Poly-N-Acetyl-β-(1-6)-Glucosamine Is a Target for Protective Immunity against Acinetobacter baumannii Infections

ABSTRACTAcinetobacter baumanniihas emerged as a highly troublesome, global pathogen. Treatment is complicated by high levels of antibiotic resistance, necessitating alternative means to prevent or treatA. baumanniiinfections. We evaluated an immunotherapeutic approach againstA. baumannii, focusing on the surface polysaccharide poly-N-acetyl-β-(1-6)-glucosamine (PNAG). We used a synthetic oligosaccharide of 9 monosaccharide units (9Glc-NH2) conjugated to tetanus toxoid (TT) to induce antibodies in rabbits. In the presence of complement and polymorphonuclear cells, antisera to 9Glc-NH2-TT mediated the killing ofA. baumanniiS1, a high-PNAG-producing strain, but not its isogenic PNAG-negative, in-frame deletion mutant strain, S1 Δpga. Complementing thepgaABCDlocus intransin the shuttle vector pBAD18kan-ori, plasmid Δpga-c, restored the high levels of killing mediated by antibody to PNAG observed with the wild-type S1 strain. No killing was observed when normal rabbit serum (NRS) or heat-inactivated complement was used. Antiserum to 9Glc-NH2-TT was highly opsonic against an additional four unrelated multidrug-resistant clinical isolates ofA. baumanniithat synthesize various levels of surface PNAG. Using two clinically relevant models ofA. baumanniiinfection in mice, pneumonia and bacteremia, antisera to 9Glc-NH2-TT significantly reduced levels ofA. baumanniiin the lungs or blood 2 and 24 h postinfection, respectively, compared to levels of control groups receiving NRS. This was true for all fourA. baumanniistrains tested. Overall, these results highlight the potential of PNAG as a vaccine component for active immunization or as a target for passive antibody immunotherapy.

Enterococcal surface protein contributes to persistence in the host but is not a target of opsonic and protective antibodies in Enterococcus faecium infection

Enterococci are important nosocomial pathogens with multiple intrinsic and acquired resistances to antibiotics. In the past, the majority of infections were caused by Enterococcus faecalis; however, an increase in Enterococcus faecium clinical isolates has been observed in recent years. The enterococcal surface protein (Esp) is expressed on the surface of most E. faecium clinical isolates and has been shown to be involved in biofilm formation. Here, E. faecium E1162 and its previously created insertion-deletion mutant of the esp gene, E. faecium E1162Δesp, were compared in a mouse bacteraemia model. Anti-Esp serum was tested for its capacity to mediate opsonophagocytic killing of E1162 in vitro and to protect against E. faecium bacteraemia. The inactivation of esp attenuated E. faecium virulence with reduced numbers of bacteria recovered from the kidneys in animals infected with the mutant compared to the wild-type strain (P=0.035). Passive immunization with rabbit polyclonal serum raised against the recombinant N-terminal Esp protein did not protect mice against E. faecium bacteraemia (P>0.05). In contrast, mice passively immunized with polyclonal antiserum raised against lipoteichoic acid (LTA) from E. faecalis had lower numbers of E. faecium E1162 in the blood compared to mice immunized with normal rabbit serum. These results suggest that Esp contributes to E. faecium persistence in the host. However, in contrast to LTA, Esp does not seem to be a target for protective antibodies in E. faecium strain E1162 in mouse bacteraemia.

295POSSIBLE DOPAMINERGIC MECHANISM REGULATING THE EQUINE CORPUS LUTEUM

Timing of embryo transfer to recipient mares is crucial to the success rate of an embryo transfer (ET) program. Recipient mares need a functional corpus luteum (CL) to maintain the early stages of pregnancy. Interference with luteal function appears to be a significant cause of failure with nonsurgical ET. Little is known about the endocrine control of luteal function in the mare, and the possibility of neuronal control of equine luteal function has not yet been studied. Dopamine (DA) has been shown to affect progesterone secretion in the bovine CL. Prior research in our laboratory suggested the possibility of dopaminergic regulation in the equine CL. The aim of this study was to document the presence of DA D1 receptor (D1r) and DA D2 receptor (D2r) within the equine CL. Immunocytochemistry (ICC) was performed on sections from 9 corpora lutea collected from a local equine abattoir. Tissues were stained using the avidin-biotin complex (ABC) method (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA, USA). Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. Tissue sections were deparaffinized and rehydrated;; endogenous peroxidase activity was quenched in 0.3% hydrogen peroxide in methanol at room temperature. To decrease nonspecific staining, tissue sections were incubated in goat serum (Vectastain ABC Elite kit). Tissue sections were then incubated overnight at 4°C with primary antibodies: Rabbit anti-Dopamine D1 receptor (Calbiochem, San Diego, CA, USA) or Rabbit anti-D2 receptor polyclonal antibody (Chemicon International, Inc, Temecula, CA, USA). Sections were then washed in PBS and incubated in biotinylated goat anti-rabbit IgG secondary antibody. After washing in PBS, sections were incubated in ABC. Sections were washed in PBS and the signal was visualized using 3-amino-9-ethylcarbazole (AEC Red, Vector Laboratories, Burlingame, CA, USA). Tissues were then counterstained with Immunomaster Hematoxylin (American Master Tech Scientific, Inc., Lodi, CA) to visualize the nuclei. As a negative control, tissues were incubated in normal rabbit serum instead of primary antibody, and as a positive control, the ICC procedure was performed on whole rat brain slices. Significant staining of luteal cells was observed using the D1r and D2r antibodies. Positive staining for D1r and D2r was seen throughout luteal cells;; however, no nuclear staining was observed. The presence of these receptors in equine CL tissue suggests a functional significance for DA in luteal function. Further research needs to be performed to determine the mechanistic function of dopamine in mare reproduction.

Cocaine- and amphetamine-regulated transcript peptide attenuates phenylephrine-induced bradycardia in anesthetized rats

The present study was undertaken to investigate the origin of cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactive (irCART) fibers observed in the nucleus of the solitary tract (NTS) and assess the role of CART peptide on phenylephrine (PE)-induced baroreflex. Immunohistochemical and retrograde tract-tracing studies showed that some of the irCART fibers observed in the NTS may have their cell bodies in the nodose ganglia. In urethane-anesthetized rats, intracisternal or bilateral intra-NTS microinjection of the CART peptide fragment 55-102 (0.1-3 nmol), referred to herein as CARTp, consistently and dose dependently attenuated PE-induced bradycardia. CARTp, in the doses used here, caused no significant changes of resting blood pressure or heart rate. Bilateral intra-NTS injections of CART antibody (1:500) potentiated PE-induced bradycardia. Injections of saline, normal rabbit serum, or concomitant injection of CARTp and CART antiserum into the NTS caused no significant changes of PE-induced baroreflex. The result suggests that endogenously released CARTp from primary afferents or exogenously administered CARTp modulates PE-induced baroreflex.

MEK inhibits secretin release and pancreatic secretion: roles of secretin-releasing peptide and somatostatin

We investigated the mechanism of action of methionine enkephalin (MEK) on HCl-stimulated secretin release and pancreatic exocrine secretion. Anesthetized rats with pancreatobiliary cannulas and isolated upper small intestinal loops were perfused intraduodenally with 0.01 N HCl while bile and pancreatic juice were diverted. The effect of intravenous MEK on acid-stimulated secretin release and pancreatic exocrine secretion was then studied with or without coinfusion of naloxone, an anti-somatostatin (SS) serum, or normal rabbit serum. Duodenal acid perfusate, which contains secretin-releasing peptide (SRP) activity, was collected from donor rats with or without pretreatment with MEK, MEK + naloxone, or MEK + anti-SS serum, concentrated by ultrafiltration, and neutralized. The concentrated acid perfusate (CAP), which contains SRP bioactivity, was infused intraduodenally into recipient rats. MEK increased plasma SS concentration and inhibited secretin release and pancreatic fluid and bicarbonate secretion dose-dependently. The inhibition was partially reversed by naloxone and anti-SS serum but not by normal rabbit serum. In recipient rats, CAP increased plasma secretin level and pancreatic secretion. CAP SRP bioactivity decreased when it was collected from MEK-treated donor rats; this was partially reversed by coinfusion with naloxone or anti-SS serum. These results suggest that in the rat, MEK inhibition of acid-stimulated pancreatic secretion and secretin release involves suppression of SRP activity release. Thus the MEK inhibitory effect appears to be mediated in part by endogenous SS.

Evidence supporting a physiological role for proANP-(1–30) in the regulation of renal excretion

The experiments, performed in pentobarbital sodium-anesthetized rats, consisted of a 1-h equilibration period followed by two 30-min control periods. Subsequently, synthetic rat pro atrial natriuretic peptide (ANP) [proANP-(1–30)] ( n = 8) was given as a bolus of 10 μg in 1 ml of 0.9% saline followed by an infusion at 30 ng/min (20 μl/min) for six additional periods. Control rats ( n = 6) received only 0.45% saline in the appropriate volumes. Mean arterial pressure, renal blood flow, and glomerular filtration rate did not change significantly in either group during the proANP-(1–30) infusion. Urine flow and potassium excretion increased ∼50% in the proANP-(1–30)-infused group only ( P < 0.05). Sodium excretion and fractional excretion of sodium, expressed as the change from their own baselines, were significantly increased by the proANP-(1–30) infusion ( P < 0.05), whereas cGMP excretion was similar in both groups. These results suggest that the rat sequence of proANP-(1–30) produces a natriuresis in the rat independent of changes in hemodynamics and renal cGMP production. In a second study, rats ( n = 8) were prepared as above and pretreated with 0.4 ml iv of rabbit serum containing an antibody directed against proANP-(1–30) (anti-proANP group). The rats were volume expanded with 3 ml of 6% albumin in Krebs and observed for 3 h to determine if the anti-proANP would attenuate the responses to volume expansion. Control rats ( n = 7) received 0.4 ml of normal rabbit serum. The elevation in potassium excretion in response to volume expansion was significantly attenuated in the anti-proANP group ( P < 0.05). Sodium excretion and urine flow responses also tended to be reduced but not significantly. These results suggest that in the rat, proANP-(1–30) plays a physiological role in regulating renal excretion.