Elmer C. Bigley

Effect of processing on recovery and variability associated with immunochemical analytical methods for multiple allergens in a single matrix: dark chocolate.

Among the major food allergies, peanut, egg, and milk are the most common. The immunochemical detection of food allergens depends on various factors, such as the food matrix and processing method, which can affect allergen conformation and extractability. This study aimed to (1) develop matrix-specific incurred reference materials for allergen testing, (2) determine whether multiple allergens in the same model food can be simultaneously detected, and (3) establish the effect of processing on reference material stability and allergen detection. Defatted peanut flour, whole egg powder, and spray-dried milk were added to cookie dough at seven incurred levels before baking. Allergens were measured using five commercial enzyme-linked immunosorbent assay (ELISA) kits. All kits showed decreased recovery of all allergens after baking. Analytical coefficients of variation for most kits increased with baking time, but decreased with incurred allergen level. Thus, food processing negatively affects the recovery and variability of peanut, egg, and milk detection in a sugar cookie matrix when using immunochemical methods.

Immunophenotype and gene expression profile of mesenchymal stem cells derived from canine adipose tissue and bone marrow.

Veterinary adult stem cell therapy is an emerging area of basic and clinical research. Like their human counterparts, veterinary mesenchymal stem cells (MSCs) offer many potential therapeutic benefits. The characterization of canine-derived MSCs, however, is poorly defined compared to human MSCs. Furthermore, little consensus exists regarding the expression of canine MSC cell surface markers. To address this issue, this study investigated characteristics of cultured canine MSCs derived from both adipose tissue and bone marrow. The canine MSCs were obtained from donors of various breeds and ages. A panel of cell surface markers for canine MSCs was selected based on current human and canine literature and the availability of canine-reactive antibodies. Using flow cytometry, canine MSCs were defined to be CD90(+)CD44(+)MHC I(+)CD14(-)CD29(-)CD34(-)MHC II(-). Canine MSCs were further characterized using real-time RT-PCR as CD105(+)CD73(+)CD14(+)CD29(+)MHC II(+)CD45(-) at the mRNA level. Among these markers, canine MSCs differed from canine peripheral blood mononuclear cells (PBMCs) by the absence of CD45 expression at the mRNA level. A novel high-throughput canine-specific PCR array was developed and used to identify changes in the gene expression profiles of canine MSCs. Genes including PTPRC, TNF, β2M, TGFβ1, and PDGFRβ, were identified as unique to canine MSCs as compared to canine PBMCs. Our findings will facilitate characterization of canine MSCs for use in research and clinical trials. Moreover, the high-throughput PCR array is a novel tool for characterizing canine MSCs isolated from different tissues and potentially from different laboratories.

Identification of Murine B-Cell and T-Cell Epitopes of Escherichia coli Outer Membrane Protein F with Synthetic Polypeptides

ABSTRACT The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. BecauseEscherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on the assumption that differences in epitope specificity could influence protective or pathologic host reactions. Because of the high level of structural homology of OmpF to porins isolated from other enteric pathogens, the identification of T- and B-cell-stimulatory determinants of E. coli OmpF may have broader application.

Ecto-5′-Nucleotidase (CD73) Regulates Host Inflammatory Responses and Exacerbates Murine Salmonellosis

Food-borne Salmonella spp., are a major cause of hospitalization and death. Adenosine, an important immune regulator of inflammation, limits tissue damage during infection. CD39 (nucleoside triphosphate dephosphorylase) combined with ecto-5′-nucleotidase (CD73) metabolizes ATP to adenosine. We studied the expressions of CD39 and CD73 in tissues, and T helper cells in mice after Salmonella infection and evaluated the role of CD73 in regulating immune responses and bacterial clearance in wild-type and CD73-deficient (CD73−/−) mice. Both CD39 and CD73 transcript levels declined in the infected wild-type mice. Compared to wild-type mice, tissues from infected CD73−/− mice had significantly higher expression of pro-inflammatory cytokines and reduced anti-inflammatory responses. CD73−/− mice were more resistant to infection and had a greater inflammatory responses and a significantly lower bacterial load in the liver compared to wild-type mice. Thus, CD73 expression attenuates inflammation during murine Salmonellosis and impairs immunity, leading to increased bacterial colonization and prolonged infection.

Diet-induced obesity precipitates kidney dysfunction and alters inflammatory mediators in mice treated with Shiga Toxin 2

Shiga Toxin (Stx)-producing E. coli (STEC) continue to be a prominent cause of foodborne outbreaks of hemorrhagic colitis worldwide, and can result in life-threatening diseases, including hemolytic uremic syndrome (HUS), in susceptible individuals. Obesity-associated immune dysfunction has been shown to be a risk factor for infectious diseases, although few studies have addressed the role of obesity in foodborne diseases. We hypothesized that obesity may affect the development of HUS through an alteration of immune responses and kidney function. We combined diet-induced obese (DIO) and HUS mouse models to look for differences in disease outcome between DIO and wild-type (WT) male and female C57 B l/6 mice. Following multiple intraperitoneal injections with endotoxin-free saline or sublethal doses of purified Stx2, we examined DIO and WT mice for signs of HUS development. DIO mice receiving Stx2 injections lost more body weight, and had significantly higher (p < 0.001) BUN, serum creatinine, and neutrophil counts compared to WT mice or DIO mice receiving saline injections. Lymphocyte counts were significantly (p < 0.05) lower in Stx2-treated obese mice compared to WT mice or saline-treated DIO mice. In addition to increased Stx2-induced kidney dysfunction, DIO mouse kidneys also had significantly increased expression of IL-1α, IL-1β, IL-6, TNF-α, MCP-1, and KC RNA compared to saline controls (p < 0.05). Serum cytokine levels of IL-6 and KC were also significantly higher in Stx2-treated mice compared to saline controls, but there were no significant differences between the WT and DIO mice. WT and DIO mice treated with Stx2 exhibited significantly higher degrees of kidney tubular dilation and necrosis as well as some signs of tissue repair/regeneration, but did not appear to progress to the full pathology typically associated with human HUS. Although the combined obesity/HUS mouse model did not manifest into HUS symptoms and pathogenesis, these data demonstrate that obesity alters kidney function, inflammatory cells and cytokine production in response to Stx2, and may play a role in HUS severity in a susceptible model of infection.

Identification of an I-Ed-Restricted T-Cell Epitope of Escherichia coli Outer Membrane Protein F

ABSTRACT A predominant T-cell epitope of Escherichia coli outer membrane protein F (OmpF) that encompasses amino acids 295 to 314 was identified in H-2d mice. BALB/c-derived T-cell hybridomas generated against this region were CD3+, CD4+, CD8−, and T-cell receptor αβ+ and secreted TH-1-associated cytokines (interleukin-2 [IL-2] and gamma interferon), but not a TH-2-associated cytokine (IL-4), when restimulated with peptide 295-314. Class II+ mouse lymphoma (A20) cells, but not class II(−) mouse mastocytoma (P815) cells, supported IL-2 secretion of hybridomas when substituted for syngeneic splenocytes as antigen-presenting cells (APCs). Antibodies specific for I-Ed blocked IL-2 secretion by hybridomas, but I-Ad-specific antiserum did not. When transfected L cells expressing I-Ad (AαAβd), I-Ed (EαEβd), or the hybrid molecule I-EαAβd were used as APCs, hybridomas recognized peptide only when presented by the I-Ed-transfected cells. When peptide 295-314 truncated at either the C or the N terminus of the sequence was used, the minimal epitope was determined. Critical residues were determined by using alanine-substituted peptide analogues. T-cell hybridomas were only stimulated by peptides that encompassed amino acids 295 to 303 (9-mer), and the core sequence required a minimum of three additional amino acids at either the amino or the carboxy terminus to induce IL-2 secretion. Critical residues were determined to be phenylalanine at position 295, threonine at position 300, and tyrosines at positions 301 and 302. This study is the first to identify a minimal T-cell epitope and major histocompatibility complex restriction element of the OmpF protein and confirms previous observations that there is considerable degeneracy in the length of peptides that can bind I-Ed and variability in the amino acid composition of the C and N termini of these peptides.