Anti-keyhole Limpet Hemocyanin Research Articles

Evaluation of immunotoxicity of sodium metavanadate following drinking water exposure in female B6C3F1/N mice in a 28-day study.

Sodium metavanadate (NaVO3 ) is a pentavalent vanadium compound used in the metal industry and dietary supplements; human exposure occurs through inhalation of fumes and dust and ingestion of NaVO3 -containing products. The objective of this study was to assess the potential immunotoxicity of NaVO3 . Female B6C3F1/N mice were exposed to 0-500 ppm NaVO3 in drinking water for 28 days and evaluated for effects on immune cell populations and innate, cellular-mediated, and humoral-mediated immunity. There was a decreasing trend in body weight (BW) and BW gain in NaVO3 exposed mice, with a decrease (p ≤ 0.05) in BW gain at ≥250 ppm, relative to control. Conversely, increasing trends in spleen weights and an increase (p ≤ 0.05) in the spleen:BW ratio at ≥250 ppm NaVO3 were observed. NaVO3 exposure altered antibody production against sheep red blood cells (SRBC). Antibody forming cells (AFC)/106 spleen cells exhibited a decreasing trend, with a decrease (p ≤ 0.05) at 500 ppm NaVO3 , concurrent with an increase in percent B cells. NaVO3 had no effect on the serum anti-SRBC IgM antibody titers or anti-keyhole limpet hemocyanin antibody production. Exposure to NaVO3 decreased the percentage of natural killer cells at all dose levels (p ≤ 0.05), with no effect on the lytic activity. NaVO3 altered T-cell populations at 500 ppm but had no effect on T-cell proliferative responses or the lytic activity of cytotoxic T cells. Collectively, these data indicate that NaVO3 exposure can adversely affect the immune system by inducing alterations in humoral-mediated immunity, specifically the AFC response, with no effect on cell-mediated or innate immunity.

Enhancement of adaptive immune responses of aged mice by dietary intake of β-glucans, with special emphasis on anti-aging activity

The naturally occurring polysaccharide, β-1,3-glucans, a well-known immunostimulant, has been highly valued for many years for their health-promoting and anti-aging properties, but its mode of action is poorly understood. In this study, we first showed that oral administration of β-1,3-glucans did not affect the general condition and physiology of male mice throughout the trial period. We then showed that dietary intake of β-1,3-glucans induced a significant increase in T helper cells (CD4+) in young, middle-aged and aged male mice. We also showed that β-1,3-glucans supplementation considerably increased the delayed-type hypersensitivity (DTH) response, a T cell-mediated immune response, in young and aged mice. In addition, we found that β-1,3-glucans supplementation remarkably promoted the production of total anti-keyhole limpet hemocyanin (KLH) immunoglobulin G (IgG), anti-KLH IgG1, and anti-KLH IgG2a in young and aged mice without disturbing immune homeostasis. These data together indicate that oral administration of β-1,3-glucans enhanced the adaptive immune responses of aged mice without disturbing their general condition and physiology, supporting the idea that β-1,3-glucans are capable of counteracting the immunosenescence in mice. They also suggest that β-1,3-glucans can be clinically useful to help the elderly generate an improved response to vaccine with stronger humoral and cell-mediated immune responses.

T-Cell-Specific Loss of the PI-3-Kinase p110α Catalytic Subunit Results in Enhanced Cytokine Production and Antitumor Response

Class IA phosphatidylinositol 3-kinase (PI3K) catalytic subunits p110α and p110δ are targets in cancer therapy expressed at high levels in T lymphocytes. The role of p110δ PI3K in normal or pathological immune responses is well established, yet the importance of p110α subunits in T cell-dependent immune responses is not clear. To address this problem, mice with p110α conditionally deleted in CD4+ and CD8+ T lymphocytes (p110α−/−ΔT) were used. p110α−/−ΔT mice show normal development of T cell subsets, but slightly reduced numbers of CD4+ T cells in the spleen. “In vitro,” TCR/CD3 plus CD28 activation of naive CD4+ and CD8+ p110α−/−ΔT T cells showed enhanced effector function, particularly IFN-γ secretion, T-bet induction, and Akt, Erk, or P38 activation. Tfh derived from p110α−/−ΔT cells also have enhanced responses when compared to normal mice, and IL-2 expanded p110α−/−ΔT CD8+ T cells had enhanced levels of LAMP-1 and Granzyme B. By contrast, the expansion of p110α−/−ΔT iTreg cells was diminished. Also, p110α−/−ΔT mice had enhanced anti-keyhole limpet hemocyanin (KLH) IFN-γ, or IL-4 responses and IgG1 and IgG2b anti-KLH antibodies, using CFA or Alum as adjuvant, respectively. When compared to WT mice, p110α−/−ΔT mice inoculated with B16.F10 melanoma showed delayed tumor progression. The percentage of CD8+ T lymphocytes was higher and the percentage of Treg cells lower in the spleen of tumor-bearing p110α−/−ΔT mice. Also, IFN-γ production in tumor antigen-activated spleen cells was enhanced. Thus, PI3K p110α plays a significant role in antigen activation and differentiation of CD4+ and CD8+ T lymphocytes modulating antitumor immunity.

Organization of glomerular territories in the olfactory bulb of post-embryonic wild chinook salmon Oncorhynchus tshawytscha.

The post-embryonic odor imprinting paradigm suggests Chinook salmon (Oncorhynchus tshawytscha) acquire memory to stream-specific amino acid olfactory odors prior to emergence as fry. Because effects of olfactory experience on development can be examined by mapping olfactory sensory neurons extending into distinct territories of glomerular neuropil in the olfactory bulb, glomerular patterning from early yolk-sac larva to fry was documented in wild salmonids, a temporal scale not yet thoroughly explored. Labeling olfactory sensory neurons with anti-keyhole limpet hemocyanin (anti-KLH) revealed seven spatially conserved glomerular territories visible at hatch and well established by the late yolk-sac larva developmental stage. Because of the responsiveness of microvillous olfactory sensory neurons to amino acids, corresponding glomeruli in the lateral bulbar region were mapped using anti-calretinin. The dorsolateral territory, distinct glomeruli of the lateral glomerular territory and the ventromedial glomeruli were immunoreactive to both KLH and calretinin. This study offers a morphological description of glomerular patterning in post-embryonic stages in wild Chinook salmon, a temporal window previously shown to be significant for olfactory imprinting. J. Morphol. 278:464-474, 2017. © 2017 Wiley Periodicals, Inc.

Short day length enhances physiological resilience of the immune system against 2-deoxy-d-glucose-induced metabolic stress in a tropical seasonal breeder Funambulus pennanti

Studies demonstrate the importance of metabolic resources in the regulation of reproduction and immune functions in seasonal breeders. In this regard, the restricted energy availability can be considered as an environmental variable that may act as a seasonal stressor and can lead to compromised immune functions. The present study explored the effect of photoperiodic variation in the regulation of immune function under metabolic stress condition. The T-cell-dependent immune response in a tropical seasonal breeder Funambulus pennanti was studied following the inhibition of cellular glucose utilization with 2-deoxy-d-glucose (2-DG). 2-DG treatment resulted in the suppression of general (e.g., proliferative response of lymphocytes) and antigen-specific [anti-keyhole limpet hemocyanin IgG titer and delayed-type hypersensitivity response] T-cell responses with an activation of the hypothalamic–pituitary–adrenal axis, which was evident from the increased levels of plasma corticosterone. 2-DG administration increased the production of inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α] and decreased the autocrine T-cell growth factor IL-2. The immunocompromising effect of 2-DG administration was retarded in animals exposed to short photoperiods compared with the control and long photoperiod-exposed groups. This finding suggested that short photoperiodic conditions enhanced the resilience of the immune system, possibly by diverting metabolic resources from the reproductive organs toward the immune system. In addition, melatonin may have facilitated the energy “trade-off” between reproductive and immune mechanisms, thereby providing an advantage to the seasonal breeders for their survival during stressful environmental conditions.

The Planorbid Snail Biomphalaria glabrata Expresses a Hemocyanin-Like Sequence in the Albumen Gland.

The parasitic flatworm Schistosoma mansoni, causative agent of human intestinal schistosomiasis in South America, relies importantly on the freshwater snail Biomphalaria glabrata as intermediate host to achieve development of cercariae that infect humans. The recommendation from the World Health Organization (WHO) to integrate snail control in efforts to counter schistosomiasis transmission provides impetus for in depth study of B. glabrata biology. Our analysis indicates that two distinct hemocyanin-like genes (hcl-1 and hcl-2) are present in B. glabrata, a snail that uses hemoglobin for oxygen transport. Characterization of BAC clones yielded the full length hcl-1 gene, which is comprised of three functional unit (FU) domains at the amino acid level. Database searches and in silico analyses identified the second hcl gene (hcl-2), composed of six FU domains. Both genes are unusual for lacking canonical residues and having fewer FU domains than typical molluscan hemocyanins that contain 7–8 FUs. Reverse transcription PCR demonstrated that Hcl-1 is expressed in a manner that correlates with reproductive maturity in the albumen gland (AG), an immune- and reproduction-relevant organ. Immune cross-reactivity with anti-keyhole limpet hemocyanin (α-KLH) antiserum and tandem-mass spectrometry validated the presence of Hcl-1 protein in the AG and egg mass fluid (EMF). The evolutionary conservation of hemocyanin-like sequences in B. glabrata in the presence of the oxygen carrier hemoglobin, combined with our results, suggest that the Hcl-1protein has a functional role in general and/or reproductive biology. Further investigations are needed to explore Hcl-1 as a potential target for snail control.

Trace metals, melanin-based pigmentation and their interaction influence immune parameters in feral pigeons (Columba livia).

Understanding the effects of trace metals emitted by anthropogenic activities on wildlife is of great concern in urban ecology; yet, information on how they affect individuals, populations, communities and ecosystems remains scarce. In particular, trace metals may impact survival by altering the immune system response to parasites. Plumage melanin is assumed to influence the effects of trace metals on immunity owing to its ability to bind metal ions in feathers and its synthesis being coded by a pleiotropic gene. We thus hypothesized that trace metal exposure would interact with plumage colouration in shaping immune response. We experimentally investigated the interactive effect between exposure to an environmentally relevant range of zinc and/or lead and melanin-based plumage colouration on components of the immune system in feral pigeons (Columba livia). We found that zinc increased anti-keyhole limpet hemocyanin (KLH) IgY primary response maintenance, buffered the negative effect of lead on anti-KLH IgY secondary response maintenance and tended to increase T-cell mediated phytohaemagglutinin (PHA) skin response. Lead decreased the peak of the anti-KLH IgY secondary response. In addition, pheomelanic pigeons exhibited a higher secondary anti-KLH IgY response than did eumelanic ones. Finally, T-cell mediated PHA skin response decreased with increasing plumage eumelanin level of birds exposed to lead. Neither treatments nor plumage colouration correlated with endoparasite intensity. Overall, our study points out the effects of trace metals on some parameters of birds' immunity, independently from other confounding urbanization factors, and underlines the need to investigate their impacts on other life history traits and their consequences in the ecology and evolution of host-parasite interactions.

Immunosuppressive CD11b+Ly6Chi monocytes in pristane-induced lupus mouse model.

Myeloid-derived suppressor cells with immunosuppressive functions have been described to be associated with one of the mechanisms by which malignant tumors escape immune surveillance. However, little is known about the role of myeloid-derived suppressor cells in autoimmunity. In the current study, when we attempted to characterize the peritoneal cells in pristane-induced lupus model, as reported previously, we observed that there were markedly increased CD11b(+)Ly6C(hi) monocytes. Surprisingly, this type of monocytes was almost phenotypically identical to the reported monocytic myeloid-derived suppressor cells. Further analysis on how these CD11b(+)Ly6C(hi) cells affected T cell response showed that they strongly suppressed T cell proliferation in vitro in a manner dependent on cell-cell contact, NO, and PGE2. In addition, we found that CD11b(+)Ly6C(hi) monocytes inhibited Th1 differentiation but enhanced development of forkhead box p3(+)CD4(+) regulatory T cells. Consistent with the in vitro experimental results, the in vivo adoptive cell transfer study showed that infusion of pristane-treated syngeneic CD11b(+)Ly6C(hi) monocytes significantly suppressed the production of anti-keyhole limpet hemocyanin antibodies induced by keyhole limpet hemocyanin immunization. In addition, we found that CD11b(+)Ly6C(hi) monocytes were also increased significantly in spleen and peripheral blood and showed immunosuppressive characteristics similar to their peritoneal counterparts. Our findings indicate that CD11b(+)Ly6C(hi) monocytes in a pristane-induced lupus mouse model are monocytic myeloid-derived suppressor cells instead of inflammatory monocytes, as demonstrated previously. To our knowledge, this is the first to describe myeloid-derived suppressor cells in a pristane-induced lupus mouse model, which may lead to a better understanding of the role of CD11b(+)Ly6C(hi) monocytes in this specific pristane-induced lupus model.

The synergistic effect of density stress during the maternal period and adulthood on immune traits of root vole (Microtus oeconomus) individuals-a field experiment.

The literature reveals that stress in early life or adulthood can influence immune function. As most studies on this are from the laboratory, there is a need for replicated studies in wild animals. This study aims to examine the effects of density stress during the maternal period and adulthood on immune traits of root vole (Microtus oeconomus) individuals. Four replicated high- and low-density parental populations were established, from which we obtained offspring and assigned each into four enclosures, two for each of the two density treatments used in establishing parental populations. The F1 offspring fecal corticosterone metabolite response to acute immobilization stress, anti-keyhole limpet hemocyanin immunoglobulin G (anti-KLH IgG) level, phytohemagglutinin (PHA)-delayed hypersensitivity and hematology at the end of the first breeding season, and prevalence and intensity of coccidial infection throughout the two breeding seasons, were tested. Density-induced maternally stressed offspring had delayed responses to acute immobilization stress. Density-stressed offspring as adults had reduced anti-KLH IgG levels and PHA responses, and the effects further deteriorated in maternally stressed offspring, leading to higher coccidial infection in the first breeding season than in the second. No correlations were found between immune traits or coccidial infection and survival over winter. These findings indicated that the combined density stresses during the maternal period and adulthood exhibited negative synergistic effects on immune traits. The synergistic effects lead to higher coccidial infection; however, this consequently reduced the risk of subsequent infection. The increased coccidial infection mediated by the synergistic effects may have an adaptive value in the context of the environment.

Plasticity of glomeruli and olfactory-mediated behavior in zebrafish following detergent lesioning of the olfactory epithelium

The zebrafish olfactory system is a valuable model for examining neural regeneration after damage due to the remarkable plasticity of this sensory system and of fish species. We applied detergent to the olfactory organ and examined the effects on both morphology and function of the olfactory system in adult zebrafish. Olfactory organs were treated once with Triton X-100 unilaterally to study glomerular innervation patterns or bilaterally to study odor detection. Fish were allowed to recover for 4–10days and were compared to untreated control fish. Axonal projections were analyzed using whole mount immunocytochemistry with anti-keyhole limpet hemocyanin, a marker of olfactory axons in teleosts. Chemical lesioning of the olfactory organ with a single dose of Triton X-100 had profound effects on glomerular distribution in the olfactory bulb at 4days after treatment, with the most significant effects in the medial region of the bulb. Glomeruli had returned by 7days post-treatment. Analysis of the ability of the fish to detect cocktails of amino acids or bile salts consisted of counting the number of turns the fish made before and after odorant delivery. Control fish turned more after exposure to both odorants. Fish tested 4 and 7days after chemical lesioning made more turns in response to amino acids but did not respond to bile salts. At 10days post-lesion, these fish had regained the ability to detect bile salts. Thus, the changes seen in bulbar innervation patterns correlated to odorant-mediated behavior. We show that the adult zebrafish brain has the capacity to recover rapidly from detergent damage of the olfactory epithelium, with both glomerular distribution and odorant-mediated behavior returning in 10days.

The effects of light stimulation during incubation on indicators of stress susceptibility in broilers

Providing light during incubation has been shown to decrease bilateral physical asymmetry of broilers posthatch, which may indicate that early light stimulation reduces later stress susceptibility. This experiment evaluated the effects of lighting during embryogenesis on other measures of stress responsiveness in broilers. Cobb 500 eggs (n = 1,404) were incubated under 0L:24D, 1L:23D, 6L:18D, or 12L:12D; the light level was 550 lx. The broilers were then raised in floor pens under a 12L:12D lighting regimen, and various stress parameters were measured during wk 3 to 6 of age. There was an effect of incubation lighting regimen on posthatch stress responses. Following 1 h of crating, the change in corticosterone (CORT) concentration was less in the 12L:12D (−0.06 ng/mL, P < 0.05) treatment than all other treatments (pooled mean = 0.24 ng/mL); however, there were no treatment differences in CORT response to adrenocorticotropic hormone administration (pooled mean pre- vs. 1 h postadministration = 17.5 ng/mL, P > 0.05). Anti-keyhole limpet hemocyanin titers were higher in the 12L:12D birds (92,395 units/mL; P < 0.05) than all other treatments (pooled mean = 68,407 units/mL) on d 1 postcrating. Additionally, composite asymmetry scores were lower in the 12L:12D treatment (0.92 mm) than all other treatments (pooled mean = 1.14 mm, P < 0.05). These results demonstrate that providing 12 h of light per day during incubation can reduce the stress susceptibility of broilers posthatch.

Promotion of anti-keyhole limpet hemocyanin IgM and IgG antibody productions in vitro by red bell pepper extract

Red bell pepper (Capsicum annuum L.), eaten as vegetables, is a rich source of vitamin C and E, provitamin A, β-carotene, and numerous phenolic compounds. Its antioxidant, anti-mutagenic and anti-inflammatory properties have been reported. However, scientific report on its immunomodulatory activities is scarce. We, for the first time, reported the promotion of polyclonal immunoglobulin (Ig) M production by red bell pepper extract (RBPE). The present study was aimed to investigate the activity of RBPE on keyhole limpet hemocyanin (KLH)-specific IgM and IgG antibody productions in vitro. Anti-KLH antibodies, anti-KLH IgM secreting cells, and viable cells were determined by ELISA, ELISPOT and MTT assays, respectively. RBPE (0.75 and 1.5mg/mL) significantly enhanced the production of anti-KLH IgM in cultured splenocytes of non-immunized (189 and 184%, respectively) and immunized mice (145% at 1.5mg/mL). The same doses promoted the formation of anti-KLH IgM secreting cells 185 and 232%, respectively. Moreover, RBPE (0.375mg/mL) augmented anti-KLH IgG production by 145 and 157% in cultured splenocytes collected from mice 1 and 3weeks after a booster injection. Thus, RBPE induced KLH-specific antibody productions. The findings of the present study suggest that consumption of RBPE or vegetables may be beneficial to strengthen humoral immune responses.

Photoperiodic induced melatonin regulates immunity and expression pattern of melatonin receptor MT1 in spleen and bone marrow mononuclear cells of male golden hamster

The pineal gland transduces day length information into chemical signal of melatonin that ultimately translates photic stimulus into season-specific immune responses to promote survival of individual from incidence of opportunistic diseases. To date, the immune adjustments being a result of photoperiodic exposures for any nocturnal seasonally breeding rodent have not been systematically examined. Therefore, we evaluated the humoral and cell mediated immune responses of photoperiodically entrained male golden hamsters. Short day induced melatonin increased the immune parameters such as spleen mass, total leukocyte (TLC) and lymphocyte count (LC), proliferation of splenocytes, peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMNCs) along with serum IL-2 and anti-Keyhole Limpet Hemocyanin (KLH) IgG production when compared with long day experienced hamsters. Short term melatonin treatment (for two weeks) to long day hamsters enhanced to some extent the proliferation of splenocytes, PBMC and TLC/LC. We also localized the melatonin membrane receptor MT1 in spleen and BMMNCs that strongly supported our western blot analysis for the expression of MT1 in spleen suggesting that different photoperiodically induced circulatory melatonin is responsible for the immunomodulation. Therefore, photoperiod can influence the peripheral melatonin level to improve immune responses of hamsters according to season for better survival.

Dietary Intake of Resveratrol Enhances the Adaptive Immunity of Aged Rats

It is well known that immune response declines with aging. Resveratrol, a polyphenol that occurs naturally in several plant species including grapevines and berries, has been shown to have potent antiaging and health-promoting activities. However, the mechanism underlying these activities remains largely unknown. Here we clearly demonstrate that: (1) Dietary intake of resveratrol induced a significant increase in T helper cells (CD4(+)) in middle-aged (12 months old) and aged (21 months old) Wistar male rats; (2) resveratrol supplementation considerably increased the delayed-type hypersensitivity response, a T cell-mediated immune response, in aged rats; and (3) reveratrol supplementation remarkably promoted the production of total anti-keyhole limpet hemocyanin (KLH) immunoglobulin G (IgG), anti-KLH IgG(1), and anti-KLH IgG(2α) in aged rats without disturbing immune homeostasis. These data together indicate that resveratrol is capable of counteracting immunosenescence, thereby leading to rejuvenation. In practice, resveratrol can be useful to help the elderly generate an improved response to vaccine with stronger humoral and cell-mediated immune responses.

No Evidence for a Trade-Off between Reproductive Investment and Immunity in a Rodent

Life history theory assumes there are trade-offs between competing functions such as reproduction and immunity. Although well studied in birds, studies of the trade-offs between reproduction and immunity in small mammals are scarce. Here we examined whether reduced immunity is a consequence of reproductive effort in lactating Brandt's voles (Lasiopodomys brandtii). Specifically, we tested the effects of lactation on immune function (Experiment I). The results showed that food intake and resting metabolic rate (RMR) were higher in lactating voles (6≤ litter size ≤8) than that in non-reproductive voles. Contrary to our expectation, lactating voles also had higher levels of serum total Immunoglobulin G (IgG) and anti-keyhole limpet hemocyanin (KLH) IgG and no change in phytohemagglutinin (PHA) response and anti-KLH Immunoglobulin M (IgM) compared with non-reproductive voles, suggesting improved rather than reduced immune function. To further test the effect of differences in reproductive investment on immunity, we compared the responses between natural large (n≥8) and small litter size (n≤6) (Experiment II) and manipulated large (11–13) and small litter size (2–3) (Experiment III). During peak lactation, acquired immunity (PHA response, anti-KLH IgG and anti-KLH IgM) was not significantly different between voles raising large or small litters in both experiments, despite the measured difference in reproductive investment (greater litter size, litter mass, RMR and food intake in the voles raising larger litters). Total IgG was higher in voles with natural large litter size than those with natural small litter size, but decreased in the enlarged litter size group compared with control and reduced group. Our results showed that immune function is not suppressed to compensate the high energy demands during lactation in Brandt's voles and contrasting the situation in birds, is unlikely to be an important aspect mediating the trade-off between reproduction and survival.

Immune responses to lipopolysaccharide challenge in a tropical rodent (Funambulus pennanti): Photoperiod entrainment and sex differences

Annual variation in day length (photoperiod) triggers changes in the immune system of seasonal breeders. The rationale behind this study was to delineate any sex differences in immune responses of photoperiodically entrained animals challenged against lipopolysaccharide (LPS)-induced inflammatory stress. We observed that photoperiodically entrained [short day, SD, 10 h light (L):14 h dark (D); long day, LD, 16 h L:8 h D; and natural day length, NDL, 12 h L:12 h D] male and female Indian palm squirrels, Funambulus pennanti, presented sexual dimorphism in immune status after LPS-induced stress. Females presented high humoral (anti-keyhole limpet hemocyanin immunoglobulin) and cellular immunity (lymphocyte proliferation) compared with the males of all photoperiodic conditions. Female squirrels showed reduced pro-inflammatory cytokine levels (interleukin-1β, interleukin-6, and tumor necrosis factor-α) than the males suggesting their high efficiency to recover from LPS-induced inflammatory stress. Increased duration of melatonin secretion and corticosterone concentration in squirrels experiencing SD evidently supported survival of squirrels as compared with control (NDL) and LD squirrels of both sexes. Decreased immune status in both sexes under LD condition might be due to a short melatonin signal mimicking the LDs of summer. Thus, we infer that photoperiodic entrainment via the levels of melatonin and corticosterone synergistically supported more the survival of female squirrels under LPS-induced stress.

Stable and Functional Lymphoid Reconstitution in Artemis-deficient Mice Following Lentiviral Artemis Gene Transfer Into Hematopoietic Stem Cells

Patients with mutations in the Artemis gene display a complete absence of T- and B lymphocytes, together with increased cellular radiosensitivity; this leads to a radiosensitive severe combined immunodeficiency (RS-SCID). Allogenic hematopoietic stem-cell (HSC) transplantation is only partially successful in the absence of an human leukocyte antigen-genoidentical donor, and this has prompted a search for alternative therapeutic approaches such as gene therapy. In this study, a self-inactivated lentiviral vector expressing Artemis was used to complement the Artemis knockout mouse (Art(-/-)). Transplantation of Artemis-transduced HSCs into irradiated Art(-/-) mice restored a stable (over a 15-month period of follow-up) and functional T- and cell repertoire that was comparable to that of control mice. The success of secondary transplantations demonstrated that the HSCs had been transduced. One of thirteen mice developed a thymoma 6 months after gene therapy. Although thymic cells were seen to be carrying two lentiviral integration sites, there was no evidence of lentivirus-driven oncogene activation. The Art(-/-) mice were found to be prone to develop T-cell lymphomas, either spontaneously or after irradiation. These data indicate that the observed lymphoproliferation was probably the consequence of the chromosomal instability associated with the Artemis-deficient background. As a whole, our work provides a basis for supporting the gene therapy approach in Artemis-deficient SCID.

Immunotoxicity Evaluation by Immune Function Tests: Focus on the T-Dependent Antibody Response (TDAR) [Overview of a Workshop Session at the 45th Annual Meeting of the Society of Toxicology (SOT) March 5–9, 2006 San Diego, CA

Increased expectations from a number of regulatory agencies, e.g., Environmental Protection Agency (EPA), Food and Drug Administration (FDA), European Medicines Agency (EMEA), and the Ministry of Health, Labour and Welfare (MHLW) of Japan, call for the evaluation of potential adverse effects on the immune system. As recently summarized in the ICH S8 guideline, the T-cell-dependent antibody response (TDAR) has been identified in a regulatory context as a main functional test of immunotoxicity. While the characterization of immunotoxic potential is pertinent to both the chemical and pharmaceutical industries, the use of immunotoxicology data for hazard identification and/or risk assessment in each case is different. Therefore, multiple approaches to immunotoxicity testing have evolved. The assays that evaluate TDAR function include both well-established tests, e.g., anti-sheep red blood cell plaque-forming cell (PFC) assay, and newer models, e.g., anti-keyhole limpet hemocyanin (KLH) antibody ELISA. These tests vary in the study design, antigen application and analytical methods. However, they all evaluate the same endpoint—a competent immune (e.g., antibody) response to an antigen. Numerous issues have been identified in the application of TDAR tests, including high animal to animal variability; differences in antigen source and potency; a lack of established “normal range” of the immune response and uncertainty about the degree of inhibition of the TDAR to be considered toxicologically important. As such, the need for a forum to discuss these issues was recognized by the immunotoxicology community, and was addressed at the 2006 Society of Toxicology (SOT) Workshop. A series of papers will summarize that forum with the ultimate objectives being to build a consensus among immunotoxicologists on the implications of these factors on using TDAR results in hazard identification and/or risk assessment, and to establish a criteria to classify compounds as immunotoxicants.

Evaluation of Acute Immunotoxicity of Aerosolized Aflatoxin B1 in Female C57BL/6N Mice

There is evidence for immunotoxicity of aflatoxin B1 (AFB1) in chronic animal feeding studies; however, little information is available as to the effects of inhalation exposure. This study evaluated the acute affects of aerosolized AFB1 on systemic immune function of female C57BL/6N mice following a single aerosol exposure. Mice were exposed in nose-only inhalation tubes to 0, 2.86, 6.59 and 10 μg AFB1 aerosol/L air for 90 minutes. A negative control group of untreated mice and a positive control group of cyclophosphamide-treated mice were included to account for day to day variation. Three days following exposure, mice were sacrificed and body, liver, lung, thymus and spleen weights, and complete blood counts and white blood cell differentials were measured. Splenocytes were isolated for flow cytometric analysis of CD4+ and CD8+ lymphocytes, CD19+ B-cells and natural killer cells (NK 1.1+). The effect of AFB1 on humoral immunity was assessed by measuring serum anti-keyhole limpet hemocyanin (KLH) IgM levels. Of the tissues examined, only the thymus weight of AFB1 exposed mice decreased significantly compared to naïve mice; however, the decrease was not dose related and was also observed in the 0 AFB1 aerosol control group. A decrease in the mean white blood cell count of treated vs. naïve mice was observed at all dose levels but was clearly not dose related and was statistically significant only in the 0 and 2.86 μg/L groups. Red blood cell and platelet counts and white blood cell differentials were not significantly affected by AFB1. The number of CD4+ (helper T-cells), CD8+ (cytotoxic T-cells) and CD19+ (B-cells) decreased in spleens of AFB1 aerosol exposed mice compared to naïve mice; however, the decrease was not dose-related and was also observed in the 0 AFB1 exposure group. Dose-related changes in the CD4+/CD8+ T-lymphocyte ratios were not observed. The IgM response to KLH was not significantly different in AFB1 compared to naïve mice, suggesting that AFB1 did not effect antigen-specific antibody production. Based on the results of this study, a single AFB1 inhalation exposure up to 10 μg/L for 90 minutes (CxT = 900 μg ·min/L) did not significantly alter the immune parameters measured in this study. The aerosol vehicle (ethanol) and/or stress could have masked subtle AFB1-dependent changes in thymus and spleen weights, and in splenic lymphocyte subpopulations. However, for other immunological parameters, such as the IgM response to KLH, there was clearly no significant effect of AFB1 aerosol exposure.

The Paradoxical Effects of Lead in Interferon-Gamma Knockout BALB/c Mice

It has been reported that lead (Pb) exposure enhances interleukin (IL)-4 and inhibits interferon-gamma (IFNgamma) production in wild-type (WT) BALB/c mice. Here, we examined Pb effects on immunity in IFNgamma knockout (KO) mice. Lead significantly enhanced serum IgG1 anti-keyhole limpet hemocyanin (KLH) levels in WT mice compared to the controls; Pb also increased serum IgG2a anti-KLH levels, but the IgG1:IgG2a ratio was greater with Pb. In addition, total serum IgE levels, but not IgE anti-KLH levels, were increased. In the KO mice, the serum IgG1, IgG2a, IgE anti-KLH, and total IgE levels were significantly lower than those of WT mice. Surprisingly, Pb significantly enhanced IgG1 and IgG2a anti-KLH levels in the KO mice. However, for these mice, unlike the WT mice, Pb caused a greater percentage change in IgG2a than in IgG1 anti-KLH, indicating less skewing toward type-2 immunoglobulins. Lead also enhanced the delayed-type hypersensitivity (DTH) response in WT mice. Not surprisingly, very low DTH occurred in the KO mice; however, Pb induced a strong KLH-specific DTH response. The in vivo Pb exposure significantly increased in vitro production of IL-4, IL-5, and IL-10, but not IFNgamma, IL-2 and IL-12, by KLH-induced WT and KO spleen cells. In contrast to KLH, dinitrofluorobenzene contact hypersensitivity (DNFB CHS) was detected in all groups, and Pb did not affect this response, which suggests that Pb has only a slight effect on CD8+ T cell-related responses. As previously reported, Pb enhances Th2 responses in WT mice; however, in the KO mice, Pb enhanced Th1-related anti-KLH production and a Th2-related DTH. The Pb enhancement of DTH in IFNgamma-deficient mice is likely due to promotion of type-2 cytokines and enhancement of major histocompatibility complex (MHC) class II expression.