Characterization of the native form of anthrax lethal factor for use in the toxin neutralization assay.

The cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

Live colonocytes in newborn stool: surrogates for evaluation of gut physiology and disease pathogenesis.

Studies of gastrointestinal pathophysiology are not feasible by biopsies in human neonates. We examined the utility of live colonocytes in stool in studying cellular markers during early neonatal life. Expression of IgA, IgG, cluster of differentiation-45 cells (CD45), and toll-like receptors-2 and 4 (TLR2 and TLR4) were analyzed by flow cytometry. Colonocyte RNA extracts were used in quantitative real-time PCR (qRT-PCR) to examine the expression of cytokeratin-19, ribosomal protein-24, and tight-junction (Tj) protein zonula occludens-1 (ZO-1). Colonocyte yield varied between 5 × 104 to 2 × 106 cells/g of stool. Meconium samples yielded a highly enriched population of viable cells. Although low, all samples showed CD45-positive cells during the initial weeks of life. Starting as early as d 2, IgA expression was observed in 69% of the cells. Low to moderate expression of IgG was observed with a linear increase as the infants grew. There was an almost total lack of TLR2 staining; however, >55% of the colonocytes showed TLR4 expression. Although high levels of IgA in gut cells may serve as a natural protectant during neonatal period, increased TLR4 may provide a niche for lipopolysaccharide (LPS)-mediated epithelial damage. Use of stool colonocytes can be a valuable noninvasive approach for studying gut pathophysiology in the neonatal period.

Markers of Inflammation and Lineage on Exfoliated Colonic Cells In Pediatric Inflammatory Bowel Disease.

OBJECTIVES: The diagnosis (endoscopy, and biopsy) and continued clinical management of Inflammatory Bowel Disease (IBD), remain highly invasive, expensive, and inconvenient for the pediatric patient. The objective of this study was to see if colonocytes obtained from stools of subjects with IBD and normal controls would demonstrate higher levels of inflammatory markers (Cox 2 in CD45+ and CD45- cells) and if the inflammatory process and treatment effects would be reflected in an altered cytokine expression in the subjects compared to controls. SETTING: Outpatient hospital based pediatric gastroenterology clinic. METHODS AND MAIN OUTCOME MEASURES: Stool samples (~ 1 gm), were obtained from 18 children between the ages of 4 and 18 diagnosed with IBD, and from a normal first degree relative. Colonocytes were isolated using the Somatic Cell Sampling Recovery (SCSR) system and assessed for the expression of COX-2, CD-45, IgA, IgG, IL6, IL18, TGF ß, TNF, and IL16ß using flow cytometry. In addition, levels of COX-2 and cytokeratin 19 transcripts were measured by microwell plate hybridization assay. RESULTS: Expression of COX-2 and co-expression of IgA and IgG were significantly higher in the IBD cases compared to the controls. In ulcerative colitis, the expression of COX-2 and co-expression of COX-2 and CD45 were greater than that in patients with Crohn's disease. In contrast, cells expressing IgA and IgG were higher in Crohn's. Subjects on immunosuppressants and/or anti-inflammatory medications, expressed significantly lower levels of COX-2 and IL-18 compared to those who were not on treatment. CONCLUSIONS: This study indicates that the use of disease markers on exfoliated colonic cells can be used for non-invasive assessment of disease status, for follow-up of response to treatment and for forecasting flare-up of disease before its symptomatic manifestations.

Exfoliated colonic epithelial cells: surrogate targets for evaluation of bioactive food components in cancer prevention.

There is significant evidence supporting the hypotheses that lifestyle, diet, and bioactive components in foods are important modifiers of cancer risk. However, our ability to assess host response noninvasively is limited. To overcome this, we have developed a technology to isolate several million viable exfoliated somatic colonic cells from a small sample of stool (0.5-1.0 g) by a procedure known as somatic cell sampling and recovery (SCSR). Orally administered carotenoids appear in these cells several days after consuming the supplement, usually showing a peak concentration between 5-7 d after their ingestion. The time lag observed for the appearance of orally administered carotenoids in SCSR cells corresponds to the turnover rate of the colonic mucosa. This is an example of how changes in cell turnover rates can be carefully assessed using the SCSR system. The specific mechanisms by which individual constituents of diet affect the cancer process are not fully understood. However, host response to dietary constituents may be investigated, noninvasively, by following the modulation of tumor-associated molecular markers in these exfoliated SCSR cells. We have demonstrated the feasibility of using SCSR cells to detect the expression of carcinoembryonic antigen, CD44, and its splice variants, c-myc, c-erbb2, and mutations in the p53 gene. In this regard, SCSR cells are a readily available surrogate cellular target that may serve to monitor changes in cell turnover, differentiation, and expression of cancer-associated biomarkers that are likely to be modulated by bioactive food components.