[Application of CD138 Immunomagnetic Bead Sorting Combined with Fluorescence in Situ Hybridization in Multiple Myeloma].

OBJECTIVE: To compare the effects of direct fluorescence in situ hybridization (D-FISH) detection without sorting and CD138 immunomagnetic bead sorting technology combined with FISH (MACS-FISH) on cytogenetic analysis of patients with multiple myeloma (MM). METHODS: FISH test results of 229 patients with initial MM were retrospectively analyzed. The patients were divided into two groups, 140 patients were tested with D-FISH and 89 patients with MACS-FISH. The combination probe was designed as P53, D13S319, RB1, 1q21, and IgH. Cytogenetic detection results were compared between the two groups. RESULTS: The total detection rate of cytogenetic abnormalities in D-FISH group was 52.9%, and that in MACS-FISH group was 79.8%. There was a significant difference in the cytogenetic abnormality rate between the two groups (P=0.020). The abnormal genes with the highest detection rate in the two groups were 1q21 and IgH, respectively, while the lowest was P53. There was no significant difference in the percentage of P53 positive cells (positive rate) between the two groups, while D13S319, RB1, 1q21, and IgH showed significant difference in positive cell rate (P=0.0002, P<0.0001, P=0.0033, P=0.0032). There was no significant correlation between the proportion of plasma cells (PC) detected by bone marrow morphology and cytogenetic abnormality rate in the D-FISH group, while there was a correlation between the proportion of PC detected by flow cytometry and cytogenetic abnormality rate (r=0.364). The PC proportion detected by bone marrow morphology and flow cytometry in the MACS-FISH group had no correlation with the cytogenetic abnormality rate and positive cell rate of the 5 genes mentioned above. Additionally, the PC proportion detected by bone marrow morphology and flow cytometry showed significant difference (P<0.0001). CONCLUSION: CD138 immunomagnetic bead sorting combined with FISH technology can significantly improve the abnormality detection rate of MM cytogenetics.

Circulating CD138 enhances disease progression by augmenting autoreactive antibody production in a mouse model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a progressive autoimmune disease characterized by high levels of antibodies directed against nuclear antigens. Elevated serum CD138, a heparan sulfate-bearing proteoglycan, correlates with increased disease activity in patients with SLE, but the contribution of CD138 to lupus disease is not known. Corroborating patient data, we detected an increase in serum CD138 in MRL/MpJ-Faslpr/J (MRL/Lpr) mice (a model for SLE disease) parallel to disease activity. Although T-cell receptor ß (TCRß)+CD138+ T cells typically expand in MRL/Lpr mice as the disease progresses, surprisingly, TCRß+CD138- cells were the primary source of circulating CD138, as the transfer of TCRß+CD138- cells, but not TCRß+CD138+ cells, to young MRL/Lpr mice resulted in higher serum CD138 in the recipients. We found that trypsin was able to cleave CD138 from TCRß+CD138+ cells, and that trypsin was highly expressed in TCRß+CD138- cells. Moreover, trypsin inhibitors, the "defined trypsin inhibitor" and leupeptin, increased CD138 expression on TCRß+CD138- cells, suggesting a contribution of cleaved CD138 to the increase in blood CD138 levels. Furthermore, soluble CD138 was able to bind "a proliferation-inducing ligand" (APRIL) and enhance APRIL-mediated plasma cell generation and autoreactive antibody production through the phosphorylation of extracellular signal-regulated kinase in B cells. The APRIL receptor "transmembrane activator, calcium modulator, and cyclophilin ligand interactor" was involved in the enhancement of APRIL activity by CD138, as the synergistic effect of APRIL and CD138 was ablated in transmembrane activator, calcium modulator, and cyclophilin ligand interactor-deficient B cells. These findings indicate a regulatory role for soluble CD138 in B-cell differentiation and autoreactive antibody production in SLE disease.

High molecular weight sodium hyaluronate improves survival of syndecan-1-deficient septic mice by inhibiting neutrophil migration.

METHODS: Sepsis was induced by cotton smoke inhalation followed by intranasal administration of Pseudomonas aeruginosa in female (> 6 months) Balb/c and syndecan-1 knockout mice. Survival of mice, lung capillary endothelial glycocalyx integrity, lung water content, and vascular hyper-permeability were determined with or without HMW-SH treatment in these mice. Effects of HMW-SH on endothelial permeability and neutrophil migration were tested in in vitro setting. RESULTS: In septic wildtype mice, we found a severely damaged pulmonary microvascular endothelial glycocalyx and elevated levels of shed syndecan-1 in the circulation. These changes were associated with significantly increased pulmonary vascular permeability. In septic syndecan-1 knockout mice, extravascular lung water content was higher, and early death was observed. The administration of HMW-SH significantly reduced mortality and lung water content in septic syndecan-1 knockout mice, but not in septic wildtype mice. In in vitro setting, HMW-SH inhibited neutrophil migration and reduced cultured endothelial cell permeability increases. However, these effects were reversed by the addition of recombinant syndecan-1 ectodomain. CONCLUSIONS: HMW-SH reduced lung tissue damage and mortality in the absence of syndecan-1 protein, possibly by reducing vascular hyper-permeability and neutrophil migration. Our results further suggest that increased shed syndecan-1 protein levels are linked with the inefficiency of HMW-SH in septic wildtype mice.

Diagnosis of chronic endometritis: How many CD138+ cells/HPF in endometrial stroma affect pregnancy outcome of infertile women?

PROBLEM: The definition of chronic endometritis (CE) differs among studies, and currently, there is no accepted consensus. This study aimed to establish the minimum number of immunohistochemical analysis of CD138+ plasma cells to identify a clinically relevant CE. METHOD OF STUDY: We performed a retrospective study on 716 infertile patients who never did CE analysis and respective antibiotic treatment before. Samples were obtained by endometrial scratching in the mid-luteal phase before IVF-ET treatment. The number and distribution of CD138+ cells were analyzed by immunohistochemistry. Thirty high-power fields (HPF) were evaluated for each sample. Patients were classified in 2 main groups: (a) CD138low (<5 CD138+ cells in all HPFs), (b) CD138high (≥5 CD138+ cells in at least one HPF). Pregnancy outcome was compared among the groups. RESULTS: In the CD138high group, ß-hCG positive rate, clinical pregnancy rate and live birth rate were significantly decreased (P = .04, P = .01, P = .04, respectively). Also after adjusting for patient age, body mass index (BMI), and clinical characteristics, the ß-hCG positive rate (P = .05), clinical pregnancy rate (P = .01) and live birth rate (P = .02) were significantly lower in the CD138high than those in the CD138low group. Within the CD138low group, these parameters were not significantly different between patients without any plasma cells and patients with up to 4 plasma cells/HPF. CONCLUSION: We conclude that immunohistochemical analysis of CD138+ cells is a reliable method to detect CE which can be identified by the presence of ≥5 plasma cells in at least one out of 30 HPF.

VIS832, a novel CD138-targeting monoclonal antibody, potently induces killing of human multiple myeloma and further synergizes with IMiDs or bortezomib in vitro and in vivo.

Therapeutically targeting CD138, a define multiple myeloma (MM) antigen, is not yet approved for patients. We here developed and determined the preclinical efficacy of VIS832, a novel therapeutic monoclonal antibody (MoAb) with differentiated CD138 target binding to BB4 that is anti-CD138 MoAb scaffold for indatuximab ravtansine (BT062). VIS832 demonstrated enhanced CD138-binding avidity and significantly improved potency to kill MM cell lines and autologous patient MM cells regardless of resistance to current standard-of-care therapies, via robust antibody-dependent cellular cytotoxicity and phagocytosis mediated by NK and macrophage effector cells, respectively. Specifically, CD38-targeting daratumumab-resistant MM cells were highly susceptible to VIS832 which, unlike daratumumab, spares NK cells. Superior maximal cytolysis of VIS832 vs. daratumumab corresponded to higher CD138 vs. CD38 levels in MM cells. Furthermore, VIS832 acted synergistically with lenalidomide or bortezomib to deplete MM cells. Importantly, VIS832 at a sub-optimal dose inhibited disseminated MM1S tumors in vivo as monotherapy (P < 0.0001), and rapidly eradicated myeloma burden in all mice concomitantly receiving bortezomib, with 100% host survival. Taken together, these data strongly support clinical development of VIS832, alone and in combination, for the therapeutic treatment of MM in relapsed and refractory patients while pointing to its potential therapeutic use earlier in disease intervention.

Host syndecan-1 promotes listeriosis by inhibiting intravascular neutrophil extracellular traps.

Heparan sulfate proteoglycans (HSPGs) are at the forefront of host-microbe interactions. Molecular and cell-based studies suggest that HSPG-pathogen interactions promote pathogenesis by facilitating microbial attachment and invasion of host cells. However, the specific identity of HSPGs, precise mechanisms by which HSPGs promote pathogenesis, and the in vivo relevance of HSPG-pathogen interactions remain to be determined. HSPGs also modulate host responses to tissue injury and inflammation, but functions of HSPGs other than facilitating microbial attachment and internalization are understudied in infectious disease. Here we examined the role of syndecan-1 (Sdc1), a major cell surface HSPG of epithelial cells, in mouse models of Listeria monocytogenes (Lm) infection. We show that Sdc1-/- mice are significantly less susceptible to both intragastric and intravenous Lm infection compared to wild type (Wt) mice. This phenotype is not seen in Sdc3-/- or Sdc4-/- mice, indicating that ablation of Sdc1 causes a specific gain of function that enables mice to resist listeriosis. However, Sdc1 does not support Lm attachment or invasion of host cells, indicating that Sdc1 does not promote pathogenesis as a cell surface Lm receptor. Instead, Sdc1 inhibits the clearance of Lm before the bacterium gains access to its intracellular niche. Large intravascular aggregates of neutrophils and neutrophil extracellular traps (NETs) embedded with antimicrobial compounds are formed in Sdc1-/- livers, which trap and kill Lm. Lm infection induces Sdc1 shedding from the surface of hepatocytes in Wt livers, which is directly associated with the decrease in size of intravascular aggregated NETs. Furthermore, administration of purified Sdc1 ectodomains or DNase inhibits the formation of intravascular aggregated neutrophils and NETs and significantly increases the liver bacterial burden in Sdc1-/- mice. These data indicate that Lm induces Sdc1 shedding to subvert the activity of Sdc1 ectodomains to inhibit its clearance by intravascular aggregated NETs.

Paving the Way toward Successful Multiple Myeloma Treatment: Chimeric Antigen Receptor T-Cell Therapy.

Despite the significant progress of modern anticancer therapies, multiple myeloma (MM) is still incurable for the majority of patients. Following almost three decades of development, chimeric antigen receptor (CAR) T-cell therapy now has the opportunity to revolutionize the treatment landscape and meet the unmet clinical need. However, there are still several major hurdles to overcome. Here we discuss the recent advances of CAR T-cell therapy for MM with an emphasis on future directions and possible risks. Currently, CAR T-cell therapy for MM is at the first stage of clinical studies, and most studies have focused on CAR T cells targeting B cell maturation antigen (BCMA), but other antigens such as cluster of differentiation 138 (CD138, syndecan-1) are also being evaluated. Although this therapy is associated with side effects, such as cytokine release syndrome and neurotoxicity, and relapses have been observed, the benefit-risk balance and huge potential drive the ongoing clinical progress. To fulfill the promise of recent clinical trial success and maximize the potential of CAR T, future efforts should focus on the reduction of side effects, novel targeted antigens, combinatorial uses of different types of CAR T, and development of CAR T cells targeting more than one antigen.

Current advances in chimeric antigen receptor T-cell therapy for refractory/relapsed multiple myeloma.

Multiple myeloma (MM), considered an incurable hematological malignancy, is characterized by its clonal evolution of malignant plasma cells. Although the application of autologous stem cell transplantation (ASCT) and the introduction of novel agents such as immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs) have doubled the median overall survival to eight years, relapsed and refractory diseases are still frequent events in the course of MM. To achieve a durable and deep remission, immunotherapy modalities have been developed for relapsed/refractory multiple myeloma (RRMM). Among these approaches, chimeric antigen receptor (CAR) T-cell therapy is the most promising star, based on the results of previous success in B-cell neoplasms. In this immunotherapy, autologous T cells are engineered to express an artificial receptor which targets a tumor-associated antigen and initiates the T-cell killing procedure. Tisagenlecleucel and Axicabtagene, targeting the CD19 antigen, are the two pacesetters of CAR T-cell products. They were approved by the US Food and Drug Administration (FDA) in 2017 for the treatment of acute lymphocytic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). Their development enabled unparalleled efficacy in combating hematopoietic neoplasms. In this review article, we summarize six promising candidate antigens in MM that can be targeted by CARs and discuss some noteworthy studies of the safety profile of current CAR T-cell therapy.

IgG4-positive Cell Quantification Distinguishes Between Inflammatory and Noninflammatory Diseases of the Orbit.

IgG4-related ophthalmic disease (IgG4-ROD) is a rare inflammatory disorder often refractory to corticosteroids and prone to recurrence. IgG4-ROD may frequently lack the characteristic histopathological features seen in other organs. Thus, the criteria for diagnosis of IgG4-ROD relies on elevated IgG4 cells seen on biopsied tissue. Proposed threshold levels of IgG4 to diagnose IgG4-ROD are currently based on a limited understanding of this cell type's presence in the orbit. This study seeks to describe the population of IgG4 in inflammatory and noninflammatory orbital tissues. A tertiary care center's pathology database was searched with keywords "orbit" or "orbital" from 1995 to 2013. Specimens meeting the selection criteria were evaluated, and regions of highest inflammation were identified and immunostained with IgG4 and CD138 antibodies. Immunohistochemical quantification proceeded as previously established by the international consensus criteria. Eighteen cases without a history of orbital inflammation were included as controls and were evaluated as above. Specimens from 68 inflammatory and 18 noninflammatory orbits met the selection criteria. Pathologist interreader correlation coefficient on quantification was >0.70 (P<0.001). The mean IgG4+/high powered field (HPF) and IgG4+/CD138 was 10.3 and 0.1 in inflammatory tissues and 0.5 and 0.01 in noninflammatory tissues, respectively. The spearman rho correlation coefficient between IgG4/HPF and IgG4+/CD138+ was >0.95 (P<0.0001). The mean IgG4/HPF in our study reached previously proposed threshold values for diagnosis of IgG4-ROD, illustrating the need for further discussion regarding diagnostic criteria of IgG4-ROD.

Current advances in chimeric antigen receptor T-cell therapy for refractory/relapsed multiple myeloma / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Multiple myeloma (MM), considered an incurable hematological malignancy, is characterized by its clonal evolution of malignant plasma cells. Although the application of autologous stem cell transplantation (ASCT) and the introduction of novel agents such as immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs) have doubled the median overall survival to eight years, relapsed and refractory diseases are still frequent events in the course of MM. To achieve a durable and deep remission, immunotherapy modalities have been developed for relapsed/refractory multiple myeloma (RRMM). Among these approaches, chimeric antigen receptor (CAR) T-cell therapy is the most promising star, based on the results of previous success in B-cell neoplasms. In this immunotherapy, autologous T cells are engineered to express an artificial receptor which targets a tumor-associated antigen and initiates the T-cell killing procedure. Tisagenlecleucel and Axicabtagene, targeting the CD19 antigen, are the two pacesetters of CAR T-cell products. They were approved by the US Food and Drug Administration (FDA) in 2017 for the treatment of acute lymphocytic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). Their development enabled unparalleled efficacy in combating hematopoietic neoplasms. In this review article, we summarize six promising candidate antigens in MM that can be targeted by CARs and discuss some noteworthy studies of the safety profile of current CAR T-cell therapy.

Autoantigen-specific B cells and plasma cells are prominent in areas of fatty infiltration in salivary glands of patients with primary Sjögren's syndrome.

Salivary and lacrimal gland involvement is a characteristic feature of primary Sjögren's syndrome (pSS), where tissue destruction is mediated by mononuclear cell infiltration, resulting in lacrimal and salivary gland impairment. We have previously shown distinct prevalence of adipose tissue replacement in the minor salivary gland tissue from pSS patients. The salivary gland microenvironment was further examined through microarray analysis, identifying signalling pathways that promoted adipose tissue development, inflammation, and lymphoma. As B cells may also contribute to disease progression, we now aimed to study the B cell pattern with regard to adipocyte development in pSS. Double immunohistochemical staining of paraffin-embedded salivary gland tissue from 22 pSS patients and 11 non-SS tissue controls was employed, using the characteristic pSS autoantigens Ro52 or Ro60, alongside CD27. Additional CD138/CD20 double staining was also performed to identify the plasma- and general B- cell pattern. Our results demonstrated CD27-positive Ro52 and Ro60 specific cells observed within and in close proximity to the adipose tissue. CD138-positive plasma cells were also seen in areas of adipose tissue replacement, while the CD20+ cells were located within focal infiltrates, forming distinct B cell zones. The quantification of CD138+ and CD20+ cells revealed elevated numbers of CD138+ cells in areas of fatty infiltration, and also interstitially, in the salivary glands of pSS patients when compared to non-SS controls. A significant increase (p < .01) in CD138+ cells close to areas of fatty infiltration, and interstitially, with increasing fatty infiltration and focus score was further observed in pSS patients. A correlation between the number of CD20+ B cell zones/mm2 of salivary gland tissue and focus score values was also witnessed in the patients (r2 = 0.6047, p < .001). In conclusion, autoantigen-specific B cells and plasma cells appear prominent in areas of fatty infiltration in salivary glands of pSS patients, where an increase in CD138+ plasma cells and CD20+ B cells, in relation to both fatty and focal infiltration, suggests their active involvement in promoting inflammation. Further studies are needed to assess whether these adipocytes are also a result of tissue repair.

Extracellular NK histones promote immune cell anti-tumor activity by inducing cell clusters through binding to CD138 receptor.

BACKGROUND: Natural killer (NK) cells are important anti-tumor cells of our innate immune system. Their anti-cancer activity is mediated through interaction of a wide array of activating and inhibitory receptors with their ligands on tumor cells. After activation, NK cells also secrete a variety of pro-inflammatory molecules that contribute to the final immune response by modulating other innate and adaptive immune cells. In this regard, external proteins from NK cell secretome and the mechanisms by which they mediate these responses are poorly defined. METHODS: TRANS-stable-isotope labeling of amino acids in cell culture (TRANS-SILAC) combined with proteomic was undertaken to identify early materials transferred between cord blood-derived NK cells (CB-NK) and multiple myeloma (MM) cells. Further in vitro and in vivo studies with knock-down of histones and CD138, overexpression of histones and addition of exogenous histones were undertaken to confirm TRANS-SILAC results and to determine functional roles of this material transferred. RESULTS: We describe a novel mechanism by which histones are actively released by NK cells early after contact with MM cells. We show that extracellular histones bind to the heparan sulfate proteoglycan CD138 on the surface of MM cells to promote the creation of immune-tumor cell clusters bringing immune and MM cells into close proximity, and thus facilitating not only NK but also T lymphocyte anti-MM activity. CONCLUSION: This study demonstrates a novel immunoregulatory role of NK cells against MM cells mediated by histones, and an additional role of NK cells modulating T lymphocytes activity that will open up new avenues to design future immunotherapy clinical strategies.

VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1.

When targeted by the tumor-promoting enzyme heparanase, cleaved and shed syndecan-1 (Sdc1) then couples VEGFR2 (also known as KDR) to VLA-4, activating VEGFR2 and the directed migration of myeloma cells. But how VEGFR2 activates VLA-4-mediated motility has remained unknown. We now report that VEGFR2 causes PKA-mediated phosphorylation of VLA-4 on S988, an event known to stimulate tumor metastasis while suppressing cytotoxic immune cells. A key partner in this mechanism is the chemokine receptor CXCR4, a well-known mediator of cell motility in response to gradients of the chemokine SDF-1 (also known as CXCL12). The entire machinery necessary to phosphorylate VLA-4, consisting of CXCR4, AC7 (also known as ADCY7) and PKA, is constitutively associated with VEGFR2 and is localized to the integrin by Sdc1. VEGFR2 carries out the novel phosphorylation of Y135 within the DRY microswitch of CXCR4, sequentially activating Gαißγ, AC7 and PKA, which phosphorylates S988 on the integrin. This mechanism is blocked by a syndecan-mimetic peptide (SSTNVEGFR2), which, by preventing VEGFR2 linkage to VLA-4, arrests tumor cell migration that depends on VLA-4 phosphorylation and stimulates the LFA-1-mediated migration of cytotoxic leukocytes.

The immunomodulatory role of tumor Syndecan-1 (CD138) on ex vivo tumor microenvironmental CD4+ T cell polarization in inflammatory and non-inflammatory breast cancer patients.

Herein, we aimed to identify the immunomodulatory role of tumor Syndecan-1 (CD138) in the polarization of CD4+ T helper (Th) subsets isolated from the tumor microenvironment of inflammatory breast cancer (IBC) and non-IBC patients. Lymphocytes and mononuclear cells isolated from the axillary tributaries of non-IBC and IBC patients during modified radical mastectomy were either stimulated with the secretome as indirect co-culture or directly co-cultured with control and Syndecan-1-silenced SUM-149 IBC cells. In addition, peripheral blood mononuclear cells (PBMCs) of normal subjects were used for the direct co-culture. Employing flow cytometry, we analyzed the expression of the intracellular IFN-γ, IL-4, IL-17, and Foxp3 markers as readout for basal and co-cultured Th1, Th2, Th17, and Treg CD4+ subsets, respectively. Our data revealed that IBC displayed a lower basal frequency of Th1 and Th2 subsets than non-IBC. Syndecan-1-silenced SUM-149 cells significantly upregulated only Treg subset polarization of normal subjects relative to controls. However, Syndecan-1 silencing significantly enhanced the polarization of Th17 and Treg subsets of non-IBC under both direct and indirect conditions and induced only Th1 subset polarization under indirect conditions compared to control. Interestingly, qPCR revealed that there was a negative correlation between Syndecan-1 and each of IL-4, IL-17, and Foxp3 mRNA expression in carcinoma tissues of IBC and that the correlation was reversed in non-IBC. Mechanistically, Syndecan-1 knockdown in SUM-149 cells promoted Th17 cell expansion via upregulation of IL-23 and the Notch ligand DLL4. Overall, this study indicates a low frequency of the circulating antitumor Th1 subset in IBC and suggests that tumor Syndecan-1 silencing enhances ex vivo polarization of CD4+ Th17 and Treg cells of non-IBC, whereby Th17 polarization is possibly mediated via upregulation of IL-23 and DLL4. These findings suggest the immunoregulatory role of tumor Syndecan-1 expression in Th cell polarization that may have therapeutic implications for breast cancer.

What is the Best Radionuclide for Immuno-PET of Multiple Myeloma? A Comparison Study Between 89Zr- and 64Cu-Labeled Anti-CD138 in a Preclinical Syngeneic Model.

Although positron emission tomography (PET) imaging with 18-Fluorodeoxyglucose (18F-FDG) is a promising technique in multiple myeloma (MM), the development of other radiopharmaceuticals seems relevant. CD138 is currently used as a standard marker for the identification of myeloma cells and could be used in phenotype tumor imaging. In this study, we used an anti-CD138 murine antibody (9E7.4) radiolabeled with copper-64 (64Cu) or zirconium-89 (89Zr) and compared them in a syngeneic mouse model to select the optimal tracers for MM PET imaging. Then, 9E7.4 was conjugated to TE2A-benzyl isothiocyanate (TE2A) and desferrioxamine (DFO) chelators for 64Cu and 89Zr labeling, respectively. 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 antibodies were evaluated by PET imaging and biodistribution studies in C57BL/KaLwRij mice bearing either 5T33-MM subcutaneous tumors or bone lesions and were compared to 18F-FDG-PET imaging. In biodistribution and PET studies, 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 displayed comparable good tumor uptake of subcutaneous tumors. On the bone lesions, PET imaging with 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 showed higher uptake than with 18F-FDG-PET. Comparison of both 9E7.4 conjugates revealed higher nonspecific bone uptakes of 89Zr-DFO-9E7.4 than 64Cu-TE2A-9E7.4. Because of free 89Zr's tropism for bone when using 89Zr-anti-CD138, 64Cu-anti-CD138 antibody had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a specific new imaging radiopharmaceutical agent in MM.

A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death.

Intracellular lipopolysaccharide (LPS) triggers the non-canonical inflammasome pathway, resulting in pyroptosis of innate immune cells. In addition to its well-known proinflammatory role, LPS can directly cause regression of some tumors, although the underlying mechanism has remained unknown. Here we show that secretoglobin(SCGB)3A2, a small protein predominantly secreted in airways, chaperones LPS to the cytosol through the cell surface receptor syndecan-1; this leads to pyroptotic cell death driven by caspase-11. SCGB3A2 and LPS co-treatment significantly induced pyroptosis of macrophage RAW264.7 cells and decreased cancer cell proliferation in vitro, while SCGB3A2 treatment resulted in reduced progression of xenograft tumors in mice. These data suggest a conserved function for SCGB3A2 in the innate immune system and cancer cells. These findings demonstrate a critical role for SCGB3A2 as an LPS delivery vehicle; they reveal one mechanism whereby LPS enters innate immune cells leading to pyroptosis, and they clarify the direct effect of LPS on cancer cells.

Protection of the endothelial glycocalyx by antithrombin in an endotoxin-induced rat model of sepsis.

INTRODUCTION: Injury and loss of the endothelial glycocalyx occur during the early phase of sepsis. We previously showed that antithrombin has a protective effect on this structure in vitro. Here, we investigated the possible protective effects of antithrombin in an animal model of sepsis. METHODS: Wistar rats were injected with endotoxin, and circulating levels of syndecan-1, hyaluronan, albumin, lactate and other biomarkers were measured in an antithrombin-treated group and an untreated control group (n = 6 in each group). Intravital microscopy was used to observe leukocyte adhesion, microcirculation, and syndecan-1 staining. RESULTS: The circulating levels of syndecan-1 and hyaluronan were significantly reduced in the antithrombin-treated group, compared with the untreated controls. Lactate levels and albumin reduction were significantly attenuated in the antithrombin-treated group. Intravital microscopic observation revealed that both leukocyte adhesion and blood flow were better maintained in the treatment group. The syndecan-1 lining was disrupted after endotoxin treatment, and this derangement was attenuated by treatment with antithrombin. CONCLUSION: Antithrombin effectively maintained microcirculation and vascular integrity by protecting the glycocalyx in a rat sepsis model.

Elevated soluble syndecan-1 levels in neuromyelitis optica are associated with disease severity.

Syndecan-1 (SDC-1) is a transmembrane member that has a profound influence on the resolution of inflammation. Soluble syndecan-1 (sSDC-1) levels have been shown to increase in many inflammatory diseases. However, it remains unknown whether sSDC-1 concentration is elevated in neuromyelitis optica (NMO) and multiple sclerosis (MS) patients. The aims of this pilot study were to investigate the relationship between sSDC-1 and disease severity in NMO and MS and whether sSDC-1 has potential as an effective marker for disease severity. We measured sSDC-1 concentrations by using an enzyme-linked immunosorbent assay (ELISA). NMO patients had significantly higher CSF sSDC-1 levels than MS patients or controls. We also found a positive correlation between the increased CSF sSDC-1 levels and increased severity in NMO disease, but not in MS. In NMO, CSF sSDC-1 concentrations were positively correlated with CSF interleukin (IL)-6, IL-8 and IL-17. Overall, we showed levels of CSF sSDC-1 were higher in NMO patients and had a positive relationship with disease severity of NMO but not with MS. CSF sSDC-1 may be an effective marker of NMO disease severity.

Syndecan-1 Regulates Psoriasiform Dermatitis by Controlling Homeostasis of IL-17-Producing γδ T Cells.

IL-17 is a potent proinflammatory cytokine that drives pathogenesis of multiple autoimmune diseases, including psoriasis. A major source of pathogenic IL-17 is a subset of γδ T cells (Tγδ17) that acquires the ability to produce IL-17 while developing in the thymus. The mechanisms that regulate homeostasis of Tγδ17 cells and their roles in psoriasis, however, are not fully understood. In this paper, we show that the heparan sulfate proteoglycan syndecan-1 (sdc1) plays a critical role in regulating homeostasis of Tγδ17 cells and modulating psoriasis-like skin inflammation in mice. sdc1 was predominantly expressed by Tγδ17 cells (but not IL-17- Tγδ cells) in the thymus, lymph nodes, and dermis. sdc1 deficiency significantly and selectively increased the frequency and absolute numbers of Tγδ17 cells by mechanisms that included increased proliferation and decreased apoptosis. Adoptive transfer experiments ruled out a significant role of sdc1 expressed on nonhematopoietic cells in halting expansion and proliferation of sdc1-deficient Tγδ17 cells. When subjected to imiquimod-induced psoriasiform dermatitis, Tγδ17 cells in sdc1KO mice displayed heightened responses accompanied by significantly increased skin inflammation than their wild-type counterparts. Furthermore, transferred sdc1-deficient γδ T cells caused more severe psoriasiform dermatitis than their sdc1-sufficient counterparts in TCR-ßδ KO hosts. The results uncover a novel role for sdc1 in controlling homeostasis of Tγδ17 cells and moderating host responses to psoriasis-like inflammation.

Synthesis of C-functionalized TE1PA and comparison with its analogues. An example of bioconjugation on 9E7.4 mAb for multiple myeloma 64Cu-PET imaging.

In view of the excellent copper(ii) and 64-copper(ii) complexation of a TE1PA ligand, a monopicolinate cyclam, in both aqueous medium and in vivo, we looked for a way to make it bifunctional, while maintaining its chelating properties. Overcoming the already known drawback of grafting via its carboxyl group, which is essential to the overall properties of the ligand, a TE1PA bifunctional derivative bearing an additional isothiocyanate coupling function on a carbon atom of the macrocyclic ring was synthesized. This led to an architecture that is comparable to that of other commercially available bifunctional copper(ii) chelators such as p-SCN-Bn-DOTA already used in clinical trials for 64Cu-immuno-PET imaging. The C-functionalization of TE1PA on one carbon atom in the ß-N position of the cyclam backbone was successfully achieved by adapting our patented methodology to the huge challenge, allowing the regiospecific mono-N-functionalization of the unsymmetrical ligand. The obtained ligand p-SCN-Bn-TE1PA was coupled to a 9E7.4 murine antibody (mAb), an IgG2a anti CD-138 for multiple myeloma (MM) targeting. The conjugation efficiency was assessed by looking at the 64Cu radiolabeling and the radiopharmaceutical 64Cu-9E7.4-p-SCN-Bn-TE1PA immunoreactivity, and in particular by comparing with 9E7.4-p-SCN-Bn-NOTA and 9E7.4-p-SCN-Bn-DOTA obtained from commercial and presumably highly efficient chelators NOTA and DOTA, respectively. The results are quite clear, showing that p-SCN-Bn-TE1PA has a coupling rate 5 times higher and an immunoreactivity 1.5 to 2 times greater than those of its two competitors. p-SCN-Bn-TE1PA also outperforms TE1PA conjugated via its carboxylic function on the same antibody. The first 64Cu-immuno-PET preclinical study in a syngeneic model of MM was performed, confirming the good in vivo properties of 64Cu-9E7.4-p-SCN-Bn-TE1PA for PET imaging, considering the high clearance even after 24 h and the particularly important tumor-to-liver ratio that was increasing at 48 h.