Neutrophil adhesion molecules in term and premature infants: normal or enhanced leucocyte integrins but defective L-selectin expression and shedding.

The functional deficits of neonatal neutrophils are well documented and are thought to contribute to the increased susceptibility of newborn infants to infection. We measured the adhesion molecules L-selectin, CD11a/CD18 and CD11b/CD18 on neutrophils from the cord blood of term (n = 22) and premature (n = 32) infants using a whole blood method with flow cytometry and quantitative bead standards to enumerate cell surface receptors. We also assayed plasma for the shed form of L-selectin (sL-selectin). Our results suggested that L-selectin expression on term infant neutrophils is lower than that on adult neutrophils (unstimulated and stimulated, both P < 0.001), but that stimulated premature infant cell express higher L-selectin than term infants (P < 0.05); it is possible that this deficiency is caused by physiological changes occurring around the normal time of parturition. We observed reduced sL-selectin in term infants (P < 0.001) compared with adults, and even lower concentrations in premature infants (P < 0.001). The sL-selectin concentrations in plasma may be a reflection of granulopoiesis, which may be reduced in premature infants. Our results showed increased resting neonatal neutrophil expression of CD11b/CD18 compared with adults, and the absence of any neonatal deficit of the ability to up-regulate CD11b/CD18 expression on stimulation. These findings are contrary to previous reports. Further studies suggested that the isolation procedures used in previous reports reduces the capability of the cells to respond to a formyl methionine leucine phenylalanine (fMLP) stimulus. This effect is more marked in neonatal neutrophils, suggesting that the previously reported deficiency is in fact due to the isolation techniques used rather than the cells' innate ability to up-regulate CD11b/CD18 expression. The results of our study lead us to propose that the adhesive function of neonatal neutrophils may be less defective than previously thought.     

Intestinal changes caused by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase

Subacute (2 week) oral or intravenous administration of DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), caused diarrhea and frequent emesis as early as 4 to 5 days in dogs (dose greater than or equal to 200 mg/kg/day). Diarrhea also occurred in monkeys after 1 week of treatment with an intravenous dose of 1000 mg/kg/day. Especially evident in the treated dogs with diarrhea were fluid loss, hemoconcentration, and decreased serum sodium and chloride which were findings totally reversible about 2 weeks after cessation of dosing. As a result of treatment with the highest intravenous dosage (1000 mg/kg/day), villous atrophy of the mucosa was observed by light and scanning electron microscopy in the canine small intestine. Transmission electron microscopy demonstrated that the most significant alterations of the canine intestinal tract involved the microvilli of epithelial cells which became shorter and were frequently less numerous or absent along focal areas of the plasma membrane. Intestinal mucosal levels of putrescine, especially in the duodenum and jejunum, were decreased as demonstrated in the monkeys following intravenous treatment with 100, 300, or 1000 mg/kg/day of DFMO. The results of this investigation are consistent with the hypothesis that the inhibition of ODC activity and subsequent altered polyamine metabolism may lead to delayed maturation of the intestinal epithelial cells and the impaired development of their microvilli, causing fluid loss due to reduced absorptive surface area.     

Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB.

Neisseria meningitidis is the etiologic agent of epidemic bacterial meningitis. Lipooligosaccharide (LOS) is a principal virulence factor associated with the organism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LOS has demonstrated that there is considerable microheterogeneity in the molecule. To begin our understanding of the nature of this heterogeneity, we identified a Tn916-generated LOS mutant of N. meningitidis NMB (serotype L3, monoclonal antibodies 3F11+, 6B4+, and 4C4-) that was designated NMB-SS3 (monoclonal antibodies 3F11-, 6B4-, and 4C4+). The transposon insertion was localized to the amino terminus of the functional copy of the UDP-Glc 4-epimerase gene (galE). UDP-Glc 4-epimerase (EC 5.1.3.2) activity was present in N. meningitidis NMB but not in NMB-SS3, indicating that the Tn916 insertion had abolished this activity. Mass spectrometric analysis of the LOS from strain NMB revealed multiple species of LOS, which is consistent with extensive microheterogeneity. While the most predominant structure was consistent with a terminal lacto-N-neotetrose structure found in other strains of N. meningitidis, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-->(GlcNAc)-->Hep2PEA-->KDO2 (where Hep is heptose, PEA is phosphoethanolamine, and KDO is 2-keto-3-deoxymannooctulosonic acid), structures containing repetitive hexoses which are not precursors of this structure were also identified. Compositional analysis of LOS from strain NMB-SS3 revealed that there were no galactoses present in the structure. Mass spectrometric analysis of O-deacylated LOS revealed the presence of multiple species, with the predominant LOS species in this mutant strain formed by the Hex-->(HexNAc)-->Hep2PEA-->KDO2 (where Hex is hexose and HexNAc is N-acetylhexosamine) structure. However, LOS structures with repetitive hexoses, e.g., Hexn-->(HexNAc)-->Hep2PEA-->KDO2 (n = 2, 3, or 4), emanating from one or both heptoses were also identified. Since this mutant cannot synthesize UDP-Gal, these structures must repetitive glucoses. These data suggest that NMB has a glycosyltransferase capable of polymerizing glucose moieties as an alternative biosynthetic pathway to the wild-type lacto-N-neotetrose structure.     

Glomerular lesions in mice transgenic for growth hormone and insulinlike growth factor-I. I. Relationship between increased glomerular size and mesangial sclerosis.

The glomeruli of mice transgenic for bovine growth hormone (GH mice) were disproportionately enlarged as a function of either kidney or body weight. Glomerular size correlated with mesangial sclerosis and the urine albumin/creatinine ratio. The glomerular lesions consisted of mesangial proliferation (4 to 5 weeks) followed by progressive mesangial sclerosis (19 weeks), resulting in complete glomerulosclerosis at 30 to 37 weeks. Albuminuria paralleled the glomerulosclerosis. In contrast, mice transgenic for insulinlike growth factor-I (IGF-I mice) did not develop glomerulosclerosis, even though glomerular size significantly increased. Glomerular hypertrophy, however, did not reach that in GH mice. These data suggest that high levels of circulating GH lead to a disproportionate increase in glomerular cellularity and volume, as well as glomerulosclerosis. This does not appear to be the result of high levels of circulating IGF-I stimulated by GH, as the serum IGF-I level in GH mice was lower than that in IGF-I mice.     

Mast cell subsets in the rat distinguished immunohistochemically by their content of serine proteinases

The specificities of antibodies raised in rabbits against rat mast cell proteinase 1 (RMCP 1) from connective tissue mast cells (CTMC), and against RMCP II from mucosal mast cells (MMC), were analysed by SDS-PAGE and Western blotting. Significant cross-reactivity was detected, and was eliminated by affinity purification and cross-absorption techniques. The resultant F(ab')2 antibodies, monospecific for each enzyme, were used for the immunohistochemical localization of RMCP I and II in rat tissues. Cells in skin, tongue, intestinal serosa and lung parenchyma which, by histochemical techniques, have been identified as CTMC, contained RMCP I exclusively. Cells in jejunal lamina propria and bronchial epithelium, previously classified as MMC, contained RMCP II. The results demonstrate the feasibility of distinguishing mast cell subsets by their content of serine proteinases.     

Improved isolation of Chlamydia trachomatis from a low-prevalence population by using polyethylene glycol.

The effect of polyethylene glycol (PEG) on the isolation of Chlamydia trachomatis was evaluated in our laboratory. Initial range-finding experiments demonstrated that the number of chlamydial inclusion bodies increased with increasing PEG concentrations. However, PEG concentrations above 10.5% became progressively more toxic to the McCoy cell monolayers. When 50 frozen clinical Chlamydia isolates were inoculated onto McCoy cell cultures with and without 7% PEG, the PEG-treated cultures produced three- to fivefold more chlamydial inclusions than cultures without PEG. This enhancement was also observed when 1,144 fresh clinical specimens from a low-prevalence population were tested. With fresh clinical specimens, PEG-treated cultures produced two- to sixfold more inclusions than standard cultures. The addition of 7% PEG to the chlamydial overlay medium significantly increased the number of inclusions in each culture, improved the sensitivity of the culture, and decreased the probability of missing a weakly positive specimen.     

Intestinal hypersensitivity reactions in the rat. I. Uptake of intact protein, permeability to sugars and their correlation with mucosal mast-cell activation.

We have confirmed previous observations that intestinal anaphylaxis induced in rats previously sensitized to ovalbumin (OVA) is associated with an increased uptake of an unrelated 'bystander' protein, bovine serum albumin (BSA) fed 1 hr previously. In this study, this enhanced protein uptake was associated with an increased lactulose/rhamnose excretion ratio after administration of these sugars, although there was no correlation between the two measurements. One hour after antigen challenge the serum levels of rat mast-cell protease II (RMCPII), a specific marker for mucosal mast-cell secretion, were significantly higher than both the pre-challenge levels and those of sham-challenged controls (P less than 0.002). There was a significant positive correlation between the serum levels of RMCPII and the lactulose/rhamnose excretion ratios (P less than 0.05), but no such correlation existed between RMCPII and BSA levels in the challenged rats. In other studies the urinary lactulose/rhamnose ratios of rats with cetrimide-induced gut damage were found to be significantly increased, although BSA uptake into the serum remained unaltered. We conclude that there is no simple correlation between gut permeation of low-molecular weight sugars and and the uptake of macromolecular proteins.     

Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury

Radioimmunoassay and immunocytochemistry were used to study the distribution of galanin, a novel 29 amino acid porcine intestinal peptide, in the central nervous system of the rat and pig. The pattern of distribution was similar in the two species, with the highest concentrations of galanin-like immunoreactivity found in the neurohypophysis, hypothalamus and sacral spinal cord. Immunocytochemical studies of these regions localized galanin-like immunoreactivity to cell bodies in the paraventricular and supraoptic nuclei of the hypothalamus, to fibres in the pars nervosa and to numerous cell bodies and fibres in the dorsal horn of the spinal cord. On both gel and high pressure liquid chromatography, galanin-like immunoreactivity in rat and pig nervous tissue eluted as a single peak in a position similar to purified procine intestinal galanin standard. Surgical and pharmacological manipulations in the rat suggest the presence of galanin in afferent fibres. An increase of galanin-like immunoreactivity was observed in the sacral spinal cord of the rat following thoracic spinal cord transection. Thus galanin-like immunoreactivity in the brain is mainly localized in the hypothalamopituitary region. The decrease of galanin-like immunoreactivity in the dorsal horn of the spinal cord, following dorsal rhizotomy and pre-treatment of rats with capsaicin, indicates that many of the fibres, which are of small diameter, may well be derived from spinal sensory neurones.     

Peptide expression is altered when afferent nerves reinnervate inappropriate tissue

Neuropeptides are found in specific subpopulations of primary afferent neurones. Peptide expression can be altered following axotomy or under the influence of nerve growth factor. Here we have examined the consequence of altering the peripheral target of afferent neurones. Many unmyelinated afferents from skin contain substance P-like immunoreactivity (SPLI) whilst those from muscle do not. We have found that fibres will innervate inappropriate tissue types. We have therefore cut and cross-anastomosed a skin and muscle hindlimb nerve in the rat and 10-12 weeks later analysed the regenerated nerves immunocytochemically for SPLI. Muscle afferents inappropriately reinnervating skin were found to contain many SPLI fibres in contrast to control nerves resutured to their own distal stumps. Conversely, skin afferents made to innervate muscle showed reduced levels of peptide staining. These results demonstrate the plasticity of peptide expression and suggest that factors in peripheral tissue or perhaps distal nerve sheaths exert a trophic influence on nervous system function.