Rhinovirus-induced PBMC responses and outcome of experimental infection in allergic subjects.

BACKGROUND: The immune response to rhinovirus (RV) infections is considered to contribute to upper respiratory symptoms and may also be an important contributor to lower airway dysfunction in patients with asthma. OBJECTIVE: This study was conducted to determine the relationship of RV-specific responses in PBMCs to the outcome of experimentally induced infection with RV16. METHODS: Twenty-two subjects with either allergic rhinitis or asthma were inoculated with RV16: virus-induced proliferation and cytokine production were determined on PBMCs obtained before and then again 7 and 28 days after inoculation. RESULTS: Several subjects had proliferative responses to RV16 before inoculation, and precold RV-specific proliferative responses were inversely correlated (r(s) = -0.62, P <. 005) with RV shedding after inoculation. In addition, there was a negative correlation (r(s) = -0.58, P = 0.01) between precold RV-induced IFN-gamma secretion ex vivo and peak RV shedding during the cold. CONCLUSIONS: Certain RV-specific lymphocyte responses before the cold (vigorous proliferation or IFN-gamma secretion) were associated with reduced viral shedding after inoculation. These findings suggest that variations in mononuclear cell responses to RV could contribute to the individual variability in viral shedding during experimentally induced, and perhaps naturally acquired, RV infections in subjects with respiratory allergy or asthma.

Characterization of CYP1B1 and CYP1A1 expression in human mammary epithelial cells: role of the aryl hydrocarbon receptor in polycyclic aromatic hydrocarbon metabolism.

CYP1B1 and CYP1A1 expression and metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) have been characterized in early-passage human mammary epithelial cells (HMECs) isolated from reduction mammoplasty tissue of seven individual donors. The level of constitutive microsomal CYP1B1 protein expression was donor dependent (<0.01-1.4 pmol/mg microsomal protein). CYP1B1 expression was substantially induced by exposure of the cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to levels ranging from 2.3 to 16.6 pmol/mg among the seven donors. Extremely low, reproducible levels of constitutive CYP1A1 expression were detectable in three donors (0.03-0.16 pmol/mg microsomal protein). TCDD inductions were larger for CYP1A1, as compared to CYP1B1, demonstrating substantial variability in the induced levels among the donors (0.8-16.5 pmol/mg). Northern and reverse transcriptase PCR analyses corroborate the donor-dependent differences in protein expression, whereby CYP1B1 mRNA (5.2 kb) was constitutively expressed and was highly induced by TCDD (33-fold). The contributions of CYP1B1 and CYP1A1 to the metabolism of DMBA were analyzed using recombinant human CYP1B1 and CYP1A1, as references, in conjunction with antibody-specific inhibition analyses (anti-CYP1B1 and anti-CYP1A1). Constitutive microsomal activity exhibited a profile of regioselective DMBA metabolism that was characteristic of human CYP1B1 (increased proportions of 5,6- and 10,11-DMBA-dihydrodiols), which was inhibited by anti-CYP1B1 (84%) but not by anti-CYP1A1. TCDD-induced HMEC microsomal DMBA metabolism generated the 8,9-dihydrodiol of DMBA as the predominant metabolite, with a regioselectivity similar to that of recombinant human CYP1A1, which was subsequently inhibited by anti-CYP1A1 (79%). A CYP1B1 contribution was indicated by the regioselectivity of residual metabolism and by anti-CYP1B1 inhibition (25%). DMBA metabolism analyses of one of three donors expressing measurable basal expression of CYP1A1 confirmed DMBA metabolism levels equivalent to that from CYP1B1. The HMECs of all donors expressed similar, very high levels of the aryl hydrocarbon receptor and the aryl hydrocarbon nuclear translocator protein, suggesting that aryl hydrocarbon receptor and aryl hydrocarbon nuclear translocator protein expression are not responsible for differences in cytochrome P450 expression. This study indicates that CYP1B1 is an important activator of polycyclic aromatic hydrocarbons in the mammary gland when environmental chemical exposures minimally induce CYP1A1. Additionally, certain individuals express low levels of basal CYP1A1 in HMECs, representing a potential risk factor of mammary carcinogenesis through enhanced polycyclic aromatic hydrocarbon bioactivation.

Isolation of human conjunctival mast cells and epithelial cells: tumor necrosis factor-alpha from mast cells affects intercellular adhesion molecule 1 expression on epithelial cells.

PURPOSE: To isolate and purify mast cells and epithelial cells from human cadaveric donor conjunctival tissue and to characterize interactions between these cell types in vitro. METHODS: Monodispersed cell suspensions obtained by enzymatic digestion of conjunctival tissue were applied to a single-density Percoll gradient. Epithelial cells obtained from the top layer of the gradient were cultured to confluence. Mast cells obtained from the pellet were equilibrated in culture medium and further purified using a two-step Percoll gradient. Using reverse transcription-polymerase chain reaction (RT-PCR), RNA from the purified mast cell preparation was probed for tumor necrosis factor-alpha (TNF alpha) message. Fluorescence activated cell sorting (FACS) analysis of intracellular immunostained mast cells was used to detect the TNF alpha protein. An examination for intercellular adhesion molecule 1 (ICAM-1) on epithelial cells was performed after 24-hour incubations with either recombinant TNF alpha supernatants from calcium ionophore A23187 (CaI)-stimulated mast cells or appropriate controls using FACS analysis. RESULTS: Highly purified human conjunctival mast cells and epithelial cells (each > 95%) were obtained from human cadaveric donor tissue. RT-PCR analysis of purified mast cell RNA revealed the expression of TNF alpha mRNA. An evaluation of mast cells for intracellular protein demonstrated positive staining for tryptase and TNF alpha. ICAM-1 was found on purified epithelial cells, and incubation of epithelial cell monolayers with supernatants from Cal-stimulated mast cells resulted in upregulation of this receptor. This upregulation was blocked by incubation with TNF alpha-neutralizing antibody. CONCLUSIONS: This work provides the methods for isolating and purifying mast cells and epithelial cells from human donor tissue and the opportunity for studying mechanisms of conjunctival inflammation by evaluating the interactions between these cells.

An E-box-mediated increase in cad transcription at the G1/S-phase boundary is suppressed by inhibitory c-Myc mutants.

To better understand the signaling pathways which lead to DNA synthesis in mammalian cells, we have studied the transcriptional activation of genes needed during the S phase of the cell cycle. Transcription of the gene encoding a pyrimidine biosynthetic enzyme, carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (cad), increases at the G1/S-phase boundary. We have mapped the growth-dependent response element in the hamster cad gene to the extended palindromic E-box sequence, CCACGTGG, which is centered at +65 in the 5' untranslated sequence. Mutation of the E box abolished growth-dependent transcription, and an oligonucleotide corresponding to the cad sequence at +55 to +75 (+55/+75) restored growth-dependent regulation to nonresponsive cad promoter mutants when placed down-stream of the transcription start site. The same oligonucleotide conferred less G1/S-phase induction when placed upstream of basal promoter elements. An analogous oligonucleotide containing the mutant E box had no effect in either location. Nuclear proteins bound the cad +55/+75 element in a cell cycle-dependent manner in electromobility shift assays; antibodies specific to USF and Max blocked the DNA-binding activity of different growth-regulated protein-DNA complexes. Expression of c-Myc mutants which have been shown to dominantly interfere with the function of c-Myc and Max significantly inhibited cad transcription during S phase but had no effect on transcription from another G1/S-phase-activated promoter, dhfr. These data support a model whereby E-box-binding proteins activate serum-induced transcription from the cad promoter at the G1/S-phase boundary and suggest that a Max-associated protein complex contributes to the serum response.

Start site selection at the TATA-less carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase promoter.

Transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, starts at a single major site. We characterized the cis-acting elements that position RNA polymerase II at the correct start site in the CAD promoter. Sequence alignment showed that the CAD promoter lacks a TATA box, but contains a consensus initiator. Mutational analysis of the CAD promoter demonstrated that the sequences between -81 and +26 were sufficient for accurate and efficient transcription in vitro and in vivo; binding sites for the transcription factor Sp1 around -70 and -49 were necessary for transcriptional activity. The binding site at -49 directed initiation about 50 base pairs downstream. A ubiquitous activator protein, Honk, bound to the CAD promoter between -30 and -12, but did not participate in start site selection. The sequences around +1, which contain the consensus initiator, contributed to promoter activity; however, the presence of a consensus initiator in this region was neither necessary nor sufficient for transcription. We concluded from these results that the Sp1 binding site at -49 substituted for the missing TATA box and played a major role in start site selection at the CAD promoter.