Effect of Calcium Supplementation and TMEM16A Inhibition on Endoplasmic Reticulum Stress Induced by Dental Fluorosis in Mice.

BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.

Stable Interstrand Cross-Links Generated from the Repair of 1,N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

Optimization and validation of a UPLC-MS/MS assay for simultaneous quantification of 2,8-dihydroxyadenine, adenine, allopurinol, oxypurinol and febuxostat in human plasma.

Adenine phosphoribosyltransferase (APRT) deficiency is a rare , hereditary disorder characterized by renal excretion of 2,8-dihydroxyadenine (DHA), leading to kidney stone formation and chronic kidney disease (CKD). Treatment with a xanthine oxidoreductase inhibitor, allopurinol or febuxostat, reduces urinary DHA excretion and slows the progression of CKD. The method currently used for therapeutic monitoring of APRT deficiency lacks specificity and thus, a more reliable measurement technique is needed. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma was optimized and validated. Plasma samples were prepared with protein precipitation using acetonitrile followed by evaporation. The chemometric approach design of experiments was implemented to optimize gradient steepness, amount of organic solvent, flow rate, column temperature, cone voltage, desolvation temperature and desolvation flow rate. Experimental screening was conducted using fractional factorial design with addition of complementary experiments at the axial points for optimization of peak area, peak resolution and peak width. The assay was validated according to the US Food and Drug Administration guidelines for bioanalytical method validation over the concentration range of 50 to 5000 ng/mL for DHA, allopurinol and febuxostat, 100 to 5000 ng/mL for adenine and 50 to 12,000 ng/mL for oxypurinol, with r2 ≥ 0.99. The analytical assay achieved acceptable performance of accuracy (-10.8 to 8.3 %) and precision (CV < 15 %). DHA, adenine, allopurinol, oxypurinol and febuxostat were stable in plasma samples after five freeze-thaw cycles at -80 °C and after storage at -80 °C for 12 months. The assay was evaluated for quantification of the five analytes in clinical plasma samples from six APRT deficiency patients and proved to be both efficient and accurate. The proposed assay will be valuable for guiding pharmacotherapy and thereby contribute to improved and more personalized care for patients with APRT deficiency.

Trans-3, 5, 4'-trimethoxystilbene restrains non-small-cell lung carcinoma progression via suppressing M2 polarization through inhibition of m6A modified circPACRGL-mediated Hippo signaling.

BACKGROUND: Non-small-cell lung carcinoma (NSCLC) accounts for ∼85% of all lung carcinomas. Trans-3,5,4'-trimethoxystilbene (TMS) shows strong anti-tumor activity and induces tumor cell apoptosis. However, its function and mechanism in NSCLC still require investigation. METHODS: PMA was used to treated THP-1 cells for macrophage differentiation. The abundance and m6A modification of circPACRGL were examined with qRT-PCR and MeRIP. Colony forming, transwell, wound healing, and Western blotting assays were applied to analyze proliferation, invasion, migration, and EMT. Macrophage polarization was determined through flow cytometry analysis of M1 and M2 markers. The interplay between circPACRGL, IGF2BP2 and YAP1 was validated by RNA pull-down and RIP assays. Mice received subcutaneous injection of NSCLC cells as a mouse model of subcutaneous tumor. RESULTS: CircPACRGL was upregulated in NSCLC cells, but it was reduced by TMS treatment. CircPACRGL depletion blocked proliferation, migration, and invasion in H1299 and H1975 cells. TMS suppressed these malignant behaviors, but it was abolished by circPACRGL overexpression. In addition, NSCLC-derived exosomes delivered circPACRGL into THP-1 cells to promote its M2 polarization, but TMS inhibited these effects by downregulating exosomal circPACRGL. Mechanically, exosomal circPACRGL bound to IGF2BP2 to improve the stability of YAP1 mRNA and regulate Hippo signaling in polarized THP-1 cells. TMS inhibited NSCLC growth via suppressing Hippo signaling and M2 polarization in vivo. CONCLUSION: TMS restrains M2 polarization and NSCLC progression by reducing circPACRGL and inhibiting exosomal circPACRGL-mediated Hippo signaling. Thus, these findings provide a novel mechanism underlying NSCLC progression and potential therapeutic targets.

Persistence of Chronic Lymphocytic Leukemia Stem-like Populations under Simultaneous In Vitro Treatment with Curcumin, Fludarabine, and Ibrutinib: Implications for Therapy Resistance.

Leukemic stem cells (LSCs) possess similar characteristics to normal hematopoietic stem cells, including self-renewal capacity, quiescence, ability to initiate leukemia, and drug resistance. These cells play a significant role in leukemia relapse, persisting even after apparent remission. LSCs were first described in 1994 by Lapidot et al. Although they have been extensively studied in acute leukemia, more LSC research is still needed in chronic lymphocytic leukemia (CLL) to understand if reduced apoptosis in mature cells should still be considered as the major cause of this disease. Here, we provide new evidence suggesting the existence of stem-like cell populations in CLL, which may help to understand the disease as well as to develop effective treatments. In this study, we identified a potential leukemic stem cell subpopulation using the tetraploid CLL cell line I83. This subpopulation is characterized by diploid cells that were capable of generating the I83 tetraploid population. Furthermore, we adapted a novel flow cytometry analysis protocol to detect CLL subpopulations with stem cell properties in peripheral blood samples and primary cultures from CLL patients. These cells were identified by their co-expression of CD19 and CD5, characteristic markers of CLL cells. As previously described, increased alkaline phosphatase (ALP) activity is indicative of stemness and pluripotency. Moreover, we used this method to investigate the potential synergistic effect of curcumin in combination with fludarabine and ibrutinib to deplete this subpopulation. Our results confirmed the effectiveness of this ALP-based analysis protocol in detecting and monitoring leukemic stem-like cells in CLL. This analysis also identified limitations in eradicating these populations using in vitro testing. Furthermore, our findings demonstrated that curcumin significantly enhanced the effects of fludarabine and ibrutinib on the leukemic fraction, exhibiting synergistic effects (combination drug index, CDI 0.97 and 0.37, respectively). Our results lend support to the existence of potential stem-like populations in CLL cell lines, and to the idea that curcumin could serve as an effective adjuvant in therapies aimed at eliminating these populations and improving treatment efficacy.

Combination Therapy with a TLR7 Agonist and a BRD4 Inhibitor Suppresses Tumor Growth via Enhanced Immunomodulation.

JQ-1 is a typical BRD4 inhibitor with the ability to directly fight tumor cells and evoke antitumor immunity via reducing the expression of PD-L1. However, problems arise with the development of JQ-1 in clinical trials, such as marked lymphoid and hematopoietic toxicity, leading to the investigation of combination therapy. SZU-101 is a TLR7 agonist designed and synthesized by our group with potent immunostimulatory activity. Therefore, we hypothesized that combination therapy of SZU-101 and JQ-1 would target innate immunity and adaptive immunity simultaneously, to achieve a better antitumor efficacy than monotherapy. In this study, the repressive effects of the combination administration on tumor growth and metastasis were demonstrated in both murine breast cancer and melanoma models. In 4T1 tumor-bearing mice, i.t. treatment with SZU-101 in combination with i.p. treatment with JQ-1 suppressed the growth of tumors at both injected and uninjected sites. Combination therapy increased M1/M2 ratio in TAMs, decreased PD-L1 expression and promoted the recruitment of activated CD8+ T cells in the TME. In summary, the improved therapeutic efficacy of the novel combination therapy appears to be feasible for the treatment of a diversity of cancers.

Renal histology of Fanconi syndrome associated with adefovir dipivoxil: A case report.

A sporadic occurrence of Fanconi syndrome associated with adefovir dipivoxil (ADV) has been reported, particularly when confirmed by renal biopsy. This study presents the case of a 53-year-old man who had been taking ADV 10 mg daily for 10 years to treat chronic hepatitis B (CHB) and subsequently developed Fanconi syndrome. The clinical manifestations included hypophosphatemic osteomalacia, glucosuria, renal tubular acidosis, low-molecular-weight proteinuria, and renal insufficiency. Renal biopsy revealed significant injury to proximal tubular epithelial cells, including vacuolar degeneration and regeneration of tubular epithelial cells. The ultrastructural pathology indicated severe morphological abnormalities of mitochondria, such as densely packed and enlarged mitochondria, with loss, blunting, and disordered arrangement of cristae. Following discontinuation of ADV and supplementation with oral phosphate, hypophosphatemia, glucosuria, and proteinuria were resolved. These findings support the previous hypothesis that ADV-induced nephrotoxicity may involve mitochondrial injury.

Anti-CD19 monoclonal antibodies for the treatment of relapsed or refractory B-cell malignancies: a narrative review with focus on diffuse large B-cell lymphoma.

PURPOSE: CD19 is a cell surface protein that is found on both healthy and malignant B cells. Accordingly, it has become an important target for novel treatments for non-Hodgkin lymphomas and B-cell leukaemia. Three anti-CD19 monoclonal antibodies with distinct mechanisms of action have been developed for the treatment of B-cell malignancies. METHODS: We reviewed the preclinical and clinical data on the development of the newly approved anti-CD19 monoclonal antibodies blinatumomab, tafasitamab and loncastuximab tesirine, and consider their place in the treatment of relapsed or refractory B-cell malignancies. RESULTS: Blinatumomab is a bispecific T-cell engager that binds to both CD19 on B cells and CD3 on T cells, facilitating antibody-dependent cytotoxicity. Blinatumomab significantly prolongs overall survival in patients with relapsed or refractory B-cell acute lymphoblastic leukaemia, although cytokine release syndrome and severe neurotoxicity may necessitate discontinuation. Tafasitamab, which has modified anti-CD19 Fab and Fc regions, has significantly enhanced affinity for both CD19 and effector cell receptors compared with unmodified anti-CD19. In L-MIND, tafasitamab plus lenalidomide provided an overall response rate (ORR) of 57.5% in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) in patients non-transplant eligible. Loncastuximab tesirine is an antibody-drug conjugate that has been studied as monotherapy and in combination with ibrutinib in 3L + relapsed or refractory DLBCL. The ORR was 48.3% in a phase II trial of loncastuximab tesirine. The optimal place of anti-CD19 monoclonal antibodies in therapy has yet to be determined, but the prospect of improved outcomes for at least some patients with treatment-resistant B-cell malignancies appears likely, particularly in those with limited therapeutic options and poor prognosis.

Effect of ibrutinib on CCR7 expression and functionality in chronic lymphocytic leukemia and its implication for the activity of CAP-100, a novel therapeutic anti-CCR7 antibody.

CAP-100 is a novel therapeutic antibody directed against the ligand binding site of human CCR7. This chemokine receptor is overexpressed in chronic lymphocytic leukemia (CLL) and orchestrates the homing of CLL cells into the lymph node. Previous studies, on a very limited number of samples, hypothesized that the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib might induce loss of surface CCR7 levels in CLL cells. CAP-100 will be evaluated in clinical trials as a therapy for relapse/refractory CLL patients, who have received at least two systemic therapies (NCT04704323). As nowadays many relapse/refractory CLL patients will have received ibrutinib as a prior therapy, we aimed to investigate in a large cohort of CLL patients the impact of this BTKi on CCR7 expression and functionality as well as on the therapeutic activity of CAP-100. Our data confirm that ibrutinib moderately down-regulates the very high expression of CCR7 in CLL cells but has no apparent effect on CCR7-induced chemotaxis. Moreover, CLL cells are perfectly targetable by CAP-100 which led to a complete inhibition of CCR7-mediated migration and induced strong target cell killing through antibody-dependent cell-mediated cytotoxicity, irrespective of previous or contemporary ibrutinib administration. Together, these results validate the therapeutic utility of CAP-100 as a next-line single-agent therapy for CLL patients who failed to ibrutinib and confirm that CAP-100 and ibrutinib have complementary non-overlapping mechanisms of action, potentially allowing for combination therapy.

An update on the progress of galidesivir (BCX4430), a broad-spectrum antiviral.

Galidesivir (BCX4430) is an adenosine nucleoside analog that is broadly active in cell culture against several RNA viruses of various families. This activity has also been shown in animal models of viral disease associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever viruses. In many cases, the compound is more efficacious in animal models than cell culture activity would predict. Based on favorable data from in vivo animal studies, galidesivir has recently undergone evaluation in several phase I clinical trials, including against severe acute respiratory syndrome coronavirus 2, and as a medical countermeasure for the treatment of Marburg virus disease.

Lymph-Node-Targeted Cholesterolized TLR7 Agonist Liposomes Provoke a Safe and Durable Antitumor Response.

Toll-like receptor (TLR) agonists as the potent stimulants of an innate immune system hold promises for applications in anticancer immunotherapy. However, most of them are limited in the clinical translation due to the uncontrolled systemic inflammatory response. In the current study, 1V209, a small molecule TLR7 agonist, was conjugated with cholesterol (1V209-Cho) and prepared into liposomes (1V209-Cho-Lip). 1V209-Cho-Lip exerted minimal toxic effects and enhanced the transportation ability in lymph nodes (LNs) compared with 1V209. 1V209-Cho-Lip treatment inhibited tumor progression in CT26 colorectal cancer, 4T1 breast cancer, and Pan02 pancreatic ductal cancer models through inducing effective DC activation and eliciting CD8+ T cell responses. Furthermore, 1V209-Cho-Lip induced tumor-specific memory immunity to inhibit cancer recurrence and metastasis. These results indicate that cholesterol conjugation with 1V209 is an effective approach to target lymph nodes and to reduce the adverse effects. This work provides a rational basis for the distribution optimization of TLR agonists for potential clinical use.

Combined ibrutinib and venetoclax treatment vs single agents in the TCL1 mouse model of chronic lymphocytic leukemia.

The covalent inhibitor of Bruton's tyrosine kinase ibrutinib and the specific Bcl-2 inhibitor venetoclax are both highly efficacious single-agent drugs in the treatment of chronic lymphocytic leukemia (CLL). Based on their complementary modes of action, ibrutinib and venetoclax are hypothesized to act in a synergistic fashion. Currently, it is unclear whether combined treatment is indeed superior to continuous single-agent treatment and what mechanisms underlie the resistance to combination treatment. In addition, the effects of such treatment on the skewed T-cell compartment characteristic of CLL are as yet unknown. In the murine Eµ-TCL1 adoptive transfer model resembling aggressive CLL, we found that combined treatment resulted in the deepest responses, with the longest duration related to a combination of decreased proliferation and increased induction of apoptosis. In addition, alterations in T-cell subsets were most prominent after combination treatment, with increased naive cells and reduced effector memory cells. Remarkably, effects of single agents but also combination treatment were eventually interrupted by relapse, and we found downregulation of BIM expression as a plausible cause of acquired drug resistance. Nevertheless, in this murine model, the combination of venetoclax and ibrutinib has increased efficacy over single agents, accompanied by a restoration of the T-cell compartment.

Sex-Specific Differences in Autophagic Responses to Experimental Ischemic Stroke.

Ischemic stroke triggers a series of complex pathophysiological processes including autophagy. Differential activation of autophagy occurs in neurons derived from males versus females after stressors such as nutrient deprivation. Whether autophagy displays sexual dimorphism after ischemic stroke is unknown. We used a cerebral ischemia mouse model (middle cerebral artery occlusion, MCAO) to evaluate the effects of inhibiting autophagy in ischemic brain pathology. We observed that inhibiting autophagy reduced infarct volume in males and ovariectomized females. However, autophagy inhibition enhanced infarct size in females and in ovariectomized females supplemented with estrogen compared to control mice. We also observed that males had increased levels of Beclin1 and LC3 and decreased levels of pULK1 and p62 at 24 h, while females had decreased levels of Beclin1 and increased levels of ATG7. Furthermore, the levels of autophagy markers were increased under basal conditions and after oxygen and glucose deprivation in male neurons compared with female neurons in vitro. E2 supplementation significantly inhibited autophagy only in male neurons, and was beneficial for cell survival only in female neurons. This study shows that autophagy in the ischemic brain differs between the sexes, and that autophagy regulators have different effects in a sex-dependent manner in neurons.

Multiregional sequencing and circulating tumour DNA analysis provide complementary approaches for comprehensive disease profiling of small lymphocytic lymphoma.

We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma-circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value.

Androgen receptor inhibition alleviated inflammation in experimental autoimmune myocarditis by increasing autophagy in macrophages.

OBJECTIVE: Experimental autoimmune myocarditis (EAM) is characterized by pronounced macrophage infiltration, cardiac necrosis, and cardiac fibrosis. Our previous studies have demonstrated that suppressed androgen receptor (AR) enables anti-inflammation to promote tissue repair by decreasing M1 macrophages and increasing M2 macrophages in an EAM model. Given that autophagy mediates inflammatory response in macrophages, we investigated whether AR inhibition executes its protective role in inflammation through the autophagy pathway in EAM. MATERIALS AND METHODS: To determine whether AR inhibition can perform its anti-inflammatory effects by upregulating autophagy, we pre-treated mice with 3-methyl adenine (3-MA), a pharmacological inhibitor of autophagy. Immunofluorescence assay and Western blot were used to detect autophagy levels and autophagy activity in five different groups. Immunofluorescence marked F4/80 and LC3 to illustrate the autophagy level in macrophages. TUNEL assays were used to detect the apoptosis level in heart tissue of five different groups. RESULTS: We demonstrated that AR inhibition resolves injury with sustained inhibition of inflammatory cytokines associated with enhanced autophagy, especially in macrophages. Increased LC3II/I expression corroborated complete autolysosome formation detected by electron microscopy and correlated with degradation of SQSTM1/p62 in the AR inhibition group by Western blot. These effects could be reversed within 3-MA, a pharmacological inhibitor of autophagy. Specifically, pharmacological inhibition of autophagy increased apoptosis and inflammation, which could be attenuated by AR inhibition. CONCLUSIONS: AR inhibition alleviates the inflammatory response and tissue apoptosis by enhancing autophagy, especially in macrophages.

Real-world Prescription Pattern, Discontinuation and Costs of Ibrutinib-Naïve Patients with Chronic Lymphocytic Leukemia: An Italian Healthcare Administrative Database Analysis.

BACKGROUND AND OBJECTIVE: In order to integrate the existing and inconsistent information from clinical trials and real-world practice on chronic lymphocytic leukemia (CLL) treated with ibrutinib, this analysis aimed to describe the prescription pattern of new users of ibrutinib affected by CLL, focusing on discontinuation, severe adverse events (AEs) and change of treatment, and to assess the integrated healthcare expenditure from the Italian National Health System (INHS) perspective. METHODS: Starting from the ReS database, adults with at least a supply of ibrutinib (ATC code L01XE27) were selected from 01/01/2016 to 12/31/2017. Those without any ibrutinib supply in the year before the index prescription were considered new users. Out of them, only patients with at least a primary or secondary in-hospital diagnosis of CLL (ICD-9-CM code 204.1*) from 01/01/2013 to 12/31/2018 were further broken down according to the ibrutinib's line treatment (first line-FL; second or later line-SLL) and analysed. They were characterized by sex and age in the selection period. Mean annual consumption (defined daily doses [DDD]), treatment discontinuation, changes of therapy, interruptions and healthcare costs in charge of the INHS were assessed during two follow-up years. RESULTS: Out of more than 5 million inhabitants of the ReS database, 69 new ibrutinib users and diagnosed with CLL in 2016 (incidence: 1.6 × 100,000) and 41 in 2017 (incidence: 0.9 × 100,000) were selected. Of these, 21 (19.1%) were FL ibrutinib users and 89 (80.9%) were SLL ones, mostly males and with mean ages (±SD) of 65 ± 14 and 70 ± 10, respectively. The mean annual consumption among FL users decreased from 222.2 DDD per patient treated to 216.0 DDD, while increased among SLL patients from 238.6 DDD to 260.1 DDD, in the first and second follow-up year, respectively. The discontinuation rate was about 40% in the first year, similarly among FL and SLL users. SLL patients discontinued more frequently (52.8% vs 20.0%) in the second year. Very few AEs were recorded. The 62.5% of FL and 55.6% of SLL users discontinuing ibrutinib in 1-year follow-up, while one SLL patient (5.3%) in the second year changed therapy. The 20.0% and 15.9% of all new users in first and second year interrupted ibrutinib. The total integrated cost of FL patients was €55,732 reducing by about €15,000, while it was €58,716 for SLL ones decreasing by €6,000, respectively, in the first and in the second year. Pharmaceuticals were the key cost driver (ibrutinib accounted for more than 77%). CONCLUSIONS: This analysis on Italian administrative data provided results about prescription patterns of ibrutinib FL and SLL new users with CLL, focusing on discontinuation, treatment change and healthcare costs over 2-year follow-up, and contributed to improve the knowledge on this hard-to-treat disease.

Successful treatment of Epstein-Barr virus-associated primary central nervous system lymphoma due to post-transplantation lymphoproliferative disorder, with ibrutinib and third-party Epstein-Barr virus-specific T cells.

Primary central nervous system lymphoma (PCNSL) occurring following organ transplantation (post-transplantation lymphoproliferative disorder [PTLD]) is a highly aggressive non-Hodgkin lymphoma. It is typically treated with high-dose methotrexate-based regimens. Outcomes are dismal and clinical trials are lacking. It is almost always Epstein-Barr virus (EBV) associated. Two patients (CA1-2) presented with EBV-associated PCNSL after renal transplant. CA1 was on hemodialysis and had prior disseminated cryptococcus and pseudomonas bronchiectasis, precluding treatment with methotrexate. CA2 was refractory to methotrexate. Both were treated off-label with the first-generation Bruton's tyrosine kinase inhibitor ibrutinib for 12 months. Cerebrospinal fluid penetration at therapeutic levels was confirmed in CA1 despite hemodialysis. Both patients entered remission by 2 months. Sequencing confirmed absence of genetic aberrations in human leukocyte antigen (HLA) class I/II and antigen-presentation/processing genes, indicating retention of the ability to present EBV-antigens. Between Weeks 10 and 13, they received third-party EBV-specific T cells for consolidation with no adverse effects. They remain in remission ≥34 months since therapy began. The strength of these findings led to an ongoing phase I study (ACTRN12618001541291).

Blockage of AMPK-ULK1 pathway mediated autophagy promotes cell apoptosis to increase doxorubicin sensitivity in breast cancer (BC) cells: an in vitro study.

BACKGROUND: Activation of autophagy flux contributed to resistance of breast cancer (BC) cells to current chemotherapeutic drugs, which seriously limited their therapeutic efficacy and facilitated BC recurrence in clinic. However, the detailed mechanisms are still not fully understood. In the present study, we identified that inactivation of AMPK-ULK1 signaling cascade mediated protective autophagy sensitized BC cells to doxorubicin in vitro. METHODS: Cell counting kit-8 (CCK-8) assay and colony formation assay were performed to evaluate cell proliferation abilities. Trypan blue staining assay was used to examine cell viability, and Annexin V-FITC/PI double staining method was conducted to determine cell apoptosis. The autophagosomes in BC cells were observed and photographed by electronic microscope (EM). Western Blot analysis was employed to examine genes expressions at protein levels. RESULTS: The parental doxorubicin-sensitive BC (DS-BC) cells were exposed to increasing concentrations of doxorubicin to establish doxorubicin-resistant BC (DR-BC) cells, and the DR-BC cells were much more resistant to high-dose doxorubicin treatment compared to the DS-BC cells. Interestingly, high-dose doxorubicin specifically increased LC3B-II/I ratio, promoted autophagosomes formation and decreased p62 expression levels to facilitate autophagy in DR-BC cells, instead of DS-BC cells, and the autophagy inhibitor 3-methyladenine (3-MA) enhanced the cytotoxic effects of high-dose doxorubicin on DR-BC cells. In addition, we proved that high-dose doxorubicin triggered protective autophagy in DR-BC cells by activating AMPK-ULK1 pathway. Functionally, high-dose doxorubicin increased the expression levels of phosphorylated AMPK (p-AMPK) and ULK1 (p-ULK1) to activate AMPK-ULK1 pathway in DR-BC cells, and the inhibitors for AMPK (compound C) and ULK1 (SBI-0206965) blocked autophagy to promote cell death and slow down cell growth in DR-BC cells treated with high-dose doxorubicin. CONCLUSIONS: Collectively, our in vitro data indicated that blockage of AMPK-ULK1 signaling cascade mediated protective autophagy might be a promising strategy to increase doxorubicin sensitivity for BC treatment.

Polygoni Multiflori Radix Preparat Delays Skin Aging by Inducing Mitophagy.

BACKGROUND: As the skin is the largest organ of the human body, it is aging inevitably and produces cosmetic and psychological problems, and even disease. Therefore, the molecular mechanisms related to the prevention of skin aging need to be further explored. METHODS: Aging models were constructed by D-galactose. Mice were administrated with polygoni multiflori radix preparat (PMRP), PMRP and 3-methyladenine, or PMRP and rapamycin intragastrically. The apparent and viscera index of aged rats was measured. Then, the physicochemical property, antioxidant ability, histological structure, mitochondrial membrane potential, ATP and ROS levels, and mitophagy of aged skins were determined. Finally, the expression of PINK1, Parkin, P62, and LC3II/I; apoptosis-related proteins; and the percentage of apoptotic cells were measured. RESULTS: PMRP relieved skin aging with reducing of thymus index, improvement of pathological damage and histological structure, increase of the expression area of fibrous tissue, the ratio of type I to type III collagen, and antioxidant ability of aged skins. Importantly, PMRP also improved mitochondrial dysfunction with an increase in the content of mitochondrial membrane potential and ATP and a decrease of ROS levels. Moreover, mitophagy was enhanced with the treatment of PMRP when observed using electron microscopy, and the expression of PINK1, Parkin, and LC3I/II was increased with PMRP treatment but P62 expression was decreased. Meanwhile, PMRP alleviated apoptosis with a decrease of apoptotic cell and the expression of Cleaved-cas3, Bax, Cyt-c, AIF, and Smac as well as an increase of Bcl-2 expression. CONCLUSION: The results demonstrated that the polygoni multiflori radix preparata may delay skin aging by inducing mitophagy.

Risk of bleeding complications and atrial fibrillation associated with ibrutinib treatment: A systematic review and meta-analysis.

The use of ibrutinib is hampered by major bleeding events and atrial fibrillation. Speculating whether randomized controlled trials might underestimate the risk of adverse events in clinical practice, we conducted a systematic review and meta-analysis studying patients treated in any setting and indication. We systematically searched the literature using MEDLINE and EMBASE databases for case series, cohort studies, or randomized controlled trials and retrieved all data in parallel. Proportions of patients with adverse events were pooled in relevant subgroups using the binominal distribution and Freeman-Tukey double arcsine transformation. Among 2'537 records screened, 85 were finally included, comprising 7'317 patients. Methodological quality according to the Newcastle-Ottawa Scale was rated as moderate to poor with regard to bleeding events and atrial fibrillation; 106 studies were excluded because of missing data at all. Reported events varied substantially between 0 % and 78 % (any bleedings), 0 % and 25 % (major bleedings), and 0 % and 38 % (new-onset atrial fibrillation). Pooled estimates were 28 % (95 % confidence interval 22 %, 34 %), 3 % (2 %, 4 %), and 8 % respectively (7 %, 10 %). The risk of events was higher in studies with an older population, high ibrutinib dosage, thrombocytopenia, antithrombotic treatment, and retrospective studies. In conclusions, reporting of bleeding events and atrial fibrillation varied substantially among studies. These observations, in combination with the estimates obtained, suggest a relevant risk in clinical practice.