Spleen Weight Research Articles

The Second Generation Type II JAK2 Inhibitor, AJ1-10502, Demonstrates Enhanced Selectivity, Improved Therapeutic Efficacy and Reduced Mutant Cell Fraction Compared to Type I JAK2 Inhibitors in Models of Myeloproliferative Neoplasms (MPNs)

Type I JAK inhibition (JAKi) represents a mainstay therapy for myelofibrosis and hydroxyurea-resistant high-risk polycythemia vera. Current type I JAKi, including ruxolitinib (RUX) and fedratinib, improve symptoms and outcomes in MPNs; however, JAK2VF allele burden remains essentially unchanged, and efficacy wanes over time. Sustained JAK/STAT signaling plays a critical role in MPN cell persistence in the setting of type I inhibition. CHZ868, a type II JAKi that binds the inactive conformation of the kinase domain, overcomes RUX persistence in vitro and reduces Jak2VF allele fraction in vivo suggesting improved JAK2 targeting might enhance clinical efficacy. However, current type II JAKi, including CHZ868, are limited by lack of kinome specificity and off-target toxicity. We therefore sought to develop novel type II JAK2i with improved potency and selectivity. Computational free energy perturbation and structure-activity relationship-based methods were used to identify lead type II inhibitor compounds. This work, followed by Absorption, Distribution, Metabolism and Excretion modeling, led to the development of AJ1-10502. Kinome selectivity profiling of AJ1-10502 revealed potent JAK2 selectivity with minimal kinase cross-reactivity compared to CHZ868, including among other JAK family tyrosine kinases (Figure 1A). Assessment of in vitro efficacy of AJ1-10502 in SET2 inhibitor naïve and SET2 RUX persistence (RUXper) cells revealed concentration-dependent inhibition of proliferation of RUXper cells comparable to that of CHZ868. The IC50 for AJ1-10502 in RUXper cells was similar to the IC50 observed in inhibitor naïve cells. We next evaluated AJ1-10502 in vivo using a novel dual Dre/Cre-recombinase Jak2VF knock-in/knock-out model allowing for the comparison of JAKi to Jak2VF genetic deletion. AJ1-10502 demonstrated reductions in leukocytosis comparable to that of RUX (K/uL: VEH 18.7 vs. RUX 10.1 vs. AJ1-10502 9.3) but dose-dependent improvements in hematocrit, platelet levels, and spleen weights superior to RUX, with reductions in spleen weights on par to that of Jak2VF deletion (mg: VEH 480 vs. RUX 255 vs. AJ1-10502 145 vs. del 107, p<0.05). A greater degree of restoration of splenic architecture was also observed with AJ1-10502 compared to type I JAKi. Critically, we observed reductions in peripheral blood (PB) and bone marrow (BM) mutant allele fraction with AJ1-10502 not observed with RUX, including within Mac1+Gr1+ myeloid cell fractions (VEH 94% vs. RUX 92% vs. AJ1-10502 78.5%, p<0.05), consistent with a mutant-biased reduction in myeloid output. In a separate series of competitive transplants using a Cre-inducible human JAK2VF transgenic mouse line, we validated the phenotypic changes in regard to leukocytosis, hematocrit, and spleen weight reduction seen with the DreCre model. Most importantly, we confirmed significant reductions in mutant cell fraction within the hematopoietic stem (HSC) compartment of BM and spleen, including among granulocytic-monocytic progenitor (GMP) and long-term HSC (LT-HSC) populations (Figure 1B). Finally, a separate study comparing AJ1-10502 to CHZ868 revealed no significant weight loss with AJ1-10502 (-0.5g) compared to CHZ868 (-2.2g, p<0.05) despite similar reductions in Jak2VF allele fraction suggesting comparable efficacy without systemic toxicity. In sum, AJ1-10502 is a potent, selective type II JAK2i with improved efficacy compared to RUX and an enhanced safety profile compared to previous non-selective type II JAKi. Most importantly, AJ1-10502 results in superior reductions in PB and BM mutant cell fraction in vivo not observed with type I JAKi. These data demonstrate the preclinical utility of type II JAKi with AJ1-10502 and inform a path to clinical development of type II JAKi for MPN patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Jak2V617F Reversible Activation Shows an Essential Requirement for Jak2V617F in Myeloproliferative Neoplasms (MPNs)

JAK inhibitors (JAKi) improve symptoms and outcomes in MPNs; however, mutant allele burden does not substantively change with chronic therapy. Alternative MAPK signaling and/or cooperating mutations are thought to contribute to MPN cell persistence; however, we hypothesized current JAKi fail to effectively abrogate mutant JAK2 signaling. To that end, we developed a conditionally inducible mouse model allowing for sequential activation, then deletion, of Jak2VF from its endogenous locus using a Dre-rox/Cre-lox (Jak2RL) dual recombinase system (Figure 1A). Dre mRNA electroporation of lineage-negative UbcCreER-Jak2RL bone marrow (BM) was used to induce Jak2VFex vivo, followed by transplant into lethally-irradiated recipient mice in competition with Cd45.1 BM. Transplanted mice developed a highly penetrant MPN characterized by leukocytosis, polycythemia, hepatosplenomegaly, and BM megakaryocytic hyperplasia. Tamoxifen administration 12 weeks post-transplant to delete Jak2VF resulted in abrupt normalization of blood cell parameters, spleen weights, and serum pro-inflammatory cytokines, with a marked prolongation in overall survival (OS) compared to MPN control mice. Reduced peripheral blood (PB) and BM Cd45.2 mutant fraction was also observed, including within hematopoietic stem/progenitor cell (HSPC) compartments and to a greater degree than ruxolitinib (RUX) therapy. Notably, treatment with CHZ868, a type II JAKi, showed improvements in regard to hematocrit and spleen weight reduction on par with deletion, as well as significant reductions in BM HSPC mutant fractions, albeit to a lesser degree than with Jak2VF reversion (Figure 1B). Transplant of Jak2VF deleted BM failed to form phenotypic disease in secondary recipient mice consistent with loss of disease-propagating MPN stem cells. RNA-Seq on sorted LSKs following Jak2VF deletion showed loss of STAT5, MAPK, and MTORC1 target gene expression, as well as negative enrichment in TGFb and TNFa/NFkB pathways consistent with reduced pro-inflammatory and proliferative output. The alterations in MAPK signaling were supported by Ybx1 RT-PCR of BM cKit+ cells and BM pERK IHC, both of which were increased with RUX but potently suppressed with deletion suggesting improved JAK2 targeting can overcome MAPK-mediated persistence. The initial proximity of Jak2RLlox sites grants Cre-mediated recombination prior to Dre-mediated Jak2VF knock-in allowing for assessment of Jak2VF oncogenic dependency alone or in the context of co-occurring mutation. Given the known effects of Tet2 loss on enhanced MPN HSC fitness, we evaluated Tet2 loss on sustaining MPN with Jak2VF reversion. UbcCreER-Jak2RL/Tet2f/f mice were pulsed with TAM to knock-out Tet2 followed by sacrifice and Dre Jak2RL knock-in 6 weeks later. Mice transplanted with UbcCreER-Jak2VF/Tet2-/- cells exhibited enhanced leukocytosis, spleen weights and PB/BM chimerism compared to Jak2VF mice; however, Jak2VF reversion resulted in similar reductions in MPN disease parameters, including Cd45.2 fraction, suggesting Tet2loss does not dramatically alter dependence on mutant JAK/STAT signaling. Notably, while Jak2VF/Tet2-/- mutant cells displayed increased serial replating capacity, this was abrogated with Jak2VF reversion suggesting despite the enhanced fitness of Tet2-/- HSCs, Jak2VF creates a unique dependency that renders double-mutant cells susceptible to eradication with Jak2VF reversion. In sum, Jak2VF deletion abrogates MPN features, extends OS, and reduces mutant cell fraction with depletion of disease-sustaining MPN HSCs, either alone or in the setting of cooperating Tet2 loss. These data suggest JAK2VF mutant-selective inhibition offers greater therapeutic potential than current JAKi and a potential curative option for the treatment of MPN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Establishment and Characterization of Mouse Models with Primary and Acquired BTK Inhibitor Resistance

Background: The second-generation Bruton's tyrosine kinase (BTK) inhibitor acalabrutinib is effective for treatment of patients with chronic lymphocytic leukemia (CLL), including high-risk and relapsed patients. Resistance to BTK inhibitors can develop not only due to on-target mutations, but also by bypassing the BTK signaling pathway. Mouse models represent useful tools in studying mechanisms of resistance as well as testing the efficacy of alternative therapy options to overcome resistance. Eµ-TCL1-transgenic (TCL1-tg) mice have the advantage of immunocompetence and resemble the human CD49d-high CLL cohort (Szenes et al., 2020). Transplantation of tumors from these mice to wild-type recipients allows acceleration of pathophysiology, selection of specific tumor clones, and more efficient testing of drug combinations. Here, we developed acalabrutinib resistance models by serial transplantation and long-term administration of the drug in drinking water, characterized these models, and compared molecular resistance signatures with those of human patient samples. Methods: Models were developed starting from four primary TCL1-tg mice at overt disease with tumor burden in spleen ranging up to 95% of lymphocytes. Serial retransplantation to wild-type C57BL/6J recipients for two to four rounds and acalabrutinib therapy pressure resulted in several drug resistant pedigrees. Mice were sacrificed at end-stage disease and tumor burden in spleen, bone marrow and blood was analyzed by flow cytometry using anti-CD5/CD19 antibodies. Purified leukemic cells were subjected to RNA-seq, real-time quantitative PCR and mutational analyses. Results: Three response patterns were identified in recipient mice: a) sustained sensitivity (models were stopped after 23 weeks of treatment), b) acquired resistance (with initial response followed by development of resistance under continuous therapy pressure and c) retransplantation-induced resistance in a case of an aggressive tumor clone that was responding to acalabrutinib in the parental mouse. The sensitive models displayed a 63% reduction in spleen weight upon treatment and a concomitant 65% reduction in blood tumor burden. In acquired or primary resistant mice, spleen weight and tumor burden was not significantly different from that of untreated mice, with blood tumor burden in the magnitude of 100,000 leukemic cells/µl and spleen tumor burden of 500-1000 million leukemic cells. Resistance could not be attributed to the mutational landscape of BTK and PLCγ2. Mutations in p53, Myc, Notch1, Atm, and Sf3b1 were detected but found to be intrinsic in individual ancestors and further present in the follow-up transplantations. RNA-seq analysis revealed 863 differentially expressed genes in mice with acquired resistance to acalabrutinib (compared to untreated, Figure 1) and 114 shared differentially expressed genes in mice with acquired or transplantation-based resistance. Gene set enrichment analysis demonstrated the presence of MAPK signaling and NF-kB signaling gene signatures within the resistant tumors. In particular, acquired resistance was accompanied by changes in the B cell receptor downstream components. We also compared the murine 863 gene signature to human samples from acalabrutinib resistant patients (progression on drug versus baseline) and found 13 genes are differently expressed in both, human and mouse. Validation for this gene expression data is ongoing and updated results will be presented. Conclusion: We have developed novel TCL1-tg based murine models of acalabrutinib resistance. Our models provide well-characterized tools to study primary as well as acquired BTK inhibitor resistance and allow the current testing of potential new therapeutic approaches to overcome resistance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Splenectomy for Immune Cytopenias: Treatment Outcomes and Predictors of Response

The pathogenesis of immune cytopenias involves diverse processes including production, inhibition, and destruction of specific blood cell types mediated by antibodies and/or T cells. Classic examples of immune-mediated cytopenias are immune thrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AIHA), and autoimmune neutropenia (AIN) as well as T cell-mediated idiopathic neutropenia and pure red cell aplasia. Both antibody and autoimmune T cell responses may often coexist, and assignment of the proper pathogenic importance, albeit difficult, is crucial for rational therapeutic approaches. Clonal processes such as T-cell large granular lymphocytic leukemia (T-LGL) or various B cell dyscrasias may also occur. While the results of splenectomy have been studied in individual conditions such as Evans syndrome (ES), AIN, and AIHA, the availability of new therapies and improvement of surgery outcomes have provided an impetus to update the older and smaller series. We retrospectively reviewed a cohort of 339 adult patients who underwent splenectomy at the Cleveland Clinic Foundation between 2002-2020 (Fig.1A), for whom the main indication for the procedure was: ITP (n= 235), AIHA (n=68), ES (n=26) and AIN (n=10). Among these subtypes, there were 15 patients with an established diagnosis of T-LGL. Our aim was to determine the hematologic response of patients who receive splenectomy for immune cytopenia following treatment failure with conventional therapies and identify factors that were associated with responses. Overall, the median age of the patients was 55 years (IQR:68-36), M:F ratio=0.98, with a median time from diagnosis to splenectomy of 11 months (IQR:31-3). Prior to the procedure, all patients had failed multiple lines of medical therapies (mode 1, range1-4). The median preoperative platelet count was 19 (IQR:44-5) x109/L, median preoperative hemoglobin (Hb) of 9 (IQR:10-8) g/dl and median preoperative ANC of 0.28 (IQR:1.19-0.23) x109/L. A total of 306 patients underwent laparoscopic splenectomy, while 33 patients had an open approach. In 16 cases (5%), we recorded an intraoperative conversion from laparoscopic to open splenectomy. The mean spleen weight was 477 gr (SD±687). The average operative time was 2.3 hours, with four days median hospital length of stay (range:1-50). The overall rate of postoperative complications with 30-day readmissions was 0.1%, with no mortality. Of note is that up to 20% of the cases had a post-operative diagnosis that was discordant with the original indication for splenectomy. These discordant diagnoses included benign conditions such as Felty's syndrome (n=2), as well as hematologic malignancies such as mature B-cell neoplasm (28), T-cell lymphoma (n=8), NK-LGL (n=1), and T-LGL (n=15). The overall response rate (ORR) to splenectomy was 74%, of which 86% complete response (CR) and 14% partial response (PR) with relapse occurring in 12% of cases. In the subset of thrombocytopenic patients, CR reached 69% of patients (P<.0001) as early as one week following the procedure. At 1-year, the mean platelet count was 246 (SD±170) x 109/L and relapse occurred in 13% of patients. Conversely, in anemic patients, a CR was observed only in 55% (P<.0001) within two weeks following splenectomy. At 1-year, the mean Hb level was 11.7 (SD±1.3) g/dl, with relapse occurring in 6% of patients. Comparatively, 80% of patients who underwent splenectomy for neutropenia responded at 26 weeks post-splenectomy with relapse in 10% of cases. In known and newly diagnosed patients with LGL (n=16), the response with regard to the cytopenia resulting in splenectomy indication was 81%. The response in Felty's syndrome was 100%. When analyzing predictors of response, failed pharmacologic therapies with either intravenous immunoglobulin (IVIg) or rituximab was predictive of non-response to splenectomy (Fig.1B). No significant differences in spleen weight between responders and non-responders were observed. Our results show a significant and durable response to splenectomy in immune-mediated cytopenia of various etiologies refractory to conventional therapies indicating that, in selected patients, splenectomy remains a rational salvage treatment with a low complication rate. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PTPN1 Deficiency Accelerates the Progression to Myelofibrosis in JAK2V617F-Driven Myeloproliferative Neoplasm

The deletion of chromosome 20q (del20q) is frequently associated with myeloid malignancies including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The del20q cytogenetic abnormality was observed most frequently in patients with myelofibrosis (MF). However, the key target genes within 20q involved in the pathogenesis of myelofibrosis has remained elusive. We previously reported that PTPN1 gene encoding protein tyrosine phosphatase non-receptor type 1 is a potential tumor suppressor within 20q in MPN/MF (Jobe et al. Leukemia 2017). The del20q involving PTPN1 deletion was more commonly found in MPN/MF in comparison to other myeloid neoplasms. We also observed significant co-occurrence of del20q involving PTPN1 deletion with JAK2V617F mutation in patients with MF. So, we hypothesize that PTPN1 deficiency may cooperate with JAK2V617F in the pathogenesis of MF. In this study, we investigated the effects of heterozygous and homozygous PTPN1 deletion in JAK2V617F-induced MPN using a conditional PTPN1 knockout allele and a JAK2V617F knock-in mouse model of MPN. We previously reported the generation of a conditional JAK2V617F knock-in mouse. Mice expressing heterozygous JAK2V617F (JAK2VF/+) exhibit a PV-like MPN characterized by increase in red blood cells (RBC), white blood cells (WBC), neutrophils and platelets in the peripheral blood. Heterozygous deletion of PTPN1 significantly increased WBC, neutrophil and platelet counts but reduced RBC and hemoglobin levels in JAK2VF/+ mice. Flow cytometric analysis showed that heterozygous deletion of PTPN1 significantly increased myeloid (Gr-1+Mac-1+) and megakaryocytic (CD41+CD61+) precursors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared with JAK2VF/+ mice. Additionally, deletion of PTPN1 significantly increased hematopoietic stem cells and myeloid progenitors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared to JAK2VF/+ mice. Spleen weight was significantly increased in JAK2VF/+ mice, while deletion of PTPN1 further enhanced splenomegaly in PTPN1-deficient JAK2VF/+ mice. Furthermore, deletion of PTPN1 significantly enhanced bone marrow fibrosis in JAK2VF/+mice. Homozygous deletion of PTPN1 resulted in more robust and accelerated development of lethal myelofibrosis in JAK2VF/+ mice. The cooperative effects of JAK2V617F and PTPN1 deficiency in myelofibrotic transformation is associated with increased production of proinflammatory cytokines including IL-1beta, IL-6 and TGF-beta. We also observed enhanced activation of STAT1, STAT3, ERK1/2 and NF-kB in PTPN1-deficient JAK2VF/+ mice bone marrow compared with JAK2VF/+ or WT mice bone marrow. CRISPR/Cas9-mediated deletion of PTPN1 also resulted in enhanced activation of these signaling molecules and increased proliferation in JAK2V617F-positive mouse and human hematopoietic cells. Furthermore, deletion of PTPN1 significantly reduced ruxolitinib sensitivity against JAK2V617F-positive hematopoietic cells, suggesting that del20q involving PTPN1 deletion may cause ruxolitinib resistance in MPN. Overall, this study demonstrates that PTPN1 is an important target in del20q-associated MPN, and deficiency of PTPN1 collaborates with JAK2V617F in the progression to myelofibrosis.

PHF6 Maintains Acute Myeloid Leukemia Via Regulating NF-Κappab Signaling Pathway

Acute myeloid leukemia (AML) is a major hematopoietic malignancy characterized by the uncontrolled expansion of the immature myeloid cells in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission and 5-year-survival rate. Many efforts have been dedicated to decipher the molecular events underlying AML transformation. PHF6 is a conserved epigenetic regulator and has been found to be frequently altered in hematologic malignancies. Previous studies demonstrated that PHF6 plays a suppressive role in T-ALL leukemogenesis. However, the functional role(s) of PHF6 in AML initiation and progression remains unknown. Here in this study, we analyzed the mRNA expression of PHF6 in blood cells from healthy donors and leukemia cells from AML patients, and found that the PHF6 expression was significantly increased in AML cells when compared with that of normal PMNs (www.Bloodspot.com) (p < 0.05). Significantly, the average survival time of AML patients with high PHF6 expression was much shorter than that of AML patients with low PHF6 expression (p = 0.022). Overexpressing of PHF6 (PHF6 OE) in Kasumi-1 or K562 leukemia cell lines showed increased growth of Kasumi-1 and K562 cells, decreased apoptosis of Kasumi-1 cells. Additionally, we knocked down (KD) PHF6 expression in AML cells and CD34+ cord blood cells, and found that PHF6 KD significantly decreased the growth of Kasumi-1 and promoted cell apoptosis, while had little effects on apoptosis of CD34+ cord blood cells. These results suggested that PHF6 is required for the growth of myeloid leukemia cells but is dispensable for normal blood cells. To further evaluate the functional role of Phf6 in leukemogenesis in vivo, we generated Phf6 KO+RUNX1-ETO9a (RE9a) and Phf6 KO+MLL-AF9 (MA9) AML mice. We found the survival time of Phf6 KO+RE9a mice was significantly longer than that of WT Phf6+RE9a mice. The Phf6 KO+RE9a mice showed much milder disease phenotypes than WT Phf6+RE9a mice, including lower counts of GFP+ leukemia cells in the PB and BM, higher body weights, lower spleen and liver weights, and lower degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked decrease in leukemia stem cell (LSC) activity in Phf6 KO+RE9a cells when compared with Phf6 WT+RE9a cells. Consistent with RE9a-induced AML mouse model, PHF6 deficiency impaired the self-renewal of LSCs and delayed the progression of MA9-induced AML, supporting a general oncogenic role of Phf6 in sustaining myeloid leukemia. To investigate the underlying molecular mechanisms of Phf6 in regulating AML development, we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 deficiency in isogenic Phf6 WT+RE9a or Phf6 KO+RE9aAML cells. Gene set enrichment analysis (GSEA) showed up-regulation of apoptosis and down-regulation of NF-κB-regulated gene expressions in Phf6 KO+RE9a cells, suggesting that Phf6 depletion might promote AML cell apoptosis by blocking the NF-κB signaling. Immunocytochemistry studies showed that PHF6 depletion partially inhibited the nuclear translocation of p50. Co-immunoprecipitation experiments revealed that PHF6 directly bound to p50 in myeloid leukemia cells, suggesting that PHF6 might activate NF-κB signaling pathways by binding with p50 and maintaining the nuclear translocation of p50. To confirm this, we treated PHF6 OE AML cells with BAY11-7082 (a selective NF-κB inhibitor), and found BAY11-7082 significantly inhibited the growth of PHF6 OE Kasumi-1 cells in vitro. In summary, we revealed that PHF6 promoted RE9a- or MA9-induced AML progression in mice by activating NF-κB signaling pathway, and promoting P50 nuclear translocation. Our study thus defined a unique role for Phf6 in regulating NF-κB signaling pathway in AMLs, and potentially can be used as a therapeutic target for treating myeloid leukemia patients.

Combination of 5-Azacytidine and Pegifna Is Able to Overcome Resistance in JAK2-V617F Positive MPN with Loss of Dnmt3a

Pegylated interferon alpha (IF) can induce molecular remissions in subset of JAK2-V617F-positive MPN patients by targeting long-term hematopoietic stem cells (LT-HSCs). Patients with additional somatic mutations in genes involved in LT-HSC self-renewal have been reported to have poorer responses to IF. We found that loss of Dnmt3a increases competitiveness and self-renewal of JAK2-V617F-positive LT-HSCs and confers resistance to IF treatment in MPN mouse models and in HSCs and progenitor cells from MPN patients. Here we examined whether the resistance of double mutant JAK2-V617F;Dnmt3aΔ/Δ hematopoietic cells to IF can be overcome by addition of arsenic trioxide (At), or 5-azacytidine (Az). To test the effects of the combination treatments in vivo, we generated cohorts of mice expressing JAK2-V617F (VF) alone, or in combination with a homozygous deletion of the Dnmt3a gene (Dnmt3aΔ/Δ). We used bone marrow cells from VF or VF;Dnmt3aΔ/Δ mice that express a GFP reporter gene mixed with a 10-fold excess of bone marrow cells from a wildtype (WT) mouse to perform competitive transplantations into lethally irradiated WT recipient mice. After 7 weeks, recipient mice were randomized into 6 treatment groups including a vehicle control group. These mice were treated for 12 weeks with IF (25µg/kg; s.c. once per week), At (5mg/kg; i.p. every second day), or Az (2mg/kg; i.p. daily for two weeks followed by a break of 2 weeks), or combinations of IF+At and IF+Az (Figure 1). Peripheral blood parameters were normalized by At and Az treatment arms in both single-mutant and double-mutant mice (Figure 1A). Spleen and liver weight was decreased in all treatment groups and genotypes, except in VF;Dnmt3aΔ/Δ mice treated with IF alone, which showed a trend towards increased spleen and liver weights. Expression of GFP allowed us to follow the contribution of cells derived from the VF or VF;Dnmt3aΔ/Δ donor mice, respectively. In VF recipient mice, a pronounced decrease in GFP chimerism of peripheral blood lineages was observed in the groups treated with a combination of IF+At, or IF+Az (Figure 1A). LT-HSC from bone marrow and spleen showed reduction in GFP chimerism upon treatment with Az alone, compared to vehicle and further reduction below 10% was observed in combination of Az and IF (Figure 1B). Double-mutant VF;Dnmt3aΔ/Δ recipient mice remained resistant to the single agent regiments, but showed a significant reduction of GFP chimerism in peripheral blood and in LT-HSCs when treated with a combination of IF+Az and to a lesser degree also when treated with IF+At (Figure 1B). Thus, a combination of pegIFNa with 5-azacytidine is a promising approach to target MPN cells carrying mutations in JAK2 and Dnmt3a genes that could be also considered as a treatment option for therapy-resistant forms of MPN in patients carrying JAK2-V617F and loss-of-function DNMT3A mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PRMT5 Inhibition Reduces Autoinflammation in a Murine Model of Secondary Hemophagocytic Lymphohistiocytosis

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare but frequently fatal autoinflammatory syndrome characterized by dysregulated immune activation, excessive production of proinflammatory cytokines and hyperactivation of T cells and macrophages leading to tissue damage. Primary HLH occurs due to predisposing genetic mutations in the perforin/granzyme pathway, while secondary HLH can be triggered by infections, rheumatologic disorders, or malignancies. HLH has a poor overall prognosis despite appropriate therapy, and new avenues targeting pathologic disease mechanisms are urgently needed. The protein arginine methyltransferase 5 (PRMT5) enzyme has been identified as an important mediator in inflammatory T cell subsets in autoimmune disease and graft versus host disease. We hypothesized that PRMT5 activity is relevant to inflammation loops that drive autoinflammation observed in HLH. Methods: We explored inhibition of type II protein arginine methyltransferase enzyme, PRMT5, in a murine model of secondary HLH. HLH was triggered in C57BL/6-J mice (Jackson Laboratories) by repeated injections of CpG 1826 (Integrated DNA Technologies) and an IL-10 receptor-blocking antibody (clone 1B1.3A; Bio X Cell) on days 0, 2, 4, and 7. Injected mice were left untreated or were treated with ruxolitinib 90 mg/kg (validated treatment of HLH) orally twice daily, starting on Day 4, or increasing doses of oral inhibitor of PRMT5, PRT382 (0.25, 1 or 2 mg/kg daily), starting on Day 0. Mice were weighed daily. On day 9, mice were euthanized and analyzed for spleen and liver weight, serum IFNγ levels and immune profiling of lymphoid and myeloid splenocyte subsets. Splenocytes were left unstimulated, or stimulated with lipopolysaccharide (LPS, myeloid cell stimulation) or CD3/CD28-directed antibodies (T cell stimulation) and analyzed by flow cytometry. Data analysis was performed by unsupervised ViSNE clustering in Cytobank software. Statistical analysis was performed by unpaired t test. Results: We observed that 1 mg/kg PRT382 lessened the hallmarks of secondary HLH, compared to the untreated HLH cohort, including weight loss (21% reduction, p-value 0.2746), the development of splenomegaly (71% reduction, p-value <0.001) and hepatomegaly (61% reduction, p-value <0.05), similarly to ruxolitinib (p-values <0.01, <0.001, and 0.0795, respectively). Furthermore, mice with untreated HLH showed a 17X increase in serum IFNγ levels compared to controls (p-value <0.01), while mice treated with ruxolitinib showed a 10X increase (p-value <0.05 compared to controls, 0.2577 compared to untreated HLH), and mice treated with 1 mg/kg PRT382 showed undetectable serum IFNγ (p-value 0.2104 compared to controls, <0.01 compared to untreated HLH). Flow cytometric analysis of mouse splenocytes showed that HLH stimulated the expansion of neutrophils (CD11b+/Ly6Clo/Ly6G+) and inflammatory monocytes (CD11b+/Ly6C+/Ly6G-). Ruxolitinib partially counteracted this expansion. PRT382 inhibited expansion of these myeloid cell subsets in a dose-dependent manner and also limited production of IFNγ and TNFα by myeloid cells in response to stimulation with LPS in presence of Golgi blockade with Brefeldin A. Furthermore, PRT382 limited the upregulation of CD25 on the surface of T cells in response to T cell stimulation, although no change in cytokine production by T cells was observed. Conclusions: PRMT5 inhibition counteracts pro-inflammatory features of pathologic hyper-inflammation driven by myeloid immune cell subsets in a model of secondary HLH. This may represent a novel avenue for urgently needed immune modulatory targeted treatment of HLH. Studies of PRMT5 expression and targeting in models of primary HLH are underway.

DEK-NUP214 Acute Myeloid Leukemia Depends on CRM1 In Vivo

Introduction: Acute myeloid leukaemia (AML) with the translocation DEK-NUP214 (t(6;9)(p23;q34)) is a rare subtype present in 1-2% of AML patients. This translocation is associated with poor clinical outcome, resistance to chemotherapy and remains poorly characterized. Among all nucleoporins, NUP214 has the highest affinity for CRM1, a major exportin protein. We hypothesized that inhibition of CRM1 would be an effective treatment against the aberrant activity of DEK-NUP214. We assessed the leukemia cell line FKH-1, which has a DEK-NUP214 translocation as its sole mutation. In parallel, we established a patient-derived xenograft (PDX) model with the intent to further characterize the disease and assess the efficacy of CRM1 inhibitor KPT-8602/eltanexor. Methods: Cell viability, induction of apoptosis, cell cycle and proliferation were measured at 24h, 72h and 96h for FKH-1 cells after single treatment with eltanexor and cytarabine. Changes in gene expression were assessed by real time PCR. Confocal immunofluorescence microscopy was used to study the localization of target proteins. Leukemic blasts from five DEK-NUP214 positive patients were injected intravenously into irradiated NSGS mice. Engraftment of human cells in peripheral blood was monitored monthly by flow cytometry, and engrafting cells were serially transplanted. The DEK-NUP214 fusion transcript was confirmed by Sanger sequencing and molecular aberrations were evaluated with a custom myeloid sequencing panel. Five weeks after transplantation, treatment with eltanexor or vehicle (CTL) was initiated in 5 mice per group at 12.5 mg/kg (for 12 weeks at 5 days/week, and 4 weeks at 4 days/week). Results: Eltanexor treated FKH-1 cells showed an increase in apoptotic cells in a dose-dependent manner (42.4% at 100 nM vs 23.4% with CTL, p=0.0008), a decrease in cell proliferation, a reduction in overall cell viability, and downregulation of the fusion gene as well as EYA3, PRDM2 and SESN1, which are putative target genes of DEK-NUP214. Although cytarabine treatment induced apoptosis in FKH-1 cells, the downstream targets were not affected, suggesting an on-target effect of eltanexor. Eltanexor inhibited cell cycle (proportion of cells in S phase for eltanexor 1.02% at 400nM vs 7.51% for CTL, p=0.018) and disrupted the co-localization of CRM1 and DEK-NUP214 in the nucleus. In addition, eltanexor reduced the formation of DEK-NUP214 nuclear bodies and reduced protein expression of CRM1 in whole cell lysates. In the first PDX model (PDX1), primary and secondary transplantations showed a mean of 12.5 and 43.9% of CD45+CD33+ cells in peripheral blood at 20-24 weeks. PDX1 mice had high white blood cell counts, low platelets and hemoglobin at time of sacrifice, as well as increased spleen weight and bone marrow blasts, which confirmed the development of AML. The engrafted cells showed a myeloid phenotype with expression of CD38, CD33 and CD14, with higher engraftment in bone marrow than spleen. Mutational analysis showed that the PDX samples retained the same mutations as observed in the patient sample (FLT3-ITD and WT1) besides the DEK-NUP214 fusion transcript. The eltanexor or CTL in vivo treatment started 5 weeks after transplantation similar mean leukemic engraftment between treatment and control groups. After sixteen weeks of treatment and additional 5 weeks of observation, CTL treated mice developed leukemia with 19% engraftment of leukemic cells in peripheral blood, while eltanexor treated mice remained negative, p=0.003. This was accompanied by a decrease in WBC, hemoglobin, and platelet count in CTL treated mice, while the eltanexor treated group retained normal counts. Forty two weeks since transplantation, four vehicle mice have succumbed to disease while eltanexor treated mice remain leukemia free. Gene set enrichment analysis (reactomeDB) from RNAseq data of treated PDX1 cells showed upregulated neutrophil degranulation, signaling by interleukins, specifically IL-10, IL-4, and IL-13, as well as reduced activation of anterior HOX genes in eltanexor vs vehicle treated mice. Conclusion: DEK-NUP214 positive FKH-1 cells are sensitive to CRM1 inhibition by eltanexor, which downregulates putative gene targets of DEK-NUP214 leukemia. We have successfully established a DEK-NUP214 PDX model and show exceptional sensitivity of these cells to eltanexor in vivo.

Disrupting MGA-MYC Driven Metabolic Reprogramming in Richter's Syndrome Pre-Clinical Models Via Novel Therapeutic Approaches

Richter's syndrome (RS) is an aggressive transition of chronic lymphocytic leukemia (CLL) to lymphoma; however, molecular mechanisms underlying CLL-to-RS transformation are poorly understood. MYC network alterations are observed in 70% of RS cases, and MGA (Max-gene-associated), a functional MYC suppressor, undergoes loss-of-function mutations in ~7-20% of RS cases. MYC overexpression induces oxidative stress via reactive oxygen species (ROS) in several B-cell lymphomas. Using CRISPR-Cas9, we recently developed a B-cell restricted CLL to RS murine model by engineering loss-of-function Mga mutations in early progenitors (LSK) in the presence of commonly occurring CLL mutations 13q deletion (Mdr) and Sf3b1K700E. We found Myc and Nme1 (Nucleoside diphosphate kinase), both common targets of Mga, were up-regulated, altering the mitochondrial structure, and increasing oxidative phosphorylation (OXPHOS), leading to mitochondrial dysregulation. Based on these observations, we sought to understand the molecular basis of mitochondrial dysregulation and determine if targeting the Mga-Myc-Nme1 axis is beneficial for RS in vivo. Dysregulated mitochondria produce reactive oxygen species (ROS), byproducts of OXPHOS that are reported to trigger oncogenic signaling. To determine the molecular basis of mitochondrial dysregulation, we used our previously established Mga KO (knockout) driven CLL to RS murine model, which develops RS upon CLL cells transplantation into either immunocompetent or immunocompromised mice with high Ki67, MYC, and CD5 loss along with aberrant changes in mitochondria. Firstly, we measured cellular and mitochondrial ROS in splenic B cells derived from CLL, RS, and healthy control mice (n=3 each). CLL and RS B cells had higher cellular ROS and mitochondrial ROS than normal B cells. RS cells showed an elevated cellular ROS compared to CLL cells (p< 0.001). Accordingly, RS mitochondria exhibited high utilization of TCA cycle substrates such as succinate and malate (Seahorse substrate oxidation test). The substrates for complex II of the mitochondrial electron transport chain were also highly utilized. To unravel the molecular mechanism underlying MGA KO-induced metabolic changes, we generated MGA KO human B cell lines [MEC1, HG3, Nalm6E (harboring SF3B1K700E)] by CRISPR-Cas9. MGA KO increased cellular ROS in all cell lines and upregulated NME1, ETC (electron transport chain) complex II protein levels, suggesting that NME1, and ETC complex II may regulate ROS. Moreover, MGA KO induced pERK1/2 (ERK) and p4E-BP1 (mTOR) activation in an NME1-dependent fashion, which was reversed by antioxidant treatment (N-acetyl cysteine), highlighting MGA KO-induced ROS as a key driver of oncogenic activation. Given that ROS activates oncogenic signaling in MGA KO B cells, we exploited mitochondrial dysregulation as a potential therapeutic approach to treating RS. Firstly, we tested the inhibitor of complex II (TTFA: Thenoyltrifluoroacetone) and CDK9 (AZ5576) in MGA KO Nalm6E cells. TTFA and CDK9 inhibitors significantly decreased the activation of pERK1/2 and p4EBP1(mTOR) with a concomitant decrease in MYC and NME1 at the protein level. We next explored these inhibitors for treating murine RS in vivo. We established murine RS cells by engrafting RS cells into NSG mice and then treated RS mice by orally given vehicle (n = 13); AZ5576 (60mg/kg) (n = 13); TTFA (25mg/kg) (n = 4); and AZ + TTFA (60 and 25mg/kg) (n = 5). AZ5576 and TTFA single treatment significantly prolonged the survival of RS mice (Log-rank test, p < 0.0001) with a reduction in total spleen weight (p=0.0007). AZ+TTFA combination had synergy in increasing the survival of RS mice by 10 days (p=0.0003) compared to a single treatment. AZ+TTFA treated murine RS cells showed reduced MYC, NME1, and p4E-BP1 protein expression, confirming the effectiveness of targeting of Mga-Myc-Nme1 axis. In summary, our murine model reveals that MGA/MYC/NME1 axis drives RS through increased mitochondrial OXPHOS and ROS accumulation. Moreover, targeting this axis provides therapeutic benefits for RS, highlighting this pathway as a novel potential target for RS treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison of intratumor and local immune response between MV X-ray FLASH and conventional radiotherapies

Background/PurposeInvestigating the antitumor effect and intratumor as well as local immune response in breast cancer-bearing mice after MV X-ray ultra-high dose rate radiotherapy (FLASH-RT) and conventional dose rate radiotherapy (CONV-RT). Materials/MethodsSix-week-old female C57BL/6 mice were inoculated subcutaneously with Py8119 and Py230 breast tumor cells in the inguinal mammary gland and administered 10 Gy abdominal 6 MV X-ray FLASH-RT (125 Gy/s) or CONV-RT (0.2 Gy/s) 15 days after tumor inoculation. Tumor and spleen tissues were obtained at different time points post-irradiation (PI) for analysis of immune cell infiltration using flow cytometry and immunohistochemical (IHC) staining. Intestine tissues were collected 3 days PI to evaluate normal tissue damage and immune cell infiltration. ResultsBoth FLASH-RT and CONV-RT significantly delayed tumor growth. Flow cytometry showed increased CD8+/CD3 + and CD8+/CD4 + ratios, and IHC confirmed a similar increased CD8 + T cell infiltration at 2 weeks PI in Py8119 tumor tissues in both irradiation groups. No statistical difference was observed between the irradiation groups in terms of tumor growth and increased T cell infiltration in the tumor. Unexpectedly, significantly smaller spleen weight and substantially higher CD8+/CD3 + and lower CD4+/CD3 + ratios were observed in the spleens of the FLASH-RT group than in the spleens of the non-irradiated control and CONV-RT groups 4 weeks PI. Pathological analysis revealed severe red pulp expansion in several spleens from the CONV-RT group, but not in the spleens of the FLASH-RT group. Reduced intestinal damage, macrophage and neutrophil infiltration were observed in the FLASH-RT group compared with CONV-RT group. ConclusionsFLASH-RT and CONV-RT effectively suppressed tumor growth and promoted CD8 + T cell influx into tumors. FLASH-RT can induce different splenic immune responses and reduce radiation-induced damage in the spleen and intestine, which may potentially enhance the therapeutic ratio of FLASH-RT.

Extraction and immunomodulatory activity of the polysaccharide obtained from Craterellus cornucopioides.

In this study, we investigated the structural features of the polysaccharide obtained from Craterellus cornucopioides (CCP2) by high-performance liquid chromatography, Fourier transform infrared spectroscopy and ion chromatography. The results showed that CCP2 was a catenarian pyranose that principally comprised of mannose, galactose, glucose, and xylose in the ratio of 1.86: 1.57: 1.00: 1.14, with a molecular weight of 8.28 × 104 Da. Moreover, the immunoregulation effect of CCP2 was evaluated both in vitro and in vivo. It displayed a remarkable immunological activity and activation in RAW264.7 cells by enhancing the phagocytosis of macrophages in a dose-dependent manner without showing cytotoxicity at the concentrations of 10-200 μg/mL in vitro. Additionally, Histopathological analysis indicated the protective function of CCP2 against immunosuppression induced by cyclophosphamide (Cy). Meanwhile, the intake of CCP2 had better immunoregulatory activity for immunosuppression BALB/c mice model. After prevention by CCP2, the spleen and thymus weight indexes of BALB/c mice model were significantly increased. The RT-qPCR and Western Blot results provided comprehensive evidence that the CCP2 could activate macrophages by enhancing the production of cytokines (IL-2, IL-6, and IL-8) and upregulating the protein expression of cell membrane receptor TLR4 and its downstream protein kinase (TRAF6, TRIF, and NF-κB p65) production of immunosuppressive mice through TLR4-NFκB p65 pathway. The results demonstrated that CCP2 could be a potential prebiotic and might provide meaningful information for further research on the immune mechanism.

Effects of combined feed additives in diets to support growth performance and intestinal health profile in nursery piglets

Two experiments were conducted to assess the effects of feed additive blends in nursery piglets diets on growth performance, apparent total tract digestibility (ATTD) of nutrients and energy, and intestinal health. In Exp. I, 144 male piglets (6.9 ± 0.04 kg) were allotted randomly into 4 treatments: 1) negative control (NC, no feed additive or antibiotic), 2) positive control (PC, amoxicilin + norfloxacin), 3) simple blend (SB) containing sodium butyrate + dried hydrolyzed yeast + zinc proteinate, and 4) complex blend (CB) containing SB + dried brewery yeast + yeast cell wall, with 12 replicates and 3 animals/pen. Diarrhea occurrence (DO) was monitored daily. On d 44, 9 animals/treatment were slaughtered to collect intestinal samples. Dietary treatments had no effect on growth performance, but piglets fed SB and CB diets showed lower DO than those fed PC diet. Lower DO was observed in starter piglets fed CB and PC diets. Pre-starter piglets fed NC diet showed greater plasma urea concentration than piglets fed PC diet. Starter piglets fed CB diet showed lower eosinophils concentration than those fed NC diet. Piglets on CB diet showed greater stomach pH. Piglets fed the NC diet had greater small intestine weight and lower spleen weight compared to piglets fed the PC and SB diets, respectively. Piglets fed CB diet showed greater jejunum villi than animals on PC and SB diets. Furthermore, greater ileum crypt depth and lower VH:CD ratio was observed in piglets fed SB diet than in CB diet. Piglets fed CB diet showed lower jejunal Enterobacteriaceae count compared to piglets fed PC diet. Goblet cells were more abundant in the jejunum of piglets fed CB diet compared to those in NC diet. Lower tissue necrosis was observed in piglets fed SB and CB diets compared to NC and PC diets. In Exp. II, 24 male piglets (20.3 ± 0.05 kg) were allotted randomly in metabolic cages with the same treatment groups as Exp. I and 6 replicates/treatment. Piglets fed PC and SB diets showed greater ATTD compared to those in CB diet. However, piglets fed SB diet had greater digestible protein and lower digestible OM was observed in piglets fed CB diet. In conclusion, SB did not improve growth performance despite favorable effects on ATTD, but CB was the most promising combination on development of the structure and function of the intestinal epithelium, no detrimental effect on growth performance in nursery piglets.

Thallium Induced Sub-chronic Toxicity Profile in Wistar Rats

Thallium (Tl) is one of the most toxic heavy metals affecting both humans and animals, yet is one of the least studied for its toxicological effects. A total of 40 male Wistar rats were randomly divided into four Groups with 10 rats in each Group. Group I received distilled water only and served as a control. Group II, III, and IV received thallium sulfate (I) at 0.48, 0.93, and 1.33 mg/kg body weight, respectively, in drinking water for 90 days. In Group IV rats, lethargy, dehydration, weakness, mild diarrhea, weight loss and 100% morality in male Wistar rat were noted. Group II and III rats showed significant reduction in cholesterol, phosphorus, triglyceride level, and absolute and relative spleen weight. Microscopically, thallium sulfate (I) found to cause lymphoid depletion in white pulp of spleen in all treatment Groups. In kidney of Group IV rats, tubular necrosis, cytoplasmic vacuolation or clearing of tubules, cast, and thrombosis were found. Dose dependent hyperkeratosis was found in non-glandular stomach in all three treated Groups. In conclusion, spleen, kidney and stomach were primarily affected organs in sub-chronic thallium toxicity.

Effect of In Ovo Vitamin C Injection against Mobile Phone Radiation on Post-Hatch Performance of Broiler Chicks.

This study aimed to investigate the effect of in ovo injection of vitamin C to reduce the harmful effects of electromagnetic waves (EMWs) emitted from mobile phones on chicken embryos. In this study, a total of 750 fertilized eggs of Ross 308 were exposed to EMWs for 1050 min during the incubation period. On the 17th day of incubation, the eggs were divided into three groups and solutions were injected into the amnion sac of embryos. The chicks were housed separately in accordance with in ovo treatments for 5 wk after hatching. An in ovo vitamin C injection resulted in a lower hatching weight. The post-hatch mortality or production efficiency factor of birds in the in ovo vitamin C injection group and the intact egg group were comparable, and were better than that of the other negative control group. In ovo vitamin C injection in eggs subjected to EMWs significantly increased their body weight gain, carcass weight, abdominal fat weight, and AST levels, but reduced spleen weight and PON-1 levels. In conclusion, an in ovo vitamin C injection in eggs subject to mobile phone EMWs improved the post-hatch performance of chicks, but low PON and high AST activities indicate an increase in oxidative damage among broiler chicks.

Toll-like receptor 3 neuroimmune signaling and behavior change: A strain comparison between Lewis and Wistar rats

There are many unanswered questions about the interaction between the immune system and behavior change, including the contributions of individual differences. The present study modeled individual differences in the immune system by comparing inbred Lewis rats, which have dysregulated stress and immune systems, to their genetically diverse parent strain, Wistar rats. The objective was to examine the consequences of an immune challenge on behavior and neuroimmune signaling in both strains. Peripheral administration of the toll-like receptor 3 (TLR3) agonist and viral memetic polyinosinic–polycytidylic acid (poly(I:C)) induced behavior changes in both strains, reducing locomotor activity and increasing avoidance behavior (time on the dark side of the light-dark box). Furthermore, poly(I:C) induced hyperarousal and increased avoidance behavior more in female Lewis than female Wistar rats. Baseline strain differences were also observed: Lewis rats had higher avoidance behavior and lower startle response than Wistars. Lewis rats also had lower levels of peripheral inflammation, as measured by spleen weight. Finally, poly(I:C) increased expression of genes in the TLR3 pathway, cytokine genes, and CD11b, a gene associated with proinflammatory actions of microglia, in the prelimbic cortex and central amygdala, with greater expression of cytokine genes in male rats. Lewis rats had lower baseline expression of some neuroimmune genes, particularly CD11b. Overall, we found constitutive strain differences in immune profiles and baseline differences in behavior, yet poly(I:C) generally induced similar behavior changes in males while hyperarousal and avoidance behavior were heightened in female Lewis rats.

Masticadienonic acid from Chios mastic gum mitigates colitis in mice via modulating inflammatory response, gut barrier integrity and microbiota

BackgroundChios mastic gum (CMG) is a traditional Greek medicine used to treat a variety of gastrointestinal disorders, including inflammatory bowel disease (IBD). However, the bioactive compounds of CMG and the mechanisms of action for controlling of IBD remain unknown. PurposeMasticadienonic acid (MDA) is one of the most abundant constituents isolated from CMG. This study aims to investigate the potential effects and underlying mechanisms of MDA in the pathogenesis of colitis. MethodsThe effects of MDA were evaluated using a dextran sulphate sodium (DSS)-induced acute colitis mouse model. The body and spleen weight and colon length and weight were measured and the clinical symptoms were analysed. Blood samples were collected to analyse the level of serum inflammatory markers. Colon tissues were processed for histopathological examination, evaluation of the epithelial barrier function, and investigation of the probable mechanisms of action. The gut microbiota composition was also studied to determine the mechanism for the beneficial effects of MDA on IBD. ResultsMDA could ameliorate the severity of IBD by increasing the body weight and colon length, reducing spleen weight, disease activity index, and histological score. MDA treatments reduce the release of serum inflammatory cytokines tumour necrosis factor-alpha (TNFα), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) via inhibiting the MAPK and NF-κB signalling pathways. MDA supplementation could also improve the intestinal barrier function by activating the NF-E2-related factor-2 (Nrf2) signalling pathway and restoring the expression of tight junction proteins zonula occludens-1 (ZO-1) and occludin. In addition, MDA administration modulates the gut microbiota composition in DSS-induced colitis mice. ConclusionThe results indicate that MDA attenuated experimental colitis by restoring intestinal barrier integrity, reducing inflammation, and modulating the gut microbiota. The present study provides novel insights into CMG-mediated remission of IBD and may facilitate the development of preventive and therapeutic strategies for IBD.

NON-CLINICAL SAFETY EVALUATION OF A NOVEL THERAPEUTIC VACCINE FOR PEANUT ALLERGY

<h3>Introduction</h3> Peanut allergy is the most common cause of fatal food-associated anaphylaxis. VLP Peanut is a novel virus-like particle (VLP)-based vaccine being developed for the treatment of peanut allergy. Prior to first in human trials, its safety was assessed in a repeat dose toxicology study. <h3>Methods</h3> VLP Peanut was administered with or without the adjuvant 1.95% MicroCrystalline Tyrosine, subcutaneously, to Wistar Han rats three times daily (TID) once every 4 weeks for 12 weeks as described (Fig 1). A maximum dose level of 3.6 mg was evaluated (anticipated maximum dose in humans). Assessment of toxicity included clinical, post-dose, and dose site observations; body weights; food consumption; ophthalmic observations; and clinical and anatomic pathology. <h3>Results</h3> Minor changes in bodyweight gains, food consumption and clinical pathology were noted in animals administered VLP Peanut ±MCT and minor, non-adverse, increases in spleen and liver weights were noted in some female animals. These changes were reversible and not adverse. Animals administered VLP Peanut ± MCT had occasional dose site observations of very slight or slight edema, more frequently noted for animals administered MCT. Macroscopic observations at subcutaneous injection site/s were noted for all test article-treated groups, which correlated with microscopic observations of inflammation and neutrophilic reactions at the subcutaneous injection site/s. Dose site observations were considered adverse but were fully reversed after a 4week recovery. <h3>Conclusion</h3> VLP Peanut administered to rats, was systemically well tolerated, with only minor, non-adverse clinical changes. Slight dose site observations were fully-reversible and did not impact on the overall health of the animals.

The targeted anti-Salmonella bacteriophage attenuated the inflammatory response of laying hens challenged with Salmonella Gallinarum

Fowl typhoid is a severe disease caused by Salmonella Gallinarum with considerable mortality and morbidity in laying hen farms. The current study has focused on controlling the infection in laying hens using anti-Salmonella spp. bacteriophage. The treatments included, PC, without challenge; NC, S. Gallinarum challenged (SGC); B5, 5 mg bacteriophage/kg+SGC; B10, 10 mg bacteriophage/kg+SGC. The Salmonella shedding, inflammatory responses, and gene expression of pro-inflammatory cytokines, toll-like receptor (TLR), and heat shock protein (HSP) in the jejunum, liver, and thigh muscle were tested in laying hens. Supplementation of bacteriophage reduced the abundance of S. Gallinarum in the excreta at d 3, 7, and 14. The abundance of S. Gallinarum was lower in the B10 than the B5 at d 7. Supplementation of bacteriophage decreased the abundance of S. Gallinarum in the oviduct, spleen, and cecum at d 14. The laying hens in the NC group showed an increased relative spleen weight compared with the PC and B10 treatments. Among the SGC treatments, the NC treatment showed higher gene expressions of IL-4 compared with the B5, higher gene expressions of interferon (IFNγ), TLR4, and tumor necrosis factor-α (TNF-α) compared with the B5 and B10, and higher gene expressions of HSP27 compared with the B10 in the jejunum. Dietary supplementation of B10 decreased the mRNA expressions of TLR4 and TNF-α compared with the B5 treatment in the jejunum. The NC treatment showed the highest gene expressions of HSP27, TLR4, and TNF-α in the liver. Dietary supplementation of B10 showed lower mRNA expressions of HSP27 compared with the B5 treatment in the liver. Moreover, the IFNγ and HSP27 were upregulated in the NC treatment compared with the B5 and B10 in the muscle. In conclusion, it can be suggested that bacteriophage is an effective supplement to control S. Gallinarum infection in laying hens and possibly lower horizontal contaminations in laying hen flocks.

Effects of heat stress on fattening performance, carcass traits, oxidant/antioxidant status, and hepatic heat shock protein 70 levels in different plumage colors of Japanese quail (Coturnix coturnix japonica).

The aim of this study was to investigate the impact of heat stress on production performance and oxidative stress in different plumage colors of Japanese quail. For this purpose, a total of 100 birds were used in this study. The 25 birds belonged to Wild-type (n=25, grey), Tuxedo (n=25, black), Golden (n=25, yellow) and Recessive white (n=25). The birds were reared for 42 days in an environmentally controlled room at 39°C and relative humidity of 60-65%. The body weight, body weight gain (g/bird/day), and feed conversion ratio were not different between the groups (p>0.05). However, the feed intake (g/bird/day) of the Wild-type had a higher value than the Tuxedo (black) group counterparts between 15 and 21 days different (p⟨0.05). There was no significant effect of heat stress on the carcass traits (p>0.05). Spleen weights were different between the groups (p⟨0.05). The yellow group had the highest spleen weight. The highest MDA level was found in the Recessive White variety, followed by Wild-type (grey), Golden (yellow) and Tuxedo (black), respectively. However, there were no statistical differences amongst the groups (p>0.05). There was also no statistical significance in glutathione (GSH) and superoxide dismutase (SOD) levels (p>0.05). The heat shock protein 70 kDa (HSP70) level was significantly different between the groups (p⟨0.001). The highest percentage was observed in the Golden (5.06%) and the lowest in the White (1.43%) variety. There was no superior color variety of Japanese quail regarding fattening performance and carcass traits. It is conceivable that when considering the stress response of the different colors, the Golden group is more sensitive to stress due to the hepatic and cellular level of HSP70.