Whole-genome CRISPR screening identifies molecular mechanisms of PD-L1 expression in adult T-cell leukemia/lymphoma.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells are still unknown. To comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression, whereas STAT3 positively did so. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it, respectively. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain patients with primary ATLL. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared with each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody avelumab and chimeric antigen receptor (CAR) T cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells.

Efficacy and Safety of Teduglutide in Infants and Children With Short Bowel Syndrome Dependent on Parenteral Support.

OBJECTIVES: Our objective was to evaluate the short- and long-term safety and efficacy of teduglutide treatment in infants and children with short bowel syndrome with intestinal failure (SBS-IF). METHODS: Two open-label phase 3 studies and 1 extension study investigated the short- and long-term safety and efficacy of teduglutide (0.05 mg/kg/day) in infants and children with SBS-IF: NCT03571516, 24-week study of infants who were randomized to receive teduglutide or standard of care (SoC); NCT02980666, 24-week study of infants and children who all received teduglutide; and NCT03268811, 24-week extension study of patients who completed NCT02980666 (patients could receive up to 48 weeks of total treatment). RESULTS: Twelve infants and 8 children enrolled in the core studies, and 2 infants and 7 children in the extension study. After 24 weeks of treatment, parenteral support (PS) requirements reduced by ≥20% from baseline for 4 infants (57.1%) and 4 children (66.7%) receiving teduglutide and for 2 infants receiving SoC (50.0%). One infant (50.0%) and 4 children (80.0%) receiving teduglutide maintained the ≥20% reduction in PS at 48 weeks of treatment. Two children receiving teduglutide achieved enteral autonomy, after 12 weeks and 28 weeks of treatment, respectively. All adverse events (AEs) were in line with known impacts of SBS-IF and adverse reactions to teduglutide. Only one serious AE (abdominal pain) was considered related to teduglutide. CONCLUSIONS: Short- and long-term treatment with teduglutide resulted in clinically meaningful reductions in PS requirements for infants and children with SBS-IF. Teduglutide was well tolerated, and efficacy improved with longer-term treatment.

Analysis and therapeutic targeting of the EP300 and CREBBP acetyltransferases in anaplastic large cell lymphoma and Hodgkin lymphoma.

Anaplastic large cell lymphoma (ALCL) and classical Hodgkin lymphoma (HL) share a similar cytological and high surface expression of CD30, and novel therapeutic strategies are needed. The EP300 and CREBBP acetyltransferases play essential roles in the pathogenesis of non-Hodgkin B cell lymphoma, but their functions in ALCL and HL are unknown. In the current study, we investigated the physiological roles of EP300 and CREBBP in both ALCL and HL, and exploited the therapeutic potential of EP300/CREBBP small molecule inhibitors that target either the HAT or bromodomain activities. Our studies demonstrated distinct roles for EP300 and CREBBP in supporting the viability of ALCL and HL, which was bolstered by the transcriptome analyses. Specifically, EP300 but not CREBBP directly modulated the expression of oncogenic MYC/IRF4 network, surface receptor CD30, immunoregulatory cytokines IL10 and LTA, and immune checkpoint protein PD-L1. Importantly, EP300/CREBBP HAT inhibitor A-485 and bromodomain inhibitor CPI-637 exhibited strong activities against ALCL and HL in vitro and in xenograft mouse models, and inhibited PD-L1 mediated tumor immune escape. Thus, our studies revealed critical insights into the physiological roles of EP300/CREBBP in these lymphomas, and provided opportunities for developing novel strategies for both targeted and immune therapies.

Genome-wide CRISPR screens identify CD48 defining susceptibility to NK cytotoxicity in peripheral T-cell lymphomas.

Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.

A phase II randomized study evaluating azacitidine versus conventional care regimens in newly diagnosed elderly Japanese patients with unfavorable acute myeloid leukemia.

A multicenter phase II study was conducted in 44 elderly (≥ 65 years) Japanese patients with newly diagnosed acute myeloid leukemia (AML) to evaluate whether azacitidine is also effective and feasible in Japanese AML patients. The 28 patients with AML with poor-risk cytogenetics and/or myelodysplasia-related changes (unfavorable AML) were randomly assigned to receive either azacitidine or conventional care regimens (CCR), and the other 16 patients without unfavorable AML received azacitidine alone. The primary endpoint was overall survival. At the median follow-up of 29 months, among the 26 evaluable patients with unfavorable AML, the median survival time (MST) of patients who received azacitidine (N = 14) was 9.6 months and that of patients who received CCR (N = 12) was 5.3 months (HR 0.73; 95% CI 0.31-1.69; log-rank P = 0.459). The MST of all 29 patients who received azacytidine, including the 15 evaluable patients without unfavorable AML, was 12.4 months. Adverse events of azacitidine were manageable and consistent with its established safety profile. Azacitidine tended to prolong survival in newly diagnosed elderly Japanese patients with AML, and was feasible as a front-line therapy for elderly AML patients.

Salvage Transplantation with Cord Blood for Graft Rejection of Peripheral Blood Stem Cells due to Donor Specific Antibody.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for various kinds of hematological malignancies and disorders. Recently, HLA-haploidentical stem cell transplantation with post-transplantation cyclophosphamide (PTCy-haplo HSCT) has been widely performed due to its safety and favorable immune recovery. However, graft rejection remains an obstacle to PTCy-haplo HSCT. Donor specific antibody (DSA) is considered to be a major factor of graft rejection of haplo HSCT. We herein present a case of graft rejection after PTCy haplo-HSCT due to DSA induced by pretransplant platelet transfusion after donor selection. The patient was dependent on platelet transfusion and had not received cytotoxic chemotherapy because he was diagnosed as myelodysplastic syndrome/myeloproliferative neoplasm-unclassifiable. We retrospectively confirmed the level of DSA just before the first transplantation and found that it was dramatically elevated, which was enough to cause graft rejection. We successfully performed cord blood transplantation of the HLA that was not the target of DSA, as salvage transplantation without any desensitization. This case illustrates that we have to confirm the presence of DSA immediately before the haplo-HSCT, particularly in high risk patients who are dependent on platelet transfusion and have no cytotoxic chemotherapy before HSCT.

[Recurrent invasive pneumococcal disease in a patient with IgG-κ smoldering multiple myeloma].

A 68-year-old female with smoldering multiple myeloma (IgG-κ type) was admitted to the hospital owing to general fatigue, fever, and pain in the right leg. On the day following admission, she developed shock, and a blood culture revealed Streptococcus pneumoniae. She was diagnosed with septic shock and invasive pneumococcal disease (IPD). She received antibiotics and intravenous immunoglobulin and improved after several days. She had a history of recurrent IPD and had received the pneumococcal polysaccharide vaccine 23 (PPSV23) 2 years earlier. Therefore, we inquired with the National Institute of Infectious Diseases if the pneumococcal serotype isolated from her present IPD contained PPSV23. The results showed that her serotype was 19F, a serotype present in PPSV23. We administered pneumococcal conjugate vaccine 13 (PCV13) ; however, she was unable to mount high enough opsonophagocytic assay titers against some serotypes, including 19F. We think she was unable to mount effective humoral immune responses to PPSV23 or PCV13 owing to her underlying disease, smoldering myeloma. It should be considered how IPD can be effectively prevented in patients with multiple myeloma.

Determination of Diphenylamine in Agricultural Products by HPLC-FL.

A method for the determination of diphenylamine in agricultural products was developed. Diphenylamine was extracted with acetonitrile from a sample under an acidic condition, passed through a C18 cartridge column, re-extracted with n-hexane, cleaned up on a PSA cartridge column, determined by HPLC with fluorescence detector and confirmed by liquid chromatography with tandem mass spectrometry. Average recoveries (n=5) from brown rice, corn, soybeans, potato, cabbage, eggplant, spinach, orange, apple and green tea were in the range from 76.7 to 94.9%, and the relative standard deviations were from 0.6 to 5.8% at concentrations equal to the maximum residue limits (MRLs). The quantification limits were 0.01 mg/kg, which is the uniform limit in the positive list system for agricultural chemical residues in food in Japan.

Urethral caruncle in a 9-year-old girl: a case report and review of the literature.

INTRODUCTION: Urethral caruncles are the most frequent benign tumors of the female urethra. Most of them are found in post-menopausal women, and they are rare in childhood. Only a few pediatric cases have been published in the literature. In this report, we present an unusual case of a pediatric patient with a urethral caruncle, along with a review of the literature. CASE PRESENTATION: A 9-year-old Mongolian girl was referred to our hospital with a 2-week history of frequent adherence of a small amount of blood to her underwear. We found a sessile smooth margin, a clear boundary and an elastic, soft red tumor over the entire circumference of the urethral meatus. At the beginning, because of the child's age, urethral prolapse was suspected. There was no response after 3 weeks of conservative treatment with steroid ointment. With the patient under general anesthesia, a partial tumor resection was performed for the purpose of histological examination. The tumor excision was limited to about 1/2 laps of the urethral meatus to prevent the development of urethral stricture. On the basis of clinical and histopathological examinations, a diagnosis of a urethral caruncle was made. Post-operatively, steroid ointment application to residual masses was continued, and these disappeared about 6 months later. Our patient was free of recurrence and had had no complications after 3 years of follow-up. CONCLUSIONS: Urethral caruncles are rare in children, and the possibility of malignancy is slight during this period. Biopsy of the mass is not required for diagnosis. It should be indicated only if the mass has other characteristics that raise suspicion of malignancy. In previously reported cases, all of the tumor was removed. However, the trigger of the caruncle in childhood is chronic inflammation. Conservative therapy with steroid ointment should be the core treatment. However, it may be necessary to proceed to treatment because caruncles take a long time to heal. The case that we describe in this report will serve as an example for similar cases in the future.

Beneficial effects of (-)-epigallocatechin gallate on ischemia-reperfusion testicular injury in rats.

BACKGROUND/PURPOSE: The purpose of the study was to determine the effect of (-)-epigallocatechin gallate (EGCG) on testicular ischemia-reperfusion injury in rats. METHODS: Forty male Wistar rats were assigned to 5 groups. A sham operation was performed on the animals in group 1. In group 2, after 4 hours of unilateral testicular ischemia, 4 hours of testicular reperfusion was performed with EGCG administered 1 hour before reperfusion. In group 3, the same surgical procedure as in group 2 was performed, but without EGCG. Serum superoxide dismutase activity, creatine kinase, and lactate dehydrogenase were then measured in blood samples from groups 1 to 3. In group 4, after 4 hours of unilateral testicular ischemia, testicular reperfusion was performed. In group 5, the same procedure as in group 4 was performed, but with EGCG administered 1 hour before reperfusion. For groups 4 and 5, bilateral orchiectomy was performed for histologic examination 4 weeks after reperfusion was started. RESULTS: Serum superoxide dismutase activity was significantly higher in group 2 than in group 3. The ratios of bilateral testicular weight, mean seminiferous tubule diameter, and germinal epithelial cell thickness were significantly higher in group 5 than in group 4. CONCLUSIONS: Therapy with EGCG before reperfusion might exert protective effects via antioxidant activities in a rat experimental model of testicular ischemia-reperfusion injury.

Glicentin inhibits internalization of enteric bacteria by cultured INT-407 enterocytes.

Glicentin, the main component of enteroglucagon, has trophic effects on intestinal mucosa. It may also have an inhibitory effect on extraintestinal invasion of enteric bacteria. We have established an in vitro bioassay system for determining the effects of recombinant human glicentin on bacterial internalization by confluent enterocytes. An INT-407 cell line was serum-deprived for 2 days and was then treated on transwell filters for 24 h with a medium containing one of the following: glicentin 100 ng-1 microg/ml, glucagons-like peptide-2 (GLP-2) 1 microg/ml, 10% fetal bovine serum (FCS), or without any growth factors. Pure cultures of Salmonella enteritidis, Escherichia coli, and Enterococcus faecalis were introduced to the upper chambers of the filter units. Following 2 h of incubation the numbers of bacteria in the lower chambers were measured. Pretreatment of enterocytes with glicentin inhibited bacterial internalization compared to untreated or GLP-2 enterocytes. Glicentin was associated with inhibition of enterocyte internalization of enteric bacteria by a mechanism that might be related to the integrity of the enterocyte adhesive junctions and tight junctions and to the production of sIgA. Glicentin seems to have a function as a barrier-sustaining agent that inhibits extraintestinal invasion of enteric bacteria.

Involvement of reactive oxygen species and SP-1 in fibronectin production by oxidized LDL.

We examined the mechanisms responsible for the production of fibronectin induced by oxidized low-density lipoprotein (oxLDL) in rat mesangial cells. oxLDL accelerated the production of fibronectin with the preceding generation of reactive oxygen species (ROS). Pretreatment with N-acetylcysteine suppressed the oxLDL-induced fibronectin production as well as ROS generation. oxLDL also elicited the activation of SP-1, nuclear factor-kappaB, and cAMP response element-binding protein, but not activator protein-1. Among these activated transcription factors, N-acetylcysteine inhibited the activation of SP-1 only. 7-Ketocholesterol, an oxidized lipid in oxLDL particles, induced the production of fibronectin and the activation of SP-1, those which were suppressed by N-acetylcysteine. Furthermore, mithramycin A, an inhibitor of SP-1, also suppressed the oxLDL- and 7-ketocholesterol-stimulated production of fibronectin. These results suggest that oxLDL stimulates fibronectin production, at least in part, through the ROS-dependent activation of SP-1 in rat mesangial cells, and further that the ROS-dependent cellular responses may be elicited by 7-ketocholesterol.

A technique using a rectus abdominis muscle flap in the treatment of adult congenital tracheoesophageal fistulas.

The authors successfully treated an adult with congenital tracheoesophageal fistula (TEF) Gross-C type by the addition of an effective pedicled muscle flap. This patient had undergone 2 thoracotomies when he was an infant at another hospital; however, the fistula recurred. After transection and closure of the fistula, a right rectus abdominis muscle flap between the 2 stumps was interposed. The current case followed a favorable postoperative course; neither fistula nor abdominal hernia occurred.

Protein kinase Calpha-dependent increase in Ca2+-independent phospholipase A2 in membranes and arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells.

We previously reported that zymosan-stimulated, protein kinase C (PKC)-dependent arachidonic acid liberation occurs with association of Ca2+-independent phospholipase A2 (iPLA2) with the membranes of macrophage-like P388D1 cells. In the present study, the possible involvement of PKC isoforms (alpha, beta, delta, and epsilon) on the increase in iPLA2 was examined. Stimulation of P388D1 cells with zymosan induced increases in iPLA2 activity and protein in the membranes and liberation of arachidonic acid. In the stimulated cells, PKCalpha, PKCdelta, and PKCepsilon, but not PKCbeta, were increased in the membranes. The zymosan-induced increase in iPLA2 activity was suppressed by pretreatment with 4beta-phorbol 12-myristate 13-acetate for 10 hr, by which PKCalpha and PKCdelta, but not PKCbeta and PKCepsilon, were depleted, and by Gö6976, a PKCalpha inhibitor, but not rottlerin, a PKCdelta inhibitor. The zymosan-induced release of arachidonic acid was also reduced by the PKC depletion and Gö6976. However, stimulation with 4beta-phorbol 12-myristate 13-acetate alone did not increase iPLA2 activity in the membranes. Furthermore, the depletion of intracellular Ca2+ also impaired the zymosan-induced increase in iPLA2 activity in the membranes. However, no increase in iPLA2 activity was observed upon stimulation with Ca2+-mobilizing agents (ionomycin or thapsigargin). Cytochalasin D, an inhibitor of actin polymerization, suppressed the zymosan-induced increases in iPLA2 activity and protein in the membranes and the release of arachidonic acid. These results suggest that zymosan stimulates an increase in iPLA2 in the membranes of P388D1 cells probably through activation of PKCalpha in concert with cytochalasin D-sensitive events.

Photochemical ion receptor based on a structurally distorted ruthenium(II) complex having a crown-ether moiety at the 3,3'-positions on the 2,2'-bipyridine ligand.

Ru(bpy)2(CE-bpy)2+ was prepared where bpy and CE-bpy were 2,2'-bipyridine and bpy having a crown-ether moiety at the 3,3'-positions, respectively. Although Ru(bpy)2(CE-bpy)2+ showed only very weak emission in acetonitrile, recognition of Na+, Li+, or K+ by the crown-ether moiety in CE-bpy resulted in increases in both the emission intensity and the lifetime of the complex, demonstrating that it acted as a photoreceptor. The results were discussed in terms of a steric hindrance between the 3,3'-substituents on CE-bpy and structural changes in both CE-bpy and the complex upon ion recognition, as studied by variable-temperature 1H-NMR and steady-state/dynamic emission spectroscopy of the complex.

Electrogenerated chemiluminescence. 68. Detection of sodium ion with a ruthenium(II) complex with crown ether moiety at the 3,3'-positions on the 2,2'-bipyridine ligand.

We show here how a Ru(bpy)2(CE-bpy) complex can be used for the ECL detection of sodium ion in both aqueous and nonaqueous media. Recognition of Na+ by the crown ether moiety in CE-bpy results in a significant increase in the ECL emission intensity of the complex.