anti-KLH IgM Research Articles

In ovo co-administration of vitamins (A and D) and probiotic lactobacilli modulates immune responses in broiler chickens

There is evidence that probiotic lactobacilli, in addition to essential vitamins, such as vitamin A and D, have immunomodulatory properties that enhance immune response of neonatal chickens against infections. The present study evaluated the effects of in ovo administration of retinoic acid (RA), 25-Hydroxyvitamin D3 (VitD), and a lactobacilli cocktail on cytokine gene expression, antibody responses and spleen cell subsets in chickens. RA (90 µmol/egg) and VitD (0.6 μg/egg) were administered in ovo, either alone or in combination with lactobacilli (107 CFU/egg), at embryonic d 18. On d 5 and 10 posthatch, gene expression and cellular composition were analyzed in the bursa of Fabricius and spleen. Birds were immunized on d 14 and 21 posthatch with 2 T-dependent antigens, sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), to assess their antibody responses. Sera were collected from the immunized chickens on d 14, 21, 28, and 35 posthatch. The results demonstrated that lactobacilli treatment increased the number of monocyte/macrophages (KUL01+) and CD3+CD4+ T cells in the spleen, and enhanced serum anti-KLH IgM and IgY on d 14 postprimary immunization (P < 0.05). RA significantly increased serum IgY and IgM titers to KLH and enhanced the expression of interferon (IFN)-α, interleukin (IL)-1β, IL-6, IL-8, IL-12, IL-13, and transforming growth factor-β (TGF-β) in the bursa of Fabricius (P < 0.05). The percentage of CD3+CD8+ T cells, and monocyte/macrophages (KUL01+) was elevated in the spleen as well (P < 0.05). These findings reveal that prehatch administration of RA improves immunocompetency of neonatal chickens by increasing the production of cytokines that regulate innate immunity and through enhancing antibody-mediated response against T-dependent antigens.

GSK2245035, a TLR7 agonist, Does Not Increase Pregnancy Loss in Cynomolgus Monkeys

GSK2245035, a small molecule Toll-like Receptor 7 (TLR7) agonist developed for immunomodulatory treatment for allergic airways disease, aimed to reduce Th2 and enhance Th1/Treg responses to aeroallergens via the local induction of type I interferons (IFNs). GSK2245035 demonstrated selectivity for potent release of type I IFNs compared to TNF-α and IL-6, with dose dependent increases in the interferon inducible chemokine, IP-10, in the nasal compartment. Implantation and parturition require pro-inflammatory processes including IFNs, Interferon Stimulated Genes, TNFα and IP-10 while pregnancy requires immune regulation to maintain maternal fetal immune tolerance, and recombinant type I IFNs induced abortions in monkeys. Due to its mechanism of action, GSK2245035 was studied at pharmacologically and clinically relevant doses in a monkey pregnancy model. Monkeys received 0, 3 or 30 ng/kg/week GSK2245035 intranasally once weekly, from Day 20 postcoitum through Day 63 postpartum. Although systemic IFN-α and IP-10 levels were approximately 14.8 or 40 -fold (respectively) above predose levels at 3 or 30 ng/kg/week, respectively, there were no effects on pregnancy and infant outcome. Non-adverse effects included increased incidence of nasal discharge, increased maternal body temperature at 30 ng/kg/week and dose-dependent increases in maternal IP-10 and IFN-α and decreased infant anti-KLH IgM and IgG titers following KLH immunization at ≥3 ng/kg/week, relative to controls. Potentially, lower IFN-α and IP-10 levels as well as once-weekly intranasal dosing vs daily subcutaneous or intramuscular dosing with recombinant type I IFNs could explain the lack of pregnancy effects; however, there was an undesired impact on offspring immune function.

A randomized controlled trial with a delayed-type hypersensitivity model using keyhole limpet haemocyanin to evaluate adaptive immune responses in man.

AimsKeyhole limpet haemocyanin (KLH) immunization is a clinical model for the evaluation of human antibody responses. The current study evaluated the anti‐KLH antibody response after KLH immunization and the delayed‐type hypersensitivity response following intradermal KLH administration, using objective imaging techniques.MethodsHealthy male subjects aged 24.5 ± 5.4 years were randomized to intramuscular immunization with 100 μg KLH (n = 12) or placebo (n = 3). Anti‐KLH antibody (Ig) M and IgG titres were determined before and every 7 days after KLH immunization for a total of 28 days. Twenty‐one days after the immunization, all subjects received 1 μg KLH intradermally. Prior to and 2 days after intradermal KLH administration, skin blood perfusion, erythema and oedema were quantified using noninvasive imaging tools. Repeated measures ANCOVAs were used to analyse data.ResultsAnti‐KLH IgM and IgG titres increased after KLH immunization compared to placebo (estimated difference [ED]: 37%, 95% confidence interval [CI]: 19–51% and ED: 68%, 95% CI: 56–76% respectively). Upon intradermal KLH administration an increase in skin blood perfusion (ED: 10.9 arbitrary units (AU), 95% CI: 1.4–20.4 AU) and erythema (ED: 0.3 AU, 95% CI: 0.1–0.5 AU) was observed in KLH‐immunized subjects compared to placebo.ConclusionKLH immunization followed by intradermal KLH administration resulted in increased anti‐KLH IgM and IgG titres and a delayed‐type hypersensitivity response quantified by an increase in skin blood perfusion and erythema. Using noninvasive imaging tools the KLH model has the potential to serve as an objective tool to study the pharmacodynamics of T‐cell‐directed immunomodulatory drugs.

Non-therapeutic administration of antimicrobial growth promoters to interrogate suppression mechanisms of innate and adaptive immune responses using an ovine model of stress

Abstract Antimicrobial growth promoters (AGPs) have been administered in non-therapeutic concentrations in livestock feed to increase weight gain per unit of feed consumed. Daily low-dose antibiotic administration is capable of modulating immunity by suppressing inflammation, potentially increasing metabolic resources that can be allocated to muscle development. It is unknown how the AGPs tylosin (TYL) and chlortetracycline (CTC) influence acquired immunity. The objective of this study was to measure acquired immune responses, assess impacts on innate immunity and to elucidate mechanisms of immunosuppression by immunizing 10-week old lambs (male Suffolk cross) with keyhole limpet hemocyanin (KLH) during AGP administration (TYL, CTC or Control diets) while enduring intermittent physical stress. Over a 4-week period, 18 lambs (n=3 per diet/stress group) were monitored. Between the third-fourth experimental week, stressed lambs gained less weight (1.50 ± 0.49 kg) than non-stressed lambs (3.06 ± 0.37 kg, P &amp;lt; 0.03). The weight of lambs fed AGPs did not surpass those on the control diet. Diet and stress did not impact serum concentrations of total IgG and IgM, or anti-KLH IgG and IgM specific titres. Endpoint hematological analysis revealed that stressed lambs had decreased red cell distribution width (RDW%, 29% vs. 31%, P&amp;lt;0.01) and peripheral lymphocyte populations (5.72 × 103/μL vs. 4.69 × 103/μL, P&amp;lt;0.02). A diet × stress interaction was observed for peripheral granulocyte population (P=0.02). The impending restriction of AGP usage in North America has generated interest in identifying AGP alternatives, and elucidation of the mechanisms of AGP function is needed to develop effective non-antibiotic alternatives that mimic their action.

Inducible T-cell co-stimulator ligand (ICOSL) blockade leads to selective inhibition of anti-KLH IgG responses in subjects with systemic lupus erythematosus

ObjectivesTo evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of single-dose and multiple-dose administration of AMG 557, a human anti-inducible T cell co-stimulator ligand (ICOSL) monoclonal antibody, in subjects...

Bovine natural antibodies in antibody-dependent bactericidal activity against Escherichia coli and Salmonella Typhimurium and risk of mastitis

Natural antibodies (NAbs) are mostly IgM antibodies produced without antigenic stimulation and serve as a first line of defence of the immune system. As both natural and specific antibodies are present in animals, NAbs are studied by determining the IgM response to naïve antigens like keyhole limpet hemocyanin (KLH). In this study, we selected cows based on high and low anti-KLH IgM titers, reflecting high and low NAb titers, and determined if the anti-KLH IgM titers were indicative for the recognition of common microbial structures (lipopolysaccharide, lipoteichoic acid and peptidoglycan) and intact bacteria (Escherichia coli and Salmonella Typhimurium). Sera with high NAbs titers showed more IgM and IgG binding to common microbial structures and S. Typhimurium bacteria than sera with low NAbs titers. The same association was observed for IgM binding to E. coli, but not for IgG binding to E. coli. Antibody-mediated complement killing of E. coli and S. Typhimurium in a newly developed bactericidal test was equal between high and low NAb cows. However, relating the outcome of the bactericidal test to the development of mastitis within one and even four years after sampling showed a significant negative correlation implying cows that were less potent in bacterial killing had a higher chance on developing mastitis. In conclusion, sera with high NAbs titers had more antibodies binding to common microbial structures and intact bacteria. Furthermore, the bactericidal test might provide a useful prognostic tool for the development of mastitis.

An Enhanced Pre- and Postnatal Development Study in Cynomolgus Monkeys with Tabalumab: A Human IgG4 Monoclonal Antibody.

Tabalumab, a human IgG4 monoclonal antibody (mAb) with neutralizing activity against both soluble and membrane B-cell activating factor (BAFF), has been under development for the treatment of autoimmune diseases. The purpose of this study was to determine the potential adverse effects of maternal tabalumab exposure on pregnancy, parturition, and lactation of the mothers and on the growth, viability, and development of the offspring through postnatal day (PND) 204. Tabalumab was administered by subcutaneous injection to presumed pregnant cynomolgus monkeys (16-19 per group) every 2 weeks from gestation day (GD) 20 to 22 until parturition at doses of 0, 0.3, or 30 mg/kg. Evaluations in mothers and infants included clinical signs, body weight, toxicokinetics, blood lymphocyte phenotyping, T-cell-dependent antibody response (infants only), antitherapeutic antibody (ATA), organ weights (infants only), and gross and microscopic histopathology. Infants were also examined for external and visceral morphologic and neurobehavioral development. There were no adverse tabalumab-related effects on maternal or infant endpoints. An expected pharmacological decrease in peripheral blood B-lymphocytes occurred in adults and infants; however, B-cell recovery was evident by PND154 in adults and infants at 0.3 mg/kg and by PND204 in infants at 30 mg/kg. At 30 mg/kg, a reduced IgM antibody response to T-cell-dependent antigen keyhole limpet hemocyanin (KLH) was observed following primary immunization. Following secondary KLH immunization, all infants in both dose groups mounted anti-KLH IgM and IgG antibody responses similar to control. Placental and mammary transfer of tabalumab was demonstrated. In conclusion, the no-observed-adverse-effect level for maternal and developmental toxicity was 30 mg/kg, the highest dose tested. Exposures at 30 mg/kg provide a margin of safety of 16× the anticipated clinical exposure.

Interaction between feed management systems and omega-3 fatty acid supplementation on bovine immunization parameters (VET1P.1116)

Abstract A trial was designed to investigate impacts of omega-3 fatty acid feeding regimes on bovine immunization responses. Dietary supplements contained either fish oil or microalgae as omega-3 fatty acid sources, and cattle were divided into 2 feed management systems; they either grazed pasture or were kept in barn tie stalls and fed total mixed rations (TMR). Cattle were immunized with keyhole limpet hemocyanin (KLH), a standard T cell dependent antigen. Dietary supplements and feeding systems influenced the response of cattle to immunization with KLH. Anti-KLH IgM, IgG and IgA antibody levels in serum were significantly higher in cattle grazing pasture than those fed TMR. Cattle consuming microalgae had consistently higher anti-KLH IgG2 and IgM antibody levels compared to cattle fed fish oil. Effects of omega-3 fatty acid supplements and TMR versus pasture-based diets were also apparent in cytokine profiles from spleen and cecal tissues. Levels of transforming growth factor-beta (TGF-β) were highest in cecal tissue of pasture-grazing cattle (P &amp;lt; 0.0001), and spleen TGF-β levels were highest in pasture-grazing cattle consuming microalgae (P = 0.0073).These findings suggest that effects of omega-3 supplements on bovine immunity are influenced by the feed management system, with heightened effects on antibody production observed with a pasture system relative to a confinement and TMR-based diet.

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.

Primary and Secondary Immune Responses to Keyhole Limpet Hemocyanin in Rats After Infusion of Hemoglobin Vesicle, an Artificial Oxygen Carrier

Hemoglobin vesicles (HbVs), artificial oxygen carriers encapsulating concentrated Hb solution on phospholipid vesicles (liposomes), are promising candidates for clinically useful transfusion. Although HbV infusion transiently suppressed the proliferative response of rat splenic T-cells to concanavalin A or keyhole limpet hemocyanin (KLH), a T-cell-dependent antigen, in ex vivo culture conditions, HbV infusion did not affect the primary IgG antibody response. We extended our assessment of the effects of HbV infusion on the systemic immune response using primary and secondary responses to KLH in rats. We observed that the generation of primary anti-KLH IgM antibody in HbV-infused rats was not suppressed but was instead higher than those in saline-infused rats. Furthermore, HbV infusion did not suppress the increase of IgG subclass of KLH antibody in secondary response. The T cell response to KLH of bulk spleen cells, as derived from 2-3 months after secondary KLH immunization, was unaffected by infusion of HbV, suggesting that HbV loading has no suppressive effect on homeostatic survival of memory T-cells against KLH. These results indicate that HbV is highly biocompatible in systemic immune responses in rats.

T-cell-dependent antibody responses in the rat: Forms and sources of keyhole limpet hemocyanin matter

The T-cell-dependent antibody response (TDAR) is a functional assay used in immunopharmacology and immunotoxicology to assess ability to mount an antibody response to immunization. Keyhole limpet hemocyanin (KLH) is extensively used as the immunogen of choice in non-clinical and clinical settings. Native KLH is comprised of high molecular weight (HMW; 4–8 MDa) assemblies of KLH subunit dimers (> 600–800 kDa). It is not known how the different forms (HMW vs subunit) and manufacturing processes (commercial sources) may impact the nature of anti-KLH immune responses (e.g. magnitude and inter-animal variability). Anti-KLH IgM and IgG responses were studied in Sprague-Dawley rats immunized with different forms and commercial sources of KLH: 100 µg of HMW KLH from two different sources or subunit KLH from three different sources. Biophysical and biochemical analyses were conducted to characterize the KLH formulations. Anti-KLH IgM and IgG responses were measured using a proprietary indirect quantitative electrochemiluminescence immunoassay. The HMW KLH preparations showed a greater number of sub-visible particles (2–150 µm size range) than the subunit KLH preparations. All HMW KLH and all subunit KLH were equivalent on SEC (hydrodynamic volume), PAGE (size and charge), and SDS-PAGE (molecular radius). Robust primary and secondary anti-KLH responses were detected for both sources of HMW KLH. The subunit KLH immunizations resulted in lower IgG and IgM responses compared to the HMW KLH, with the exception of Stellar Biotechnologies subunit KLH that produced both robust primary and secondary responses, which approached the HMW KLH responses. Inter-animal variability for IgM and IgG responses was lower with HMW KLH than with subunit KLH. In conclusion, different forms and commercial sources of KLH were associated with different magnitudes and inter-animal variability in IgM and IgG responses, a critical finding to take into consideration when designing TDAR studies for robust immunotoxicology or immunopharmacology testing.

A novel assay to measure B cell responses to keyhole limpet haemocyanin vaccination in healthy volunteers and subjects with systemic lupus erythematosus.

The aim of the study was to characterize performance of a complementary set of assays to measure antigen-specific immune responses in subjects immunized with a neoantigen. Healthy volunteers (HV) (n = 8) and patients with systemic lupus erythematosus (SLE) (n = 6) were immunized with keyhole limpet haemocyanin (KLH) on days 1 and 29. Serum antibodies were detected using a flow cytometric bead array (CBA) that multiplexed the KLH response alongside pre-existing anti-tetanus antibodies. Peripheral blood mononuclear cells were studied by B cell ELISPOT. These assays were built upon precedent assay development in cynomolgus monkeys, which pointed towards their utility in humans. Primary anti-KLH IgG responses rose to a mean of 65-93-fold above baseline for HV and SLE patients, respectively, and secondary responses rose to a mean of 260-170-fold above baseline. High levels of anti-tetanus IgG were detected in pre-immunization samples and their levels did not change over the course of study. Anti-KLH IgG1-4 subclasses were characterized by a predominant IgG1 response, with no significant differences in subclass magnitude or distribution between HV and SLE subjects. Anti-KLH IgM levels were detectable, although the overall response was lower. IgM was not detected in two SLE subjects whodid generate an IgG response. All subjects responded to KLH by B cell ELISPOT, with no significant differences observed between HV and SLE subjects. The CBA and B cell ELISPOT assays reliably measured anti-KLH B cell responses, supporting use of this approach and these assays to assess the pharmacodynamic and potential safety impact of marketed/investigational immune-therapeutics.

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

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

Immunotoxic effects of cyclophosphamide and cyclosporine in the dog

Limited non-clinical immunotoxicity data are available in the dog, although this is a major non-rodent species in regulatory safety studies. The present study aimed to test whether widely accepted immunotoxicity endpoints including lymphocyte subset immunophenotyping, the anti-KLH TDAR assay, and histological examination of the main lymphoid organs were reliable to detect immunosuppression induced by cyclosporine and cyclophosphamide in dogs and could, therefore, be used for non-clinical immunotoxicity evaluation in this species. Male and female Beagle dogs were treated orally from Day 1 for 4 weeks with 25 mg/kg cyclosporine daily, or with 2 mg/kg cyclophosphamide on 4 consecutive days each week, or the same volume of drinking water daily. Blood samples were withdrawn pre-test and on Days 11, 18, and 23 to measure standard hematology parameters and analyze lymphocyte subsets. All animals received an intramuscular injection of 5 mg KLH on Day 11. Sandwich ELISA assays were used to quantify anti-KLH IgM and anti-KLH IgG levels in blood samples taken pre-test, on Days 18 and 23, and pre-test, on Days 23 and 28, respectively. At the end of the treatment period, all animals were submitted to histological examination of lymphoid organs, liver, and kidneys. No signs of marked toxicity were observed. No changes in lymphocyte subsets, but markedly decreased primary anti-KLH IgM and IgG responses, and a slightly-to-markedly increased cortex/medulla ratio in the thymus were observed in cyclosporine-treated dogs. Lower total WBC counts correlating with lower total and B-lymphocyte subset and decreased germinal center development in mesenteric lymph nodes, but no changes in primary anti-KLH IgM and IgG responses were observed in cyclophosphamide-treated dogs. These results demonstrate that widely accepted immunotoxicity endpoints can adequately detect the effects of known immunosuppressive drugs in the dog and support the conclusion that it is a relevant animal species for immunotoxicity evaluation.

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

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

Multiparametric immunotoxicity screening in mice during early drug development

Evaluation of potential adverse effects on the immune system should be incorporated into drug development prior to phase III clinical trials. In addition to standard toxicity results, T-dependent antibody response (TDAR) assays are widely used to evidence impaired immune function. The present study was aimed at validating a multiparametric screening approach in mice to investigate exaggerated pharmacologic or unintended immunosuppressive effects in early drug development. Male CD1 mice injected with a single IV dose of 2mg KLH displayed a robust anti-KLH IgM response that peaked on day +5. Anti-KLH IgM response, standard haematology parameters, and thymus/spleen weight and histology were examined in mice treated once daily for 4 days with cyclophosphamide (CY; 5–20mg/kg/day), cyclosporine (CS; 10–90mg/kg/day), dexamethasone (DX; 5–20mg/kg/day), prednisolone (PR; 3–30mg/kg/day) or chlorpromazine (CZ; 10–30mg/kg/day). CY and CS decreased anti-KLH IgM response at all dose levels. CY induced a marked decrease in WBC count and thymus/spleen weight with histological changes in both lymphoid organs. CS mainly decreased thymus weight (highest dose), which was associated with lymphoid depletion, without relevant effects on haematology parameters. Neither DX nor PR nor CZ induced significant changes in anti-KLH IgM response. DX and PR decreased lymphocyte counts and thymus/spleen weight, and induced histological changes in both lymphoid organs. CZ (higher doses) decreased lymphocyte count and thymus weight, and induced consistent histological changes in the thymus. This multiparametric study was able to detect 5 human drugs with variable immunosuppressive potency and thus may prove to be a useful early screening tool for predicting drug immunotoxicity.

Development and application of a species-specific anti-KLH ELISA in Beagle dogs

Limited nonclinical immunotoxicity data are available in the dog, although this is a major nonrodent species in regulatory safety studies. The aim of the present study was to measure primary anti-KLH IgM and IgG responses in Beagle dogs treated with the reference immunosuppressive drugs cyclosporine and cyclophosphamide, using a dog-specific sandwich ELISA assay jointly developed in-house by CiToxLAB France and Atlanbio.

Anti-streptococcus IgM antibodies induce repetitive stereotyped movements: Cell activation and co-localization with Fcα/μ receptors in the striatum and motor cortex

Group A beta-hemolytic streptococcus (GABHS) infections are implicated in neuropsychiatric disorders associated with an increased expression of repetitive stereotyped movements. Anti-streptococcus IgG presumably cross-reacts with elements on basal ganglia cells, modifies their function, and triggers symptoms. IgM may play a unique role in precipitating behavioral disturbances since variations in cortico-striatal activity occur in temporal congruity with peak IgM titers during an orchestrated immune response. We discovered in Balb/c mice that single subcutaneous injections of mouse monoclonal IgM antibodies to streptococcus group A bacteria induce marked dose-dependent increases in repetitive stereotyped movements, including head bobbing, sniffing, and intense grooming. Effects were antibody- and antigen-specific: anti-streptococcus IgG stimulated ambulatory activity and vertical activity but not these stereotypies, while anti-KLH IgM reduced activity. We suggest that anti-streptococcus IgM and IgG play unique roles in provoking GABHS-related behavioral disturbances. Paralleling its stereotypy-inducing effects, anti-streptococcus IgM stimulated Fos-like immunoreactivity in regions linked to cortico-striatal projections involved in motor control, including subregions of the caudate, nucleus accumbens, and motor cortex. This is the first evidence that anti-streptococcus IgM antibodies induce in vivo functional changes in these structures. Moreover, there was a striking similarity in the distributions of anti-streptococcus IgM deposits and Fos-like immunoreactivity in these regions. Of further importance, Fcα/μ receptors, which bind IgM, were present- and co-localized with anti-streptococcus IgM in these structures. We suggest that anti-streptococcus IgM-induced alterations of cell activity reflect local actions of IgM that involve Fcα/μ receptors. These findings support the use of anti-streptococcus monoclonal antibody administration in Balb/c mice to model GABHS-related behavioral disturbances and identify underlying mechanisms.

Cardiovascular exercise intervention improves the primary antibody response to keyhole limpet hemocyanin (KLH) in previously sedentary older adults

Based upon a prior cross-sectional study, we hypothesized that an aerobic exercise intervention in sedentary older adults would improve a primary T cell-dependent immune response. Participants were a subset of older subjects from a large, ongoing exercise intervention study who were randomly assigned to either an aerobic exercise (Cardio, n = 30, 68.9 + 0.8 years) or flexibility/balance (Flex, n = 20, 69.9 + 1.2 years) intervention. The intervention consisted of either three aerobic sessions for 30–60 min at 55–70% VO 2max or two 60 min flexibility/balance sessions weekly for 10 months. Eight months into the intervention, samples were collected before intramuscular administration of KLH (125 μg), followed by sampling at 2, 3, and 6 weeks post-KLH. Serum anti-KLH IgM, IgG1, and IgG2 was measured by ELISA. Physiological and psychosocial measures were also assessed pre- and post-intervention. While there was no difference in the anti-KLH IgG2 response between groups, Cardio displayed significantly ( p < 0.05) higher anti-KLH IgG1 (at weeks 2, 3, and 6 post) and IgM responses when compared to Flex. Despite cardiovascular intervention-induced improvement in physical fitness (∼11% vs. 1% change in VO 2peak in Cardio vs. Flex, respectively), we found no relationship between improved fitness and enhanced anti-KLH antibody responses. Optimism, perceived stress, and affect were all associated with enhanced immune response. We have shown for the first time that cardiovascular training in previously sedentary elderly results in significantly higher primary IgG1 and IgM antibody responses, while having no effect on IgG2 production.

Neonatal-Age Treatment with Vitamin A Delays Postweaning Vitamin A Deficiency and Increases the Antibody Response to T-cell Dependent Antigens in Young Adult Rats Fed a Vitamin A-Deficient Diet

Vitamin A supplementation for infants and young children is recommended by WHO/UNICEF for countries with a high prevalence of vitamin A deficiency, and vitamin A is often administered at immunization contacts. Using a rat model, we tested whether supplementation with vitamin A or other retinoids at the time of neonatal immunization has prospective benefit in terms of preventing postweaning vitamin A deficiency and promoting antibody responses to T-cell dependent (TD) antigens administered at the neonatal stage and at the young adult stage. Rats were treated orally on postnatal d 6–8 with oil (placebo control), vitamin A, retinoic acid, or a combination of both (VARA) (n ≥ 12/group), and immunized with tetanus toxoid (TT) on d 7. The primary anti-TT response was measured on d 21, after which weanling rats were fed the vitamin A-deficient diet until ∼10 wk. At 8 wk, rats were immunized again with TT to determine the recall response, and with a novel TD antigen, keyhole limpet hemocyanin (KLH), to assess the adult primary response. None of the supplements affected the plasma titer of anti-TT immunoglobulin G (IgG) on d 21 (P = 0.25). However, neonatal-age supplementation with vitamin A or VARA at the young adult stage resulted in: >5 times higher anti-TT IgG recall response (P < 0.01); 5- and 9-times higher anti-KLH primary IgM and IgG responses, respectively (P < 0.05), and plasma retinol in the normal range (∼1.0 μmol/L vs. ∼0.35 μmol/L in retinoic acid-treated and control groups, P < 0.0001). We conclude that early-life supplementation with vitamin A or VARA can prospectively benefit the primary and recall antibody responses to TD antigens administered at the young adult stage, which may involve the maintenance of normal plasma retinol levels.