Decreased inflammatory pain due to reduced carrageenan-induced inflammation in mice lacking adenosine A3 receptors.

Mice with a targeted disruption of adenosine A(3) receptor (A(3)AR) gene were assessed for their nociceptive threshold and for their localized inflammatory response following carrageenan injected into the hindpaw. Under basal conditions no difference was seen between A(3)AR knock-out (A(3)AR(-/-)) and wild-type (A(3)AR(+/+)) mice in nociceptive response to mechanical or heat stimuli. The antinociceptive response to the intrathecal adenosine analogue R-phenylisopropyl adenosine (R-PIA) was also unchanged in the A(3)AR(-/-) mice. In contrast, heat hyperalgesia, plasma extravasation and edema following carrageenan-induced inflammation in the hind paw were significantly reduced in A(3)AR(-/-) mice compared to the A(3)AR(+/+) controls. Thus, mice lacking A(3)AR had deficits in generating the localized inflammatory response to carrageenan, supporting a pro-inflammatory role of A(3)AR in peripheral tissues. However, no evidence for a role of A(3)AR in nociception and the antinociceptive effect of R-PIA was found.

Targeted deletion of A(3) adenosine receptors improves tolerance to ischemia-reperfusion injury in mouse myocardium.

A(3) adenosine receptors (A(3)ARs) have been implicated in regulating mast cell function and in cardioprotection during ischemia-reperfusion injury. The physiological role of A(3)ARs is unclear due to the lack of widely available selective antagonists. Therefore, we examined mice with targeted gene deletion of the A(3)AR together with pharmacological studies to determine the role of A(3)ARs in myocardial ischemia-reperfusion injury. We evaluated the functional response to 15-min global ischemia and 30-min reperfusion in isovolumic Langendorff hearts from A(3)AR(-/-) and wild-type (A(3)AR(+/+)) mice. Loss of contractile function during ischemia was unchanged, but recovery of developed pressure in hearts after reperfusion was improved in A(3)AR(-/-) compared with wild-type hearts (80 +/- 3 vs. 51 +/- 3% at 30 min). Tissue viability assessed by efflux of lactate dehydrogenase was also improved in A(3)AR(-/-) hearts (4.5 +/- 1 vs. 7.5 +/- 1 U/g). The adenosine receptor antagonist BW-A1433 (50 microM) decreased functional recovery following ischemia in A(3)AR(-/-) but not in wild-type hearts. We also examined myocardial infarct size using an intact model with 30-min left anterior descending coronary artery occlusion and 24-h reperfusion. Infarct size was reduced by over 60% in A(3)AR(-/-) hearts. In summary, targeted deletion of the A(3)AR improved functional recovery and tissue viability during reperfusion following ischemia. These data suggest that activation of A(3)ARs contributes to myocardial injury in this setting in the rodent. Since A(3)ARs are thought to be present on resident mast cells in the rodent myocardium, we speculate that A(3)ARs may have proinflammatory actions that mediate the deleterious effects of A(3)AR activation during ischemia-reperfusion injury.

Cytomegalovirus (CMV)-specific CD4+ T lymphocyte immune function in long-term survivors of AIDS-related CMV end-organ disease who are receiving potent antiretroviral therapy.

To better understand the relation of cytomegalovirus (CMV)-specific CD4+ T lymphocyte immunity and clinical outcome in AIDS-related CMV end-organ disease, 2 patient groups were prospectively studied: patients recently diagnosed with active CMV end-organ disease and survivors of CMV retinitis who had responded to highly active antiretroviral therapy and had quiescent retinitis when anti-CMV therapy was discontinued. Most patients with active CMV disease had negative CMV-specific CD4+ T lymphocyte responses at diagnosis, as measured by lymphoproliferation (7/7) or cytokine flow cytometry (3/5) assays. In contrast, all 10 subjects with quiescent retinitis and >150 absolute CD4+ T lymphocytes/microL whose anti-CMV therapy was discontinued during 6 months of follow-up had positive CMV-specific immune responses at least once by each assay. However, 6 of these 10 subjects also had negative CMV-specific immune responses > or =1 time. Such patients may be at risk for future CMV disease progression and should be closely monitored.

Targeted deletion of the A3 adenosine receptor confers resistance to myocardial ischemic injury and does not prevent early preconditioning.

We used mice with genetic disruption of the A3 adenosine receptor (AR) gene (A3AR(-/-)mice) to assess the in vivo role of the A3AR in modulating myocardial ischemia/reperfusion injury and preconditioning (PC). Surprisingly, infarct size induced by 30 min of coronary artery occlusion and 24 h of reperfusion was 35% smaller in A3AR(-/-)compared to wild-type mice (A3AR(+/+)). The reduction in infarct size was not the result of differences in heart rate, body temperature or increased cardiac expression of A1ARs. However, neutrophil infiltration within infarcted regions was less in A3AR(-/-)mice. Furthermore, ischemic PC induced by either a single episode (one 5 min occlusion) or multiple episodes (six 4 min occlusions) of ischemia produced equivalent reductions in infarct size in A3AR(-/-)and A3AR(+/+)mice. These results indicate that, in the mouse, (i) A3ARs play an injurious role during acute myocardial ischemia/reperfusion injury, possibly by exacerbating the inflammatory response, and (ii) A3ARs are not necessary for the development of the early phase of ischemic PC.

Invasive fungal sinusitis and meningitis due to Arthrographis kalrae in a patient with AIDS.

We report the first described case of Arthrographis kalrae pansinusitis and meningitis in a patient with AIDS. The patient was initially diagnosed with Arthrographis kalrae pansinusitis by endoscopic biopsy and culture. The patient was treated with itraconazole for approximately 5 months and then died secondary to Pneumocytis carinii pneumonia. Postmortem examination revealed invasive fungal sinusitis that involved the sphenoid sinus and that extended through the cribiform plate into the inferior surfaces of the bilateral frontal lobes. There was also an associated fungal meningitis and vasculitis with fungal thrombosis and multiple recent infarcts that involved the frontal lobes, right caudate nucleus, and putamen. Post mortem cultures were positive for A. kalrae.

Adenosine and inosine increase cutaneous vasopermeability by activating A(3) receptors on mast cells.

Adenosine has potent effects on both the cardiovascular and immune systems. Exposure of tissues to adenosine results in increased vascular permeability and extravasation of serum proteins. The mechanism by which adenosine brings about these physiological changes is poorly defined. Using mice deficient in the A(3) adenosine receptor (A(3)AR), we show that increases in cutaneous vascular permeability observed after treatment with adenosine or its principal metabolite inosine are mediated through the A(3)AR. Adenosine fails to increase vascular permeability in mast cell-deficient mice, suggesting that this tissue response to adenosine is mast cell-dependent. Furthermore, this response is independent of activation of the high-affinity IgE receptor (FcepsilonR1) by antigen, as adenosine is equally effective in mediating these changes in FcepsilonR1 beta-chain-deficient mice. Together these results support a model in which adenosine and inosine induce changes in vascular permeability indirectly by activating mast cells, which in turn release vasoactive substances. The demonstration in vivo that adenosine, acting through a specific receptor, can provoke degranulation of this important tissue-based effector cell, independent of antigen activation of the high-affinity IgE receptor, supports an important role for this nucleoside in modifying the inflammatory response.

Disruption of the A(3) adenosine receptor gene in mice and its effect on stimulated inflammatory cells.

The A(3) adenosine receptor (A3AR) is one of four receptor subtypes for adenosine and is expressed in a broad spectrum of tissues. In order to study the function of A3AR, a mouse line carrying a mutant A(3) allele was generated. Mice homozygous for targeted disruption of the A3AR gene, A3AR(-/-), are fertile and visually and histologically indistinguishable from wild type mice. The lack of a functional receptor in the A3AR(-/-) mice was confirmed by molecular and pharmacological analyses. The absence of A3AR protein expression in the A3AR(-/-) mice was demonstrated by lack of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine binding to bone marrow-derived mast cell membranes that were found to express high levels of A3AR in wild type mice. In A3AR(-/-) mice, the density of A(1) and A(2A) adenosine receptor subtypes was the same as in A3AR(+/+) mice as determined by radioligand binding to brain membranes. Additionally, A(2B) receptor transcript expression was not affected by ablation of the A3AR gene. A3AR(-/-) mice have basal heart rates and arterial blood pressures indistinguishable from A3AR(+/+) mice. Functionally, in contrast to wild type mice, adenosine and the A3AR-specific agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2-Cl-IB-MECA), elicit no potentiation of antigen-dependent degranulation of bone marrow-derived mast cells from A3AR(-/-) mice as measured by hexosaminidase release. Also, the ability of 2Cl-IB-MECA to inhibit lipopolysaccharide-induced tumor necrosis factor-alpha production in vivo was decreased in A3AR(-/-) mice in comparison to A3AR(+/+) mice. The A(2A) adenosine receptor agonist, 2-p-(2-carboxyethyl)phenylamino)-5'-N-ethylcarboxamidoadenosine, produced inhibition of lipopolysaccharide-stimulated tumor necrosis factor-alpha production in both A3AR(-/-) and A3AR(+/+) mice. These results show that the inhibition in vivo can be mediated by multiple subtypes, specifically the A(3) and A(2A) adenosine receptors, and A3AR activation plays an important role in both pro- and anti-inflammatory responses.

Effect of antiviral drugs used to treat cytomegalovirus end-organ disease on subsequent course of previously diagnosed Kaposi's sarcoma in patients with AIDS.

OBJECTIVE: To evaluate the association of ganciclovir (GCV) and foscarnet (PFA) therapy with the outcome of previously diagnosed Kaposi's sarcoma (KS) after initiating antiviral therapy for cytomegalovirus (CMV) end-organ disease. DESIGN: Retrospective study. METHODS: KS progression was defined as a clinically obvious increase in size of baseline cutaneous or mucosal lesions, a new diagnosis of visceral KS, or initiation of a new systemic antineoplastic regimen or radiation therapy to treat KS. Multivariate analyses of risk of KS progression were calculated for prior duration of KS before initiating CMV treatment, treatment with PFA or GCV, number of weeks treated with PFA or GCV, absolute CD4 lymphocyte count at time of CMV-related disease diagnosis, diagnosis of KS prior to 1991, visceral KS, prior systemic chemotherapy, and prior radiation therapy. RESULTS: Among 66 patients who received > or = 14 days PFA (N=20) or only GCV (N=46), median time to progression of KS was 211 days (95% confidence interval [CI], 46-578) for patients who received PFA versus 22 days (95% CI, 15-41) for those who received only GCV (p < .001). In the stepwise multivariate analysis, only prior visceral KS (rate ratio [RR]=2.80; 95% CI, 1.07-7.35) and foscarnet therapy (RR = 0.24; 95% CI, 0.11-0.53) were significantly associated with risk of KS progression. CONCLUSION: PFA may be an effective therapy for AIDS-related KS; prospective trials are indicated.

Transient vitreous inflammatory reactions associated with combination antiretroviral therapy in patients with AIDS and cytomegalovirus retinitis.

PURPOSE: To report the observation that a transient vitreous inflammatory reaction may develop in the eyes of patients with acquired immunodeficiency syndrome (AIDS), cytomegalovirus retinitis, and an increased CD4+ T-lymphocyte count during treatment with antiretroviral therapy including a protease inhibitor. METHODS: We reviewed the medical records of eight patients with AIDS and cytomegalovirus retinitis who developed vitreous inflammatory reactions greater than those usually seen with this disease. RESULTS: Vitreous inflammatory reactions obscured the view of the posterior pole in all patients. No iris nodules, synechiae, glaucoma, or cystoid macular edema were observed. Six patients had unilateral cytomegalovirus retinitis, and, in each, the inflammation occurred only in the eye with cytomegalovirus retinitis. The vitreous inflammatory reactions were associated with clinically inactive cytomegalovirus retinitis in six patients, with disease reactivation in one patient, and were present at diagnosis of active disease in one patient. Cytomegalovirus retinitis has not recurred in any of these patients since their episodes of vitreous inflammation. Vitreous inflammation developed in all eight patients after a substantial increase in CD4+ T-lymphocyte counts caused by combination antiretroviral therapy. Five patients had CD4+ T-lymphocyte counts of greater than 100 cells per microl at the time the vitreous inflammatory reaction developed. No other causes of uveitis were found. CONCLUSIONS: Patients with AIDS and cytomegalovirus retinitis may develop transient intraocular inflammation associated with combination antiretroviral therapy. We believe that this inflammation reflects an improved immune response against cytomegalovirus.

Filgrastim prevents severe neutropenia and reduces infective morbidity in patients with advanced HIV infection: results of a randomized, multicenter, controlled trial. G-CSF 930101 Study Group.

OBJECTIVE: To assess the effect of filgrastim treatment on the incidence of severe neutropenia in patients with advanced HIV infection, and the effect of initial filgrastim treatment on prevention of infectious morbidity. DESIGN: Randomized, controlled, open-label, multicenter study. SETTING: Outpatient centers and physician offices. PATIENTS: Men and women aged > 13 years, who were HIV antibody-positive, and had a CD4 cell count < 200 x 10(6)/l, absolute neutrophil count (ANC) 0.75-1.0 x 10(9)/l, and platelet count > or = 50 x 10(9)/l within 7 days of randomization were eligible. Two hundred and fifty-eight patients entered and 201 completed the study. INTERVENTION: Daily filgrastim (starting at 1 microg/kg daily, adjusted up to 10 microg/kg daily) or intermittent filgrastim (starting at 300 microg daily one to three times per week to a maximum of 600 microg daily 7 days weekly) was administered to maintain an ANC between 2 and 10 x 10(9)/l. Patients in the control group received filgrastim if severe neutropenia developed. MAIN OUTCOME MEASURES: Incidence of severe neutropenia (ANC < 0.5 x 10(9)/l) or death, incidence of bacterial and fungal infections, duration of hospitalization and intravenous antibacterial use, and safety. RESULTS: The primary endpoint of severe neutropenia or death was less frequent in patients who received daily (12.8%) or intermittent (8.2%) filgrastim compared with control patients (34.1%; P<0.002 and P<0.0001 for comparison with daily and intermittent groups, respectively). Filgrastim-treated patients developed 31% fewer bacterial infections and 54% fewer severe bacterial infections than control patients, required 26% less hospital days including 45% fewer hospital days for bacterial infections, and needed 28% fewer days of intravenous antibacterials. Filgrastim was not associated with an increase in HIV-1 plasma RNA level in a subset of patients in whom this was measured or any new or unexpected adverse events. CONCLUSION: Filgrastim was safe and effective in preventing severe neutropenia in patients with advanced HIV infection, and may reduce the incidence and duration of bacterial infections, incidence of severe bacterial infections, duration of hospital days for infections, and days of intravenous antibacterial agents.

Adenosine A3 receptor expression and function in eosinophils.

The A3 adenosine receptor is widely expressed in human tissues with the most abundant expression in the lung and liver, but the predominant cellular localization and functions of this receptor in humans are unknown. Since adenosine influences the activation of circulating and resident inflammatory cells within the lung and leads to exaggerated airway narrowing in individuals with inflammatory airway disorders, we hypothesized that A3 receptor gene expression is localized to inflammatory cells and that gene expression is upregulated in airway inflammation. Lung and airway tissue were obtained at thoracotomy from nonsmoking subjects and subjects with inflammatory airway disorders associated with tobacco smoke or asthma. In situ hybridization identified A3 receptors in mesenchymal cells and eosinophils within the lamina propria of the airways and the adventitia of blood vessels, but not in mast cells. A3 receptor transcripts were highly expressed in peripheral blood eosinophils purified from atopic donors (6.36 +/- 0.60 pg/microg total RNA) in comparison with neutrophils (0.26 +/- 0.06 pg/microg) or mononuclear cells (0.9 +/- 0.15 pg/microg). Mean A3 receptor transcript abundance was greater in lung tissue from subjects with airway inflammation (0.33 +/- 0.04 pg/microg total RNA) than in normal lung (0.24 +/- 0.03 pg/microg total RNA, P = 0.035). The A3 receptor agonist N6-(4-amino-3-iodobenzyl)adenosine dose-dependently inhibited platelet activating factor-induced eosinophil chemotaxis to a maximum of 41%. This inhibitory effect was completely abolished by addition of the A3 receptor selective antagonist 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine. We conclude that A3 receptors are primarily expressed on eosinophils in human lung, where they mediate inhibition of eosinophil chemotaxis. Specific A3 receptor ligands may be useful agents in the treatment of eosinophil-dependent diseases such as asthma and rhinitis.

Adenosine A2a receptor activation delays apoptosis in human neutrophils.

Adenosine has both pro- and anti-inflammatory effects on neutrophils. Exposure of cultured neutrophils to 2-chloroadenosine or 5'-N-ethylcarboxamidoadenosine (NECA) decreased apoptosis after 16 h, with half-maximal responses for NECA and 2-chloroadenosine of 7.1 +/- 7.7 and 59.0 +/- 32.0 nM, respectively. Adenosine receptor agonists exhibited a rank order of potency for decreasing apoptosis of 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) > NECA > or = 2-chloro-N6-cyclopentyladenosine >> 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide, which is consistent with the affinity order profile established for human A2a receptors. The reduction in apoptosis in cultured neutrophils at 16 h by CGS 21680 was due to a delay in apoptosis. The addition of CGS 21680 (100 nM) increased the half-life for the appearance of apoptosis from 10.9 +/- 3.1 to 21.0 +/- 1.0 h. Addition of the non-xanthine phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazalidinone (Ro-20-1724; 1 microM) enhanced the effects of CGS 21680 at all agonist concentrations. PGE1 (10 microM), PGE2 (0.1-10 microM), and dibutyryl cAMP (5-500 microM) all decreased apoptosis in cultured neutrophils. The enhancement of the effect of adenosine by a phosphodiesterase inhibitor and the similar actions of PGE2, PGE1, and dibutyryl cAMP suggest that this decrease in apoptosis may be mediated by a cAMP-dependent pathway.

Interleukin-12 administered in vivo decreases human NK cell cytotoxicity and antibody-dependent cellular cytotoxicity to human immunodeficiency virus-infected cells.

Persons infected with human immunodeficiency virus (HIV) have cellular cytotoxicity defects. Interleukin (IL)-12 is a potent stimulator of cytotoxicity. Fifteen HIV-infected patients were studied in a phase 1, single-dose escalation trial of human recombinant IL-12. One day after subjects received an IL-12 dose of 300 or 1000 ng/kg, they had a reduction in absolute lymphocyte count and peripheral blood mononuclear cell recovery. In evaluable patients 24 h after IL-12 administration, there was a 31% reduction overall in NK cell cytotoxicity (NKC) to HIV-infected cells at all doses and a 52% reduction in antibody-dependent cellular cytotoxicity (ADCC) at doses of 300 and 1000 ng/kg. In vitro incubation of patients' cells with IL-12 (before IL-12 administration) for 24 h increased NKC but had no effect on ADCC. The paradoxic acute reduction in cell number and cytotoxicity in vivo may be due to NK cell trafficking or regulatory cytokine mechanisms not apparent in vitro.

Stimulation by alkylxanthines of chloride efflux in CFPAC-1 cells does not involve A1 adenosine receptors.

A series of 8-substituted derivatives of 1,3,7-alkylxanthines was synthesized as potential activators of chloride efflux from a human epithelial cell line (CFPAC) expressing the cystic fibrosis transmembrane regulator (CFTR) delta F508 mutation. Their interactions with rat brain A1 and A2a receptors were also studied in radioligand binding experiments. Substitution was varied at the xanthine 1-, 3-, 7- and 8-positions. 1,3-Dipropyl-8-cyclopentylxanthine (CPX) stimulated Cl- efflux in the 10(-8) M range, with a maximal effect reaching 200% of control and diminishing at higher concentrations. The potent adenosine antagonist 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]- 1,3-dipropylxanthine, nonselective at human A1 and A2a receptors, was inactive in Cl- efflux. 1,3-Diallyl-8-cyclohexylxanthine (DAX) was highly efficacious in stimulating chloride efflux with levels reaching > 300% of control, although micromolar concentrations were required. 1,3,7-Trimethyl-8-(3-chlorostyryl)xanthine, an A2a-selective adenosine antagonist, was only weakly active. Caffeine, which acts as an nonselective adenosine antagonist in the range of 10(-5) M, was active in Cl- efflux in the low nanomolar range but with low efficacy. Thus, among the xanthine derivatives of diverse structure, there was no correlation between potency in Cl- efflux and adenosine antagonism. Poly(A)+ RNA isolated from CFPAC-1 cells showed no hybridization to a human A1 receptor cDNA probe, excluding this receptor as a mediator of CPX-elicited Cl- efflux. Thus, this action of xanthines in stimulating Cl- efflux in CFPAC cells, which express a defective CFTR, represents a novel site of action apparently unrelated to adenosine receptors.

Cloning and chromosomal localization of the human A2b adenosine receptor gene (ADORA2B) and its pseudogene.

To determine the chromosomal localization of the human A2b adenosine receptor, the corresponding genomic clone was isolated and used as a probe for fluorescence in situ hybridization to metaphase chromosomes. Partial sequence analysis of the A2b gene (AD-ORA2B) revealed an intron that interrupted the coding region corresponding to the second intracellular loop similar to that reported for A1 and A2a adenosine receptor genes. A pseudogene for the A2b receptor was also identified; it exhibited 79% identity to the A2b adenosine receptor cDNA coding sequence and contained multiple deletions, point mutations, and frame shifts and two in-frame stops. These changes would result in the inability to encode a functional receptor. The genomic clones were utilized to localize the A2b receptor to chromosome 17p12 and the A2b pseudogene to chromosome 1q32.

Relationship between plasma concentrations of 3'-deoxy-3'-fluorothymidine (alovudine) and antiretroviral activity in two concentration-controlled trials.

Two concentration-controlled trials (CCTs) defined the relationship between plasma concentrations of 3'-deoxy-3'-fluorothymidine (alovudine) and changes in surrogate markers of antiretroviral activity. In an initial open-label CCT involving 14 subjects infected with human immunodeficiency virus (HIV), unacceptable hematologic toxicity occurred when the area under the concentration-time curve during a 12-h dosing interval (AUC12) was > or = 300 ng*h/mL. Consequently, 46 subjects were assigned to AUC12s of 50, 100, or 200 ng*h/mL for up to 16 weeks in a prospective, randomized, double-blind CCT. Alovudine caused a concentration-dependent reduction in p24 antigen and peripheral blood mononuclear cell HIV titers within 4 weeks of start of treatment. The AUC12 producing a 50% reduction in p24 (108 ng*h/mL) had a trough concentration identical to the reported IC50 of alovudine in HIV-infected H9 cells. It may be possible to predict the antiretroviral activity of certain nucleoside analogues as a function of plasma drug concentration.

Molecular cloning and characterization of the human A3 adenosine receptor.

The human A3 adenosine receptor was cloned from a striatal cDNA library using a probe derived from the homologous rat sequence. The cDNA encodes a protein of 318 amino acids and exhibits 72% and 85% overall identity with the rat and sheep A3 adenosine receptor sequences, respectively. Specific and saturable binding of the adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine [125I]ABA was measured on the human A3 receptor stably expressed in Chinese hamster ovary cells with a Kd = 10 nM. The potency order for adenosine receptor agonists was N-ethylcarboxamidoadenosine (NECA) > or = (R)-N6-phenyl-2-propyladenosine [(R)-PIA] > N6-cyclopentyladenosine (CPA) > (S)-N6-phenyl-2-propyladenosine [(S)-PIA]. The human receptor was blocked by xanthine antagonists, most potently by 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine (I-ABOPX) with a potency order of I-ABOPX > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amino congener >> 1,3-dipropyl-8-cyclopentylxanthine. Adenosine, NECA, (R)- and (S)-PIA, and CPA inhibited forskolin-stimulated cAMP accumulation by 30-40% in stably transfected cells; I-ABA is a partial agonist. When measured in the presence of antagonists, the dose-response curves of NECA-induced inhibition of forskolin-stimulated cAMP accumulation were right-shifted. Antagonist potencies determined by Schild analyses correlated well with those established by competition for radioligand binding. The A3 adenosine receptor transcript is widespread and, in contrast to the A1, A2a, and A2b transcripts, the most abundant expression is found in the lung and liver. The tissue distribution of A3 mRNA is more similar to the widespread profile found in sheep than to the restricted profile found in the rat. This raises the possibility that numerous physiological effects of adenosine may be mediated by A3 adenosine receptors.

Expression, purification and characterization of multigram amounts of a recombinant hybrid HV1-HV2 hirudin variant expressed in Saccharomyces cerevisiae.

Hirudin (HIR), derived from leeches, and tick anticoagulant peptide (TAP) are polypeptide protease inhibitors of thrombin and coagulation factor Xa (fXa), respectively, and they have both shown utility in vitro and in vivo as potent antithrombotic agents. A thorough side-by-side comparison of the in vivo efficacy of factor Xa inhibition compared to thrombin inhibition by TAP and HIR, respectively, required purification and characterization of multigram amounts of hirudin. Therefore, a recombinant Saccharomyces cerevisiae strain was developed using a plasmid containing the gene encoding the MF alpha 1 preproleader, a synthetic hybrid HV1-HV2 HIR gene, and a galactose-inducible promoter which directed the secretion of 44 mg/liter of recombinant HIR (rHIR) after induction. rHIR was purified by a process that consisted of two chromatographic steps and decolorization. Total yield for the purification process was 3.6 g, or 41%. This process gave 59-fold purification of rHIR that was judged to be > 96% pure with regard to polypeptide content by capillary zonal electrophoresis and reversed-phase high-performance liquid chromatography. Single, unique N- and C-termini were obtained by sequencing and were identical to those predicted from the deduced sequence of the cDNA. Determination of the dissociation constant, by thrombin:hirudin inhibition reaction, and anticoagulant activity, by the activated partial thromboplastin time, demonstrated that the hybrid rHIR HV1-HV2 protein discussed in this report was essentially equipotent with rHIR preparations HV1 and HV2 reported by others.

Decreased serum dehydroepiandrosterone is associated with an increased progression of human immunodeficiency virus infection in men with CD4 cell counts of 200-499.

Dehydroepiandrosterone (DHEA) and its interconvertible sulfate derivative (DHEA-S) are human androgenic steroids that have been reported to inhibit viral expression and have been associated with a decreased risk of cancer. The relationship between serum DHEA and DHEA-S levels and subsequent progression to AIDS was investigated in a sample of human immunodeficiency virus (HIV)-infected men from the San Francisco Men's Health Study followed prospectively since 1984. Among 108 men seropositive for HIV at study entry and with CD4 lymphocyte counts of 200-499 microliters 24 months later, serum DHEA levels below the lower limit of normal (less than 180 ng/dl) at this later date were predictive of subsequent progression to AIDS (relative hazard = 2.34; 95% confidence interval = 1.18-4.63; P = .01) after controlling for hematocrit, age, and log absolute CD4 cell number in a Cox proportional hazards model. This is the first large prospective cohort in which an endocrinologic variable has been observed to independently predict progression to AIDS. These observations, in addition to recent in vitro data, suggest that DHEA might have a protective effect in HIV infection.

Morbidity and mortality of patients with AIDS and first-episode Pneumocystis carinii pneumonia unaffected by concomitant pulmonary cytomegalovirus infection.

To determine the significance of cytomegalovirus (CMV) pulmonary coinfection with Pneumocystis carinii pneumonia in AIDS, we examined the association of long- and short-term survival and morbidity (as defined by length of hospital stay) with recovery of CMV from bronchoscopy specimens and an indirect measure of virus titer in bronchoalveolar lavage fluid (the time to develop CMV cytopathology in culture) in 111 patients diagnosed with a first episode of P. carinii pneumonia. Compared with 57 individuals from whom CMV was not isolated, the 54 individuals from whom CMV were isolated did not differ in baseline characteristics, long-term survival (213 versus 275 days, p = 0.97), acute death rate (19% in both, p = 1.0), or length of hospital stay (19.7 versus 21.1 days, p = 0.68). Also, the time to develop CMV cytopathology in culture did not correlate with acute or long-term survival. Our observations thus do not support the use of CMV-specific antiviral therapy in AIDS patients with P. carinii pneumonia who also have evidence of pulmonary CMV infection.