Facilitating cholangiocarcinoma inhibition by targeting CD47.

Immune evasion is one of the mechanisms by which cancer cells acquire immunity during cancer development and progression. One of these is the increased expression of cluster of differentiation 47 (CD47), a transmembrane glycoprotein that protects cells from phagocytic elimination. The interaction between CD47 and signal regulatory protein alpha (SIRPα) on macrophages alleviates the phagocytic signal. The present group previously reported high CD47 expression in cholangiocarcinoma (CCA), a major health problem in Thailand and East Asia, and that blocking CD47 using anti-CD47 antibodies promoted the removal of CCA. However, the mechanism through which CD47 inhibition attenuates CCA growth remains unclear. This study explored the clinical significance of targeting CD47 in CCA. Expression levels of CD47 and the macrophage marker CD68 were determined in CCA tissues by immunohistochemistry and correlated with clinical parameters. The role of CD47 in CCA cells was established using CD47-deficient KKU-213A CCA clones in vitro and in vivo. The results showed that CD47 was highly expressed in CCA tissues and significantly correlated with lymph node metastasis (P = 0.038). Moderate-to-dense CD68-positive infiltrating cells in CCA tissues were significantly associated with shorter survival of patients (P = 0.019) and were an independent prognostic factor of CCA patients as determined by the Cox proportional hazard model (hazard ratio, 2.040; 95 % confidence interval, 1.109-3.752; P = 0.022). Three CD47-deficient KKU-213A clones (#19, #23, and #28) were generated. The elimination of CD47 did not affect cell proliferation but increased monocyte-derived macrophage-mediated phagocytosis in vitro. Decreased tumor weights and volumes were observed in mice injected with CD47-deficient CCA clones. This revealed a significant role for CD47 in CCA, with a focus on protecting cancer cells from macrophage phagocytosis.

Rare finding of mucorales sporangia and chlamydospores in tissue.

Most elements of filamentous fungi seen in human tissue by pathologists are hyphae, and encountering other elements may interfere with diagnosis. Sporangia and chlamydospores are such elements that have been described in only a few case reports. We present an autopsy case with the extremely rare coexistence of Mucorales sporangia and chlamydospores in the lung. These fungal elements must be recognized and identified accurately because they can easily be mistaken for other fungi, microorganisms, or degenerated tissue structures.

Carcinogenesis caused by transcription-coupled DNA damage through GANP and other components of the TREX-2 complex.

Perturbation of genes is important for somatic hypermutation to increase antibody affinity during B-cell immunity; however, it may also promote carcinogenesis. Previous studies have revealed that transcription is an important process that can induce DNA damage and genomic instability. Transciption-export-2 (TREX-2) complex, which regulates messenger RNA (mRNA) nuclear export, has been studied in the budding yeast Saccharomyces cerevisiae; however, recent studies have started investigating the molecular function of the mammalian TREX-2 complex. The central molecule in the TREX-2 complex, that is, germinal center-associated nuclear protein (GANP), is closely associated with antibody affinity maturation as well as cancer etiology. In this review, we focus on carcinogenesis, lymphomagenesis, and teratomagenesis caused by transcription-coupled DNA damage through GANP and other components of the TREX-2 complex. We review the basic machinery of mRNA nuclear export and transcription-coupled DNA damage. We then briefly describe the immunological relationship between GANP and the affinity maturation of antibodies. Finally, we illustrate that the aberrant expression of the components of the TREX-2 complex, especially GANP, is associated with the etiology of various solid tumors, lymphomas, and testicular teratoma. These components serve as reliable predictors of cancer prognosis and response to chemotherapy.

DNA Damage, DNA Repair, and Cancer: Second Edition.

Following our first Special Issue, we are pleased to present this Special Issue in the International Journal of Molecular Sciences, titled 'DNA Damage, DNA Repair, and Cancer: Second Edition' [...].

Testicular teratomagenesis from primordial germ cells with overexpression of germinal center-associated nuclear protein.

Testicular teratomas are the major histologic type of testicular germ cell tumors and their incidence continues to grow. Moreover, teratomas can develop from undifferentiated cells in induced pluripotent stem (iPS) cell transplantation therapy, seriously hampering the progress of regenerative medicine. Germinal center-associated nuclear protein (GANP) is thought to be important to the biogenetic control of primordial germ cells and is among the genes susceptible to testicular germ cell tumors. Thus, we analyzed the expression of GANP in human testicular postpubertal-type teratomas and established a novel mouse model to reveal the association between GANP and teratomagenesis. We analyzed 31 cases of human testicular postpubertal-type teratomas and, in all cases, GANP was overexpressed. The aberrant expression was also detected in germ cell neoplasia in situ accompanied by the teratoma. GANP expression was particularly high in the epithelia of the epidermis, cutaneous appendages, and trachea-like ciliated epithelium. To further clarify the association between GANP and teratomagenesis, we established a novel teratomagenesis mouse model (CAG-ganpTg mice). In the GANP-teratoma mice, GANP-overexpressing teratomas were more frequent at the testes and the middle portion of the uterus than has been seen in the previously established mouse models. In conclusion, GANP is overexpressed in testicular postpubertal-type teratomas and is an essential teratomagenic factor. We also found that CAG-ganpTg mice are useful mouse models of teratomagenesis that mimics human midline teratomas and that teratomas may originate from the overexpression of GANP in primordial germ cells.

An autopsy case report of fulminant myocarditis: Following mRNA COVID-19 vaccination.

There have been few case reports on fatal outcomes in patients with acute myocarditis after mRNA coronavirus disease 2019 (COVID-19) vaccination. In most cases of myocarditis after mRNA COVID-19 vaccination, the myocarditis is mild, and the prognosis is good. Here we report an autopsy case of fulminant myocarditis following mRNA COVID-19 vaccination. Learning objective: The global distribution of the mRNA coronavirus disease 2019 vaccine requires consideration of appropriate treatment for postvaccination myocarditis. Eosinophil-mediated immunological injury to cardiomyocytes can be involved in the cause of fulminant inflammation from the pathological findings of postvaccination myocarditis.

Histology of Cardiac Sarcoidosis with Novel Considerations Arranged upon a Pathologic Basis.

Sarcoidosis is a rare disease of isolated or diffuse granulomatous inflammation. Although any organs can be affected by sarcoidosis, cardiac sarcoidosis is a fatal disorder, and it is crucial to accurately diagnose it to prevent sudden death due to dysrhythmia. Although endomyocardial biopsy is invasive and has limited sensitivity for identifying granulomas, it is the only modality that yields a definitive diagnosis of cardiac sarcoidosis. It is imperative to develop novel pathological approaches for the precise diagnosis of cardiac sarcoidosis. Here, we aimed to discuss commonly used diagnostic criteria for cardiac sarcoidosis and to summarize useful and novel histopathologic criteria of cardiac sarcoidosis. While classical histologic observations including noncaseating granulomas and multinucleated giant cells (typically Langhans type) are the most important findings, others such as microgranulomas, CD68+ CD163- pro-inflammatory (M1) macrophage accumulation, CD4/CD8 T-cell ratio, Cutibacterium acnes components, lymphangiogenesis, confluent fibrosis, and fatty infiltration may help to improve the sensitivity of endomyocardial biopsy for detecting cardiac sarcoidosis. These novel histologic findings are based on the pathology of cardiac sarcoidosis. We also discussed the principal histologic differential diagnoses of cardiac sarcoidosis, such as tuberculosis myocarditis, fungal myocarditis, giant cell myocarditis, and dilated cardiomyopathy.

Increased chemosensitivity via BRCA2-independent DNA damage in DSS1- and PCID2-depleted breast carcinomas.

Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations.

The critical role of germinal center-associated nuclear protein in cell biology, immunohematology, and hematolymphoid oncogenesis.

Germinal center-associated nuclear protein (GANP) is a unique and multifunctional protein that plays a critical role in cell biology, neurodegenerative disorders, immunohematology, and oncogenesis. GANP is an orthologue of Saccharomyces Sac3, one of the components of the transcription export 2 (TREX-2) complex and a messenger RNA (mRNA) nuclear export factor. GANP is widely conserved in all mammals, including humans. Although GANP was originally discovered as a molecule upregulated in the germinal centers of secondary lymphoid follicles in peripheral lymphoid organs, it is expressed ubiquitously in many tissues. It serves numerous functions, including making up part of the mammalian TREX-2 complex; mRNA nuclear export via nuclear pores; prevention of R-loop formation, genomic instability, and hyper-recombination; and B-cell affinity maturation. In this review, we first overview the extensive analyses that have revealed the basic functions of GANP and its ancestor molecule Sac3, including mRNA nuclear export and regulation of R-loop formation. We then describe how aberrant expression of GANP is significantly associated with cancer development. Moreover, we discuss a crucial role for GANP in B-cell development, especially affinity maturation in germinal centers. Finally, we illustrate that overexpression of GANP in B cells leads to lymphomagenesis resembling Hodgkin lymphoma derived from germinal center B cells, and that GANP may be involved in transdifferentiation of B cells to macrophages, which strongly affects Hodgkin lymphomagenesis.

A Novel Cytological Model of B-Cell/Macrophage Biphenotypic Cell Hodgkin Lymphoma in Ganp-Transgenic Mice.

Hodgkin lymphoma (HL) is one of the most difficult neoplasms in terms of cytopathological research owing to the lack of established cytological murine models. Although HL is believed to be of lymphoid germinal center B-cell origin, HL cells exhibit unique biphenotypic characteristics of B cells and macrophages. B-cell/macrophage biphenotypic cells have also been identified in the spleen of Lyn-deficient mice. Moreover, Lyn-targeting germinal center-associated nuclear protein (GANP)-transgenic mice (Ig-ganpTg mice) spontaneously develop a lymphoid tumor. We aimed to investigate whether the lymphoid tumor developed in Ig-ganpTg mice exhibit biphenotypic characteristics of B cells/macrophages that correspond to human HL. Here, we demonstrated GANP overexpression in human HL cells and found that it may regulate transdifferentiation between B cells and macrophages. We also demonstrated that tumors were comparable with B-cell/macrophage biphenotypic Hodgkinoid lymphomas. The tumor cells expressed macrophage-related F4/80, CD68, and CD204 as well as cytoplasmic B220 and µ-/κ-chains; in addition, these cells exhibited phagocytic activity. These cells also expressed transcripts of CD30; c-fms; and the cytokines monocyte chemoattractant protein (MCP)-1, MCP-5, RANTES, tumor necrosis factor-α and thrombopoietin associated with macrophages as well as granulocyte/macrophage colony-stimulating factor, interleukin (IL)-4, IL-10, IL-12, and IL-13. Ig-ganpTg mice represent a novel cytological model for the study of cytopathological etiology and oncogenesis of HL.

Impact of germinal center-associated nuclear protein polymorphisms on breast cancer risk and prognosis in a Japanese population.

BACKGROUND: Germinal center-associated nuclear protein (GANP) is a phosphoprotein involved in mRNA export and regulation of DNA recombination. Although GANP expression in human breast cancer tissue is associated with breast cancer prognosis, the association between the genetic background of GANP and susceptibility and prognosis of breast cancer is unclear. METHODS: We selected 694 breast cancer cases and 1376 age- and menopausal status-matched non-cancer controls from the Hospitable-based Epidemiologic Research Program, conducted at Aichi Cancer Center between 2001 and 2005. We evaluated the impact of two polymorphisms at the GANP locus (rs2839178 and rs11702450) on the susceptibility and prognosis of breast cancer. Reference alleles were defined as the A allele for rs2839178 and G allele for rs11702450. RESULTS: The GG genotype of rs2839178 was statistically significantly associated with breast cancer risk (odds ratio [OR] 0.48, 95% confidence interval [CI] 0.30-0.76, P = 0.002). In prognostic analysis, compared to those with AA genotype of rs2839178, patients with AG or GG genotypes had longer disease-free survival (DFS) (hazard ratio [HR] 0.71, 95% CI 0.49-1.04 and HR 0.42, 95% CI 013-1.42, respectively, P for trend = 0.04). eQTL analysis indicated that association with rs2839178 can be explained by the effect of rs2839173 on expression of GANP/MCM3AP. CONCLUSIONS: The G allele of rs2839178 at the GANP locus was significantly associated with reduced breast cancer risk and longer DFS in breast cancer patients, showing a consistent direction in the association between susceptibility and clinical outcome. GANP is, therefore, important for the occurrence and progression of sporadic breast cancer.

Attenuation of CD47-SIRPα Signal in Cholangiocarcinoma Potentiates Tumor-Associated Macrophage-Mediated Phagocytosis and Suppresses Intrahepatic Metastasis.

The involvement of chronic inflammation in cholangiocarcinoma (CCA) progression is well established. Cluster of differentiation 47 (CD47) is mutually expressed in various cancers and serves as a protective signal for phagocytic elimination. CD47 signaling blockage is a recent treatment strategy; however, little is known regarding CD47 in CCA. Therefore, the potential use of CD47 targeting in CCA was focused. CD47 was highly expressed in CCA compared to hepatocellular carcinoma (HCC). Disturbance of CD47-signal regulatory protein-α (SIRPα) interaction by blocking antibodies promoted the macrophage phagocytosis. The therapeutic potential of anti-CD47 therapy was demonstrated in liver metastatic model; alleviation of cancer colonization together with dense macrophage infiltrations was observed. The usefulness of anti-CD47 was emphasized by its universal facilitating macrophage activities. Moreover, increased production of inflammatory cytokines, such as IL-6 and IL-10, in macrophage exposed to CCA-conditioned media suggested that CCA alters macrophages toward cancer promotion. Taken together, interfering of CD47-SIRPα interaction promotes macrophage phagocytosis in all macrophage subtypes and consequently suppresses CCA growth and metastasis. The unique overexpression of CD47 in CCA but not HCC offers an exceptional opportunity for a targeted therapy. CD47 is therefore a novel target for CCA treatment.

Classic Hodgkin lymphoma with osseous involvement mimicking Langerhans cell histiocytosis in a child.

Hodgkin lymphoma (HL) commonly presents superficial lymphadenopathy. In addition, HL cells can arise in various organs including the liver and spleen as an extranodal lymphoma. HL in bone is unusual at the initial diagnosis, although some cases show late-stage localization of lymphoma cells to bone. We report the rare case of a young patient with cranial bone classic HL, presumably originating from the skull without any involvement of lymph nodes. As the main clinical manifestation was only tumor mass in the skull without osteoscopic pain, the tentative diagnosis of Langerhans cell histiocytosis was histologically confirmed by an excisional biopsy. Before the final pathological diagnosis as classic HL, we noticed several small lesions in extranodal regions through systemic surveys, suggesting that the cranial lesion appeared antecedent to those lesions. This is a rare and instructive case of cranial bone HL for which a histological diagnosis has been meticulously made.

Immunodominant SARS Coronavirus Epitopes in Humans Elicited both Enhancing and Neutralizing Effects on Infection in Non-human Primates.

Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) and has the potential to threaten global public health and socioeconomic stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro and in non-human primates clouds the prospects for a safe vaccine. Using antibodies from SARS patients, we identified and characterized SARS-CoV B-cell peptide epitopes with disparate functions. In rhesus macaques, the spike glycoprotein peptides S471-503, S604-625, and S1164-1191 elicited antibodies that efficiently prevented infection in non-human primates. In contrast, peptide S597-603 induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (ESD) mechanism. This peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (S597-603 and S604-625) and a long-term human B-cell memory response with antisera from convalescent SARS patients. Thus, peptide-based vaccines against SARS-CoV could be engineered to avoid ADE via elimination of the S597-603 epitope. We provide herein an alternative strategy to prepare a safe and effective vaccine for ADE of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection.

GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development.

Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse-free survival, hazard ratio = 2.37, 95% confidence interval, 1.27-4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42-5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap-cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland-specific GANP-deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP-heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance.

JNK regulatory molecule G5PR induces IgG autoantibody-producing plasmablasts from peritoneal B1a cells.

Peritoneal B1a cells expressing CD5 and CD11b generate autoantibody-producing precursors in autoimmune-prone mice. Previous studies show reduced JNK signaling in peritoneal B1a cells of female New Zealand Black mice and an abnormal increase of protein phosphatase 2A subunit G5PR that regulates BCR-mediated JNK signaling as a cause of autoimmunity. To investigate the mechanism regulating B1a differentiation into autoantibody-secreting plasmablasts (PBs), we applied an in vitro culture system that supports long-term growth of germinal center (GC) B cells (iGB) with IL-4, CD40L, and BAFF. Compared with spleen B2 cells, B1a cells differentiated into GC-like B cells, but more markedly into PBs, and underwent class switching toward IgG1. During iGB culture, B1a cells expressed GC-associated aicda, g5pr, and bcl6, and markedly PB-associated prdm1, irf4, and xbp1. B1a-derived iGB cells from New Zealand Black × New Zealand White F1 mice highly differentiated into autoantibody-secreting PBs in vitro and localized to the GC area in vivo. In iGB culture, JNK inhibitor SP600125 augmented the differentiation of C57BL/6 B1a cells into PBs. Furthermore, B1a cells from G5PR transgenic mice markedly differentiated into IgM and IgG autoantibody-secreting PBs. In conclusion, JNK regulation is critical to suppress autoantibody-secreting PBs from peritoneal B1a cells.

Efficacy of anti-CD47 antibody-mediated phagocytosis with macrophages against primary effusion lymphoma.

BACKGROUND: Recently, the critical role of CD47 on the surface of resistant cancer cells has been proposed in their evasion of immunosurveillance. Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma that shows serous lymphomatous effusion in body cavities, especially in advanced acquired immunodeficiency syndrome (AIDS). PEL is resistant to conventional chemotherapy and has a poor prognosis. In this study, we evaluated the effect of anti-CD47 antibody (Ab) on PEL in vitro and in vivo. METHODS: Surface CD47 of PEL cell lines was examined by flow cytometry. Efficacy of knocking down CD47 or anti-CD47 Ab-mediated phagocytosis against PEL was evaluated using mouse peritoneal macrophages and human macrophages in vitro. Primary PEL cells were injected intraperitoneally into NOD/Rag-2/Jak3 double-deficient (NRJ) mice to establish a direct xenograft mouse model. RESULTS: Surface CD47 of PEL cell lines was highly expressed. Knocking down CD47 and anti-CD47 Ab promoted phagocytic activities of macrophages in a CD47 expression-dependent manner in vitro. Treatment with anti-CD47 Ab inhibited ascite formation and organ invasion completely in vivo compared with control IgG-treated mice. CONCLUSION: CD47 plays the pivotal role in the immune evasion of PEL cells in body cavities. Therapeutic antibody targeting of CD47 could be an effective therapy for PEL.

Pcid2 inactivates developmental genes in human and mouse embryonic stem cells to sustain their pluripotency by modulation of EID1 stability.

Self-renewal and differentiation are the hallmarks of embryonic stem cells (ESCs). However, it is largely unknown about how the pluripotency is regulated. Here we demonstrate that Pcid2 is required for the maintenance of self-renewal both in mouse and human ESCs. Pcid2 plays a critical role in suppression of ESC differentiation. Pcid2 deficiency causes early embryonic lethality before the blastocyst stage. Pcid2 associates with EID1 and is present in the CBP/p300-EID1 complex in the ESCs. We show that MDM2 is an E3 ligase for K48-linked EID1 ubiquitination for its degradation. For the maintenance of self-renewal, Pcid2 binds to EID1 to impede the association with MDM2. Then EID1 is not degraded to sustain its stability to block the HAT activity of CBP/p300, leading to suppression of the developmental gene expression. Collectively, Pcid2 is present in the CBP/p300-EID1 complex to control the switch balance of mouse and human ESCs through modulation of EID1 degradation.

Histopathological evaluation of the diversity of cells susceptible to H5N1 virulent avian influenza virus.

Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage.