Burden In Spleen Research Articles

Hypertonic Saline Induces Host Protective Immune Responses against Brucella abortus Infection in Mice.

Hypertonic saline (HTS) resuscitation can enhance immune responses against various pathogens, however, the effect of HTS on brucellosis is yet to be defined. In this study, we found that HTS inhibited Brucella infection in mice by augmenting Th1 immunity. HTS treatment enhanced the serum cytokines production and the expression of nitric oxide synthase (NOS2) and nuclear factor kappa B (NF-ĸB) p50 and p65, crucial anti-Brucella effectors in splenocytes. In addition, HTS treatment also inhibited the phosphorylation of MAPK signaling, accompanied by the down-regulation of the autophagy marker LC3B-II. Due to directing an appropriate immune response, HTS treatment substantially decreased bacterial burden in spleen and liver tissues. In summary, corroborating previous studies showing the antimicrobial effects of HTS, our findings indicate that HTS treatment triggers a protective immune response against Brucella infection. Additionally, these results provide promising evidence of the immunomodulatory role of HTS in controlling bacterial infections.

Lactobacillus casei displaying MCP2α and FlaC delivered by PLA microspheres effectively enhances the immune protection of largemouth bass (Micropterus salmoides) against LMBV infection

Largemouth bass ranavirus (LMBV) seriously affects the development of largemouth bass (Micropterus salmoides) industry and causes huge economic losses. Oral vaccine can be a promising method for viral disease precaution. In this study, MCP2α was identified as a valuable epitope region superior to MCP and MCP2 of LMBV by neutralizing antibody experiments. Then, recombinant Lactobacillus casei expressing the fusion protein MCP2αC (MCP2α as antigen, C represents flagellin C from Aeromonas hydrophila as adjuvant) on surface was constructed and verified. Further, PLA microsphere vaccine loading recombinant MCP2αC L. casei was prepared. The PLA microspheres vaccine were observed by scanning electron microscopy and showed a smooth, regular spherical surface with a particle size distribution between 100 and 200 μm. Furthermore, we evaluated the tolerance of PLA-MCP2αC vaccine in simulated gastric fluid and simulated intestinal fluid, and the results showed that PLA-MCP2αC can effectively resist the gastrointestinal environment. Moreover, the protective effect of PLA-MCP2αC against LMBV was evaluated after oral immunization and LMBV challenge. The results showed that PLA-MCP2αC effectively up-regulated the activity of serum biochemical enzymes (T-SOD, T-AOC, LZM, complement C3) and induced the mRNA expression of representative immune genes (IL-1β, TNF-α, IFN-γ, MHC-IIα, Mx, IgM) in spleen and head kidney tissues. The survival rate of largemouth bass vaccinated with PLA-MCP2αC increased from 24 % to 68 %. Meanwhile, PLA-MCP2αC inhibited the LMBV burden in spleen, head kidney and liver tissues and attenuated tissue damage in spleen. These results suggested that PLA-MCP2αC can be used as a candidate oral vaccine against LMBV infection in aquaculture.

The Integrin Adaptor Kindlin-3 Is a Gatekeeper for Germinal Center Responses and the Development of Chronic Lymphocytic Leukemia

Background: High affinity antibody production occurs in the germinal centers (GCs), which are segregated into a dark zone (DZ) and a light zone (LZ). GC B cells proliferate in the DZ and encounter follicular dendritic cells in the LZ. Cell trafficking between the two zones is regulated by CXCR4 (DZ) and CXCR5 (LZ) signaling. Integrins may regulate B cell positioning and B cell immune synapse formation. Recently, we have shown that the integrin adaptor kindlin-3 is not only required for integrin activation in B cells, but unexpectedly also acts as a brake on CXCR5 responsiveness. Kindlin-3 thereby prevents B cell hyperactivation and differentiation into GC-like B cells in absence of antigen. 1 Here we investigate the impact of kindlin-3 on GC responses upon antigen challenging and on malignant transformation. Methods: We developed a B cell-specific knockout mouse model (mb1Cre Fermt3fl/fl (K3ΔB)) in which kindlin-3 ( Fermt3) was knocked out in all B cells. K3ΔB and control wild-type (WT) mice were challenged with sheep red blood cells (SRBC). GC formation and immunoglobulin (Ig) production were analyzed by flow cytometry. Ex vivo B cell proliferation and Ig class switching formation assays were performed. We investigated the immune synapse formation of WT and kindlin-3-deficient B cells using a biomimetic model involving a planar artificial lipid bilayer containing GPI-linked integrin ligands and bound fluorescently labeled surrogate antigens. The formation of immune synapse and central antigen aggregation in the immune synapse were analyzed by real-time interference reflection microscopy and laser scanning confocal microscopy. To investigate the contribution of kindlin-3 in a model of mature B cell malignancy, we used TCL1 transgenic (TCL1) mice resembling chronic lymphocytic leukemia (CLL) to generate the mb1Cre Fermt3fl/fl Tcl1 (TCL1-K3ΔB) mouse model. Tumor burden in TCL1-K3ΔB and TCL1 control mice was monitored by flow cytometry over time and at sacrifice using CD19/CD5 antibodies. B cell receptor (BCR) signaling of CLL cells was analyzed using Phosphoflow. Results: K3∆B mice immunized with SRBC exhibited defective GC responses (30% reduction in GC-B cells compared to WT) and antibody production (49% reduction in SRBC-specific IgG2b production in serum compared to WT) on day 7 after immunization. Ex vivo, kindlin-3-deficient B cells were unable to form immune synapses (Figure 1A), to proliferate, and to switch antibody classes. The tumors in TCL1 mice started in the peritoneal cavity and subsequently spread to the spleen, blood and bone marrow. TCL1-K3ΔB mice presented with an almost complete abolished leukemia onset in the peritoneal cavity (92% less tumor burden in TCL1-K3ΔB compared to control TCL1 mice) in 4-month-old mice (Figure 1B i). Moreover, tumor burden in blood (Figure 1B ii), spleen, and bone marrow (Figure 1B iii) was strongly reduced at the late stage of disease (11.5-month-old mice), with an 82% lower tumor burden in spleen and a 90% lower tumor burden in bone marrow of TCL1-K3ΔB mice compared to TCL1 control mice. In addition, kindlin-3-deficient CLL cells exhibited impaired phosphoAkt and phosphoSyk signaling upon BCR engagement. Summary/Conclusion: In summary, kindlin-3 maintains the balance between a state of B cell homeostasis and an effective antigenic response by acting as a gatekeeper of GC formation, and is substantial for leukemic onset in CLL. Reference: 1. Härzschel A, Li L, Krenn PW, et al. Kindlin-3 maintains marginal zone B cells but confines follicular B cell activation and differentiation. J Leukoc Biol. 2022 Apr;111(4):745-758.

Retrospective Real-World Analysis of Baseline Characteristics and Posttreatment Hematologic Outcomes in Patients with Myelofibrosis Receiving Ruxolitinib

Introduction: Myelofibrosis (MF) is a chronic myeloproliferative neoplasm characterized by the key hallmarks of constitutional symptoms, splenomegaly, and cytopenias such as anemia and thrombocytopenia. While Janus kinase (JAK) inhibitors have become a mainstay of MF treatment, improving symptom and spleen burden in many patients (pts), some approved JAK inhibitors such as ruxolitinib (RUX) may worsen or fail to adequately address anemia and thrombocytopenia. Dose reduction may mitigate the myelosuppressive effects of RUX, but this strategy and resultant effects on real-world clinical outcomes are not well characterized. Here we describe real-world baseline characteristics and posttreatment hematologic outcomes in a US pt population with MF treated with RUX, including those with red blood cell (RBC) transfusion burden at baseline. Methods: This was a retrospective analysis of adult pts in the US Flatiron Health electronic health record-derived deidentified database with evidence of MF ( International Classification of Diseases, Ninth Revision or Tenth Revision diagnosis) on or after January 1, 2013, and ≥1 administration of an antineoplastic agent; the data cutoff was March 1, 2023. Pts were required to have ≥1 order or administration of RUX after MF diagnosis. Follow-up was from initiation of RUX (index date) to earliest of end of clinical activity, data availability, or death; only pts with index dates in April 2013 or later were included to allow for a 3-month baseline period. Baseline characteristics were summarized at index date (or closest available visit ≤3 months prior) and included demographics, disease characteristics, hematologic measures, transfusion status, and prior treatment. Incidence of anemia, thrombocytopenia, and either anemia or thrombocytopenia (defined as a negative hematologic outcome), based on laboratory values (hemoglobin [Hb] levels <10 g/dL, platelet counts <150×10 9/L) or the presence of diagnostic codes, were evaluated in time-to-event analyses over the follow-up period. Subgroup analyses in pts who were transfusion dependent (TD; ≥4 RBC units transfused in any 8-week period, or ≥1 Hb level <8 g/dL at any point, within the previous 12 weeks) at baseline were also performed. Transfusion status at specific time points during follow-up was summarized. Results: A total of 383 pts with MF treated with RUX in the real-world database met the criteria for analysis. The mean age was 72.0 years, 44.4% were female, and 72.4% were White. A total of 58.2% had primary MF; mean time from diagnosis to index was 8.7 months, and 22.2% had prior non-JAK inhibitor therapy before RUX initiation. Cytopenias were common at baseline; 45.7% had anemia and 24.5% had thrombocytopenia, based on laboratory assessment or diagnostic codes. Among pts with available Hb laboratory values, 13.4% had severe (<8 g/dL) and 38.5% had moderate (≥8 to <10 g/dL) anemia. A total of 20.9% were TD, with a mean 0.6 RBC units transfused per month. In time-to-event analyses, among the 175 pts who did not have either anemia or thrombocytopenia at baseline, 149 (85%) had an incident negative hematologic outcome (ie, anemia or thrombocytopenia) event during follow-up. The 6- and 12-month event rates were 63.5% and 72.7%; median time to event was 2.7 months (95% CI, 2.1-4.9 months) (Figure). In the respective subpopulations without anemia (n=208) or thrombocytopenia (n=289) at baseline, median time to anemia was 3.8 months (95% CI, 2.4-6.7 months), and median time to thrombocytopenia was 10.9 months (95% CI, 7.7-19.7 months); 12-month event rates were 66.8% and 51.8%. At 3 months after index date, 41.3% of pts were TD. Among those who were TD at baseline, 88.8% remained TD after 3 months of RUX treatment, with a mean increase in transfusion intensity of 0.4 units per month. Among pts who were not TD at baseline, 24.4% became TD after 3 months of RUX treatment, with a mean increase in transfusion intensity of 0.6 units per month. Conclusions: This retrospective analysis provides insights into hematologic outcomes under the current first-line treatment paradigm for MF in clinical practice. Many pts who initiated RUX were anemic and/or thrombocytopenic at baseline, and incidences of anemia and thrombocytopenia as well as RBC transfusion burden increased over time. These results highlight the continued need for treatments that address the underlying hematologic burden, including anemia and thrombocytopenia, of pts with MF.

Immunoregulatory and Anti-cancer Activities of Combination Treatment of Novel Four-Herb Formula and Doxorubicin in 4T1-Breast Cancer Bearing Mice.

To investigate the in vivo immunomodulatory and anti-tumor mechanisms of the combined treatment of novel Four-Herb formula (4HF) and doxorubicin in triple-negative breast cancer (TNBC). Murine-derived triple-negative mammary carcinoma cell line, 4T1 cells, was cultured and inoculated into mouse mammary glands. Sixty-six mice were randomly assigned into 6 groups (n=11 in ench): naïve, control, LD 4HF (low dose 4HF), HD 4HF (high dose 4HF), LD 4HF + D (low dose and doxorubicin), and D (doxorubicin). Apart from the naïve group, each mouse received subcutaneous inoculation with 5 × 105 4T1 cells resuspended in 100 µL of normal saline in the mammary fat pads. Starting from the day of tumor cell inoculation, tumors were grown for 6 days. The LD and HD groups received daily oral gavage of 658 and 2,630 mg/kg 4HF, respectively. The LD 4HF+D group received daily oral gavage of 658 mg/kg 4HF and weekly intraperitoneal injection of doxorubicin (5 mg/kg). The D group received weekly intraperitoneal injections of doxorubicin (5 mg/kg). The treatment naïve mice received daily oral gavage of 0.2 mL double distilled water and 0.1 mL normal saline via intraperitoneal injection once a week. The control group received daily oral gavage of 0.2 mL double-distilled water. The treatment period was 30 days. At the end of treatment, mice organs were harvested to analyze immunological activities via immunophenotyping, gene and multiplex analysis, histological staining, and gut microbiota analysis. Mice treated with the combination of 4HF and doxorubicin resulted in significantly reduced tumor and spleen burdens (P<0.05), altered the hypoxia and overall immune lymphocyte landscape, and manipulated gut microbiota to favor the anti-tumor immunological activities. Moreover, immunosuppressive genes, cytokines, and chemokines such as C-C motif chemokine 2 and interleukin-10 of tumors were significantly downregulated (P<0.05). 4HF-doxorubicin combination treatment demonstrated synergetic activities and was most effective in activating the anti-tumor immune response (P<0.05). The above results provide evidence for evaluating the immune regulating mechanisms of 4HF in breast cancer and support its clinical significance in its potential as an adjunctive therapeutic agent or immune supplement.

Efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates.

The aim of this study was to evaluate the efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates. Initially, 24-1 fractional experimental design was carried out to obtain an optimized formulation of liposomes containing CPO (CPO-LipoC), which were then used to prepare stealth liposomes (CPO-LipoS). Liposomal formulations were characterized by their mean size diameter, polydispersity index (PDI), and drug encapsulation efficiency (EE%). Immunosuppressed mice were exposed to CPO-LipoS at 0.5mg/kg/day for 14days to verify possible histopathological alterations in the liver and kidneys. Immunosuppressed mice infected with C. neoformans were treated with CPO-LipoS at 0.5mg/kg/day for 14days to quantify the fungal burden in spleen, liver, lungs, and brain. CPO-LipoS presented a mean size diameter, PDI, and EE% of 101.4 ± 0.7nm, 0.307, and 96.4 ± 0.9%, respectively. CPO-LipoS was non-toxic for the liver and kidneys of immunosuppressed mice. At the survival curve, all infected animals submitted to treatment with CPO-LipoS survived until the end of the experiment. Treatment with CPO-LipoS reduced C. neoformans cells in the spleen (59.3 ± 3.4%), liver (75.0 ± 3.6%), lungs (75.7 ± 6.7%), and brain (54.2 ± 3.2%). CPO-LipoS exhibit antifungal activity against C. neoformans, and the encapsulation of CPO into stealth liposomes allows its use as a systemic drug for treating cryptococcosis.

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

Microbial uptake in oral mucosa-draining lymph nodes leads to rapid release of cytotoxic CD8+ T cells lacking a gut-homing phenotype.

The gastrointestinal (GI) tract constitutes an essential barrier against ingested microbes, including potential pathogens. Although immune reactions are well studied in the lower GI tract, it remains unclear how adaptive immune responses are initiated during microbial challenge of the oral mucosa (OM), the primary site of microbial encounter in the upper GI tract. Here, we identify mandibular lymph nodes (mandLNs) as sentinel lymphoid organs that intercept ingested Listeria monocytogenes (Lm). Oral Lm uptake led to local activation and release of antigen-specific CD8+ T cells that constituted most of the early circulating effector T cell (TEFF) pool. MandLN-primed TEFF disseminated to lymphoid organs, lung, and OM and contributed substantially to rapid elimination of target cells. In contrast to CD8+ TEFF generated in mesenteric LN (MLN) during intragastric infection, mandLN-primed TEFF lacked a gut-seeking phenotype, which correlated with low expression of enzymes required for gut-homing imprinting by mandLN stromal and dendritic cells. Accordingly, mandLN-primed TEFF decreased Lm burden in spleen but not MLN after intestinal infection. Our findings extend the concept of regional specialization of immune responses along the length of the GI tract, with CD8+ TEFF generated in the upper GI tract displaying homing profiles that differ from those imprinted by lymphoid tissue of the lower GI tract.

Trained Innate Immunity Induced by Vaccination with Low-Virulence Candida Species Mediates Protection against Several Forms of Fungal Sepsis via Ly6G+ Gr-1+ Leukocytes.

ABSTRACTWe recently discovered a novel form of trained innate immunity (TII) induced by low-virulence Candida species (i.e., Candida dubliniensis) that protects against lethal fungal/bacterial infection. Mice vaccinated by intraperitoneal (i.p.) inoculation are protected against lethal sepsis following Candida albicans/Staphylococcus aureus (Ca/Sa) intra-abdominal infection (IAI) or Ca bloodstream infection (BSI). The protection against IAI is mediated by long-lived Gr-1+ leukocytes as putative myeloid-derived suppressor cells (MDSCs) and not by prototypical trained macrophages. This study aimed to determine if a similar TII mechanism (Gr-1+ cell-mediated suppression of sepsis) is protective against BSI and whether this TII can also be induced following intravenous (i.v.) vaccination. For this, mice were vaccinated with low-virulence Candida strains (i.p. or i.v.), followed by lethal challenge (Ca/Sa i.p. or Ca i.v.) 14 days later, and observed for sepsis (hypothermia, sepsis scoring, and serum cytokines), organ fungal burden, and mortality. Similar parameters were monitored following depletion of macrophages or Gr-1+ leukocytes during lethal challenge. The results showed that mice vaccinated i.p. or i.v. were protected against lethal Ca/Sa IAI or Ca BSI. In all cases, protection was mediated by Ly6G+ Gr-1+ putative granulocytic MDSCs (G-MDSCs), with no role for macrophages, and correlated with reduced sepsis parameters. Protection also correlated with reduced fungal burden in spleen and brain but not liver or kidney. These results suggest that Ly6G+ G-MDSC-mediated TII is induced by either the i.p. and i.v. route of inoculation and protects against IAI or BSI forms of systemic candidiasis, with survival correlating with amelioration of sepsis and reduced organ-specific fungal burden.

Pre-Clinical Studies of Allogeneic Anti-CD123 CAR T-Cells for the Therapy of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Pre-Clinical Studies of Allogeneic Anti-CD123 CAR T-Cells for the Therapy of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Combinatorial Inhibition of Focal Adhesion Kinase and BCL-2 in AML

Focal adhesion kinase (FAK), activated by integrins and cytokines, promotes cell growth, migration and metastasis. We previously reported that a selective FAK inhibitor, VS-4718, inhibited cell growth and induced cell death in leukemia cell lines in vitro, effectively reduced leukemia burden in peripheral blood and tissues including bone marrow, spleen, liver and lung, and significantly prolonged survival time in a human xenograft murine model (Carter BZ et al., MCT, 2017), 16(6):1133).Although BCL-2 inhibition by the BCL-2 inhibitor ABT-199 (Venetoclax) has demonstrated antileukemia activities, an acquired drug resistance frequently ensues. When combined with ABT-199, VS-4718 significantly potentiated ABT-199-induced apoptosis in AML cell lines and primary AML cells. VS-4718 treatment of Molm14 AML cells decreased levels of p-STAT5, MCL-1, and BCL-XL (Wang et al., ASH 2016) that play important roles in the acquired resistance to ABT-199.To further examine the effect of VS-4718 as a single agent and in combination with ABT-199 on primary AML cells in vivo, we generated patient-derived xenografts (PDX) from AML patient bone marrow cells expanded in NSGS (NOD-SCID IL2Rgnull-3/GM/SF, NSG-SGM3) mice. The xenograft used AML cells from a patient who had failed multiple chemotherapies and had NPM1, FLT3-ITD, TET2, DNMT3A, and WT1 gene mutations and a complex karyotype. The combination of VS-4718 and ABT-199 synergistically killed the PDX cells in vitro even under MSC co-culture conditions. Once PDX cells were engrafted into NSGS mice, animals were randomized into 4 groups (N=10/group) and treated with vehicle, VS-4718 (75 mg/kg, twice daily), ABT-199 (100 mg/kg, once per day), or the combination via oral gavage for 29 days. We observed that ABT-199 had a stronger anti-leukemic effect on peripheral blood than on tissue-resident cells, while VS-4718 was more effective in tissues. At the end of the treatments, VS-4718 significantly reduced circulating human CD45+ cells (P=0.0003), and ABT-199 alone (P=5.90E-07) and the ABT-199/VS-4718 combination (P=5.38E-07) were ever more effective. VS-4718 alone or in combination with ABT-199 reduced leukemia burden in spleen and liver and effectively reduced splenomegaly, while ABT-199 alone did not. VS-4718-treated mice survived significantly longer than the untreated controls (median survival 72 vs. 36 days, P=0.0002), while ABT-199 did not prolong survival (median survival 35 days). Also, the combination of VS-4718 with ABT-199 significantly prolonged survival (median survival 65.5 days, P=0.011 vs. control and P=0.0072 vs. ABT-199). No statistical difference in survival was found between VS-4718 and the combination treatment groups (P=0.33) (Fig. 1). All the mice had large spleen at death except the combination group: the 1st dead mouse had small and the 2nd to 4th ones had slightly enlarged spleen and only the last two had large spleen as others suggesting that additional reasons such as toxicity at the dose used may have contributed to the death of the 1st 4 mice in the combination group, hence shortened the median survival in this group.We determined expression of FAK and BCL-2 family proteins in cells from mouse spleens at the end of treatment and found that the combination of VS-4718 and ABT-199 decreased protein levels of FAK. Consistent with our in vitro results, the increased level of MCL-1 by ABT-199 was antagonized by the combination with VS-4718 in vivo suggesting the combination potentially be more effective.Conclusions: These results suggest a potential clinical benefit of combining a FAK inhibitor with Venetoclax for patients with AML. [Display omitted] DisclosuresPachter:Verastem, Inc.: Employment. Weaver:Verastem, Inc.: Employment. Carter:Daiichi Sankyo: Research Funding; novartis: Research Funding.

Splicing Modulation Perturbs Key Survival Pathways and Sensitizes Chronic Lymphocytic Leukemia to Venetoclax Treatment

Splicing Modulation Perturbs Key Survival Pathways and Sensitizes Chronic Lymphocytic Leukemia to Venetoclax Treatment

Targeting of the Interleukin-7 Receptor Alpha-Chain (IL-7Rα) Blocks B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Development and Reduces Leukemic Infiltration to the Central Nervous System (CNS)

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has a favorable prognosis when treated according to contemporary protocols. Nevertheless, patients subjected to intensive chemotherapy suffer from significant short- and long-term toxicity, which supports the need for novel targeted therapies. Here, we study the role of IL-7Rα in BCP-ALL development in preclinical model and show gene expression data of patient cohorts suggesting that targeting IL-7Rα may be beneficial for treating ALL patients, especially those with CNS involvement.IL-7Rα is expressed in almost all BCP-ALL patients with particularly high expression levels in E2A-PBX1 leukemias. Therefore, we analyzed mRNA levels of IL-7Rα in diagnostic BM of 61 E2A-PBX1+ pediatric BCP-ALL patients at initial diagnosis. High IL-7Rα expression was associated with an increased white blood cell count (p= 0.0206), which is a poor prognostic indicator in patients. Importantly, high IL-7Rα expression was also associated with CNS positivity (p= 0.0162). There were no correlations between IL-7Rα expression and sex, age, prednisone response or risk group. To further investigate the role of IL-7Rα in CNS infiltration, we determined IL-7Rα expression levels in an expanded BCP-ALL cohort of 98 patients. The cohort was selected to contain 26 CNS-positive (only 1 patient was E2A-PBX1 +) and 72 CNS-negative patients. IL-7Rα expression was significantly elevated in CNS-positive as compared to CNS-negative patients (p= 0.0284). There were no statistical differences in sex, age, prednisone response and MRD risk groups between the CNS-negative and CNS-positive patients. These data suggest that IL-7Rα expression is associated with CNS infiltration in both E2A-PBX1 positive and negative BCP-ALL patientsTo study the effect of inhibiting IL-7Rα signaling in xenograft models, we injected E2A-PBX1 + primary BCP-ALL cells into NSG mice. We monitored the survival of recipient mice under ruxolitinib treatment, which targets IL-7Rα downstream signaling via JAK1/2. Interestingly, ruxolitinib treated mice showed a significantly prolonged survival as compared to untreated mice (median survival 76 vs. 64 days, respectively, p= 0.0002). However, there were no significant differences in leukemic burden in spleen, bone marrow or CNS. Ruxolitinib abrogates IL-7Rα signaling via JAK1/2, but not via JAK3. Therefore, we investigated whether interfering with IL-7Rα expression directly would block infiltration more efficiently than ruxolitinib treatment. For that purpose, the E2A-PBX1 + cell line 697 was lentivirally transduced with specific shRNAs against IL-7Rα (shIL-7Rα) or a non-targeting green fluorescent protein (shGFP) control. The cells were then injected intravenously into NSG mice, which were sacrificed when leukemia symptoms developed. Leukemic bone marrow and splenic infiltration was significantly reduced in shIL-7Rα animals as compared to the control group (median % blasts 64% vs. 21%, p= 0.0167 and 52% vs. 9%, p= 0.0238, respectively). Similarly, immunohistochemical scoring of CNS infiltration revealed that 6/6 animals (100%) in the shGFP group and only 1/6 animals (17%) in the shIL-7Rα group were CNS-positive (p= 0.0152). This suggests that interfering with IL-7Rα expression impairs the proliferation as well as the CNS-positive phenotype of 697 cells in vivo . To test if treatment with anti-IL-7Rα antibody can also interfere with leukemia development, NSG mice bearing 697 cells were treated with anti-human IL-7Rα antibody in the presence or absence of ruxolitinib. Spleen volumes of mice treated with anti-IL-7Rα antibody were significantly smaller than controls (median size 0.1130 cm3 vs. 0.0885 cm3, respectively, p= 0.0079) whereas mice treated with ruxolitinib alone were not significantly different from controls. Similarly, leukemic infiltration in spleen and bone marrow was significantly reduced in mice treated with antibody or with both antibody and ruxolitinib compared to control mice or mice treated with ruxolitinib alone (Figure 1). These data suggest that anti-IL-7Rα therapy is highly efficient in abrogating leukemia development in xenograft mice and outperforms ruxolitinib blockade.Altogether, we show that expression of IL-7Rα is a hallmark of primary BCP-ALL and associated with CNS leukemia. Importantly, we propose the use of IL-7Rα antibodies as a potential therapeutic alternative for treating BCP-ALL. [Display omitted] DisclosuresSchrappe:JAZZ Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; Medac: Consultancy, Research Funding.

Probiotic Bacteria and Their Cell Walls Induce Th1-Type Immunity Against Salmonella Typhimurium Challenge.

Probiotics have been associated with a variety of health benefits. They can act as adjuvant to enhance specific immune response. Bacterial cell wall (CW) molecules are key structures that interact with host receptors promoting probiotic effects. The adjuvant capacity underlying this sub-cellular fraction purified from Lactobacillus casei CRL431 and L. paracasei CNCMI-1518 remains to be characterized. We interrogated the molecular and cellular events after oral feeding with probiotic-derived CW in addition to heat-inactivated Salmonella Typhimurium and their subsequent protective capacity against S. Typhimurium challenge. Intact probiotic bacteria were orally administered for comparison. We find that previous oral feeding with probiotics or their sub-cellular fraction reduce bacterial burden in spleen and liver after Salmonella challenge. Antibody responses after pathogen challenge were negligible, characterized by not major changes in the antibody-mediated phagocytic activity, and in the levels of total and Salmonella-specific intestinal sIgA and serum IgG, respectively. Conversely, the beneficial effect of probiotic-derived CW after S. Typhimurium challenge were ascribed to a Th1-type cell-mediated immunity which was characterized by augmentation of the delayed-type hypersensitivity response. The cell-mediated immunity associated with the oral feeding with probiotic-derived CW was accompanied with a Th1-cell polarizing cytokines, distinguished by increase IFN-γ/IL-4 ratio. Similar results were observed with the intact probiotics. Our study identified molecular events associated with the oral administration of sub-cellular structures derived from probiotics and their adjuvant capacity to exert immune modulatory function.

Prostaglandin I2 (PGI2) inhibits Brucella abortus internalization in macrophages via PGI2 receptor signaling, and its analogue affects immune response and disease outcome in mice

To date, the implications of prostaglandin I2 (PGI2), a prominent lipid mediator for modulation of immune responses, has not been clearly understood in Brucella infection. In this study, we found that cyclooxygenase-2 (COX-2) was significantly expressed in both infected bone marrow-derived macrophages (BMMs) and RAW 264.7 cells. Prostaglandin I2 synthase (PTGIS) expression was not significantly changed, and PGI2receptor (PTGIR) expression was downregulated in BMMs but upregulated in RAW 264.7 macrophages at late infection. Here, we presented that PGI2, a COX-derived metabolite, was produced by macrophages during Brucella infection and its production was regulated by COX-2 and IL-10. We suggested that PGI2 and selexipag, a potent PGI2 analogue, inhibited Brucella internalization through IP signaling which led to down-regulation of F-actin polymerization and p38α MAPK activity. Administration with selexipag suppressed immune responses and resulted in a notable reduction in bacterial burden in spleen of Brucella-challenged mice. Taken together, our study is the first to characterize PGI2 synthesis and its effect in evasion strategy of macrophages against Brucella infection.

Development of a novel T cell-oriented vaccine using CTL/Th-hybrid epitope long peptide and biodegradable microparticles, against an intracellular bacterium.

Antigen-specific CD8+ T-lymphocytes (cytotoxic T-lymphocytes: CTL), as well as CD4+ T-lymphocytes (helper T-lymphocytes: Th), simultaneously play an important role in the elimination of intracellular bacteria such as Mycobacterium tuberculosis and Listeria monocytogenes. Administration of T-cell epitope short peptide needs large numbers of peptides for effective vaccination due to its easily degradable nature in vivo. In this respect, biocompatible and biodegradable microparticles combined with CTL/Th-hybrid epitope long peptide (long peptide) have been used to diminish the degradation of loaded peptide. The aim of this study is to develop a novel T cell-oriented vaccine against intracellular bacteria that is composed of long peptide and poly (lactic-co-glycolic acid) (PLGA) microparticles. Mouse bone marrow-derived dendritic cells (BMDCs) were loaded with L. monocytogenes listeriolysin O (LLO)-derived or ovalbumin (OVA)-derived long peptide/PLGA or other comparative antigens. The antigen-loaded BMDCs were injected subcutaneously into the flank of mice twice, and then, the spleens were collected and lymphocyte proliferation and interferon-γ production were evaluated. The median diameter of the PLGA spheres was 1.38 μm. Both LLO- and OVA-long peptide/PLGA showed significantly more robust CTL and Th proliferations with higher interferon-γ production than the long peptide alone or CTL and Th short peptides/PLGA vaccination. Furthermore, the LLO-long peptide/PLGA vaccination showed a significantly lower bacterial burden in spleens compared with the long peptide alone or the CTL and Th short peptides/PLGA vaccination after the challenge of lethal amounts of L. monocytogenes. These results suggest that the novel vaccine taking advantages of CTL/Th-hybrid epitope long peptide and PLGA microparticle is effective for protection against intracellular bacteria.

The TRPM2 ion channel regulates the inflammatory response of neutrophils during Listeria monocytogenes infection

Abstract Calcium signaling is required for most antimicrobial functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating calcium mobilization, cell migration and oxidative stress in neutrophils. However, the precise mechanism by which TRPM2 activation may exert a pro- or anti-inflammatory function remains unclear. In this study, we used a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils’ function. We found that infected Trpm2−/− mice were associated with high bacterial burden in spleen and liver indicating increased susceptibility to Listeria infection. Pronounced migration rates of neutrophils and monocytes to the liver and spleen characterized the inflammatory response to Listeria during the first 18h. In the Trpm2−/− mice, the acute phase infection resulted in septic shock, defined by increased serum levels of TNF-α, IL-6, and IL-10, but no decreased levels of IFN-γ or IL-12. Furthermore, depletion of neutrophils demonstrated the critical role of these cells in regulating acute inflammation by conferring resistance of Trpm2−/− mice to Listeria infection. Gene expression and inflammatory cytokine analyses of infected tissues further confirmed the hyperinflammatory profile of Trpm2−/− neutrophils. Finally, the increased inflammatory properties of Trpm2−/− neutrophils correlated with cellular calcium overloading and potentiated membrane depolarization, in response to Listeria infection. In conclusion, our findings suggest that TRPM2 channel plays critical roles in the regulation of the inflammatory properties of neutrophils during Listeria monocytogenes infection.

Immunization With a Secreted Esterase Protects Mice Against Multiple Serotypes (M1, M3, and M28) of Group A Streptococcus.

Streptococcal secreted esterase (Sse) is a platelet-activating factor acetylhydrolase that is critical for Group A Streptococcus (GAS) skin invasion and innate immune evasion. There are two Sse variant complexes that share >98% identity within each complex but display about 37% variation between the complexes in amino acid sequences. Sse immunization protects mice against lethal infection and skin invasion in subcutaneous infection with the hypervirulent CovRS mutant strain, MGAS5005. However, it is not known whether Sse immunization provides significant protection against infection of GAS with functional CovRS and whether immunization with Sse of one variant complex provides protection against infection of GAS that produces Sse of another variant complex. This study was designed to address these questions. Mice were immunized with recombinant Sse of M1 GAS (SseM1) and challenged with MGAS5005 (serotype M1, CovS mutant, and Sse of variant complex I), MGAS315 (M3, CovS mutant, and Sse of variant complex I), MGAS2221 (M1, wild-type CovRS, and Sse of variant complex I), and MGAS6180 (M28, wild-type CovRS, and Sse of variant complex II). SseM1 immunization significantly increased survival rates of mice in subcutaneous MGAS5005 and intraperitoneal MGAS6180 challenges and showed consistently higher or longer survival in the other challenges. Immunized mice had smaller skin lesion and higher neutrophil responses in subcutaneous infections and lower GAS burdens in spleen, liver, and kidney in most of the challenge experiments than control mice. SseM1 immunization enhanced proinflammatory responses. These data suggest that Sse immunization has a broad benefit against GAS infections that can vary in extent from strain to strain and that the benefit may be due to the immunization-enhanced proinflammatory responses. In particular, immunization with SseM1 can provide protection against M28 GAS infection even though its Sse and SseM1 have significant variations.

Long Non-coding RNA LincRNA-EPS Inhibits Host Defense Against Listeria monocytogenes Infection.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of gene expression in several biological systems. The long intergenic RNA-erythroid pro-survival (lincRNA-EPS) has been shown to play a critical role in restraining inflammatory gene expression. However, the function of lincRNA-EPS during bacterial infections remains unknown. Here, we demonstrate that following infection with the intracellular bacterium Listeria monocytogenes, both mouse macrophages and dendritic cells lacking lincRNA-EPS exhibit an enhanced expression of proinflammatory cytokine genes, as well as an increased expression of the inducible nitric oxide synthase (iNos) and nitric oxide (NO) production. Importantly, we found that lincRNA-EPS−/− mice intraperitoneally infected with L. monocytogenes exhibit lower bacterial burdens in spleen and liver and produce more NO than control mice. Furthermore, lincRNA-EPS−/− mice are less susceptible to a lethal dose of L. monocytogenes than wild type (WT) mice. Collectively these findings show that lincRNA-EPS suppresses host protective NO expression and impairs the host defense against L. monocytogenes infection.

ROR1 Targeted Immunoliposomal Delivery of OSU-2S Show Selective Cytotoxicity in t(1;19) Translocated B-ALL

The receptor tyrosine kinase ROR1 is uniquely expressed on and required for many hematological malignancies such as t(1;19) positive acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). The t(1;19) is one of the most frequent translocations in B-ALL, observed in both adult and pediatric patients. The translocation has intermediate prognosis on its own, but is associated with a poor prognosis in the unbalanced der(19)t(1;19) form in pediatric ALL, and in the context of hyperdiploid B-ALL. While leukemic cell dependence on ROR1 is known, ROR1 lacks kinase activity making it difficult to target therapeutically. However, we have previously shown that ROR1 can be targeted to deliver therapeutic payload specifically to leukemic cells in CLL, sparing the normal cells from toxic side effects. This encouraged us to develop ROR1 directed immunoliposomal nanoparticles encapsulating a novel small molecule OSU-2S. OSU-2S is a non-immunosuppressive derivative of the sphingosine analogue FTY720, with potent anti-tumor activity against multiple hematological malignancies including CLL, mantle cell lymphoma (MCL) and canine B-cell lymphoma. OSU-2S demonstrated potent dose dependent cytotoxicity in patient derived B-ALL samples with different cytogenetic backgrounds including translocations t(4;11), t(9;22) and t(1;19) as well as hyperdiploid, hypodiploid and normal cytogenetic background [n=7, p= 0.0032 (0 vs 2.5µM), mean decrease in relative viability= 44.51±12.12%] as assessed by Annexin V/Propidium Iodide staining. We confirmed ROR1 expression on t(1;19) translocated patient samples by flow cytometry, and synthesized ROR1 targeted OSU-2S immunoliposomal nanoparticles (2A2-OSU-2S-ILP) (mean size= 186.9 +/- 0.8 nm, mean concentration= 1.38*1013 particles/ml). 2A2-OSU-2S-ILP was selectively cytotoxic to t(1;19) translocated ALL, including unbalanced der(19)t(1;19), from relapsed patients aged 29-37, but not ROR1-ve, t(1;19) non translocated ALL, as compared to control IgG-OSU-2S-ILP, or 2A2/IgG immunoliposomes without OSU-2S [n=3, p= 0.04, mean decrease in relative viability (IgG-OSU-2S-ILP vs 2A2-OSU-2S-ILP)= 35.14±7.36%]. Similar results were seen in ROR1+ve 697 cells, a B-ALL cell line carrying t(1;19) translocation, where 2A2-OSU-2S-ILP showed selective cytotoxicity [n=8, p=0.004, mean decrease in relative viability (IgG-OSU-2S-ILP vs 2A2-OSU-2S-ILP)= 61.62±14.63%]. To assess the effect of 2A2-OSU-2S-ILP on t(1;19) positive ALL in-vivo, we used a disseminated cell line derived xenograft model. Immunocompromised NSG mice were engrafted with 697 cells, treated with 2A2-OSU-2S-ILP or IgG-OSU-2S-ILP for 14 days and tumor burden was assessed in the spleen and bone marrow. 2A2-OSU-2S-ILP treatment significantly reduced the number of human CD45+CD19+ cells in the bone marrow as compared to IgG-OSU-2S-ILP cohort (n=6 per cohort, p=0.022, mean decrease in 697 cells in marrow= 1.751 ± 0.6372 million cells/ femur). There was also a trend towards decreased tumor burden in spleen (mean decrease in 697 cells in spleen= 1.883 ± 0.9729 million cells). Together, these data show the ability of ROR1 targeted liposomal nanoparticles to selectively deliver its payload to leukemic cells in t(1;19) translocated B-ALL, sparing toxicity to the normal cells. Ongoing studies are directed towards understanding the mechanistic basis of OSU-2S mediated therapeutic benefit in B-ALL in-vitro and in-vivo. [This work was supported by NIH-R01-CA197844-01. SG is supported by Pelotonia Graduate Fellowship] Disclosures Baskar: NIH: Patents &amp; Royalties: ROR1 mAb 2A2. Rader:NIH: Patents &amp; Royalties: ROR1 mAb 2A2. Byrd:Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents &amp; Royalties: OSU-2S; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau. Bhatnagar:Novartis and Astellas: Consultancy, Honoraria; Cell Therapeutics, Inc.: Other: Research support; Karyopharm Therapeutics: Other: Research support. Muthusamy:Ohio State University: Patents &amp; Royalties: OSU-2S.