Variability of human milk neutral oligosaccharides in a diverse population.

BACKGROUND: A complex array of free oligosaccharides is a distinctive compositional feature of human milk. Although these oligosaccharides have been studied for several years, their variability and distribution have not been systematically studied, and their nutritional and functional roles have not been elucidated. This report describes a study in which a large number of human milk samples were analyzed for the presence and content of nine neutral oligosaccharides. The resultant data were used to probe for distribution trends by donor groups and stage of lactation. METHODS: Milk samples from 435 women residing in 10 countries were analyzed using a simple preparation procedure, gel filtration, and high-performance anion-exchange chromatography. RESULTS: All samples contained structures based on lacto-N-neotetraose and lacto-N-tetraose. This contrasts with the fucosyloligosaccharides tested, none of which was detected in 100% of the samples. Unexpected distribution trends were observed. For example, 100% of the samples from Mexico (n = 156) contained 2'-fucosyllactose, whereas only 46% of the samples from the Philippines (n = 22) contained this structure. Concentration ranges for the analyzed oligosaccharides revealed quantitative and qualitative distribution trends. CONCLUSIONS: The oligosaccharide composition of human milk varied among samples. The geographical origin of the donors was one of the factors that accounted for this variability. This can be explained by genetically determined traits that are not uniformly distributed. Results indicated that further systematic studies are needed to ascertain the effect of other factors, such as lactation stage or diet.

Modulation of the immune system by human milk and infant formula containing nucleotides.

OBJECTIVE: To determine whether human milk and nucleotides added to infant formula at levels present in human milk enhance development of the immune system during infancy. METHODS: A 12-month, controlled, randomized and blinded, multisite feeding trial was conducted on two infant formulas: iron-fortified, milk-based control formula (Control) or the same formula fortified with nucleotides (Nucleotide). The level (72 mg/L) and ratio of individual nucleotides selected were patterned after those available in human milk. A third group fed human milk exclusively for 2 months and then human milk or Similac with iron until 12 months of age also was studied. Response to immunizations was chosen to assess development of the immune system. Infants followed the immunization schedule recommended by the American Academy of Pediatrics in 1991. OUTCOME VARIABLES: Antibody responses were determined at 6, 7, and 12 months of age to Haemophilus influenzae type b polysaccharide (Hib), to diphtheria and tetanus toxoids, and to oral polio virus (OPV) immunizations. RESULTS: Of 370 full-term, healthy infants enrolled, 311 completed the study (107 Control, 101 Nucleotide, 103 human milk/Similac with iron). Intake, tolerance, and growth of infants were similar in all three groups. Compared with the Control group 1 month after the third immunization (7 months of age), the Nucleotide group had a significantly higher Hib antibody concentration (geometric mean concentrations of 7.24 vs 4.05 micrograms/mL, respectively), and a significantly higher diphtheria antibody concentration (geometric mean of 1.77 vs 1.38 U/mL). The significantly higher Hib antibody response in the Nucleotide group persisted at 12 months. The antibody responses to tetanus and OPV were not enhanced by nucleotide fortification. There also was an effect of breastfeeding on immune response. Infants who breastfed had significantly higher neutralizing antibody titers to polio virus than either formula-fed group (1:346 vs 1:169 and 1:192 in the Control and Nucleotide groups, respectively) at 6 months of age. CONCLUSION: Infant formula fortified with nucleotides enhanced H influenzae type b and diphtheria humoral antibody responses. Feeding human milk enhanced antibody responses to OPV. Dietary factors play a role in the antibody response of infants to immunization.

Differential absorption of amoxicillin from the human small and large intestine.

Differences in extent of amoxicillin absorption from various regions of the gastrointestinal tract were determined and compared with the same dose administered orally. Nine healthy men were intubated at a proximal (duodenum or jejunum) or distal (ileum or colon) site with use of a 15-foot double lumen nasointestinal tube. Amoxicillin solutions (375 mg in 120 ml water) were delivered on 2 successive days as a bolus or a 4-hour infusion. Subjects were reintubated at another site and amoxicillin administration was repeated. Subjects with colonic intubation received only infusions. Finally, all subjects received an oral dose of amoxicillin solution. Plasma samples were obtained at 16 time points over a 10-hour period and assayed for amoxicillin by use of an HPLC method. Area under the concentration-time curve and the maximum plasma concentration were computed to evaluate amoxicillin absorption. Amoxicillin absorption was rate and site dependent in the gastrointestinal tract. The drug was well absorbed in the duodenum and jejunum, with no significant differences in absorption when administered as a bolus or 4-hour infusion, but absorption was decreased and rate dependent in the ileum, where more drug was absorbed as an infusion compared with a bolus. Amoxicillin was unabsorbed when infused in all colonic regions.

Human immune response to cationized proteins. I. Characterization of the in vitro response to cationized diphtheria toxoid.

Cationization of proteins, i.e., increasing net positive charge by the substitution of carboxyl groups with positively charged residues, has been reported to enhance protein immunogenicity in animal model systems. In the present study, we have investigated the effect of cationization on the in vitro cell-mediated immune response of human mononuclear cells to diphtheria toxoid. A series of cationized DT preparations were generated by covalent modification with ethylenediamine, with pIs ranging from 4.6 to > 9.3, and tested for their ability to induce proliferation of normal human peripheral blood mononuclear cells. Cationized DT (cDT) was found to induce an antigen-specific, augmented proliferative response, relative to native antigen, which was directly proportional to the degree of cationization. Further characterization of the response to cDT demonstrated that (1) proliferative responses could be detected considerably earlier, and typically at much lower antigen concentrations, than the response to native DT; (2) the response was dependent on HLA-DR; (3) production of a number of cytokines, sp. IL-1 beta, IL-2, and IFN-gamma, was also elevated in cDT-stimulated cultures; and (4) the enhanced proliferative response to cDT could be attributed to CD4+ helper T cells. These results demonstrate that cationization of proteins enhances the ability to generate a cell-mediated immune response in humans and suggest that cationization may have utility in the design of more effective carrier proteins for human vaccines.

Human immune response to cationized proteins. II. Characterization of interaction of cationized diphtheria toxoid with human mononuclear cells.

Cationized diphtheria toxoid (cDT) has previously been shown to be more effective than the native protein as an inducer of human antigen-specific T cell responses. In the present study, biotin-labeled antigen and flow cytometric analysis were used to examine the possibility that enhanced immunogenicity of cDT may be a consequence of preferential binding to antigen-presenting cells. Strong binding of cDT, relative to native antigen, was noted for both monocytes and B cells. Characteristics of binding were similar for both cell types, including rapid saturation, temperature independence, and inhibition by unlabeled cationized proteins. Although both B cells and monocytes bound cDT, only monocytes were effective in triggering T cell proliferation, possibly as a result of slow internalization of bound antigen by B cells. Definition of the target structures of cationized proteins may allow for the design of more efficient vaccines, which would be specifically targeted to antigen-presenting cells in vivo.

Antibiotic management of complicated appendicitis.

A prospective, randomized, double-blind clinical trial was undertaken comparing gentamicin, ampicillin, and clindamycin (GAC) to gentamicin, ampicillin, and placebo (GAP) in children with complicated appendicitis. Of the 64 patients enrolled in this study, 33 were assigned to the GAC group and 31 received GAP. A single GAC patient (3%) was considered a therapeutic failure in comparison to seven children (23%) in the GAP group (P less than 0.05). Duration of fever was significantly prolonged in the GAP patients (4.7 +/- .8 days versus 2.9 +/- .5 days) when compared to the clindamycin treated children (P less than 0.05). Duration of leukocytosis was 3.2 +/- .4 days for GAC patients and 4.9 +/- .9 days for those on the GAP protocol (P = 0.08). On the basis of this experience the routine use of gentamicin, ampicillin, and clindamycin is recommended for all children with complicated appendicitis.

Identification of long chain dicarboxylic acids in the serum of two patients with Reye's syndrome.

Sera from two patients with Reye's Syndrome were analysed by computerized capillary gas chromatography--mass spectrometry profiling techniques. The most striking abnormalities were the accumulation of long chain dicarboxylic acids. Four saturated dicarboxylic acids (dodecanedioic, tetradecanedioic, hexadecanedioic, and octadecanedioic), and six unsaturated long chain dicarboxylic acids (dodecenedioic, tetradecenedioic, tetradecadienedioic, hexadecenedioic, octadecadienedioic, and octadecenedioic) were identified. The C16 and C13 dicarboxylic acids have never been reported for Reye's Syndrome or any other dicarboxylic acidemias. The data might reflect marked increase of extramitochondrial omega-oxidation of long chain fatty acids or impaired metabolism of omega-dicarboxylic acids formed in Reye's patients.

Decreased absorption of cefaclor in short bowel syndrome.

Oral cefaclor is rapidly absorbed in patients with a shortened gastrointestinal tract (23 cm). A case is reported of a 27-month-old female child who had received oral cefaclor for five days in treatment of otitis media. The child had had a bowel resection at birth. She did not respond to oral therapy with cefaclor and was admitted to the hospital, where she received cefamandole intramuscularly. The patient recovered in five days. Further investigation using cefaclor with this patient demonstrated serum concentrations below the minimum inhibitory concentration of Hemophilus influenzae, pneumococcus, and many other organisms. When oral antibiotics are prescribed, it is necessary to monitor serum concentrations to ensure absorption.

Antibody response to herpes simplex virus type 1 polypeptides and glycoproteins in primary and recurrent infection.

Sequential sera from a patient with primary Herpes Simplex Virus type I (HSV-1) encephalitis and a patient with HSV-1 recurrent oral lesions were collected. Sera were analyzed quantitatively by radioimmunoassay and qualitatively by electrophoresis and autoradiography of immune precipitates to determine the sequence of antibody production to specific radiolabeled HSV-1 polypeptide and glycoprotein antigens. The major antibody response in both primary and recurrent sera was against HSV-1 envelope antigens and the major capsid polypeptide. Sequential sera showed a significant correlation between neutralizing antibody titers and quantitative antibody to HSV-1 glycoproteins. Qualitative electrophoretic analysis of primary infection sera showed sequential appearance of antibodies to increasing numbers of HSV-1 polypeptides by the fourteenth day of infection. A corresponding qualitative antibody response to glycoproteins was not seen. Sequential sera obtained before, during, or after a recurrent lip lesion in another patient showed no significant quantitative or qualitative changes in antibodies to either HSV-1 glycoproteins or polypeptides.

Effect of intravenous flow rate and injection site on in vitro delivery of chloramphenicol succinate and in vivo kinetics.

The delivery rates of chloramphenicol succinate from a standard pediatric intravenous infusion set were studied in vitro at varying flow rates and injection sites of the infusion set. The pharmacokinetic properties of CAPS and chloramphenicol were then studied in 15 children given intravenous injections of CAPS via the infusion set at the flashball and Buretrol sites in a crossover fashion on successive days. In vitro, the actual times required for 95% delivery of CAPS from the infusion set were two- to fourfold longer than the predicted infusion times at flow rates of 5, 15, and 29 ml/min and at all three available injection sites. In vivo, flashball injections vs Buretrol injections resulted in significantly higher mean peak serum concentrations of CAPS and CAP, with peaks occurring significantly sooner after the beginning of the intravenous infusion. These results suggest a need for considering characteristics of CAPS infusion when monitoring and interpreting serum concentration values.

Glycerol: a review of its pharmacology, pharmacokinetics, adverse reactions, and clinical use.

Glycerol is a potent osmotic dehydrating agent with additional effects on brain metabolism. In doses of 0.25-2.0 g/kg glycerol decreases intracranial pressure in numerous disease states, including Reye's syndrome, stroke, encephalitis, meningitis, pseudotumor cerebri, central nervous system tumor, and space occupying lesions. It is also effective in lowering intraocular pressure in glaucoma and shrinking the brain during neurosurgical procedures. Hyperosmolality with rebound cerebral overhydration is of concern, especially in patients with altered blood brain barriers. They may be avoided if glycerol is administered on an intermittent rather than a continuous basis. Intravascular hemolysis does not occur with oral use. When administered intravenously, hemolysis can be minimized by using glycerol 10% in dextrose 5% with normal saline at rates of 6 mg/kg/min or less. However, intravenous doses of 1-2 g/kg every 2 hr can be administered safely in severe cases of elevated ICP. In such patients, glycerol serum concentration, serum osmolality and ICP monitoring are required to optimize glycerol therapy.

Survival following myocarditis and myocardial calcification associated with infection by Coxsackie virus B-4.

A Coxsackie virus B-4 was isolated from a neonate with clinical sepsis and clinical evidence of myocardial dysfunction. Radiographs demonstrated the presence and subsequent resolution of myocardial calcification. Electrocardiograms showed a left bundle branch block, presumably caused by calcification in the corresponding segment of the bundle of His. This case demonstrates an unusual complication of neonatal Coxsackie virus B infection.

Variations in glycerol kinetics in Reye's syndrome.

Glycerol kinetics and adverse affects were studied in nine patients with Reye's syndrome. Glycerol was given by continuous infusion over 2 hr, half over the first 0.5 hr and the remainder over the next 1.5 hr. The dose was adjusted to keep intracranial pressure less than or equal to 15 mmHg. At steady state, serial blood samples were collected during glycerol infusion and analyzed by an enzymatic assay specific for glycerol. At 0.75 to 1.75 gm/kg/2 hr glycerol doses, the serum levels ranged from 1.48 to 5.83 mg/ml. Total body clearance ranged from 1.99 to 5.1 ml/kg/min. Glycerol clearance was not related to serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), or serum ammonia levels. Glycerol provided effective control of intracranial pressure in all patients. Temporary elevation of serum creatinine and blood urea nitrogen (BUN) and presence of hemolysis in two patients, appeared to be related to glycerol.

Bone scan patterns in acute osteomyelitis.

Sixteen patients with acute hematogenous osteomyelitis underwent serial Tc-99m-diphosphonate bone scanning on admission and at intervals of four to seven days thereafter. Scans were analyzed by computer-assisted determination of the ratio of activity in the lesion to that in a corresponding area of normal bone. Two patterns were observed. In seven children this ratio was low initially and increased during hospitalization despite clinically adequate therapy. In nine children the ratio was high initially and decreased during therapy. Lack of focal uptake within bone on the initial scan performed within the first three days of the onset did not exclude the diagnosis of osteomyelitis.

Acute septic arthritis caused by Neisseria meningitidis serogroup W-135.

Neisseria meningitidis, serogroup W-135 was isolated from blood cultures of a 22-month-old child with acute septic arthritis of the left knee. Recovery was complete after treatment with penicillin G. Acute septic arthritis may be the initial presentation of disease caused by Neisseria meningitidis serogroup W-135.