Detection of the cancer-associated T antigen using an Arachis hypogaea agglutinin biosensor.

An impedimetric biosensor was developed for the selective detection of the cancer-associated T antigen, using the lectin from Arachis hypogaea (peanut agglutinin, PNA) as the recognition element. The increase in the biosensor's impedance after sample incubation was indicative of lectin recognition and complex formation between PNA and glycoproteins containing T antigen. When using asialofetuin as model glycoprotein, a minimum amount of 100 ng of glycoprotein could be detected, generating an increase in impedance of 7.2%. Albumin did not cause interference in the detection of T-carrying glycoproteins up to a concentration of 0.01 mg ml-1. The biosensor was used to evaluate the T-antigen expression in serum samples and was able to discriminate between control samples (of individuals without cancer) and case samples from patients with diverse types of carcinomas (skin, colon, breast, prostate, stomach, kidney, lung, liver and rectum) in which an increase in the expression of T antigen is well-known. The same samples were analyzed with a Vicia villosa agglutinin biosensor that has specificity for the cancer-associated Tn antigen, to compare the expression of both antigens in the diverse carcinomas. The results were different for both biosensors, confirming that the use of different lectins allows to monitor different antigen expression. Furthermore, combining different lectins, glycosylation profiles for each carcinoma type can be obtained. This work demonstrates the feasibility of employing PNA to selectively recognize the T epitope in glycoproteins and the proposed biosensor could be used for high-throughput, label-free profiling of the cancer-associated T antigen in serum samples.

Merkel Cell Carcinomas Arising in Autoimmune Disease Affected Patients Treated with Biologic Drugs, Including Anti-TNF.

Purpose: The purpose of this investigation was to characterize Merkel cell carcinomas (MCC) arisen in patients affected by autoimmune diseases and treated with biologic drugs.Experimental Design: Serum samples from patients with MCC were analyzed for the presence and titer of antibodies against antigens of the oncogenic Merkel cell polyomavirus (MCPyV). IgG antibodies against the viral oncoproteins large T (LT) and small T (ST) antigens and the viral capsid protein-1 were analyzed by indirect ELISA. Viral antigens were recombinant LT/ST and virus-like particles (VLP), respectively. MCPyV DNA sequences were studied using PCR methods in MCC tissues and in peripheral blood mononuclear cells (PBMC). Immunohistochemical (IHC) analyses were carried out in MCC tissues to reveal MCPyV LT oncoprotein.Results: MCPyV DNA sequences identified in MCC tissues showed 100% homology with the European MKL-1 strain. PBMCs from patients tested MCPyV-negative. Viral DNA loads in the three MCC tissues were in the 0.1 to 30 copy/cell range. IgG antibodies against LT/ST were detected in patients 1 and 3, whereas patient 2 did not react to the MCPyV LT/ST antigen. Sera from the three patients with MCC contained IgG antibodies against MCPyV VP1. MCC tissues tested MCPyV LT-antigen-positive in IHC assays, with strong LT expression with diffuse nuclear localization. Normal tissues tested MCPyV LT-negative when employed as control.Conclusions: We investigated three new MCCs in patients affected by rheumatologic diseases treated with biologic drugs, including TNF. A possible cause-effect relationship between pharmacologic immunosuppressive treatment and MCC onset is suggested. Indeed, MCC is associated with MCPyV LT oncoprotein activity. Clin Cancer Res; 23(14); 3929-34. ©2017 AACR.

Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects.

Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18-65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses.

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.

Prognostic value of antibodies to Merkel cell polyomavirus T antigens and VP1 protein in patients with Merkel cell carcinoma.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is the main aetiological agent of Merkel cell carcinoma (MCC). Serum antibodies against the major MCPyV capsid protein (VP1) are detected in the general population, whereas antibodies against MCPyV oncoproteins (T antigens) have been reported specifically in patients with MCC. OBJECTIVES: The primary aim was to assess whether detection of serum antibodies against MCPyV proteins at baseline was associated with disease outcome in patients with MCC. The secondary aim was to establish whether evolution of these antibodies during follow-up was associated with the course of the disease. METHODS: Serum T-antigen and VP1 antibodies were assessed by enzyme-linked immunosorbent assay using recombinant proteins in a cohort of 143 patients with MCC, including 84 patients with serum samples available at baseline. RESULTS: Low titres of VP1 antibodies at baseline (< 10 000) were significantly and independently associated with increased risk of recurrence [hazard ratio (HR) 2·71, 95% confidence interval (CI) 1·13-6·53, P = 0·026] and death (HR 3·74, 95% CI 1·53-9·18, P = 0·004), whereas T-antigen antibodies were not found to be associated with outcome. VP1 antibodies did not differ between patients in remission and those with recurrence or progression during follow-up. However, T-antigen antibodies were more frequently detected in patients with recurrence or progression at 12 months (P = 0·020) and 24 months (P = 0·016) after diagnosis. CONCLUSIONS: VP1 antibodies constitute a prognostic marker at baseline, whereas T-antigen antibodies constitute a marker of disease recurrence or progression if detected > 12 months after diagnosis.

Exposure of Thomsen-Friedenreich antigen in Streptococcus pneumoniae infection is dependent on pneumococcal neuraminidase A.

Pneumococcal hemolytic uremic syndrome is recognized in a small portion of otherwise healthy children who have or have recently had Streptococcus pneumoniae infections, including severe pneumonia, meningitis, and bacteremia. As in other types of hemolytic uremic syndrome (HUS), pneumococcal HUS is characterized by microangiopathic hemolytic anemia, and thrombocytopenia, usually with extensive kidney damage. Although not demonstrated in vivo, the pathogenesis of pneumococcal HUS has been attributed to the action pneumococcal neuraminidase exposing the usually cryptic Thomsen-Friedenreich antigen (T-antigen) on red blood cells (RBC), and kidney glomeruli. We evaluated the effect of pneumococcal infection on desialylation of RBC and glomeruli during pneumococcal infections in mice. Following intravenous infection with capsular type 19F pneumococci, CFU levels exceeding 1000 CFU/mL blood by the third day were significantly more likely to result in exposed T-antigen on RBC than lower levels of bacteremia. In a pneumonia model, significantly more T-antigen was exposed on RBC in mice treated with penicillin than in those receiving mock treatment. Utilizing mutant pneumococci, we demonstrated that neuraminidase A but not neuraminidase B was necessary for exposure of T-antigen on RBC in vivo. Thus, pneumococcal neuraminidase A is necessary for the exposure of T-antigen in vivo and treatment with penicillin increases this effect. Interestingly, NanA(-) pneumococci were found in the blood in higher numbers and caused more deaths than wild type, NanB(-), or the NanA(-)/NanB(-) pneumococci.

Variations in glycosylation of von Willebrand factor with O-linked sialylated T antigen are associated with its plasma levels.

The glycosylation profile of von Willebrand factor (VWF) is known to strongly influence its plasma levels. VWF contains several carbohydrate structures, including O-linked glycans that primarily consist of sialylated T antigen (NeuAc(alpha2-3)Gal-(beta1-3)-[NeuAc(alpha2-6)]GalNAc). It is not yet known whether O-linked carbohydrates affect VWF levels. We developed an immunosorbent assay based on neuraminidase incubation allowing subsequent binding of peanut agglutinin (PNA) to desialylated O-linked T antigen on VWF. An inverse relation was found between PNA binding and VWF antigen levels in healthy individuals (n = 111; Pearson rank = -0.43; P < .001). A similar inverse association was observed in randomly selected plasma samples from our diagnostic laboratory: 252% +/- 125% for VWF levels less than 0.5 U/mL (n = 15); 131% +/- 36% for VWF levels between 0.5 and 1.5 U/mL (n = 32); and 92% +/- 40% for VWF levels more than 1.5 U/mL (n = 19). Reduced or increased PNA binding was also observed in patients with increased (liver cirrhosis) or reduced (von Willebrand disease [VWD] type 1) VWF antigen levels, respectively. VWD type 1 patients further displayed increased ratios of propeptide over mature VWF antigen levels (0.38 +/- 0.18 versus 0.17 +/- 0.03 for patients and controls, respectively; P < .001), which is indicative of reduced VWF survival in these patients. Of interest, a linear relation between PNA binding and propeptide/VWF ratio was observed (Spearman rank = 0.47), suggesting a potential association between O-linked glycosylation and VWF survival. Finally, we detected a marked decrease in PNA binding in post-DDAVP (1-deamino-8-D-arginine vasopressin) samples from various patients, indicating that the O-linked glycosylation profile of VWF stored in endothelial storage organelles may differ from circulating VWF.

Detection of CD8+ T cells sensitized to BK virus large T antigen in healthy volunteers and kidney transplant recipients.

BK virus (BKV) infections after renal transplantation are increasingly recognized. Development of immune monitoring strategies against BKV requires definition of antigenic epitopes. Hence, T cells from HLA-A02-positive healthy subjects and kidney transplant recipients were stimulated by BKV lysate pulsed on mature autologous dendritic cells and screened against four different T antigen peptides or against BKV lysate. IFN-gamma production was measured by ELISPOT assays. The peptide BKV362-371 (MLTERFNHIL) was naturally processed and recognized by five of six healthy subjects (39 +/- 11 IFN-gamma spots/100,000 cells) and five of seven kidney transplant recipients (21 +/- 12 IFN-gamma spots). Less frequent and weaker CD8+ T-cell responses were detected against three other peptides. Thus, BKV large T antigen is a target for CD8+ T-cell immunity. T-antigen-specific T-cytotoxic cells circulate in healthy blood donors, implying that transient expression of T antigen presumably occurs at sites of viral latency and helps maintain a constant pool of circulating CD8+ T memory cells.

Prevalence of 52-kd and 60-kd Ro/SS-A autoantibodies in Japanese patients with polymyositis/dermatomyositis.

To determine the prevalence of 52-kd and 60-kd Ro/SS-A antibodies in Japanese patients with polymyositis/dermatomyositis, we examined serum samples from 61 patients with PM/DM, 10 patients with primary Sjögren's syndrome, and 25 healthy control subjects. Six serum samples possessed anti-Ro/SS-A antibodies and were positive for anti-Ro52, anti-Ro60, or both. Two reacted with both Ro52 and Ro60, and 4 reacted with Ro52 alone. The results suggest that Ro52 is the main antigen of anti-Ro/SS-A antibodies in patients with polymyositis/dermatomyositis and that its coexistence with other defined antibodies suggests the existence of a subgroup of patients with various serologic abnormalities.

Multiple conformational epitopes are recognized by natural and induced immunity to the E7 protein of human papilloma virus type 16 in man.

The reactivity of sera from patients with cervical cancer with the E7 protein of human papilloma virus type 16 (HPV16) was estimated using a novel non-radioactive immunoprecipitation assay and four established protein- and peptide-based immunoassays. Six of 14 sera from patients with cervical cancer and 1 of 10 sera from healthy laboratory staff showed repeated reactivity with E7 in at least one assay. Four of the 7 reactive sera were consistently reactive in more than one assay, but only one was reactive in all four assays. Following immunization with E7, 2 of 5 patients with cervical cancer had increased E7-specific reactivity, measurable in one or more assays. No single assay was particularly sensitive for E7 reactivity, or predictive of cervical cancer. Mapping of E7 reactivity to specific E7 peptides was unsuccessful, suggesting that natural or induced E7 reactivity in human serum is commonly directed to conformational epitopes of E7. These results suggest that each assay employed in this study measures a different aspect of E7 reactivity, and that various reactivities to E7 may manifest following HPV infection or immunization. This finding is of significance for monitoring of E7 immunotherapy and for serological screening for cervical cancer.

Expression of the T antigen on a T-lymphoid cell line, supT1.

We have measured glycosyltransferase activities of SupT1 cells, a T-lymphoid cell line shown to react with autoantibodies in the sera of many HIV patients. Since considerable alpha-N-acetylgalactosaminyl-transferase and beta 1, 3 galactosyltransferase activities were found in SupT1 cells, at least the O-glycan core 1 structure can probably be synthesized. FACS analysis using an anti-T monoclonal antibody showed expression of the T antigen (Gal beta 1-3 GalNAc). Glycoproteins with the T antigen were isolated by immunoprecipitation with the anti-T antibody from a SupT1 cell lysate labelled metabolically with 3H-glucosamine and then analysed by SDS-PAGE. It was revealed that the precipitate contained a glycoprotein with a molecular weight corresponding to that of leukosialin. O-glycans were prepared from the immunoprecipitate by alkaline-borohydride treatment and then fractionated on Bio-Gel P-2, GalNAcOH and Gal-GalNAcOH being identified inter alia. These results suggest that an anti-T antibody may be included in the autoantibodies found in HIV-1 infected individuals.

IgA/VCA as a follow-up marker in the monitoring of nasopharyngeal carcinoma.

Titers of IgA/VCA from 92 nasopharyngeal carcinoma (NPC) patients were monitored for 3 to 11 years from the time of diagnosis. The fluctuations in the IgA/VCA titers during follow-up did not correlate with the clinical status of the patients, suggesting that IgA/VCA is of marginal significance in the monitoring of NPC patients during follow-up. In addition, the frequency of recurrence of NPC was independent of presence or absence of elevated IgA/VCA at diagnosis.

Investigation of the pathogenesis of massive hemolysis in a case of Clostridium perfringens septicemia.

Massive hemolysis is a rare, usually fatal complication of Clostridium perfringens septicemia. Of all toxins produced by the bacterium, phospholipase C (PLC) is believed to be the most likely cause of hemolysis. An influence of neuraminidase has often been suspected. In the present study, a case of C. perfringens septicemia with acute massive intravascular hemolysis is described. It led to death within 4 h of admission to the hospital. While the course of events was comparable to previously reported cases, we succeeded in gaining deeper insight into the pathogenesis by monitoring serum anti-T titer and quantifying serum PLC activity during the course of the disease. We excluded an effect of neuraminidase by a negative direct antiglobulin test, a negative anti-T lectin test, and a steady serum anti-T titer of 1 in 32. Serum PLC activity, on the other hand, showed a nearly fivefold increase (6.0 to 27.3 U/l), which is consistent with the hypothesized dominant role of this enzyme.

[Liberation of the T-antigen in bacteremia]. / Freilegung des T-Antigens bei Bakteriämie.

Many microorganisms are able to produce neuraminidase, which can uncover the T antigen on red blood cells and cause hemolysis. We studied T activation in 224 patients with positive blood cultures. Only those patients were included who had real bacteremia according to clinical parameters and microbiological results. None of our patients showed T transformation. We conclude that T activation is a rare or a very passing phenomenon which has less importance in routine diagnosis of sepsis.

TAF test in primary esophageal carcinoma: comparison with other tumor markers.

An ELISA method for the determination of circulating specific HSV-TAA antibodies has recently become available (TAF test). The presence of TAF was tested in serum of 154 patients with primary esophageal carcinoma, collected in three institutions. The overall TAF-test positivity rate was 57.1%, being significantly lower in stage IV than in stage III patients. The concordance rate between TAF and CEA, ferritin, TPA, SCC and TATI was low, suggesting that TAF is probably independent of the other tumor markers evaluated. The clinical role of TAF-test determination in patients with esophageal carcinoma is currently under evaluation.