Sheep Red Blood Cells Research Articles

Eliminating extracellular autoinducing peptide signals inhibits the Staphylococcus aureus quorum sensing agr system

An accessory gene regulator (agr) in the quorum sensing (QS) system in Staphylococcus aureus contributes to host infection, virulence factor production, and resistance to oxidative damage. Artificially maintaining the inactive state of agr QS impedes the host infection strategy of S. aureus and inhibits toxin production. The QS system performs intercellular signal transduction, which is activated by the mature autoinducer peptide (AIP). It is released from cells after AgrD peptide processing as an intercellular signal associated with increased bacterial cell density. This study evaluated the effectiveness of inhibiting agr QS wherein AIP trap carriers were made to coexist when culturing Staphylococcus aureus. Immersing a nitrocellulose (NC) membrane in Staphylococcus aureus ATCC 12600 culture inhibited QS-dependent α-hemolysin production, which significantly reduced the hemolysis ratio of sheep red blood cells by the culture supernatant. A quartz crystal microbalance analysis supported AIP adsorption onto the NC membrane. Adding the NC membrane during culture was found to maintain the expression levels of the agr QS gene agrA and α-hemolysin gene hla lower than that when it was not added. Eliminating extracellular AIP signals allowed agr QS to remain inactive and prevented QS-dependent α-hemolysin expression. Isolating intercellular signals secreted outside the cell is an effective strategy to suppress gene expression in bacterial cells that collaborate via intercellular signaling.

Immunoregulatory and antioxidant properties of tryptophan in quail chick

A dose-response assay was carried out to investigate the effects of graded levels of dietary tryptophan (Trp) on blood variables, immunity, and meat quality in quail chicks during the last two weeks of the growing period. A total of 420 21-day-old quail chicks were randomly distributed across the seven experimental groups (i.e., 2.12, 2.25, 2.38, 2.51, 2.64, 2.77, and 2.90 g Trp/kg of diet) with four pen replicates of 15 birds each. Blood variables, including uric acid (UA), albumin (ALB), and triglycerides (TG), responded inversely to increasing dietary Trp (P < 0.001). The concentration of aspartate aminotransferase (AST) in serum, the relative weight of bursa of Fabricius (BF), immunoglobulin G (IgG), water holding capacity (WHC), and antigen production against the sheep red blood cells (SRBC) increased with increasing dietary Trp (P < 0.001). In contrast, the concentration of malondialdehyde (MDA) and drip loss in meat samples decreased with increasing dietary Trp (P < 0.001). The best models for optimal dietary Trp were identified based on a statistical merit basis known as the model accuracy index (δ). The estimated dietary Trp for optimizing ALP, UA, total protein (TP), TG, SRBC, IgG, BF, drip loss, WHC, and MDA were obtained at 2.347, 2.371, 2,372, 2.485, 2,691, 2.738, 2.306, 2.359, 2.247, and 2.500 g/kg of diet, respectively. Principal component analysis showed that UA, TG, IgG, and drip loss had a higher association with dietary Trp rather than other responses. Considering the high δ and eigenvalues of the models, the best estimation of dietary Trp level required for the optimization of the studied traits in quail chicks would be 2.738 g Trp/kg of diet, which was significantly higher than that recommended for the quail performance by NRC (1994).

Effects of replacing Na selenite in laying hen feed with selenized glucose on production performance, egg quality, egg selenium content, microbial population, immunological response, antioxidant enzymes, and fatty acid composition

This study aimed to explore the effects of selenized glucose (SeGlu) and Na selenite supplementation on various aspects of laying hens such as production performance, egg quality, egg Se concentration, microbial population, antioxidant enzymes activity, immunological response, and yolk fatty acid profile. Using a 2 × 2 factorial design, 168 laying hens at 27-wk of age were randomly divided into 4 treatment groups with 7 replications. Se source (Na selenite and SeGlu) and Se level (0.3 and 0.6 mg/kg) were used as treatments. When 0.3 mg SeGlu/kg was compared to 0.3 mg Na selenite/kg, the interaction findings revealed that 0.3 mg SeGlu/kg increased egg production percent and shell ash (P < 0.05). When compared to 0.3 mg Na selenite/kg, dietary supplementation with 0.3 and 0.6 mg SeGlu/kg resulted in an increase in albumen height, Haugh unit, and yolk color of fresh eggs (P < 0.05). SeGlu enhanced albumen height, Haugh unit, shell thickness (P < 0.01), albumen index, yolk share, specific gravity, shell ash (P < 0.05) of fresh eggs and shell thickness (P < 0.05) of stored eggs as compared to Na selenite. The interaction showed that 0.6 mg SeGlu/kg enhanced yolk Se concentration while decreasing malondialdehyde levels in fresh egg yolk (P < 0.05). SeGlu enhanced Se concentration in albumen and glutathione peroxidase activity in plasma (P < 0.05) as compared to Na selenite. 0.6 mg Se/kg increased lactic acid bacteria, antibody response to sheep red blood cells, and lowered ∑n-6 PUFA/ ∑n-3 PUFA ratio (P < 0.05). As a result, adding SeGlu to the feed of laying hens enhanced egg production, egg quality, egg Se concentration, fresh yolk lipid oxidation, and glutathione peroxidase enzyme activity.

Immune stimulatory effect of Nigella sativa in healthy animal models: A systematic review and meta-analysis

The immune-modulatory effects of black seeds (Nigella sativa seeds, NSS) are well documented, but the overall in vivo impact of this important natural medicinal product on immune system function has yet to be established. Here we systematically reviewed and meta-analyzed the effects of NSS on humoral [serum titers of immunoglobulins including IgG, IgM, anti-Newcastle virus disease (anti-NDV), and sheep red blood cell antigen (anti-SRBC)] and cellular immunity [total white blood cell (WBC) count and percentages of monocytes, lymphocytes, basophils, neutrophils, and eosinophils] in healthy animals. The PubMed, ScienceDirect, Web of Science, and Scopus databases were searched according to predefined eligibility criteria. Meta-analyses were performed to estimate the final effect size using RevMan software. Seventeen animal studies were eligible for analysis. For humoral immunity, the overall pooled effect size (ES) of NSS on serum titers of IgM and anti-NVD antibodies was not significantly different [mean difference (MD) 75.27, 95% CI: −44.76 to 195.30, p = 0.22 (I2 = 89%, p = 0.003), and −0.01, 95% CI: −0.27 to 0.25, p = 0.94 (I2 = 74%, p = 0.02), respectively]. However, NSS significantly increased serum titers of IgG and anti-SRBC antibodies [MD 3.30, 95% CI: 2.27 to 4.32, p = 0.00001 (I2 = 0%, p = 0.97), and 1.15, 95% CI: 0.74 to 1.56, p = 0.00001 (I2 = 0%, p = 0.43), respectively]. For cellular immunity, the ES of NSS on WBCs, monocytes, and lymphocytes were not significantly different [MD 0.29, 95% CI: −0.55 to 1.13, p = 0.50, (I2 = 14%, p = 0.32), - 0.01, 95% CI: −0.45 to 0.44, p = 0.97 (I2 = 0%, p = 0.77), and 4.73, 95% CI: −7.13 to 16.59, p = 0.43, (I2 = 99%, p = 0.00001), respectively]. In conclusion, black seeds enhance humoral immunity in healthy animals but do not affect cellular immunity.

Some productive and reproductive performance, eggshell quality, serum metabolitesand immune responses due to L-threonine supplementation in Japanese quail breeders'diet.

A dose-response experiment was conducted to evaluate the effects of graded levels of dietary digestible threonine (dThr) during the first laying cycle on productive and reproductive performance, egg qualityand immune responses of Japanese quail breeders (Coturnix coturnix japonica). Also, dThr requirements were determined based on nutrient dose-response data. A total of 450 (360 females and 90 males) 11-week-old breeders were allocated to five increment (+0.03%) levels of dThr (0.49%, 0.52%, 0.55%, 0.58%, 0.61%and 0.64%) with five replicates per treatment and 15 (12 females and three males) birds each. The experiment lasted for 12 weeks. In response to increasing dietary dThr levels, egg production, egg mass, feed efficiency, egg specific gravity, eggshell relative weight, eggshell thickness, egg fertility (EF) and immune response against sheep red blood cell (SRBC) inoculation were improved with quadratic trends and egg hatchability as set eggs was improved with linear trends. Japanese quail breeders fed a diet with 0.58% dThr concentration (threonine/lysine ratio of 59%) showed the productive performance traits, EF, eggshell qualityand immune response against SRBC inoculation in the highest values. However, feed intake, egg weight, egg albumen and yolk relative weight, egg shape index, haugh unitand egg composition were not affected by increasing dietary dThr level. Based on the broken-line regression model, the dThr requirements to optimize productive performance, eggshell quality, EFand immune response against SRBC inoculation were estimated at 159-188, 169-183, 175and 178 mg/bird per day, respectively. It is concluded, in the Japanese quail breeders during the first laying phase a daily dThr intake of 188 mg/bird, dietary dThr concentration at 0.58% (threonine/lysine ratio of 59%) is adequate for optimized productive and reproductive performance, eggshell qualityand immune responses. The estimated requirements depend on what production parameter is taken into considered for optimization.

Effects of Combinations of Dietary Vitamin C and Acetylsalicylic Acid on Growth Performance, Carcass Traits and, Serum and Immune Response Parameters in Broilers.

This study aimed to investigate the combined effects of ascorbic acid (VC) and acetylsalicylic acid (ASA) on broiler health and performance. A total of 200 Ross 308 male broilers were divided into five groups, each receiving different dietary combinations of ASA and VC (ASA: 50 or 100 mg/kg; VC: 200 or 400 mg/kg). The 42-day trial assessed parameters such as feed intake, average daily gain (ADG), feed conversion ratio (FCR), carcass characteristics, serum parameters, immune response and cecal microbial flora. The results indicate significant treatment effects on feed intake and growth performance, with a higher feed intake of ADG and FCR in treatment groups (p < 0.05). Serum lipid parameters were unaffected, but creatine kinase increased with ASA and VC intake (p < 0.05). Changes in sheep red blood cell titers and influenza antibodies were noted (p < 0.05). The combination of ASA and VC positively influenced carcass traits, reducing abdominal fat and altering the ratio of immune response organs to body weight (p < 0.05). Additionally, the cecal E. coli count decreased with treatment (p < 0.05). This study underscores the intricate interactions between ASA and VC supplementation, growth performance and carcass composition and immune response in broilers. Further research is warranted to explore dosage nuances and variations under specific stress conditions.

The effect of different levels of vitamin C on performance, carcass characteristics, digestive organs, immunity, blood parameters, liver enzymes, cecal microflora, evaluation of meat taste and fatty acid profile of breast meat of broilers

The objective of this experiment was to investigate the effect of different levels of vitamin C on performance, carcass characteristics, immunity, blood parameters, cecal microbial flora, evaluation of meat taste and fatty acid profile of Arbor Acres commercial strain broilers. In order to investigate the different levels of vitamin C, 120 one-day-old male broilers of the Arbor Acres commercial strain were used in a completely randomized design. In this way, three different levels of vitamin C (0, 250 and 50 mg/kg diet) were tested with 4 repetitions with 10 chicks in each repetition for 42 days. Average daily weight gain, average daily feed intake and feed conversion ratio were determined. At the age of 42 days, 2 chicks were randomly selected from each replication and the characteristics of carcass components, blood parameters, liver enzymes, taste evaluation and fatty acid profile of breast meat were measured. The evaluation of the immune system was evaluated by measuring the antibody titers against the Newcastle vaccine, anvian influenza and sheep red blood cells (SRBC) and were analyzed with SPSS statistical software. The results showed that in the whole period, no statistical difference was observed between the treatment groups in terms of feed consumption and weight gain and feed conversion ratio (P&gt;0.05), The effect of vitamin C on carcass and intestinal characteristics was not significant, except for the relative weight of the breast (P&gt;0.05). The effect of vitamin C on blood parameters, except for HDL and total cholesterol which were increased were not affected by experimental treatments. Liver enzymes including creatine phosphokinase and lactate dehydrogenase in broilers receiving experimental treatments were increased by higher levels of vitamin C in the diet (P&lt;0.05). The examination of the immune system of broilers showed that the comparison of the average amount of white and red blood cells, hemoglobin, HTC, MCV, MCH and MCHC was not affected by different levels of vitamin C in the diet (P&gt;0.05). However, the percentage of lymphocytes and heterophils was affected by different levels of vitamin C (P&lt;0.05). The amount of antibody titer against Newcastle virus, influenza and SRBC and organs related to the immune system was not affected by experimental treatments (P&gt;0.05).The effect of vitamin C on taste evaluation traits showed that general acceptance and chewing ability were different in all three treatment groups (P&lt;0.05), but other traits were not affected by different levels of vitamin C (P&gt;0.05). The effect of vitamin C on the fatty acid profile of breast meat showed C12:0 was unchanged in the 3 treatment groups, but C14:0, C16:0, C18:0, C18:3 decreased with increasing consumption of vitamin C. The present research suggests that use of 250 mg of vitamin C per kg of diet of broilers of the Arbor Acres strain may have potential economic benefits.

Differences in the microbiome of the small intestine of Leghorn lines divergently selected for antibody titer to sheep erythrocytes suggest roles for commensals in host humoral response.

For forty generations, two lines of White Leghorn chickens have been selected for high (HAS) or low (LAS) antibody response to a low dose injection of sheep red blood cells (SRBCs). Their gut is home to billons of microorganisms and the largest number of immune cells in the body; therefore, the objective of this experiment was to gain understanding of the ways the microbiome may influence the differential antibody response observed in these lines. We achieved this by characterizing the small intestinal microbiome of HAS and LAS chickens, determining their functional microbiome profiles, and by using machine learning to identify microbes which best differentiate HAS from LAS and associating the abundance of those microbes with host gene expression. Microbiome sequencing revealed greater diversity in LAS but statistically higher abundance of several strains, particularly those of Lactobacillus, in HAS. Enrichment of microbial metabolites implicated in immune response such as lactic acid, short chain fatty acids, amino acids, and vitamins were different between HAS and LAS. The abundance of several microbial strains corresponds to enriched host gene expression pathways related to immune response. These data provide a compelling argument that the microbiome is both likely affected by host divergent genetic selection and that it exerts influence on host antibody response by various mechanisms.

Endogenous complement-activating IgM is not required for primary antibody responses but promotes plasma cell differentiation and secondary antibody responses to a large particulate antigen in mice.

Lack of complement factor C1q of the classical pathway results in severely impaired primary antibody responses. This is a paradox because antibodies, especially IgM, are the most efficient activators of the classical pathway and very little specific IgM will be present at priming. A possible explanation would be that natural IgM, binding with low affinity to the antigen, may suffice to activate complement. In support of this, mice lacking secretory IgM have an impaired antibody response, which can be rescued by transfer of non-immune IgM. Moreover, passive administration of specific IgM together with antigen enhances the antibody response in a complement-dependent fashion. To test the idea, we have used a knock-in mouse strain (Cμ13) carrying a point mutation in the IgM heavy chain, rendering the IgM unable to activate complement. Mutant mice backcrossed to BALB/c or C57BL/6 background were primed and boosted with a low dose of sheep red blood cells. Confirming earlier data, no impairment in early, primary IgM- or IgG-responses were seen in either of the Cμ13 strains. However, in one of the mutant strains, late primary IgG responses were impaired. A more pronounced effect was observed after boost, when the IgG response, the number of germinal center B cells and antibody secreting cells as well as the opsonization of antigen were impaired in mutant mice. We conclude that complement activation by natural IgM cannot explain the role of C1q in primary antibody responses, but that endogenous, specific, wildtype IgM generated after immunization feedback-enhances the response to a booster dose of antigen. Importantly, this mechanism can only partially explain the role of complement in the generation of antibody responses because the IgG response was much lower in C3- or complement receptor 1 and 2-deficient mice than in Cμ13 mice.

Investigation of The Effect of Metal-based Medicine Arsenicum album on Humoral Immune Response in SRBC-immunized Mice.

In complementary and alternative medicinal systems, the Arsenicum album in ultra-high dilution was used in various therapeutic conditions, considering its effects on the body's immune system, including the COVID-19 pandemic. However, scientific evidence regarding its immunomodulatory effects is insufficient. The current study aimed to investigate the immunomodulatory effects of Arsenicum album in an experimental mouse model. Immunomodulatory activity of potentized dilutions of Arsenicum album i.e., 6C, 30C, 200C in BALB/c mice was evaluated by humoral antibody titer and delayed- type hypersensitivity assays wherein a fixed concentration (0.5 ml of 1× 109 cells/ml) of freshly prepared sheep RBC was administered as a foreign antigen to generate primary and secondary antibodies. Arsenicum album showed significant immunomodulatory activity by increasing primary antibody titer evaluated on day 21 of the treatment in all the dilutions as compared to SRBC and vehicle control group in humoral immune response assay without showing any effect on delayed-type hypersensitivity. The results of this preliminary study indicate that oral administration of Arsenicum album has the potential to augment primary humoral response at all dilutions. Hence, the possibility of using the Arsenicum album could be explored to treat immunological conditions, infections, etc., as an alternative therapy alongwith modern medicines.

Tissue cytokines in chickens from lines selected for high or low humoral antibody responses, given supplemental Limosilactobacillus reuteri and challenged with Histomonas meleagridis.

Histomonas meleagridis, a protozoan parasite, induces blackhead disease (histomoniasis) in poultry. During hatching, chicks from lines divergently selected for high (HAS) and low (LAS) antibody responses to sheep red blood cells were divided into two groups, each of HAS and LAS, and placed in pens with wood shavings as litter. Feed and water were allowed ad libitum. Half of the chicks from each line had Limosilactobacillus reuteri (L. reuteri) inoculated to their drinking water. On day 18, all chicks were given a transcloacal inoculation of 100,000 H. meleagridis cells. Then, 10days later, they were euthanized, followed by collection of tissues from the brain, cecal tonsil, ceca, liver, thymus, and spleen for qPCR analyses of cytokines involved in immunological development. Changes in cytokine expressions were most numerous in the cecal tonsil, ceca, and liver. In the absence of a functional medication for control of histomoniasis, L. reuteri and/or its secretory product, reuterin, might serve, in some genetic populations, as a means to reduce the impact of histomoniasis in chickens. The data demonstrate that L. reuteri treatment had tissue specificity between the two genetic lines, in which the effects were targeted primarily toward the cecal tonsil, ceca, and liver, which are the primary tissue targets of the parasite (H. meleagridis), as well as the thymus and spleen. However, interactions among main effects reflect that responses to inflammatory markers observed in tissues for one genetic line may not be observed in another.

Pathological effects of feeding aflatoxin-contaminated feed on immune status and reproductive performance of juvenile white leghorn males and its mitigation with ∝-tocopherol and Moringa oleifera.

This study was planned to detect the adverse pathological consequences of aflatoxin B1 in White Leghorn (WLH) layer breeder males. Eight-week-old male layer cockerels were separated into six experimental categories: A group was kept as negative control, offered with normal feed only; group B was fed with 400ppb amount of aflatoxin, while groups F and D fed with normal feed and supplemented with vitamin E 100ppm and 1% Moringa oleifera, respectively, whereas groups E and C were fed with 400ppb aflatoxin containing feed and ameliorated with vitamin E 100ppm and 1% Moringa oleifera, respectively. This study was continued for 2months and immunologic disorders and reproductive parameters were observed during the trial. To find out immunological status lymphoproliferative response to phytohemagglutinin-P (PHA-P), antibody titers against sheep red blood cells (SRBCs) and carbon clear assay were performed by collecting samples from five birds from each group. The whole data was measured by ANOVA test, and group means were compared by DMR test by using M-Stat C software. Regarding the reproductive status, spermatogenesis, blood testosterone level, testes weight, testes histology, sperm motility, and morphology were negatively affected by aflatoxins, but these deviations positively ameliorated by vitamin E and Moringa. Vitamin E and Moringa found advantageous in boosting the immune status of affected bird.All the immunological parameters including antibody titers against sheed red blood cells, lymphoproliferativeresponse to avian tuberculin and phagocytic potential of macrophages were suppressed by AFB1 however incontrol, Moringa and vitamin E groups these immunological responses were significantly higher.

Multi-strain probiotic improved feed conversion ratio and selected health indicators in Japanese quail*

Abstract Use of multi-strain probiotics in poultry nutrition provides advantages, including promoting microbial diversity in the birds’ gut. Less investigated, as compared to broilers, is how application of probiotics affects performance and health of Japanese quails. Up to date effects of either probiotics based on a single microorganism or using a single dose of probiotic were investigated. The aim of the study was to investigate the effect of gradual levels of multi-strain probiotic applied in the Japanese quails’ diet on their performance, carcass, microbiological and immunological parameters. Total of 240 Japanese quail mixed sex chicks were randomly selected into four treatments: 0.15, 0.10, 0.05 g/l of the probiotic and control group not provided with probiotic. Body weight gain (BWG), feed intake (FI), feed conversion ratio (FCR), carcass characteristics, selected gut microbiome species and sheep red blood cell (SRBC) immunoglobulin IgT, IgG and IgM antibodies were determined. Repeated measures ANOVA was performed on parameters related to productive performance of the birds, while one-way ANOVA on all other parameters using SAS software v 9.4. FI was the highest in the control group (16.84±2.19) and did not differ significantly between other groups (from 16.53±2.12 to 15.18±1.86). FCR was the highest in the control and the lowest dose group (3.15±0.28 and 3.07±0.27), as compared to two other groups of birds which received multi-strain probiotic (2.8±0.23 and 2.77±0.24, respectively). Birds in the control group and birds which received the lowest dose of the probiotic had lower counts of IgT (5.63±0.26 and 5.75±0.25, respectively) and IgG (4.00±0.38 and 4.13±0.3, respectively), as compared to two other treatment groups which received the multi-strain probiotic. Birds which did not receive the multi-strain probiotic had significantly lower levels of Lactobacillus (6.98±0.01), as compared to other birds, while those in the control group and which received lowest amount of probiotic had significantly higher levels of E.Coli (6.67±0.01). Multi-strain probiotic had overall positive effect on the selected parameters of Japanese quails’ performance and health, while efficacy varied depending on dosage.

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

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

SWI/SNF Chromatin Remodeling Complex Orchestrates Sequential Binding of Key Transcription Factors in B Cells and Restricts Aggressive Lymphoma

Mutations in the SWI/SNF chromatin remodeling complex BAF are a recurring feature in many cancers, with a high prevalence in follicular lymphoma (FL; &amp;gt;19%) and diffuse large B-cell lymphoma (DLBCL; &amp;gt;34%). Despite this, the mechanism that links mutations in ARID1A, most frequently mutated BAF subunit, to the development of lymphoma is still not understood. To this aim, we crossed Arid1a-floxed mice with Cγ1-Cre mice to yield offspring with a conditional (cKO) Arid1a deletion in germinal center (GC) B cells, FL and DLBCL cell-of-origin. We immunized WT/WT ( Arid1a+/+; Cγ1Cre/+) and cKO/WT ( Arid1a+/-; Cγ1Cre/+) mice with sheep red blood cells and analyzed 10 days later at the peak of the GC reaction. Upon Arid1a deletion, we found a decrease in the total number of GC B cells relative to total B cells (WT/WT vs. cKO/WT; p&amp;lt;0.001). However, we observed skewing of GC polarity manifesting as increased proportions of proliferating centroblasts (CB) vs. centrocytes (CC) (WT/WT vs. cKO/WT; p=0.004). These observations imply a substantial dysfunction at the CC stage, which could account for the overall reduced number of GC B-cells. To investigate this, we performed a detailed molecular characterization of these cells. We performed the ATAC-seq assay on sorted CB and CC from our mouse model and observed an extensive loss of chromatin accessibility. Changes in chromatin accessibility are thought to be dependent on the recruitment of the BAF complex by specific transcription factors. We discovered strong enrichment for DNA motifs of PU.1 and NF-kB factors in closing chromatin (p-value &amp;lt;0.001), indicating their dependence on the BAF complex. Furthermore, we noted that this chromatin closing occurred in proximity to canonical GC exit programs, including genes induced by the CD40, NF-kB signaling, IRF4, STAT3, IL2, IL4, IL6 and Notch pathways (hypergeometric p-value &amp;lt;0.001), indicating GC-exit perturbation. Expanding upon our findings, we leveraged a combined single-cell RNA and ATAC assay (Multiome) to scrutinize subtle shifts within GC populations. Determining the chromatin accessibility within the pseudo-time trajectory of GC transitions, we found that PU.1 and NF-kB factors are chronologically dependent on ARID1A with PU.1 accessibility initially compromised, followed by a subsequent decrease at binding sites for NF-kB factors. This sequence reveals ARID1A's unrecognized function as a regulator of temporal dynamics of these key transcription factors. Strikingly, our Multiome data revealed expansion of the pre-memory B cells accompanied by a decrease in the proportion of pre-plasma cells upon Arid1a deletion (p-value &amp;lt;0.001), suggesting that Arid1a loss favors GC exit towards the memory cell fate. To validate our findings, we performed immunophenotyping and observed an increase in memory B cells in cKO/WT mice (p=0.019), and a decrease in long-lived plasma cells (p=0.012) upon NP-KLH immunization. Furthermore, we observed that the absence of ARID1A tilts GC cell-fate towards immature IgM+CD80-PDL2- memory B cells (p=0.0016), known for their potential to re-enter new GCs. Linking this critical role of ARID1A in chromatin regulation to lymphomagenesis, we observed a reduction in overall survival for mice carrying VavP-Bcl2; Arid1a+/- allele (compared to VavP-Bcl2; Arid1a+/+; p=0.0087). Remarkably, we further show that FL patients with ARID1A-inactivating mutations display an immature memory B-cell-like state with increased transformation risk to aggressive disease (p=0.0391). These observations offer mechanistic understanding into the emergence of both indolent and aggressive lymphomas in ARID1A-mutant patients through formation of immature memory-like clonal precursors.

CTCF Is a Haploinsufficient Tumor Suppressor in B-Cell Lymphoma

Alteration of epigenetic regulators is a hallmark of diffuse large B cell lymphoma (DLBCL), the most common type of non-Hodgkin B cell malignancy. It has been recently shown that haploinsufficiency of the cohesin complex ATPase subunit Smc3 cooperates with the oncogene IμBcl6 to induce lymphoma in mice, by a mechanism that involves decreased intra-topological associating domain (TAD) chromosomal interactions and downregulation of tumor suppressor genes. The transcription factor CTCF is an obligate partner of the cohesin complex in defining TADs and regulatory gene loops, acting as the boundaries of TADs and restricting the interaction of promoters and enhancers. Alterations in CTCF have been shown in several cancer types, including lymphomas. In this study, we aim to define the role of CTCF during B-cell lymphoma and lymphomagenesis. We generated germinal center (GC) B cell-specific conditional knockout mice for CTCF by crossing the CTCF floxed mice to the Ighg1-Cre (Cγ1-Cre) allele. In these experiments, we compared haploinsufficient CTCF conditional knockout mice-in which only one allele of CTCF was knocked out-versus wild-type mice expressing the Cγ1-Cre. Eight days after immunization with the T-cell-dependent antigen sheep red blood cells, we excised spleens and surveyed B cell populations by flow cytometry and immunohistochemistry. Remarkably, CTCF haploinsufficient mice contained a similar percentage of follicular GC B cells compared to wild-type mice (Live B220 +CD95 +GL7 + or live B220 +CD95 +CD38 DIM splenocytes). However, CTCF haploinsufficient GCs displayed a bias in the GC subpopulations, namely an increased percentage of light zone centrocytes (LZ: GC + markers CXCR4 loCD86 hi) and decreased dark zone centroblasts (DZ: GC + markers CXCR4 hiCD86 lo). Because biases in GCs were an early indication of malignant transformation in previous studies, we wondered whether CTCF haploinsufficiency would cooperate with lymphoma oncogenes to induce B-cell lymphoma. To address the lymphomagenic potential of CTCF haploinsufficiency, we crossed CTCF conditional knockout mice to the vavP-Bcl2 mice that develop GC-derived follicular-like lymphoma with long latency. We harvested bone marrow blood cell precursors from Cγ1-Cre (“WT”), Cγ1-Cre;CTCF +/- (“CTCF”), vavP-BCL2 +;Cγ1-Cre (“BCL2”), and vavP-BCL2 +;Cγ1-Cre;CTCF +/- (“BCL2/CTCF”) and transplanted them into lethally irradiated C57BL6/J (30 per group). Mice that survived more than two weeks after irradiation and bone marrow transplant were considered stably engrafted and monitored for any signs of disease. Lethargic mice were sacrificed and macroscopically inspected for the presence of tumors. Survival analysis by Kaplan-Meier showed that BCL2/CTCF mice had the shortest survival from all four groups, having a median survival of 230 days, while survival for the BCL2 alone group did not fall below 50% up to day 370. Immunohistochemistry against B220 + was performed in tissue sampling from dying mice to confirm the cause of death. Curiously, CTCF haploinsufficiency accelerated BCL2-induced lymphomagenesis but did not affect the survival of IμBCL6-induced lymphoma. To explore the mechanistic basis of CTCF haploinsufficiency-induced lymphoma in mice, we FACS sorted DZ and LZ GC B-cells from 8-day immunized mice and explored global transcriptomics by RNA-sequencing. While the transition from DZ to LZ involves the regulation of around ~1,500 genes, we failed to detect changes in gene expression between wild-type and CTCF haploinsufficiency cells. ATAC-seq assay on the same cells showed little to no change in chromatin accessibility between cells from CTCF haploinsufficient and wild-type mice. TOBIAS footprinting analysis, which identifies biases in Tn5 transposase activity due to protein binding, showed that CTCF haploinsufficient cells maintained a deep depletion in CTCF binding. Hi-C chromosomal conformation analysis identified an overlapping set of gene regulatory loops that lose interactions and showed decreased signal by gene set enrichment analysis. In this study, we characterized the role of CTCF haploinsufficiency phenotypically and are currently exploring the molecular mechanisms that contribute to the acceleration of the lymphomagenesis induced by the BCL2 oncogene.

Isolation of a novel pentasaccharide Ebaliose from immunostimulant fraction of buffalo milk oligosaccharide

Buffalo milk processed by method of Kobata and Ginsburg for isolation of its oligosaccharide contents showed significant stimulation of anti-body, delayed type hypersensitivity response to sheep red blood cells in BALB/c mice. This oligosaccharide mixture also stimulated non-specific immune response in terms of MMI. In continuation to our previous studies of oligosaccharide contents of buffalo milk another novel pentasaccharide Ebaliose was isolated from immunostimulant oligosaccharide fraction of buffalo milk. Ebaliose was procured in its purest form, by combining the different chromatographic techniques like Gel filtration, Silica Gel column chromatography, and HPLC. The structure of novel oligosaccharide was determined by 1H,13C, HSQC, TOCSY, COSY, HMBC, ESI-MS, chemical transformation, and chemical degradation. The stereoscopic structure of this novel pentasaccharide was established as under.

The Genotyping Diversity and Hemolytic Activity of Cronobacter spp. Isolated from Plant-Based Food Products in Poland.

The present study aimed to determine the genotyping diversity and hemolytic properties of 24 strains of Cronobacter spp. (15 Cronobacter sakazakii, 6 Cronobacter malonaticus, 2 Cronobacter turicensis, and 1 Cronobacter condimenti) isolated from commercial ready-to-eat leaf vegetables, sprouts, nuts, and dried fruits. The multilocus sequence typing (MLST) method was used to determine the sequence types (ST) and clonal complexes (CC) of these strains. The study demonstrated the high genotypic diversity of the Cronobacter genus bacteria isolated from plant-based foods. Five novel sequence types (804, 805, 806, 807, and 808) and the presence of novel alleles in the ppsA, gltB, gyrB, and infB loci were detected. In total, 16 of the 24 strains were assigned to the sequence types ST99, ST258, ST17, ST648, ST21, ST494, and ST98. One C. sakazakii strain (s12) isolated from alfalfa sprouts was assigned to the clonal complex CC4, which encompasses strains often associated with severe infections leading to meningitis in infants. In addition, 87.5% and 16.7% of the Cronobacter spp. strains showed β-hemolysis of equine and sheep red blood cells, respectively. The presence of the pathogenic species C. sakazakii, C. malonaticus, and C. turicensis in ready-to-eat plant-derived food products shows they are potential sources of infection, especially to those with compromised immunity, which substantiates their further multi-faceted characterization. The significance of this study may prove useful not only in epidemiological investigations, but also in assessing the risk of infections caused by the presence of Cronobacter.

Immunomodulation by tramadol combined with acetaminophen or dexketoprofen: In vivo animal study

Among other functions, macrophages remove foreign particles, including medications, from the circulation, making them an important target for immunomodulatory molecules. Currently, growing evidence suggests that analgesics affect the activity of immune cells not directly related to pain, and thus may induce unwanted immunosuppression in patients at risk. However, the immunomodulatory effects resulting from macrophage targeting by these drugs are understudied. Therefore, the current study investigated the immune effects induced in healthy mice by repeated administration of tramadol alone or in combination with acetaminophen or dexketoprofen. We observed that drug administration decreased the percentage of infiltrating macrophages in favor of resident macrophages in peritoneal exudates. While all drugs reduced the number of infiltrating macrophages that phagocytosed sheep red blood cells (SRBC), their administration increased the effectiveness of phagocytosis, and treatment with acetaminophen with or without tramadol elevated the expression of MHC class II by Mac3+ macrophages. Interestingly, SRBC-pulsed macrophages from mice treated with tramadol combined with acetaminophen potently activated SRBC-specific B cells in humoral response, and administration of these drugs to recipients of contact hypersensitivity effector cells augmented the resulting cellular immune response. In addition, tramadol administered alone or with dexketoprofen enhanced the spontaneous release of pro-inflammatory cytokines by macrophages. Our current research findings demonstrate that tramadol therapy in combination with acetaminophen or dexketoprofen has a relatively low risk of causing immunosuppressive side effect because the drugs slightly reduce the inflammatory reaction of macrophages but do not impair their ability to activate the adaptive immune responses.

The effect of different levels of vitamin C and chromium on growth performance, carcass characteristics, digestive organs, immunity, blood constituents, liver enzymes, cecal microflora, meat sensory taste and fatty acid profile of breast meat in broilers.

Antioxidants such as vitamin C (VC) and chromium (Cr), which effectively scavenge free radicals, may improve functional characteristics of the intestine and may reduce intestinal diseases. Cr absorption increases in the presence of VC. In poultry, VC is mainly derived from glucose; hence, Cr is an important component for glucose tolerance. We evaluated the synergistic effects of these two antioxidants together. This study aims to investigate two levels of VC and two levels of Cr and their interaction on growth performance, carcass characteristics, digestive organs, immunity, blood constituents, liver enzymes, cecal microflora, meat sensory taste and fatty acid profile of breast meat in broilers. Two levels of VC (250 and 500mg/kg dry matter [DM]) and two levels of Cr (700 and 1400μg/kg DM) were added to a basic diet for 42 days in five treatments. The 2 × 2 plus 1 (control group) factorial experiment was performed in a completely randomised design for 42 days using 360 one-day-old male chicks. Very low-density lipoprotein, high-density lipoprotein, low-density lipoprotein, total cholesterol and triglyceride level, liver enzymes, antibody titer against sheep red blood cells, fat content, odour, chewing ability, elasticity and oral sensation traits were affected by combination of VC and Cr. Combination of VC and Cr can increase unsaturated fatty acids and decrease saturated fatty acids, as well as improve cecal microbial flora, and may be useful as antioxidant compounds and non-antimicrobial stimulants for economic growth. The use of 250mg/kg of VC and 700μg/kg of Cr is recommended in broiler diets.