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PubMed1982

A standardized quantitative skin-test assay of allergen potency and stability: studies on the allergen dose-response curve and effect of wheal, erythema, and patient selection on assay results.

A quantitative skin-test assay of allergenic potency was developed and tested on 28 allergic patients. The best-fit linear regression line was calculated from the sum of erythema or wheal diameters obtained from four intradermal threefold serial dilutions near the endpoint. Each line must have a correlation coefficient greater than 0.85 and the slopes of reference and test extract lines must not be significantly different. The relative potency can be calculated from the horizontal distance between the reference and test lines. The between-assay reproducibility was determined by comparing references against themselves. The 95% confidence limits were 54% to 186% when erythema was used and 27% to 367% when wheal was used. The narrower limits for the assay using erythema maybe due to the 5.5 times steeper slope of erythema lines than wheal lines. Potency results on five commercial extracts were highly correlated with potency results by RAST, AgE assay, or rye I assay. Extracts with isolectric focusing patterns dissimilar from the references were of significantly lower potency by in vivo and in vitro assays. The measured potency of two low-potency short ragweed extracts varied significantly with patient sensitivity to heat-stable and heat-labile ragweed allergens, indicating that patient selection may significantly affect assay results. Dose-response lines using either histamine or short ragweed by puncture (P) and intradermal (ID) techniques in the same patient showed that P/ID dose for equal erythema response was 909 for histamine and 31,250 for ragweed. This quantitative skin-test assay is highly re[rpdicob;e, yields potency data comparable to those of in vitro tests, can be applied to studies of extract stability, and is suitable as a primary bioassay of allergenic activity.

Allergens↗
PubMed1985

Parasympathetic neurotrophic activity in the rat iris: determination after different denervations.

The iris of the adult rat contains one or several neurotrophic factors that enhance the survival of dissociated parasympathetic neurons (from the embryonic chick ciliary ganglion) in culture. To assay survival activity, iris homogenates were serially diluted with culture medium and the percentage of neurons surviving for 2 days in a collagen matrix in culture determined. The extract induced survival curves that were similar for denervated and normal irides. Similarly no differences in fibre outgrowth from cultured whole ciliary ganglia were found. The results suggest that the apparent level of parasympathetic growth factor(s) is not under strict control of the innervation of the iris.

Animals↗
PubMed2000

The effect of minocycline on the metabolism of androgens by human oral periosteal fibroblasts and its inhibition by finasteride.

The antimicrobial minocycline has matrix-stimulatory effects on connective tissue and bone. The aim here was to study the effect of minocycline on 5alpha reduction of androgen substrates to 5alpha-dihydrotestosterone (DHT) in periosteal fibroblasts and the influence of the antiandrogen finasteride on this conversion. Confluent cultures of periosteal fibroblasts established from oral periosteum isolated from the bone surface were incubated in duplicate in multiwell dishes with two androgen substrates, [(14)C]-testosterone/[(14)C]-4-androstenedione, in the presence or absence of serial concentrations of minocycline or the antiandrogen finasteride or the two in combination for 24 h. The metabolites formed were solvent-extracted with ethyl acetate, separated by thin-layer chromatography and quantified using a radioisotope scanner. Both androgen substrates were metabolized to DHT and 4-androstenedione or testosterone. Minocycline stimulated the synthesis of DHT from these substrates by 75-83% at 20-30 microg/ml (n=4; p<0.01). Finasteride inhibited the 5alpha-reductase activity of these substrates by 3-5-fold at 1 microg/ml and 40-80% at 0.01 and 0.1 microg/ml (n=4; p<0.01), with little change in 17beta-hydroxysteroid dehydrogenase activity. Minocycline and finasteride in combination showed an intermediate response with one substrate. As finasteride inhibits the type 2, 5alpha-reductase isoenzyme associated with anabolic functions, these findings demonstrate target-tissue androgen metabolic activity in periosteal fibroblasts at baseline and in response to minocycline. This has implications for the reparatory potential of the diseased periodontium during adjunctive treatment with minocycline.

17-Hydroxysteroid Dehydrogenases↗
PubMed1997

The use of short and long PCR products for improved detection of prunus necrotic ringspot virus in woody plants.

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of prunus necrotic ringspot virus (PNRSV) in dormant peach and almond trees by the application of two different pairs of primers yielding a short and a long product, respectively. The relative amount of the short (200 base pair, bp) product was higher than the longer (785 bp) product. PNRSV was detected better in plant tissues with a low virus concentration (e.g. dormant trees) by amplification of the short PCR product, whereas the long product was product was produced at higher virus titers. Simultaneous amplification of both short and long products was demonstrated using a three-primer mixture in a single reaction tube. In this assay, amplification of either PCR product indicated the presence of PNRSV-specific sequences in the plant tissue examined, thus covering a wide range of virus concentrations in a single test. Dilution of the RNA extracted from infected plant material resulted in a steep decline in the amplification of both short and long PCR products. In contrast, serial dilutions of the intermediate cDNA template differentially affected the amplification patterns: the relative amount of the short product increased whereas that of the long product decreased. These results may explain the preferential amplification of the short PCR product observed in samples containing low virus concentrations.

DNA, Complementary↗
PubMed2002

Real-time sequence-specific primer polymerase chain reaction amplification of HLA class II alleles: a novel approach to analyze microchimerism.

The careful assessment of microchimerism is essential to investigate the effects of donor bone marrow-derived cells in transplantation. We have developed a protocol to assess microchimerism based on the HLA mismatch between the recipient and the donor. Our approach combines real-time polymerase chain reaction (PCR) with sequence-specific primer PCR (SSP-PCR) to selectively amplify and measure the abundance of donor HLA alleles in DNA samples extracted from the recipient after transplant. To optimize and validate the reliability of this method at different levels of microchimerism, we tested serial dilutions of donor DNA into recipient DNA. We demonstrate that donor alleles can be readily detected and reliably measured at concentrations as low as 0.1%. This method is simple and rapid and could find practical application in the assessment of microchimerism in patients receiving organ or cellular transplants in conjunction with donor bone marrow cells infusion.

Alleles↗
PubMed2001

A comparative molecular analysis of developing mouse forelimbs and hindlimbs using serial analysis of gene expression (SAGE).

The analysis of differentially expressed genes is a powerful approach to elucidate the genetic mechanisms underlying the morphological and evolutionary diversity among serially homologous structures, both within the same organism (e.g., hand vs. foot) and between different species (e.g., hand vs. wing). In the developing embryo, limb-specific expression of Pitx1, Tbx4, and Tbx5 regulates the determination of limb identity. However, numerous lines of evidence, including the fact that these three genes encode transcription factors, indicate that additional genes are involved in the Pitx1-Tbx hierarchy. To examine the molecular distinctions coded for by these factors, and to identify novel genes involved in the determination of limb identity, we have used Serial Analysis of Gene Expression (SAGE) to generate comprehensive gene expression profiles from intact, developing mouse forelimbs and hindlimbs. To minimize the extraction of erroneous SAGE tags from low-quality sequence data, we used a new algorithm to extract tags from -analyzed sequence data and obtained 68,406 and 68,450 SAGE tags from forelimb and hindlimb SAGE libraries, respectively. We also developed an improved method for determining the identity of SAGE tags that increases the specificity of and provides additional information about the confidence of the tag-UniGene cluster match. The most differentially expressed gene between our SAGE libraries was Pitx1. The differential expression of Tbx4, Tbx5, and several limb-specific Hox genes was also detected; however, their abundances in the SAGE libraries were low. Because numerous other tags were differentially expressed at this low level, we performed a 'virtual' subtraction with 362,344 tags from six additional nonlimb SAGE libraries to further refine this set of candidate genes. This subtraction reduced the number of candidate genes by 74%, yet preserved the previously identified regulators of limb identity. This study presents the gene expression complexity of the developing limb and identifies candidate genes involved in the regulation of limb identity. We propose that our computational tools and the overall strategy used here are broadly applicable to other SAGE-based studies in a variety of organisms. [SAGE data are all available at GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession nos. GSM55 and GSM56, which correspond to the forelimb and hindlimb raw SAGE data.]

Animals↗
PubMed1978

A report on clinical aspects and histopathology of sweet itch.

Sweet itch is an intensely pruritic dermatitis of horses recurring annually in Ireland from April to November. The tissue changes of sweet itch have similarities to immediate-type hypersensitivity reactions which occur in skin sensitised to the saliva of bloodsucking insects. There was subepidermal oedema, and marked eosinophilia; the blood vessels were tortuous and enlarged. Microfilaria were not found in serial sections of lesions of 5 affected horses. The histopathology of the immediate dermal remal reaction to the intradermal injection of Culicoides extract shows dermal vasodilation and eosinophil diapedesis. Seven horses challenged intradermally with whole extract of Culicoides, Stomoxys calcitrans, Tabanidae and saline, gave immediate reactions to Culicoides, 3 gave immediate reactions to Stomoxys calcitrans. Immediate and delayed reactions only occurred at the sites of challenge with Culicoides extract. Three normal ponies when challenged intradermally with extracts of biting flies, showed minimal dermal reactions.

Animals↗
PubMed2006

Development and validation of a real-time PCR method to quantify rumen protozoa and examination of variability between entodinium populations in sheep offered a hay-based diet.

PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (epsilon) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R2= 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (10(0) and 10(6) entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.

Animals↗
PubMed1987

Effect of repeated in vitro transfer of Pasteurella haemolytica A1 on encapsulation, leukotoxin production, and virulence.

Pasteurella haemolytica serotype 1 was transferred daily for 128 serial passages on both unsupplemented brain heart infusion agar and the same basal medium supplemented with bovine blood, horse serum, and yeast extract. Repeatedly transferred cultures were shown to retain the ability to produce both capsular material and leukotoxin. Furthermore, intact organisms were found to be as toxic in vitro for bovine leukocytes and as virulent for mice as unpassaged cultures. These results indicate that the precaution of using only freshly isolated cultures in the study of this organism may not be necessary.

Animals↗
PubMed1997

Characterization of multiple promoters directing tissue-specific expression of the human gonadotropin-releasing hormone gene.

Two promoters directing tissue-specific expression of GnRH gene in neuronal and reproductive tissues were characterized by functional analyses of GnRH promoter-luciferase (LUC) constructs in transfected placental cells (JEG) and hypothalamic neuronal cells (GT1-7). Results indicate that the downstream promoter directs the expression in a neuronal cell-specific manner, whereas the upstream promoter is fully active in the nonneural placental cell line. Transfection studies carried out in several tumor cell lines derived from human reproductive tissues verified that the upstream GnRH promoter construct was much more active in directing luciferase expression in reproductive tissue. The use of both upstream and downstream promoters in various human tumor cell lines derived from reproductive tissues were demonstrated by RT-PCR. Our studies also demonstrate that the reproductive tissue-specific messenger RNA transcribed from upstream promoter is capable of directing synthesis of preproGnRH protein. Serial deletion studies localized a cell-specific upstream promoter region that directs reproductive tissue expression. DNase I footprint analysis using nuclear extract obtained from the JEG cells indicated DNA/protein interactions in four specific sequence elements of the upstream promoter region. The interaction between nuclear binding proteins present in the JEG cells (but not the GT1-7 cells) and the four specific sequences in the upstream promoter region was confirmed by gel mobility shift analysis.

Animals↗
PubMed1986

Persistence of fetal zone function in the infant rhesus monkey adrenal gland.

Plasma dehydroepiandrosterone sulfate (DHAS) concentrations increase markedly in the rhesus monkey fetus at the end of gestation. A further increase occurs in the infant. To determine whether the changes in plasma concentration between the fetus and infant represent maintenance of DHAS production by the infant adrenal gland, we measured the t1/2, distribution volume (VD), MCR, and production rate of DHAS in the late gestation rhesus monkey fetus (129-155 days gestation; term is 165 days) and infant (14-42 days of age). A single bolus dose of [3H]DHAS was injected into five fetuses and four infants, and blood samples were collected serially from 5 min to 24 h after the injection. The amount of [3H]DHAS in the circulation was measured after solvolysis, extraction, and Celite chromatography. The concentration of DHAS in each sample was measured by RIA. DHAS was cleared significantly more rapidly in the fetus than in the infant [MCR in fetus, 2.4 +/- 0.4 (+/- SE); MCR in infant, 0.6 +/- 0.2 liters day-1 kg-1]. The t1/2 of DHAS was shorter in the fetus than in the infant (1.0 +/- 0.1 vs. 3.3 +/- 0.7 h). Absolute VD values were larger in the fetus than in the infant (231 +/- 29 and 143.8 +/- 11.6 ml kg-1); however, they were similar when the fetal VD was calculated including placental weight as a component of fetal weight. The production rate of DHAS, calculated as the product of MCR and integrated plasma DHAS concentration for the duration of the experiment, was not significantly different between the fetus and the infant (1.0 +/- 0.2 and 3.3 +/- 1.2 mg kg-1 day-1) in spite of the marked differences in plasma DHAS concentrations (445.8 +/- 103.8 ng ml-1 in the fetus and 5165 +/- 1296 ng ml-1 in the infant). These results indicate that the adrenal of the infant rhesus monkey continues to secrete DHAS at a rate at least as high as that in the late gestation fetus. Since the infant maintains DHAS production similar to that of the fetus in the absence of the placenta, a corollary of these studies is that the elevated DHAS secretion in the rhesus infant is independent of the placenta or the hormonal milieu of pregnancy. The maintenance of a functional fetal zone in the adrenal gland makes the rhesus infant a suitable model to use in studying the regulation of DHAS secretion and fetal zone morphology.

Adrenal Glands↗
PubMed1986

Serologic follow-up study in neurocysticercosis patients by ELISA after praziquantel treatment.

/A total of 69 patients of confirmed neurocysticercosis was followed serologically by ELISA up to 22 months after praziquantel treatment. The intervals and numbers of follow-up were variable by patients. Serially collected samples of serum and CSF were examined simultaneously for their specific IgG antibody levels by ELISA, using cystic fluid, saline extracts of bladder wall and scolex as antigen. Within 4 months after praziquantel treatment, the antibody levels were elevated temporarily in both serum and CSF in most patients. In some cases antibody levels exhibited steady declining tendency after the treatment. Concomitant administration of dexamethasone appeared to suppress the elevation of antibody levels. The rate of mean absorbance of antibody changed more in serum than in CSF. The rate of elevation was greater in antibodies to parenchymal antigens than that to cystic fluid, but absolute difference of antibody levels was greater in anitbody to cystic fluid. Previously negative samples for IgG antibody may become positive after praziquantel treatment, which could be used as a complementary tool(provocation test) in serodiagnosis. One month was considered to be sufficient interval for the follow-up test for that purpose. In the follow-up of up to 22 months, only few cases of chronic neurocysticercosis showed declining tendency of IgG antibody levels below negative range. During acute encephalitic attacks in chronic patients, IgG antibody to parenchymal antigen were elevated in CSF temporarily. These results indicated that serologic follow-up of every year was recommendable to differentiate the cured patients from chronic patients with slowly calcifying lesions.

Journal Article↗
PubMed2004

Isolation of bovine viral diarrhea virus 1, a pestivirus from autopsied lamb specimen from Tamil Nadu, India.

An epizootic of febrile illness among the Madras red breed of sheep had occurred in 1994 in Verrapuram, Chennai, India. The epizootic was suspected as Rift Valley fever (RVF)-like sickness based on clinical features. However, its etiological agent could neither be isolated nor implicated conclusively. During the post-epizootic period a male lamb died of similar clinical features and the spleen was immediately collected. Inoculation of spleen suspension in infant mouse brain yielded a virus that was serially passaged in infant mice and rhabdomyosarcoma (RD) cells. Electron microscopic observations revealed virus particles resembling flaviviruses. RT-PCR performed on extracted total RNA from infected cells and mouse brains with flavivirus-specific or RVF-specific primers gave negative results. However, an amplicon of 280 bp was obtained with pestivirus-specific primers from the 5'-UTR. Further, a nested PCR yielded a product of 157 bp. Nucleotide sequencing of the 157 bp product showed 100% homology to BVDV-1. Western blot analysis with a flavivirus envelope protein-specific MAb revealed three proteins of 33 K, 45 K and 55 K. Further studies suggested that the 33 K and 55 K proteins were glycosylated. This is the first report of isolation of BVDV-1 from a lamb in India.

Animals↗
PubMed1988

Report on skin test standardization. The Committee on Skin Test Standardization of The Netherlands Society of Allergology.

The decision to set up a Committee on Skin Test Standardization was taken in 1985 at the spring meeting of the Netherlands Society of Allergology. The committee's terms of reference were to make recommendations about: (i) the standardization of skin-test techniques; (ii) ensuring the quality of skin-test techniques; (iii) determining whether tests should be carried out using serial dilutions and, if so, under what conditions; (iv) the adaptation of standard series or the use of minimum numbers of allergen extracts with adults and children. This committee cannot be viewed separately from the previously established Committee on the Standardization of Allergen Extracts. The availability of allergen extracts that are as highly standardized as possible is an essential condition for good skin-test diagnostics.

Adult↗
PubMed2001

IgA cross-reactivity between a nuclear autoantigen and wheat proteins suggests molecular mimicry as a possible pathomechanism in celiac disease.

Celiac disease patients display IgA antibody reactivity to wheat as well as to human proteins. We used serum IgA from celiac patients and, for control purposes, from patients with Crohn's disease, ulcerative colitis and from healthy individuals to identify celiac disease-specific IgA autoantigens in nitrocellulose-blotted extracts from various human cell types (epithelial, endothelial, intestinal cells, fibroblasts). The pattern, recognition intensity and time course of IgA autoreactivity was monitored using serial serum samples obtained from celiac children before and under gluten-free diet. By immunoblot inhibition and subcellular (cytosolic, nuclear) cell fractionation we identified a 55 kDa nuclear autoantigen expressed in intestinal, endothelial cells and in fibroblasts which was recognized by IgA antibodies of approximately half of the celiac disease patients and cross-reacted with wheat proteins. IgA reactivity to the 55 kDa autoantigen disappeared during gluten-free diet and was inhibited after pre-absorption of sera with wheat proteins but not with tissue transglutaminase, previously reported as the unique celiac disease-specific autoantigen. In conclusion, we defined a novel 55 kDa celiac disease-specific nuclear IgA autoantigen which shares epitopes with wheat proteins and which is different from tissue transglutaminase and calreticulin. Although the newly defined autoantigen was recognized much less frequently than tissue transglutaminase, our data suggest molecular mimicry between wheat and human proteins as a possible pathomechanism for the induction and/or maintenance of mucosal tissue damage in celiac disease.

Adolescent↗
PubMed1971

Quantitation of extrathyroidal conversion of L-thyroxine to 3,5,3'-triiodo-L-thyronine in the rat.

Studies of the rate of extrathyroidal conversion of thyroxine (T4) to 3,5,3'-triiodo-L-thyronine (T3) were carried out in rats. Total body homogenates were prepared and extracted with ethanol 48, 72, and 96 hr after the intravenous injection of (125)I-T4. (131)I-T3 was added, and the paper chromatographic purification of T3 was effected by serial elution and rechromatography in three paper and one thin-layer cycles. The ratio of (131)I-T3 and (125)I-T3 counting rates in the final chromatograms, which was identical in three different paper chromatography systems, was used to calculate the proportion of (125)I-T3 to (125)I-T4 in the original homogenates. In order to discount the effects of in vitro monodeiodination of T4 during extraction and chromatography, we killed control animals immediately after injection of (125)I-T4 and processed them in a similar fashion to the experimental groups. The average ratio of (125)I-T3 to (125)I-T4 in carcass extracts of animals killed between 48 and 96 hr after isotopic injection was 0.08 whereas the average ratio of (125)I-T3 to (125)I-T4 in chromatograms of control animals was 0.01. On the basis of the proposed model, calculations indicated that about 17% of the secreted T4 was converted to T3. Assuming values cited in the literature for the concentration of nonradioactive T3 in rat plasma, these findings would suggest that about 20% of total body T3 is derived by conversion from T4. Moreover, since previous estimates have suggested that in the rat, T3 has about 3 to 5 times greater biologic activity than T4, these results also raise the possibility that the hormonal activity of T4 may be dependent in large part on its conversion to T3.A necessary assumption in calculating T4 to T3 conversion in this and other studies is that the 3' and 5' positions are randomly labeled with radioiodine in phenolic-ring iodine-labeled T4. Evidence supporting this assumption was obtained in the rat by comparing the amount of labeled T3 produced after injection of phenolic and nonphenolic-ring iodine-labeled T4.

Animals↗
PubMed1998

Measurements of human breast cancer using magnetic resonance spectroscopy: a review of clinical measurements and a report of localized 31P measurements of response to treatment.

A review of the literature has shown that in human breast tumours, large signals from phosphomonoesters (PME) and phosphodiesters (PDE) are evident. In serial measurements in 19 patients with breast cancer, a decrease in PME was significantly associated with a stable or responding disease (p = 0.017), and an increase in PME was associated with disease progression. Extract studies have shown PME to comprise of phosphoethanolamine (PEth) and phosphocholine (PCho), with the PEth to PCho ratio ranging from 1.3 to 12. The PCho content of high grade tumours was found to be higher than low grade tumours. In some animal models, changes in PCho have been shown to correlate with indices of cellular proliferation, and spheroid studies have shown a decrease in PCho content in spheroids with smaller growth fractions. A serial study of 25 patients with advanced primary breast tumours undergoing hormone, chemotherapy or radiotherapy treatments, showed that in this heterogenous group there were significant changes in metabolites that were seen during the first 3 weeks (range 2-4 weeks) of treatment, that correlated with volume change over this period, employed here as a measure of response. Changes in PME (p = 0.003), total phosphate (TP) (p = 0.008) and total nucleoside tri-phosphate (TNTP) (p = 0.02) over 3 (+/-1) weeks were significantly associated with response, as were the levels of PME (p<0.001), PDE (p = 0.01), TP (p = 0.001) and TNTP (p = 0.007) at week 3 (+/-1). PME at week 3 (+/-1) was also significantly associated with the best volume response to treatment (p = 0.03). A reproducibility analysis of results from the observation of normal breast metabolism in four volunteers showed a mean coefficient of variation of 25%, after correcting for changes resulting from the menstrual cycle. Reproducibility studies in four patients with breast cancer showed a mean coefficient of variation of 33%, with the reproducibility being better in patients measured on different days (difference in TP was -6%) compared with those measured on the same day (difference in TP was -29%).

Adult↗
PubMed1987

Surfactant displacement by meconium free fatty acids: an alternative explanation for atelectasis in meconium aspiration syndrome.

Meconium, an ether extract of meconium, and the major free fatty acids of meconium (palmitic, stearic, and oleic acids) were all found to increase the surface tension minimum of dog lung extract in a Wilhelmy balance. Each of these fractions was instilled into the lungs of dogs (15 experimental, eight saline solution controls), and cardiac output, venous and arterial blood gases, pulmonary, atrial, and systemic pressures, airway pressure, and static lung compliance were serially monitored for 2 hours. Mean airway pressure increased and static lung compliance decreased significantly in all of the experimental groups. Although arterial pH and PaCO2 and the various hemodynamic measurements did not change during the experiment, PaO2 decreased significantly and did not return to baseline in all experimental groups. Extracts from atelectatic portions of experimental dog lung had a surface tension minimum of greater than 20 dynes/cm, whereas airway foam had a surface tension minimum of less than 10 dynes/cm, suggesting that the free fatty acids of meconium are able to strip surfactant from the alveoli.

Animals↗