Sheep Red Blood Cells Immunization Research Articles

Impact of cow, buffalo, goat or camel milk consumption on oxidative stress, inflammation and immune response post weaning time

Milk is a whitish liquid that is secreted from mammary glands; and considered as the primary source of nutrition for newborns since they are not able to digest solid food. However, it contains primary nutrients, as well as growth and immune factors. Early weaning is a critical issue that face women and their babies in developing countries. To avoid infant malnutrition, they tend to use other milk types instead of baby formula. Therefore, the present study aimed to evaluate the impact of cow, buffalo, goat or camel milk consumption on oxidative stress, inflammation and immune response in male and female Sprague Dawley rats post weaning time. The amino acids, fatty acids, minerals and vitamins in the tested milk types were evaluated. Animals were divided into 5 groups (control, cow, buffalo, goat and camel milk administrated groups) (10 rats/group); each animal was administrated by 3.4 ml/day. Rats were administered with milk for 6 weeks; at the end of the 5th week, five animals of each group were isolated and the remaining five animals were immunized with sheep red blood cells (SRBCs) and kept for another week to mount immune response. The effect of different milk types on rats’ immune response towards SRBCs was evaluated through pro-inflammatory cytokines, antioxidants, ESR and CRP measurement; together, with the histopathological examination of spleen samples and hemagglutination assay. Camel milk consumption reduced oxidative stress and inflammation in spleen that resulted from SRBCs immunization; in addition to, B cell stimulation that was apparent from the high level of anti-SRBCs antibodies. Camel milk is recommended for newborn consumption, due to its high-water content, unsaturated fatty acids, and vitamin C, as well as low lactose and fat content.

Smarca4 (Brg1) Controls Germinal Center B-Cell Development and Is a Haploinsufficient Tumor Suppressor in Lymphoma

SMARCA4 encodes one of two mutually exclusive ATPases present in BAF (aka SWI/SNF) chromatin remodeling complexes. SMARCA4 is dynamically expressed during normal B-cell development, with high expression in germinal center B (GCB)-cells, and is targeted by heterozygous inactivating mutations in GCB-derived lymphomas including 34% of pediatric Burkitt lymphoma (BL), 17% of adult BL, 4% of GCB-DLBCL. However, the functional role of SMARCA4 in GCB cell development and malignant transformation has not been thoroughly investigated. We generated a novel floxed Smarca4 transgenic mouse model and crossed to the Aid-Cre line for GC-specific conditional knock-out (cKO). Following immunization with sheep red blood cells (SRBCs) to induce a GC reaction, Smarca4+/- mice had a significant increase in GCB-cell abundance compared to Smarca4+/+ littermates (p<0.001) due to both an increased average GC size and number. Notably, Smarca4-/- mice displayed a significantly reduced frequency of GC B-cells (p<0.001) and sorting of residual cells revealed retention of 1 or more copies of the floxed allele, suggesting that biallelic cKO of Smarca4 is lethal to GCB cells. We therefore focused subsequent studies on comparisons between Smarca4+/- and Smarca4+/+ mice. Further analysis revealed significant polarization towards a centroblastic phenotype in Smarca4+/- mice (p<0.001), in line with the centroblastic morphology of human BL. Analysis of competitive advantage with the same GC using bone marrow chimera models showed a significant advantage of Smarca4+/- GCB cells compared to Smarca4+/+, associated with a significant increase in proliferation measured by Edu incorporation and reduction of apoptosis measured by Annexin-B. We extended upon this using single cell RNA-seq (scRNA-seq) of GCB cells from Smarca4+/- and Smarca4+/+ mice, which confirmed an increased centroblastic polarization of Smarca4+/- GCB cells and identified a significant increase in light zone to dark zone recycling cells that may underly this polarization. We validated this using the Myc-GFP allele, which confirmed increased Myc+ recycling cells in GCs from Smarca4+/- mice compared to Smarca4-/- mice. BAF complexes drive ATP-dependent mobilization and eviction of nucleosomes to convert chromatin from inactive to active states. Brg1 deficiency may therefore alter distinct BAF complexes and transcription factor programs. We therefore explored the role of Smarca4 in regulating chromatin accessibility and gene expression using ATAC-seq and RNA-seq in Smarca4+/- and Smarca4+/+ murine GCB-cells. Supervised analysis revealed predominant loss of chromatin accessibility associated with repression of nearby genes. Smarca4+/- cells showed significant loss of accessibility of genes upregulated in centrocytes, and a preferential loss of NF-ĸB target genes (GSEA FDR<0.01). MYC translocations are pathognomonic for BL, in which SMARCA4 mutations are most frequent. We therefore crossed our Smarca4 cKO mice with a cre-inducible Myc knock-in (Myc-KI) allele to mimic combined Smarca4 loss-of-function and Myc activation. The addition of Myc-KI did not lead to a significant increase in GCB cell frequency following a single SRBC immunization. However, serial re-challenge of mice with SRBCs led to lymphadenopathy, splenomegaly and infiltration of highly proliferative B-cells into the major organs (lung, liver, and kidney) in Smarca4+/- + Myc-KI mice following 3 immunizations that was not observed with Smarca4+/- or Myc-KI alleles alone. Furthermore, long term follow-up of serially-challenged mice showed a significant reduction in overall survival in Smarca4+/- + Myc-KI mice compared to Smarca4+/- or Myc-KI mice (p=0.010). Collectively, these data demonstrate a competitive advantage of GCB cells with monoallelic Smarca4 cKO, and a centroblastic GC polarization driven in part by increased recycling. Mechanistically, this is associated with loss of chromatin accessibility and gene expression of genes that are expressed in centrocytes, including NFkB target genes. Monoallelic Smarca4 cKO cooperates with Myc over-expression to drive expansion and extranodal dissemination of GCB cells following serial re-challenge, leading to reduced survival. These data provide the first functional and mechanistic insight into the role of SMARCA4 in GCB cell development and lymphomagenesis.

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.

Efforts to overcome hypoxia condition in Balb/c mouse macrophages after intraperitoneal SRBC immunization

BACKGROUND Activated macrophages require increased oxygen to destroy foreign bodies, leading to an increase in the levels of reactive oxygen species (ROS). Therefore, macrophages would experience hypoxic and oxidative stress conditions at the same time. Thus, this study was aimed to evaluate the mechanism of the activated macrophages to overcoming this dual condition.METHODS The activated macrophages were harvested from the intraperitoneal cavities of 18 BALB/c mice immunized with 2% sheep red blood cells (SRBCs). The macrophage suspension was divided into four groups: control, 24, 48, and 72 hours after-immunization groups. The expressions of hypoxia-inducible factor (HIF)-1α, HIF-2α, and cytoglobin (Cygb), as markers for hypoxic condition, were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), whereas peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) protein as a marker for mitochondrial biogenesis and aerobic metabolism was measured with ELISA. The analysis of oxidative stress was conducted with the water-soluble tetrazolium salt test.RESULTS The HIF-1α mRNA expression was the highest at 24 hours, whereas the HIF-2α mRNA showed no increased expression during the observation. The Cygb mRNA decreased after 24 hours. The highest expressions of HIF-1α and HIF-2α proteins were detected at 72 hours, whereas the Cygb protein expression increased since 24 hours. The PGC-1α protein expression increased at 72 hours. The WST test showed the highest ROS level at 24 hours.CONCLUSIONS The macrophages were activated by SRBCs underwent dual hypoxia and oxidative stress condition simultaneously to overcome the foreign bodies. The macrophages overcame these stress conditions by increasing their aerobic metabolism.

Interleukin-21 receptor blockade inhibits secondary humoral responses and halts the progression of preestablished disease in the (NZB × NZW)F1 systemic lupus erythematosus model.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is driven in part by chronic B and T lymphocyte hyperresponsiveness to self antigens. A deficiency of interleukin-21 (IL-21) or IL-21 receptor (IL-21R) in mice dramatically reduces inflammation and B and T cell activation in models of autoimmunity, including SLE. However, whether IL-21 is essential for the maintenance and amplification of preestablished inflammation has not been widely examined in various animal models. The purpose of this study was to examine the impact of novel mouse IL-21R neutralizing antibodies on recall responses to antigen challenge and on disease progression in the (NZB × NZW)F1 (NZB/NZW) mouse model of SLE. Humoral and cellular immune responses to immunization with sheep red blood cells (SRBCs) were measured in mice dosed with IL-21R blocking antibodies. Progression of nephritis and markers of immune activation was monitored in NZB/NZW mice following different anti-IL-21R treatment regimens. IL-21R blockade specifically inhibited secondary IgG responses to SRBC immunization. In NZB/NZW mice, IL-21R blockade completely inhibited the onset of nephritis, which was associated with dramatic reductions in splenomegaly and in B cell and T cell activation. When administered to mice with preexisting disease, anti-IL-21R antibody halted the disease progression and mortality and reversed the nephritis in a subset of mice. Furthermore, treatment cessation was not followed by rapid reemergence of disease. Our results highlight the importance of IL-21 in promoting humoral recall responses and in sustaining autoimmune inflammation.

PPARγ negatively regulates T cell activation to prevent follicular helper T cells and germinal center formation.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates lipid and glucose metabolism. Although studies of PPARγ ligands have demonstrated its regulatory functions in inflammation and adaptive immunity, its intrinsic role in T cells and autoimmunity has yet to be fully elucidated. Here we used CD4-PPARγKO mice to investigate PPARγ-deficient T cells, which were hyper-reactive to produce higher levels of cytokines and exhibited greater proliferation than wild type T cells with increased ERK and AKT phosphorylation. Diminished expression of IκBα, Sirt1, and Foxo1, which are inhibitors of NF-κB, was observed in PPARγ-deficient T cells that were prone to produce all the signature cytokines under Th1, Th2, Th17, and Th9 skewing condition. Interestingly, 1-year-old CD4-PPARγKO mice spontaneously developed moderate autoimmune phenotype by increased activated T cells, follicular helper T cells (TFH cells) and germinal center B cells with glomerular inflammation and enhanced autoantibody production. Sheep red blood cell immunization more induced TFH cells and germinal centers in CD4-PPARγKO mice and the T cells showed increased of Bcl-6 and IL-21 expression suggesting its regulatory role in germinal center reaction. Collectively, these results suggest that PPARγ has a regulatory role for TFH cells and germinal center reaction to prevent autoimmunity.

Akt activation by CD40 controls plasma cell and memory B cell differentiation but not germinal center formation by autoreactive B cells (P4032)

Abstract A hallmark of adaptive immunity is the formation of germinal centers that generate long-lived plasma cells and memory B cells. These events are undesirable by autoreactive B cells since they would lead the production of pathogenic autoantibodies and memory B cells that would accrue over a lifetime, but is unknown at a molecular level how this is achieved. To address this question we examined the ability of B cells specific for the SLE-specific self-antigen Sm to respond to antigen in vivo in the presence of functional T cells. Sheep red blood cells (SRBC) have an antigen that binds anti-Sm B cells and therefore immunization of anti-Sm mice with SRBCs provides antigen for anti-Sm B cell activation and activation of functional cognate T cells. We find that anti-Sm B cells from germinal centers in response to SRBC immunization, but fail to generate splenic or bone marrow plasma cells. Secondary, immunization shows the absence of memory B cell formation. BCR and CD40 signaling appears normal in anti-Sm B cells with the exception of an Akt signal by CD40. Our data suggest that Akt activation by CD40 signaling controls the formation of plasma cells and memory B cells by autoreactive B cells of the type activated in murine and human SLE. Experiments are underway to test this possibility.

The effects of florfenicol on lymphocyte subsets and humoral immune response in mice

Florfenicol is a broad-spectrum bacteriostatic antibiotic used in domestic animals. The aim of the study was to determine the effect of florfenicol on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the peripheral lymphatic organs in non-immunized mice and humoral immune response in sheep red blood cells (SRBC)-immunized mice. Florfenicol was administered orally at a dose of 30 mg/kg six times at 24 h intervals to non-immunized mice and four or seven times at 24 h intervals to SRBC-immunized mice. SRBC was injected 2 hours prior to the first dose of the drug. Florfenicol increased the percentage of CD4CD8- thymocytes and the absolute number of CD4+ and CD8+ thymocytes on day 7. The increased percentage and absolute number of CD3+, CD4+ and CD8+ lymphocytes in mesenteric lymph nodes and decreased percentage of lymphocytes B were also observed 24 hours from the last administration of florfenicol. Florfenicol administered after SRBC immunization reduced the number of plaque forming cells (PFC) and the production of anti-SRBC antibodies on days 4 and 7 after priming.

Effects of Administration of a Monoclonal Antibody against Mouse Tumor Necrosis Factor Alpha during Pregnancy and Lactation on the Pre- and Postnatal Development of the Mouse Immune System

Monoclonal antibodies directed against tumor necrosis factor alpha (TNFalpha) are currently employed in the treatment of various immune-mediated diseases. These studies were designed to evaluate potential effects of anti-TNFalpha treatment in mice during pregnancy and lactation on the development of the immune system in the F1 generation. Pregnant CD-1 mice were treated with vehicle or with 10 or 40 mg/kg of an anti-mouse TNFalpha monoclonal antibody (mAb) (cV1q) on days 6, 12, and 18 of gestation and on days 3, 9, and 15 of lactation. Evaluation of immune system functionality was conducted in F1 generation mice at 11 weeks of age. Immune function was evaluated by splenocyte phenotyping, immunoglobulin M (IgM) antibody response to sheep red blood cells (SRBCs), spleen cell proliferative response to anti-CD3, and natural killer cell activity. Treatment of pregnant mice with cV1q produced no adverse effects in the dams and no adverse effects in the F1 generation. In general, the functioning of the immune system of the F1 generation did not appear to be adversely affected following exposure to cV1q in utero and during lactation. The only statistically significant change was a slight (approximately 20%) reduction in the spleen cell expansion in response to SRBC immunization in the female F1 mice from the 40 mg/kg cV1q treatment group. In conclusion, administration of a monoclonal antibody against mouse TNFalpha during pregnancy and lactation had little or no effect on selected immune parameters in mice, with only a possible minor attenuation of spleen cell response to immunization noted in the female F1 generation at 11 weeks of age.

Use of Whole Sheep Red Blood Cells in ELISA to Assess Immunosuppression In Vivo

An enzyme-linked immunosorbent assay (ELISA) using whole sheep red blood cells (SRBC) has been reported as one of the methods for detecting a T-lymphocyte-dependent antibody response. However, it has not been widely used because of SRBC problems such as the weak attachment to ELISA plates, specificity and short-term stability. The objectives of this study were to address these issues and to validate the SRBC-specific antibody response assay. Male Sprague–Dawley rats were bled after 6 days of SRBC immunization. In our new procedure, glutaraldehyde was added before discarding the supernatant of inoculated SRBC suspension to attach SRBC firmly to the plate, while in the original method it was added after discarding. As a result, the attached SRBC was maintained throughout the ELISA procedures. No interference was observed in the titration curve of IgM and IgG antibodies in rats and IgM-antibody in mice when control sera were analyzed to evaluate specificity of this method. The short-term stability of SRBC was overcome by using the different lots of SRBC. They provided antibody titers, which were consistent with those measured using the same lot for immunization. In addition, cyclophosphamide, cyclosporine, prednisolone and methotrexate, well-known immunosuppressive agents, were tested to confirm the applicability of the improved ELISA method to detect the T-lymphocyte-dependent antibody response. All four compounds inhibited the IgM antibody responses dose-dependently. These results demonstrate that the improved whole SRBC-ELISA method provides reproducible and reliable results in the T-lymphocyte-dependent antibody response assay.

Enhancement of the Humoral Immune Response by Echinacea purpurea in Female Swiss Mice

Various preparations of the plant Echinacea purpurea have been investigated for their potential to enhance immune function, primarily through activation of innate immune responses. Few studies have examined the potential for enhancement of humoral immunity. Using female Swiss mice we administered a volumetric dose of a glycerine extract of E. purpurea by oral gavage, to evaluate effects on the IgM specific antibody forming cell (AFC) response. Four days of treatment following immunization with sheep red blood cells (SRBC) produced a significant enhancement over naïve controls at doses of 0.4 and 0.8 mL/kg/day. A few clinical trials and anecdotal reports have suggested that the greatest efficacy for E. purpurea occurs in acute use following onset of illness. A time course study, using the time of SRBC immunization to mimic the onset of illness, examined the effects of 8 and 4 days of E. purpurea treatment at 0.6 mL/kg/day. Only in the 4‐day administration, with dosing beginning 1 hour after SRBC immunization, was there an observed enhancement of the antibody forming cell response. This supports the acute use of E. purpurea as suggested by anecdotal reports, and demonstrates the potential for enhancement of humoral immune responses as well as innate immune responses.

CHARACTERIZATION OF AN APPROACH TO DEVELOPMENTAL IMMUNOTOXICOLOGY ASSESSMENT IN THE RAT USING SRBC AS THE ANTIGEN

Although developmental immunotoxicology is an area of emerging importance, few data exist that compare profiles of activity in neonatal and adult rodents. One of the factors contributing to the paucity of comparative results is that it is unclear whether immunotoxicological test procedures optimized to evaluate the immune system of adults can be integrated into studies of younger animals. Therefore,the following objectives were addressed in this study for both male and female Crl:CD(SD)BR rat pups and weanlings: (1) to quantify baseline values for absolute and relative splenic lymphocyte populations using flow cytometry in nonimmunized animals; (2) to optimize and establish baseline values for the primary humoral immune response to sheep red blood cells (SRBC) using either an enzyme-linked immunosorbant assay (ELISA) or the plaque-forming cell (PFC) assay; and (3) to determine the impact of SRBC immunization on the histology of the spleen. Responses for each age group were compared both withinandbetween l...

POSSIBLE FUNCTIONAL IMMUNOTOXICITY OF ACRYLONITRILE (VCN)

Acrylonitrile (vinyl cyanide, VCN), an environmental pollutant, has been shown to be an animal and human carcinogen particularly for the GIT. In a previous work done in our laboratory, VCN induced immunosuppressive effects as indicated by a decrease in plaque forming cell (PFC) response to SRBCs (sheep red blood cell) immunization, a marked depletion of spleen lymphocyte subsets by flow cytometric analysis as well as bacterial translocation of the normal flora leading to brachial lymph node abscess. This work was carried out to evaluate the systemic and/or local immunotoxic potential of VCN. Acrylonitrile (2.7 mg kg−1day−1) was given to CD-1 mice once daily for 5, 10 and 15 days. Immunohistochemical assessment of the number of cells capable of producing IgA in different intestinal compartments (duodenum, jejunum and ileum) revealed a significant decrease following VCN treatment. On the contrary, Bromodeoxyuridine (BrdU) incorporation in gut epithelial cells (duodenum and ileum) showed a significant increase in the same VCN-treated groups of animals. On the other hand, [3H]thymidine uptake was significantly decreased in splenocytes stimulated with phytohemaglutinin (PHA), Concanavalin-A (Con-A) and Lipopolysaccharide (LPS) and derived from animals treated with VCN. The effects of VCN were started after 5 days and increased up to 15 days of daily treatment in most of the investigated parameters. The results suggested that VCN has a profound immunosuppressive effect either systemically or locally which could be a contributing factor in its GIT carcinogenicity.

In vivo indomethacin reverse exercise-induced immunosuppression in rats

The effect of oral indomethacin on the immunosuppressive effect of exercise was examined in exercised untrained female Wistar rats immunized with sheep red blood cell (SRBC) antigens. Intensity of the 1 h exercise was controlled by 0–50 kPa air pressure, generated by a compressor located at the bottom of a water tank, during continuous swimming of the rats, previously immunized with SRBC. After 48–72h, depending on the ip (intraperitoneal) or iv (intravenous) route of SRBC immunization, the exercise suppressed humoral PFC response and augmented phagocytosis of peritoneum macrophages. These effects occurred only when exercise was performed at 48 h after antigen injection. Animals receiving indomethacin, however, did not show any exercise-related suppression of the PFC response. The data suggest a relationship between exercise-induced immunosuppression and possible increased in vivo prostaglandin synthesis during the intense exercise. Overall, exercise-related suppression of humoral PFC response was dependent on the intensity of the exercise, was time specific, and was reversible by pharmacological blockade of the cyclooxygenase pathway of prostaglandin synthesis.

Influence of arecoline on immune system: III. Suppression of B cell-mediated immune response in mice after short-term exposure.

Arecoline, a major alkaloid of arecanut was examined to explore its modulatory influence on B cell-mediated immune response in a murine model system. The in vivo and in vitro effects were evaluated at sub-toxic concentrations of arecoline. The number of primary antibody forming cells (AFC) and hemagglutinating and hemolysis antibody titers to Sheep Red Blood Cells (SRBC) were evaluated in male mice. Arecoline exposure for a week invoked dose-dependent effect on primary antibody forming cells to SRBC with a maximum reduction at the dosage of 20 mg/kg bw, a moderate reduction at 10 mg/kg bw and no effect at 5 mg/kg bw dose level. HA and HL antibody titers to SRBC were suppressed markedly at arecoline dosage of 20 mg/kg bw and moderately at a dose of 10 mg/kg bw, given daily for 1, 2 or 3 weeks. The inhibitory effect of arecoline was not dependent on the duration of treatment. Like the primary antibody response, the secondary HA and HL antibody titers were also decreased after arecoline exposure. The administration of arecoline dosages 10 and 20 mg/kg bw daily for 4 days following SRBC immunization also, exerted dose-dependent suppression of primary antibody response. Similarly, when treated after 12 h following immunization, significant reduction in response was observed with arecoline dosage of 20 mg/kg bw. While moderate suppression of antibody response was noticed at the dose level of 10 mg/kg bw, there was no alteration in response at a dosage of 5 mg/kg bw.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation into the immunological effects of miltefosine, a new anticancer agent under development

Miltefosine is the prototype of alkylphosphocholines, a new class of anticancer agents related to alkyl-lysophospholipids. These agents were considered to possess potent immunomodulatory properties. Miltefosine was highly active against the human KB tumour xenograft in nude mice, leading to growth inhibition as well as regression of large established tumours, which suggested that its mode of action was not mediated by the T cell system. In vivo natural killer cell activity was measured by chromium release of YAC-1 cells using spleen cells from treated animals as effector cells. Miltefosine had no significant effect on YAC-1 cytolysis. Similarly, the compound did not induce cytotoxic spleen cells against KB target cells. The results were identical when spleen cells from tumour-bearing animals were used. Humoral antibody production in rats following sheep red blood cell immunization was not changed by miltefosine pretreatment. Finally, the in vitro phagocytic activity of mouse bone marrow macrophages was not stimulated but rather inhibited in a dose-dependent manner. In conclusion, there is no experimental evidence that the miltefosine action is mediated by the host immune system and no major immunotoxicity was observed.

Effect of saikosaponin on the immune responses in mice

The in vivo effects of saikosaponin, isolated from Bupleurum radix, on the immune responses are still poorly understood. We have already shown that saikosaponin-d increases phagocytic activities of murine peritoneal macrophages such as spreading activity, phagocytosis, lysosomal enzyme activity and intracellular killing activity of living yeast. This work extends these observations by showing that treatment with saikosaponin also increased the antibody response in plaque-forming cell numbers after in vivo immunization with sheep red blood cells (SRBC) and an augmentation of spleen cell proliferation responses to stimulation with T- or B-cell mitogens both before and after immunization. Furthermore, after SRBC immunization, the macrophages from mice treated with saikosaponin-d revealed significant increases in spreading activity and lysosomal enzyme activity. The chemiluminescences of the macrophages from mice treated with saikosaponin-d stimulated by opsonized zymosan and PMA were enhanced and interleukin-1 production by the cells was increased in a dose-dependent manner. These results demonstrate that saikosaponin-d may stimulate in vivo immunological lymphocyte functions, partly by activating some macrophage functions.

Influence of Arecoline on Immune System: II. Suppression of Thymus-Dependent Immune Responses and Parameter of Non-specific Resistance After Short-Term Exposure

Arecoline, a major alkaloid of arecanut was screened to explore its modulatory influence on cell-mediated immune response in a murine model system. The in vivo and in vitro effects were evaluated at subtoxic concentrations of arecoline. Delayed type hypersensitivity (DTH) reactions to sheep red blood cells (SRBC) were evaluated in male mice. When treated subcutaneously with 20 mg/kg bw (1/5 of LD50) dose of arecoline for 1, 2 or 3 weeks, the DTH reactions were significantly suppressed. At arecoline concentration of 10 mg/kg bw, there was a moderate reduction in DTH response, while no appreciable change was observed at a dosage of 5 mg/kg bw. The effects were not dependent on the duration of treatment. In contrast, treating with arecoline continuously for 4 days following SRBC immunization showed significant suppression in DTH reactions at both 10 and 20 mg/kg bw doses. When treated after 12 h following immunization with 20 mg/kg bw arecoline, significant reduction in DTH reactions were seen. While moderate reduction in response was observed with arecoline dosage of 10 mg/kg bw, there was no alteration in response at the dose level of 5mg/kg bw. Recovery experiments in mice revealed that arecoline mediated effects are of a reversible nature. Arecoline treatment did not appreciably alter the host resistance to endotoxin shock. In vitro experiments revealed both dose-dependent and time-dependent cytotoxic effects of arecoline when spleen cells were incubated with varying concentrations of arecoline. Concomitant exposure of arecoline at concentrations of 10(-6) - 10(-4) M with con A, markedly suppressed both 3H-thymidine incorporation and interleukin-2 production of splenic cells. In contrast, concomitant exposure of arecoline with IL-2 did not alter 3H-thymidine incorporation in the IL-2 dependent cytolytic T-lymphocyte line (CTLL), except at the concentration of 10(-4) M arecoline. From these studies it is concluded that the dose-dependent suppressive effects of arecoline on DTH response to SRBC and on certain in vitro lymphocyte functions are more clear than the host resistance to endotoxin shock.

Evaluation of the Miniature Swine for Use in Immunotoxicity Assessment: I. Quantitation of Humoral Responses to Sheep Red Blood Cells and Effects of Cyclophosphamide

T-cell-dependent and t-independent humoral responses as well as cell-mediated re-sponses(1) were assessed with groups of young, Hormel-Hanford miniature swine. Test antigens established in various immunotoxicity studies with rodents were used. Results with the widely used t-dependent test antigen, sheep red blood cells (SRBC), are presented in this report. Groups of swine were immunized with varying dosages of SRBC to evaluate basal responses. Serum antibody titers were estimated by the standardized hemolysin (HLY) visual endpoint (EP) assay and a quantitative spectro-photometric (SP) variation using highly purified immunoglobulin (Ig) M and Ig G standards. The sensitivity of the EP HLY assay is about 0.5-1.0 μg for Ig G anti-SRBC; the SP variation is 10 times more sensitive. Peak responses were obtained 7 days after either the first or second injection for both males and females. Peak responses for females, however, were two to four times greater than those in males. Ig M antibodies had a three times higher specific HLY titer than those in the Ig G class. Use of purified antibody standards in either assay variation permitted conversion of dilution titers to specific amounts of antibody. The latter is a more meaningful parameter for various comparisons in either inter-or intraspecies evaluations in immunotoxicity studies. To evaluate SRBC as a test antigen, low and intermediate doses of the immunotoxicant, cyclophosphamide, were given i.p. starting 2 days before SRBC injection. Antibody levels were reduced correspondingly to approximately 25 and 60% immunosuppression for the two dose levels in both primary and secondary responses. After 2 weeks of rest, animals recovered from treatment with CP. These studies demonstrate that SRBC immunization can be used in the FDA, Hormel-Hanford miniature swine to evaluate the immunotoxicity potential of chemicals without adjuvants or apparent harmful effects to the animal.

Deficiency in immunocompetence of mice cured from large MOPC-315 plasmacytomas by melphalan therapy.

Mice cured from large MOPC-315 tumors by a single dose of melphalan, 7.5 mg/kg, were examined for up to 60 days after the drug treatment (71 days after the tumor inoculation) for their ability to respond to mitogenic stimulation, specific and nonspecific antigenic stimulation and for their susceptibility to inoculation with an unrelated tumor, L10 lymphoma. The response of spleen cells from cured mice to mitogenic stimulation by phytohemagglutinin or concanavalin A was slightly depressed at an early stage after the drug treatment. The allogeneic response against C57BL spleen cells and the antibody response against sheep red blood cells (SRBC) of spleen cells from cured mice remained below normal levels during the whole observation period. The deficiency in response to antigenic stimulation was found to be due to impairment in T-cell function. Cured mice were also deficient in their response to SRBC immunization (antibody and delayed-type hypersensitivity responses) and were more susceptible to inoculation with an unrelated tumor, L10 lymphoma, than normal, noninoculated mice. On the other hand, spleen cells of cured mice developed a highly specific cytotoxic response against target MOPC-315 tumor cells and the cured mice were resistant to challenge with an otherwise highly tumorigenic dose of MOPC-315. Thus, cured mice remained deficient for a long period of time in their response to MOPC-315-unrelated antigens but, at the same time, they showed a potent specific antitumor immunity potential in vivo and in vitro.