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The importance of genetic diagnosis for patients with hemophilia has been recently demonstrated. However, the pathological variant cannot be identified in some patients. Here, we aimed to identify the pathogenic intronic variant causing hemophilia A using induced pluripotent stem cells (iPSCs) from patients and genome editing. We analyzed siblings with moderate hemophilia A and without abnormalities in the F8 exon. Next-generation sequencing of the entire F8 revealed 23 common intron variants. Variant effect predictor software indicated that the deep intronic variant at c.5220-8563A>G (intron 14) might act as a splicing acceptor. We developed iPSCs from patients and used genome editing to insert the elongation factor 1α promoter to express F8 messenger RNA (mRNA). Then, we confirmed the existence of abnormal F8 mRNA derived from aberrant splicing, resulting in a premature terminal codon as well as a significant reduction in F8 mRNA in iPSCs due to nonsense-mediated RNA decay. Gene repair by genome editing recovered whole F8 mRNA expression. Introduction of the intron variant into human B-domain-deleted F8 complementary DNA suppressed factor VIII (FVIII) activity and produced abnormal FVIII lacking the light chain in HEK293 cells. Furthermore, genome editing of the intron variant restored FVIII production. In summary, we have directly proven that the deep intronic variant in F8 results in aberrant splicing, leading to abnormal mRNA and nonsense-mediated RNA decay. Additionally, genome editing targeting the variant restored F8 mRNA and FVIII production. Our approach could be useful not only for identifying causal variants but also for verifying the therapeutic effect of personalized genome editing., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Gene therapy using adeno-associated virus (AAV)-based vectors has become a realistic therapeutic option for hemophilia. We examined the potential of a novel engineered liver-tropic AAV3B-based vector, AAV.GT5, for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, another bioengineered vector used in a clinical trial. However, liver transduction following intravenous injection of these vectors was similar in mice with a humanized liver and in macaques. This discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the capsid. Bypassing systemic clearance with the intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced liver transduction in pigs and macaques. AAV.GT5 did not develop neutralizing antibodies (NAbs) in two of four animals, while AAV-Spark100 induced serotype-specific NAbs in all macaques tested (4 of 4). The NAbs produced after AAV-Spark100 administration were relatively serotype specific, and challenge with AAV.GT5 through the hepatic artery successfully boosted liver transduction in one animal previously administered AAV-Spark100. In summary, AAV.GT5 showed different vector kinetics and NAb induction compared with AAV-Spark100, and intra-hepatic vascular administration may minimize the vector dose required and vector dissemination., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: Intravenous administration of adeno-associated virus (AAV) vectors is a promising gene therapy approach for monogenic diseases. However, re-administration of the same AAV serotype is impossible because of the induction of anti-AAV neutralizing antibodies (NAbs). Here, we examined the feasibility of re-administrating AAV vector serotypes different from the initial AAV vector serotype., Methods: Liver-targeting AAV3B, AAV5, and AAV8 vectors were intravenously injected in C57BL/6 mice, and the emergence of NAbs and the transduction efficacy following re-administration were evaluated., Results: For all serotypes, re-administration of the same serotype was not possible. Although the highest neutralizing activity of NAb was induced by AAV5, anti-AAV5 NAbs did not react with other serotypes, resulting in successful re-administration with the other serotypes. AAV5 re-administration was also successful in all mice treated with AAV3B and AAV8. Effective secondary administration of AAV3B and AAV8 was observed in most mice initially administrated AAV8 and AAV3B, respectively. However, few mice developed NAbs cross-reacting with the other serotypes, especially those with close sequence homology., Conclusions: In summary, AAV vector administration induced NAbs relatively specific to the administrated serotype. Secondary administration of AAVs targeting liver transduction could be successfully achieved by switching AAV serotypes in mice., (© 2023 John Wiley & Sons Ltd.)

Background: Base editing via CRISPR-Cas9 has garnered attention as a method for correcting disease-specific mutations without causing double-strand breaks, thereby avoiding large deletions and translocations in the host chromosome. However, its reliance on the protospacer adjacent motif (PAM) can limit its use. We aimed to restore a disease mutation in a patient with severe hemophilia B using base editing with SpCas9-NG, a modified Cas9 with the board PAM flexibility., Methods: We generated induced pluripotent stem cells (iPSCs) from a patient with hemophilia B (c.947T>C; I316T) and established HEK293 cells and knock-in mice expressing the patient's F9 cDNA. We transduced the cytidine base editor (C>T), including the nickase version of Cas9 (wild-type SpCas9 or SpCas9-NG), into the HEK293 cells and knock-in mice through plasmid transfection and an adeno-associated virus vector, respectively., Results: Here we demonstrate the broad PAM flexibility of SpCas9-NG near the mutation site. The base-editing approach using SpCas9-NG but not wild-type SpCas9 successfully converts C to T at the mutation in the iPSCs. Gene-corrected iPSCs differentiate into hepatocyte-like cells in vitro and express substantial levels of F9 mRNA after subrenal capsule transplantation into immunodeficient mice. Additionally, SpCas9-NG-mediated base editing corrects the mutation in both HEK293 cells and knock-in mice, thereby restoring the production of the coagulation factor., Conclusion: A base-editing approach utilizing the broad PAM flexibility of SpCas9-NG can provide a solution for the treatment of genetic diseases, including hemophilia B., (© 2023. The Author(s).)

Purpose: Evaluate the frequency of benign versus malignant masses within the prestyloid parapharyngeal space (PPS) and determine if tumor margins on preoperative cross-sectional imaging can predict malignancy status., Materials and Methods: The electronic health record at UC Davis Medical Center was searched for PPS masses surgically resected between 2015 and 2021. Cases located centrally within the prestyloid PPS with confirmed histologic diagnosis were included and separated into either benign or malignant groups. Margins of the tumors were categorized as "well defined" or "infiltrative" on preoperative cross-sectional imaging. Statistical analysis was performed to evaluate relationships between malignancy status and tumor margins., Results: A total of 31 cases met the inclusion criteria. Fourteen separate histologic diagnoses were observed. Benign cases comprised 77% (24/31) and the remaining 23% (7/31) were malignant. Pleomorphic adenoma was the most common overall diagnosis at 48% (15/31). Adenoid cystic carcinoma 6% (2/31) was the most common malignant diagnosis. Well-defined tumor margins were seen in 81% (25/31) of cases. A benign diagnosis was found in 96% (24/25) of the cases with well-defined margins. Infiltrative tumor margins were displayed in 19% (6/31) of cases, all were malignant. The sensitivity and specificity of infiltrative tumor margins for malignancy were 85.7% and 100%, respectively. The negative predictive value of infiltrative margins for malignancy was 96%., Conclusion: Infiltrative tumor margins on preoperative imaging demonstrate high specificity and negative predictive value for malignant histology in prestyloid PPS masses. Margins should therefore be considered when determining clinical management for newly diagnosed PPS tumors.

Adeno-associated virus (AAV) vectors are promising modalities of gene therapy to address unmet medical needs. However, anti-AAV neutralizing antibodies (NAbs) hamper the vector-mediated therapeutic effect. Therefore, NAb prevalence in the target population is vital in designing clinical trials with AAV vectors. Hence, updating the seroprevalence of anti-AAV NAbs, herein we analyzed sera from 100 healthy individuals and 216 hemophiliacs in Japan. In both groups, the overall seroprevalence against various AAV serotypes was 20%-30%, and the ratio of the NAb-positive population increased with age. The seroprevalence did not differ between healthy participants and hemophiliacs and was not biased by the concomitant blood-borne viral infections. The high neutralizing activity, which strongly inhibits the transduction with all serotypes in vitro , was mostly found in people in their 60s or of older age. The multivariate analysis suggested that "60s or older age" was the only independent factor related to the high titer of NAbs. Conversely, a large proportion of younger hemophiliacs was seronegative, rendering them eligible for AAV-mediated gene therapy in Japan. Compared with our previous study, the peak of seroprevalences has shifted to older populations, indicating that natural AAV exposure in the elderly occurred in their youth but not during the last decade., Competing Interests: All authors have no competing financial interests to declare., (© 2022 The Author(s).)

IκBζ is a transcriptional regulator that augments inflammatory responses from the Toll-like receptor or interleukin signaling. These innate immune responses contribute to the progression of nonalcoholic fatty liver disease (NAFLD); however, the role of IκBζ in the pathogenesis of NAFLD remains elusive. We investigated whether IκBζ was involved in the progression of NAFLD in mice. We generated hepatocyte-specific IκBζ-deficient mice (Alb-Cre; Nfkbiz fl/fl ) by crossing Nfkbiz fl/fl mice with Alb-Cre transgenic mice. NAFLD was induced by feeding the mice a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). CDAHFD-induced IκBζ expression in the liver was observed in Nfkbiz fl/fl mice, but not in Alb-Cre; Nfkbiz fl/fl mice. Contrary to our initial expectation, IκBζ deletion in hepatocytes accelerated the progression of NAFLD after CDAHFD treatment. Although the increased expression of inflammatory cytokines and apoptosis-related proteins by CDAHFD remained unchanged between Nfkbiz fl/fl and Alb-Cre; Nfkbiz fl/fl mice, early-stage steatosis of the liver was significantly augmented in Alb-Cre; Nfkbiz fl/fl mice. Overexpression of IκBζ in hepatocytes via the adeno-associated virus vector attenuated liver steatosis caused by the CDAHFD in wild-type C57BL/6 mice. This preventive effect of IκBζ overexpression on steatosis was not observed without transcriptional activity. Microarray analysis revealed a correlation between IκBζ expression and the changes of factors related to triglyceride biosynthesis and lipoprotein uptake. Our data suggest that hepatic IκBζ attenuates the progression of NAFLD possibly through the regulation of the factors related to triglyceride metabolism., (© 2022. The Author(s).)

Background: Patient-reported outcomes are the primary measurement of breast reconstruction success, but results may be affected by nontechnical factors such as socioemotional determinants. Third-party observers provide an independent assessment of aesthetic outcomes. Factors associated with disparity between patient and observer perceptions of outcomes are not well understood., Methods: One hundred forty-seven patients underwent breast reconstruction at the authors' institution between 2009 and 2011, completed the BREAST-Q, and had photographs graded by a diverse panel using the Validated Breast Aesthetic Scale. Patient satisfaction with breasts scores that aligned with observer scores were categorized as group 2; patient satisfaction that exceeded observer scores were group 1; and those lower than observer scores were group 3. Statistical analysis was performed using SPSS, with values of p < 0.05 considered statistically significant., Results: Twenty-eight patients (19 percent) were categorized as group 1, 93 (63 percent) in group 2, and 26 (18 percent) in group 3. Median overall appearance was highest in group 3 (median, 4.0; interquartile range, 4 to 4) and lowest in group 1 (median, 3.0; interquartile range, 2 to 3) ( p < 0.001). Psychosocial, sexual, and physical well-being were significantly associated with disparity (group 1 or 3 status) ( p < 0.01). Satisfaction with outcomes, nipples, abdomen, and breasts were significantly associated with disparity. Factors not significantly associated with disparity include age, body mass index, autologous or implant-based, adjuvant therapies, and timing of reconstruction., Conclusions: Incongruously high patient satisfaction with breast reconstruction aesthetics relative to third-party perception of aesthetic outcomes is associated with high quality-of-life scores. Incongruously low patient satisfaction with breast cosmesis compared with higher third-party perceptions was associated with low quality-of-life scores., Clinical Question/level of Evidence: Risk, II., (Copyright © 2022 by the American Society of Plastic Surgeons.)

Background: Breast aesthetics impacts patients' quality of life after breast reconstruction, but patients and surgeons frequently disagree on the final aesthetic evaluation. The need for a comprehensive, validated tool to evaluate breast aesthetics independently from the patient motivated this study., Methods: The 13-item Validated Breast Aesthetic Scale was developed after several internal meetings, and worded to be understood by a nonspecialist. Three items are common for both breasts, with the remaining being side-specific. To test the internal consistency of the scale subitems, postoperative photographs after different breast reconstruction techniques were graded by a six-member panel. To test interrater and intrarater correlation across time, four physicians evaluated the results of abdominally based breast reconstructions following nipple-sparing mastectomies., Results: Graded aesthetic outcomes of 53 patients showed that the Cronbach alpha of the subitems of the scale was 0.926, with no single item that, if excluded, would increase it. Twenty-two patients underwent aesthetic outcomes grading at four different time points. The mean overall appearance was 3.71 ± 0.62. The mean grade for overall nipple appearance was 4.0 ± 0.57. The coefficient alpha of the panel overall aesthetic grade across different time points was 0.957; whereas intragrader reliability for graders 1 through 4 individually showed alpha coefficients of 0.894, 0.9, 0.898, and 0.688, respectively. Similar results were found for the other items of the scale., Conclusions: The proposed aesthetic scale evaluates different aspects of the breast reconstruction aesthetic result with excellent internal consistency among its subitems. Grading by a gender-balanced, diverse four-member panel using postoperative photographs showed higher reliability and reproducibility compared to single graders., (Copyright © 2022 by the American Society of Plastic Surgeons.)

Coagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31 high , CD146 high , and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1) + . EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin - and C-type lectin-like receptor-2 (CLEC-2) + . In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31 high , CD146 high , Lyve1 + , CLEC-2 + , and podoplanin - in liver sinusoidal endothelial cells., (© 2021. The Author(s).)

Most gene therapy clinical trials that systemically administered adeno-associated virus (AAV) vector enrolled only patients without anti-AAV-neutralizing antibodies. However, laboratory tests to measure neutralizing antibodies varied among clinical trials and have not been standardized. In this study, we attempted to improve the sensitivity and reproducibility of a cell-based assay to detect neutralizing antibodies and to determine the detection threshold to predict treatment efficacy. Application of the secreted type of NanoLuc and AAV receptor-expressing cells reduced the multiplicity of infection (MOI) for AAV transduction and improved the sensitivity to detect neutralizing antibodies with a low coefficient of variation, whereas the detection threshold could not be improved by the reduction of MOI to <100. After human immunoglobulin administration into mice at various doses, treatment with high-dose AAV8 vector enabled evasion of the inhibitory effect of neutralizing antibodies. Conversely, gene transduction was slightly influenced in the mice treated with low-dose AAV8 vector, even when neutralizing antibodies were determined to be negative in the assay. In conclusion, we developed a reliable and sensitive cell-based assay to measure neutralizing antibodies against AAV and found that the appropriate MOI to detect marginal neutralizing antibodies was 100. Other factors, including noninhibitory antibodies, marginally influence in vivo transduction at low vector doses., Competing Interests: T.O. received grant support from Novo Nordisk outside the scope of the study. M.H. received a research grant from Bristol-Myers Squibb outside the scope of this study. The remaining authors declare no competing interests., (© 2021 The Author(s).)

Introduction: The use of acellular dermal matrix (ADM) for breast reconstruction continues to change in both single- and two-stage reconstruction. Determining optimal outcomes clinically, aesthetically, financially as well as for the patient's quality of life has become a priority., Methods: A retrospective review of implant-based reconstructions was performed at a single center from 2010 to 2016, with patients blindly matched 1:1:1 into three cohorts based on reconstruction type: 1) single stage direct to implant with ADM, 2) two-stage tissue expander to implant (TE/I) without ADM, and 3) two-stage TE/I with ADM. Relative cost between groups, esthetic outcomes, and quality of life within each group was analyzed., Results: Group 1 was more likely to be older and use intraoperative angiography, but with fewer overall surgeries and postoperative visits (p<0.001). There was no statistically significant difference in reconstructive success among all three groups (p = 0.85). Cost was significantly higher for group 3 relative to groups 1 and 2. Overall appearance was higher in groups 1 and 3 relative to group 2, with radiation therapy the only independent factor. Group 1 had higher scores using Breast-Q for the physical well-being domain (p = 0.01)., Conclusion: This is the first study to incorporate clinical outcomes, esthetic visual grading, and patient-reported quality within the same cohort of individuals, considering both use of ADM and staging. Despite the added ADM cost, it is proven safe, eliminates time and cost associated with tissue expanders, decreases post-operative visits and can lead to equally as functional and aesthetically pleasing outcomes in single- and two-stage breast reconstructions., Competing Interests: Declaration of competing interest This study was supported by funding from Allergan. No other conflict of interest among authors., (Copyright © 2020. Published by Elsevier Ltd.)

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

Psychoneuroimmunological studies have clearly demonstrated that both cellular and humoral immunity are related to major depression. Soluble ST2 is regarded as a key molecule regulating immune system as well as cell proliferation. Indeed, soluble ST2 is reported to reduce IL-33-induced IL-6 and TNF-α production in macrophages and IL-33-induced IL-5 and IL-13 production in type 2 innate lymphoid cells. Elevated serum concentrations of soluble ST2 have been reported in patients with neuropsychiatric disorders, suggesting pathophysiological roles of soluble ST2 in behavioral phenotypes. Nevertheless, the relation between soluble ST2 and depressive behavior remain to be uncovered. To complement this point, we performed broad behavioral phenotyping, utilizing transgenic mice with a high concentration of serum ST2 in the present study. Soluble ST2 overexpression mice (ST2 Tg mice) were generated on a C3H/HeJ background. ST2 Tg mice crossed onto the BALB/c genetic background were used. Before starting tests, each mouse was observed in a clean cage for a general health check and neurological screening tests. In Experiment I, comprehensive behavioral phenotyping was performed to reveal the role of soluble ST2 on sensorimotor functions, anxiety-like behaviors, depression-like behaviors, social behaviors, and learning and memory functions. In Experiment II, to confirm the role of soluble ST2 on depression-like behaviors, a depression test battery (two bottle choice test, forced swimming test, and tail suspension test) was applied. The general health check indicated good general health and normal gross appearance for ST2 Tg mice. Further, the neurological reflexes of all the mice were normal. We found that soluble ST2 overexpression resulted in decreased social interaction. Moreover, depression-like behaviors of ST2 Tg mice were observed in two well-established behavioral paradigms, the forced swimming test and the tail suspension test. Nevertheless, hedonic reaction to sucrose was observed in ST2 Tg mice similar to WT mice. These results suggest the depression in the ST2 Tg mice. In conclusion, through a series of experiments, we established the animal model for assessing role of soluble ST2 in neuropsychiatric disorders, and revealed the possible involvement of soluble ST2 in depressive behavior.

Background: The most important purpose of reconstruction is to increase or restore the patient's quality of life (QOL). The purpose of our study was to evaluate the QOL and aesthetic outcomes of patients after autologous versus implant-based breast reconstruction., Methods: Patients who underwent breast reconstruction between 2009 and 2011 were included. The Breast-Q, a validated breast reconstruction QOL questionnaire, was used along with postoperative photographs panel analyses using a multiparameter breast-specific aesthetic outcome scale and retrospective evaluation of demographic and treatment data., Results: Of 820 patients, 261 complete questionnaires were evaluated. On the multivariable linear regression, the "satisfaction with breasts" was positively influenced by autologous and bilateral reconstructions, whereas radiation therapy (RTx), the time between the reconstruction and the questionnaire, and the number of surgeries due to complications were negative factors (adjusted R = 0.183; P < 0.001). The same factors influenced the "satisfaction with the outcomes." The mean "overall breast appearance" was also positively influenced by autologous and bilateral reconstructions, and RTx and the total number of surgeries were negative predictive factors (adjusted R = 0.311, P < 0.001)., Conclusions: The aesthetic result and QOL after breast reconstruction for breast cancer treatment are positively influenced by the use of autologous tissue and bilaterality. Factors that negatively influenced the aesthetic result and the QOL include use of RTx, a higher number of surgeries needed for the reconstruction, reoperations due to complications, higher body mass index, and a longer time elapsed between reconstruction and the questionnaire.

IκBζ (encoded by the Nfkbiz ) is a member of the nuclear IκB family, which is involved in the expression of secondary response genes based on signals from TLR or IL-1R. ST2L, an IL-33R, is a member of the IL-1R family and abundantly expressed in tissue-resident immune cells, such as mast cells and innate lymphoid cells; however, its downstream signaling pathway remains unelucidated. In this study, we examined the role of IκBζ in ST2L-mediated cytokine and chemokine production in mast cells. Murine bone marrow cells were differentiated ex vivo into bone marrow-derived mast cells (BMMCs). The treatment of BMMCs with IL-33 transiently induced robust IκBζ expression. Of the 40 cytokines and chemokines examined using a cytokine and chemokine array, the concentrations of IL-6, IL-13, CCL2, CCL3, and TNF-α in the supernatant were augmented by IL-33. The deletion of IκBζ in BMMCs resulted in a significant reduction of the production of these mediators and the expression of their mRNA. NF-κB p50 but not p65 translocated to the nucleus by IL-33 and was not affected by the deletion of IκBζ. However, induction of IκBζ and the resultant cytokine and chemokine productions were significantly inhibited by pretreatment with an NF-κB inhibitor. The deletion of IκBζ did not affect the phosphorylation of ERK, p38 MAPK, or JNK by IL-33, and the treatment with inhibitors of these mitogen-activated kinases failed to abolish the expression of Nfkbiz Our findings suggest that IκBζ augments IL-33-dependent cytokine and chemokine production in BMMCs through the action of NF-κB., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Background: Chemokines and chemokine receptors are potential targets for the prevention and treatment of graft-versus-host disease (GVHD). The objective of the current study is to determine the clinical relevance of xenogeneic transplantation models in terms of host and donor chemokine profiles and, if this is the case, to assess the clinical efficacy of C-C chemokine receptor (CCR) 5 antagonist maraviroc for the prevention of GVHD using this model., Methods: Xenogeneic GVHD was induced by intravenous injection of 5 × 10 human pan T cells into NOD/Shi-scid-IL2rγ (NOG) mice or MHC class I/II-deficient NOG mice in the presence or absence of total body irradiation before transplantation., Results: Extensive tissue destruction with human T-cell infiltration was observed throughout the body, particularly in lungs and liver, but relatively mild in gut. Consistent with this finding, quantitative polymerase chain reaction confirmed the upregulation of mouse CXC chemokine ligand (CXCL) 9 and CXCL10 in lungs and CCL4 in lungs and liver but not in gut. The addition of total body irradiation (1) led to the early release of mouse CCL4 and CXCL10, (2) upregulated a number of chemokine-related genes in human T cells, (3) induced higher expression of CCR5 on human CD4 and CD8 T cells and CXCR3 on human CD4 T cells, and (4) promoted their migration and proliferation in organs, resulting in more severe tissue damage. In this context, pharmacological CCR5 blockade neither ameliorated GVHD nor prolonged survival in NOG mice., Conclusions: Our experimental data do not demonstrate clinical benefit of CCR5 antagonist for the prevention of GVHD in a myeloablative setting.

Objective: The objectives of this study are to evaluate incidence, duration, and quality of life (QOL) impact of early tympanostomy tube otorrhea and tube patency when comparing topical ciprofloxacin versus normal saline use in the perioperative period., Methods: Overall, 200 patients undergoing tube placement between November 19, 2015, and September 12, 2016, were randomized to intraoperative plus 5 days of either topical ciprofloxacin or normal saline. Parents or caregivers reported the incidence, duration, and QOL impact of early otorrhea via 4 weekly surveys. In addition, the patient's otorrhea history and tube patency were evaluated at a 4- to 6-week postoperative visit., Results: Survey and in-office follow-ups were completed on 128 patients. The overall otorrhea incidence was 23.9% for normal saline and 16.7% for ciprofloxacin ( P = .32). The week-by-week otorrhea incidence was not statistically different. The percentage of days otorrhea was present, likewise, was not statistically different (normal saline 4.5%, ciprofloxacin 2.8%; P = .74). The QOL impact was not statistically different (normal saline 1.2, ciprofloxacin 1.5; P = .71). Tube patency was not statistically different, with only 1 of 280 ears occluded at follow-up., Conclusion: We find no difference in the incidence, duration, and QOL impact of early tympanostomy tube otorrhea or tube patency between ciprofloxacin and normal saline. This supports the option to substitute normal saline for ciprofloxacin in ears without an active ear infection at the time of tube placement, which would reduce both cost and unnecessary antibiotic use., Level of Evidence: 1b.

Objective- Inhibition of HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is atheroprotective primarily by decreasing plasma LDL (low-density lipoprotein)-cholesterol. However, it is unknown whether inhibition of HMGCR in myeloid cells contributes to this atheroprotection. We sought to determine the role of myeloid HMGCR in the development of atherosclerosis. Approach and Results- We generated mice with genetically reduced Hmgcr in myeloid cells ( Hmgcr m- /m - ) using LysM (Cre) and compared various functions of their macrophages to those of Hmgcr fl/fl control mice. We further compared the extent of atherosclerosis in Hmgcr m-/ m- and Hmgcr fl/fl mice in the absence of Ldlr (LDL receptor). Hmgcr m-/ m- macrophages and granulocytes had significantly lower Hmgcr mRNA expression and cholesterol biosynthesis than Hmgcr fl/fl cells. In vitro, Hmgcr m-/ m- monocytes/macrophages had reduced ability to migrate, proliferate, and survive compared with Hmgcr fl/fl monocytes/macrophages. However, there was no difference in ability to adhere, phagocytose, store lipids, or polarize to M1 macrophages between the 2 types of macrophages. The amounts of plasma membrane-associated small GTPase proteins, such as RhoA (RAS homolog family member A), were increased in Hmgcr m-/ m- macrophages. In the setting of Ldlr deficiency, Hmgcr m-/ m- mice developed significantly smaller atherosclerotic lesions than Hmgcr fl/fl mice. However, there were no differences between the 2 types of mice either in plasma lipoprotein profiles or in the numbers of proliferating or apoptotic cells in the lesions in vivo. The in vivo migration of Hmgcr m-/ m- macrophages to the lesions was reduced compared with Hmgcr fl/fl macrophages. Conclusions- Genetic reduction of HMGCR in myeloid cells may exert atheroprotective effects primarily by decreasing the migratory activity of monocytes/macrophages to the lesions.

Background: Natural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined., Methods: Tumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed., Results: Cultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab., Conclusions: Tumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.

Background: The ability of radiation to enhance antitumor immunity under specific experimental conditions is well established. Here, we explore preclinical data and the rationale for combining different radiation doses and fractions with immune checkpoint blockade immunotherapy., Methods: We conducted a review of the literature., Results: The ability of high-dose or hypofractionated radiation to enhance antitumor immunity resulting in additive or synergistic tumor control when combined with checkpoint blockade is well studied. Whether low-dose daily fractionated radiation does the same is less well studied and available data suggests it may be immunosuppressive., Conclusion: Although daily fractionated radiation is well established as the standard of care for the treatment of patients with head and neck cancer, how this radiation schema alters antitumor immunity needs further study. If the radiation doses and fractions alter antitumor immunity differently can have profound implications in the rational design of clinical trials investigating whether radiation can enhance response rates to immune checkpoint blockade., (© 2018 Wiley Periodicals, Inc.)

Preclinical evidence suggests that high-dose hypofractionated ionizing radiation (IR) can enhance anti-tumor immunity and result in significant tumor control when combined with immune checkpoint blockade (ICB). However, low-dose daily fractioned IR used for many tumor types including head and neck squamous cell carcinoma results in lymphopenia and may be immunosuppressive. We compared immune correlates, primary tumor and abscopal tumor control rates following the addition of PD-1 mAb to either high-dose hypofractioned (8Gyx2) or low-dose daily fractionated (2Gyx10) IR in syngeneic models of cancer. When compared to 2Gyx10 IR, 8Gyx2 IR preserved peripheral and tumor-infiltrating CD8 + T-lymphocyte accumulation and activation and reduced peripheral and tumor gMDSC accumulation. Regulatory T-lymphocytes were largely unaltered. Type I and I IFN levels and expression of IFN-responsive MHC class I and PD-L1 was enhanced in tumors treated with 8Gyx2 compared to 2Gyx10 IR. Functionally, tumor-specific CD8 + T-lymphocyte IFN responses within tumor draining lymph nodes were enhanced following 8Gyx2 IR but suppressed following 2Gyx10 IR. When combined with PD-1 mAb, reversal of adaptive immune resistance and subsequent enhancement of CD8+ cell dependent primary and abscopal tumor control was observed following 8Gyx2 but not 2Gyx10 IR. These data strongly support that compared to daily fractionated low-dose IR, high-dose hypofractionated IR preserves or enhances anti-tumor immunity and, when combined with PD-1 mAb to reverse adaptive immune resistance, promotes anti-tumor immunity to control primary and distant tumors. These data critically inform the rational design of trials combining IR and ICB.

Objectives: Determine if direct tumor cell cytotoxicity, antigen release, and susceptibility to T-lymphocyte killing following radiation treatment is dose-dependent., Materials and Methods: Mouse oral cancer cells were engineered to express full-length ovalbumin as a model antigen. Tumor antigen release with uptake and cross presentation of antigen by antigen presenting cells with subsequent priming and expansion of antigen-specific T-lymphocytes following radiation was modeled in vitro and in vivo. T-lymphocyte mediated killing was measured following radiation treatment using a novel impedance-based cytotoxicity assay., Results: Radiation treatment induced dose-dependent induction of executioner caspase activity and apoptosis in MOC1 cells. In vitro modeling of antigen release and T-lymphocyte priming demonstrated enhanced proliferation of OT-1 T-lymphocytes with 8Gy treatment of MOC1ova cells compared to 2Gy. This was validated in vivo following treatment of established MOC1ova tumors and adoptive transfer of antigen-specific T-lymphocytes. Using a novel impedance-based cytotoxicity assay, 8Gy enhanced tumor cell susceptibility to T-lymphocyte killing to a greater degree than 2Gy., Conclusion: In the context of using clinically-relevant doses of radiation treatment as an adjuvant for immunotherapy, 8Gy is superior to 2Gy for induction of antigen-specific immune responses and enhancing tumor cell susceptibility to T-lymphocyte killing. These findings have significant implications for the design of trials combining radiation and immunotherapy., (Published by Elsevier Ltd.)

The evolutionarily conserved protein kinase p38 mediates innate resistance to environmental stress and microbial infection. Four p38 isoforms exist in mammals and may have been co-opted for new roles in adaptive immunity. Murine T cells deficient in p38α, the ubiquitously expressed p38 isoform, showed no readily apparent cell-autonomous defects while expressing elevated amounts of another isoform, p38β. Mice with T cells simultaneously lacking p38α and p38β displayed lymphoid atrophy and elevated Foxp3 + regulatory T cell frequencies. Double deficiency of p38α and p38β in naïve CD4 + T cells resulted in an attenuation of MAPK-activated protein kinase (MK)-dependent mTOR signaling after T cell receptor engagement, and enhanced their differentiation into regulatory T cells under appropriate inducing conditions. Pharmacological inhibition of the p38-MK-mTOR signaling module produced similar effects, revealing potential for therapeutic applications., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Type 2 innate lymphoid cells (ILC2) in lungs produce interleukin (IL)-5 and IL-13 in response to IL-33 and may contribute to the development of allergic diseases such as asthma. However, little is known about negative regulators and effective inhibitors controlling ILC2 function. Here, we show that soluble ST2, a member of the IL-1 receptor family, suppresses the effect of IL-33 on lung ILC2 in vitro . Stimulation with IL-33 to naïve ILC2 induced morphological change and promoted cell proliferation. In addition, IL-33 upregulated expression of cell surface molecules including IL-33 receptor and induced production of IL-5 and IL-13, but not IL-4. Pretreatment with soluble ST2 suppressed IL-33-mediated responses of ILC2. The results suggest that soluble ST2 acts as a decoy receptor for IL-33 and protects ILC2 from IL-33 stimulation.

Cell-membrane lipid composition can greatly influence biophysical properties of cell membranes, affecting various cellular functions. We previously showed that lipid synthesis becomes altered in the membranes of resistant breast cancer cells (MCF-7/ADR); they form a more rigid, hydrophobic lipid monolayer than do sensitive cell membranes (MCF-7). These changes in membrane lipids of resistant cells, attributed to epigenetic aberration, significantly affected drug transport and endocytic function, thus impacting the efficacy of anticancer drugs. The present study's objective was to determine the effects of the epigenetic drug, 5-aza-2'-deoxycytidine (DAC), delivered in sustained-release nanogels (DAC-NGs), on the composition and biophysical properties of membrane lipids of resistant cells. Resistant and sensitive cells were treated with DAC in solution (DAC-sol) or DAC-NGs, and cell-membrane lipids were isolated and analyzed for lipid composition and biophysical properties. In resistant cells, we found increased formation of cholesterol-sphingomyelin (CHOL-SM) rafts with culturing time, whereas DAC treatment reduced their formation. In general, the effect of DAC-NGs was greater in changing the lipid composition than with DAC-sol. DAC treatment also caused a rise in levels of certain phospholipids and neutral lipids known to increase membrane fluidity, while reducing the levels of certain lipids known to increase membrane rigidity. Isotherm data showed increased lipid membrane fluidity following DAC treatment, attributed to decrease levels of CHOL-SM rafts (lamellar beta [Lβ] structures or ordered gel) and a corresponding increase in lipids that form lamellar alpha-structures (Lα, liquid crystalline phase). Sensitive cells showed marginal or insignificant changes in lipid profile following DAC-treatment, suggesting that epigenetic changes affecting lipid biosynthesis are more specific to resistant cells. Since membrane fluidity plays a major role in drug transport and endocytic function, treatment of resistant cells with epigenetic drugs with altered lipid profile could facilitate anticancer drug transport to overcome acquired drug resistance in a combination therapy.

The cytokine interleukin-4 (IL-4) exerts pleiotropic effects on macrophages as it plays a key role in the immune response to infectious agents, allergens, and vaccines. Macrophages exposed to IL-4 drastically change their gene expression and metabolic state to adjust to new functional requirements. IL-4 also induces macrophages to fuse together and form multinucleated giant cells (MGCs). MGC formation is associated with chronic inflammation resulting from persistence of pathogenic microorganisms or foreign materials in tissues. Very little is known, however, about the mechanisms regulating IL-4-induced macrophage-to-MGC conversion. We observed a dramatic increase in β-catenin protein but not mRNA amount in mouse macrophages following exposure to IL-4. To investigate the role of β-catenin in macrophages, we generated mice with a myeloid cell-specific deletion of the β-catenin gene. Ablation of β-catenin expression did not affect the viability of macrophages or impair expression of known IL-4-inducible genes. Intriguingly, β-catenin-deficient macrophages incubated with IL-4 formed MGCs with markedly greater efficiency than wild-type macrophages. Similar increases in multinucleated cell formation were detected in the peritoneal cavity of myeloid cell-specific β-catenin knockout mice injected with chitin, which is known to induce endogenous IL-4 production. Our findings reveal β-catenin as a novel regulator of macrophage responses to IL-4, and suggest that therapeutic modulation of its expression or function may help enhance the effectiveness or ameliorate the pathology of IL-4-driven immune responses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Interleukin-33 (IL-33) is a dual-function molecule that regulates gene expression in nuclei and, as a cytokine, conveys proinflammatory signals from outside of cells via its specific receptor ST2L. There are still a lot of questions about localization and processing of IL-33 gene products. In the course of re-evaluating human IL-33 gene, we found distinct promoter usage depending on the cell type, similar to the case in the ST2 gene. Furthermore, we found a novel exon 2E in the conventional intron 2 whose open reading frame corresponded to a transmembrane protein of 131 amino acids. Dependence of exon 2E expression on differentiation of HUVEC cells is of great interest in relation to human IL-33 function., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nitrogen-containing bisphosphonates (NCBPs) have been widely used as standard supportive therapy to reduce skeletal-related events (SREs) in myeloma patients through suppression of osteoclast activity. In various prospective randomized trials that were performed following preliminary reports concerning efficacy, NCBPs have shown a significant beneficial effect on myeloma bone disease through both suppression of bone resorption and direct antimyeloma activity. Thus, NCBPs have an influence on many types of human cells. In this study, we examined the effect of an NCBP (YM-175) on an apoptosis of a monocytic cell line and of human native monocytes/macrophages and dendritic cells (DCs). We confirmed that monocytes, monocyte-derived macrophages, DCs, and a monoblastic cell line (THP-1) showed dose-dependent and time-dependent apoptosis related to the activation of caspases after exposure to YM-175 at concentrations below that at which the apoptosis of myeloma cell lines was induced. Such apoptosis of monocytic cells was suppressed by the addition of farnesol or geranylgeraniol. These findings suggest that the inhibition of monocyte-lineage cells or DCs by NCBPs might interfere with phagocytic activity or pathogen-presenting activity., (Copyright © 2012 Wiley Periodicals, Inc.)

The kinase p38α, originally identified because of its endotoxin- and cytokine-inducible activity and affinity for antiinflammatory compounds, has been posited as a promising therapeutic target for various immune-mediated disorders. In clinical trials, however, p38α inhibitors produced adverse skin reactions and other toxic effects that often outweighed their benefits. Such toxicity may arise from a perturbation of physiological functions unrelated to or even protective against the disease being treated. Here, we show that the effect of interfering with p38α signaling can be therapeutic or adverse depending on the targeted cell type. Using a panel of mutant mice devoid of p38α in distinct cell types and an experimental model of allergic skin disease, we find that dendritic cell (DC)-intrinsic p38α function is crucial for both antigen-specific T-cell priming and T-cell-mediated skin inflammation, two independent processes essential for the immunopathogenesis. By contrast, p38α in other cell types serves to prevent excessive inflammation or maintain naïve T-cell pools in the peripheral lymphoid tissues. These findings highlight a dilemma in the clinical use of p38α inhibitors, yet also suggest cell-selective targeting as a potential solution for improving their therapeutic index.

IL-33, a member of the IL-1 family, activates MAPK and NF-kappaB through its receptor ST2L and IL-1RAcP. ST2, a member of the IL-1R superfamily, is a secreted form of ST2 gene products, which has been shown to act as a decoy receptor for IL-33 and to inhibit the IL-33/ST2L/IL-1RAcP signaling pathway. In this work, we generated ST2 transgenic mice. In control mice, intraperitoneal administration of IL-33 caused an increased number of eosinophils in blood and in peritoneal cavity, an increased number of peritoneal M Phi, splenomegaly, accumulation of periodic acid-Schiff-positive material in the lung, and high concentrations of serum IL-5 and IL-13. However, these alterations were hardly detectable in ST2 Tg mice. In peritoneal M Phi from IL-33-stimulated mice, mRNA expression of M2 M Phi marker genes were increased compared with thioglycollate-elicited peritoneal M Phi. The IL-33-stimulation also increased the secretion of IL-6 from M Phi. However, when the IL-33 was preincubated with ST2 prior to its addition to the M Phi cultures, the secretion of IL-6 was attenuated. These data suggest that, though IL-33 induced the Th2-type immune responses and infiltration of M2 type M Phi into the peritoneal cavity, ST2 can downregulate these reactions both in vivo and in vitro.

ST2 gene products that are members of IL-1 receptor family are expressed in various cells such as growth-stimulated fibroblasts and Th2 helper T-cells, and recently, IL-33, which belongs to IL-1 family, was identified as the ligand for ST2L, the receptor type product of the ST2 gene. Subsequently, IL-33 and ST2L have been reported to be involved in Th2 immunity and inflammation, however, their functions on non-immunological cells are still obscure. Among non-immunological adhesive cells, vascular endothelial cells were reported to express both ST2 gene products and IL-33, therefore, we investigated the expression manner of the ST2 gene in vascular endothelial cells and the effect of IL-33 on endothelial cells. ST2 gene was expressed in each of the vascular endothelial cell types tested, and the expression was growth-dependent and down-regulated when the cells were differentiated to form vascular structures on the extracellular membrane matrix. IL-33 scarcely affected the growth and tube formation of the endothelial cells, but induced IL-6 and IL-8 secretion from endothelial cells with the rapid activation of extracellular signal-regulated kinase (ERK) 1/2, so IL-33 is supposed to involve in inflammatory reaction of vascular endothelial cells through its receptor, ST2L.