Victor G. Sendra

Pathological lymphangiogenesis is modulated by galectin-8-dependent crosstalk between podoplanin and integrin-associated VEGFR-3

Lymphangiogenesis plays a pivotal role in diverse pathological conditions. Here, we demonstrate that a carbohydrate-binding protein, galectin-8, promotes pathological lymphangiogenesis. Galectin-8 is markedly upregulated in inflamed human and mouse corneas, and galectin-8 inhibitors reduce inflammatory lymphangiogenesis. In the mouse model of corneal allogeneic transplantation, galectin-8-induced lymphangiogenesis is associated with an increased rate of corneal graft rejection. Further, in the murine model of herpes simplex virus keratitis, corneal pathology and lymphangiogenesis are ameliorated in Lgals8−/− mice. Mechanistically, VEGF-C-induced lymphangiogenesis is significantly reduced in the Lgals8−/− and Pdpn−/− mice; likewise, galectin-8-induced lymphangiogenesis is reduced in Pdpn−/− mice. Interestingly, knockdown of VEGFR-3 does not affect galectin-8-mediated lymphatic endothelial cell (LEC) sprouting. Instead, inhibiting integrins α1β1 and α5β1 curtails both galectin-8- and VEGF-C-mediated LEC sprouting. Together, this study uncovers a unique molecular mechanism of lymphangiogenesis in which galectin-8-dependent crosstalk among VEGF-C, podoplanin and integrin pathways plays a key role.

Detection and isolation of auto-reactive human antibodies from primary B cells

The isolation of human monoclonal antibodies (hmAb) has emerged as a versatile platform in a wide variety of contexts ranging from vaccinology to therapeutics. In particular, the presence of high titers of circulating auto-antibodies is implicated in the pathology and outcome of autoimmune diseases. Therefore, the molecular characterization of these hmAb provides an avenue to understanding the pathogenesis of autoimmune diseases. Additionally, the phenotype of the auto-reactive B cells may have direct relevance for therapeutic intervention. In this report, we describe a high-throughput single-cell assay, microengraving, for the screening, characterization and isolation of anti-citrullinated protein antibodies (ACPA) from peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis (RA) patients. Stimulated B cells are profiled at the single-cell level in a large array of sub-nanoliter nanowells (∼10(5)), assessing both the phenotype of the cells and their ability to secrete cyclic-citrullinated peptide (CCP)-specific antibodies. Single B cells secreting ACPA are retrieved by automated micromanipulation, and amplification of the immunoglobulin (Ig) heavy and light chains is performed prior to recombinant expression. The methodology offers a simple, rapid and low-cost platform for isolation of auto-reactive antibodies from low numbers of input cells and can be easily adapted for isolation and characterization of auto-reactive antibodies in other autoimmune diseases.

Single-cell Characterization of Autotransporter-mediated Escherichia coli Surface Display of Disulfide Bond-containing Proteins

Background: The ability of autotransporter (AT) to translocate polypeptides with multiple disulfide bonds is controversial. Results: Surface display of functional chymotrypsin (4 S-S) and M18 scFv (2 S-S) was quantitatively characterized. Conclusion: Surface display of functional recombinant protein with multiple disulfide bonds can be achieved using AT system. Significance: Displaying recombinant proteins with disulfide bonds enhances utility of ATs. Autotransporters (ATs) are a family of bacterial proteins containing a C-terminal β-barrel-forming domain that facilitates the translocation of N-terminal passenger domain whose functions range from adhesion to proteolysis. Genetic replacement of the native passenger domain with heterologous proteins is an attractive strategy not only for applications such as biocatalysis, live-cell vaccines, and protein engineering but also for gaining mechanistic insights toward understanding AT translocation. The ability of ATs to efficiently display functional recombinant proteins containing multiple disulfides has remained largely controversial. By employing high-throughput single-cell flow cytometry, we have systematically investigated the ability of the Escherichia coli AT Antigen 43 (Ag43) to display two different recombinant reporter proteins, a single-chain antibody (M18 scFv) that contains two disulfides and chymotrypsin that contains four disulfides, by varying the signal peptide and deleting the different domains of the native protein. Our results indicate that only the C-terminal β-barrel and the threaded α-helix are essential for efficient surface display of functional recombinant proteins containing multiple disulfides. These results imply that there are no inherent constraints for functional translocation and display of disulfide bond-containing proteins mediated by the AT system and should open new avenues for protein display and engineering.

Immune response to Thomsen-Friedenreich disaccharide and glycan engineering.

Cancer‐associated mucins show frequent alterations of their oligosaccharide chain profile, with a switch to unmask normally cryptic O‐glycan backbone and core regions. Epithelial tumour cells typically show overexpression of the uncovered Galβ1‐3GalNAcα‐O‐Ser/Thr (Core 1) structure, known as the T antigen or the Thomsen–Friedenreich antigen, the oligosaccharide chain of which is called the Thomsen–Friedenreich disaccharide (TFD). T antigen expression has been associated with immunosuppression, metastasis dissemination, and the proliferation of cancer cells. Several different strategies have been used to trigger a specific immune response to TFD. Natural T antigen and synthetic TFD residues have low immunodominance. In the T antigen, flexibility of the glycosidic bond reduces the immunogenicity of the sugar residue. Enhanced rigidity should favour certain glycan conformations and thereby improve TFD immunotargeting. We propose the term ‘glycan engineering’ for this approach. Such engineering of TFD should reduce the flexibility of its glycan moiety and thereby enhance its stability, rigidity and immunogenicity.

Delivery of Bone Marrow-Derived Mesenchymal Stem Cells Improves Tear Production in a Mouse Model of Sjögren’s Syndrome

The purpose of the present study was to test the potential of mouse bone marrow-derived mesenchymal stem cells (BD-MSCs) in improving tear production in a mouse model of Sjögrens syndrome dry eye and to investigate the underlying mechanisms involved. NOD mice (n = 20) were randomized to receive i.p. injection of sterile phosphate buffered saline (PBS, control) or murine BD-MSCs (1 × 106 cells). Tears production was measured at baseline and once a week after treatment using phenol red impregnated threads. Cathepsin S activity in the tears was measured at the end of treatment. After 4 weeks, animals were sacrificed and the lacrimal glands were excised and processed for histopathology, immunohistochemistry, and RNA analysis. Following BD-MSC injection, tears production increased over time when compared to both baseline and PBS injected mice. Although the number of lymphocytic foci in the lacrimal glands of treated animals did not change, the size of the foci decreased by 40.5% when compared to control animals. The mRNA level of the water channel aquaporin 5 was significantly increased following delivery of BD-MSCs. We conclude that treatment with BD-MSCs increases tear production in the NOD mouse model of Sjögrens syndrome. This is likely due to decreased inflammation and increased expression of aquaporin 5.

Staining tumor cells with biotinylated ACL-I, a lectin isolated from the marine sponge, Axinella corrugata

Abstract Axinella corrugata lectin 1 (ACL-1) was purified from aqueous extracts of the marine sponge, Axinella corrugata. ACL-1 strongly agglutinates native rabbit erythrocytes. The hemagglutination is inhibited by N-acetyl derivatives, particularly N, N’, N”-triacetylchitotriose, N-acetyl-D- glucosamine, N-acetyl-D-mannosamine and N-acetyl-D-galactosamine. We investigated the capacity of biotinylated ACL-1 to stain several transformed cell lines including breast (T-47D, MCF7), colon (HT-29), lung (H460), ovary (OVCAR-3) and bladder (T24). ACL-I may bind to both monosaccharides and oligosaccharides of tumor cells, N-acetyl-D-galactosamine, and N-acetyl-D- glucosamine glycan types. The lectins are useful, not only as markers and diagnostic parameters, but also for tissue mapping in suspicious neoplasms. In addition, they provide a better understanding of neoplasms at the cytological and molecular levels. Furthermore, the use of potential metastatic markers such as lectins is crucial for developing successful tools for therapy against cancer. We observed that biotinylated ACL-I stains tumor cells and may hold potential as a probe for identifying transformed cells and for studying glycan structures synthesized by such cells.

Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.

Anti-idiotypic antibody mimicking a T-antigen-specific lectin inhibits human epithelial tumor cell proliferation

Cancer‐associated mucins show frequent alterations of oligosaccharide chain profile. Terminal structures may be deleted, thereby exposing normally ‘cryptic’ structures such as Tn (GalNAcα‐O‐Ser/Thr) and T antigen (Galβ1‐3GalNAcα‐O‐Ser/Thr). Overexpression of these commonly hidden glycoforms, and reduced level of naturally occurring anti‐T or anti‐Tn antibodies, is associated with epithelial tumor progression and aggressiveness. The lectin from the common edible mushroom Agaricus bisporus (ABL) shows high affinity binding to T antigen, and reversible noncytotoxic inhibitory effect on epithelial tumor cell proliferation. The aim of this study was to induce immune response with tumor‐associated glycan specificity and biological activity similar to those of ABL. An anti‐idiotypic (Id) antibody strategy was developed using ABL as first template. ABL was purified by affinity chromatography and assayed as immunogen in rabbit. Rabbit IgG was purified from anti‐ABL serum using a protein G column, and specific anti‐ABL IgG was obtained by affinity chromatography using immobilized ABL. Affinity‐purified anti‐ABL IgG contained an antibody fraction that recognizes the carbohydrate‐binding site of ABL. This IgG was used as immunogen in mouse to yield anti‐Id antibody recognizing tumor‐associated glycans such as Tn and T antigen. Competitive assays showed that α‐anomeric GalNAc is the main binding subsite of anti‐Id antibody in glycan recognition. Anti‐Id antibody bound human epithelial tumor cells, as shown by cell enzyme‐linked immunosorbent assay and immunofluorescence. Anti‐Id antibody raised by immunization with affinity‐purified anti‐ABL IgG had antiproliferative effect on human epithelial tumor cells through apoptosis induction similar to that of ABL. The anti‐Id immune response developed here has potential application in cancer therapy.

Glycan bioengineering in immunogen design for tumor T antigen immunotargeting.

Bioengineering of Galbeta3GalNAcalpha, known as Thomsen-Friedenreich disaccharide (TFD), is studied to promote glycan immunogenicity and immunotargeting to tumor T antigen (Galbeta3GalNAcalpha-O-Ser/Thr). Theoretical studies on disaccharide conformations by energy minimization of structures using MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlalphaTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope. Antibodies produced by glycan bioengineering recognize HT29, T47D, MCF7, and CT26 epithelial tumor cells. Epithelial tumor cell adhesion to T antigen-binding lectins and endothelial cells was lower in the presence of antibodies raised against the engineered immunogen. The immune response directed to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens for immunotargeting purposes.