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Research Letters

Zinc fights diarrhoea in HIV-1-infected children: in-vitro evidence to link clinical data and pathophysiological mechanism

Canani, Roberto Berni; Ruotolo, Serena; Buccigrossi, Vittoria; Passariello, Annalisa; Porcaro, Francesco; Siani, Maria Concetta; Guarino, Alfredo

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doi: 10.1097/QAD.0b013e328011849a

Abstract

A beneficial reduction of watery diarrhoea-related morbidity induced by zinc supplementation in HIV-1-infected children has recently been demonstrated [1]. In the past few years zinc has emerged as a major therapeutic and preventive strategy against diarrhoea [2–4]. These data now open new perspectives for its use, suggesting that zinc could be a safe, simple and cost-effective tool to reduce morbidity and mortality in HIV-1-infected children [1]. Even though zinc is recommended for the treatment of childhood diarrhoea by WHO/UNICEF [3], several important questions remain to be answered, in particular in the HIV-1-infected child, because the study by Bobat et al. [1] did not provide evidence on the mechanisms elicited by zinc to limit diarrhoea in these particular patients.

The aetiology of HIV-1-related severe watery diarrhoea is certainly multifactorial and still largely unknown [5]. We have recently provided evidence for a primary role of Tat (the transactivating peptide produced by HIV-1 and essential for its replication) in the pathogenesis of diarrhoea in AIDS patients. Functioning as an enterotoxin, like Vibrio cholerae toxin, Tat stimulates active fluid secretion from the serosal to the luminal side of human enterocytes in the classical Ussing chamber in-vitro model used to investigate transepithelial ion transport [6,7]. A similar mechanism has been reported [8] for Cryptosporidium parvum, the most frequent and dangerous opportunistic enteric pathogen in AIDS patients. These findings suggest that HIV-1-infected children are at a high risk of secretory diarrhoea. In addition, we have demonstrated that Tat is able to inhibit sodium ion/glucose symporter activity, a major mechanism to absorb fluid at the intestinal level, further determining the occurrence of diarrhoea [7]. Interestingly, the Tat effects on intestinal ion transport were dose dependent, with a maximal effective dose of 0.1 nmol, which is well within the range of what is generally measured in the sera of patients with HIV-1 infection, suggesting that effective Tat concentrations may well be reached in vivo[9,10]. On the other hand, zinc is directly active on transepithelial ion transport at the intestinal level, as direct zinc–enterocyte interaction results in net ion absorption, thereby counteracting active secretion, such as that induced by V. cholerae toxin in the same experimental model [11]. To determine whether zinc was effective in inhibiting Tat-induced ion secretion, we incubated human derived Caco-2 intestinal cells with chemical synthesized, high-pressure liquid chromatography 96% pure, HIV-1 Tat, (Tecnogen, Piana di Monteverna, Italy), in the presence or absence of zinc (ZnSO4; Sigma Chemical Co., St Louis, Missouri, USA), using the Ussing chamber experimental model [6]. As shown in Figure 1, the pre-incubation of human enterocytes with zinc (35 μmol) resulted in the almost total inhibition of Tat-induced ion secretion, as reflected by the intensity of the short circuit current. These results suggest that zinc is able to prevent intestinal fluid secretion induced by Tat, and is able to interact directly with a specific mechanism of HIV-1-related diarrhoea, explaining well the results obtained in South Africa by Bobat and co-workers [1], and supporting the usefulness of the ‘zinc approach’ in adjunct to specific antiretroviral therapy in HIV-1-infected children.

Fig. 1
Fig. 1:
Zinc inhibitory effect against HIV-1 Tat protein-induced intestinal ion secretion. (a) Time course of the effect of HIV-1 Tat protein and ZnSO4 addition, alone or in combination, on transepithelial ion transport in human enterocyte (Caco-2 cell) monolayer mounted in Ussing chambers (i.e. an established in-vitro model to study ion transport). Tat addition to the enterocytes serosal side (S) induced an increase in the intensity of short circuit current (Isc), indicating the presence of active chloride ion secretion. ZnSO4 determined a pure pro-absorptive effect on transepithelial chloride ion transport (i.e. a decrease in Isc). Pre-incubation for 20 min with ZnSO4 to the mucosal side (M) was able significantly to reduce the secretory response elicited by Tat at intestinal level. The arrows indicate the time of addition either of Tat or ZnSO4. (b) Maximal Isc modifications after Tat and ZnSO4 addition, alone or in combination, to human enterocytes mounted in Ussing chambers. Data are expressed as mean ± SE, and significance was evaluated by the non-parametric, two-tailed Mann–Whitney U test. *P < 0.001 Tat alone versus ZnSO4 plus Tat. ♦ Tat to S; ▪ ZnSO4 to M plus Tat to S; ▴ ZnSO4 to M.

An emphasis on the costs and economic benefits of an alternative therapy is an important aspect of health services research. The cost savings and the attractive cost-effectiveness, disposability and thermostability of zinc indicates the need to assess further the role of these micronutrients in the prevention and treatment of diarrhoea in AIDS patients.

References

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