Comparison of Interleukin-33 Serum Levels in Patients with Breast Cancer and Idiopathic Granulomatous Mastitis.

BACKGROUND: Breast cancer (BC) is the main cause of cancer death in women. Idiopathic granulomatous mastitis (IGM), a rare chronic disease that clinically mimics breast carcinoma, and is associated with high mortality and morbidity, but an immediate and accurate diagnosis can substantially decrease these rates. Expressed by numerous human tissues, interleukin-33 (IL-33) has an inductive role in the network of pro-inflammatory cytokines. The aim of this study was to investigate the serum levels of IL-33 in BC and IGM patients in comparison with healthy women. MATERIALS AND METHODS: This descriptive-analytical study was carried out on 28 patients with BC and 25 patients with IGM as the patient groups and 25 healthy volunteers with normal screening reports as the control group. Histopathological pattern of BC and IGM were confirmed by specialized pathologists. The serum concentration of IL-33 was measured using enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. RESULTS: The mean age of the patients with BC and IGM and the control group was 49.1, 37.1, and 36.8 years, respectively. There was no significant difference in IL-33 expression among the participants with regard to age, marital status, body mass index (BMI), and menopausal status. IL-33 assay indicated a significant difference between the BC (P=0.011) and IGM (P=0.031) groups compared to the controls, although no substantial differences were observed between the IGM and BC groups. CONCLUSION: IL-33 can be considered a significant factor distinguishing IGM and BC patients from controls, although it cannot be applied to diagnose and differentiate BC from IGM patients.
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Inner retinal injury in experimental glaucoma is prevented upon AAV mediated Shp2 silencing in a caveolin dependent manner.

SH2 domain containing tyrosine phosphatase 2 (Shp2; PTPN11) regulates several intracellular pathways downstream of multiple growth factor receptors. Our studies implicate that Shp2 interacts with Caveolin-1 (Cav-1) protein in retinal ganglion cells (RGCs) and negatively regulates BDNF/TrkB signaling. This study aimed to investigate the mechanisms underlying the protective effects of shp2 silencing in the RGCs in glaucomatous conditions. Methods: Shp2 was silenced in the Cav-1 deficient mice and the age matched wildtype littermates using adeno-associated viral (AAV) constructs. Shp2 expression modulation was performed in an acute and a chronic mouse model of experimental glaucoma. AAV2 expressing Shp2 eGFP-shRNA under a strong synthetic CAG promoter was administered intravitreally in the animals' eyes. The contralateral eye received AAV-eGFP-scramble-shRNA as control. Animals with Shp2 downregulation were subjected to either microbead injections or acute ocular hypertension experimental paradigm. Changes in inner retinal function were evaluated by measuring positive scotopic threshold response (pSTR) while structural and biochemical alterations were evaluated through H&E staining, western blotting and immunohistochemical analysis of the retinal tissues. Results: A greater loss of pSTR amplitudes was observed in the WT mice compared to Cav-1-/- retinas in both the models. Silencing of Shp2 phosphatase imparted protection against inner retinal function loss in chronic glaucoma model in WT mice. The functional rescue also translated to structural preservation of ganglion cell layer in the chronic glaucoma condition in WT mice which was not evident in Cav-1-/- mice retinas. Conclusions: This study indicates that protective effects of Shp2 ablation under chronic experimental glaucoma conditions are dependent on Cav-1 in the retina, suggesting in vivo interactions between the two proteins.

Phosflow assessment of PDGFRA phosphorylation state: A guide for tyrosine kinase inhibitor targeted therapy in hypereosinophilia patients.

Clonal eosinophilia is a hematologic disorder caused by translocation in growth factor receptor (GFR) genes. Despite the identified molecular mechanisms underlying clonal hypereosinophilia, the distinction between clonal and reactive eosinophilia has remained challenging due to the diversity of partner genes for translocated GFRs. This study aimed to examine the feasibility of phosphoflow cytometry in the diagnosis of clonal hypereosinophilia through evaluating the level of platelet-derived growth factor receptor alpha (PDGFRA) phosphorylation and its correlation with PDGFRA genetic aberration. Blood samples were collected from 45 hypereosinophilia patients and 10 healthy controls. Using phosphoflow cytometry method, the phosphorylation state of PDGFRA was assessed. The specificity of phosflow results was confirmed by western blotting and eventually compared with qRT-PCR expression analysis of 3'-region of PDGFRA. To detect the genetic aberration of PDGFRA, 5'-rapid amplification of cDNA ends (5'-RACE) was performed. Phosflow analysis illustrated that 9 of 45 hypereosinophilic patients had higher level of PDGFRA phosphorylation while sequence analysis of 5'-RACE-PCR fragments confirmed that in seven cases of them, there was a PDGFRA-FIP1L1 fusion. We also verified that two of nine patients with hyperposphorylated PDGFRA hold ETV6-PDGFRA and STRN-PDGFRA rearrangements. Importantly, nine cases also had significantly higher levels of PDGFRA mRNA expression when compared with healthy controls, and cases with no PDGFRA rearrangement. These findings highlight a robust correlation between hyperphosphorylation state of PDGFRA and aberrant PDGFRA gene fusions. This implicates phosflow as an efficient and reliable technique raising an intriguing possibility that it could replace other genomic and cDNA-amplification-based diagnostic approaches with limited effectiveness.

GDF-15 negatively regulates excess erythropoiesis and its overexpression is involved in erythroid hyperplasia.

Growth differentiation factor-15 (GDF-15) is a member of TGF-ß superfamily. Among hematopoietic cells, this factor is mainly produced by erythroid series and is recently considered a biomarker of ineffective erythropoiesis (IE). Whether IE induces enhanced GDF-15 expression or is prompted by it, has remained elusive. In this study we investigated how high levels of GDF-15 contribute to IE-associated erythroid dysplasia. We assessed mRNA levels of GDF-15 during erythroid maturation as well as in patients with IE using qRT-PCR. Later, the erythroid colony-forming capacity of GDF-15-treated hematopoietic stem cells (HSCs) was evaluated by CFC assay. Any effect of elevated levels of GDF-15 on erythroid maturation was ultimately examined by expression analysis of erythroid-associated transcription factors and flow cytometry analysis of CD235a expression. GDF-15 mRNA expression increased during erythroid differentiation and also in ß-thalassemia and MDS patients which was directly correlated with erythropoiesis severity. Treating the cells with high GDF-15 concentration (50 ng/ml) resulted in an approximate 30% decline in the capacity of erythroid colony formation of HSCs and CD235a positive cells. Additionally, erythroid-specific transcription factors showed significant down-regulation in the early stages of erythroid differentiation. According to the expression level of GDF-15 and the role it plays in the erythroid system, high-levels of this factor could be an auto-modulatory mechanism to control the excessive production of erythroid cells.

Loss of Shp2 Rescues BDNF/TrkB Signaling and Contributes to Improved Retinal Ganglion Cell Neuroprotection.

Glaucoma is characterized by the loss of retinal ganglion cells (RGC), and accordingly the preservation of RGCs and their axons has recently attracted significant attention to improve therapeutic outcomes in the disease. Here, we report that Src homology region 2-containing protein tyrosine phosphatase 2 (Shp2) undergoes activation in the RGCs, in animal model of glaucoma as well as in the human glaucoma tissues and that Shp2 dephosphorylates tropomyosin receptor kinase B (TrkB) receptor, leading to reduced BDNF/TrkB neuroprotective survival signaling. This was elucidated by specifically modulating Shp2 expression in the RGCs in vivo, using adeno-associated virus serotype 2 (AAV2) constructs. Shp2 upregulation promoted endoplasmic reticulum (ER) stress and apoptosis, along with functional and structural deficits in the inner retina. In contrast, loss of Shp2 decelerated the loss of RGCs, preserved their function, and suppressed ER stress and apoptosis in glaucoma. This report constitutes the first identification of Shp2-mediated TrkB regulatory mechanisms in the RGCs that can become a potential therapeutic target in both glaucoma and other neurodegenerative disorders.

Bexarotene Modulates Retinoid-X-Receptor Expression and Is Protective Against Neurotoxic Endoplasmic Reticulum Stress Response and Apoptotic Pathway Activation.

Retinoid X-receptors (RXRs) are members of the ligand-dependent transcription factor family of nuclear receptors that have gained recent research focus as potential targets for neurodegenerative disorders. Bexarotene is an RXR pharmacological agonist that is shown to be neuroprotective through its effects in promoting amyloid beta (Aß) uptake by the glial cells in the brain. This study aimed to evaluate the dose-dependent effects of bexarotene on RXR expression in SH-SY5Y neuroblastoma cells and validate the drug effects in the brain in vivo. The protein expression studies were carried out using a combination of various drug treatment paradigms followed by expression analysis using Western blotting and immunofluorescence. Our study demonstrated that bexarotene promoted the expression of RXR α, ß and γ isoforms at optimal concentrations in the cells and in the mice brain. Interestingly, a decreased RXR expression was identified in Alzheimer's disease mouse model and in the cells that were treated with Aß. Bexarotene treatment not only rescued the RXR expression loss caused by Aß treatment (p < 0.05) but also protected the cells against Aß-induced ER stress (p < 0.05) and pro-apoptotic BAD protein activation (p < 0.05). In contrast, higher concentrations of bexarotene upregulated the ER stress proteins and led to BAD activation. Our study revealed that these downstream neurotoxic effects of high drug concentrations could be prevented by pharmacological targeting of the TrkB receptor. The ER stress and BAD activation induced by high concentrations of bexarotene were rescued by the TrkB agonist, 7,8 dihydroxyflavone (p < 0.05) while TrkB inhibitor CTX-B treatment further exacerbated these effects. Together, these findings suggest a cross-talk of TrkB signalling with downstream effects of bexarotene toxicity and indicate that therapeutic targeting of RXRs could prevent the Aß-induced molecular neurotoxic effects.

PTPN11 induces endoplasmic stress and apoptosis in SH-SY5Y cells.

PTPN11 is associated with regulation of growth factor signaling pathways in neuronal cells. Using SH-SY5Y neuroblastoma cells, we showed that adeno-associated virus (AAV)-mediated PTPN11 upregulation was associated with TrkB antagonism, reduced neuritogenesis and enhanced endoplasmic reticulum (ER) stress response leading to apoptotic changes. Genetic knock-down of PTPN11 on the other hand leads to increased TrkB phosphorylation in SH-SY5Y cells. ER stress response induced by PTPN11 upregulation was alleviated pharmacologically by a TrkB agonist. Conversely the enhanced ER stress response induced by TrkB receptor antagonism was ameliorated by PTPN11 suppression, providing evidence of cross-talk of PTPN11 effects with TrkB actions. BDNF treatment of neuronal cells with PTPN11 upregulation also resulted in reduced expression of ER stress protein markers. This study provides evidence of molecular interactions between PTPN11 and the TrkB receptor in SH-SY5Y cells. The results reinforce the role played by PTPN11 in regulating neurotrophin protective signaling in neuronal cells and highlight that PTPN11 dysregulation promotes apoptotic activation. Based on these findings we suggest that blocking PTPN11 could have potential beneficial effects to limit the progression of neuronal loss in neurodegenerative disorders.