Abacavir Induced T Cell Reactivity from Drug Na?ve Individuals Shares Features of Allo-Immune Responses

Abacavir hypersensitivity is a severe hypersensitivity reaction which occurs exclusively in carriers of the HLA-B*57∶01 allele. In vitro culture of PBMC with abacavir results in the outgrowth of abacavir-reacting CD8⁺ T cells, which release IFNγ and are cytotoxic. How this immune response is induced and what is recognized by these T cells is still a matter of debate. We analyzed the conditions required to develop an abacavir-dependent T cell response in vitro. The abacavir reactivity was independent of co-stimulatory signals, as neither DC maturation nor release of inflammatory cytokines were observed upon abacavir exposure. Abacavir induced T cells arose in the absence of professional APC and stemmed from naïve and memory compartments. These features are reminiscent of allo-reactivity. Screening for allo-reactivity revealed that about 5% of generated T cell clones (n = 136) from three donors were allo-reactive exclusively to the related HLA-B*58∶01. The addition of peptides which can bind to the HLA-B*57∶01-abacavir complex and to HLA-B*58∶01 during the induction phase increased the proportion of HLA-B*58∶01 allo-reactive T cell clones from 5% to 42%. In conclusion, abacavir can alter the HLA-B*57∶01-peptide complex in a way that mimics an allo-allele (‘altered self-allele’) and create the potential for robust T cell responses.     

Relaxation Effect of Abacavir on Rat Basilar Arteries

Background

The use of abacavir has been linked with increased cardiovascular risk in patients with human immunodeficiency virus infection; however, the mechanism involved remains unclear. We hypothesize that abacavir may impair endothelial function. In addition, based on the structural similarity between abacavir and adenosine, we propose that abacavir may affect vascular contractility through endogenous adenosine release or adenosine receptors in blood vessels.

Methods

The relaxation effect of abacavir on rat basilar arteries was studied using the myograph technique. Cyclic GMP and AMP levels were measured by immunoassay. The effects of abacavir on nucleoside transporters were studied using radiolabeled nucleoside uptake experiments. Ecto-5′ nucleotidase activity was determined by measuring the generation of inorganic phosphate using adenosine monophosphate as the substrate.

Results

Abacavir induced the relaxation of rat basilar arteries in a concentration-dependent manner. This relaxation was abolished when endothelium was removed. In addition, the relaxation was diminished by the nitric oxide synthase inhibitor, L-NAME, the guanylyl cyclase inhibitor, ODQ, and the protein kinase G inhibitor, KT5820. Abacavir also increased the cGMP level in rat basilar arteries. Abacavir-induced relaxation was also abolished by adenosine A2 receptor blockers. However, abacavir had no effect on ecto-5’ nucleotidase and nucleoside transporters. Short-term and long-term treatment of abacavir did not affect acetylcholine-induced relaxation in rat basilar arteries.

Conclusion

Abacavir induces acute endothelium-dependent relaxation of rat basilar arteries, probably through the activation of adenosine A2 receptors in endothelial cells, which subsequently leads to the release of nitric oxide, resulting in activation of the cyclic guanosine monophosphate/protein kinase G-dependent pathway in vascular smooth muscle cells. It is speculated that abacavir-induced cardiovascular risk may not be related to endothelial dysfunction as abacavir does not impair relaxation of blood vessels. The most likely explanation of increased cardiovascular risk may be increased platelet aggregation as suggested by other studies.     

Modulation of heme and myristate binding to human serum albumin by anti-HIV drugs. An optical and NMR spectroscopic study

Human serum albumin (HSA) has an extraordinary ligand-binding capacity, and transports Fe(III)heme and medium- and long-chain fatty acids. In human immunodeficiency virus-infected patients the administered drugs bind to HSA and act as allosteric effectors. Here, the binding of Fe(III)heme to HSA in the presence of three representative anti-HIV drugs and myristate is investigated. Values of the dissociation equilibrium constant K(d) for Fe(III)heme binding to HSA were determined at different myristate concentrations, in the absence and presence of anti-HIV drugs. Nuclear magnetic relaxation dispersion profiles of HSA-Fe(III)heme were measured, at different myristate concentrations, in the absence and presence of anti-HIV drugs. Structural bases for anti-HIV drug binding to HSA are provided by automatic docking simulation. Abacavir and nevirapine bind to HSA with K(d) values of 1 x 10(-6) and 2 x 10(-6) M, respectively. Therefore, at concentrations used in therapy (in the 1-5 x 10(-6) M range) abacavir and nevirapine bind to HSA and increase the affinity of heme for HSA. In the presence of abacavir or nevirapine, the affinity is not lowered by myristate. FA7 should therefore be intended as a secondary binding site for abacavir and nevirapine. Binding of atazanavir is limited by the large size of the drug, although preferential binding may be envisaged to a site positively coupled with FA1 and FA2, and negatively coupled to FA7. As a whole, these results provide a foundation for the comprehension of the complex network of links modulating HSA-binding properties.     

Abacavir modulates peroxynitrite-mediated oxidation of ferrous nitrosylated human serum heme-albumin

Human serum albumin (SA) is best known for its extraordinary ligand-binding capacity. Here, kinetics of peroxynitrite-mediated oxidation of SA-heme(II)-NO is reported. Peroxynitrite reacts with SA-heme(II)-NO leading to SA-heme(III) and ()NO by way of the transient SA-heme(III)-NO species. Abacavir facilitates peroxynitrite-mediated oxidation of SA-heme(II)-NO, in the absence and presence of CO2. Values of the second order rate constant for peroxynitrite-mediated oxidation of SA-heme(II)-NO are (6.5+/-0.9) x 10(3) M(-1) s(-1) in the absence of CO2 and abacavir, (1.3+/-0.2) x 10(5) M(-1) s(-1) in the presence of CO2, (2.2+/-0.2) x 10(4) M(-1) s(-1) in the presence of abacavir, and (3.6+/-0.3) x 10(5) M(-1) s(-1) in the presence of both CO2 and abacavir. The value of the first-order rate constant for *NO dissociation from the SA-heme(III)-NO complex (=(1.8+/-0.3) x 10(-1) s(-1)) is CO2- and abacavir-independent, representing the rate-limiting step. Present data represent the first evidence for the allosteric modulation of SA-heme reactivity by heterotropic interaction(s).     

Productive infection maintains a dynamic steady state of residual viremia in human immunodeficiency virus type 1-infected persons treated with suppressive antiretroviral therapy for five years

To provide insight into the dynamics and source of residual viremia in human immunodeficiency virus (HIV) patients successfully treated with antiretroviral therapy, 14 intensely monitored patients treated with indinavir and efavirenz sustaining HIV RNA at <50 copies/ml for >5 years were studied. Abacavir was added to the regimen of eight patients at year 5. After the first 9 months of therapy, HIV RNA levels had reached a plateau ("residual viremia") that persisted for over 5 years. Levels of residual viremia differed among patients and ranged from 3.2 to 23 HIV RNA copies/ml. Baseline HIV DNA was the only significant pretreatment predictor of residual viremia in regression models including baseline HIV RNA, CD4 count, and patient age. In the four of five patients with detectable viremia who added abacavir to their regimen after 5 years, HIV RNA levels declined rapidly. The estimated half-life of infected cells was 6.7 days. Decrease in activated memory cells and a reduction in gamma interferon production to HIV Gag and p24 antigen in ELISpot assays were observed, consistent with a decrease in HIV replication. Thus, in patients treated with efavirenz plus indinavir, levels of residual viremia were established by 9 months, were predicted by baseline proviral DNA, and remained constant for 5 years. Even after years of highly suppressive therapy, HIV RNA levels declined rapidly after the addition of abacavir, suggesting that productive infection contributes to residual ongoing viremia and can be inhibited with therapy intensification.     

Abacavir-Reactive Memory T Cells Are Present in Drug Na?ve Individuals

BackgroundFifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population.MethodsTo determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling.ResultsAbacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells.ConclusionsWe propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection.     

Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase

To examine the role of the mitochondrial polymerase (Pol gamma) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU), beta-L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol gamma holoenzyme in single turnover kinetic studies to measure K(d) (K(m)) and k(pol) (k(cat)) to estimate the specificity constant (k(cat)/K(m)) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2',3'-didehydro-2',3'-dideoxythymidine (d4T) > (+)3TC > (-)3TC > PMPA > azidothymidine (AZT) > Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC approximately equal to (-)3TC > CBV > AZT > PMPA approximately equal to d4T > ddA (ddI) > ddC. Removal of ddC was too slow to measure (<0.00002 s(-1)). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s(-1)) faster than it is removed (0.06 s(-1)) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.     

Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k_off) is reported. In the absence of drugs, the value of k_off is (1.3 ± 0.2) × 10⁻⁴ s⁻¹. Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k_off value increases to (8.6 ± 0.9) × 10⁻⁴ s⁻¹. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NO (i.e., K = (1.2 ± 0.2) × 10⁻³ M and (6.2 ± 0.7) × 10⁻⁵ M, respectively) were determined. The increase of k_off values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow’s site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.     

Transcervical artificial insemination of Australian Merino ewes with frozen-thawed semen

We compared conventional methods for laparoscopic and cervical artificial insemination (AI) to a transcervical AI procedure (Guelph System for Transcervical AI; GST-AI) for use with frozen semen in Merino ewes. The GST-AI procedure was performed by an experienced operator in Experiment 1 (771 ewes) and by 2 inexperienced operators in Experiment 2 (555 ewes). In Experiment 1, intrauterine insemination by GST-AI was achieved in 76% of the ewes. The pregnancy rate at Day 70 for ewes inseminated by laparoscopy (48%, 120 251 ) was higher (P<0.01) than for ewes inseminated by either intrauterine GST-AI (32%, 64 201 ) or cervical AI (9%, 24 256 ). The overall (intrauterine and intracervical) pregnancy rate for GST-AI was 26% (68 264 ) and was unaffected by depth of insemination within the cervix. Pregnancy rates were unaffected by ram or day of insemination. In Experiment 2, the operators achieved intrauterine inseminations by GST-AI in 43% (78 182 ) of the ewes, with a significant operator effect (P<0.01) on depth of cervical penetration. The pregnancy rate to intrauterine GST-AI (40%, 31 78 ) did not differ from that to laparoscopic insemination. The total pregnancy rate for GST-AI in Experiment 2 (19%, 34 182 ) was lower (P<0.05) than that for laparoscopic AI (39%, 72 187 ) but superior (P<0.05) to that for cervical AI (1%, 1 186 ). The GST-AI pregnancy rates were affected by depth of AI (P<0.01) and by operator (P<0.05). It is concluded that GST-AI is superior to cervical AI, and may have application in Merinos if cervical penetration rates can be improved.     

Arginine 177 is involved in Mn(II) binding by manganese peroxidase.

Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K(m) values for Mn(II) were approximately 20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K(m) values for the substrates, H(2)O(2) and ferrocyanide, and the k(cat) values for Mn(II) and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn(II) were approximately 10-fold lower than for wild-type MnP. In addition, the K(D) values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn(II) for the mutant enzymes were approximately 22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn(II) were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn(II) to the oxidized heme apparently is not affected.     

Enzyme elements involved in the interconversion of L-carbamylaspartate and L-dihydroorotate by dihydroorotase from Clostridium oroticum

Enzyme elements that are involved in the reversible cyclization of L-carbamylaspartate to L-dihdroorotate catalyzed by dihydroorotase (EC 3.5.2.3) from Clostridium oroticum (ATCC 25750) have been studied. Removal of Zn(II) from the enzyme by chelators followed by incubation of apoenzyme with Co(II) results in replacement of two to three of the four Zn(II) ions per molecule by Co(II). The catalytic properties of the Zn(II)Co(II) dihydroorotase are different from those of native enzyme. The Vmax is increased for both the synthesis and hydrolysis of L-dihydroorotate. The Km for L-dihydroorotate is unchanged, while the Km for L-carbamylaspartate is increased more than twofold. On the other hand, the kinetic properties of Zn(II)-reconstituted dihydroorotase are indistinguishable from those of native enzyme. The pH dependence of Vmax is also altered by the Co(II) substitution. For both Zn(II)- and Zn(II)Co(II)-dihydroorotase, this pH dependence is well described by a single ionization and the pK's for L-dihydroorotate synthesis and hydrolysis are different. Substitution with Co(II) increases the pK for both reaction directions to different extents. These results strongly support a role for the tightly bound metals in the catalytic mechanism. In addition, diethylpyrocarbonate rapidly inactivates the enzyme. The inactivation is prevented by L-dihydroorotate. This result is consistent with a role for at least one histidine in catalysis. The possibility that C. oroticum dihydroorotase may be useful model for the more complex mammalian enzyme is considered.     

Microbial reduction of technetium by Escherichia coli and Desulfovibrio desulfuricans: enhancement via the use of high-activity strains and effect of process parameters

Escherichia coli and Desulfovibrio desulfuricans reduce Tc(VII) (TcO(4)(-)) with formate or hydrogen as electron donors. The reaction is catalyzed by the hydrogenase component of the formate hydrogenlyase complex (FHL) of E. coli and is associated with a periplasmic hydrogenase activity in D. desulfuricans. Tc(VII) reduction in E. coli by H(2) and formate was either inhibited or repressed by 10 mM nitrate. By contrast, Tc(VII) reduction catalyzed by D. desulfuricans was less sensitive to nitrate when formate was the electron donor, and unaffected by 10 mM or 100 mM nitrate when H(2) was the electron donor. The optimum pH for Tc(VII) reduction by both organisms was 5.5 and the optimum temperature was 40 degrees C and 20 degrees C for E. coli and D. desulfuricans, respectively. Both strains had an apparent K(m) for Tc(VII) of 0.5 mM, but Tc(VII) was removed from a solution of 300 nM TcO(4)(-) within 30 h by D. desulfuricans at the expense of H(2). The greater bioprocess potential of D. desulfuricans was shown also by the K(s) for formate (>25 mM and 0.5 mM for E. coli and D. desulfuricans, respectively), attributable to the more accessible, periplasmic localization of the enzyme in the latter. The relative rates of Tc(VII) reduction for E. coli and D. desulfuricans (with H(2)) were 12.5 and 800 micromol Tc(VII) reduced/g biomass/h, but the use of an E. coli HycA mutant (which upregulates FHL activities by approx. 50%) had a similarly enhancing effect on the rate of Tc reduction. The more rapid reduction of Tc(VII) by D. desulfuricans compared with the E. coli strains was also shown using cells immobilized in a hollow-fiber reactor, in which the flow residence times sustaining steady-state removal of 80% of the radionuclide were 24.3 h for the wild-type E. coli, 4.25 h for the upregulated mutant, and 1.5 h for D. desulfuricans.