Ribonucleotides: conditionally essential nutrients shown to enhance immune function and reduce diarrheal disease in infants.

It remains a goal of pediatric nutrition to provide optimal nourishment for infants who are not fed human milk. Investigators have attempted to emulate the composition and functionality of human milk, the gold standard for infant nutrition. These efforts began with the analysis of milk components and continued with assessments of biological effects that culminated in clinical studies in infants. This chapter summarizes the path that researchers followed to study ribonucleotides and their role in infant nutrition. Based on analytical methods for the quantification of ribonucleotides in human milk, investigators assessed their potential impact on the immune systems of infants and looked for concomitant mechanistic explanations. These inquiries evolved into clinical trials in which ribonucleotide-supplemented formula performance was compared with that of non-supplemented formulas and with human milk. This chapter intends to summarize an area of pediatric nutrition that has yielded both enlightening evidence and seemingly contradictory data.

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.

Bis(bioreductive) alkylating agents: synthesis and biological activity in a nude mouse human carcinoma model.

Chemical investigations leading to the construction of bis(bioreductive) alkylating agents having both conformationally restricted and mobile spacer regions are described. Two targets having the conformationally mobile ethylene spacer group, namely, 2,2'-ethylenebis[6-(hydroxymethyl)-p-benzoquinone] diacetate (3b) and 2,2'-ethylenebis[6-(bromomethyl)-p-benzoquinone] (3c), were studied in vivo and in vitro using an established epithelial/Burkitt lymphoma hybrid cell line (D98/HR1) previously shown to induce carcinomas in nude mice. Inactivity of both test compounds in vitro, the relative resistance of these cells to test drugs in vitro, and the selective antitumor properties of the bis(bromomethyl) analogue in vivo lead to the proposal that this compound undergoes bioreduction to an alkylating species in the hypoxic core of the tumor, thereby exerting its action.

Induction of retinal degeneration in cats by methylnitrosourea and ketamine hydrochloride.

Persistent mydriasis seen in cats used in an oncology study apparently was not related to neoplasia. Ophthalmoscopically, the cats had severely atrophic retinas and clinically observable visual impairment. These findings were confirmed by electroretinographic and histologic examination. Cats with these retinal lesions had received combinations of methylnitrosourea, ketamine hydrochloride, and feline leukemia virus. Retinopathy was not seen in ketamine-anesthetized cats receiving feline leukemia virus. To test the nature of this phenomenon, four cats were given both drugs and three received methylnitrosourea alone. The four cats developed severe generalized retinal degeneration by day 5, whereas the three cats given methylnitrosourea alone had normal retinas. Histologic changes in the affected cats were extensive loss of rods and cones, and of the outer nuclear layer. The electroretinographic responses to white light were depressed or extinguished. Retinal degeneration, therefore, appeared to be dependent upon administration of both methylnitrosourea and ketamine hydrochloride.

Infection of feline embryo adherent cells with feline leukemia virus: feline oncornavirus-associated cell membrane antigen expression and morphologic transformation.

Feline embryo adherent cells were infected with the Richard or Kawakami-Theilen strains of feline leukemia virus (FeLV) and examined for feline oncornavirus-associated cell membrane antigen (FOCMA), viral group-specific antigen (gsa) production, and in vitro evidence of transformation. As early as 10 days after infection, when more than half of the infected cells were gsa positive, FOCMA was detected on 5-10 percent of the cells. Transitory morphologic alterations (epithelioid appearance and rounding) were first noted in most cultures around 20-30 days post infection. At this time, approximately 50% of the cells in infected cultures expressed FOCMA. Morphologic characteristics of transformed fibroblastic cells (rounded shape, disordered alignment, and low adhesion to substratum), as well as enhanced agglutinability by plant lectins and ability to grow in agar, were demonstrated in one of four FeLV-infected, FOCMA-positive cultures. Findings showed that FOCMA may be expressed in FeLV-infected monolayer cells independent of transformation as assessed by in vitro criteria.

The effects of methylnitrosourea on the immune system and hematopoietic system of adult specific pathogen free cats.

The effects of a single non-carcinogenic dose of 15 mg/kg methylnitrosourea (MNU) on the immune and hematopoietic systems of adult specific-pathogen-free (SPF) cats were determined. The cell-mediated-immune (CMI) system was markedly suppressed, as evidenced by: (i) Prolonged cutaneous allograft retention time (41-84 days); (ii) Decreased lymphocyte blast transformation response to mitogens (2% of pretreatment response to pokeweed mitogen or concanavalin A) and antigen (12% of untreated control cat response to keyhole limpet hemocyanin); (iii) Reduced number of absolute erythrocyte-rosetting T-cells in the peripheral blood. This immunosuppression lasted at least 3 months, the duration of the experiment. Suppression of the hematopoietic system was also noted as evidenced by: (i) Peripheral lymphopenia lasting 3 months and neutropenia lasting 3 weeks; (ii) Bone marrow hypocellularity lasting 3 weeks; (iii) Hypoplasia of neutrophilic precursors lasting 3 weeks and erythroid precursors lasting 4 days. It was concluded that a single non-carcinogenic dose of MNU induces a prolonged suppression of the CMI system and a brief suppression of hematopoiesis in adult SPF cats. The immunosuppression may in part be responsible for the previously observed increased susceptibility to feline leukemia virus infection and disease of adult SPF cats treated with MNU.

Enhancement of feline leukemia virus-induced leukemogenesis in cats exposed to methylnitrosourea.

The effect of methylnitrosourea (MNU), a potent resorptive carcinogen, was evaluated for its influence on the susceptibility of adult cats to infection and induction of oncornavirus disease by feline leukemia virus (FeLV). Young adult cats at an age previously demonstrated to be highly resistant to FeLV, were injected intravenously with moderately toxic doses (15-20 mg/kg) of MNU alone or with infections FeLV (Rickard strain). Following exposure to virus and chemical, cats were monitored for antibody to the feline oncornavirus-associated cell membrane antigen (FOCMA), viremia by direct infectivity and the presence of gsa in peripheral blood leukocytes, and for toxic effects of MNU by hemogram analyses on peripheral blood. Of 8 cats injected with MNU + FeLV, 6 developed persistent viremia, 5 of which became debilitated from thymic lymphoma. Only 1 of 6 non-MNU-treated and infected cats of the same age became transiently viremic. FOCMA antibody development was markedly depressed in MNU + FeLV inoculated cats compared with cats inoculated with FeLV alone. Results show that MNU was apparently responsible for the obliteration of age-related susceptibility in cats to FeLV infection and induction of FeLV-related disease, and suggest that in nature exposure to toxic chemical carcinogens may act as factors which determine susceptibility to feline oncornaviruses in the cat.

Immunosuppressive properties of a virion polypeptide, a 15,000-dalton protein, from feline leukemia virus.

The 15,000-molecular-weight polypeptide (p15) of feline leukemia virus (FeLV) was shown to impair normal lymphocyte function in vitro and to abrogate immunity to feline oncornavirus disease in vivo. FeLVp15 suppressed concanavalin A-induced blast transformation of normal feline lymphocytes by 68%, while other virion proteins had no effect. p15 suppression was not due to toxicity, nor was p15 a competitive inhibitor of concanavalin A binding. Capping of receptors for concanavalin A on normal feline lymphocytes also was inhibited by either inactivated FeLV or FeLV p15. Groups of cats were immunized with either killed feline oncornavirus-associated cell membrane antigen bearing tumor cells or tumor cells plus FeLV p15. After challenge with feline sarcoma virus, three of four p15-treated cats developed progressive fatal fibrosarcoma as compared to one of five non-p15-treated cats. The cats receiving p15 also had lower cytotoxic antibody titers against feline oncornavirus-associated cell membrane antigen (mean peak titer, 1:6) than did the non-p15 group (1:74). These data support the hypothesis that the immunosuppression in cats infected with FeLV is mediated by FeLV p15.

Modifications of the immunofluorescence assay for feline leukemia virus group-specific antigens.

Observations and minor modifications are presented concerning the immunofluorescence assay for feline leukemia virus (FeLV) group-specific antigens (GSA) in blood cells of cats. Data are given regarding absorption of goat FeLV GSA antiserum in vivo in cats, absorption of the antiserum in vitro with feline blood cells, and the comparative efficacy of various chemical fixatives in preservation of FeLV GSA for immunofluorescent staining. The best results were obtained with in vitro absorption of antiserum and methanol fixation of FeLV GSA in blood smears.

Increased susceptibility to feline leukemia virus infection in cats exposed to methylnitrosourea.

Exposure of adult specific-pathogen-free cats to methylnitrosourea resulted in increased susceptibility to infection by feline leukemia virus. A greater proportion of cats exposed to methylnitrosourea and feline leukemia virus (69%) became persistently viremic than those exposed to feline leukemia virus alone (17%). Segmented neutrophils were reduced by 90 to 99% within 3 days following exposure to methylnitrosourea, (15 to 20 mg/kg) whereas the effects on lymphocytes and erythrocytes, although less obvious, were also detected.

Detection and evaluation of feline oncornavirus-induced cell surface antigen(s) shed from cells in vitro.

A method for preparation of soluble feline oncornavirus-induced cell surface antigens was described. This technique relied upon the natural release of antigen(s) from FL-74 feline lymphoblastoid cells during their maintenance at 37 degrees in serum-deficient medium. When concentrated and clarified spent medium from 4-day cultures was tested for its antigen content by inhibition of humoral cytotoxicity, it was found that this natural production of soluble antigen provided more feline oncornavirus-associated cell membrane antigen per cell than did a solubilization procedure in which papain was used. The shed antigen preparation was immunogenic in cats, eliciting humoral antibody that was reactive with the surface of FL-74 Cells and feline sarcoma virus-transformed nonproducer mink cells but was not reactive with feline leukemia virus in a virus neutralization assay.

Relationship between feline leukemia virus antigen expression and viral infectivity in blood, bone marrow, and saliva of cats.

Correlation was greater than 90% between feline leukemia virus (FeLV), group-specific antigen (GSA) in leukocytes, and viral infectivity (VI) in serum or plasma from 132 cats infected with either the Rickard strain of FeLV, the Snyder-Theilen strain of feline sarcoma virus, or field strains of FeLV. Detection of GSA in blood cells was at least as sensitive as detection of VI in serum. In 45% of FeLV GSA-positive cats inoculated with FeLV-Rickard strain, VI was detected in saliva. No saliva samples from GSA-negative cats had VI. Sequential bone marrow biopsies from 34 cats inoculated with Snyder-Theilen feline sarcoma virus indicated that the correlation between FeLV GSA in bone marrow cells and blood cells was virtually 100%. FeLV GSA appeared in bone marrow leukocyte precursors 1 week before its appearance in peripheral blood leukocytes in 50% of the cats. The FeLV GSA-positive state was transient (3 to 6 weeks) in 34% of the Snyder-Theilen feline sarcoma virus-inoculated cats.

Abrogation of resistance to feline oncornavirus disease by immunization with killed feline leukemia virus.

Four-week-old specific-pathogen-free cats were immunized with a combined vaccine composed of killed feline leukemia virus and killed feline oncornavirus-associated cell membrane antigen-containing tumor cells. Immunization induced feline oncornavirus-associated cell membrane antigen antibody titers ranging from 1:32 to 1:256 but did not elicit detectable virus-neutralizing antibody titers. Kittens immunized with tumor cells alone developed higher feline oncornavirus-associated cell membrane antigen antibody titers (ranging from 1:512 to 1:2048) than those given the combined vaccine. All kittens were challenged with virulent Dynder-Theilen feline sarcoma virus at 12 weeks of age. Seventy-five % of the kittens vaccinated with combined vaccine and 67% of unvaccinated control kittens developed progressive fibrosarcomas after challenge. By contrast, none of the kittens vaccinated with killed tumor cells alone developed progressive fibrosarcomas after challenge. The combined vaccine did not, however, inhibit the induction of feline leukemia virus viremia.