Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9.

CD46, a transmembrane protein known for protecting cells from complement­mediated damage, is frequently dysregulated in various types of cancer. Its overexpression in bladder cancers safeguards the cancer cells against both complement and antibody­mediated cytotoxicity. The present study explored a new role of CD46 in facilitating cancer cell invasion and metastasis, examining its regulatory effect on matrix metalloproteases (MMPs) and their effect on the metastatic capability of bladder cancer cells. Specifically, CD46 alteration positively influenced MMP9 expression, but not MMP2, in several bladder cancer cell lines. Furthermore, CD46 overexpression triggered phosphorylation of p38 MAPK and protein kinase B (AKT), leading to enhanced activator protein 1 (AP­1) activity via c­Jun upregulation. The inhibition of p38 or AKT pathways attenuated the CD46­induced MMP9 and AP­1 upregulation, indicating that the promotion of MMP9 by CD46 involved activating both p38 MAPK and AKT. Functionally, the upregulation of MMP9 by CD46 translated to increased migratory and invasive capabilities of bladder cancer cells, as well as enhanced in vivo metastasis. Overall, the present study revealed a novel role for CD46 as a metastasis promoter through MMP9 activation in bladder cancers and highlighted the regulatory mechanism of CD46­mediated MMP9 promotion via p38 MAPK and AKT activation.

Toward the understanding of DSG2 and CD46 interaction with HAdV-11 fiber, a super-complex analysis.

IMPORTANCE: The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there's still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.

The CD46 ectodomain participates in human cytomegalovirus infection of epithelial cells.

Human cytomegalovirus (HCMV) primary infections are typically asymptomatic in healthy individuals yet can cause increased morbidity and mortality in organ transplant recipients, AIDS patients, neonates, and the elderly. The successful, widespread dissemination of this virus among the population can be attributed in part to its wide cellular tropism and ability to establish life-long latency. HCMV infection is a multi-step process that requires numerous cellular and viral factors. The viral envelope consists of envelope protein complexes that interact with cellular factors; such interactions dictate virus recognition and attachment to different cell types, followed by fusion either at the cell membrane or within an endocytic vesicle. Several HCMV entry factors, including neuropilin-2 (Nrp2), THBD, CD147, OR14I1, and CD46, are characterized as participating in HCMV pentamer-specific entry of non-fibroblast cells such as epithelial, trophoblast, and endothelial cells, respectively. This study focuses on characterizing the structural elements of CD46 that impact HCMV infection. Infectivity studies of wild-type and CD46 knockout epithelial cells demonstrated that levels of CD46 expressed on the cell surface were directly related to HCMV infectivity. Overexpression of CD46 isomers BC1, BC2, and C2 enhanced infection. Further, CD46 knockout epithelial cells expressing CD46 deletion and chimeric molecules identified that the intact ectodomain was critical for rescue of HCMV infection in CD46 knockout cells. Collectively, these data support a model that the extracellular domain of CD46 participates in HCMV infection due to its surface expression.

Knockdown of membrane-bound complement regulatory proteins suppresses colon cancer growth in mice through inducing tumor cell apoptosis.

CD46, CD55 and CD59 are membrane-bound complement regulatory proteins (mCRPs) and highly expressed in many tumor tissues. Our analysis by RNA sequencing and qRT-PCR revealed that the expression of mCRPs was significantly elevated in cancer tissues of 15 patients with colon cancer. To further investigate the role of mCRPs in the development of colon cancer, we suppressed the expression of mCRPs by CD46-shRNA, CD55-shRNA and CD59-shRNA in colon cancer cell lines, SW620 and HT-29 cells. The results indicated that CD46-shRNA, CD55-shRNA and CD59-shRNA effectively reduced the expression of mCRPs, accompanied with the increased LDH release and the percentage of Annexin V + 7-AAD- early phase of apoptotic cells. The similar cytotoxic effects were also observed in the cells treated with CD46 neutralizing antibody (aCD46), associated with the increased C5b-9 deposition, cleaved caspase-3 and Bax expression in the treated cells. The cytotoxic effects by mCRPs knock-down were potentiated in the cells co-treated with doxorubicin (Dox). In addition, STAT3, STAT6, and p38 MAPK inhibitors, including C188-9, AS1517499 and SB203580 effectively reduced the expression of CD46 in the treated colon cells, associated with increased cell apoptosis and LDH release. Further study with mouse model revealed that mCRPs knockdown by mCRPs-shRNA significantly reduced colon cancer growth, associated with increased expression of Bax, cleaved caspase-3 and C5b-9 deposition, but reduced expression of Bcl-2, IL-6 and IL-1beta in tumor tissues of nude mice transplanted with SW620 cells. Thereby, mCRPs expression in human colon cancer cells were upregulated by STAT3/STAT6/p38 MAPK signaling and mCRPs knockdown reduced colon cancer growth in mice through inducing tumor cell apoptosis.

miR-132-3p regulates antibody-mediated complement-dependent cytotoxicity in colon cancer cells by directly targeting CD55.

The overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells helps them survive complement attacks by suppressing antibody-mediated complement-dependent cytotoxicity (CDC). Consequently, mCRP overexpression limits monoclonal antibody drug immune efficacy. CD55, an mCRP, plays an important role in inhibiting antibody-mediated CDC. However, the mechanisms regulating CD55 expression in tumour cells remain unclear. Here, the aim was to explore CD55-targeting miRNAs. We previously constructed an in vitro model comprising cancer cell lines expressing α-gal and serum containing natural antibodies against α-gal and complement. This was used to simulate antibody-mediated CDC in colon cancer cells. We screened microRNAs that directly target CD55 using LoVo and Ls-174T colon cell lines, which express CD55 at low and high levels, respectively. miR-132-3p expression was dramatically lower in Ls-174T cells than in LoVo cells. miR-132-3p overexpression or inhibition transcriptionally regulated CD55 expression by specifically targeting its mRNA 3'-untranslated regions. Further, miR-132-3p modulation regulated colon cancer cell sensitivity to antibody-mediated CDC through C5a release and C5b-9 deposition. Moreover, miR-132-3p expression was significantly reduced, whereas CD55 expression was increased, in colon cancer tissues compared to levels in adjacent normal tissues. CD55 protein levels were negatively correlated with miR-132-3p expression in colon cancer tissues. Our results indicate that miR-132-3p regulates colon cancer cell sensitivity to antibody-mediated CDC by directly targeting CD55. In addition, incubating the LoVo human tumour cell line, stably transfected with the xenoantigen α-gal, with human serum containing natural antibodies comprises a stable and cheap in vitro model to explore the mechanisms underlying antibody-mediated CDC.

[Silencing CD46 and DSG2 in host A549 cells inhibits entry of human adenovirus type 3 and type 7 and reduces interleukin-8 release].

OBJECTIVE: To investigate the effect of silencing CD46 and desmoglein 2 (DSG2) in host A549 cells on the entry of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and host cell secretion of inflammatory cytokines. METHODS: RNA interference technique was use to silence the expression of CD46 or DSG2 in human epithelial alveolar A549 cells as the host cells of HAdV-3 or HAdV-7. The binding of the viruses with CD46 and DSG2 were observed with immunofluorescence staining at 0.5 and 1 h after viral infection. The viral load in the host cells was determined with qRT-PCR, and IL-8 secretion level was measured using ELISA. RESULTS: In infected A549 cells, immunofluorescent staining revealed colocalization of HAdV-3 and HAdV-37 with their receptors CD46 and DSG2 at 0.5 h and 2 h after infection, and the copy number of the viruses increased progressively after the infection in a time-dependent manner. In A549 cells with CD46 silencing, the virus titers were significantly lower at 2, 6, 12 and 24 h postinfection in comparison with the cells without gene silencing; the virus titers were also significantly decreased in the cells with DSG2 silencing. The secretion level of IL-8 increased significantly in A549 cells without siRNA transfection following infection with HAdV-3 and HAdV-7 (P < 0.0001), but decreased significantly in cells with CD46 and DSG2 silencing (P < 0.0001). CONCLUSION: HAdV-3 and HAdV-7 enter host cells by binding to their receptors CD46 and DSG2, and virus titer and cytokines release increase with infection time. Silencing CD46 and DSG2 can inhibit virus entry and cytokine IL-8 production in host cells.

Silencing EGFR-upregulated expression of CD55 and CD59 activates the complement system and sensitizes lung cancer to checkpoint blockade.

The complement system is a critical immune component, yet its role in tumor immune evasion and CD8+ T cell activation is not clearly defined. Here, we demonstrate that epidermal growth factor receptor (EGFR)/Wnt signaling induces ß-catenin-mediated long noncoding RNA (lncRNA) LINC00973 expression to sponge CD55-targeting miR-216b and CD59-targeting miR-150. The consequently upregulated CD55/CD59 expression suppresses the complement system and cytokine secretion required for CD8+ T cell activation. CD55/CD59-neutralizing antibody treatment or mutation of the LINC00973 promoter activates the complement and CD8+ T cells, inhibiting tumor growth. Importantly, combined anti-CD55/CD59 and anti-programmed death 1 (anti-PD-1) antibody treatments elicit a synergistic tumor-inhibiting effect. In addition, CD55/CD59 levels are inversely correlated with infiltration of M1 macrophages and CD8+ T cells in human lung cancer specimens and predict patient outcome. These findings underscore the critical role of EGFR/Wnt/ß-catenin-upregulated CD55/CD59 expression in inhibiting the complement and CD8+ T cell activation for tumor immune evasion and immune checkpoint blockade resistance and identify a potential combination therapy to overcome these effects.

[CD36 gene deletion reduces muscle insulin sensitivity in mice by up-regulating PTP1B expression].

OBJECTIVE: To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. METHODS: Wild-type (WT) mice and systemic CD36 knockout (CD36-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. RESULTS: CD36-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36-/- mice (P>0.05). In the muscle tissue of CD36-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36-/- mice. CONCLUSION: CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.

Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26).

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV-1. However, its primary receptor portfolio remains controversial, potentially including sialic acid, coxsackie and adenovirus receptor (CAR), integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned on the basis of the inability to cocrystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domain CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild-type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (i.m.) injection but not after intranasal (i.n.) administration. Ad26 transduction was 10-fold lower than Ad5 transduction after intratumoral (i.t.) injection of CD46-expressing tumors. Ad26 transduction of liver was 1,000-fold lower than that ofAd5 after intravenous (i.v.) injection. These data demonstrate the use of CD46 by Ad26 in certain situations but also show that the receptor has little consequence by other routes of administration. Finally, i.v. injection of high doses of Ad26 into CD46 mice induced release of liver enzymes into the bloodstream and reduced white blood cell counts but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during coronavirus disease 2019 (COVID-19) vaccination with this serotype of adenovirus. IMPORTANCE The human species D Ad26 is being investigated as a low-seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection and its role in virus biology, vaccine efficacy, and, importantly, vaccine safety.

New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source.

Transplantation of xenogenic porcine chondrocytes could represent a future strategy for the treatment of human articular cartilage defects. Major obstacles are humoral and cellular rejection processes triggered by xenogenic epitopes like α-1,3-Gal and Neu5Gc. Besides knockout (KO) of genes responsible for the biosynthesis of respective epitopes (GGTA1 and CMAH), transgenic expression of human complement inhibitors and anti-apoptotic as well as anti-inflammatory factors (CD46, CD55, CD59, TNFAIP3 and HMOX1) could synergistically prevent hyperacute xenograft rejection. Therefore, chondrocytes from different strains of single- or multi-genetically modified pigs were characterized concerning their protection from xenogeneic complement activation. Articular chondrocytes were isolated from the knee joints of WT, GalTKO, GalT/CMAH-KO, human CD59/CD55//CD46/TNFAIP3/HMOX1-transgenic (TG), GalTKO/TG and GalT/CMAHKO/TG pigs. The tissue-specific effectiveness of the genetic modifications was tested on gene, protein and epitope expression level or by functional assays. After exposure to 20% and 40% normal human serum (NHS), deposition of C3b/iC3b/C3c and formation of the terminal complement complex (TCC, C5b-9) was quantified by specific cell ELISAs, and generation of the anaphylatoxin C5a by ELISA. Chondrocyte lysis was analyzed by Trypan Blue Exclusion Assay. In all respective KO variants, the absence of α -1,3-Gal and Neu5Gc epitope was verified by FACS analysis. In chondrocytes derived from TG animals, expression of CD55 and CD59 could be confirmed on gene and protein level, TNFAIP3 on gene expression level as well as by functional assays and CD46 only on gene expression level whereas transgenic HMOX1 expression was not evident. Complement activation in the presence of NHS indicated mainly effective although incomplete protection against C3b/iC3b/C3c deposition, C5a-generation and C5b-9 formation being lowest in single GalTKO. Chondrocyte viability under exposure to NHS was significantly improved even by single GalTKO and completely preserved by all other variants including TG chondrocytes without KO of xenoepitopes.

Membrane cofactor protein (MCP; CD46): deficiency states and pathogen connections.

Membrane cofactor protein (MCP; CD46), a ubiquitously expressed complement regulatory protein, serves as a cofactor for serine protease factor I to cleave and inactivate C3b and C4b deposited on host cells. However, CD46 also plays roles in human reproduction, autophagy, modulating T cell activation and effector functions and is a member of the newly identified intracellular complement system (complosome). CD46 also is a receptor for 11 pathogens ('pathogen magnet'). While CD46 deficiencies contribute to inflammatory disorders, its overexpression in cancers and role as a receptor for some adenoviruses has led to its targeting by oncolytic agents and adenoviral-based therapeutic vectors, including coronavirus disease of 2019 (COVID-19) vaccines. This review focuses on recent advances in identifying disease-causing CD46 variants and its pathogen connections.

A genetic variant in the promoter of CD46 is associated with the risk and prognosis of hepatocellular carcinoma.

CD46 (also known as membrane cofactor protein), which is a member of the membrane-bound complement regulatory protein family, has been reported to cause cancer cells to escape complement-dependent cytotoxicity. However, the association between CD46 polymorphisms and the risk of hepatocellular carcinoma (HCC) has not been investigated. This two-stage association study was conducted to assess the relationship between the tagging single nucleotide polymorphisms (tagSNPs) of CD46 and HCC risk and prognosis. A series of functional analyses were performed to study the underlying mechanisms. Among the eight tagSNPs, rs2796267 (P = .003) and rs2796268 (P = .011) were found to modify HCC risk in the discovery set. Only rs2796267 (P < .0001) was confirmed to be associated with HCC susceptibility in the validation set. Compared with the wild-type AA genotype, the GG genotype significantly increased the HCC risk (adjusted odds ratio [OR] = 2.03; 95% confidence interval [CI], 1.34-3.08; P = .001). Moreover, subgroups analysis suggested a positive correlation among male and younger patients, especially among drinkers, smokers, and hepatitis B surface antigen-positive individuals. In functional analyses, we found that the rs2796267 G allele in the promoter region of CD46 could increase the expression of CD46 by affecting the binding affinity of STAT5a. Furthermore, Cox regression analysis revealed that the rs2796267 AG/GG genotype was significantly associated with worse prognosis of resected patients with HCC (hazard ratio = 2.27; 95% CI, 1.27-4.05; P = .006). These results suggest that the CD46 rs2796267 polymorphism may contribute to susceptibility and prognosis of HCC by altering promoter activity.

Curative in vivo hematopoietic stem cell gene therapy of murine thalassemia using large regulatory elements.

Recently, we demonstrated that hematopoietic stem/progenitor cell (HSPC) mobilization followed by intravenous injection of integrating, helper-dependent adenovirus HDAd5/35++ vectors resulted in efficient transduction of long-term repopulating cells and disease amelioration in mouse models after in vivo selection of transduced HSPCs. Acute innate toxicity associated with HDAd5/35++ injection was controlled by appropriate prophylaxis, making this approach feasible for clinical translation. Our ultimate goal is to use this technically simple in vivo HSPC transduction approach for gene therapy of thalassemia major or sickle cell disease. A cure of these diseases requires high expression levels of the therapeutic protein (γ- or ß-globin), which is difficult to achieve with lentivirus vectors because of their genome size limitation not allowing larger regulatory elements to be accommodated. Here, we capitalized on the 35 kb insert capacity of HDAd5/35++ vectors to demonstrate that transcriptional regulatory regions of the ß-globin locus with a total length of 29 kb can efficiently be transferred into HSPCs. The in vivo HSPC transduction resulted in stable γ-globin levels in erythroid cells that conferred a complete cure of murine thalassemia intermedia. Notably, this was achieved with a minimal in vivo HSPC selection regimen.

A CRISPR/Cas9 Generated Bovine CD46-knockout Cell Line-A Tool to Elucidate the Adaptability of Bovine Viral Diarrhea Viruses (BVDV).

Bovine viral diarrhea virus (BVDV) entry into a host cell is mediated by the interaction of the viral glycoprotein E2 with the cellular transmembrane CD46 receptor. In this study, we generated a stable Madin-Darby Bovine Kidney (MDBK) CD46-knockout cell line to study the ability of different pestivirus A and B species (BVDV-1 and -2) to escape CD46-dependent cell entry. Four different BVDV-1/2 isolates showed a clearly reduced infection rate after inoculation of the knockout cells. However, after further passaging starting from the remaining virus foci on the knockout cell line, all tested virus isolates were able to escape CD46-dependency and grew despite the lack of the entry receptor. Whole-genome sequencing of the escape-isolates suggests that the genetic basis for the observed shift in infectivity is an amino acid substitution of an uncharged (glycine/asparagine) for a charged amino acid (arginine/lysine) at position 479 in the ERNS in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the ERNS resulted in a loss of ERNS dimerization that is likely to influence viral cell-to-cell spread. In general, the CD46-knockout cell line is a useful tool to analyze the role of CD46 for pestivirus replication and the virus-receptor interaction.

Bovine pestivirus is a new alternative virus for multiple myeloma oncolytic virotherapy.

BACKGROUND: The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients' antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. METHODS: We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. RESULTS: Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. CONCLUSIONS: Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

Human Desmoglein-2 and Human CD46 Mediate Human Adenovirus Type 55 Infection, but Human Desmoglein-2 Plays the Major Roles.

Human adenovirus type 55 (HAdV55) represents an emerging respiratory pathogen and causes severe pneumonia with high fatality in humans. The cellular receptors, which are essential for understanding the infection and pathogenesis of HAdV55, remain unclear. In this study, we found that HAdV55 binding and infection were sharply reduced by disrupting the interaction of viral fiber protein with human desmoglein-2 (hDSG2) but only slightly reduced by disrupting the interaction of viral fiber protein with human CD46 (hCD46). Loss-of-function studies using soluble receptors, blocking antibodies, RNA interference, and gene knockout demonstrated that hDSG2 predominantly mediated HAdV55 infection. Nonpermissive rodent cells became susceptible to HAdV55 infection when hDSG2 or hCD46 was expressed, but hDSG2 mediated more efficient HAd55 infection than hCD46. We generated two transgenic mouse lines that constitutively express either hDSG2 or hCD46. Although nontransgenic mice were resistant to HAdV55 infection, infection with HAdV55 was significantly increased in hDSG2+/+ mice but was much less increased in hCD46+/+ mice. Our findings demonstrate that both hDSG2 and hCD46 are able to mediate HAdV55 infection but hDSG2 plays the major roles. The hDSG2 transgenic mouse can be used as a rodent model for evaluation of HAdV55 vaccine and therapeutics.IMPORTANCE Human adenovirus type 55 (HAdV55) has recently emerged as a highly virulent respiratory pathogen and has been linked to severe and even fatal pneumonia in immunocompetent adults. However, the cellular receptors mediating the entry of HAdV55 into host cells remain unclear, which hinders the establishment of HAdV55-infected animal models and the development of antiviral approaches. In this study, we demonstrated that human desmoglein-2 (hDSG2) plays the major roles during HAdV55 infection. Human CD46 (hCD46) could also mediate the infection of HAdV55, but the efficiency was much lower than for hDSG2. We generated two transgenic mouse lines that express either hDSG2 or hCD46, both of which enabled HAd55 infection in otherwise nontransgenic mice. hDSG2 transgenic mice enabled more efficient HAdV55 infection than hCD46 transgenic mice. Our study adds to our understanding of HAdV55 infection and provides an animal model for evaluating HAdV55 vaccines and therapeutics.

Isolated thrombotic microangiopathy of the small intestine in a patient with atypical hemolytic uremic syndrome - a case report.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by systemic thrombotic microangiopathy (TMA) reflected by hemolysis, anemia, thrombocytopenia and systemic organ injury. The optimal management of aHUS-patients when undergoing kidney transplantation to prevent recurrence in the allograft is eculizumab, an approved recombinant antibody targeting human complement component C5. CASE PRESENTATION: A 39 year-old woman presented with severe abdominal pain, diarrhea and emesis for 3 days. In her past medical history she had experienced an episode of aHUS leading to end stage renal disease (ESRD) in 2007 and a genetic workup revealed a heterozygous mutation in the membrane cofactor protein gene. In 2014 she underwent cadaveric kidney transplantation. Four years later she had to go back on hemodialysis due to allograft failure following a severe systemic cytomegalovirus infection resulting in transplant failure. At presentation she still received calcineurin-inhibitor therapy and reported subfebrile temperatures and pain projecting over the transplant prior to the current symptoms. A contrast enhanced CT-scan of the abdomen revealed inflammatory wall thickening of the small intestine. Diagnostic endoscopy discovered fresh blood in the small intestine without a clear source of bleeding. Histopathology of the small intestine biopsies showed severe thrombotic microangiopathy. Of note, the patient persistently had no signs of systemic hemolysis. Since the TMA of the small intestine was most likely due to aHUS, eculizumab treatment was initiated which abolished the symptoms. CONCLUSION: Here we report a patient with thrombotic microangiopathy with predominant manifestation in a single organ, the small intestine, due to aHUS with absence of systemic signs and symptoms. aHUS patients usually require a secondary trigger for the disease to manifest. In this case, the trigger may be attributed to the dysfunctional renal transplant, which was subsequently explanted. Histology of the explanted kidney showed severe inflammation due to purulent nephritis and signs of cellular rejection. After nephrectomy, we continued eculizumab therapy until the patient completely recovered. No signs of TMA recurred after discontinuation of eculizumab, further supporting the concept of the renal transplant as the main trigger of TMA of the small intestine in our patient.

Chimeric oncolytic Ad5/3 virus replicates and lyses ovarian cancer cells through desmoglein-2 cell entry receptor.

Despite new therapies, the estimated 229 875 women living with ovarian cancer have a 5-year survival rate of 47.6%. This cavity-localized cancer lends itself to local administration of modalities, such as the oncolytic adenovirus (Ad) Ad5/3-D24-granulocyte-macrophage colony-stimulating factor virus (ONCOS-102). Its repeated administration to a patient with chemotherapy-refractory ovarian cancer induced CD8+ antitumor immune responses with the overall survival reaching 40 months. Here we probe the dominant receptor used by ONCOS-102 in four established epithelial ovarian cancer cell lines. Ad3 can use the desmoglein-2 (DSG2) and CD46 receptors on susceptible cells. DSG2 was nearly absent in A2780 cells but was expressed in more than 90% of OAW42, OVCAR3, and OV-90 cells. After 96 hours, ONCOS-102 treatment showed significant oncolytic activity (≧50%) in OAW42, OVCAR3, and OV-90 cells, but minimal activity in A2780 cells, suggesting DSG2 as the dominant receptor for ONCOS-102. Furthermore, retrospective analyses of phase I clinical trial of ONCOS-102 treatment of 12 patients with varied tumors indicated a correlation between viral genomes in blood and DSG2 RNA expression. These data support the role of DSG2 expression on cancer cells in virus infectivity and the continued development of ONCOS-102 for ovarian cancer treatment.

Suicide gene­armed measles vaccine virus for the treatment of AML.

Virotherapy comprises a novel therapeutic approach to selectively eliminate cancer cells. Preclinical, as well as clinical data have demonstrated the efficacy of tumor­selective (oncolytic) viruses in hematological malignancies. In this study, we infected AML cell lines and primary AML cells from patients with measles vaccine virus either expressing GFP or armed with super cytosine deaminase, which converts the prodrug, 5­fluorocytosine, into the chemotherapeutic compound, 5­fluorouracil. Target cell density of the measles entry receptor, CD46, infection rates of targeted leukemic cells, tumor cell viability, and apoptotic rates were determined. We found that measles vaccine virus infected the leukemic blasts and profoundly diminished the number and viability of leukemic cells via the induction of apoptosis. The conversion of 5­fluorocytosine to 5­fluorouracil exerted a potent additive tumoricidal effect. This was also observed in cases when leukemic cells displayed only moderate susceptibility to the oncolytic virus and hence direct oncolysis. Taken together, in this study, we provide a first characterization of the combinatorial use of measles vaccine virus and 5­fluorouracil for treatment of AML. Our approach to site­specifically produce the active drug and combine this agent with the direct lytic effect of virotherapy may overcome present limitations and constitutes a feasible method with which to introduce 5­fluorouracil in the treatment of AML.

Life-supporting Kidney Xenotransplantation From Genetically Engineered Pigs in Baboons: A Comparison of Two Immunosuppressive Regimens.

BACKGROUND: The aims of this study were to evaluate the efficacy of US Food and Drug Administration-approved drugs in genetically engineered pig-to-baboon kidney xenotransplantation and compare the results with those using an anti-CD40 monoclonal antibody (mAb)-based regimen. METHODS: Ten life-supporting kidney transplants were carried out in baboons using α1,3-galactosyltransferase gene-knockout/CD46 pigs with various other genetic manipulations aimed at controlling coagulation dysregulation. Eight transplants resulted in informative data. Immunosuppressive therapy consisted of induction with antithymocyte globulin and anti-CD20mAb, and maintenance based on either (1) CTLA4-Ig and/or tacrolimus (+rapamycin or mycophenolate mofetil) (GroupA [US Food and Drug Administration-approved regimens], n = 4) or (2) anti-CD40mAb + rapamycin (GroupB, n = 4). All baboons received corticosteroids, interleukin-6R blockade, and tumor necrosis factor-α blockade. Baboons were followed by clinical and laboratory monitoring of kidney function, coagulation, and immune parameters. At euthanasia, morphological and immunohistochemical studies were performed on the kidney grafts. RESULTS: The median survival in GroupB was 186 days (range 90-260), which was significantly longer than in GroupA; median 14 days (range 12-32) (P < 0.01). Only GroupA baboons developed consumptive coagulopathy and the histopathological features of thrombotic microangiopathic glomerulopathy and interstitial arterial vasculitis. CONCLUSIONS: Recognizing that the pig donors in each group differed in some genetic modifications, these data indicate that maintenance immunosuppression including anti-CD40mAb may be important to prevent pig kidney graft failure.