Evaluation of the systemic innate immune response and metabolic alterations of nonlactating cows with diet-induced subacute ruminal acidosis.

Subacute ruminal acidosis (SARA) increases lipopolysaccharide endotoxin in the rumen, which might translocate into the systemic circulation, triggering a cascade of clinical and immunological alterations. The objective of this study was to characterize the clinical immune and metabolic responses to ruminal-derived lipopolysaccharide in nonlactating cows induced with SARA using 2 challenges, a grain-based SARA challenge (GBSC) or an alfalfa-pellet SARA challenge (APSC). Six dry, nonlactating Holstein cows were used in a 3 × 3 Latin square arrangement of treatments with 4-wk experimental cycles. All cows received the control diet containing 70% forage and 30% mixed concentrates (dry matter basis) for 3 wk. In wk 4, cows received a control diet, GBSC (38% wheat-barley pellets, 32% other mixed concentrate, and 30% forages), or APSC (45% mixed concentrate, 32% alfalfa pellets, and 23% other forages). Total plasma proteins and immunology-related proteins, acute phase proteins, blood cells, serum chemistry, mRNA gene expression of peripheral blood cell surface markers, and selected proinflammatory cytokines were evaluated. Ruminal pH was lower in both groups with induced SARA compared with a control group. Ruminal endotoxins were higher in GBSC; however, plasma endotoxin was not detected in any study group. No significant differences in feed intake, rectal temperature, white blood cell counts, or differentials were found between control and SARA challenge groups; changes in glucose, urea, Ca, and Mg were observed in SARA groups. Total plasma proteins were lower in both SARA groups, and acute phase proteins were higher in GBSC. The expression of CD14, MD2, and TLR4 mRNA in peripheral blood leukocytes was not affected by SARA induction. The induction of SARA as a result of GBSC or APSC challenge was successful; however, LPS was not detected in plasma. Changes in clinical, metabolic, and inflammatory responses were not observed in the SARA-challenged cows, suggesting that, in this study, SARA was not associated with a systemic response to inflammation.

Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers.

Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)(3)KC to the end of PMOs. In strains with an intact outer membrane, (KFF)(3)KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)(3)KC-PMO conjugate targeted to lacI mRNA induced beta-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)(3)KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce beta-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)(3)KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets.