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Background: Type 1 conventional dendritic cells (cDC1s) are characterized by their ability to induce potent CD8 + T cell responses. In efforts to generate novel vaccination strategies, notably against cancer, human cDC1s emerge as an ideal target to deliver antigens. cDC1s uniquely express XCR1, a seven transmembrane G protein-coupled receptor. Due to its restricted expression and endocytic nature, XCR1 represents an attractive receptor to mediate antigen-delivery to human cDC1s., Methods: To explore tumor antigen delivery to human cDC1s, we used an engineered version of XCR1-binding lymphotactin (XCL1), XCL1(CC3). Site-specific sortase-mediated transpeptidation was performed to conjugate XCL1(CC3) to an analog of the HLA-A*02:01 epitope of the cancer testis antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1). While poor epitope solubility prevented isolation of stable XCL1-antigen conjugates, incorporation of a single polyethylene glycol (PEG) chain upstream of the epitope-containing peptide enabled generation of soluble XCL1(CC3)-antigen fusion constructs. Binding and chemotactic characteristics of the XCL1-antigen conjugate, as well as its ability to induce antigen-specific CD8 + T cell activation by cDC1s, was assessed., Results: PEGylated XCL1(CC3)-antigen conjugates retained binding to XCR1, and induced cDC1 chemoattraction in vitro. The model epitope was efficiently cross-presented by human cDC1s to activate NY-ESO-1-specific CD8 + T cells. Importantly, vaccine activity was increased by targeting XCR1 at the surface of cDC1s., Conclusion: Our results present a novel strategy for the generation of targeted vaccines fused to insoluble antigens. Moreover, our data emphasize the potential of targeting XCR1 at the surface of primary human cDC1s to induce potent CD8 + T cell responses., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Human induced pluripotent stem cells (hiPSCs) are useful starting materials for the generation of cell therapy products, due to their pluripotency and ability to self-renew. Quality control of hiPSCs is extremely important in creating a stable supply of hPSC-derived products. Previously we identified an hiPSC-specific lectin probe, rBC2LCN, which binds specifically to α1,2-fucosylated glycan and recognizes podocalyxin (PODXL) as a glycoprotein ligand. In this study, we produced monoclonal antibodies (mAbs) specific for α1,2-fucosylated PODXL expressed on hiPSCs. PODXL was recombinantly expressed in fucosyltransferase 1 (FUT1)-transfected HEK293, followed by immunization into mice. Monoclonal antibodies, which bind to PODXL/FUT1-transfected cells, but not to cells transfected with only one of PODXL or FUT1, were screened by flow cytometry. The two mAbs generated (179-6B8C9 and 179-7E12E10), termed α1,2-fucosylated PODXL-specific mAbs (FpMabs), showed binding specificity to PODXL/FUT1-transfected cells. The FpMabs bound to hiPSCs but never to human adipose-derived mesenchymal stem cells, human dermal fibroblasts, or hiPSC-derived mesoderm. Altogether, FpMabs are highly specific probes for hiPSCs, which might be a powerful tool for the characterization of hiPSCs used in regenerative medicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The success of new targeted cancer therapies has been dependent on the identification of tumor-specific antigens. Podocalyxin (Podxl) is upregulated on tumors with high metastatic index and its presence is associated with poor outcome, thus emerging as an important prognostic and theragnostic marker in several human cancers. Moreover, in human tumor xenograft models, Podxl expression promotes tumor growth and metastasis. Although a promising target for immunotherapy, the expression of Podxl on normal vascular endothelia and kidney podocytes could hamper efforts to therapeutically target this molecule. Since pathways regulating post-translational modifications are frequently perturbed in cancer cells, we sought to produce novel anti-Podxl antibodies (Abs) that selectively recognize tumor-restricted glycoepitopes on the extracellular mucin domain of Podxl., Methods: Splenic B cells were isolated from rabbits immunized with a Podxl-expressing human tumor cell line. Abs from these B cells were screened for potent reactivity to Podxl + neoplastic cell lines but not Podxl + primary endothelial cells. Transcripts encoding heavy and light chain variable regions from promising B cells were cloned and expressed as recombinant proteins. Tumor specificity was assessed using primary normal tissue and an ovarian cancer tissue microarray (TMA). Mapping of the tumor-restricted epitope was performed using enzyme-treated human tumor cell lines and a glycan array., Results: One mAb (PODO447) showed strong reactivity with a variety of Podxl+ tumor cell lines but not with normal primary human tissue including Podxl+ kidney podocytes and most vascular endothelia. Screening of an ovarian carcinoma TMA (219 cases) revealed PODO447 reactivity with the majority of tumors, including 65% of the high-grade serous histotype. Subsequent biochemical analyses determined that PODO447 reacts with a highly unusual terminal N-acetylgalactosamine beta-1 (GalNAcβ1) motif predominantly found on the Podxl protein core. Finally, Ab-drug conjugates showed specific efficacy in killing tumor cells in vitro ., Conclusions: We have generated a novel and exquisitely tumor-restricted mAb, PODO447, that recognizes a glycoepitope on Podxl expressed at high levels by a variety of tumors including the majority of life-threatening high-grade serous ovarian tumors. Thus, tumor-restricted PODO447 exhibits the appropriate specificity for further development as a targeted immunotherapy., Competing Interests: Competing interests: The authors are inventors of a pending patent application on PODO447 and methods of using the same (US20180296673A1). MRH, PB, IS, CBG, KM and CR are inventors of a pending patent application on PODO83 and methods of using the same (US20190367606A1). KM and CR possess an awarded patent on podocalyxin as a prognostic marker in cancer (US9309323B2)., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Enhancing the therapeutic efficacy of drugs for inflammatory diseases is of high demand. One possible approach is targeting drugs to the extracellular matrix of the inflamed area. Here, we target collagens in the matrix, which are inaccessible in most tissues yet are exposed to the bloodstream in the inflamed area because of vascular hyperpermeability. We conferred collagen affinity to anti-tumor necrosis factor-α (α-TNF) antibody by conjugating a collagen-binding peptide (CBP) derived from the sequence of decorin. CBP-α-TNF accumulated in the inflamed paw of the arthritis model, and arthritis development was significantly suppressed by treatment with CBP-α-TNF compared with the unmodified antibody. Similarly, CBP-anti-transforming growth factor-β (α-TGF-β) accumulated in the inflamed lung of pulmonary fibrosis model and significantly suppressed pulmonary fibrosis compared with the unmodified antibody. Together, collagen affinity enables the anticytokine antibodies to target arthritis and pulmonary fibrosis accompanied by inflammation, demonstrating a clinically translational approach to treat inflammatory diseases., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Isolation of specific single chain antibodies (scFvs) against key epitopes of cancer markers are applied for cancer immunotherapy and diagnosis. In this study following the prediction of the 3D structure of the DSP part of Dentin sialophosphoprotein (DSPP), the epitope was chosen using in silico programs. Panning process was applied to isolate specific human scFv against the epitope. PCR and DNA fingerprinting differentiated the specific clones, which were evaluated by phage ELISA. Following DNA sequencing, the 3D structure of isolated scFv was modeled and Docked on DSP. Results demonstrated the selection of a specific anti-DSPP scFv with 40% frequency, which reacted significantly with the predicted epitope and PCa patients' urines in ELISA tests (P-value < 0.05). The VH and VL of the isolated scFv were from VH1 and VL3 gene families with several amino acid changes in CDRs and FRs domains. The scFv tightly bound to the DSP epitope with the lowest energy level by hydrogen bonds, cation-pi, hydrophobic and ionic interactions demonstrating the specificity of Ag-Ab interactions. The anti-DSPP scFv selected in this study with significant specificity to DSPP antigen offers a promising new agent for both PCa early detection and treatment of cancers with DSPP expression., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The chemokine receptor XCR1 is known to be selectively expressed by cross-presenting dendritic cells (DCs), while its ligand XCL1/lymphotactin is mainly produced by activated CD8 + T cells and natural killer cells. Recent studies have shown that XCL1-antigen fusion proteins efficiently induce CD8 + T cell responses by preferentially delivering antigens to XCR1 + DCs. However, XCL1 per se was found to be a poor adjuvant for induction of CD8 + T cell responses. XCL1 is unique because of its lack of one of the two disulfide bonds commonly conserved in all other chemokines and thus has an unstable structure with a relatively weak chemokine activity. In the present study, we generated a variant form of murine XCL1 termed mXCL1-V21C/A59C that contained a second disulfide bond to stabilize its chemokine structure. We confirmed that mXCL1-V21C/A59C had much more potent chemotactic and calcium mobilization activities than the wild type XCL1 (mXCL1-WT). Intradermal injection of mXCL1-V21C/A59C, but not that of mXCL1-WT, significantly increased the accumulation of XCR1 + CD103 + DCs in the injection site, and most of the accumulated XCR1 + CD103 + DCs were found to take up co-injected ovalbumin (OVA). Furthermore, recruited XCR1 + CD103 + DCs efficiently migrated to the draining lymph nodes and stayed for a prolonged period of time. Consequently, mXCL1-V21C/A59C strongly induced OVA-specific CD8 + T cells. The combination of OVA and mXCL1-V21C/A59C well protected mice from E.G7-OVA tumor growth in both prophylactic and therapeutic protocols. Finally, memory CTL responses were efficiently induced in mice immunized with OVA and mXCL1-V21C/A59C. Although intradermal injection of OVA and polyinosinic-polycytidylic acid (poly(I:C)) as an adjuvant also induced CD8 + T cell responses to OVA, poly (I:C) poorly recruited XCR1 + CD103 + DCs in the injection site and failed to induce significant memory CTL responses to OVA. Collectively, our findings demonstrate that a highly active form of XCL1 is a promising vaccine adjuvant for cross-presenting DCs to induce antigen-specific effector and memory CD8 + T cells.

Esophageal cancer is one of the highly malignant cancers. It comprises two of the most common histological tumor types: squamous cell carcinoma (SCC) and adenocarcinoma. SCC accounts for about 90% of esophageal cancers. Despite developments in treatment strategies, the prognosis and survival rate remain poor. Podocalyxin (PODXL) is a highly glycosylated type-I transmembrane protein. It is expressed in normal tissues such as kidney, heart, breast, and pancreas. Upregulation of PODXL correlates with tumor progression, invasion, and metastasis. Therefore, this glycoprotein could be a potential biomarker for predicting the prognosis of some cancers, for instance, brain, colorectal, oral, lung, bladder, prostate, and ovarian cancers. We previously developed a specific and sensitive anti-PODXL monoclonal antibody (mAb), PcMab-47 (mouse IgG 1 , kappa) and its mouse IgG 2a -type (47-mG 2a ). We showed their utility in immunohistochemical analysis of oral cancers. Herein, we demonstrate that PcMab-47 and 47-mG 2a can also be used to detect esophageal squamous cell carcinoma (ESCC) with this technique. These two antibodies, respectively, stained 123/130 (94.6%) and 127/130 (97.7%) ESCC cases, indicating that they can detect PODXL with high sensitivity in this carcinoma. Of more than 3+ cases, 47-mG 2a was more effective than PcMab-47, respectively, staining 56/127 (44.1%) and 41/123 (33.3%). Therefore, 47-mG 2a can be used for the detection of PODXL in ESCC using immunohistochemical analysis.

Lung cancer is one of the leading causes of cancer-related deaths in the world. Regardless of the advances in lung cancer treatments, the prognosis is still poor. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is expressed in normal tissues, including the heart, pancreas, and breast. It is also found and used as a diagnostic marker in many cancers, such as renal, brain, breast, oral, and lung cancers. We previously developed specific and sensitive anti-PODXL monoclonal antibodies, PcMab-47 (mouse IgG 1 , kappa) and its mouse IgG 2a -type (47-mG 2a ), both of which were suitable for immunohistochemical analyses of oral cancers. In this study, we investigated the utility of PcMab-47 and 47-mG 2a for the immunohistochemical analyses of lung cancers. PcMab-47 stained 51/70 (72.9%) cases of lung cancer, whereas 47-mG 2a stained 59/70 (84.3%) cases, indicating that the latter antibody is more sensitive and is useful for detecting PODXL in lung cancers.

T cell functions are regulated by multiple signaling cascades, including the MKK4-JNK (c-Jun NH 2 terminal kinase) pathway. However, the mechanism regulating the MKK4-JNK axis in T cells remains unclear. Herein, we demonstrated that protein acetylation modulates JNK activity induced by T cell receptor (TCR) activation. The acetyltransferase, CREB-binding protein (CBP), is transported from the nucleus to the cytoplasm in response to TCR cross-linking. To investigate the role of CBP in TCR signaling, we overexpressed CBP in the cytoplasm of Jurkat cells, a human T lymphocyte line. Enforced expression of cytoplasmic CBP led to MKK4 acetylation and interfered with MKK4-mediated JNK phosphorylation. Insufficient JNK activity decreased the activity of the transcription factor, AP-1. In contrast, other transcription factors, NF-κB and NFAT, stimulated with anti-CD3 and anti-CD28 antibodies were activated normally in the presence of cytoplasmic-CBP. These results provide valuable insights into the role of acetylation in MKK4-JNK signaling in T cells., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

Podocalyxin (PODXL) is expressed in several cancers, including brain tumors and colorectal cancers. PODXL overexpression is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human PODXL, which was produced using LN229 glioblastoma cells, and produced a clone PcMab-47 that could be used for investigating PODXL expression by flow cytometry and immunohistochemical analysis. Herein, we produced a human-mouse chimeric PcMab-47 (chPcMab-47) and investigated its antitumor activity against PODXL-expressing tumors. chPcMab-47 reacted with LN229, LN229/PODXL, and Chinese hamster ovary (CHO)/PODXL cells, but it did not react with CHO-K1 or PODXL-knockout LN229 cell line (PDIS-13). chPcMab-47 exerted antitumor activity against a mouse xenograft model using CHO/PODXL. Furthermore, chPcMab-47 was reactive with colorectal cancer cell lines such as HCT-15, Caco-2, HCT-8, and DLD-1. chPcMab-47 also exhibited antitumor activity against a mouse xenograft model using HCT-15. These results suggest that chPcMab-47 could be useful for antibody therapy against PODXL-expressing cancers.

Podocalyxin (PODXL) is a CD34-related sialomucin and a well-known marker of embryonic stem cells. PODXL is expressed in many types of tumors including colorectal cancers, breast cancers, and brain tumors. Overexpression of PODXL is an independent predictor of progression, metastasis, and poor outcome. PODXL is also expressed in many normal cells such as renal podocytes and endothelial cells (ECs). However, high-sensitive and high-specific anti-PODXL monoclonal antibodies (mAbs) have not been established. Herein, we immunized mice with recombinant human PODXL, which was produced using LN229 glioblastoma cells. The anti-PODXL mAb, PcMab-47, reacted with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in flow cytometry. Immunohistochemical analysis showed that PcMab-47 detected PODXL-expressing normal cells such as podocytes of kidney or ECs. Furthermore, PcMab-47 stained PODXL-expressing cancer cells of colon or breast cancers. These results suggest that PcMab-47 could be useful for investigating the expression and function of PODXL in cancers and normal tissues.

Podocalyxin-like protein (PODXL) is a member of CD34 family proteins. It is the protein that carries many post-translational epitopes responsible for various pluripotent surface markers including TRA-1-60, TRA-1-81, GCTM2, GP200, and mAb84. However, PODXL has not attracted the attention of stem cell biologists. Here, we report several features of PODXL mRNA and protein in pluripotent stem cells. Similar to the modification-dependent pluripotent epitopes, PODXL transcripts and carrier protein are also features of pluripotency. PODXL is highly expressed in early human embryos from oocytes up to four-cell stages. During reprogramming of human cells to pluripotency, in contrast to TRA-1-60 and TRA-1-81, PODXL is activated by KLF4 at a very early time of reprogramming. Although TRA-1-60 and TRA-1-81 are completely lost upon differentiation, a residual PODXL(+) population exists even after extended differentiation and they were identified by the universal human PODXL epitope 3D3. Unlike TRA-1-60 and TRA-1-81 epitopes that are unique to primate pluripotent stem cells (PSCs), PODXL carrier protein can be used as a murine surface marker. Most importantly, antibody to 3D3 epitope causes massive necrosis and apoptosis of human PSCs (hPSCs). We suggest that 3D3 antibody could be employed to eliminate the tumorigenic pluripotent cells in hPSC-derived cells for cell transplantation.

Podocalyxin-like protein 1 (PCLP1), a CD34-related sialomucin involved in the regulation of cellular morphology and adhesion, is expressed by a number of normal cells and various tumor cells. In breast malignancies PCLP1 overexpression has been associated with the most aggressive, metastatic cancers and poor prognosis. These observations suggest that PCLP1 expression could provide a mechanism to evade the immune response, thereby promoting metastatic progression of cancer. In the present work, we aimed to determine the effect of PCLP1 overexpressed in MCF7 breast cancer cells on natural killer (NK) cell cytotoxicity, dendritic cell maturation, and agonist-induced T cell proliferation. The results showed that PCLP1 expressed in MCF7 breast cancer cells confers resistance to NK cell-mediated cytolysis and impairs T cell proliferation. Furthermore, PCLP1 decreased the levels of NK cell activating receptors NKG2D, NKp30, NKp44, NKp46, DNAM-1, and CD16 on cell surface in a contact-dependent manner. Moreover, NK cells acquired PCLP1 from MCF7 cells by a process known as trogocytosis. These data reveal a new function of PCLP1 expressed on tumor cells as an immunomodulatory molecule, which may represent a mechanism to evade the immune response., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Poultry are highly susceptible to the immunotoxic effects of the food-borne mycotoxin aflatoxin B1 (AFB1). Exposure impairs cell-mediated and humoral immunity, limits vaccine efficacy, and increases the incidence of costly secondary infections. We investigated the molecular mechanisms of AFB1 immunotoxicity and the ability of a Lactobacillus-based probiotic to protect against aflatoxicosis in the domestic turkey (Meleagris gallopavo). The spleen transcriptome was examined by RNA sequencing (RNA-seq) of 12 individuals representing four treatment groups. Sequences (6.9 Gb) were de novo assembled to produce over 270,000 predicted transcripts and transcript fragments. Differential expression analysis identified 982 transcripts with statistical significance in at least one comparison between treatment groups. Transcripts with known immune functions comprised 27.6 % of significant expression changes in the AFB1-exposed group. Short exposure to AFB1 suppressed innate immune transcripts, especially from antimicrobial genes, but increased the expression of transcripts from E3 ubiquitin-protein ligase CBL-B and multiple interleukin-2 response genes. Up-regulation of transcripts from lymphotactin, granzyme A, and perforin 1 could indicate either increased cytotoxic potential or activation-induced cell death in the spleen during aflatoxicosis. Supplementation with probiotics was found to ameliorate AFB1-induced expression changes for multiple transcripts from antimicrobial and IL-2-response genes. However, probiotics had an overall suppressive effect on immune-related transcripts.

Podoplanin (PDPN/Aggrus/T1α/gp36/OTS-8), a type I transmembrane sialoglycoprotein, is involved in platelet aggregation, cell invasion, and cancer metastasis. Podoplanin expression in cancer cells or cancer-associated fibroblasts was reported to be involved in poor prognosis of several cancers. Furthermore, podoplanin is expressed in lymphatic endothelial cells or lung type I alveolar cells. Although many anti-podoplanin monoclonal antibodies (MAbs), such as NZ-1 and D2-40, have been established, almost all anti-podoplanin MAbs are produced against a platelet aggregation-inducing (PLAG) domain. In this study, we produced and characterized a novel anti-podoplanin monoclonal antibody, LpMab-3, the epitope of which is a sialylated glycopeptide of podoplanin. We identified the minimum epitope of LpMab-3 as Thr76-Glu81 of human podoplanin, which is different from PLAG domain, using Western blot analysis and flow cytometry. Immunohistochemical analysis showed that LpMab-3 is useful for detecting lung type I alveolar cells and lymphatic endothelial cells. Because LpMab-3 detects only sialylated podoplanin, it could be useful for uncovering the physiological function of sialylated human podoplanin.

Background: Podocalyxin (PODXL) is a transmembrane sialomucin, whose aberrant expression and/or allelic variation associates with poor prognosis and unfavourable clinicopathological characteristics in different cancers. Membranous expression of PODXL has been suggested to be an independent marker of poor prognosis in colorectal cancer (CRC), and previously by an in-house monoclonal antibody, we showed that also cytoplasmic overexpression of PODXL predicts poor prognosis. The aim of this study was to compare two PODXL antibodies with different epitopes case-by-case in CRC patients., Methods: Of 840 consecutively operated CRC patients from Helsinki University Central Hospital, PODXL expression by polyclonal HPA 2110 antibody was evaluated from 780. Associations of PODXL expression with clinicopathological parameters and the impact of PODXL expression on survival were assessed. Kappa-value was used to assess the comparability of the two antibodies., Results: Membranous PODXL expression associated with unfavourable clinicopathological parameters and with higher risk for disease-specific death from CRC within 5 years (unadjusted hazard ratio (HR) = 1.90; 95% confidence interval (CI) (1.32-2.75); adjusted HR = 1.64; 95% CI (1.11-2.43)). The comparability of expressions by the two antibodies was low (kappa =0.219, standard error 0.060, p < 0.0001). Combination of two antibodies identified a group of patients with even worse prognosis (unadjusted HR = 6.00; 95% CI (3.27-13.0); adjusted HR = 2.14; 95% CI (1.12-4.07))., Conclusion: Membranous expression by the polyclonal PODXL antibody and cytoplasmic overexpression by the monocolonal PODXL antibody are both independent markers of poor prognosis, but they recognise different groups of patients, both of which have poor prognosis. The combined use of the antibodies reveals a group with an even worse prognosis. The biological reasons for the difference between antibodies warrant further studies.

In this issue of Blood, Sreeramkumar and colleagues report that E-selectin ligand-1 (ESL-1) is a highly selective ligand for E-selectin on hematopoietic progenitors with unexpected important contributions to their trafficking.

Beyond its well-established roles in mediating leukocyte rolling, E-selectin is emerging as a multifunctional receptor capable of inducing integrin activation in neutrophils, and of regulating various biological processes in hematopoietic precursors. Although these effects suggest important homeostatic contributions of this selectin in the immune and hematologic systems, the ligands responsible for transducing these effects in different leukocyte lineages are not well defined. We have characterized mice deficient in E-selectin ligand-1 (ESL-1), or in both P-selectin glycoprotein-1 (PSGL-1) and ESL-1, to explore and compare the contributions of these glycoproteins in immune and hematopoietic cell trafficking. In the steady state, ESL-1 deficiency resulted in a moderate myeloid expansion that became more prominent when both glycoproteins were eliminated. During inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation, and extravasation of mature neutrophils, but only the combined deficiency in PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly, we find that the levels of ESL-1 were strongly elevated in hematopoietic progenitor cells. These elevations correlated with a prominent function of ESL-1 for E-selectin binding and for migration of hematopoietic progenitor cells into the bone marrow. Our results uncover dominant roles for ESL-1 in the immature compartment, and a functional shift toward PSGL-1 dependence in mature neutrophils.

Background: The Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens characterized by decreased IL-2 synthesis. Trypanosoma cruzi mucin (Tc Muc) has been implicated in this phenomenom. These molecules contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via N-acetylglucosamine residues., Methodology/principal Findings: In this study, we evaluated the ability of Tc Muc to modulate the activation of CD4(+) T cells. Our data show that cross-linking of CD3 on naïve CD4(+) T cells in the presence of Tc Muc resulted in the inhibition of both cytokine secretion and proliferation. We further show that the sialylated O-Linked Glycan residues from tc mucin potentiate the suppression of T cell response by inducing G1-phase cell cycle arrest associated with upregulation of mitogen inhibitor p27(kip1). These inhibitory effects cannot be reversed by the addition of exogenous IL-2, rendering CD4(+) T cells anergic when activated by TCR triggering. Additionally, in vivo administration of Tc Muc during T. cruzi infection enhanced parasitemia and aggravated heart damage. Analysis of recall responses during infection showed lower frequencies of IFN-γ producing CD4(+) T cells in the spleen of Tc Muc treated mice, compared to untreated controls., Conclusions/significance: Our results indicate that Tc Muc mediates inhibitory efects on CD4(+) T expansion and cytokine production, by blocking cell cycle progression in the G1 phase. We propose that the sialyl motif of Tc Muc is able to interact with sialic acid-binding Ig-like lectins (Siglecs) on CD4(+) T cells, which may allow the parasite to modulate the immune system.

The possible role of sialic acids in host cells-fungi interaction and their association with glycoproteins were evaluated using a clinical isolate of the dimorphic fungus Mucor polymorphosporus. Lectin-binding assays with spores and yeast cells denoted the presence of surface sialoglycoconjugates containing 2,3- and 2,6-linked sialylglycosyl groups. Western blotting with peroxidase-labeled Limulus polyphemus agglutinin revealed the occurrence of different sialoglycoprotein types in both cell lysates and cell wall protein extracts of mycelia, spores, and yeasts of M. polymorphosporus. Sialic acids contributed to the surface negative charge of spores and yeast forms as evaluated by adherence to a cationic substrate. Sialidase-treated spores were less resistant to phagocytosis by human neutrophils and monocytes from healthy individuals than control (untreated) fungal suspensions. The results suggest that sialic acids are terminal units of various glycoproteins of M. polymorphosporus, contributing to negative charge of yeasts and spore cells and protecting infectious propagules from destruction by host cells.

The article presents a sandwich enzyme linked immunosorbent assay (ELISA) for identification of edible bird's nest. The characteristic sialoglycoproteins were found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified by liquid-phase isoelectric focusing (LIEF). According to the analysis, the molecular weight was 106-128 kDa and the isoelectric point was ≤pH 3.0. Two anti-characteristic sialoglycoprotein monoclonal antibodies were produced. The monoclonal antibodies were examined by western-blot assay. One of the monoclonal antibody was used as coating and the other as the enzyme-labeled antibody after being coupled to horseradish peroxidase (HRP). Based on the optimized ELISA condition, the method was established with IC(50) of 1.5 ng/mL, and low cross-reactivity with various fake materials (<0.01%). ELISA provided a suitable means for screening of a large number of samples. The coefficients of variation were between 2.9% and 5.8%.

Aims/objective: Nephropathy, a major complication of diabetes, is the leading cause of end-stage renal disease. Recent studies have demonstrated that podocyte injury is involved in the onset of and progression to renal insufficiency. Here, we describe a novel, highly sensitive ELISA for detecting urinary podocalyxin, a glycoconjugate on the podocyte apical surface that indicates podocyte injury, particularly in the early phase of diabetic nephropathy., Methods: Urine samples from patients with glomerular diseases (n = 142) and type 2 diabetes (n = 71) were used to quantify urinary podocalyxin by ELISA. Urine samples were obtained from 69 healthy controls for whom laboratory data were within normal values. Podocalyxin was detected in urine by immunofluorescence, immunoelectron microscopy and western blotting., Results: Morphologically, urinary podocalyxin was present as a vesicular structure; western blotting showed it as a positive band at 165-170 kDa. Levels of urinary podocalyxin were elevated in patients with various glomerular diseases and patients with diabetes. In patients with diabetes, urinary podocalyxin was higher than the cut-off value in 53.8% patients at the normoalbuminuric stage, 64.7% at the microalbuminuric stage and 66.7% at the macroalbuminuric stage. Positive correlations were observed between urinary podocalyxin levels and HbA(1c), urinary β(2) microglobulin, α(1) microglobulin and urinary N-acetyl-β-D-glucosaminidase, although urinary podocalyxin levels were not correlated with other laboratory markers such as blood pressure, lipid level, serum creatinine, estimated GFR or proteinuria., Conclusions/interpretation: Urinary podocalyxin may be a useful biomarker for detecting early podocyte injury in patients with diabetes.

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial-and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.

Reversible interactions of glycoconjugates on leukocytes with P- and E-selectin on endothelial cells mediate tethering and rolling of leukocytes in inflamed vascular beds, the first step in their recruitment to sites of injury. Although selectin ligands on hematopoietic precursors have been identified, here we review evidence that PSGL-1, CD44, and ESL-1 on mature leukocytes are physiologic glycoprotein ligands for endothelial selectins. Each ligand has specialized adhesive functions during tethering and rolling. Furthermore, PSGL-1 and CD44 induce signals that activate the β2 integrin LFA-1 and promote slow rolling, whereas ESL-1 induces signals that activate the β2 integrin Mac-1 in adherent neutrophils. We also review evidence for glycolipids, CD43, L-selectin, and other glycoconjugates as potential physiologic ligands for endothelial selectins on neutrophils or lymphocytes. Although the physiologic characterization of these ligands has been obtained in mice, we also note reported similarities and differences with human selectin ligands.

Aim: To obtain purificated his-XCL1 recombinant protein of porcine in vitro and prepare the associated polyclonal antibody; To study how the recombinant protein affects on lymphocytes proliferation., Methods: Purify the recombinant protein using HiTrap(TM); Chelating HP chromatographic column; Check the purified product using SDS-PAGE; Detect the expression of the recombinant protein using Western blot; Immunize the experimental animals with the purified fusion protein to prepare the serum containing the associated polyclonal antibody. The serum will then undergo double immnodiffusion test and indirect ELISA test to determine the polyclonal antibody titer. Then, test the condition of lymphocytes proliferation by MTT., Results: Single target strip could be seen under conducting the SDS-PAGE electrophoresis when the concentration of the binding buffer is 40 mmol/L and the concentration of the elution buffer is 500 mmol/L; Western blot test showed that the recombinant protein could be successfully expressed; Double immunodiffusion test showed the antigen-antibody binding ratio to be 1:8, and the titre of antibodies was 1:12 800 detected by indirect ELISA. The result of MTT showed that both the native XCL1 and the recombinant protein could stimulate lymphocytes proliferation, and this stimulating effect could be effectively blocked by the polyclonal antibody we prepared., Conclusion: To conclude, this recombinant protein has biological activity and this research can provide basic material for further investigation of the function of XCL1 in swine.

We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell-specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19xTCR bispecific DART molecule revealed equivalent potency with the CD19xCD3 DART molecule, demonstrating flexibility of the DART structure to support T-cell/B-cell associations for redirected T cell-killing applications. The enhanced level of killing mediated by DART molecules was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19(-) cells. Cell-association studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19xTCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART molecules for the treatment of B-cell malignancies.

Although interleukin-10 (IL-10) is commonly regarded as an immunosuppressive cytokine, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating antitumor properties. Previous studies demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. In this study, we explored the tumorigenesis of EG7 cells transduced with IL-10 gene. In vivo, IL-10 gene transfer reduced tumorigenic capacity of EG7 cells and prolonged survival of the EG7 tumor-bearing mice. It was found that the cytotoxicities of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) were enhanced. Assessment of the immune status of the animals showed prevalence of a systemic and tumor-specific Th2 response (high levels of IL-4 and IL-10). To improve the therapeutic efficacy, we combined with intratumoral injection of adenovirus-mediated lymphotactin (Ad-Lptn) into the overestablished EG7 tumor model. More significant inhibition of tumor growth were observed in EG7 tumor-bearing mice that received combined treatment with IL-10 and Lptn gene than those of mice treated with IL-10 or Lptn gene alone. The highest NK cells and CTL activity was induced in the combined therapy group, increasing the production of IL-2 and interferon-γ (IFN-γ) significantly but decreasing the expression of immune suppressive cells (CD4(+)Foxp3(+) Treg cells and Gr1(+)CD11b(+) MDSCs). The necrosis of tumor cells was markedly observed in the tumor tissues, accompanying with strongest expression of Mig (monokine induced by interferon-gamma) and IP-10 (interferon-inducible protein 10), weakest expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2). In vivo, depletion analysis demonstrated that CD8(+) T cells and NK cells were the predominant effector cell subset responsible for the antitumor effect of IL-10 or Lptn gene. These findings may provide a potential strategy to improve the antitumor efficacy of IL-10 and Lptn.

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with β-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc+Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcβ-nLc₄Cer² sequence., (Copyright © 2011 Elsevier Inc. All rights reserved.)

D2-40 is a recently developed monoclonal antibody that reacts with a 40 kDa O-linked sialoglycoprotein and has been used for the assessment of lymphatic invasion in tumor specimens. We have evaluated the diagnostic usefulness of D2-40 and association of its immunopositivity with clinicopathological parameters in adenocarcinoma and squamous cell carcinoma of the lung. We investigated 97 cases of surgically resected adenocarcinoma or squamous cell carcinoma of the lung for the determination of D2-40 positivity in tumor cells and peritumoral lymphatic vessel density (LVD) using an immunostaining method. D2-40 immunoreactivity in tumor cells was invariably negative in adenocarcinoma but 47% of squamous cell carcinomas were positive. D2-40 positivity in the tumor was significantly associated with high LVD in squamous cell carcinoma (P < 0.006). There was no significant association between peritumoral LVD and clinicopathologic parameters, including lymphatic vessel invasion, lymph node metastasis, and survival in adenocarcinoma and squamous cell carcinoma. These results suggest that D2-40 immunoreactivity in tumor cells can be used for distinguishing between adenocarcinoma and squamous cell carcinoma and that the reactivity of tumor cells with D2-40 is positively correlated with LVD in squamous cell carcinoma but not with lymph node metastasis in adenocarcinoma and squamous cell carcinoma.

Glycophorin A (GPA, CD235a) is a major membrane glycoprotein and marker of cells of the erythroid lineage. It is also the target of Plasmodium falciparum and of influenza virus. We describe a novel series of 10 antibodies towards GPA, recognizing four extra- and intracellular peptide epitopes of this molecule (defined by epitope mapping) and one mixed peptide/carbohydrate epitope. All antibodies bind better to the desialylated than to the fully sialylated molecule, including those specific for the intracellular epitope. For some of the antibodies (representing all five epitopes) functional binding constants were determined by Surface Plasmon Resonance. The new panel complements the already known anti-glycophorin antibodies and offers several potential applications for, e.g., differential diagnosis of erythroleukemias, lineage analyses of erythroid cells, isolation of senescent erythrocytes, or a highly sensitive neuraminidase assay., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In addition to Ag recognition, some Abs are capable of killing target organisms in the absence of phagocytes and complement. In this study, we report that an anti-Pseudomonas aeruginosa O6ad LPS IgG(1), tobacco-expressed human S20 IgG(1) (te-hS20), as well as its recombinant Fab and single-chain variable fragment (scFv) fragments have cellular- and complement-independent bactericidal activity. te-hS20 and its Fab and scFv significantly reduced viability of P. aeruginosa O6ad in dose- and time-dependent manners in vitro and also showed lower levels of bactericidal activity against P. aeruginosa PAO1, but had no activity against P. aeruginosa O10, Escherichia coli TG1, and Streptococcus agalactiae. The H chain and its Fd fragment both had significant Ag-binding and bactericidal activities against P. aeruginosa O6ad. Bactericidal activity was completely inhibited with specific LPS Ag, suggesting that Ag binding is involved in the bactericidal mechanism. Live/dead cell staining and electron microscopic observations indicate that the bactericidal effect was due to disruption of the cell wall and suggest inhibition of cell division. In addition to te-hS20, the Fab and scFv were also protective in vivo, as leukopenic mice had prolonged and improved survival after administration of these Ab fragments followed by challenge with P. aeruginosa O6ad cells at 80-90% lethal dose, supporting a bactericidal mechanism independent of phagocytes and complement. Understanding of the bactericidal mechanism will allow assessment of the potential for therapeutic application of these Abs.

Background: Despite the recognition that lymphatic and blood vessel invasion is an important prognostic factor in lung cancer, there is no common definition for pathological evaluation of vessel invasion. The aim of the present study was to determine whether D2-40 immunostaining can increase the accuracy of detection of lymphatic vessel invasion and whether our new grading system of vessel invasion by "degree" could be used instead of conventional evaluation by "presence" for pathological stage IA non-small cell lung cancer (NSCLC)., Methods: The vessel invasion classification was re-evaluated in 221 recent paraffin-embedded sections of p-stage IA NSCLC stained by Hematoxylin-Eosin (HE), Elastica-Van-Gieson (EVG), and D2-40., Results: After re-assessment using D2-40 immunostaining, 41.2% (31 of 75) of ly1 cases by HE/EVG changed to ly0, and 14.9% (17 of 114) of ly0 cases by HE/EVG changed to ly1. Overall, 4 of 28 ly2 cases on conventional staining were changed to ly1, and 2 were changed to ly0 using D2-40 immunostaining. When the patients were divided into two groups by the presence of vessel invasion (v/ly0 vs. 1, 2, 3), there was no significant difference in cancer-specific survival (p=0.1107, 0.0875, respectively), while when they were divided according to degree of vessel invasion (v/ly0, 1 vs. 2, 3), there was a statistically significant difference (p=0.0038, p=0.0002, respectively). On multivariate analysis, lymphatic vessel invasion had a significant impact on cancer-specific survival (p=0.0061)., Conclusion: Our results suggest that D2-40 immunostaining provides a precise diagnosis of lymphatic vessel invasion, and our new grading system of vessel invasion by "degree" is accurate and has prognostic value in early lung cancer.

Coxsackievirus B3 (CVB3) is a gastrointestinal virus causing myocarditis in human and mice. An ideal vaccine for CVB3-myocarditis requires both humoral and cellular immunity at systemic and mucosal compartments. We described here an enhancing strategy for chitosan-pVP1 vaccine by co-immunizing with lymphotactin (LTN) gene, a T cell-attractive-chemokine, encapsulated in chitosan particle to provide more protection against CVB3. Mice were intranasally co-immunized with 4 doses of chitosan-DNA vaccines separately encapsulating VP1 and LTN plasmids by 2 week-intervals and challenged with CVB3 4 weeks after the last immunization. Compared with chitosan-pVP1 alone, co-immunization with chitosan-pLTN significantly increased high-avidity-neutralizing antibody levels in serum and in intestinal mucosa, and promoted systemic and mucosal Th1 and CD8(+)CTL immune responses. Accordingly, enhanced resistance to CVB3-myocarditis was evidenced by reduced myocardial viral load, profound subsidence of myocarditis and increased survival rate. This strategy represents a promising platform for Th1 polarization and protection against mucosal infectious pathogens.

Background/aims: Morphological change of podocytes is closely correlated with the development of proteinuria. Podocalyxin is a major sialoglycoprotein of the podocytes and is thought to be involved in the maintenance of the foot processes structure. Our aim was to examine the mechanism by which podocalyxin contributes to the morphological change of podocytes., Methods: We searched protein(s) which coprecipitate with podocalyxin using rat glomerular lysate. Localization of podocalyxin and the coprecipitated protein, cortactin, was studied in a model of puromycin aminonucleoside (PAN) nephrosis by immunocytochemistry. Tyrosine phosphorylation of cortactin was examined. Association of the podocalyxin/cortactin complex with the actin cytoskeleton was evaluated by extraction with Triton-X and immunoblotting., Results: Cortactin was found to be co-immunoprecipitated with podocalyxin. Immunocytochemical analysis revealed that these 2 proteins colocalized in the apical side of podocytes. In PAN nephrosis, localization of cortactin was altered after the onset of proteinuria, with increased tyrosine phosphorylation. Simultaneously, the dissociation of the podocalyxin/cortactin complex from the actin cytoskeleton was induced., Conclusions: These results indicate that cortactin mediates interaction between podocalyxin and actin filaments in podocytes and that alteration of this interaction may play a role in the process of morphological change of podocytes.

Background/aims: The vasculature has become an important target in the development of therapies for increasing numbers of human diseases, yet there are few reliable markers available to identify the endothelium in rodent models. We have characterized the expression, subcellular localization and accessibility of the rat pan-endothelial marker podocalyxin (podxl) using a newly developed monoclonal antibody (mAb), G278., Methods: podxl expression and accessibility to binding by G278 were determined in the rat by a variety of experimental approaches., Results: mAb G278 reliably immunostained blood vessels of all types and of every size in fresh-frozen, fixed-frozen and paraffin-embedded sections of all tissues, but did not stain lymphatic vessels. Western blotting, in vivo imaging and biodistribution analyses demonstrated that the highest levels of endothelial podxl were found in the lung and heart. We also determined that podxl is not enriched in caveolae and that its expression can be modulated in the tumor microenvironment., Conclusion: Our study shows that podxl is a better identifier of rat endothelia than are some of the more commonly used markers and that mAb G278 is a robust antibody for use not only in identifying rat blood vessels but also as a tool to elucidate podxl function.

Hemangioblasts are common progenitors of hematopoietic and angiogenic cells, which have been demonstrated in the mouse to possess a unique cell surface marker, podocalyxin-like protein 1 (PCLP1) (Hara, T. et al., Immunity, 11: 567-578. 1999). In this study, we prepared a novel monoclonal antibody against human PCLP1 (hPCLP1) and attempted to isolate human hematopoietic progenitor cells from umbilical cord blood and peripheral blood using nano-sized bacterial magnetic particles (BacMPs) coupled with the anti-hPCLP1 antibody. Flow cytometric analysis demonstrated that the purity of separated hPCLP1-positive cells from peripheral blood was approximately 95% whereas peripheral blood mononuclear cells contained only 0.1% PCLP1+ cells. Umbilical cord blood was demonstrated to be a better source for PCLP1+ cells than peripheral blood. These results suggest that the separation of human PCLP1+ cells using BacMPs with anti-hPCLP1 were extremely effective and may be useful as a means to prepare human hematopoietic progenitor cells.

Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one antibody (mAb 84) is cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complement-independent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications. Disclosure of potential conflicts of interest is found at the end of this article.

The cattle V antigen from the FV blood group system was characterized. Hemolytic as well as immunochemical analyses with monoclonal antibody (MAb) IVA-41 found that V antigen of bovine red blood cells is a membrane-bound, papain- and pronase-sensitive, trypsin- and chymotrypsin-resistant N-glycosylated sialoglyco-protein with molecular weight of 64, 56, and 50 kDa under no reduction and 23 kDa under reduction conditions. In contrary to some human blood group antigens, the expression of bovine blood group V antigen is restricted to the erythrocyte membrane.

In murine models, transgenic chemokine-cytokine tumor vaccines overcome many of the limitations of single-agent immunotherapy by producing the sequence of T-cell attraction followed by proliferation of tumor antigen-activated clones. The safety and immunologic effects of this approach in humans were tested in 7 patients with relapsed or refractory neuroblastoma. They each received up to 8 subcutaneous injections of a vaccine combining lymphotactin--and interleukin-2 (IL-2)--secreting autologous neuroblastoma cells in a dose-escalating scheme. Adverse events were limited to grade 1 or 2 localized reactions in all patients, pain in 3 patients, and fever in 3 patients. Injection site biopsies revealed increased cellularity caused by infiltration of CD4 and CD8 lymphocytes, eosinophils, and dendritic cells with a decrease in dendritic cells from the first to the second vaccination. Systemically, vaccine was associated with increased tumor recognition as measured by enzyme-linked immunosorbent spot assays. Two patients had interferon-gamma predominant responses and 3 had IL-5 predominant responses. Only 1 patient received all 8 injections, 1 patient stopped the study early, and all other patients progressed before completion of the study. Hence, autologous tumor cell vaccines combining transgenic lymphotactin with IL-2 seem to have little toxicity in humans and can induce an antitumor immune response. In this setting, the immune response was insufficient to overcome active recurrent neuroblastoma.

Background and Aim: The development of autoantibodies against antigens of the pancreatic islet cells is a typical phenomenon in patients with type 1 diabetes. The expression of densin, recently shown to be present in kidney podocytes, was explored in the pancreas. Additionally, we studied whether densin and filtrin, another molecule shared between the kidney podocytes and pancreatic islet cells, can act as autoantigens and whether autoantibodies against these can be detected in patients with type 1 diabetes., Methods: Expression of pancreatic densin was studied with reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence. Children and adolescents (n = 66) with type 1 diabetes and control subjects were analysed for densin autoantibodies (DAA) and filtrin autoantibodies (FAA) using radioimmunoprecipitation assay. The serum samples were obtained at the time of diagnosis and after a duration of 2, 5 and 10 years., Results: Densin expression was observed in the pancreas, localising to the beta cells. DAA were detected in 33% of the patients and the positivity was typically seen already at diagnosis. FAA were observed in 11% of the patients. The proportion of islet cell antibody (ICA) positive, GADA positive and protein tyrosine phosphatase-related islet antigen 2 antibody (IA-2A)-positive patients decreased during the follow-up period, and a similar trend was seen for DAA but not for FAA. Among the 14 patients with signs of renal injury, four tested positive for DAA and two for FAA., Conclusions: Densin is a novel molecule shared by the kidney glomerular podocytes and pancreatic islet cells. Densin and filtrin can act as autoantigens, and autoantibodies against these can be detected in patients with type 1 diabetes., (Copyright 2006 John Wiley & Sons, Ltd.)

Conventional treatment of recurrent and metastasized prostate cancer (CaP) remains inadequate; this fact mandates development of alternative therapeutic modalities, such as specific active or passive immunotherapy. Previously, we reported the identification of a novel highly immunogenic HLA-A*0201-restricted Prostatic Acid Phosphatase-derived peptide (PAP-3) by a two-step in vivo screening in an HLA-transgenic (HHD) mouse system. In the present study we aimed at elucidating the efficiency of PAP-3-based vaccine upon active antitumor immunization. To this end we established preventive and therapeutic carcinoma models in HHD mice. The 3LL murine Lewis lung carcinoma clone D122 transduced to express HLA-A*0201 and PAP served as a platform for these models. The HLA-A*0201-PAP-3 complex specific recombinant single chain scFV-PAP-3 antibodies were generated and used to confirm an endogenous PAP processing resulting in PAP-3 presentation by HLA-A*0201. PAP-3 based vaccines significantly decreased tumor incidence in a preventive immunization setting. Therapeutic vaccination of HHD mice with PAP-3 led to rejection of early established tumors and to increase of mouse survival. These results strongly support a therapeutic relevance of the identified CTL epitope upon active antitumor immunization. The newly established carcinoma model presented herein might be a useful tool for cancer vaccine design and optimization.

Objective: To assess the significance of urinary podocyte and its possible implication as a marker of activity of lupus nephritis., Methods: The presence of podocytes in urinary sediment was detected with immunochemical staining using anti-podocalyxin antibody. The correlation of the number of urinary podocytes with activity index of renal pathological lesions, hematuria, and proteinuria was analyzed respectively. The proliferating podocytes in renal biopsy tissue and urine from patients with class IV lupus nephritis were examined with double immunohistochemical staining., Results: Thirty-one patients with lupus nephritis undergoing renal biopsy were enrolled into the study. Renal pathological findings of the patients could be classified into WHO class III (25.8%), class IV (64.5%) and class V (9.7%). 90% of the patients had positive urinary podocytes. The number of urinary podocytes was strongly and positively correlated with the severity of hematuria (r=0.639, P=0.000) and glomerular pathological activity index (r=0.487, P=0.014) in patients of class III and class IV. The amount of proteinuria was not correlated with pathological activity index, even though all the patients had proteinuria. Furthermore, the number of urinary podocytes, the severity of hematuria and the amount of proteinuria were all decreased after treatment with methyl prednisone, cyclophosphamide or mycophenolate mofetil. Interestingly, the urinary podocytes could disappear even before the remission of hematuria and proteinuria after treatment. Proliferative podocytes were observed both in biopsied kidney tissue and urinary sediments in patients of class IV., Conclusion: The presence and the number of urinary podocytes can be used as a valuable marker to grade the activity of lupus nephritis and to evaluate the efficacy of steroid therapy.

A significant percentage of allergic diseases (e.g. certain cases of intrinsic asthma, chronic idiopathic urticaria, and atopic dermatitis) cannot be explained by the classical mechanisms of IgE/allergen-mediated activation of basophils and mast cells. We found that protein Fv, an endogenous protein synthesized in the human liver and increased during viral hepatitis, act as a superallergen by binding to IgE of the VH3 family and activating human basophils and mast cells. Similarly, envelope gp120 of HIV-1 and protein A of Staphylococcus aureus are viral and bacterial superallergens, respectively, because they interact with IgE VH3+. Protein L binds to the V domain of he kappa light chains of IgE. Our results demonstrate that endogenous, viral and bacterial products activate primary effector cells of allergic disorders to release proinflammatory mediators and cytokines thereby acting as immunoglobulin superantigens (superallergens).

The aim of this study was to investigate the combined effect of zoledronic acid and an antibody against bone sialoprotein II (BSPII) on proliferation and osteolytic activity of MDA-MB-231(GFP) breast cancer cells. For this purpose, the cells were exposed to zoledronic acid (10-20 microg/ml [25-50 microgM]) and an anti-BSPII IgY (10-100 microg/ml) for up to 5 days alone or in combination. The combined treatment showed synergistic antiproliferative effects at the higher dose of zoledronic acid. Following inoculation of 1 x 10(5) MDA-MB-231 (GFP) breast cancer cells into a branch of the femoral artery of nude rats, lytic lesions developed in the tibia, femur or fibula of the injected hind leg after approximately 30 days. The appearance and development of these lesions were monitored radiographically. Rats with lytic lesions were treated with zoledronic acid (60 microg/kg/week sc x 8; n = 10), zoledronic acid and an anti-BSPII IgY antibody (60 microg/kg/week sc x 8 + 10 mg/kg/week sc x 8; n = 10), or left untreated (n = 20). In addition, rats were treated for 4 weeks (n = 10) with both regimens starting right after tumor cell inoculation. Finally, ten rats were treated with zoledronic acid for 2 weeks before tumor cell inoculation (60 microg/kg/week sc x 2). The antiosteolytic effect of zoledronic acid was high as shown by inhibition of osteolytic growth. Addition of the anti-BSPII IgY further decreased the incidence of femoral osteolytic lesions (40% reduction), indicating remineralization, and reduced periosteal defects of cortical bone (20% reduction). These observations favor using the IgY-antibody in addition to zoledronic acid in order to stimulate osteoblast-induced remineralization.

Rabbit is one of several species that depend on development of B lymphocytes in gut-associated lymphoid tissues for primary immunoglobulin-repertoire diversification. The rabbit appendix is an important site of early B-lymphocyte development. We previously reported that peripheral lymph node addressin detected by monoclonal antibody (mAb) MECA-79 played a role in recruitment of immature blood-borne B cells into neonatal rabbit appendix. Here, we report expression of an approximately 127 000 MW O-linked sulphated proteoglycan on developing B cells in appendix and Peyer's patches recognized by the mAb MECA-79. Binding of the mAb to B lymphocytes was sensitive to enzyme treatment with O-sialoglycoprotease and expression was partially inhibited by sodium chlorate, a metabolic inhibitor of sulphation. The proportions of MECA-79(+) B lymphocytes gradually increased from < 0.5% at 3 days to > 70% at 6 weeks in appendix and Peyer's patches. The proportions of MECA-79(+) B lymphocytes in spleen and peripheral blood were very low (0.5-2%). However, the MECA-79 determinant was detected on B cells in splenic germinal centres after immunization. In situ labelling of appendix cells showed that the MECA-79 determinant was expressed on fluorescein-labelled B lymphocytes that migrated from appendix into mesenteric lymph nodes. B-cell MECA-79 may be involved in interactions with T cells and/or dendritic cells. Alternatively, because we found that lymphatic endothelium in the thymus-dependent area of appendix, a site for lymphocyte exit, expressed P-selectin (CD62P), interaction of the MECA-79 determinant on B cells with CD62P may have a role in the exit of B lymphocytes from rabbit appendix.

Using noncompetitive methodologies comparing CD43(+/+) and CD43(-/-) mice, it has been reported that CD43(-/-) leukocytes exhibit reduced recruitment efficiency to sites of inflammation. More recent analyses demonstrate that CD43 on activated T cells can function as an E-selectin ligand (E-SelL) in vitro, suggesting that CD43 might promote rolling interactions during recruitment of leukocytes and account for the reported recruitment deficits in CD43(-/-) T cells and neutrophils in vivo. Internally controlled competitive in vivo methods using fluorescent tracking dyes were applied to compare recruitment efficiency of CD43(+/+) vs CD43(-/-) activated T cells to inflamed skin and of peripheral blood neutrophils to inflamed peritoneum. A simple CFSE perfusion method was developed to distinguish arterial/venous vasculature and confirm appropriate extravasation through venules in a Con A-induced cutaneous inflammation model. In vivo recruitment of peripheral blood neutrophils to inflamed peritoneum was core 2 GlcNAcT-I dependent, but recruitment efficiency was not influenced by absence of CD43. There were also no significant differences in core 2 GlcNAcT-I-dependent, selectin-dependent, cutaneous recruitment of activated T cells from CD43(+/+) and congenic CD43(-/-) mice in either B6 or P-selectin(-/-) recipients despite biochemical confirmation that a CD43-specific E-SelL was present on activated T cells. We conclude that recruitment of neutrophils and activated T cells in these in vivo models is not influenced by CD43 expression and that if CD43 on activated T cells performs an E-SelL function in vivo, it contributes in a limited physiological context.

Podocalyxin (podxl) is a protein with a peptide bone of approximately 55.5 kDa that undergoes a post-translational glycosylation, yielding a final molecular mass from approximately 145 to approximately 200 kDa. This protein is normally found covering the vascular side of the epithelial glomerular cells, the podocytes, and its presence is essential to maintain a normal renal function. It has also been reported in other cells and tissues although its function has not been yet clarified. The carboxy-terminal intracellular domain of podxl is nearly 100% identical in most species; however, the ectodomain shows considerable variations although the cysteine residues are conserved. Detection of this protein is elusive, most likely due to differences in post-translational modifications. We aimed at producing murine monoclonal antibodies against human podxl. Immunization with Chinese hamster ovarian -hpodxl-green fluorescence protein live cells yielded five different monoclonal antibodies that were characterized by enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis/western blot, flow cytometry, immunohistochemistry, and immunoprecipitation. The different behavior of these antibodies suggests that some of them may react against epitopes masked by different glycosylated protein moieties.

Currently available treatments for rheumatoid arthritis (RA) are limited in terms of their long-term effects and their abilities to control disease progression. Interleukin-1 receptor antagonist (IL-1Ra) is a natural inhibitor of the biologic actions of IL-1, which is known to promote inflammation and degeneration of the joint. In this study, we investigated whether human IL-1Ra gene transfer is effective at treating an established experimental arthritis model. A recombinant adenovirus carrying the gene that encode human hIL-1Ra and GFP (Ad.hIL-1Ra/GFP) was administered by intra-articular injection into the ankle joints of the mice with established the IL-1Ra-deficient Balb/cA mice (IL-1Ra(-/-)), which develop spontaneously chronic inflammatory arthropathy. The effects of two injections of Ad.hIL-1Ra/GFP or control virus with no inserted target gene (Ad.GFP) were compared with the effects of PBS injection with respect to the clinical characteristics of arthritis, as determined by articular index scores, histopathological and immunological assays. We further divided the outcomes of Ad.hIL-1Ra/GFP gene therapy in IL-1Ra(-/-) mice according arthritis stage; early stage and chronic stage corresponding to 8 and 15 weeks of age, respectively. Intra-articular injections of Ad.hIL-1Ra/GFP reduced arthritis severity and footpad swelling compared with control groups treated with Ad.GFP or PBS in early stage IL-1Ra(-/-) mice. Moreover, the histopathology of the ankle joints of IL-1Ra(-/-) mice treated with Ad.hIL-1Ra/GFP showed a significant decrease in synovial proliferation and inflammatory cell infiltration, and preserved proteoglycan levels in the joints of early stage IL-1Ra(-/-) mice compared with the control mice. Moreover, Ad.hIL-1Ra/GFP treated mice showed reduced levels of inflammatory T helper type 1 (Th1) driven IgG2a antibodies to collagen type II but increased levels Th2 driven IgG1 antibody. These results suggest that adenovirus-mediated gene transfer of IL-1Ra may be a promising therapeutic option in the early stage of autoimmune arthritis.

Objective: To determine the safety profile of anakinra after extended exposure in a diverse clinical trial population of patients with rheumatoid arthritis., Methods: A six month, randomised, double blind phase comparing anakinra (100 mg/day) with placebo was followed by open label anakinra treatment for up to three years in patients with rheumatoid arthritis. Concomitant non-steroidal anti-inflammatory drugs, corticosteroids, and other disease modifying antirheumatic drugs were permitted., Results: In all 1346 patients with rheumatoid arthritis received anakinra for up to three years. Patients had varying levels of disease severity, concomitant drug use, and comorbid conditions. Cumulative, exposure adjusted event (EAE) rates for all adverse events (AEs), serious AEs, and deaths were similar during each year of anakinra treatment; the overall rate (0 to 3 years) was similar to that observed for controls during the blinded phase. The most frequent AEs were injection site reactions (122.26 events/100 patient-years), rheumatoid arthritis progression (67.80 events/100 patient-years), and upper respiratory infections (26.09 events/100 patient-years). The EAE rate of serious infections was higher for patients treated with anakinra for 0 to 3 years (5.37 events/100 patient-years) than for controls during the blinded phase (1.65 events/100 patient-years). However, if the patient was not receiving corticosteroid treatment at baseline, the serious infection rate was substantially lower (2.87 event/100 patient-years). The overall incidence of malignancies was consistent with expected rates reported by SEER. Neutralising antibodies developed in 25 patients, but appeared to be transient in 12; neutralising antibody status did not appear related to occurrence of malignancies or serious infections. There were no clinically significant trends in laboratory data related to anakinra., Conclusion: Anakinra is safe and well tolerated for up to three years of continuous use in a diverse population of patients with rheumatoid arthritis.