Phosphatidylserine and Tyro3-Axl-Mertk Receptor Tyrosine Kinase level detection in plasma and on plasma-derived extracellular vesicle surface in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disorder characterized by monoclonal B cell proliferation. Studies carried out in recent years suggest that extracellular vesicles (EVs) may be a potential biomarker in cancer. Tyro3-Axl-Mertk (TAM) Receptor Tyrosine Kinases (RTKs) and Phosphatidylserine (PS) have crucial roles in macrophage-mediated immune response under normal conditions. In the tumor microenvironment, these molecules contribute to immunosuppressive signals and prevent the formation of local and systemic antitumor immune responses. Based on this, we aimed to evaluate the amount of PS and TAM RTK in plasma and on the surface of EVs in CLL patients and healthy volunteers in this study. In this study, 25 CLL (11 F/14 M) patients in the Rai (O-I) stage, newly diagnosed or followed up without treatment, and 15 healthy volunteers (11 F/4 M) as a control group were included. For all samples, PS and TAM RTK levels were examined first in the plasma and then in the EVs obtained from the plasma. We detected a significant decrease in plasma PS, and TAM RTK levels in CLL patients compared to the control. Besides, we determined a significant increase in TAM RTK levels on the EV surface in CLL, except for PS. In conclusion, these receptor levels measured by ELISA in plasma may not be effective for the preliminary detection of CLL. However, especially TAM RTKs on the surface of EVs may be good biomarkers and potential targets for CLL therapies.

Gas6/TAM system as potential biomarker for multiple sclerosis prognosis.

Introduction: The protein growth arrest-specific 6 (Gas6) and its tyrosine kinase receptors Tyro-3, Axl, and Mer (TAM) are ubiquitous proteins involved in regulating inflammation and apoptotic body clearance. Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the central nervous system leading to progressive and irreversible disability if not diagnosed and treated promptly. Gas6 and TAM receptors have been associated with neuronal remyelination and stimulation of oligodendrocyte survival. However, few data are available regarding clinical correlation in MS patients. We aimed to evaluate soluble levels of these molecules in the cerebrospinal fluid (CSF) and serum at MS diagnosis and correlate them with short-term disease severity. Methods: In a prospective cohort study, we enrolled 64 patients with a diagnosis of clinical isolated syndrome (CIS), radiological isolated syndrome (RIS) and relapsing-remitting (RR) MS according to the McDonald 2017 Criteria. Before any treatment initiation, we sampled the serum and CSF, and collected clinical data: disease course, presence of gadolinium-enhancing lesions, and expanded disability status score (EDSS). At the last clinical follow-up, we assessed EDSS and calculated MS severity score (MSSS) and age-related MS severity (ARMSS). Gas6 and TAM receptors were determined using an ELISA kit (R&D Systems) and compared to neurofilament (NFLs) levels evaluated with SimplePlex™ fluorescence-based immunoassay. Results: At diagnosis, serum sAxl was higher in patients receiving none or low-efficacy disease-modifying treatments (DMTs) versus patients with high-efficacy DMTs (p = 0.04). Higher CSF Gas6 and serum sAXL were associated with an EDSS <3 at diagnosis (p = 0.04; p = 0.037). Serum Gas6 correlates to a lower MSSS (r2 = -0.32, p = 0.01). Serum and CSF NFLs were confirmed as disability biomarkers in our cohort according to EDSS (p = 0.005; p = 0.002) and MSSS (r2 = 0.27, p = 0.03; r2 = 0.39, p = 0.001). Results were corroborated using multivariate analysis. Conclusions: Our data suggest a protective role of Gas6 and its receptors in patients with MS and suitable severity disease biomarkers.

Serum AXL is a potential molecular marker for predicting COVID-19 progression.

Background: The severity, symptoms, and outcome of COVID-19 is thought to be closely linked to how the virus enters host cells. This process involves the key roles of angiotensin-converting enzyme 2 (ACE2) and the Tyrosine protein kinase receptor UFO (AXL) receptors. However, there is limited research on the circulating levels of ACE2 and AXL and their implications in COVID-19. Methods: A control group of 71 uninfected individuals was also included in the study. According to the Guidance for Corona Virus Disease 2019 (10th edition), a cohort of 358 COVID-19 patients were categorized into non-severe and severe cases. Serum ACE2/AXL levels in COVID-19 patients were detected by enzyme-linked immunosorbent assay (ELISA) at different time points post-COVID-19 infection, including days 0-7, 8-15, 31-179 and >180 days. Serum SARS-CoV-2 IgG/IgM antibodies in COVID-19 patients at the same intervals were assessed by using an iFlash 3000 Chemiluminescence Immunoassay Analyzer. The receiver operating characteristic (ROC) curves were used to assess the diagnostic value of the biological markers, and the association between laboratory parameters and illness progression were explored. Results: Compared with the uninfected group, the levels of ACE2 and AXL in the COVID-19 group were decreased, and the SARS-COV-2 IgG level was increased. AXL (AUC = 0.774) demonstrated a stronger predictive ability for COVID-19 than ACE2. In the first week after infection, only the level of AXL was statistically different between severe group and non-severe group. After first week, the levels of ACE2 and AXL were different in two groups. Moreover, in severe COVID-19 cases, the serum ACE2, AXL, and SARS-COV-2 IgM levels reached a peak during days 8-15 before declining, whereas serum SARS-COV-2 IgG levels continued to rise, reaching a peak at day 31-180 days before decreasing. In addition, the AXL level continued to decrease and the SARS-COV-2 IgG level continued to increase in the infected group after 180 days compared to the uninfected group. Conclusions: The levels of serum ACE2 and AXL correlate with COVID-19 severity. However, AXL can also provide early warning of clinical deterioration in the first week after infection. AXL appears to be a superior potential molecular marker for predicting COVID-19 progression.

Serum GAS6, sAXL, IL-10, NO, and BCL-2 levels are decreased in patients with Behçet's disease.

PURPOSE: Behçet's disease (BD) is an autoimmune chronic systemic inflammatory disease characterized by a versatile clinical spectrum. Growth arrest specific protein 6 (GAS6)/soluble AXL (sAXL) signaling pathway draws attention in the resolution of inflammation, and its deficiency is associated with chronic inflammatory, autoimmune diseases, as well as clearance of apoptotic cells by phagocytes - efferocytosis. In this study, it was aimed to investigate whether GAS6/sAXL, interleukin (IL)-10, nitric oxide (NO), and BCL-2 levels were associated with inflammation and efferocytosis contributes to the pathogenesis of BD. METHODS: A total of 37 Behçet patients with ocular involvement and 30 healthy control subjects were included in this study. GAS6, sAXL, IL-10, NO, and BCL-2 levels were quantified using enzyme-linked immunosorbent assay (ELISA) method. RESULTS: Serum GAS6, sAXL, IL-10, NO, and BCL-2 levels were significantly lower in patients with BD compared to the controls (P < 0.005, P < 0.001, P < 0.001, P < 0.001, and P < 0.001, respectively). In correlation analysis, research parameters decreased in patients with BD was significantly correlated with each other: GAS6-IL-10 (r = 0.585, P < 0.001), GAS6-BCL-2 (r = 0.541, P < 0.001), sAXL-BCL-2 (r = 0.696, P < 0.001), IL-10-NO (r = 0.717, P < 0.001), IL-10-BCL-2 (r = 0.759, P < 0.001), and NO-BCL-2 (r = 0.541, P < 0.001). CONCLUSION: In conclusion, decreased serum BCL-2 level may be an indicator of increased apoptosis in these patients and decreased levels of GAS6/sAXL, IL-10, and NO may indicate insufficient clearance of apoptotic bodies released as a result of increased apoptosis in BD patients.

Distinguishing placenta accreta from placenta previa via maternal plasma levels of sFlt-1 and PLGF and the sFlt-1/PLGF ratio.

INTRODUCTION: Our study aimed to distinguish patients with placenta accreta (crete, increta, and percreta) from those with placenta previa using maternal plasma levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PLGF) and the sFlt-1/PLGF ratio. METHODS: We obtained maternal plasma from 185 women in late pregnancy and sorted them into three groups: 72 women with normal placental imaging results (control group), 50 women with placenta previa alone (PP group), and 63 women with placenta previa and placenta accreta (PAS group). The concentrations of sFlt-1 and PLGF in the maternal plasma were measured using ELISA kits and the sFlt-1/PLGF ratio was calculated. RESULT: The median (min-max) sFlt-1 levels and the sFlt-1/PLGF ratio in the PAS group (12.8 ng/ml, 3.8-34.2 ng/ml) (133, 14-361) were lower than in the PP group (28.7 ng/ml, 13.1-60.3 ng/ml) (621, 156-2013) (p < 0.0001 and P < 0.0001, respectively). The median (min-max) PLGF levels in the PAS group (108 pg/ml, 38-679 pg/ml) was higher than that in the PP group (43 pg/ml, 12-111 pg/ml) (p < 0.0001 and p < 0.0001, respectively). The area under the ROC of the sFlt-1 levels, PLGF levels, and sFlt-1/PLGF ratio were 0.91, 0.90, and 0.99, respectively; the cut-off values were 18.9 ng/ml, 75.9 pg/ml, and 229.5, respectively. The concentration of sFlt-1 and sFlt-1/PLGF ratio were associated with the volume of blood loss (-.288*, -.301*). DISCUSSION: The concentrations of sFlt-1 and PLGF and ratio of plasma sFlt-1/PLGF may distinguish patients with placenta accreta from those with placenta previa.

Up-regulated serum levels of TAM receptor tyrosine kinases in a group of Egyptian autistic children.

TAM receptor family belongs to receptor tyrosine kinases (TAMRTKs). It includes three receptors; Tyro-3, Axl and Mer. TAMRTKs has a great role in resolution of inflammation due to their role in clearance of apoptotic cells by macrophages. Dysregulated TAM signaling pathways are associated with many autoimmune diseases and chronic inflammatory disorders. Autism may be an autoimmune disease in some patients. This work was the first study that investigated serum levels of the soluble ectodomain shed TAMRTKs in a group of autistic children. Serum levels of TAMRTKs were measured by ELISA in 30 autistic children aged between 3.5 and 11 years and 30 age and sex-matched healthy control children. Serum levels of TAMRTKs were significantly higher in autistic children than healthy control children (P < 0.001). Patients with severe autism had significantly higher serum levels of TAMRTKs than patients with mild to moderate autism (P < 0.01). In addition, there were significant positive correlations between scores of the Childhood Autism Rating Scale (CARS) and serum levels of TAMRTKs in autistic patients, (P < 0.01). In conclusions, serum levels of TAMRTKs were up-regulated in autistic children with significant positive correlations with the degree of the disease severity. This initial report requires further studies to investigate the relationship between TAMRTKs and autism.

Increased serum AXL is associated with radiographic knee osteoarthritis severity.

OBJECTIVE: To investigate the expression and clinical significance of serum soluble AXL in patients with radiographic knee osteoarthritis (KOA). METHODS: There were 183 patients with KOA who were selected and divided based on the Kellgren-Lawrence (KL) score into KL 0 subgroups (n = 42), KL I-II subgroups (n = 90), and KL III-IV subgroups (n = 51). Healthy volunteers (n = 170) in our hospital were selected with matched age and gender as the control group. AXL level in serum was detected by enzyme-linked immunosorbent assay. The correlation between serum AXL with severity and clinical indicators of osteoarthritis was analyzed. RESULTS: The level of serum AXL was significantly higher in the osteoarthritis group than that in the control group (P < .05). In the osteoarthritis patients, serum AXL level was increased with the increase of KL score. Serum AXL level was positively correlated with age, body mass index, erythrocyte sedimentation rate, serum C-reactive protein, cartilage oligomeric protein, matrix metalloproteinase-13, and transforming growth factor-ß1 levels. The cut-off value for serum AXL was determined as 33.375 ng/mL by receiver operating curve analysis. CONCLUSION: The level of serum AXL in patients with osteoarthritis is significantly higher than in healthy controls, and is closely related to the severity of radiographic osteoarthritis.

Vitamin K Effects on Gas6 and Soluble Axl Receptors in Intensive Care Patients: An Observational Screening Study.

Growth arrest-specific gene 6 protein (Gas6) is avitamin K-dependent tissue bound protein. Gas6 has been shown to promote growth and therapy resistance among different types of cancer as well as thromboembolism. The aim of this prospective screening study: ClinicalTrials.gov; Identifier: NTC3782025, was to evaluate the effects of intravenously administered vitamin K1 on Gas6 and its soluble (s)Axl receptor plasma levels in intensive care patients. Vitamin K1 was intravenously injected in non-warfarin treated patients with prolonged Owren prothrombin time international normalized ratio (PT-INR) > 1.2 and blood samples were retrieved before and 20-28 h after injection. Citrate plasma samples from 52 intensive care patients were analysed for different vitamin K dependent proteins. There was a significant, but small increase in median Gas6. Only one patient had a large increase in sAxl, but overall, no significant changes in sAxl Gas6 did not correlate to PT-INR, thrombin generation assay, coagulation factors II, VII, IX and X, but to protein S and decarboxylated matrix Gla protein (dp-ucMGP). In conclusion, there was a small increase in Gas6 over 20-28 h. The pathophysiology and clinical importance of this remains to be investigated. To verify a true vitamin K effect, improvement of Gas6 carboxylation defects needs to be studied.

Cholinergic hyperactivity in patients with myasthenia gravis with MuSK antibodies: A neurophysiological study.

OBJECTIVE: Patients with myasthenia gravis associated with muscle-specific tyrosine kinase antibodies (MuSK-MG) often manifest signs of cholinergic hyperactivity with standard doses of acetylcholinesterase inhibitors (AChE-Is). Aim of the study was to investigate whether repetitive compound muscle action potential (R-CMAP), the neurophysiological correlate of cholinergic hyperactivity, was present in MuSK-MG irrespective of AChE-I treatment. METHODS: Patients with confirmed diagnosis of MuSK-MG were consecutively enrolled during follow-up visits, from January 2019 to April 2020. All these subjects underwent the same neurophysiological protocol, including motor nerve conduction studies and repetitive nerve stimulation. In patients taking pyridostigmine, neurophysiological testing was performed at least 12 hours after the last dose. For comparison, the presence of R-CMAP was investigated in 20 consecutive acetylcholine receptor antibody positive myasthenia gravis (AChR-MG) patients. RESULTS: We enrolled 25 MuSK-MG patients (20 females), aged 16-79 years at the study time, with disease duration ranging 0.6-48.8 years (median: 17.7 years). R-CMAP was detected in 12/25 (48%) MuSK-MG cases and in none of the AChR-MG controls (p = 0.0003). In the MuSK-MG population, a history of muscle cramps and fasciculations, during low-dose pyridostigmine therapy, was significantly more frequent in R-CMAP positive than in R-CMAP negative patients (100% vs 31%, p = 0.001). At the time of the study, the proportion of patients still symptomatic for MG was higher among R-CMAP positive cases (92% vs 23%, p = 0.0005). CONCLUSIONS: Cholinergic hyperactivity is a relatively common finding in MuSK-MG patients, independent of AChE-I treatment, and may constitute an intrinsic feature of the disease. SIGNIFICANCE: R-CMAP detection can represent a useful diagnostic clue for MuSK-MG and predicts poor tolerance to AChE-Is.

Cross-sectional study of plasma Axl, ferritin, IGFBP4 and sTNFR2 as biomarkers of disease activity in childhood-onset SLE: A study of the Pediatric Nephrology Research Consortium.

OBJECTIVE: To evaluate the performance of 4 plasma protein markers for detecting disease activity in childhood-onset systemic lupus erythematosus (SLE) patients. METHODS: Eighty-three consecutive pediatric patients fulfilling ≥4 ACR criteria for SLE and twenty-five healthy controls were prospectively recruited for serological testing of 4 protein markers identified by antibody-coated microarray screen, namely Axl, ferritin, IGFBP4 and sTNFR2. SLE disease activity was assessed using SLEDAI-2000 score. Fifty-seven patients had clinically active SLE (SLEDAI score ≥4, or having a flare). RESULTS: The plasma concentrations of Axl and ferritin were significantly higher in patients with active SLE than inactive SLE. Plasma Axl levels were significantly higher in active renal versus active non-renal SLE patients. Levels of Axl, ferritin and IGFBP4 correlated significantly with SLEDAI scores. Levels of Axl, IFGBP4 and sTNFR2 inversely correlated with plasma complement C3 levels. Only plasma Axl and ferritin levels correlated with degree of proteinuria. These markers were more specific, but less sensitive, in detecting concurrent SLE activity than elevated anti-dsDNA antibody titer or decreased C3. Ferritin and IGFBP4 levels were more specific for concurrent active lupus nephritis than anti-dsDNA or C3. Plasma ferritin was the best monitor of global SLE activity, followed by C3 then Axl, while both Axl and C3 were best monitors of clinical lupus nephritis activity. CONCLUSION: In childhood-onset SLE patients, plasma ferritin and Axl perform better than traditional yardsticks in identifying disease activity, either global or renal. The performance of these plasma markers should be explored further in longitudinal cohorts of SLE patients.

Axl is related to inflammation in hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients often develop chronic inflammation, which is associated with an increased risk of cardiovascular complications and mortality. Axl and its ligand, growth arrest 6 (Gas6), have been reported to play key roles in regulating the immune response. However, the function of Axl in HD patients has not been clarified. METHODS: In the present study, we enrolled 130 HD patients and 117 normal controls (NCs) and evaluated the levels of inflammatory markers, soluble Axl (sAxl), membrane Axl (mAxl), and Gas6 in all participants. The potential downstream cascades of Gas6-Axl signaling in HD patients were identified by quantitative real time polymerase chain reaction and western blotting. RESULTS: The levels of inflammatory cytokines-tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)-plasma sAxl, and Gas6, were significantly increased in HD patients compared to NCs. Additionally, sAxl was positively associated with the inflammatory factor, interleukin-6 (IL-6), in HD patients. Moreover, we found that mAxl in CD14+ mononuclear cells and CD19+ B cells was increased upon HD. Furthermore, we discovered that the metalloproteinase ADAM17, also called TACE, contributed to the cleavage of mAxl into sAxl, and not ADAM10, in the peripheral blood mononuclear cells (PBMCs) of HD patients. The upregulation of Gas6-mAxl signaling caused the activation of the STAT1-SOCS3 pathway in the PBMCs of HD patients. After two years follow-up, patients with lower sAxl levels had longer survival time than those with higher sAxl levels. CONCLUSION: Our results suggest that Axl may play a significant role in systemic inflammation in HD patients.

Therapeutic and Diagnostic Challenges in Myasthenia Gravis.

"Myasthenia gravis (MG) is the most common autoimmune neuromuscular disorder. This article highlights several cases that the practicing neurologist may encounter in the treatment of MG. Diagnostic uncertainty continues to be an issue in patients who are seronegative to the 2 most common antibodies, acetylcholine receptor and muscle-specific tyrosine kinase (MuSK). Specific populations of patients with MG including MuSK MG, thymomatous MG, refractory MG, and pregnant women also require special consideration. This article reviews specific cases and an update on current management."

AChR myasthenia gravis switching to MuSK or double antibody positive myasthenia gravis in two children and literature review.

Muscle-specific tyrosine kinase antibody (MuSK-Ab) and acetylcholine receptor antibody (AChR-Ab) coexistence in myasthenia gravis (MG) is very rare. In this report, two children with AChR-Ab switching to double antibody positive MG (DP-MG) or MuSK-Ab positive MG (MuSK-MG) are described. Six similar cases were found in the literature via online database search. Therefore, this study describes eight patients in total, six female and two male. The average age of onset was 7.25 ± 5.95 years. Four AChR-MG patients switched to DP-MG with no known precipitating factor and four switched after thymectomy (two to MuSK-MG and two to DP-MG). After the serological switch, the patients transitioned to the phenotype of MuSK-MG and responded poorly to cholinesterase inhibitors and well to corticosteroids and plasma exchange.

Role of Gas6 and TAM Receptors in the Identification of Cardiopulmonary Involvement in Systemic Sclerosis and Scleroderma Spectrum Disorders.

BACKGROUND: Few biomarkers are available for early identification of pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD) in systemic sclerosis (SS) and scleroderma spectrum disorders (SSD). AIMS: To evaluate Gas6, sAxl, and sMer as biomarkers for cardiopulmonary complications of SS and SSD. METHODS: In a cross-sectional observational study, we recruited 125 consecutive patients, affected by SS and SSD and referred to a tertiary-level pulmonary hypertension outpatient clinic. All patients underwent a comprehensive evaluation for identification of PAH and ILD. Gas6, sMer, and sAxl concentrations were measured with ELISA protocols, and concentrations were compared according to PAH or ILD. RESULTS: Nineteen subjects had pulmonary hypertension (PH) (14 PAH), and 39 had ILD (6 severe). Plasma sMer was increased in PAH (18.6 ng/ml IQR [11.7-20.3]) with respect to the absence (12.4 [8.0-15.8]) or other form of pulmonary hypertension (9.6 [7.4-12.5]; K-W variance p < 0.04). Conversely, Gas6 and sAxl levels were slightly increased in mild ILD (25.8 ng/ml [19.5-32.1] and 24.6 [20.1-32.5]) and reduced in severe ILD (16.6 [15.0-22.1] and 15.5 [14.9-22.4]) in comparison to no evidence of ILD (23.4 [18.8-28.1] and 21.6 [18.1-28.4]; K-W, p ≤ 0.05). Plasma sMer ≥ 19 ng/ml has 50% sensitivity and 92% specificity in PAH identification (area under the ROC curve (AUC) 0.697, p < 0.03). Values of Gas6 ≤ 24.5 ng/ml and of sAxl ≤ 15.5 ng/ml have 100% and 67% sensitivity and 47% and 86% specificity, respectively, in identifying severe ILD (Gas6 AUC 0.787, p < 0.001; sAxl AUC 0.705, p < 0.05). CONCLUSIONS: The assay of Gas6 sAxl and sMer may be useful to help in the identification of PAH and ILD in SS and SSD patients. The Gas6/TAM system seems to be relevant in cardiopulmonary complications of SS and SSD and merits further investigations.

Soluble Axl Is a Novel Diagnostic Biomarker of Hepatocellular Carcinoma in Chinese Patients with Chronic Hepatitis B Virus Infection.

PURPOSE: The purpose of this study was to evaluate the diagnostic value of soluble Axl (sAxl) in hepatocellular carcinoma (HCC) in comparison with serum α-fetoprotein (AFP). MATERIALS AND METHODS: Eighty HCC patients, 80 liver cirrhosis patients (LC), 80 patients with hepatitis B virus (HBV) infection, and 80 healthy controls (HC) were enrolled. sAxl levels were measured by an enzyme-linked immunosorbent assay, serum AFP levelswere measured by an electrochemiluminescence immunoassay. Receiver operating characteristic (ROC) curves were used to evaluate diagnostic performances. RESULTS: The results show that levels of sAxl were high expression in patients with HCC (p < 0.05), varied with disease state as follows: HCC > LC > HC > HBV. Logistic regression and ROC curve analysis identified the optimal cut-off for sAxl in differentiating all HCC and non-HCC patients was 1,202 pg/mL (area under the receiver operating characteristic [AUC], 0.888; 95% confidence interval [CI], 0.852 to 0.924) with sensitivity 95.0%, specificity 73.3%. Furthermore, differential diagnosis of early HCC with non-HCC patients for sAxl showed the optimal cut-off was 1,202 pg/mL (AUC, 0.881; 95% CI, 0.831 to 0.931; sensitivity, 94.1%; specificity, 73.3%). Among AFP-negative HCC patients with non-HCC patients, the cut-off was 1,301 pg/mL (AUC, 0.898; 95% CI, 0.854 to 0.942) with a sensitivity of 84.6%, a specificity of 76.3%. The optimal cut-off for sAxl in differentiating all HCC and chronic liver disease patients was 1,243 pg/mL (AUC, 0.840; 95% CI, 0.791 to 0.888) with sensitivity 93.8%, specificity 61.9%. The combination of AFP and sAxl increased diagnostic value for HCC. CONCLUSION: sAxl outperforms AFP in detecting HCC, especially in early HCC and in AFP-negative HCC. Combination sAxl with AFP improved the specificity for early HCC diagnosis. In summary, sAxl is a candidate serum marker for diagnosing HCC.

Expression of Receptor Tyrosine Kinases on Peripheral Blood Mononuclear Cells and Tumor-Infiltrating Lymphocytes in Patients with Renal Cell Carcinoma and Healthy Donors.

BACKGROUND: Very little is known about receptor tyrosine kinase (RTK) expression on peripheral blood mononuclear cells (PBMC) in humans including renal cell carcinoma (RCC) patients. OBJECTIVES: The primary objective of this study was to evaluate expression levels of major RTKs on PBMC and tumor-infiltrating lymphocytes (TIL) isolated from RCC patients. The secondary aim was to compare levels of RTK expression in RCC patients before surgery and on the 180th day after surgery (lymphocyte lifetime) and to compare them with the expression in healthy donors. In addition, we compared RTK and PD-L1 expression in TIL. METHODS: Tumor and blood samples were obtained from 20 patients with primary RCC immediately after surgical resection. Blood samples were collected from 20 healthy donors. Tumors were harvested into RPMI 1640 medium (Gibco) and processed within 4 h. TIL isolation was performed using a modified protocol [Baldan et al. Br J Cancer. 2015;112:1510-18]. Expression of RTKs was evaluated with NovoExpress Software. Twenty tumors from the same patients were stained with PD-L1 IHC assay (clone SP142; Ventana). RESULTS: PBMC and TIL express RTKs in humans. The RTK expression level was significantly lower on peripheral blood cells in patients with RCC (mean 41%, range 27.1-62.6%) as compared with healthy donor PBMC (mean 77.1%, range 72.1-80.1%, all p < 0.05). Furthermore, RTK expression was significantly downregulated on intratumoral cells (mean 40%, range 23.2-52.3%) in comparison with healthy donor PBMC. There was no significant recovery of RTK expression on the 180th day except for VEGFR2. Five of 20 (25%) patients were PD-L1 positive. PD-L1 expression on TIL was strongly associated with downregulated expression of PDGFRα (p = 0.017) and PDGFRß (p = 0.024). CONCLUSIONS: PBMC and TIL had similar low RTK expression levels in RCC patients. PBMC of healthy humans had a significantly higher expression of RTK. PD-L1 and PDGFRα-ß expression could correlate. Comprehensive basic and clinical studies will be needed to define a biological role of RTKs on different lymphocyte subsets and correlations between clinical outcomes and expression levels.

Possible Association of Elevated Plasma Levels of Growth Arrest-Specific Protein 6 and the Soluble Form of Tyrosine Kinase Receptor Axl with Low Hepatitis C Viral Load in Patients with Type 2 Diabetes Mellitus.

This study aimed to investigate the plasma levels of Gas6 and soluble Axl (sAxl) in patients with chronic hepatitis C virus (HCV) infection with and without type 2 diabetes mellitus (T2DM). The study involved four groups; 50 patients with chronic HCV, 50 patients with T2DM, 50 patients with chronic HCV and T2DM, and 31 age- and sex-matched healthy controls. T2DM was diagnosed according to American Diabetes Association criteria, HCV antibodies were detected by enzyme-linked immunosorbent assays (ELISA) and confirmed by real-time-polymerase chain reaction. Plasma Gas6 and sAxl levels were assayed in all groups by ELISA. Significant low levels of GAS 6 in HCV/T2DM group versus HCV group were detected (7.92 ± 5.18 vs. 16.09 ± 7.36, respectively, p = 0.000), but higher than T2DM and control groups (p ≥ 0.05), although nonsignificant. HCV load was higher in the HCV group than the HCV/T2DM group (1,888,300 ± 5,595,070 vs. 1,417,900 ± 4,066,460 copies/mL, respectively, p = 0.632). Among HCV group, significant positive correlations were detected between Gas6 and sAxl levels with HCV viral load (r = 0.48, p = 0.000 and r = 0.43, p = 0.002, respectively), while among HCV/T2DM group, significant negative correlations were detected (r = -0.29, p = 0.04 and r = -0.34, p = 0.014, respectively). Significant negative correlations were detected between Gas6/sAxl levels and glycated hemoglobin (r = -0.36, p = 0.01 and r = -0.4, p = 0.003, respectively) in T2DM despite the positive correlations detected in HCV/T2DM (r = 0.27, p = 0.053 and r = 0.55, p = 0.000, respectively). In conclusion, Gas6/Axl system in combined HCV/T2DM diseases may affect the pathogenesis and can alter the biomarkers and complications of both diseases in a manner that differs from a solitary disease.

Soluble AXL as a marker of disease progression and survival in melanoma.

Receptor tyrosine kinase AXL is a one-pass transmembrane protein upregulated in cancers and associated with lower survival and therapy resistance. AXL can be cleaved by the A Disintegrin and Metalloproteinases (ADAM)10 and ADAM17, yielding a soluble version of the protein. Elevated soluble AXL (sAXL) has been reported to be associated with disease progression in hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases. In the present work, we analyzed sAXL levels in blood from melanoma patients and showed that sAXL increases with disease progression. Additionally, increased sAXL levels were found correlated with shorter two-year survival in stage IV patients treated with ipilimumab. Furthermore, we showed that sAXL levels were related to the percentage of cells expressing AXL in resected melanoma lymph node metastases. This finding was verified in vitro, where sAXL levels in the cell media corresponded to AXL expression in the cells. AXL inhibition using the small-molecular inhibitor BGB324 reduced sAXL levels, while the cellular expression was elevated through increased protein stability. Our findings signify that quantification of sAXL blood levels is a simple and easily assessable method to determine cellular AXL levels and should be further evaluated for its use as a biomarker of disease progression and treatment response.

Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.

Infectious complications in patients with cirrhosis frequently initiate episodes of decompensation and substantially contribute to the high mortality. Mechanisms of the underlying immuneparesis remain underexplored. TAM receptors (TYRO3/AXL/MERTK) are important inhibitors of innate immune responses. To understand the pathophysiology of immuneparesis in cirrhosis, we detailed TAM receptor expression in relation to monocyte function and disease severity prior to the onset of acute decompensation. TNF-α/IL-6 responses to lipopolysaccharide were attenuated in monocytes from patients with cirrhosis (n = 96) compared with controls (n = 27) and decreased in parallel with disease severity. Concurrently, an AXL-expressing (AXL+) monocyte population expanded. AXL+ cells (CD14+CD16highHLA-DRhigh) were characterised by attenuated TNF-α/IL-6 responses and T cell activation but enhanced efferocytosis and preserved phagocytosis of Escherichia coli Their expansion correlated with disease severity, complications, infection, and 1-yr mortality. AXL+ monocytes were generated in response to microbial products and efferocytosis in vitro. AXL kinase inhibition and down-regulation reversed attenuated monocyte inflammatory responses in cirrhosis ex vivo. AXL may thus serve as prognostic marker and deserves evaluation as immunotherapeutic target in cirrhosis.