The microRNA-9/B-lymphocyte-induced maturation protein-1/IL-2 axis is differentially regulated in progressive HIV infection.

The fine control of T-cell differentiation and its impact on HIV disease states is poorly understood. In this study, we demonstrate that B-lymphocyte-induced maturation protein-1 (Blimp-1/Prdm1) is highly expressed in CD4(+) T cells from chronically HIV-infected (CHI) patients compared to cells from long-term nonprogressors or healthy controls. Stimulation through the T-cell receptor in the presence of IL-2 induces Blimp-1 protein expression. We show here that Blimp-1 levels are translationally regulated by microRNA-9 (miR-9). Overexpression of miR-9 induces Blimp-1 repression, restoring IL-2 secretion in CD4(+) T cells via reduction in the binding of Blimp-1 to the il-2 promoter. In CHI patients where IL-2 expression is reduced and there is generalized T-cell dysfunction, we show differential expression of both miR-9 and Blimp-1 in CD4(+) cells compared with levels in long-term nonprogressors. These data identify a novel miR-9/Blimp-1/IL-2 axis that is dysregulated in progressive HIV infection.

Differential regulation of the Let-7 family of microRNAs in CD4+ T cells alters IL-10 expression.

MicroRNAs (miRNAs) are ∼22-nt small RNAs that are important regulators of mRNA turnover and translation. Recent studies have shown the importance of the miRNA pathway in HIV-1 infection, particularly in maintaining latency. Our initial in vitro studies demonstrated that HIV-1-infected HUT78 cells expressed significantly higher IL-10 levels compared with uninfected cultures. IL-10 plays an important role in the dysregulated cytotoxic T cell response to HIV-1, and in silico algorithms suggested that let-7 miRNAs target IL10 mRNA. In a time course experiment, we demonstrated that let-7 miRNAs fall rapidly following HIV-1 infection in HUT78 cells with concomitant rises in IL-10. To show a direct link between let-7 and IL-10, forced overexpression of let-7 miRNAs resulted in significantly reduced IL-10 levels, whereas inhibition of the function of these miRNAs increased IL-10. To demonstrate the relevance of these results, we focused our attention on CD4(+) T cells from uninfected healthy controls, chronic HIV-1-infected patients, and long-term nonprogressors. We characterized miRNA changes in CD4(+) T cells from these three groups and demonstrated that let-7 miRNAs were highly expressed in CD4(+) T cells from healthy controls and let-7 miRNAs were significantly decreased in chronic HIV-1 infected compared with both healthy controls and long-term nonprogressors. We describe a novel mechanism whereby IL-10 levels can be potentially modulated by changes to let-7 miRNAs. In HIV-1 infection, the decrease in let-7 miRNAs may result in an increase in IL-10 from CD4(+) T cells and provide the virus with an important survival advantage by manipulating the host immune response.

Impact of treatment with raltegravir during primary or chronic HIV infection on RNA decay characteristics and the HIV viral reservoir.

BACKGROUND: Eradication of HIV-1 is prevented by the formation of viral reservoirs in peripheral blood, lymphoid tissues and other sanctuary sites. In most patients, rebound upon treatment cessation is prompt. We assessed whether early treatment with raltegravir can impact on the formation of the viral reservoir. METHODS: We conducted an open-label, nonrandomized study, and assessed in detail the decay characteristics of HIV-1 RNA in plasma, HIV DNA in CD4 T cells and colon tissue biopies (CTBs) in 16 treatment-naive patients during either primary (PHI, n = 8) or chronic (CHI, n = 8) HIV-1 infection after treatment with raltegravir and Truvada for 52 weeks. RESULTS: HIV-1 RNA decreased rapidly with treatment in all patients; first and second phase levels were lower in PHI patients with no appreciable difference in residual viremia between the two groups at 52 weeks. Episomal HIV-1 DNA increased sharply in both groups with peak levels at 3-4 weeks. Total HIV-1 DNA levels were reduced in both groups with similar kinetics, but were markedly lower in PHI patients after 52 weeks. Integrated HIV-1 DNA levels were significantly lower at baseline in PHI patients and this difference widened on treatment. Finally, total HIV-1 DNA decayed substantially in both groups in CTB. CONCLUSION: Treatment with raltegravir resulted in a large number of abrogated integration events, reflected by the increase of episomal HIV-1 DNA after treatment initiation. Levels of total and integrated HIV-1 DNA were lower in PHI patients at the end of the study period.

Activation of NK cells by ADCC responses during early HIV infection.

Partial control of HIV occurs during acute infection, although the mechanisms responsible are poorly understood. We studied the ability of antibody-dependent cellular cytotoxicity (ADCC) antibodies in serum to activate natural killer (NK) cells in longitudinal samples from 8 subjects with well-defined early HIV infection who controlled viremia to low levels. NK cell activation by ADCC antibodies to gp140 Env proteins was detected in half of the subjects at the first time point studied, a mean of 111 d after the estimated time of infection. In contrast, ADCC-mediated NK cell activation in response to linear HIV peptides evolved more slowly, over the first 2 y of infection. Our studies suggest that HIV-specific ADCC responses to conformational epitopes occur early during acute HIV infection, and broaden to include linear epitopes over time. These findings have implications for the immune control of HIV.

HIV immune escape at an immunodominant epitope in HLA-B*27-positive individuals predicts viral load outcome.

The CTL response in HLA-B*27(+) HIV-infected individuals is characterized by an immunodominant response to a conserved epitope in gag p24 (aa 263-272, KRWIILGLNK; KK10). Mutations resulting in substitution of the arginine (R264) at position 2 of this epitope have been identified as escape mutations. Nineteen HLA-B*27(+) long-term nonprogressors were identified from an Australian cohort with an average follow-up of 16 y following infection. Viral and host genetic factors impacting on disease progression were determined at multiple time points. Twelve of 19 had wild-type sequences at codon 264 at all time points; 7 of 19 carried CTL escape variants. Median viral load and CD4(+) T cell counts were not significantly different between these groups at enrollment. Viral load, as judged by levels at their last visit (1,700 and 21,000 RNA copies/ml, respectively; p = 0.01) or by time-weighted area under the curve was higher in the escape group (p = 0.02). Escape mutants at other HLA-B*27-restricted epitopes were uncommon. Moreover, host polymorphisms, such as CCR5Δ32, CCR2-64I, and SDF1-3'A, or breadth of TCR repertoire responding to KK10 did not segregate to wild-type or escape groups. Host and viral factors were examined for a relationship to viral load. The only factor to affect viral load was the presence of the R264 escape mutations at the immunodominant epitope. CTL escape at R264 in the KK10 epitope is a major determinant of subsequent viral load in these HLA-B*27(+) individuals.

Antihypernociceptive synergy between ibuprofen, paracetamol and codeine in rats.

We investigated the effects of intraperitoneal injections of a combination of two cyclo-oxygenase inhibitors, ibuprofen and paracetamol, with a weak opiate, codeine, on nociception in Sprague Dawley rats. Administration of paracetamol (11, 44, and 88 mg/kg), ibuprofen (8.75, 35, and 140 mg/kg) or codeine (0.44, 1.75, and 3.5mg/kg) alone caused a dose-dependent inhibition of reperfusion hypernociception. Administration of a combination of 0.44 mg/kg codeine+8.75 mg/kg ibuprofen+11 mg/kg paracetamol, drug doses that did not significantly reduce reperfusion hypernociception when administered individually or in pairs, abolished reperfusion hypernociception, such that the antihypernociceptive efficacy of the combination was approximately 2.5-fold greater than that of the sum of the antihypernociceptive efficacy of the individual drugs. Coordinated motor function, tested using a rotarod, was not impaired at the doses we used. Thus, we have demonstrated that codeine, paracetamol and ibuprofen act synergistically to induce antihypernociception in rats at doses which do not affect motor function.

Trends in antiretroviral treatment use and treatment response in three Australian states in the first decade of combination antiretroviral treatment.

OBJECTIVES: To determine if there were any differences in antiretroviral treatment (ART) use across the three eastern states of Australia, New South Wales (NSW), Victoria and Queensland, during the period 1997 to 2006. METHODS: We used data from a clinic-based cohort, the Australian HIV Observational Database (AHOD), to determine the proportion of HIV-infected patients on ART in selected clinics in each state and the proportion of treated patients with an undetectable viral load. Data from the national Highly Specialised Drugs program and AHOD were used to estimate total numbers of individuals on ART and the proportion of individuals living with HIV on ART nationally and by state. Data from the HIV Futures Survey and the Gay Community Periodic Survey were used to determine the proportion of community-based men who have sex with men on ART. The proportion of patients with primary HIV infection (PHI) who commenced ART within 1 year of diagnosis was obtained from the Acute Infection and Early Disease Research Program (AIEDRP) CORE01 protocol and Primary HIV and Early Disease Research: Australian Cohort (PHAEDRA) cohorts. RESULTS: We estimated that the numbers of individuals on ART increased from 3181 to 4553 in NSW, 1309 to 1926 in Victoria and 809 to 1615 in Queensland between 2000 and 2006. However, these numbers may reflect a lower proportion of individuals living with HIV on ART in NSW compared with the other states (37% compared with 49 and 55% in 2000). We found similar proportions of HIV-positive men who have sex with men participants were on ART in all three states over the study period in the clinic-based AHOD cohort (81-92%) and two large, community-based surveys in Australia (69-85% and 49-83%). Similar proportions of treated patients had an undetectable viral load across the three states, with a consistently increasing trend over time observed in all states. We found that more PHI patients commenced treatment in the first year following HIV diagnosis in NSW compared with Victoria; however, the sample size was very small. CONCLUSIONS: For the most part, patterns of ART use were similar across NSW, Victoria and Queensland using a range of available data from cohort studies, community surveys and national prescription databases in Australia. However, there may be a lower proportion of individuals living with HIV on ART in NSW compared with the other states, and there is some indication of a more aggressive treatment approach with PHI patients in NSW compared with Victoria.

Epidemiology of primary HIV-1 infection.

PURPOSE OF REVIEW: To review recent studies reporting epidemiological and public health aspects of primary and recently acquired HIV infection, with a particular emphasis on patterns of occurrence, relationship to onward transmission, diagnostic strategies and risk factors. RECENT FINDINGS: Diagnosis of primary HIV infection remains a relatively infrequent occurrence. Clinical and demographic characteristics may be helpful indicators to guide the decision to offer testing. The high levels of viraemia associated with primary HIV infection represent a particular risk of onward transmission, as recently demonstrated through studies using genotyping methods to link newly acquired cases. Diagnostic strategies involving nucleic acid detection have been increasingly used to identify cases prior to the development of antibodies. Serological tests for early infection are valid for epidemiological purposes but are not generally viewed reliable enough for individual diagnosis. Prospective studies continue to be a useful means of identifying factors associated with the risk of newly acquired HIV infection, and can guide the implementation and evaluation of prevention strategies. SUMMARY: The occurrence of primary HIV infection is an event of public health importance. Understanding of the extent of primary HIV infection and its risk factors in populations can guide the development and evaluation of preventive interventions.

Modality-specific hypersensitivity of dorsal horn convergent neurones during reperfusion of their receptive fields on the rat's tail.

In rats anaesthetised with enflurane, we examined the responses of convergent neurones in the dorsal horn of the spinal cord to noxious thermal and mechanical stimulation and to innocuous brushing, during reperfusion of their receptive fields on the tail, following transient ischaemia. Neurones were included if they responded, before induction of ischaemia, to both pinching and brushing of receptive fields restricted to the tail. Ischaemia was induced by occluding the blood supply to the tail for 30 min using a tourniquet. Compared to their own responses before ischaemia, during reperfusion almost all the neurones (17 of 20) exhibited significantly increased activity to noxious pinching and innocuous brushing of their receptive fields, following 30 and 60 min of reperfusion. Receptive field size increased markedly in 16 of 20 of the neurones tested. Only 13 of 35 of the neurones responded to noxious thermal stimulation of the tail before induction of ischaemia, and of these only two exhibited enhanced sensitivity to thermal stimulation during reperfusion. Our results indicate that there is a population of convergent neurones that demonstrates hypersensitivity to mechanical stimulation of the rat's tail, but not to noxious thermal stimulation, during reperfusion of their receptive fields following transient ischaemia.

Intracerebroventricular micro-injections of non-steroidal anti-inflammatory drugs abolish reperfusion hyperalgesia in the rat's tail.

Prostaglandins are mediators of reperfusion hyperalgesia; their site of action may be in the periphery, in the central nervous system, or both. We have investigated whether prostaglandins play a role in the central nervous system during reperfusion hyperalgesia, by intracerebroventricular (i.c.v.) micro-injection of non-steroidal anti-inflammatory drugs (NSAID), to inhibit local prostanoid synthesis. We induced tail ischaemia in conscious rats by applying an inflatable cuff at the base of the tail. The cuff was released at the first signs of co-ordinated escape behaviour. Responses to a noxious thermal stimulus were assessed, by measuring tail flick latency following immersion of the tail in water at 49 degrees C, prior to and immediately after release of the tourniquet. Tail flick latency decreased significantly following ischaemia, that is there was post-ischaemic reperfusion hyperalgesia. Intracerebroventricular micro-injection of NSAID prior to applying the tourniquet had no effect on the co-ordinated escape behaviour during ischaemia or on the tail flick latency after application of a sham tourniquet (uninflated cuff). However all the drugs abolished the hyperalgesia evident during reperfusion. Doses required to abolish hyperalgesia were 0.001 mg/kg indomethacin, 0.08 mg/kg dipyrone, 0.09 mg/kg ibuprofen, 0.2 mg/kg diclofenac sodium and 0.2 mg/kg paracetamol. These doses are 2-3 orders of magnitude less than those necessary to abolish reperfusion hyperalgesia when the same drugs are administered systemically. Our results indicate that the development of reperfusion hyperalgesia of the rat's tail depends on the synthesis of prostanoids within the central nervous system.

Role of N-methyl-D-aspartate and opiate receptors in nociception during and after ischaemia in rats.

We have investigated the effects of systemic administration of two N-methyl-D-aspartate (NMDA) receptor antagonists and two opiate agonists on nociception during and after tail ischaemia in conscious rats. The two NMDA receptor antagonists, D-2-amino-5-phosphonovalerate (APV) and ketamine hydrochloride, did not alter tail flick latencies in rats not subjected to ischaemia but inhibited post-ischaemic hyperalgesia (PIH) in a dose-dependent manner. Neither of these agents impaired motor function of the rats, as assessed by rotarod performance, suggesting a purely sensory antinociceptive effect. The antinociceptive effect of APV during reperfusion following ischaemia was not antagonised by the mu-opiate receptor antagonist naloxone (1 mg/kg). The two opiate receptor agonists, morphine and pethidine, increased tail flick latencies in rats not subjected to ischaemia, inhibited PIH in a dose-dependent manner, and also caused significant motor malfunction, all in naloxone-reversible fashion. We conclude that the role of the NMDA receptor in mediating afferent nociceptive traffic is confined to its involvement in neuronal events mediating hyperalgesia.

Behavioral and thalamic nociceptive responses in rats following noxious ischaemia of the tail.

In rats anaesthetized with urethane, we have investigated the response of neurones in the ventrobasal complex of the thalamus to noxious ischaemia of the tail, and to graded noxious thermal stimulation of the tail before and after ischaemia. In behavioural experiments conscious rats were exposed to the same experimental procedure. After ischaemia the threshold tail temperature required to elicit both a neuronal response and aversive behaviour in conscious rats, to thermal stimulation, was decreased significantly (P less than 0.01 paired t tests). Threshold temperatures for the neuronal response and the behavioural response were not significantly different, either before or after ischaemia. The time course of recovery to pre-ischaemic threshold temperatures was the same for both the behavioural and neuronal responses. Most thalamic neurones responding to noxious thermal stimulation of the tail also increased firing rate during ischaemia. The latency of response of the thalamic neurones to ischaemia was 12.1 +/- 1.8 min and the latency of the behavioural response to the same stimulus was 11.9 +/- 2.1 min. Ventrobasal thalamic neurones, therefore, which responded to noxious thermal stimulation of the tail also responded to noxious ischaemia, and exhibited a neuronal correlate of post-ischaemic hyperalgesia which paralleled behavioural responses closely.

Injectable aspirin and mepyramine abolish post-ischaemic hyperalgesia in rats.

We have investigated the effects of mepyramine, an H1 receptor antagonist, and lysine acetylsalicylate, a cyclo-oxygenase inhibitor, on post-ischaemic hyperalgesia in rats. We induced tail ischaemia in conscious rats by applying a tourniquet until the rats exhibited coordinated escape behaviour, when we released the tourniquet. We assessed hyperalgesia, by measuring tail flick latency following tail immersion in water at 49 degrees C, immediately after releasing the tourniquet and then at 30 min intervals for 2 h. After pretreatment with the drug vehicles, tail flick latency decreased significantly following ischaemia. Pretreatment with mepyramine maleate (3 mg/kg), or lysine acetylsalicylate (400 mg/kg), injected subcutaneously, abolished the decrease. We conclude that both histamine release and prostanoid synthesis are involved in the post-ischaemic hyperalgesia.

Hyperalgesia following ischaemia of the rat's tail.

We have investigated the effects of ischaemia on responses to a subsequent noxious stimulus in rats. Tail flick latencies to a noxious thermal stimulus were determined by immersing the tail in water at temperatures ranging from 39 to 49 degrees C. We then produced ischaemia by occluding the blood supply to the tail; ischaemia was terminated at the first signs of an escape response. Tail flick latencies were recorded immediately after termination of ischaemia and at 30 min intervals for another 2 h. Each rat acted as its own control. Tail flick latency decreased after ischaemia; we found a decrease of about 39% immediately after ischaemia, at immersion temperatures above 39 degrees C. The duration of the hyperalgesia increased with increasing water temperatures. Thus noxious ischaemia of the rat tail induced hyperalgesia to subsequent noxious thermal stimuli. The hyperalgesia could have arisen through either central or peripheral mechanisms.