Effect of Proximity to a Cattle Feedlot on Escherichia coli O157:H7 Contamination of Leafy Greens and Evaluation of the Potential for Airborne Transmission

The impact of proximity to a beef cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens was examined. In each of 2 years, leafy greens were planted in nine plots located 60, 120, and 180 m from a cattle feedlot (3 plots at each distance). Leafy greens (270) and feedlot manure samples (100) were collected six different times from June to September in each year. Both E. coli O157:H7 and total E. coli bacteria were recovered from leafy greens at all plot distances. E. coli O157:H7 was recovered from 3.5% of leafy green samples per plot at 60 m, which was higher (P < 0.05) than the 1.8% of positive samples per plot at 180 m, indicating a decrease in contamination as distance from the feedlot was increased. Although E. coli O157:H7 was not recovered from air samples at any distance, total E. coli was recovered from air samples at the feedlot edge and all plot distances, indicating that airborne transport of the pathogen can occur. Results suggest that risk for airborne transport of E. coli O157:H7 from cattle production is increased when cattle pen surfaces are very dry and when this situation is combined with cattle management or cattle behaviors that generate airborne dust. Current leafy green field distance guidelines of 120 m (400 feet) may not be adequate to limit the transmission of E. coli O157:H7 to produce crops planted near concentrated animal feeding operations. Additional research is needed to determine safe set-back distances between cattle feedlots and crop production that will reduce fresh produce contamination.     

Proposed standard nomenclature for the alpha- and beta-globin gene families

The globin family of genes and proteins has been a recurrent object of study for many decades. This interest has generated a vast amount of knowledge. However it has also created an inconsistent and confusing nomenclature, due to the lack of a systematic approach to naming genes and failure to reflect the phylogenetic relationships among genes of the gene family. To alleviate the problems with the existing system, here we propose a standardized nomenclature for the alpha and beta globin family of genes, based on a phylogenetic analysis of vertebrate alpha and beta globins, and following the Guidelines for Human Gene Nomenclature.     

Involvement of Lysophosphatidic Acid,Sphingosine 1-Phosphate and Ceramide 1-Phosphate in the Metabolization of Phosphatidic Acid by Lipid Phosphate Phosphatases in Bovine Rod Outer Segments

The aim of the present research was to evaluate the generation of [2-3H]diacylglycerol ([2-3H]DAG) from [2-3H]-Phosphatidic acid ([2-3H]PA) by lipid phosphate phosphatases (LPPs) at different concentrations of lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (C1P) in purified ROS obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. Western blot analysis revealed the presence of LPP3 exclusively in all membrane preparations. Immunoblots of entire ROS and depleted ROS did not show dark-light differences in LPP3 levels. LPPs activities were diminished by 53% in BLROS with respect to DROS. The major competitive effect on PA hydrolysis was exerted by LPA and S1P in DROS and by C1P in BLROS. LPPs activities in depleted ROS were similar to the activity observed in entire DROS and BLROS, respectively. LPA, S1P and C1P competed at different extent in depleted DROS and BLROS. Sphingosine and ceramide inhibited LPPs activities in entire and depleted DROS. Ceramide also inhibited LPPs activities in entire and in depleted BLROS. Our findings are indicative of a different degree of competition between PA and LPA, S1P and C1P by LPPs depending on the illumination state of the retina.     

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W., Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525–532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, V.I., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66, 331–341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1' in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high β sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae.     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

Cytokine mRNA expression in unilateral ischemic-reperfused rat lung with salt solution supplemented with low-endotoxin or standard bovine serum albumin

Our aim was to determine whether cytokine mRNA expression is induced by experimental manipulation including artificial perfusate or ischemia-reperfusion (I/R) in an isolated, perfused rat lung model. Constant pulmonary flow [Krebs-Henseleit solution supplemented with low-endotoxin (LE) or standard (ST) bovine serum albumin 4%, 0.04 ml/g body wt] and ventilation were maintained throughout. Right and left pulmonary arteries were isolated, and the left pulmonary artery was occluded for 60 min and then reperfused for 30 min. Analysis of tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-10, and IFN-gamma mRNA expression by RT-PCR and evaluation of vascular permeability by bronchoalveolar lavage (BAL) fluid albumin content were conducted separately in right and left lung. Both LE and ST groups (each 12 rats) showed increases in vascular permeability by I/R (BAL fluid albumin content: 5.53 +/- 1.55 vs. 15.63 +/- 8.87 and 4.76 +/- 2.71 vs. 16.72 +/- 4.85 mg.ml BAL fluid-1.g lung dry wt-1, mean +/- SD; right vs. left lung in LE and ST groups, P < 0.05 between right and left). Cytokine mRNA expression was significantly higher in the I/R lung than in the control lung in the LE group, whereas it was higher in the control lung in the ST group (P < 0.05). mRNAs of not only proinflammatory but also anti-inflammatory cytokines were expressed in I/R lung, which are expected to aggravate I/R injury. The reversed pattern of cytokine mRNA expression in the ST group was possibly due to the longer perfusion of control lung with perfusate containing endotoxin, which caused no lung damage without I/R.     

The Diamine Oxidase Gene Is Associated with Hypersensitivity Response to Non-Steroidal Anti-Inflammatory Drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) are the drugs most frequently involved in hypersensitivity drug reactions. Histamine is released in the allergic response to NSAIDs and is responsible for some of the clinical symptoms. The aim of this study is to analyze clinical association of functional polymorphisms in the genes coding for enzymes involved in histamine homeostasis with hypersensitivity response to NSAIDs. We studied a cohort of 442 unrelated Caucasian patients with hypersensitivity to NSAIDs. Patients who experienced three or more episodes with two or more different NSAIDs were included. If this requirement was not met diagnosis was established by challenge. A total of 414 healthy unrelated controls ethnically matched with patients and from the same geographic area were recruited. Analyses of the SNPs rs17740607, rs2073440, rs1801105, rs2052129, rs10156191, rs1049742 and rs1049793 in the HDC, HNMT and DAO genes were carried out by means of TaqMan assays. The detrimental DAO 16 Met allele (rs10156191), which causes decreased metabolic capacity, is overrepresented among patients with crossed-hypersensitivity to NSAIDs with an OR  = 1.7 (95% CI  = 1.3–2.1; Pc  = 0.0003) with a gene-dose effect (P = 0.0001). The association was replicated in two populations from different geographic areas (Pc  = 0.008 and Pc  = 0.004, respectively).

Conclusions and implications

The DAO polymorphism rs10156191 which causes impaired metabolism of circulating histamine is associated with the clinical response in crossed-hypersensitivity to NSAIDs and could be used as a biomarker of response.     

Toll-like receptor 4 gene polymorphism influences dendritic cell in vitro function and clinical outcomes in vaccinated melanoma patients

Toll-like receptor 4 (TLR4) is expressed on dendritic cells (DCs), sensing environmental danger molecules that induce their activation and maturation. Recently, we reported a method for the production of therapeutic DCs against melanoma, called tumor antigen-presenting cells (TAPCells), using a heat-shocked allogeneic melanoma cell lysate (TRIMEL) as an activation factor and antigen provider. Since TRIMEL contains endogenous TLR4 ligands, we evaluated the role of TLR4 in TAPCells differentiation by antibody neutralization and the association of a Tlr4 polymorphism (896A/G) (Asp299Gly), determined by PCR–RFLP, with the in vitro activation capacity and the clinical outcome of TAPCells-vaccinated patients. Antibody blocking of monocyte TLR4 inhibited surface expression, determined by flow cytometry, of the major histocompatibility complex class I, CCR7, CD80, CD83 and CD86 on TAPCells, reduced interleukin (IL)-6 and tumor necrosis factor -α gene expression evaluated by qRT-PCR, and also inhibited the TAPCells-mediated interferon-γ (IFN-γ) secretion of melanoma-specific CD8⁺ T cells determined by ELISpot (p?<?0.01). Moreover, CD8⁺ T-cell activation capacity was significantly reduced in TAPCells bearing the TLR4 Asp299Gly receptor (p?<?0.05). Finally, TAPCells-vaccinated stage-IV melanoma patients bearing the Tlr4 896G allele showed a shortened post-therapy median survival rate compared with those carrying the Tlr4 896A allele (p?<?0.05; log-rank test). Our results indicate that TLR4 is a key receptor for the tumor lysate-mediated in vitro generation of clinically efficient antigen-presenting cells. Further analysis of patients included in different vaccine protocols is necessary for definitively establishing a role for TLR4 polymorphism in clinical responses.