Mass Spectrometry Proteomics Characterization of Plasma Biomarkers for Colorectal Cancer Associated With Inflammation.

Background: Colorectal cancer (CRC) prognosis is determined by the disease stage with low survival rates for advanced stages. Current CRC screening programs are mainly using colonoscopy, limited by its invasiveness and high cost. Therefore, non-invasive, cost-effective, and accurate alternatives are urgently needed. Objective and design: This retrospective multi-center plasma proteomics study was performed to identify potential blood-based biomarkers in 36 CRC patients and 26 healthy volunteers by high-resolution mass spectrometry proteomics followed by the validation in an independent CRC cohort (60 CRC patients and 44 healthy subjects) of identified selected biomarkers. Results: Among the 322 identified plasma proteins, 37 were changed between CRC patients and healthy volunteers and were associated with the complement cascade, cholesterol metabolism, and SERPIN family members. Increased levels in CRC patients of the complement proteins C1QB, C4B, and C5 as well as pro-inflammatory proteins, lipopolysaccharide-binding protein (LBP) and serum amyloid A4, constitutive (SAA4) were revealed for first time. Importantly, increased level of C5 was verified in an independent validation CRC cohort. Increased C4B and C8A levels were correlated with cancer-associated inflammation and CRC progression, while cancer-associated inflammation was linked to the acute-phase reactant leucine-rich alpha-2-glycoprotein 1 (LRG1) and ceruloplasmin. Moreover, a 4-protein signature including C4B, C8A, apolipoprotein C2 (APO) C2, and immunoglobulin heavy constant gamma 2 was changed between early and late CRC stages. Conclusion: Our results suggest that C5 could be a potential biomarker for CRC diagnosis. Further validation studies will aid the application of these new potential biomarkers to improve CRC diagnosis and patient care.

Plasma protein changes reflect colorectal cancer development and associated inflammation.

Introduction: Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of death worldwide. Efficient non-invasive blood-based biomarkers for CRC early detection and prognosis are urgently needed. Methods: To identify novel potential plasma biomarkers, we applied a proximity extension assay (PEA), an antibody-based proteomics strategy to quantify the abundance of plasma proteins in CRC development and cancer-associated inflammation from few µL of plasma sample. Results: Among the 690 quantified proteins, levels of 202 plasma proteins were significantly changed in CRC patients compared to age-and-sex-matched healthy subjects. We identified novel protein changes involved in Th17 activity, oncogenic pathways, and cancer-related inflammation with potential implications in the CRC diagnosis. Moreover, the interferon γ (IFNG), interleukin (IL) 32, and IL17C were identified as associated with the early stages of CRC, whereas lysophosphatidic acid phosphatase type 6 (ACP6), Fms-related tyrosine kinase 4 (FLT4), and MANSC domain-containing protein 1 (MANSC1) were correlated with the late-stages of CRC. Discussion: Further study to characterize the newly identified plasma protein changes from larger cohorts will facilitate the identification of potential novel diagnostic, prognostic biomarkers for CRC.

Proteomics approaches to characterize the immune responses in cancer.

Despite the dynamic development of cancer research, annually millions of people die of cancer. The human immune system is the major 'guard' against tumor development. Unfortunately, cancer cells have the ability to evade the immune system and continue to grow. The proper understanding of the intricate immune response in tumorigenesis remains the holy grail of cancer immunology and designing effective immunotherapy. To decode the immune responses in cancer, in recent years, proteomics studies have received considerable attention. Proteomics studies focus on the detection and quantification of proteins, which are the effectors of biological functions, and as such, are proven to reflect the cell state more accurately, in comparison to genomic or transcriptomic studies. In this review, we discuss the proteomics studies applied to characterize the immune responses in cancer and tumor immune microenvironment heterogeneity. Further, we describe emerging single-cell proteomics approaches that have the potential to be applied in cancer immunity studies.

The effects of post-translational modifications on Th17/Treg cell differentiation.

Regulatory T (Treg) cells and Th17 cells are subsets of CD4+ T cells which play an essential role in immune homeostasis and infection. Dysregulation of the Th17/Treg cell balance was shown to be implicated in the development and progression of several disorders such as autoimmune disease, inflammatory disease, and cancer. Multiple factors, including T cell receptor (TCR) signals, cytokines, metabolic and epigenetic regulators can influence the differentiation of Th17 and Treg cells and affect their balance. Accumulating evidence indicates that the activity of key molecules such as forkhead box P3 (Foxp3), the retinoic acid-related orphan receptor gamma t (RORγt), and signal transducer and activator of transcription (STAT)s are modulated by the number of post-translational modifications (PTMs) such as phosphorylation, methylation, nitrosylation, acetylation, glycosylation, lipidation, ubiquitination, and SUMOylation. PTMs might affect the protein folding efficiency and protein conformational stability, and consequently determine protein structure, localization, and function. Here, we review the recent progress in our understanding of how PTMs modify the key molecules involved in the Th17/Treg cell differentiation, regulate the Th17/Treg balance, and initiate autoimmune diseases caused by dysregulation of the Th17/Treg balance. A better understanding of Th17/Treg regulation may help to develop novel potential therapeutics to treat immune-related diseases.

Abdominal aortic aneurysm and virus infection: A potential causative role for cytomegalovirus infection?

An abdominal aortic aneurysm (AAA) is a multifactorial disease with a variety of genetic and environmental risk factors, but the exact mechanism of AAA formation and progression is still not well understood. The present study investigated the frequency of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and papillomavirus types 6 and 11 (HPV6 and HPV11), their impact on clinical manifestations of cardiovascular diseases, and their possible association with inflammation in patients with AAA and healthy volunteers. Genotyping of CMV UL75, EBV LMP-1, and HPV6, and HPV11 E6 was performed by polymerase chain reaction (PCR), while the viral DNA loads were measured by quantitative real-time PCR. Cytokine levels were determined by enzyme-linked immunosorbent assays. The CMV UL75 was detected more frequently in the blood of patients with AAA than in the blood of healthy volunteers (32.7% vs. 6.3%, p < .0001). Neither EBV LMP-1 nor HPV6 E6 was found in blood and aortic wall biopsies, while the HPV11 E6 was detected in 36.4% of AAA walls. The CMV infection in patients with AAA was associated with an increased risk of hypertension and coronary artery disease (OR, 9.057; 95% CI, 1.141-71.862; p = .037; and OR, 2.575; 95% CI, 1.002-6.615; p = .049, respectively). Additionally, CMV-infected patients with AAA had higher tumor necrosis factor-α levels compared with noninfected subjects (p = .017). Our findings suggest that CMV infection can stimulate local inflammation in the aorta but is not a direct cause of most abdominal aortic aneurysms.

Polymorphisms in the IL-6 and TNF-α gene are associated with an increased risk of abdominal aortic aneurysm.

BACKGROUND: An abdominal aortic aneurysm (AAA) is a complex disease of the aging population that is associated with inflammation and the cellular immune response. To investigate the influence of interleukin (IL)-6 and tumor necrosis factor (TNF)-α single nucleotide polymorphisms (SNPs) on the risk of AAA formation and progression, the frequency of AAA and its associated risk factors were determined. METHOD: Four SNPs in the IL-6 (-174G/C, rs1800795; -572G/C, rs1800796) and TNF-α (-238G/A, rs361525; -308G/A, rs1800629) genes were studied by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in patients with AAA and healthy volunteers. The mRNA expression and plasma IL-6 and TNF-α levels were also determined. RESULTS: A mutation detected in at least one allele of the IL-6 -174G/C SNP was associated with a 2-fold increased risk of AAA occurrence (OR: 2.08; 95% CI: 1.15-3.76; p = 0.014, in the dominant model). An increased risk of AAA incidence among heterozygous carriers of the TNF-α - 308G/A genotype was observed (OR: 2.06; 95% CI: 1.17-3.62; p = 0.011, in the overdominant model). The wild-type genotypes of the IL-6 -174G/C and the TNF-α -308G/A SNPs coexisted more frequently in healthy subjects than in AAA patients and was associated with decreased risk of AAA (p < 0.001). Moreover, elevated levels of IL-6 and TNF-α were associated with an increased risk of hypertension (p < 0.001 and p = 0.022, respectively). CONCLUSIONS: The IL-6 -174G/C and the TNF-α -238G/A gene polymorphisms are associated with an increased risk of abdominal aortic aneurysm development.

Insight into the expression of RIG-I-like receptors in human third trimester placentas following ex vivo cytomegalovirus or vesicular stomatitis virus infection.

A viral infection is detected through germline-encoded pattern-recognition receptors (PRRs) leading to the production of interferons (IFNs) and proinflammatory cytokines. The objective of this study was to investigate the expression of retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) in response to viral infection and the selected cytokine responses in the human term placenta. Placental villi and decidual explants were infected with human cytomegalovirus (CMV) or vesicular stomatitis virus (VSV) and cultured ex vivo to study viral infection. To evaluate DDX58 (RIG-I), IFIH1 (MDA5), and DHX58 (LGP2) expression, quantitative real-time PCR (qRT-PCR) was used. The expression of RLRs was detected by Western blotting. Cytokine and chemokine production, as well as RLR protein levels, were quantified using ELISA. The increased expression of both RIG-I and MDA5 and the enhanced secretion of IFN-ß were observed in response to VSV infection compared to mock-infected tissues. CMV infection resulted in higher transcript levels of DDX58 and IFIH1, while no changes in the cytokine production were observed. Our results indicate that RIG-I and MDA5 are specifically expressed in chorionic villi and deciduae in response to VSV infection. These findings suggest that RLRs may play a key role in pathogen recognition and the immune response against intrauterine viral transmission.

TLR4 896A/G and TLR9 1174G/A polymorphisms are associated with the risk of infectious mononucleosis.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and activate innate and adaptive immune responses. Single nucleotide polymorphisms (SNPs) within the TLR genes may influence host-pathogen interactions and can have an impact on the progression of infectious diseases. The present study aimed to investigate the genotype distribution of TLR2 (2029C/T, rs121917864; 2258G/A, rs5743708), TLR4 (896A/G, rs4986790), and TLR9 (- 1237T/C, rs5743836; - 1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) polymorphisms in 149 children and adolescents with infectious mononucleosis (IM) and 140 healthy individuals. The potential association of TLR SNPs with the clinical manifestations of EBV infection was also studied. The presence of TLR2, TLR4, and TLR9 SNPs was identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). EBV DNA loads were detected by quantitative real-time PCR assay. The TLR4 896 GG and the TLR9 1174 GA genotypes were associated with an increased risk of EBV-related IM in examined patients (p = 0.014 and p = 0.001, respectively). The heterozygous genotype of the TLR4 896A/G SNP was associated with an increased risk of elevated liver enzyme levels and leukocytosis (p < 0.05). Our preliminary study revealed that the TLR4 896A/G and the TLR9 1174G/A polymorphisms seem to be related to the course of acute EBV infection in children and adolescents.

Insight into the expression of toll-like receptors 2 and 4 in patients with abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is a relatively common, life-threatening disease prevalent in persons over the age of 65. In recent years, an increasing number of studies have suggested that pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), may serve as important regulators in the development of AAAs. In this study, we evaluated the TLR2 and TLR4 expression in the aortic wall and blood of patients with AAA. The TLR2 and TLR4 mRNA expression were significantly higher in the blood of patients with AAA than in the blood of healthy volunteers (p = 0.009 and p = 0.010, respectively). The expression of TLR2 and TLR4 transcripts was also higher in the blood compared with the aortic wall tissue of AAA patients (p = 0.001 for both). Higher TLR2 protein expression was observed in the aortic wall of AAA patients compared with the blood (p = 0.026). A significantly higher concentration of TNF-α and IL-4 in patients with AAA than in healthy volunteers (p < 0.001 for both) was noticed. This study suggests that TLR2 may play a role in the inflammatory response in the aorta, both locally and systemically, in patients with AAA.

Detection and genotyping of CMV and HPV in tumors and fallopian tubes from epithelial ovarian cancer patients.

Viral and bacterial infections are detected in epithelial ovarian cancer (EOC) tissues. Since the fallopian tubes are often affected by pelvic inflammatory disease (PID) and the majority of serous EOCs appear to originate from dysplastic lesions in the distal tube, it is relevant to consider the potential role that infectious agents may play in ovarian carcinogenesis. We sought to analyze the prevalence of human papillomavirus (HPV) and cytomegalovirus (CMV) in EOC tissue and fallopian tube specimens obtained at tumor resection. Ovarian cancer and fallopian tube tissue samples obtained from patients with EOC were analyzed by both qualitative and quantitative PCR to detect and quantify viral DNA. The presence of CMV and HPV DNA was detected in 70% and 74% cancerous ovarian tissues, respectively, and was significantly higher in EOC than in benign tumor cases (P ≤ 0.01). CMV or HPV infection was observed also in the fallopian tube samples. Infection with HPV16 was determined in 70% of EOC cases. Almost two thirds of EOC patients demonstrated coinfection with CMV and HPV in the pathological samples. The results revealed that the presence of CMV and HPV in EOC samples is common. CMV and HPV infections can be potential risks for EOC development.

Distribution of the CMV glycoprotein gH/gL/gO and gH/gL/pUL128/pUL130/pUL131A complex variants and associated clinical manifestations in infants infected congenitally or postnatally.

Human cytomegalovirus (CMV) is a major cause of morbidity in fetuses following intrauterine infection. The glycoprotein (g) envelope trimeric gH/gL/gO and pentameric gH/gL/pUL128/pUL130/pUL131A complexes are required for CMV entry into fibroblasts and endothelial/epithelial cells, respectively, and both are targets for neutralizing antibodies. The role of sequence variability among viral strains in the outcome of congenital CMV infection is controversial. Variation in the CMV UL75 gene encoding glycoprotein H (gH), the UL115 (gL), the UL74 (gO), and the UL128 locus (UL128L) encoding three structural proteins (pUL128, pUL130, and pUL131A) was determined in 82 newborns with congenital CMV infection and 113 infants with postnatal or unproven congenital CMV infection. Genotyping was performed by sequencing analysis of PCR-amplified fragments and the PCR-restriction fragment length polymorphism (RFLP) method, and the viral load was measured by quantitative real-time PCR. The obtained results demonstrated that (1) different CMV variants and mixed CMV infections can be detected in newborns infected congenitally; (2) the gH1 genotype, UL130 variant 6, and UL131A variant 1 were associated with some signs/symptoms within cohort of pediatric patients, mainly consisting of infants with symptomatic CMV infection. The results revealed that pUL130, pUL131A, and gH polymorphisms seemed to be associated with the outcome of CMV infection in infants.

Analysis of host Toll-like receptor 3 and RIG-I-like receptor gene expression in patients with abdominal aortic aneurysm.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a vascular disease relatively common in the elderly population. Although some events that contribute to the development and progression of AAA are known, there are limited data examining the association of Toll-like receptor 3 (TLR3) and RIG-I-like receptor expression with the pathogenesis of AAAs. In this study, we investigated the gene and protein expression of TLR3 and RIG-I-like receptors (RIG-I and MDA5) in aortic wall and blood of AAA patients and examined the relationship between their expression and immune response. METHODS: Total RNA was extracted from aortic wall tissues and blood samples collected from 20 patients with AAA and blood samples of 17 healthy volunteers without aortic aneurysm. To evaluate the DDX58 (RIG-I), IFIH1 (MDA5), and TLR3 gene expression level, quantitative real-time polymerase chain reaction was used. Extracellular cytokine and pattern recognition receptor levels were quantified by enzyme-linked immunosorbent assays. RESULTS: TLR3, RIG-I, and MDA5 were constitutively expressed in both aortic tissues and blood samples from AAA patients and healthy volunteers. In patients with AAA, higher TLR3 expression in aortic tissues than in blood was found (P = .004). The DDX58 messenger RNA expression was higher in blood of patients with AAA compared with healthy subjects (P = .021). A significantly higher level of plasma interleukin 4 was noticed in patients with AAA than in healthy individuals (P = .008). CONCLUSIONS: This study suggests that RIG-I and TLR3 seem to be important factors in the pathogenesis of AAA.

Cytomegalovirus alpha-chemokine genotypes are associated with clinical manifestations in children with congenital or postnatal infections.

Human cytomegalovirus (HCMV) is the leading cause of congenital infections. The aim of our study was to determine the prevalence of genotypes based on the highly polymorphic UL146 and UL147 HCMV genes and the relationship between the genotype and symptoms or viral load. We analyzed samples from 121 infants with symptomatic HCMV infection, including 32 congenitally infected newborns. The G7 and G5 genotypes were predominant in postnatal infection, whereas the G1 genotype was prevalent in congenital infection. Central nervous system (CNS) damage and hepatomegaly were detected more frequently among children infected with the G1 genotype than in those infected by other genotypes. An association between the viral genotype and viruria level was found. There was a strong correlation between HCMV genotypes determined through the UL146 and UL147 sequences (ĸ=0.794). In conclusion, we found that certain vCXCL genotypes are associated with clinical sequelae following HCMV infection.

[Role of the RIG-I-like receptors in antiviral response]. / Rola receptorów RIG-I-podobnych w odpowiedzi przeciwwirusowej.

The innate nonspecific immunity is the first line of defense against viral infection. Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are two main receptor families detecting viral nucleic acid. So far, three RLR family members were characterized: RIG-I, MDA5 and LGP2. RLR constitute a family of cytoplasmic helicases, which recognized intracellular single-stranded and double-stranded RNA that is introduced to cytosol during viral infection and replication. In this work we review the current knowledge about the mechanisms of viral recognition by RIG-I-like receptors and their signaling pathways for the activation of type I interferons and pro-inflammatory cytokines synthesis.

Cytomegalovirus glycoprotein H genotype distribution and the relationship with hearing loss in children.

Cytomegalovirus (CMV) is a leading cause of congenital infection and a leading infectious cause of hearing loss in children. The ORF UL75 gene encodes envelope glycoprotein H (gH), which is essential for CMV entry into host cells and the target of the immune response in humans. However, the distribution of gH variants and the relationship between the viral genotype, viral load, and sequelae in children infected with CMV is debated. The UL75 genetic variation of CMV isolates from 42 newborns infected congenitally with CMV and 93 infants with postnatal or unproven congenital CMV infection was analyzed. Genotyping was performed by analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. There were no differences in the distribution of gH genotypes in the children infected congenitally and postnatally. Mixed-genotype infections with both gH1 and gH2 variants were detected in approximately 25% of the examined patients. No relationship between UL75 gene polymorphisms and the symptoms at birth was observed. The results suggest that the infection with gH2 genotype diminishes the risk of hearing loss in children (P = 0.010). In addition, sensorineural hearing loss was associated with CMV gH1 genotype infection in infants (P = 0.032) and a high viral load in urine (P = 0.005). In conclusion, it was found that the gH genotype does not predict clinical sequelae in newborn infants following congenital CMV infection. However, these results suggest that the gH genotype might be associated with hearing loss in children.

Distribution of cytomegalovirus gN variants and associated clinical sequelae in infants.

BACKGROUND: Human cytomegalovirus (HCMV) is the most widespread cause of congenital infection. The effects of various viral strains and viral loads on the infection outcome have been under debate. OBJECTIVES: To determine the distribution of gN variants in HCMV strains isolated from children with congenital or postnatal infection and to establish the relationship between the viral genotype, the viral load, and the sequelae. STUDY DESIGN: The study population included congenitally HCMV-infected newborns and children with postnatal or unproven congenital HCMV infection. The genotyping was performed by RFLP analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. RESULTS: Our results demonstrated that the HCMV genotypes gN3b, gN4b, and gN4c were prevalent in the patients examined. There were no differences in the distributions of gN genotypes in the congenitally and postnatally infected children. Multiple HCMV strains were detected in both groups of children. A significant association between the HCMV gN4 genotype and the incidence of neurological disorders was observed (p=0.045). Our results suggest that the detection of the gN2 or the gN4 genotype may be indicative of serious manifestations in children. In contrast, the gN3b and the gN1 genotypes represent less pathogenic HCMV strains. The HCMV load in urine was significantly higher in children with congenital infection compared with children with postnatal infection. No correlation was found between the viral load and the genotype. CONCLUSION: Our results suggest that the gN genotype may be a virological marker of symptomatic HCMV infection in newborns.

Granulomatous pneumonia and hepatitis associated with Providencia rettgeri infection in a crocodile monitor lizard (Varanus salvadorii).

The present report describes a case of granulomatous pneumonia and hepatitis in a male crocodile monitor lizard (Varanus salvadorii). During the necropsy of the monitor lizard, multifocal to coalescing pale yellow lesions were observed in both lung lobes, as well as similar, though milder, changes in the liver, and an ulcerative lesion on the food pad of the right hindlimb. Histopathologically, the presence of multiple necrotising, chronic granulomas containing bacterial clumps were observed in the parenchyma of the lung and the liver. By microbiological examination of the pathologically altered lung tissues, Providencia rettgeri was identified. Altogether, our findings indicate that the bacterial infection resulting in extensive chronic necrotising granulomatous inflammation was the primary cause of the reptile's death. To our knowledge, this is the first report of Providencia rettgeri-associated granulomatous pneumonia and hepatitis in the monitor lizard.