concentration of a specific protein in a purified preparation demonstrated that our new mass spectrometric method can be used to measure apolipoproteins and other specific proteins without the use of epitopic immunoassay methods.

Analysis of benzoylecgonine in dried blood spots by liquid chromatography--atmospheric pressure chemical ionization tandem mass spectrometry.

Residual samples from blood spots (i.e., whole blood spotted onto filter paper) are a useful source for epidemiological screening studies involving newborns. However, the small volume of blood available from residual blood spots complicates the assay. A method for analyzing benzoylecgonine (BZE; the primary metabolite of cocaine) in blood spots, in which the blood spot is eluted with aqueous ammonium acetate-methanol containing N-methyl trideuterated-BZE as an internal standard, followed by high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry using multiple reaction monitoring, has been developed. This approach provides a rapid, direct, sensitive (limit of detection, approximately 2 ng/mL, based on a 12-microL sample size), and highly specific means of determining BZE concentrations in blood spots. We have applied this method for confirmatory analyses in a large epidemiological study of the prevalence of cocaine use during late pregnancy.

Two-dimensional protein electrophoresis and multiple hypothesis testing to detect potential serum protein biomarkers in children with fetal alcohol syndrome.

Fetal alcohol syndrome (FAS) surveillance and intervention efforts are hampered by the lack of a specific biochemical test for diagnosis of the syndrome. Based on the hypothesis that abnormalities in growth and development (key features of FAS) involve altered protein metabolism, we analyzed serum proteins by two-dimensional gel electrophoresis and image analysis to search for potential protein biomarkers of FAS. Serum samples from 12 participants in whom FAS had been diagnosed and 8 sex- and age-matched participants whose mothers did not consume alcohol were analyzed in duplicate to determine whether the integrated intensities of matched proteins are significantly altered in children with FAS. Multiple hypothesis testing on 34 of the gels consisting of more than 1700 spots per gel revealed 21 proteins that we classified as potential protein biomarkers of FAS on the basis of significant t-test differences at p < 0.02. We classified 8 of the proteins as candidate biomarkers on the basis of significant concentration differences between case and control subjects at p < 0.01. One of the proteins is clearly an isoform of retinol binding protein; two appear in the area of the gel where alcohol dehydrogenase is expected to appear; one appears to be an isoform of alpha-1-antitrypsin; three appear to be isoforms of the beta-chain of haptoglobin; three may be forms of immunoglobulin light chains; and several others have not been associated with known proteins. No single protein differentiated all case subjects from control subjects, but stepwise canonical discriminant analyses revealed four groups of spots that distinguished between FAS case and control subjects with no misclassifications.(ABSTRACT TRUNCATED AT 250 WORDS)

Method-dependent variations in the stability of apolipoprotein B in a stabilized liquid reference material.

Using accelerated Arrhenius-type short-term and long-term temporal studies, we evaluated the storage life of a stabilized, liquid-frozen reference material (SLRM) for human apolipoprotein B (apo B) developed by the International Federation of Clinical Chemistry. As measured by our candidate reference RIA, the concentrations of immunoreactive apo B in the SLRM showed pronounced degradation with exposure to increasing temperatures over time. The SLRM was stable for as long as 1 year when stored at - 70 degrees C, but its immunoreactive apo B declined by < 10% when stored at 4 degrees C for 10 months. Using radial immunodiffusion and an ELISA to assess the equivalency of measured mass for the accelerated thermal stability of the SLRM, we found a loss of immunoreactive apo B similar to that measured by RIA. Analyzing the same samples by liquid immunoprecipitation (nephelometry) resulted in the amount of apo B present being overestimated, especially in samples held for long periods. By using different immunological methods to evaluate this thermally aged SLRM, we demonstrated that its measured behavior varies depending on the method of quantitation.

International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B. IV. Comparability of apolipoprotein B values by use of International Reference Material.

We performed temporal and thermal stability studies on SP3-07, a liquid-stabilized reference material for apolipoprotein (apo) B, selected during the previous phase of the International Federation of Clinical Chemistry project on standardization of apolipoprotein measurements. Results indicate that SP3-07 stored at -70 degrees C has the long-term stability required for a reference material. We assigned an accuracy-based apo B value of 1.22 g/L to SP3-07, using a nephelometric method that was calibrated with freshly isolated low-density lipoprotein for which the apo B mass value was determined by a standardized sodium dodecyl sulfate-Lowry procedure. Using a common protocol, the study participants transferred the assigned mass value from SP3-07 to the individual calibrators of the analytical systems and measured the apo B concentration of 20 fresh-frozen samples obtained from individual donors and covering a clinically relevant range of apo B values. The among-laboratory CV on these samples, analyzed by 25 analytical systems, ranged from 3.1% to 6.7%. These results demonstrate the lack of matrix effects of SP3-07 and its ability to provide accurate and comparable apo B values in a variety of immunochemical methods. On the basis of the outcome of these studies, the World Health Organization has endorsed SP3-07 as the International Reference Material for Apolipoprotein B.

International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B. III. Comparability of apolipoprotein A-I values by use of international reference material.

In the third phase of the International Federation of Clinical Chemistry (IFCC) study for the standardization of apolipoprotein (apo) measurements, the preparation SP1-01, selected as the candidate international reference material for apo A-I, was investigated for its ability to transfer an accuracy-based value to the immunoassay calibrators and to produce comparability of the values for patients' samples. An apo A-I value of 1.50 g/L (SD 0.08 g/L) was assigned to SP1-01 by a highly standardized RIA calibrated with purified apo A-I for which the mass value had been determined by amino acid analysis. According to a common detailed protocol, the participants transferred the mass value from SP1-01 to the calibrator of each method. To confirm that uniformity of calibration ensures comparability of the values over a wide range of apo A-I values, each laboratory analyzed 50 fresh-frozen samples from individual donors, using an approach similar to that adopted by the Cholesterol Reference Laboratory Network. The consensus mean value for each sample was in excellent agreement with the value assigned by the Northwest Lipid Research Laboratories, with the average absolute bias between assigned and consensus value being 0.01 g/L. The among-laboratory CV on each of the 50 samples ranged from 2.1% to 5.6% (mean 3.6%), demonstrating that comparable apo A-I results can be obtained by a variety of immunochemical methods through the use of certified reference material. Based on the results obtained in these studies, SP1-01 has been approved as Apolipoprotein A-I International Reference Material by the World Health Organization.

Radioimmunoassay screening of dried blood spot materials for benzoylecgonine.

Residual samples of blood spots, which are routinely collected on almost all newborns in the United States, can be used to determine seroprevalence information on newborns and maternal exposures to various substances, including drugs of abuse. By modifying a commercial radioimmunoassay (RIA) kit for urinary samples, one can use blood spotted on filter paper as a matrix to quantitate the cocaine metabolite benzoylecgonine (BE). BE is stable for long periods of time in blood spots and we were able to quantitatively extract it with aqueous buffer. There were no matrix effects of the blood spot eluate on the RIA, and excess lipid in the blood did not alter measurement of BE. By using standards made up of BE in negative blood spot eluate and calibrators of blood that were spiked with BE and then spotted on filter paper to determine extraction efficiency, low levels of BE in blood could be measured. The limit of detection was 5 ng/mL, and the limit of quantitation was 10 ng/mL. Levels of BE in blood collected at autopsy in eluates of blood spots were measured, and they established excellent correlation (r2 = 0.93) with gas chromatography/mass spectrometry measurements. To test this technology, residual blood spots on 545 infants from three states were analyzed for BE.