Sheep Red Blood Cells Research Articles

Effect of dietary L-tryptophan supplementation and‏ ‏light-emitting diodes on growth ‎and immune response of broilers ‎.

Light-emitting diodes (LEDs) lights are more energy-efficient and provide adequate illumination compared to compact fluorescent (CFL) lamps and incandescent light (ICD) bulbs. However, as new light sources, the LED lights may have a stress effect on broiler chickens. Thus, this study aimed to determine the effects of dietary L-tryptophan (Trp), as an anti-stress agent and different color temperatures of light-emitting diodes on immune responses and growth performance of male broiler chickens. Four hundred and eighty day-old Ross 308 male chicks were used from day 1 to 42. The chicks were randomly distributed into six treatment groups in a 2 × 3 factorial arrangement [0 or 1 g Trp per kg diet along with neutral-white (4286 K), warm-white (2990 K), and incandescent (2790 K) light bulbs] with four replicates of 20 chicks each. Results showed that dietary Trp and Trp×light interaction did not affect growth performance, immune responses, a total number of leukocytes, and different leukocytes count (heterophil, eosinophil, monocyte, and lymphocyte) of male broiler chickens. However, LEDs’ different color temperatures significantly affected the feed conversion ratio (FCR) and primary antibody of sheep red blood cell (SRBC). The FCR was the lowest in the warm-white light, and primary SRBC antibody titers of the chicks were the highest. In conclusion, although adding Trp to male broiler diets did not affect the growth performance and immune responses of chickens, the warm-white light improved the FCR and primary SRBC.

A T-dependent antibody response evaluation in CD-1 mice after an acute whole-body inhalation exposure to nickel (II) chloride hexahydrate

Nickel (Ni) in ambient air may vary regionally with contributions from both natural processes and anthropogenic activities. Exposure to Ni compounds in ambient air above a certain level is associated with acute adverse effects, such as upper respiratory tract irritation, pneumonitis, and chronic adverse effects, such as respiratory cancer. Inhalation reference exposure standards are enacted in different jurisdictions to minimize exposures to ambient Ni above levels that can elicit adverse effects. This paper reports a guideline-/GLP-compliant study designed for setting inhalation exposure standards to protect from immunological effects associated with acute exposure to Ni. Female CD-1 mice were exposed via whole-body inhalation to aerosolized nickel chloride hexahydrate for 24-hr at nominal (vs. mean analyzed) concentrations of 20 (16), 50 (44) and 100 (81) µg Ni/m3. Host T-cell antibody immunological responses to intravenously-injected sheep red blood cells were then measured ex vivo in an Antibody-Forming Cell (AFC) assay. Exposure to the Ni substance significantly decreased spleen cell levels by 33%, but this was within biological variability for outbred mice. No concurrent decreases in spleen, thymus, or body weights were noted. No immunosuppression was observed with the Ni substance in the context of Total Spleen Activity [IgM AFC/spleen (× 103)] and Specific Activity [IgM AFC/spleen cells (× 106)]. Significant concentration-independent increases in Total Spleen Activity and Specific Activity seen with the nickel chloride hexahydrate were normal and within biological variability for outbred mice. In contrast, cyclophosphamide (positive control) significantly decreased spleen cell numbers, spleen and thymus weights, and abolished Specific Activity and Total Spleen Activity. Based on results here, an NOAEC of 81 µg Ni/m3 for immunosuppressive effects from inhaled nickel chloride hexahydrate was identified. It is hoped this value can be used to derive a reference standard for human exposure to ambient Ni.

Maintenance of liver fluke, Dicrocoelium dendriticum, outside the body of its native host

Aims: In vitro cultivation of digenea would help the development of effective treatments and studies of the biology of the parasites. The goal of the present study was to optimize culture conditions for the maintenance liver fluke, Dicrocoelium dendriticum. Materials and Methods: Forty fresh D. dendriticum were collected from the sheep liver and washed three times with warm Roswell Park Memorial Institute (RPMI) 1640 Medium. The collected worms were transferred to 24-well Nunc-Immuno plates containing RPMI media supplemented with 50% of fetal bovine serum (FBS), 2% of sheep red blood cells (RBCs), 50 IU/ml of penicillin, and 50 mg/ml streptomycin. The mobility of the live/dead worms was observed by inverted microscope. The mean and median survival time was calculated by Kaplan–Meier model, and survival and hazard function graphs were also analyzed. Results: D. dendriticum was lived in vitro only for long periods of about 25 dyes. The 1st day of maintaining in culture media, one worm was dead and the number of dead worms was raised to 40 after 25 days of incubation. On the one hand, the mean survival time was 392 h with a confidence interval (CI) of 95% (384.8–400.03). On the other hand, the median survival time was 420 h with a CI of 95% (406.9–433.09). D. dendriticum was able to be alive in RPMI 1640 media for at least 25 days. Conclusion: RPMI 1640 supplemented with FBS, and RBCs can be used as short-term maintenance for the in vitro culture of D. dendriticum. The outcomes of the current study could be useful for many aspects of parasitological analysis.

Pathogenesis and shedding of Usutu virus in juvenile chickens

ABSTRACT Usutu virus (USUV; family: Flaviviridae, genus: Flavivirus), is an emerging zoonotic arbovirus that causes severe neuroinvasive disease in humans and has been implicated in the loss of breeding bird populations in Europe. USUV is maintained in an enzootic cycle between ornithophilic mosquitos and wild birds. As a member of the Japanese encephalitis serocomplex, USUV is closely related to West Nile virus (WNV) and St. Louis encephalitis virus (SLEV), both neuroinvasive arboviruses endemic in wild bird populations in the United States. An avian model for USUV is essential to understanding zoonotic transmission. Here we describe the first avian models of USUV infection with the development of viremia. Juvenile commercial ISA Brown chickens were susceptible to infection by multiple USUV strains with evidence of cardiac lesions. Juvenile chickens from two chicken lines selected for high (HAS) or low (LAS) antibody production against sheep red blood cells showed markedly different responses to USUV infection. Morbidity and mortality were observed in the LAS chickens, but not HAS chickens. LAS chickens had significantly higher viral titers in blood and other tissues, as well as oral secretions, and significantly lower development of neutralizing antibody responses compared to HAS chickens. Mathematical modelling of virus-host interactions showed that the viral clearance rate is a stronger mitigating factor for USUV viremia than neutralizing antibody response in this avian model. These chicken models provide a tool for further understanding USUV pathogenesis in birds and evaluating transmission dynamics between avian hosts and mosquito vectors.

Comparison of performance and carcass composition of a novel slow-growing crossbred broiler with fast-growing broiler for chicken meat in Australia

Slow-growing broilers offer differentiation in the chicken meat market for consumers who have distinct preferences based on perceived higher welfare indices and willingness to pay a higher price for the product. Although breeding for slow-growing broilers is relatively advanced in Europe and the United States, it is limited in Australia. Crossbreeding is one of the approaches taken to developing slow-growing broiler strains. Thus, the aim of this study was to compare performance, immune response, leg health, carcass characteristics, and meat quality of a novel crossbred slow-growing broiler breed (SGB) with the conventional, fast-growing Cobb 500 broiler (CB) to assess their suitability as an alternative for chicken meat production in Australia. A total of 236 one-day-old broiler chicks (116 SGB and 120 fast-growing CB) were reared on standard commercial diet in an intensive production system. Birds and feed were weighed on a weekly basis and feed intake and feed conversion ratio calculated. At 21 d of age, a 2% suspension of sheep red blood cells was injected subcutaneously into 8 broilers of each breed to compare their antibody response. Birds from both breeds were grown to a final live weight of 2.0–2.2 kg, before a latency-to-lie (LTL) test, carcass analysis and apparent metabolizable energy (AME) assay were performed. The SGB reached the target weight at 55 d of age compared with 32 d in CB. However, SGB stood for longer during LTL, had higher thigh, drumstick, and wing yields (as a percentage of carcass weight) as well as darker and redder meat in comparison with the CB. The CB had better feed conversion efficiency, higher antibody (IgM) production, higher AME, heavier breast yield, and lower meat drip loss than the SGB. Although fast-growing CB outperformed the SGB for traditional performance parameters, the crossbred in this study was comparable with other slow-growing broiler breeds and strains across different countries and is thus a suitable candidate for a slow-growing alternative in Australia.

Immune response kinetics in Nigerian indigenous chickens challenged with attenuated Salmonella

Salmonella infection is a major cause of death in chickens and this disease can be transferred through meat and egg to humans. The use of antibiotics and vaccines in the prevention and control of this infection are not totally efficient and also have their side effects. Therefore, genetic control is a better approach in finding a lasting solution. One hundred and ten chickens (4th generation) comprising the three genotypes of Nigerian indigenous chickens(NIC) that had shown divergent response (high vs low) to sheep red blood cells antigens were used in this study to investigate the immune response kinetics in NIC after challenged with attenuated Salmonella. There were six classes namely, high titre normal, low titre normal, high titre frizzle, low titre frizzle, high titre naked neck and low titre naked neck. Haematological parameters: packed cell volume (PCV), haemoglobin concentration (Hb),red blood cells count (RBC), white blood cells count (WBC), heterophils (H), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M) and heterophils-lymphocytes ratio (HLR) were used to assess the immune responses of the chickens and data collected were subjected to General Linear Model procedure of SAS 9.2 version. The results showed that genotypeantibody titre had significant effect (p<0.05) on the immune response parameters but sex had no significant effect (p>0.05), while sex by genotype-antibody titre interaction had significant effect (p<0.05). The immune response kinetics of the chickens after challenge with attenuated Salmonella was shown by the significance of the effect of days of blood collection after challenge. High titre naked neck chickens recorded the highest PCV, Hb and RBC values while genotype-antibody titre had no significant effect (p>0.05) on the rest of the parameters. Low titre naked neck males recorded the highest PCV, HB and RBC values while the rest of the parameters were not significant (p>0.05). Generally, days had significant effect (p<0.05) on all the immune response parameters except on H, L and HLR. The study showed that the high titre naked neck males were least affected by the Salmonella vaccine in terms of decrease in packed cell volume, haemoglobin concentration and red blood cell count while the low titre normal females were most affected.

Functional Characterization of Cells Separated From Suspensions of Hodgkin Disease Tumor Cells in an Isokinetic Gradient

AbstractSuspensions of cells from Hodgkin disease tumors were separated by velocity sedimentation in an isokinetic Ficoll gradient. The separated cells were cultured with mitogens. The modal population of lymphocytes from the tumor contained 50%-75% of all lymphocytes separated from the tumor of each studied patient. Despite the fact that these cells appeared to be mature lymphocytes, the majority of which formed nonimmune E rosettes with sheep red blood cells, they showed a lack of responsiveness to phytohemagglutinin which was almost complete in all but one patient. Lymphocytes which had been obtained in suspension and purified by identical methods from tonsils and spleens without tumors did respond to stimulation by phytohemagglutinin.

Effects of early nutrition and sanitary conditions on antibody levels in early and later life of broiler chickens

Immune maturation of broiler chickens may be affected by management, such as early life feeding strategy (early versus delayed nutrition) or by low or high sanitary conditions (LSC versus HSC). We compared systemic maternal (MAb), natural (NAb), natural auto- (NAAb), and antigen specific antibody (SpAb) levels (IgM, IgY) between broilers (n = 48 per treatment) that received early (EN) or delayed nutrition for 72 h (DN) housed in either low (LSC) or high sanitary conditions (HSC) between 7 and 35 d of age. We found minimal interactions between feeding strategy and sanitary conditions. At 7 d of age, broilers receiving EN compared with DN, had elevated levels of IgM binding keyhole limpet hemocyanin (KLH), phosphoryl-conjugated ovalbumin (PC-OVA), and muramyl dipeptide (MDP), whereas effects of feeding strategy diminished at later ages. In LSC compared with HSC broilers, levels of NAb agglutinating RRBC and sheep red blood cells (SRBC) were already elevated from 14 d of age onwards. At 33 d of age, antibody levels (NAb, NAAb, anti-LPS, anti-MDP) were all elevated in LSC, compared with HSC broilers, for both IgM and IgY, but not IgM against KLH. Western blotting revealed different binding patterns of NAAb against chicken liver homogenate, which may indicate that the NAAb repertoire is affected by antigenic pressure. Our data suggest that antibody levels are affected for an important part by environmental conditions (feeding strategy and sanitary conditions), but minimally by their interaction. However, it remains to be further studied whether the enhanced levels of antibodies as initiated by EN and LSC contribute to enhanced resistance to infectious diseases.

Age-related features of the immune status associated with the development of arterial hypertension in employees of an oil production enterprise

Introduction. Arterial hypertension (AH) is a multifactorial disease, the main risk factors of which are environmental determinants, the duration and level of their exposure, as well as the age of the examined population. The conditions of the production environment are characterized by a more pronounced effect of technogenic factors compared to the environment, which creates prerequisites for the development of adaptation disorders and an increased risk of hypertension among employees of the oil production enterprise. The aim of study was to analyze age - related features of the immune status associated with the development of AH in employees of an oil production enterprise in the Perm region. Materials and methods. 150 oil production operators exposed to chronic exposure to harmful production factors were examined. The observation group consisted of 67 people over 40 years of age with more than 10 years of work experience who had episodes of increased blood pressure (BP). The comparison group consisted of 73 men under 45 years of age with less than 10 years of experience and no cardiovascular pathology. Determination of the total IgE, IgG to benzene, and VEGF content was performed by the ELISA method. The content of CD3+CD25+, CD127-, CD3+CD95+, and Bax was determined by flow cytometry. Phagocytic activity of white blood cells was studied using formalized sheep red blood cells. The relationship between age and seniority with the development of episodes of increased blood pressure was evaluated using the odds - OR ratio indicator. Statistical processing of the results was performed using the Statistica 6.0 software package. Differences between groups were considered significant at p<0.05. Results. Episodes of increased blood pressure in employees of an oil production enterprise in the Perm region are significantly associated with work experience of more than 10 years when they reach the age of 40 years (p<0.05). The observation group is characterized by an increased (p<0.05) level of General (IgE General) and specific (IgG to benzene) sensitization. A decrease (p<0.05) in the CD3+CD25+ content relative to the comparison group was found against the background of an increase (p<0.05) in the level of regulatory CD127 lymphocytes. Episodes of increased blood pressure in the observation group are associated (p<0.05) with a reduced (p<0.05) level of the Bax protein and CD95+-receptor of lymphocytes relative to the comparison group. At the same time, signs of phagocytosis activation (p<0.05) were established according to the criteria of phagocytosis percentage, phagocytic number, and absolute phagocytosis relative to the comparison group. Conclusions. Detected changes in immune regulation: an increase in the level of General (IgE General) and specific (IgG to benzene) sensitization, suppression of the immune response (CD3+CD25+, CD127-) and apoptosis (CD3+CD95+, Bax) against the background of phagocytosis activation characterize the development of hypertension in employees of an oil production enterprise older than 40 years with more than 10 years of experience in this industry.

Antibody Response and Haematological Changes of Nigerian Locally Adapted Turkeys to Sheep Red Blood Cells (SRBC)

Haemagglutination assay and haematological analysis of 143 poults generated as F1 individuals by artificial insemination from randomly selected turkeys of White, Black and Lavender genotypes which are classified by antibody titre was carried out so as to confirm their antibody titre levels in response to sheep red blood cells (SRBC). Results showed that mean values obtained for high and low antibody titres were 7.31 and 2.67 respectively, resulting in the classification of the turkeys into Black high and low, Lavender high and black, and White high and low antibody titres. The genotype’s titre had significant (P <0.05) effect on the packed cell volume (PCV), haemoglobin (Hb), red blood cells (RBC), white blood cells (WBC) and basophil (BAS) of the basal haematological parameters. Genotype’s titre had no significant (P >0.05) effect on the primary response haematological parameters. Meanwhile, the primary response haematological parameters to SRBC antigen varied along the genotypes with the WBC increasing drastically in all the genotypes, signifying the presence of an antigen. The study concluded that the F1 turkey poult population studied diverged along the high and low antibody titre in response to SRBC. Thus, the F1 generation of the high antibody titre genotypes (Black high, White high and Lavender high) can be used as foundation stock for selection of local turkeys for high antibody titre.

Evaluation of cottonseed bioactive peptides on growth performance, carcase traits, immunity, total antioxidant activity of serum and intestinal morphology in broiler chickens

The objective of the present experiment was to evaluate the effect of bioactive peptides derived from cottonseed (BPC) on chicken performance, immunity, the total antioxidant activity of serum and intestinal morphology. A total of 280 one-day-old male broiler chicks (Ross 308) were randomly allocated into 1 of the following 7 experimental treatments (5 replicates per treatment with 8 broilers per pen). Five diets were formulated to contain 0 (control), 3, 4, 5 and 6 g BPC/kg of diet in comparison with control + 50 U excessive dietary vitamin E and control + 2 mg lincomycin. At 40 d the BW tended to improve in broilers supplemented with an antibiotic, 3, 5 and 6 g BPC/kg groups (p > .05). In the whole trial, supplementing 5 g BPC/kg increased feed intake of broilers in comparison to other groups (p < .05). In the whole trial broilers fed diets supplemented with 6 g BPC/kg had a significantly better FCR value (p < .001). Supplementation of 3 g BPC/kg increased antibody titres against Newcastle disease virus and sheep red blood cell (p < .01). Dietary supplementation of vitamin E, antibiotic, 3, 4 and 5 g BPC/kg significantly (p < .001) increased total antioxidant activity of serum compared with those fed the basal diet. In conclusion, the results indicated that supplementation of 6 g BPC/kg in broiler diets could induce favourable influences on growth performance, immune responses and total antioxidant activity of serum and it could be used in broiler diets as an alternative to antibiotics. HIGHLIGHTS Broilers fed diets supplemented with 6 g bioactive peptides (BPC)/kg had better FCR. Supplementation of BPC increased humoral immune responses.

Evaluation of various levels of sweet almond meal as a source of protein on the production variables and immune response of broiler chickens.

This study was conducted to evaluate effects of a sweet almond meal (SAM) as a source of protein on performance and immune responses of broiler chickens. Treatments consisted of different levels of SAM (7%, 14%, 21% and 28% of diet) and control diet that was performed in a completely randomized design in 42 days. Body weight gain (BWG) and feed intake (FI) were recorded weekly. Carcass characteristics and blood variables were measured on the 42 day of age. Immune response to sheep red blood cell (SRBC) was assessed at d 35 and d 42 (7 days after each injection). White blood cell count was done at d 28. The results showed that chickens fed 28% of SAM had lowest FI during the experiment. During grower period and also whole period of experiment, BWG was highest in chickens that consumed 7% SAM. Feed conversion ratio (FCR) significantly increased in chickens fed 28% SAM compared to the other treatments during grower period and also whole period of the experiment. The relative weight of thigh and breast significantly decreased and the relative weight of GIT increased by increasing levels of SAM in the diet. The concentration of cholesterol and LDL significantly decreased in chickens that consumed SAM in the diet. However, the highest glucose and HDL concentration were observed in chickens fed 28% SAM in the diet. Treatments did not have a significant effect on the immune response of broilers (primary and secondary SRBC antibody titres, relative weights of spleen and bursa of fabricius and white blood cell count) (p > 0.05). The results of this study show that a diet supplemented with SAM at the level of 14%, due to the improved FCR and decreased blood cholesterol and LDL concentration, can be a good replacement for soybean meal in broiler chicken's nutrition.

The Loss of Kdm6a in B-Cell Development Causes Germinal Center Hyperplasia and Impedes the B-Cell Immune Response in a Specific Manner

Putative loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell malignancies, including B-cell non-Hodgkin lymphoma. How the KDM6A in normal B cell development and function, as well as the mechanism(s) by which its loss contributes lymphomagenesis has not been defined. To address this issue, we generated a conditional knockout mouse of the Kdm6a gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate Kdm6a in hematopoietic stem/progenitor cells. Our previous data have shown young Kdm6a-null mice have a myeloid skewing in the bone marrow, spleen and peripheral blood. These changes became more pronounced with age and were specific to the female, homozygous Kdm6a knockout mice. Early B-cell development is also altered in female Kdm6a-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female Kdm6a-null mice bone marrow. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D. The GC B-cells are thought to be the cell-of-origin of diffuse large B-cell lymphoma (DLBCL). To determine if the loss of Kmd6a could impact the mature B cells undergo germinal center (GC) reaction, we immunized the young, female Kdm6a-null mcie and wildtype littermates with T cell-dependent antigen sheep red blood cell (SRBC). Mice were scrificed 14 days after immunization, spleen cells were examined by flow cytometry. As expected, we observed a significant increase in the percentage of GC B cells (B220+GL7+CD95+) from female Kdm6a-null mice compared to control mice. We also observed differences in the percentage of other B-cell subsets between these mice, including an increase in plasma cells (B220-CD138+) and memory B cells (B220+CD19+CD27+), concomitant with an increase trend towards the elevated marginal zone B cells (B220+CD23loCD21+) and transitional B cells (B220+CD23-CD21-). In contrast, there was a decrease in the follicular zone B cells (B220+CD23-CD21-) and plasmablast (B220+CD138+). To analyze the levels of SRBC-specific Abs from immunized mice, serum was collected from blood at day 14. A flow cytometry-based assay was performed to detect the fluorescent-labeled SRBC-specfic Abs for immunoglobulin. Results showed that the abundance of non-class-switched anti-SRBC IgM level was significantly increased in female Kdm6a-null mice serum compared with control mice. In contrast, these mice had significantly decreased anti-SRBC IgA, IgG, IgG1, IgG3 and IgE levels indicating a isotype class switch defect. The aberrant GC phenotype induced by SRBC indeicated that kdm6a loss results in expansion of GC B cells, which subsequently enhances the plasma cell generation. This finding prompted us to investigate if the Kdm6a impairs the immunoglobulin affinity maturation. Therefore, we analyzed the ability of female Kdm6a-null mice and wildtype littermates to generate specific Abs against another T cell-dependent antigen NP-Chicken Gamma Globulin (NP-CGG). Mice were immunized with NP-CGG (29) and serum were collected weekly up to 8 weeks total. ELISA analysis of serum revealed that NP-specfic total Ig level were similar for both groups of mice over time. However, consistent with the SRBC immunization results, we did observed a sinificant reduction in the titers of NP-specific IgA and IgG1 Abs in female Kdm6a-null mice compared with control mice at each time point, while these mice had a sinificant increase in NP-specific IgM Abs, which indicating the loss of Kdm6a disrupts the balance between non-class-switched and class-switched NP-specific Abs isotypes (Figure 1A-D). Likewise, we also observed an increase in the percentage of GC B cells and plasma cells 8 weeks after NP-CGG immunization by flow cytometry. Again, our findings indicate the loss of Kdm6a causes germinal center hyperplasia, enhances plasma cell differentiation, and likely impairs class switch recombination (CSR). Taken together, our data shows that Kdm6a plays an important, but complex, role in B-cell transiting in the GC reaction and that loss of Kdm6a causes germinal center hyperplasia and impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies. Disclosures Wartman: Novartis: Consultancy; Incyte: Consultancy.

NSD2-E1099K Mutation Leads to Glucocorticoid-Resistant B Cell Lymphocytic Leukemia in Mice

Background: NSD2 (nuclear receptor binding SET domain protein 2) is a histone methyltransferase specific for dimethylation of histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. In pediatric acute lymphoblastic leukemia (ALL), particularly at relapse, a gain of function mutation (E1099K) of NSD2 is found in 10-15% of cases. The NSD2 mutation is found in addition to fusion proteins such as E2A-PBX and ETV6-RUNX1. The mutation can be subclonal at diagnosis and dominant at relapse, suggesting a link to therapeutic resistance. The NSD2-E1099K mutation affects gene expression through an increase in H3K36me2 and a decrease in H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines, we found that NSD2-E1099K mutation drove oncogenic programming by altering chromatin architecture, gene expression and enhancing cell growth, migration and infiltration to the central neural system (CNS). NSD2 mutation caused resistance of ALL cells to glucocorticoids (GC) by blocking genome wide binding of the glucocorticoid receptor (GR, encoded by NR3C1 gene) preventing GC-mediated induction of pro-apoptotic genes. NR3C1 levels were depressed in NSD2-E1099K cells and GC failed to induce autoactivation of NR3C1. While H3K27me3 was globally decreased by NSD2-E1099K, increased H3K27me3 was noted at the promoter of NR3C1, suggesting a novel role of polycomb repressive complex 2 as a therapeutic target for relapsed ALL with NSD2 mutation. While NSD2 is highly expressed in B cells and NSD2 knockout causes defects in B cell development, how the NSD2 mutation affects B cell development and leukemia occurrence in vivo is uncertain. Aims: To determine the role of NSD2 mutation in the pathogenesis of lymphocytic malignancies and GC resistance in a mouse model. Methods: We generated a conditional NSD2-E1099K knock-in mouse model in which the NSD2-E1099K allele was placed in the Rosa26 locus and expressed in B cells under the control of Cd19-Cre (Cd19+/-NSD2E1099K/WT). The resulting phenotype was characterized through peripheral blood counts, cellular morphology and histology of blood smears, bone marrow (BM), spleen and liver, flow cytometric analysis, germinal center B cells (GCB) immunization, BM transplantation, and hematopoiesis analysis in a CD3-/- background. We further established mouse leukemia cell lines with NSD2 mutation for functional analysis. RNA-Seq, real time PCR, immunoblotting, and apoptosis analysis (Annexin V/PI staining) following GC treatment were performed to demonstrate the effects of NSD2 mutation on histone modifications, transcriptome and GC resistance. Results: The NSD2-E1099K mutation increased H3K36me2 and decreased H3K27me3 in isolated B cells from mouse BM and spleen. Mice were aged and did not develop signs of malignancy and RNA-sequencing showed few differences between B cells with or without the NSD2 mutation. However, after immunizing the mice with sheep red blood cells (SRBC), more GCBs were seen in the spleen of NSD2 mutant mice, suggesting mutant NSD2 stimulated germinal center hyperplasia. Transplantation of BM cells from mice expressing NSD2-E1099K into lethally irradiated recipients lead to an expansion of B cells while myeloid and T cells and life span of the recipients impaired. The NSD2 knock-in mouse model was crossed with Cd3-/- mice to create Cd19+/-Cd3-/-NSD2E1099K/WT mice, which within 2 months of birth developed a disease resembling an immature B lymphocytic leukemia (B220+CD19+IgM+IgD-CD5-) with infiltration of the spleen, liver and CNS and a median survival of 4.8 months. These tumors could be transplanted into immunodeficient mice but not immunocompetent mice. RNA seq analysis of these cells revealed 6,815 genes (3,295 upregulated and 3,520 downregulated) differentially expressed in NSD2 mutant B cells compared to normal B cells. The upregulated genes were related to abnormal immunoglobulin level , B cell activation, T-helper 1 physiology, and decreased B cell apoptosis. Importantly, the NSD2 mutant leukemic cells displayed depressed level of NR3C1 gene expression and GC resistance. Conclusions: The NSD2 mutation alters B cell development, particularly in an immunodeficient background and causes B cells to become resistant to glucocorticoids. The inability of the mutation to generate disease on its own except in an immunodeficient background suggests genes that collaborate with NSD2 in ALL may play a role in immune escape. Disclosures No relevant conflicts of interest to declare.

Incomplete Complement Inhibition in Patients with PNH on Eculizumab - 5 Year Experience from the National PNH Service Leeds

Background Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired disorder characterised by intravascular hemolysis and thrombosis. Patients with symptomatic PNH are commenced on the complement inhibitor eculizumab (600mg weekly for 5 weeks then 900mg 2 weekly). This monoclonal antibody targets C5 in the complement cascade, halting terminal complement activation thus inhibiting intravascular hemolysis. In some patients intravascular hemolysis is not adequately controlled on the standard regimen. Patient symptoms, transfusion requirements and raised Lactate dehydrogenase (LDH) levels are indicators for suboptimal control of PNH and review of eculizumab dosing. The 50% hemolytic complement (CH50) test is a functional assay assessing capability of serum complement components of the classical pathway to lyse sheep red blood cells pre-coated with rabbit anti-sheep red blood cell antibody. Patients with complement inhibited PNH should demonstrate absent lysis. As the test is expensive and difficult to organise, we tested if incomplete complete blockade as determined by CH50 activity would be better to confirm under-dosing than LDH value. Methods The Leeds (UK) PNH National Service reviewed patients who underwent CH50 assay between January 2015 and March 2020. All patients were on eculizumab with clinical concerns regarding suboptimal control of PNH. Patients receive eculizumab infusions intravenously every 14 days and routine follow up from the PNH Service. Serum samples were obtained 24 hours prior to infusions for CH50 assay; LDH values were routinely collected. Complete complement blockade was defined by &amp;lt;10% CH50 activity; intravascular hemolysis was indicated by LDH value &amp;gt;1.5x upper limit of normal (ULN). Confidence intervals were set at 95% and significance set at p&amp;lt;0.05. Results In the study period, 327 tests (median 2, range 1 - 8) were carried out in 146 patients (median age 54 years, range 16 - 89; 74 female). 81% (265) were successful; 19% (62) were unsuccessful due to processing errors. Of the successful tests, 74% (197 in 127 patients) indicated complete complement blockade and 26% (68 in 38 patients) indicated incomplete blockade. Of the patients with incomplete blockade, 68% (26) demonstrated complete blockade on repeat testing and 32% (12) had their eculizumab dose increased. Clinical symptoms of under-dosing in the 12 patients requiring a dose increase included increased transfusion requirements and/or breakthrough hemolysis (7), pregnancy (2; both returned to 900mg post pregnancy) and significant lethargy (3). Of the patients requiring a dose increase, 3 were on 1200mg before 2015; their dose was increased to 1500mg. Repeat testing was carried out in 10/12 patients after dose increase; 8 indicated complete blockade; 2 patients were incompletely blocked at 1200mg and received a further dose increase to 1500mg. Further testing indicated complete blockade in 1 patient; 1 required a 3rd dose increase to 1800mg due to incomplete blockade and ongoing transfusion requirement. Corresponding LDH values were analysed; median LDH for the complete blockade group was 1.16xULN (range 0.54 - 2.16) and 1.28xULN (range 0.76 - 2.38) for the incomplete blockade group. LDH values were not significantly higher in the incomplete blockade group compared to the complete blockade group, p=0.08. There was no significant difference in LDH values pre- and post-dose increase, p=0.38 (Figure 1); median pre-dose increase LDH 1.14xULN; median post-dose increase LDH 1.13xULN. Correlation coefficient shows that CH50 activity was positively correlated with LDH value, r(123)=0.18, p=0.04. Conclusion We report the effective utilisation of CH50 analysis where there is clinical concern of suboptimal control of PNH. All patients demonstrating hemolytic activity on CH50 assays indicated subsequent complement blockade following increase of eculizumab dose. Increasing eculizumab is costly requiring robust evidence of suboptimal complement inhibition; a positive correlation between CH50 activity and LDH values was shown however this is not sufficient to guide clinical decisions. LDH values of the incomplete blockade group were not significantly higher than those with complete blockade, suggesting the use of LDH values as an assessment of complement inhibition in patients with ongoing symptoms or transfusion requirements is not sufficient to guide eculizumab dose increases. Disclosures Munir: Alexion: Honoraria; F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Pike:Apellis: Research Funding. Arnold:Alexion Pharmaceuticals: Honoraria. Hillmen:AstraZeneca: Consultancy, Speakers Bureau; Alexion: Consultancy, Research Funding, Speakers Bureau; Acerta: Other: Financial or material support; Roche: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; Pharmacyclics: Other: Financial or material support, Research Funding; Janssen: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; Apellis: Consultancy, Research Funding, Speakers Bureau; Gilead: Other: Financial or material support, Research Funding. Griffin:Alexion Pharmaceuticals: Honoraria, Other: Conference Support; Biocryst: Membership on an entity's Board of Directors or advisory committees.

A Novel pH Dependent C5 Monoclonal Antibody with Better Developability

Introduction: Dysregulation of complement system is the driver of many immune disorder diseases, including paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and etc. All pathways of complement activation converge at the cleavage of C5, which leads to the generation of membrane attack complex (MAC). The approval of Eculizumab, anti-C5 monoclonal antibody, opened the complement-inhibitor era and proved C5 to be a feasible therapeutic target with manageable risk. Eculizumab and Crovalimab are currently approved for treatment of patients with PNH, yet treatment limitations include hemolysis breakthrough for non-sufficient C5 inhibition, lack of efficacy in patients with C5 mutational variants, and the burden of regular intravenous infusions. The next-generation C5 inhibitor emerges as a new/existing C5 monoclonal antibody engineered to have pH dependence (to reduce dosing frequency), and other new approached to target C5 in complement system. Here we report the results of a novel pH dependent C5 monoclonal antibody-LP-005, to prevent the hemolysis of red blood cells (RBCs) from PNH patients in vitro experiment. Methods: The sequence of Ravulizumab and Crovalimab were obtained from INN. LP-005 was generated using hybridoma method and multidimentionally optimized in variable and content regions. All antibodies were expressed by NHK293 cells. For antibody affinity test, Fortebio Octet K2 was used, different C5 antibodies were first loaded on the AHC sensors and serial diluted recombinant human C5 have been run for global-fit analysis. For antibody biological function test, antibody-sensitized sheep RBCs hemolysis (classic pathway/CP activity) inhibition assay have been run in 1% normal human serum (NHS) with antibodies serial diluted by GVB++ buffer. Antibody-sensitized chicken RBC hemolysis (CP activity) inhibition assay have been run in 6.25% NHS with antibodies serial diluted by GVB++ buffer. Ex vivo inhibition of PNH patients (with more than 70% CD59 deficiency) RBCs hemolysis by anti-C5 antibodies was performed using ABO blood group-compatible serum (20%) under conditions of robust alternative pathway (AP) specific activation (GVB0 + MgEGTA, pH7.3). CD59 deficiency on RBCs of PNH patients has been analyzed by flow cytometry using APC-conjugated anti-human CD59 (negative) and PE-conjugated anti-human CD47 antibodies (positive). Antibody stability test was conducted, antibodies were treated at 66℃ and 67.5℃ for 1hr, and C5 binding activity was then tested using by ELISA. Results: In the present study, we report a novel humanized anti-C5 monoclonal antibody, named LP-005. LP-005, Ravulizumab and Crovalimab exhibits different binding kinetics to C5 (Table 1), however, there is no clear correlation between their binding affinity and complement inhibition activity (NHS-induced CP and AP hemolysis). In antibody-sensitized sheep RBCs hemolysis inhibition assay (CP), LP-005 shows an IC50 of 0.41 nM, whereas Ravulizumab and Crovalimab has IC50 of 0.99 nM and 0.4 nM respectively (Figure 1). For AP induced helmolysis assay, PNH patient's RBCs with more than 70% CD59 absence was used, however, little difference was found between LP-005 and Ravulizumab hemolysis inhibition activity. Moreover, Crovalimab showed incomplete and less efficient inhibition compared with Ravulizumab and LP-005 (Figure 2). The thermal stability study showed that LP-005 is comparable to Crovalimab and is more stable than Ravulizumab (Figure 3). Conclusion: In summary, LP-005 is a novel anti-C5 monoclonal antibody with higher bioactivity in CP induced hemolysis compared to Ravulizumab, and comparable activity in AP induced hemolysis using PNH patient's RBCs. LP-005 is also engineered to increase FcRn binding property as well as thermal stability, which has the potential to be a good drug candidate with improved pharmacokinetic properties of decreasing doses frequency during clinical practice as well as a more robust manufacture process during drug development. Disclosures He: Celgene: Research Funding; furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland: Other: Medical writing support; Celgene, Novartis, Amgen, Ra Pharma, Alexion, Achilleon, Biocryst, Akari, F. Hoffmann-La Roche: Speakers Bureau; Celgene, Novartis, Amgen, Ra Pharma, Alexion, Achilleon, Biocryst, Akari, F. Hoffmann-La Roche: Honoraria; Celgene, Novartis, Amgen, Ra Pharma, Alexion, Achilleon, Biocryst, Akari, Apellis, F. Hoffmann-La Roche: Consultancy.

Comparative evaluation of immune-responsiveness in indigenous and exotic breeds of chicken

In this research we conducted a comparative study of immune responsive traits for the two exotic chicken breeds viz. Australorp and Cornish with one of the indigenous Kadaknath chicken breeds. Immune response to Sheep Red Blood Cells (SRBC) was measured in terms of Haemagglutination Titre (HA) and Haemagglutination Inhibition titre (HI) at five stages viz; 0 (pre inoculation), 3rd , 6th , 9th and 12th days post primary inoculation (PPI) of SRBC. The HA titre was significantly (P

The composition of peripheral immunocompetent cell subpopulations and cytokine content in the brain structures of mutant Disc1-Q31L mice.

The DISC1 (disrupted in sсhizophrenia 1) gene is associated with brain dysfunctions, which are involved in a variety of mental disorders, such as schizophrenia, depression and bipolar disorder. This is the first study to examine the immune parameters in Disc1-Q31L mice with a point mutation in the second exon of the DISC1 gene compared to mice of the C57BL/6NCrl strain (WT, wild type). A flow cytometry assay has shown that intact Disc1- Q31L mice differ from the WT strain by an increase in the percentage of CD3+ T cells, CD3+CD4+ Т helper cells and CD3+CD4+CD25+ T regulatory cells and a decrease in CD3+CD8+ T cytotoxic/suppressor cells in the peripheral blood. A multiplex analysis revealed differences in the content of cytokines in the brain structures of Disc1-Q31L mice compared to WT mice. The content of pro-inflammatory cytokines was increased in the frontal cortex (IL-6, IL- 17 and IFNγ) and striatum (IFNγ), and decreased in the hippocampus and hypothalamus. At the same time, the levels of IL-1β were decreased in all structures being examined. In addition, the content of anti-inflammatory cytokines IL-4 was increased in the frontal cortex, while IL-10 amount was decreased in the hippocampus. Immune response to sheep red blood cells analyzed by the number of antibody-forming cells in the spleen was higher in Disc1-Q31L mice at the peak of the reaction than in WT mice. Thus, Disc1-Q31L mice are characterized by changes in the pattern of cytokines in the brain structures, an amplification of the peripheral T-cell link with an increase in the content of the subpopulations of CD3+CD4+ T helpers and CD3+CD4+CD25+ T regulatory cells, as well as elevated immune reactivity to antigen in the spleen.

The Selenoprotein MsrB1 Instructs Dendritic Cells to Induce T-Helper 1 Immune Responses.

Immune activation associates with the intracellular generation of reactive oxygen species (ROS). To elicit effective immune responses, ROS levels must be balanced. Emerging evidence shows that ROS-mediated signal transduction can be regulated by selenoproteins such as methionine sulfoxide reductase B1 (MsrB1). However, how the selenoprotein shapes immunity remains poorly understood. Here, we demonstrated that MsrB1 plays a crucial role in the ability of dendritic cells (DCs) to provide the antigen presentation and costimulation that are needed for cluster of differentiation antigen four (CD4) T-cell priming in mice. We found that MsrB1 regulated signal transducer and activator of transcription-6 (STAT6) phosphorylation in DCs. Moreover, both in vitro and in vivo, MsrB1 potentiated the lipopolysaccharide (LPS)-induced Interleukin-12 (IL-12) production by DCs and drove T-helper 1 (Th1) differentiation after immunization. We propose that MsrB1 activates the STAT6 pathway in DCs, thereby inducing the DC maturation and IL-12 production that promotes Th1 differentiation. Additionally, we showed that MsrB1 promoted follicular helper T-cell (Tfh) differentiation when mice were immunized with sheep red blood cells. This study unveils as yet unappreciated roles of the MsrB1 selenoprotein in the innate control of adaptive immunity. Targeting MsrB1 may have therapeutic potential in terms of controlling immune reactions.

Sex-based trade-offs in the innate and acquired immune systems of Sternotherus minor.

Longevity patterns in most vertebrates suggest that females benefit most from maintenance investment. A reversed longevity pattern in loggerhead musk turtles (Sternotherus minor) allowed us to test trade-offs between maintenance and survivorship. We tested the hypothesis that the sex with greater longevity has greater maintenance than the sex with shorter longevity. We also compared the following parameters between sexes: Bactericidal ability (BA) and heterophil:lymphocyte ratios (HLR). Baseline blood samples were collected from turtles in the field; a subset of turtles was returned to a laboratory for experiments of acquired immune responses to sheep red blood cells (SRBC). We found no support for the original hypothesis of reversal in sex-dependent immune trade-offs (difference between sex SRBC titers: p = .102; interaction between treatment and sex: p = .177; difference between treatments: p < .001; effect of sex on BA: p = .830; effect of sex on HLR: p = .717). However, we did find support for sex-dependent differences in immunity in the relationship between HLR and body condition (BCI) (effect of BCI on HLR: p = .015). In field conditions, we found that males with higher body condition indices express stressed phenotypes more than males with lower body condition indices (p = .002). However, females expressed similar stress loads across all body conditions (p = .900). Testosterone concentrations were assayed in free-living turtles and were not related to any of the immune parameters. Our results suggest that the immune systems play an important role in balancing sex-specific responses to different selective pressures in S. minor.