Specific detection of Salmonella enterica and Escherichia coli strains by using ELISA with bacteriophages as recognition agents.

The use of bacteriophages, instead of antibodies, in the ELISA-based detection of bacterial strains was tested. This procedure appeared to be efficient, and specific strains of Salmonella enterica and Escherichia coli could be detected. The sensitivity of the assay was about 10(5) bacterial cells/well (10(6)/ml), which is comparable with or outperforms other ELISA tests detecting intact bacterial cells without an enrichment step. The specificity of the assay depends on the kind of bacteriophage used. We conclude that the use of bacteriophages in the detection and identification of bacteria by an ELISA-based method can be an alternative to the use of specific antibodies. The advantages of the use of bacteriophages are their environmental abundance (and, thus, a possibility to isolate various phages with different specificities) and the availability of methods for obtaining large amounts of phage lysates, which are simple, rapid, cheap, and easy.

Identifying studies for systematic reviews of diagnostic tests was difficult due to the poor sensitivity and precision of methodologic filters and the lack of information in the abstract.

BACKGROUND AND OBJECTIVES: Methods to identify studies for systematic reviews of diagnostic accuracy are less well developed than for reviews of intervention studies. This study assessed (1) the sensitivity and precision of five published search strategies and (2) the reliability and accuracy of reviewers screening the results of the search strategy. METHODS: We compared the results of the search filters with the studies included in two systematic reviews, and assessed the interobserver reliability of two reviewers screening the list of articles generated by a search strategy. RESULTS: In the first review, the search strategy published by van der Weijden had the greatest sensitivity, and in the second, four search strategies had 100% sensitivity. There was "substantial" agreement between two reviewers, but in the first review each reviewer working on their own would have missed one paper eligible for inclusion in the review. Ascertainment intersection techniques indicate that it is unlikely that further papers have been missed in the screening process. CONCLUSION: Published search strategies may miss papers for reviews of diagnostic test accuracy. Papers are not easily identified as studies of diagnostic test accuracy, and the lack of information in the abstract makes it difficult to assess the eligibility for inclusion in a systematic review.

Polymacron enzyme immunoassay system for detection of naturally contaminating Salmonella in foods, feeds, and environmental samples.

A simple dot blot enzyme immunoassay was developed to screen enrichment broth cultures for the presence of Salmonella. This unique system utilizes macroporous polyester cloth (Polymacron) with an inexpensive hemoglobin coating to provide a high-affinity adsorbent for lipopolysaccharide (LPS) antigens in test samples. Bound LPS antigens are then detected using a monoclonal antibody conjugate recognizing a core oligosaccharide epitope common to all salmonellae frequently found in foods and related samples. The entire test (not including enrichment culture) could be completed in less than 1 h. The performance of this assay was evaluated in the analysis of enrichment broth cultures from a variety of egg and dairy products, chicken carcasses, animal feeds, and food-processing plant environmental samples for the presence of Salmonella.

Characterization of serum antibodies to Porphyromonas gingivalis in individuals with and without periodontitis.

Although Porphyromonas gingivalis is a defined pathogen in periodontal disease, many subjects control the infection without experiencing loss of attachment. Differences in host susceptibility to the disease may be reflected in the pattern of humoral antibodies against specific P. gingivalis antigens. The aim of this study was to determine the presence of antibodies against immunodominant P. gingivalis antigens as well as the isotype and subclass of anti-P. gingivalis antibodies against outer membrane antigens in four groups of patients: P. gingivalis-positive, 1) with and 2) without periodontitis, and P. gingivalis-negative, 3) with and 4) without periodontitis. Antigens of molecular weight 92, 63, and 32 kDa and lipopolysaccharide were found to be immunodominant. Group 1 subjects showed a significantly higher response to the 92 and 63 kDa antigens compared with other groups. The response to lipopolysaccharide was significantly higher in group 1, and lower in group 4 than in groups 2, 3. Immunoglobulin G1 (IgG1), IgG2 and IgM antibodies against P. gingivalis outer membrane were present in all subjects, while only some subjects were seropositive for IgG3, IgG4 and IgA. There were no differences in concentrations for IgG1, IgG3 and IgM. The IgG2 concentration in group 4 was significantly higher than in groups 1 and 2, while the IgG4 concentration in group 4 was significantly lower than in other groups. The frequency of seropositivity for IgG4 and IgA was lowest in group 4, while IgG3 seropositivity was almost exclusively seen in healthy patients in groups 2, 4. These findings suggest that the presence of IgG3 may reflect non-susceptibility to the disease, while lack of IgG4 may be indicative of periodontal health and lack of infection.

CAST-ELISA test--a new diagnostic tool in pollen allergy.

We have applied a recently developed method called CAST-ELISA to evaluate the degree of leukocyte stimulation by specific allergen. This method is based on the measurement of sulfidoleukotriene levels in supernatants taken from previously stimulated peripheral blood leukocytes by specific allergen in the presence of interleukin 3.23 patients with pollinosis entered the study. All of them received no medication during 2 weeks before the test. Leukocytes were isolated by dextran sedimentation followed by single centrifugation. After removal of platelet rich plasma the cells were suspended in stimulation buffer and divided into portions incubated with or without specific allergen for 40 minutes at 37 degrees C. After the incubation, the cells were centrifuged and the evaluation of sulfidoleukotrienes in supernatant was performed. The results were expressed in pg/ml after subtraction of the value of spontaneous sulfidoleukotriene production in portions incubated without allergen. The concentration higher than 200 pg/ml of sulfidoleukotrienes above spontaneous production was regarded as a positive result. We have observed a large spectrum of the leukocyte response upon allergen stimulation. In the initial part of our study we established the optimal allergen concentration. The concentration of sulfidoleukotrienes in supernatants ranged from 10 to 5130 pg/ml. The mean sulfidoleukotriene concentration in the whole group was 1671.69. Positive results were observed in 20 persons. In 3 persons the results of allergen stimulation were negative. We conclude that CAST-ELISA is a reliable method to determine the allergic status in persons with pollinosis.

Expression of the E-selectin gene in human gingival epithelial tissue.

E-selectin is an adhesion molecule, expressed by cytokine-activated endothelial cells, that participates in the binding of neutrophils. Recent studies in our laboratory documented binding of the E-selectin-specific monoclonal antibody H4/18 to keratinocytes in inflamed human oral mucosa, particularly gingival epithelium. To determine whether this immunoreactivity was due to expression of authentic E-selectin, the presence of E-selectin mRNA in gingival epithelium was analysed using the polymerase chain reaction (PCR). Reverse transcription of epithelial RNA and amplification of cDNA with E-selectin-specific primers resulted in the formation of a 178 nucleotide PCR product identical to that obtained from cytokine-activated endothelial cells. Sequencing of the PCR product revealed 100% homology between epithelial and endothelial E-selectin fragments. Epithelial preparations did not contain mRNA for von Willebrand factor, excluding the possibility of contamination by endothelial cells. These results confirm immunohistochemical studies of E-selectin immunoreactivity in human oral mucosa and demonstrate that E-selectin expression is not confined to endothelium.

Human gingival keratinocytes express E-selectin (CD62E).

OBJECTIVES: E-selectin (CD62E) is an adhesion molecule that participates in the binding of leukocytes to activated blood vascular endothelium. The present study was undertaken to characterise the pattern of E-selectin expression on epithelial cells of the gingival crevice and oral epithelium. METHODS: A panel of six anti-E-selectin monoclonal antibodies was reacted with cryosections of human gingiva and cytospins of cultured gingival keratinocytes. RESULTS: Three antibodies raised against leukocyte binding epitopes of the molecule (H4/18, H18/7, 1.2B6) were reactive with gingival keratinocytes, three were negative (4D10, BBA1, BBA2), while all six reacted with endothelial cells. Staining with H18/7 and 1.2B6 was weaker and more variable than with H4/18. In cell culture, levels of E-selectin expression reduced slowly, and were only modestly increased by treatment with exogenous TNF alpha, a known inducer of E-selectin on endothelium. CONCLUSIONS: E-selectin epitopes are expressed on gingival keratinocytes. These binding epitopes may be involved in the traffic of leukocytes in this tissue compartment.

Differential reactivity of anti-E-selectin monoclonal antibodies in human gingival tissue.

E-selectin is a cytokine-inducible endothelial glycoprotein which participates in the binding of leukocytes to vascular endothelium. Variable levels of expression of E-selectin have been reported in gingivitis and periodontitis, and two differing concepts of its significance have emerged: either gingival blood vessels express E-selectin constitutively, or are continuously activated by inflammatory mediators arising from the gingival environment. A range of monoclonal antibodies reacting with different epitopes of E-selectin are available commercially. The present study explored the possibility that the choice of antibody could affect estimation of the level of expression of E-selectin in vivo. Five monoclonal antibodies were used to investigate E-selectin expression in serial cryosections of human gingival tissue. While E-selectin-positive vascular endothelial cells were detected with all antibodies, the number of positively staining endothelial cells varied, with BBA1 > H4/18 = H18/7 = 4D10 > 1.2B6. The frequency of strong staining in tissue specimens was BBA1 > 4D10 > H4/18 = H18/7 > 1.2B6, while the frequency of weak staining showed the reverse trend. Additionally, with antibodies H18/7 and 1.2B6, 17 and 36% of the specimens were E-selectin negative. The occurrence of what appear to be false positives and false negatives confounds estimation of the level of E-selectin expression. Based on these findings, patterns of endothelial E-selectin expression in gingivitis and periodontitis should be re-evaluated.

Antibodies made against a formaldehyde-protein adduct cross react with an acetaldehyde-protein adduct. Implications for the origin of antibodies in human serum which recognize acetaldehyde-protein adducts.

Acetaldehyde, the major metabolite of ethanol, reacts with lysine and other free amino groups on proteins to form acetaldehyde-protein adducts. The presence of antibodies which recognize such acetaldehyde-protein adducts in sera from alcoholics has been attributed to an immune response to such adducts. Complicating this conclusion is the finding that sera from non-alcoholic control subjects also contain antibodies which recognize acetaldehyde-protein adducts. In the current research we sought to determine whether antibodies which recognize epitopes formed by the reaction of a protein with acetaldehyde can be formed in response to a protein modified with a structurally related protein adduct. We modified lysine residues on apolipoprotein (apo) B-100 with acetaldehyde and formaldehyde under reducing conditions, to form epsilon-N-methyl- and epsilon-N-ethyl-lysine residues, and with acetic anhydride to form epsilon-N-acetyl-lysine residues, and made antibodies against these modified proteins in guinea-pigs. In ELISA assays antibodies made against methylated apoB-100 (Me-apoB) cross-reacted effectively with ethylated apoB-100 (Et-apoB), while antibodies made against acetic anhydride-modified apoB-100 did not cross-react. We conclude that methyl-lysine shares one or more immunoreactive epitopes with ethyl-lysine, and that antibodies which recognize acetaldehyde-modified proteins can be formed in response to formaldehyde-modified proteins. We demonstrate that sera from both alcoholics and non-drinkers contain antibodies which recognize Me-apoB and Et-apoB and that the titres of these antibodies are comparable. These data raise the possibility that some human serum antibodies which recognize acetaldehyde-modified protein epitopes may have been made against formaldehyde-modified protein epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)

TGF-beta 2 gene and protein expression in maternal and fetal tissues at various stages of murine development.

The transforming growth factor beta family of peptides have diverse actions on the reproductive tracts of primates and rodents. In this study we report the expression of high levels of mRNA of one member of this superfamily, TGF-beta 2, in the pregnant mouse uterus. Using Northern blot analysis and in situ hybridization techniques, we have examined the pattern of expression of TGF-beta 1, TGF-beta 2 and colony-stimulating factor (CSF-1) in mouse maternal and fetal tissue at specific days of gestation. We report here that TGF-beta 2 is synthesized primarily in maternal decidual and uterine epithelial tissues. We observed a shift in the major site of synthesis from decidua to uterus between days 8.5 and 10.5 of gestation. These data demonstrate that the expression of TGF-beta 2 is differentially regulated in the decidua and uterine epithelial cells at various times during gestation. Small amounts of TGF-beta 2 mRNAs were detected in the fetus, and none was detected in placenta, yolk sac, or amniotic membrane. The uterus is likely the major site of synthesis of the TGF-beta 2 found in mouse amniotic fluid. TGF-beta 1 mRNAs are expressed in the uterus at markedly lower levels when compared to TGF-beta 2 mRNAs in both the decidua and uterus. Our results suggest that there is a unique regulation of TGF-beta 2 during pregnancy which may depend on pregnancy hormone(s) and differentiates it from the other mammalian isoforms of the TGF-beta s. TGF-beta 2 may play an important, albeit unknown, role at the maternal/fetal interface.

Interaction of chronic ethanol consumption and aging on brain muscarinic cholinergic receptors.

It has been proposed that ethanol and aging may interact synergistically to impair brain function through effects on central muscarinic receptors. Previous studies have investigated the effect of either chronic ethanol treatment or aging, but not both factors simultaneously, on brain muscarinic receptors. We have studied brain muscarinic receptors in animals treated with ethanol for up to 25 months. Ethanol consumption for 3 and 9 months resulted in increased density of quinuclidinyl benzilate (QNB) binding sites in cortex, coinciding with an increase in high-affinity pirenzepine binding sites and low-affinity carbachol binding sites. Upregulation of QNB binding sites in striatum and hippocampus became obvious after further ethanol treatment (15 and 21 months, respectively). Affinity of QNB binding sites and carbachol binding sites was not altered by ethanol treatment. However, there was an ethanol-related decrease in affinity of low-affinity pirenzepine binding sites in cerebral cortex. Density of QNB binding sites and low-affinity pirenzepine binding sites decreased with age in three brain areas investigated. There were age-related changes in receptor affinity in hippocampus and striatum, but not in cortex. Ethanol-related upregulation of muscarinic receptors was superimposed on age-related loss of receptors. We conclude that acceleration of the aging process associated with ethanol abuse is unlikely to be explained on the basis of alterations in receptor density or affinity.

Chronic ethanol administration alters binding of [(35)S]t-butylbicyclophosphorothionate to the GABA-benzodiazepine receptor complex in rat brain.

Chronic treatment of male Wistar rats with ethanol (15% v/v) in drinking fluid for a period of 3 months affected the binding of the chloride channel antagonist, [(35)S]TBPS, to well-washed synaptic membranes and slide-mounted brain slices, the affinity for [(35)S]TBPS in brains of ethanol-treated animals was significantly decreased in comparison to controls while receptor density was increased (P < 0.001). However, other well described treatments, viz. an ethanol-containing liquid diet and chronic inhalation of ethanol failed to demonstrate changes in the binding of [(35)S]TBPS in brain preparations. Our findings suggest that long-term administration of ethanol can induce alterations in the characteristics of the ionophore component of the GABA-benzodiazepine receptor complex. This may have relevance to ethanol-induced neuronal damage.

Effects of chronic ethanol treatment and aging on brain phosphoinositide turnover and adenylate cyclase activity.

Inositol phosphate accumulation and adenylate cyclase activity were investigated in the cortex of young and aged ethanol-treated rats. Three months of ethanol treatment of young rats decreased maximal stimulation of inositol phosphate accumulation by carbachol by 26%, from 494 +/- 76% of basal turnover in control animals to 396 +/- 54% in ethanol-treated animals (mean +/- SD). In aged rats ethanol-related changes were no longer observed but age-related changes were evident. EC(50) was significantly higher than in young animals and maximal stimulation was significantly lower. Basal adenylate cyclase activity in cortical membranes of all groups of animals was not different. Forskolin-stimulated adenylate cyclase activity was not affected by ethanol treatment, but was higher in aged animals. The activity of forskolin-stimulated adenylate cyclase in the presence of carbachol was higher in both young and aged ethanol-treated animals, when compared to young controls. These results suggest that both ethanol and aging impair the efficiency of receptor/effector coupling.

Plasticity of brain muscarinic receptors in aging rats: the adaptative response to scopolamine and ethanol treatment.

Young and aged rats were treated chronically with ethanol or scopolamine. Muscarinic receptors were measured in cerebral cortex, hippocampus and striatum. Following scopolamine treatment muscarinic receptor density in cerebral cortex, hippocampus and striatum of young rats increased by 34, 57 and 27%, respectively; in brains of aged rats the increase was 41% in cerebral cortex, 43% in hippocampus and nil in striatum. Affinity of muscarinic receptors was not changed by scopolamine treatment. Following chronic ethanol administration there was a 48% increase in cortical muscarinic receptor density in young, but not aged rats. The density of muscarinic receptors in hippocampus and striatum of both young and aged rats was not affected by ethanol treatment. Affinity of receptors in hippocampus of aged, ethanol-treated rats was increased compared to age-matched controls. Adaptative responses of the muscarinic receptor/transducer system to neurotransmitter availability are present in both young and aged rats, both the ethanol-induced response is present only in young animals. This suggests differences in the mechanism of action of ethanol and receptor agonists and antagonists in modulating receptor plasticity.

The effect of chronic ethanol consumption on [14C]deoxyglucose uptake in rat brain in vivo.

The uptake of [14C]deoxyglucose by brains of rats that were given alcohol in drinking water for 7 months was investigated. There was a general, approximately 50%, increase in deoxyglucose uptake in brains of ethanol-treated rats with areas of the limbic system being particularly affected.

A feeding regime for the study of the interaction of ethanol and aging.

A suitable model for the study of the interaction of the aging process and prolonged treatment with ethanol is described. Animals were given 15% ethanol in the drinking water for up to 25 months. There was little disruption in the growth of the animals. Ethanol treatment resulted in a number of neurological and morphological changes some of which persisted throughout the treatment period. Both young and old animals displayed metabolic tolerance by the criteria of resistance to ethanol-induced hypothermia and increased metabolism of [14C]ethanol. The regime used has proved useful in the study of the interaction of ethanol and aging in the rat.

Abnormally banded region in a poorly differentiated sarcoma is not correlated with amplification of c-MYC or c-MOS protooncogenes.

A poorly differentiated sarcoma in a 32-year-old female revealed a large, abnormally banded region in one chromosome #8 in all metaphases. The modal karyotype was 46,XX, -8, +mar. Southern blot hybridization was performed with probes for two protooncogenes located in chromosome 8q (c-MYC and c-MOS). No amplification or rearrangement was observed to account for the cytogenetic abnormality.

The human int-1 gene is located at chromosome region 12q12-12q13 and is not rearranged in myxoid liposarcoma with t(12;16) (q13;p11).

The mouse cellular oncogene int-1 is often transcriptionally activated as a consequence of nearby proviral insertions in mouse mammary tumors. A highly conserved sequence has been found in the human genome, called int-1 gene, the role of which in human tumors is not known. By somatic hybrids, the human int-1 gene has been assigned to the segment 12q14-12pter. Using a genomic DNA clone containing the fourth exon of the human int-1 gene we have mapped the human int-1 gene to 12q12-12q13. To determine whether this gene, which is located close to the 12q13 breakpoint associated with myxoid liposarcoma, is rearranged in these tumors, we have performed Southern blot analysis of DNA from myxoid liposarcomas carrying the translocation t(12;16) (q13;p11). In the two tumors investigated, the translocation does not disrupt the int-1 gene.

Chronic consumption of ethanol increases glucose oxidation in isolated cerebral tissue of rat.

Tolerance to ethanol was induced in male Wistar rats by adding 15% ethanol to the drinking water for 3 and 9 months. The oxidation of [U-14C]glucose was measured in cerebral cortex prisms. 14CO2 production in prisms for ethanol-treated animals was markedly increased when compared to controls. Neither a single injection of a low dose of ethanol nor a comparable concentration of ethanol in vitro had this stimulatory effect.