Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging.

BACKGROUND: Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging. METHODS: We designed a 13 kDa ß-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1. RESULTS: Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors. CONCLUSIONS: Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.

IL-6 and its role in IgA nephropathy development.

IL-6 is considered one of the well characterized cytokines exhibiting homeostatic, pro- and anti-inflammatory activities, depending on the receptor variant and the induced intracellular cis- or trans-signaling responses. IL-6-activated pathways are involved in the regulation of cell proliferation, survival, differentiation, and cell metabolism changes. Deviations in IL-6 levels or abnormal response to IL-6 signaling are associated with several autoimmune diseases including IgA nephropathy (IgAN), one of most frequent primary glomerulonephritis worldwide. IgAN is associated with increased plasma concentration of IL-6 and increased plasma concentration of aberrantly galactosylated IgA1 immunoglobulin (Gd-IgA1). Gd-IgA1 is specifically recognized by autoantibodies, leading to the formation of circulating immune complexes (CIC) with nephritogenic potential, since CIC deposited in the glomerular mesangium induce mesangial cells proliferation and glomerular injury. Infection of the upper respiratory or digestive tract enhances IL-6 production and in IgAN patients is often followed by the macroscopic hematuria. This review recapitulates general aspects of IL-6 signaling and summarizes experimental evidences about IL-6 involvement in the etiopathogenesis of IgA nephropathy through the production of Gd-IgA1 and regulation of mesangial cell proliferation.

Glucocorticoids Reduce Aberrant O-Glycosylation of IgA1 in IgA Nephropathy Patients.

BACKGROUND/AIMS: IgA nephropathy is associated with aberrant O-glycosylation of IgA1, which is recognized by autoantibodies leading to the formation of circulating immune complexes. Some of them, after deposition into kidney mesangium, trigger glomerular injury. In patients with active disease nonresponding to angiotensin-converting enzyme inhibitors or angiotensin II blockers, corticosteroids are recommended. METHODS: The relationship between the corticosteroid therapy and serum levels of IgA, aberrantly O-glycosylated IgA1, IgA-containing immune complexes and their mesangioproliferative activity was analyzed in IgA nephropathy patients and disease and healthy controls. RESULTS: Prednisone therapy significantly reduced proteinuria and levels of serum IgA, galactose-deficient IgA1, and IgA-IgG immune complexes in IgA nephropathy patients and thus reduced differences in all of the above parameters between IgAN patients and control groups. A moderate but not significant reduction of mesangioproliferative potential of IgA-IgG immune complexes and IgA sialylation was detected. CONCLUSION: The prednisone therapy reduces overall aberrancy in IgA1 O-glycosylation in IgA nephropathy patients, but the measurement of IgA1 parameters does not allow us to predict the prednisone therapy outcome in individual patients.

[IgA nephropathy - research-generated questions]. / IgA nefropatie - otázky, které nastolil výzkum.

IgA nephropathy (IgAN) is the most common type of glomerulonephritis. Its etiology involves an increased production of polymeric immunoglobulin A1 with an abnormal composition of some carbohydrate chains. The reaction of these abnormal forms of IgA1 with specific autoantibodies while circulating immune complexes arise and settle in the renal mesangium with subsequent inflammatory activation of mesangial cells which in up to 50% of cases results in end-stage kidney failure. Pathogenesis involves an interplay of genetic predisposition and environmental effects, mainly of microbial nature. Current therapy is not sufficiently effective and lacks the focus on the cause of the disease, therefore more efficient and specific ways of therapy are being sought to target the individual stages of the pathogenetic process of IgAN development. With the accumulation of knowledge, new questions arise, concerning detailed mechanisms of the pathological processes, as discussed in the text.Key words: autoimmunity - glycosylation of IgA hinge region - IgA nephropathy - immunoglobulin IgA - IgA1 hinge region.

Origin of galactose-deficient immunoglobulin g in gingival crevicular fluid in periodontitis.

BACKGROUND: Periodontitis is a chronic inflammatory disease initiated by a synergistic and dysbiotic microbial community that elicits a gingival inflammatory response leading to tissue breakdown. Periodontitis shares many characteristics with other chronic inflammatory diseases, including abnormal glycosylation of immunoglobulin (Ig)G. The current authors have previously demonstrated that IgG from gingival crevicular fluid (GCF) of patients with chronic periodontitis contains galactose (Gal)-deficient IgG. METHODS: The origin of the aberrantly glycosylated IgG was determined by measuring levels of Gal-deficient IgG in GCF and serum from patients with periodontitis and non-periodontitis controls using lectin enzyme-linked immunosorbent assay. The Ig-producing cells and the proportion of cells producing Gal-deficient IgG were immunohistochemically determined in gingival tissues from patients with periodontitis by fluorescence microscopy. The results were statistically evaluated and correlated with clinical data. RESULTS: The results indicate that GCF of patients with periodontitis had higher levels of Gal-deficient IgG compared with controls (P = 0.002). In gingival tissues, IgG was the dominant isotype among Ig-producing cells, and 60% of IgG-positive cells produced Gal-deficient IgG. Moreover, the proportion of Gal-deficient IgG-producing cells directly correlated with clinical parameters of probing depth and clinical attachment loss (AL). CONCLUSION: These results suggest that the presence of Gal-deficient IgG is associated with gingival inflammation and may play a role in the worsening of clinical parameters of periodontitis, such as AL.

Cell-cycle regulation in embryonic stem cells: centrosomal decisions on self-renewal.

Embryonic stem cells seem to have the intriguing capacity to divide indefinitely while retaining their pluripotency. This self-renewal is accomplished by specialized mechanisms of cell-cycle control. In the last few years, several studies have provided evidence for a direct link between cell-cycle regulation and cell-fate decisions in stem cells. In this review, we discuss the peculiarities of embryonic stem cell-cycle control mechanisms, implicate their involvement in cell-fate decisions, and distinguish centrosomes as important players in the self-renewal versus differentiation roulette.

DNA damage-induced degradation of Cdc25A does not lead to inhibition of Cdk2 activity in mouse embryonic stem cells.

Cyclin-dependent kinase two (Cdk2) is the major regulator of the G1/S transition and the target of an activated G1 checkpoint in somatic cells. In the presence of DNA damage, Cdk2 kinase activity is abrogated by a deficiency of Cdc25A phosphatase, which is marked by Chk1/Chk2 for proteasomal degradation. Embryonic stem cells (ESCs) lack a G1 checkpoint response. In this study, we analyzed the G1 checkpoint pathways in mouse ESCs (mESCs) in the presence of DNA double-strand breaks evoked by ionizing radiation (IR). We show that checkpoint pathways, which operate during G1 phase in somatic cells, are activated in mESCs after IR; however, Cdk2 activity is not abolished. We demonstrate that Cdc25A is degraded in mESCs, but this degradation is not regulated by Chk1 and Chk2 kinases because they are sequestered to the centrosome. Instead, Cdc25A degradation is governed by glycogen synthase kinase-3beta kinase. We hypothesize that Cdc25A degradation does not inhibit Cdk2 activity because a considerable proportion of Cdk2 molecules localize to the cytoplasm and centrosomes in mESCs, where they may be sheltered from regulation by nuclear Cdc25A. Finally, we show that a high Cdk2 activity, which is irresponsive to DNA damage, is the driving force of the rapid escape of mESCs from G1 phase after DNA damage.

Cdk2 inhibition prolongs G1 phase progression in mouse embryonic stem cells.

Embryonic stem cells (ESCs) proliferate rapidly and have a unique cell-cycle structure with a very short G1 phase. Previous reports suggested that the rapid G1 phase progression of ESCs might be underpinned by high and precocious Cdk2 activity and that Cdk2 activity might be crucial for both cell-cycle regulation and cell-fate decisions in human ESCs. However, the actual role of Cdk2 in cell-cycle progression of mouse ESCs (mESCs) has not been elucidated. In this study, we investigated the effects of down-regulation of Cdk2 activity by olomoucine II in 2 mESC lines. Olomoucine II treatment significantly increased the G1 phase cell numbers, decreased the S phase cell numbers, and inhibited DNA replication in mESCs. In nocodazole-synchronized mESCs, we show that specific down-regulation of Cdk2 activity prolongs G1 phase progression. In addition, down-regulation of Cdk2 activity in mESCs established a somatic cell-like cell cycle and induced expression of differentiation markers. Our results suggest that high Cdk2 activity is essential for rapid G1 phase progression and establishment of ESC-specific cell-cycle structure in mESCs and support the hypothesis of a link between cell-cycle regulation and pluripotency maintenance in ESCs. This study reveals olomoucine II to be an effective tool for manipulation of the cell cycle and pluripotency in ESCs and very likely also for the manipulation of other stem cell types, including cancer stem cells.