Inflammatory Blood Biomarkers Are Associated with Long-Term Clinical Disease Severity in Parkinson's Disease.

An altered immune response has been identified as a pathophysiological factor in Parkinson's disease (PD). We aimed to identify blood immunity-associated proteins that discriminate PD from controls and that are associated with long-term disease severity in PD patients. Immune response-derived proteins in blood plasma were measured using Proximity Extension Technology by OLINK in a cohort of PD patients (N = 66) and age-matched healthy controls (N = 52). In a selection of 30 PD patients, we evaluated changes in protein levels 7-10 years after the baseline and assessed correlations with motor and cognitive assessments. Data from the Parkinson's Disease Biomarkers Program (PDBP) cohort and the Parkinson's Progression Markers Initiative (PPMI) cohort were used for independent validation. PD patients showed an altered immune response compared to controls based on a panel of four proteins (IL-12B, OPG, CXCL11, and CSF-1). The expression levels of five inflammation-associated proteins (CCL23, CCL25, TNFRSF9, TGF-alpha, and VEGFA) increased over time in PD and were partially associated with more severe motor and cognitive symptoms at follow-up. Increased CCL23 levels were associated with cognitive decline and the APOE4 genotype. Our findings provide further evidence for an altered immune response in PD that is associated with disease severity in PD over a long period of time.

Modulation of the Tumor Microenvironment by Ellagic Acid in Rat Model for Hepatocellular Carcinoma: A Potential Target against Hepatic Cancer Stem Cells.

The resistance to therapy and relapse in hepatocellular carcinoma (HCC) is highly attributed to hepatic cancer stem cells (HCSCs). HCSCs are under microenvironment control. This work aimed to assess the systemic effect of ellagic acid (EA) on the HCC microenvironment to decline HCSCs. Fifty Wistar rats were divided into six groups: negative control (CON), groups 2 and 3 for solvents (DMSO), and (OVO). Group 4 was administered EA only. The (HCC-M) group, utilized as an HCC model, administered CCL4 (0.5 mL/kg in OVO) 1:1 v/v, i.p) for 16 weeks. HCC-M rats were treated orally with EA (EA + HCC) 50 mg/kg bw for five weeks. Biochemical, morphological, histopathological, and immunohistochemical studies, and gene analysis using qRT-PCR were applied. Results revealed elevated liver injury biomarkers ALT, AST, ALP, and tumor biomarkers AFP and GGT, and marked nodularity of livers of HCC-M. EA effectively reduced the biomarkers and restored the altered structure of the livers. At the mRNA level, EA downregulated the expression of TGF-α, TGF-ß, and VEGF, and restored p53 expression. This induced an increase in apoptotic cells immunostained with caspase3 and decreased the CD44 immunostained HCSCs. EA could modulate the tumor microenvironment in the HCC rat model and ultimately target the HCSCs.

Efficacy of Supra-HFR in Removing FGF23 and Cytokines: A Single Session Analysis.

BACKGROUND/AIM: Supra hemodiafiltration with reinfusion of the endogenous ultrafiltrate (Supra-HFR) is a dialysis technique used to improve uremic toxin removal in the range of the middle molecular weight molecules. Supra-HFR does not require the preparation and online infusion of high-purity dialysis water because it allows the production of an endogenous ultrafiltrate that undergoes detoxification through an adsorbing resin. PATIENTS AND METHODS: We investigated the ability of Supra-HFR to remove fibroblast growth factor 23 (FGF23), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 8 (IL-8), and transforming growth factor alpha (TGF-alpha) after a single session dialysis in nine patients affected by end stage renal disease (ESRD). The same patients underwent a single session of online hemodiafiltration (OL-HDF) to evaluate possible differences in FGF23 and IL-6 levels. RESULTS: A significant reduction in FGF23 was observed with both Supra-HFR (p=0.001) and OL-HDF. As for TNF-alpha and TGF-alpha, which were measured using Supra-HFR only, their percentage values were significantly lower at the end of dialysis than at the start (p=0.0028 and p=0.03, respectively). This did not change with post-dialysis rebound. Supra-HFR was found to have no effect on IL-6 and IL-8. Interestingly, the removal rate for FGF23 and IL-6 was similar to that observed with OL-HDF. CONCLUSION: Supra-HFR was not superior to OL-HDF, with suboptimal convective volume in the removal of the molecules tested, especially FGF23, which is considered a large middle molecular weight uremic toxin.

Tetraspanin 6 is a regulator of carcinogenesis in colorectal cancer.

Early stages of colorectal cancer (CRC) development are characterized by a complex rewiring of transcriptional networks resulting in changes in the expression of multiple genes. Here, we demonstrate that the deletion of a poorly studied tetraspanin protein Tspan6 in Apcmin/+ mice, a well-established model for premalignant CRC, resulted in increased incidence of adenoma formation and tumor size. We demonstrate that the effect of Tspan6 deletion results in the activation of EGF-dependent signaling pathways through increased production of the transmembrane form of TGF-α (tmTGF-α) associated with extracellular vesicles. This pathway is modulated by an adaptor protein syntenin-1, which physically links Tspan6 and tmTGF-α. In support of this, the expression of Tspan6 is frequently decreased or lost in CRC, and this correlates with poor survival. Furthermore, the analysis of samples from the epidermal growth factor receptor (EGFR)-targeting clinical trial (COIN trial) has shown that the expression of Tspan6 in CRC correlated with better patient responses to EGFR-targeted therapy involving Cetuximab. Importantly, Tspan6-positive patients with tumors in the proximal colon (right-sided) and those with KRAS mutations had a better response to Cetuximab than the patients that expressed low Tspan6 levels. These results identify Tspan6 as a regulator of CRC development and a potential predictive marker for EGFR-targeted therapies in CRC beyond RAS pathway mutations.

NRF2 Enables EGFR Signaling in Melanoma Cells.

Receptor tyrosine kinases (RTK) are rarely mutated in cutaneous melanoma, but the expression and activation of several RTK family members are associated with a proinvasive phenotype and therapy resistance. Epidermal growth factor receptor (EGFR) is a member of the RTK family and is only expressed in a subgroup of melanomas with poor prognosis. The insight into regulators of EGFR expression and activation is important for the understanding of the development of this malignant melanoma phenotype. Here, we describe that the transcription factor NRF2, the master regulator of the oxidative and electrophilic stress response, mediates the expression and activation of EGFR in melanoma by elevating the levels of EGFR as well as its ligands EGF and TGFα. ChIP sequencing data show that NRF2 directly binds to the promoter of EGF, which contains a canonical antioxidant response element. Accordingly, EGF is induced by oxidative stress and is also increased in lung adenocarcinoma and head and neck carcinoma with mutationally activated NRF2. In contrast, regulation of EGFR and TGFA occurs by an indirect mechanism, which is enabled by the ability of NRF2 to block the activity of the melanocytic lineage factor MITF in melanoma. MITF effectively suppresses EGFR and TGFA expression and therefore serves as link between NRF2 and EGFR. As EGFR was previously described to stimulate NRF2 activity, the mutual activation of NRF2 and EGFR pathways was investigated. The presence of NRF2 was necessary for full EGFR pathway activation, as NRF2-knockout cells showed reduced AKT activation in response to EGF stimulation compared to controls. Conversely, EGF led to the nuclear localization and activation of NRF2, thereby demonstrating that NRF2 and EGFR are connected in a positive feedback loop in melanoma. In summary, our data show that the EGFR-positive melanoma phenotype is strongly supported by NRF2, thus revealing a novel maintenance mechanism for this clinically challenging melanoma subpopulation.

Human blood serum can donor-specifically antagonize effects of EGFR-targeted drugs on squamous carcinoma cell growth.

Many patients fail to respond to EGFR-targeted therapeutics, and personalized diagnostics is needed to identify putative responders. We investigated 1630 colorectal and lung squamous carcinomas and 1357 normal lung and colon samples and observed huge variation in EGFR pathway activation in both cancerous and healthy tissues, irrespectively on EGFR gene mutation status. We investigated whether human blood serum can affect squamous carcinoma cell growth and EGFR drug response. We demonstrate that human serum antagonizes the effects of EGFR-targeted drugs erlotinib and cetuximab on A431 squamous carcinoma cells by increasing IC50 by about 2- and 20-fold, respectively. The effects on clonogenicity varied significantly across the individual serum samples in every experiment, with up to 100% differences. EGF concentration could explain many effects of blood serum samples, and EGFR ligands-depleted serum showed lesser effect on drug sensitivity.

Study of the biologic behavior of odontogenic keratocyst and orthokeratinaized odontogenic cyst using TGF-alpha and P53 markers.

Odontogenic keratocyst (OKC) is an aggressive cyst, and its recurrence rate is higher than that of other odontogenic cysts. Orthokeratinized odontogenic cyst (OOC) is less aggressive than OKC, but bears the probability of carcinomatous changes. In this study, we evaluated the expression and intensity of P53 and TGF-alpha in order to compare the biologic behavior or probable carcinomatous changes of these two cysts. In this cross-sectional study, 15 OKC and 15 OOC were stained immunohistochemically for P53 and TGF-alpha using the Novolink polymer method. Then, all slides were examined by an optical microscope with 400× magnification, and the stained cells in the basal and parabasal layers were counted. Finally, the results were analyzed by the Mann-Whitney and Wilcoxon tests (P-value<0.05). The difference between the expression of P53 and TGF alpha in the basal layer of OKC and OOC was not statistically significant (P-value>0.05), but the expression of P53 and TGF-alpha in the parabasal layer in OKC was statistically higher compared to OOC (P<0.05). Considering the known role of P53 and TGF-alpha in malignant changes and the higher expression of P53 and TGF-alpha in OKC compared to those in OOC, the probability of carcinomatous changes was higher in OKC than in OOC.

Submandibular glands and the regulation of gastric proliferation and TGFα distribution during rat postnatal development

Rodent gastric mucosa grows and differentiates during suckling-weaning transition. Among the molecules in rat milk, EGF and TGFβ are important peptides in the control of cell proliferation, and together with TGFα, they are also produced by submandibular glands. We aimed to determine the effect of saliva and milk on epithelial cell proliferation in the stomach of rat pups. We also examined the distribution of TGFα in the gastric mucosa after sialoadenectomy (SIALO) and fasting in order to determine whether this growth factor is affected by the deprivation of molecules derived from saliva and milk. SIALO was performed at 14 days and fasting was induced 3 days later. Cell proliferation was evaluated through metaphasic index and TGFα was detected by immunohistochemistry. We observed that whereas SIALO did not alter cell division, since the metaphasic index (MI) was unchanged, fasting stimulated cell proliferation (P < 0.05). After SIALO and fasting, MI was reduced when compared to the fasted group (P < 0.05). We found that TGFα is distributed along gastric gland and SIALO did not interfere in the localization and number of immunolabeled cells, but fasting increased their density when compared to the control (P < 0.05). The association of SIALO and fasting reduced TGFα immunostaining (P < 0.05). Therefore, during fasting, high MI was parallel to increased TGFα in gastric epithelium, but interestingly, this effect was found only in the presence of submandibular glands. We suggest that during suckling, peptides derived from saliva and milk are important to regulate gastric growth.

Comparative study of TGF-alpha and P53 markers' expression in odontogenic keratocyst and orthokeratinaized odontogenic cyst.

BACKGROUND: Odontogenic keratocyst (OKC) is an aggressive cyst and its recurrence is higher than other odontogenic cysts, orthokeratinized odontogenic cyst (OOC) is a cyst with moderate biological behavior in comparison with OKC, but with the probability of carcinomatous changes. The present study aims to evaluate the quantity and intensity of the expression of P53 protein and transforming growth factor alpha (TGF-alpha) in OKC and OOC in order to compare the biologic behavior of these two cysts. MATERIALS AND METHODS: This is a cross-sectional study. The samples include 30 cysts (15 OKC and 15 OOC), all stained immunohistochemically for P53 protein and TGF-alpha by the Novolinke polymer method. Then, all the cases were examined with an optical microscope with Χ400 magnification and the stained cells were counted in the basal and parabasal layers. Finally the results were analyzed by the Mann-and Wilcoxon tests (P value < 0.05). RESULTS: The difference between the expression of P53 protein in the basal layer in OKC and OOC was not statistically significant (P value = 0.076). The difference between the expression of P53 protein in the parabasal layer in OKC and OOC was statistically significant (P value = 0.003); moreover, the difference between the expression of TGF-alpha in the basal layer in OKC and OOC was not statistically significant (P value = 0.284). The difference between the expression of TGF-alpha in the parabasal layer in OKC and OOC was statistically significant (P value = 0.015). CONCLUSION: Since there was a higher expression of P53 protein and TGF-alpha in OKC compared to those in OOC, the probability of carcinomatous changes was at least theoretically higher in OKC than in OOC.