Adam Bartos

N‐acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: A randomized, double‐blind, placebo‐controlled trial

OBJECTIVE Systemic lupus erythematosus (SLE) patients exhibit T cell dysfunction, which can be regulated through mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione (GSH). This randomized, double-blind, placebo-controlled study was undertaken to examine the safety, tolerance, and efficacy of the GSH precursor N-acetylcysteine (NAC). METHODS A total of 36 SLE patients received either daily placebo or 1.2 gm, 2.4 gm, or 4.8 gm of NAC. Disease activity was evaluated monthly by the British Isles Lupus Assessment Group (BILAG) index, the SLE Disease Activity Index (SLEDAI), and the Fatigue Assessment Scale (FAS) before, during, and after a 3-month treatment period. Mitochondrial transmembrane potential and mTOR were assessed by flow cytometry. Forty-two healthy subjects matched to patients for age, sex, and ethnicity were studied as controls. RESULTS NAC up to 2.4 gm/day was tolerated by all patients, while 33% of those receiving 4.8 gm/day had reversible nausea. Placebo or NAC 1.2 gm/day did not influence disease activity. Considered together, 2.4 gm and 4.8 gm NAC reduced the SLEDAI score after 1 month (P = 0.0007), 2 months (P = 0.0009), 3 months (P = 0.0030), and 4 months (P = 0.0046); the BILAG score after 1 month (P = 0.029) and 3 months (P = 0.009); and the FAS score after 2 months (P = 0.0006) and 3 months (P = 0.005). NAC increased Δψm (P = 0.0001) in all T cells, profoundly reduced mTOR activity (P = 0.0009), enhanced apoptosis (P = 0.0004), reversed expansion of CD4-CD8- T cells (mean ± SEM 1.35 ± 0.12-fold change; P = 0.008), stimulated FoxP3 expression in CD4+CD25+ T cells (P = 0.045), and reduced anti-DNA production (P = 0.049). CONCLUSION This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes.

Mechanistic Target of Rapamycin Activation Triggers IL-4 Production and Necrotic Death of Double-Negative T Cells in Patients with Systemic Lupus Erythematosus

The mechanistic target of rapamycin (mTOR) is recognized as a sensor of mitochondrial dysfunction and effector of T cell lineage development; however, its role in autoimmunity, including systemic lupus erythematosus, remains unclear. In this study, we prospectively evaluated mitochondrial dysfunction and mTOR activation in PBLs relative to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) during 274 visits of 59 patients and 54 matched healthy subjects. Partial least square–discriminant analysis identified 15 of 212 parameters that accounted for 70.2% of the total variance and discriminated lupus and control samples (p < 0.0005); increased mitochondrial mass of CD3+/CD4−/CD8− double-negative (DN) T cells (p = 1.1 × 10−22) and FOXP3 depletion in CD4+/CD25+ T cells were top contributors (p = 6.7 × 10−7). Prominent necrosis and mTOR activation were noted in DN T cells during 15 visits characterized by flares (SLEDAI increase ≥ 4) relative to 61 visits of remission (SLEDAI decrease ≥ 4). mTOR activation in DN T cells was also noted at preflare visits of SLE patients relative to those with stable disease or healthy controls. DN lupus T cells showed increased production of IL-4, which correlated with depletion of CD25+/CD19+ B cells. Rapamycin treatment in vivo blocked the IL-4 production and necrosis of DN T cells, increased the expression of FOXP3 in CD25+/CD4+ T cells, and expanded CD25+/CD19+ B cells. These results identify mTOR activation to be a trigger of IL-4 production and necrotic death of DN T cells in patients with SLE.

Genetic Polymorphisms Associated with Hearing Threshold Shift in Subjects during First Encounter with Occupational Impulse Noise

Noise-induced hearing loss (NIHL) is the most significant occupational health issue worldwide. We conducted a genome-wide association study to identify single-nucleotide polymorphisms (SNPs) associated with hearing threshold shift in young males undergoing their first encounter with occupational impulse noise. We report a significant association of SNP rs7598759 (p < 5 x 10-7; p = 0.01 after permutation and correction; Odds Ratio = 12.75) in the gene coding for nucleolin, a multifunctional phosphoprotein involved in the control of senescence and protection against apoptosis. Interestingly, nucleolin has been shown to mediate the anti-apoptotic effect of HSP70, a protein found to prevent ototoxicity and whose polymorphisms have been associated with susceptibility to NIHL. Increase in nucleolin expression has also been associated with the prevention of apoptosis in cells undergoing oxidative stress, a well-known metabolic sequela of noise exposure. To assess the potential role of nucleolin in hearing loss, we tested down-regulation of nucleolin in cochlear sensory cells HEI-OC1 under oxidative stress conditions and report increased sensitivity to cisplatin, a chemotherapeutic drug with ototoxic side effects. Additional SNPs were found with suggestive association (p < 5 x 10-4), of which 7 SNPs were located in genes previously reported to be related to NIHL and 43 of them were observed in 36 other genes previously not reported to be associated with NIHL. Taken together, our GWAS data and in vitro studies reported herein suggest that nucleolin is a potential candidate associated with NIHL in this population.

New biotin derivatives for labeling and solubilizing IgG peptides

In this article, we describe the synthesis of a new class of oligoethylene‐glycol based water‐soluble biotin derivatives for labeling of peptides with limited solubility in aqueous solution. First 4,7,10‐trioxa‐1,13‐tridecanediamine was mono‐acetylated by succinic anhydride (Ttds) followed by the introduction of N‐Fmoc‐protecting group using Fmoc‐N‐hydroxysuccinimide ester. The resulting compound (Fmoc‐Ttds) was used for the preparation of 4,7,10‐trioxa‐1,13‐tridecanediamine di‐ and trimers on solid phase using Wang resin by carbodiimide coupling method. After attachment of Fmoc‐Ttds to the solid support, the Fmoc‐blocking group was removed and the Ttds‐modified resin was repeatedly acylated by Fmoc‐Ttds or by biotin using PyBOP/HOBt active ester reaction. Finally the product [Fmoc‐(Ttds)n or biotinyl‐(Ttds)n (where n = 1, 2 or 3)] was removed from the resin by trifluoroacetic acid in the presence of water. After appropriate HPLC purification and characterization (MS) biotinyl‐(Ttds)n (where n = 1, 2 or 3) were introduced to the N‐terminal of poorly soluble oligopeptides by solid phase peptide synthesis. We found that this new class of biotinylating reagent could be prepared easily and in good yield. Comparative solubility measurements suggest that the incorporation of these moieties–depending on the number of Ttds unit–could enhance water solubility.