Mucosal immunity in the brushtail possum (Trichosurus vulpecula): detection of antibody in serum and at female reproductive sites after intranasal immunization

Vaccination strategies for the brushtail possum, which rely upon stimulation of mucosal immunity, are being developed for biocontrol purposes. As little is known about how to stimulate possum immune responses via a mucosal site, groups of possums were immunized intranasally with keyhole limpet haemocyanin (KLH) alone or in combination with known or novel mucosal adjuvants. Antigen-specific antibody titres in female reproductive secretions were measured by ELISA and compared with antibody titres in the serum. Antigen-induced lymphocyte proliferative responses were measured as an indicator of cell-mediated responses. Intranasal immunization with KLH alone stimulated a weak serum antibody response that was significantly increased when KLH was given with cholera toxin subunit B (CTB), recombinant possum tumour necrosis factor alpha (TNF alpha) or live Mycobacterium bovis bacillus Calmette Guerin (BCG). Antibody titres in secretions from ovarian follicles and the uterus were very low in animals administered KLH alone. Significantly higher antibody titres to KLH were present in the reproductive secretions of possums immunized with KLH plus CTB, BCG or heat-killed Mycobacterium vaccae. Antibody titres were lower in mucosal secretions than in the serum, but there was a significant correlation between the two. In addition, coadministration of live BCG with KLH produced a strong antigen-specific cell-mediated response to KLH. This study has shown that an immune response to a protein antigen can be stimulated in possums by intranasal immunization and that antigen-specific antibodies can be detected in secretions from the female reproductive tract.     

KLH单独刺激不影响黑线仓鼠的静止代谢率

根据生活史理论,免疫功能与产热和繁殖等其他生理学功能类似,具有能量代价。然而,关于提高免疫能力的能量学代价的实验例证很少,且此类代价可能受所选择的免疫学参数和实验测定的影响。为探究提高小型哺乳动物免疫能力的能量学代价,以匙孔血蓝蛋白（Keyhole limpet haemocyanin,KLH）作为外源抗原刺激,在注射前（第0天）、注射后第5天和第10天,分别测定非繁殖期雌、雄性黑线仓鼠（Cricetulus barabensis）体重和静止代谢率（Resting metabolic rate, RMR）的变化,通过眼眶静脉丛采血技术和ELISA分析,分别测定第5天（抗IgM抗体）和第10天（抗IgG抗体）动物血清抗体含量的变化。结果显示：（1）雄鼠的体重高于雌鼠,雌、雄鼠实验组和对照组之间的体重均无差异且不随时间而增加;（2）无论雌鼠还是雄鼠,注射KLH组血清中抗IgM和抗IgG抗体的含量均高于注射生理盐水的对照组动物;（3）注射KLH前后,RMR既无性别差异,也无组间差异,同一组动物的RMR均不随取样时间点而变化（KLH注射后第5天和第10天）。结果表明,单一的KLH刺激激活了黑线仓鼠的体液免疫,但在能量不受限的情况下不影响体重和能量收支的变化。有两种可能的解释：黑线仓鼠增强的体液免疫可能以免疫系统内另一免疫组分活动的降低作为代价,或它对体液免疫应答具有构建能力,温和的免疫刺激不伴随额外的能量学代价。     

Song predicts immunocompetence in male European starlings (Sturnus vulgaris)

According to the immunocompetence handicap hypothesis, sexually selected characteristics predict immune function and this relationship is mediated by testosterone. In the present study, we investigated whether bird song could predict immunity in European starlings (Sturnus vulgaris). We recorded the singing and reproductive behaviours of 16 adult male starlings in an outdoor aviary and then assessed their cell-mediated and humoral immunity in vivo. The males were observed in groups of four for 2 h each day over a 4-day period. For each male, the number of songs produced was recorded and the average song-bout length was computed. Next, cell-mediated and humoral immunity were assessed via cutaneous swelling responses to the T-cell mitogen phytohaemagglutinin and antibody responses to a novel antigen, keyhole limpet haemocyanin. Song rate and song-bout length were positively correlated with cell-mediated and humoral immunity, respectively. Additionally, a negative relationship between plasma testosterone concentration and antibody response was observed. These data demonstrate that male starling song can be used as a predictor of immunocompetence, with more robust singers exhibiting enhanced immunity. Whether this relationship is mediated directly by testosterone requires further investigation.     

Social interactions unmask sex differences in humoral immunity in voles

Sex differences in immune function are well established among laboratory rodents, with males typically having lower immunity than females. This sex difference may reflect the suppressive effects of testosterone on immune function. Because polygynous males generally have higher circulating testosterone concentrations than monogamous males, sex differences in immune function are hypothesized to be more pronounced among polygynous as compared to monogamous species. Sex differences in immune function have not been consistently observed among individually housed Microtus in the laboratory; thus, social interactions are hypothesized to be necessary for the expression of sex differences in immune function. We assessed the effect of differential housing conditions on humoral immunity and steroid hormone concentrations in polygynous meadow voles Microtus pennsylvanicus, and monogamous prairie voles M. ochrogaster. We examined humoral immunity by immunizing voles with keyhole limpet haemocyanin (KLH) and measuring antibody production 5, 10, 15 and 30 days postimmunization. Overall, meadow voles mounted higher anti-KLH immunoglobulin (Ig)M and IgG responses than prairie voles, regardless of the housing condition. Sex differences in antibody production were only observed among meadow voles housed in pairs, in which females had higher anti-KLH IgM and IgG responses than males. Sex differences in antibody production were not observed among prairie voles or meadow voles housed individually. Sex and species differences in circulating oestradiol, testosterone, and corticosterone concentrations were not related to differences in humoral immunity. These data suggest that sex differences in immune function are more pronounced among polygynous species than monogamous species, but may be context dependent. Copyright 1999 The Association for the Study of Animal Behaviour.     

Selenium Supplementation Restores Innate and Humoral Immune Responses in Footrot-Affected Sheep

Dietary selenium (Se) alters whole-blood Se concentrations in sheep, dependent upon Se source and dosage administered, but little is known about effects on immune function. We used footrot (FR) as a disease model to test the effects of supranutritional Se supplementation on immune function. To determine the effect of Se-source (organic Se-yeast, inorganic Na-selenite or Na-selenate) and Se-dosage (1, 3, 5 times FDA-permitted level) on FR severity, 120 ewes with and 120 ewes without FR were drenched weekly for 62 weeks with different Se sources and dosages (30 ewes/treatment group). Innate immunity was evaluated after 62 weeks of supplementation by measuring neutrophil bacterial killing ability. Adaptive immune function was evaluated by immunizing sheep with keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. At baseline, FR-affected ewes had lower whole-blood and serum-Se concentrations; this difference was not observed after Se supplementation. Se supplementation increased neutrophil bacterial killing percentages in FR-affected sheep to percentages observed in supplemented and non-supplemented healthy sheep. Similarly, Se supplementation increased KLH antibody titers in FR-affected sheep to titers observed in healthy sheep. FR-affected sheep demonstrated suppressed cell-mediated immunity at 24 hours after intradermal KLH challenge, although there was no improvement with Se supplementation. We did not consistently prevent nor improve recovery from FR over the 62 week Se-treatment period. In conclusion, Se supplementation does not prevent FR, but does restore innate and humoral immune functions negatively affected by FR.     

Transfer of humoural immunity over two generations in urban pigeons

Maternal antibodies (MatAb) are known to provide passive protection early in life for young vertebrates but their effects on the development of offspring immune response across generations are still unknown. Here, we investigated the effects of antigen exposure (keyhole limpet haemocyanin, KLH) experienced by urban pigeon (Columba livia) females on the amount of antigen-specific antibodies (Abs) transferred into the egg yolk of their daughters and on the humoural immune response towards this same antigen in their grandchildren. We found that chicks from KLH-injected maternal grandmothers had a higher humoural response than chicks from sham-injected grandmothers. However, we did not detect a significant effect of female KLH exposure on the ability of their daughters to transmit anti-KLH Abs into their eggs. These results suggest that antigen exposure at one generation may shape the immune profile of offspring over two next generations, although the underlying mechanisms remain to be investigated.     

Reductions in total body fat decrease humoral immunity

Mounting an immune response requires substantial energy, and it is well known that marked reductions in energy availability (e.g. starvation) can suppress immune function, thus increasing disease susceptibility and compromising survival. We tested the hypothesis that moderate reductions in energy availability impair humoral immunity. Specifically, we examined the effects of partial lipectomy (LIPx) on humoral immunity in two seasonally breeding rodent species, prairie voles (Microtus ochrogaster) and Siberian hamsters (Phodopus sungorus). Animals received bilateral surgical removal of epididymal white adipose tissue (EWATx), inguinal white adipose tissue (IWATx) or sham surgeries and were injected with the antigen keyhole limpet haemocyanin (KLH) either four or 12 weeks after surgery. In prairie voles, serum anti-KLH immunoglobulin G (IgG) did not differ significantly at four weeks. At 12 weeks, serum IgG was significantly reduced in IWATx, but not EWATx animals, compared with sham-operated animals. In Siberian hamsters, both IWATx and EWATx animals reduced serum IgG at four weeks. At 12 weeks, EWATx hamsters displayed a significant compensatory increase in IWAT pad mass compared with sham-operated hamsters, and serum IgG no longer differed from sham-operated animals. There was no significant increase in EWAT in IWATx hamsters compared with sham animals and IgG remained significantly reduced in IWATx hamsters. These results suggest that reductions in energy availability can impair humoral immunity.     

Synthesis and Evaluation of Protein Conjugates of GM3 Derivatives Carrying Modified Sialic Acids as Highly Immunogenic Cancer Vaccine Candidates

GM3, a sialylated trisaccharide antigen expressed by a number of tumors, is an attractive target in the design of therapeutic cancer vaccines. However, a serious problem associated with GM3 is that it is poorly immunogenic. To overcome this problem for the development of GM3-based cancer vaccines, four GM3 derivatives, including 5'-N-p-methylphenylacetyl, 5'-N-p-methoxyphenylacetyl, 5'-N-p-acetophenylacetyl and 5'-N-p-chlorophenylacetyl GM3, were synthesized and then coupled to a carrier protein, keyhole limpet haemocyanin (KLH). The resultant glycoconjugates were evaluated as vaccines in mouse and compared to the KLH conjugate of 5'-N-phenylacetyl GM3 (GM3NPhAc), a highly immunogenic GM3 derivative that was previously investigated as a vaccine candidate. All of the four new GM3 derivatives were proved to be more immunogenic than GM3NPhAc and elicit very strong T cell-dependent immune responses desirable for cancer immunotherapy. It was concluded that the new GM3 derivatives can form promising vaccine candidates that may be used to combine with cell glycoengineering for cancer immunotherapy.     

雌性黑线仓鼠免疫功能的季节变化

温带地区动物的免疫功能常表现出季节性变化.本研究以捕获于2014年秋季、冬季和2015年春季、夏季的雌性黑线仓鼠为研究对象,通过注射植物血球凝集素(PHA)后足垫的肿胀程度反映细胞免疫能力;注射抗原匙孔血蓝蛋白(KLH)后,利用酶联免疫吸附(EHSA)方法检测血清免疫球蛋白IgG和IgM的浓度,反映体液免疫功能;检验冬...     

食物限制对黑线仓鼠免疫功能的影响

温带地区小型哺乳动物经常面临着食物资源的波动。食物对动物的免疫功能具有重要影响。将19只成年雄性黑线仓鼠(Cricetulus barabensis)随机分为自由取食组(n=9)和限食组(n=10)。注射植物血球凝集素(PHA)来测定细胞介导的免疫反应,用匙孔血蓝蛋白(KLH)免疫动物,然后测定抗KLH抗体的浓度以反映其体液免疫功能。旨在检验食物限制是否会抑制黑线仓鼠的细胞免疫和体液免疫功能。结果发现,与对照组相比,限食组黑线仓鼠具有较低的体重、体脂、脾脏鲜重、血清瘦素水平、免疫球蛋白Ig G和Ig M浓度。而限食对胸腺鲜重、白细胞数、皮质酮水平以及PHA反应没有显著影响。结果表明黑线仓鼠免疫系统的不同成分对限食反应存在差异,在食物资源短缺时,黑线仓鼠防御细胞外病原体的能力降低,从而导致生存能力的下降。     

长爪沙鼠免疫功能、体脂含量和器官重量的季节变化

小型哺乳动物的体脂含量与免疫功能有关,并受环境条件的影响。为了进一步理解野生长爪沙鼠对环境的生存适应策略,我们于2004 年夏季(7～8月)和2005年冬季(1～3月)测定了野生长爪沙鼠的体重、体脂含量和脏器重量,以及由匙孔血蓝蛋白(KLH)对雄鼠所介导的体液免疫反应。结果发现:雄鼠的胴体干重和体脂含量都显著高于雌鼠,其他各项指标无性别差异。冬季雌雄沙鼠的体重、胴体干重、体脂含量和褐色脂肪组织的湿重,以及雄鼠的睾丸湿重都显著高于夏季。脾脏的重量冬季趋于降低。处理组(注射KLH )动物血清中抗KLH 抗体的含量在冬季和夏季都显著高于对照组(注射生理盐水),且冬季处理组显著高于夏季处理组。本研究结果为动物种群调节的“冬季免疫增强假说”提供了一个新的野外例证。没有检测到免疫器官和产热器官、免疫器官和繁殖器官之间的权衡关系。     

Two-dimensional crystallization,transmission electron microscopy and image processing of keyhole Limpet Haemocyanin (KLH)

KLH, the respiratory protein haemocyanin of the keyhole limpet Megathura crenulata, has been obtained as a high g pellet from the cell-free haemolymph and purified by gel permeation chromatography on a Biogel A15m column. The leading major protein peak eluted has been found to contain haemocyanin multi-decamers, followed by a second major peak containing single di-decamers, with small amounts of decamer and partly dissociated material following in the later fractions. The purified KLH di-decamer has been for two- dimensional crystallization studies with the negative staining-carbon film technique, in the presence of polyethylene glycol. In the side-on orientation, KLH has been found to produce two-dimensional crystals with a half-molecule linear displacement in consecutive rows. This is shown to be due to a specific association and two-dimensional crystal nucleation of the molecules in this arrangement. When oriented end-on, KLH has been found to form close- packed hexagonal monomolecular arrays which are not truly crystalline. This is because the five-fold rotational symmetry of the cylindrical macromolecule is not readily compatible with the hexagonal molecular packing. Computer-processed averaged images have been produced from the side-on KLH two-dimensional crystals and the end-on arrays, which reveal the principal molecular features of this homo-oligomeric protein complex to a resolution of ca 2.7 nm.     

Influence of age and physical activity on the primary in vivo antibody and T cell-mediated responses in men.

The aging immune system is characterized by the progressive decline in the antibody and T cell-mediated responses to antigen. Little is known, however, about the benefits of exercise in aging on the generation of a primary immune response to antigen and the subsequent antibody and memory T cell-mediated response. Most in vivo immune research to date has utilized vaccines or recall antigens to elicit an immune response. Therefore, the purpose of this experiment was to examine the association of aging and physical activity on the primary antibody and T cell response to the novel protein antigen keyhole-limpet hemocyanin (KLH). Forty-six physically active and sedentary, young (20-35 yr) and older (60-79 yr) men were recruited. Subjects were intramuscularly immunized with 100 microg of KLH, and blood samples were collected at days 0, 7, 14, 21, and 28. Samples were measured for anti-KLH IgM, IgG, IgG1, and IgG2 by ELISA. On day 21 after intramuscular KLH administration, subjects received an intradermal injection with 1 microg of KLH of inflammation recorded at 24, 48, 72, 96, and 120 h to assess anti-KLH delayed-type hypersensitivity response. There was a significant reduction in all anti-KLH measures with aging except for anti-KLH IgG2. The physically active older group had significantly higher anti-KLH IgM, IgG, IgG1, and delayed-type hypersensitivity responses, but not IgG2 compared with the sedentary older group. In conclusion, regular physical activity in older men is associated with a more robust immune response to novel antigenic challenge.     

Factors of the local immune defence of the reproductive tract in pregnant women with vaginosis

Clinico-immunological examination of 99 pregnant women with diagnosed vaginosis and 132 pregnant women without genital infection as a control group, was carried out. The immunological factors of cervical and vaginal secretions in pregnant women before and after local treatment were studied. The conclusion was made that the established changes in the cell-mediated and humoral factors of the immune resistance of the reproductive system could probably play some pathogenetic role in the development of vaginosis and its relapses in pregnant women. As found in this study, more pronounced changes in the local factors of immune protection (the signs of the functional irritation of neutrophils in combination with the prevalence of sIgA and lysozyme simultaneously with a decrease in the level of IgM and IgG in cervical slime) developed in patients with subsequent relapses of vaginosis, these changes remaining after local treatment. The defects of cell-mediated and humoral factors of cervical and humoral secretions, together with some clinical parameters, were shown to be prognostically unfavorable with respect to the relapses of vaginosis in pregnant women.     

Adaptive immune responses are linked to the mating system of arvicoline rodents

ABSTRACT Males generally exhibit reduced immune responses and greater susceptibility to disease than females. The suppressive effect of testosterone on immune function is hypothesized to be one reason why males have lower immune responses than females. Presumably, this effect of testosterone should be more pronounced among polygynous than monogamous species because circulating testosterone is higher among polygynous than monogamous males. The present study examined the extent to which sex differences in specific humoral immunity are related to the endocrine status and mating system of two arvicoline rodents. Humoral immunity was evaluated among polygynous meadow voles (Microtus pennsylvanicus) and monogamous prairie voles (Microtus ochrogaster) by challenging them with the novel antigen keyhole limpet hemocyanin (KLH) and assessing specific immune responses 5, 10, and 15 d following immunization. Overall, meadow voles mounted higher anti-KLH IgM and IgG responses than prairie voles did. Sex differences were also apparent for anti-KLH IgM responses; male meadow voles mounted higher antibody responses than conspecific females, whereas female prairie voles mounted greater responses to KLH than did conspecific males. Male meadow voles had significantly higher testosterone concentrations and reproductive organ mass than male prairie voles did but had elevated immune responses, suggesting that testosterone may not be the primary factor involved in the observed sex and species differences in immune responses. Species and sex differences in corticosterone concentrations were also evident and may contribute to the observed differences in immune function. The influence of extrinsic factors on immune function is also discussed. Taken together, these data provide evidence that the mating system may influence endocrine-immune interactions.     

Differential diagnosis of schistosomiasis mekongi and trichinellosis in human.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.     

Murine defense mechanism against Candida albicans infection. I. Collaboration of cell-mediated and humoral immunities in protection against systemic C. albicans infection

Mice immunized with viable C. albicans cells demonstrated a high incidence of cell-mediated and a low incidence of humoral immune response. There was good agreement between the final survival rate of C. albicans infected mice and the rate of simultaneous cell-mediated and humoral immune response acquisition. Immunized mice with positive delayed hypersensitivity (DTH) against C. albicans crude antigen showed significant protection against intravenous challenge with C. albicans. Furthermore, the transfer of immunoglobulins from rabbit anti-C. albicans serum to DTH-positive mice enhanced protection, while it did not protect control mice against a subsequent challenge with C. albicans. These results suggest that cell-mediated immunity plays a major role and humoral immunity a side role in the defense mechanism(s) of C. albicans infected mice.     

Effects of housing conditions during the rearing and laying period on adrenal reactivity, immune response and heterophil to lymphocyte (H/L) ratios in laying hens

This study was conducted to evaluate the effect of early rearing conditions on physiological, haematological and immunological responses relevant to adaptation and long-term stress in white Leghorn hens with intact beaks housed in furnished cages (FC) or conventional cages (CC) during the laying period. Pullets were cage reared (CR) or litter floor reared (FR). From 16 to 76 weeks of age, hens were housed in FC (eight hens per cage) or in CC (three hens per cage). As measures of long-term stress at the end of the laying period, adrenal reactivity was quantified by assessing corticosterone responses to adrenocorticotropin challenge, and immune response was assessed by measuring antibody responses after immunization with sheep red blood cells (SRBC) and keyhole limpet haemocyanin (KLH). Heterophil to lymphocyte (H/L) ratio was employed as an indicator of stress. Rearing conditions significantly affected anti-SRBC titres (P < 0.0001) and tended to affect H/L ratios (P = 0.07), with the highest values found in FR hens. Layer housing affected H/L ratio (P < 0.01); the highest ratio was found in FR birds housed in FC during the laying period. This study shows that early rearing environment affects immunological indicators that are widely used to assess stress in laying hens. However, while results on H/L ratio indicated that FR birds experienced more stress particularly when they were housed in FC during the laying period, the immune responses to SRBC in FR hens was improved, indicating the opposite. This contradiction suggests that the effects on immune response may have been associated with pathogenic load due to environmental complexity in FR and FC hens rather than stress due to rearing system or housing system per se.     

A Novel Immunomodulatory Hemocyanin from the Limpet Fissurella latimarginata Promotes Potent Anti-Tumor Activity in Melanoma

Hemocyanins, the huge oxygen-transporting glycoproteins of some mollusks, are used as immunomodulatory proteins with proven anti-cancer properties. The biodiversity of hemocyanins has promoted interest in identifying new anti-cancer candidates with improved immunological properties. Hemocyanins promote Th1 responses without known side effects, which make them ideal for long-term sustained treatment of cancer. In this study, we evaluated a novel hemocyanin from the limpet/gastropod Fissurella latimarginata (FLH). This protein has the typical hollow, cylindrical structure of other known hemocyanins, such as the keyhole limpet hemocyanin (KLH) and the Concholepas hemocyanin (CCH). FLH, like the KLH isoforms, is composed of a single type of polypeptide with exposed N- and O-linked oligosaccharides. However, its immunogenicity was significantly greater than that of KLH and CCH, as FLH induced a stronger humoral immune response and had more potent anti-tumor activity, delaying tumor growth and increasing the survival of mice challenged with B16F10 melanoma cells, in prophylactic and therapeutic settings. Additionally, FLH-treated mice demonstrated increased IFN-γ production and higher numbers of tumor-infiltrating CD4⁺ lymphocytes. Furthermore, in vitro assays demonstrated that FLH, but not CCH or KLH, stimulated the rapid production of pro-inflammatory cytokines (IL-6, IL-12, IL-23 and TNF-α) by dendritic cells, triggering a pro-inflammatory milieu that may explain its enhanced immunological activity. Moreover, this effect was abolished when deglycosylated FLH was used, suggesting that carbohydrates play a crucial role in the innate immune recognition of this protein. Altogether, our data demonstrate that FLH possesses increased anti-tumor activity in part because it activates a more potent innate immune response in comparison to other known hemocyanins. In conclusion, FLH is a potential new marine adjuvant for immunization and possible cancer immunotherapy.     

Darker female pigeons transmit more specific antibodies to their eggs than do paler ones

Melanin‐based coloration is widespread among vertebrates, yet the adaptive significance of such pigments remains elusive, particularly with regard to the link between melanin and immune‐mediated maternal effects. The aim of this study was to investigate whether melanin‐based coloration could signal the ability of mothers to mount a humoral response and to transfer maternal antibodies (Ab) to their young. We injected differently coloured (pale and dark) female feral pigeons (Columba livia) with Chlamydiae (a natural antigen) and Keyhole Limpet Haemocyanin (KLH, an artificial antigen), and found no significant difference in humoral response between differently coloured females. However, darker females transferred more Ab against Chlamydiae into their eggs than paler ones, despite similar circulating levels of Ab. In addition to this, melanin‐based coloration showed a high heritability value. This suggests that a genetically based coloured trait might be linked to the ability of females to transfer specific Ab against Chlamydiae (but not against KLH) to their offspring, independent of their ability to produce Ab. This suggests that transmission of maternal Ab is antigen dependent, and that melanin‐based coloration might signal female ability to transmit specific Ab against natural pathogens. © 2013 The Linnean Society of London