Neonatal Rat Serum Research Articles

Hydrolytic biotransformation of the bumetanide ester prodrug DIMAEB to bumetanide by esterases in neonatal human and rat serum and neonatal rat brain-A new treatment strategy for neonatal seizures?

The loop diuretic bumetanide has been proposed previously as an adjunct treatment for neonatal seizures because bumetanide is thought to potentiate the action of γ-aminobutyric acid (GABA)ergic drugs such as phenobarbital by preventing abnormal intracellular accumulation of chloride and the subsequent "GABA shift." However, a clinical trial in neonates failed to demonstrate such a synergistic effect of bumetanide, most likely because this drug only poorly penetrates into the brain. This prompted us to develop lipophilic prodrugs of bumetanide, such as the N,N-dimethylaminoethyl ester of bumetanide (DIMAEB), which rapidly enter the brain where they are hydrolyzed by esterases to the parent compound, as demonstrated previously by us in adult rodents. However, it is not known whether esterase activity in neonates is sufficient to hydrolyze ester prodrugs such as DIMAEB. In the present study, we examined whether esterases in neonatal serum of healthy term infants are capable of hydrolyzing DIMAEB to bumetanide and whether this activity is different from the serum of adults. Furthermore, to extrapolate the findings to brain tissue, we performed experiments with brain tissue and serum of neonatal and adult rats. Serum from 1- to 2-day-old infants was capable of hydrolyzing DIMAEB to bumetanide at a rate similar to that of serum from adult individuals. Similarly, serum and brain tissue of neonatal rats rapidly hydrolyzed DIMAEB to bumetanide. These data provide a prerequisite for further evaluating the potential of bumetanide prodrugs as add-on therapy to phenobarbital and other antiseizure drugs as a new strategy for improving pharmacotherapy of neonatal seizures.

Brain Distribution and Metabolism of Flupirtine, a Nonopioid Analgesic Drug with Antiseizure Effects, in Neonatal Rats.

Flupirtine, a nonopioid analgesic drug, is effective in treating neonatal seizures. However, its brain delivery and pharmacokinetics are unknown in neonatal mammals. The purpose of this study was to determine the pharmacokinetics of flupirtine and the formation of its active metabolite D-13223 in various tissues such as brain in neonate animals. On postnatal day 7, rat pups received 25 mg/kg of flupirtine intraperitoneally. Liver; heart; kidney; lung; spleen; retina; serum; and brain regions hippocampus, cortex, and the remaining brain (devoid of cerebellum) were harvested up to 24-h postdosing. An LC-MS/MS assay was developed to quantify flupirtine and D-13223. Flupirtine was delivered to all tissues assessed, with the highest area under the concentration vs. time curve (AUC0–24h) in liver (488 µg·h/g tissue) and the lowest in spleen (82 µg·h/g tissue). Flupirtine reached the brain, including the hippocampus and cortex, within 1 h of dosing and persisted at 24 h. Flupirtine AUC in various brain regions was approximately 195 µg·h/g tissue. The half-life of flupirtine in various tissues ranged from 3.1 to 5.2 h. D-13223 was formed in vivo and detected in all tissues assessed, with the concentrations being the highest in the liver. Incubation of isolated neonatal rat liver, heart, kidney, lung, spleen, whole eye, serum, or whole brain with flupirtine for 3 h at 37 °C formed D-13223 in all tissues, except serum. D-13223 formation was the highest in isolated liver tissue. Tissue partition coefficients based on isolated tissue uptake correlated well with in vivo tissue:serum drug exposure ratios. Thus, flupirtine reaches the target brain tissues from the systemic route in neonatal rats, and brain tissue forms the active metabolite D-13223.

Neuroprotective Effect of ω-3 Polyunsaturated Fatty Acids on Bilirubin Encephalopathy In Vitro and In Vivo.

BackgroundBilirubin encephalopathy is a serious complication in neonatal jaundice and is associated with high mortality and disability in newborns. The present study aimed to investigate the neuroprotective effects of omega-3 polyunsaturated fatty acids (ω-3 PUFA) on bilirubin encephalopathy in vitro and in vivo.Material/MethodsThe cytotoxicity of unconjugated bilirubin (UCB) to neurons and neuroprotection of ω-3 PUFA were investigated using MTT assays and apoptosis evaluations. Superoxide dismutase (SOD) and catalase (CAT) enzyme activity were measured to investigate the anti-oxidative effect of ω-3 PUFA. The differences between eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also compared. The in vivo neuroprotective effect of DHA was demonstrated in neonatal rats with bilirubin encephalopathy by bilirubin monitoring, neuron-specific enolase (NSE) monitoring, H&E staining of brain tissue, and apoptosis rate evaluations.ResultsOmega-3 PUFA reduced the rate of apoptosis induced by UCB and increased SOD and CAT enzyme activity for anti-oxidation. DHA did not reduce the bilirubin in the serum of neonatal rats with bilirubin encephalopathy, but did reduce the damage caused by bilirubin with decreased NSE and apoptosis rate as well as improved neuron morphology.ConclusionsOmega-3 PUFA, particularly DHA, can reduce neurological damage in neonatal rats with bilirubin encephalopathy by increasing anti-apoptosis and anti-oxidation effects against UCB, providing a theoretical basis for the clinical treatment of bilirubin encephalopathy in newborns.

The cyanobacterial amino acid β-N-methylamino-l-alanine perturbs the intermediary metabolism in neonatal rats

The neurotoxic amino acid β-N-methylamino-l-alanine (BMAA) is produced by most cyanobacteria. BMAA is considered as a potential health threat because of its putative role in neurodegenerative diseases. We have previously observed cognitive disturbances and morphological brain changes in adult rodents exposed to BMAA during the development. The aim of this study was to characterize changes of major intermediary metabolites in serum following neonatal exposure to BMAA using a non-targeted metabolomic approach. NMR spectroscopy was used to obtain serum metabolic profiles from neonatal rats exposed to BMAA (40, 150, 460mg/kg) or vehicle on postnatal days 9–10. Multivariate data analysis of binned NMR data indicated metabolic pattern differences between the different treatment groups. In particular five metabolites, d-glucose, lactate, 3-hydroxybutyrate, creatine and acetate, were changed in serum of BMAA-treated neonatal rats. These metabolites are associated with changes in energy metabolism and amino acid metabolism. Further statistical analysis disclosed that all the identified serum metabolites in the lowest dose group were significantly (p<0.05) decreased. The neonatal rat model used in this study is so far the only animal model that displays significant biochemical and behavioral effects after a low short-term dose of BMAA. The demonstrated perturbation of intermediary metabolism may contribute to BMAA-induced developmental changes that result in long-term effects on adult brain function.

Ontogeny of VEGF, IGF-I, and GH in Neonatal Rat Serum, Vitreous Fluid, and Retina from Birth to Weaning

Vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-I, and growth hormone (GH) are major regulators of physical growth, as well as normal and pathologic retinal development. Ocular tissues are protected by the blood-ocular barrier. This study was conducted to test the hypothesis that the ontogenic profiles of VEGF, IGF-I, and GH in the rat serum, vitreous fluid, and retina are compartment specific, and that the vitreous is a reservoir for retinal growth factors. Sprague-Dawley rat pups were killed at birth (postnatal day [P]0) and at P7, P14, and P21. At death, serum, vitreous fluid, and retinal homogenates were analyzed for ontogeny of VEGF, IGF-I, and GH. VEGF levels were 10 times higher in the vitreous than in serum at all stages of development. Vitreous and serum VEGF levels progressively declined, with lowest concentrations at P21. Retinal VEGF levels increased with the highest concentration at P21. IGF-I levels in the vitreous decreased from P7 through P21. IGF-I levels in serum and retinal homogenates increased with advancing postnatal age. Although IGF-I levels were four times higher in the vitreous than in the retina at P0, equilibration was achieved at P21. GH levels in the vitreous were 10 times lower than serum levels, were decreased at P14 and P21, and remained unchanged from P0 through P21 in the retina. VEGF and IGF-I act in concert to promote retinal development with the vitreous fluid as a reservoir. The ontogenic profiles of VEGF, IGF-I and GH in the serum and ocular compartments are specific. These differences should be considered when therapies for ROP are proposed.

Identification of 14-hydroxy- retro-retinol and 4-hydroxy-retinol as endogenous retinoids in rats throughout neonatal development

14-Hydroxy- retro-retinol was previously described as an in vivo and in vitro metabolite of retinol. Furthermore, the retinoid 4-hydroxy-retinol was identified as an endogenous occurring retinoid in the amphibian organism and an in vitro metabolite of retinol. We describe in the present study that 14-hydroxy- retro-retinol and 4-hydroxy-retinol are present in normal neonatal rat serum as endogenous occurring retinoids in normal non-vitamin A supplemented mammals (rats). Both retinoids were detected in serum and liver of neonatal rats at days 3 and 11 after birth. The respective concentrations at day 11 after birth were 41.8 ± 2.8 ng/ml (serum)/ 104 ± 6 ng/g (liver) for 4-hydroxy-retinol and 23 ± 4.6 ng/ml (serum)/ 285 ± 5 ng/g (liver) for 14-hydroxy- retro-retinol. Both retinoids could not be detected in adult rat serum and liver. From our experiments important physiological functions of these retinoids during postnatal development could be postulated.

Nonspecific Human IgG Reduces Survival in Neonatal Rats Infected with Escherichia coli

Human intravenous IgG (IVIG) containing specific antibodies protects neonatal rats from septic death. However, IVIG has immunosuppressive properties and clinical trials of IVIG in neonates at risk for sepsis have yielded conflicting results. This study was designed to test the hypothesis that nonspecific antibodies in IVIG reduce survival in neonatal rats infected with Escherichia coli. Specific antibodies were adsorbed from IVIG with E. coli to produce IVIG/anti-E. coli-. After transthoracic administration of E. coli, survival was determined in neonatal rats injected intraperitoneally with phosphate-buffered saline, IVIG/anti-E. coli- (500 mg/kg) or IVIG containing anti-E. coli antibodies (IVIG/anti-E. coli+). Complement-mediated hemolytic activity of neonatal rat serum was quantified using sensitized sheep erythrocytes. Compared with placebo, intraperitoneal IVIG/anti-E. coli- reduced neonatal survival after E. coli infection. In contrast, IVIG/anti-E. coli+ protected infected animals. Both IVIG/anti-E. coli- and IVIG/anti-E. coli+ impaired the complement-mediated hemolytic activity of neonatal rat serum. IVIG contained (1) nonspecific antibodies that reduced survival in neonatal rats infected with E. coli and (2) protective anti-E. coli antibodies that enhanced survival in neonatal rats infected with E. coli. We speculate that in clinical trials of IVIG to treat or prevent neonatal sepsis, inconsistent results may be caused, in part, by lot-to-lot variations in the ratio of immunosuppressive, nonspecific antibodies to protective, specific antibodies.

Complement component C9 enhances the capacity of beta-lactam antibiotics to kill Escherichia coli in vitro and in vivo.

Complement component C9 is required for rapid complement-mediated killing of Escherichia coli. In this report, the influence of supplemental C9 on the bactericidal and protective effects of beta-lactam antibiotics in neonates was assessed. By rocket immunoelectrophoresis, the intrinsic C9 concentrations of pooled serum from both human and rat neonates was less than 20% of adult levels. Supplemental C9 purified from human plasma enhanced the capacity of ampicillin-treated serum from human neonates to impair the survival of E coli O7:K1:NM (P < 0.02). Similarly, supplemental C9 enhanced the capacity of cefotaxime-treated neonatal rat serum to impair the survival of E coli O1:K1:NM (P < 0.05). Moreover, the intraperitoneal administration of C9 enhanced the survival of cefotaxime-treated neonatal rats that were septic with E coli (P < 0.05). These observations may contribute to the development of new strategies, such as augmentation of complement component serum concentrations, to reduce the morbidity and mortality of neonatal E coli sepsis.

Complement Component C9 Enhances the Capacity of Beta-Lactam Antibiotics to Kill Escherichia coli In Vitro and In Vivo

Complement component C9 is required for rapid complement-mediated killing of Escherichia coli. In this report, the influence of supplemental C9 on the bactericidal and protective effects of beta-lactam antibiotics in neonates was assessed. By rocket immunoelectrophoresis, the intrinsic C9 concentrations of pooled serum from both human and rat neonates was less than 20% of adult levels. Supplemental C9 purified from human plasma enhanced the capacity of ampicillin-treated serum from human neonates to impair the survival of E coli O7:K1:NM (P < 0.02). Similarly, supplemental C9 enhanced the capacity of cefotaxime-treated neonatal rat serum to impair the survival of E coli O1:K1:NM (P < 0.05). Moreover, the intraperitoneal administration of C9 enhanced the survival of cefotaxime-treated neonatal rats that were septic with E coli (P < 0.05). These observations may contribute to the development of new strategies, such as augmentation of complement component serum concentrations, to reduce the morbidity and mortality of neonatal E coli sepsis.

The administration of complement component C9 enhances the survival of neonatal rats with Escherichia coli sepsis.

To determine the significance of neonatal C9 deficiency, an animal model was developed in the rat. By rocket immunoelectrophoresis, the concentration of C9 in pooled adult rat serum was 224 +/- 7.2 microg/mL. In contrast, the concentration of C9 in pooled serum from 1-d-old rats was only 43 +/- 3.8 microg/mL and increased during the first 3 wk of life to 170 +/- 20 microg/mL. Similarly, the capacities of neonatal rat serum to kill two pathogenic strains of Escherichia coli and to lyse sensitized sheep erythrocytes were diminished compared with adult serum but increased during the first 3 wk of life. Supplemental human C9 significantly enhanced the bactericidal and hemolytic activity of neonatal rat serum. The capacity of neonatal rats to survive after the intrapulmonary injection of E. coli was positively correlated with the serum C9 concentration, bactericidal activity, and hemolytic activity. In 2-d-old rats infected with E. coli, the intraperitoneal administration of human C9 significantly enhanced survival and also enhanced the protective effect of intraperitoneal human IgG antibodies. The data indicate that C9 deficiency predisposed neonatal rats to invasion by E. coli. The neonatal rat appears to be a suitable model with which to investigate the significance of C9 deficiency.

Characterization of rat serum insulin-like growth factor-binding proteins by two-dimensional gel electrophoresis: identification of a potentially novel form.

The insulin-like growth factor-binding proteins (IGFBPs) in rat serum were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western ligand or immunoblotting. A ligand blot of adult female rat serum revealed at least 20 proteins that bound labeled IGF-I specifically. They ranged in size from about 24-50K, and their pI values ranged from approximately 5.5-8.0. Immuno- and ligand blots showed that IGFBP-3 appeared as a broad band composed of numerous individual spots of about 40-45K, with pI values ranging from about 5.5-7.7. Immunoblots showed that IGFBP-4 resolved into at least three spots of approximately 29K, with pI values of about 6.3-6.7 and an approximately 24K nonglycosylated form that usually appeared as a single spot with a pI of about 7, but occasionally it resolved into a doublet. Immunoblots of neonatal rat serum using antiserum to IGFBP-2 resulted in immunoreactivity of two sets of proteins: approximately 33K forms composed of two proteins and approximately 30K forms comprising at least five different variants with pI values of about 7.0-7.5. However, ligand analysis showed that only the 30K forms had binding activity. IGFBP-1, -5, and -6 were not detectable by immunoblotting. However, a set of about 29K IGFBPs with pI values of approximately 5.9, which appears in significant amounts in the serum of pups and diabetic rats and in placental tissue-conditioned medium, has been tentatively identified as IGFBP-1 by ligand blotting. One finding of particular interest is an approximately 50K BP with a pI of about 6 that comigrates with IGFBP-3. However, unlike IGFBP-3, which is glycosylated and undergoes proteolysis during gestation, this approximately 50K IGFBP is not susceptible to endoglycosidase-F treatment and persists throughout pregnancy. Immunoblotting analysis revealed weak cross-reactivity between the approximately 50K IGFBP and antiserum to IGFBP-4. These results show that the rat serum IGFBPs are more heterogeneous than was previously realized. The two-dimensional system will allow changes in these different forms to be evaluated critically in different physiological and experimental states.

Differential removal of insulin-like growth factor binding proteins in rat serum by solvent extraction procedures

Solvent extraction of serum and other biological fluids at an acidic pH is a convenient method to remove the insulin-like growth factor binding proteins (IGFBPs); however, an incomplete removal of IGFBPs can occur and this can potentially interfere with the radioimmunoassay of insulin-like growth factors (IGFs). This study compared the removal of IGFBPs from normal adult rat serum and 5-day old neonatal rat serum by acid-gel filtration, and three solvent extraction methods, i.e., acid-ethanol (AE), acid-cryo-ethanol (ACE) and formic acid-acetone (FAA) treatments by western ligand blotting and slot-blotting analysis. In adult rat serum all three extraction methods removed nearly 75% of total IGFBPs present. For the neonatal serum, AE and FAA were very inefficient in eliminating the IGFBPs, while ACE was somewhat better, as it removed nearly 30% of IGFBPs. Ligand blots of extracted samples showed that IGFBPs of lower size range, 24 to 32 kDa (IGFBP-4, IGFBPs-1 and -2), were resistant to solvent extraction. Acid-gel filtration, in contrast, eliminated > 95% of IGF-binding components in both sera. Determination of IGF-I concentrations in samples after gel filtration and extraction methods revealed lower IGF-I values in neonatal serum in acid extracted samples. These data caution against using solvent extractions for IGFBP removal in fetal/neonatal serum.

Evidence for pituitary regulation of somatic growth, insulin-like growth factors-I and -II, and their binding proteins in the fetal rat.

We investigated pituitary regulation of late-gestation fetal growth in the spontaneous dwarf rat, a strain with an autosomal recessive mutation (gene symbol dr) in the growth hormone (GH) gene resulting in complete isolated GH deficiency. GH-normal/GH-deficient (Dr/dr) females were crossed with Dr/dr or dr/dr males, producing both GH-deficient and GH-normal fetuses within the same litter. Pups were killed within 3 h after birth to approximate the developmental state of a late-gestation fetus. The body weight of GH-deficient fetuses was inhibited by 14% in comparison to GH-normal animals, but tail length remained unaffected. The brain and lungs were the only organs whose growth appeared to be pituitary-independent. Other organs showed moderate pituitary dependence in proportion to body weight. Serum IGF-I and IGF-II were reduced by 73% and 52%, respectively, in the absence of GH. The major IGF-binding proteins (IGFBP) were analyzed by Western ligand blot. The predominant 26- to 30-kD IGFBP band normally seen in neonatal rat serum was greatly increased in GH-deficient sera, to 250% of GH-normal sera as measured by densitometry. However, addition of alpha-Hec 1 antibody to IGFBP-2, which has been used to identify IGFBP-2 as the major neonatal IGFBP, resulted in immunoprecipitation of only a small amount of the 26- to 30-kD band from the GH-deficient fetuses, suggesting the presence of an additional IGFBP. Northern analysis of GH-deficient livers did not reveal any visible increase in IGFBP-1, IGFBP-2, or IGFBP-4 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of myosin and overall protein degradation in mouse C2 skeletal myotubes

We compared the breakdown of total cellular protein with that of the contractile protein, myosin, in cultured C2 mouse skeletal myotubes. The degradation of long-lived cellular proteins (which comprise the vast majority of myotube proteins) was inhibited by serum, insulin, and rat insulin-like growth factor-2. A physiological concentration of insulin was effective, but most of the effect of insulin occurred at concentrations well above the physiological range. IGF-2 inhibited protein breakdown at concentrations well within the range of total IGF-2 known to be present in the serum of fetal and neonatal rats. The breakdown of short-lived proteins was not altered by insulin or serum. We measured myosin degradation using a monoclonal antibody directed against myosin heavy chain. The half-life of myosin was 27 hours, and myosin breakdown was not altered by serum withdrawal applies to certain proteins, but not to others.

The E-domain peptide of rat pro-insulin-like growth factor II (proIGF-II): Properties of the peptide in serum and production by rat cell lines

We previously identified a naturally occurring peptide fragment derived from the carboxyl terminal region of the E-domain of pro-insulin-like growth factor II (proIGF-II 117–156) in medium conditioned by cultured BRL-3A rat liver cells. In the present study we utilized a radioimmunoassay (RIA) for this peptide to measure physiological concentrations of the peptide in media and serum. Serum levels of the E-domain peptide were very high in the 5-day neonatal rat and declined thereafter to reach low levels in adult rat serum. Chromatography of adult rat serum on Sephadex G-75 in 1 M acetic acid yielded a single broad peak of E-peptide immunoreactivity that coeluted with a synthetic E-peptide standard. However, chromatography of day 5 neonatal rat serum on Sephadex G-75 yielded two peaks of immunoreactivity. One of the peaks coeluted with a synthetic E-peptide standard, whereas the other peak eluted in a region where higher molecular weight proteins typically elute. Experiments aimed at determining whether adult rat serum contained a binding protein for the E-domain peptide revealed that: (1) serum contains little, if any, binding protein for the E-domain peptide, (2) serum contains a proteinase activity that degrades the E-domain peptide, and (3) the proteinase activity can be eliminated by acetic acid/ethanol extraction. Of several rat cell lines tested (BRL-3A, rat embryo fibroblasts (REF), hepatoma cell lines (H4, HTC), GH 3 pituitary tumor cells, and normal rat kidney fibroblasts (NRK)), only BRL-3A and REF cells secreted measurable E-domain peptide into the medium. In addition, it was found that some component(s) of serum could stimulate secretion of E-domain peptide from BRL-3A and REF cells. Chromatography of the immunoreactivity from BRL-3A and REF-conditioned media on Sephadex G-75 in 1 M acetic acid yielded a single peak that coeluted with a synthetic E-domain peptide standard. Since secretion of the E-domain peptide parallels the expression of IGF-II, the RIA for the proIGF-II E-domain peptide may be useful for studies of the biosynthesis and secretion of IGF-II under different physiological conditions. The RIA for the IGF-II E-domain peptide has two technical advantages over the RIA for IGF-II, namely, the lack of interference by IGF binding proteins and the relative ease with which large quantities of pure antigen can be synthesized.

Tissue, Developmental, and Metabolic Regulation of Messenger Ribonucleic Acid Encoding a Rat Insulin-Like Growth Factor-Binding Protein*

Insulin-like growth factor-II (IGF-II) is the predominant insulin-like growth factor in fetal and neonatal rat serum and tissues. In serum, it occurs complexed to a 30-kDa nonglycosylated IGF-binding protein (IGFBP) that is immunologically related to the IGFBP in BRL-3A rat liver cells (rIGFBP-2). Levels of rIGFBP-2 and IGF-II decrease in rat serum after birth. Using a recently isolated cDNA clone for rIGFBP-2 as hybridization probe, we now compare the expression of rIGFBP-2 and IGF-II in fetal tissues and the effects of hypophysectomy and fasting on the abundance of these mRNAs in adult rat liver. rIGFBP-2 mRNA is expressed at high levels in term gestation liver and at lower levels in other tissues. The ratio of rIGFBP-2 to IGF-II mRNAs in stomach, kidney, and lung is similar to that seen in liver, whereas IGF-II mRNA is more abundant than rIGFBP-2 mRNA in muscle, intestine, heart, and skin. Both mRNAs are more abundant in fetal tissues than in the corresponding tissues from adult rats. Dexamethasone treatment of 4-day-old rats for 4 days caused a greater (90%) decrease in hepatic IGF-II mRNA than in rIGFBP-2 mRNA (50%), suggesting subtle differences in the developmental regulation of the two mRNAs. Even more striking differences were observed in the regulation of the two mRNAs in adult rats after hypophysectomy or fasting. Hepatic rIGFBP-2 mRNA was increased 10- to 20-fold compared to age-matched control rats, whereas IGF-II mRNA was not increased. A parallel increase in serum rIGFBP-2 was observed, suggesting that this regulation may result at least in part from the increased abundance of rIGFBP-2 mRNA. Thus, in addition to modulating the stimulation of growth and differentiation by IGF-II in fetal tissues, rIGFBP-2 may play a homeostatic role during catabolic states in the adult rat.

An IgA monoclonal antibody directed against type III antigen on group B streptococci acts as an opsonin.

We have investigated the mechanisms by which a murine IgA mAb directed against the type III Ag (IgA anti-III mAb) of group B streptococci (GBS) protects neonatal rats from lethal infection with these organisms. Purified IgA anti-III mAb enhanced phagocytosis of type III GBS by rat peritoneal macrophages in vitro by fourfold compared with phagocytosis of buffer-treated GBS. In the absence of antibody, neonatal rat serum did not promote phagocytosis, but addition of neonatal rat serum to GBS opsonized with IgA anti-III led to a sevenfold increase in phagocytosis. Heat inactivation of C destroyed the ability of neonatal rat serum to enhance phagocytosis in the presence of IgA. C3 deposition was observed when GBS coated with IgA anti-III mAb were incubated in untreated neonatal rat serum or in serum treated with Mg/EGTA. This latter observation suggested that C3 deposition occurred through activation of the alternative pathway. The control IgA mAb MOPC 315 did not enhance GBS ingestion or C3 deposition on GBS. Depletion of C in vivo by using cobra venom factor abolished the protective effect of IgA anti-III mAb in the neonatal rat model. These data suggest that the ability of this IgA to activate C further enhances its opsonic activity and may be essential for its protective effect in vivo.

Ontogeny of Serum Insulin-Like Growth Factor Binding Proteins in the Rat*

Insulin-like growth factors (IGF-I and -II) are peptide growth factors that may be important for neonatal development. Specific high affinity IGF binding proteins (BPs) have been characterized in serum and extracellular fluids. The major serum binding complex in the adult has an apparent Mr of 150 K, while the predominant BP in the neonate is approximately 30 K. In the rat, the transition from the neonatal BP to the adult form occurs during the third postnatal week, concomitant with an increase in serum IGF-I and a decrease in serum IGF-II concentrations. Using specific RIAs and Western ligand blot analyses we have characterized the changes in serum IGF and IGF BPs, respectively, during the early postnatal period. Seven BPs were identified in serum with apparent Mr values of 42, 41, 40, 38, 28, 26, and 22 K. After deglycosylation, the 42, 41, 40, and 38 K BPs were reduced to two bands with apparent Mr values of 35 and 32 K, while the 28, 26, and 22 K BP were unchanged. In the neonate, the 28, 26, and 22 K BPs were present, with the 28 K BP in highest concentration. With increasing age, the 28 K BP decreased and the 42, 41, 40, and 38 K BPs appeared at approximately 19 days of age. Comparison of Western ligand blots of neonatal serum, BRL-3A conditioned media, rat amniotic fluid, and rat cerebrospinal fluid (CSF) demonstrated that all contained a prominent 28 K BP. A polyclonal antibody (alpha Hec 1) developed against the 31 K human IGF-BP (hBP-31) immunoprecipitated the 28 K BP from neonatal rat serum, BRL-3A media, rat amniotic fluid, and rat CSF, but did not react with adult rat serum. These findings suggest that, in the rat, the predominant neonatal serum BP is structurally and immunologically similar to the major BRL-3A, amniotic fluid, and CSF BPs, but distinct from the predominant adult serum BP.

The Effects of Altered Nutritional Status upon Insulin-Like Growth Factors and their Binding Proteins in Neonatal Rats

To investigate the role of nutrition in the regulation of IGFs during the perinatal period, 10-d-old rats were infused intravenously with various concentrations of nutrients for 24 h. Breast-fed litter mates served as controls. The effect of caloric intake on concentrations of IGF-I and IGF-II as well as IGF-binding proteins in serum, liver, and brain of neonatal rats was studied. A total of 45 rats from 10 litters was infused with solutions ranging from a caloric intake of 0 (saline) to 75% (glucose, amino acids, and lipids) of the estimated intake of control rats. In serum, both IGF-I and -II concentrations fell markedly in response to fasting. Serum IGF-II levels were linearly related to caloric intake in the pooled data from all groups. Concentrations of IGF-II, but not IGF-I, in liver and brain were depressed by caloric restriction. In contrast to the fall in IGF concentrations, activity of IGF-associated binding proteins rose in serum and in liver cytosol 2- to 4-fold in response to decreased nutrient intake. In serum, but not liver, the rise in binding protein activity was inversely related to to caloric intake. In liver, cytosol, but not serum, the rise in binding protein activity was inversely related to total serum amino acid concentration. Thus, IGF concentrations in preweanling rats change in response to alterations of nutrient intake. The fasting induced decrements in IGF levels, as well as the elevations in IGF-associated binding protein activity, may serve as a protective mechanism to depress growth in times of caloric restriction.

Immunologic regulation of E. coli K1 by serum from neonatal rats is enhanced following intraperitoneal administration of human IgG.

The effect of 1500 mg/kg intraperitoneal human IgG on the capacity of neonatal rat serum to opsonize and to kill Escherichia coli K1 and to deposit IgG and C3 onto this organism was investigated. Unlike serum from neonatal rats injected with albumin, serum from neonatal rats injected with human IgG opsonized and killed E. coli K1 efficiently. Heat treatment abolished the bactericidal effect of serum from IgG recipients, suggesting a role for complement. A radioimmunobinding assay demonstrated that the capacity of neonatal rat serum to deposit IgG and C3 onto E. coli K1 was impaired. However, intraperitoneal human IgG enabled serum from neonatal rats to deposit human IgG onto the bacteria and enhanced C3 deposition to a level equivalent to that observed with adult rat serum. Therefore, neonatal rats have a functionally competent classic pathway of complement. Human IgG ameliorates the opsonic and bacteriolytic inadequacies of neonatal rat serum that result primarily from a deficiency of antibodies to E. coli.