Nitric oxide in HIV-1 perinatally infected children treated with highly active antiretroviral therapy

Reflection & Reaction Nitric oxide in HIV-1 perinatally infected children treated with highly active antiretroviral therapy We read with interest the review by Torre and colleagues1 of the role of nitric oxide (NO) in the pathogenesis of HIV-1 infection. The authors conclude that NO production is clearly increased in both adults2 and children3 with HIV-1 infection, but that the issue of whether NO acts principally as inducer or inhibitor of viral replication, and the mechanisms of increased NO production in HIV-1 infection remain unclear. Torre et al1 speculate that NO increases viral replication, predominantly in primary infections and in late stages of the disease when NO production is higher. However, NO might show beneficial antiviral effects during successful highly active antiretroviral therapy (HAART), when production is low. To our knowledge, no study has investigated concentrations of NO in children before and after switching antiretroviral treatment to HAART. We contribute to the debate by reporting data from eight HIV-1 perinatally infected children (four boys, four girls; median age 12·1 years, range 2·5–14·9), who were investigated before starting and after 12 weeks of HAART. The 12-week period is indicated by the Italian guidelines for antiretroviral therapy in children as appropriate to verify response to therapy.4 The children were switched from double combination antiretroviral therapy with two nucleoside reverse transcriptase inhibitors (NRTIs) or one NRTI

and one non-nucleoside reverse transcriptase inhibitor (NNRTI) to HAART on the basis of clinical and/or immunological and/or virological deterioration.4 HAART included two NRTIs (abacavir [16 mg/day], didanosine [180 mg/m2/day], lamivudine [8 mg/kg/day], stavudine [2 mg/kg/day], or zidovudine [480 mg/m2/day]), or one NRTI and one NNRTI (nevirapine [200 mg/m2/day]), plus one protease inhibitor (PI) including nelfinavir (90 mg/kg/day), ritonavir or lopinavir (800 mg/m2/day), (24 mg/kg/day). Informed consent for the study was given by the parents or guardians of the children. The study received local ethics committee approval. Compliance with therapy was assessed as described.4 The Centers for Disease Control and Prevention criteria were adopted in defining the children’s infection status and clinical condition.5 Immunological response to therapy was defined as an increase in CD4+ T-lymphocyte counts above 10 centiles for age (if baseline percentage was >15%) or over 5 centiles for age (if baseline percentage was ⭐15%) after 12 weeks of therapy. Virological failure was defined as a reduction of viral load of less than 1·0 log10 RNA copies/mL after 12 weeks of therapy.4 At baseline and after 12 weeks of HAART, serum samples were obtained for total white cell and lymphocyte counts, CD4+ T-lymphocyte subset analyses, plasma viral load determinations, and

Viral load, CD4+ T-lymphocyte counts, and nitrite/nitrate serum concentrations before and after 12 weeks of HAART in HIV-1 perinatally infected children

Entire data set (n=8) Log10 HIV-1 RNA (copies /mL) CD4+ lymphocytes (%) CD4+ lymphocytes (cells/mL) Nitrite/nitrate (␮mol/L)

Baseline (mean and standard deviation) 4·7 (0·4) 8·0 (4·1) 152 (93) 26·0 (3·2)

12-week (mean and standard deviation) 3·1 (0·7) 15·0 (4·0) 303 (112)

p

<0·001 0·004 0·011

20·5 (2·8)

0·003

4·6 (0·4)

2·5 (0·6)

<0·001

12·0 (4·1)

21·2 (4·0)

0·01

Virological responders (n=5) Log10 HIV-1 RNA (copies/mL) CD4+ lymphocytes (%) CD4+ lymphocytes (cells/mL) Nitrite/nitrate levels (␮mol/L)

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217 (92) 25·0 (3·1)

410 (101) 14·2 (2·6)

0·01 <0·001

nitrite/nitrate concentration measurements, as end-products of NO production. All patients were in clinical category B or C.5 No child had current opportunistic infections; one had HIV-1-encephalopathy. All had good compliance to therapy. Plasma viral load was evaluated quantitatively by the Amplicor HIV Monitor test (Roche Diagnostic Systems Inc, Branchburg, NJ, USA) and results were expressed as log10 HIV-1 RNA copies/mL. Lymphocyte subsets were analysed by FACScan after double staining with phycoerythrinconjugated CD3 and fluorescent isothiocyanate-conjugated CD4 monoclonal antibodies (Becton Dickinson), by means of a FACScan Lysis II analysis program (Becton Dickinson, San Jose, CA, USA). Serum nitrite/nitrate concentrations were measured in duplicate by an enzymelinked immunosorbent assay (Oxis Inc, Portland, OR, USA), according to the manufacturer’s recommendations. Reference values for nitrite/nitrate concentrations considered in this study were 14·2 (4·6) ␮mol/L, obtained by analysis of blood samples from eight healthy age-matched control children undergoing elective surgery, consent for which was obtained from the parents. The paired t-test was used to analyse differences in continuous variables over time. Linear regression analysis was used to investigate the association between nitrite/nitrate concentrations and CD4+ T-lymphocyte counts, or viral loads. A p<0·05 was considered statistically significant. Statistical analyses were done with the SPSS software package (SPSS 8.0; Chicago, IL, USA). Baseline mean (SD) nitrite/nitrate concentrations were significantly higher in HIV-1-infected children than in healthy age-matched controls (26·0 [3·2] vs 14·2 [4·6] ␮mol/L; p<0·0001). Patients did not show current opportunistic or recurrent bacterial infections but they failed the previous combined therapy, possibly explaining this finding. After 12 weeks

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Reflection & Reaction of therapy, all of the eight children showed immunological response to therapy. Five presented associated virological response, while three had virological failure to therapy. During the same period, a significant decrease in viral load (p<0·001) and a significant increase in CD4+ T lymphocyte percentages and absolute counts (p=0·004 and p=0·011, respectively) were evident (table). Mean nitrite/nitrate concentrations were significantly decreased after 12 weeks of therapy (p=0·003) (table). Nitrite/nitrate serum concentrations were markedly reduced in all the children with virological response to therapy (mean=–10·8 ␮mol/L), but not in those with virological failure (mean=–3·4 ␮mol/L), including the child with HIV-1 encephalopathy. A significant direct correlation between nitrite/nitrate serum concentration and viral load, both at baseline (r=0·9; p=0·002) and after 12 weeks of therapy (r=0·8; p=0·004), was present. No correlation was seen between nitrite/nitrate concentrations and CD4+ T-lymphocyte percentage and absolute counts (p=0·22 and p=0·16 before HAART; p=0·21 and p=0·21 after HAART, respectively). Torre points out that NO may upregulate HIV-1 replication,6,7 but other studies report the opposite result.8 In children, whose viral dynamics are substantially different from those of adults,9 we noted an association between nitrite/nitrate and viral load, which might support the

former hypothesis. A marked reduction in nitrite/nitrate concentrations was seen only in the subgroup of children who had a positive virological response to HAART. In adults, NO overproduction has been associated with opportunistic infections that may activate the synthesis of proinflammatory cytokines and subsequently induce NO synthase.1 In our children, mechanisms other than opportunistic infections seem to be responsible for increased nitrite/nitrate concentrations, a finding supported by experiments in animal models that showed a NO production significantly higher in neonatal than in adult macrophages.10 Our results in HIV-1-infected children suggest that (1) nitrite/nitrate concentrations in children, as in adults, are higher than in healthy age-matched controls; (2) high nitrite/nitrate concentrations are found in the absence of opportunistic infections; (3) there is no association between nitrite/nitrate concentrations and CD4+ T-lymphocyte counts; (4) by contrast, nitrite/nitrate concentrations correlate with HIV-1 load; and (5) virological effectiveness of HAART may be associated with decreased nitrite/nitrate concentrations. Obviously, this is a pilot study with a small data set and may stimulate further studies. Elena Chiappini, Luisa Galli, Chiara Azzari, and Maurizio de Martino

EC, LG, and MM are at the Pediatric Clinic and Division of Infectious Diseases and CA is at the Pediatric Clinic III, Department of Pediatrics, University of Florence, Florence, Italy. Correspondence: Professor Maurizio de Martino, Division of Pediatrics, Infectious Diseases, HIV & Cystic Fibrosis, Anna Meyer Children’s Hospital, via Luca Giordano 13, I-50132 Florence, Italy. Tel +39 055 5662494; fax +39 055 570380; email [email protected] Acknowledgments

We are indebted to Sergio Nanni for his excellent technical assistance. References 1

Torre D, Pugliese A, Speranza F. Role of nitric oxide in HIV-1 infection: friend or foe? Lancet Infect Dis 2002; 2: 273–80. 2 Groenveld PHP, Kroon FP, Nibbering PH, Bruisten SM, Van Sweten P, Van Furth R. Increased production of nitric oxide correlates with viral load and activation of mononuclear phagocytes in HIVinfected patients. Scand J Infect Dis 1996; 28: 341–45. 3 Torre D, Ferrario G, Speranza F, Martegani R, Zeroli C. Increased levels of nitrite in the sera of children infected with human immunodeficency virus type I. Clin Infect Dis 1996; 22: 605–53. 4 de Martino M, Tovo PA, Giaquinto C, de Rossi A, Galli L, Gabiano C. Italian guidelines for antiretroviral therapy in children with human immunodeficiency virus-type 1 infection. Acta Paediatr 1999; 88: 228–32. 5 Centers for Disease Control. 1994 revised classification system for human immunodeficiency virus infection in children less than 13 years of age. MMWR Recomm Rep 1994; 43: 1–10. 6 Jimenez JL, Gonzalez-Nicolas J, Alvarez S, Fresno M, Munoz-Fernandez MA. Regulation of human immunodeficiency virus type 1 replication in human T lymphocytes by nitric oxide. J Virol 2001; 75: 4655–63. 7 Blond D, Raoul H, Le Grand R, Dormont D. Nitric oxide enhances human immunodeficiency virus replication in primary human macrophages. J Virol 2000; 74: 8904–12. 8 Mannik JB, Stamler JS, Teng E, et al. Nitric oxide modulates HIV-1 replication. J Acquir Immune Defic Syndr 1999; 22: 1–9. 9 de Martino M, Galli L, Moriondo M, et al. Dissociation of responses to highly active antiretroviral therapy notwithstanding virologic failure and virus drug resistance, both CD4+ and CD8+ T lymphocytes recover in HIV-1 perinatally infected children. J Acquir Immune Defic Syndr 2001; 26: 196–97. 10 Sherman MP, Wong VZ. Nitric oxide production by alveolar macrophages from newborn versus infant and adult rats: American Pediatric Society 104th annual meeting and Society for Pediatric Research 63rd annual meeting; Seattle, WA; May 2–5,1994 (abstr).

Author’s reply Chiappini et al have shed new insight on the role of nitric oxide (NO) in HIV-1 infection, in particular on the effects of NO in stimulating viral replication. The authors have clearly shown that highly active antiretroviral therapy (HAART) decreases serum concentrations of nitrite/nitrate in treated paediatric patients and, more importantly, that there is a significant correlation between reduction of HIV-RNA concentrations and decrease of nitrite/nitrate concentrations after HAART.

In our review,1 we pointed out that the dichotomous effect of NO is that its high production—seen especially in the primary infection, in the advanced stage of the disease, and probably in patients who did not respond to HAART—leads to active replication of the HIV-1. By contrast, low NO production may cause a reduction or inhibition of HIV-1 replication, especially during the symptomless stage of the disease or during treatment with HAART.

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Indeed, the results of Chiappini’s study may highlight a proinflammatory activity of NO that is able to stimulate HIV-1 replication. Several findings further support the proviral activity of NO in HIV-1 infection. First, Groenveld et al2 have reported increased production of NO, which correlated with viral load and activation of mononuclear phagocytes in HIV-infected patients who were afebrile and without symptoms of active opportunistic infections. More recently, Gonzales-Nicolas3 saw higher

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