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Biomedical subjects

R H Yolken

Publications and source records attributed to R H Yolken.

At least 19 recordsLinked to original sources
PubMed1992

Cell surface ligands for rotavirus: mouse intestinal glycolipids and synthetic carbohydrate analogs.

Rotaviral binding to receptors on epithelial cells in the small intestine is thought to be a key event in the infection process and may be carbohydrate-mediated. Strain SA11 of rotavirus bound in vitro both to glycolipids isolated from mouse small intestine and to authentic glycolipids using thin layer chromatography overlay and microtiter well adsorption assays. Neutral mouse intestinal glycolipids which bound rotavirus were GA1 (Gal beta 1----3GalNAc beta 1---4Glc beta 1----4Glc beta 1----1-ceramide) and pentaosylceramides with terminal N-acetylgalactosamine, while acidic lipids which bound rotavirus included cholesterol 3-sulfate and two compounds termed bands 80 and 81. Digestion with ceramide glycanase suggested that bands 80 and 81 have lactosyl ceramide cores and an unidentified acidic moiety(s). No sialic-acid-containing glycolipids tested were active in viral binding. Band 81, which may have a ganglio core, bound rotavirus with greatest avidity, followed by GA1. Of authentic glycolipids assayed, only GA1 and GA2 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1-ceramide) displayed rotaviral binding. A phosphatidylethanolamide dipalmitoyl-containing neoglycolipid analog of GA2 bound rotavirus with avidity similar to native GA2. Substitution of beta 1----4-linked GlcNAc or beta 1----3-linked GalNAc for terminal GalNAc of GA2 neoglycolipid supported rotaviral binding, while other substitutions abrogated it. These findings suggest that a carbohydrate epitope similar to that of GA2 is sufficient for in vitro rotaviral binding, although binding may be enhanced by galactose and/or an acidic moiety in a secondary epitope.

Animals↗
PubMed1992

Baculovirus expression of pestivirus non-structural proteins.

Bovine viral diarrhoea virus (BVDV) belongs to the pestivirus group, a genus within the Flaviviridae family. It possesses a positive-sense ssRNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. Here we report the continuation of our studies of pestivirus protein biogenesis, involving expression from the viral non-structural protein-encoding region. The 3'-terminal 60% of the BVDV ORF was cloned into a plasmid transfer vector which was then used to construct a recombinant baculovirus. Infection of Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of the expected mature viral proteins. Polyprotein processing by the BVDV p80 proteinase appeared to be nearly identical to that observed in authentic BVDV-infected bovine cells, and as previously shown to occur when expression of the same region was studied in a mammalian cell transient expression system. However, one viral proteolytic cleavage did not occur in the baculovirus-infected insect cells; the viral p80 proteinase failed to act at its own N terminus. This recombinant baculovirus/insect cell expression system provides an abundant source of BVDV non-structural proteins. Therefore we explored the utility of the proteins produced in this system for the detection of anti-BVDV antibodies in bovine sera. In preliminary experiments using these antigens in an ELISA we found a positive correlation between the presence of ELISA-reactive antibody and virus-neutralizing activity.

Animals↗
PubMed1992

Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis.

Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens.

Adult↗
PubMed1992

A human milk factor inhibits binding of human immunodeficiency virus to the CD4 receptor.

Perinatal transmission of human immunodeficiency virus (HIV) from infected mothers to their children occurs at rates reported as 20-50%. The role of breast feeding in perinatal transmission of viral infections has not been well established. We studied 34 milk and colostral samples obtained from HIV-seropositive and HIV-seronegative women to determine if they contained anti-HIV activity. We found that all the samples contained a factor that inhibited the binding of HIV epitope-specific MAb to recombinant CD4 receptor molecules. The titers of inhibitory activity ranged from 1:200 to 1:10,000 and did not differ between HIV-seropositive and HIV-seronegative mothers. This milk factor also inhibited the binding of gp120 to CD4. Neither human sera nor bovine milk exhibited appreciable inhibitory activity. Fractionation of human milk indicated that the inhibitory activity was confined to the macromolecular fraction; little activity was found in isolated milk lipids or oligosaccharides. Chromatographic procedures indicated that the active macromolecule has an isoelectric point of 9.3-9.6. The active material did not bind to concanavalin A; however, the activity was partially destroyed by chemical and enzymatic treatments that removed sulfated residues. The active material may thus be a sulfated protein, glycoprotein, mucin, or glycosaminoglycan that inhibits the binding of CD4 to HIV envelope glycoproteins. The role of this factor in the natural history of HIV infection in infants and children should be the subject of additional investigations.

Antiviral Agents↗
PubMed1991

The polymerase chain reaction: a new tool for the understanding and diagnosis of HIV-1 infection at the molecular level.

The polymerase chain reaction (PCR) is at present the most powerful analytical tool for detection of specific nucleic acid sequences. The method is based on the in vitro amplification of DNA segments before detection with conventional hybridization techniques or visualization following electrophoresis and staining. The current diagnostic methods for HIV-1 do not allow easy identification of subgroups of infected patients including infants born to seropositive mothers, individuals with delayed serological responses to the virus, infected patients with indeterminate serology results, and patients with dual retroviral infections. Furthermore, response to antiviral therapy cannot be evaluated with serological assays. The rationale for applying PCR in those situations is elaborated here. The applications of this technique for HIV-1 as a diagnostic test and for the understanding of the pathogenesis of this retrovirus are described. Potential limitations of this technique for diagnostic purposes include mainly the possibility of false-positive results due to contamination and false-negative reactions caused by Taq polymerase inhibition. Non-isotopic means for detection of amplified products have been described and should allow for a wider application of this technology. Modifications of PCR which make use of internal standards seem promising for quantitative analysis of nucleic acids. PCR has great potential for viral diagnosis but still requires further studies and better characterization.

Acquired Immunodeficiency Syndrome↗
PubMed1979

Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus.

The possibility of immunizing human infants against rotaviruses, which cause severe dehydrating diarrheal disease, may depend on the use of a related rotavirus, derived from another animal species, as a source of antigen. To test the feasibility of this approach, calves were infected in utero with a bovine rotavirus and challenged with bovine or human type 2 rotavirus shortly after birth. Infection in utero with bovine rotavirus induced resistance to diarrheal disease caused by the human virus as well as the homologous bovine virus. These data suggest that the bovine virus is sufficiently related antigenically to the human type 2 virus to warrant further evaluation of the former as a source of vaccine.

Animals↗
PubMed1979

Ultrasensitive enzymatic radioimmunoassay: application to detection of cholera toxin and rotavirus.

Rotavirus and enterotoxin-producing bacteria are major causes of diarrheal disease in humans. A method of rapid diagnosis, ultrasensitive enzymatic radioimmunoassay, has been developed to quantitatively detect cholera toxin and rotavirus. The method uses features of both enzyme-linked immunosorbent assay and radioimmunoassay; however, the sensitivity of the assay is 100- to 1000-fold more sensitive than the two parent assays. Ultrasensitive enzymatic radioimmunoassay should also be useful in measuring other biologically important agents such as drugs and hormones.

Alkaline Phosphatase↗
PubMed1979

Role of Norwalk virus in outbreaks of nonbacterial gastroenteritis.

Twenty-five separate outbreaks of nonbacterial gastrointestinal illnesses were studied serologically for evidence of infection with the Norwalk virus and the rotaviruses that affect humans. Eight of 25 outbreaks appeared to be related to the Norwalk virus. In one of the 25 outbreaks, there was evidence of rotavirus infection. These observations suggest that the Norwalk virus or serologically related agents play an important role in epidemic nonbacterial gastroenteritis in adults and older children.

Antibodies, Viral↗
PubMed1979

Common exposure outbreak of gastroenteritis due to type 2 rotavirus with high secondary attack rate within families.

A sharp outbreak of gastroenteritis associated with human rotavirus type 2 involved not only all of nine infants and young children in a playgroup but also seven of 10 parents and grandparents studied. The source of the outbreak appeared to be two non-playgroup siblings. Six of 11 individuals studied shed human rotavirus type 2, and each of seven from whom paired sera were obtained developed a type 2 sero-response. Overall, evidence of infection with rotavirus type 2 was demonstrated in 10 of 11 individuals by detection of virus in stools and/or a serologic response in an enzyme-linked immunosorbent assay.

Antibodies, Viral↗
PubMed1979

Comparative epidemiology of two rotavirus serotypes and other viral agents associated with pediatric gastroenteritis.

Human rotavirus (HRV) type 1 or 2, adenovirus, or non-cultivatable 27 nm virus-like particles were demonstrated by electron microscopy and/or rotavirus ELISA in fecal samples from 45.5% of 604 gastroenteritis inpatients, 25.0% of 200 gastroenteritis outpatients and 6.0% of 812 control subjects, all sampled at Children's Hospital National Medical Center. Washington, DC. Rotaviruses were the most common pathogens detected as 39% and 22% of gastroenteritis inpatients and outpatients, respectively, shed HRV. About three-fourths of the rotaviruses were type 2, which was prevalent during five successive epidemic years from January, 1974, through June, 1978. HRV type 1 was detected in the last four successive epidemic years and represented nearly half of the HRV infections observed among gastroenteritis inpatients during the year 1977--1978. Both rotavirus serotypes were detected most often in the month of January, when 71% of 123 gastroenteritis inpatients and 62% of 34 gastroenteritis outpatients shed one of these viruses. Uncultivatable adenoviruses were detected significantly more frequently in stools from patients with gastroenteritis (3.9%) than from control subjects (0.6%), suggesting that these viruses played a role in acute enteric disease. The frequency of detection of 27 nm particles was not significantly different in gastroenteritis and control patients. Numerically, HRV infection was detected most often in gastroenteritis inpatients who were 10 through 12 months of age. The group of gastroenteritis inpatients with the highest percentage of HRV infection was 13 through 15 months of age. The excess of type 2 HRV infection relative to type 1 infection was especially large in those aged 7 through 24 months. Lower socioeconomic status or greater crowding appeared to be associated with the occurrence of rotavirus infection earlier in life and earlier in the epidemic year.

Adenoviridae↗
PubMed1979

S-IgA cholera toxin and rotavirus antibody in human colostrum.

S-IgA antibodies against cholera toxin and rotavirus were assayed in 43 colostral samples by means of an enzyme-linked immunosorbent assay (ELISA). All specimens contained specific S-IgA antibodies against both antigens. Significant antibody titres to the antigens were demonstrated in almost all colostral samples.

Adolescent↗
PubMed1979

Enzyme-linked fluorescence assay: Ultrasensitive solid-phase assay for detection of human rotavirus.

Enzyme-linked immunosorbent assay (ELISA) has proven to be a useful assay system for the direct detection of infectious agents. However, when the usual color-producing substrates are employed, relatively large amounts of substrate must be hydrolyzed by the bound enzyme before detection can be achieved. We attempted to improve the sensitivity of ELISA by utilizing a substrate that yields a fluorescent product on enzyme action. The enzyme-linked fluorescence assay (ELFA) based on this principle was approximately 100 times more sensitive than the corresponding ELISA or radioimmunoassay for the detection of human rotavirus in a standard stool suspension. In addition, the ELFA for human rotavirus was capable of detecting antigen in six specimens that were negative by ELISA. Five of these specimens were obtained late in the course of confirmed rotavirus infections. ELFA provides a simple, reliable, ultrasensitive method for the rapid detection of viral antigen.

Antigens, Viral↗
PubMed1979

Analysis of nonspecific reactions in enzyme-linked immunosorbent assay testing for human rotavirus.

Solid-phase enzyme immunoassays can be utilized to detect antigens directly in clinical specimens. However, a small number of stools which we tested for human rotavirus by enzyme-linked immunosorbent assay (ELISA) were found to have nonspecific activity in the absence of rotaviral antigen. Similar nonspecific activity was found in eight of eight sera which contained rheumatoid factor. This nonspecific activity was markedly reduced by pretreatment of the specimens with reducing agents, normal goat serum, and anti-human immunoglobulin M (IgM). Thus, it is likely that these specimens contain an IgM antibody capable of reacting nonspecifically with the other components of the assay. Although pretreatment with the mild reducing agent N-acetylcysteine markedly reduced this nonspecific activity, such treatment did not reduce the specific ELISA activity due to rotavirus. Other treatments did produce a reduction in specific activity. Thus pretreatment with N-acetylcysteine offers a practical means to increase the specificity of ELISA systems without reducing their sensitivity.

Acetylcysteine↗
PubMed1979

Diarrhea associated with rotavirus in rural Guatemala: a longitudinal study of 24 infants and young children.

A population of 24 infants and young children followed prospectively during the first 3 years of life was studied for the occurrence of rotavirus infection by using enzyme-linked immunosorbent assay to detect virus in stools. Infection with rotavirus was associated with 26 (14.2%) of 183 selected diarrheal episodes. Twenty of the 24 infants and young children had diarrhea associated with rotavirus on at least one occasion and six had two such episodes. Rotavirus infection was documented in over 50% of the dehydrating episodes studied, thus further indicating the importance of rotavirus in this population.

Child, Preschool↗