Alternative Explanations for Negative Findings in the Community Popular Opinion Leader Multisite Trial and Recommendations for Improvements of Health Interventions Through Social Network Analysis

To the Editors: Mosoian et al identified in macrophages that prothymosin-alpha (ProTa) has a potent postentry HIV-1–inhibitory activity, which is dependent on type I interferon (IFN) induction. They proposed that ProTa may provide, therefore, therapeutic leads for IFN-sensitive viral infections. Interestingly, Ueda et al has recently suggested that ProTa may have neuroprotective roles in brain stroke or traumatic damage because it is a unique cell death regulatorymolecule that converts the intractable cell death necrosis into controllable apoptosis that can be inhibited therapeutically by growth factors. Human prothymosin-alpha is a small 109 amino acid highly acidic protein that it was initially described as the precursor molecule for several peptides originally regarded as thymic factors/hormones, including its N-terminal region (D1–29) thymosin-apha1 (Ta1). Many of them have been used as therapeutics in late-stage clinical trials in cancer and immunodeficiency disorders, including ProTa. Whether ProTa, a nuclear protein involved in transcription and chromatin decondensation and in the apoptotic process, was secreted itself (it can be found in human serum) and/or served as precursor of Ta1 was disputed for several years. It is well established that a lot of intracellular molecules, including DNA and RNA, can be secreted via several alternative (nonclassical) secretion pathways. Endogenous molecules that are released in response to injury, infection, or other inflammatory stimuli and initiate inflammatory responses are so-called damage-associated molecular patterns and/or alarmins. It has been shown that under apoptotic stimuli, ProTa is released from the nucleus to the cytosol, where inhibits apoptosome formation. Very recently, Ueda et al described how necrosis-inducing stress induces an extracellular release of ProTa from neurons and astrocytes in a nonvesicular manner, whereas apoptosis-inducing stress does not, through interaction with cargo molecule for extracellular release S100A13, a member of the Ca2-binding S100 family. It is probable that virusinfected CD8+ T cells secrete ProTa in a similar way. ProTa exerts its effect on macrophages via TLR4-dependent signal transduction and Mosoian et al demonstrated that ProTa activity is lost in the absence of TLR4. Because our laboratory and others had observed many immunomodulatory activities for ProTa, we looked for cell surface ProTa receptors on cells from the immune system and were the first group to report their existence. The presence of a cell surface ProTa receptor has also been confirmed in cortical neurons. Although we failed to identify TLR4 at the time, reevaluating these previously reported results under the light of new data, a model that may help to design new ProTarelated therapeutic compounds that can fight against IFN-sensitive viral infections, including HIV-1, is suggested. We looked for ProTa cell-surface receptors by using a direct approach, radiolabeling the ligand and analyzing steady state binding data. Important data in which our results agree with those found byMosoian et al is the fact that Ta1 neither displays anti-HIV-1 activity nor competes with ProTa for its receptors. Therefore, the ProTa binding to TLR4 is through its non–N-terminal part. ProTa mutants that lacked the N-terminal region (D1–29), including Ta1, or the C-terminal region (D102–112), including the nuclear localization signal TKKQKK, also retained the original antinecrosis activity of ProTa. Ta1 has shown a variety of effects on cells and pathways of the immune system by modulating dendritic cell (DC) function via the TLR9/MyD88-dependent viral recognition signal transduction mechanism. Our results and those of Mosoian et al also agree in that ProTa effects are not unique to macrophages because it induced IFNb in DCs as well and it bound sites in the natural killer (NK) cell–derived YT cell line and T lymphocytes, these subsets expressing TLR4 at least in some cases andwhere ProTa canmodulate several pathways. This means that the physiological context of a clinical trial with ProTa as therapy will be more complex, although not necessary for worse, than the ProTa effects described on macrophages treated in vitro. Incardinated with this issue is the fact that only one binding site was found on the NK-like cells 19 versus two sites, with high and low affinity, on T cells and monocytes. Furthermore, although there were binding sites with higher affinity in T cells, there were more lower affinity sites per cell in monocytes (macrophage precursors). Equilibrium Binding Data Analysis, Ligand, and Kinetic programs did not detect cooperation between both binding sites and both 1 receptor/2 chains and the 2 receptor model fitted with our data. These data support the role of the participation of TLR4 with its extracellularly associated MD-2 protein (they may form TLR4/MD-2 complexes that dimerize in the presence of ligands), with lipopolysaccharide (LPS) binding protein or with CD14, in ProTa’s anti– HIV-1 activity. Although the participation of LPS binding protein or CD14 cannot be totally excluded, the big difference between LPS and ProTa sizes, and certain data about the MW of the ProTa binding molecule (around 30 KDa), support MD2. However, the formation of different types of complexes, and their number, in each cell type may explain the diverse affinities of ProTa. If neuron cell surface ProTa receptor is confirmed as TLR4, the existence of different complexes may also explain that ProTa survival activity is mediated by a Gai/o-coupled receptor that activates phospholipase C and PKCbII, inducing GLUT1/4 transport to membranes, whereas ProTa apoptotic effect is mediated by PKCbI and PKCbII activation, leading to I-kB kinase complex and necrosis factor kB activation. These signal transduction pathways can be mediated by one of the major TLR4 signaling adaptors, MyD88, whereas type I IFN induction is dependent of the other main downstreamTLR4 adaptor, TRIF. Perhaps, TRIF is not expressed in neurons, or the adaptor may respond to the type of extracellular complex formed pending on the type of ligand bound.