Membrane-associated Glycoprotein Research Articles

Novel TROP2-targeted CAR T cells with high specificity and sustained efficacy for solid tumors in animal models.

e14563 Background: CAR T cell therapy, successful in hematological malignancies, encounters challenges against solid tumors due to immune exhaustion and senescence within the tumor microenvironment. The imperative need for an optimal tumor target antigen is evident in advancing CAR T therapy. TROP2, a membrane-associated glycoprotein, is highly expressed in diverse cancers, including cervical, bladder, esophageal, ovarian, and endometrial cancers. TROP2 overexpression, approaching 100% in cervical and hormone-resistant prostate cancer, correlates inversely with prognosis and survival, presenting TROP2 as a promising CAR T therapy target. This study engineered the novel TROP2 antibody R4702, featuring a distinct binding epitope from Sacituzumab and Datopotamab, to develop TROP2-specific CAR T cells and evaluated their efficacy against a spectrum of solid tumors. Methods: Expansion of CAR T Cells and Cytotoxicity: CAR T cells were created through lentivirus-mediated genetic engineering with the TROP2 antibody, followed by 10 days of expansion. T cell differentiation was evaluated, and luciferase-encoding target cells incubated with varying CAR T cell ratios overnight were quantified luciferase activity. In Vivo Efficacy and Persistence: CAR T cells were adoptively transferred into ASID mice with tumors. In vivo tumor imaging used luciferase activity, and calipers monitored tumor size weekly. CAR T cell persistence assessed their ability to suppress rechallenged tumor growth. Results: R4702, a novel TROP2-specific antibody, displayed high binding affinity to a distinct epitope. Post- ex vivo expansion, R4702 CAR T cells showed a substantial increase (100 to 200-fold) over 10 days, with a high expression level (50~80%) of R4702 CAR on T cells. Over 60% of R4702 CAR T cells displayed a less differentiated status, characterized by a CD62L+ population comprising TSCM and TCM. In vitro cytotoxicity revealed potent and specific efficacy against various TROP2-positive cancer cell lines, with no observed killing activity against TROP2-negative cells. In vivo efficacy of R4702 CAR T cells demonstrated 100% tumor growth inhibition within 30 days post-treatment across diverse xenograft models. Importantly, R4702 CAR T cells exhibited enduring anti-tumor efficacy against successive tumor challenges in xenograft models of pancreatic, gastric, NSCLC, TNBC, and ovarian cancers. In a head-to-head comparison with Sacituzumab and Datopotamab CAR T, R4702 demonstrated superior anti-tumor efficacy to Sacituzumab and comparable efficacy with Datopotamab in the BxPC-3 CDX model of pancreatic cancer. Conclusions: A novel TROP2-specific R4702 CAR T cell, developed, showcased robust tumor-killing capabilities. The anti-tumor activity persisted upon repeated tumor challenges across various xenograft models, underscoring the potential of R4702 CAR T cells as a promising therapeutic strategy.

Peptide Binder to Glypican-3 as a Theranostic Agent for Hepatocellular Carcinoma.

Glypican-3 (GPC3) is a membrane-associated glycoprotein that is significantly upregulated in hepatocellular carcinomas (HCC) with minimal to no expression in normal tissues. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy to treat HCC, a leading cause of cancer-related deaths worldwide. Methods: DOTA-RYZ-GPC3 (RAYZ-8009) comprises a novel macrocyclic peptide binder to GPC3, a linker, and a chelator that can be complexed with different radioisotopes. The binding affinity was determined by surface plasma resonance and radioligand binding assays. Target-mediated cellular internalization was radiometrically measured at multiple time points. In vivo biodistribution, monotherapy, and combination treatments with 177Lu or 225Ac were performed on HCC xenografts. Results: RAYZ-8009 showed high binding affinity to GPC3 protein of human, mouse, canine, and cynomolgus monkey origins and no binding to other glypican family members. Potent cellular binding was confirmed in GPC3-positive HepG2 cells and was not affected by isotope switching. RAYZ-8009 achieved efficient internalization on binding to HepG2 cells. Biodistribution study of 177Lu-RAYZ-8009 showed sustained tumor uptake and fast renal clearance, with minimal or no uptake in other normal tissues. Tumor-specific uptake was also demonstrated in orthotopic HCC tumors, with no uptake in surrounding liver tissue. Therapeutically, significant and durable tumor regression and survival benefit were achieved with 177Lu- and 225Ac-labeled RAYZ-8009, as single agents and in combination with lenvatinib, in GPC3-positive HCC xenografts. Conclusion: Preclinical invitro and invivo data demonstrate the potential of RAYZ-8009 as a theranostic agent for the treatment of patients with GPC3-positive HCC.

Loss of CDH16 expression is a strong independent predictor for lymph node metastasis in Middle Eastern papillary thyroid cancer

Papillary Thyroid Cancer (PTC) is the most common type of thyroid cancer. The membrane-associated glycoprotein cadherin-16 (CDH16) plays a significant role in the embryonal development of thyroid follicles and cell adhesion. Previous studies have indicated a substantial downregulation of CDH16 in PTC. However, its role in Middle Eastern PTC has not been elucidated. We analyzed a tissue microarray comprising 1606 PTC and 240 normal thyroid tissues using immunohistochemistry to assess CDH16 expression and determine its clinico-pathological associations. We also conducted BRAF and TERT mutations analyses through Sanger sequencing. Disease-free survival (DFS) was assessed using Kaplan–Meier curves. CDH16 immunostaining was seen in 100% of normal thyroid tissues but only in 9.4% of PTC tissues (p < 0.0001). The loss of CDH16 expression was associated with aggressive PTC characteristics including bilaterality, multifocality, extrathyroidal extension, tall cell variant, lymph node metastasis (LNM) and distant metastasis. Additionally a correlation between loss of CDH16 expression and BRAF and TERT mutations was identified. Intriguingly, upon conducting multivariate logistic regression analysis, CDH16 was determined to be an independent predictor for LNM (Odds ratio = 2.46; 95% confidence interval = 1.60–3.79; p < 0.0001). Furthermore, CDH16 loss was associated with a shorter DFS (p = 0.0015). However, when we further subdivided CDH16 negative patients based on the co-existence of TERT and/or BRAF mutations, we found that patients with both CDH16 negative expression and TERT mutation exhibited the shortest DFS (p < 0.0001). In conclusion, our results suggest that CDH16 protein expression could serve as a valuable diagnostic tool for PTC. Furthermore, these findings demonstrate that the loss of CDH16 expression is an independent predictor of LNM and may contribute to the aggressiveness of PTC. Therefore, downregulation of CDH16 in PTC might be a potential target for designing novel therapeutic strategies to treat PTC.

Structure of engineered hepatitis C virus E1E2 ectodomain in complex with neutralizing antibodies

Hepatitis C virus (HCV) is a major global health burden as the leading causative agent of chronic liver disease and hepatocellular carcinoma. While the main antigenic target for HCV-neutralizing antibodies is the membrane-associated E1E2 surface glycoprotein, the development of effective vaccines has been hindered by complications in the biochemical preparation of soluble E1E2 ectodomains. Here, we present a cryo-EM structure of an engineered, secreted E1E2 ectodomain of genotype 1b in complex with neutralizing antibodies AR4A, HEPC74, and IGH520. Structural characterization of the E1 subunit and C-terminal regions of E2 reveal an overall architecture of E1E2 that concurs with that observed for non-engineered full-length E1E2. Analysis of the AR4A epitope within a region of E2 that bridges between the E2 core and E1 defines the structural basis for its broad neutralization. Our study presents the structure of an E1E2 complex liberated from membrane via a designed scaffold, one that maintains all essential structural features of native E1E2. The study advances the understanding of the E1E2 heterodimer structure, crucial for the rational design of secreted E1E2 antigens in vaccine development.

Cadherin-16 (CDH16) Immunohistochemistry: A Novel Diagnostic Tool for Renal Cell Carcinoma and Papillary Carcinomas of the Thyroid

Abstract Introduction/Objective Introduction: Cadherin-16 (CDH16), also termed kidney specific cadherin (ksp-cadherin), is a membrane-associated glycoprotein with a role in the embryonal development of tubules in kidney and thyroid. Downregulation of CDH16 RNA was found in papillary carcinomas of the thyroid. Methods/Case Report Methods: A set of tissue microarrays containing 14,978 samples from 149 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry to determine the expression of CDH16 in cancer and to assess the diagnostic utility of immunohistochemical CDH16 analysis. Results (if a Case Study enter NA) Results: Among normal tissues, a membranous CDH16 immunostaining predominated in thyroid, kidney, cauda epididymis, and in mesonephric remnants. In the thyroid, CDH16 staining was seen in 100% of normal samples, 83% of follicular adenomas, 58% of follicular carcinomas, but in only 9% of papillary carcinomas (p&amp;lt;0.0001). Among non-thyroidal tumors, CDH16 positivity was particularly frequent in nephrogenic adenomas (100%), oncocytomas (98%), chromophobe (97%), clear cell (85%), and papillary (76%) renal cell carcinomas (RCCs), clear cell (56%), mucinous (36%), and endometroid (16%) carcinomas as well as carcinosarcomas (18%) of the ovary, adenocarcinomas of the cervix uteri (40%), serous (33%), clear cell (33%), and endometroid carcinomas (18%) of the endometrium and in various subtypes of neuroendocrine neoplasms (4-26%). Conclusion Given the massive loss of CDH16 expression in &amp;gt;90% of papillary carcinomas of the thyroid, CDH16 is a highly useful diagnostic marker for these tumors. CDH16 immunohistochemistry is also useful for the identification of nephrogenic adenomas and the distinction of renal cell carcinomas from other neoplasms.

Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate

SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

High Prevalence of 5T4/Trophoblast Glycoprotein in Soft Tissue Sarcomas.

Simple SummarySoft tissue sarcoma (STS) is a heterogeneous group of hard-to-treat malignancies from mesenchymal or connective tissues. The treatment options in case the cancer relapses after surgery or is at an advanced state are limited, and in all cases the response rates are low. Targeted therapy with antibody drug conjugates may provide an alternative. To this end, molecules that are specifically (over)expressed on the surface of the tumor cells are needed. In this study, we show that 5T4/trophoblast glycoprotein, a molecule with limited expression in healthy tissues, is prominently expressed on the cell surface of many STS subtypes and can be used for such an approach.The expression of 5T4/trophoblast glycoprotein was evaluated in several histological subtypes of soft tissue sarcoma (STS) to determine whether the prevalence and level of expression of this membrane-associated glycoprotein is sufficient for use in targeted therapies. Tumor tissue microarrays containing cores from different histological subtypes of STS were stained using a standardized immunohistochemical staining method to detect 5T4; the level of staining was assessed using a semi-quantitative scoring method. No 5T4 staining was seen in the angiosarcomas and liposarcomas investigated in this study. 5T4 staining in the other STS subtypes was seen in more than 50% of cases, warranting further investigation into whether this antigen could evoke an anti-tumor immune response or can be used as target for the delivery of more potent toxins through antibody drug conjugates.

Herpes Simplex Virus Type 2 Mucin-Like Glycoprotein mgG Promotes Virus Release from the Surface of Infected Cells.

The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by ~2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs ~fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.

Activated IL-6 signaling contributes to the pathogenesis of, and is a novel therapeutic target for, CALR-mutated MPNs

Calreticulin (CALR), an endoplasmic reticulum–associated chaperone, is frequently mutated in myeloproliferative neoplasms (MPNs). Mutated CALR promotes downstream JAK2/STAT5 signaling through interaction with, and activation of, the thrombopoietin receptor (MPL). Here, we provide evidence of a novel mechanism contributing to CALR-mutated MPNs, represented by abnormal activation of the interleukin 6 (IL-6)-signaling pathway. We found that UT7 and UT7/mpl cells, engineered by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to express the CALR type 1–like (DEL) mutation, acquired cytokine independence and were primed to the megakaryocyte (Mk) lineage. Levels of IL-6 messenger RNA (mRNA), extracellular-released IL-6, membrane-associated glycoprotein 130 (gp130), and IL-6 receptor (IL-6R), phosphorylated JAK1 and STAT3 (p-JAK1 and p-STAT3), and IL-6 promoter region occupancy by STAT3 all resulted in increased CALR DEL cells in the absence of MPL stimulation. Wild-type, but not mutated, CALR physically interacted with gp130 and IL-6R, downregulating their expression on the cell membrane. Agents targeting gp130 (SC-144), IL-6R (tocilizumab [TCZ]), and cell-released IL-6 reduced proliferation of CALR DEL as well as CALR knockout cells, supporting a mutated CALR loss-of-function model. CD34+ cells from CALR-mutated patients showed increased levels of IL-6 mRNA and p-STAT3, and colony-forming unit–Mk growth was inhibited by either SC144 or TCZ, as well as an IL-6 antibody, supporting cell-autonomous activation of the IL-6 pathway. Targeting IL-6 signaling also reduced colony formation by CD34+ cells of JAK2V617F-mutated patients. The combination of TCZ and ruxolitinib was synergistic at very low nanomolar concentrations. Overall, our results suggest that target inhibition of IL-6 signaling may have therapeutic potential in CALR, and possibly JAK2V617F, mutated MPNs.

Characterization of rVSVΔG-ZEBOV-GP glycoproteins using automated capillary western blotting

Ebolavirus (EBOV) entry to host cells requires membrane-associated glycoprotein (GP). A recombinant vesicular stomatitis virus vector carrying Zaire Ebola virus glycoprotein (rVSV-ZEBOV) was developed as a vaccine against ebolaviruses. The VSV glycoprotein gene was deleted (rVSVΔG) and ZEBOV glycoprotein (GP) was inserted into the deleted VSV glycoprotein open reading frame (ORF) resulting in a live, replication-competent vector (rVSVΔG-ZEBOV-GP). Automated capillary westerns were used to characterize the rVSVΔG-ZEBOV-GP vaccine (ERVEBO®) manufacturing process with regards to glycoprotein (GP) structure and variants. The method shows a unique electropherogram profile for each process step which could be used to monitor process robustness. rVSVΔG-ZEBOV-GP encodes GP (GP1-GP2), secreted GP (sGP), and small secreted GP (ssGP) variants. Furthermore, a TACE-like activity was observed indirectly by detecting soluble GP2Δ after virus precipitation by ultracentrifugation. Capillary western blotting techniques can guide process development by evaluating process steps such as enzyme treatment. In addition, the technique can assess GP stability and process lot-to-lot consistency. Finally, capillary western-based technology was used to identify a unique biochemical profile of the rVSVΔG-ZEBOV-GP vaccine strain in final product. Virion membrane-bound GP1-GP2 is critical to vaccine-elicited protection by providing both neutralizing antibodies and T-cell response.

Generation of CART cells targeting oncogenic TROP2 for the elimination of epithelial malignancies

Background & Aim TROP2 (also referred to as Tacstd2) is a membrane-associated glycoprotein highly expressed in many epithelial cancers, including oral, head-and-neck, thyroid, lung, esophageal, gastric, colorectal, pancreatic, breast, renal, uterine, cervical, ovarian, prostate, and glioma, and expression of TROP2 negatively correlates with prognosis and survival in many of these malignancies. TROP2 signaling supports tumor growth and progression through the induction of pathways controlling cell cycle progression, invasion and metastasis, proliferation, angiogenesis, cell growth and survival. In addition, tumor release of TROP2-containing exosomes can promote the migration of TROP2-negative tumor cells. Here, we generated several TROP-2-targeting CAR molecules and evaluated their activity as CART cells against various epithelial tumor cell lines. Methods, Results & Conclusion Single-chain variable fragments targeting TROP2 were produced through domain sequencing of hybridomas and through other publically available sources and cloned into lentiviral transfer plasmids containing second-generation CAR backbones. Transduction of human T cells routinely leads to the expression of >50% CAR+ cells, including the generation of TROP2-CART cells from T cells of a mTNBC patient. We evaluated interferon-gamma production as well as cytotoxicity of TROP2-CART cells cultured with TROP2+ breast, prostate and pancreatic tumor cells to choose a lead TROP2-CAR. In addition, we generated an isogenic TROP2- prostate cancer through Cas9-based gene editing and observed loss of cytotoxicity, demonstrating specificity of the TROP2-CART. However, TROP2-CART can kill TROP2- cells in the presence of TROP2+ cells, a feature also observed by CART19 when cultured with CD19+ and CD19- cells, consistent prior reports that activated CART cells induce tumor apoptosis through death receptors. Prior reports demonstrated that blockade of death receptor ligands abolished the non-specific cytotoxicity of CART19 cells, but this approach provided only partial blockade of TROP2-CART cytotoxicity. Instead, we describe and demonstrate a new mechanism by which transfer of TROP2+ tumor exosomes to TROP2- tumor cells increases the proportion of tumor targetable by TROP2-CART and may allow TROP2-CART to overcome the challenge of antigen heterogeneity in solid tumors.

The Ebola-Glycoprotein Modulates the Function of Natural Killer Cells.

The Ebola virus (EBOV) uses evasion mechanisms that directly interfere with host T-cell antiviral responses. By steric shielding of human leukocyte antigen class-1, the Ebola glycoprotein (GP) blocks interaction with T-cell receptors (TCRs), thus rendering T cells unable to attack virus-infected cells. It is likely that this mechanism could promote increased natural killer (NK) cell activity against GP-expressing cells by preventing the engagement of NK inhibitory receptors; however, we found that primary human NK cells were less reactive to GP-expressing HEK293T cells. This was manifested as reduced cytokine secretion, a reduction in NK degranulation, and decreased lysis of GP-expressing target cells. We also demonstrated reduced recognition of GP-expressing cells by recombinant NKG2D and NKp30 receptors. In accordance, we showed a reduced monoclonal antibody-based staining of NKG2D and NKp30 ligands on GP-expressing target cells. Trypsin digestion of the membrane-associated GP led to a recovery of the recognition of membrane-associated NKG2D and NKp30 ligands. We further showed that membrane-associated GP did not shield recognition by KIR2DL receptors; in accordance, GP expression by target cells significantly perturbed signal transduction through activating, but not through inhibitory, receptors. Our results suggest a novel evasion mechanism employed by the EBOV to specifically avoid the NK cell immune response.

Potential of serum soluble CD93 as a biomarker for asthma in an ovalbumin-induced asthma murine model

Background: CD93 is a membrane-associated glycoprotein, which can be released in a soluble form (sCD93) into the serum. CD93 has received renewed attention as a candidate biomarker of inflammation in various inflammatory and immune-mediated diseases, including asthma.Objective: We aimed to evaluate the effects of airway inflammation on CD93 levels in murine models.Methods: We established an ovalbumin (OVA)-induced acute asthma murine model (OVA model) and a lipopolysaccharide (LPS)-induced airway inflammation murine model (LPS model). Dexamethasone was administered by gavage to attenuate the airway inflammation.Results: The OVA model demonstrated typical allergic asthma features with increased airway hyper-responsiveness, inflammatory cell infiltration, increased Th2 cytokine levels, compared to the control group. CD93 levels were decreased in lung homogenates and, respiratory epithelial cells, whereas serum sCD93 levels were increased in the OVA model, as compared to the control group. Dexamethasone reversed these effects of OVA. In contrast, in the LPS model, CD93 levels were not affected in neither respiratory epithelial cells nor serum.Conclusions: Our findings demonstrate the potential of using sCD93 as a biomarker for allergic asthma.

CD317 Signature in Head and Neck Cancer Indicates Poor Prognosis

Targeted therapy using monoclonal antibodies (mAbs) has emerged as a widely used form of immunotherapy in head and neck squamous cell carcinoma (HNSCC). Membrane-associated glycoprotein CD317 has been preferentially overexpressed by multiple myeloma cells, and its humanized mAb has been previously used in clinical trials. However, overexpression of CD317 in HNSCC and its correlation with tumor immunity is still uncertain. Here, the immunoreactivity of CD317 was detected in human HNSCC tissue microarrays, which contained 43 oral mucosa samples, 48 dysplasia samples, and 165 primary HNSCC. We found that CD317 expression was up-regulated in HNSCC tumor cells, and the CD317 expression level was independent of the histological grade, tumor size, and lymph node metastasis. Moreover, Kaplan–Meier survival curve analysis showed that patients with high expression of CD317 had a poor prognosis compared with patients with low expression. Furthermore, CD317 overexpression in HNSCC was correlated with immune checkpoint molecules PD-L1, B7-H3, and B7-H4 and tumor-associated macrophage markers (CD68 and CD163). We also observed that CD317 was overexpressed in immunocompetent mouse HNSCC tissue compared with normal tissue. Taken together, our findings demonstrate that CD317 overexpression indicates poor prognosis and is correlated with immune-related components in this patient cohort. CD317 may serve as a potential target for effective immunotherapy of HNSCC.

Production of Recombinant Rabies Virus Glycoprotein by Insect Cells in a Single-Use Fixed-Bed Bioreactor.

A single-use fixed-bed bioreactor (iCELLis nano) can be used for cultivating non adherent insect cells, which can be then recovered for scaling up or for harvesting a membrane-associated viral glycoprotein with high quality in terms of preserved protein structure and biological function. Here, we describe the procedures for establishing genetically modified Drosophila melanogaster Schneider 2 (S2) cell cultures in the iCELLis nano bioreactor and for quantifying by ELISA the recombinant rabies virus glycoprotein (rRVGP) synthesized. By using the described protocol of production, the following performance can be regularly achieved: 1.7±0.6 × 1E10 total cells; 2.4±0.8 × 1E7 cells/mL and 1.2±0.9μg of rRVGP/1E7 cells; 1.5±0.8mg of total rRVGP.

Mucin 1 protects against severe Streptococcus pneumoniae infection

ABSTRACT Streptococcus pneumoniae is a bacterial pathogen that commonly resides in the human nasopharynx, typically without causing any disease. However, in some cases these bacteria migrate from the nasopharynx to other sites of the body such as the lungs and bloodstream causing pneumonia and sepsis, respectively. This study used a mouse model of infection to investigate the potential role of Mucin 1 (MUC1), a cell membrane-associated glycoprotein known for playing a key barrier role at mucosal surfaces, in regulating this process. Wildtype (WT) and MUC1-deficient (Muc1−/−) mice were infected intranasally with an invasive strain of S. pneumoniae and bacterial loads in the nasopharynx, lungs, and blood were analyzed. Lungs were graded histologically for inflammation and cytokine profiles in the lungs analyzed by ELISA. While there was no difference in pneumococcal colonization of the nasopharynx between WT and Muc1−/− mice, infected Muc1−/− mice showed high pneumococcal loads in their lungs 16 hours post-infection, as well as bacteremia. In contrast, infected WT mice cleared the pneumococci from their lungs and remained asymptomatic. Infection in Muc1−/− mice was associated with an elevation in lung inflammation, with cellular recruitment especially of monocytes/macrophages. While MUC1-deficiency has been shown to increase phagocytosis of Pseudomonas aeruginosa, macrophages from Muc1−/− mice exhibited a reduced capacity to phagocytose S. pneumoniae indicating diverse and bacterial-specific effects. In conclusion, these findings indicate that MUC1 plays an important role in protection against severe pneumococcal disease, potentially mediated by facilitating macrophage phagocytosis.

Plasmablast Response to Primary Rhesus Cytomegalovirus (CMV) Infection in a Monkey Model of Congenital CMV Transmission.

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide and the leading infectious cause of neurologic deficits and hearing loss in newborns. Development of a maternal HCMV vaccine to prevent vertical virus transmission is a high priority, yet protective maternal immune responses following acute infection are poorly understood. To characterize the maternal humoral immune response to primary CMV infection, we investigated the plasmablast and early antibody repertoire using a nonhuman primate model with two acutely rhesus CMV (RhCMV)-infected animals-a CD4+ T cell-depleted dam that experienced fetal loss shortly after vertical RhCMV transmission and an immunocompetent dam that did not transmit RhCMV to her infant. Compared to the CD4+ T cell-depleted dam that experienced fetal loss, the immunocompetent, nontransmitting dam had a more rapid and robust plasmablast response that produced a high proportion of RhCMV-reactive antibodies, including the first identified monoclonal antibody specific for soluble and membrane-associated RhCMV envelope glycoprotein B (gB). Additionally, we noted that plasmablast RhCMV-specific antibodies had variable gene usage and maturation similar to those observed in a monkey chronically coinfected with simian immunodeficiency virus (SIV) and RhCMV. This study reveals characteristics of the early maternal RhCMV-specific humoral immune responses to primary RhCMV infection in rhesus monkeys and may contribute to a future understanding of what antibody responses should be targeted by a vaccine to eliminate congenital HCMV transmission. Furthermore, the identification of an RhCMV gB-specific monoclonal antibody underscores the possibility of modeling future HCMV vaccine strategies in this nonhuman primate model.

Membrane-Associated Kaposi Sarcoma-Associated Herpesvirus Glycoprotein B Promotes Cell Adhesion and Inhibits Migration of Cells via Upregulating IL-1β and TNF-α

Objectives: Kaposi sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is expressed on the viral envelope as well as on the cytoplasmic membrane of infected cells. In the current study, we aimed to decipher the impact of membrane-associated gB on adhesion and migration of cells via modulating the expression of cytokines. Methods: A combination of polymerase chain reaction array, cell adhesion assay, and wound-healing migration assay was conducted to study the influence of the gB-induced cytokines on cell adhesion and migration. Results: Membrane-associated gB was demonstrated to significantly upregulate the expression of IL-1β and TNF-α. Elevated levels of these cytokines were observed in conditioned medium (CM) collected from gB-expressing cells (gB-CM) compared to CM collected from untransfected cells or cells transfected with empty vector. KSHV gB-induced IL-1β and TNF-α play a role in the ability of gB-CM to mediate cell adhesion while inhibiting migration. Conclusion: Our results provide novel evidence that demonstrates full-length gB expressed on cell membrane to mediate adhesion and inhibit migration of cells not only by autocrine mechanism mediated by RGD-based interactions [Hussein et al.: BMC Cancer 2016; 16: 148], but also by paracrine mechanism mediated by gB-induced IL-1β and TNF-α.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Glycosylation is crucial for a proper catalytic site organization in human glucocerebrosidase.

Gaucher disease, an autosomal recessive disorder, is caused by a deficiency of glucocerebrosidase (GCase) enzyme, a peripheral membrane-associated glycoprotein that hydrolyses glucosylceramide in lysosomes. Glycosylation is essential for the development of a catalytically active enzyme, specifically in the first site, located at Asn19. However, both the molecular basis of the relevance of N-glycosylation over GCase activity and the effects of glycosylation over its structure and dynamics are still not fully understood. Thus, the present work evaluated GCase enzyme in increasing glycosylation content using triplicate unbiased molecular dynamics simulations. Accordingly, the N-linked glycan chains caused local conformational stabilization effects over the protein, as well as in regions flanking the enzyme catalytic dyad. In the case of the Asn19-linked glycan, it also occurred around region 438-444, where one of the most prevalent GCase mutations is found. Markedly, an increasing catalytic dyad organization was related to increasing glycosylation contents, offering the first atomic-level explanation for the experimental observation that GCase activity is controlled by glycosylation, especially at Asn19.