Expression of autophagy related genes in peripheral blood cells in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and affects dopaminergic neurons. Autophagy often shows a circadian rhythm pattern under physiological conditions across 24 h. Abnormal autophagy and circadian dysfunction are two characteristics of PD. Whether the rhythm of autophagy is altered in PD has not yet been reported. Therefore, in this study, we collected peripheral blood samples at 6:00 h and 18:00 h from PD patients and age-matched controls, and analyzed the mRNA expressions of ULK1, BECN1, LAMP2, AMPK, and SNCA using real-time quantitative PCR. Blood samples analysis found that BECN1 and LAMP2 levels were decreased in patients with PD. Simultaneously, the rhythm of autophagy in PD is not consistent with that in the Control group, which may be a manifestation of the abnormal biological rhythm of PD.

Alterations of serum levels of plasminogen, TNF-α, and IDO in granulomatosis with polyangiitis patients.

BACKGROUND: Granulomatosis with polyangiitis (GPA) is a representative of vasculitides associated with anti-neutrophil cytoplasmic autoantibodies. "Classical" antibodies directed against proteinase 3 are involved in the pathogenesis and are part of the GPA diagnosis at the same time. Along with them, however, antibodies against Lysosomal-Associated Membrane Protein-2 (LAMP-2) and antibodies directed against plasminogen have been described in GPA.Objectives and methodology: We performed a cross-sectional study enrolling 34 patients diagnosed with GPA. Our study was aimed at looking for correlations between serum levels of LAMP-2 and plasminogen and the clinical manifestations of the GPA. Furthermore, we examined serum levels of tumor necrosis factor-alpha (TNF-α) and its associated indoleamine-pyrrole 2,3-dioxygenase (IDO), as well as we looked for a correlation between these cytokines and the clinical manifestations of GPA. RESULTS: The results showed that in GPA, serum plasminogen levels were negatively associated with renal involvement (receiver operating characteristic (ROC) area under the curve (AUC) of 0.78) (95% CI 0.53-0.91), p = 0.035, and the extent of proteinuria, Spearman's Rho = -0.4, p = 0.015. Increased levels of TNF-α and IDO correlated with disease activity, Spearman's Rho =0.62, p = 0.001 and Spearman's Rho = 0.4, p = 0.022, respectively, whereas only TNF-α was increased in severe forms of GPA with lung involvement (ROC AUC of 0.8) (95% CI 0.66-0.94), p = 0.005. CONCLUSIONS: In this study, we demonstrate the alteration of soluble factors, which play an important role in the pathogenesis of GPA and their relationship with the clinical manifestations of the disease. Our main results confirm the associations of increased secretory TNF-α and some clinical manifestations, and we describe for the first time decreased serum plasminogen levels and their association with renal involvement.

Treating neutropenia and neutrophil dysfunction in glycogen storage disease type Ib with an SGLT2 inhibitor.

Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)-deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.

Autoantibodies Against Lysosome Associated Membrane Protein-2 (LAMP-2) in Pediatric Chronic Primary Systemic Vasculitis.

Background: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a small vessel vasculitis in adults and children that commonly affects the kidneys. Although the frequent antigenic, and presumed pathogenic, targets of ANCA in AAV are proteinase-3 (PR3) and myeloperoxidase (MPO), ANCA against lysosome associated membrane protein-2 (LAMP-2), a lesser known ANCA antigen that is expressed on the glomerular endothelium, are present in some adults with AAV-associated renal disease. LAMP-2-ANCA has not been assessed in children with chronic systemic vasculitis, and, if present, would be a potentially valuable biomarker given that treatment decisions for these pediatric patients at diagnosis are largely informed by kidney function. Methods: A custom ELISA, using commercially available reagents, was designed to detect autoantibodies to human LAMP-2 in serum. Sera obtained from 51 pediatric patients at the time of diagnosis of chronic primary systemic vasculitis (predominantly AAV) were screened. LAMP-2-ANCA titers were evaluated for correlation with clinical metrics of disease activity (pediatric vasculitis activity score [pVAS], C-reactive protein [CRP] concentration, and erythrocyte sedimentation rate [ESR]), MPO- and PR3-ANCA titers, and renal function (glomerular filtration rate [GFR], renal-specific pVAS, and serum creatinine concentration). Results: LAMP-2-ANCA (>1,000 ng/ml) were detected in 35% (n = 18) of pediatric systemic vasculitis patients, of which, 10 (20% of all patients) were found to have high positive titers (>1,500 ng/ml). Undetectable or negative titres (<500 ng/ml) were identified in 12% (n = 6) of patients, those with titers between 500 and 1,000 ng/ml were considered low with unknown clinical relevance (53%, n = 27). Although LAMP-2-ANCA titers did not significantly differ between patients with AAV versus ANCA-negative vasculitis, only AAV patients had high concentrations (>1,500 ng/ml) of LAMP-2-ANCA. LAMP-2-ANCA titers did not correlate with measures of disease activity (pVAS, CRP, or ESR) at the time of diagnosis. In contrast, for patients with 12-month post diagnosis follow-up, a negative correlation was observed between the change in GFR (from diagnosis to 12-month follow-up) and LAMP-2-ANCA titer at diagnosis. Conclusions: Moderate to high LAMP-2-ANCA titers were detected in 35% (18/51) of children with chronic systemic vasculitis affecting small-to-medium vessels. Although the highest concentrations of LAMP-2-ANCA in this population were observed in individuals positive for classic ANCA (MPO- or PR3-ANCA), similar to previous reports on adult patients, LAMP-2-ANCA titers do not correlate with classic ANCA titers or with overall disease activity at diagnosis. Renal disease is a common manifestation in systemic small-medium vessel vasculitis (both in adults and children, though more severe in children) and our preliminary data suggest LAMP-2-ANCA at diagnosis may be a risk factor for more severe renal disease.

Study on the association of serum pentraxin-3 and lysosomal-associated membrane protein-2 levels with disease activity in Chinese Takayasu's arteritis patients.

OBJECTIVES: To evaluate the association between disease activity and serum levels of pentraxin-3 (PTX-3) and lysosomal-associated membrane protein-2 (LAMP-2) as well as the acute reactants in Chinese Takayasu's arteritis (TAK) patients. METHODS: The serum PTX-3 and LAMP-2 levels were tested in 98 TAK patients and 40 age- and gender-matched healthy controls. The disease activity of these TAK patients was assessed according to the National Institute of Health (NIH) and Abatacept in giant cell arteritis and Takayasu's arteritis (AGATA) criteria, respectively. RESULTS: Among the 98 TAK patients, 45 and 52 patients had active disease according to the NIH criteria and AGATA criteria respectively. The total agreement rate between these two criteria was 90.82% (κ=0.817, p<0.001). Both serum PTX-3 and LAMP-2 levels were elevated in TAK patients compared with those in healthy controls (PTX- 3: 0.32±0.03 ng/ml vs. 0.18±0.02 ng/ ml, p=0.001; LAMP-2: 4.40±0.14 ng/ ml vs. 3.30±0.20 ng/ml, p<0.001). TAK patients with active disease had higher serum PTX-3 levels compared with those who had inactive disease (NIH criteria: 0.42±0.06 ng/ml vs. 0.25±0.02 ng/ml, p=0.004; AGATA criteria: 0.38±0.05 ng/ml vs. 0.27±0.02 ng/ml, p=0.049). However, serum LAMP-2 levels did not differ between patients with active and inactive disease according to both NIH and AGATA criteria. A cutoff value of PTX-3 with 0.30 ng/ml maximised the ability of disease activity assessment with a sensitivity/specificity of 57.10%/73.10% and 47.90%/71.10% according to the NIH and AGATA criteria, respectively. CONCLUSIONS: Serum PTX-3 and LAMP- 2 levels are elevated in Chinese TAK patients. However, serum PTX-3 but not LAMP-2 level is associated with active disease.

Serum lysosomal-associated membrane protein-2 levels are increased in small and medium-vessel vasculitis, especially in polyarteritis nodosa.

OBJECTIVES: Lysosomal-associated membrane protein-2 (LAMP-2) is a highly glycosylated type I glycoprotein ex- pressed on the membranes of neutrophils, endothelial cells and other cells, which are closely linked to subsets of systematic vasculitis. The aim of this study was to investigate whether serum LAMP-2 can be used as a biomarker in small and medium vessel vasculitis (SMVV). METHODS: Serum samples from 39 patients with SMVV (including ANCA-associated vasculitis (AAV) and polyarteritis nodosa (PAN)) confirmed by angiography and/or biopsy and 78 healthy controls (HC) were collected. Serum LAMP-2 levels were determined by enzyme-linked immunosorbent assay. RESULTS: Serum LAMP-2 levels in SMVV patients were increased compared with HC (p<0.001). Serum LAMP-2 levels were significantly different between patients with active stage and those with inactive stage (p=0.024). Patients with renal involvement had higher LAMP-2 levels than patients with non-renal involvement at presentation (p=0.022). Furthermore, serum LAMP-2 levels were correlated with Birmingham Vasculitis Activity Score (BVAS), C-reactive protein (CRP), hypersensitive CRP (Hs-CRP), serum creatinine (Scr) and 24-hour proteinuria (all p<0.05). Among SMVV subsets, serum LAMP-2 levels were signi cantly higher in PAN compared with AAV (p=0.003). In PAN patients, serum LAMP-2 levels were correlated with BVAS and Hs-CRP (all p<0.05). CONCLUSIONS: Serum LAMP-2 levels can reflect the disease activity and renal involvement of SMVV. Furthermore, serum LAMP-2 levels were significantly higher in PAN compared with AAV, and associated with disease activity. LAMP-2 might be a potential biomarker for SMVV, especially in PAN.

Circulating Levels of LAMP2 in Coronary Artery Disease: Association with Serum Lipid Profile.

Whereas several in vitro and in vivo studies have described the role of lysosomal associated membrane protein 2 (LAMP2) in lipid homeostasis, there is no study addressing LAMP2 serum concentration and its association with lipid profiles in the context of coronary artery disease (CAD). We aimed to determine the LAMP2 serum concentration and its association with serum lipid profiles as well as the gene expression of LAMP2 in peripheral blood mononuclear cells (PBMCs) of CAD patients and control group. Circulating levels of LAMP2 were quantified by enzyme-linked immunosorbent assay (ELISA) in CAD patients (n=85) and control group (n=65) and correlation to lipid parameters was assessed. Gene expression analysis was performed by quantitative real-time PCR. Mean LAMP2 serum concentration adjusted for drug consumption, age and gender was not significantly different between the CAD and control groups (p>0.05). However, LAMP2 serum concentration showed independent significant association with lipid profiles including triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) (all p<0.05). Furthermore, increased expression of LAMP2 has been observed in PBMCs of CAD patients compared to the control group (p<0.05). Our findings supported the previous observations showing the contribution of LAMP2 in lipid homeostasis and pathogenesis of CAD.

A decline of LAMP- 2 predicts ursodeoxycholic acid response in primary biliary cirrhosis.

Biochemical response to ursodeoxycholic acid (UDCA) in patients with primary biliary cirrhosis (PBC) is variable. We have previously reported that augmented expression of lysosome-associated membrane protein 2 (LAMP-2) was correlated with the severity of PBC. This study aimed to determine whether serum LAMP-2 could serve as a predictor of biochemical response to UDCA. The efficiency of serum LAMP-2 to predict biochemical response was assessed after 1 year of UDCA treatment in PBC patients by a retrospective analysis. We found that the basal serum LAMP-2 level was increased in PBC, especially in patients with stage III-IV (p = 0.010) or TBIL > 1 mg/dL (p = 0.014). Baseline serum LAMP-2 was higher in non-responders than that in responders, but the difference was statistically insignificant. However, after UDCA treatment, serum LAMP-2 level decreased prominently in the first 3 months, which was more obvious in responders. Further studies showed that the 35% decline of LAMP-2 after treatment for 3 months could be stated as an indicator of UDCA response with the sensitivity of 62.9% and specificity of 75.0% by Paris criteria. Meanwhile the specificity and sensitivity were identified as 63.5% and 64.1% by Barcelona criteria. Together, a decline in LAMP-2 might help to predict the response to UDCA.

Reproducibility and efficiency of serum-derived exosome extraction methods.

OBJECTIVES: Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum. DESIGN AND METHODS: We used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick™. Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight™ technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94. RESULTS: Correlation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick™ is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG. CONCLUSION: ExoQuick™ is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes.

Activation of lymphocyte autophagy/apoptosis reflects haemodynamic inefficiency and functional aerobic impairment in patients with heart failure.

Lymphocytopenia is associated with an adverse prognosis in heart failure (HF). The present study investigated whether lymphocytopenia results from activated lymphocyte autophagy/apoptosis, which reflects haemodynamic inefficiency and functional aerobic impairment in patients with HF. One hundred and twenty-seven patients with HF were divided into three groups: HF with non- (lymphocytes ≥2000 cells/µl; n=45), mild (lymphocytes between ≥1500 cells/µl and <2000 cells/µl; n=39) and severe (lymphocytes <1500 cells/µl; n=43) lymphocytopenia. Lymphocyte autophagy/apoptosis, ventilatory/haemodynamic efficiencies and generic/disease-specific quality of life were analysed in these patients with HF and 35 normal counterparts. The results demonstrated that patients with HF with severe lymphocytopenia had (i) increased G-protein-coupled receptor kinase-2 (GRK-2) levels, (ii) lower mammalian target of rapamycin (mTOR) levels with higher lysosome-associated membrane protein-2 (LAMP-2) expression and Acridine Orange (AO) staining, (iii) lower mitochondrial transmembrane potential with higher caspase-3 activation and phosphatidylserine (PS) exposure, and (iv) greater extents of adrenaline (epinephrine)-induced apoptosis in lymphocytes, and higher plasma noradrenaline (norepinephrine)/adrenaline, myeloperoxidase and interleukin-6 concentrations than patients with HF without lymphocytopenia and normal counterparts did. Moreover, lymphocyte caspase-3 activation was an effect modifier, which modulated the correlation status between lymphocyte count and GRK-2 level. Lymphocyte count was positively correlated with peak cardiac output and peak oxygen consumption (VO2peak) in patients with HF. In addition, HF with lymphocytopenia was accompanied by lower Short Form-36 physical/mental component scores and increased Minnesota Living with Heart Failure Questionnaire scores. Therefore, we conclude that increased sympathetic activation and oxidative stress/pro-inflammatory status cause lymphocytopenia by activating programmed lymphocyte death in patients with HF. Moreover, a low lymphocyte count correlates with reduced haemodynamics and aerobic capacity, which reflects poor generic/disease-specific quality of life in patients with HF.

High basal level of autophagy in high-altitude residents attenuates myocardial ischemia-reperfusion injury.

OBJECTIVE: Hypoxia can induce autophagy, which plays an important role in cardioprotection. The present study tested the hypothesis that patients with congenital heart disease living at a high altitude could resist ischemia-reperfusion injury better than those at a low altitude, through elevated basal autophagy by chronic hypoxia. METHODS: Twelve Tibetan patients residing at a high altitude of >3000 m and 12 Han patients residing at a low altitude of <500 m with simple atrial or ventricular septal defects were prospectively recruited. All patients underwent cardiopulmonary bypass, maintaining a flow rate of approximately 2.4 to 2.8 L/min/m2 and mean arterial pressure of ≥40 to 60 mm Hg. Myocardial ischemia-reperfusion injury between the 2 groups was compared using cardiac troponin I, brain natriuretic peptide, hematoxylin eosin staining, and the terminal deoxynucleotidyl transferase dUTP nick end labeling test. Autophagy-related proteins microtubule-associated protein 1 light chain 3 II (LC3II), Beclin1, and lysosomal-associated membrane protein 2 (LAMP2) and their upstream protein BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) were evaluated with Western blotting. RESULTS: The maximal cardiac troponin I concentration and increasing x-fold of brain natriuretic peptide in the high-altitude group were obviously lower than those in the low-altitude group (3.10±0.77 vs 7.10±2.28 ng/mL and 2.51±0.94 vs 14.66±6.83, respectively). The preoperative and postoperative levels of LC3II, LAMP2, and upstream Bnip3 in the high-altitude group were obviously greater. No difference was found in the Beclin1 level between the 2 groups at baseline or ischemia-reperfusion. CONCLUSIONS: Patients living at a high altitude with congenital heart disease resisted ischemia-reperfusion injury during cardiac surgery better than those at a low altitude, possibly through elevated basal autophagy induced by chronic hypoxia.

Reduced expression of the chaperone-mediated autophagy carrier hsc70 protein in lymphomonocytes of patients with Parkinson's disease.

Chaperone-mediated autophagy (CMA) impairment is recognized to play a pathogenetic role in Parkinson's disease (PD). A reduced expression of lysosomal-associated membrane protein (lamp) 2A and heat shock cognate (hsc) 70 protein, the two key regulators of CMA, has been reported in brains of PD patients. To verify the existence of a possible systemic CMA dysfunction in PD, in this study the expression of hsc70 and lamp2A was assessed in peripheral blood mononuclear cells (PBMC) of patients with sporadic PD and compared to healthy subjects. The expression of myocyte enhancer factor 2D (MEF2D), a transcriptional factor implicated in neuronal survival and specific substrate of CMA, was also evaluated. Protein and gene expression was assessed by Western blot and real-time PCR, respectively, in PBMC obtained from 53 sporadic PD patients and 53 healthy subjects. A significant reduction of hsc70 levels was observed in PBMC of PD patients, both under basal conditions and after autophagy induction obtained with serum deprivation. No difference emerged in lamp2A and MEF2D expression between patients and controls. No influence of the clinical characteristics of patients emerged on hsc70, lamp2A and MEF2D expression. These results, despite being not suggestive of the existence of a CMA impairment in PBMC of PD patients, identify a systemic hsc70 reduction in PD patients. Further studies on specific mechanisms and biological significance of such alteration are needed to corroborate this finding that could lead to the identification of a new trait biomarker for PD.

Lysosomal-associated membrane protein-2 plays an important role in the pathogenesis of primary cutaneous vasculitis.

OBJECTIVES: Recent research suggests that lysosomal-associated membrane protein-2 (LAMP-2) could be one of the target antigens in the pathogenesis of vasculitides. We established a transgenic rat model, env-pX rats, with various vasculitides including cutaneous vasculitis. Human primary cutaneous vasculitis includes cutaneous polyarteritis nodosa (CPN) and Henoch-Schönlein purpura (HSP). We measured serum anti-LAMP-2 antibody levels in morbid env-pX rats and injected anti-LAMP-2 antibody into premorbid env-pX rats. We further measured serum anti-LAMP-2 antibody levels in patients with CPN and HSP. METHODS: Cutaneous vasculitis was observed in ∼30% of 6-month-old morbid env-pX rats. In contrast, these findings were rare in premorbid env-pX rats under 3 months old. We also examined 85 patients with CPN and 36 adult patients with HSP. Serum anti-LAMP-2 antibody levels were determined using ELISA. Premorbid env-pX rats under 3 months old were given an i.v. injection of anti-LAMP-2 antibody at day 0 and day 7. At day 14, these rats underwent histopathological and direct immunofluorescence examination. Cell surface LAMP-2 expression of rat neutrophils was examined by flow cytometry. RESULTS: Serum anti-LAMP-2 antibody levels were significantly higher in morbid env-pX rats than in wild-type normal rats. In addition, the levels in the cutaneous vasculitis group of morbid env-pX rats were significantly higher than the no cutaneous vasculitis group. Intravenous anti-LAMP-2 antibody injection into premorbid env-pX rats under 3 months old induced infiltration of neutrophils into cutaneous small vessels. Anti-LAMP-2 antibody-binding neutrophils were detected there. LAMP-2 expression on the cell surface of neutrophils in premorbid env-pX rats under PMA stimulation was higher compared with controls. Serum anti-LAMP-2 antibody levels in CPN and HSP were significantly higher than those of healthy controls. CONCLUSION: These data support a positive relationship between anti-LAMP-2 antibody and cutaneous vasculitis.