Recovery of effective HIV-specific CD4+ T-cell activity following antiretroviral therapy in paediatric infection requires sustained suppression of viraemia.

BACKGROUND: The success of increasing access to antiretroviral therapy (ART) in paediatric HIV infection prompts the question of the potential for eradication of HIV infection in this age group. 'Shock-and-kill' HIV cure approaches, currently in development, may depend upon an effective antiviral T-cell response to eradicate virus-infected cells. METHOD: We here investigate the ability of HIV-infected children receiving ART from early childhood (median 24 months' age) to generate effective HIV-specific CD4 and CD8 T-cell immune responses that would facilitate future immune-based cure therapies. RESULTS: Initial analysis of ART-naive HIV-infected children demonstrated that maintenance of normal-for-age absolute CD4 T-cell counts was strongly linked to high IL-2 production and polyfunctional HIV-specific CD4 T-cell responses (P < 0.0001 in each case). Low viral load was, similarly, strongly associated with markedly low IFN-γ and high IL-2 HIV-specific CD4 T-cell responses (P < 0.0001). In children receiving ART, establishment of this immune profile (high IL-2 and low IFN-γ HIV-specific T-cell production) was strongly related to the duration of viraemic suppression. Failure to suppress viraemia on ART, and even the successful suppression of viraemia interrupted by the occurrence of transient viraemia of more than 1000 HIV copies/ml, was associated with an immune profile of high IFN-γ and low IL-2 HIV-specific T-cell responses and low polyfunctionality. CONCLUSION: These data are consistent with recovery of functional CD4 T-cell responses in ART-treated children, in contrast to relative lack of CD4 T-cell function recovery described in ART-treated adults. However, the challenges of achieving long-term suppression of viraemia in ART-treated children through adolescence remain daunting.

[An approach to the patients with cryopyrin-associated periodic syndrome (CAPS) : a new biologic response modifier, canakinumab].

Cryopyrin-associated periodic syndrome (CAPS) comprises a group of rare, but severe, autoinflammatory syndrome, and includes 3 distinct conditions, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (MONID). These syndromes are characterized by urticarial-like rash, periodic fever, central nervous system inflammation, an arthropathy, and the risk of amyloidosis. About 20% die by age 20 years in the most severe cases. The disease is associated with mutations in the NLRP3 gene that encodes for the protein cryopyrin, a component of the inflammasome complex that regulates the production and secretion of IL-1ß. Canakinumab is a human IgG monoclonal antibody targeting IL-1ß. The clinical trials of canakinumab for patients with CAPS in both western countries and Japan were well-tolerated in most patients, and provided significant advantages over existing competitive therapies. Although no serious adverse effects have been reported, the frequencies of common infectious diseases including nasopharyngitis, upper respiratory tract infections, and gastroenteritis were reported presumably due to the blockade of proinflammatory cytokine, IL-1ß. For us pediatrician, it will be important to be more careful for infectious diseases to provide the maximum safety of canakinumab for these patients.

Oxidation of aminonitrotoluenes by 2,4-DNT dioxygenase of Burkholderia sp. strain DNT.

Aminonitrotoluenes form rapidly from the reduction of dinitrotoluenes (DNTs) which are priority pollutants and animal carcinogens. For example, 4-amino-2-nitrotoluene (4A2NT) and 2A4NT accumulate from the reduction of 2,4-DNT during its aerobic biodegradation. Here, we show that 2,4-DNT dioxygenase (DDO) from Burkholderia sp. strain DNT oxidizes the aminonitrotoluenes 2A3NT, 2A6NT, 4A3NT, and 5A2NT to 2-amino-3-nitrobenzylalcohol, 2-amino-4-nitro-m-cresol and 3-amino-5-nitro-p-cresol, 4-amino-3-nitrobenzylalcohol and aminonitrocresol, and 2-amino-5-nitro-o-cresol, respectively. 2A5NT and 3A4NT are oxidized to aminonitrocresols and/or aminonitrobenzylalcohols, and 4A2NT is oxidized to aminonitrocresol. Only 2A4NT, a reduced compound derived from 2,4-DNT, was not oxidized by DDO or its three variants. The alpha subunit mutation I204Y resulted in two to fourfold faster oxidization of the aminonitrotoluenes. Though these enzymes are dioxygenases, they acted like monooxygenases by adding a single hydroxyl group, which did not result in the release of nitrite.

Protein engineering of the archetypal nitroarene dioxygenase of Ralstonia sp. strain U2 for activity on aminonitrotoluenes and dinitrotoluenes through alpha-subunit residues leucine 225, phenylalanine 350, and glycine 407.

Naphthalene dioxygenase (NDO) from Ralstonia sp. strain U2 has not been reported to oxidize nitroaromatic compounds. Here, saturation mutagenesis of NDO at position F350 of the alpha-subunit (NagAc) created variant F350T that produced 3-methyl-4-nitrocatechol from 2,6-dinitrotoluene (26DNT), that released nitrite from 23DNT sixfold faster than wild-type NDO, and that produced 3-amino-4-methyl-5-nitrocatechol and 2-amino-4,6-dinitrobenzyl alcohol from 2-amino-4,6-dinitrotoluene (2A46DNT) (wild-type NDO has no detectable activity on 26DNT and 2A46DNT). DNA shuffling identified the beneficial NagAc mutation G407S, which when combined with the F350T substitution, increased the rate of NDO oxidation of 26DNT, 23DNT, and 2A46DNT threefold relative to variant F350T. DNA shuffling of NDO nagAcAd also generated the NagAc variant G50S/L225R/A269T with an increased rate of 4-amino-2-nitrotoluene (4A2NT; reduction product of 2,4-dinitrotoluene) oxidation; from 4A2NT, this variant produced both the previously uncharacterized oxidation product 4-amino-2-nitrocresol (enhanced 11-fold relative to wild-type NDO) as well as 4-amino-2-nitrobenzyl alcohol (4A2NBA; wild-type NDO does not generate this product). G50S/L225R/A269T also had increased nitrite release from 23DNT (14-fold relative to wild-type NDO) and generated 2,3-dinitrobenzyl alcohol (23DNBA) fourfold relative to wild-type NDO. The importance of position L225 for catalysis was confirmed through saturation mutagenesis; relative to wild-type NDO, NDO variant L225R had 12-fold faster generation of 4-amino-2-nitrocresol and production of 4A2NBA from 4A2NT as well as 24-fold faster generation of nitrite and 15-fold faster generation of 23DNBA from 23DNT. Hence, random mutagenesis discovered two new residues, G407 and L225, that influence the regiospecificity of Rieske non-heme-iron dioxygenases.

Cytochrome P450 1A1 (CYP1A1) inhibitor alpha-naphthoflavone interferes with UDP-glucuronosyltransferase (UGT) activity in intact but not in permeabilized hepatic microsomes from 3-methylcholanthrene-treated rats: possible involvement of UGT-P450 interactions.

The effects of cytochrome P450 (P450, CYP) ligands and permeabilization of microsomes on 3-hydroxybenzo(a)pyrene [3-OH-B(a)P] glucuronidation mediated by rat hepatic microsomes were studied. While the UDP-glucuronosyltransferase (UGT) activity with non-permeabilized microsomes from 3-methylcholanthrene (MC)-treated rats was markedly reduced by alpha-naphthoflavone (NF), this inhibitor had hardly any effect when permeabilized microsomes were used in which the inhibitor was expected to have easy access to UGT. Kinetic analysis indicated that the inhibitory effect of alpha-NF is competitive. These results suggest that a UGT isoform(s) involved in 3-OH-B(a)P glucuronidation is interfered by a CYP1A inhibitor via a mechanism dependent on the intact nature of microsomal membranes in MC-treated rats. It is likely that P450 functions as a substrate transporter for some isoforms of UGT via possible interactions between UGT and P450.