CRP not only correlated with NYHA, but also with the two BNP and NT-proBNP following depletion, as schematically presented on Determine 7.In this study, for the very first time, we shown that efficient immuno-depletion of the precursor proBNP1-108 (up to 96% of theTRH Acetate circulating proBNP1-108) revealed a crystal clear cross-reactivity of proBNP in BNP assays and in a lesser extent of NTproBNP in schedule scientific assays in HF management. The BNP ranges are overestimated by additional than 50% in absence of depletion of proBNP1-108. Next, we confirm that intact proBNP1-108 is correlated to HF phase and raises in situation of renal impairment (as well as BNP or NT-proBNP). CRP seems to be correlated the two to purposeful classes and natriuretic peptides soon after depletion justifies to be stated, as this correlation is not always so strong in latest scientific tests [eleven], and as this could explain -at least partly- the progression of the ailment [twelve]. From a medical position-of-view, the simultaneous measurement of two peptides symbolizing on the one particular hand Figure three. Correlations among BNP and NTproBNP assays just before and soon after depletion of proBNP1-108. A: BNP. Correlation in between the two assays is extremely powerful, in spite of the deep reduction of the values: n=178, r=.ninety seven p<0.0001. B: NT-proBNP. Correlation between the two assays is very strong, in spite of the mild reduction of the values N=178, r=0.99 p<0.0001.Figure 5. Distribution according to NYHA classes of: BNP before depletion (A), after depletion (B) or NTproBNP before depletion (C), after depletion (D), and proBNP1-108 before depletion (E). All of these Natriuretic peptides are strictly correlated with the functional classes, with p<0.001 (specific values after Kruskal-Wallis analyses are indicated).Figure 7. Correlations between CRP, NYHA and BNP or NTproBNP after depletion of proBNP1-108. A: CRP is correlated with NYHA. CRP levels increase significantly when the NYHA class becomes higher (P<0.0001). B: BNP after depletion is correlated with CRP levels. BNP levels are considered in quartiles. P=0.0003. C: NT-proBNP after depletion is correlated with CRP levels. BNP levels are considered in quartiles. P<0.0001.Figure 6. Distribution according to renal function (estimated by CKD-EPI) of: BNP before depletion (A), after depletion (B) or NTproBNP before depletion (C), after depletion (D), and proBNP1-108 before depletion (E). All of these Natriuretic peptides are strictly correlated with the renal function, with p<0.001 (specific values after Kruskal -Wallis analyses are indicated)the biologically inactive intact precursor proBNP1-108 and on the other hand the active derived BNP peptide, could allow clinicians to follow better the status of the patient, especially in pathological conditions. For instance, in ambulatory patients with chronic HF, combined assessment of BNP and proBNP provided additional information with regards to risk of adverse clinical outcomes [13]. In these patients, this combined measurement was particularly promising in patients with low levels of BNP, in which the clinicians could be falsely reassured. Clinical evaluation of combinations of highly specific assays for the assessment of BNP peptides are still needed [14] especially in patients with chronic renal failure since the half-life of the 3 peptides and/or their respective renal clearance is poorly understood [15]. Secondly, our findings rise concerns about the currently available methods for assessing natriuretic peptides: either to encourage more specific methods, such as the new process presented here, or to redefine cut-off values (especially as regards the "grey zone" even if this was not specifically the point of interest in the present study), taking into account the lack of specificity of the currently used methods. Dries et al showed indeed that simultaneous assessment of unprocessed proBNP1-108 in addition to processed BNP32 could improve identification of high-risk ambulatory patients with HF [13]. Our method could provide similar information, so that clinical implications deserve to be clarified. Surprisingly, the clinical signification of BNP overestimation remains unclear as the sample pre-treatment technique did not allow us to better stratify the HF patients and did not contribute to a better patient management care. At least two reasons could be proposed. First, the two different methods for assessment of peptides before or after depletion seem to provide similar clinical information on this HF cohort of patients, as suggested by the very strong correlations between them (at least for BNP). Secondly, BNP peptides are already powerful tools to stratify HF patients, so that it could be difficult to improve significantly the clinical meaning in a small population or in a real-life population: only class III patient for instance should have been included to get a more specific population with better predefined endpoints, and a long follow-up. This hypothesis is consistent with the study on a specific not commercially available test in a large cohort [16]. Disappointingly, although proBNP1-108 was a sensitive (78.8%) and specific (86.1%) biomarker for detecting left ventricular systolic dysfunction, this biomarker was comparable but not better than BNP and was even less informative than NT-proBNP1-76 [16], at least in this HF cohort. In addition, in a recent cohort of 187 Class III-IV HF patients, pro-BNP1-108, standard assay BNP and troponin T (cTnT) were investigated in relation to the primary endpoint of death or cardiac transplantation [17]. Both elevated proBNP1-108 and BNP were associated with increased clinical events. More importantly, elevated levels of proBNP1-108 or BNP identified by serial monitoring similarly predicted events. The second surprising result of this study is that no significant benefit for immunodepletion could be observed on the NT-proBNP molecule measurement itself (5% decrease for NT-proBNP1-76 versus 50% decrease for BNP). Knowing that an equimolar ratio is expected in the proBNP1-108 cleavage by furin/ corin, one could guess to observe a significant decrease in the NT-proBNP1-76 molecule while using the immunodepletion method. To understand this surprising effect, we recognized that O-glycosylated forms of proBNP in heart failure patients were reported, along with uncleaved proBNP [8,18-20] all reported an overestimation of BNP1-32 and NT-proBNP1-76 due to the cross-reactivity of antibodies towards the proBNP1-108. We therefore hypothesized that the hyperglycosylation of proBNP 1-108at the 1-76 part [18,19,21] might prevent the accessibility of the anti-hinge antibody during the immunodepletion process and therefore decrease its efficiency. We therefore suggest verifying that by using O-glycosidases, neuraminidase and galactosidase enzymes [21], the immunodepletion will stay the same or will benefit from a better access of the anti-hinge antibody. Furthermore, this limited cross-reactivity of the NT-proBNP assay with the proBNP could result from the impact of glycosylation on the detection of NTproBNP: NT-proBNP could be underdetected because of the glycosylation of the NT-proBNP [22], which could prevent the antibodies from an accurate recognition. These findings demonstrate that the "BNP" which is supposed to be measured in various clinical settings is not really the true BNP pool Only few publications have already showed the cross-reactivity among natriuretic peptide assays. Luckenbill et al studied pooled samples of healthy donors with established concentrations and demonstrated that BNP assays crossreact with NT-proBNP or proBNP, and calculated the various differences between 5 commercial BNP and 3 commercial NT-proBNP assays [8]. Hawkridge et al, used an immunoaffinity purification assay to isolate endogenous BNP specifically in the plasma of 4 patients ranged NYHA class IV dedicated to subsequent analysis by nano-liquid chromatography, to evidence the absence of circulating BNP in advanced-stage HF patients and they suggested the existence of altered forms of BNP [23]. Here, in a real-life cohort of 180 patients with HF, we established a similar cross-reactivity corroborating that the true BNP pool could represent less than 50% of what is measured by a currently used commercial assay. Furthermore, it could explain at least partly discrepancies among commercial kits, as specificity could vary from one to another, hence a lack of alignment and difficulties to follow patients when different commercial assays are run in different laboratories.These findings have to be confirmed in a larger population, although this cohort of 180 patients with HF represents a reallife situation. Secondly, it will be of critical importance to test different platforms to demonstrate the general impact of proBNP depletion on BNP levels. Here is provided first technical proof of concept of the impact of proBNP depletion on BNP testing in a pilot study. In this population, taking into consideration the cross reactivity did not modify the classification of patients. It remains unclear whether considering these true values simply drives all values to 50% lower levels or on the contrary could elicit a profound impact in the classification for specific patients (BNP grey zone, renal impairment, elderly etc). Further larger studies will address this concern. Finally, it could be suggested that since diagnostic and prognostic performances of BNP and NT-proBNP are roughly similar, if NT-proBNP measurement is not affected by the presence of proBNP, only NT-proBNP and not BNP could be suggested to be used. Nevertheless, as performances are similar, our results could not by themselves support such conclusions. They should on the contrary suggest to develop more specific assays to better understand what is currently assayed (other interactions could obviously be searched as regards NT-proBNP as previously discussed) and propose perhaps more accurate cut-offs.Routinely used BNP assays are not specific to BNP molecule but also cross react with the proBNP1-108 precursor. By using an immunodepletion method, we were able to really assess the true BNP measurement. Assessing accurately each of the three major natriuretic peptides could be of interest to better characterize patients and to address appropriate therapy especially in case of decompensated heart failure patients. This could open new fields of interest to better understand unclear medical situations and above all to enlighten the grey zone, in order to better stratify patients and to perform a better drug monitoring. Clinical implications of these new methods remain to be investigated.Classical HH signaling initiates when the soluble HH ligands, Sonic (SHH), Desert (DHH) or Indian (IHH) HH bind their transmembrane receptor Patched (Ptch), thereby releasing the transmembrane protein, Smoothened (Smo) from Ptch-mediated inhibition. Smo subsequently activates the GLI family of transcription factors that regulate HH target genes. The GLI family of transcription factors includes GLI1, GLI2 and GLI3. By virtue of a C-terminal activator and N-terminal repressor domains, GLI2 and GLI3 have context-dependent activator or repressor activity. GLI1 lacks the repressor domain and functions predominantly as an activator [1,2]. GLI2 has a C-terminal activator and N-terminal repressor domains [3]. GLI2 is reported to be the initial mediator of HH signaling events, which then induces the expression GLI1, which further increases HH target gene expression [4]. When the HH signaling pathway is active, the latent cytoplasmic GLI proteins translocate to the nucleus where they bind the GACCACCCA-like elements on the promoters of the HH-target genes [5,6]. HH signaling regulates cellular events by modulating specific target genes. During normal embryonic development, HH signaling activity is essential, being regulated spatially and temporally resulting in normal tissue patterning and differentiation. Coordinated HH signaling is also involved in cellular proliferation and survival, maintenance of stemness and determination of cell fate [6]. Aberrantly activated HH signaling is involved in multiple human cancers and it regulates cancer cell proliferation, survival, cancer stem cell functions, epithelial to mesenchymal transition and metastasis [6]. We have reported that HH signaling is critical for the survival of human colon cancer cells, while blocking these signals induces rapid DNA damage, culminating in extensive cytotoxicity [7,8,9,10]. Unlimited replication potential of cancer cells is closely associated with cancer cell survival, however, the role of HH signaling in the replication potential of cancer cells is not known. Replication potential of human somatic cells is limited by special heterochromatic structures known as telomeres at the ends of linear chromosomes [11]. Mammalian telomeres are comprised of tandem repeats of TTAGGG sequences that are subjected to shortening with every DNA replication cycle [12]. Conventional DNA polymerases are not capable of fully replicating the ends of linear DNA molecules hence, telomeric DNA is expected to shorten with every DNA replication cycle. Critically shortened telomeres fail to protect chromosomal ends resulting in irreversible growth arrest and limited cellular lifespan. Hence, telomere homeostasis is critical for cell proliferation and survival. Telomerase, a ribonucleoprotein comprised of a RNA component (TR) and a reverse transcriptase catalytic subunit (TERT), replenishes the telomere repeats and hence regulates cellular replicative potential [13]. 24900421In most adult cells, TR is constitutively present but TERT expression is repressed, resulting in limited proliferation potential and cellular life span [14,15]. In actively proliferating cells such as stem cells and cancer cells, TERT expression is upregulated resulting in unlimited replicative potential and immortality of these cells [16]. Human TERT (hTERT) expression and activity has been evidenced in .75% of human colorectal cancer cells, but only 35% of normal mucosa and surrounding non-cancerous cells [17]. In concert with its importance in cancer cell survival, hTERT is stringently regulated with multiple activators and repressors, of which several have been identified. Here we demonstrate for the first time that HH signaling trancriptionally upregulates hTERT. Suppression of GLI1/GLI2 reduced hTERT protein levels in human colon, prostate and brain cancer cells. Overexpression of GLI2DN increased the levels of hTERT mRNA, protein and hTERT promoter-driven luciferase (luc) activity in colon cancer cells. Blocking GLI1/2 activity reduced hTERT mRNA expression and the direct interaction between GLI1/GLI2 proteins and the hTERT promoter in human colon cancer cells. In contrast, GLI1/GLI2DN expression in non-cancerous 293T cells did not alter the levels of hTERT mRNA, protein or hTERT promoter-luc activity. Abrogating HH signaling in cancer cells decreased the telomerase activity, which was increased by GLI2DN expression.
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