Hepatic arterial infusion chemotherapy following simultaneous metallic stent placement and iodine-125 seed strands for advanced cholangiocarcinoma causing malignant obstructive jaundice: a propensity score matching study.

OBJECTIVE: This study aims to evaluate the effectiveness and safety of hepatic arterial infusion chemotherapy (HAIC) following the simultaneous placement of self-expandable metallic stent (SEMS) and iodine-125 (125I) seed strands for the management of advanced cholangiocarcinoma (CCA) patients presenting with malignant obstructive jaundice (MOJ). METHODS: Data from 74 patients with MOJ caused by advanced CCA treated with stent placement with 125I seed strands with or without HAIC between November 2015 and October 2020 were analysed retrospectively. Eighteen patients received 5 sessions of HAIC after SEMS placement with 125I seed strands (HAIC group), and 56 patients only underwent SEMS placement with 125I seed strands and served as controls (control group). HAIC consisted of infusions of gemcitabine (600-1000 mg/m2 given over 30 min) followed by oxaliplatin (60-100 mg/m2 given over 2 h), with an interval of 4 weeks. Propensity score matching (PSM) analysis was used to adjust for differences in the baseline characteristics of the groups (including age, total bilirubin, and serum alanine aminotransferase level). Overall survival (OS), stent patency, and adverse events were compared between the two groups. RESULTS: OS and stent patency were significantly better in patients in the HAIC group than in those in the control group (median survival time: before PSM, 362 vs. 185 days, p = 0.005; after PSM, 357 vs. 183 days, p = 0.012; median duration of stent patency: before PSM, 294 vs. 156 days, p = 0.001; after PSM, 287 vs. 183 days, p = 0.039). All adverse reactions were controllable by temporary symptomatic treatment. Serious complications and treatment-related deaths were not observed. CONCLUSION: Our preliminary study showed that HAIC following SEMS placement with 125I seed strands is effective and safe for the management of advanced CCA patients presenting with MOJ and could improve stent patency and patient survival.

FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

SHLD2 promotes class switch recombination by preventing inactivating deletions within the Igh locus.

The newly identified shieldin complex, composed of SHLD1, SHLD2, SHLD3, and REV7, lies downstream of 53BP1 and acts to inhibit DNA resection and promote NHEJ. Here, we show that Shld2-/- mice have defective class switch recombination (CSR) and that loss of SHLD2 can suppress the embryonic lethality of a Brca1Δ11 mutation, highlighting its role as a key effector of 53BP1. Lymphocyte development and RAG1/2-mediated recombination were unaffected by SHLD2 deficiency. Interestingly, a significant fraction of Shld2-/- primary B-cells and 53BP1- and shieldin-deficient CH12F3-2 B-cells permanently lose expression of immunoglobulin upon induction of CSR; this population of Ig-negative cells is also seen in other NHEJ-deficient cells and to a much lesser extent in WT cells. This loss of Ig is due to recombination coupled with overactive resection and loss of coding exons in the downstream acceptor constant region. Collectively, these data show that SHLD2 is the key effector of 53BP1 and critical for CSR in vivo by suppressing large deletions within the Igh locus.

Early-life programming of mesenteric lymph node stromal cell identity by the lymphotoxin pathway regulates adult mucosal immunity.

Redundant mechanisms support immunoglobulin A (IgA) responses to intestinal antigens. These include multiple priming sites [mesenteric lymph nodes (MLNs), Peyer's patches, and isolated lymphoid follicles] and various cytokines that promote class switch to IgA, even in the absence of T cells. Despite these backup mechanisms, vaccination against enteric pathogens such as rotavirus has limited success in some populations. Genetic and environmental signals experienced during early life are known to influence mucosal immunity, yet the mechanisms for how these exposures operate remain unclear. Here, we used rotavirus infection to follow antigen-specific IgA responses through time and in different gut compartments. Using genetic and pharmacological approaches, we tested the role of the lymphotoxin (LT) pathway-known to support IgA responses-at different developmental stages. We found that LT-ß receptor (LTßR) signaling in early life programs intestinal IgA responses in adulthood by affecting antibody class switch recombination to IgA and subsequent generation of IgA antibody-secreting cells within an intact MLN. In addition, early-life LTßR signaling dictates the phenotype and function of MLN stromal cells to support IgA responses in the adult. Collectively, our studies uncover new mechanistic insights into how early-life LTßR signaling affects mucosal immune responses during adulthood.

Fibroblastic reticular cells initiate immune responses in visceral adipose tissues and secure peritoneal immunity.

Immune protection of the body cavities depends on the swift activation of innate and adaptive immune responses in nonclassical secondary lymphoid organs known as fat-associated lymphoid clusters (FALCs). Compared with classical secondary lymphoid organs such as lymph nodes and Peyer's patches, FALCs develop along distinct differentiation trajectories and display a reduced structural complexity. Although it is well established that fibroblastic reticular cells (FRCs) are an integral component of the immune-stimulating infrastructure of classical secondary lymphoid organs, the role of FRCs in FALC-dependent peritoneal immunity remains unclear. Using FRC-specific gene targeting, we found that FRCs play an essential role in FALC-driven immune responses. Specifically, we report that initiation of peritoneal immunity was governed through FRC activation in a myeloid differentiation primary response 88 (MYD88)-dependent manner. FRC-specific ablation of MYD88 blocked recruitment of inflammatory monocytes into FALCs and subsequent CD4+ T cell-dependent B-cell activation and IgG class switching. Moreover, containment of Salmonella infection was compromised in mice lacking MYD88 expression in FRCs, indicating that FRCs in FALCs function as an initial checkpoint in the orchestration of protective immune responses in the peritoneal cavity.

The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity.

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.

Kin17 facilitates multiple double-strand break repair pathways that govern B cell class switching.

Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR.

Hematopoietic LTßR deficiency results in skewed T cell cytokine profiles during a mucosal viral infection.

The lymphotoxin signaling pathway plays an important role in the homeostasis and function of peripheral and mucosal dendritic cells, and dendritic cell-intrinsic lymphotoxin ß receptor expression is required for optimal responses to opportunistic intestinal bacteria. However, it is unknown whether dendritic cell-intrinsic lymphotoxin ß receptor signaling is required for responses to intestinal viral infections. We explored this question by orally administrating murine rotavirus to chimeric mice that lack lymphotoxin ß receptor signaling in the myeloid compartment but retain lymphoid tissues. We found that although clearance of rotavirus was unimpaired in the lymphotoxin ß receptor(-/-) → wild-type chimeric mice compared with wild-type → wild-type chimeric mice, IFN-γ-producing CD8(+) and CD4(+) T cells were significantly increased in the small intestinal lamina propria of lymphotoxin ß receptor(-/-) → wild-type chimeric mice. In contrast, IL-17-producing CD4(+) T cells were reduced in lymphotoxin ß receptor(-/-) → wild-type chimeric mice in the steady state, and this reduction persisted after rotavirus inoculation. In spite of this altered cytokine profile in the small intestinal lamina propria of lymphotoxin ß receptor(-/-) → wild-type chimeric mice, the local production of rotavirus-specific IgA was unperturbed. Collectively, our results demonstrate that lymphotoxin ß receptor signaling in radio-sensitive myeloid cells regulates the balance of IFN-γ and IL-17 cytokine production within the small intestinal lamina propria; however, these perturbations do not affect mucosal antiviral IgA responses.

Maternal anti-platelet ß3 integrins impair angiogenesis and cause intracranial hemorrhage.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against ß3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-ß3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-ß3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-ß3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-ß3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

AID and caspase 8 shape the germinal center response through apoptosis.

Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC, the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda(-/-) GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda(-/-) GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition, we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation, demonstrating the importance of apoptosis in GC homeostasis and clonal selection.

AID-expressing germinal center B cells cluster normally within lymph node follicles in the absence of FDC-M1+ CD35+ follicular dendritic cells but dissipate prematurely.

Upon activation with T-dependent Ag, B cells enter germinal centers (GC) and upregulate activation-induced deaminase (AID). AID(+) GC B cells then undergo class-switch recombination and somatic hypermutation. Follicular dendritic cells (FDC) are stromal cells that underpin GC and require constitutive signaling through the lymphotoxin (LT) ß receptor to be maintained in a fully mature, differentiated state. Although it was shown that FDC can be dispensable for the generation of affinity-matured Ab, in the absence of FDC it is unclear where AID expression occurs. In a mouse model that lacks mature FDC, as well as other LT-sensitive cells, we show that clusters of AID(+)PNA(+)GL7(+) Ag-specific GC B cells form within the B cell follicles of draining lymph nodes, suggesting that FDC are not strictly required for GC formation. However, later in the primary response, FDC-less GC dissipated prematurely, correlating with impaired affinity maturation. We examined whether GC dissipation was due to a lack of FDC or other LTß receptor-dependent accessory cells and found that, in response to nonreplicating protein Ag, FDC proved to be more critical for long-term GC maintenance. Our study provides a spatial-temporal analysis of Ag-specific B cell activation and AID expression in the context of a peripheral lymph node that lacks FDC-M1(+) CD35(+) FDC and other LT-sensitive cell types, and reveals that FDC are not strictly required for the induction of AID within an organized GC-like environment.

Plant food delphinidin-3-glucoside significantly inhibits platelet activation and thrombosis: novel protective roles against cardiovascular diseases.

Delphinidin-3-glucoside (Dp-3-g) is one of the predominant bioactive compounds of anthocyanins in many plant foods. Although several anthocyanin compounds have been reported to be protective against cardiovascular diseases (CVDs), the direct effect of anthocyanins on platelets, the key players in atherothrombosis, has not been studied. The roles of Dp-3-g in platelet function are completely unknown. The present study investigated the effects of Dp-3-g on platelet activation and several thrombosis models in vitro and in vivo. We found that Dp-3-g significantly inhibited human and murine platelet aggregation in both platelet-rich plasma and purified platelets. It also markedly reduced thrombus growth in human and murine blood in perfusion chambers at both low and high shear rates. Using intravital microscopy, we observed that Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for vessel occlusion. Dp-3-g also significantly inhibited thrombus growth in a carotid artery thrombosis model. To elucidate the mechanisms, we examined platelet activation markers via flow cytometry and found that Dp-3-g significantly inhibited the expression of P-selectin, CD63, CD40L, which reflect platelet α- and δ-granule release, and cytosol protein secretion, respectively. We further demonstrated that Dp-3-g downregulated the expression of active integrin αIIbß3 on platelets, and attenuated fibrinogen binding to platelets following agonist treatment, without interfering with the direct interaction between fibrinogen and integrin αIIbß3. We found that Dp-3-g reduced phosphorylation of adenosine monophosphate-activated protein kinase, which may contribute to the observed inhibitory effects on platelet activation. Thus, Dp-3-g significantly inhibits platelet activation and attenuates thrombus growth at both arterial and venous shear stresses, which likely contributes to its protective roles against thrombosis and CVDs.

[Effect of paeoniflorin on hepatic immunopathogenesis in mice with Schistosoma japonicum infection].

OBJECTIVE: To investigate the mechanism of paeoniflorin in preventing hepatic granuloma formation and fibrosis in mice infected with Schistosoma japonicum. METHODS: Model of hepatic granuloma and fibrosis was established by infecting mice with S. japonicum cercariae. The infected mice were randomly divided into 4 groups: group A as model (infected control) group (15 mice), and paeoniflorin being given before, simultaneously and after praziquantel treatment as groups B, C and D. Each of the groups B, C and D was subdivided into 3 subgroups (15 mice each): low dose (paeoniflorin 2 ml, 30 mg/(kg x d) x 30 d), high dose(paeoniflorin 2 ml, 120 mg/(kg x d) x 30 d) and control (2 ml, 0.5% sodium carboxymethylcellulose x 30 d). In group B, paeoniflorin or sodium carboxymethylcellulose was orally administrated on 12 d after infection. In groups C and D, paeoniflorin or sodium carboxymethylcellulose was administrated on 42 d or 72 d after infection. Each of group B, C and D was orally given praziquantel 2 ml (500 mg/(kg x d) x 2 d) on 42 d after infection. On the 102nd day after infection, all animals were sacrificed by cervical dislocation. Serum hyaluronic acid (HA) was detected by radioimmunoassay; area of egg granuloma and degree of hepatic fibrosis were observed via HE and Masson stainings; the expression of transforming growth factor beta1 (TGF-beta1), alpha smooth muscle actin (alpha-SMA and collagen I (Col I) protein were measured by immunohistochemical method. RESULTS: In group B, the level of HA (0.719 +/- 0.239 microg/ml, 0.721 +/- 0.182 microg/ml) in low or high dose subgroups was significantly lower (F = 9.429, P < 0.01) than the control subgroup (1.049 +/- 0.286 microg/ml); the area of granuloma (0.066 +/- 0.005 mm2, 0.064 +/- 0.004 mm2) or the degree of hepatic fibrosis (2.067 +/- 0.458, 1.967 +/- 0.399) in low or high dose subgroups was significantly greater (F = 862.540, F = 29.738, P < 0.01) than the control (0.141 +/- 0.008 mm2, 3.467 +/- 0.834); the expression of alpha-SMA positive cells (2.933 +/- 0.594, 3.000 +/- 0.535) in low or high dose subgroups was significantly lower (F = 12.323, P < 0.01, P < 0.01) than its control (4.800 +/- 1.859); the expression of TGF-beta1 (0.256 +/- 0.057, 0.274 +/- 0.054) in low or high dose subgroups was significantly lower (F = 148.990, P < 0.01) than its control (0.552 +/- 0.047); the content of Col I (0.334 +/- 0.041, 0.339 +/- 0.042) in low or high dose subgroups was significantly lower (F = 180.881, P < 0.01) than its control (0.601 +/- 0.049). In groups C & D, no significant difference was found between the low or high dose subgroups or between the subgroups and their corresponding controls. CONCLUSION: Paeoniflorin can significantly reduce hepatic granuloma formation and fibrosis due to schistosome eggs, and decrease the expression of TGF-beta1, alpha-SMA in mice when it is given before praziquantel administration, which may associate with the activation of hepatic stellate cells and the expression of TGF-beta1 in liver tissue.

[Effect of paeoniflorin on secretion of TGF-beta1 from macrophages in mice].

OBJECTIVE: To explore the effect of paeoniflorin (PAE) on the production of transforming growth factor beta1 (TGF-beta1) from peritoneal macrophages(PMs) stimulated by soluble egg antigen (SEA) of Schistosoma japonicum. METHODS: SEA was prepared by trituration and added into culture plank, flask and dish containing PMs which were cultured for 24 h. TGF-beta1 secreted from PMs was measured by ELISA. TGF-beta1 mRNA and protein produced from PMs were evaluated by RT-PCR and Western blotting, respectively. SEA (10 mg/L) 5 ml was added into culture flask and dish containing PMs. PMs were cultured for 12 h, and PAE at different concentrations (0, 7.5, 15, 30, 60, 120 mg/L) was added into the culture flask and dish, and PMs were cultured consecutively for another 12 h and 24 h, respectively. TGF-beta1 mRNA and protein from PMs stimulated by SEA were evaluated by RT-PCR and Western blotting, respectively. RESULTS: TGF-beta1 (235.86 +/- 3.43 ng/L) was produced from PMs under stimulation of SEA at 10 mg/L, and the expression of TGF-beta1 mRNA and protein in PMs were depressed significantly by PAE in a concentration-dependent manner (r = -0.827, P < 0.01; r = -0.952, P < 0.01, respectively). CONCLUSION: PAE inhibits the production of TGF-beta1 from PMs stimulated by SEA.

Toxoplasma gondii: siRNA can mediate the suppression of adenosine kinase expression.

Adenosine kinase (AK) is one of the most important enzymes in the Toxoplasma gondii purine salvage pathway. Three siRNAs specific to the AK gene were designed in the present study. At 24h following electroporation, two of them (siRNA786 and siRNA1200) significantly reduced the mRNA level compared with mock electroporation (P <0.05). The ability to incorporate [3H]-adenosine in the parasites electroporated with 4 microM siRNA786 or 4 microM siRNA1200 was decreased to 39+/-11% and 39+/-7% of the mock electroporation, respectively. At the 48th hour of electroporation, the enzyme's activity was still significantly lower than that of mock electroporation. The data show the siRNAs transfected into cells can work efficiently to regulate gene expression in T. gondii. The application of siRNA in interrupting gene expression in T. gondii would be useful for elucidating gene function as a step toward development of anti-toxoplasmasis vaccines and therapeutic reagents.