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Rapid serological profiling by enzyme-linked immunosorbent assay. I. Measurement of antibody activity titer against Newcastle disease virus in a single serum dilution.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure specific antibody activity in sera of chickens exposed to Newcastle disease virus (NDV). A near-linear relationship existed between the log of the corrected absorbance of antisera at a single working dilution and the corresponding observed serum titers as determined by a standard serial-dilution method. Regression analysis was used to construct a standard curve and extract an equation from this relationship. The equation was used to convert corrected absorbance readings of the single working dilution directly into predicted ELISA antibody activity titers. In a comparative study, a correlation (P less than 0.01) was found between ELISA and hemagglutination-inhibition (HI) antibody titers to NDV. ELISA titers were as much as 160 times greater than the HI titers. ELISA was also able to detect much lower levels of antibody activity than the HI test.

Animals↗

PubMed1995

A comparative study of peanut agglutinin and amaranthin binding to human urinary bladder tumor glycoproteins.

The reactivity of two T-antigen specific lectins, Amaranthin (ACA) and Peanut Agglutinin (PNA), was compared by immunohistochemical staining of serial sections of human bladder tumors and by Western Blot analysis of glycoproteins extracted from human bladder tumors prior to and after sialic acid removal. In low grade tumors ACA reacted prior to neuraminidase treatment. PNA on the other hand only showed positive reaction in these tumors after neuraminidase treatment. The two lectins showed identical staining patterns after neuraminidase treatment in tumors. In Western blottings both lectins detected a 28 kD glycoprotein. PNA also reacted with several other bands after neuraminidase treatment. The reactivity of ACA was not enhanced after neuraminidase treatment. In monosaccharide inhibition tests ACA- and PNA-binding to different T-antigens was most efficiently inhibited by GalNAc and Gal respectively. PNA binding was also inhibited by Glc, GlcNAc and GalNAc at higher concentrations, while the binding of ACA was only scarcely affected by Glc, GlcNAc and Gal.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed1998

Effect of vitamin D receptor gene alleles on bone loss in early rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a polygenic disease characterized by localized joint destruction and generalized osteoporosis resulting in increased fracture risk. The pathogenetic mechanisms that determine the severity of generalized bone loss in RA are poorly understood. Polymorphisms in the vitamin D receptor (VDR) gene have been described as a significant determinant of bone turnover and mass. In this prospective study we describe VDR gene allele effects on bone loss in patients with early RA. METHODS: We recruited 232 patients with early RA. Bone mineral density measurements were repeated in 167 patients. Serial clinical and laboratory measures were recorded during the period of followup. DNA extraction, polymerase chain reaction amplification, and restriction fragment length polymorphism analysis of VDR alleles were performed using standard techniques. Presence of the Taq restriction site for both alleles was denoted "tt", and absence "TT". RESULTS: In women with RA the tt genotype group lost bone more rapidly than subjects with TT genotype at both the lumbar spine (-0.1 vs -4.9% p.a, respectively; p < 0.05) and femoral neck (-3.9 vs -9.6%, respectively; p < 0.01). The effect was independent of other disease characteristics. CONCLUSION: The presence of the VDR gene "t" allele in female patients with RA was associated with accelerated bone loss.

Alleles↗

PubMed1997

[Using polymerase chain reaction to detect Helicobacter heilmannii in gastric biopsy materials].

Helicobacter heilmannii is one of the species of Helicobacter other than Helicobacter pylori noticed in human gastric mucosa. In our study, a polymerase chain reaction (PCR) was designed for the detection of Helicobacter heilmannii in gastric biopsy specimens. Two broad-range bacterial 16S rDNA primers and one Helicobacter genus-specific 16S rDNA primer were matched for detecting Helicobacter genus bacteria. A Hp-specific primer pair was used simultaneously to exclude the presence of Hp. The PCR assay detected 11 out of 12 Hh strains which were confirmed by smears, but did not amplify DNA extracts from some other enteric bacteria such as E. coli, C. jejuni, bifidobacteria. Serial dilution experiments revealed the detection of as little as 0.1 pg DNA by PCR and 0.01 pg by nested PCR. Our data showed that PCR is a rapid, accurate and sensitive method for the detection of Hh and is thought valuable in the epidemiological investigation of Helicobacter and follow-up studies after treatment.

Biopsy↗

PubMed1985

The distribution of corticotrophin-releasing factor immunoreactivity in various ovine tissues.

The presence of CRF in various ovine tissues has been quantified by a radioimmunoassay using an antibody raised against ovine CRF synthesized in vitro. Acetic acid extracts of ovine hypothalamus, cerebral cortex, cerebellum, pituitary and a number of visceral organs assayed in serial dilutions gave inhibition curves parallel to those of synthetic CRF. The highest concentration of CRF immunoreactivity (CRF-I) was found in the pituitary (84.6 +/- 8.77 ng/g wet weight) and the second highest in the hypothalamus (79.1 +/- 2.77 ng/g wet weight). CRF-I in the cerebral cortex and visceral organs (stomach, duodenum, jejunum, ileum, pancreas and liver) was about 60 and 30% of that in the hypothalamus, respectively. Chromatography of extracted CRF-I from these tissues on Bio-Gel P-10, equilibrated and eluted with 0.1 M acetic acid, revealed a single immunoreactive peak corresponding to synthetic ovine CRF. These findings indicate that CRF is generally distributed throughout ovine tissues including visceral organs, although its function remains to be clarified.

Adrenal Glands↗

PubMed1986

Measurement of sebum output using a lipid absorbent tape.

A sebum absorbent tape is introduced as a reproducible and convenient method for estimation of sebaceous gland output. We have tested the reproducibility of this method by serial measurements of sebum excretion rates (SER) of 10 individuals over a 6-week period, and in addition we have correlated this method with the conventional hexane extraction technique. The sebum absorbent tapes gave consistent values for the SERs, and within subjects variation over the 6-week period was statistically nonsignificant. A coefficient of variation for the tapes was calculated as 16.25 +/- 6.78% based on these serial measurements. Furthermore, the amount of total lipid collected using this technique (n = 16) correlated well with the hexane extraction technique, r = 0.89. Free fatty acids (r = 0.87), triglycerides (r = 0.92), wax and cholesterol esters (r = 0.83), and squalene (r = 0.88) also showed a good correlation. Cholesterol occasionally suffered from incomplete separation on thin-layer chromatograms; however, a sample cleanup procedure was developed for tape extracts that removed interfering materials and allowed complete separation of all sebum components.

Absorption↗

PubMed2005

Omission of serial arterial blood sampling in neuroreceptor imaging with independent component analysis.

We have previously proposed a statistical method for extracting a plasma time-activity curve (pTAC) from dynamic PET images, named EPICA, for kinetic analysis of cerebral glucose metabolism. We assumed that the dynamic PET images consist of a blood-related component and a tissue-related component which are spatially independent in a statistical sense. The aim of this study is to investigate the utility of EPICA in imaging total distribution volume (DVt) and binding potential (BP) with Logan plots in a neuroreceptor mapping study. We applied EPICA to dynamic [(11)C]MPDX PET images in 25 subjects, including healthy subjects and patients with brain diseases, and validated the estimated pTACs. [11C]MPDX is a newly developed radiopharmaceutical for mapping cerebral adenosine A1 receptors. EPICA successfully extracted pTAC for all 25 subjects. Parametric images of DVts were estimated by applying Logan plots with the EPICA-estimated pTAC and then used to define a reference region. The BPs estimated using EPICA were evaluated in 18 subjects by ROI-based comparison with those obtained using the nonlinear least squares method (NLSM). The calculated BPs were identical to the estimates using NLSM in each subject. We conclude that EPICA is a promising technique that generates parametric images of DVt and BP in neuroreceptor mapping without requiring arterial blood sampling.

Algorithms↗

PubMed1976

Paper point transfer of minimal numbers of microorganisms from a prepared canal in vitro.

A strain of Str. faecalis resistant to 2 mg./c.c. streptomycin was developed, and serial dilutions of known quantities of the organism were introduced into prepared root canals of extracted teeth. The canals were cultured with sterile dry paper points, incubated in tubes of thioglycollate media containing 2 mg./c.c. streptomycin at 37 degrees C. These tubes were checked for growth at 24-hour intervals for 7 days. Some tubes yielded growth on the first day, while those with greater dilutions of sample took 7 days. All samples with bacteria did yield growth by the seventh day. At the time of transfer of the organism into the prepared root canals, an identical sample of suspended organisms were spread on pour plates of brain-heart infusion broth containing 2 mg./c.c. streptomycin. After incubation at 37 degrees C. for 24 hours, the numbers of colonies were counted in order to determine the exact number of organisms in each serial dilution. Taking a culture after introduction of sterile distilled water into a canal and evaluation of the culture at the time of the scheduled filling appointment are recommended clinical procedures.

Dental Instruments↗

PubMed1993

The effects of inhalation of grain dust extract and endotoxin on upper and lower airways.

To characterize the short-term effects of grain dusts on pulmonary function, mucosal inflammation, and systemic responses, four women and three men inhaled nebulized corn and soybean dust extracts, endotoxin diluted with Hanks' balanced salt solution (HBSS), and HBSS. Subjects were volunteers recruited via newspaper advertisement and were required to be healthy, nonasthmatic, nonatopic never-smokers. The mean age was 26.9 years (range, 19 to 36 years). Using a randomized, double-blind, crossover design, each subject was challenged with each of the 4 substances with at least 10 days between challenges. Serial spirometry, peripheral blood leukocyte and differential cell counts, and 24-h postchallenge nasal lavages were performed. Extracts were produced by mixing 3 g of the corn or soybean dust with 30 ml HBSS followed by shaking for 60 min, centrifugation, then filter sterilization. The endotoxin solution was produced by mixing lyophilized Escherichia coli endotoxin (serotype 0111:B4) with HBSS to attain a final concentration of 7 mg/L, which was the same as the concentration of endotoxin in both grain dust solutions. The pH of all solutions and unmixed HBSS was adjusted to 5.8, which was the native pH of the soybean dust extract. Subjects were challenged with 0.08 ml/kg of each substance, resulting in a range of endotoxin doses of 30 to 60 micrograms, similar to that which a worker might inhale over the course of one workshift. The lowest mean percentage baseline FEV1 (+/- SD) after inhalation challenge was 99.2 +/- 2.1 for HBSS, and it was significantly lower for endotoxin (90.1 +/- 8.5, p = 0.03), corn dust extract (93.1 +/- 4.3, p = 0.02), and soybean dust extract (96.2 +/- 3.7, p = 0.03). In addition, a peripheral blood leukocytosis developed after exposure to all three endotoxin-containing solutions (p < 0.05), yet a lower peripheral blood lymphocyte count was found only after inhalation of corn dust extract (p = 0.02). Interestingly, this was associated with a higher nasal lavage lymphocyte count after inhalation of corn dust extract (p = 0.03). Neither the decrease in peripheral blood lymphocytes nor the increase in nasal lymphocytes were found after inhalation of soybean dust extract or endotoxin. Our results indicate that extracts of grain dusts have physiologic effects similar to endotoxin. However, in spite of the same endotoxin levels, the effects of corn dust extract appear to have different biologic activity than either soybean dust extract or endotoxin.

Administration, Inhalation↗

PubMed1986

Serial analysis of zein by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis.

Zein, the major storage protein of maize (Zea mays L.) endosperm, was extracted from a number of inbreds with alcohol plus a reducing agent. Isoelectric focusing (IEF) separated total zeins into 41 components, while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated total zeins into about 15 components. Each procedure gave characteristic patterns of zein bands for a number of maize inbreds. IEF and SDS-PAGE were used serially so that each band separated by IEF could be assayed as an individual SDS-PAGE sample. Some IEF bands revealed only a single band after SDS-PAGE, while others revealed two or more bands. A nomenclature system is presented which integrates the two separation systems with information about chromosome locations of zein genes, maize mutations which affect zein synthesis, and inbred sources for different zeins. SDS-PAGE of zein gives apparent molecular masses which vary widely according to the standards used and the properties of the gels, therefore an artificial nomenclature for identifying zein bands after SDS-PAGE is presented. The new nomenclature provides a flexible system which is useful and can be conveniently used in different laboratories.

Journal Article↗

PubMed1995

Prediction of visual function after cataract surgery. A prospectively validated model.

OBJECTIVE: To develop a model to predict visual functional improvement after cataract extraction with intraocular lens implantation based on preoperative data. DESIGN: A prospective study with serial evaluations of visual function preoperatively and at 3 and 12 months after surgery. SETTING: The General Eye Service of the Massachusetts Eye and Ear Infirmary. Boston, Mass, and 33 ophthalmology practices in Boston. PATIENTS: Patients (N = 426; ages, > or = 65 years) who were undergoing cataract surgery. METHODS: Twelve-month improvement in visual function was measured by using the Activities of Daily Vision Scale (ADVS). Ordinal logistic regression was used to identify correlates of improved ADVS scores in 281 patients (derivative set). Potential factors included the preoperative visual acuity, preoperative ADVS score, four chronic ocular diseases, eight medical conditions, and demographic characteristics. Five predictors were identified and used to construct a prediction rule. The accuracy of the prediction rule was evaluated in an independent group of 145 patients (validation set). RESULTS: Postoperatively, 40% of the 281 patients in the derivative set had substantial improvement in their ADVS scores, and 53 (19%) had some improvement. Predictors of improvement included younger age (P < .001), a poorer preoperative ADVS score (P < .001), posterior subcapsular cataract (P = .09), and absence of age-related cataract (P = .09), and absence of age-related macular degeneration (P = .07) and/or diabetes (P = .006). When applied to the independent sample of 145 patients, these five characteristics classified the patients into three groups in which the probabilities of substantial improvement were 85%, 34%, and 3%, thus verifying the discriminatory power of the prediction rule. CONCLUSIONS: Preoperative data can identify patients who are likely to have improvements in visual function after cataract surgery. Such findings may be useful in the selection of patients for this high-volume procedure.

Activities of Daily Living↗

PubMed2005

Heavy metal release from different ashes during serial batch tests using water and acid.

Most ashes contain a significant amount of heavy metals and when released from disposed or used ash materials, they can form a major environmental concern for underground waters. The use of water extracts to assess the easily mobilisable content of heavy metals may not provide an appropriate measure. This study describes the patterns of heavy metal release from ash materials in context with results from the German standard extraction method DIN-S4 (DIN 38 414 S4). Samples of four different ashes (municipal solid waste incineration ash, wood ash, brown coal ash and hard coal ash) were subjected to a number of serial batch tests with liquid renewal, some of which involved the addition of acid to neutralize carbonates and oxides. Release of heavy metals showed different patterns depending on the element, the type of material, the method of extraction and the type of the extractant used. Only a small fraction of the total heavy metal contents occurred as water soluble salts; of special significance was the amount of Cr released from the wood ash. The reaction time (1, 24 or 72 h between each extraction step with water) had only a small effect on the release of heavy metals. However, the release of most of the heavy metals was governed by the dissolution processes following proton inputs, indicating that pH-dependent tests such as CEN TC 292 or others are required to estimate long-term effects of heavy metal releases from ashes. Based on the chemical characteristics of ash materials in terms of their form and solubility of heavy metals, recommendations were made on the disposal or use of the four ash materials.

Carbon↗

PubMed1999

In vitro susceptibility of opportunistic Fusarium spp. to essential oils.

The inhibitory effects of essential oils extracted from 10 Indian plants were evaluated against five fungi. The plants used for extraction of essential oils were six species of the genus Eucalyptus and Ocimum basilicum, Prosopis cineraria and Derris indica. The fungi used in the experiments were Fusarium solani, F. oxysporum, F. pallidoroseum, F. acuminatum and F. chlamydosporum. The susceptibility of the Fusarium species was tested by the paper disc method and the serial dilution technique. The results were compared with the inhibitory effects of miconazole on the fungi. The essential oils extracted from the Eucalyptus species markedly inhibited fungal growth. Prosopis cineraria did not show inhibiting properties. Among the fungi, F. oxysporum proved to be the most resistant species.

Antifungal Agents↗

PubMed1986

Detection of human papillomavirus DNA by the nucleic acid sandwich hybridization method from cervical scraping.

Cervical scrapes collected from 100 consecutive patients participating in a prospective follow-up study for cervical human papillomavirus (HPV) infections were tested for the presence of HPV 11 DNA by the nucleic acid sandwich hybridization method, which allows testing the specimens in a crude form. Part of each specimen was processed through phenol extraction and DNA purification to a dot blot hybridization assay. The dot blots were serially hybridized with HPV 6, 11, 16, and 18 probes as well as with an Alu-repeat probe to estimate the number of cells in the specimen. In PAP smears, HPV-infection was suspected in 63 patients whereas in 37 patients the smear was negative. In the first group, the dot blot assay revealed three cases of HPV 11, two of HPV 16, and one of HPV 18 infection. In the second group with normal PAP smear, one additional HPV 18 infection was found. The sandwich hybridization assay detected 5 HPV 11 infections, including the three mentioned above. All HPV DNA-positive samples contained at least 1.6 X 10(6) cells. Since we considered this a prerequisite for successful diagnosis, only 25 specimens in the first group and 15 in the second were adequate specimens. Thus the HPV-DNA detection rate was 32% (8/25) in the first group and 1/15 in the second. This study demonstrates that sandwich hybridization, detecting 1-3 X 10(5) HPV 11 molecules is a reliable diagnostic method. Cervical scrape is a valuable alternative to punch biopsy, but the number of cells collected is critical for the outcome of the assay.

Cervix Uteri↗

PubMed1982

Effect of operation on cell-mediated immune response to autologous tumor extract in cancer patients.

Utilizing lymphocyte proliferation assay, effects of operation on cell-mediated immune response to autologous tumor extract were studied in 33 patients with gastric or colon carcinoma. To evaluate the lymphocyte proliferation response to autologous tissue extract preoperatively, the lymphocytes were left in culture for 24-30 hours before addition of the extract while 3 M KCl extract of autologous tissue was being processed. A significant level (P less than 0.001) of correlation was observed between stimulation index values to autologous tumor extract added at the initiation of culture and after a 24-30-hour period of incubation. Using this type of assay system, lymphocyte proliferation response to autologous tumor extract was examined immediately before operation and 1, 2, and 4 weeks after operation serially. Data comparing the responses of 20 patients who had curative resection and 13 patients who underwent noncurative resection appeared to fall into three categories in each group. The clinical courses of these patients in each category might clarify the significance of changes in the response following operation.

Colonic Neoplasms↗

PubMed1997

Quantitation of ERCC-2 gene expression in human tumor cell lines by reverse transcription-polymerase chain reaction in comparison to northern blot analysis.

Excision repair cross-complementing rodent repair deficiency genes (ERCC) are human genes implicated in nucleotide excision repair. ERCC-2 has been implicated in the repair of DNA damaged by chemotherapeutic agents, and may thus play an important role in anticancer drug resistance. ERCC-2 gene expression is low in primary tumor samples rendering it difficult to quantitate. We have developed a semiquantitative method to measure ERCC-2 gene expression utilizing reverse transcription-polymerase chain reaction (RT-PCR). Total RNA extracted from established human tumor cell lines was reverse-transcribed to obtain cDNA. Serially diluted reference ERCC-2 DNA fragment was amplified by PCR to obtain a 617-bp fragment. A standard curve was then created using densitometry readings of the 617-bp bands on agarose gel. A fixed amount of sample cDNA from each cell line was amplified at the same time and the resultant PCR product was read by densitometer. Using the standard curve, ERCC-2 gene expression in a given amount of total RNA was quantitated and normalized to beta-actin expression. There was minimal variation in three repeated experiments with PCR amplification. ERCC-2 gene expression determined by this semiquantitative PCR was also correlated to ERCC-2 quantitation by Northern blot analysis, with a significant concordance (r = 0.912, P = 0.0002). We also successfully applied this sensitive method to quantify five clinical glioma samples.

Actins↗

PubMed2001

Qualitative and quantitative assessment of intratubular dentin formation in human natural carious lesions.

It was the aim of this study to investigate the scanning electron microscopy (SEM) features and the element content of calcium (Ca) phosphorus (P), magnesium (Mg), oxygen (O), and carbon (C) of sound dentin, demineralized dentin, secondary dentin, intratubular and peritubular de novo dentin formation due to caries progression and to compare the element content with chemically pure hydroxyapatite. Eighteen extracted teeth with deep dentin carious lesions were embedded in Technovit 9100 (Kulzer), and serial sections of 80 microm thickness were made. These sections were then investigated with polarized light microscopy to identify the lesions. Two sections of each lesion were then coated with carbon and studied with a scanning electron microscope. Of the 18 teeth, 8 showed intratubular dentin formation. The element content was measured using energy dispersive X-ray analysis (EDX). About 75% of all involved dentin tubules showed intratubular de novo dentin formation. The Ca/P ratio in sound dentin, demineralized dentin, peritubular dentin, and secondary dentin was within the range of hydroxyapatite, whereas in intratubular dentin the Ca/P ratio was different than that of hydroxyapatite. The element content for Ca was statistically highly significantly different (P < 0.01) among sound dentin/intratubular dentin, sound dentin/peritubular dentin, and sound dentin/ secondary dentin, but not between sound dentin/ demineralized dentin and sound dentin/hydroxyapatite. For the other measured elements varying statistical differences were found. Our results indicate that intratubular dentin does not occlude the dentin tubules completely and mineralizes different than that of sound dentin and may therefore not be true hydroxyapatite.

Dental Caries↗

PubMed2005

Outcome of isolated antenatal hydronephrosis: a systematic review and meta-analysis.

Idiopathic antenatal hydronephrosis (IAHN), defined as antenatal hydronephrosis not associated with other morphologic renal tract abnormalities, is the most common abnormality detected by antenatal ultrasound. We performed a systematic review and meta-analysis to determine the outcome of IAHN. We screened three databases and bibliographies to identify English-language original peer-reviewed papers that reported serial postnatal renal ultrasonography in children with IAHN. Patients who stabilized and/or improved were extracted and pooled according to the individual grading systems used by each study. A systematic analysis of data extracted from 25 articles revealed overall resolution of pelviectasis in milder cases of IAHN (Society of Fetal Urology [SFU] grade 1-2; anterior posterior pelvic diameter [APPD]<12 mm). In contrast, IAHN of higher severity (grades 3-4; APPD>12 mm) resolved with a lower frequency. Meta-analysis of data extracted from seven papers showed stabilization of pelviectasis in 98% of patients with grades 1-2 (95% confidence interval [CI] 0.93-1.0; p =0.0008) and in 51% of patients with grades 3-4 (95% CI 0.34-0.68; p <0.00001). Grades 1-2 pelviectasis was five times more likely to stabilize than grades 3-4 pelviectasis (odds ratio [OR] 4.69; 95% CI 1.73-12.76; p =0.002). We conclude that in patients with IAHN and lesser degrees of pelvic dilatation, pelvic diameter decreases to the normal range or does not worsen with the vast majority of patients. Further studies are needed to define outcomes, particularly in more severe forms of IAHN.

Fetal Diseases↗