Parturition and the perinatal period: can mode of delivery impact on the future health of the neonate?

Caesarean section and instrumental delivery rates are increasing in many parts of the world for a range of cultural and medical reasons, with limited consideration as to how 'mode of delivery' may impact on childhood and long-term health. However, babies born particularly by pre-labour caesarean section appear to have a subtly different physiology from those born by normal vaginal delivery, with both acute and chronic complications such as respiratory and cardio-metabolic morbidities being apparent. It has been hypothesized that inherent mechanisms within the process of labour and vaginal delivery, far from being a passive mechanical process by which the fetus and placenta are expelled from the birth canal, may trigger certain protective developmental processes permissive for normal immunological and physiological development of the fetus postnatally. Traditionally the primary candidate mechanism has been the hormonal surges or stress response associated with labour and vaginal delivery, but there is increasing awareness that transfer of the maternal microbiome to the infant during parturition. Transgenerational transmission of disease traits through epigenetics are also likely to be important. Interventions such as probiotics, neonatal gut seeding and different approaches to clinical care have potential to influence parturition physiology and improve outcomes for infants.

Efficacy of antilipopolysaccharide and anti-tumor necrosis factor monoclonal antibodies in a neutropenic rat model of Pseudomonas sepsis.

Monoclonal antibodies (MAb) directed against bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF) provide partial protection in experimental models of septic shock. To determine if additional benefit accrues from a combination of anti-TNF and anti-LPS MAb in the treatment of septic shock, a neutropenic rat model was developed to study active infection with Pseudomonas aeruginosa 12.4.4. Animals were treated intravenously with an irrelevant MAb (group 1); anti-TNF MAb (group 2); MAb directed against P. aeruginosa 12.4.4 LPS (group 3); or a combination of anti-TNF and anti-LPS MAb (group 4). None of the control animals in group 1 survived the 7-d period of neutropenia (0/16). In contrast, the survival rate was 44% in group 2 (P less than 0.02); 37% in group 3 (P less than 0.05); and 75% in group 4 (P less than 0.0002). The combination of monoclonal antibodies provided greater protection than either MAb given alone (P less than 0.05). Serum TNF levels during infection were significantly greater in groups 1 and 3 (20.1 +/- 3.3 U, mean +/- SE) than in groups 2 and 4 (0.9 +/- 0.8 U, P less than 0.0001). These results indicate that a combination of monoclonal antibodies to LPS and TNF have additive benefit in experimental Pseudomonas aeruginosa sepsis. This immunotherapeutic approach may be of potential utility in the management of serious, gram-negative bacterial infection in neutropenic patients.

Pressure pain tolerance at different sites on the quadriceps femoris prior to and following eccentric exercise.

Downhill running, particularly for the untrained subject, is a mode of eccentric exercise that produces delayed-onset muscle soreness (DOMS) in the quadriceps femoris muscle which is maximal between 24 and 72 h after the exercise. It is not clear whether sensitivity to pain is uniform over the surface of the muscle, or whether some locations become more sensitive following eccentric exercise. The purpose of this investigation was to compare pressure pain tolerance (PPTO) at various sites on the quadriceps femoris muscle on 2 days prior to exercise, immediately after, and at 24, 48 and 72 h following a bout of eccentric exercise. Fifteen untrained female subjects performed a 40 min downhill run on a motorized treadmill with a gradient of -12%, where running speed was adjusted to elicit a heart rate of approximately 60% of age-related maximum heart rate reserve, and were measured for PPTO at seven sites on the right thigh. Sites were visited sequentially three times and repeated on each of 6 days. Pressure pain tolerance as an index of tenderness was determined using a strain gauge algometer. Two sites were close to the distal myotendinous junction, three sites were located on the mid belly of the muscle and two sites were located at the proximal myotendinous junction. There was a significant difference (p<0.01) in PPTO between muscle sites prior to eccentric exercise (Days 1 and 2), and a significant difference between sites following eccentric exercise (p<0.01). Sites close to the distal and proximal myotendinous junction were most sensitive to pain (p<0.01). There was no difference in PPTO at any site across the belly of the muscle. These results suggest that the belly of the quadriceps femoris is the most suitable area for measurement of PPTO.

An established case of dentatorubral pallidoluysian atrophy (DRPLA) with unusual features on muscle biopsy.

Dentatorubral pallidoluysian atrophy (DRPLA) belongs to the group of autosomal dominant ataxias. Central nervous system pathology and inheritance are both well characterized, although the illness is rare. The presentation of a European child affected by this illness is described. He presented at 9 years of age with intractable progressive myoclonus epilepsy against a background of learning difficulties and developed progressive hypertonicity and dementia before his death at 15 years of age. Significant histological changes in a muscle biopsy were found. There was an absence of type IIB fibres and a predominance of type I fibres. Mean fibre diameter of all the fibre types was markedly reduced. All type I fibres showed an increase in lipid droplets. No previous descriptions exist of muscle histology in DRPLA. Although at least five adult family members have symptoms consistent with a diagnosis of DRPLA, their condition had not been recognized. We therefore describe the clinical picture and histological findings.

2-(Cyclohexylamino)-1-(4-cyclopentylpiperazin-1-yl)-2-methylpropan-1-one, a novel compound with neuroprotective and neurotrophic effects in vitro.

Focusing on development of novel drug candidates for the treatment of neurodegenerative diseases, we developed and synthesized a new compound, 2-(cyclohexylamino)-1-(4-cyclopentylpiperazin-1-yl)-2-methylpropan-1-one (amido-piperizine 1). The compound demonstrated robust neuroprotective properties after both glutamate excitotoxicity and peroxide induced oxidative stress in primary cortical cultures. Furthermore, amido-piperizine 1 was found to significantly induce neurite outgrowth in vitro which could suggest central reparative and regenerative potential of the compound. With these potential beneficial effects in CNS, the ability of the amido-piperizine 1 to penetrate the blood-brain barrier was tested using MDR1-MDCK cells. Amido-piperizine 1 was found not to be a P-gp substrate and to have a high blood-brain barrier penetration potential, indicating excellent availability to the CNS. Moreover, amido-piperizine 1 had a fast metabolic clearance rate in vitro, suggesting that parenteral in vivo administration seems preferable. As an attempt to elucidate a possible mechanism of action, we found that amido-piperizine 1 bound in nano-molar range to the sigma-1 receptor, which could explain the observed neuroprotective and neurotrophic properties, and with a 100-fold lower affinity to the sigma-2 receptor. These results propose that amido-piperizine 1 may hold promise as a drug candidate for the treatment of stroke/traumatic brain injury or other neurodegenerative diseases.

Bacteria-phagocyte interactions: emerging tactics in an ancient rivalry.

Although phagocytes appear to have a redundancy of both oxidative and non-oxidative killing mechanisms, nevertheless, bacterial pathogens are still able to evade these defenses in vivo and cause lethal infection. As the mechanisms by which phagocytes function have become detailed at the molecular level, both the recognition of specific bacterial virulence determinants and their effects at specific sites in the phagocyte are also being identified. Knowledge of these interactions may permit the use of immunomodulators either to neutralize these virulence determinants or to enhance the bactericidal capabilities of the phagocyte.

Interleukin-6 is a better marker of lethality than tumor necrosis factor in endotoxin treated mice.

We established a mouse model to differentiate between a lethal and non-lethal presentation of endotoxic shock. The model involved injecting different amounts of Escherichia coli LPS into C3H/HeN mice which had been 'primed' with BCG. We found that the mice receiving non-lethal and lethal doses of LPS could not be differentiated in terms of their physical symptoms for the first 8 h post-injection. Tumour necrosis factor (TNF) was detected at concentrations 2-9-fold greater in mice receiving lethal doses of LPS when compared with non-lethally injected mice. However, given that (i) the successful detection of this differential was dependent on the time of sampling and (ii) that TNF was only detected in the first 3-4 h post LPS challenge, we suggest that TNF may not be very useful as a prognostic marker in endotoxic shock. In contrast, circulating IL-6 appeared to mirror the symptoms of the endotoxic mice. The relative disappearance of IL-6 after 10 h in the non-lethally injected mice corresponded with their symptomatic recovery, while IL-6 continued to circulate up to the time of death in the lethally injected mice. Furthermore, there appeared to be a good correlation between the levels of injected LPS and the levels of IL-6 induced into the circulation. Our results suggest that IL-6, rather than TNF, may serve as a prognostic marker for endotoxic shock.

Differential induction of tumor necrosis factor by bacteria expressing rough and smooth lipopolysaccharide phenotypes.

The lipid A portion of the lipopolysaccharide (LPS) molecule of gram-negative bacteria has the ability to turn on the production of tumor necrosis factor (TNF) in macrophage cells. The question addressed in this paper was whether the presence of the polysaccharide moiety on the LPS molecule had any bearing on this ability. The question was asked (i) by using isolated LPS from a series of Salmonella mutants having progressively less polysaccharide attached to the lipid A portion of the molecule and (ii) by using whole bacteria expressing alternatively the smooth or rough LPS phenotype. Isolated LPS and bacteria were examined for their abilities to induce bioactive TNF in the mouse macrophage cell line RAW 264.7. The results indicated that the presence of long- or short-chain polysaccharide moieties had no bearing on the ability of the isolated LPS molecule to induce TNF. However, the presence of long-chain polysaccharides attached to the lipid moiety on the intact smooth bacterium was associated with a decreased ability to induce TNF. To test whether the bacteria were inducing TNF by a cell (bacterium)-to-cell (macrophage) contact mechanism or through a releasable product, the bacteria were removed from direct contact with the macrophage cells by using a Transwell filter insert. Under these conditions the rough bacteria continued to induce TNF, while the smooth bacteria were no longer capable of doing so. When filtrates from the bacteria were examined in the Limulus amebocyte lysate assay, the results showed that the rough bacteria were releasing approximately a log order more Limulus amebocyte lysate activity than the smooth bacteria. The results of this study suggest that rough bacteria may be superior inducers of TNF compared with their smooth counterparts because of a greater propensity to shed their LPS.

Antiendotoxin activity of cationic peptide antimicrobial agents.

The endotoxin from gram-negative bacteria consists of a molecule lipopolysaccharide (LPS) which can be shed by bacteria during antimicrobial therapy. A resulting syndrome, endotoxic shock, is a leading cause of death in the developed world. Thus, there is great interest in the development of antimicrobial agents which can reverse rather than promote sepsis, especially given the recent disappointing clinical performance of antiendotoxin therapies. We describe here two small cationic peptides, MBI-27 and MBI-28, which have both antiendotoxic and antibacterial activities in vitro and in vivo in animal models. We had previously demonstrated that these peptides bind to LPS with an affinity equivalent to that of polymyxin B. Consistent with this, the peptides blocked the ability of LPS and intact cells to induce the endotoxic shock mediator, tumor necrosis factor (TNF), upon incubation with the RAW 264.7 murine macrophage cell line. MBI-28 was equivalent to polymyxin B in its ability to block LPS induction of TNF by this cell line, even when added 60 min after the TNF stimulus. Furthermore, MBI-28 offered significant protection in a galactosamine-sensitized mouse model of lethal endotoxic shock. This protection correlated with the ability of MBI-28 to reduce LPS-induced circulating TNF by nearly 90% in this mouse model. Both MBI-27 and MBI-28 demonstrated antibacterial activity against gram-negative bacteria in vitro and in vivo against Pseudomonas aeruginosa infections in neutropenic mice.

Acetamide broth for isolation of Pseudomonas aeruginosa from patients with cystic fibrosis.

The recovery of Pseudomonas aeruginosa was enhanced by incubating specimens in acetamide broth before subculture on cetrimide agar. This finding is of particular value in screening pediatric patients with cystic fibrosis for carriage of P. aeruginosa.

Surface characteristics of Pseudomonas aeruginosa grown in a chamber implant model in mice and rats.

Pseudomonas aeruginosa PAO1 was grown in vivo in chambers implanted into the peritoneums of mice and rats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of bacterial cells taken from the chambers and washed to remove loosely bound host proteins revealed the presence of the major outer membrane proteins D2, E, F, G, and H2. Western immunoblotting with specific antisera confirmed the presence of porin protein F and lipoprotein H2. However, there was no apparent induction of the phosphate starvation-inducible porin P or the divalent cation starvation-inducible protein H1. Small amounts of proteins with molecular weights similar to those of the iron-regulated outer membrane proteins were found in cells grown in vivo; however, their presence could not be confirmed immunologically. The presence of pili and flagella on the cells grown in vivo was demonstrated by electron microscopy and Western immunoblotting. A consistent alteration in the lipopolysaccharide banding pattern was observed after growth in vivo. Compared with cells of strain PAO1 grown in vitro, cells grown in vivo appeared to lack a series of high-molecular-weight O-antigen-containing lipopolysaccharide bands and gained a new series of lower-molecular-weight lipopolysaccharide bands. This alteration in the lipopolysaccharide after growth in vivo did not affect the O-antigen serotype or the resistance of the bacteria to serum.

Stimulation by fibronectin of macrophage-mediated phagocytosis of Pseudomonas aeruginosa.

In a previous investigation it was determined that Pseudomonas aeruginosa cells taken directly from a mouse in vivo growth system were significantly more susceptible to nonopsonic phagocytosis by macrophages than were similar cells after being washed in buffer (N. M. Kelly, J. L. Battershill, S. Kuo, J. P. Arbuthnott, and R. E. W. Hancock, Infect. Immun. 55:2841-2843, 1987). It was demonstrated that a phagocytosis-promoting factor was found in the supernatant obtained from chambers incubated in the peritoneal cavities of laboratory mice or rats. The phagocytosis-promoting factor was effective with both strains of P. aeruginosa tested, using both unelicited mouse peritoneal macrophages and the P388D1 mouse macrophage cell line as the phagocytic cells. Phagocytosis enhancement was observed with in vivo-grown bacteria and with bacteria grown in vitro on agar plates, but not with bacteria grown in vitro with rapid agitation. Supernatants from mice and rats were fractionated using a fast pressure liquid chromatography gel exclusion column. The phagocytosis-promoting factor copurified with fibronectin. Furthermore, antifibronectin sera negated the phagocytosis-promoting activities of in vivo chamber supernatant, while commercial bovine fibronectin was itself capable of promoting phagocytosis. The concentrations of fibronectin increased in both rat and mouse peritoneal chambers with time, coincident with the ability of chamber supernatants to promote phagocytosis. It was concluded that fibronectin was the phagocytosis-promoting factor of chamber supernatants. Bacterial presence in the peritoneal chambers was not required to elicit fibronectin uptake into the chambers.

Determinants of the efficacy of tobramycin therapy against isogenic nonmucoid and mucoid derivatives of Pseudomonas aeruginosa PAO1 growing in peritoneal chambers in mice.

Mice which were supporting the growth of Pseudomonas aeruginosa in chambers implanted in their peritoneums were given two intramuscular injections of tobramycin (15 mg/kg of body weight each) at an interval of 8 h. Three hours later, chambers were removed and their contents were assessed for viable counts. Controls revealed that tobramycin levels in the chambers were 3.8 micrograms/ml 15 min after injection of 15 mg of tobramycin per kg and remained above the in vitro MICs (0.5 to 1 microgram/ml) for the tested strains for 8 h. It was demonstrated that tobramycin therapy was less effective with higher inocula and with longer delay before administration. Thus, in vivo, the concentration of bacteria in the chambers at the time of the first tobramycin injection had a profound effect on the bactericidal efficacy of tobramycin therapy. No such concentration dependence was observed in mock in vitro therapy experiments. A phage-selected mucoid derivative of P. aeruginosa PAO1 showed only a marginal increase in in vitro aminoglycoside susceptibility and no major alteration in in vivo susceptibility compared with its isogenic nonmucoid parent strain.

A differential response to treatment with divalproex sodium in patients with intractable headache.

We consecutively recruited 75 patients with intractable headache syndromes, divided them into three groups (frequent migraine = FM, transformed migraine = TM, and tension type headache = TT) based on their headache symptoms and treated all 75 with divalproex sodium. Thirty-six patients (48%) reported a > or = 50% reduction in headache frequency. We noted significantly different treatment response rates in the three patient groups, with FM patients reporting the highest rate of improvement (11/18 = 61%), TM patients an intermediate rate (22/43 = 51%), and TT patients the lowest response rate (3/14 = 21%). These data suggest that prophylactic therapy with divalproex may be effective in selected patients with intractable headache syndromes and that identification of clinically distinct headache subtypes may assist in predicting response to treatment.

Colonial dissociation and susceptibility to phagocytosis of Pseudomonas aeruginosa grown in a chamber implant model in mice.

Pseudomonas aeruginosa strains were grown in 1-cm plastic chambers sealed at both ends with porous Millipore filters and implanted in the peritonea of mice. Mucoid and nonmucoid strains of P. aeruginosa isolated from a patient with cystic fibrosis largely retained their phenotypes when grown for up to 1 year in this in vivo system, although colonial dissociation occurred, as observed in chronic lung infections of patients with cystic fibrosis. In the absence of added opsonins, P. aeruginosa M2 cells taken directly from the in vivo system were significantly more susceptible to phagocytosis than were the same P. aeruginosa cells after being washed in buffer. Phagocytosis of in vivo-grown P. aeruginosa cells could be further enhanced by using a porin protein F-specific monoclonal antibody.

Pseudomonas aeruginosa pili as ligands for nonopsonic phagocytosis by fibronectin-stimulated macrophages.

Fibronectin is capable of activating macrophages for enhanced nonopsonic phagocytosis of Pseudomonas aeruginosa grown in vivo in rats or mice or in vitro on nutrient agar plates. In this study it was determined that while fibronectin was able to significantly increase phagocytosis of organisms grown in static broth, uptake of agitated bacteria could not be promoted. Agitated P. aeruginosa cultures were proven to lack surface pili expression, as assessed by electron microscopic studies. A pilus-deficient pilA::Tn501 mutant of P. aeruginosa PAO was constructed by gene replacement techniques. Phagocytosis of this mutant could not be enhanced by fibronectin regardless of growth conditions. Furthermore, 60 micrograms of exogenously added Pseudomonas pili per ml was capable of abrogating the enhanced phagocytosis of the wild-type strain observed with fibronectin-stimulated macrophages. It is concluded that Pseudomonas pili were the bacterial ligands required for attachment to fibronectin-stimulated macrophages in the initial stages of nonopsonic phagocytosis.

Comparison of the outer membrane protein and lipopolysaccharide profiles of mucoid and nonmucoid Pseudomonas aeruginosa.

Laboratory-derived mucoid variants of Pseudomonas aeruginosa were selected by plating the standard PAO1 laboratory strain with bacteriophage. These mucoid variants formed two distinct groups of strains on the basis of phage typing. The first group had the same phage-typing pattern as the parent PAO1 strain, while the second group had a distinctly different phage-typing pattern. One strain from each group was assessed along with the parent PAO1 strain for its outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles by sodium dodecyl sulfate-gel electrophoresis followed by appropriate staining. The mucoid derivatives were found to differ from the parent PAO1 nonmucoid strain in having lost a high-molecular-weight LPS species. Furthermore, the reversion of the mucoid strains to the nonmucoid phenotype was accompanied by a return of the missing high-molecular-weight LPS species. No observable difference between the mucoid derivatives and the parent nonmucoid strain was noted in the OMP profiles. The opposite was found in the case of four isolates of mucoid P. aeruginosa from patients with cystic fibrosis. Two OMP bands (of approximately 55 and 25 kilodaltons) were present in the mucoid isolates but missing in their sister nonmucoid strains. In the case of the cystic fibrosis isolates, no difference in the LPS profiles within mucoid-nonmucoid pairs was noted.

Fibronectin as an enhancer of nonopsonic phagocytosis of Pseudomonas aeruginosa by macrophages.

Fibronectin is capable of enhancing uptake by macrophages of Pseudomonas aeruginosa grown in vivo in rats or mice or in vitro on nutrient agar plates. It was demonstrated that concentrations as low as 27 nM fibronectin produced significant enhancement of macrophage phagocytosis. Washing of fibronectin-treated macrophages did not prevent phagocytosis enhancement, but washing of fibronectin-treated bacteria did. The tetrapeptide arginine-glycine-aspartic acid-serine, which comprises the eucaryotic cell-binding domain of fibronectin, was also capable of promoting bacterial uptake, whereas the control tetrapeptide tetraglycine was not. Fibronectin caused depolarization of the mouse macrophage cell line P388D1, plasma membrane, as demonstrated by using a polarization-sensitive fluorescent probe. These data indicate that promotion by fibronectin of nonopsonic phagocytosis is mediated by the action of fibronectin on the macrophages.

Does pseudomonas cross-infection occur between cystic-fibrosis patients.

Over a 12-months period respiratory Pseudomonas aeruginosa isolated from CF patients were typed by serology and pyocin production to determine whether cross-infection was occurring. Results of typing were interpreted in relation to the degree of contact patients had with each other. One strain appeared in 4 unrelated patients. However, since none of these patients had been in contact with each other the strains considered to have been acquired from the environment. Each of six pairs of siblings shared the same strain, but the pairs of strains were distinct from each other. These results suggest that the environment is the most important source of Pseudomonas strains for CF patients and that for cross-infection to occur prolonged intimate contact is required.

Efficacy of a monoclonal antibody directed against tumor necrosis factor in protecting neutropenic rats from lethal infection with Pseudomonas aeruginosa.

A monoclonal antibody directed against murine tumor necrosis factor-alpha (TNF) was studied in a neutropenic rat model to determine its efficacy in protecting animals from lethal infection with Pseudomonas aeruginosa. Anti-TNF monoclonal antibody at a dose of 20 mg/kg given intravenously at 0 and 120 h resulted in a 53% survival rate (8/15) compared with no survival in control animals (0/15) (P less than .005). The combination of anti-TNF monoclonal antibody and oral ciprofloxacin at a suboptimal dose of 2.5 mg/kg/day resulted in a 100% survival rate in neutropenic animals (16/16), while ciprofloxacin alone produced only a 67% survival rate (10/15) during the 7-day period of neutropenia (P less than .05). Thus anti-TNF monoclonal antibody alone or in addition to antimicrobial agents improved survival in neutropenic animals after infection with P. aeruginosa.