Inter-alpha-trypsin Inhibitor Heavy Chain 4 Research Articles

Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 (ITIH4) as a Compensatory Protease Inhibitor in Hereditary Angioedema (HAE)

BackgroundHereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1-INH) activity. C1-INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE. ObjectiveInvestigate ITIH4 activation in HAE, establish it as a potential biomarker, and explore its involvement in HAE-associated proteolytic pathways. MethodsSpecific immunoassays for non-cleaved ITIH4 (Intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (Total ITIH4) were developed. We initially tested serum samples from HAE patients (n=20), ACEI-induced edema patients (ACEI) (n=20), and unknown HAE patients (U-HAE) (n=20). Validation involved an extended cohort of 80 HAE patients (60 type I, 20 type II), including samples taken at attack and quiescent disease periods, as well as 100 healthy controls. ResultsIn 63% of HAE patients, the Intact ITIH4 assay showed lower signals than the Total ITIH4 assay. This difference was not observed in ACEI and U-HAE patients. Western blotting confirmed cleaved ITIH4 in low-Intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4 suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both HAE type 1 and type 2 patients compared to controls, with consistently low Intact/Total ITIH4 ratios during clinical HAE attacks. ConclusionThe disease-specific low intact ITIH4 levels highlight its unique nature in HAE. The results suggest that ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.

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.

Gaining knowledge about biomarkers of the immune system and inflammation in the saliva of pigs: The case of myeloperoxidase, S100A12, and ITIH4

An assay for the measurement of myeloperoxidase (Mpx) in porcine saliva was developed and validated, and factors influencing Mpx and another two biomarkers of inflammation and immune system, the protein S100A12 and the inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), were studied. The spectrophotometric method for Mpx measurement validated in this assay showed an adequate analytical performance including precision and accuracy. When a group of twenty healthy pigs was sampled every 4 h from 8 a.m. until 8 p.m., Mpx and S100A12 showed significant increases at 4 p.m., whereas ITIH4 concentration showed a significant decrease at 12 a.m. Increases were also seen in salivary Mpx, S100A12, and ITIH4 levels 24 h after the intramuscular administration of Escherichia coli lipopolysaccharide in five pigs; whereas in a non-septic inflammation after the subcutaneous administration of turpentine oil to five pigs changes were seen in S100A12 at 3 h and in ITIH4 at 48 h. When a stressful situation consisting of the transportation and stay of 4 h to a slaughterhouse of 24 pigs was performed, all analytes were increased after 4 h of lairage in the slaughterhouse compared with the values that were obtained the day before at the same time of the day. Mpx can be measured in the saliva of pigs with the automated assay described in this report. Mpx, S100A12, and ITIH4 salivary levels can change depending on the hour of the day in which the sample is taken, and increases can be produced due to sepsis, non-septic inflammation and stress.

Changes in salivary biomarkers of stress, inflammation, redox status, and muscle damage due to Streptococcus suis infection in pigs

BackgroundStreptococcus suis (S. suis) is a Gram-positive bacteria that infects pigs causing meningitis, arthritis, pneumonia, or endocarditis. This increases the mortality in pig farms deriving in severe economic losses. The use of saliva as a diagnostic fluid has various advantages compared to blood, especially in pigs. In this study, it was hypothesized that saliva could reflect changes in different biomarkers related to stress, inflammation, redox status, and muscle damage in pigs with S. suis infection and that changes in these biomarkers could be related to the severity of the disease.ResultsA total of 56 growing pigs from a farm were selected as infected pigs (n = 28) and healthy pigs (n = 28). Results showed increases in biomarkers related to stress (alpha-amylase and oxytocin), inflammation (haptoglobin, inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), total protein, S100A8-A9 and S100A12), redox status (advanced oxidation protein producs (AOPP)) and muscle damage (creatine kinase (CK), CK-MB, troponin I, lactate, aspartate aminotransferase, and lactate dehydrogenase). An increase in adenosine deaminase (ADA), procalcitonin, and aldolase in infected animals were also observed, as previously described. The grade of severity of the disease indicated a significant positive correlation with total protein concentrations, aspartate aminotransferase, aldolase, and AOPP.ConclusionsThis report revealed that S. suis infection caused variations in analytes related to stress, inflammation, redox status, and muscle damage in the saliva of pigs and these can be considered potential biomarkers for this disease.

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.

Inter-alpha-trypsin inhibitor heavy chain 4: A serologic marker relating to disease risk, activity, and treatment outcomes of rheumatoid arthritis.

ObjectiveInter‐alpha‐trypsin inhibitor heavy chain 4 (ITIH4) regulates immunity and inflammation, but its clinical role in rheumatoid arthritis (RA) patients remains unclear. Hence, this study was conducted to explore the association of circulating ITIH4 with disease risk, clinical features, inflammatory cytokines, and treatment outcomes of RA.MethodsAfter the enrollment of 93 active RA patients and 50 health controls (HCs), their serum ITIH4 level was analyzed by enzyme‐linked immunosorbent assay (ELISA). For RA patients only, serum ITIH4 level at week (W) 6 and W12 after treatment was also analyzed. Besides, serum tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, IL‐6, and IL‐17A at baseline of RA patients were also detected by ELISA.ResultsITIH4 was downregulated in RA patients (151.1 (interquartile range (IQR): 106.2–213.5) ng/mL) than in HCs (306.8 (IQR: 238.9–435.1) ng/mL) (p < 0.001). Furthermore, ITIH4 was negatively related to C‐reactive protein (CRP) (rs  = −0.358, p < 0.001) and 28‐joint disease activity score using erythrocyte sedimentation rate (DAS28‐ESR) (rs  = −0.253, p = 0.014) in RA patients, but not correlated with other clinical features (all p > 0.05). Besides, ITIH4 was negatively linked with TNF‐α (rs  = −0.337, p = 0.001), IL‐6 (rs  = −0.221, p = 0.033), and IL‐17A (rs  = −0.368, p < 0.001) in RA patients, but not correlated with IL‐1β (rs  = −0.195, p = 0.061). Moreover, ITIH4 was gradually elevated in RA patients from baseline to W12 after treatment (p < 0.001). Additionally, the increment of ITIH4 at W6 and W12 was linked with treatment response and remission in RA patients (all p < 0.05).ConclusionCirculating ITIH4 possesses clinical utility in monitoring disease risk, inflammation, disease activity, and treatment outcomes of RA.

Serum inter-alpha-trypsin inhibitor heavy chain 4 in patients with inflammatory bowel disease: correlation with disease risk, inflammation, activity, and its variation after treatment

Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) plays vital roles in inflammatory and auto-immune diseases, but its correlations with disease risk and clinical features in inflammatory bowel disease (IBD) need further investigation. The present study intended to explore the correlation of ITIH4 with disease activity and inflammation, as well as its change after treatment in IBD patients. Totally, 40 active Crohn's disease (A-CD) patients, 40 clinical-remission CD (R-CD) patients, 40 active ulcerative colitis (A-UC) patients, 40 clinical-remission UC (R-UC) patients, and 40 health controls (HCs) were enrolled. ITIH4 in serum was assessed by ELISA. ITIH4 was lower in A-CD, R-CD, A-UC, and R-UC patients than in HCs (P < 0.001). Notably, ITIH4 reduced in A-CD patients than in R-CD patients (P = 0.017), and in A-UC patients compared with R-UC patients (P = 0.010). Besides, in A-CD patients, ITIH4 negatively correlated with tumor necrosis factor-alpha (TNF-α), interleukin (IL)-17A, IL-1β, C-reactive protein (CRP), and clinical disease activity index score (all P < 0.05). In A-UC patients, ITIH4 negatively correlated with TNF-α, IL-17A, IL-1β, IL-6, CRP, and Mayo score (all P < 0.05). However, in R-CD and R-UC patients, these correlations were less obvious than in A-CD and A-UC patients. ITIH4 was increased after treatment (all P < 0.05), and its expression at W12 after treatment was higher in response patients compared with no response patients in A-CD (P = 0.022) and A-UC groups (P = 0.038). ITIH4 correlates with IBD susceptibility, active risk, inflammation level, and its elevation after treatment relates to clinical response in IBD patients.

ITIH4 is a novel serum biomarker for early gastric cancer diagnosis

BackgroundGastric cancer (GC) is one of the most lethal forms of cancer due to the absence of tools for its early detection. Here, we explored critical biomarkers for early diagnosis. Materials and methodsKey biomarkers in serum from patients with early gastric cancer (EGC) and healthy controls (HCs) were identified via mass spectrometry and the expression of inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) was evaluated using several methods. Furthermore, ITIH4 expression in sera and exosomes from patients with EGC, advanced GC (AGC), low grade intraepithelial neoplasia (LGN), chronic superficial gastritis with Helicobacter pylori infection (Hpi), other systemic malignant tumors (OSTs), and healthy controls was also evaluated. ResultsITIH4 was identified as a key biomarker in patients with EGC. Its expression level in serum from the EGC group, which showed the highest specificity (94.44%), was significantly higher than those in sera from other GC groups as well as the control. Western blot analysis, immunohistochemical staining, and exosome analysis also confirmed ITIH4 expression in sera from patients with GC, but not in those from healthy individual. ConclusionITIH4 is a key biomarker in serum from patients with EGC and has potential as a high value diagnostic marker for EGC.

[Proteomic analysis of lacrimal gland adenoid cystic carcinoma with high-grade transformation].

Objective: To analyze the protein expression differences of lacrimal gland adenoid cystic carcinoma (LACC) with high-grade transformation (HGT). Methods: Experimental study. A total of 8 paraffin tissue samples were collected in Tianjin Medical University Eye Hospital from December 2012 to January 2019. According to pathological examination, the samples were divided into the LACC group and the LACC-HGT group, with 4 cases in each group. The LACC group included 2 male samples and 2 female samples, with an average age of 53 years. The LACC-HGT group included 2 male samples and 2 female samples, with an average age of 44 years. Primary cells were cultured from fresh tumor tissue. Isobaric tags for relative and absolute quantification techniques were used to screen the differentially expressed proteins between the two groups, and bioinformatics analysis was conducted for the differentially expressed proteins. Microarray was used to screen differentially expressed mRNAs between LACC and LACC-HGT primary cells. The mass spectrum data were intersected with mRNA microarray data, and quantitative real-time (qRT) PCR was performed to verify the results. Proteomics and microarray data were compared using the independent sample t test. The qRT-PCR data were compared pairwise by one-way analysis of variance. Results: A total of 105 HGT-related differential proteins were detected in this study, including 50 up-regulated proteins and 55 down-regulated proteins. The significantly up-regulated proteins included hemoglobin subunit beta, hemoglobin subunit alpha 1, and collagen type Ⅵ alpha 2 chain; the significantly down-regulated proteins included Cereblon, adenosylhomocysteinase like 2, and ribosomal protein L39 pseudogene 5. Gene ontology analysis results showed that the LACC-HGT differential proteins were mainly located in the cytoplasm, vesicle cavity, and extracellular matrix, had organic acid binding and molecular carrier activity, and participated in the regulation of extracellular matrix composition, immunity, inflammation, apoptosis, and other biological processes. Pathway analysis showed that the LACC-HGT differential proteins were mainly involved in signal pathways such as mitogen-activated protein kinase signal pathway and extracellular matrix proteoglycans and glycan metabolism signal pathway. Protein complex prediction analysis screened out 4 up-regulated protein complexes and 1 down-regulated protein complex. There were 15 LACC-HGT differential proteins that overlapped with mRNA chip differential genes, of which 6 were tumor-related proteins including collagen type XIV alpha 1 chain (COL14A1), EMAP like 4 (EML4), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), NDRG family member 2 (NDRG2), osteoglycin (OGN) an Ras homolog family member C (RhoC). The main function was the movement and migration of tumor cells. The qRT-PCR results showed that the relative expression levels of COL14A1, EML4, ITIH4, NDRG2, OGN, and RhoC in primary LACC-1, LACC-2, LACC-HGT-1, and LACC-HGT-2 cells were significantly different (F=1 675.98, 38.53, 27.37, 16.47, 13.38, 25.22, all P<0.01). For example, the relative expression of COL14A1 in primary LACC-HGT-1 (16.09±0.51) and LACC-HGT-2 (9.96±0.34) cells was significantly higher than that in primary LACC-1 (1.00±0.13) and LACC-2 (0.67±0.08) cells (all P<0.05). Conclusion: There are differentially expressed proteins between LACC-HGT and LACC, among which COL14A1, EML4, ITIH4, NDRG2, OGN, and RhoC may play an important role in LACC-HGT and can be used as potential targets of LACC-HGT in further study. (Chin J Ophthalmol, 2021, 57: 531-539).

Alveolar epithelial inter-alpha-trypsin inhibitor heavy chain 4 deficiency associated with senescence-regulated apoptosis by air pollution.

Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is a type II acute-phase protein; however, the role of pulmonary ITIH4 after exposure to air pollution remains unclear. In this study, we investigated the role of ITIH4 in the lungs in response to air pollution. ITIH4 expression in bronchoalveolar lavage fluid (BAL) of 47 healthy human subjects and of Sprague-Dawley rats whole-body exposed to air pollution was determined, and the underlying antiapoptotic and matrix-stabilizing pathways in alveolar epithelial A549cells induced by diesel exhaust particles (DEPs) as well as ITIH4-knockdown were investigated. We found that an interquartile range (IQR) increase in PM2.5 was associated with a decrease of 2.673ng/mL in ITIH4, an increase of 1.104pg/mL of 8-isoprostane, and an increase of 6.918pg/mL of interleukin (IL)-6 in human BAL. In rats, increases in 8-isoprostane, IL-6, and p53 and a decrease in sirtuin-1 (Sirt1) in the lungs and decreases in ITIH4 in the BAL, lungs, and serum were observed after PM2.5 and gaseous exposure. ITIH4 levels in lung lysates were correlated with levels in BAL samples (r=0.377, p<0.01), whereas ITIH4 levels in BAL were correlated with IL-6 levels (r=-0.420, p<0.01). ITIH4 expression was significantly reduced in alveolar epithelial A549cells by DEP in a dose-dependent manner. A decrease in Sirt1 and increases in phosphorylated extracellular signal-regulated kinase (p-ERK) and caspase-3 were observed after DEP exposure and ITIH4-knockdown. In conclusion, air pollution decreased ITIH4 expression in the lungs, which was associated with alveolar epithelial cell senescence and apoptosis. ITIH4 could be a vital protein in regulating alveolar cell destruction and its inhibition after exposure to air pollution.

Measurement of ITIH4 and Hp levels in bitches with pyometra using newly developed ELISA methods

Measurement of acute phase proteins (APPs) as biomarkers in canine medicine is in increasing demand. In the present study, the development and validation of two ELISA methods for the quantification of canine inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and haptoglobin (Hp) are shown. The adequate imprecision and accuracy and wide analytical range make the developed methods appropriate to quantify ITIH4 and Hp in serum samples. The inter- and intra-assay CVs were lower than 10 %, and the assays maintained linearity under dilution and showed analytical equivalence with the method of radial immunodiffusion. The measurement of CRP, Hp and ITIH4 in sera from bitches affected by pyometra allowed us to determine that ITIH4 behaves as a moderate APP in dogs. The group of bitches affected by pyometra showed very high levels of CRP (147 ± 91 mg/L), corresponding to a strong inflammatory process, which resulted in a moderate increase in the concentrations of Hp (7 times) and ITIH4 (3 times) compared to the control group.

Identifying Plasma Biomarkers with high specificity for major depressive disorder: A multi-level proteomics study

BackgroundThere are currently no objective diagnostic biomarkers for major depressive disorder (MDD) due to the biological complexity of the disorder. The existence of blood-based biomarkers with high specificity would be convenient for the clinical diagnosis of MDD. MethodsA comprehensive plasma proteomic analysis was conducted in a highly homogeneous cohort [7 drug-naïve MDD patients and 7 healthy controls (HCs)], with bioinformatics analysis combined with machine learning used to screen candidate proteins. Verification of reproducibility and specificity was conducted in independent cohorts [60 HCs and 74 MDD, 42 schizophrenia (SZ) and 39 bipolar I disorder (BD-I) drug-naïve patients]. Furthermore, verification of consistency was accomplished by proteomic analysis of postmortem brain tissue from 16 MDD patients and 16 HCs. Results: Levels of C-reactive protein (CRP), antithrombin III (ATIII), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and vitamin D-binding protein (VDB) were significantly higher in MDD patients, both in the discovery cohort and independent replication cohort. In comparison with SZ or BD-I patients, two proteins (VDB and ITIH4) were significantly elevated only in MDD patients. In addition, increased VDB and ITIH4 were observed consistently in both plasma and postmortem dorsolateral prefrontal cortex tissues of MDD patients. Furthermore, a panel consisting of all four plasma proteins was able to distinguish MDD patients from HCs or SZ or BD-I patients with the highest accuracy. ConclusionPlasma ITIH4 and VDB may be potential plasma biomarkers of MDD with high specificity. The four-protein panel is more suitable as a potential clinical diagnostic marker for MDD.

Sgp120 and the contact system in hereditary angioedema: A diagnostic tool in HAE with normal C1 inhibitor

Mutations in Factor XII, plasminogen gene, angiopoietin-1 gene and kininogen 1 gene have been found in some patients with hereditary angioedema with normal C1 inhibitor (HAE-nl-C1inh), but the underlying disease mechanisms remain unclear. Additionally, there are no accepted biomarkers for this disease. Because the contact system has been implicated in hereditary angioedema with C1 inhibitor deficiency (HAE-C1inh), we studied the fragmentation patterns of serum glycoprotein 120 (sgp120), a protein that is highly susceptible to cleavage by kallikrein, in 31 HAE-C1inh and 13 HAE-nl-C1inh patient plasma samples. Compared to normal controls, the majority of plasma samples from patients with HAE-C1inh contained fragmented sgp120. These samples also showed increased kallikrein amidolytic activity indicating spontaneous contact system activation. In contrast, most samples from HAE-nl-C1inh patients exhibited intact sgp120. However, if these samples were incubated at 4 °C in plastic, significant sgp120 fragmentation and spontaneous contact system activation were observed. Concurrently, there was C1 inhibitor fragmentation that generated the nonfunctional 94 kD fragment and a reduction in C1 inhibitor function. Normal samples did not show sgp120 or C1 inhibitor fragmentation after incubation. We sequenced sgp120 and found it to be identical to inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4). These results suggest that sgp120 or ITIH4 is cleaved when the contact system is activated and that this cleavage could be used as a biomarker in patients with HAE-nl-C1inh.

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.

Elevated levels of circulating ITIH4 are associated with hepatocellular carcinoma with nonalcoholic fatty liver disease: from pig model to human study

BackgroundNoninvasive biomarkers are urgently needed for optimal management of nonalcoholic fatty liver disease (NAFLD) for the prevention of disease progression into nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). In order to identify the biomarkers, we generated the swine hepatocellular carcinoma (HCC) model associated with NAFLD and performed serum proteomics on the model.MethodsMicrominipigs were fed a high-fat diet to induce NAFLD and a normal diet as the control. To induce HCC, diethylnitrosamine was intraperitoneally administered. Biopsied liver samples were histopathologically analyzed every 12 weeks. Serum proteins were separated by blue native two-dimensional gel electrophoresis and proteins of interest were subsequently identified by MALDI-TOF MS/MS. Human serum samples were analyzed to validate the candidate protein using antibody-mediated characterization.ResultsIn the NAFLD pigs, hepatic histology of nonalcoholic steatohepatitis (NASH) was observed at 36 weeks, and HCC developed at 60 weeks. Among serum proteins identified with MALDI-TOF MS/MS, serum inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), an acute response protein which is secreted primarily by liver, was identified as the most characteristic protein corresponding with NAFLD progression and HCC development in the NAFLD pigs. With immunoassay, serum ITIH4 levels in the NAFLD pigs were chronologically increased in comparison with those in control animal. Furthermore, immunohistochemistry showed ITIH4 expression in hepatocytes also increased in both the cancer lesions and parenchyma as NAFLD progressed. Human study is also consistent with this observation because serum ITIH4 levels were significantly higher in HCC-NAFLD patients than in the simple steatosis, NASH, and virus-related HCC patients. Of note, HCC-NAFLD patients who had higher serum ITIH4 levels exhibited poorer prognosis after hepatectomy.ConclusionsWe established an HCC pig model associated with NAFLD. Serum proteomics on the swine HCC with NAFLD model implicated ITIH4 as a non-invasive biomarker reflecting NAFLD progression as well as subsequent HCC development. Most importantly, the results in the swine study have been validated in human cohort studies. Dissecting speciation of serum ITIH4 promises to have clinical utility in monitoring the disease.

Acute-phase inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) levels in serum and milk of cows with subclinical mastitis caused by Streptococcus species and coagulase-negative Staphylococcus species

The aim of the study was to investigate the concentrations of acute-phase inter-α-trypsin inhibitor heavy chain 4 (ITIH4) in serum and milk of cows with subclinical mastitis caused by Streptococcus spp. (STR) and coagulase-negative Staphylococcus spp. (CNS) and healthy cows. The blood and milk samples were obtained from 60 mid-lactation, multiparous Holstein-Friesian cows from 7 herds in the Lublin region of Poland. In the milk samples from 40 cows with subclinical mastitis, Streptococcus spp. and CNS were isolated. The ITIH4 was significantly higher in serum of cows with subclinical mastitis caused both by STR and CNS compared with healthy cows. One hundred percent of animals infected with Streptococcus spp. and 89% of animals infected with Staphylococcus spp. showed ITIH4 concentration in sera higher than 0.5 mg/mL. The concentration of ITIH4 in milk also was significantly higher in cows with subclinical mastitis caused by Streptococcus spp. and Staphylococcus spp. compared with the control group. Seventy percent of cows infected by STR and CNS showed ITIH4 concentration in milk higher than 2.5 μg/mL. Milk ITIH4 concentration higher than 5 µg/mL was found in 55% of animals infected with Streptococcus spp. and in 40% of animals infected with Staphylococcus spp. No statistically significant differences were observed in ITIH4 concentrations both in serum and in milk between the studied unhealthy animal groups. These results suggest that ITIH4 may be used in the future as a novel diagnostic marker in serum and in milk of subclinical mastitis in cows.

Prediction of Outcome in Diabetic Acute Ischemic Stroke Patients: A Hospital-Based Pilot Study Report

Background: Demographic and clinical characteristics are known to influence the outcome in acute ischemic stroke (AIS) patients. Purpose: This study is aimed at evaluating short- and long-term outcomes in diabetic AIS patients. In addition, the study also evaluates the impact of diabetes on the performance of indigenously reported biomarker, inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and known biomarkers, neuron-specific enolase (NSE) and glial-derived S-100 beta beta protein (S-100ββ). Methods: This study was performed on 29 diabetes and 75 non-diabetes AIS patients. Outcome of AIS patients was analyzed by using modified Rankin scale at discharge, then at 12 and 18 months after discharge. Based on the obtained scores, patients were classified as improved group (scales 1-3) and dependent/expired group (scales 3-6). Blood samples were collected during admission and at discharge/expired time. Levels of NSE, S100ββ, and ITIH4 were analyzed in all samples. Results: On discharge, frequencies of dependent/expired outcome were 4/29 (14%) and 19/75 (17%) in diabetic and non-diabetic AIS patients. However, follow-up outcome at 12 and 18 months showed higher dependent/expired cases of 43 and 41% among diabetic AIS patients compared to 27 and 21% in non-diabetic patients. Multivariate analysis revealed that diabetes is an independent risk factor for dependent/expired outcome in AIS patients (OR 0.484 (at discharge); 1.307 (at 12 months) and 1.675 (at 18 months)). NSE, S100ββ, and ITIH4 showed a differential expression in both the outcome groups of AIS patients, irrespective of diabetes. Conclusion: Diabetes increases the risk of dependent/expired outcome in AIS patients. Also, serum NSE, S100ββ, and ITIH4 are independent biomarkers for prognosis of outcome in AIS patients, irrespective of diabetes.

Serum Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 (ITIH4) in Children with Chronic Hepatitis C: Relation to Liver Fibrosis and Viremia.

Liver fibrosis and viremia are determinant factors for the treatment policy and its outcome in chronic hepatitis C virus (HCV) infection. We aimed to investigate serum level of inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and its relation to liver fibrosis and viremia in children with chronic HCV. ITIH4 was measured by ELISA in 33 treatment-naive children with proved chronic HCV and compared according to different clinical, laboratory and histopathological parameters. Liver histopathological changes were assessed using Ishak score and compared with aspartate transaminase-to-platelet ratio (APRI) and FIB-4 indices as simple noninvasive markers of fibrosis. ITIH4 was measured in a group of 30 age- and sex-matched healthy controls. ITIH4 was significantly higher in patients than in controls (54.2 ± 30.78 pg/mL versus 37.21 ± 5.39 pg/mL; P = 0.021). ITIH4, but not APRI or FIB-4, had a significant direct correlation with fibrosis stage (P = 0.015, 0.961, and 0.389, resp.), whereas, the negative correlation of ITIH4 with HCV viremia was of marginal significance (P = 0.071). In conclusion, ITIH4 significantly correlated with higher stages of fibrosis indicating a possible relation to liver fibrogenesis. The trend of higher ITIH4 with lower viremia points out a potential antiviral properties and further studies in this regard are worthwhile.

Effects of Occupational Exposure to Tobacco Smoke: Is There a Link Between Environmental Exposure and Disease?

In a previous study, evidence was provided that indoor secondhand tobacco smoke (SHS) air pollution remains high in Lisbon restaurants where smoking is allowed, regardless of the protective measures used. The aim of this study was to determine in these locations the levels of polycyclic aromatic hydrocarbons (PAH) associated with the particulate phase of SHS (PPAH), a fraction that contains recognized carginogens, such as benzo[a]pyrene (BaP). Data showed that restaurant smoking areas might contain PPAH levels as high as 110 ng/m3, a value significantly higher than that estimated for nonsmoking areas (30 ng/m3) or smoke-free restaurants (22 ng/m3). The effective exposure to SHS components in restaurant smoking rooms was confirmed as cotinine levels found in workers' urine. Considering that all workers exhibited normal lung function, eventual molecular changes in blood that might be associated with occupational exposure to SHS and SHS-associated PPAH were investigated by measurement of two oxidative markers, total antioxidant status (TAS) and 8-hydroxyguanosine (8-OHdG) in plasma and serum, respectively. SHS-exposed workers exhibited higher mean levels of serum 8-OHdG than nonexposed workers, regardless of smoking status. By using a proteomics approach based on 2D-DIGE-MS, it was possible to identify nine differentially expressed proteins in the plasma of SHS-exposed nonsmoker workers. Two acute-phase inflammation proteins, ceruloplasmin and inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), were predominant. These two proteins presented a high number of isoforms modulated by SHS exposure with the high-molecular-weight (high-MW) isoforms decreased in abundance while low-MW isoforms were increased in abundance. Whether these expression profiles are due to (1) a specific proteolytic cleavage, (2) a change on protein stability, or (3) alterations on post-translational modification pattern of these proteins remains to be investigated. Considering that these events seem to precede the first symptoms of tobacco-related diseases, our findings might contribute to elucidation of early SHS-induced pathogenic mechanisms and constitute a useful tool for monitoring the effects of SHS on occupationally exposed individuals such as those working in the hospitality industry.

Proteomic biomarkers apolipoprotein A1, truncated transthyretin and connective tissue activating protein III enhance the sensitivity of CA125 for detecting early stage epithelial ovarian cancer

Objective The low prevalence of ovarian cancer demands both high sensitivity (> 75%) and specificity (99.6%) to achieve a positive predictive value of 10% for successful early detection. Utilizing a two stage strategy where serum marker(s) prompt the performance of transvaginal sonography (TVS) in a limited number (2%) of women could reduce the requisite specificity for serum markers to 98%. We have attempted to improve sensitivity by combining CA125 with proteomic markers. Methods Sera from 41 patients with early stage (I/II) and 51 with late stage (III/IV) epithelial ovarian cancer, 40 with benign disease and 99 healthy individuals, were analyzed to measure 7 proteins [Apolipoprotein A1 (Apo-A1), truncated transthyretin (TT), transferrin, hepcidin, ß-2-microglobulin (ß2M), Connective Tissue Activating Protein III (CTAPIII), and Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4)]. Statistical models were fit by logistic regression, followed by optimization of factors retained in the models determined by optimizing the Akaike Information Criterion. A validation set included 136 stage I ovarian cancers, 140 benign pelvic masses and 174 healthy controls. Results In a training set analysis, the 3 most effective biomarkers (Apo-A1, TT and CTAPIII) exhibited 54% sensitivity at 98% specificity, CA125 alone produced 68% sensitivity and the combination increased sensitivity to 88%. In a validation set, the marker panel plus CA125 produced a sensitivity of 84% at 98% specificity (P = 0.015, McNemar's test). Conclusion Combining a panel of proteomic markers with CA125 could provide a first step in a sequential two-stage strategy with TVS for early detection of ovarian cancer.