Burden In Spleen Research Articles

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 & Royalties: ROR1 mAb 2A2. Rader:NIH: Patents & 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 & 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 & Royalties: OSU-2S.

Anti-MiR-126 Therapy for Inv(16) Acute Myeloid Leukemia

Inv(16)(p13q22) or t(16;16)(p13.1;q22) [hereafter inv(16)], a chromosomal rearrangement occurred in 5-12% acute myeloid leukemia (AML) patients, disrupts the core-binding factor (CBF) transcription complex and creates a leukemogenic fusion gene CBFb-MYH11 (CM). Only about 50% inv(16) patients achieve long-term survival with standard chemotherapy. Therefore, more effective therapies are necessary to improve outcomes. MicroRNAs (miRNAs) are small non-coding RNA molecules that inhibit multiple target gene expression. High miR-126 expression is a miRNA hallmark of inv(16) AML. However, the function and mechanism of miR-126 dysregulation in inv(16) AML remains elusive. Our results indicate that miR-126 levels are significantly higher in inv(16) AML CD34+ and CD34- blasts compared to normal counterparts. We have previously generated a conditional Cbfb-MYH11 (CM) knock-in (KI) mouse model (Cbfb+/56M/Mx1-Cre), in which lethal AML develops in 4-6 months after CM induction. We observed an overtime-increased miR-126 in the peripheral blood relative to control (log fold change = 4.11; p< 0.0001) as well as in multiple progenitor subpopulations, including LSK, pre-megakaryocyte/erythrocyte (Pre-Meg/E), pre-granulocyte-macrophage (Pre-GM), and granulocyte-macrophage progenitors (GMP) in CM preleukemic and leukemic mice. Expression of CM in 32D cells significantly upregulated miR-126, pri-miR-126, pre-miR-26 and the host gene Egfl7 compared to vector control or WT CBFb, suggesting that transcriptional activation of miR-126 is induced by CM. But, is the aberrant expression of miR-126 necessary for leukemia growth in CM AML? To address this question, we crossed the conditional CM KI mice with a miR-126-floxed model (miR-126f/f). CM/miR-126△/△ mice showed significantly reduced AML incidence (3 out of 10) and prolonged disease-free survival compared to CM mice (p < 0.0001), indicating that high miR-126 promotes AML growth. Deletion of miR-126 significantly reduced the expansion of preleukemic (6 weeks after induction) stem/progenitor populations (LSK, Pre-GM, and Pre-Meg/E). In addition, we observed significantly increased radiation-induced apoptosis in LSK (CM 11.61±2.277% vs. CM/miR-126△/△16±1.386%, p=0.03) and Pre-Meg/E (CM 8.885±1.607% vs. CM/miR-126△/△25.5±3.961%, p=0.0081), which represent leukemia-initiating populations in CM mice (Cai et al, Blood 2016). Furthermore, miR-126 knockdown in human inv(16) AML patient samples led to significantly increased apoptosis in all AML stem/progenitor cell subsets (shCtrl 7.469±1.085% vs. shmiR-126 13.8±1.585% in CD34+, p=0.0045; shCtrl 11.59±1.723% vs. shmiR-126 17.97±1.461% in CD34-, p=0.0122) and reduced quiescence/increased cycling in more primitive subsets (e.g, CD34+CD38-, CD34+CD38+). These results indicate that high miR-126 contributes to CM leukemia initiation and maintenance by antagonizing stress-induced apoptosis and maintaining the quiescence of stem/progenitor cells. Thus, miR-126 may be a novel therapeutic target in this subtype AML. To target aberrantly expressed miR-126 in CM-AML, we designed a CpG-anti-miR-126 oligonucleotide (ODN) inhibitor (named miRisten, Bin Zhang et al, Nat Med 2018) that is efficiently (60-90%) taken up and achieved 50-80% reduction of miR-126 in AML LSK and blasts. Compared with a scramble ODN control (Ctrl), in vivo miRisten treatment (20 mg/kg i.v. daily for 3 weeks) significantly reduced CM-AML burden in spleen (Ctrl 71.23±3.756% vs. miRisten 51.91±5.788%, p=0.0103) and bone morrow (Ctrl 66.07±3.203% vs. miRisten 50.9±2.999%, p=0.0038), and reduced the frequency of LSK (Ctrl 1.72±0.6176% vs. miRisten 0.2316±0.03894%, p=0.0482). Importantly, miRisten treatment significantly reduced the leukemia-initiating capacity with prolonged survival in secondary transplants compared to Ctrl (median survival 95.5 days vs. 84.5 days; n=10, p=0.0004). When we combined miRisten with chemotherapy, adopting a regimen consisted of Cytarabine (Ara-C; 50 mg/kg i.p. daily for 5 days) and daunorubicin (DNR; 1.5 mg/kg i.v. every other day for 3 days), we observed a further reduction of AML burden and prolonged survival compared to chemotherapy alone (median survival 102 days vs. 84 days, n=10-11, p<0.0001). Therefore, miRisten is a novel targeted therapeutics that effectively targets miR-126 and mediates elimination of AML blasts and leukemia-initiating stem cells. Disclosures No relevant conflicts of interest to declare.

Type I interferons differentially modulate maternal host immunity to infection by Listeria monocytogenes and Salmonella enterica serovar Typhimurium during pregnancy.

IFN-alpha receptor deficiency (IFNAR-/- ) enhances immunity to Listeria monocytogenes (LM) and Salmonella enterica serovar Typhimurium (ST) in the non-pregnant state by inhibiting pathogen-induced immune cell death. However, the roles of IFNAR signaling in modulating immunity to infection during pregnancy are not well understood. C57BL/6J wild-type (WT) and IFNAR-/- mice were infected systemically with LM or ST. Bacterial burden in spleen and individual placentas was enumerated at day 3 post-infection. Immune cell numbers and percentages were quantified in spleen and individual placentas, respectively, through flow cytometry. Cytokine expression in serum, spleen, and individual placentas was measured through cytometric bead array. IFNAR-/- mice exhibited decreased splenic monocyte numbers in non-pregnant and pregnant state, and an altered distribution of placental immune cell types in the non-infected state. IFNAR-/- mice controlled LM infection more effectively than WT mice even during pregnancy. This correlated with enhanced serum IL-12 expression, despite reduced splenic monocyte numbers relative to WT controls. In contrast, pregnant IFNAR-/- mice unlike their non-pregnant counterparts exhibited increased susceptibility to ST infection, which was associated with decreased serum IL-12 expression. Type I IFN responses differentially impact host resistance to LM and ST infection during pregnancy through modulation of immune cell distribution and cytokine responses.

The impact of the absence of Toll-like receptor-2 during Sporothrix brasiliensis infection

Sporothrix brasiliensis, a member of the Sporothrix schenckii complex, is a major cause of epidemic outbreaks of sporotrichosis due to its greater virulence and ability to evade the immune system. The absence of studies about this species led to this study, with the aim to evaluate the importance of Toll-like receptor-2 (TLR-2) during S. brasiliensis infection. In vitro assays were performed using bone marrow-derived macrophages from both wild-type (C57BL/6) and TLR-2 knockout (-/-) mice. In vivo assays were also performed, on which the mice (C57BL/6 and TLR-2-/-) were intraperitoneally infected with S. brasiliensis yeast American Type Culture Collection MYA-4831 and euthanized on days 7, 14 and 28 post infection. The following parameters were then evaluated: fungal burden in spleen, liver, kidney and brain; the production of cytokines TNF-α, IFN-γ, IL-4, IL-2, IL-6 and IL-10. The in vitro results showed that the absence of TLR-2 resulted in impaired phagocytosis, microbicide mechanisms utilizing the production of nitric oxide, and the cytokine production (TNF-α, IL-6 and IL-10). The in vivo results demonstrated that the absence of TLR-2 during experimental S. brasiliensis infection promoted increased dissemination after 14 and 28 days and suggests a polarized Th17 response in an attempt to control the infection. TLR-2 signalling appears to be important in the innate immune response against S. brasiliensis.

Abstract 4129: RUNX2 as a regulator of high-risk T-ALL

Abstract For patients with T-cell lineage acute lymphoblastic leukemia (T-ALL) who fail induction or relapse, event free survival is <10%. Mixed lineage leukemia gene rearrangements (KMT2A-R) are recurrent translocations found in ~3-5% of T-ALL and are determinants of high-risk disease. However, the roles of downstream targets of KMT2A chimeras such as Runt-related transcription factor 2 (RUNX2) remain uclear. We assessed RUNX2 mRNA levels in 100 pediatric T-ALL samples derived from the COG AALL0434 study. RUNX2 expression was upregulated in patients harboring KMT2A-R (p <0.0005) and those who failed induction therapy (p <0.005). Therefore, we hypothesized that RUNX2 is a critical determinant of high-risk T-ALL. To investigate the relationship between RUNX2 and KMT2A chimeras we utilized the chromatin immunoprecipitation assay to assess primary T-ALL samples with KMT2A-R (n = 3). We found that RUNX2 targets KMT2A-R (p < 0.0005) and conversely, that KMT2A chimeras target the promoter region of RUNX2 (p < 0.0005). We next demonstrated that KMT2A-MLLT4 induces RUNX2 expression. To further test the effects of RUNX2 on KMT2A-R, we performed shRNA mediated silencing of RUNX2 expression in primary T-ALL cells (n =3). RUNX2 silencing decreased KMT2A levels, suggesting the functional cross talk between KMT2A-R and RUNX2. To test the regulatory role of RUNX2 on T-ALL signal transduction we knocked down RUNX2 in T-ALL primary samples and cell lines (Loucy and Jurkat). RUNX2 silencing led to a decrease in cellular proliferation (p < 0.0005) indicating that RUNX2 positively regulates T-ALL cell growth in vitro. We next utilized flow-based assays to examine the effects of RUNX2 on cell death and cell cycle. RUNX2 inactivation induced apoptotic cell death and delayed cell cycle progression as demonstrated by increased G1 and decreased S/G2/M phases of the cell cycle. Apoptotic cell death was associated with increased expression of cleaved caspase 3, 7 and 9. We next examined the effects of RUNX2 on T-ALL cell survival and found that RUNX2 depletion reduced activation of Akt, active β-catenin and survivin levels but had no effect on p53 and Bcl-2. Similar experiments showed that RUNX2 inactivation leads to decreased expression of c-Myc and Ras. In addition, treatment with the specific allosteric Akt inhibitor, MK2206 (10 μM, 24 hrs) reduced RUNX2, active β-catenin, and survivin protein levels suggesting reciprocal activation between RUNX2 and Akt. To study the role of RUNX2 in leukemia burden, Jurkat cells transduced with RUNX2 shRNA and control plasmid were transplanted intravenously into the NSG mice (5 mice/group). RUNX2 silencing significantly decreased leukemia burden in spleen (p <0.001) and bone marrow (p <0.01) of the tested animals compared to the negative control group. We propose upregulation of RUNX2 expression as a novel mechanism governing high-risk T-ALL. We postulate that the Akt/β-catenin/survivin axis serves as a functional driver of RUNX2-mediated T-ALL survival. Citation Format: Nitesh Devinarayan Sharma, Christian K. Nickl, Stuart S. Winter, Huining Kang, Ksenia Matlawska Wasowska. RUNX2 as a regulator of high-risk T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4129.

Transcriptional Profiling of Immune-Related Genes in Leishmania infantum-Infected Mice: Identification of Potential Biomarkers of Infection and Progression of Disease.

Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. In this large-scale analysis, differential expression of 112 immune-related genes has been analyzed using high-throughput qPCR in spleens of infected and naïve Balb/c mice at four different time points. This analysis revealed that early response against Leishmania infection is characterized by the upregulation of Th1 markers and M1-macrophage activation molecules such as Ifng, Stat1, Cxcl9, Cxcl10, Ccr5, Cxcr3, Xcl1, and Ccl3. This activation doesn't protect spleen from infection, since parasitic burden rises along time. This marked difference in gene expression between infected and control mice disappears during intermediate stages of infection, probably related to the strong anti-inflammatory and immunosuppresory signals that are activated early upon infection (Ctla4) or remain activated throughout the experiment (Il18bp). The overexpression of these Th1/M1 markers is restored later in the chronic phase (8 wpi), suggesting the generation of a classical “protective response” against leishmaniasis. Nonetheless, the parasitic burden rockets at this timepoint. This apparent contradiction can be explained by the generation of a regulatory immune response characterized by overexpression of Ifng, Tnfa, Il10, and downregulation Il4 that counteracts the Th1/M1 response. This large pool of data was also used to identify potential biomarkers of infection and parasitic burden in spleen, on the bases of two different regression models. Given the results, gene expression signature analysis appears as a useful tool to identify mechanisms involved in disease outcome and to establish a rational approach for the identification of potential biomarkers useful for monitoring disease progression, new therapies or vaccine development.

Effects of nanoemulsions prepared with essential oils of copaiba- and andiroba against Leishmania infantum and Leishmania amazonensis infections

Plant products are an important source of bioactive agents against parasitic diseases, including leishmaniasis. Among these products, vegetable oils have gained ground in the pharmaceutical field. Here we report the development of nanoemulsions as a delivery system for copaiba and andiroba oils (nanocopa and nanoandi) in order to test their effects on Leishmania infantum and L. amazonensis. The nanocopa and nanoandi had an average particle size of 76.1 and 88.1, respectively with polydispersity index 0.14 to 0.16 and potential zeta −2.54 to −3.9. The data indicated toxic activity of nanocopa and nanoandi against promastigotes of both Leishmania species ultrastructural analyses by scanning electron microscopy revealed that exposition to nanoemulsions induced oval cell shape and retracted flagella. The treatment with nanocopa and nanoandi led to a reduction in L. infantum and L. amazonensis infection levels in macrophage cultures. The nanoemulsions treatment have significant beneficial effects on all the parameters evaluated in lesions induced by L. amazonensis (lesion size, parasite burden and histopathology) on BALB/c mice. The treatment of L. infantum-infected BALB/c mice with nanoemulsions also showed promising results reducing parasite burden in spleen and liver and improving histopathological features.

Constitutively Photomorphogenic 1 Reduces the Sensitivity of Chronic Lymphocytic Leukemia Cells to Fludarabine Through Promotion of Ubiquitin-Mediated P53 Degradation

Background/Aims: Chronic Lymphocytic leukemia (CLL) is characterized by accumulation of cells in the G0/G1 phase of the cell cycle and resistance to apoptosis due to gene mutation or abnormal gene expression. In our previous study, constitutively photomorphogenic 1 (COP1) was shown to be upregulated in Binet C-phase CLL patients. Based on the negative regulation of COP1 in the repair of DNA damage, we further studied the function of COP1 in CLL cell apoptosis induced by fludarabine in vitro and in vivo. Methods: We analyzed the sensitivity of primary CLL cells to the fludarabine by CCK-8, and detected the expression of p53 in cells after drug treatment by western blot. Next, we constructed COP1 overexrpessing CLL cell line HG3, and analyzed the effect of COP1 overexpression on the HG3 cell’s apoptosis, and HG3 transplant mice survival with drug treatment. Results: Here, we found that primary CLL cells with high expression of COP1 showed low sensitivity to the drug and presented delayed enrichment of p53 protein than cells with low COP1 expressed. COP1 overexpression reduced HG3 cell sensitivity to the fludarabine treatment and inhibited cell apoptosis, and also retarded itself via autoubiquitination. The further study showed that COP1 promoted ubiquitin-dependent p53 degradation, which further disrupts the formation of the p53-Brn-3a complex and activation of Bcl-2 transcription. Moreover, mice engrafted with cells overexpressing COP1 showed a shortened survival, increased tumor cells burden in spleen and bone marrow (BM), and reduced tumor cell apoptosis even when fludarabine combined cyclophosphamide (F+C) therapy was administered. Conclusion: This study demonstrates that COP1 contributes to drug resistance of CLL cells to the fludarabine treatment in vitro and in vivo.

Dectin-1 expression by macrophages and related antifungal mechanisms in a murine model of Sporothrix schenckii sensu stricto systemic infection.

The available information about the role of Dectin-1 in sporotrichosis is scarce. Hence, we aimed to assess Dectin-1 expression by macrophages and the activation of some related antifungal mechanisms during the Sporothrix schenckii sensu stricto infection as a first attempt to elucidate the role of this receptor in sporotrichosis. Balb/c mice were intraperitoneally infected with S.schenckii sensu stricto yeast ATCC 16345 and euthanized on days 5, 10 and 15 post-infection, when the following parameters were evaluated: fungal burden in spleen, Dectin-1 expression and nitric oxide (NO) production by peritoneal macrophages, as well as IL-1β, TNF-α and IL-10 exvivo secretion by these same cells. Peritoneal macrophages were exvivo challenged with either the alkali-insoluble fraction (F1) extracted from the S.schenckii cell wall, a commercially available purified β-1,3-glucan or whole heat-killed S.schenckii yeasts (HKss). Additionally, a Dectin-1 antibody-mediated blockade assay was performed on day 10 post-infection to assess the participation of this receptor in cytokine secretion. Our results showed that Dectin-1 expression by peritoneal macrophages was augmented on days 10 and 15 post-infection alongside elevated NO production and exvivo secretion of IL-10, TNF-α and IL-1β. The antibody-mediated blockade of Dectin-1 inhibited cytokine production in both infected and non-infected mice, mainly after β-1,3-glucan stimulation. Our results suggest a role for Dectin-1 in triggering the immune response during S.schenckii infection.

Transgenic West Nile Virus virus-specific CD8+ T cells provide insights on activation patterns and protective role of cytotoxic T cells during infection

Abstract West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. Currently, there are no vaccines or therapeutics approved for use in humans. Virus-specific CD8+ T cells are essential in mediating protective immunity during WNV infection. However, the mechanisms that promote protective CD8+ T cell immunity against WNV are not well understood. In this study, we characterized a novel transgenic mouse encoding CD8+ T cells to the immunodominant epitope located within the WNV NS4b protein (WNV-I). Through adoptive transfer studies in mice, we evaluated the expansion, migration, and functional properties of WNV-I CD8+ T cells during infection. In the spleen, WNV-I CD8+ T cell numbers increased on day 7 post-infection followed by contraction in cell numbers through day 15 post-infection. We observed a robust infiltration of virus-specific CD8+ T cells to the brain on day 7 concomitant with an increase in virus replication within the CNS. Interestingly, we observed clearance of infectious virus from the brain, however, WNV-I CD8+ T cells remained elevated in the brain through day 15 post-infection. Functionally, WNV-I CD8+ T cells secrete both IFN-γ and TNF-α, with peak responses in the spleen and in the brain on days 7 and 11 post-infection, respectively. Furthermore, Rag-1−/− mice that received WNV-I CD8+ T cells had lower viral burden in spleens and serum at day 7 of infection. Thus, WNV-I cells are functionally active and can mediate protective immunity against WNV infection. Further studies are underway to better understand how CD8+ T cells mediate WNV clearance in the central nervous system.

Development of high efficacy peptide coated iron oxide nanoparticles encapsulated amphotericin B drug delivery system against visceral leishmaniasis

Visceral leishmaniasis, a protozoan disease transmitted by phlebotomine sandflies which affect mostly in Indian sub-continent. The treatment for visceral leishmaniasis (VL) caused by Leishmania donovani are limited and unsatisfactory. Currently available drug against such as miltefosine and polymer based drugs amBisome has high efficacy against VL but found serious side effects and poor absorbance. To overcome this, we developed peptide (glycine) coated iron oxide (Fe3O4) nanoparticles (GINPs) encapsulated amphoterecin B (AmB) drug against visceral leishmaniasis. Synthesis of GINPs was carried out and different characterization technique used to confirm the synthesis and size of GINPs GINPs-AmB showed that particle size in the range of 10–15nm and closed to spherical in shaped. GINPs-AmB showed release rate of AmB is higher at lower pH. Significantly two fold higher efficacies of GINP encapsulated AmB during in vitro study. There was a substantial reduction in the total parasite burden in spleen in treated groups (GINPs encapsulate AmB), compare to AmB alone. The results obtained from this study revealed that AmB loaded GINPs is twofold effective than AmB and therefore, it opens a new avenue for use of AmB loaded GINPs against VL.

Inhaled Liposomal Ciprofloxacin Protects against a Lethal Infection in a Murine Model of Pneumonic Plague.

Inhalation of Yersinia pestis can lead to pneumonic plague, which without treatment is inevitably fatal. Two novel formulations of liposome-encapsulated ciprofloxacin, ‘ciprofloxacin for inhalation’ (CFI, Lipoquin®) and ‘dual release ciprofloxacin for inhalation’ (DRCFI, Pulmaquin®) containing CFI and ciprofloxacin solution, are in development. These were evaluated as potential therapies for infection with Y. pestis. In a murine model of pneumonic plague, human-like doses of aerosolized CFI, aerosolized DRCFI or intraperitoneal (i.p.) ciprofloxacin were administered at 24 h (representing prophylaxis) or 42 h (representing treatment) post-challenge. All three therapies provided a high level of protection when administered 24 h post-challenge. A single dose of CFI, but not DRCFI, significantly improved survival compared to a single dose of ciprofloxacin. Furthermore, single doses of CFI and DRCFI reduced bacterial burden in lungs and spleens to below the detectable limit at 60 h post-challenge. When therapy was delayed until 42 h post-challenge, a single dose of CFI or DRCFI offered minimal protection. However, single doses of CFI or DRCFI were able to significantly reduce the bacterial burden in the spleen compared to empty liposomes. A three-day treatment regimen of ciprofloxacin, CFI, or DRCFI resulted in high levels of protection (90–100% survival). This study suggests that CFI and DRCFI may be useful therapies for Y. pestis infection, both as prophylaxis and for the treatment of plague.

Peptide-Receptor Radiotherapy with CXCR4-Targeting Pentixather Reduces Leukemia Burden in Acute Leukemia PDX and Patients

Introduction:The G-protein coupled CXC-motif Chemokine Receptor 4 (CXCR4) and its ligand CXCL12 are master regulators of cell migration, organogenesis and maintenance of the hematopoietic stem/progenitor cell (HSPC) niche. However, CXCR4 also drives survival, proliferation and metastasis of cancer cells and its expression is associated with adverse prognosis in a broad range of malignancies, including acute myeloid and lymphoblastic leukemia (AML, ALL). Despite of high rates of complete remissions after induction chemotherapy, AML, and to a lesser extend ALL, frequently relapse with a more aggressive phenotype and require highly active therapies to reduce leukemic burden before allogeneic stem cell transplantation (alloSCT). We recently showed that CXCR4-directed PET imaging with 68Ga-Pentixafor is feasible in AML patients, providing first evidence for the potential of CXCR4-directed theranostics (Herhaus, Habringer et al., Haematologica 2016).Methods:We used patient-derived xenograft (PDX) and cell line xenograft mouse models of AML and ALL to evaluate the efficacy and toxicity of a CXCR4-targeted peptide receptor radiotherapy (PRRT) theranostic approach with the CXCR4-binding PET tracer 68Ga-Pentixafor and its b-emitting therapeutic counterpart 177Lu-Pentixather. We analyzed bone marrow (BM), spleen, blood (PB) and liver of treated PDX mice by flow cytometry, immunohistochemistry and radioactive biodistribution assays. The toxicity to the murine BM HSPC and the hematopoietic niche was assessed via flow cytometry, colony forming unit assays, isolation and differentiation of BM mesenchymal stem cells (MSCs) and co-culture experiments. We provide first evidence for this highly innovative CXCR4-directed theranostic approach in patients with AML who relapsed after alloSCT and had no other established treatment options.Results:We generated PDX models of acute leukemia patients in NSG mice that required intact CXCR4 signaling for disease initiation and progressively infiltrated spleen, BM and PB. 68Ga-Pentixafor PET imaging enabled visualization of CXCR4-positive leukemic burden in spleen and BM of acute leukemia PDX and cell line xenografts. In ALL PDX, CXCR4-directed PRRT with 177Lu-Pentixather rapidly distributed to leukemia-harboring organs, which lead to significant accumulation of radioactivity in spleen and BM. 177Lu-Pentixather therapy resulted in efficient eradication or significant reduction of leukemic infiltration in PB, spleen and BM. Spleen size was reduced dramatically as early as 24hr after initiation of PRRT. Treated mice suffered severe suppression of BM function as evidenced by therapy-induced severe cytopenia affecting mature CD45+ blood cells and colony-forming unit potential of progenitors in the BM. To assess the damage to the BM niche, we isolated MSCs of 177Lu-Pentixather treated mice compared to control. We found that MSCs from 177Lu-Pentixather-treated mice were still viable and proliferated in vitro. Importantly, treated MSCs were still capable of supporting normal lineage-marker negative murine HSPC and induced their differentiation into mature leukocytes in co-culture. Finally, we treated patients with refractory AML after alloSCT and multiple failed treatment regimens, who had no further established treatment options with CXCR4 PRRT followed by a conventional conditioning regimen and second alloSCT. Our data indicate that CXCR4 targeting and the inevitable and desired cross-fire effect of 177Lu-Pentixather PRRT could be a highly efficient means to eradicate leukemia and to induce myeloablation. This approach could serve as a valuable addition to conditioning protocols in alloSCT.Conclusion:In conclusion, our work provides first evidence for the efficacy of the novel CXCR4-directed agent 177Lu-Pentixather in acute leukemia PDX models and in a proof-of-concept individual treatment approach in one patient relapsed after standard alloSCT. Importantly, these findings can directly be translated into clinical studies in patients and provide crucial information regarding efficacy and toxicity. A phase I/II study to integrate CXCR-directed PRRT into conditioning regimens is planned. DisclosuresPeschel:MophoSys: Honoraria. Wester:Scintomics GmbH: Employment, Other: CEO.

In vitro and in vivo susceptibility of Leishmania major to some medicinal plants

ObjectiveTo evaluate the efficacy of some medicinal plants and systemic glucantime in a comparative manner against the causative agent of cutaneous leishmaniasis both in vitro and in BALB/c mice. MethodsFor in vivo testing, inbred mice were challenged with Leishmania major parasites and the resultant ulcers were treated with extract based-ointments applied topically two times per day for a period of 20 days. A group of 56 mice were randomly divided into 7 subgroups. The control group received the ointment void of extracts, whereas the reference group received glucantime only. The efficacy of treatments was evaluated by measuring ulcer diameter, parasite burden and NO production. ResultsOur results indicated that plant extract based-ointments were effective in reducing ulcer size and parasite burden in spleens, but their effects did not differ significantly from that of glucantime. The plant extracts tested in this study were able to increase NO production that helped parasite suppression. ConclusionsOur findings indicate that the tested plant extracts are effective against Leishmania major both during in vitro and in vivo experiments, but further researches are required to recommend a potential plant extract as an alternative drug.

Oleanolic acid loaded poly lactic co- glycolic acid- vitamin E TPGS nanoparticles for the treatment of Leishmania donovani infected visceral leishmaniasis

Oleanolic acid (OA) has low aqueous solubility and low permeability, which results poor bioavailability. To surmount the inadequacy, our aim was to fabricate oleanolic acid loaded poly lactic co- glycolic acid (PLGA)- d-α- tocopheryl polyethylene glycol 1000 succinate (TPGS) nanoparticles, which could be efficacious for the treatment of Leishmania donovani mediated visceral leishmaniasis (VL). OA loaded PLGA- TPGS nanoparticles were prepared by emulsion solvent evaporation technique. Cellular uptake was investigated. In vitro cumulative drug release study was carried out. In vitro susceptibility was confirmed against the amastigotes of Leishmania donovani AG83 wild and drug resistant strains. In vivo antileishmanial activity was evaluated against wild type amastigotes of L. donovani. OA loaded nanoparticles were successfully formulated. The highest drug loading was found to be 11.08%±0.35%. 85.66%±0.56% was the highest in vitro OA release for 30days among the formulations. In vivo study revealed that, 98.82±1.92% amastigote burden in spleen of BALB/c mice were suppressed by the polymeric nanoformulation of OA. Experimental OA nanoparticle formulation proved itself as an attractive carrier for OA which was significantly efficacious against both in vitro and in vivo amastigote form of Leishmania donovani than pure OA for chemotherapeutic intervention of visceral leishmaniasis.

Lack of IL-1 Receptor-Associated Kinase-4 Leads to Defective Th1 Cell Responses and Renders Mice Susceptible to Mycobacterial Infection.

The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1β secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections.

Development of a Therapeutic to Preserve Vaccine Memory Following Ionizing Radiation Exposure

Abstract We have demonstrated that in a mouse model of Listeria monocytogenes (LM) infection, revaccination within 3 days after exposure to sub-lethal ionizing radiation prevents the death of memory effector T lymphocytes and preserves vaccine memory. Although revaccination immediately after radiation exposure circumvents an increased risk of infection, it is impractical to revaccinate victims of radiation exposure to the innumerable agents to which they have developed immunity. Thus, we are seeking to develop a single therapeutic or a combination that mimic the effects of T cell activation via revaccination. We have selected our array of potential therapeutics based on their differential targets within T cell survival pathways. Broad spectrum therapeutics, stimulating numerous cell types such as Nicotine, Cimetidine, and Valproic Acid activate AKT and inhibit GSK3-beta to prevent subsequent apoptotic signals. Common gamma chain cytokines such as IL-15 and IL-21 are also candidates because of their ability to potently and specifically promote lymphocyte activation and survival. In addition, we have included Lovastatin to increase levels of P21 by proteasome inhibition. P21 may improve survival through temporary cycle arrest and enhanced DNA repair in activated T cells. We also utilized supplements to our therapeutics such as the FOXP3 inhibiting peptide- P60 and the checkpoint inhibitors anti-CTLA4 and anti-CD25 since regulatory T cells are a radio-resistant population that hinder the immune response to infection. We show, through comparisons of Listeria burden in spleens, that these candidates, either alone or in combination, are able to preserve vaccine memory following radiation exposure.

A Versatile Imaging and Therapeutic Platform Based on Dual-Band Luminescent Lanthanide Nanoparticles toward Tumor Metastasis Inhibition.

Upconversion (UC) luminescent lanthanide nanoparticles (LNPs) are expected to play an important role in imaging and photodynamic therapy (PDT) in vitro and in vivo. However, with the absorption of UC emissions by photosensitizers (PSs) to generate singlet oxygen ((1)O2) for PDT, the imaging signals from LNPs are significantly weakened. It is important to activate another imaging route to track the location of the LNPs during PDT process. In this work, Nd(3+)-sensitized LNPs with dual-band visible and near-infrared (NIR) emissions under single 808 nm excitation were reported to address this issue. The UC emissions in green could trigger covalently linked rose bengal (RB) molecules for efficient PDT, and NIR emissions deriving from Yb(3+) and magnetic resonance imaging (MRI) were used for imaging simultaneously. Notably, the designed therapeutic platform could further effectively avoid the overheating effect induced by the laser irradiation, due to the minimized absorption of biological media at around 808 nm. TdT-mediated dUTP nick end labeling (TUNEL) assay showed serious cell apoptosis in the tumor after PDT for 2 weeks, leading to an effective tumor inhibition rate of 67%. Benefit from the PDT, the tumor growth-induced liver and spleen burdens were largely attenuated, and the liver injury was also alleviated. More importantly, pulmonary and hepatic tumor metastases were significantly reduced after PDT. The Nd(3+)-sensitized LNPs provide a multifunctional nanoplatform for NIR light-assisted PDT with minimized heating effect and an effective inhibition of tumor growth and metastasis.

Innate signaling mechanisms controlling Mycobacterium chelonae-mediated CCL2 and CCL5 expression in macrophages

Mycobacterium chelonae (Mch) is an atypical rapidly growing mycobacterium (RGM) that belongs to the M. chelonae complex, which can cause a variety of human infections. During this type of mycobacterial infection, macrophage-derived chemokines play an important role in the mediation of intracellular communication and immune surveillance by which they orchestrate cellular immunity. However, the intracellular signaling pathways involved in the macrophage-induced chemokine production during Mch infections remain unknown. Thus, the present study aimed to determine the molecular mechanisms by which Mch activates the gene expressions of chemokine (C-C motif) ligand 2 (CCL2) and CCL5 in murine bone marrow-derived macrophages (BMDMs) and in vivo mouse model. Toll-like receptor 2 (TLR2)-deficient mice showed increased bacterial burden in spleen and lung and decreased protein expression of CCL2 and CCL5 in serum. Additionally, Mch infection triggered the mRNA and protein expression of CCL2 and CCL5 in BMDMs via TLR2 and myeloid differentiation primary response gene 88 (MyD88) signaling and that it rapidly activated nuclear factor (NF)-κB signaling, which is required for the Mch-induced expressions of CCL2 and CCL5 in BMDMs. Moreover, while the innate receptor Dectin-1 was only partly involved in the Mch-induced expression of the CCL2 and CCL5 chemokines in BMDMs, the generation of intracellular reactive oxygen species (ROS) was an important contributor to these processes. Taken together, the present data indicate that the TLR2, MyD88, and NF-κB pathways, Dectin-1 signaling, and intracellular ROS generation contribute to the Mch-mediated expression of chemokine genes in BMDMs.

Safety and immunogenicity of an attenuated Salmonella enterica serovar Paratyphi A vaccine candidate

Enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased in recent years and became a global health issue. Currently licensed typhoid vaccines do not confer adequate cross-immunoprotection against S. Paratyphi A infection. Therefore, vaccines specifically against enteric fever caused by S. Paratyphi A are urgently needed. In the present study, an attenuated vaccine strain was constructed from S. Paratyphi A CMCC50093 by the deletions of aroC and yncD. The obtained strain SPADD01 showed reduced survival within THP-1 cells and less bacterial burden in spleens and livers of infected mice compared with the wild-type strain. The 50% lethal doses of SPADD01 and the wild-type strain were assessed using a murine infection model. The virulence of SPADD01 is approximately 40,000-fold less than that of the wild-type strain. In addition, SPADD01 showed an excellent immunogenicity in mouse model. Single intranasal inoculation elicited striking humoral and mucosal immune responses in mice and yielded effective protection against lethal challenge of the wild-type strain. A high level of cross-reactive humoral immune response against LPS of Salmonella enterica serovar Typhi was also detected in immunized mice. However, SPADD01 vaccination only conferred a low level of cross-protection against S. Typhi. Our data suggest that SPADD01 is a promising vaccine candidate against S. Paratyphi A infection and deserves further evaluation in clinical trial. To date, no study has demonstrated a good cross-protection between serovars of S. Typhi and S. Paratyphi A, suggesting that the dominant protective antigens of both serovars are likely different and need to be defined in future study.