Inter-alpha-trypsin Inhibitor Research Articles

Longitudinal Fluctuations in Protein Concentrations and Higher-Order Structures in the Plasma Proteome of Kidney Failure Patients Subjected to a Kidney Transplant.

Using proteomics and complexome profiling, we evaluated in a year-long study longitudinal variations in the plasma proteome of kidney failure patients, prior to and after a kidney transplantation. The post-transplant period was complicated by bacterial infections, resulting in dramatic changes in the proteome, attributed to an acute phase response (APR). As positive acute phase proteins (APPs), being elevated upon inflammation, we observed the well-described C-reactive protein and Serum Amyloid A (SAA), but also Fibrinogen, Haptoglobin, Leucine-rich alpha-2-glycoprotein, Lipopolysaccharide-binding protein, Alpha-1-antitrypsin, Alpha-1-antichymotrypsin, S100, and CD14. As negative APPs, being downregulated upon inflammation, we identified the well-documented Serotransferrin and Transthyretin, but added Kallistatin, Heparin cofactor 2, and interalpha-trypsin inhibitor heavy chain H1 and H2 (ITIH1, ITIH2). For the patient with the most severe APR, we performed plasma complexome profiling by SEC-LC-MS on all longitudinal samples. We observed that several plasma proteins displaying alike concentration patterns coelute and form macromolecular complexes. By complexome profiling, we expose how SAA1 and SAA2 become incorporated into high-density lipid particles, replacing largely Apolipoprotein (APO)A1 and APOA4. Overall, our data highlight that the combination of in-depth longitudinal plasma proteome and complexome profiling can shed further light on correlated variations in the abundance of several plasma proteins upon inflammatory events.

Relationships between haptoglobin and inter-alpha trypsin inhibitor heavy chain 4 levels inlocal and peripheral blood and systemic inflammatory response syndrome in bitches withpyometra.

We aimed to assess the usefulness of monitoring inter-alpha trypsin inhibitor heavy chain 4 (ITIH4) and haptoglobin (Hp) in peripheral and local blood in canine pyometra, and evaluation the relationships among acute phase proteins (APPs), systemic inflammatory response syndrome (SIRS) and the presence of bacteria. The material was collected from bitches with pyometra and from healthy ones. Blood was taken from the cephalic and uterine veins. APPs levels were quantified by ELISA. In the peripheral circulation, the Hp was higher in animals with open-cervix pyometra (OCP) than in the closed-cervix pyometra (CCP) and the control group. The Hp concentration was not correlated with age, with the presence of SIRS or with the type of bacteria (Gram-negative, Gram-positive or mixed flora). The ITIH4 concentrations in the peripheral blood did not differ significantly in the cases of pyometra. The Hp concentration in the local circulation increased in the OCP but not in the CCP groups, although the histopathological changes in the endometrium were similar. Peripheral Hp concentrations may be a useful tool in differentiating between the types of pyometra.

Changes in S100A8/A9 and S100A12 and Their Comparison with Other Analytes in the Saliva of Pigs with Diarrhea Due to E. coli.

The family of calgranulins includes S100A8 (calgranulin A), S100A9 (calgranulin B), which can appear as a heterodimer known as S100A8/A9 or calprotectin, and S100A12 (calgranulin C). These proteins are related to different inflammatory conditions, immune-mediated diseases, and sepsis and are considered biomarkers of potential interest. This study aims to evaluate if S100A8/A9 and A12 could change in pigs with diarrhea due to E. coli and to compare the changes of S100A8/A9 and A12 with other analytes in order to explore the possible causes or mechanisms involved. For this purpose, a panel integrated by analytes related to inflammation (haptoglobin, inter-alpha trypsin inhibitor 4 (ITIH4), and total protein); immune system (adenosine deaminase, ADA); stress (alpha-amylase); tissue damage (lactate and lactate dehydrogenase (LDH)); sepsis (aldolase) and redox status (ferric-reducing ability of saliva (FRAS) and advanced oxidation protein products (AOPP)) was evaluated. S100A8/A9 and A12 and the other analytes measured in this study showed increases in the saliva of pigs with diarrhea due to E. coli. S100A8/A9 and/or A12 showed a significant correlation of different magnitude with some of the other analytes evaluated. Further studies should be conducted to gain knowledge about the possible practical applications as biomarkers of the measurements of S100A8/A9 and A12 in the saliva of pigs.

Enteral protease inhibition reduces systemic enzymatic activity and proteolysis after experimental trauma-hemorrhagic shock

Trauma-hemorrhagic shock (T-HS) is a major cause of multiorgan failure and mortality world-wide. Our previous work demonstrates that uncontrolled proteolysis in T-HS is associated with poor outcomes and that enteral inhibition of proteolytic activity may be beneficial. However, the enzymes involved, their degradation products, and the systemic consequences of protease activity in T-HS are not well understood. Our aim was to identify plasma enzymatic activity and the resulting peptidome in experimental T-HS, and determine the changes in this activity following enteral protease inhibition. Anesthetized male Wistar rats (~400g) were subjected to T-HS via laparotomy followed by blood removal to achieve a mean blood pressure of ~35-40mmHg for 90 min. An orogastric catheter was passed through the mouth into the proximal duodenum and Golytely (Shock-control, n=8) or Golytely+gabexate mesilate, 10mg/kg (GM-treated, n=8) was enterally infused starting 30 minutes into the shock period. Animals were resuscitated with Lactated Ringers solution (LR) and monitored for 120 min after reperfusion. The gabexate mesilate treatment improved mean arterial pressure after reperfusion (57.6±4.3 mmHg) compared to Shock-control group (43.7±4.5mmHg, p<0.0001) requiring significantly less resuscitation fluid (14±3ml vs 25.7±2.3 ml, respectively, p<0.0001). Systemic proteolysis, as measured by the plasma peptidome, increased by 630±212% in the Shock-control group compared to baseline, and was mitigated by treatment with gabexate mesilate to 203±70% (p=0.02). Using tagged fluorescent substrates, trypsin-like activity in plasma increased with T-HS to 937±90% of baseline (p<0.0001) and was reduced by enteral protease inhibition to 321±72% of baseline (p=0.0321 vs. baseline, p=0.0078 vs. Shock-control). The most common proteins to be degraded were structural (myosin, actin, and collagen), coagulation-related (inter-alpha trypsin inhibitor, kininogen, complement C4, and fibrinogen) and metabolic in function (apolipoprotein E, fructose-bisphosphate aldolase, and carbonyl reductase). These proteins were cleaved after T-HS 1.5-4 times more in the Shock-control compared to the GM-treated group. In conclusion, we were able to identify trypsin-like activity in plasma from animals subjected to T-HS. Enteral inhibition of serine proteases was able to significantly decrease this activity and improve outcomes, suggesting that the gut is a driver of uncontrolled proteolysis and resulting morbidity in T-HS. Enteral serine protease inhibition may be an effective clinical strategy for decreasing inappropriate systemic proteolysis and improving outcomes in T-HS. This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs and the Defense Health Agency J9, Research and Development Directorate under Award No. W81XWH-17-2-0047. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteomic profiling of cerebrospinal fluid in pediatric myelin oligodendrocyte glycoprotein antibody-associated disease

BackgroundMyelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an autoimmune demyelinating disorder of the central nervous system.MethodsExtracted proteins from 34 cerebrospinal fluid (CSF) samples [patients with MOGAD (MOG group, n = 12); healthy controls (HC group, n = 12); patients with MOG seronegative and metagenomics next-generation sequencing-negative inflammatory neurological diseases (IND group, n = 10)] were processed and subjected to label-free quantitative proteomics. Supervised partial least squares-discriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) models were also performed based on proteomics data. Functional analysis of differentially expressed proteins (DEPs) was performed using Gene Ontology, InterPro, and Kyoto Encyclopedia Genes and Genomes. An enzyme-linked immunosorbent assay was used to determine the complement levels in serum from patients with MOGAD.ResultsFour hundred and twenty-nine DEPs (149 upregulated and 280 downregulated proteins) were identified in the MOG group compared to the HC group according to the P value and fold change (FC). Using the O-PLS-DA model, 872 differentially abundant proteins were identified with variable importance projection (VIP) scores > 1. Five proteins (gamma-glutamyl hydrolase, cathepsin F, interalpha-trypsin inhibitor heavy chain 5, latent transforming growth factor beta-binding protein 4 and leukocyte-associated immunoglobulin-like receptor 1) overlapping between the top 30 DEPs with top-ranked P value and FC and top 30 proteins in PLS-DA VIP lists were acquired. Functional analysis revealed that the dysregulated proteins in the MOG group were primarily involved in complement and coagulation cascades, cell adhesion, axon guidance, and glycosphingolipid biosynthesis compared to the HC group.ConclusionThe proteomic alterations in CSF samples from children with MOGAD identified in the current study might provide opportunities for developing novel biomarker candidates.

Urine inter-alpha-trypsin inhibitor family-related proteins may serve as biomarkers for disease activity of lupus.

BackgroundSystemic lupus erythematosus (SLE) is a chronic inflammatory disease involving multiple tissues. Inter‐Alpha‐Trypsin Inhibitor (ITI) family proteins have a role in maintaining tissue homeostasis, but their possible clinical significance in the SLE patients has not been reported. The aim of this study was to analyze and verify the expression of ITI‐related proteins in the urine of SLE patients, further explore the features of these proteins in disease activity.MethodsBased on label‐free proteomics technology and bioinformatics technology, we analyzed the expression of ITI family‐related proteins in the urine of lupus. Subsequently, Western‐blot and targeted proteomics were used to qualitatively and quantitatively verify the expression of these proteins, respectively.ResultsA total of seven ITI family‐related proteins were screened and identified; and six of these proteins were differentially expressed in the urine of SLE patients. Further quantitative analysis showed that the expressions of ITIH2, ECM1, and ITIH5 in urine between active SLE group and stable SLE group were consistent with the preliminary screening results. The expression of ITIH2 and ECM1 in the renal damage group were also consistent with the screening results. Moreover, ITIH2 and ECM1 have a good correlation with disease activity and have a certain correlation with renal damage.ConclusionsIn this exploratory study, we evaluated the expression of ITI family‐related proteins in the urine of SLE and found that urine ITIH2 and ECM1 were closely related to SLE activity, especially kidney damage, providing an experimental basis for further exploration of the potential roles in monitoring lupus and lupus nephritis activity.

Glycomic Analysis Reveals a Conserved Response to Bacterial Sepsis Induced by Different Bacterial Pathogens.

Sepsis is an extreme inflammatory response to infection that occurs in the bloodstream and causes damage throughout the body. Glycosylation is known to play a role in immunity and inflammation, but the role of glycans in sepsis is not well-defined. Herein, we profiled the serum glycomes of experimental mouse sepsis models to identify changes induced by 4 different clinical bacterial pathogens (Gram-positive: Streptococcus pneumoniae and Staphylococcus aureus, Gram-negative: Escherichia coli and Salmonella Typhimurium) using our lectin microarray technology. We observed global shifts in the blood sera glycome that were conserved across all four species, regardless of whether they were Gram positive or negative. Bisecting GlcNAc was decreased upon sepsis and a strong increase in core 1/3 O-glycans was observed. Lectin blot analysis revealed a high molecular weight protein induced in sepsis by all four bacteria as the major cause of the core 1/3 O-glycan shift. Analysis of this band by mass spectrometry identified interalpha-trypsin inhibitor heavy chains (ITIHs) and fibronectin, both of which are associated with human sepsis. Shifts in the glycosylation of these proteins were observed. Overall, our work points toward a common mechanism for bacterially induced sepsis, marked by conserved changes in the glycome.

Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 Plays an Important Role in the Development and Reproduction of Nilaparvata lugens.

Simple SummaryThe brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae), is a destructive insect pest of rice. It causes reductions in rice yield and great economic losses. In this study, we used RNAi to explore the function of the inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) gene in the development and reproduction of the brown planthopper. Our results revealed that ITIH4 influences the survival, ovarian development, egg production, and egg hatching of this insect, indicating that ITIH4 plays important roles in development and reproduction. Considering the importance of ITIH4 in the brown planthopper, it may be a potential target for pest management.The brown planthopper, Nilaparvata lugens, is a difficult-to-control insect pest affecting rice yields in Asia. As a structural component of the inter-alpha-trypsin inhibitor (ITI), the inter-alpha-trypsin inhibitor heavy chain (ITIH) has been reported to be involved in various inflammatory or malignant disorders, ovarian development, and ovulation. To reveal the function of ITIH4 in N. lugens, the gene encoding N. lugens ITIH4 (NlITIH4) was cloned and characterized. NlITIH4 contains a signal peptide, a vault protein inter-alpha-trypsin domain, and a von Willebrand factor type A domain. qPCR analysis showed that NlITIH4 was expressed at all developmental stages and in all tissues (fat body, ovary, and gut), with the highest expression in the fat body. Double stranded NlITIH4 (dsNlITIH4) injection clearly led to an RNAi-mediated inhibition of the expression of NlITIH4 and resulted in reduced survival, delayed ovarian development, and reduced egg production and egg hatching. These results indicate that NlITIH4 plays an important role in the development and reproduction of N. lugens.

A clinical model including protein biomarkers predicts radiographic knee osteoarthritis: a prospective study using data from the Osteoarthritis Initiative

We aimed to provide a model to predict the prospective development of radiographic KOA (rKOA). Baseline sera from 333 non-radiographic KOA subjects belonging to OA Initiative (OAI) who developed or not, rKOA during a follow-up period of 96 months were used in this study. The exploratory cohort included 200 subjects, whereas the replication cohort included 133. The levels of inter-alpha trypsin inhibitor heavy chain 1 (ITIH1), complement C3 (C3) and calcyclin (S100A6), identified in previous large proteomic analysis, were analyzed by using sandwich immunoassays on suspension bead arrays. The association of protein levels and clinical covariates with rKOA incidence was assessed by combining logistic regression analysis, Receiver Operating Characteristic (ROC) analysis, Integrated Discrimination Improvement (IDI) analysis and Kaplan-Meier curves. Levels of ITIH1, C3 and S100A6 were significantly associated with the prospective development of rKOA, showing an area under the curve (AUC) of 0.713 (0.624-0.802), 0.708 (0.618-0.799) and 0.654 (0.559-0.749), respectively to predict rKOA in the replication cohort. The inclusion of ITIH1 in the clinical model (age, gender, BMI, previous knee injury and WOMAC pain) improved the predictive capacity of the clinical covariates (AUC=0.754 [0.670-0.838]) producing the model with the highest AUC (0.786 [0.705-0.867]) and the highest IDI index (9%). High levels of ITIH1 were also associated with an earlier onset of the disease. A clinical model including protein biomarkers that predicts incident rKOA has been developed. Among the tested biomarkers, ITIH1 showed potential to improve the capacity to predict rKOA incidence in clinical practice.

Protein corona profile of graphene oxide allows detection of glioblastoma multiforme using a simple one-dimensional gel electrophoresis technique: a proof-of-concept study

Glioblastoma multiforme (GBM) is the most aggressive form of gliomas. The development of supplementary approaches for glioblastoma diagnosis, limited to imaging techniques and tissue biopsies so far, is a necessity of clinical relevance. In this context, nanotechnology might afford tools to enable early diagnosis. Upon exposure to biological media, nanoparticles are coated with a layer of proteins, the protein corona (PC), whose composition is individual and personalized. Here we show that the PC of graphene oxide nanosheets has a capacity to detect GBM using a simple one-dimensional gel electrophoresis technique. In a range of molecular weights between 100 and 120 kDa, the personalized PC from GBM patients is completely discernible from that of healthy donors and that of cancer patients affected by pancreatic adenocarcinoma and colorectal cancer. Using tandem mass spectrometry, we found that inter-alpha-trypsin inhibitor (ITI) heavy chain H4 is enriched in the PC of all tested individuals but not in the GBM patients. Overall, if confirmed on a larger cohort series, this approach could be advantageous at the first level of investigation to decide whether to carry out more invasive analyses and/or to follow up patients after surgery and/or pharmacological treatment.

Development and validation of an ELISA for the quantification of bovine ITIH4 in serum and milk

Inter alpha trypsin inhibitor heavy chain 4 (ITIH4) is a serum protein belonging to the Inter alpha trypsin inhibitor (ITI) family, which was previously characterized by our group as a new APP in cattle. This protein was firstly described in pigs where is known to be a major acute phase protein, also denominated Pig-MAP. Increases of ITIH4 of up to 12 times the pre-infection values were previously reported in the serum of heifers with experimentally induced summer mastitis. ITIH4 was detected in the milk of cows with mastitis by western blot, but the method previously used to quantify this protein, radial immunodiffusion, was not sensitive enough to quantify it in milk samples. In this study we developed an ELISA method which allows the quantification of bovine ITIH4 in serum and milk samples. Previously developed antibodies were used to perform the assay, including anti bovine ITIH4 polyclonal antibodies and a monoclonal antibody against pig ITIH4 that also recognizes the bovine homologous protein. The ELISA developed showed an adequate precision, with inter and intra- assay coefficients of variation lower than 10% for serum and milk samples. The assay keeps linearity under dilution for both serum and milk samples. A good agreement was observed between the values measured by ELISA and radial immunodiffusion in serum samples.

MP80-19 URINE PROTEOMIC ANALYSIS IN CYSTINURIC PATIENTS WITH RENAL STONES

You have accessJournal of UrologyStone Disease: Evaluation II1 Apr 2015MP80-19 URINE PROTEOMIC ANALYSIS IN CYSTINURIC PATIENTS WITH RENAL STONES Larisa Kovacevic, Hong Lu, Yegappan Lakshmanan, Joseph A. Caruso, and David Goldfarb Larisa KovacevicLarisa Kovacevic More articles by this author , Hong LuHong Lu More articles by this author , Yegappan LakshmananYegappan Lakshmanan More articles by this author , Joseph A. CarusoJoseph A. Caruso More articles by this author , and David GoldfarbDavid Goldfarb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2855AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The pathogenesis of cystinuric renal stones is not well understood. Using a proteomic approach, we aimed to assess (1) the differences in the function of urinary proteins between patients with cystinuria and renal stone (CYS), patients with renal stone and no cystinuria (RS), and healthy controls (HC); (2) the gender differences in the proteomic profile within CYS group;(3) the presence of diagnostic biomarkers for CYS. METHODS We compared urinary proteomes of 10 patients with CYS (35.7±10.6 years, 5 males), 10 age- and gender-matched patients with RS, and 10 age- and gender-matched HC, using liquid chromatography-mass spectrometry (LC-MS/MS). Proteins of interest were selected based on relative fold-differences in abundance compared to controls, and were expressed as ratios. The pathway analysis using Gene Ontology (GO) annotation was done on the top 10% of proteins that exhibited the more pronounced differences in abundance between the groups. These significantly expressed proteins were used to analyze the group differences for biological processes, cellular components, and molecular function. RESULTS Of the 2219 proteins identified by proteomic analysis, 146 proteins were up-regulated in CYS and 80 proteins were up-regulated in HC. GO analysis in CYS showed a significant number of peptides involved in signal transduction (68 in males and 80 in females), and cell-cell signaling (17 in males and 21 in females). However, the signaling pathway was different in CYS males (Peroxisome Proliferator-Activated Receptor, PPAR) than in CYS females (NOTCH3). Proteins involved in fibrosis were found predominantly in CYS males (Table). Inter-alpha-trypsin inhibitor (ITIH), paraoxonase (PON1) and ceruloplasmin (CP) were more abundant in male CYS than female CYS and their matched HC. CONCLUSIONS Patients with cystinuria and renal stones have a different urinary polypeptide profile compared to healthy controls. Fibrosis is more common in male CYS, and an aggressive approach may be warranted. Additionally, we identified ITIH and CP as potential diagnostic biomarkers and novel therapeutic targets in CYS. These unique proteins merit further investigation. Accession Number∗ Protein MW (kDa) No. of assigned peptides∗∗ No. of unique peptides∗∗ Sequence coverage CYS/HC CYS/HC (%)∗∗∗ ITIH2 Inter-alpha-trypsin inhibitor 106 100 100 43.5 PON1 Paraoxonase 40 41.22 40.67 58.9 CP Ceruloplasmin 122 26.8 26.5 80.6 VIME Vimentin 54 8.35 8.28 23.8 IGFBP-6 Insulin-like growth factor-binding protein 6 25 3.35 3.3 4.17 ACTG Actin 42 3.28 4.11 66.1 MMP9 Matrix metalloproteinase 9 78 3.13 3.09 15.9 VCAM Vascular cell adhesion protein 81 2.12 2.1 35.3 ∗ Human. ∗∗ Values presented as CYS/HC ratios in males. ∗∗∗ % of sequence that has been identified for each protein. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e1026-e1027 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Larisa Kovacevic More articles by this author Hong Lu More articles by this author Yegappan Lakshmanan More articles by this author Joseph A. Caruso More articles by this author David Goldfarb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Difference Quantity of Urinary Peptides between Two Groups of Type 2 Diabetic Patients with or without Coronary Artery Disease.

Objectives. We aim to explore urinary biomarkers that could monitor CAD in type 2 diabetic patients. Materials and Methods. Urine samples from two groups, twenty-eight type 2 diabetic patients with coexisting CAD and thirty type 2 diabetic patients without CAD, were purified by MB-WCX and then analyzed by MALDI-TOF-MS. Subsequently, we compared the urinary peptide signatures of the two groups by use of ClinProTools2.1 and evaluated the potential ability of the differently expressed peptides to distinguish type 2 diabetic patients with coexisting CAD from type 2 diabetic patients without CAD by ROC analysis. Finally, the differently expressed peptides were identified by nanoliquid chromatography-tandem mass spectrometry. Results. There were six differently expressed peptides (m/z 1305.2, 1743.9, 2184.9, 2756.1, 3223.2, and 6196.1) between the two groups of subjects, and they were identified as fragments of isoform 1 of fibrinogen alpha chain precursor, prothrombin precursor, and interalpha-trypsin inhibitor heavy chain H4. The diagnostic efficacy of m/z 2756.1 and m/z 3223.2 was better than the other peptides. Area under ROC of the m/z 2756.1, and m/z 3223.2 was 0.98 and 0.93, respectively. Conclusions. These urinary peptides are potential urinary biomarkers for monitoring of type 2 diabetic patients with CAD.

Function and regulation of inter-alpha-trypsin inhibitor heavy chain 5 (ITIH5) in normal human skin, inflammatory skin diseases, constitutive knock-out mice and murine 3D-skin models

Function and regulation of inter-alpha-trypsin inhibitor heavy chain 5 (ITIH5) in normal human skin, inflammatory skin diseases, constitutive knock-out mice and murine 3D-skin models

Early diagnostic protein biomarkers for breast cancer: how far have we come?

Many studies have used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to search for blood-based proteins that are related to the presence of breast cancer. We review the biomarkers discovered or targeted measured by these methods and discuss the strengths and weaknesses of these studies. We highlight two proteins that were most often related to breast cancer: C3a des-arginine anaphylatoxin (C3adesArg) (molecular weight: 8,938 Da) and fragments of inter-alpha trypsin inhibitor heavy chain H4 (ITIH4). In addition, we elaborate on three important methodological aspects related to these studies: protein identification, specificity of the markers, and disease heterogeneity. Finally, we propose some points to be addressed in future studies. These include the use of other analytical measurement techniques, need of protein identification, the importance of identical sample handling protocols for cases and controls, and the stratification of the results according to molecular subtypes and stages of breast cancer. Ultimately this may lead to the discovery of new and valid breast cancer specific biomarkers.

Prospective Validation of a Chronic GvHD-Specific Proteome Pattern (cGvHD-MS14) Post Allogeneic Hematopoietic Stem Cell Transplantation

Abstract 1970 Introduction:Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for many hematologic malignancies and non-malignant hematopoietic disorders, but is associated with significant morbidity and mortality with focus on acute and chronic graft-versus-host disease (GvHD). Chronic GvHD (cGvHD) occurs with increasing frequency, hampering quality of life of patients post-allogeneic HSCT and leading to increased morbidity and mortality even years after allogeneic HSCT. Diagnosis of chronic GvHD is based on clinical features and histology. Here we present the generation of cGvHD-specific proteomic pattern (cGvHD-MS14) using capillary electrophoreses and mass spectrometry to analyze urine sample collected prospectively after allogeneic HSCT. Methods:A proteomic pattern (cGvHD-MS14) was developed in order to diagnose cGvHD, to differentiate acute versus cGvHD, and to predict onset and severity of cGvHD prior to clinical diagnosis of cGVHD as a non-invasive, unbiased laboratory test for diagnosis of cGvHD. This pattern was prospectively evaluated on 329 patients (1034 urine samples) after allogeneic HSCT at MHH and 3 collaborating transplant centers. The majority of the patients had acute leukemias prior to transplantation (n=210) and were transplanted from matched unrelated or related donors (MUD n=134; MRD n=125). Reduced intensity conditioning regimens were used in about 75% of all patients and the majority (80%) received ATG (anti-thymocyte globulin) as GVHD-prophylaxis prior to transplantation and a calcineurin-inhibitor based prophylaxis afterwards. Results:Prospective and blinded evaluation revealed the correct classification of patients developing cGvHD with a sensitivity 78% and specificity of about 71% at time of diagnosis. Differentiation between late onset acute GvHD and chronic GvHD was achieved in 3 patients in this validation set. Acute GvHD prior to day 100 is not recognized by cGvHD-MS14, since aGvHD-specific peptides had been excluded during cGvHD-pattern generation. The pattern consists of 14 differentially excreted peptides, differentiating chronic GvHD from tolerant patients. Four of 14 peptides have been sequenced to date, 2 are fragments from collagen 1, 1 is from inter-alpha trypsin inhibitor heavy chain 4 and 1 is a fragment from the fibrinogen ß-chain. Conclusions:The proteomic pattern of urine proteomics enables diagnosis of cGvHD as well as differentiation of acute versus chronic GvHD. Further prospective evaluation of the cGvHD-specific pattern cGvHD-MS14 for organ specificity as well as severity prediction is currently ongoing.Taken together our results indicate that diagnosis of cGvHD is possible using CE/MS analysis of prospectively collected urine samples with high sensitivity and specificity. Disclosures:Metzger:mosaiques-diagnostics GmbH: Employment. Krons:mosaiques-diagnostics GmbH: Employment.

Inhibition of proteasomal proteolysis affects expression of extracellular matrix components and steroidogenesis in porcine oocyte-cumulus complexes

Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway–regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.

Searching for early breast cancer biomarkers by serum protein profiling of pre-diagnostic serum; a nested case-control study

BackgroundSerum protein profiles have been investigated frequently to discover early biomarkers for breast cancer. So far, these studies used biological samples collected at or after diagnosis. This may limit these studies' value in the search for cancer biomarkers because of the often advanced tumor stage, and consequently risk of reverse causality. We present for the first time pre-diagnostic serum protein profiles in relation to breast cancer, using the Prospect-EPIC (European Prospective Investigation into Cancer and nutrition) cohort.MethodsIn a nested case-control design we compared 68 women diagnosed with breast cancer within three years after enrollment, with 68 matched controls for differences in serum protein profiles. All samples were analyzed with SELDI-TOF MS (surface enhanced laser desorption/ionization time-of-flight mass spectrometry). In a subset of 20 case-control pairs, the serum proteome was identified and relatively quantified using isobaric Tags for Relative and Absolute Quantification (iTRAQ) and online two-dimensional nano-liquid chromatography coupled with tandem MS (2D-nanoLC-MS/MS).ResultsTwo SELDI-TOF MS peaks with m/z 3323 and 8939, which probably represent doubly charged apolipoprotein C-I and C3a des-arginine anaphylatoxin (C3adesArg), were higher in pre-diagnostic breast cancer serum (p = 0.02 and p = 0.06, respectively). With 2D-nanoLC-MS/MS, afamin, apolipoprotein E and isoform 1 of inter-alpha trypsin inhibitor heavy chain H4 (ITIH4) were found to be higher in pre-diagnostic breast cancer (p < 0.05), while alpha-2-macroglobulin and ceruloplasmin were lower (p < 0.05). C3adesArg and ITIH4 have previously been related to the presence of symptomatic and/or mammographically detectable breast cancer.ConclusionsWe show that serum protein profiles are already altered up to three years before breast cancer detection.

Proteomic biomarkers in combination with CA 125 for detection of epithelial ovarian cancer using prediagnostic serum samples from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial

When epithelial ovarian cancer is detected at an early stage (I-II), the 5-year survival rate is between 70% and 90%; whereas, when it is detected in late stages (III-IV), the 5-year survival rate slips to <30%. In a previous report, the authors observed that proteomic biomarkers and cancer antigen 125 (CA 125) exhibited a sensitivity of 84% at a specificity of 98% for identifying sera from patients who had stage I disease at the time of surgery, significantly improving the sensitivity of CA 125 alone. The challenge, however, is to detect ovarian cancer before clinical diagnosis. The current study was part of a large effort to compare different multimarker biomarker panels for the early detection of ovarian cancer. Several biomarkers were evaluated alone and in combination with CA 125 in prediagnostically collected sera from women in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Proximal prediagnostic sera from 118 women with ovarian cancer (cases) and from 951 age-matched women (controls) (8 controls per case, including 4 randomly selected from the general population, 2 with CA 125 levels ≥ 35 U/mL, and 2 with a positive family history of breast/ovarian cancer) were analyzed using the CA 125 immunoassay and surface-enhanced laser desorption and ionization time-of-flight mass spectrometry to measure 7 proteins (apolipoprotein A1, truncated transthyretin, transferrin, hepcidin, β-2 microglobulin, connective tissue activating protein III), and interalpha-trypsin inhibitor heavy-chain 4). Data were analyzed by 2 statistical strategies that combined the 7 markers and CA 125 into 1 predictive score for disease classification. CA 125 levels were elevated (≥ 35 U/mL) in 61.5% of 65 patients who had CA 125 data available from samples that were collected <12 months before cancer diagnosis; however, levels of the additional 7 biomarkers were not different between cases and the 3 control groups individually or combined. Two panels that combined CA 125 and the 7 biomarkers failed to improve the sensitivity of CA 125 alone. In contrast to earlier findings from analyzes of postdiagnostically collected sera, the addition of 7 biomarkers to CA 125 did not improve sensitivity for preclinical diagnosis beyond CA 125 alone.

Sialylated Lewis x Antigen Bearing Glycoproteins in Human Plasma

Recent studies have shown that antibodies targeting Lewis x (Le(x)) antigen are a valuable tool in the isolation and identification of glycoproteins in plasma. A focus of this study was to determine whether sialylated Lewis x (sLe(x)) antigen carrying glycoproteins occur in human plasma and whether an antibody targeting this antigen could be used to isolate and identify glycoproteins bearing this antigen. An additional objective was to determine the degree to which proteins conjugated to Le(x) and sLe(x) antigens are similar in structure. A specific anti-sLe(x) antibody (anti-sLe(x)Ab), CHO-131, immobilized in an immunoaffinity column was used to select a set of specific sLe(x) bearing proteins from human plasma, after which they were identified by either of two analytical strategies. One approach was to further resolve the affinity selected proteins by reversed phase chromatography (RPC), tryptic digest the RPC fractions, and identify peptide fragments by MALDI-MS/MS. The second was to tryptic digest the affinity selected protein fraction, further resolve the tryptic fragments by RPC, and identify peptides from RPC fractions by MALDI-MS/MS. Histidine-rich glycoprotein, plasminogen, apolipoprotein A-I, vitronectin, proteoglycan-4, clusterin, Ig gamma-2 chain C region, Ig mu chain C region, and interalpha-trypsin inhibitor heavy chain H4 were found to change three folds or more in association with breast cancer. Fifty percent of the glycoproteins carrying either sLe(x) antigen from CHO-131 selection, Le(x) antigen from selection with TG-1 antibody, or both were found to be changed three folds or more in concentration in breast cancer plasma relative to controls.