Plaque-forming Cell Response Research Articles

The NE Atlantic European hake: A neglected high exposure risk for zoonotic parasites in European fish markets

A sampling programme to understand the factors affecting zoonotic parasite presence and infection intensity of Atlantic European hake populations was conducted from 2013 to 2015. Commercial fish samples comprising 430 hake specimens from northern (Grand Sole) and southern (Atlantic Iberian Peninsula) fishing grounds were sampled, recorded and inspected. We also analysed 75 additional samples collected around Scotland. Parasites were microscopically identified to genus level. Fish biometric measurements were statistically evaluated as categorical predictors of parasite recruitment into fish stocks. Stable isotope composition was determined in another sample batch of 501 hakes from the NE Atlantic (Grand Sole and Galician grounds) to explore trophic indicators as potential predictors of parasite burden. Partial sequences from mitochondrial and nuclear marker genes (mtDNA cox2 and EF1 α-1 nDNA) from a set of 915 Anisakis larvae confirmed the northern stock to be parasitized by A. simplex (s.s.) while off the Iberian Peninsula hake there was a mixed infection pattern with larvae of A. simplex s.s. (67.4% of identified larvae), A. pegreffii (31.9%) and the F1hybrid (0.7%). The observed burden of Anisakis spp. larvae differed markedly between fishing areas. The presence of very high Anisakis spp. burdens in a comparatively small proportion of individual fish was well-illustrated with characteristic tensegrity grid architecture for parasite aggregation derived from a plaque-forming cell response observed in the abdominal cavity of larger fish specimens. Demographic infection values were remarkably high in Grand Sole and off the Iberian Peninsula, with 100% prevalence and mean abundances in the fillets ranging from 247 (A. simplex) in hake from Grand Sole to 60 A. simplex and 20 A. pegreffii in southern European populations. Infections were less prevalent and less intense in the hake sample from Scottish waters. Fish size was the most important factor affecting Anisakis spp. prevalence and abundance, with larger fish containing more Anisakis larvae. Anisakis abundance within individual fish was also positively correlated with the condition index although this does not rule out negative effects on the host, and with trophic level, even after fish size was accounted for. These results suggest that higher Anisakis burden is associated with a higher food intake and with eating larger prey. The risk profile for zoonotic parasites herein described for European hake populations underlines the urgent need for adopting a contingency plan that minimizes the risk exposure.

Hypoglycemic and Immunostimulating Activities of Exopolysaccharides from Ganoderma sp. Mushroom in Streptozotocin-Induced Diabetes in Rats

The incidence of diabetes mellitus is increasing dramatically worldwide. Different studies support an association between diabetes and decreased function of the immune system. On the other hand, Ganoderma mushroom, and its bioactive compounds, is widely used and recommended by physicians for its supporting effects on immune system. Therefore, this work aimed to test the hypoglycemic and immunomodulating activities of Ganoderma sp. exopolysaccharides (EPS) in streptozotocin (STZ)-induced diabetes in rats. Animals were divided into control, diabetic and Ganoderma EPS-treated (10, 50 or 100 mg/kg BW) diabetic groups, administered with Ganoderma EPS orally for consecutive 3 weeks. Glucose concentrations were measured using test strips, while the immunomodulating activity was determined by measuring the delayed-type hypersensitivity (DTH) reaction, rosette-forming cells (RFCs) and plaque-forming cells (PFCs) responses as well as the in vitro proliferative response of murine splenocytes. The present results showed that induction of diabetes elicited a marked increase in blood glucose concentrations, a reduction in DTH, RFCs, PFCs immunological responses, as well as a decrease in splenocytes proliferation. However, treatment of diabetic rats with Ganoderma EPS alleviated all the above mentioned effects in a dose dependent manner. The present study concluded that treatment with Ganoderma EPS may have the ability to alleviate diabetes by reducing blood glucose concentrations and by enhancing immunity in STZ-diabetic rats sug-gesting its potential application as an antidiabetic agent in pharmaceutical and medical studies.

Effects of selenium-chitosan on blood selenium concentration, antioxidation status, and cellular and humoral immunity in mice.

One hundred and eighty Kunming mice were allotted to three groups in a randomized complete block design, including two treatments and one control. Mice in group 1 were fed a basal diet as control, while mice in groups 2 and 3 were fed the basal diet supplemented with 0.2mg/kg selenium as sodium selenite (SS) or selenium-chitosan (SC), respectively. On day 28 of the experiment, blood selenium concentration, glutathione peroxidase (GPx) activity, plasma superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and Con A-induced splenocyte proliferation were determined, and plasma interleukin-2 (IL-2) and interferon-γ (IFN-γ) concentrations, splenic plaque-forming cell (PFC) responses, serum hemolysis level (HC50), and delayed-type hypersensitivity (DTH) responses were determined on day 15 of the experiment. The results showed that blood selenium concentration, GPx activity, splenic PFC response, and plasma IL-2 and IFN-γ concentrations in SC group were higher than those in the control and SS groups (P < 0.01 or P < 0.05), respectively. Plasma SOD activity, Serum hemolysis level, DTH responses, and Con A-induced splenocyte proliferation in SC group were higher than those in control (P < 0.01 or P < 0.05). Plasma SOD activity, serum hemolysis level, DTH responses, and Con A-induced splenocyte proliferation in SC group were also higher than those in SS group, while there was no significant difference between SC and SS groups (P > 0.05). Plasma MDA content in SC group was lower than those in the control and SS groups (P < 0.01 or P < 0.05). It is concluded that SC supplement can increase blood selenium concentration, antioxidation status, and cellular and humoral immunity, and SC has better biological activity than SS in mice.

Immunotoxicity of the organochlorine pesticide methoxychlor in female ICR, BALB/c, and C3H/He mice

Several types of pesticides, including organochlorines, are known to suppress or modulate immune responses. The present study evaluated the immunotoxicity of the organochlorine pesticide methoxychlor (MXC) in female BALB/c, C3H/He, and ICR mice. Mice were given oral MXC doses of 0, 30, 100, and 300 mg/kg each day for 7 consecutive days. On day 4, the mice also received an intravenous injection of sheep red blood cells (SRBC). The splenic plaque-forming cell (PFC) IgM response and the serum anti-SRBC IgM antibody titer were evaluated while splenic lymphocytes were counted by flow cytometry and the spleen underwent histopathological analysis. Significant decreases in IgM PFC responses were seen in BALB/c, C3H/He, and ICR mice that received MXC doses of 100 and 300 mg/kg. Similar changes in serum anti-SRBC IgM antibody titers occurred in three strain mice. Flow cytometric analysis revealed significantly decreased splenic T-cell (CD3+) populations in a dose dependent manner in BALB/c mice, and in the 300 mg/kg of MXC-treated group of C3H/He mice. Germinal center (GC) B-cell (CD19+PNA+) populations were significantly decreased in the 300 mg/kg of MXC-treated groups of all three mouse strains and in the 30 and 100 mg/kg of MXC-treated groups of BALB/c and C3H/He strain mice. Histopathological analysis revealed decreased cellularity of the periarteriolar lymphoid sheath (PALS; T-cell area) and decreased GC development in all three strains of mice treated with 300 mg/kg MXC. These results suggest that MXC has an immune-suppressive effect in mice, and that our protocol may be useful for rapidly detecting immunosuppression induced by environmental chemicals.

Influence of peroral application of a herbal immunomodulator on the antibody production of Peyer's patches cells.

The influence of the peroral administration of the macromolecular components of a herbal immunomodulator isolated from an aqueous-ethanolic extract of the mixed herbal drugs Thujae summitates, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix on the function of Peyer's patches cells was investigated in mice. Peyer's patches cells isolated from mice which had received oral administration of the macromolecular fraction of the plant extract developed a significantly enhanced plaque-forming cell (PFC) response to sheep red blood cells after incubation with these cells in the presence of lipopolysaccharide or the extract fraction for 7 days in vitro. These results show that after oral administration of the herbal immunomodulator, the immunologically active macromolecules can contact the cells of the gut-associated lymphoid tissue and modulate the mucosal immune response.

Immunomodulatory Activities of &lt;i&gt;Dendrophthoe falcata&lt;/i&gt; (L.f) Ettingsh in Experimental Animals: &lt;i&gt;In vitro&lt;/i&gt; and &lt;i&gt;In vivo&lt;/i&gt; Investigations

In the present study, an attempt was made to screen immunomodulatory activity of the hydroalcoholic extract (HEDF) of Dendrophthoe falcata (L.f.) Ettingsh (Loranthaceae), an Indian Ayurvedic plant, on different arms of the immune system. HEDF was evaluated for immunological function by studying delayed type hypersensitivity (DTH) to sheep RBCs, nitric oxide (NO) release from murine peritoneal macrophages, phagocytic activity of polymorphonuclear (PMN) cells in vitro and reticuloendothelial system in vivo, plaque forming cell response of splenic lymphocytes to sheep erythrocytes, haemagglutination antibody titer and neutrophil adhesion test. Significant increase in NO production by mouse peritoneal macrophages was detected in culture supernatants indicated increased phagocytic activity of macrophages. After post oral administration of HEDF in three doses of 250, 475 and 950 mg/kg body weight, a significant increase in phagocytic activity of PMN cells/reticuloendothelial system, stimulation of neutrophil function and splenic antibody secreting cells, were also noticed. Stimulation of humoral immune response was further observed with elevation in haemagglutination antibody titer. Heightened DTH reaction suggested convincing evidence for activation of cellular immune system. Present study thus confirms the immunomodulatory activity of the hydroalcoholic extract of D. falcata and the immunomodulatory responses were found to be dose dependent manner.Keywords: Dendrophthoe falcata; Antibody titer; Neutrophil adhesion; Phagocytic activity.© 2011 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved.doi:10.3329/jsr.v3i3.7655 J. Sci. Res. 3 (3), 629-640 (2011)

Alterations in immune function and CYP450 activity of adult male deer mice following exposure to either 7,12-dimethylbenz[a]anthracene or benzo[b]fluoranthene

Adult male deer mice were exposed every other day for a period of 11 days to either 7,12-dimethylbenzanthracene (DMBA; CAS# 57-97-6) or benzo[b]fluoranthene (BbF; CAS# 205-99-2) (0, 0.3, 1, 3, 10, or 30 mg kg−1). Immune endpoints assessed were lymphocyte proliferation, macrophage pinocytosis, and the antibody plaque-forming cell (PFC) response. Cytochrome P450 (CYP450) activity was assessed using ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD). Macrophage pinocytosis was not altered by either compound. Both T- and B-cell proliferations were significantly increased by DMBA at 0.3 or 1 mg kg−1 and by BbF at 10 or 30 mg kg−1, but decreased by DMBA at 30 mg kg−1. Sheep red blood cell (SRBC)-specific-IgM production, as measured by the PFC response, was the most striking adverse immune effect observed and was significantly suppressed compared to control at all treatment concentrations for both compounds. EROD activity was markedly induced by DMBA at 30 mg kg−1, while BbF produced induction at 1, 10, or 30 mg kg−1. No marked effect on PROD activity was noted following DMBA treatment, but BbF-induced PROD activity at 1, 10, or 30 mg kg−1. Unexpectedly, four of six mice in the 30 mg DMBA kg−1 group did not survive to the end of the experiment, and one animal died in both the 3 and 10 mg kg−1 treatments. The calculated LD50 was 20.8 mg DMBA kg−1. The PFC response in deer mice was a more sensitive endpoint than CYP450 activity, suggesting that utilization of CYP450 endpoints in risk assessment without assessment of immune function, specifically antibody production, might possibly underestimate the risk to wild rodents environmentally exposed to polycyclic aromatic hydrocarbons.

Chronic effects of perfluorooctanesulfonate exposure on immunotoxicity in adult male C57BL/6 mice

A paucity of data exists to corroborate the few studies that report immune suppression after exposure to perfluorooctanesulfonate (PFOS). In this study, adult male C57BL/6 mice were exposed to PFOS daily via gavage for 60 days [0, 0.5, 5, 25, 50, or 125 mg/kg total administered dose (TAD)]. The results showed that liver mass was significantly increased at > or =5 mg PFOS/kg TAD and in a dose-dependent manner. Lymphocyte proliferation and natural killer cell activity were altered in male mice. Plaque forming cell (PFC) response was suppressed beginning at 5 mg/kg TAD. Based on the liver mass and PFC response, the no observed adverse effect level and lowest observed adverse effect level for male mice exposed PFOS for 60 days was 0.5 and 5 mg/kg TAD, respectively. Measured PFOS serum concentrations at these dose levels were 0.674 +/- 0.166 and 7.132 +/- 1.039 mg/l, respectively. These results indicate that PFOS exposure can affect the immunity function in mice at levels approximately 50-fold for highly exposed human populations.

N,N,-Diethyl-m-Toluamide (DEET) Suppresses Humoral Immunological Function in B6C3F1 Mice

N,N-diethyl-meta-toluamide (DEET) is a particularly effective broad-spectrum insect repellent used commonly in recreational, occupational and military environments. Due to its widespread use and suggested link to Gulf War Illness, this study examined the immunotoxicity of DEET. Adult female B6C3F1 mice were injected sc for 14 days with DEET at 0, 7.7, 15.5, 31, or 62 mg/kg/day. Due to differences in the dermal absorption of DEET between mice and humans, this study eliminated this confounding factor by utilizing sc injection and measured circulating blood levels of DEET to assess bioavailability from sc administration. Effects on lymphocyte proliferation, natural killer cell activity, thymus and spleen weight and cellularity, the antibody plaque-forming cell (PFC) response, and thymic and splenic CD4/CD8 lymphocyte subpopulations were assessed 24 h after the last dose. No effect was observed in lymphocyte proliferation, natural killer cell activity, thymic weight, splenic weight, thymic cellularity, or splenic cellularity. Significant decreases were observed in the percentage of splenic CD4-/CD8- and CD4+/CD8- lymphocytes but only at the 62 mg DEET/kg/day treatment level and not in absolute numbers of these cells types. Additionally, significant decreases in the antibody PFC response were observed following treatment with 15.5, 31, or 62 mg DEET/kg/day. Pharmacokinetic (PK) data from the current study indicate 95% bioavailability of the administered dose. Therefore, it is likely that DEET exposure ranges applied in this study are comparable to currently reported occupational usage. Together, the evidence for immunosuppression and available PK data suggest a potential human health risk associated with DEET in the occupational or military environments assuming similar sensitivity between human and rodent responses.

Chromium(III) Nanoparticles Affect Hormone and Immune Responses in Heat-Stressed Rats

This study was conducted to evaluate the effects of chromium nanoparticles (CrNano) on the hormone and immune responses of rats in heat stress condition. A total of 80 male Sprague-Dawley rats were randomly assigned to four dietary treatment groups (n = 20). The first group was offered a basal diet as a control. The second, third, and fourth groups received basal diet supplemented with 150, 300, and 450 microg/kg Cr, respectively, in the form of CrNano. At the end of the 8-week trial, growth performance, food utilization, and sera concentrations of hormones, immunoglobulins, and alexins were determined. Lymphocyte proliferation activity, antibody response to injected sheep red blood cells (SRBCs), and phagocytosis of peritoneal macrophages were determined by (3)H-thymidine uptake method, plaque-forming cells (PFC) assay, and ingesting chicken red blood cells test, respectively. The results indicated that rats that received CrNano exhibited no changes in growth rate and food efficiency compared to the control group. However, dietary supplementation of 150, 300, and 450 microg/kg Cr from CrNano significantly decreased serum concentrations of insulin and cortisol, increased sera levels of insulin-like growth factor I and immunoglobulin G, and enhanced the lymphoproliferative response, anti-SRBC PFC response, and phagocytic activity of peritoneal macrophages. These results suggest that dietary supplementation of Cr as CrNano affects hormone and immune status in heat-stressed rats.

Immunotoxic changes associated with a 7-day oral exposure to perfluorooctanesulfonate (PFOS) in adult male C57BL/6 mice

Perfluorooctanesulfonate (PFOS) is a widespread contaminant in the environment, as well as in wildlife and in humans. Toxicity tests in rodents have raised concerns about potential developmental, reproductive, and systemic effects of PFOS. However, there is little information about the effect of PFOS on immune system. In this study, adult male C57BL/6 mice were given by gavage 0, 5, 20 or 40 mg PFOS/kg day(-1) for 7 days. The results showed that PFOS exposure decreased food intake and body weight and increased liver mass and serum corticosterone levels in a dose-dependent manner. Flow cytometry analysis showed that the number of lymphocytic subpopulation cells decreased significantly in 20 or 40 mg PFOS/kg day(-1) group in comparison with normal C57BL/6 mice. Treatment with PFOS also markedly depressed the natural killer (NK) cell activity, lymphocyte proliferation and the plaque-forming cell (PFC) response. These results indicate that PFOS exposure can affect the immunity function in mice.

Adenosine-deaminase-associated immunodeficiency. I. Differential sensitivities of lymphocyte subpopulations exposed to 2-deoxycoformycin in vivo.

In order to obtain a better understanding of the degree of immune dysfunctions caused by the absence of adenosine deaminase, we gave a single i.p. injection of 2'-deoxycoformycin (2-dcf), a potent inhibitor of the enzyme ADA at various doses into adult Syrian hamsters. These animals were examined for their ability to mount primary in vivo antibody responses to helper T cell dependent (Th-d) and helper T cell independent (Th-ind) antigens. Hamsters treated with 0.5 mg/kg of 2-dcf mounted enhanced splenic plaque-forming cell (PFC) responses to sheep erythrocytes, a Th-d antigen, and to pneumococcal polysaccharide type III (SIII), a Th-ind antigen. Treatment of animals with 1.0 mg/kg of 2-dcf resulted in a significantly depressed (P less than 0.001) PFC response to Th-d antigen, but a further enhanced response to Th-ind antigen. One mechanism which may be responsible for such a dichotomous response to these two types of antigens was selective dysfunction of T cell subpopulations. At higher doses (1.5-4.0 mg/kg), PFC responses to both types of antigens were significantly suppressed. Immunoenhancement at low doses of 2-def was attributed to an increased susceptibility of T suppressor cells to 2-dcf. This hypothesis was confirmed by priming the 2-dcf-treated animals with low-dose Th-ind antigens. These animals failed to induce low-dose tolerance by stimulation of antigen-specific suppressor T cell subsets. At low doses, B cells and T helper cell functions were found to be intact, as further confirmed by priming the animals with the carrier keyhole limpet haemocyanin (KLH) and challenging with trinitrophenyl-KLH. This dose-dependent selective susceptibility of various T cell subpopulations and B cells may explain the heterogeneity of clinical, biochemical and immunological parameters observed in children with ADA deficiency severe combined immunodeficiency.

Immunosuppressive activity of FK-506 in rats: flow cytometric analysis of lymphocyte populations in blood, spleen and thymus during treatment and following drug withdrawal

Rats were immunized systemically with sheep red blood cells (SRBC) and given either FK-506 (1 mg/kg) or drug vehicle by i.m. injection for 7 days. In animals receiving FK-506, there was suppression (87%) of the splenic plaque-forming cell response on day 4 and marked reductions in the serum antibody titre throughout the 3-week period following immunization. Sequential flow cytometric analyses of blood lymphocytes revealed statistically significant attenuation, by FK-506, of the increase in relative numbers of OX-12+ (B) cells between days 4 and 7. Following drug withdrawal, OX-12 values remained elevated, whereas in control animals a decline was observed. These changes were reflected in concomitant increases in the relative numbers of OX-19+ (CD3+), W3/25+ (CD4+) and OX-8+ (CD8+) T cells; however, due to an overall reduction in lymphocytes by day 7, absolute values were not significantly affected compared with controls. The pattern of changes in OX-6+ (MHC class II+) cells in blood was similar to that observed for B cells. FK-506 also suppressed increases in the small proportion of blood-borne OX-40+ (activated CD4+) cells and OX-39+ (interleukin-2 receptor+) cells in the 7 day period following immunization; thereafter values for activation marker expression between treatment and control groups were similar. In the spleen, there were fewer significant differences between FK-506 and control groups in the incidences of cells expressing the above markers. OX-8+ cells, however, were significantly higher in drug-treated animals on day 7, and there were also reductions in the small proportions of OX-39+ and OX-40+ cells when compared with controls. In the thymus, reversible medullary atrophy induced by FK-506 was accompanied on day 7 by increases in the incidence of CD4+ and CD8+ cells and by a concomitant reduction in OX-44+ mature, medullary thymocytes. Two weeks after drug withdrawal, the phenotypic marker expression profile had been restored to normal in blood, spleen and thymus. These data provide new information on the apparent capacity of FK-506 to interfere with T cell maturation and its influence on lymphocyte activation in vivo.

Suppression of Humoral Immunity in Mice following Exposure to Perfluorooctane Sulfonate

Adult male and female B6C3F1 mice were exposed to perfluorooctane sulfonate (PFOS) daily via gavage for 28 days (0, 0.005, 0.05, 0.1, 0.5, 1, or 5 mg/kg total administered dose [TAD]). Following exposure, various immune parameters were assessed and serum PFOS concentrations were determined. Lymphocyte proliferation was not altered in either gender. Natural killer cell activity was increased compared with control at 0.5, 1, and 5 mg/kg TAD in male mice but was not altered in female mice. At these treatment levels, splenic T-cell immunophenotypes were minimally altered in females, but all T-cell subpopulations were significantly modulated in males beginning at 0.1 mg/kg TAD. The sheep red blood cell (SRBC) plaque-forming cell (PFC) response was suppressed in male mice beginning at 0.05 mg/kg TAD and in females at 0.5 mg/kg TAD. Serum trinitrophenyl (TNP)-specific IgM titers were also decreased by PFOS after TNP-LPS (TNP conjugated to lipopolysacharide) challenge suggesting that the humoral immune effects may be attributed to the B-cell rather than T-cell because both T-dependent (SRBC) and T-independent (TI) (TNP-LPS) antigens result in suppressed IgM production. Based on the PFC response, the low observed effect level (LOEL) for males was 0.05 mg/kg TAD (ED(50) = 0.021 mg/kg TAD) and for females was 0.5 mg/kg TAD (ED(50) = 0.59 mg/kg TAD). Measured PFOS serum concentrations at these dose levels were 91.5 +/- 22.2 ng/g and 666 +/- 108 ng/g (mean +/- SD), respectively. The male LOEL serum level was approximately 14-fold lower than reported mean blood levels from occupationally exposed humans and fell in the upper range of concentrations reported for the general population. Overall, this study provides a profile of PFOS immunotoxicity showing effects at levels reported in humans and identifies the B-cells as a potential target.

Alterations in Immune Function and CYP450 Activity in Adult Male Deer Mice (Peromyscus maniculatus) Following Exposure to Benzo[a]pyrene, Pyrene, or Chrysene

Polycyclic aromatic hydrocarbons (PAHs) are among the most common classes of chemical contaminants found at hazardous waste sites. Deer mice (Peromyscus maniculatus) exhibit a wide geographic distribution throughout North America and have been suggested as a terrestrial biomonitoring species to facilitate comparisons between superfund sites. Chemicals tested were benzo[a]pyrene (BaP; CAS number 50-32-8), pyrene (Pyr; CAS number 129-00-0), and chrysene (Chr; CAS number 218-01-9). Adult male deer mice were exposed via intraperitoneal (ip) injection every other day for 11 d to the PAHs (0.3, 1, 3, 10, or 30 mg/kg) or a corn oil carrier control. Both BaP and Chr suppressed the plaque-forming cell (PFC) response at all treatment levels. Pyr exposure (1–30 mg/kg) also resulted in suppression of this response. Macrophage pinocytosis was suppressed only by Chr (3, 10, and 30 mg/kg). Concanavalin A-induced proliferation was stimulated by BaP at all dose levels, by Pyr at 1–30 mg/kg, and by Chr at 30 mg/kg. Chr did not affect pokeweed mitogen (PWM)-induced proliferation; however, BaP (1–30 mg/kg) and Pyr (0.3–30 mg/kg) produced stimulation of this response as compared to respective controls. BaP and Chr stimulated cytochrome P-450 1A1 (CYP1A1) activity (3, 10, or 30 mg/kg) as measured by ethoxyresorufin O-deethylase (EROD) activity, but Pyr did not. These results indicate that immune function endpoints appear to be more sensitive to these PAHs than measured hepatic CYP450 activity.

Analysis of Immunotoxicity by Enumeration of Antibody‐Producing B Cells

The plaque-forming cell (PFC) assay measures the humoral immune response mediated by the concerted actions of antigen-presenting cells, T lymphocytes, and B lymphocytes. The most common form of the plaque method is used for the detection of murine primary IgM antibodies directed against the T cell-dependent sheep red blood cell (sRBC) surface antigens. Research has shown that the PFC response to sRBC is not only an excellent monitor of the primary effector function of the B cell, but that it may be the most sensitive immune parameter currently available to identify chemical perturbation. Several modifications and variations of the PFC assay are described in detail in this unit.

Recovery of age‐dependent immunological deterioration in old mice by thyroxine treatment

On the basis that multiple interactions exist between thyroid hormones and immune system, and ageing is accompanied by changes in thyroid hormone secretion, it seems possible that thyroid hormones may be involved in the age-related immune dysfunction. The present study was conducted to evaluate in vivo and in vitro effects of thyroxine (T(4)) treatment on both cell-mediated and humoral immune responses of aged mice. In a trial to improve age-associated immune dysfunction, T(4) (0.2, 1.0 and 5.0 microg) was subcutaneously supplemented to BALB/c mice (over 18 months old) for 30 consecutive days. The present results showed that exogenous treatment of aged mice with T(4) was associated with a marked increase in serum T(4) level, and the total number of peripheral blood leukocytes as well as the total cellularity of thymus, spleen, peripheral lymph nodes (PLNs), mesenteric lymph nodes (MLNs) and bone marrow (BM). T(4) treatment also caused a significant increase in the total and differential numbers of peritoneal exudate cells (PECs), while it caused a slight increase in macrophages' phagocytic activity of PEC. Moreover, T(4) treatment elicited a statistically significant increase in both plaque-forming cell and rosette-forming cell responses. In vitro results showed that the addition of T(4) at concentrations of 0.001, 0.005 and 0.025 microg/well substantially potentiated the ability of splenocytes from aged mice to proliferate in the presence of concanavalin-A mitogen. Histological examination of thymuses from T(4)-treated aged mice revealed that the cortex was preferentially enlarged and repopulated with immature thymocytes. The present study postulates that thyroid hormones may be involved in the observed decrease in the immune responsiveness during ageing, and that T(4) treatment to aged mice is able to restore the age-related decline of the immune efficiency.

Splenic macrophages and B cells mediate immunosuppression following abrupt withdrawal from morphine

We have previously shown that abrupt withdrawal (AW) from morphine induces greater than 80% immunosuppression in murine spleen cells, as assessed by the capacity to mount an in vitro plaque-forming cell response to sheep red blood cells. Present studies about the mechanisms of immunosuppression following AW showed that addition of highly enriched (CD11b+) splenic macrophages (obtained by cell sorting or magnetic separation) from AW mice to cultures of normal, unfractionated spleen cells suppressed immune responses. Further, addition of highly enriched (CD19+) B cells (but not T cells) from AW mice to normal cells was also immunosuppressive. B cells from AW mice were also able to inhibit the proliferative response of normal spleen cells to concanavalin A but not to lipopolysaccharide. Overall, the data suggest that immunosuppression by AW spleen cells is a result of active suppression by macrophages and B cells.

Novel sulfated polysaccharide derived from red-tide microalga Gyrodinium impudicum strain KG03 with immunostimulating activity in vivo

The high-sulfate-containing exopolysaccharide p-KG03 is produced by the red-tide microalga Gyrodinium impudicum strain KG03. The immunostimulatory effects of this sulfated exopolysaccharide were investigated by isolating peritoneal macrophages from mice 10 or 20 days after they had received a single dose of p-KG03 (100 or 200 mg/kg body weight). The cytotoxicity of the isolated macrophages for B16 tumor cells was tested, as B16 tumor cells are sensitive to tumor necrosis factor alpha (TNF-alpha) and nitric oxide. The activities of natural killer cells from the p-KG03-treated mice against YAC-1 mouse lymphoma cells were also tested. The nonspecific immune functions mediated by natural killer cells and macrophages were increased by treatment with p-KG03 in vivo. These results suggest that p-KG03 has immunostimulatory effects and enhances the tumoricidal activities of macrophages and NK cells in vivo. In addition, p-KG03 treatment increased the plaque-forming cell response to sheep red blood cells, as well as the levels of IgM and IgG Exposure to p-KG03 also increased the production by macrophages of cytokines, such as interleukins -1beta and -6, and TNF-alpha. This is the first report of a marine microalgal sulfated polysaccharide having immunostimulatory activities. The p-KG03 polysaccharide may be useful for the development of biotechnological and pharmaceutical products that incorporate bioactive marine exopolysaccharides.

Cytochrome P450 1B1 Is Required for 7,12-Dimethylbenz(a)-anthracene (DMBA) Induced Spleen Cell Immunotoxicity

7,12-Dimethylbenz(a)anthracene (DMBA) is a potent carcinogen that induces immunosuppression of both humoral and cell-mediated immunity in mice and other species. Previous studies have shown that CYP1B1 is required for bone marrow toxicity produced by DMBA in mice. Therefore, the purpose of these studies was to determine whether CYP1B1 was required for spleen cell immunotoxicity. Female C57BL/6N wild-type (WT) and CYP1B1 knockout (-/-) mice were treated with 0, 17, 50, or 150 mg/kg (cumulative dose) DMBA in corn oil by oral gavage once a day for five days. Several immunotoxicological assays were used to assess the effects of DMBA on systemic immunity. These included the in vitro T-dependent antibody response to sheep red blood cells (SRBC) measured using a direct plaque forming cell (PFC) assay, T- and B-cell mitogenesis induced by Con A and LPS, and nonspecific cell-mediated immunity was evaluated using an NK cytotoxicity assay. In addition, lymphocyte subpopulations were measured by flow cytometry using specific cell surface markers. Following five days of DMBA treatment, the body weights and spleen cell surface markers of the WT and CYP1B1 (-/-) mice showed no significant changes. A decrease in NK activity was found at the 50 mg/kg DMBA dose in WT mice, but not in the CYP1B1 (-/-) mice. Interestingly, at the 150 mg/kg dose of DMBA, CYP1B1 null mice had decreased NK activity, whereas WT mice did not. The SRBC PFC response demonstrated that the IgM antibody response was suppressed by DMBA in WT mice in a dose-dependent manner (significant at 50 and 150 mg/kg). However, there were no changes in the SRBC PFC responses in any DMBA test group in the CYP1B1 (-/-) mice. Similarly, while DMBA suppressed B- and T-cell mitogenesis at the 50 and 150 mg/kg dose levels in C57BL/6N WT mice, no effect was seen in CYP1B1 (-/-) mice. Thus, CYP1B1 appears to be critical for the immunosuppression of DMBA in mice, suggesting a role for bioreactive metabolites in the spleen cell immunotoxicity produced by DMBA.