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Biomedical subjects

S Rigby

Publications and source records attributed to S Rigby.

At least 19 recordsLinked to original sources
PubMed2006

Worries and concerns of patients with multiple sclerosis: development of an assessment scale.

This study examined the nature of worry in adult patients with multiple sclerosis (MS) in the UK. A measure of worry in MS was developed and the relationship between worry and standard scale measures of anxiety and depression was examined. Thirty-nine patients with MS and 40 controls completed the new worry scale (WQMS) that was psychometrically evaluated, together with the Penn State Worry Questionnaire (PSWQ), the Hospital Anxiety and Depression Scale (HAD) and a previously developed scale to assess self-efficacy in MS (SESMS). The scores for both anxiety (t = 2.34; P < 0.05) and depression (t = 5.52; P <0.01) were higher in MS patients than controls, as was the new scale for worry--the WQMS. A factor analysis (explaining 65.73% of the variance) suggests that the worries of patients may be dichotomized into those concerned with the physical effects of the disease and those relating to the impact on social interaction, family relationships and daily activity in the home and/or work. Worry in patients with MS was associated with a decreased sense of being able to produce positive activities or effect positive outcomes (low self-efficacy). The questionnaire provides a framework for investigation in clinic of specific concerns and level of worry they engender.

Adult↗
PubMed2003

Osmic acid revisited: factors that predict a favourable response.

OBJECTIVE: Chemical synovectomy with osmic acid has been used for many years in the treatment of chronic synovitis that is unresponsive to systemic treatment and intra-articular corticosteroid. Our aims were to compare the safety and the efficacy of this procedure with that of alternative treatment modalities, and to identify any factors that may predict the response to treatment. METHODS: A retrospective review of 103 osmic acid injections was performed in 65 patients with chronic knee synovitis, between 1992 and 1998. After a clinical review 6 weeks after injection, the length of remission was determined by telephone survey. Thirty-six months of follow-up was available for 96 injections, with a minimum of 12 months for the remainder. Remission was defined as complete absence of pain and swelling. RESULTS: Sixty-nine (67.0%) knee joints remained completely free of pain and swelling at 6 months, falling to 52 (50.5%) at 12 months, 32 (31.1%) at 24 months, and 19 (18.4%) at 36 months. Knees with mild radiological changes experienced significantly better results compared with those with moderate or severe changes (P=0.006 and 0.046 respectively). In patients undergoing bilateral injections, there was a correlation between the duration of remission achieved for each of the two knees (r=0.83, P < 0.01). A correlation was also observed between the responses obtained after first and repeated synovectomies of the same knee (r=0.62, P < 0.05). Eighty-six per cent of injections were uncomplicated. Side-effects included pain, which followed 13 injections (12.4%), and two skin burns. CONCLUSIONS: Long-term complete remission was achieved in 18.4% of knees. This treatment may be best reserved for joints with less severe radiological changes. The response to a first osmic acid injection has a strong predictive value when considering further treatment of the same or the contralateral joint.

Adult↗
PubMed2002

Insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -5 in equine seminal plasma: association with sperm characteristics and fertility.

The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P > or = 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.

Animals↗
PubMed2001

Rapid detection, identification, and enumeration of Escherichia coli by fluorescence in situ hybridization using an array scanner.

A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes and an array scanner for rapid detection, identification, and enumeration of Escherichia coli is described. The test utilizes Cy3-labeled peptide nucleic acid (PNA) probes complementary to a specific 16S rRNA sequence of E. coli. Samples were filtered and incubated for 5 h, the membrane filters were then analyzed by fluorescence in situ hybridization and results were visualized with an array scanner. Results were provided as fluorescent spots representing E. coli microcolonies on the membrane filter surface. The number of fluorescent spots correlated to standard colony counts up to 100 colony-forming units per membrane filter. Above this level, better accuracy was obtained with PNA FISH due to the ability of the scanner to resolve neighboring microcolonies, which were not distinguishable as individual colonies once they were visible by eye.

Colony Count, Microbial↗
PubMed2001

Identification of indicator microorganisms using a standardized PNA FISH method.

A standardized fluorescent in situ hybridization (FISH) method using Peptide Nucleic Acid (PNA) probes for analysis of gram-negative and gram-positive bacteria, as well as yeast, has been developed. Fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas aeruginosa, Staphyloccocus aureus, Salmonella were designed, as well as PNA probes targeting eubacteria and eucarya. These PNA probes were evaluated by PNA FISH using 27 bacterial and 1 yeast species, representing both phylogenetically closely related species, as well as species important to both clinical and industrial settings. The S. aureus and P. aeruginosa PNA probes did not cross react with any of the organisms tested, whereas the E. coli PNA probe, as expected from sequence data, also detected Shigella species. The Salmonella PNA probe reacted with all of the 13 Salmonella strains, representing the 7 subspecies of Salmonella, however, it is also complementary to a few other bacterial species. The eubacteria- and eucarya-specific PNA probes detected all bacterial species and one yeast species, respectively. The general applicability of the PNA FISH method made simultaneous identification of multiple species, both gram-negative and gram-positive, in a mixed population an attractive possibility never accomplished using DNA probes. Four color images using differently labeled PNA probes showed simultaneous identification of E. coli, P. aeruginosa, S. aureus and Salmonella, thereby demonstrating the potential of multiplex FISH for various diagnostic applications within both clinical and industrial microbiology.

Gram-Negative Bacteria↗
PubMed2001

CD4+CD25+ regulatory cells in acquired MHC tolerance.

Tolerance to self-antigens is an ongoing process that begins centrally during T-cell maturation in the thymus and continues throughout the cell's life in the periphery by a network of regulated restraints. Remaining self-reactive T-cells that escape intrathymic deletion may be silenced within the peripheral immune system by specialized regulatory CD4+ cells. By analogy, regulatory CD4+ cells that control immunity to "acquired self" should arise in circumstances where the immune system acquires tolerance to foreign MHC, such as the tolerance that develops following the exposure to foreign MHC antigens during the neonatal period. We have used this classic model of neonatal tolerance to examine the role of regulatory CD4+ cells in acquired tolerance to disparate class I and class II MHC. Adoptive transfer of unfractionated but not CD4+-depleted spleen cells from neonatal tolerant mice into SCID recipients inhibited skin graft rejection by immunocompetent CD8+ T cells. Using 5-bromo-2'-deoxyuridine incorporation, standard cytotoxic T-lymphocyte assays, short-term interferon-gamma ELISPOT, and intracellular FACS analysis to study CD8+ T-cell effector function, we demonstrated that neonatal tolerant mice contain CD4+CD25+ cells that suppress the development of anti-donor CD8+ T-cell responses in vitro. We conclude that regulatory CD4+CD25+ cells initiate and/or maintain tolerance by preventing the development of CD8+ T-cell alloreactivity.

Animals↗
PubMed2000

Traffic of L-selectin-negative T cells to sites of inflammation.

T cells responding to antigen in vivo down-regulate L-selectin, the lymph node homing receptor, as they develop into activated effector cells. The concomitant up-regulation of the proinflammatory adhesion molecules LFA-1, CD44, and VLA-4 suggests that, after their release into the circulation, they traffic to sites of antigen deposition and inflammation. Previous evidence, however, has suggested a role for L-selectin in the recruitment of both neutrophils and lymphocytes into sites of inflammation, which would indicate that these L-selectin(-) effector cells could not be the precursors of inflammatory cells. We therefore directly tested whether L-selectin(-) T cells activated in vivo are capable of homing to model inflammatory sites. L-selectin(-) cells isolated from mice primed with alloantigen or with a contact sensitizer migrated to inflammation markedly better than L-selectin(+) cells from the same animals. Furthermore, the analogous population of CD44(hi)integrin(hi) cells from intravenously primed L-selectin knockout mice traffic efficiently to inflammatory sites and reject allogeneic skin grafts with normal kinetics. These data demonstrate that the previously described L-selectin(-) population of T cells that differentiate into effectors in spleen and lymph nodes subsequently traffic to inflammatory sites, due in part to their increased expression of other proinflammatory adhesion molecules.

Animals↗
PubMed2000

Effect of intrauterine treatment with prostaglandin E2 prior to insemination of mares in the uterine horn or body.

Two trials were conducted to investigate the effects of intrauterine infusion of PGE2 and uterine horn insemination on pregnancy rates in mares achieved by breeding with a suboptimal number of normal spermatozoa. Estrus was synchronized and mares were teased daily with a stallion to detect estrus. Mares in estrus were examined by transrectal palpation and ultrasonography to monitor follicular status. On the first day a 35-mm diameter follicle was present, hCG (1500 IU, iv) was administered and the mares were bred the next day. Mares (Trial 1, n = 34; Trial 2, n = 28) were inseminated with 25 million total spermatozoa from either a stallion with good semen quality (Trial 1) or poor semen quality (Trial 2). In each trial, mares were assigned to 1 of 4 treatment groups as follows: Group PGE-HI - infusion of 0.25 mg PGE2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; Group PGE-BI - infusion of 0.25 mg PGE2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body; Group SAL-HI - infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; or Group SAL-BI - infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body. After breeding, mares were examined daily by transrectal ultrasonography to confirm ovulation, and were re-examined 14 to 16 d after ovulation for pregnancy status. Data were analyzed by Chi-square. Overall pregnancy rates were 59% for stallion 1 and 29% for stallion 2. Group pregnancy rates did not differ for mares bred by either stallion (P > 0.10). Pregnancy rates were not altered by horn insemination for either stallion (P > 0.10). Intrauterine infusion of PGE2 improved pregnancy rate in mares bred by the stallion with good quality semen (P < 0.05), but did not alter pregnancy rate in mares bred by the stallion with poor quality semen (P > 0.10). Further research is warranted to determine if intrauterine infusion of PGE2 will enhance spermatozoal colonization of the oviduct and pregnancy rates in mares, and if PGE-treatment will improve pregnancy rates achieved by subfertile stallions.

Animals↗
PubMed1999

The prevalence of asthma symptoms, bronchial hyperresponsiveness and atopy in New Zealand adults.

AIMS: To examine the prevalence of asthma symptoms, bronchial hyperresponsiveness (BHR) and atopy in a random population sample of New Zealand adults. METHODS: A random sample of 2004 adults, aged 20-44 years, in Hawkes Bay, Wellington and Christchurch, were selected from respondents to a one-page respiratory screening questionnaire and invited to take part in further testing. Subjects attending the testing centres' laboratories underwent a detailed respiratory symptom questionnaire, Phazet testing to eleven common allergens, blood samples for total and specific IgE, and measurement of bronchial hyperresponsiveness. Subjects who did not wish to participate were encouraged to complete the questionnaire by telephone. RESULTS: A participation rate of 67% (1257 of 1877 eligibles) was achieved. We found a high prevalence for all measures of asthma in the previous 12 months: wheezing was reported by 28.5%, waking with shortness of breath by 7.7%, a physician diagnosis of asthma by 15.9% and asthma medications were used by 8.5%. Bronchial hyperresponsiveness was found in 24.9%, atopy in 34.8% and elevated serum IgE levels in 30.5%. Asthma symptoms (in the past 12 months) and atopy decreased with increasing age, whereas bronchial hyperresponsiveness increased with age. Females reported higher prevalences of waking with coughing (45.9%), nasal allergies (43.5%) and skin allergies (48.8%) compared to males (30.5%, 31.9% and 37.0%, respectively). There were no significant regional differences. CONCLUSIONS: Asthma symptoms, bronchial hyperresponsiveness and atopy are all common in adult New Zealanders. Their prevalence is associated with age, gender and current smoking but there are no significant regional differences between Hawkes Bay, Wellington and Christchurch.

Adult↗
PubMed1999

Administration of oxytocin immediately after insemination does not improve pregnancy rates in mares bred by fertile or subfertile stallions.

It is probable that reduced pregnancy rates in mares bred to subfertile stallions is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both uterine and oviductal contractility. The hypothesis that oxytocin may enhance sperm transport to/into the oviducts, and thereby increase pregnancy rates, was tested in 2 trials. For both trials, fertile estrous mares with follicles > or = 35 mm in diameter were inseminated once at 24 h after administration of 1500 to 2000 U hCG. The inseminate dose was limited to 100 million spermatozoa in order to lower pregnancy rates and thus increase the chance of detecting a treatment effect. Pregnancy status was determined by transrectal ultrasound examination 14 to 16 d after insemination. In Trial 1, 49 mares were inseminated with 4 mL extended semen from 1 of 3 stallions (1 fertile and 2 subfertile males). Immediately after insemination, the mares were administered either 20 U oxytocin or 1 mL saline intravenously. In Trial 2, 51 mares were inseminated with 4 mL extended semen from 1 of 4 stallions (1 fertile and 1 subfertile male used in Trial 1, and 2 additional fertile males). Immediately after insemination, and again 30 min later, mares were administered either 5 U oxytocin or 0.25 mL saline intramuscularly. To test for effects of treatment with oxytocin and for the interaction between semen quality and treatment, a generalized linear mixed regression model was used that accounted for the split-plot design (treatment within stallions), the random effect of stallion, the fixed effect of semen quality, the binary outcome of a single breeding trial, and the varying number of trials per stallion/treatment groups. Three treatment protocols or regimens were used: placebo, 5 U oxytocin injected twice intramuscularly, and 20 units oxytocin injected twice intravenously. Semen was classified as high (fertile stallions) or low (subfertile stallions) quality. No interaction between semen quality and treatment was detected (P > 0.10). The pregnancy rate of mares treated with oxytocin immediately after insemination was 30% (15/50) compared with 50% (25/50) for mares treated with saline immediately after breeding. Administration of oxytocin did not affect pregnancy rates (P > 0.10).

Animals↗
PubMed1999

Clonally unrelated BCR-ABL-negative acute myeloblastic leukemia masquerading as blast crisis after busulphan and interferon therapy for BCR-ABL-positive chronic myeloid leukemia.

We report a patient with Philadelphia (Ph)-positive, BCR-ABL rearrangement positive, chronic myeloid leukemia (CML) with a prolonged chronic phase of 24 years who was first prescribed alpha-2 interferon 22 years after initial diagnosis. This therapy was tolerated poorly on account of thrombocytopenia, but an eventual major cytogenetic response was followed soon afterwards by transformation to terminal acute myeloid leukemia (AML). Cytogenetic studies indicated that the transformed myeloblasts were karyotypically normal and Ph negative. Although polymerase chain reaction (PCR) analysis of total leukemic mRNA remained BCR-ABL positive, other molecular studies, including Southern blotting and fluorescent in situ hybridization (FISH) analyses, showed that myeloblasts were BCR-ABL rearrangement negative. PCR-based clonality studies using an X-chromosome-linked restriction fragment polymorphism within the phosphoglycerate kinase gene (PGK1) further showed that the Ph-negative blast cells had a different clonal origin from the Ph-positive clone of chronic phase. We suggest that cases of underlying Ph-negative leukemic transformation in Ph-positive CML warrant further study and should be considered for trial of intensive remission induction therapy as appropriate for acute leukemia.

Acute Disease↗
PubMed1998

Use of prostaglandin E2 to ripen the cervix of the mare prior to induction of parturition.

Eleven light-breed pregnant mares (335 to 347 d gestaton) were used to evaluate the use of prostaglandin E2 as a cervical ripening agent prior to induction of parturition during the months of April and May. Six hours prior to induction, each mare's cervix was examined per vagina for softness and dilation. Each mare was then assigned to 1 of 2 treatment groups: Group PGE mares (n = 7) received 2.0 to 2.5 mg prostaglandin E2 deposited intracervically; Group SAL mares (n = 4) received 0.5 mL of sterile NaCl deposited intracervically. Six hours later, the mares were readied for parturition by wrapping the tail, scrubbing and rinsing the perineum and udder, and examining the cervix as previously described. Each mare was then administered 15 U, i.v. oxytocin at 15-min intervals until the chorioallantois ruptured. Intervals from initial oxytocin injection until rupture of the chorioallantois, from initial oxytocin injection until delivery of the foal, from delivery of the foal until the foal stood unassisted, and from delivery of the foal until the foal suckled were recorded. Mean cervical dilation immediately prior to induction of parturition tended to be greater in Group PGE mares (3.9 +/- 1.7 cm) than in Group SAL mares (1.9 +/- 1.9 cm; P = 0.10). Mean change in cervical dilation over the 6-h period prior to induction (3.4 +/- 1.9 cm vs 1.5 +/- 2.1 cm), mean number of injections of oxytocin required until the chorioallantois ruptured (1.9 +/- 0.7 vs 2.5 +/- 1.0), and mean intervals from initial injection of oxytocin to rupture of the chorioallantois (20 +/- 10 min vs 28 +/- 19 min) and delivery of the foal (28 +/- 7 min vs 34 +/- 22 min) were not different between Group PGE and SAL mares, respectively (P > 0.10). The proportion of foals that stood within 1 h of birth also did not differ between Group PGE foals (6/7; 86%) and Group SAL foals (3/4; 75%; Chi-square = 0.17; P > 0.10). The proportion of foals that nursed within 2 h of birth was higher in Group PGE foals (6/7; 86%) than in Group SAL foals (1/4; 25%; Chi-square = 4.02; P < 0.05). Premature separation requiring manual rupture of the chorioallantois at the vulvar labia occurred in 1 Group PGE mare (cervical dilation of 1.5 cm at time of induction) and 1 Group SAL mare (cervix closed and firm at time of induction). Foals born from the 2 mares with premature placental separation had the longest intervals from initial oxytocin injection to delivery, delivery to ability to stand unassisted, and delivery to suckling within their respective treatment groups. In summary, it appears that cervical ripening prior to induction of parturition favors shorter deliveries and foal vigor. Intracervical administration of prostaglandin E2 may prove useful for ripening the cervix of the mare prior to induction of parturition. Further studies are indicated to determine optimal dosage and method of administration of prostaglandin E2.

Allantois↗
PubMed1998

Homing of transgenic gammadelta T cells into murine vaginal epithelium.

The vaginal epithelium of normal mice contains lymphocytes of fetal thymic origin that express an invariant Vgamma4/Vdelta1 TCR. The apparent lack of other gammadelta TCR species suggests that a selection mechanism might operate to regulate the localization of gammadelta T cells at this anatomical site. Selection might be connected to the Vgamma4/Vdelta1 TCR or to some homing characteristic of the fetal thymic lineage that appears at day 17-18 of embryonic life. In the present studies, we investigated whether transgenic gammadelta cells expressing a TCR species characteristic of the subpopulation of gammadelta T cells found in the blood, spleen and lymph would translocate to the vaginal epithelium. We found that the transgenic Vgamma2 TCR+ cells did accumulate in the vagina of transgenic mice. Furthermore, like normal vaginal gammadelta T cells, the transgenic vaginal gammadelta T cells expressed the phenotype of recently activated memory/effector T cells (CD44(hi), CD62L-, CD45RB(lo), CD69+). Vaginal gammadelta T cells in normal mice do not express the CD2 and CD28 antigens, but both of these markers are present on transgenic vaginal gammadelta T cells. We observed that a small fraction of splenic transgenic gammadelta T cells had the same surface phenotype as the vaginal transgenic gammadelta T cells, raising the possibility that the gammadelta T cells present in the vaginal epithelium of transgenic mice originated from the peripheral lymphoid organs. Data in support of this possibility came from experiments in which co-incubation of splenic transgenic gammadelta T cells with vaginal epithelial cell suspensions induced the vaginal gammadelta phenotype on the splenic gammadelta T cells. The finding of transgenic gammadelta T cells in the vaginal epithelium suggests that homing of gammadelta T cells to this site is not restricted to gammadelta T cells that express the V4/NS1 invariant TCR. Furthermore, these findings imply that retention of gammadelta T cells in the vaginal epithelium of normal mice is affected by a Vgamma4/Vdelta1-specific mechanism. The finding of a significant level of apoptosis in the transgenic vaginal gammadelta T cells, but not in the normal vaginal gammadelta T cells, could reflect that the mechanism of retention of Vgamma4/Vdelta1 + in the vaginal epithelium involves selective survival at the site.

Animals↗
PubMed1997

Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay.

We have adapted an assay for the direct detection of Mycobacterium tuberculosis using a prototype automated instrument platform in which probes are amplified with Q-beta replicase. The assay was based on amplification of specific detector probe following four cycles of background reduction (reversible target capture) in a closed disposable pack. The assay signal was the time required for fluorescence to exceed background levels (response time [RT]). RT was inversely related to the number of M. tuberculosis rRNA target molecules in the sample. Equivalent signals and noises were observed in assays containing either sputum or buffer. All mock samples containing > or = 10 CFU of M. tuberculosis responded in the assay (average RT, 13.91 min), while most (83%) samples containing as many as 10(7) CFU of Mycobacterium avium gave no response during a 25-min amplification reaction. The samples containing M. avium which did respond had an average RT of 17.04 min. Seventy-five percent (167 of 223) of samples containing no target gave no responses; the remaining 25% had an average RT of 15.53 min. Eighty-three frozen sputum samples were tested to develop a candidate cutoff RT for the assay prior to more extensive clinical testing. After resolution of discrepant results and with a 14-min RT cutoff, 30 of 38 M. tuberculosis-positive samples were positive by the assay; 1 of 45 negative samples responded within 14 min. Assay sensitivity, specificity, and positive and negatives predictive values in this pilot study were 79, 98, 97, and 85%, respectively.

DNA Probes↗
PubMed1997

Performance of an automated Q-beta replicase amplification assay for Mycobacterium tuberculosis in a clinical trial.

We present data from a clinical trial study in which an automated version (Galileo) of a previously described Q-Beta replicase-amplified probe assay (J. S. Shah et al., J. Clin. Microbiol. 33:1435-1441, 1995) was used for the direct detection of Mycobacterium tuberculosis complex in sputum. The assay was designed to target specific regions of 23S rRNA found in M. tuberculosis, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium microti and had a sensitivity ranging from approximately <10 to 300 CFU. The assay was tested for cross-hybridization by using large numbers (e.g., 10(5)to 10(10) CFU/assay) of 133 other organisms commonly found in respiratory tract samples, including non-M. tuberculosis Mycobacterium spp., other bacteria, fungi, and viruses. All of these competitors tested negative by the assay. Automated assay results for 780 respiratory tract samples (sputum or bronchoalveolar lavage specimens) collected and tested at three trial sites in the United States) were compared with the results of culture and acid-fast microscopy. Aliquots of conventionally digested and decontaminated sputum pellets were heated at 100 degrees C and mechanically disrupted prior to hybridization and background reduction, amplification, and detection in a closed disposable test pack. Pertinent elements of individual patient histories relating to tuberculosis exposure, previous active disease, antituberculosis therapy status, etc., were considered in the resolution of discrepant results for 48 (assay false-positive) samples. Seventy-one of 90 (78.9%) culture-positive samples were positive when tested in the Galileo assay, while 7% of culture-negative samples were assay positive, corresponding to a sensitivity of 79% and a specificity of 93%. Following resolution of discrepant results by chart review, the sensitivity and specificity for the Q-Beta replicase amplification assay with the Galileo analyzer were 84 and 97%, respectively. A total of 69.2% of smear-negative (culture positive) samples were detected by the assay. Ten test packs at a time were automatically processed by the Galileo analyzer without operator intervention following loading of samples. The first result was reported in approximately 3 h, and the last result was available in 6.5 h. To our knowledge, this is the first report of a clinical study with a fully automated amplification probe hybridization assay for the detection of pathogens directly from a clinical specimen.

Bronchoalveolar Lavage Fluid↗
PubMed1997

Tissue and differentiation specific expression on the endogenous retrovirus ERV3 (HERV-R) in normal human tissues and during induced monocytic differentiation in the U-937 cell line.

ERV3 (HERV-R) is a complete, single copy human endogenous retrovirus located on the long arm of chromosome 7. The open reading frame in its envelope gene has been conserved during evolution but the gag and pol genes contain in-frame termination codons. To find a suitable experimental model system for analysis of the functions of the ERV3 genome, an extensive screening study of different normal and neoplastic human tissues was performed. Most tissues express low levels of the ERV3 env mRNA although high expression levels are observed in placenta, sebaceous glands, adrenals, testis, bronchial, epithelium and the monocytic cell line U-937. In U-937 cells the ERV3 env expression varied in a manner related to the differentiation status of the cells; being highest in the terminally differentiated non proliferating cells. U-937 cells can be induced to differentiate from the monoblastic to the mature monocyte/macrophage stage upon stimulation by several substances such as phorbolesters (TPA), Vitamin D3, Retinoic Acid (RA) and combinations of some cytokines. We conclude that the ERV3 locus is expressed in a tissue and differentiation specific way and that the U-937 cell line is a suitable model system to further analyze the proposed functions of ERVs such as immunomodulation, cell fusion and protection against exogenous retroviral infections.

Cell Differentiation↗
PubMed1996

Expression of the endogenous retrovirus ERV3 (HERV-R) during induced monocytic differentiation in the U-937 cell line.

ERV3 (HERV-R) is a complete human endogenous retrovirus located on the long arm of chromosome 7. LTR-env-gene-spliced mRNA of 9 and 3.5 Kb is widely expressed in human tissues and cells, but gag-pol mRNA has not been found. Further, the env gp70 gene contains an open reading frame throughout its length and its expression has recently been detected as a full-length protein. The highest expression of ERV3 detected so far is in placenta and the lowest in cytotrophoblasts and choriocarcinoma cell lines. In this report we have studied ERV3 mRNA and protein expression in the human monoblastic cell line U-937 during differentiation into monocytes/macrophages. Differentiation of U-937 cells was induced by 1,25a-dihydroxyvitamin D3 (vitD3), retinoic acid (RA), gamma interferon (IFN-gamma) and phorbol-myristate-acetate (PMA-TPA). The expression of ERV3 env mRNA was found to be differentiation-associated, with high expression detected in the late stages of monocytic development. Using TPA, the expression of ERV3 env was detected as 9- and 3.5-kb transcripts by Northern blotting, as mRNA by in situ hybridization and as a cytoplasmic 65-kDa protein by immunofluorescence and Western blots. Low levels of basal expression were found, with up-regulation of both message and protein at 24 to 48 hr after addition of TPA. Induction with vitD3, IFN-gamma and RA produced higher levels of mRNA at earlier time points. It is concluded that the U-937 cell line represents an excellent model system for further studies to study the relationship between ERV3 expression and cellular differentiation.

Calcitriol↗