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Immune response to biologics treatment, while widely reported, yet fails to correlate with clinical outcomes and assay to assay comparison is often not possible. Hence, we developed a new peptide based-detection assay to stratify pediatric patients with juvenile idiopathic arthritis (JIA) or chronic non-infectious uveitis (CNU) and monitor anti-drug antibodies (ADAbs) formed as part of an immune response to treatment with the fully human monoclonal therapeutic antibody Adalimumab. Adalimumab derived synthetic peptides were optimized for maximum immunogenicity and were tested by SP-ELISA on a development cohort of 18 JIA and CNU treated patients. The two best performing peptides able to differentiate patient groups were selected for evaluation with a larger scale ELISA testing on a total of 29 sera from pediatric patients with JIA or CNU. The results of this peptide-based assay were compared to an in-house developed SPR biosensor ADAbs assay and a commercially available bridging ELISA. The first peptide, termed HC3, was able to positively detect ADAbs in 7 out of the 29 sera, while the second peptide, called LC3, was able to detect ADAbs in 11 out of 29 sera in the evaluation group. Following statistical data evaluation, it has been found that the detection of ADAbs using the peptide-based ELISA assay positively correlates with disease progression and remission. Two synthetic peptides derived from Adalimumab may provide a beneficial tool to clinicians for monitoring patient response to such treatment and taking informed decisions for treatment alternatives.

Background Mitochondria play a role in type 1 diabetes (T1D) particularly in the treatment and prevention of disorder consequences. Due to their demonstrated role in diabetes pathology, mitochondrial proteins can be an interesting starting point to study candidate antigens in T1D. We investigated the role of relevant post-translational modifications (PTM) on a synthetic mitochondrial peptide as putative antigen. Methods The antibody response in T1D was evaluated by solid phase-ELISA using a collection of synthetic peptides bearing different PTMs. We investigated the role of lipoylation, phosphorylation, and glycosylation. The PTMs were introduced at position 173 of the mitochondrial pyruvate dehydrogenase E2 complex peptide PDC-E2(167–184) and at position 7 of a structure-based designed β-turn peptide as an irrelevant sequence to investigate the role of the specific PDC-E2 peptide sequence. Results IgM titres in 31 T1D patients were higher than IgGs to all the synthetic PTM peptides. Results demonstrated the crucial role of lysine lipoamide, serine O-phosphorylation, and O-glycosylation into the PDC-E2(167–184) peptide sequence for IgM antibody recognition. Conclusions Results highlight the importance of immune dysregulation in T1D, furthermore, if confirmed in a large number of patients, they will contribute to add novel diagnostic markers for the understanding the physiopathology of the disease.

Cell line PER-423 was derived from the cells of a patient with an immature acute T-lymphoblastic leukaemia and the growth of this human cell line is strictly dependent on interleukin-2 (IL-2). PER-423 cells express the p75 (beta) subunit of the IL-2 receptor (IL-2R beta), while the p55 chain (IL-2R alpha) is not detectable by immunofluorescence. The analysis of the IL-2R revealed that it is of intermediate affinity and the median effective IL-2 concentration for PER-423 cells (EC50 value) was determined to be 1.44 +/- 0.29 nM. Chemical crosslinking studies showed that the receptor consists of one polypeptide of approximately 95 kDa as well as a doublet of 70 kDa and 60 kDa and does not include the IL-2R alpha-chain. The steady-state mRNA level for the p75 subunit was similar to that present in a cell line expressing an IL-2R alpha+ beta+, while only traces for the alpha-chain were detectable. PER-423 cells can be induced to express the alpha-chain of the IL-2R on the cell surface, concomitant with a much reduced EC50 level. Since cell line PER-423 is functionally dependent on IL-2, it provides an ideal model for IL-2 signal transduction studies and for investigations focusing on the requirements for ligand binding vs activation.

An immature human T cell line, PER-117, can be induced to secrete interleukin-2 (IL-2). In contrast to mature T cells or the Jurkat cell line, PER-117 cells require interleukin-1 (IL-1) for optimal IL-2 secretion, in addition to calcium ionophore and phorbol 12-myristate 12-acetate (PMA). These requirements mirror the conditions reported to be optimal for normal immature T cell receptor (TCR) negative thymocytes. IL-1 did not substitute for either of the other signals required for IL-2 production, i.e., calcium ionophore or PMA, suggesting that IL-1 activates pathways different from those elicited by the two other stimuli. For optimal effect, IL-1 needed to be provided at the same time as the two other signals. Significantly, a signal provided by a low concentration of PMA (not leading to IL-2 induction by itself in the presence of calcium ionophore) was necessary. The studies reported here provide the first evidence that three signals are required for optimal IL-2 production in a human immature T cell line. PER-117 cells produce substantial levels of IL-2 and thus provide a model to study stage-specific signal transduction and transcriptional activation of the IL-2 gene in IL-1 responsive immature thymocytes.

Cell line PER-117 is a T-cell receptor negative human T-cell line that can be induced to express a functional interleukin-2 receptor (IL-2R). Recombinant interleukin-1 (IL-1) as well as certain combinations of inducer substances could be shown to stimulate the expression of the p55 (alpha)-chain of the IL-2R in PER-117 cells. The synergistic increases in IL-2R alpha expression were demonstrated at the cell surface as well as at the mRNA level. The results suggested that in PER-117 cells IL-1 appears to induce expression of the alpha-chain by pathways that are different to activation via protein kinase C (PKC), and that drug-induced cyclic AMP (cAMP) activation did not substitute for IL-1. We found that the regulation of mRNA for IL-2R beta (p75) differed significantly from that seen for IL-2R alpha. Moreover, the requirements for IL-2R alpha induction determined for this cell line differ from other human cell lines, which may reflect that there are distinct requirements for activation depending on the stage of differentiation and/or lineage of the cells. The PER-117 cell line provides a unique model to examine further the mechanism leading to induction of a functional IL-2R at an early stage of human T-cell differentiation.

The metabolism of steroid hormones has been investigated in 10 workers exposed to lead and in 10 non-exposed subjects to determine whether lead interferes with the first or second phase reactions of steroid hormone biotransformation, or both. In the exposed workers blood lead concentrations (PbB) ranged from 45 to 69 micrograms/100 ml; in the controls PbB was less than 25 micrograms/100 ml. No statistical differences were found for the total amount of the urinary hormone metabolites, but a drop of about 50% was observed for the sulphated portion. It is suggested that lead interferes with the mechanisms of sulphoconjugation through an effect on the cytosol enzymes sulphotransferase and sulphokinase.

After reviewing the principal studies on the interferences of lead on the endocrine system, this paper reports the results of a survey carried out on 30 subjects in which the effects of lead on serum FSH, LH and testosterone and on urinary steroid hormones excretion were examined. Statistically significant differences were not demonstrated between lead exposed subjects and control group for serum LH and testosterone (total and free) and for urinary steroids. A significant increase of FSH has been detected in lead exposed workers even if this increase remains between the "normal limits" of the laboratory which performed the determinations. The results are discussed in the light of the more recent knowledge about the endocrine effects of xenobiotics in general and heavy metals in particular and some interpretations to justify the detected FSH increase are proposed.