Detection of pyrimethamine resistance in Plasmodium falciparum by mutation-specific polymerase chain reaction.

The point mutation at nucleotide 323 within the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum, which distinguishes pyrimethamine-sensitive from drug-resistant isolates, can be discriminated by the polymerase chain reaction using mutation-specific primers. The technique makes use of the principle that short oligonucleotides with a perfect match at their 3' ends, complementary to the mutation to be detected, will initiate the polymerization by Taq polymerase far more efficiently than primers with a single mismatch in this position. The Asn-108 codon was detected using a primer of 17 nucleotides with an adenosine at its 3' end, the Thr-108 codon with a 14-mer primer ending with a cytosine and the Ser-108 codon with a 16-mer containing guanidine at the critical 3' end. By selecting appropriate counterprimers, the size of the amplification products is either indicative of pyrimethamine-resistant parasites of the 7G8 type, or of pyrimethamine-sensitive parasites of the FCR-3 type or 3D7 type. The fragments obtained can be easily separated in a single lane after agarose gel electrophoresis. Coded P. falciparum DNA samples were typed unambiguously using these primers as were reconstituted parasitized blood samples stored as high salt lysates. Sensitivity, speed and specificity make this assay a realistic alternative to in vitro drug testing to monitor the resistance of P. falciparum to inhibitors of the dihydrofolate reductase.

Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum.

The dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional complex from pyrimethamine-sensitive (3D7) and drug-resistant (HB3 and 7G8) clones from Plasmodium falciparum was purified to homogeneity. A modified sequence of purification steps with a 10-formylfolate affinity column at its center, allows the isolation of the enzyme complex with a 10-fold higher yield than previously reported, irrespective of the pyrimethamine resistance of the parasites. Titration of the homogenous DHFR-TS complex with the inhibitor revealed a 500-fold lower affinity of the enzyme from clone 7G8 for the drug than found with the enzyme from clone 3D7. Direct comparison of the homogenous enzyme preparations on SDS-PAGE revealed no difference in the molecular mass of the DHFR-TS from the 3 clones, nor could a reproducible difference be detected in the peptide patterns obtained after digesting the DHFR-TS complex with various proteases. The amplification of segments from the DHFR-TS coding region of the 3 clones and 7 isolates of P. falciparum by polymerase chain reaction resulted in fragments of the predicted length without any size heterogeneity. The DNA sequence of the DHFR coding region from FCR-3, 3D7, HB3 and 7G8 differs in a total of 4 nucleotides. One point mutation changes amino acid residue 108 from threonine (FCR-3) or serine (3D7) to asparagine (HB3 and 7G8). The presence of asparagine-108 appears to be the molecular basis of pyrimethamine resistance of HB3 and 7G8. The degree of resistance is associated with a point mutation affecting the codon for amino acid 51 in 7G8.

A Plasmodium falciparum-specific reverse target capture assay.

Plasmodium falciparum DNA is detected with an assay modeled according to the reverse target capture assay described by Morrissey et al. [19] for the detection of Listeria cells. A poly(A)-tailed oligonucleotide (pWZ34), derived from the partial sequence of a 4-kb repetitive unit of P. falciparum, functions as a capture probe and the labelled 21-bp repetitive units specific for P. falciparum serve as a reporter probe. Both probes are complementary to non-overlapping regions of the target DNA and in the presence of high concentration of chaotropic salts, hybridization efficiently takes place at relatively low temperatures (15 min. 37 degrees C). The addition of poly(dT)-derivatized ferromagnetic beads allows the formation of A:T base pairing between the tailed beads and the tailed capture probe. Upon applying magnetic force, the target-capture-reporter-probe complex attached to the beads is removed from the reaction mixture, leaving the bulk of unreacted reporter molecules behind. Subsequent washings of the immobilized complex reduces the amount of non-specifically bound reporter probe. After elution of the complex from the beads a new cycle of capture, washing and release of the target-capture-reporter-probe complex is initiated by the additions of unused (dT)-tailed beads. After 3 cycles, the signal-to-noise ratio with 0.1 pg of P. falciparum DNA as a target was as high as 21-27, with a background of 8-10 cpm. The assay is unique in its speed, well suited for large sample numbers, and allows the manipulation of the background at will by simply increasing the number of capture rounds.

Inhibition of VCAM-1 expression in human bronchial epithelial cells by glucocorticoids.

We have demonstrated previously that cytokines induce surface expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on BEAS-2B bronchial epithelial cells in vitro. The present studies demonstrate glucocorticoid inhibition of cytokine-induced VCAM-1 expression as detected using flow cytometry and Northern blot analysis. Several commonly used inhaled glucocorticoids were tested for their ability to inhibit VCAM-1 and ICAM-1 expression. All glucocorticoids tested inhibited VCAM-1 expression in a dose-dependent manner. No inhibition of ICAM-1 expression was observed. The most potent of the glucocorticoids tested for inhibition of VCAM-1 expression were mometasone furoate and fluticasone propionate (FP), which had IC50 values (i.e., concentrations at which each glucocorticoid produced 50% inhibition) of under 10 pM. Budesonide, triamcinolone acetonide, and beclomethasone dipropionate (BDP) had intermediate potency, and hydrocortisone and the BDP metabolite beclomethasone-17-monopropionate were the least potent of the steroids tested. Kinetic analysis of the ability of FP to inhibit VCAM-1 expression revealed that preincubation with FP for 3 h completely inhibited VCAM-1 expression induced by tumor necrosis factor-alpha (TNF-alpha). FP inhibited VCAM-1 expression by 50% even when added as late as 6 h after stimulation with TNF-alpha. Using Northern blot analysis, we confirmed inhibition of VCAM-1 and ICAM-1 messenger RNA (mRNA) expression by FP. Pretreatment with FP (10(-11) M to about 10(-7) M, 24 h) inhibited TNF-alpha-induced VCAM-1 mRNA expression in BEAS-2B in a dose-dependent manner, but did not inhibit expression of ICAM-1 mRNA. Studies with actinomycin D indicate that FP treatment accelerated the degradation of TNF-alpha-induced VCAM-1 mRNA. FP (10(-7) M) also inhibited VCAM-1 mRNA expression induced by TNF-alpha in primary human bronchial epithelial cells as assessed by reverse transcription-polymerase chain reaction. These results suggest that suppression of epithelial VCAM-1 expression by glucocorticoids may contribute to their anti-inflammatory effects.

Phenotyping and cytokine regulation of the BEAS-2B human bronchial epithelial cell: demonstration of inducible expression of the adhesion molecules VCAM-1 and ICAM-1.

Airway epithelium may actively participate in inflammatory responses, such as occur in asthma. The presence and regulation of surface molecules on the airway epithelium, however, is incompletely understood. We have determined the phenotype of the human bronchial epithelial cell line BEAS-2B by flow cytometry. We confirmed previous observations that human bronchial epithelial cells constitutively express CD29, CD44, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54 (ICAM-1), CD61, and HLA class 1. BEAS-2B cells were also found to constitutively express CD9, CD13, CD15, CD15s, CD23, CD33, CD36, CD40, CD41b, CD42b, CD48, CD50, CD71, and CD102 (ICAM-2). Culture of BEAS-2B cells with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta (1 ng/ml) was found to enhance intercellular adhesion molecule-1 (ICAM-1) expression (several fold) and induce de novo CD106 [vascular cell adhesion molecule-1 (VCAM-1)] expression. TNF-alpha or IL-1beta did not change the expression of CD9, CD13, CD16, CD23, CD29, CD31, CD32, CD35, CD45, CD61, or CD64 in BEAS-2B cells. IL-4 (1 ng/ml) also induced expression of VCAM-1 (1.5-fold) but not ICAM- expression while interferon-gamma (1 ng/ml) enhanced only ICAM-1 expression (2-fold). Maximal VCAM-1 expression was obtained with the combination of TNF-alpha and IL-4 (8-fold). Using Northern blot hybridization analysis, ICAM-1 and VCAM-1 mRNA was detected in BEAS-2B cells stimulated with cytokines. VCAM-1 on stimulated BEAS-2B was functionally active as determined by adhesion of purified eosinophils and blockade with specific antibodies. Primary isolates of bronchial epithelial cells produced detectable levels of VCAM-1 protein and mRNA as detected by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. These results suggest that cytokine activation induces expression of ICAM-1 and VCAM-1 on airway epithelium, an event which may influence leukocyte infiltration and activation.

Expression of interleukin-16 by human epithelial cells. Inhibition by dexamethasone.

Production of chemoattractants by bronchial epithelial cells may contribute to the local accumulation of inflammatory cells in patients with bronchial asthma and other pulmonary diseases. Recently, interleukin (IL)-16 (lymphocyte chemoattractant factor) was reported to be a potent chemotactic stimulus for CD4(+) T lymphocytes and eosinophils, the types of leukocyte found in the proximity of bronchial epithelium in asthmatic individuals. To test the possibility that bronchial epithelial cells produce IL-16, we analyzed RNA and culture supernatants from the human bronchial epithelial cell line BEAS-2B, using reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. BEAS-2B constitutively expressed IL-16 messenger RNA (mRNA) and protein; IL-16 expression was significantly upregulated in a concentration-dependent manner within 24 h by stimulation with histamine, IL-1beta, or tumor necrosis factor (TNF)-alpha whereas interferon-gamma did not significantly increase IL-16. Findings in BEAS-2B cells were confirmed in primary bronchial epithelial cells. Using TA cloning, IL-16 was cloned from BEAS-2B airway epithelial cells. Sequence analysis confirmed its near identity with lymphocyte-derived IL-16. The combination of IL-1beta and TNF-alpha had an additive effect on IL-16 expression. This combination of cytokines also had a priming effect on histamine-induced IL-16 mRNA expression, which was observed within 24 h and which increased to at least 48 h after stimulation. The IL-16 expression induced by histamine and combined cytokines was significantly inhibited by pretreatment with the protein synthesis inhibitor cycloheximide (10 microg/ml). Pretreatment with dexamethasone also significantly suppressed the expression of IL-16, in a concentration-dependent manner. Sputum samples from asthmatic subjects were found to have higher levels of IL-16 than were samples from subjects with other pulmonary inflammatory diseases. These findings suggest that bronchial epithelial cells have the capacity to produce IL-16 after stimulation with histamine, IL-1beta, and TNF-alpha, and raise the possibility that epithelium-derived IL-16 may play a role in recruitment of eosinophils and CD4(+) T lymphocytes in the airways. Downregulation of IL-16 expression by dexamethasone suggests that glucocorticoids may inhibit airway inflammation partly by suppressing the synthesis of inflammatory cytokines including IL-16.

Expression of the C-C chemokine receptor CCR3 in human airway epithelial cells.

Chemokine-induced eosinophil chemotaxis is mediated primarily through the C-C chemokine receptor, CCR3. We have now detected CCR3 immunoreactivity on epithelial cells in biopsies of patients with asthma and other respiratory diseases. CCR3 mRNA was detected by Northern blot analysis after TNF-alpha stimulation of the human primary bronchial epithelial cells as well as the epithelial cell line, BEAS-2B; IFN-gamma potentiated the TNF-alpha-induced expression. Western blots and flow cytometry confirmed the expression of CCR3 protein. This receptor is functional based on studies demonstrating eotaxin-induced intracellular Ca(2+) flux and tyrosine phosphorylation of cellular proteins. The specificity of this functional response was confirmed by blocking these signaling events with anti-CCR3 mAb (7B11) or pertussis toxin. Furthermore, (125)I-eotaxin binding assay confirmed that CCR3 expressed on epithelial cells have the expected ligand specificity. These studies indicate that airway epithelial cells express CCR3 and suggest that CCR3 ligands may influence epithelial cell functions.

Interleukin-13 upregulates eotaxin expression in airway epithelial cells by a STAT6-dependent mechanism.

Interleukin (IL)-13 is a T helper 2-derived cytokine that has recently been implicated in allergic airway responses. We hypothesized that IL-13 may regulate expression of eotaxin in airway epithelium. We found that IL-13 upregulated eotaxin messenger RNA and protein synthesis in the airway epithelial cell line BEAS-2B; this effect showed synergy with tumor necrosis factor (TNF)-alpha and also was inhibited by the glucocorticoid budesonide. To establish the mechanisms of eotaxin upregulation by IL-13, cells were transfected with an eotaxin promoter-luciferase reporter plasmid and transcription was activated by IL-13 (1.7-fold) and TNF-alpha (2.8-fold). The combination of IL-13 and TNF-alpha additively activated the promoter constructs (4.1-fold). Activation of signal transducer and activator of transcription (STAT) 6 by IL-13 was confirmed by nuclear protein binding to a DNA probe derived from the eotaxin promoter. Activation of eotaxin transcription by IL-13 and the additive effect with TNF-alpha were lost in plasmids mutated at a putative STAT6 binding site. Cotransfection with a wild-type STAT6 expression vector significantly enhanced activation of the eotaxin promoter after IL-13 stimulation (6-fold induction). A significant increase of eotaxin protein secretion in the supernatant of STAT6 wild-type-transfected cells was observed after IL-13 stimulation. Cotransfection with a dominant negative STAT6 mutant expression vector inhibited activation of the eotaxin promoter by IL-13. These results indicate that IL-13 stimulates eotaxin expression in airway epithelial cells and that STAT6 plays a pivotal role in this response.

Activation of eotaxin gene transcription by NF-kappa B and STAT6 in human airway epithelial cells.

The C-C chemokine eotaxin is a potent chemoattractant for eosinophils and probably plays an important role in the pathogenesis of asthma, although the mechanisms of its regulation are not well known. Airway epithelial cells express eotaxin mRNA and protein after stimulation with a variety of cytokines. We focused on the molecular mechanisms of eotaxin gene regulation by TNF-alpha and IL-4 in the airway epithelial cell line, BEAS-2B. Cells were transfected with luciferase reporter plasmids, which contained up to 1363 bp of the eotaxin promoter. Eotaxin promoter activity was increased by TNF-alpha (2.5-fold) and IL-4 (1.5-fold), respectively. The combination of TNF-alpha and IL-4 produced 3.6-fold activation of the eotaxin promoter. The eotaxin promoter contains overlapping consensus binding sites for transcription factors, NF-kappa B and STAT6, which are known to mediate responses to TNF-alpha and IL-4, respectively. Electrophoretic mobility shift assays revealed NF-kappa B binding after TNF-alpha stimulation and STAT6 binding after IL-4 stimulation using a DNA probe derived from the eotaxin promoter. Mutant plasmids were generated to define the roles of these transcription factors in eotaxin promoter activity. TNF-alpha stimulation, but not IL-4 stimulation, was lost in plasmids mutated at the NF-kappa B binding site, whereas IL-4 stimulation, but not TNF-alpha stimulation, was lost in plasmids mutated at the STAT6 binding site. When both sites were mutated, all transcriptional activation was lost. These results imply that TNF-alpha and IL-4 stimulate expression of the eotaxin gene by activating NF-kappa B and STAT6.

Atopic dermatitis is associated with a functional mutation in the promoter of the C-C chemokine RANTES.

Up-regulation of C-C chemokine expression characterizes allergic inflammation and atopic diseases. A functional mutation in the proximal promoter of the RANTES gene has been identified, which results in a new consensus binding site for the GATA transcription factor family. A higher frequency of this allele was observed in individuals of African descent compared with Caucasian subjects (p < 0.00001). The mutant allele was associated with atopic dermatitis in children of the German Multicenter Allergy Study (MAS-90; p < 0.037), but not with asthma. Transient transfections of the human mast cell line HMC-1 and the T cell line Jurkat with reporter vectors driven by either the mutant or wild-type RANTES promoter showed an up to 8-fold higher constitutive transcriptional activity of the mutant promoter. This is the first report to our knowledge of a functional mutation in a chemokine gene promoter. Our findings suggest that the mutation contributes to the development of atopic dermatitis. Its potential role in other inflammatory and infectious disorders, particularly among individuals of African ancestry, remains to be determined.