2-Hour cytomegalovirus pp65 antigenemia assay for rapid quantitation of cytomegalovirus in blood samples

Of 109 blood samples tested for cytomegalovirus (CMV) antigenemia, 18 (16.5%) were positive. CMV Brite detected 13 and CMV Brite Turbo detected 16 of the 18 positives. There was no significant difference in the number of positive cells detected per sample. The seven discrepant samples contained a median of only one positive cell.     

Instrumental learning within the rat spinal cord: localization of the essential neural circuit

Following spinal transection of the upper thoracic spinal cord, male Sprague-Dawley rats given legshock whenever a hindlimb is extended learn to maintain the leg in a flexed position. The region of the cord that mediates this instrumental learning was isolated using neuroanatomical tracing, localized infusion of lidocaine, and surgical transections. DiI and Fluoro-Gold microinjection at the site of shock application labeled motor neuron bodies of lamina IX in the lower lumbar region. Local application of the Na-super++ channel blocker lidocaine disrupted learning when it was applied over a region extending from the lower lumbar (L3) to upper sacral (S2) cord. The drug had no effect rostral or caudal to this region. Surgical transections as low as L4 had no effect on learning. Learning also survived a dual transection at L4 and S3, but not L4 and S2. The results suggest that the essential neural circuit lies between L4 and S3.     

Rolling Circle Amplification : A New Approach to Increase Sensitivity for Immunohistochemistry and Flow Cytometry

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immunological and molecular probes. RCA-mediated signal amplification was successfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracellular molecules (vimentin and prostate-specific antigen) within a variety of routinely fixed specimens, as well as samples prepared for flow cytometry. RCA technology, which has an intrinsically wide dynamic range, is a robust and simple procedure that can provide a universal platform for the localization of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emerging information from genomics and proteomics into cell- and tissue-based analyses.     

Transplacental passage of mifepristone and its influence on maternal and fetal steroid concentrations in the second trimester of pregnancy

The maternal and fetal endocrine effects of the maternal administration of the anti-progestin mifepristone in mid-pregnancy have been investigated. Mifepristone and the metabolite RU 42,633 were detected in the fetal circulation and in the amniotic fluid 4, 24 and 48 h after oral ingestion. Maximum fetal plasma concentrations of mifepristone occurred 4 h after treatment indicating rapid placental transfer of the drug. No significant changes in progesterone, cortisol, oestradiol or aldosterone concentrations were detected in the maternal circulation after mifepristone treatment. No significant changes occurred in the fetal progesterone, oestradiol or cortisol concentrations, but a significant increase in fetal aldosterone occurred 4 and 24 h after treatment. The significance of these results is discussed in relation to the possible therapeutic uses of mifepristone for inducing labour.     

Dietary and mechanically induced rabbit iliac-femoral atherosclerotic lesions: a chemical and morphologic evaluation

The effect of combining chronic endothelial injury and intermittent meal feeding of a high and low cholesterol, coconut oil, peanut oil diet on plama lipid and lipoprotein content and on the formation of atherosclerotic lesions within the iliac-femoral artery of rabbits was studied. Alternate feeding of a 1 or 0.1% cholesterol, 3% coconut oil, 3% peanut oil diet for 3 to 14 weeks resulted in a 4- to 11-fold increase in plasma cholesterol with 59 to 79% of the plasma cholesterol eluting in a molecular weight fraction comparable to human low density lipoproteins (LDL). In the iliac-femoral artery, an atherosclerotic intimal lesion with an average cross-sectional area of 0.452 mm2 was present in 98% of the animals. The lesion was typically eccentric in location and contained both superficial- and deep-intimal lipid-filled monocyte-macrophages, extracellular lipid, smooth muscle cells, and extracellular connective tissue matrix. The relative percent lipid composition of the iliac-femoral lesion was 62% cholesteryl ester, 21% free cholesterol, and 17% phospholipid. Thus, we conclude that the combination of meal feeding a cholesterol/fat diet, dietary regimen and chronic mild endothelial injury in the rabbit results in (1) a diet-induced hypercholesterolemia in which LDL appear to be the predominant lipoprotein; and (2) a lesion within the iliac-femoral artery comparable in histologic and chemical composition to a human fatty streak.     

Immunogenicity and antigenicity of chimeric picornaviruses which express hepatitis A virus (HAV) peptide sequences: evidence for a neutralization domain near the amino terminus of VP1 of HAV.

We evaluated the antigenic characteristics of chimeric picornaviruses created by inserting peptide sequences from hepatitis A virus (HAV) capsid proteins into the B-C loop of VP1 of Sabin strain type 1 poliovirus (PV-1). Fifteen viable chimeras were generated. Each retained the ability to be neutralized by polyclonal PV-1 antisera. Two chimeras (H15 and H2) stimulated production of low levels of HAV neutralizing antibodies in immunized rabbits or mice, although in both cases only a small fraction of immunized animals produced this response. The H15 chimera, which contains residues 13-24 of HAV VP1, elicited HAV neutralizing antibodies in three of nine rabbits and at least one of seven immunized mice. These results indicate that a neutralization domain exists in this region of VP1. However, human sera with high titers of antibodies to HAV failed to neutralize or immunoprecipitate this chimera, suggesting the absence of a significant antibody response to this neutralization domain following natural infection. Sera from rabbits immunized with H15 that did not develop HAV neutralizing antibodies contained antibodies reactive with the HAV peptide segment expressed by the H15 virus, indicating substantial differences in the specificities of antibodies elicited by this peptide segment among individual immunized rabbits. The H15 peptide insert was an effective antigen, as indicated by a high level of sensitivity of the H15 chimera to neutralization by a related anti-peptide antibody which was itself devoid of HAV neutralizing activity. One of 16 rabbits immunized with the H2 chimera (residues 101-108 of HAV VP1) developed HAV neutralizing antibodies, confirming both the presence and the highly conformational nature of a neutralization antigenic site involving these residues of HAV.     

Transplant arteriosclerosis in a rat aortic model.

Transplant arteriosclerosis (TA) has emerged as an obstacle to the long-term survival of transplanted organs, especially cardiac transplants. The animal models that have been used to study TA have not been fully characterized with regard to features such as the time course of cell proliferation and the sequence of cell types arriving in the developing intimal lesion. We present a model of TA based on a transplanted segment of abdominal aorta that helps address these questions. Two strains of rats (PVG x DA) underwent orthotopic aortic transplantation without immunosuppression and were killed at 14, 20, 40, and 60 days after transplantation. The within-strain control group displayed minimal evidence of cellular rejection with minimal to absent intimal lesions. In contrast, the allograft group showed a linearly increasing intimal lesion, up through 60 days after transplantation. The mechanism of intimal thickening was by an increase in cell number at the earlier time points with the later deposition of extracellular matrix. The early intimal lesion consisted mostly of mononuclear inflammatory cells (45%) with gradually increasing presence of smooth muscle cells (SMC) in the intima between 20 and 60 days. Conversely, the media showed gradual infiltration by macrophage-type cells with virtual loss of all SMC from the media by 40 days. The proliferative index showed a peak of 6% and 8% at 20 days in both the intima and media, respectively, and was preceded by the presence of macrophages. In fact, most of the proliferating cells at the earlier time points were either monocytes/macrophages, or were immediately adjacent to monocyte-/macrophage-rich regions. This straight artery segment model of transplant arteriosclerosis provides an easily quantifiable system in which the effects of different interventions (e.g., immunosuppressive regimens) can be tested.     

Staphylococcal exopolysaccharides inhibit lymphocyte proliferative responses by activation of monocyte prostaglandin production.

The glycocalyx (exopolysaccharides) of Staphylococcus epidermidis has been reported to inhibit a variety of host defense mechanisms. We have examined the inhibitory effects of glycocalyx on the proliferation of human peripheral blood mononuclear cells (PBMC) and the mechanism of this inhibition. Glycocalyx isolated and partially purified under endotoxin-free conditions from defined liquid medium cultures of S. epidermidis and Staphylococcus lugdunensis inhibited the proliferative response of PBMC when added to cultures at 10 to 100 micrograms/ml. Glycocalyx-mediated inhibition of phytohemagglutinin-stimulated proliferation of PBMC required the presence of plastic-adherent peripheral blood monocytes. Culture supernatants of monocytes stimulated with glycocalyx contained a soluble factor that inhibited the proliferation of monocyte-depleted PBMC. This soluble inhibitory factor was not produced in the absence of glycocalyx or in the presence of both glycocalyx and indomethacin. Analysis of the supernatants of cultures of adherent monocytes revealed that glycocalyx from S. epidermidis and from S. lugdunensis could activate monocyte production of prostaglandin E2 (PGE2), human interleukin-1, and tumor necrosis factor alpha. The addition of purified PGE2, at the same levels of PGE2 (greater than or equal to 10(-9) M) generated in the monocyte cultures, to PBMC cultures resulted in a similar inhibition of proliferative responses. It is concluded that, contrary to previous suggestions, the bacterial glycocalyx does not have a direct inhibitory effect on T lymphocytes. However, it does appear that glycocalyx from coagulase-negative staphylococci can activate monocyte PGE2 production and that it is this activity that in turn contributes to the inhibition of T-cell proliferation.     

Gliadin presented via the gut induces oral tolerance in mice.

When an antigen is first presented via the gut, either priming or suppression of the systemic immune response may result. Many factors influence the outcome, including physico-chemical properties of the antigen. The aim of this study is to establish if wheat gliadin behaves as an oral immunogen or tolerogen. Mice reared on a gluten-free diet were fed gliadin, either as wheat flour in a standard rodent diet or as the purified molecule. Immune status (tolerant or sensitized) was then defined by measuring specific systemic immune responses after parenteral immunization of gliadin-fed and control mice. A single feed of 25 or 125 mg of purified gliadin resulted in a dose-dependent suppression of both cell-mediated and humoral immune responses. Similar oral tolerance was achieved by feeding mice with a gluten-containing diet for a week. Finally, mice reared on a normal, gluten-containing diet showed evidence of established oral tolerance, with significantly lower systemic immune response to gliadin than mice reared on gluten-free diet. These results indicate that gliadin is an effective oral tolerogen. In vivo studies on the immunogenicity of gliadins should be conducted in animals from gluten-free colonies.     

Perceptions of commercial snack food and beverages for infant and young child feeding: A mixed‐methods study among caregivers in Kathmandu Valley,Nepal

Ensuring nutritious complementary feeding is vital for child nutrition. Prior research in Kathmandu Valley found high consumption rates of commercially produced snack foods among young children, which are often energy‐dense/nutrient poor. This mixed‐methods study was conducted to elicit Nepali caregivers' perceptions of commercial snack foods and beverages and factors influencing their use for young child feeding. Seven facilitated focus group discussions (FGD) were conducted with Kathmandu Valley caregivers of children 12–23 months, and a survey of 745 primary caregivers of children 12–23 months of age was then conducted. During the FGD, caregivers reported commonly providing commercial food and beverage products to their children as snacks, and 98.6% of caregivers participating in the survey reported feeding their child such a food in the previous week. Because of processing and packaging, snack foods were not trusted by many FGD participants and considered as “junk foods” and not healthy for children. However, commercial snack foods were consistently ranked highly on convenience, both because of minimal preparation and ease of feeding; 48.5% of all surveyed caregivers reported providing a snack food because of convenience. Other family members' diets or provision of snack foods as treats also influenced children's consumption of these snack foods and beverages. This study indicates that caregivers of young children prefer snack options that are nutrient rich; however, this may conflict with preferences for foods that require minimal preparation and are appealing to young children. Such findings carry programmatic implications for interventions aiming to address children's diet quality in urban Nepal.