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Biomedical subjects

M Brown

Publications and source records attributed to M Brown.

At least 307 records · Page 17Linked to original sources
PubMed1995

Differential activation of peroxisome proliferator-activated receptors by eicosanoids.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate gene transcription in response to peroxisome proliferators and fatty acids. PPARs also play an important role in the regulation of adipocyte differentiation. It is unclear, however, what naturally occurring compounds activate each of the PPAR subtypes. To address this issue, a screening assay was established using heterologous fusions of the bacterial tetracycline repressor to several members of the peroxisome proliferator-activated receptor (PPAR) family. This assay was employed to compare the activation of PPAR family members by known PPAR activators including peroxisome proliferators and fatty acids. Interestingly, the activation of PPARs by fatty acids was partially inhibited by the cyclooxygenase inhibitor indomethacin, which prevents prostaglandin synthesis. Indeed, prostaglandins PGA1 and 2, PGD1 and 2, and PGJ2-activated PPARs, while a number of other prostaglandins had no effect. We also screened a variety of hydroxyeicosatetraenoic acids (HETEs) for the ability to activate PPARs. 8(S)-HETE, but not other (S)-HETEs, was a strong activator of PPAR alpha. Remarkably, PPAR activation by 8(S)-HETE was stereoselective. In addition, 8(S)-HETE was able to induce differentiation of 3T3-L1 preadipocytes. These results indicate that PPARs are differentially activated by naturally occurring eicosanoids and related molecules.

3T3 Cells↗
PubMed1995

Polarity-specific activities of retinoic acid receptors determined by a co-repressor.

Retinoic acid receptors (RARs) and retinoid-X receptors (RXRs) activate or repress transcription by binding as heterodimers to DNA-response elements that generally consist of two direct repeat half-sites of consensus sequence AGGTCA. On response elements consisting of direct repeats spaced by five base pairs (DR + 5 elements), RAR/RXR heterodimers activate transcription in response to RAR-specific ligands, such as all-trans-retinoic acid (RA). In contrast, on elements consisting of direct repeats spaced by one base pair (DR + 1 elements), RAR/RXR heterodimers exhibit little or no response to activating ligands and repress RXR-dependent transcription. Here we show that ligand-dependent transactivation by RAR on DR + 5 elements requires the dissociation of a new nuclear receptor co-repressor, N-CoR, and recruitment of the putative co-activators p140 and p160. Surprisingly, on DR + 1 elements, N-CoR remains associated with RAR/RXR heterodimers even in the presence of RAR ligands, resulting in constitutive repression. These observations indicate that DNA-response elements can allosterically regulate RAR-co-repressor interactions to determine positive or negative regulation of gene expression.

Allosteric Regulation↗
PubMed1995

Enhancement and destruction of antibody function by somatic mutation: unequal occurrence is controlled by V gene combinatorial associations.

We examined the positive and negative effects of somatic mutation on antibody function using saturation mutagenesis in vitro to mimic the potential of the in vivo process to diversify antibodies. Identical mutations were introduced into the second complementarity determining region of two anti-phosphocholine antibodies, T15 and D16, which share the same germline VH gene sequence. T15 predominates in primary responses and does not undergo affinity maturation. D16 is representative of antibodies that co-dominate in memory responses and do undergo affinity maturation. We previously reported that > 50% of T15 mutants had decreased antigen binding capacity. To test if this high frequency of binding loss was unique to T15 or a consequence of random point mutations applicable to other combining sites, we analyzed the same mutations in D16. We show that D16 suffers a similar loss of function, indicating an equally high potential for B-cell wastage. However, only D16 displayed the capacity for somatic mutation to improve antigen binding, which should enhance its persistence in memory responses. Mutation of residues contacting the haptenic group, as determined by molecular modeling, did not improve binding. Instead, productive mutations occurred in residues that either contacted carrier protein or were distant from the antigen binding site, possibly increasing binding site flexibility through long-range effects. Targeting such residues for mutation should aid in the rational design of improved antibodies.

Amino Acid Sequence↗
PubMed1995

The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis.

Cytotoxic T lymphocytes (CTL) are thought to contribute to viral clearance and liver cell injury during hepatitis B virus (HBV) infection. Using a strategy involving the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with HBV-derived synthetic peptides containing HLA-A2.1, -A31, and -Aw68 binding motifs, we have previously described CTL responses to several epitopes within the HBV nucleocapsid and envelope antigens in patients with acute hepatitis. In this study we define six HLA-A2-restricted CTL epitopes located in the highly conserved reverse transcriptase and RNase H domains of the viral polymerase protein, and we show that the CTL response to polymerase is polyclonal, multispecific, and mediated by CD8+ T cells in patients with acute viral hepatitis, but that it is not detectable in patients with chronic HBV infection or uninfected healthy blood donors. Importantly, the peptide-activated CTL recognize target cells that express endogenously synthesized polymerase protein, suggesting that these peptides represent naturally processed viral epitopes. DNA sequence analysis of the viruses in patients who did not respond to peptide stimulation indicated that CTL nonresponsiveness was not due to infection by viral variants that differed in sequences from the synthetic peptides. CTL specific for one of the epitopes were unable to recognize several naturally occurring viral variants, except at high peptide concentration, underlining the HBV subtype specificity of this response. Furthermore, CTL responses against polymerase, core, and envelope epitopes were detectable for more than a year after complete clinical recovery and seroconversion, reflecting either the persistence of trace amounts of virus or the presence of long lived memory CTL in the absence of viral antigen. Finally, we demonstrated that wild type viral DNA and RNA can persist indefinitely, in trace quantities, in the serum and PBMC after complete clinical and serological recovery, despite a concomitant, vigorous, and sustained polyclonal CTL response. Since viral persistence is not due to escape from CTL recognition under these conditions, the data suggest that HBV may retreat into immunologically privileged sites from which it can seed the circulation and reach CTL-inaccessible tissues, thereby maintaining the CTL response in apparently cured individuals and, perhaps, prolonging the liver disease in patients with chronic hepatitis.

Acute Disease↗
PubMed1995

Selective proteolitic activation and degradation of ETs and big ETs in parenchymal strips of the guinea-pig lung.

Human and porcine big ET-1 and big ET-2 are similarly potent in contracting parenchymal strips of the guinea-pig lung while big ET-3 is inactive, suggesting that the endothelin-converting enzyme (ECE) which converts big ET-3 is not present and that at least two distinct ECE activities exist, one selective for big ET-1 and big ET-2 and one for big ET-3. Metalloendoprotease inhibitors (phosphoramidon and DL-thiorphan), but not captopril, inhibited the contractions elicited by human big ET-1 and big ET-2 but DL-thiorphan was less active, suggesting that a non-selective enzymatic process is involved in conversion of big ET-1 and big ET-2 in addition to a phosphoramidon-sensitive ECE. Big ET-1 and big ET-2 induced much higher contractions than their corresponding mature peptides. Both metalloendoprotease inhibitors, but not captopril, similarly potentiated contractions induced by ET-1, ET-2 or ET-3 to the level of those evoked by big ET-1 and big ET-2, indicating that only mature ET isopeptides and not their precursors are susceptible to degradation by metalloendoproteases.

Animals↗
PubMed1995

Dose escalation trial of cyclophosphamide with Sargramostim in the treatment of central nervous system (CNS) neoplasms.

We conducted a dose escalation trial of cyclophosphamide plus Sargramostim in the therapy of patients with newly diagnosed or recurrent central nervous system tumors. Cyclophosphamide was administered at doses ranging between 1.0 and 2.5 g/m2 daily for two doses. Sargramostim was administered at a fixed dose of 250 micrograms/m2 subcutaneously twice a day beginning 24 hours after the second cyclophosphamide dose and continuing through the leukocyte nadir until the absolute neutrophil count (ANC) was > 1,000 cells/microliters for two consecutive days. The MTD for patients who had not received any prior chemotherapy and who had received either no radiotherapy or radiotherapy confined to the cranium was 2.0 g/m2 daily for two doses. The MTD for patients previously treated with chemotherapy or neuraxis radiotherapy was also 2.0 g/m2 daily for two doses. Responses were seen in patients with medulloblastoma (8/9), glioblastoma multiforme (2/13), germinoma (1/1), and pineoblastoma (1/2).

Adolescent↗
PubMed1995

Cyclophosphamide in combination with sargramostim for treatment of recurrent medulloblastoma.

Thirteen patients with recurrent medulloblastoma were treated with cyclophosphamide in association with Sargramostim. Cyclophosphamide was given at doses ranging between 1.0-2.5 g/m2 daily for two doses. Sargramostim was given at a fixed dose of 250 micrograms/m2 subcutaneously twice a day beginning 24 hours after the second cyclophosphamide dose and continuing through the leukocyte nadir until the ANC was more than 1,000 cells/microliters for two consecutive days. A total of 33 courses were given with toxicity consisting of grade 4 neutropenia in all courses and grade 3-4 thrombocytopenia in 10 of 13 patients. There were no deaths related to infection or bleeding. Four patients were taken off study because of prolonged myelosuppression. Three of these patients were at the 2.5 g/m2 level, and of these three, two developed lung toxicity (grades 2 and 4, respectively). One patient developed an allergic reaction following the first injection of Sargramostim and was also taken off study. Of 10 evaluable patients, there were 9 PR and 1 SD. We conclude that cyclophosphamide at a dose of 2.0 g/m2/day x 2 days q 4 weeks in association with Sargramostim demonstrates marked activity with acceptable toxicity in patients with recurrent medulloblastoma.

Adolescent↗
PubMed1995

Microvascularity in benign prostatic hyperplasia.

The vascular density of benign prostatic hyperplasia (BPH) has not been characterized. Previously we reported vessel density (vv/mm2) in prostatic carcinoma was twice that of normal prostate [Bigler et al.: Hum Pathol 24:220-226 1993]. To further characterize vessel density in benign prostate tissue we examined 15 cases of BPH obtained by open prostatectomy. Vessels were stained with antibodies to Factor VIII-related antigen, and vessel density was measured using computer-assisted image analysis. Vessel density was analyzed between various histologic tissue types. Mean vessel density in all transition zone tissue was 70.2 vv/mm2. Vessel density in epithelial hyperplastic nodules (mean 99.3, SD 40.7) exhibited density levels similar to those found in prostatic carcinoma (mean 101.4, SD 35.6). Vessel density in epithelial nodules was significantly higher than in non-nodular epithelial tissue (mean 76.7, SD 23.1; P < 0.001, ratio = 1.3). Higher vessel densities were found in hypercellular stromal nodules (mean 64.7, SD 19.1) than in adjacent stromal areas (mean 36.5, SD 15.3; P < 0.001, ratio = 1.8). Overall, vessel density in BPH was higher than previously found in benign tissue measured in radical prostatectomy specimens, especially in areas of nodular morphology.

Capillaries↗
PubMed1995

Phase I studies of treatment of malignant gliomas and neoplastic meningitis with 131I-radiolabeled monoclonal antibodies anti-tenascin 81C6 and anti-chondroitin proteoglycan sulfate Me1-14 F (ab')2--a preliminary report.

The advent of monoclonal antibody (MAb) technology has made Ehrlich's postulate of the 'magic bullet' an attainable goal. Although specific localization of polyvalent antibodies to human gliomas was demonstrated in the 1960s, the lack of specific, high affinity antibody populations and of defined target antigens of sufficient density precluded therapeutic applications. Not until the identification of operationally specific tumor-associated antigens (present in tumor tissue but not normal central nervous system tissue); production of homogeneous, high affinity MAbs to such antigens; and the use of compartmental administration (intrathecal or intracystic), has the promise of passive immunotherapy of primary and metastatic central nervous system neoplasms been recognized. We report here preliminary data from Phase I studies of the compartmental administration of the anti-tenascin MAb 81C6 and F(ab2)2 fragments of MAb Me1-14, which recognizes the proteoglycan chondroitin sulfate-associated protein of gliomas and melanomas, to patients with primary central nervous system tumors or tumors metastatic to the central nervous system. Phase I dose escalation studies of intracystically administered 131I-labeled anti-tenascin MAb 81C6 to either spontaneous cysts of recurrent gliomas or surgically created cystic resection cavities have resulted in striking responses. Of five patients with recurrent cystic gliomas treated, four had partial responses, clinically or radiographically. Similarly, in patients with surgically created resection cavities, a partial response at the treatment site and extended stable disease status has been obtained following intracystic administration of 131I-labeled 81C6. No evidence of hematologic or neurologic toxicity has been observed in either patient population, with the exception of transient exacerbation of a pre-existing seizure disorder in a single patient. Dosimetry calculations indicated high intracystic retention for four to six weeks with little or no systemic dissemination; estimated total doses intracystically ranged from 12,700-70,290 rad. Intrathecal administration of labeled MAbs to patients with neoplastic meningitis is more difficult to assess in terms of clinical responsiveness. Of patients so treated with either 131I-labeled 81C6 or 131I-labeled Me1-14 (F(ab)2, cerebrospinal fluid and radiographic responses have been achieved, and survival prolongation through maintenance of stable disease has been observed in several cases. Initial results from pHase I dose escalation trials are encouraging in terms of the proportion of cases of disease stabilization and partial and complete responses obtained. Importantly, neurotoxicity has been virtually nonexistent, and hematologic toxicity rare and rapidly responsive to treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1995

Dose dependent effects of S-20098, a melatonin agonist, on direction of re-entrainment of rat circadian activity rhythms.

The chronobiotic properties of melatonin are well documented. For example, following an 8-h phase advance of the light-dark cycle daily injections of melatonin administered at the pre-shift dark onset alter the direction of re-entrainment of rat activity rhythms. Using this 8-h phase advance paradigm, the effects of the melatonin agonist S-20098 (1 mg/kg and 3 mg/kg) on the rat circadian system were compared with those of melatonin. S-20098 altered the direction of re-entrainment in the same manner as melatonin. A study using lower doses of S-20098 showed that the effect on direction of re-entrainment was dose-dependent, with 100% of rats responding at a dose of 100 micrograms/kg. S-20098 may, therefore, have therapeutic potential as a chronobiotic in the treatment of circadian disorders in humans.

Acetamides↗
PubMed1995

Dietary sodium intake modulates systemic but not forearm norepinephrine release.

INTRODUCTION: Sodium intake has profound effects on systemic and renal sympathetic activity, but its effects on sympathetic activity in skeletal muscle vascular beds, a site at which local regulatory mechanisms could alter vascular tone directly, are unclear. METHODS: To determine the effect of dietary sodium intake on basal and isoproterenol-stimulated systemic and forearm norepinephrine kinetics, we studied seven healthy male volunteers twice, 4 weeks apart, while they were receiving a low-sodium (10 mmol sodium/24 hours) diet and a high-sodium diet (250 mmol sodium/24 hours). Forearm blood flow, measured by plethysmography, and systemic and forearm norepinephrine spillover, measured by radioisotope dilution, were determined before and after intra-arterial infusion of 60 and 400 ng/min isoproterenol. RESULTS: Baseline (before isoproterenol) systemic norepinephrine spillover was higher when subjects received the low-sodium diet (448.1 +/- 55.7 ng/min) compared with the high-sodium diet (269.7 +/- 42.7 ng/min; p < 0.05). In contrast, sodium intake did not affect local forearm norepinephrine spillover, either at baseline (low-sodium diet, 2.05 +/- 0.48 ng/min versus high-sodium diet, 2.63 +/- 0.79 ng/min; p = 0.50) or after stimulation with isoproterenol in doses of 60 ng/min (low-sodium diet, 8.84 +/- 2.2 ng/min versus high-sodium diet, 6.1 +/- 1.9 ng/min; p = 0.38) or 400 ng/min (low-sodium diet, 16.4 +/- 4.5 ng/min versus high-sodium diet, 16.7 +/- 2.5 ng/min; p = 0.93). CONCLUSIONS: Under conditions of low sodium intake, systemic norepinephrine spillover was increased but forearm norepinephrine spillover was not, suggesting that alteration in sodium intake may produce a differential effect on norepinephrine spillover in different tissues but that decreased local sympathetic activity in skeletal muscle is not the likely mechanism by which a low-sodium diet may lower blood pressure or attenuate stress-induced pressor responses.

Adrenergic beta-Agonists↗
PubMed1995

Multicenter trial of octreotide in patients with refractory acquired immunodeficiency syndrome-associated diarrhea.

BACKGROUND/AIMS: Diarrhea is a significant problem in patients with acquired immunodeficiency syndrome (AIDS). The aim of this study was to determine octreotide effectiveness in refractory AIDS-associated diarrhea. METHODS: In a 3-week protocol, 129 patients with a stool weight of > 500 g/day despite standard antidiarrheal therapy were randomized to receive octreotide or placebo (3:2 ratio). Octreotide dose was increased 100 micrograms weekly to a maximum of 300 micrograms three times a day based on weekly 72-hour stool collections. Subsequently, patients received open-label octreotide at doses of up to 500 micrograms three times a day. RESULTS: A 30% decrease in stool weight defined response. After 3 weeks, 48% of octreotide- and 39% of placebo-treated patients had responded (P = 0.43). At 300 micrograms three times a day, 50% of octreotide- and 30.1% of placebo-treated patients responded (P = 0.12). At a baseline stool weight of 1000-2000 g/day, 57% of octreotide- and 25% of placebo-treated patients responded (P = 0.06). Response rates based on CD4 counts, diarrhea duration, body weight, human immunodeficiency virus risk factor, and presence or absence of pathogens showed no benefit of octreotide. Adverse events were more frequent in the octreotide-treated group. CONCLUSION: In the doses studied, octreotide was not more effective than placebo in patients with refractory AIDS-associated diarrhea. This lack of effectiveness may be attributable to inadequate sample size, doses, and duration of study treatment.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Sialyl-Lewis(x) and related carbohydrate antigens in the prostate.

Alteration of cell surface carbohydrate antigens during malignant transformation is a well-known phenomenon observed in various tumors. In prostatic carcinoma, nearly total deletion of normally occurring ABO and type I-based Lewis antigens, Le(a) and Leb, has been observed in several studies. We studied expression of the closely related type II antigens Le(x), Le(y), and sialyl-Lewis(x) (SLe(x)) using monoclonal antibodies. Thirty formalin-fixed specimens obtained from radical prostatectomy, containing prostatic carcinoma as well as benign tissue, were evaluated by immunohistochemistry. In both cancer and benign tissue, Le(x) expression was minimal or absent. In benign tissue, Le(y) was expressed in ducts and in the basal layer of glandular epithelium. In tumor tissue, Le(y) expression was greatly increased and extensive staining was observed in 26 of 30 cases. The SLe(x) expression in benign tissue was observed only in larger ducts, never in glandular secretory epithelial cells. In carcinoma, rare cells positive for SLe(x) were present in 8 of 30 cases, and stronger expression with focal to patchy distribution was observed in 14 of 30 cases. The results suggest an alteration in glycosyl transferase activity in prostatic carcinoma, with preserved or increased activity of enzymes responsible for the synthesis of the type II core sequence. This sequence is further glycosylated and expressed as the difucosylated compound Le(y) or the monofucosyl, monosialyl compound SLe(x). For prostate, Le(y) and SLe(x) are the only blood group-related antigens known to be minimal or absent in benign secretory epithelial cells that are more highly expressed in malignant tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers, Tumor↗
PubMed1995

High-voltage galvanic stimulation on wound healing in guinea pigs: longer-term effects.

The purpose of this investigation was to determine the effects of high-voltage stimulation (HVS) on wound tensile strength properties and wound closure (histology). Eighteen mature guinea pigs with full-thickness incisions were treated with HVS for 45 minutes daily for 2 weeks; 9 animals were studied after the 14 days of treatment and the remaining 9 were studied 2 weeks later. Five animals (10 wounds) served as controls at each time period. After 2 or 4 weeks, treated and untreated skin was harvested, tested to failure, and prepared for histological examination. Two-week-treated and control wounds had comparable values for peak force to failure, elongation, and energy absorbed to failure. Epithelialization was more advanced in treated animals at 14 days (p < .05). There was a trend (p = .068) toward stronger wounds in 4-week-treated animals (maximum load to failure), but not differences were observed between controls and treated groups for elongation or energy absorbed to failure. Dermal healing appeared to be more advanced in treated animals at 30 days. Although peak force to failure was almost 500g higher for treated guinea pigs after 2 weeks of treatment and more than 700g higher than controls after 4 weeks, mean data were highly variable, so the hypothesis that HVS augments wound strength could not be accepted. It is difficult, however, not to assign clinical significance to the findings.

Animals↗
PubMed1995

Weight-bearing effects on skeletal muscle during and after simulated bed rest.

The detrimental consequences of bed rest include a rapid loss of muscle mass and strength. Yet, the utility of treatment to offset the effects of bed rest has not been well established. It was the purpose of this study to examine the effects of therapeutic intervention on simulated bed rest in rats. Simulated bed rest was accomplished by unweighting the hindlimbs of rats for 2 weeks via a suspension apparatus attached to the tail and midriff. Weight-bearing (eg, standing, walking) effects on hindlimb unweighting (HLU) were investigated under three conditions: (1) 1 hour of weight bearing per day during HLU; (2) a week of natural cage recovery after HLU; and (3) a combination of 1 hour of weight bearing per day during HLU and a week of recovery after HLU. Muscle contractile function and fiber atrophy were examined in the soleus (SOL), a postural muscle, and the extensor digitorum longus (EDL), a nonpostural muscle. The unweighted SOL showed a 37% loss in wet weight and a 61% decline in peak tetanic tension (P0). The SOL in rats allowed 1 hour of weight bearing per day lost 22% of its mass and 38% of its P0, a 38% attenuation. One week of cage recovery after HLU resulted in a SOL wet weight that was 26% less and P0 that was 42% less than controls. Animals that received the combination of 1 hour of weight bearing per day and 1 week of cage activity nearly recovered muscle mass, but P0 still was 19% less than controls. The EDL was much less affected by HLU than the SOL.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1995

Effect of acetazolamide on intracellular pH and bicarbonate transport in bovine corneal endothelium.

Carbonic anhydrase inhibitors are known to inhibit fluid transport by the corneal endothelium. This phenomenon could be due to a combination of effects involving disruption of intracellular pH regulation, reduced gradients for diffusion of CO2, substrate limitation to HCO3- transport systems or direct inhibition of membrane HCO3- transport. We examined the effects of the carbonic anhydrase inhibitor, Acetazolamide (ACTZ), on intracellular pH (pHi) and HCO3- transport in cultured bovine corneal endothelium. The pHi was measured utilizing the pH sensitive fluorescent dye, BCECF. Na+:HCO3- cotransport and Cl-/HCO3- exchange activities were studied by measuring the HCO3(-)-dependent flux of Na+ and Cl-, respectively. Na+ and Cl- fluxes were measured using the ion-sensitive dyes SBFI and SPQ, respectively. Application of 100 or 500 microM ACTZ to cells perfused under HCO3(-)-rich conditions, significantly reduced steady-state pHi by 0.06 +/- 0.01 (n = 14, P < 0.05). ACTZ also eliminated rapid pHi transients due to CO2 diffusion, significantly slowed the initial rate of pHi changes (50 +/- 10% of control, n = 7, P < 0.05) secondary to Na+:HCO3- cotransport or Cl-/HCO3- exchange (37 +/- 1% of control, P < 0.05, n = 7). However, the flux of the cotransported ions, Na+ and Cl-, and the steady-state levels of these ions were not affected by ACTZ. We conclude that the drop in steady-state pHi, the elimination of CO2 induced pHi transients and the slowed pHi changes secondary to HCO3- transport were due to inhibition of cytosolic carbonic anhydrase by ACTZ, i.e. slowing the equilibrium among CO2, HCO3- and H+ and not due to limitation of substrate availability or direct inhibition of the membrane transporters.

Acetazolamide↗
PubMed1995

How about another piece of pie: the allusional pretense theory of discourse irony.

The allusional pretense theory claims that ironic remarks have their effects by alluding to a failed expectation. In normal conversation, this is accomplished by violating pragmatic rules of discourse, usually the maxim of sincerity. Such violations simultaneously draw a listener's attention to the failed expectation and express the speaker's attitude (normally but not necessarily negative) toward the failed expectation. Using a variety of utterance types, 3 experiments tested the theory. The first experiment, using 4 speech act types, showed that both insincerity and allusion were perceived far more frequently in ironically intended utterances than in literally intended ones. The second experiment demonstrated that the negative attitudes frequently expressed with ironic utterances are a function of the relative frequency of positive versus negative expectations and not an intrinsic characteristic of discourse irony per se. The third experiment found that over-polite requests are more likely to be used ironically than under-polite ones, presumably because the former can serve a speaker's politeness considerations while simultaneously conveying both an intended request and the speaker's attitude. It was concluded that irony is used primarily to express a speaker's attitude toward the referent of the ironic utterance, while simultaneously fulfilling other goals as well, such as to be humorous, to make a situation less face threatening, and to serve politeness considerations.

Communication↗