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.

The immunoassay of cytokines and growth factors in biological fluids.

There are a number of problems associated with the development of standards suitable for use in the most commonly used assays to detect cytokines in biological fluids. These problems include: (i) the failure of some MoAbs used in immunoassays to detect all different <> of recombinant or natural material; (ii) the use of many different MoAbs, with different specificities, in different immunoassay kits, and (iii) the detection of non-active cytokines (fragments, inhibitors, receptor antagonists, etc.) in these immunoassays. As a result, it is possible to have biologically active material which is not detected in these immunoassays. Alternatively, biologically inactive material can be detected in these assays and is indistinguishable from biologically active material. In addition, the use of different antibodies with different specificities, affinities and avidities in different kits designed to detect the same biological materials results in markedly different sensitivities and specificities. Many of these same concerns can be raised for the use of bioassays for detection of molecules in biological fluids. The solution will not be simple (if possible at all). In most cases, the immunoassay kits are designed to detect <> material in biological fluids, but are made with MoAbs against recombinant material. Because of the markedly different specificities, affinities, etc. of the MoAbs in these kits, their standardization is possible only with a highly purified preparation of natural material. For the assay of recombinant materials, immunoassays should be specifically designed with the recombinant material in mind (i.e. the MoAbs made specifically against the recombinant material to be detected or shown to bind effectively with the recombinant material). Importantly, it should be made clear to investigators using different immunoassays that: (i) the reporting of biological material detected using immunoassays can only be made in units of weight (i.e. ng/ml); (ii) because of the detection of biologically active and inactive material using immunoassay kits these assays cannot be directly compared to bioassays or their results represented as <>; (iii) because of the difference in specificity and sensitivity of the different reagents used in different immunoassays, the results from different assays cannot be directly compared, and (iv) because of these same considerations, comparison of different > of materials within a single immunoassay is also not possible. The use of specific immunoassays for recombinant material in combination with bioassays and the use of cytokine standards, made from highly purified natural material, would help to standardize the results in this field.

Safety evaluation of recombinant human interleukin-4. I. Preclinical studies.

Recombinant human IL-4 (rhuIL-4) has been evaluated in a series of preclinical studies. These studies have demonstrated that rhuIL-4 is a very potent cytokine with a wide range of pharmacologic and toxicologic effects. Target systems/organs included the cardiovascular system, liver, spleen, and bone marrow. The incidence and severity of effects correlated strongly with both the dose level and the duration of rhuIL-4 administration. The major dose-limiting toxicities identified included death, cardiac inflammation and necrosis, hepatitis, and hepatic necrosis and occurred at sc doses > or = 25 micrograms/kg/day, while a sc dose of 5 micrograms/kg/day was the highest tested that did not result in major dose-limiting toxicity. Clinical trials in humans have demonstrated that sc administration of Escherichia coli-derived rhuIL-4 is safe and well tolerated at doses up to and including 5 micrograms/kg/day and up to 10 micrograms/kg when administered 3 times/week.

Safety evaluation of recombinant human interleukin-4. II. Clinical studies.

The safety and tolerability of Escherichia coli-derived recombinant human interleukin-4 (rhuIL-4) have been evaluated in phase I and phase II studies in human patients with a variety of malignancies. Clinical trials have demonstrated that subcutaneous administration of rhuIL-4 is safe and well tolerated at doses as high as 5 micrograms/kg/day and as high as 10 micrograms/kg when administered 3 times/week. Although preclinical safety studies in cynomolgus monkeys demonstrated a number of adverse effects following repeated daily dosing with rhuIL-4, similar effects have generally not been observed in human patients.