Secondary Humoral Immune Response Research Articles

Evaluation of immunomodulatory effect of aqueous extract of aloe vera in Wistar albino rat models

Aim of the study wasto evaluate the immunomodulatory effects of Aqueous extracts of Aloe vera in Wistar albino rats using humoral immune response (antibody titre), a cellular immune response, and a neutrophil adhesion test after oral administration. : 24 healthy Wistar albino rats of either sex were divided into 4 different groups containing 6 rats for each immunomodulatory model. Group I ,the control group received gum acacia suspended in normal saline; Group II rats were treated with dexamethasone (1 mg/kg bw)whereas Groups III & IV received AloeVera aqueous extract at a dose of 125 mg/kg and 250 mg/kg, respectively. Each rat was antigenically challenged by injecting 0.1 ml of 0.5 × 10 SRBCs (suspended in normal saline) intraperitoneally. The study of humoral immune response was seen by measurement of antibody titre obtained on 20 day (7 day of Ag challenge) and 27 day (14 day of challenge) with sRBC considered the primary and secondary humoral immune response, respectively. For the cellular immune response, foot pad edema was calculated due to a hypersensitivity reaction after injection of sRBC into the .rat hind paw. A test of neutrophil adhesion is used to evaluate immunomodulatory activity. Aloe vera aqueous extracts at 250 mg/kg significantly improved a significant increase in paw edema volume indicated increased cell-mediated immunity, and elevated Ab titre on the 20th and 27th days served as a marker of increased humoral immune response. Additionally, AVE 250 mg/kg caused an increase in neutrophil adhesion, demonstrating its immunomodulatory effects. Aloe vera aqueous extracts at a dose of 250 mg/kg demonstrated immunomodulatory activity in a model using Wistar albino rats.Aloe vera extract at a dose of 250 mg/kg significantly increased hemagglutination antibody titers compared to the control group, indicating enhanced humoral immunity. This study demonstrates the immunostimulant properties of orally administered Aloe vera aqueous extract in Wistar albino rats. The extract increased antibody production, enhanced cell-mediated immunity, and improved neutrophil function. These findings support the potential of Aloe vera as a natural immunomodulator and warrant further exploration of its therapeutic applications.

POS0185 BELIMUMAB DISRUPTS MEMORY B-CELL TRAFFICKING IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

BackgroundBelimumab (BEL), a recombinant human monoclonal antibody directed against B-cell activating factor (BAFF), is the first approved biological agent for patients with active systemic lupus erythematosus (SLE) and lupus nephritis (LN).1 BEL inhibits primary humoral immune responses by depleting naïve B cells that are dependent on BAFF for their survival while secondary humoral immune responses by memory B cells (MBCs) remain intact. Indeed, some studies reported an increase of circulating MBCs following neutralisation of BAFF.2-4 So far, these effects of BEL on the MBC compartment in SLE patients have not been investigated.ObjectivesThis study aimed to establish the dynamics of circulating MBCs in patients with SLE treated with BEL and to perform an in-depth analysis of the impact of BEL on the MBC compartment.MethodsFirst, a retrospective meta-analysis was performed by pooling individual patient MBC flow cytometry data from 1245 patients with SLE treated with BEL 10 mg/kg IV or placebo (PBO) from four randomised clinical trials (NCT00071487, NCT00410384 [BLISS-76],3NCT01632241 [EMBRACE],5NCT01649765 [PLUTO]6). Second, extensive B-cell subset phenotyping was performed prospectively by employing high-sensitivity flow cytometry (HSFC) based on EuroFlow protocols7 in patients with active SLE (from the BLISS-BELIEVE trial [NCT03312907])8 and with severe SLE/LN (from the SynBioSe-2 trial [NCT03747159])9 treated with BEL. Additionally, in-depth characterisation of surging MBCs in circulation was performed by single-cell RNA sequencing (scRNA-seq).ResultsBy comparing BEL-treated with PBO-treated patients with SLE, a substantial increase in circulating MBC counts was established 4 weeks after BEL initiation, gradually returning to baseline by Week 52. The increase of MBCs was most prominent in BEL-treated patients with higher SLE disease activity (SLE Disease Activity Index >9), serologically active patients (dsDNA positive and/or low complement levels) and with younger age (below 18 years). HSFC established that the increase was non-specific and observed in a broad range of MBC subclasses peaking as early as 2 weeks after BEL initiation. Subsequent scRNA-seq analysis of the emerging MBCs revealed a non-proliferating phenotype with a prominent decrease in activation status. In these circulating MBCs, a large amount of migration and adhesion genes were downregulated suggesting that the accumulation of MBCs following BEL treatment was related to their impaired cell-cell adhesion, disrupting cell-trafficking and preventing extravasation.ConclusionAfter initiation of BEL treatment, a substantial increase of circulating MBCs was firmly established and was most notable in patients with severe, serologically active SLE/LN. The surge of circulating MBCs appeared to be associated with disrupted lymphocyte trafficking of MBCs, thereby suggesting a new potential therapeutic mechanism of BEL on MBCs in SLE. These findings have important implications to our understanding and consequent improvement of B-cell targeted treatment strategies in patients with active SLE and LN, as MBC accumulation in circulation might allow for more efficient targeting of the B-cell compartment.

MO246: Belimumab Disrupts Memory B Cell Trafficking in Patients with Systemic Lupus Erythematosus

Abstract BACKGROUND AND AIMS Belimumab (BEL), a recombinant human monoclonal antibody directed against B-cell activating factor (BAFF), is the first approved biological agent for patients with systemic lupus erythematosus (SLE) and a high level of disease activity or lupus nephritis (LN). BEL inhibits primary humoral immune responses by depleting naive B-cells that are dependent on BAFF for their survival while secondary humoral immune responses by memory B cells (MBCs) remain intact. Indeed, some studies reported an increase of circulating MBCs following neutralization of BAFF [1–3]. So far these effects of BEL on the MBC compartment in SLE patients have not been investigated. This study aimed to establish the dynamics of circulating MBCs in patients with SLE treated with BEL and to perform an in-depth analysis of the impact of BEL on the MBC compartment. METHOD First, extensive B cell subset phenotyping was performed prospectively by employing high-sensitivity flow cytometry (HSFC) based on EuroFlow protocols [4] in severe SLE/LN patients treated with BEL [5]. Additionally, in-depth characterisation of surging MBCs in circulation was performed by single-cell RNA sequencing (scRNA-seq). RESULTS HSFC established that the increase in MBCs was non-specific and observed in a broad range of MBC immunoglobulin subclasses peaking as early as 2 weeks after BEL initiation. Subsequent scRNA-seq analysis of the emerging MBCs revealed a non-proliferating phenotype with a prominent decrease in activation status. In these circulating MBCs, a large amount of migration and adhesion genes were downregulated suggesting that the accumulation of MBCs by BEL initiation was related to their impaired cell–cell adhesion disrupting cell-trafficking and preventing extravasation. CONCLUSION After initiation of BEL treatment, a substantial increase of circulating MBCs was firmly established in patients with SLE/LN. The surge of circulating MBCs appeared to be associated with disrupted lymphocyte trafficking of MBCs, thereby suggesting a new potential therapeutic mechanism of BEL on MBCs in SLE. These findings have important implications to our understanding and consequent improvement of B-cell targeted treatment strategies in patients with active SLE and LN as MBC accumulation in circulation might allow for more efficient targeting of the B-cell compartment.

The immunosenescence-related factor DOCK11 is involved in secondary immune responses of B cells

BackgroundMemory B cells are an antigen-experienced B-cell population with the ability to rapidly differentiate into antibody-producing cells by recall responses. We recently found that dedicator of cytokinesis 11 (DOCK11) contributes to the expansion of antigen-specific populations among germinal center B cells upon immunization. In comparison, limited information is available on the contribution of DOCK11 to secondary humoral immune responses.ResultsIn this study, effects of the DOCK11 deficiency in B cells were examined on secondary immune responses to protein antigen. The lack of DOCK11 in B cells resulted in the impaired induction of antibody-producing cells upon secondary immunization with protein antigen. DOCK11 was dispensable for the recall responses of antigen-experienced B cells, as demonstrated by the comparable induction of antibody-producing cells in mice given transfer of antigen-experienced B cells with no DOCK11 expression. Instead, the lack of DOCK11 in B cells resulted in the impaired secondary immune responses in a B cell-extrinsic manner, which was recovered by the adoptive transfer of cognate T cells.ConclusionsWe addressed that intrinsic and extrinsic effects of DOCK11 expression in B cells may contribute to secondary humoral immune responses in manner of the induction of cognate T-cell help.

Synthesis, Physicochemical Characteristics and Plausible Mechanism of Action of an Immunosuppressive Isoxazolo[5,4-e]-1,2,4-Triazepine Derivative (RM33).

Previous studies demonstrated strong anti-inflammatory properties of isoxazolo[5,4-e]-1,2,4-triazepine (RM33) in vivo. The aim of this investigation was to describe synthesis, determine physicochemical characteristics, evaluate biological activities in murine and human in vitro models, as well as to propose mechanism of action of the compound. The compound was devoid of cell toxicity up to 100 μg/mL against a reference A549 cell line. Likewise, RM33 did not induce apoptosis in these cells. The compound stimulated concanavalin A (ConA)-induced splenocyte proliferation but did not change the secondary humoral immune response in vitro to sheep erythrocytes. Nevertheless, a low suppressive effect was registered on lipopolysaccharide (LPS)-induced splenocyte proliferation and a stronger one on tumor necrosis factor alpha (TNFα) production by rat peritoneal cells. The analysis of signaling pathways elicited by RM33 in nonstimulated resident cells and cell lines revealed changes associated with cell activation. Most importantly, we demonstrated that RM33 enhanced production of cyclooxygenase 2 in LPS-stimulated splenocytes. Based on the previous and herein presented results, we conclude that RM33 is an efficient, nontoxic immune suppressor with prevailing anti-inflammatory action. Additionally, structural studies were carried out with the use of appropriate spectral techniques in order to unequivocally confirm the structure of the RM33 molecule. Unambiguous assignment of NMR chemical shifts of carbon atoms of RM33 was conducted thanks to full detailed analysis of 1H, 13C NMR spectra and their two-dimensional (2D) variants. Comparison between theoretically predicted chemical shifts and experimental ones was also carried out. Additionally, N-deuterated isotopologue of RM33 was synthesized to eliminate potentially disturbing frequencies (such as NH, NH2 deformation vibrations) in the carbonyl region of the IR (infrared) spectrum to confirm the presence of the carbonyl group.

4-Methylpseudoproline analogues of cyclolinopeptide A: Synthesis, structural analysis and evaluation of their suppressive effects in selected immunological assays

The synthesis of new analogues of cyclolinopeptide A (CLA) and their linear precursors modified with (R)- and (S)-4-methylpseudoproline in the Pro3-Pro4 fragment are presented. The peptides were tested in comparison with cyclosporine A (CsA) in concanavalin A (Con A) and pokeweed mitogen (PWM)-induced mouse splenocyte proliferation and in secondary humoral immune response in vitro to sheep erythrocytes (SRBC). Their effects on expression of selected signaling molecules in the Jurkat T cell line were also determined. In addition, the structural features of the peptides, applying nuclear magnetic resonance and circular dichroism, were analyzed.The results showed that only peptides 7 and 8 modified with (R)-4-methylpseudoproline residue (c(Leu1-Val2-(R)-(αMe)Ser(ΨPro)3-Pro4-Phe5-Phe6-Leu7-Ile8-Ile9) and c(Leu1-Val2-Pro3-(R)-(αMe)Ser(ΨPro)4-Phe5-Phe6-Leu7-Ile8-Ile9), respectively) strongly suppressed mitogen-induced splenocyte proliferation and the humoral immune response, with peptide 8 being more potent. Likewise, peptide 8 more strongly elevated expression of Fas, a proapoptotic signaling molecule in Jurkat cells. We postulate that the increased biological activity of peptide 8, compared to the parent molecule and other studied peptides, resulted from its more flexible structure, found on the basis of both CD and NMR studies. CD and NMR spectra showed that replacement of Pro3 by (R)-(αMe)Ser(¬Pro) caused much greater conformational changes than the same replacement of the Pro4 residue. Such a modification could lead to increased conformational freedom of peptide 8, resulting in a greater ability to adopt a more compact structure, better suited to its putative receptor. In conclusion, peptide 8 is a potent immune suppressor which may find application in controlling immune disorders.

Inclusion of Hermetia illucens larvae reared on fish offal to the diet of broiler quails: Effect on immunity and caecal microbial populations

Hermetia illucens (black soldier fly, BSF) larvae meal has shown to be a good protein source in monogastric animal diets, but published data regarding its immunomodulatory properties is limited. For this purpose, a study has been conducted to evaluate the effects of larvae meal on selected immune parameters and caecal bacterial counts of broiler quails. Hermetia illucens larvae were reared on two substrates (100% chicken feed, BSF-M, or 50% chicken feed + 50% fish offal, BSF-F) in order to manipulate the fatty acid profile of larvae meal by increasing the long-chain omega-3 (n-3) polyunsaturated fatty acids which are known for their immunomodulatory properties. For immunological purposes, a total of 60 birds were randomly allocated to three dietary treatment groups (n = 20/treatment): control quail diet (CON), or quail diets including 10% of BSF-F or BSF-M larvae meal. Blood was collected 27 and 37 days after the trial commenced to determine the humoral immune response, serum lysozyme concentrations, serum bactericidal activity and protein fractions of the serum. The dietary inclusion of BSF-M meal resulted in an increased secondary humoral immune response compared to the CON treatment group (P < 0.01). Quails in the BSF-F treatment had significantly higher serum lysozyme activity compared to quails in the CON and BSF-M treatment group. Both larvae meals significantly increased cell-mediated immunity on day 37 (P < 0.001) but they had no effect on serum bactericidal activity. Both larvae meal sources resulted in higher α2-globulin levels on both sampling days, whereas γ-globulin levels were significantly lower in quails with the BSF-F treatment on day 27. Dietary treatments had no significant effect on caecal bacterial counts. In conclusion, the present study showed that the larvae meal had immunostimulatory effects in quails, but the rearing substrate of larvae strongly influenced the immunostimulatory properties of the larvae meal.

Melatonin enhances responsiveness to Dichelobacter nodosus vaccine in sheep and increases peripheral blood CD4 T lymphocytes and IgG-expressing B lymphocytes

The immunomodulatory functions mediated by melatonin support its use as vaccine adjuvant. Previously, we have demonstrated that melatonin enhances antibody responses in sheep vaccinated against Dichelobacter nodosus. Here, we analyze the effect of melatonin on T and B lymphocyte subsets in peripheral blood of sheep vaccinated against D. nodosus. We also compare the use of melatonin in implants and in injections. Melatonin administration either as implants or by injection produced higher antibody titers against A1 and C serotypes compared to those animals that received only the vaccine. These results support the use of melatonin as an adjuvant in vaccination against D. nodosus. Firstly, melatonin induces higher antibody titer than the vaccine alone, secondly, melatonin increase IgG+ B lymphocytes and CD4+ T lymphocytes in vaccinated sheep. These results suggest that melatonin enhances T CD4 cell activation and subsequently secondary humoral immune responses. Further studies are required to determine the mechanism underlining the immunomodulatory role of melatonin in the context of vaccination.

Measurement of the IgM and IgG Autoantibody Immune Responses in Human Serum has High Predictive Value for the Presence of Colorectal Cancer

IntroductionColorectal cancer is a major public health issue, with incidences continuing to rise owing to the growing and aging world population. Current screening strategies for colorectal cancer diagnosis suffer from various limitations, including invasiveness and poor uptake. Consequently, there is an unmet clinical need for a minimally invasive, sensitive, and specific method for detecting the presence of colorectal cancer and pre-malignant lesions. Patients and MethodsAn indirect enzyme-linked immunosorbent assay was used to measure the primary (IgM) and secondary (IgG) adaptive humoral immune responses to a panel of previously identified cancer antigens in the sera of normal and adenoma samples, and sera from patients with colorectal cancer. ResultsAn optimal panel of 7 biomarkers capable of identifying patients with colorectal cancer as distinct from both normal and adenoma samples is identified. The cumulative sensitivity and specificity of the assay are 70.8% and 86.5%, respectively. The positive and negative predictive values of the cohort are 77.3% and 82.1%. This assay was not able to accurately discriminate between normal and adenoma samples. Patients whose serum was positive for the presence of anti-ICLN IgM autoantibodies had a significantly poorer 5-year survival than patients whose serum was negative (P = .004). ConclusionThis study describes a novel minimally invasive enzyme-linked immunosorbent assay-based method, capable of identifying patients with colorectal cancer as distinct from both normal and adenoma samples. Patients are likely to be far more amenable to a blood-based test such as the one described herein, rather than a fecal-based test, likely leading to increased patient uptake.

Comparison of growth performance and immune responses of broiler chicks reared under heat stress, cold stress and thermoneutral conditions

This study was conducted to compare the effects of thermal stress on growth performance and some immunity variables of broiler chickens. Birds were randomly assigned to one of three thermal treatments as follows: cold stress (CS, 12±1°C), (b) heat stress (HS, 33±3 °C) and (c) thermoneutral (TN, 24±2 °C). Body weight gain (BWG), feed intake (FI), water intake (WI) and feed conversion ratio (FCR) were recorded. In order to evaluate the primary and secondary humoral immune responses, two birds per replicate were intravenously administrated with a suspension of 7% sheep red blood cell (SRBC) at 28 and 35 days. The heat-stressed broiler chickens had lower FI (-14.90%), BWG (-25.71%) and higher FCR (+13.06%) in comparison to broiler chickens reared under TN condition (p<0.001) from 1 to 42 days of age. The cold-stressed broiler chickens showed lower FI (-22.05%), BWG (-38.32%) and higher FCR (+22.47%) in comparison to birds reared under TN conditions (p<0.001). Stressed birds (CS and HS) showed decreased antibody titer against SRBC, lymphocyte count and the relative weights of lymphoid organs and increased heterophil count, heterophil to lymphocyte ratio and the serum concentration of corticosterone, in comparison to birds in TN group (p<0.001). In conclusion, HS and CS conditions have similar negative effects on performance and immunity of broiler chickens.

Low-Dose Methotrexate Prevents Primary and Secondary Humoral Immune Responses and Induces Immune Tolerance to a Recombinant Immunotoxin.

Recombinant immunotoxins (RITs) are chimeric proteins being developed for cancer treatment. They are composed of an Ab fragment that targets a cancer Ag and a cytotoxic portion of Pseudomonas exotoxin A. They are effective for patients with hematologic malignancies with defective immunity, but their efficacy against solid tumors is limited by anti-drug Ab (ADA) responses in immune-competent patients. Pre-existing Abs or immune memory owing to previous toxin exposure represent additional hurdles because they induce rapid and strong ADA responses. Here, we evaluated the efficacy of methotrexate (MTX) to prevent ADA formation against the mesothelin-targeting RIT LMB-100 in naive mice and in mice with pre-existing Abs. We found that low-dose MTX combined with LMB-100 completely suppressed the formation of ADAs in a dose- and frequency-dependent manner. Suppression of the immune response restored blood levels of LMB-100 and prevented its neutralization. Furthermore, combination of MTX with LMB-100 did not compromise the immune response against a second Ag given after stopping MTX, indicating specific immune tolerance. Adoptive transfer of splenocytes suppressed Ab responses to LMB-100 in recipient mice, indicating a durable immune tolerance. We conclude that combination of MTX and LMB-100 is effective at preventing immune responses in a durable, Ag-specific manner. We propose combining low-dose MTX in immune-competent cancer patients receiving RIT therapy to prevent immunogenicity. This approach could be applied to other immunogenic therapeutic agents and to proteins for which there is pre-existing immunity.

Effects of in Ovo Injection and Inclusion a Blend of Essential Oils and Organic Acids in High NSPs Diets of Broiler Breeders on Performance of Them and Their Offspring.

Two factorial completely randomized design trials 2×2 and 2×2×2 were conducted to evaluate the effect of a blend of essential oils and organic acids (Biacid™) in broiler breeder diets at two levels, two dietary non-starch polysaccharides (NSPs) levels and in ovo injection of Biacid™ on their progenies performance, respectively. 240 broiler breeders of Ross 308 strain were fed from the age of week 44th for 12 weeks in four groups. 120 produced eggs from each group were divided in two groups of 60 eggs for injecting by 0.5 ml of Biacid™ or distilled water. Injection was done during transferring from setter to hatcher in day 18th of incubation. Twenty-five cockerels from each of 8 treatments were housed into separate pens. Using Biacid™ and high NSPs in broiler breeders' ration affected hatchability, embryo mortality, weight of day old chicks and progenies' carcass yield significantly (p<0.05) whereas in ovo injection of Biacid™ did not show significant effects in this regards (p≥0.05). Offspring's abdominal fat was neither affected by broiler breeders' rations nor in ovo injection of Biacid™ (p≥0.05). Biacid™ and high NSPs content in broiler breeders' ration affected all primary and secondary humoral immune responses of progenies against sheep red blood cells (p<0.05). In ovo injection of Biacid™ increased the primary IgG, primary IgT and secondary IgG responses (p<0.05). The interaction of the effects of Biacid™ and high NSPs in broiler breeders' ration and also in ovo injection of Biacid™ affected progenies' weight gain, feed intake, feed conversion ratio and European production index significantly (p<0.05). It seems that using Biacid™ in broiler breeders' diet can modify the undesirable effects of high NSPs content of breeders' ration on performance of their offspring.

Possible Association of IFN-γ Gene -316A/G SNP with Humoral Immune Response to Killed H5N1 HPAI Vaccine in a Red Junglefowl Population.

To understand the role of interferon (IFN)-γ gene (IFNG) single-nucleotide polymorphisms (SNPs) in the resistance to H5N1 highly pathogenic avian influenza (HPAI), we determined the SNPs, the log2-transformed specific antibody titers, and ex vivo recall antigen-specific IFN-γ production by peripheral blood mononuclear cells (PBMCs) in 62 red junglefowls (Gallus gallus) immunized twice with inactivated H5N1 HPAI vaccine. Consequently, 52 SNPs were detected in the amplified 1137-bp length covering the promoter region and all exons, with +2133A/T SNP in the coding DNA sequence that caused a missense mutation and was identified in G. gallus for the first time. For -316A/G SNP genotypes, the chi-squared test showed that the bird sample was in the Hardy-Weinberg equilibrium (P=0.369>0.05), and the least squares analysis revealed an increasing tendency in the antibody titers with G to A substitution so that a significant difference occurred between the AA genotype (8.031±0.247) and the GG genotype (6.571±0.528) (P=0.015<0.05, 95% confidence interval [CI]: 0.0004-0.0866), as basically consistent with the antigen-induced IFN-γ protein expression, which indicated possible association of the -316A/G SNP with a secondary humoral immune response to the HPAI vaccine in the bird population. These findings may help to improve genetic resistance using cross-breeding and enhance HPAI vaccine-induced immunity in chicken production.

CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen.

Chemokine-dependent localization of specific B cell subsets within the immune microarchitecture is essential to ensure successful cognate interactions. Although cognate interactions between T cells and memory B cells (B(mem)) are essential for the secondary humoral immune responses, the chemokine response patterns of B(mem) cells are largely unknown. In contrast to naive B cells, this study shows that Ag-specific B(mem) cells have heightened expression of CCR6 and a selective chemotactic response to the CCR6 ligand, CCL20. Although CCR6 appears be nonessential for the initial clonal expansion and maintenance of B(mem), CCR6 is essential for the ability of B(mem) to respond to a recall response to their cognate Ag. This dependency was deemed intrinsic by studies in CCR6-deficient mice and in bone marrow chimeric mice where CCR6 deficiency was limited to the B cell lineage. Finally, the mis-positioning of CCR6-deficient B(mem) was revealed by immunohistological analysis with an altered distribution of CCR6-deficient B(mem) from the marginal and perifollicular to the follicular/germinal center area.

Immunomodulatory effects of Stachytarpheta cayennensis leaf extract and its synergistic effect with artesunate.

BackgroundThe leaves of Stachytarpheta cayennensis C. Rich. (Verbenaceae) have been reported to possess potent anti-inflammatory and anti-malarial activities. Due to close association between inflammatory and immune responses, we evaluated the immunomodulatory activity of leaves extract of S. cayennensis. The combined effects of the leaves extract and artesunate, a standard antimalarial agent with immunomodulatory effects, were also evaluated.MethodsThe immunomodulatory activity of the methanol extract of the leaves of S. cayennensis (MESC) was evaluated in mice using the Delayed-Type hypersensitivity response (DTHR), primary and secondary humoral immune responses and the in vivo leucocyte mobilization tests. The immunomodulatory effect of artesunate and the combined effects of MESC and artesunate were evaluated using the phagocytic activity of polymorphonuclear neutrophils. Acute toxicity and lethality test in addition to the preliminary phytochemical studies of MESC were also performed.ResultsThe MESC exhibited 64.21% inhibition of DTHR at 500 mg/kg dose and evoked 139.64% of phagocytic stimulation at 100 μg/ml dose. Also MESC significantly (p < 0.05) showed dose related stimulation of humoral immunity and a highest percentage leucocyte mobilization of 10.15% at 250 mg/kg dose. Artesunate offered a non-significant (p > 0.05) percentage phagocytic stimulation (PPS) while the combined effect of artesunate and MESC exhibited a significant (p < 0.05) dose dependent PPS with highest PPS of 393.77% at 100 μg/ml. The LD50 of the MESC was estimated to be greater than 5000 mg/kg since there were no lethality and signs of acute intoxication after 48 h observation. Preliminary phytochemical analysis revealed the presence of carbohydrates, glycosides, flavonoids, saponins, alkaloids, terpenoids and steroids.ConclusionsThe results of this study indicated that MESC possesses immunostimulatory action with significant synergistic effects with artesunate, and can therefore, offer immune boosting activities in disorders of immune suppression.

Chemokines and chemokine receptors: positioning cells for host defense and immunity.

Chemokines are chemotactic cytokines that control the migratory patterns and positioning of all immune cells. Although chemokines were initially appreciated as important mediators of acute inflammation, we now know that this complex system of approximately 50 endogenous chemokine ligands and 20 G protein-coupled seven-transmembrane signaling receptors is also critical for the generation of primary and secondary adaptive cellular and humoral immune responses. Recent studies demonstrate important roles for the chemokine system in the priming of naive T cells, in cell fate decisions such as effector and memory cell differentiation, and in regulatory T cell function. In this review, we focus on recent advances in understanding how the chemokine system orchestrates immune cell migration and positioning at the organismic level in homeostasis, in acute inflammation, and during the generation and regulation of adoptive primary and secondary immune responses in the lymphoid system and peripheral nonlymphoid tissue.

The effect of carbohydrate moiety structure on the immunoregulatory activity of lactoferrin in vitro.

The aim of this study was to evaluate the immunoregulatory effects of recombinant human lactoferrin (rhLF) in two in vitro models: (1) the secondary humoral immune response to sheep erythrocytes (SRBC); and (2) the mixed lymphocyte reaction (MLR). We compared the non-sialylated glycoform of rhLF as expressed by glycoengineered Pichia pastoris with one that was further chemically sialylated. In an earlier study, we showed that sialylated rhLF could reverse methotrexate-induced suppression of the secondary immune response of mouse splenocytes to SRBC, and that the phenomenon is dependent on the interaction of lactoferrin (LF) with sialoadhesin (CD169). We found that the immunorestorative activity of sialylated rhLF is also dependent on its interaction with the CD22 antigen, a member of the immunoglobulin superfamily that is expressed by B lymphocytes. We also demonstrated that only sialylated rhLF was able to inhibit the MLR reaction. MLR was inhibited by bovine lactoferrin (bLF), a glycoform that has a more complex glycan structure. Desialylated bLF and lactoferricin, a bLF-derived peptide devoid of carbohydrates, did not express such inhibitory activity. We showed that the interaction of LF with sialic acid receptors is essential for at least some of the immunoregulatory activity of this glycoprotein.

Time Course and Metabolic Costs of a Humoral Immune Response in the Little Ringed Plover Charadrius dubius

Despite host defense against parasites and pathogens being considered a costly life-history trait, relatively few studies have assessed the energetic cost of immune responsiveness. Knowledge of such energetic costs may help to understand the mechanisms by which trade-offs with other demanding activities occur. The time course and associated metabolic costs of mounting a primary and secondary humoral immune response was examined in little ringed plovers Charadrius dubius challenged with sheep red blood cells. As was expected, the injection with this antigen increased the production of specific antibodies significantly, with peaks 6 d postinjection in both primary and secondary responses. At the peak of secondary antibody response, the antibody production was 29% higher than that observed during the primary response, but the difference was nonsignificant. Mounting the primary response did not significantly increase the resting metabolic rate (RMR) of birds, whereas the secondary response did by 21%, suggesting that the latter was more costly in terms of RMR. In spite of the fact that the primary response did not involve an increase in RMR, birds significantly decreased their body mass. This could imply an internal energy reallocation strategy to cope with the induced immune challenge. Last, we found that RMR and antibody production peaks were not coupled, which could help to conciliate the variable results of previous studies. Collectively, the results of this study support the hypothesis that humoral immunity, especially the secondary response, entails energetic costs that may trade-off with other physiological activities.

Evaluation of canine T-cell dependent antibody response to the primary and secondary immunization with keyhole limpet hemocyanin

T-cell dependent antibody response (TDAR) incorporating both primary and secondary responses to keyhole limpet hemocyanin (KLH) in canine models have not yet been fully understood. To develop a practical dog TDAR model, we characterized primary and secondary antibody responses by intravenous or intramuscular immunization of KLH twice at intervals of 8 days during a 28-day course of study. Primary immunization with KLH by both routes induced a maximum IgM response on 6 to 8 days after the treatment, whereas the IgG response started 6 to 8 days after the treatment with relatively low levels. Remarkable increases in anti-KLH IgG levels (about 10-times compared with the primary response) were produced 5 to 7 days after the secondary KLH immunization by both routes. These results indicate that IgM-predominant and IgG-predominant responses were respectively induced by the primary and secondary immunization. Furthermore, the intravenous route showed higher baseline titers of primary and secondary anti-KLH IgM responses, suggesting that intravenous immunization of KLH might be a more suitable method for immunotoxicity evaluation. No remarkable inter-individual variability was noted in our canine models. Treatment with cyclophosphamide at 2 mg/kg/day for a consecutive 28 days significantly suppressed primary and secondary anti-KLH IgM and IgG responses induced by KLH injection on Days 15 and 23 of CPA treatment. These results demonstrate that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in dogs when exposed to potential immunomodulatory drugs.

Evaluation of primary and secondary responses to a T-cell-dependent antigen, keyhole limpet hemocyanin, in rats

To develop a rat T-cell-dependent antibody response (TDAR) model evaluating both primary and secondary antibody responses, keyhole limpet hemocyanin (KLH) was used to immunize rats twice during a 14-day course of study, a pattern closely linked to that of a short-term general toxicity study. Female rats of four representative strains (e.g., Sprague-Dawley, Wistar, Fischer, and Lewis) were immunized twice with intravenous administrations of KLH (300 µg/rat) on Days 5 and 9 during a 14-day treatment regimen with cyclophosphamide (CPA) at 1, 3, or 6 mg/kg/day. The primary and secondary immunizations of KLH markedly elevated serum anti-KLH IgM and IgG levels in all strains on Days 9 and 15. Remarkable higher levels of anti-KLH IgG (≈ 1000 µg/ml) were noted in all strains, which were more than 4-times compared with those of anti-KLH IgM levels at Day 9, indicating that predominant IgG reactions were induced by the dual immunizations. A large inter-individual variability in KLH-specific IgM and IgG production was observed in all strains. However, levels of the KLH-specific antibodies were considered sufficient for the evaluation, even in Sprague-Dawley and Wistar rats reported as strains with a wide range of variability since immunosuppression of CPA on responses in both anti-KLH IgM and IgG were observed in all strains to the same extent. In addition, the sensitivity of the KLH-ELISA assay system detecting the immunosuppressive effects of CPA was comparable to other assay systems with PFC assay or ELISA using SRBC. The results here demonstrated that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in rats when exposed to potential immunomodulatory drugs. Furthermore, the design of the presented TDAR study would support comprehensive evaluation together with the outcome of the conventional general toxicity study.