Load In Spleen Research Articles

Development of a Multi-Organ Radiation Injury Model with Precise Dosimetry with Focus on GI-ARS.

The goal of this study was to establish a model of partial-body irradiation (PBI) sparing 2.5% of the bone marrow (BM2.5-PBI) that accurately recapitulates radiological/nuclear exposure scenarios. Here we have reported a model which produces gastrointestinal (GI) damage utilizing a clinical linear accelerator (LINAC) with precise dosimetry, which can be used to develop medical countermeasures (MCM) for GI acute radiation syndrome (ARS) under the FDA animal rule. The PBI model (1 hind leg spared) was developed in male and female C57BL/6 mice that received radiation doses ranging from 12-17 Gy with no supportive care. GI pathophysiology was assessed by crypt cell loss and correlated with peak lethality between days 4 and 10 after PBI. The radiation dose resulting in 50% mortality in 30 days (LD50/30) was determined by probit analysis. Differential blood cell counts in peripheral blood, colony forming units (CFU) in bone marrow, and sternal megakaryocytes were analyzed between days 1-30, to assess the extent of hematopoietic ARS (H-ARS) injury. Radiation-induced GI damage was also assessed by measuring: 1. bacterial load (16S rRNA) by RT-PCR on days 4 and 7 after PBI in liver, spleen and jejunum, 2. liposaccharide binding protein (LBP) levels in liver, and 3. fluorescein isothiocyanate (FITC)-dextran, E-selectin, sP-selectin, VEGF, FGF-2, MMP-9, citrulline, and serum amyloid A (SAA) levels in serum. The LD50/30 of male mice was 14.3 Gy (95% confidence interval 14.1-14.7 Gy) and of female mice was 14.5 Gy (95% confidence interval 14.3-14.7 Gy). Secondary endpoints included loss of viable crypts, higher bacterial loads in spleen and liver, higher LBP in liver, increased FITC-dextran and SAA levels, and decreased levels of citrulline and endothelial biomarkers in serum. The BM2.5-PBI model, developed for the first time with precise dosimetry, showed acute radiation-induced GI damage that is correlated with lethality, as well as a response to various markers of inflammation and vascular damage. Sex-specific differences were observed with respect to radiation dose response. Currently, no MCM is available as a mitigator for GI-ARS. This BM2.5-PBI mouse model can be regarded as the first high-throughput PBI model with precise dosimetry for developing MCMs for GI-ARS under the FDA animal rule.

The Function and Mechanism Research of NLRP6 in Bone Marrow Microenvironment in Myeloproliferative Neoplasms

Since the important role of inflammation in myeloproliferative neoplasms (MPN) progression, inflammasome is an important regulator of inflammation involved in the development of tumor. Here, we aimed to investigate the function of NLRP6 in bone marrow (BM) microenvironment in MPN in the hope of getting better treatment for MPN. We first analyzed that NLRP6 was highly expressed in BM mononuclear cells from individuals newly diagnosed with ET, PV, PMF patients (with or without JAK2 V617Fmutation). Similar results were obtained in BM cells and BM stromal cells of MPL W515L transduced C57BL/6 mice (C57BL/6 MPN). The results were validated that NLRP6 of the BM microenvironment was involved in the development of MPN. We thus hypothesized that the absence of NLRP6 in BM microenvironment affects the development of MPN. NLRP6 -/- mice and C57BL/6 mice MPN models were established by the same method. The experiments demonstrated that overall survival of NLRP6 -/- MPN mice was significantly prolonged compared to C57BL/6 MPN mice. Loss of NLRP6 in BM microenvironment interfered with hematopoiesis of BM, but it had no effect on the extramedullary hematopoiesis (EMH) in spleen. Pathological analysis showed that the destruction of spleen structure in NLRP6 -/- MPN mice and the infiltration of atypical megakaryocytes were less than C57BL/6 MPN mice. The tumor load in spleen was significantly reduced and the structure of spleen was intact, the hematopoietic function of the BM returned to normal and the tumor load in the BM was significantly reduced at 5 weeks in NLRP6 -/- MPN mice after sequential transplantation. It suggested that knockout of NLRP6 in the BM microenvironment significantly attenuated the tumorigenicity of malignant HSCs, delayed or even inhibited the development of MPN disease. The expression of inflammatory cytokines, abnormal megakaryocytes and the phenomenon of aggregation and reticular fibers in BM microenvironment were significantly reduced in NLRP6 -/- MPN group compared with the control group. After sequential transplantation, the destruction of BM structure was alleviated, megakaryocytes aggregation and heteromorphic megakaryocytes in BM were reduced, and reticular fibers were significantly diminished. Furthermore, the cytokines promoting disease progression in BM microenvironment were significantly reduced in NLRP6 -/- MPN group, suggesting that knockout of NLRP6 can improve the BM microenvironment and alleviate the progression of myelofibrosis. Finally, to gain better insight into the role of NLRP6 inflammasome in MPN pathogenesis, we sought to dissect the changes with scRNA-seq and pathway identification based on available data and bioinformatic pathway analysis in NLRP6 -/- mice. The results of scRNA-seq analysis showed that C18 population of malignant HSCs of recipients in primary transplantation was added, C16, C13 and C9 cell populations disappeared after deletion of NLRP6 in BM microenvironment, while C5, C6, C7, C10 and C12 cell populations increased significantly. Regulon of various subpopulations of malignant HSCs changed significantly, mainly involving cell development and differentiation, immune response, inflammatory BM microenvironment, vascular endothelial homeostasis and tumor related genes. Our results indicate that knockout of NLRP6 in the BM microenvironment may affect the progression of MPN by altering some malignant HSCs subpopulations and regulons, participating in immune, inflammatory, tumor metabolism and other signaling pathways to regulate the functional characteristics of tumor stem cells of MPN.Our data here provide new evidence for NLRP6 as potential target in the treatment of MPN.

Aeromonas hydrophila causes ferroptotic damage via its secreted effectors targeting splenic macrophages in teleost

Aeromonas hydrophila (A. hydrophila) is a well-known pathogen leading to huge economic losses in aquaculture. Using grass carp, a commercially important fish species, as a model, we found that A. hydrophila decreased fish survival, caused ferrugination and induced lipid peroxidation damage especially in spleen. Further investigations revealed that the bacteria particularly utilized secretory effectors to trigger ferroptosis by specially targeting splenic macrophages in which A. hydrophila effectors significantly elevated intracellular Fe2+ levels, initiated lipid peroxidation, and dampened mitochondrial membrane potential. Mechanistically, bacterial effectors could degrade ferritin and ferroportin1 via autophagy and proteasome pathways respectively, leading intracellular iron accumulation. Moreover, the effectors promoted glutathione (GSH) depletion via their stimulatory effects on ROS generation in splenic macrophages, suggesting that A. hydrophila effectors might suppress GSH-dependent cellular antioxidant system. Finally, an in vivo study uncovered that ferroptosis inhibitors significantly improve fish survival, ameliorated oxidative damage, and reduced bacterial load in spleen. Our data highlight the ferroptotic damage of A. hydrophila via its effectors in teleost and suggest ferroptosis is a potential target for fish against A. hydrophila infection.

Antimicrobial resistance, molecular characteristics, virulence and pathogenicity of bla NDM-1-positive Enterobacter cloacae.

Introduction. The bla NDM-1 -positive Enterobacter cloacae has led to limited therapeutic options for clinical treatment.Hypothesis/Gap Statement. Analysing the antimicrobial resistance and molecular typing of bla NDM-1-positive E. cloacae is of great significance. Meanwhile, the effect of the bla NDM-1 gene on the virulence and pathogenicity of E. cloacae remains unclear and should be assessed.Aim. To understand bla NDM-1-positive E. cloacae from different perspectives.Methodology. The PCR was used to screen bla NDM-1-positive E. cloacae, then, antimicrobial susceptibility tests and multilocus sequence typing (MLST) were performed on them; sixty-nine strains of bla NDM-1-negative E. cloacae were collected as the controls, 28 pairs of virulence-related genes' carriage and biofilm-forming ability were detected for preliminary evaluation of the virulence phenotype of the strains; to gain insight into the effect of the bla NDM-1 gene on the virulence and pathogenicity of E. cloacae, the bla NDM-1-positive E. cloacae T2 (NDM-1), the T2 bla NDM-1 knockout strain (ΔNDM-1) and ATCC13047 (ST) were studied, compared the motility, anti-serum killing ability, and virulence to cells. Then, the mice intraperitoneal infection model was established, the survival curve, histopathological characteristics, bacterial load in spleen and the contents of cytokines were compared.Results. (1) Thirty-five bla NDM-1-positive E. cloacae exhibited multidrug resistance. MLST distinguished 12 STs, ST74 was the most common clonal type (11/35), followed by ST114 (10/35). (2) The detection rates of virulence genes clpB, icmf, VasD/Lip and acrA in the bla NDM-1-positive E. cloacae were significantly higher than those in bla NDM-1-negative E. cloacae (P<0.05), while there was no significant difference in the amount of biofilm formation between two groups. (3) The presence of bla NDM-1 gene attenuated the motility diameter of E. cloacae, but had no significant effect on their ability to resist serum killing, and the virulence to cells. The survival rate, histopathological changes, bacterial load in spleen and inflammatory cytokines were not significantly affected.Conclusions. (1) The bla NDM-1-positive E. cloacae exhibited multidrug resistance, and the MLST typing was mainly ST74 and ST114, with a small-scale clonal spread of the ST114 strain in the hospital NICU ward. (2) The bla NDM-1 gene did not affect the virulence and pathogenicity of E. cloacae.

Deletion of MIF gene from live attenuated LdCen−/− parasites enhances protective CD4+ T cell immunity

Vaccination with live attenuated Leishmania parasites such as centrin deleted Leishmania donovani (LdCen−/−) against visceral leishmaniasis has been reported extensively. The protection induced by LdCen−/− parasites was mediated by both CD4+ and CD8+ T cells. While the host immune mediators of protection are known, parasite determinants that affect the CD4+ and CD8+ T cell populations remain unknown. Parasite encoded inflammatory cytokine MIF has been shown to modulate the T cell differentiation characteristics by altering the inflammation induced apoptosis during contraction phase in experimental infections with Leishmania or Plasmodium. Neutralization of parasite encoded MIF either by antibodies or gene deletion conferred protection in Plasmodium and Leishmania studies. We investigated if the immunogenicity and protection induced by LdCen−/− parasites is affected by deleting MIF genes from this vaccine strain. Our results showed that LdCen−/−MIF−/− immunized group presented higher percentage of CD4+ and CD8+ central memory T cells, increased CD8+ T cell proliferation after challenge compared to LdCen−/− immunization. LdCen−/−MIF−/− immunized group presented elevated production of IFN-γ+ and TNF-α+ CD4+ T cells concomitant with a reduced parasite load in spleen and liver compared to LdCen−/−group following challenge with L. infantum. Our results demonstrate the role of parasite induced factors involved in protection and long-term immunity of vaccines against VL.

Longitudinal analysis of changes in the gut microbiota of zebrafish following acute spring viremia of carp virus infection

The gut microbiota of vertebrates plays a critical role in resistance to invading pathogens. Spring viremia of carp (SVC) is one of the most severe viral diseases in aquaculture. The causative agent is the SVC virus (SVCV), resulting in devastating damage to cyprinid. Yet, knowledge regarding the relationship between the gut microbes and disease is still lacking, particularly the dynamic alteration of the gut microbiota in fish following SVCV infection. To understand the longitudinal effects of SVCV infection on the microbiota of the zebrafish intestine, an intraperitoneal injection zebrafish model mimicking acute SVCV infection in fish with typical virologic and pathologic features was used. Intestinal microbes from both control (CON) and SVCV-infected fish were characterized at various time points by 16S ribosomal RNA gene V3-V4 region sequencing. Viral load in spleen, kidney, and intestine and the total bacterial load in the gut were detected by qPCR.The results showed that SVCV infection was associated with alterations of the gut microbiota in zebrafish as indicated by both alpha and beta diversity indexes. Compared with the CON group, SVCV-infected fish had significantly reduced bacterial diversity at 4 to 6 days post-infection (dpi). It has been found that a number of bacteria were enriched in opportunistic pathogens and depleted of beneficial commensals, at the time of onset of obvious symptoms or even death and at the time of complete disappearance of symptoms. A noticeably higher relative abundance of opportunistic pathogens, such as Clostridium_XlVa, Vibrio, Aeromonas, Mycoplasmataceae_unclassified, and Rothia; and a lower relative abundance of beneficial symbionts were found in the gut microbiome at 4, 7, 10 days post-infection. Five biomarkers showed high accuracy for distinguishing SVCV-infected fish from CON with an area under the curve (AUC) up to 0.78, 0.80, 0.79, 0.89, and 0.84, respectively. The relative abundance of Enterobacteriaceae_unclassified showed a positive correlation with viral loads of SVCV and the relative abundance of Cetobacterium and Aeromonas were positively related to the bacterial load in the gut, while the same results were observed for the corresponding two phyla (Fusobacteria and Proteobacteria). SVCV causes long-term gut microbiome dysbiosis, which is linked to viral load in target organs and total intestinal bacterial count. The results of our study suggest the potential value of the gut microbiota as a diagnostic biomarker and therapeutic target for SVC, but further validation is required.

Emetic Tartar-Loaded Liposomes as a New Strategy for Leishmaniasis Treatment.

Emetic tartar (ET), was used in the treatment of leishmaniasis but its use was discontinued due to its low therapeutic index. Liposomes have been shown to be a promising strategy for delivery of bioactive substances in the region of interest, in order to reduce and/or eliminate undesirable effects. In the present study, liposomes containing ET were prepared and characterized to evaluate acute toxicity as well as their leishmanicidal action using BALB/c mice with an inoculum of Leishmania (Leishmania) infantum. Liposomes were composed of egg phosphatidylcholine and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, with an average diameter of 200 nm, zeta potential of +18 mV, and ET encapsulated into liposomes at a concentration near 2 g/L. Healthy mice were treated with ET or liposome containing ET (Lip-ET) in a single dose of 16 mg/kg of Sb3+ intravenously and observed for 14 days. The death of two animals in the ET-treated group and no deaths in the Lip-ET-treated group was observed. Higher hepatic and cardiac toxicity were observed in animals treated with ET when compared to animals treated with Lip-ET, blank liposomes (Blank-Lip) and PBS. The study of antileishmanial efficacy was conducted by intraperitoneal administration of Lip-ET, for ten consecutive days. It was observed by limiting dilution that treatments with liposomal formulations containing ET, as well as Glucantime®, led to a significant reduction in parasitic load in spleen and liver (p < 0.05) when compared to the untreated control group.

Is the production of reactive oxygen and nitrogen species by macrophages associated with better infectious control in female mice with experimentally disseminated and pulmonary mucormycosis?

Different levels of resistance against Rhizopus oryzae infection have been observed between inbred (BALB/c) and outbred (Swiss) mice and are associated with the genetic background of each mouse strain. Considering that macrophages play an important role in host resistance to Rhizopus species, we used different infectious outcomes observed in experimental mucormycosis to identify the most efficient macrophage response pattern against R. oryzae in vitro and in vivo. For this, we compared BALB/c and Swiss macrophage activity before and after intravenous or intratracheal R. oryzae infections. The production of hydrogen peroxide (H2O2) and nitric oxide (NO) was determined in cultures of peritoneal (PMΦ) or alveolar macrophages (AMΦ) challenged with heat-killed spores of R. oryzae. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) were measured to confirm our findings. Naïve PMΦ from female BALB/c mice showed increased production of H2O2, TNF-α, and IL-10 in the presence of heat-killed spores of R. oryzae. Naïve PMΦ from female Swiss mice were less responsive. Naïve AMΦ from the two strains of female mice were less reactive to heat-killed spores of R. oryzae than PMΦ. After 30 days of R. oryzae intravenous infection, lower fungal load in spleen from BALB/c mice was accompanied by higher production of H2O2 by PMΦ compared with Swiss mice. In contrast, AMΦ from BALB/c mice showed higher production of NO, TNF-α, and IL-10 after 7 days of intratracheal infection. The collective findings reveal that, independent of the female mouse strain, PMΦ is more reactive against R. oryzae upon first contact than AMΦ. In addition, increased PMΦ production of H2O2 at the end of disseminated infection is accompanied by better fungal clearance in resistant (BALB/c) mice. Our findings further the understanding of the parasite-host relationship in mucormycosis.

Dietary supplementation with nanoparticle CMCS-20a enhances the resistance to GCRV infection in grass carp (Ctenopharyngodon idella)

Combination of antimicrobial proteins and nanomaterials provides a platform for the development of immunopotentiators. Oral administration of immunopotentiators can significantly enhance the immunity of organisms, which provides ideas for disease prevention. In this study, we confirmed that nanoparticles CMCS-20a can efficiently prevent grass carp reovirus (GCRV) infection. Firstly, we verified that CiCXCL20a is involved in the immune responses post GCRV challenge in vivo and alleviates the cell death post GCRV challenge in CIK cells. Then, we prepared nanoparticles CMCS-20a using carboxymethyl chitosan (CMCS) loaded with grass carp (Ctenopharyngodon idella) CXCL20a (CiCXCL20a). Meanwhile, we confirmed nanoparticles CMCS-20a can alleviate the degradation in intestine. Subsequently, we added it to the feed by low temperature vacuum drying method and high temperature spray drying method, respectively. Grass carp were oral administration for 28 days and challenged by GCRV. Low temperature vacuum drying group (LD-CMCS-20a) significantly improve grass carp survival rate, but not high temperature spray drying group (HD-CMCS-20a). To reveal the mechanisms, we investigated the serum biochemical indexes, intestinal mucus barrier, immune gene regulation and tissue damage. The complement component 3 content, lysozyme and total superoxide dismutase activities are highest in LD-CMCS-20a group. LD-CMCS-20a effectively attenuates the damage of GCRV to the number of intestinal villous goblet cells and mucin thickness. LD-CMCS-20a effectively regulates mRNA expressions of immune genes (IFN1, Mx2, Gig1 and IgM) in spleen and head kidney tissues. In addition, LD-CMCS-20a obviously alleviate tissue lesions and viral load in spleen. These results indicated that the nanoparticles CMCS-20a can enhance the disease resistance of fish by improving their immunity, which provides a new perspective for fish to prevent viral infections.

Immunoprotective characterization of egg yolk immunoglobulin raised to loop 3 of outer membrane protein 34 (Omp34) in a murine model against Acinetobacter baumannii

Acinetobacter baumannii is one of the most notorious nosocomial pathogens with high mortality rates. Recently, egg yolk antibody (IgY), has been considered as a promising biomolecule against pneumonia caused by this bacterium. Loop 3 of outer membrane protein 34 (Omp34) was predicted as a highly exposed immunogenic peptide. However, its immunogenicity remains to be experimentally elucidated. In the current study, a construct composed of 5 copies of loop3 of Omp34 labeled as Omp34L3X5 was designed. This construct as well as the recombinant Omp34 were expressed, purified, and injected into laying hens to raise specific antibodies. The specific IgYs were extracted from hyperimmune egg yolks. The Omp34L3X5 and whole cells (WC) of A. baumannii served as antigens in indirect ELISA to assess the purified IgYs reactivity. These antibodies were administered to neutropenic mice 1 h before the challenge with 10 × LD50 of a clinical isolate of A. baumannii. The specific IgYs recognized recombinant Omp34 (P < 0.0001) as well as WC of A. baumannii (P < 0.05). The survival rate of mice that received anti- Omp34L3X5 or anti-Omp34 IgYs was 83.33 % and 100 % respectively. All control mice died within 24 h while mice that received non-specific IgYs died within 72 h. After 24 h, bacterial load in the lung of mice received non-specific IgYs, anti-Omp34L3X5 or anti-Omp34 IgYs were 2.03 × 108, 2.2 × 108, and 1.93 × 108 CFU/organ respectively. Bacterial load in the spleen of mice received non-specific IgYs, anti-Omp34L3X5 or anti-Omp34 IgYs were 1.03 × 108, 7.8 × 107, and 6.3 × 107 CFU/organ respectively. Bacterial load in lung and spleen of control mice were 3.03 × 109 and 1.45 × 108 CFU/organ respectively.

Protective cellular and humoral immune responses to Edwardsiella tarda in flounder (Paralichthys olivaceus) immunized by an inactivated vaccine

Protection is crucial for a vaccine. In this study, flounder was inoculated with an inactivated Edwardsiella tarda for 5 weeks then challenged with E. tarda, after that, the protective cellular and humoral immune responses were studied using specific antibodies against CD4, CD8 and IgM, respectively, previously produced in our lab. And then gene transcription, relative percent survival (RPS), innate enzyme activities and histopathology were investigated. The results showed that, in the vaccine group, the percentage of CD4+ T and CD8+ T lymphocytes increased significantly, peaking on 7th post challenge day. The percentage of IgM+ B lymphocytes increased significantly first and peaked at 2nd post challenge week. Total and specific antibodies significantly increased and peaked at 3rd week. The mRNA expressions of MHCIα, MHCIIα, CD4–1 and CD8α were significantly upregulated. In the meantime, alkaline phosphatase, acid phosphatase, superoxide dismutase and lysozyme significantly rose and reached to the maximum. The RPS was 54.76 ± 1.92%. The bacterial load in spleen and head kidney was lower in the vaccine group than that of PBS group. These data illustrated that T lymphocyte subsets, B lymphocytes, immune enzymes were induced strong protection against E. tarda for flounder. These results revealed a strong support protection of the vaccine from four aspects, and provided a new perspective for the evaluation of fish vaccines.

Evaluation of Th17 immune responses of recombinant DNA vaccine encoding GRA14 and ROP13 genes against Toxoplasma gondii in BALB/c mice.

Toxoplasma gondii, a worldwide opportunistic parasite, causes serious diseases in both humans and fetuses with defective immune systems. The development of an effective vaccine is urgently required to prevent and control the spread of toxoplasmosis, caused by the apicomplexan parasite Toxoplasma gondii which is one of the most damaging zoonotic diseases of global importance. Plasmid DNA vaccination is a promising procedure for vaccine development and following the previous studies, pcROP13+pcGRA14 cocktail DNA vaccine was evaluated for Th17 immune responses. Four groups of BALB/c mice were immunized intramuscularly three times at 2-week intervals. Subsequently, the production of anti- T. gondii antibodies and serum levels of cytokines IL-17, and IL-22 were evaluated against the RH strain of T. gondii. In addition, both the reactive oxygen species (ROS) and parasite load were assessed using ELISA and Q-PCR, respectively. The results of this study showed that high levels of IgG were found in mice immunized with cocktail DNA vaccine (p<0.05). The cytokines level of Th17, IL-17, and IL-22, increased remarkably in the immunized mice (p<0.05). Also, significant induction (p<0.05) was observed in ROS. In addition, immunization with pcROP13+GRA14 resulted in a considerable decrease in parasite load compared to the control groups (p<0.05). Based on the results, the pcROP13+GRA14 cocktail DNA vaccine induced Th17 related cytokines and decreased the parasite load in spleen and brain tissues. Hence, pcGRA14+pcROP13 cocktails are suitable candidates for DNA-based vaccines and due to the development of protective immune responses against T. gondii infection, future studies may yield promising results using these antigens in vaccine design.

Synergistic Activity of BH3-Mimetics By Combined Targeting of Anti-Apoptotic Regulators in B-Cell Precursor ALL

In acute lymphoblastic leukemia (ALL), the most frequent malignancy in children and adolescents, deregulated cell death pathways contribute to leukemia development and therapy failure. Apoptosis (programmed cell death) is controlled at the mitochondrial level by different pro- and anti-apoptotic regulators. Molecules of the BCL-2 family are key in regulating intrinsic apoptosis signaling. Pro-apoptotic BH3-only proteins such as BIM and BID activate pro-death proteins like BAX and BAK leading to cell death, while anti-apoptotic BCL-2 family members including BCL-2, MCL-1 and BCL-XL sequester pro-apoptotic molecules, thereby preventing pro-death protein activation and apoptosis induction. Small molecule inhibitors have been developed, which bind to the anti-apoptotic molecules BCL-2, MCL-1 and BCL-XL leading to cell death induction. In particular inhibition of BCL-2, a key regulator of survival in B-cell malignancies including BCP-ALL, by the specific inhibitor venetoclax (VEN) has shown substantial, clinical anti-tumor activity. However, in BCP-ALL heterogeneous sensitivities for VEN have been described suggesting that other BCL-2 family members like MCL-1 and BCL-XL interfere with BCL-2 inhibition thereby counteracting VEN activity leading to poor treatment response.In this study, we compared the effects of inhibition of the anti-apoptotic BCL-2 family members BCL-2 (venetoclax, VEN), MCL-1 (S63845) and BCL-XL (A-1331852), investigated molecular mechanisms and determinants of inhibitor sensitivity, and addressed potential synergistic activity upon simultaneous blockage of BCL-2 together with MCL-1 or BCL-XL in BCP-ALL.First, we investigated the activity of the single inhibitors to induce cell death (positive staining for propidium iodide) in 7 BCP-ALL cell lines and a series of 27 BCP-ALL patient-derived xenograft samples (PDX). Titrating increasing concentrations, we estimated half-maximal effective concentrations (EC 50) for each inhibitor showing heterogenous responses of individual samples to the different inhibitors. Importantly, sensitivities to either inhibitor were not associated with leukemia characteristics including recurrent genetic alterations described in BCP-ALL. We also did not observe similar sensitivities to the inhibitors of individual samples, however some VEN insensitive samples showed sensitivity to MCL-1 and/or BCL-XL-inhibition, suggesting functional substitution of the anti-apoptotic regulators.Next, we investigated anti-apoptotic addictions of BCP-ALL cells to BCL-2 family proteins upon VEN treatment. Analyzing apoptosis signaling after exposure to VEN and BH3-peptides, we identified that ALL cells adapt their anti-apoptotic addiction to MCL-1 and BCL-XL as an escape strategy from VEN-induced cell death. Analyzing protein complexes by co-immunoprecipitation, we found that exposure of ALL cells to VEN rapidly led to reduced BIM/BCL-2 and compensatory increased BIM/MCL-1 complexes. Conversely, S63845 reduced BIM/MCL-1 complexes and led to increased BIM/BCL-2 binding. Importantly, both protein complexes could be effectively disrupted by combination treatment with VEN and S63845, which resulted in release of BIM and promotion of apoptosis signaling.Based on our mechanistic findings, we evaluated BH3-mimetic combinations for cell death induction using multi-dose matrix assays to calculate synergy metrics based on the Bliss independence model. Analyzing combination effects in seven BCP-ALL cell lines and four PDX samples, we identified positive mean synergy scores of VEN with S63845 and A-1331852 in all cases. However, the most synergistic area of the multi-dose-response matrices differed among different leukemias, indicating different extents of addictions and thereby synergies at different drug concentrations.Finally, combined BCL-2 and MCL-1 inhibition was evaluated in vivo in a PDX model of KMT2A-ENL positive pro-B ALL. Combination treatment of VEN with S63845 led to reduced leukemia loads in spleen, bone marrow and CNS as compared to single agent treatment.Taken together, we found heterogeneous responses of BCP-ALL samples to BH3-mimetics antagonizing BCL-2, MCL-1 and BCL-XL. The ability of leukemia cells to adapt their anti-apoptotic dependency from BCL-2 to MCL-1 or BCL-XL can be used as target for combination therapy, demonstrating synergistic activity in PDX samples ex vivo and in vivo. DisclosuresNo relevant conflicts of interest to declare.

Prevalence, Characterization, and Pathogenicity of Salmonella enterica Subspecies enterica Serovar Derby from Yaks in the Aba Tibetan Autonomous Prefecture, China.

Simple SummarySalmonella spp. is a very important pathogen in the livestock industry and public health, which poses a major threat to global public health. Yaks and their by-products have a significant economic status in the Qinghai–Tibetan Plateau. The aim of this study was to investigate the prevalence of salmonella in yak farms and to conduct a molecular characterization and tests on its pathogenicity in mice with the use of salmonella isolated from yaks with diarrhea as well as from drinking water samples. The prevalence of salmonella was 19.75% of 162 samples collected from yak farms, and all isolates were found to belong to the serovar of Salmonella Derby and ST40. All Salmonella Derby isolates from both fecal and drinking water samples from 13 farms were clonally related based on SNP alignment. Salmonella Derby was still detected positively in the feces of model mice on day 24 post-injection. This study reports the prevalence of Salmonella Derby in yaks with diarrhea and in their drinking water. In addition, the pathogenicity of the S. Derby in mice was investigated. Findings suggest that Salmonella Derby excreted by diarrheic yaks is a source of contamination for other yaks and the environment and is highly pathogenic to mice. Seeing that Salmonella Derby has become one of the most common Salmonella serovars, this situation gives rise to further risk from the potential spread of food-borne diseases.Salmonella enterica subsp. enterica serovar Derby (S. Derby) is one of the numerous non-typhoidal Salmonella serovars and has been recognized as a food-borne pathogen. In 2019, outbreaks of salmonellosis were reported in 13 yak farms in the Aba Tibetan Autonomous Prefecture, China. A total of 32 salmonella strains were isolated from 162 fecal samples of yaks with diarrhea as well as from drinking water samples. The isolates were subjected to serovar identification, animal experiments, and whole-genome sequencing (WGS) analyses. The serovar of all the isolates was S. Derby, and the sequence types (STs) were ST40. The analysis of the differences of single-nucleotide polymorphisms (SNPs) showed that the salmonella strains isolated from 13 farms were clonally related. Animal experiments showed that the lethal dose (LD50) was 4.57 × 107 CFU (colony-forming units); the shedding time of S. Derby in mice was 24 days; the bacterial loads in spleen were higher than those in other organs (ileum, liver, and cecum). Pathological analyses by hematoxylin and eosin (H&E) staining revealed obvious damage in the spleen, liver, and intestine. These results indicate that the S. Derby from yaks can cause infection in mice.

A novel NK-lysin in hybrid crucian carp can exhibit cytotoxic activity in fish cells and confer protection against Aeromonas hydrophila infection in comparison with Carassius cuvieri and Carassius auratus red var

NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection.

Evaluation of CVC1302 for Improved Efficacy of FMD-Inactivated Vaccine in Oxidative Stressed Mice Generated with PCV2 Infection.

This study aimed to explore the effect of the immunopotentiator CVC1302 on foot-and-mouth disease (FMD) vaccination in animals placed under oxidative stress. We established oxidative stress models using porcine circovirus type 2 (PCV2)-infected PK-15 cells and mice model both in vitro and in vivo, respectively. The efficacy of CVC1302 on PK-15 cells or in addition to the FMD vaccine was evaluated by quantitative real-time polymerase chain reaction, histopathological and enzyme-linked immunosorbent assay (ELISA) analysis. CVC1302 affected apoptosis of PCV2-infected PK-15 cells and significantly inhibited PCV2 replication, while it had no effect on the viability for blank PK-15 cell in vitro test with varying dilutions of CVC1302. Results showed that PCV2 induced a strong oxidative stress response in mice. CVC1302 reduced the viral load in spleen of PCV2-infected mice and ameliorated the pathological injury of spleen. Furthermore, CVC1302 significantly increased IgG antibody titer, cytokine expression, superoxide dismutase activity, catalase concentrations, and glutathione content in mice immunized with FMD vaccine. In conclusion, CVC1302 inhibits PCV2 replication and regulates oxidative stress in PCV2-infected mice, which can improve the immune efficacy of the FMD vaccine, providing a safe and effective immune enhancement.

Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

In canine leishmaniosis caused by the protozoan Leishmania infantum, little is known about how co-infections with or co-seropositivities for other pathogens can influence aggravation of this disease. Therefore, the objectives of this study were to evaluate the frequency of co-infections with or co-seropositivities for certain pathogens in dogs seropositive for L. infantum and their relationship with clinical signs, histological changes and L. infantum load. Sixty-six L. infantum-seropositive dogs were submitted to clinical examination, collection of blood and bone marrow, culling, and necropsy. Antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and Toxoplasma gondii and Dirofilaria immitis antigens were investigated in serum. Samples from different tissues were submitted to histopathology and immunohistochemistry for the detection of Leishmania spp. and T. gondii. Quantitative real-time PCR was used to assess the L. infantum load in spleen samples. For detection of Coxiella burnetii, conventional PCR and nested PCR were performed using bone marrow samples. All 66 dogs tested positive for L. infantum by qPCR and/or culture. Fifty dogs (76%) were co-seropositive for at least one pathogen: T. gondii (59%), Ehrlichia spp., (41%), and Anaplasma spp. (18%). Clinical signs were observed in 15 (94%) dogs monoinfected with L. infantum and in 45 (90%) dogs co-seropositive for certain pathogens. The L. infantum load in spleen and skin did not differ significantly between monoinfected and co-seropositive dogs. The number of inflammatory cells was higher in the spleen, lung and mammary gland of co-seropositive dogs and in the mitral valve of monoinfected dogs. These results suggest that dogs infected with L. infantum and co-seropositive for certain pathogens are common in the region studied. However, co-seropositivities for certain pathogens did not aggravate clinical signs or L. infantum load, although they were associated with a more intense inflammatory reaction in some organs.

A Potent Inhibitor of IL-10/STAT3 Axis Signaling Modulates Anti-Inflammatory Responses and Boosts Anti-Tuberculosis Immunity in rBCG30 Immunized Mice

Aims and Objectives: In the present study we tried to improve anti-TB immunity offered by 2nd generation rBCG30 vaccine. We attempted to bolster rBCG30 invoked anti-TB immunity in mice through modulation of the IL-10/STAT3 interaction mediated anti-inflammatory program. The study was aimed towards generating optimum activated APCs, early either after vaccination (with rBCG30) or infection (with Mtb), which can process and present mycobacterial antigens more efficiently to T cells. It was envisage that downmodulating IL-10/STAT3 signaling may improve immunity and protection offered by rBCG30 vaccine. Methods: A small molecule immunomodulator aimed at disrupting anti-inflammatory signaling orchestrated by IL-10/STAT3 axis was administered in rBCG30 immunized mice. The comparative expansion of pathogen permissive Alternatively Activated Monocytes/Macrophages/DCs (AAMs/AADCs) and Classically Activated Monocytes/Macrophages/DCs (CAMs/CADCs), the cells that resist establishment of successful Mtb infection, was profiled in both peritoneal and splenic compartment. To assess innate and adaptive T cell responses, splenocytes cytokine production was checked ex vivo using sandwich ELISA. We also checked polyfunctional T cell response, T cell memory response and comparative Th17/Treg cells expansion using flow cytometry. Finally, to assess the real time efficacy of the employed immunotherapeutic strategy, in vivo protection study was undertaken in BALB/c mice challenge with aerosolized Mtb (H37 Rv). Results: The small molecule mediated inhibition of IL-10/STAT3 signaling was resulted in improved pro inflammatory immune responses in both post-vaccination and post-infection immunotherapy schedules. The improved immune profile in rBCG30 vaccinated and immunotherapy administered mice was accompanied with enhanced expansion of CAMs/CADCs, elevated production of polyfunctional T cells producing signature Th1 cytokines, long lasting CD4+ and CD8+ memory T cell response and balanced Th17/Treg cells ratio. Interestingly, post-infection, and not post-vaccination immunotherapy, was able to significantly reduce mycobacterial loads in spleen and lungs of experimentally infected mice. Conclusions: Small molecule mediated transient disruption of anti-inflammatory effectors, such as IL-10/STAT3, during vaccination or post infection with Mtb, may serve to enhance the efficacy of existing and forthcoming TB vaccines. The strategy can also be tested concomitantly with standard chemotherapy (either in vaccinated or unvaccinated host) to enhance the efficacy of therapeutic regimen and to reduce the treatment length.

Neuroinvasive Listeria monocytogenes infection triggers accumulation of brain CD8+ tissue-resident memory T cells in a miR-155-dependent fashion

BackgroundBrain inflammation is a key cause of cognitive decline after central nervous system (CNS) infections. A thorough understanding of immune responses to CNS infection is essential for developing anti-inflammatory interventions that improve outcomes. Tissue-resident memory T cells (TRM) are non-recirculating memory T cells that provide surveillance of previously infected tissues. However, in addition to protecting the brain against reinfection, brain TRM can contribute to post-infectious neuroinflammation. We hypothesized that accumulation of CD8+TRM in the brain could be reduced by inhibiting microRNA (miR)-155, a microRNA that influences development of cytotoxic CD8+ T lymphocytes during infection.MethodsC57BL/6J mice were infected by intraperitoneal injection with a lethal inoculum of Listeria monocytogenes (Lm) then treated with antibiotics. Flow cytometry was used to quantify specific populations of brain leukocytes 28–29 days (d) post-infection (p.i.). To test the degree to which miR-155 altered leukocyte influxes into the brain, infected mice were injected with a miR-155 inhibitor or locked nucleic acid (LNA) scramble control 2d, 4d, 6d, and 8d p.i. along with antibiotic treatment. Bacterial loads in spleen and liver and body weights were measured up to 7d p.i. Brain leukocytes were analyzed 14d and 28d p.i. Confirmatory studies were performed in mutated mice lacking miR-155 (miR-155−/−)ResultsLm infection significantly increased the numbers of brain CD3+CD8+ lymphocytes at 28d p.i. These cells were extravascular, and displayed markers characteristic of TRM, with the predominant phenotype of CD44+CD62L-CD69+CX3CR1−. Further analysis showed that > 75% of brain TRM also expressed CD49a, PD-1, Ly6C, CD103, and CD127. Mice injected with miR-155 inhibitor lost less weight through 7d p.i. than did control mice, whereas bacterial loads in brain, liver, and spleen were not different from controls. By 28d p.i., the numbers of brain CD8+ TRM cells were significantly decreased in mice treated with the inhibitor compared with controls. Similarly, miR-155−/− mice showed significantly reduced numbers of brain CD8+TRM cells by 28d p.i.ConclusionsBrain CD8+ TRM populations are established during neuroinvasive Lm infection. Accumulation of brain CD8+ TRM cells is reduced by blocking miR-155 and in miR-155−/− mice, indicating that this molecule has a critical role in development of these specialized cells. Administering anti-miR-155 during infection could provide a novel avenue for reducing post-infectious neuroinflammation.

Immunization efficacy and safety of Brucella 104M against aerosol challenge in BALB/c mice

Objective: To evaluate the protective efficacy and safety of Brucella 104M against aerosol challenge in BALB/c mice and characterize its immunological effects. Methods: Female mice of 6-8 weeks old were immunized with Brucella abortus strain 104M by intratracheal aerosol delivery or intranasal instillation or subcutaneous injection route. Six mice of each group were sacrificed at 4, 8, 16, 24 weeks after immunization. At each time point, the clinical manifestations of mice were investigated, the serum, spleen and lung samples of mice were collected, body weight, spleen weight, bacteria loads in spleens, the anti-Brucella antibodies titers in serum and the cytokines concentrations of IFN-γ, IL-18 in serum or lung homogenate of the mice were detected. Twenty two weeks after immunization, all the mice were challenged with Brucella A19 through intratracheal aerosol delivery. Results: Compared with the control group, neither abnormal clinical symptoms nor significant changes in body weight were found in 104M immunization groups, at each time point when immunized through either nose dropping route, subcutaneous injection or aerosol routes; and the spleen weight of immunization groups were lower than control group after challenge (P<0.05): *M1 (0.26±0.16)g<M2 (0.40±0.19)g; *M3(0.21±0.11)g<M4(0.28±0.19)g; *M5(0.14±0.02)g<M6 (0.30±0.18)g. The spleen bacterium load of the mice in 104M immunization groups decreased until 20 weeks later, the cells were completely clear out, 2 weeks after challenge (24 weeks), the bacterium load increased again, the bacterium load in immunization groups were significantly lower than that in control group (P<0.05): *M1(4.49±0.13)log(10) (Colony-Forming Units) CFU/g<M2(6.90±0.46)log(10)CFU/g; *M3(3.59±1.06)log(10)CFU/g<M4(7.08±0.14) log(10)CFU/g; *M5(3.00±2.03)log(10)CFU/g<M6(6.81±0.34)log(10)CFU/g. The high titer of Brucella 104M specific antibodies were detected at week 4, and the peak was reached at week 8 of immunization groups. At each time point, the serum antibody titers IgG 2a was higher than that of IgG 1; before challenge, the cytokines concentrations of IFN-γ and IL-18 in all immunization groups were significantly higher than that in control group (P<0.05), after challenge, cytokines concentrations of IFN-γ and IL-18 in the serum were lower in all immunization groups than in control group (P<0.05). At each time point, IFN-γ, IL-1βand IL-18 concentrations in lung homogenate were higher in immunization groups than in control group (P<0.05). There were no significant differences among the three immunization routes in all sample detection indexes (P<0.05). Conclusions: Intratracheal aerosol delivery is a safe and effective immunization route in BALB/c mice. Both humoral immunity and cellular immunity were stimulated by Brucella 104M, showing obvious protective efficacy against aerosol challenge in BALB/c mice. No significant weight loss was detected. However, the colonizing of Brucella 104M in the spleen of mice was too long (20 weeks), and slighter spleen swelling of the mice were detected, which illustrated that the attenuated strain 104M has residual virulence as a vaccine strain in mice.