Longitudinal study of intracellular T cell cytokine production in infants compared to adults.

Intracellular cytokine production in lymphocytes obtained longitudinally from 325 healthy infants aged 2-12 months was compared with adult lymphocytes using four-colour flow cytometry. Peripheral blood samples (180 microlitres) were stimulated with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A to induce production and intracellular accumulation of cytokines. The method was validated by assessing reproducibility, repeatibility, ruggedness (i.e. fresh versus day-old blood samples), precision, linearity and sensitivity. Among infants, the number and percentage of T lymphocytes (helper/inducer T cell subsets and cytotoxic/suppressor T cell subsets) producing IFN-gamma (type 1) and IL4 (type 2) increased over the first year of life but remained significantly lower than levels found in adults. In both infants and adults more CD4- T cells than CD4+ T cells were induced to make IFN-gamma. Infant Th1/Th2 ratios revealed modest Th1-skewed (predominant) profiles compared to adults, which were 5-10 times higher. Infant Tc1/Tc2 ratios revealed Tc1-skewed responses which were equal to adult ratios by age 12 months. At 12 months infant Th2 responses were closer to adult levels than were Th1 cells. Intracellular cytokine detection by flow cytometry is a rapid, sensitive, rugged and precise method to characterize immune status changes over time.

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.

Prevention of human rotavirus-induced diarrhea in gnotobiotic piglets using bovine antibody.

The efficacy of passively administered bovine antibody for preventing human rotavirus (HRV)-induced diarrhea was investigated using a gnotobiotic pig model. Cows were immunized with inactivated HRV serotypes 1 (Wa) and 2 (S2) and simian rotavirus serotype 3 (SA11), and immune colostrum and milk were collected. Antibody concentrates derived from these materials were fed to germ-free piglets that were subsequently inoculated with HRV Wa. Both viral shedding and diarrhea were effectively reduced or eliminated in a dose-dependent manner as a result of HRV immune antibody feeding. A quantitative virus-neutralizing (VN) antibody method permitted assessment of the functional antibody dose required to achieve a 50% reduction of disease (PD50). PD50 dose levels of 15.8 and 19.5 x 10(6) VN antibody units were determined for inhibition of diarrhea and viral shedding, respectively. Studies reported here provide new information on the quantitative relationship between protective antibody dose and diarrheal disease response.

Distinctive characteristics of crude interferon from virus-infected guinea-pig embryo fibroblasts.

Crude interferon preparations from primary guinea-pig embryo cells infected with vesicular stomatitis virus strain T1026R1 were shown to be more sensitive to heat (37 degrees C), pH 2.0, and SDS than crude mouse interferon. Since the proportion of antiviral activity lost after each treatment was nearly the same, the existence of a single fraction of antiviral activity sensitive to all three treatments was suggested. Support for this possibility was given by the finding that subjecting this guinea-pig interferon to any one of the treatments rendered it insensitive to the effects of the other two.

Improved conditions for the production and detection of interferon from guinea pig embryo cells.

Primary guinea pig embryo (GPE) fibroblasts were assessed as potential sources of guinea pig interferon (IFN). GPE cells proved to be excellent in vitro producers of guinea pig IFN, although the actual amounts produced were only detectable when sample irradiation under ultraviolet light (to inactivate inducing viruses) was substituted for overnight sample treatment at pH 2. Thus, the rapid spontaneous inactivation of large proportion of the antiviral activity after overnight exposure to 4 degrees C, regardless of pH, was avoided. IFN was induced using Newcastle disease virus (NDV), Sindbis virus, and a genetic variant of vesicular stomatitis virus, VSV T1026R1. Each virus exhibited different dose response kinetics, with VSV T1026R1 proving the most efficient inducer of the three. Optimal IFN production depended largely on virus multiplicity and cell age. All the antiviral activity produced by GPE fibroblasts had the classical properties of species specificity, susceptibility to trypsin, and a broad range of antiviral activity.

Host restriction property of a vesicular stomatitis virus mutant isolated from carrier cultures.

Carrier cultures of L cells infected with wild-type vesicular stomatitis virus (VSV0) were initiated without the use of defective-interfering particles or homologous interferon. The cloned viruses recovered from such carrier cultures after passage 21 were characterized as temperature-sensitive. Furthermore, these clones of the mutant showed restricted replication at permissive temperature in HEp-2 cell cultures as compared to the wild-type VSV0. This restrictive replication of the mutant in HEp-2 cells was not due to a defect in the expression of virion-associated primary transcriptase activity in vivo, but due to the marked reduction in virus-specific characterized may govern the synthesis of mutant virus-specific amplified RNA in HEp-2 cells.

A sensitive method for quantification of vesicular stomatitis virus defective interfering particles: focus forming assay.

A focus-forming assay for the quantification of defective interfering (DI) particles of vesicular stomatitis virus (VSV) is described. This assay is based on the procedures described for lymphocytic choriomeningitis (LCM) virus by Popescu et al. (1976). Under appropriate conditions this focus-forming assay can quantify fewer than 100 DI particles/ml in a preparation containing a large number of infectious particles.

Interferon induction by viruses. VI. Reovirus: virion genome dsRNA as the interferon inducer in aged chick embryo cells.

The interferon-inducing particle (IFP) activity of avian and human reoviruses in aged chick embryo cells was determined by analyzing dose (multiplicity)-response (interferon yield) curves. These curves fit best a model in which each cell infected with greater than or equal to 1 IFP produces a quantum yield of interferon. Avian reovirus stocks contained as many as 60 times more IFP than plaque-forming particles (PFP). Upon UV-irradiation the ratio of IFP:PFP became 197, suggesting that virtually every physical particle of avian reovirus could function as an interferon-inducing particle. Thus, about one-third of the non-infectious particles were intrinsically IFP and the other two-thirds could be converted to IFP status at an optimal dose of UV radiation, the equivalent of 9.4 lethal hits, i.e., 8000 ergs/mm2. UV-irradiated avian reovirus induced about twice the usual yield of interferon on a per cell basis. Wildtype human reovirus (type 3) and mutants ts201(A,RNA+) and ts447(C,RNA-) were excellent inducers of interferon, but only about 1 in 3 infectious particles functioned as an interferon-inducing particle, meaning that virtually all physical particles failed to function as IFP, UV-irradiation of human reoviruses resulted in a slight loss of IFP activity. Our data support the hypothesis that virion genome dsRNA constitutes the interferon inducer moiety of avian reoviruses and that in its permissive host cell the processing of genome dsRNA from most particles to a putative recognition site in the cytoplasm occurs naturally with a high probability. For human reovirus this is a much rarer event which may be intrinsic only to infectious virus, and may require limited transcription for expression. The sensitivity of the avian reovirus-aged chick embryo cell system recommends it for further study on the mechanism of interferon induction by virions containing pre-existing dsRNA.