Epitomics: Analysis of Plasma C9 Epitope Heterogeneity in the Plasma of Lung Cancer Patients and Control Subjects.

The human proteome is more complex than the genetic code predicts it to be. Epitomics, or protein epitome profiling, is a tool for understanding sub-protein level variation. With the ultimate goal to explore C9 proteoforms and their relevance to lung cancer, here we report plasma C9 epitope-associated molecular heterogeneity in plasma samples of lung cancer patients and control subjects. We show three C9 epitopes (BSI0449, BSI0581, BSI0639) with markedly different association with lung cancer ("unaltered", "upregulated" and "downregulated"). In order to exclude confounding effects, we show first that the three epitope-defining mAbs recognize C9 in purified form and in the natural context, in the human plasma. Then, we present data demonstrating the lack of major epitope interdependence or overlap. The next experiments represent a quest toward the understanding of the molecular basis of apparent disparate association with lung cancer. Using immunochemistry, SDS PAGE and LC-MS/MS technologies, we demonstrate that epitope-specific immunoprecipitates of plasma C9 seem identical regarding peptide sequence. However, we found epitope-specific posttranslational modification and coprecipitated protein composition differences with respect to control and lung cancer plasma. Epitope profiling enabled the classification of hypothetical C9 proteoforms through differential association with lung cancer.

Multiplexed MRM-Based Proteomics Identified Multiple Biomarkers of Disease Severity in Human Heart Failure.

Heart failure (HF) is a complex disease due to the intricate interplay of several mechanisms, which therefore implies the need for a multimarker strategy to better personalize the care of patients with HF. In this study, we developed a targeted mass spectrometry approach based on multiple reaction monitoring (MRM) to measure multiple circulating protein biomarkers, involved in cardiovascular disease, to address their relevance in the human HF, intending to assess the feasibility of the workflow in the disease monitoring and risk stratification. In this study, we analyzed a total of 60 plasma proteins in 30 plasma samples from eight control subjects and 22 age- and gender- matched HF patients. We identified a panel of four plasma proteins, namely Neuropilin-2, Beta 2 microglobulin, alpha-1-antichymotrypsin, and complement component C9, that were more abundant in HF patients in relation to disease severity and pulmonary dysfunction. Moreover, we showed the ability of the combination of these candidate proteins to discriminate, with sufficient accuracy, HF patients from healthy subjects. In conclusion, we demonstrated the feasibility and potential of a proteomic workflow based on MRM mass spectrometry for the evaluation of multiple proteins in human plasma and the identification of a panel of biomarkers of HF severity.

Evaluation of Serum Glycoprotein Biomarker Candidates for Detection of Esophageal Adenocarcinoma and Surveillance of Barrett's Esophagus.

Esophageal adenocarcinoma (EAC) is thought to develop from asymptomatic Barrett's esophagus (BE) with a low annual rate of conversion. Current endoscopy surveillance of BE patients is probably not cost-effective. Previously, we discovered serum glycoprotein biomarker candidates which could discriminate BE patients from EAC. Here, we aimed to validate candidate serum glycoprotein biomarkers in independent cohorts, and to develop a biomarker candidate panel for BE surveillance. Serum glycoprotein biomarker candidates were measured in 301 serum samples collected from Australia (4 states) and the United States (1 clinic) using previously established lectin magnetic bead array (LeMBA) coupled multiple reaction monitoring mass spectrometry (MRM-MS) tier 3 assay. The area under receiver operating characteristic curve (AUROC) was calculated as a measure of discrimination, and multivariate recursive partitioning was used to formulate a multi-marker panel for BE surveillance. Complement C9 (C9), gelsolin (GSN), serum paraoxonase/arylesterase 1 (PON1) and serum paraoxonase/lactonase 3 (PON3) were validated as diagnostic glycoprotein biomarkers in lectin pull-down samples for EAC across both cohorts. A panel of 10 serum glycoprotein biomarker candidates discriminated BE patients not requiring intervention (BE± low grade dysplasia) from those requiring intervention (BE with high grade dysplasia (BE-HGD) or EAC) with an AUROC value of 0.93. Tissue expression of C9 was found to be induced in BE, dysplastic BE and EAC. In longitudinal samples from subjects that have progressed toward EAC, levels of serum C9 were significantly (p < 0.05) increased with disease progression in EPHA (erythroagglutinin from Phaseolus vulgaris) and NPL (Narcissus pseudonarcissus lectin) pull-down samples. The results confirm alteration of complement pathway glycoproteins during BE-EAC pathogenesis. Further prospective clinical validation of the confirmed biomarker candidates in a large cohort is warranted, prior to development of a first-line BE surveillance blood test.

iTRAQ based investigation of plasma proteins in HIV infected and HIV/HBV coinfected patients - C9 and KLK are related to HIV/HBV coinfection.

OBJECTIVES: Human immunodeficiency virus (HIV) and hepatitis B virus (HBV) share similar routes of transmission, and rapid progression of hepatic and immunodeficiency diseases has been observed in coinfected individuals. Our main objective was to investigate the molecular mechanism of HIV/HBV coinfections. METHODS: We selected HIV infected and HIV/HBV coinfected patients with and without Highly Active Antiretroviral Therapy (HAART). Low abundance proteins enriched using a multiple affinity removal system (MARS) were labeled with isobaric tags for relative and absolute quantitation (iTRAQ) kits and analyzed using liquid chromatography-mass spectrometry (LC-MS). The differential proteins were analyzed by Gene Ontology (GO) database. RESULTS: A total of 41 differential proteins were found in HIV/HBV coinfected patients as compared to HIV mono-infected patients with or without HAART treatment, including 7 common HBV-regulated proteins. The proteins involved in complement and coagulation pathways were significantly enriched, including plasma kallikrein (KLK) and complement component C9 (C9). C9 and KLK were verified to be down-regulated in HIV/HBV coinfected patients through ELISA analysis. CONCLUSION: The present iTRAQ based proteomic analyses identified 7 proteins that are related to HIV/HBV coinfection. HBV might influence hepatic and immune functions by deregulating complement and coagulation pathways. C9 and KLK could potentially be used as targets for the treatment of HIV/HBV coinfections.

Association between the plasma proteome and serum ascorbic acid concentrations in humans.

Vitamin C has been associated with a reduced risk of chronic diseases, but the biological pathways regulated by vitamin C are not all known. The objective was to use a proteomics approach to identify plasma proteins associated with circulating levels of ascorbic acid. Men and women (n=1022) 20-29 years of age from the Toronto Nutrigenomics and Health Study completed a general health and lifestyle questionnaire and a 196-item food frequency questionnaire and provided a fasting blood sample. Circulating ascorbic acid was analyzed by high-performance liquid chromatography, and a mass-spectrometry-based multiple reaction monitoring method was used to measure 54 proteins abundant in plasma that are involved in numerous physiologic pathways. Mean protein concentrations were compared across tertiles of serum ascorbic acid using analysis of covariance adjusted for sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma α-tocopherol. A Bonferroni significance level of P<.0009 was applied, and analyses were adjusted for multiple comparisons using the Tukey-Kramer procedure. Levels of complement C9, ceruloplasmin, alpha-1-anti-trypsin, angiotensinogen, complement C3, vitamin D binding protein and plasminogen were inversely associated with levels of ascorbic acid. The inverse association between ascorbic acid and vitamin D binding protein was highest in those with higher levels of serum 25-hydroxyvitamin D. In conclusion, several plasma proteins from various physiologic pathways are significantly associated with circulating levels of ascorbic acid. These findings suggest that vitamin C may have novel physiological effects.

Necrotic epithelial cells in proximal renal tubules of 2nd trimester fetuses: is this "acute tubular necrosis"?

PURPOSE: The aim of this study is to describe the occurrence of necrotic tubular cells in kidneys of non-macerated fetuses. METHODS: Description of histology and immunostaining results using C9 immunostain of proximal tubular epithelium of kidneys from 30 consecutive non-macerated fetuses' autopsies. RESULTS: the gestational age ranged from 13 to 22 weeks. The mean gestational age was 18.6 weeks; the cause of death was acute chorioamnionitis in 13 cases (43.3%), termination of pregnancy for fetal anomalies in 13 (43.3%) and other causes in 4 (13.3%). Histology of the kidneys revealed vacuolation of proximal tubule epithelial cells (100%), dilatation of tubules (93.4%) and tubular cell necrosis (53.4%). C9 immunostaining was positive in 24 cases (80%) and was seen in all gestational ages. CONCLUSIONS: These results indicate that tubular cell necrosis is not an uncommon finding in the kidneys of 2(nd) trimester fetuses and may represent acute tubular necrosis (ATN). C9 is a helpful marker in confirming this diagnosis. Future studies may further explore this preliminary observation.

Plasma biomarker discovery and validation for colorectal cancer by quantitative shotgun mass spectrometry and protein microarray.

The development of a new plasma biomarker for early detection would be necessary to improve the overall outcome of colorectal cancer. Here we report the identification and validation of the ninth component of complement (C9) as a novel plasma biomarker for colorectal cancer by cutting-edge proteomic technologies. Plasma proteins were enzymatically digested into a large array of peptides, and the relative quantity of a total of 94,803 peptide peaks was compared between 31 colorectal cancer patients and 59 age/sex-matched healthy controls using 2D image-converted analysis of liquid chromatography and mass spectrometry. The selected biomarker candidates were validated in 345 subjects (115 colorectal cancer patients and 230 age/sex-matched healthy controls) using high-density reverse-phase protein microarrays. Plasma levels of Apo AI and C9 in colorectal cancer patients significantly differed from healthy controls with P values of 7.94×10(-4) and 1.43×10(-12) (Student's t-test), respectively. In particular, C9 was elevated in patients with colorectal cancer, including those with stage-I and -II diseases (P=3.01×10(-3) and P=1.13×10(-5) , respectively). Although the significance of the present study must be validated in an independent clinical study, the increment of plasma C9 may be applicable to the early detection of colorectal cancer.

The utility of C4d, C9, and troponin T immunohistochemistry in acute myocardial infarction.

CONTEXT: Full activation and involvement of the complement pathway follows acute myocardial infarction. Complement fragment C4d is a stable, covalently bound marker of complement activation. Troponin T is specific for cardiomyocytes. OBJECTIVES: To determine the specificity of C4d, C9, and troponin T immunoreactivity in necrotic myocytes and to establish whether they can be used to delineate acute myocardial infarction. DESIGN: Twenty-six autopsy cases with a total of 54 myocardium areas of infarction were reviewed retrospectively. Immunohistochemistry for C4d, C9, and troponin T was used on paraffin sections of formalin-fixed tissue. Controls consisted of 5 cases without evidence of infarction, and histologically normal myocardium functioned as an internal control. RESULTS: C4d and C9 antibodies reacted strongly and diffusely with necrotic myocytes in all samples of infarctions for up to 2 days (19 of 19; 100%). Adjacent histologically normal myocytes were nonreactive, resulting in a clear delineation between damaged and viable myocardium. Reactivity declined with increased duration and was absent in scars. Troponin T showed loss of staining in preinflammatory lesions (8 of 13; 62%); however, nonspecific patchy loss of staining was present in negative controls and in viable myocardium. Immunostains provided new diagnoses in 2 cases, including evidence of reinfarction and a newly diagnosed acute myocardial infarction. CONCLUSIONS: C4d and C9 have comparable reactivity and specificity for necrotic myocytes. C4d and C9 staining of necrotic myocytes is apparent before the influx of inflammatory cells, demonstrating utility in early myocardial infarction. Patchy loss of Troponin T in some cases of histologically normal myocardium limited its usefulness as a sole marker of infarction.

Glomeruli of Dense Deposit Disease contain components of the alternative and terminal complement pathway.

Dense Deposit Disease (DDD), or membranoproliferative glomerulonephritis type II, is a rare renal disease characterized by dense deposits in the mesangium and along the glomerular basement membranes that can be seen by electron microscopy. Although these deposits contain complement factor C3, as determined by immunofluorescence microscopy, their precise composition remains unknown. To address this question, we used mass spectrometry to identify the proteins in laser microdissected glomeruli isolated from paraffin-embedded tissue of eight confirmed cases of DDD. Compared to glomeruli from five control patients, we found that all of the glomeruli from patients with DDD contain components of the alternative pathway and terminal complement complex. Factor C9 was uniformly present as well as the two fluid-phase regulators of terminal complement complex clusterin and vitronectin. In contrast, in nine patients with immune complex-mediated membranoproliferative glomerulonephritis, glomerular samples contained mainly immunoglobulins and complement factors C3 and C4. Our study shows that in addition to fluid-phase dysregulation of the alternative pathway, soluble components of the terminal complement complex contribute to glomerular lesions found in DDD.

Ephedra sinica inhibits complement activation and improves the motor functions after spinal cord injury in rats.

The activation of complement system has been known as an important and significant reaction against the secondary injury after spinal cord injury (SCI). In the present study, we investigated the effect of Ephedra sinica to the inflammation or complement system of injured spinal cord and the influence to the functional recovery after spinal cord injury in rats. We prepared the complement-inhibiting component from E. sinica. Contusive spinal cord injury was induced to Sprague-Dawley rats. We administrated the product from E. sinica to E. sinica group, while distilled water was administered to the control group by gavage after SCI. Complement hemolytic activity (CH50), expression of C3 and C9, myeloperoxidase activity, and motor function were evaluated in E. sinica group and control group. The CH50, complement depositions, and myeloperoxidase activity in the E. sinica group were significantly reduced as compared to the control group. The motor function of E. sinica group was significantly improved from the 7th day as compared with the control group. The results demonstrated that E. sinica might reduce inflammation and improve motor function in rats after spinal cord injury by inhibiting complement activation. The present study has shown that complement system is playing an important role in spinal cord injury, and the possibility of a new therapy strategy, inhibiting or controlling the complement activation and inflammation, for spinal cord injury.

Deficiency of the human complement regulatory protein factor H associated with low levels of component C9.

We identified a 4-year-old Brazilian boy from a family of Japanese descent and history of consanguinity, who suffered from severe recurrent pneumonia. He carries factor H (FH) deficiency associated with reduced levels of component C9 and low serum levels of C3 and factor B. His mother also presented low levels of these proteins and factor I, while his father and sister had only lower levels of FH. Western blot assays confirmed the complete absence of FH and FHL-1 polypeptides in this patient. Sequencing of the proband's FH cDNA revealed a homozygous G453A substitution, encoding an Arg(127)His change. His mother, father and sister are heterozygous for this substitution. Despite the absence of FH in the plasma, this protein was detected in the patient's fibroblasts, suggesting that Arg(127) may be important for FH secretion. Low concentrations of C9 were detected in the proband serum but no mutations in the patient's C9 gene or promoter have been identified, suggesting that this is a consequence of uncontrolled complement activation and high C9 consumption.

[Immunohistochemical diagnostic management of early stages of myocardial infarction for the purposes of postmortem medicolegal examinations]. / Diagnostyka immunohistochemiczna wczesnego zawalu miesnia sercowego dla celów posmiertnego badania sadowo-lekarskiego.

A postmortem diagnosis of a "well developed" myocardial infarction is not difficult. In the initial stages of infarction, however, at the stage of gross examination it is impossible to discriminate between the infarcted zone and the undamaged cardiac muscle. When a cardiogenic cause of death is suspected, this fact determines the method of material collection and the amount to be collected, as well as further examinations to be performed. The histopathological diagnosis of a "fresh" myocardial infarction employing a basic routine staining method (hematoxylin/eosin) is not sufficient either. This makes it necessary to use additional special staining techniques to resolve diagnostic problems, thus verifying the precise diagnosis. The aim of this research was to introduce the immunohistochemical C9 staining technique as a postmortem diagnostic method of detecting recent myocardial infarctions for the purpose of postmortem medicolegal examinations, to develop the principles of result interpretation and to compare the said technique with the previously used Nielsen-Selye staining method. The specimens examined were collected from the heart muscle in 90 autopsy cases: cases of myocardial infarction positively diagnosed during autopsies and verified by routine staining examination (5); clinically positively diagnosed cases of myocardial infarction, without autopsy verification and examined microscopically using the routine staining method (5); sudden deaths with symptoms of acute circulatory failure due to cardiac causes (50) and the controls (30). The investigation demonstrated the specificity of the immunohistochemical C9 staining method in cases of myocardial damage associated with focal ischemia. It also confirmed the higher usefulness of the immunohistochemical method as compared to the Nielsen-Selye staining method in postmortem diagnostics of recent stages of myocardial infarction.

Loss of aquaporin 4 in lesions of neuromyelitis optica: distinction from multiple sclerosis.

Neuromyelitis optica (NMO) is an inflammatory and necrotizing disease clinically characterized by selective involvement of the optic nerves and spinal cord. There has been a long controversy as to whether NMO is a variant of multiple sclerosis (MS) or a distinct disease. Recently, an NMO-specific antibody (NMO-IgG) was found in the sera from patients with NMO, and its target antigen was identified as aquaporin 4 (AQP4) water channel protein, mainly expressed in astroglial foot processes. However, the pathogenetic role of the AQP4 in NMO remains unknown. We did an immunohistopathological study on the distribution of AQP4, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), activated complement C9neo and immunoglobulins in the spinal cord lesions and medulla oblongata of NMO (n = 12), MS (n = 6), brain and spinal infarction (n = 7) and normal control (n = 8). The most striking finding was that AQP4 immunoreactivity was lost in 60 out of a total of 67 acute and chronic NMO lesions (90%), but not in MS plaques. The extensive loss of AQP4 accompanied by decreased GFAP staining was evident, especially in the active perivascular lesions, where immunoglobulins and activated complements were deposited. Interestingly, in those NMO lesions, MBP-stained myelinated fibres were relatively preserved despite the loss of AQP4 and GFAP staining. The areas surrounding the lesions in NMO had enhanced expression of AQP4 and GFAP, which reflected reactive gliosis. In contrast, AQP4 immunoreactivity was well preserved and rather strongly stained in the demyelinating MS plaques, and infarcts were also stained for AQP4 from the very acute phase of necrosis to the chronic stage of astrogliosis. In normal controls, AQP4 was diffusely expressed in the entire tissue sections, but the staining in the spinal cord was stronger in the central grey matter than in the white matter. The present study demonstrated that the immunoreactivities of AQP4 and GFAP were consistently lost from the early stage of the lesions in NMO, notably in the perivascular regions with complement and immunoglobulin deposition. These features in NMO were distinct from those of MS and infarction as well as normal controls, and suggest that astrocytic impairment associated with the loss of AQP4 and humoral immunity may be important in the pathogenesis of NMO lesions.

[Myocardial ischemia of the driver as a cause of a traffic road accident. Immunohistochemical C9 staining method in diagnostics of early myocardial infarction]. / Niedotlenienie miesnia sercowego u kierowcy przyczyna wypadku drogowego. Metoda immunohistochemiczna C9 w diagnostyce wczesnego zawalu miesnia sercowego.

The authors presented a case of verification of pathological lesions as a cause of traffic accident where the driver--the culprit--was a fatal victim due to multiple injuries. Histopathological examination of postmortem samples of myocardium was conducted, using the hematoxylin-eosin, Nielsen-Selye and immunohistochemical C9 staining methods in order to verify the hypothesis about a possible myocardial ischemia triggering the accident. The results of "routine" (H&E) and--especially--immunohistochemical C9 staining showed myocardial damage due to ischemia, which was a morphological indicator evidencing the cause of "misbehavior" of the driver.

Complement-dependent apoptosis and inflammatory gene changes in murine lupus cerebritis.

The role of complement activation in the brains of MRL/lpr lupus mice was determined using the potent C3 convertase inhibitor, CR1-related y (Crry), administered both as an overexpressing Crry transgene and as Crry-Ig. Prominent deposition of complement proteins C3 and C9 in brains of MRL/lpr mice was indicative of complement activation and was significantly reduced by Crry. Apoptosis was determined in brain using different independent measures of apoptosis, including TUNEL staining, DNA laddering, and caspase-3 activity, all of which were markedly increased in lupus mice and could be blocked by inhibiting complement with Crry. Complement activation releases inflammatory mediators that can induce apoptosis. The mRNA for potentially proinflammatory proteins such as TNFR1, inducible NO synthase, and ICAM-1 were up-regulated in brains of lupus mice. Crry prevented the increased expression of these inflammatory molecules, indicating that the changes were complement dependent. Furthermore, microarray analysis revealed complement-dependent up-regulation of glutamate receptor (AMPA-GluR) expression in lupus brains, which was also validated for AMPA-GluR1 mRNA and protein. Our results clearly demonstrate that apoptosis is a prominent feature in lupus brains. Complement activation products either directly and/or indirectly through TNFR1, ICAM-1, inducible NO synthase, and AMPA-GluR, all of which were altered in MRL/lpr mouse brains, have the potential to induce such apoptosis. These findings present the exciting possibility that complement inhibition is a therapeutic option for lupus cerebritis.

Contrast in the efficacy of hDAF mouse hearts between ex vivo perfusion and transplantation into primates.

In recent experiments, in which we compared hDAF transgenic rat hearts perfused with 15% human serum in the Langendorff device and hDAF rat hearts transplanted into cynomolgus monkeys, we demonstrated that in the ex vivo heart perfusion model both homozygous and heterozygous hDAF hearts survived longer as nontransgenic controls. Surprisingly, we found that only homozygous hDAF hearts were protected against hyperacute rejection in vivo. The first aim of this study was to determine whether perfusion of mouse hearts with higher human serum concentrations or human blood might explain some of the differences found in survival time of the recently performed experiments with rat heart xenografts. Secondly, we investigated whether the observed differences in survival times of rat xenografts between in vivo and ex vivo transplantation would also hold for mouse hearts transgenic for hDAF. An ex vivo model was used to perfuse hDAF mouse hearts and controls with human serum or blood, and hDAF transgenic hearts and controls were transplanted into cynomolgus monkeys. hDAF transgenic mouse hearts survived significantly longer than their controls when perfused with 15% human serum, but no difference was found when 30% human serum was used, or when these hearts were transplanted into cynomolgus monkeys. However, in both the in vivo and ex vivo models the amount of PMNs adhering to the vascular endothelium was significantly lower in hDAF transgenes as compared with their controls. In conclusion, in the ex vivo situation, the efficacy of hDAF transgenesis in preventing HAR is limited by serum complement concentration.

CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: further regulation by CD55 and CD59.

Complement-dependent cytotoxicity is thought to be an important mechanism of action of the anti-CD20 monoclonal antibody rituximab. This study investigates the sensitivity of freshly isolated cells obtained from 33 patients with B-cell chronic lymphocytic leukemia (B-CLL), 5 patients with prolymphocytic leukemia (PLL), and 6 patients with mantle cell lymphoma (MCL) to be lysed by rituximab and complement in vitro. The results showed that in B-CLL and PLL, the levels of CD20, measured by standard immunofluorescence or using calibrated beads, correlated linearly with the lytic response (coefficient greater than or equal to 0.9; P <.0001). Furthermore, the correlation remained highly significant when the 6 patients with MCL were included in the analysis (coefficient 0.91; P <.0001), which suggests that CD20 levels primarily determine lysis regardless of diagnostic group. The role of the complement inhibitors CD46, CD55, and CD59 was also investigated. All B-CLL and PLL cells expressed these molecules, but at different levels. CD46 was relatively weak on all samples (mean fluorescence intensity less than 100), whereas CD55 and CD59 showed variability of expression (mean fluorescence intensity 20-1200 and 20-250, respectively). Although CD55 and CD59 levels did not permit prediction of complement susceptibility, the functional block of these inhibitors demonstrated that they play an important role in regulating complement-dependent cytotoxicity. Thus, lysis of poorly responding B-CLL samples was increased 5- to 6-fold after blocking both CD55 and CD59, whereas that of high responders was essentially complete in the presence of a single blocking antibody. These data demonstrate that CD20, CD55, and CD59 are important factors determining the in vitro response to rituximab and complement and indicate potential strategies to improve the clinical response to this biologic therapy.

Rhabdomyolysis, acute renal failure, and death after monensin ingestion.

We report a case of human monensin intoxication; to our knowledge, this is the first reported case in the medical literature. The patient took a dose of monensin three times higher than a dose considered lethal for cattle and developed a clinical picture similar to that reported in veterinary medicine. There was an early and extremely severe rhabdomyolysis followed by acute renal failure, heart failure, and death. The main changes observed at autopsy were extensive skeletal muscle necrosis, complement deposition at the myocardial level, pulmonary edema, and acute tubular damage.