Alternative Splicing of the Angiogenesis Associated Extra-Domain B of Fibronectin Regulates the Accessibility of the B-C Loop of the Type III Repeat 8

Background

Fibronectin (FN) is a multi-domain molecule involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. The biological activities of FN are mediated by exposed loops located mainly at the interdomain interfaces that interact with various molecules such as, but not only, integrins. Different FN isoforms arise from the alternative splicing of the pre-mRNA. In malignancies, the splicing pattern of FN pre-mRNA is altered; in particular, the FN isoform containing the extra-domain B (ED-B), a complete FN type III repeat constituted by 91 residues, is undetectable in normal adult tissues, but exhibits a much greater expression in fetal and tumor tissues, and is accumulated around neovasculature during angiogenic processes, thus making ED-B one of the best markers and targets of angiogenesis. The functions of ED-B are still unclear; however, it has been postulated that the insertion of an extra-domain such as ED-B modifies the domain-domain interface and may unmask loops that are otherwise cryptic, thus giving FN new potential activities.

Methodology

We used the mAb C6, which reacts with ED-B containing FN, but not with ED-B-free FN and various recombinant FN fragments containing mutations, to precisely localize the epitopes recognized by the mAb C6.

Conclusion

We formally demonstrated that the inclusion of the alternatively spliced angiogenesis-associated ED-B leads to the unmasking of the FNIII 8 B-C loop that is cryptic in FN molecules lacking ED-B. Thus, the mAb C6, in addition to providing a new reagent for angiogenesis targeting, represents a new tool for the study of the potential biological functions of the B-C loop of the repeat FNIII 8 that is unmasked during angiogenic processes.     

The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence.

We have previously reported an anti-fibronectin monoclonal antibody (mAb) (BC-1) which reacts with an ED-B-containing beta-galactosidase-fibronectin fusion protein but not with an identical beta-galactosidase-fibronectin fusion protein in which the ED-B sequence is omitted. In further experiments aimed at localizing more precisely the epitope recognized by this mAb, we demonstrate that 1) the mAb BC-1 is indeed specific for ED-B-containing fibronectin (FN) molecules even though the epitope recognized by this mAb is localized on the type III homology repeat 7 (the one which precedes the ED-B sequence) and 2) in fibronectin molecules lacking the ED-B sequence, this epitope is masked. We further demonstrate that, to mask the epitope recognized by the mAb BC-1, the presence of at least half of the FN type III homology repeat 9 is necessary. We also report the production of the mAb IST-6 which recognizes only FN molecules in which the ED-B sequence is lacking. These data clearly demonstrate that the presence of the ED-B sequence within FN molecules generates conformational modification in the central part of the molecules that unmasks previously cryptic sequences and masks others.     

Expression of fibronectin variants in vascular and visceral smooth muscle cells in development

Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied--aorta, esophagus, stomach, and jejunum. By 20-25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice--during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo.     

NMR structure of the human oncofoetal fibronectin ED-B domain, a specific marker for angiogenesis

BACKGROUND: The process of angiogenesis (i.e. the formation of new blood vessels from pre-existing ones) is fundamental to physiological processes such as reproduction, development and repair, as well as to pathological conditions such as tumor progression, rheumathoid arthritis and ocular disorders. The oncofoetal ED-B domain, a specific marker of angiogenesis, consists of 91 amino acid residues that are inserted by alternative splicing into the fibronectin (FN) molecule. RESULTS: The NMR structure of the ED-B domain is reported and reveals important differences from other FN type III domains. A comparison of the ED-B domain with the crystal structure of a four-domain FN fragment shows the novel features of ED-B to be located in loop regions that are buried at interdomain interfaces, and which therefore largely determine the global shape of the FN molecule. The negatively charged amino acids in this highly acidic protein are uniformly distributed over the molecular surface, with the sole exception of a solvent-exposed hydrophobic patch that represents a potential specific recognition site. Epitope mapping with 82 decapeptides that span the ED-B sequence revealed that three ED-B-specific monoclonal antibodies, which selectively target newly forming blood vessels in tumor-bearing mice, bind to adjacent regions on the ED-B surface. CONCLUSIONS: The NMR structure enables the identification of a large surface area of the ED-B domain that appears to be accessible in vivo, opening up new diagnostic and therapeutic opportunities. Furthermore, the mapping of specific monoclonal antibodies to the three-dimensional structure of the ED-B domain, and their use in angiogenesis inhibition experiments, provides a basis for further investigation of the role of the ED-B domain in the formation of new blood vessels.     

Endothelins: regulators of extracellular matrix protein production in diabetes

Fibronectin (FN), a key extracellular matrix protein, is upregulated in target organs of diabetic angiopathy and in cultured cells exposed to high levels of glucose. FN has also been reported to undergo alternative splicing to produce the extra domain-B (ED-B) containing isoform, which is exclusively expressed during embryogenesis, tissue repair, and tumoral angiogenesis. The present study was aimed at elucidating the role and mechanism of endothelins (ETs) in FN and ED-B FN expression in diabetes. We investigated vitreous samples for ED-B FN expression from patients undergoing vitrectomy for proliferative diabetic retinopathy. Our results show increased FN and ED-B FN expression in the vitreous of diabetic patients in association with augmented ET-1. Using an antibody specific to the ED-B segment of FN, we show an increase in serum ED-B FN levels in patients with diabetic retinopathy and nephropathy. We further examined retinal tissues, as well as renal and cardiac tissues, from streptozotocin-induced diabetic rats. Diabetes increased FN and ED-B FN in all three organs, which was prevented by ET antagonist bosentan. To provide insight into the mechanism of glucose-induced and ET-mediated ED-B FN upregulation, we assayed endothelial cells (ECs). Inhibition of mitogen-activated protein kinase with pharmacological inhibitors and protein kinase B with dominant negative transfections prevented glucose- and ET-1-mediated FN and ED-B FN expression. Furthermore, treatment of cells exposed to high levels of glucose with ET antagonist prevented the activation of all signaling pathways studied and normalized glucose-induced ED-B FN expression. We then determined the functional significance of ED-B in ECs and show that ED-B FN is involved in vascular endothelial growth factor expression and cellular proliferation. These studies show that glucose-induced and ET-mediated FN and ED-B FN expressions involve complex interplays between signaling pathways and that ET may represent an ideal target for therapy in chronic diabetic complications.     

Structure-function studies of bacteriorhodopsin XV. Effects of deletions in loops B-C and E-F on bacteriorhodopsin chromophore and structure

Bacteriorhodopsin mutants containing deletions in loop B-C, delta Thr67-Glu74 or delta Gly65-Gln75 or a deletion in the loop E-F, delta Glu161-Ala168, were prepared. Following their expression in Escherichia coli, the mutant proteins were purified to homogeneity and refolded with retinal in detergent-phospholipid mixtures. The mutants containing deletions in the loop B-C were normal at 4 degrees C but showed the following changes at 20 degrees C. 1) The lambda max shifted from 540 to below 510 nm; 2) the rates of bleaching by hydroxylamine in the dark increased; and 3) the rate and steady state of proton pumping decreased. Deletion of the eight amino acids in loop E-F did not affect wild-type behavior. However, all the mutant proteins were more prone to thermal and sodium dodecyl sulfate denaturation than the wild-type bacteriorhodopsin. These observations show that the structures of the B-C and E-F loops are not essential for correct folding of bacteriorhodopsin, but they contribute to the stability of the folded protein.     

Engineering an L-cell line that expresses insulin under the control of the glucagon-like peptide-1 promoter for diabetes treatment

Background

Ligand-targeted approaches have proven successful in improving the therapeutic index of a number of drugs. We hypothesized that the specific targeting of TNF-alpha antagonists to inflamed tissues could increase drug efficacy and reduce side effects.

Results

Using uteroglobin (UG), a potent anti-inflammatory protein, as a scaffold, we prepared a bispecific tetravalent molecule consisting of the extracellular ligand-binding portion of the human TNF-alpha receptor P75 (TNFRII) and the scFv L19. L19 binds to the ED-B containing fibronectin isoform (B-FN), which is expressed only during angiogenesis processes and during tissue remodeling. B-FN has also been demonstrated in the pannus in rheumatoid arthritis. L19-UG-TNFRII is a stable, soluble homodimeric protein that maintains the activities of both moieties: the immuno-reactivity of L19 and the capability of TNFRII to inhibit TNF-alpha. In vivo bio-distribution studies demonstrated that the molecule selectively accumulated on B-FN containing tissues, showing a very fast clearance from the blood but a very long residence time on B-FN containing tissues. Despite the very fast clearance from the blood, this fusion protein was able to significantly improve the severe symptomatology of arthritis in collagen antibody-induced arthritis (CAIA) mouse model.

Conclusions

The recombinant protein described here, able to selectively deliver the TNF-alpha antagonist TNFRII to inflamed tissues, could yield important contributions for the therapy of degenerative inflammatory diseases.     

Residues 39-56 of Stem Cell Factor Protein Sequence Are Capable of Stimulating the Expansion of Cord Blood CD34+ Cells

Background

Stem cell factor (SCF) can stimulate hematopoietic stem cell (HSC) expansion; however, the specific structural region(s) of SCF protein that are critical for this function are still unknown. A novel monoclonal antibody (named 23C8) against recombinant human SCF (rhSCF) was previously found to inhibit the ability of rhSCF to enhance HSC expansion, making it possible to identify the relevant active region to HSC.

Methods

Eleven polypeptides were synthesized, which were designed to cover the full-length of rhSCF, with overlaps that are at least 3 amino acids long. ELISA was used to identify the polypeptide(s) that specifically react with the anti-SCF. The effects of the synthetic polypeptides on human HSC expansion, or on the ability of the full-length rhSCF to stimulate cell proliferation, were evaluated ex vivo. Total cell number was monitored using hemocytometer whereas CD34⁺ cell number was calculated based on the proportion determined via flow cytometry on day 6 of culture.

Results

Of all polypeptides analyzed, only one, named P0, corresponding to the SCF protein sequence at residues 39–56, was recognized by 23C8 mAb during ELISA. P0 stimulated the expansion of CD34⁺ cells derived from human umbilical cord blood (UCB). Addition of P0 increased the numbers of total mononucleated cells and CD34⁺ cells, by ~2 fold on day 6. P0 also showed partial competition against full-length rhSCF in the ex vivo cell expansion assay.

Conclusion

Residues 39–56 of rhSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34⁺ cells. The peptide P0 is a potential candidate for further development as a synthetic substitute for rhSCF in laboratory and clinical applications.     

Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay

Background

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored.

Methodology/Principal Findings

We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer.

Conclusions/Significance

Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.     

Development of a humanized antibody with high therapeutic potential against dengue virus type 2

Background

Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control.

Methodology/Principal Findings

We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials.

Conclusions/Significance

We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.     

HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses

Background

Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response.

Methods

In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4⁺ T-cell lines derived from RV144 volunteers.

Results

Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4⁺ T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial.

Conclusions

Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4⁺T cell and antibody responses in the RV144 vaccinees.     

Allogeneic Bone Marrow Transplant from MRL/MpJ Super-Healer Mice Does Not Improve Articular Cartilage Repair in the C57Bl/6 Strain

Background

Articular cartilage has been the focus of multiple strategies to improve its regenerative/ repair capacity. The Murphy Roths Large (MRL/MpJ) “super-healer” mouse demonstrates an unusual enhanced regenerative capacity in many tissues and provides an opportunity to further study endogenous cartilage repair. The objective of this study was to test whether the super-healer phenotype could be transferred from MRL/MpJ to non-healer C57Bl/6 mice by allogeneic bone marrow transplant.

Methodology

The healing of 2mm ear punches and full thickness cartilage defects was measured 4 and 8 weeks after injury in control C57Bl/6 and MRL/MpJ “super-healer” mice, and in radiation chimeras reconstituted with bone marrow from the other mouse strain. Healing was assessed using ear hole diameter measurement, a 14 point histological scoring scale for the cartilage defect and an adapted version of the Osteoarthritis Research Society International scale for assessment of osteoarthritis in mouse knee joints.

Principal Findings

Normal and chimeric MRL mice showed significantly better healing of articular cartilage and ear wounds along with less severe signs of osteoarthritis after cartilage injury than the control strain. Contrary to our hypothesis, however, bone marrow transplant from MRL mice did not confer improved healing on the C57Bl/6 chimeras, either in regards to ear wound healing or cartilage repair.

Conclusion and Significance

The elusive cellular basis for the MRL regenerative phenotype still requires additional study and may possibly be dependent on additional cell types external to the bone marrow.     

Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays

Background

Most antibodies recognize conformational or discontinuous epitopes that have a specific 3-dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD) by using a silicon-based peptide array and computational methods.

Methods

Using a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes.

Results

Using a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity.

Conclusions

These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultra-high-density peptide microarray described here would be broadly useful to develop high-fidelity diagnostic tests and explore pathogenesis.     

Dynamics and Determinants of Pneumococcal Antibodies Specific against 13 Vaccine Serotypes in the Pre-Vaccination Era

Introduction

Introduction of pneumococcal conjugate vaccines (PCVs) for infants decreased overall invasive pneumococcal disease (IPD), while non-vaccine serotype IPD increased. To fully understand this serotype replacement, knowledge about serotype dynamics in the pre-vaccine era is needed. In addition to IPD surveillance and carriage studies, the serotype replacement can be investigated by serosurveillance studies. The current study compared the results of two Dutch serosurveillance studies conducted in 1995–1996 (PIENTER1) and 2006–2007 (PIENTER2).

Methods

Participants in these studies donated a blood sample and completed a questionnaire. Pneumococcal antibodies of serotypes included in PCV13 were measured with a fluorescent-bead based multiplex immunoassay. Geometric mean antibody concentrations (GMCs) and determinants of pneumococcal antibody levels were investigated.

Results

GMCs were higher in PIENTER2 for serotypes 1, 6A, 6B, 9V, 18C, 19F and 23F and lower for 3 and 5. Age, day care attendance, household size, vaccination coverage, and urbanisation rate were associated with pneumococcal antibodies in children. Education level, ethnicity, age, low vaccination coverage sample, urbanisation rate, and asthma/COPD were associated with pneumococcal antibodies in elderly. The determinants significantly associated with pneumococcal IgG were slightly different for the elderly in PIENTER1 compared to the elderly in PIENTER2.

Conclusion

Although most of the serotype antibody levels remained stable, some of the serotype-specific antibody levels varied during the pre-vaccine era, indicating that exposure of certain serotypes changes without interference of PCVs.     

Collagen Type III and VI Turnover in Response to Long-Term Immobilization

Background

Muscle mass and function are perturbed by immobilization and remobilization. When muscle mass changes, the quality and quantity of the extracellular matrix protein, particularly the collagens, change with it. In this study, we investigated the temporal profile of three peptide biomarkers derived from turnover of collagen type III and type VI in a long-term immobilization and remobilization study. We also compared individual biomarker levels with Lean body Mass (LBM) and changes therein, hypothesizing that these biomarkers would be biomarkers of the remodeling processes associated with immobilization and/or remobilization.

Methods

In the Berlin bed rest study, 20 young men were recruited and randomly assigned to 8-week’s strict bed rest with or without resistive vibration exercise countermeasure. We measured three neo-epitope ELISA kits in the serum samples of this study: Pro-C3, measured the synthesis of collagen type III; Pro-C6, measured the synthesis of collagen type VI; and C6M measured the degradation of collagen type VI induced by MMP-2 and MMP-9 cleavage.

Results

Pro-C3 and Pro-C6 biomarkers are up-regulated with both immobilization and remobilization, whereas C6M is hardly affected at all. We found that Pro-C3 and C6M levels are related to LBM at baseline and that high levels of Pro-C6 are associated with smaller changes in muscle mass during both immobilization and remobilization.

Conclusion

The Pro-C3 and–C6 biomarkers change likely reflect remodeling changes in response to unloading or reloading, whereas C6M does not appear to respond to unloading. Pro-C3 and C6M levels correlate with LBM at baseline, while Pro-C6 is related to the anabolic and catabolic responses to unloading and reloading.     

Salivary Alterations in Rats with Experimental Chronic Kidney Disease

Objective

This study aimed to analyze changes in saliva composition and salivary secretion process of rats with chronic kidney disease induced by 5/6 nephrectomy to set the foundation for salivary studies related to CKD.

Methods

CKD was induced in Wistar rats via 5/6 nephrectomy. Blood and saliva samples were collected from Control, Sham and CKD groups at 8 and 12 weeks after the surgery. Salivation was stimulated via intraperitoneal injections of pilocarpine (1.0 mg/Kg body weight) or isoproterenol (5.0 mg/Kg body weight). Saliva was collected and immediately stored at -80°C until analysis. The salivary flow rate, total protein, amylase and peroxidase activities, and urea concentrations were measured. The blood urea nitrogen (BUN) and serum creatinine concentrations were also evaluated.

Results

Increases in BUN and serum creatinine concentrations were observed in the CKD groups. Amylase activity was significantly reduced in response to both stimuli in the CKD groups at 8 weeks and increased in the CKD groups at 12 weeks in response to isoproterenol stimulus. The peroxidase activities of the CKD groups were significantly reduced in response to isoproterenol stimulation and were increased at 12 weeks in response to pilocarpine stimulation. Salivary urea was significantly increased in the CKD groups at 8 weeks in response to the isoproterenol stimuli and at 12 weeks in response to both salivary agonists.

Conclusions

The pattern of alterations observed in this experimental model is similar to those observed in patients and clearly demonstrates the viability of 5/6 nephrectomy as an experimental model in future studies to understand the alterations in salivary compositions and in salivary glands that are elicited by CKD.     

An Anti-Ubiquitin Antibody Response in Transitional Cell Carcinoma of the Urinary Bladder

Background

To use combinatorial epitope mapping (“fingerprinting”) of the antibody response to identify targets of the humoral immune response in patients with transitional cell carcinoma (TCC) of the bladder.

Methods

A combinatorial random peptide library was screened on the circulating pool of immunoglobulins purified from an index patient with a high risk TCC (pTa high grade plus carcinoma in situ) to identify corresponding target antigens. A patient cohort was investigated for antibody titers against ubiquitin.

Results

We selected, isolated, and validated an immunogenic peptide motif from ubiquitin as a dominant epitope of the humoral response. Patients with TCC had significantly higher antibody titers against ubiquitin than healthy donors (p<0.007), prostate cancer patients (p<0.0007), and all patients without TCC taken together (p<0.0001). Titers from superficial tumors were not significantly different from muscle invasive tumors (p = 0.0929). For antibody response against ubiquitin, sensitivity for detection of TCC was 0.44, specificity 0.96, positive predictive value 0.96 and negative predictive value 0.41. No significant titer changes were observed during the standard BCG induction immunotherapy.

Conclusions

This is the first report to demonstrate an anti-ubiquitin antibody response in patients with TCC. Although sensitivity of antibody production was low, a high specificity and positive predictive value make ubiquitin an interesting candidate for further diagnostic and possibly immune modulating studies.     

Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells

Background

Previous data have proven that microvesicles derived from hypoxia-induced mesenchymal stem cells (MSC-MVs) can be internalized into endothelial cells, enhancing their proliferation and vessel structure formation and promoting in vivo angiogenesis. However, there is a paucity of information about how the MSC-MVs are up-taken by endothelial cells.

Methods

MVs were prepared from the supernatants of human bone marrow MSCs that had been exposed to a hypoxic and/or serum-deprivation condition. The incorporation of hypoxia-induced MSC-MVs into human umbilical cord endothelial cells (HUVECs) was observed by flow cytometry and confocal microscopy in the presence or absence of recombinant human Annexin-V (Anx-V) and antibodies against human CD29 and CD44. Further, small interfering RNA (siRNA) targeted at Anx-V and PSR was delivered into HUVECs, or HUVECs were treated with a monoclonal antibody against phosphatidylserine receptor (PSR) and the cellular internalization of MVs was re-assessed.

Results

The addition of exogenous Anx-V could inhibit the uptake of MVs isolated from hypoxia-induced stem cells by HUVECs in a dose- and time-dependent manner, while the anti-CD29 and CD44 antibodies had no effect on the internalization process. The suppression was neither observed in Anx-V siRNA-transfected HUVECs, however, addition of anti-PSR antibody and PSR siRNA-transfected HUVECs greatly blocked the incorporation of MVs isolated from hypoxia-induced stem cells into HUVECs.

Conclusion

PS on the MVs isolated from hypoxia-induced stem cells is the critical molecule in the uptake by HUVECs.