Direct intramuscular injection (IM) of adeno-associated virus (AAV) has been proven a safe and potentially efficient procedure for gene therapy of many genetic diseases including hemophilia B. It is, however, contentious whether high antigen level induces tolerance or immunity to coagulation factor IX (FIX) following IM of AAV. We recently reported induction of FIX-specific immune tolerance by IM of AAV serotype one (AAV1) vector in mice. We hypothesize that the expression of high levels of FIX is critical to induction of FIX tolerance. In this study, we investigated the correlation among AAV dose, FIX expression, and tolerance induction. We observed that induction of immune tolerance or immunity to FIX was dependent on the dose of AAV1-human FIX (hFIX) given and the level of FIX antigen expressed in both normal and hemophilia mice. We then defined the minimum AAV1-hFIX dose and the lowest level of FIX needed for FIX tolerance. Different from hepatic AAV-hFIX gene transfer, we found that FIX tolerance induced by IM of AAV1 was not driven by regulatory T cells. These results provided further insight into the mechanism(s) of FIX tolerance, contributing to development of hemophilia gene therapy, and optimization of FIX tolerance induction protocols.

, SEM ± 35.7) and hFIX-specific immune tolerance in C57BL/6 mice. Conclusions: A single i.m. of AAV1 can result in efficient expression of therapeutic levels of hFIX and induction of hFIX tolerance in hemostatically normal and hemophilic B mice. Our results substantiate the prospect of i.m. of AAV1 for hemophilia B gene therapy and FIX tolerance induction.

Interactions of human natural killer (NK) cell inhibitory receptors with polymorphic HLA-A, -B and -C molecules educate NK cells for immune surveillance against tumor cells. The KIR A haplotype encodes a distinctive set of HLA-specific NK cell inhibiting receptors having strong influence on immunity. We observed higher frequency of KIR A homozygosity among 745 healthy Chinese Southern Han than 836 adult patients representing three types of leukemia: ALL (OR = 0.68, 95% CI = 0.52–0.89, p = 0.004), AML (OR = 0.76, 95% CI = 0.59–0.98, p = 0.034), and CML (OR = 0.72 95% CI = 0.51–1.0, ns). We observed the same trend for NHL (OR = 0.47 95% CI = 0.26–0.88 p = 0.017). For ALL, the protective effect of the KIR AA genotype was greater in the presence of KIR ligands C1 (Pc = 0.01) and Bw4 (Pc = 0.001), which are tightly linked in East Asians. By contrast, the C2 ligand strengthened protection from CML (Pc = 0.004). NK cells isolated from KIR AA individuals were significantly more cytotoxic toward leukemic cells than those from other KIR genotypes ( p < 0.0001). These data suggest KIR allotypes encoded by East Asian KIR A haplotypes are strongly inhibitory, arming NK cells to respond to leukemogenic cells having altered HLA expression. Thus, the study of populations with distinct KIR and HLA distributions enlightens understanding of immune mechanisms that significantly impact leukemia pathogenesis.

Acute myeloid leukemia (AML) is a hematological malignancy with a low survival rate. Curcumin, which is a multi-targeted anticancer agent, has been shown to exert anti‑oxidant, anti‑inflammatory, anti‑mutagenic and anti‑carcinogenic activities. Naringenin is extracted from citrus fruits and exerts anti‑mutagenic and anti‑carcinogenic activities in various types of cancer cells. However, the effects of curcumin and naringenin in combination in AML cells have yet to be studied. The present study aimed to investigate the combination effects of curcumin and naringenin on the viability, cell cycle distribution and apoptosis rate of THP‑1 cells using cell viability assays, flow cytometry, and western blotting. Naringenin enhanced curcumin‑induced apoptosis and cell viability inhibition. In addition, curcumin and naringenin induced cell cycle arrest at S phase and G2/M phase. Numerous pathways, including p53, c‑Jun N‑terminal kinases (JNK), Akt and extracellular signal‑regulated kinases (ERK)1/2 pathways were markedly altered following treatment of THP‑1 cells with curcumin and naringenin. These results indicated that naringenin may enhance curcumin‑induced apoptosis through inhibiting the Akt and ERK pathways, and promoting the JNK and p53 pathways.

Background:The mechanistic association ofHLA-DRB1alleles that code a “shared epitope” (SE) with rheumatoid arthritis (RA) is not yet clear. Previous data has suggested the carriage of SE is associated with the production of cyclic citrullinated peptide antibodies (anti-CCP)1and severe RA2-4. The interrelationship among SE, anti-citrullinated protein antibody (ACPA) positivity and disease outcomes is not fully understood.Objectives:To assess the RA prognosis associated with the carriage of SE, in relation to ACPA positivity.Methods:Pts enrolled in a large RA registry, Brigham and Women’s Hospital RA Sequential Study between March 2003 to June 2018, with known SE and ACPA status were included in the analysis. HLA-DRB1 SE status was determined by allele-specific polymerase chain reaction and DNA sequencing for most of the subjects and by GWAS-based imputation for the rest. Disease activity (DA) was measured at baseline (BL) and 1-year follow-up by DAS28(CRP), CDAI and SDAI. Pts were stratified by SE+ (1 or 2 SE alleles) and SE- (0 alleles) and ACPA status. We analyzed the relationship of SE with ACPA positivity and change in DA by a linear regression model separately. A mediation analysis was used to examine the mediating effect of ACPA on association between SE and change in DA.Results:Out of 926 pts included in the analysis, 65.1% were SE+, of whom 75.6% were ACPA+. In comparison, 51.7% were ACPA+ in SE- pts. SE+ pts were similar with SE- pts in age, gender, BMI and smoking status, but had longer disease duration, were more likely to be rheumatoid factor positive, have erosive disease and higher comorbidity burden irrespective of ACPA status. The differences were more pronounced if the pts were also ACPA+. Adjusting for BL differences, pts with SE 1 and 2 alleles (vs 0) had an odd ratio of 1.97 (95% CI:1.36-2.84; p=0.0003) and 3.82 (95% CI: 2.44-5.98; p<.0001) to be ACPA +, respectively. The regression analysis suggests that SE+ (vs SE-) pts had an average increase in DAS28 (CRP) of 0.22 (p=0.033), CDAI of 2.07 (p=0.045) and SDAI of 2.43 (p=0.029) over a year (Fig 1). Using a mediation analysis, the direct effect of SE+ account for 78.8% to 81.0% of total effect in the increase in DAS28 (CRP), CDAI and SDAI, and the indirect effect mediated by ACPA account for 19.0% to 21.2% (Table 1).Table 1.Mediation Analysis for SE and ACPA Association with Change in DAParameterChange in DAS28 CRP (N=666)Change in CDAI (N=653)Change in SDAI (N=629)EstimateP-valueEstimateP-valueEstimateP-valueTotal Effect of SE on DA change0.220.0342.050.0472.400.030Direct effect of SE on DA change excluding mediation of ACPA0.170.1011.570.1401.890.098Indirect effect of SE on DA change due to ACPA mediation and interaction0.040.1830.480.1330.510.143The model is adjusted with other covariates: Age, Gender, Charlson comorbidity score; baseline biologic use, Smoking status, baseline DA, Interaction term (ACPA*SE)Figure 1.Linear Regression Model for SE Association with Change in Disease Activity *Estimates, p-values are shown as data labels on the graphs; Change in disease activity (DA) = (follow-up DA- baseline DA); The above model is adjusted for age, gender, CCI, baseline DA, baseline biologic use, SE status and smoking statusConclusion:SE is strongly related to ACPA and a greater burden of disease in RA pts. In pts receiving standard treatments including biologics, SE is predictive of a greater increase in DA, which is partially mediated by the presence of ACPA.References:[1] Dayan I, et al.,Arch of Rheumatology, 2010;25:012-018.[2] Gregerson PK, et al,Arthritis Rheum. 1987;30:1205-1213.[3] Turesson C, et al.Arthritis Res Ther. 2005;7:R1386-1393.[4] Moreno I, et al.J Rheumatol. 1996;23:6-9.Disclosure of Interests:Joe Zhuo Shareholder of: Bristol-Myers Squibb, Employee of: Bristol-Myers Squibb, Joshua Bryson Shareholder of: I own shares of Bristol-Myers Squibb Company, Employee of: I am a paid employee of Bristol-Myers Squibb Company, Qian Xia Shareholder of: I own shares of Bristol-Myers Squibb Company, Employee of: I am a paid employee of Bristol-Myers Squibb Company, Niyati Sharma Consultant of: I work as a consultant for Bristol-Myers Squibb Company, Sheng Gao Shareholder of: Bristol-Myers Squibb, Employee of: Bristol-Myers Squibb, Sonie Lama Shareholder of: I own shares of Bristol-Myers Squibb Company., Employee of: I am a paid employee of Bristol-Myers Squibb Company., Michael E. Weinblatt Grant/research support from: BMS, Amgen, Lilly, Crescendo and Sonofi-Regeneron, Consultant of: Horizon Therapeutics, Bristol-Myers Squibb, Amgen, Abbvie, Crescendo, Lilly, Pfizer, Roche, Gilead, Nancy Shadick Grant/research support from: Mallinckrodt, BMS, Lilly, Amgen, Crescendo Biosciences, and Sanofi-Regeneron, Consultant of: BMS