Circulating Markers of Necroptosis in Acute Pancreatitis.

OBJECTIVES: Necroptosis, a programmed inflammatory cell death, is involved in the pathogenesis of acute pancreatitis (AP). We compared levels of interleukin (IL)-33 (released upon necroptosis), sST2 (soluble IL-33 receptor), MLKL, RIPK1 and RIPK3 (necroptosis executioner proteins), and proinflammatory cytokines IL-6, TNF and IL-1ß at various severity categories and stages of AP. METHODS: Plasma from 20 patients with early mild AP (MAP) (symptom onset < 72 h), 7 with severe AP (SAP) without and 4 with persistent organ failure (OF) at sampling, 8 patients with late SAP and 20 healthy controls (HC) were studied by ELISAs. RESULTS: Early sST2 and IL-6 levels predicted the development of SAP and were higher in both MAP and early and late SAP than in HC. RIPK3 levels were higher than in HC in the patients who had or would later have SAP. MLKL levels were associated with the presence of OFs, particularly in the late phase, but were also higher in MAP than in HC. CONCLUSIONS: sST2, RIPK3 and IL-6 levels may have prognostic value in AP. Elevated MLKL levels are associated with OF in AP. Better understanding of necroptosis in AP pathophysiology is needed to evaluate whether inhibiting and targeting necroptosis is a potential therapeutic option in AP.

Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease.

BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. METHODS: C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. RESULTS: When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in ß-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. CONCLUSION: The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. LAY SUMMARY: There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

Receptor-Interacting Protein Kinase 3 (RIPK3) mRNA Levels Are Elevated in Blood Mononuclear Cells of Patients with Poor Prognosis of Acute-on-Chronic Hepatitis B Liver Failure.

Necroptosis refers to a programmed form of necrosis, which involves the receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). In this study, to investigate the role of necroptosis in the pathogenesis of acute-on-chronic hepatitis B liver failure (ACHBLF), we retrospectively analyzed 122 patients with ACHBLF, 131 patients with chronic hepatitis B (CHB), and 35 healthy controls (HCs). Using quantitative real-time polymerase chain reaction (RT-qPCR), we measured RIPK3 mRNA levels in peripheral blood mononuclear cells (PBMCs). ELISA was performed to measure the serum levels of MLKL, TNF-α and caspase-8. We found that RIPK3 mRNA levels were significantly higher in patients with ACHBLF than those with CHB or HCs. RIPK3 mRNA levels in patients with ACHBLF were positively correlated with serum levels of TNF-α or MLKL and negatively correlated with caspase-8 levels. Univariate and multivariate analysis revealed that RIPK3 mRNA level was predictive of 3-month mortality of ACHBLF. The area under receiver operating characteristic curve (AUC) of RIPK3 mRNA levels was 0.810 (95% CI: 0.729-0.876), which was higher than that of MELD scores (0.766, 95% CI: 0.681-0.838). The optimal cut-off point of 8.81 was determined for RIPK3 mRNA levels, which showed a sensitivity of 80.7% and a negative predictive value of 80.4%. These results indicate that elevated RIPK3 mRNA levels in PBMCs are associated with poor prognosis of ACHBLF. We thus propose that necroptosis may play an important role in pathogenesis of ACHBLF.

Excessive training induces molecular signs of pathologic cardiac hypertrophy.

Chronic exercise induces cardiac remodeling that promotes left ventricular hypertrophy and cardiac functional improvement, which are mediated by the mammalian or the mechanistic target of rapamycin (mTOR) as well as by the androgen and glucocorticoid receptors (GRs). However, pathological conditions (i.e., chronic heart failure, hypertension, and aortic stenosis, etc.) also induce cardiac hypertrophy, but with detrimental function, high levels of proinflammatory cytokines and myostatin, elevated fibrosis, reduced adenosine monophosphate-activated protein kinase (AMPK) activation, and fetal gene reactivation. Furthermore, recent studies have evidenced that excessive training induced an inflammatory status in the serum, muscle, hypothalamus, and liver, suggesting a pathological condition that could also be detrimental to cardiac tissue. Here, we verified the effects of three running overtraining (OT) models on the molecular parameters related to physiological and pathological cardiac hypertrophy. C57BL/6 mice performed three different OT protocols and were evaluated for molecular parameters related to physiological and pathological cardiac hypertrophy, including immunoblotting, reverse transcription polymerase chain reaction, histology, and immunohistochemistry analyses. In summary, the three OT protocols induced left ventricle (LV) hypertrophy with signs of cardiac fibrosis and negative morphological adaptations. These maladaptations were accompanied by reductions in AMPKalpha (Thr172) phosphorylation, androgen receptor, and GR expressions, as well as by an increase in interleukin-6 expression. Specifically, the downhill running-based OT model reduced the content of some proteins related to the mTOR signaling pathway and upregulated the ß-isoform of myosin heavy-chain gene expression, presenting signs of LV pathological hypertrophy development.

Low Necroptosis Process Predicts Poor Treatment Outcome of Human Papillomavirus Positive Cervical Cancers by Decreasing Tumor-Associated Macrophages M1 Polarization.

BACKGROUND/AIM: The aim of this study was to assess the functions of the necroptosis process on the prognosis of high-risk human papillomavirus (HR-HPV)-related cervical cancer. METHODS: PCR and western blotting were used to demonstrate the expression of the necroptosis marker, mixed lineage kinase domain-like protein (MLKL), in whole blood and peripheral blood mononuclears (PBMCs) of 89 cervical cancer patients and 15 healthy volunteers. Necroptosis levels and M1 polarization were determined in tumor co-cultured macrophages. RESULTS: We found that MLKL expressions were significantly increased in cervical cancer patients in both whole blood and PBMC samples compared to the expressions in the healthy controls. Low MLKL expression was significantly associated with decreased survival rate in overall survival and disease-free survival. Co-culture cervical cancer cells decrease the necroptosis process of macrophage, together with the proinflammatory factors (M1 markers) downregulation, and this negative regulation was exacerbated in HPV-positive cases. Necroptosis enhancer RIPK3 overexpression showed reversed regulation of these M1 markers, suggesting that co-culture cervical cancer cells decrease the macrophage M1 polarization partly through necroptosis downregulation. CONCLUSION: Our study revealed that necroptosis process could be a relevant marker for the determination of the prognosis in cervical cancer patients, which might be because of its role in regulating macrophage polarization.

p38MAPK is involved in apoptosis development in apheresis platelet concentrates after riboflavin and ultraviolet light treatment.

BACKGROUND: Pathogen inactivation (PI) accelerates the platelet (PLT) storage lesion, including apoptotic-like changes. Proteomic studies have shown that phosphorylation levels of several kinases increase in PLTs after riboflavin and UV light (RF-PI) treatment. Inhibition of p38MAPK improved in vitro PLT quality, but the biochemical basis of this kinase's contribution to PLT damage requires further analysis. STUDY DESIGN AND METHODS: In a pool-and-split design, apheresis PLT concentrates were either treated or kept untreated with or without selected kinase inhibitors. Samples were analyzed throughout 7 days of storage, monitoring in vitro quality variables including phosphatidylserine exposure, degranulation, and glucose metabolism. Changes in the protein expression of Bax, Bak, and Bcl-xL and the activities of caspase-3 and -9 were determined by immunoblot analysis and flow cytometry, respectively. RESULTS: The expression levels of the proapoptotic proteins Bax and Bak, but not the antiapoptotic protein Bcl-xL, were significantly increased after the RF-PI treatment. This trend was reversed in the presence of p38MAPK inhibitor SB203580. As a result of increasing proapoptotic protein levels, caspase-3 and -9 activities were significantly increased in RF-PI treatment during storage compared with control (p < 0.05). Similarly, p38MAPK inhibition significantly reduced these caspase activities compared with vehicle control after RF-PI treatment (p < 0.05). CONCLUSION: These findings revealed that p38MAPK is involved in signaling leading to apoptosis triggered by RF-PI. Elucidation of the biochemical processes influenced by PI is a necessary step in the development of strategies to improve the PLT quality and ameliorate the negative effects of PI treatment.

Phosphorylation of protein S by platelet kinases enhances its activated protein C cofactor activity.

Protein S (PS) is a multifunctional plasma protein of the hemostatic and inflammatory pathways, although mechanisms for its regulation are poorly understood. Since certain plasma proteins are regulated through extracellular phosphorylation, we investigated whether the anticoagulant activity of PS is regulated through phosphorylation by platelet-secreted kinases. PS was phosphorylated on exposure to activated platelets or their releasates, as judged by immunoblotting for phospho-amino acids and PS. PS phosphorylation was reduced by specific inhibitors of casein kinase 1 (CK1) and casein kinase 2 (CK2) (10 µM D4476, 100 µM CK2-inhibitory peptide YNLKSKSSEDIDESS). Involvement of CKs in PS phosphorylation was confirmed using purified CK1/CK2. Phosphorylation of PS by purified CK1 did not affect its activated protein C (APC) cofactor activity in activated partial thromboplastin time assays in PS-depleted plasma. However, phosphorylation of PS by CK2 or by CK1/CK2 increased PS cofactor activity ∼1.5-fold (158.7±4.8%, P<0.01) or ∼2-fold (191.5±6.4%, P<0.0001), respectively. The APC cofactor activity of PS in PS-depleted plasma exposed to platelet-secreted kinases was enhanced, while CK2 but not CK1 inhibitors reduced APC cofactor activity. Mass spectrometry revealed a phosphorylated CK2 site at Thr37 within the N-terminal Gla-domain. Thus, platelet-mediated extracellular phosphorylation of PS is a potential mechanism by which its activity is regulated.

Serum HMGB-1 released by ferroptosis and necroptosis as a novel potential biomarker for systemic lupus erythematosus.

High mobility group box proterin-1 (HMGB-1) is a multifunctional protein that can be released by various programmed cell deaths (PCDs), such as necroptosis and ferroptosis. PCDs play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). However, the role of HMGB-1 in the process of SLE remains unclear. This study aims to demonstrate the potential diagnosing role of serum HMGB-1 in SLE that released by necroptosis and ferroptosis. We found that the serum levels of HMGB-1, receptor-interacting protein kinase 3 (RIPK3) /mixed lineage kinase domain-like protein (MLKL) related with necroptosis, and metabolites associated with ferroptosis were significantly upregulated in SLE patients compared to HC individuals. These serum levels were positively correlated with SLE disease activity. Additionally, the serum level of HMGB-1 showed a strong positive correlated with the levels of RIPK3/MLKL and ferroptosis metabolites. Moreover, the serum level of HMGB-1 was correlated with renal involvement and high-antinuclear antibodies (ANA) titer. After SLE serum and interferon γ (IFN-γ) treatment in vitro, the level of necroptosis and ferroptosis markers were activated and HMGB1 was released both in HEK293 and HK2 cells. Clinically, HMGB-1 was considered as a significant independent risk factor in SLE serum by binary logistic assay. Notably, HMGB-1 exhibited outstanding diagnostic ability for SLE by the area under the curve (AUC) in receiver operating characteristic (ROC) curve analysis. Taken together, our study indicates that the serum level of HMGB-1 is a promising biomarker for the diagnosis and monitoring of SLE.

[Plasma proteomic research on obesity subtype and non-obesity subtype of T2DM].

OBJECTIVE: With the increasingly serious epidemic situation of diabetes, plasma proteomic method and OFFGEL electrophoresis have been applied for screening different proteins between obese and non-obese T2DM patients, which may be used to further explain the mechanism of T2DM. METHODS: Twenty male T2DM volunteers (Obesity Subtype: 10; Non-obesity Subtype: 10) with the age of 18-44 years have been selected. The control group has been matched considering the factors of age, gender, etc. Albumin and IgG were removed from the plasma samples with highly specific immune-affinity method. Then the peptide-mixed samples were separated by pI with OFFGEL electrophoresis after solution digestion. Further separation and identification were performed by Nano HPLC-Chip-MS/MS system. Comparing the three groups, the differences were obtained and annotated on functions and its mechanism. RESULTS: 391, 415 and 371 proteins have been identified in the experimental groups and control group, respectively. The different proteins in groups and their annotations showed that adiponectin was down-regulated in obesity subtype of T2DM group, while STIM1 (stromal interaction molecule 1) was up-regulated. There were six protein kinases high expression in non-obesity DM patients, such as Serine-protein kinase ATM, Serine/threonine-protein kinase WNK1. CONCLUSION: Adiponectin, STIM1 and protein kinases may act in different roles on the pathogenesis of obesity subtype and non-obesity subtype of T2DM.

Serum protein levels of brain-derived neurotrophic factor and tropomyosin-related kinase B in bipolar disorder: effects of mood stabilizers.

AIM: In this study, we investigated serum protein levels of brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) in patients with bipolar disorder. METHODS: Over a 2-year period, 26 patients with bipolar I disorder (manic episode) and 56 healthy controls were recruited. The Young Mania Rating Scale scores of patients with bipolar mania were >26. Serum BDNF and TrkB protein levels were measured with ELISA kits. RESULTS: Using ANCOVA with age adjustment, we found that there were no significant differences in serum BDNF protein levels between patients with bipolar mania and healthy controls (p = 0.582). In contrast, the serum TrkB protein level was significantly higher in bipolar mania patients than in healthy controls (p = 0.001), especially in women (p = 0.001). Of 26 patients with bipolar mania, 21 underwent a second measurement of serum BDNF and TrkB protein levels after a 4-week treatment with mood stabilizers. There were no significant changes in serum BDNF or TrkB protein levels. CONCLUSION: These findings suggest that serum TrkB protein levels may play an important role in the psychopathology of bipolar mania. However, a larger sample size is needed to confirm these results.

Platelet reactivity in chronic venous insufficiency.

BACKGROUND: Chronic venous insufficiency (CVI) is a common medical problem that may result in significant morbidity and mortality. Platelets are key players in haemostasis and thrombosis, but their role in the development of venous thrombosis is more controversial. AIM: The purpose of this study was to investigate platelet properties in CVI and their interaction with the venular endothelium. METHODS: Human peripheral venules were explanted during leg surgery of patients with CVI and of healthy subjects (C); concurrently, the platelets were isolated from blood samples collected. The techniques used were: fluorescence and electron microscopy and Western-blotting. RESULTS: Compared with the C group, the platelets of patients with CVI are activated, as demonstrated by: (i) cellular modifications, such as alteration of the discoidal shape by the development of extended cytoplasmic filopodia and changes of the cells normal ultrastructure, (ii) biochemical modifications, such as the enhanced protein levels of FAK, p85 PI3K, Akt and src, accounting for activation of alphaIIbbeta3 outside-in signaling, and (iii) apparent higher adhesion to the venular endothelium. We demonstrate in addition, that CVI is accompanied by severe modifications of the ultrastructure of the cells within the venular wall. CONCLUSIONS: In CVI, platelets circulate in an activated state and may contribute to the altered dysfunctional response of the venous wall and to the development of this pathology.

Higher tumor protein kinase D1 correlates with increased tumor size, BCLC stage, CA199 level, AFP level and worse overall survival in hepatocellular carcinoma patients.

OBJECTIVE: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Protein kinase D1 (PKD1) is recognized as a key regulator in the progression in several solid cancers, while its clinical role in HCC is unclear. This study aimed to evaluate the correlation of PKD1 with clinical features and prognosis in HCC patients. METHODS: A total of 218 HCC patients who underwent resection were retrospectively enrolled. PKD1 expression in tumor (N = 218) and adjacent (N = 110) tissues was detected by immunohistochemical staining, scored by a semi-quantitative scoring method ranging from 0 to 12, and further classified as PKD1-, PKD1+, PKD1++ and PKD1+++ for analysis. Meanwhile, patients' clinical features and survival data were acquired from the database. RESULTS: PKD1 was elevated in tumor tissues compared with adjacent tissues. Meanwhile, higher tumor PKD1 was correlated with elevated tumor size, Barcelona Clinic Liver Cancer (BCLC) stage, carbohydrate antigen 199 (CA199) level and alpha fetoprotein (AFP) level; while no correlation was found in tumor PKD1 with patients' basic features or liver function indexes. Moreover, higher tumor PKD1 was correlated with worse overall survival (OS) in HCC patients, then further validated as an independent predictive factor for worse OS by multivariate Cox's regression model analysis. Additionally, in Child-Pugh stage A, Child-Pugh stage B, BCLC stage 0/A, and BCLC stage B subgroups, higher tumor PKD1 was also correlated with worse OS. CONCLUSION: Higher PKD1 in tumor tissues correlates with elevated BCLC stage, bigger tumor size, increased CA199 level, higher AFP level and worse OS in HCC patients.

Potential of PINK1 and PARKIN Proteins as Biomarkers for Active Multiple Sclerosis: A Japanese Cohort Study.

Background: Mitochondrial dysfunction has been suggested to play an important role in all stages of multiple sclerosis (MS). Objective: To determine the expression of two mitophagy-related proteins, PTEN-induced kinase 1 (PINK1) and PARKIN, in a cohort of Japanese patients with different neuroinflammatory disorders. Methods: Protein concentrations were measured using commercial ELISA in paired cerebrospinal fluid (CSF) and serum samples from patients with multiple sclerosis (MS), neuromyelitis optica spectrum disorders (NMOSD), and myelin oligodendrocyte glycoprotein antibody disorders (MOGAD), and from age- and sex-matched controls. Results: CSF and serum concentrations of PINK1 were higher in patients with MS than in patients with NMOSD (p = 0.004 and p < 0.001, respectively), MOGAD (p = 0.008 and p = 0.011, respectively), and controls (p = 0.021 and p = 0.002, respectively). CSF and concentrations of PARKIN were elevated in patients with MS in comparison with those in controls (p = 0.016 and p = 0.05, respectively). Conclusions: Our study highlighted the importance of mitophagy in MS and suggested the potential application of PINK1 and PARKIN as biomarkers to predict disease activity.

Altered Transcriptional Profile of Mitochondrial DNA-Encoded OXPHOS Subunits, Mitochondria Quality Control Genes, and Intracellular ATP Levels in Blood Samples of Patients with Parkinson's Disease.

Mitochondrial dysfunctions are significant contributors to neurodegeneration. One result or a cause of mitochondrial dysfunction might be the disruption of mtDNA transcription. Limited data indicated an altered expression of mtDNA encoded transcripts in Alzheimer's disease (AD) or Parkinson's disease (PD). The number of mitochondria is high in cells with a high energy demand, such as muscle or nerve cells. AD or PD involves increased risk of cardiomyopathy, suggesting that mitochondrial dysfunction might be systemic. If it is systemic, we should observe it in different cell types. Given that, we wanted to investigate any disruption in the regulation of mtDNA encoded gene expression in addition to PINK1, PARKIN, and ATP levels in peripheral blood samples of PD cases who are affected by a neurodegenerative disorder that is very well known by its mitochondrial aspects. Our results showed for the first time that: 1) age of onset > 50 PD sporadic (PDS) cases: mtDNA transcription and quality control genes were affected; 2) age of onset <50 PDS cases: only mtDNA transcription was affected; and 3) PD cases with familial background: only quality control genes were affected. mtDNA copy number was not a confounder. Intracellular ATP levels of PD case subgroups were significantly higher than those of healthy subjects. We suggest that a systemic dysregulation of transcription of mtDNA or mitochondrial quality control genes might result in the development of a sporadic form of the disease. Additionally, ATP elevation might be an independent compensatory and response mechanism. Hyperactive cells in AD and PD require further investigation.

Low-temperature induction of calcium-dependent protein phosphorylation in blood platelets.

Exposure to low temperature causes platelets to change shape in a manner similar to the shape change that precedes secretagogue-induced serotonin release. Previous studies have shown that two proteins, of approximately 20,000 and approximately 40,000 Mr, become phosphorylated before secretion. We have investigated whether low temperature can induce phosphorylation of these proteins and/or serotonin secretion. The data indicate that low-temperature-induced shape change has no requirement for extracellular calcium, whereas phosphorylation of the two proteins and subsequent serotonin release both have strong calcium requirements. Because cold treatment is thought to influence platelet shape through an effect on microtubules, the events in the shape change-release sequence would seem to be ordered as follows: microtubule disassembly leads to shape change leads to protein phosphorylation leads to secretion.

Peripheral proteins and smooth membrane from erythrocyte ghosts. Segregation of ATP-utilizing enzymes into smooth membrane.

Erythrocytes and their isolated membranes display ATP-dependent endocytosis. To localize the enzymes responsible for this phenomenon, the erythrocyte membranes (ghosts) were fractionated under conditions which retained ATPase activity. Fractionation of the ghosts resulted in three fractions: spectrin-actin, the peripheral proteins soluble in high salt, and the smooth membrane containing integral proteins. On the average, 87% of the protein and 88% of the phosphorus of the original ghosts were recovered in these fractions, and all of the kinds of ATP-splitting activities of the membrane were recovered in the smooth membrane. A tiny ATPase activity, detectable by special methodology in spectrinactin, could have been due to contamination with membranous material. Although the purified spectrin-actin did not have a significant ATPase of its own, it stimulated the Ca2+, Mg2+-ATPase of the smooth membrane significantly, suggesting a cooperative interaction between these two fractions. This segregation of the ATPase activities into the smooth membrane, combined with the energy dependence of endocytosis, showed that the smooth membrane must be involved in the energy production for endocytosis. The possibility that the spectrin-actin filaments cooperate with a myosinlike ATPase in the membrane to generate membrane movements is discussed.

Biophysical properties and microfilament assembly in neutrophils: modulation by cyclic AMP.

The microfilament lattice, composed primarily of filamentous (F)-actin, determines in large part the mechanical (deformability) properties of neutrophils, and thus may regulate the ability of neutrophils to transit a microvascular bed. Circulating factors may stimulate the neutrophil to become rigid and therefore be retained in the capillaries. We hypothesized that cell stiffening might be attenuated by an increase in intracellular cAMP. A combination of cell filtration and cell poking (mechanical indentation) was used to measure cell deformability. Neutrophils pretreated with dibutyryl cAMP (db-cAMP) or the combination of prostaglandin E2 (PGE2, a stimulator of adenylate cyclase) and isobutylmethylxanthine (IBMX, an inhibitor of phosphodiesterase) demonstrated significant inhibition of the n-formyl-methionyl-leucyl-phenylalanine (fMLP)-inducing stiffening. The inhibition of cell stiffening was associated with an increase in intracellular cAMP as measured by enzyme-linked immunoassay (EIA) and an increase in the activity of the cAMP-dependent kinase (A-kinase). Treatment with PGE2 and IBMX also resulted in a decrease in the F-actin content of stimulated neutrophils as assayed by NBD-phallacidin staining and flow cytometry or by changes in right angle light scattering. Direct addition of cAMP to electropermeabilized neutrophils resulted in attenuation of fMLP-induced actin assembly. Neutrophils stimulated with fMLP demonstrated a rapid redistribution of F-actin from a diffuse cortical location to a peripheral ring as assessed by conventional and scanning confocal fluorescence microscopy. Pretreatment of neutrophils with the combination of IBMX and PGE2 resulted in incomplete development and fragmentation of the cortical ring. We conclude that assembly and redistribution of F-actin may be responsible for cell stiffening after exposure to stimulants and that this response was attenuated by agents that increase intracellular cAMP, by altering the amount and spatial organization of the microfilament component of the cytoskeleton.

Myocardial 123metaiodobenzylguanidine uptake in genetic Parkinson's disease.

Myocardial (123)Metaiodobenzylguanidine (MIBG) enables the assessment of postganglionic sympathetic cardiac innervation. MIBG uptake is decreased in nearly all patients with Parkinson's disease (PD). Our objective was to evaluate MIBG uptake in patients with genetic PD. We investigated MIBG uptake in 14 patients with PD associated with mutations in different genes (Parkin, DJ-1, PINK1, and leucine-rich repeat kinase 2 -LRRK2), in 15 patients with idiopathic PD, and 10 control subjects. The myocardial MIGB uptake was preserved in 3 of the 4 Parkin-associated Parkinsonisms, in 1 of the 2 patients with DJ-1 mutations, in 1 of the 2 brothers with PINK1 mutations, in 3 of the 6 unrelated patients with Gly2019Ser mutation in the LRRK2 gene, whereas it was impaired in all patients with idiopathic PD. MIBG was preserved in all control subjects. Our study shows that myocardial MIGB uptake was normal in 8 of 14 patients with genetic PD, suggesting that cardiac sympathetic denervation occurs less frequently in genetic PD than in idiopathic PD. Our findings also demonstrate that MIGB uptake has a heterogeneous pattern in genetic PD, because it was differently impaired in patients with different mutations in the same gene or with the same gene mutation.

Early diagnosis of maple syrup urine disease using polymerase chain reaction-based mutation detection.

BACKGROUND: Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defective activity of the branched-chain alpha-ketoacid dehydrogenase enzyme complex. Early diagnosis and management of MSUD are imperative for preventing permanent neurological impairments. In the Philippines, a 4.7 kb deletion in the dihydrolipoamide branched-chain transacylase E2 (DBT) gene has been commonly identified in MSUD patients. Polymerase chain reaction (PCR) amplification of a junction fragment between intron 10 and exon 11 has been used to detect this deletion. The purpose of the present paper was to use PCR-based mutation detection of the deletion mutation to diagnose MSUD in neonates in order to provide proper diagnosis and effective treatment. METHODS: A region encompassing exon 11 and the junction fragment of the E2 (DBT) gene was PCR amplified from genomic DNA prepared from two neonates at risk for MSUD. RESULTS: PCR amplification of both exon 11 and the junction fragment from one of the neonates demonstrated that this case was a heterozygous carrier of the deletion. Thus, normal feeding was started. For the other neonate, PCR amplification of the junction fragment was successful, whereas the region encompassing exon 11 was not amplified. This neonate was genotyped as homozygous for the deletion, and treatment for MSUD was provided immediately. CONCLUSION: Examination of the deletion mutation in the E2 (DBT) gene facilitated early MSUD diagnosis and was beneficial for the determination of the proper course of treatment.

Elevated Serum Levels of Mixed Lineage Kinase Domain-Like Protein Predict Survival of Patients during Intensive Care Unit Treatment.

Mixed lineage kinase domain-like (MLKL), a crucial regulator of necroptotic cell death, was shown to play a role in inflammatory diseases. However, its role as a biomarker in critical illness and sepsis is currently unknown. We analyzed serum levels of MLKL in 136 critically ill patients at admission to the intensive care unit (ICU) and after three days of ICU treatment. Results were compared with 36 healthy controls and correlated with clinical and laboratory patients' data. MLKL serum levels of critically ill patients at admission to the ICU were similar compared to healthy controls. At ICU admission, MLKL serum concentrations were independent of disease severity, presence of sepsis, and etiology of critical illness. In contrast, median serum levels of MLKL after three days of ICU treatment were significantly lower compared to those at admission to the ICU. While serum levels of MLKL at admission were not predictive for short-term survival during ICU treatment, elevated MLKL concentrations at day three were an independent negative predictor of patients' ICU survival. Thus, elevated MLKL levels after three days of ICU treatment were predictive for patients' mortality, indicating that sustained deregulated cell death is associated with an adverse prognosis in critical illness.