Design of short peptides to block BTLA/HVEM interactions for promoting anticancer T-cell responses.

Antibody based immune-checkpoint blockade therapy is a major breakthrough in oncology, leading to clinical benefit for cancer patients. Among the growing family of inhibitory receptors, the B and T lymphocyte attenuator (BTLA), which interacts with herpes virus entry mediator (HVEM), is a promising target for immunotherapy. Indeed, BTLA inhibits T-cell proliferation and cytokine production. The crystal structure of the BTLA/HVEM complex has shown that the HVEM(26-38) fragment is directly involved in protein binding. We designed and analyzed the capacity of several analogs of this fragment to block the ligation between BTLA and HVEM, using competitive ELISA and cellular assay. We found that the HVEM(23-39) peptide can block BTLA/HVEM ligation. However, the blocking ability was due to the Cys encompassed in this peptide and that even free cysteine targeted the BTLA protein and blocked its interaction with HVEM. These data highlight a Cys-related artefact in vitro, which should be taken in consideration for future development of BTLA/HVEM blocking compounds.

Characteristics of C-terminal, ß-amyloid peptide binding fragment of neuroprotective protease inhibitor, cystatin C.

Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aß and the hCC-induced inhibition of Aß fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C-terminal fragment hCC(101-117) was identified as the Aß-binding region. The fragment of Aß peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aß(17-24). Structures and affinities of both Aß and hCC binding sites were characterized by the enzyme-linked immunosorbent assay-like assay, by surface plasmon resonance, and by nano-ESI-FTICR MS of the hCC-Aß-binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101-117), revealed the highest relative inhibitory effect toward Aß-fibril formation. Herein, we present further studies on molecular details of the hCC-Aß complex. With Ala substitution, affinity experiments, and enzyme-linked immunosorbent assay-like assays for the Aß-binding fragment, hCC(101-117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101-117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd.

The association of bone turnover markers with pro- and anti-inflammatory adipokines in patients with gestational diabetes.

OBJECTIVE: The aim of the study was to assess differences in circulating osteocalcin (OC) and osteoprotegerin (OPG), as well as in their expression in subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and placental tissue obtained from patients with gestational diabetes mellitus (GDM) and normal glucose tolerance (NGT). MATERIALS AND METHOD: Serum levels of OC, OPG and soluble nuclear factor-kB ligand (sRANKL) were measured in 49 women with GDM and 30 subjects with NGT between weeks 24-32 of gestation, and three months after childbirth. OC and OPG mRNA expression was measured in 23 patients with GDM and 23 women with NGT at term, using quantitative real-time RT-PCR. RESULTS: The patients with GDM had decreased OC mRNA expression in SAT (p=0.015), lower adiponectin mRNA expression in VAT (p=0.039), and a lower circulating adiponectin level (p=0.04). Multiple regression analysis revealed that serum adiponectin was significantly associated with OC mRNA expression in SAT (b=0.49, p=0.03). Three months postpartum, the OPG/sRANKL ratio was markedly higher in the subjects with prior GDM (p=0.03) and correlated positively with HbA1c (R=0.33; p=0.04), fasting insulin (R=0.35; p=0.03) and HOMA-IR (R=0.34; p=0.04). CONCLUSIONS: In the patients with GDM decreased OC mRNA expression in SAT might be associated with a reduced stimulatory effect on adiponectin expression in adipose tissue. On the other hand, higher OPG/sRANKL ratio suggests a better protection against bone loss in the subjects with prior GDM.

The expression of suppressor of cytokine signaling 1 and 3 in fat and placental tissue from women with gestational diabetes.

The suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of signaling pathways induced by cytokines, hormones and growth factors. In the present study we measured the expression of SOCS1, SOCS3, interleukin-6 (IL-6), IL-6 receptor, IL-8 and leptin mRNA in paired samples of subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and placental tissue obtained from 18 pregnant women with normal glucose tolerance (NGT) and 20 subjects with gestational diabetes mellitus (GDM), using quantitative RT-PCR. The patients with GDM had significantly higher IL-8 mRNA expression in VAT than the women with NGT (p = 0.007), whereas the expression of SOCS1, SOCS3 and other genes study did not differ significantly between the two groups. Stepwise regression analysis revealed that SOCS1 mRNA expression in VAT was significantly associated with prepregnancy BMI (ß = -0.68, p = 0.03) and IL-8 mRNA expression (ß = 0.66, p = 0.03), whereas SOCS3 mRNA expression in VAT was independently predicted by IL-6 mRNA expression (ß = 0.94, p = 0.0002, R(2) = 0.88). In conclusion, our results did not show significant differences in SOCS1 and SOCS3 mRNA expression in adipose and placental tissue obtained from pregnant women with and without GDM.

The expression of transcription factor 7-like 2 (TCF7L2) in fat and placental tissue from women with gestational diabetes.

In the present study we showed that the expression of transcription factor 7-like 2 (TCF7L2) mRNA in visceral adipose tissue obtained from 20 women with gestational diabetes was lower than in 18 pregnant women with normal glucose tolerance (p = 0.02), however after adjusting for BMI values, the difference was not significant.