Identification and target-size analysis of the leukotriene D4 receptor in the human THP-1 cell line

The human acute monocytic leukemia cell line THP-1 has been identified, by radioligand binding, as expressing the leukotriene D4 receptor at a high level (4000 binding sites per cell), without the need for further cell differentiation. [3H]Leukotriene D4-specific binding to THP-1 cell membranes was of high affinity (KD = 0.47 nM) and saturable, enhanced by divalent cations but inhibited by both monovalent cations and non-hydrolyzable GTP analogs. The cysteinyl leukotrienes competed for [3H]leukotriene D4-specific binding with the following rank order of potency: leukotriene D4 > leukotriene E4 > leukotriene C4. In addition, leukotriene D4-receptor antagonists from two structural classes, the quinolines MK-571 and L-697,008, and the indole ICI 204,219, displayed nanomolar potency in [3H]leukotriene D4 competition assays. These data show that [3H]leukotriene D4-specific binding to THP-1 cell membranes fulfils the criteria for binding to a leukotriene D4 receptor regulated through interaction with a G protein. Several novel features of the THP-1 leukotriene D4 receptor were investigated. Culture of THP-1 cells in the presence of tunicamycin, an inhibitor of N-glycosylation, resulted in a 6-fold decrease in the number of detectable [3H]leukotriene D4-specific binding sites. Target-size analysis by radiation inactivation estimated a molecular mass of 65 kDa for the [3H]leukotriene D4 specific binding site(s) present in THP-1 cell membranes. Together, these results suggest that the human THP-1 cell leukotriene D4 receptor is a glycosylated protein with a molecular mass of approx. 65 kDa within the membrane environment.     

Pertussis in Missouri: evaluation of nasopharyngeal culture, direct fluorescent antibody testing, and clinical case definitions in the diagnosis of pertussis

No diagnostic test for pertussis in routine use in the United States has both high sensitivity and high specificity. During a statewide increase in the incidence of pertussis in Missouri, we studied the clinical features of 153 patients with suspected pertussis in the Greater St. Louis area from whom a specimen for pertussis culture had been taken between 15 May and 19 September 1989. In this cross-sectional study, nasopharyngeal cultures were more likely to be positive for persons whose specimens were collected < 21 days after cough onset (adjusted rate ratio [RRa] and 95% confidence interval = 3.4; 1.5-8.0) and who were not receiving erythromycin/sulfamethoxazole prior to the culture [RRa = 5.8; 0.8-40.6], who had received fewer than three prior doses of pertussis vaccine [RRa = 1.8; 0.8-4.2], and whose specimen was in transit to the laboratory for < 4 days [RRa = 2.0; 0.8-5.5]. Among children < 5 years of age, spasmodic cough plus a lymphocytosis of > 10,000/mm3 was the acute symptom complex associated with the highest predictive value for a positive culture result (67%). Cough for > or = 14 days plus whoop was sensitive (81%) and specific (58%) for identifying children with culture-confirmed pertussis. Direct fluorescent antibody staining performed well as a screening test for pertussis but requires substantial commitment of personnel and resources. In the absence of a positive culture result, clinical case definitions should be used for decision making (e.g., initiation of antimicrobial therapy and routine case reporting).     

Quantitation of matrix metalloproteinases in cultured rat astrocytes using the polymerase chain reaction with a multi-competitor cDNA standard

Matrix metalloproteinases (MMPs) are a family of Zn2+ endopeptidases that are expressed in many inflammatory conditions and that contribute to connective tissue breakdown and the release of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). There is emerging evidence that MMPs have a role in inflammatory disorders of the central nervous system (CNS) such as multiple sclerosis. However, little is known about the expression of MMPs by inflamed tissue within the CNS or by the glia, neurones, and leucocytes which participate in the inflammatory response. To address this issue we have developed a polymerase chain reaction (PCR)-based method for the quantitation of rat MMP mRNA levels, which we have applied to astrocyte cultures with and without inflammatory stimulation. The technique relies on a competition reaction in which a synthetic standard cDNA is co-amplified with the target cDNA in the same PCR reaction. Standard multi-competitor cDNAs, containing priming sites for nine MMPs, and two housekeeping genes were constructed. We have shown that MMP activity is increased over three-fold in neonatal rat astrocyte cultures following stimulation with lipopolysaccharide (LPS). At the mRNA level, MT-MMP-1, 72 kDa gelatinase, and stromelysin-3 were constitutively expressed and unaffected by LPS treatment, whereas 92 kDa gelatinase, and stromelysin-1 were strongly induced (1,000-fold). Stromelysin-2, rat collagenase, and macrophage metalloelastase were modestly upregulated by LPS treatment. Matrilysin was not expressed. This technique is suitable for quantifying MMP expression in the cells which contribute to inflammation in the CNS and could also be applied directly to tissue samples from animal models of disease.     

First trimester maternal serum concentrations of fetal antigen 2 in normal pregnancies and those affected by trisomy 21

Serum concentrations of fetal antigen 2 (FA-2), the amino-propeptide of the alpha1 chain of collagen type I, were measured in peripheral blood from women with normal (n = 234) and trisomy 21 affected (n = 14) pregnancies between 9 and 11 weeks gestation. Serum FA-2 concentrations were seen to be stable throughout this period, and though raised FA-2 concentrations were seen at the 10th week of gestation, a statistically significant difference between normal and trisomy 21 affected pregnancies was not found overall. Therefore it seems unlikely that FA-2 has a role in first trimester screening for trisomy 21, despite the fact that significantly higher FA-2 concentrations in trisomy 21 and significantly lower concentrations in trisomy 18 had been previously demonstrated in amniotic fluid in the second trimester.     

The in-vitro effect of continuous and pulsed magnetic field on the colony-forming ability of bone marrow cells from hematologic patients

The investigation was concerned with the effect of magnetic field on the colony- and cluster-forming potential of myelo- and monocytopoietic precursor-cells in different blood diseases. Bone marrow from 72 patients was exposed in vitro to low continuous magnetic field and to intermittent continuous and pulsed one--from another 22 patients. The ADMT-Magnipulse source was used. Magnetic field proved a biologically-active factor capable of changing the clone-forming properties, proliferative and, possibly, differentiating potential of hemopoietic precursor-cells.