Lumpy skin disease (LSD) is a severe viral disease of cattle. Circumstantial evidence suggests that the virus is transmitted mechanically by blood-feeding arthropods. We compared the importance of transmission via direct and indirect contact in field conditions by using mathematical tools. We analyzed a dataset collected during the LSD outbreak in 2006 in a large dairy herd, which included ten separated cattle groups. Outbreak dynamics and risk factors for LSD were assessed by a transmission model. Transmission by three contact modes was modelled; indirect contact between the groups within a herd, direct contact or contact via common drinking water within the groups and transmission by contact during milking procedure. Indirect transmission was the only parameter that could solely explain the entire outbreak dynamics and was estimated to have an overall effect that was over 5 times larger than all other possible routes of transmission, combined. The R0 value induced by indirect transmission per the presence of an infectious cow for 1 day in the herd was 15.7, while the R0 induced by direct transmission was 0.36. Sensitivity analysis showed that this result is robust to a wide range of assumptions regarding mean and standard deviation of incubation period and regarding the existence of sub-clinically infected cattle. These results indicate that LSD virus spread within the affected herd could hardly be attributed to direct contact between cattle or contact through the milking procedure. It is therefore concluded that transmission mostly occurs by indirect contact, probably by flying, blood-sucking insects. This has important implications for control of LSD.

Defining sleep organization on the basis of transitions between states proved useful for identifying risk and resilience indicators in neonatal behavior to predict trajectories of neurobehavioral, emotional, and cognitive growth.

How do we build multiagent algorithms for agent interactions with human adversaries? Stackelberg games are natural models for many important applications that involve human interaction, such as oligopolistic markets and security domains. In Stackelberg games, one player, the leader, commits to a strategy and the follower makes their decision with knowledge of the leader's commitment. Existing algorithms for Stackelberg games efficiently find optimal solutions (leader strategy), but they critically assume that the follower plays optimally. Unfortunately, in real-world applications, agents face human followers (adversaries) who -because of their bounded rationality and limited observation of the leader strategy -may deviate from their expected optimal response. Not taking into account these likely deviations when dealing with human adversaries can cause an unacceptable degradation in the leader's reward, particularly in security applications where these algorithms have seen real-world deployment. To address this crucial problem, this paper introduces three new mixed-integer linear programs (MILPs) for Stackelberg games to consider human adversaries, incorporating: (i) novel anchoring theories on human perception of probability distributions and (ii) robustness approaches for MILPs to address human imprecision. Since these new approaches consider human adversaries, traditional proofs of correctness or optimality are insufficient; instead, it is necessary to rely on empirical validation. To that end, this paper considers two settings based on real deployed security systems, and compares 6 different approaches (three new with three previous approaches), in 4 different observability conditions, involving 98 human subjects playing 1360 games in total. The final conclusion was that a model which incorporates both the ideas of robustness and anchoring achieves statistically significant better rewards and also maintains equivalent or faster solution speeds compared to existing approaches.

Summary The L265P somatic mutation in the Myeloid Differentiation Primary Response 88 (MYD88) gene is a recurrent mutation in chronic lymphocytic leukaemia (CLL). This mutation has functional effects in various haematological malignancies but its role in CLL remains to be fully elucidated. Here, we report that MYD88 L265P mutations are associated with mutated immunoglobulin heavy‐chain gene (IGHV‐M) status and that among IGHV‐M patients, the presence of MYD88 L265P is associated with younger age at diagnosis. Using microarray and RNA‐Seq gene expression analysis, we further observe that the MYD88 L265P mutation is associated with a distinctive gene expression signature that predicts both failure‐free survival and overall survival. This association was validated in an independent cohort of patients. To determine whether MYD88 L265P mutations can be therapeutically exploited in CLL, we treated primary cells with an inhibitor of interleukin 1 receptor‐associated kinase 4 (IRAK4), a critical effector of the MYD88 pathway. IRAK4 inhibition decreased downstream nuclear factor‐κB signalling and cell viability in CLL cells, indicating the potential of the MYD88 pathway as a therapeutic target in CLL.

We have developed a bioinformatic method based on a maximum likelihood analysis of phylogenetic lineage trees to estimate the parameters of a B cell clonal expansion model, which includes somatic hypermutation with the possibility of lethal mutations. Lineage trees are created from clonally related B cell receptor DNA sequences. Important links between tree shapes and underlying model parameters are identified using mutual information. Parameters are estimated using a likelihood function based on the joint distribution of several tree shapes, without requiring a priori knowledge of the number of generations in the clone (which is not available for rapidly dividing populations in vivo). A systematic validation on synthetic trees produced by a mutating birth-death process simulation shows that our estimates are precise and robust to several underlying assumptions. These methods are applied to experimental data from autoimmune mice to demonstrate the existence of hypermutating B cells in an unexpected location in the spleen.

In chronic lymphocytic leukemia (CLL), mutations in the NOTCH1 receptor occur in 4-10% of newly diagnosed patients and 15–20% of multiply relapsed patients. Using next-generation sequencing, our group previously reported NOTCH1 p.P2514fs mutations in 15 CLL patients (9.4%) in an initial cohort of 160 CLL patients in which NOTCH1 mutations were associated with IGHV unmutated (UM) CLL (p=0.0001). Further analysis using a three-group comparison (NOTCH1 mut, IGHV UM vs. NOTCH1 wild-type [wt] IGHV UM vs. NOTCH1 wt IGHV mut) showed that NOTCH1 mutations associated with both trisomy 12 (p=0.049) and 17p deletion (p=0.0008) and poor overall survival (HR 2.99, p=0.008). Given that targeting activating mutations has proven an effective therapeutic strategy in many cancers, we explored the therapeutic potential of a Notch1 inhibitor, PF-03084014, in CLL. Previous studies in T-cell acute lymphoblastic leukemia cells harboring NOTCH1 mutations have shown that gamma secretase inhibitors can induce apoptosis by blocking Notch1 receptor activation. When we tested the gamma secretase inhibitor (GSI) PF-03084014 in 18 CLL samples with NOTCH1 mutations, it consistently induced apoptosis of all CLLs after 48 hours in culture across all cytogenetic groups tested (13.3-47.2% death with 5 μM GSI, p<0.0001)(Figure A). The induction of apoptosis was similar (GSI vs. ibrutinib, p=ns) to that of ibrutinib (n=11,11.9-74.4% death with 5 μM ibrutinib, p<0.0001). In contrast, GSI treatment only induced apoptosis in some (n=10), but not all NOTCH1 wt CLLs (n=6) (p=0.0137). We next tested the effect of GSI PF-03084014 in the context of a stromal environment. Co culture of CLL cells with CD40L-expressing fibroblasts partially mimics the lymph node and bone marrow microenvironments, which are known sites of drug-resistance and proliferation of CLL in vivo. We tested whether the interaction with stromal cells protects CLL cells from Notch1 inhibitor-induced apoptosis, as seen with other drugs including ibrutinib. We found that co culture with CD40L-expressing 3T3s decreased GSI-induced apoptosis in NOTCH1 mutant CLLs (p=0.0006) and in the majority of the NOTCH1 wt CLLs that responded to the GSI (Figure A). Since NOTCH1 mutations have been reported to be an independent marker of aggressive disease in CLL, we tested whether CLL cells with NOTCH1 mutations were more proliferative in vitro compared to NOTCH1 wt CLL cells. We showed that CLL cells upregulate Ki67 expression in co culture with 3T3-CD40L cells and in a cohort of 10 NOTCH1 mutants and 11 NOTCH1 wt, we found the NOTCH1 mutants to be more proliferative than the NOTCH1 wt (median of 7.6% vs. 2.3%, p=0.015). To then address whether blocking of the Notch1 pathway decreases proliferation, we treated CLL cells in co culture with 3T3-CD40L cells with 5 μM GSI for 7 days. GSI treatment decreased the percentage of Ki67+ CLL cells in all but one NOTCH1 mutant (median decrease 28.3%, p=0.044) as wells as in the majority of NOTCH1 wt samples (median decrease 38.7%, p=0.037). Having established that inhibition of Notch1 can reduce proliferation and induce apoptosis in CLL cells in vitro, we were interested in determining the downstream genes that may be the effectors of this activity. We therefore compared the gene expression profiles (GEP) of NOTCH1 mut vs. NOTCH1 wt CLLs, and found upregulation of genes involved in the Notch1 pathway, in apoptosis and in chemokine signaling in the NOTCH1 mutants. Furthermore, comparing GEP of high Ki67 vs. low Ki67 expressing CLL cells revealed higher expression of a range of both upstream and downstream Notch1 pathway genes in high Ki67 expressing CLL cells. In conclusion, we show that PF-03084014 induces apoptosis and decreases proliferation in both NOTCH1 mutant and wt CLL cells. We find NOTCH1 mutant CLL cells to be more proliferative than NOTCH1 wt and show upregulation of Notch1 pathway genes in NOTCH1 mutants compared to wt CLL cells and in high Ki67 expressing compared to low Ki67 expressing CLL cells. Taken together, these results emphasize the important role of Notch1 signaling in CLL in general, perhaps particularly in proliferative compartments like lymph nodes, and demonstrate that Notch1 pathway inhibitors are worthy of therapeutic investigation in CLL. Figure A. GSI-induced apoptosis in NOTCH1 mut vs. wt CLL cells at 48 h. CLL cells naked or in co culture with 3T3-CD40L cells +/- 5 µM PF-03084014. Survival was assessed by CD19 and Annexin V/PI staining. Figure A. GSI-induced apoptosis in NOTCH1 mut vs. wt CLL cells at 48 h. CLL cells naked or in co culture with 3T3-CD40L cells +/- 5 µM PF-03084014. Survival was assessed by CD19 and Annexin V/PI staining. Disclosures No relevant conflicts of interest to declare.

The L265P somatic mutation in the Myeloid Differentiation Primary Response 88 (MYD88) gene is recurrently observed in CLL; although this mutation has been demonstrated to have functional effects in multiple hematologic malignancies, its role in CLL is largely unknown. To address this gap in knowledge, we examined the clinical and biological impact of MYD88 L265P mutations in CLL by analysis of gene expression, cell viability and Toll-like Receptor 9 (TLR9)-induced signaling and cytokine production. Out of 160 CLL patient samples subjected to whole-exome sequencing and previously reported by our group, 10 were found to harbor MYD88 L265P mutations, all of which possessed mutated immunoglobulin heavy chain variable (IGHV) regions (p = 0.006). While IGHV mutated patients are generally known to exhibit better prognosis compared to IGHV unmutated patients, the presence of MYD88 L265P within the IGHV mutated subset was associated with earlier age of disease onset (p = 0.04) and worse overall survival (OS; p = 0.00017), comparable to IGHV unmutated samples with wild-type (WT) MYD88. No association with the presence of chromosome 13q deletions (p = 0.26) or prior treatment at the time of sampling (p = 0.10) was observed. Gene expression microarray analysis restricted to the IGHV mutated subset (MYD88 WT: n = 76; MYD88 L265P: n=10) and conducted using a PAM-based approach demonstrated that MYD88 L265P mutation was associated with differential expression of 28 genes, whose expression was then examined across all CLL samples with available gene expression data (n = 150). Using Cox modeling, a composite gene signature score was determined for each patient, who were subsequently dichotomized based on median signature. This method was able to predict both OS and event free survival (EFS) in a univariable analysis (OS: p = 1.2E-06; EFS: p = 7.6E-13). Statistical significance was maintained when a multivariable analysis was conducted, adjusting for known CLL risk factors including age, IGHV status, ZAP70 expression, cytogenetics and prior treatment (p < 0.0001 for OS and EFS). The univariable (OS: p = 1.6E-05; EFS: p = 5.7E-10) and multivariable findings (p < 0.003 for OS and EFS) were further confirmed in an independent validation cohort (n = 87). To identify a more parsimonious gene set, we applied a L1 penalized proportional hazards model to the discovery and validation cohorts, separately. This approach identified 5 overlapping genes (BCAT1, BMP6, CHAD, IKZF2, and TRIO) between the two cohorts that appear to be the main drivers of the predictive signature. To inhibit MYD88 signaling in CLL cells, we treated MYD88 L265P and WT cells (n = 6/group, matched for clinical characteristics: IGHV, ZAP70, cytogenetic, and treatment status) in vitro with a highly-selective small molecule IRAK4 inhibitor, ND-2158 (Nimbus Therapeutics). ND-2158 significantly reduced cell viability in a dose dependent manner in both MYD88 WT and L265P primary CLL cells, either alone or in combination with a fixed concentration of the B-cell receptor (BCR) pathway inhibitor, ibrutinib. The TLR9 agonist CpG was used to stimulate signaling through the MYD88 pathway in vitro. ND-2158 inhibition of CpG-induced IRAK4 activation in CLL cells (n = 3/group, matched for clinical characteristics) blocked IRAK1 and IκBα degradation and led to a dose-dependent decrease in the ratio of phospho/total proteins. No significant differences were noted between MYD88 WT and L265P samples, consistent with our cell viability results. CLL-secreted levels of IL-6, IL-10 and CCL3 were measured in culture supernatants treated with ND-2158+/- CpG stimulation (n = 4/group, matched for clinical characteristics). CpG stimulated cytokine levels (p < 0.0001 for all cytokines+/- CpG) were significantly inhibited in a dose-dependent manner by ND-2158. Again, no significant differences were observed between MYD88 WT and L265P CLL with respect to cytokine production, either at baseline or in CpG-stimulated DMSO treated control cells. In conclusion, the differences in clinical outcome and gene expression observed between MYD88 WT and L265P IGHV mutated CLLs indicate a functional role for MYD88 L265P in CLL. The inferior clinical outcome in IGHV mutated CLL with L265P mutation suggests that MYD88 signaling may be a relevant target in CLL. ND-2158 inhibits signaling in the MYD88 pathway, suggesting potential therapeutic utility of IRAK4 inhibitors in CLL. Disclosures Chaudhary: Nimbus Therapeutics: Equity Ownership. Miao:Nimbus Therapeutics: Employment. Westlin:Nimbus Therapeutics: Employment.