Plasma Proteins In Patients Research Articles

Early Diagnosis of Herpes Zoster Neuralgia: A Narrative Review.

Early intervention reduces the incidence of postherpetic neuralgia (PHN). Typical shingles are easy to diagnose; however, there is no clear diagnostic method for neuralgia symptoms manifested before the onset of the rash, which can easily cause misdiagnosis. This not only increases the patient's pain, medical expenses, and mental burden, but more importantly, delays the valuable time for early treatment of shingles, and increases the probability of complications and PHN. In this paper, the diagnostic methods of preherpetic neuralgia were summarized and analyzed, and the current challenges were put forward to provide directions for the early diagnosis of herpes zoster (HZ) in the future. PubMed, and China National Knowledge Infrastructure (CNKI) libraries were searched using the terms "herpes zoster," "before the blistering," "diagnosis," and "neuralgia." Clinical trials, reviews, and case reports were collected and reviewed. The period of literature search is from 1 January 1980 to 1 October 2022. The early diagnosis of herpes zoster neuralgia can reduce misdiagnosis and mistreatment, and timely and effective intervention can significantly reduce the incidence of PHN. The body may possess a mechanism that limits the local breakthrough of the virus in the skin, causing blistering later than the onset of pain. Changes in the plasma proteins of patients with varicella-zoster virus shingles neuralgia may be used as an early diagnostic indicator in patients with HZ neuralgia before eruption. Early diagnosis of HZ neuralgia before eruption can facilitate timely targeted treatment, thereby reducing the incidence of PHN. Proteomic quantitative analysis and validation results can serve as a simple, micro, rapid, and accurate diagnostic method.

Plasma host protein signatures correlating with Mycobacterium tuberculosis activity prior to and during antituberculosis treatment

There is a need for rapid non-sputum-based tests to identify and treat patients infected with Mycobacterium tuberculosis (Mtb). The overall objective of this study was to measure and compare the expression of a selected panel of human plasma proteins in patients with active pulmonary tuberculosis (ATB) throughout anti-TB treatment (from baseline to the end of treatment), in Mtb-infected individuals (TBI) and healthy donors (HD) to identify a putative host-protein signature useful for both TB diagnosis and treatment monitoring. A panel of seven human host proteins CLEC3B, SELL, IGFBP3, IP10, CD14, ECM1 and C1Q were measured in the plasma isolated from an HIV-negative prospective cohort of 37 ATB, 24 TBI and 23 HD. The protein signatures were assessed using a Luminex xMAP® to quantify the plasmatic levels in unstimulated blood of the different clinical group as well as the protein levels at baseline and at three timepoints during the 6-months ATB treatment, to compare the plasma protein levels between culture slow and fast converters that may contribute to monitor the TB treatment outcome. Protein signatures were defined using the CombiROC algorithm and multivariate models. The studied plasma host proteins showed different levels between the clinical groups and during the TB treatment. Six of the plasma proteins (CLEC3B, SELL, IGFBP3, IP10, CD14 and C1Q) showed significant differences in normalised median fluorescence intensities when comparing ATB vs HD or TBI groups while ECM1 revealed a significant difference between fast and slow sputum culture converters after 2 months following treatment (p = 0.006). The expression of a four-host protein markers (CLEC3B-ECM1-IP10-SELL) was significantly different between ATB from HD or TBI groups (respectively, p < 0.05). The expression of the same signature was significantly different between the slow vs the fast sputum culture converters after 2 months of treatment (p < 0.05). The results suggest a promising 4 host-plasma marker signature that would be associated with both TB diagnostic and treatment monitoring.

Altered Plasma Proteins in Myogenous Temporomandibular Disorders.

The aims of this study were (1) to compare the levels and interactions of several plasma proteins in patients with myogenous temporomandibular disorders (TMDM) compared to healthy and pain-free controls, (2) to compare the levels and interactions in two TMDM subgroups, myalgia (MYA) and myofascial pain (MFP), and (3) to explore associations between the proteins and clinical data. Thirty-nine patients with TMDM (MFP, n = 25, MYA, n = 14), diagnosed according to the diagnostic criteria for TMD (DC/TMD), aged 38 years, and sex-matched pain-free controls completed an extended DC/TMD Axis II questionnaire and the plasma concentration of 87 biomarkers were analyzed. Nine proteins separated TMDM from controls (p = 0.0174) and 12 proteins separated MYA from MFP (p = 0.019). Pain duration, characteristic pain intensity, pain catastrophizing, perceived stress, and insomnia severity were significantly associated with protein markers (p < 0.001 to p < 0.022). In conclusion, several plasma proteins were upregulated in TMDM and either upregulated or downregulated in MYA compared to MFP. Some proteins in TMDM were associated with pain variables, sleep disturbance, and emotional function. These results show that systemic differences in protein expression exist in patients with TMDM and that altered levels of specific plasma proteins are associated with different clinical variables.

Using the Plasma Proteome for Risk Stratifying Patients with Pulmonary Arterial Hypertension.

Rationale: NT-proBNP (N-terminal pro-brain natriuretic peptide), a biomarker of cardiac origin, is used to risk stratify patients with pulmonary arterial hypertension (PAH). Its limitations include poor sensitivity to early vascular pathology. Other biomarkers of vascular or systemic origin may also be useful in the management of PAH. Objectives: Identify prognostic proteins in PAH that complement NT-proBNP and clinical risk scores. Methods: An aptamer-based assay (SomaScan version 4) targeting 4,152 proteins was used to measure plasma proteins in patients with idiopathic, heritable, or drug-induced PAH from the UK National Cohort of PAH (n = 357) and the French EFORT (Evaluation of Prognostic Factors and Therapeutic Targets in PAH) study (n = 79). Prognostic proteins were identified in discovery-replication analyses of UK samples. Proteins independent of 6-minute-walk distance and NT-proBNP entered least absolute shrinkage and selection operator modeling, and the best combination in a single score was evaluated against clinical targets in EFORT. Measurements and Main Results: Thirty-one proteins robustly informed prognosis independent of NT-proBNP and 6-minute-walk distance in the UK cohort. A weighted combination score of six proteins was validated at baseline (5-yr mortality; area under the curve [AUC], 0.73; 95% confidence interval [CI], 0.63-0.85) and follow-up in EFORT (AUC, 0.84; 95% CI, 0.75-0.94; P = 9.96 × 10-6). The protein score risk stratified patients independent of established clinical targets and risk equations. The addition of the six-protein model score to NT-proBNP improved prediction of 5-year outcomes from AUC 0.762 (0.702-0.821) to 0.818 (0.767-0.869) by receiver operating characteristic analysis (P = 0.00426 for difference in AUC) in the UK replication and French samples combined. Conclusions: The plasma proteome informs prognosis beyond established factors in PAH and may provide a more sensitive measure of therapeutic response.

Hepatitis B e antigen loss in adults and children with chronic hepatitis B living in North America: A prospective cohort study.

Hepatitis B e antigen (HBeAg) is a soluble viral protein in plasma of patients with hepatitis B virus infection. HBeAg loss is an important first stage of viral antigen clearance. We determined the rate and predictors of HBeAg loss in a North American cohort with chronic hepatitis B viral infection (CHB). Among children and adults with CHB and without HIV, HCV or HDV co-infection enrolled in the Hepatitis B Research Network prospective cohort studies, 819 were HBeAg positive at their first assessment (treatment naïve or >24weeks since treatment). Of these, 577 (200 children, 377 adults) were followed every 24-48weeks. HBeAg loss was defined as first HBeAg-negative value; sustained HBeAg loss was defined as ≥2 consecutive HBeAg-negative values ≥24weeks apart. During a median follow-up of 1.8years, 164 participants experienced HBeAg loss, a rate of 11.4 (95% CI, 9.8-13.3) per 100 person-years. After adjustment for confounders, HBeAg loss rate was significantly higher in males than females, in older than younger individuals, in Whites or Blacks than Asians, in those with genotype A2 or B versus C, and in those with basal core promoter/pre-core mutations versus wild type. Additionally, during follow-up, an ALT flare and a lower quantitative HBsAg, quantitative HBeAg or HBV DNA level predicted higher rates of HBeAg loss. The majority (88%) with HBeAg loss had sustained HBeAg loss. In conclusion, a number of specific demographic, clinical and viral characteristics impacted rate of HBeAg loss and may prove useful in design and interpretation of future therapeutic studies.

A Plasma Protein Biomarker Strategy for Detection of Small Intestinal Neuroendocrine Tumors

Background: Small intestinal neuroendocrine tumors (SI-NETs) are difficult to diagnose in the early stage of disease. Current blood biomarkers such as chromogranin A (CgA) and 5-hydroxyindolacetic acid have low sensitivity (SEN) and specificity (SPE). This is a first preplanned interim analysis (Nordic non-interventional, prospective, exploratory, EXPLAIN study [NCT02630654]). Its objective is to investigate if a plasma protein multi-biomarker strategy can improve diagnostic accuracy (ACC) in SI-NETs. Methods: At the time of diagnosis, before any disease-specific treatment was initiated, blood was collected from patients with advanced SI-NETs and 92 putative cancer-related plasma proteins from 135 patients were analyzed and compared with the results of age- and sex-matched controls (n = 143), using multiplex proximity extension assay and machine learning techniques. Results: Using a random forest model including 12 top ranked plasma proteins in patients with SI-NETs, the multi-biomarker strategy showed SEN and SPE of 89 and 91%, respectively, with negative predictive value (NPV) and positive predictive value (PPV) of 90 and 91%, respectively, to identify patients with regional or metastatic disease with an area under the receiver operator characteristic curve (AUROC) of 99%. In 30 patients with normal CgA concentrations, the model provided a diagnostic SPE of 98%, SEN of 56%, and NPV 90%, PPV of 90%, and AUROC 97%, regardless of proton pump inhibitor intake. Conclusion: This interim analysis demonstrates that a multi-biomarker/machine learning strategy improves diagnostic ACC of patients with SI-NET at the time of diagnosis, especially in patients with normal CgA levels. The results indicate that this multi-biomarker strategy can be useful for early detection of SI-NETs at presentation and conceivably detect recurrence after radical primary resection.

Characterization of differentially expressed plasma proteins in patients with acute myocardial infarction

Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality worldwide. Novel biomarkers are needed to identify NSTEMI in AMI patients. The study objective was to use proteomics to identify novel plasma biomarkers for STEMI and NSTEMI patients. iTRAQ analysis was performed on pooled samples from 8 healthy controls and 12 STEMI and 12 NSTEMI patients. Bioinformatics analysis identified 95 differentially expressed proteins that were differentially expressed in the plasma of AMI patients and healthy controls; 28 of these proteins were found in STEMI/Con (22 upregulated and 6 downregulated), 48 in NSTEMI/Con (12 upregulated and 36 downregulated), and 44 in NSTEMI/STEMI (11 upregulated and 33 downregulated). Protein network analysis was then performed using STRING software. Functional analysis revealed that the identified plasma proteins were mainly involved with carbon metabolism, toll-like receptor signaling pathway, and hypertrophic cardiomyopathy. Nine of the proteins (SSA1, MDH1, FCN2, GPI, S100A8, LBP, vinculin, VDBP, and RBP4) that changed levels during AMI progression were further validated by ELISA. The constructed plasma proteome could reflect the AMI pathogenesis molecular mechanisms and provide a method for the early identification of NSTEMI in AMI patients. SignificanceThe aim of this study was to use proteomics to identify novel predictive plasma biomarkers for patients with acute myocardial infarction (AMI), which would allow for either identification of individuals at risk of an infarction, and early identification of NSTEMI in patients with AMI. Using an approach that combined iTRAQ with LC-MS/MS, we found 95 proteins that showed significant differences in expression levels among the AMI patients and healthy controls. The proteins SSA1, MDH1, FCN2, GPI, S100A8, LBP, vinculin, VDBP, and RBP4 were found to play crucial roles in the pathogenesis of AMI. Using bioinformatics analysis, we found that dysregulation of carbon metabolism, toll-like receptor signaling pathway, and hypertrophic cardiomyopathy may be the major driving forces for cardiac damage during myocardial infarction. However, further investigations are needed to verify the mechanisms involved in the development of AMI especially NSTEMI. Taken together, our findings lay the foundation for understanding the molecular mechanisms underlying the pathogenic processes of AMI, and suggest potential applications for specific biomarkers in early diagnosis and determination of prognosis.

Comparative proteomics analysis of plasma protein in patients with neuropsychiatric systemic lupus erythematosus.

BackgroundThe aim of this study was to evaluate serum biomarkers of systemic lupus erythematous (SLE) patients, with and without neuropsychiatric (NP) manifestation by high-resolution proteomic analysis.MethodsSLE patients with NP (NPSLE, n=9), SLE patients without NP (non-NPSLE, n=9) and healthy controls (HC, n=9) were prospectively enrolled in this study, and their plasma samples were collected and pooled into 3 NPSLE, 3 non-NPSLE and 3 HC samples for discovery profile. The TMT-LC-MS/MS-based proteomics approach was used to identify the differential proteome among the three matched groups, and the data were analyzed by bioinformatics tools, including Gene Ontology (GO) categories, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, to explore canonical pathways and networks involved in the pathogenesis of NPSLE. To validation of differentially expressed proteomics results, four proteins were measured by ELISA.ResultsThere were altogether 223 differentially expressed proteins in NPSLE groups compared with healthy controls (HC), of which 96 proteins increased while 127 proteins decreased. Compared with non-NPSLE, there were only 49 differentially expressed proteins in NPSLE groups, of which 37 proteins increased while 12 proteins decreased. The significantly changed pathway that those proteins are involved in was complement and coagulation cascades in NPSLE group compared with health controls. However, we didn’t find significantly changed pathway between NPSLE group and non-NPSLE group. Five proteins were found significantly changed in all group-comparisons with consistent tendencies using Venn analysis, including Vitamin D binding protein (VDBP), C-reactive protein (CRP), KRT16, IGHV4-4 and CTRP3. Four proteins including CTRP3, VDBP, PAPPA and TRYP2 were selected to estimate the validity of the proteomics approach by ELISA. The expression levels of CTRP3 and TRYP2 were significantly changed in NPSLE patients compared with either HC or non-NPSLE patients.ConclusionsOur research has successfully established serum protein profiles of NPSLE and non-NPSLE patients through TMT technology and screened out five proteins significantly changed in group-comparisons with consistent tendencies. The pathway of complement and coagulation cascades may participate in pathogenesis of NPSLE and non-NPSLE.

Proteomic screening of plasma identifies potential noninvasive biomarkers associated with significant/advanced fibrosis in patients with nonalcoholic fatty liver disease.

Noninvasive biomarkers are clinically useful for evaluating liver fibrosis stage in patients with nonalcoholic fatty liver disease (NAFLD). The aim of the present study was to compare plasma proteins in patients with early nonalcoholic steatohepatitis (NASH) (F0-F1) versus NASH with significant/advanced fibrosis (F2–F4) to determine whether candidate proteins could be used as potential noninvasive biomarkers. Nineteen biopsy-proven NAFLD patients including ten early NASH patients and nine NASH patients with significant/advanced fibrosis were enrolled in the present study. High-resolution proteomics screening of plasma was performed with the SCIEX TripleTOF 5600 System. Proteins were quantified using two different software platforms, Progenesis Qi and Scaffold Q+, respectively. Progenesis Qi analysis resulted in the discovery of 277 proteins compared with 235 proteins in Scaffold Q+. Five consensus proteins (i.e. Complement component C7; α-2-macroglobulin; Complement component C8 γ chain; Fibulin-1; α-1-antichymotrypsin) were identified. Complement component C7 was three-fold higher in the NASH group with significant/advanced fibrosis (F2–F4) compared with the early NASH (F0-F1) group (q-value = 3.6E-6). Complement component C7 and Fibulin-1 are positively correlated with liver stiffness (P=0.000, P=0.002, respectively); whereas, Complement component C8 γ chain is negatively correlated (P=0.009). High levels of Complement C7 are associated with NASH with significant/advanced fibrosis and Complement C7 is a perfect classifier of patients included in this pilot study. Further studies will be needed in a larger validation cohort to confirm the utility of complement proteins as biomarkers or mechanistic determinants of NASH with significant/advanced fibrosis.

ITRAQ-based high-throughput proteomics analysis reveals alterations of plasma proteins in patients infected with human bocavirus.

Human bocavirus (HBoV) is a member of the genus Bocavirus, family Parvoviridae, and subfamily Parvovirus and was first identified in nasopharyngeal aspirates of Swedish children with acute respiratory tract infection (ARTI) in 2005. It is the causative agent of nasopharyngeal aspirate disease and death in children. The HboV genomic structure is a linear single-stranded DNA (ssDNA). Its clinical pathogenic characteristics have been extensively studied, however, at present the molecular mechanism underlying the pathogenesis of HBoV infection is not completely clear. In this study, a total of 293 differentially expressed proteins (DEPs) between ARTI cases and healthy plasma samples were characterized using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled bioinformatics analysis, among which 148 were up-regulated and 135 were down-regulated. Gene Ontology (GO) and Cluster of Orthologous Groups of proteins (COG) annotated an enrichment of DEPs in complement activation and biological processes like immunity, inflammation, signal transduction, substance synthesis, and metabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis enriched DEPs mainly in the Wnt signaling pathway (ko04310), PPAR signaling pathway (ko03320), intestinal immune network for IgA production (ko04672), complement and coagulation cascades (ko04610), Toll-like receptor signaling pathway (ko04620) and B cell receptor signaling pathway (ko04662). Further, expression levels of three candidate proteins (upregulated PPP2R1A and CUL1, and downregulated CETP) were validated using western blotting. Our investigation is the first analysis of the proteomic profile of HBoV-infected ARTI cases using the iTRAQ approach, providing a foundation for a better molecular understanding of the pathogenesis of ARTI in children.

Plasma Biomarkers: Potent Screeners of Alzheimer's Disease.

Alzheimer's disease (AD), a neurological disorder, is as a complex chronic disease of brain cell death that usher to cognitive decline and loss of memory. Its prevalence differs according to risk factors associated with it and necropsy performs vital role in its definite diagnosis. The stages of AD vary from preclinical to severe that proceeds to death of patient with no availability of treatment. Biomarker may be a biochemical change that can be recognized by different emerging technologies such as proteomics and metabolomics. Plasma biomarkers, 5-protein classifiers, are readily being used for the diagnosis of AD and can also predict its progression with a great accuracy, specificity, and sensitivity. In this review, upregulation or downregulation of few plasma proteins in patients with AD has also been discussed, when juxtaposed with control, and thus serves as potent biomarker in the diagnosis of AD.

Plasma FGF-19 Levels are Increased in Patients with Post-Bariatric Hypoglycemia.

Hypoglycemia is an increasingly recognized complication of bariatric surgery. Mechanisms contributing to glucose lowering remain incompletely understood. We aimed to identify differentially abundant plasma proteins in patients with post-bariatric hypoglycemia (PBH) after Roux-en-Y gastric bypass (RYGB), compared to asymptomatic post-RYGB. Proteomic analysis of blood samples collected after overnight fast and mixed meal challenge in individuals with PBH, asymptomatic RYGB, severe obesity, or overweight recruited from outpatient hypoglycemia or bariatric clinics. The top-ranking differentially abundant protein at 120min after mixed meal was fibroblast growth factor 19 (FGF-19), an intestinally derived hormone regulated by bile acid-FXR signaling; levels were 2.4-fold higher in PBH vs. asymptomatic post-RYGB (mean + SEM, 1094 ± 141 vs. 428 ± 45, P < 0.001, FDR < 0.01). FGF-19 ELISA confirmed 3.5-fold higher concentrations in PBH versus asymptomatic (360 ± 70 vs. 103 ± 18, P = 0.025). To explore potential links between increased FGF-19 and GLP-1, residual samples from other human studies in which GLP-1 was modulated were assayed. FGF-19 levels did not change in response to infusion of GLP-1 and PYY in overweight/obese individuals. Infusion of the GLP-1 receptor antagonist exendin 9-39 in recently operated asymptomatic post-RYGB did not alter FGF-19 levels after mixed meal. By contrast, GLP-1 receptor antagonist infusion yielded a significant increase in FGF-19 levels after oral glucose in individuals with PBH. While plasma bile acids did not differ between PBH and asymptomatic post-RYGB, these data suggest unique interrelationships between GLP-1 and FGF-19 in PBH. Taken together, these data support FGF-19 as a potential contributor to insulin-independent pathways driving postprandial hypoglycemia in PBH.

Soluble programmed death-ligand 1 are highly expressed in peripheral T-cell lymphoma: a biomarker for prognosis

ABSTRACTPurposeTo investigate the role of soluble programmed cell death ligand 1 (sPD-L1) protein in plasma of patients with Peripheral T-cell lymphoma (PTCL).MethodsIn total, 80 patients with newly diagnosed PTCL and 75 healthy controls were enrolled. Levels of sPD-L1 were measured by ELISA at diagnosis and after 3–8 courses of chemotherapy. The expression of PD-L1 in tumor tissues from nine PTCL patients was also detected.ResultssPD-L1 was higher in PTCL patients at diagnosis compared to healthy subjects (P < 0.0001). Patients in the intermediate/high-risk disease group had a higher level of sPD-L1 than patients in the low-risk group (P = 0.0003). Elevated sPD-L1 (≥176.30 pg/ml) was the only biomarker for PTCL that retain statistical significance in multivariate analysis. Patients in the low-risk group with sPD-L1 ≥ 176.30 pg/ml had an adverse prognosis. Histological analysis showed that the expression of PD-L1 in tissues was positively correlated with sPD-L1 levels in plasma (Spearman = 0.9177, P = 0.0013).ConclusionssPD-L1 is a sensitive biomarker predicting clinical outcomes in PTCL.

Dynamics of oxidative modification of blood plasma proteins in patients with acute epididymitis under the influence of treatment

У нашому дослідженні була виконана спроба з'ясування діагностичної та прогностичної значимості рівня окисної модифікації білків (ОМБ) у плазмі крові хворих на гострий епідидиміт (ГЕ) протягом лікування. У всіх обстежених пацієнтів до лікування виявили підвищення рівня окисної модифікації білків. У період надходження до стаціонару у всіх хворих на ГЕ рівні ОМБ виявились високими, що свідчить про високий окисний стрес при даному захворюванні. Протягом лікування ці показники поступово знижувалися до норми. Отримані дані можуть бути використані як для моніторингу лікування, так і одужання хворих.

Exploration of differentially expressed plasma proteins in patients with lung adenocarcinoma using iTRAQ-coupled 2D LC-MS/MS.

Lung adenocarcinoma is characterized by early asymptomatic progression and metastasis. Appearance of respiratory symptoms usually means the disease is aggravated. The aim of this study was to identify the protein profile in plasma of lung adenocarcinoma of stages I-IV, and look for novel diagnostic biomarkers. Isobaric tags for relative and absolute quantification (iTRAQ) coupled with two dimensional liquid chromatography - tandem mass spectrometry technology (2D LC-MS/MS) was applied to separate and identify differential expression of proteins in plasma specimens from 10 healthy individuals, 10 patients with pneumonia, 7 patients with lung adenocarcinoma of stages I-II, respectively, and 10 patients with lung adenocarcinoma of stages III-IV, then analyze the functions of the differential expression of proteins by bioinformatics. ADAMTS-like protein 4, Fibrinogen-like protein 1 precursor, secretoglobin family 3A member 2 and haptoglobin were up-regulated in patients with lung adenocarcinoma by proteomics analysis, and the expression levels of SCGB3A2 and Hp by ELISA were consistent. Pathway analysis of identified differential expression of proteins showed they were mainly involved in chemokine/p53/TGF-beta signaling pathway. Plasma SCGB3A2 is a potential maker for diagnosis of primary pulmonary adenocarcinoma. Abnormal post-translational protein modification may be associated with the progression of lung cancer.

Evaluation of hemostasis parameters and the role of the oxidative damage to plasma proteins in the modulation of hemostasis in patients with nephrolithiasis before and after extracorporeal shock wave lithotripsy.

PurposeExtracorporeal shock wave lithotripsy (ESWL) is a commonly-used method in urology, which may modulate hemostasis and may induce lipid peroxidation in patients with nephrolithiasis. However, previous studies only examine changes occurring in patients 30–240 min after ESWL. The main aim of the present study was to determine whether oxidative stress may modulate the hemostatic activity of plasma in patients with nephrolithiasis before ESWL and the day after treatment ESWL. This will be performed by measuring selected parameters of hemostasis in these patients, both before ESWL and the following day, and assessing the level of oxidative damage to plasma proteins in these patients by measuring two biomarkers.MethodsTwelve patients with nephrolithiasis and 10 healthy participants were included. The following parameters of hemostasis were measured: the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of plasma, the level of fibrinogen, the level of D-dimer and blood platelet count. In addition, two selected biomarkers of oxidative stress were measured: protein carbonylation level and the number of protein thiol groups.ResultsNo difference was observed between patients with nephrolithiasis before and after ESWL and healthy controls with regard to PT, TT or APTT. Fibrinogen concentration and blood platelet count were lower in the nephrolithiasis patients in the period after ESWL than before ESWL. The nephrolithiasis patients demonstrated elevated D-dimer concentration after ESWL. However, although oxidative damage was observed in the plasma proteins in the nephrolithiasis patients, this was not influenced by ESWL.ConclusionOxidative stress may induce changes of hemostasis in patients with nephrolithiasis, both before and after ESWL. In addition, changes of hemostasis parameters such as fibrinogen, blood platelet count and D-dimer level can be observed in these patients, especially after ESWL, and this may suggest that ESWL modulates hemostasis. By having a better understanding of the influence of ESWL on hemostasis, this could lead to modifying patient care for those patients at increased risk of bleeding.

P 039 - Mass Spectrometric Identification of Collagen Alpha-1 (III) Chain and Chondroitin Sulfate Proteoglycan-4 Nitration in Patients with Acute Pulmonary Embolism: A Preliminary Study

Pulmonary embolism (PE)-induced oxidative stress can lead to the accumulation of nitroproteins that may play a role in disease progression. We used a proteomic approach to analyze nitrated plasma proteins in patients diagnosed with acute PE. Nitrotyrosine (NO2Tyr)-containing proteins were immunoprecipitated with a NO2Tyr affinity sorbent. Precipitated proteins were separated by SDS-PAGE and visualized by either Coomassie Blue staining or western blotting with mouse monoclonal anti-NO2Tyr antibody. Immunoreactive bands observed in disease patients were in-gel digested and analyzed by MALDI-TOF mass spectrometry. Mass fingerprint data sets obtained from the 138 kDa peptide fragment ions matched human collagen alpha-1 (III) chain (CO3A1)with Mascot algorithm analysis giving a score of 65 (p

Personalized protein corona on nanoparticles and its clinical implications.

It is now well understood that once in contact with biological fluids, nanoscale objects lose their original identity and acquire a new biological character, referred to as a protein corona. The protein corona changes many of the physicochemical properties of nanoparticles, including size, surface charge, and aggregation state. These changes, in turn, affect the biological fate of nanoparticles, including their pharmacokinetics, biodistribution, and therapeutic efficacy. It is progressively being accepted that even slight variations in the composition of a protein source (e.g., plasma and serum) can substantially change the composition of the corona formed on the surface of the exact same nanoparticles. Recently it has been shown that the protein corona is strongly affected by the patient's specific disease. Therefore, the same nanomaterial incubated with plasma proteins of patients with different pathologies adsorb protein coronas with different compositions, giving rise to the concept of personalized protein corona. Herein, we review this concept along with recent advances on the topic, with a particular focus on clinical relevance.

Circulating Exosomes Carrying an Immunosuppressive Cargo Interfere with Adoptive Cell Therapy in Acute Myeloid Leukemia

Introduction: Adoptive immunotherapy, including transfer of activated NK cells (aNK), is currently under active investigation for patients with refractory/relapsed acute myeloid leukemia (AML). However, the highly immunosuppressive microenvironment in patients with AML may create a major barrier for adoptive immunotherapy. Exosomes, virus-size (30-100 nm) membrane-bound vesicles found in all body fluids, are produced in abundance by leukemic blasts and are strongly immunosuppressive. The presence of exosomes in the plasma of relapsed/refractory AML patients could impair anti-leukemia activity of adoptively transferred aNK cells. To test this hypothesis, we evaluated ex vivo effects of exosomes isolated from plasma of AML patients on the activity of aNK cells which were used for adoptive immunotherapy in a Phase 1 clinical trial (NCT00900809) for patients with relapsed/refractory AML performed at our institution.Methods: Venous blood (20-50 mL) was obtained from patients with refractory/relapsed AML (n=7) prior to and after therapy with adoptively-transferred aNK cells. Exosomes were isolated from the patients’ plasma as well as normal controls’ (NC) plasma by size exclusion chromatography on Sepharose 2B. Exosome numbers and size (qNano), morphology (TEM) and protein levels were determined. The presence of exosome-related markers (Tsg101, CD81), myeloid cell-surface markers associated with AML (CD123, CLL-1, CD44, CD96) and of immunosuppressive proteins (CD39, CD73, PD-1/ PDL-1, Fas/FasL, TGF-β1) in exosomes was studied by Western blots. The AML plasma-derived exosomes were co-incubated with normal human NK cells or aNK cells used for therapy. Up-take of PKH26-labeled exosomes by recipient aNK cells was monitored by confocal microscopy and changes in NKG2D expression levels on aNK cells or in NK-cell cytotoxicity by multiparameter flow cytometry. Exosome-induced signaling via receptors (A2AR, TGFβR, CD95 or PD-1) on aNK cells was studied by Western blots ± inhibitors of each relevant pathway.Results: The protein levels of exosome fractions in plasma of refractory/relapsed AML patients prior to adoptive therapy with aNK cells were significantly higher (70 ± 15 μg protein/mL) than those isolated from plasma of NCs (35±2.7 μg protein/mL) (P<0.05). Exosome protein levels remained persistently elevated after therapy with the aNK cells. The molecular profile of exosomes in AML patients’ plasma was distinct from that of exosomes in plasma of NCs; in addition to leukemia-associated antigens (LAA), AML exosomes were enriched in TGF-β1 (LAP), CD39 and CD73 ectoenzymes, PD-L1 and FasL. Co-incubation of these AML exosomes with the aNK cells that express cognate surface receptors for the above listed ligands or ectoenzymes down-regulated survival and lytic functions of aNK cells. Pharmacologic inhibitors or blocking anti-PD1 mAbs and anti-TGF-β1 mAbs prevented apoptosis and partly abrogated dysfunction of aNK cells. Inhibitory in vivo effects of the high plasma exosome levels in plasma of AML patients was demonstrated by the absence of evidence for up-regulation of NK cytotoxicity in the peripheral blood of the patients receiving adoptive therapy with aNK cells.Conclusions: Persistently elevated levels of biologically-active immunosuppressive exosomes carrying leukemia-associated antigens and various immunosuppressive proteins in plasma of patients with relapsed/refractory AML interfere with anti-leukemia activity of adoptively transferred aNK cells. Blast-derived exosomes emerge as a significant deterrent to a success of adoptive immunotherapies with activated immune cells. DisclosuresNo relevant conflicts of interest to declare.

Assessment of oxidative/nitrative modifications of plasma proteins, selected ROTEM parameters and kinetics of fibrinogen polymerization in patients with multiple myeloma at diagnosis.

Patients with multiple myeloma (MM) are at increased risk of thrombosis. Growing evidence indicates that oxidative and nitrative modifications of proteins, including fibrinogen, may lead to changes in hemostasis. The study compares samples from patients with MM at diagnosis and healthy volunteers with regard to the oxidative/nitrative modifications of proteins, ROTEM and thrombin-catalyzed fibrin polymerization. The content of carbonyl groups in plasma proteins of patients with MM was significantly higher than in controls (2.981 vs. 1.807nmol/mg of protein, p=0.005), while no differences were seen in the concentrations of nitrated proteins. Maximum clot firmness readings were significantly higher in the samples of patients than in controls according to FIBTEM test (23.5 vs. 15mm, p=0.006). The lag time of the fibrin polymerization process and the velocity of clot lysis (V Lys) were found to be significantly higher in the group of MM patients than controls. In contrast, no marked differences were identified between studied groups in reference to maximal velocity of fibrin polymerization process (V max), maximal absorbance (A max) and plasmin amidolytic activity values. In conclusion, our study demonstrates that at the time of diagnosis, patients with MM demonstrated greater oxidative stress than healthy volunteers, which is reflected in a higher amount of carbonylated proteins. Some prothrombotic features found in ROTEM tests in MM patients were not confirmed by turbidimetry.