Iron injections in mice increase skeletal muscle iron content, induce oxidative stress and reduce exercise performance

Iron accelerates the production of reactive oxygen species (ROS). Excessive levels of ROS are thought to accelerate skeletal muscle fatigue and contribute to the loss of skeletal muscle mass and function with age. Patients with an iron overload disease frequently report symptoms of weakness and fatigue, which is attributed to reduced cardiac function. The contribution of skeletal muscle to these symptoms is unknown. Using a mouse model of iron overload, we determined the extent of iron accumulation in skeletal muscle and the concentrations of the iron storage protein ferritin. The level of oxidative stress, changes in antioxidant enzymes and exercise performance were also assessed. Compared with control mice, the iron overloaded mice had elevated levels of iron in the tibialis anterior muscle and a fourfold increase in ferritin light chain. The oxidative stress product malondialdehyde was increased in the iron group compared with the control group, as was the antioxidant enzyme activity of glutathione reductase and glutathione peroxidase. The iron group performed less work on an endurance test and produced less force in a strength test. Body weight and skeletal muscle weight were lower in the iron group following the intervention. Iron loading reduced the weight of the fast-twitch extensor digitorum longus muscle more than the slow-twitch soleus muscle. In summary, iron accumulation in skeletal muscle may play a significant role in the reduced exercise capacity seen in iron overload disorders and in ageing, and may play an underlying role in skeletal muscle atrophy.     

Allergens induce apoptosis in lymphocytes from atopic patients.

Repeated stimulation of immune cells may induce an "activation-induced cell death" (AICD) program. Allergy is characterized by the cyclic activation of allergen-reactive immune cells. To study the effects of allergen stimulation in cell proliferation and apoptosis in atopic subjects, peripheral blood mononuclear cells (PBL) from 40 atopic patients with positive reactivity to the allergens Olea Europaea (OE) and Lollium Perenne (LP) (20 without immunotherapy and 20 with specific immunotherapy) and 10 normal subjects were cultured with the allergens OE and LP. PBL from atopic patients proliferate more vigorously than cells from normal subjects after culture in vitro with both allergens, although PBL from atopic subjects without immunotherapy proliferate more than PBL from atopic subjects with immunotherapy. The study of cell proliferation shows that in atopic patients PBL mainly exhibit the CD4/CD45RO phenotype. This preferential proliferation is more evident in PBL from atopic patients treated without immunotherapy. Cell culture with specific allergens induces apoptosis in PBL from atopic patients. The percentage of apoptosis increased when atopic patients had been previously treated with immunotherapy. In addition to the observed increase in cell proliferation, apoptosis mainly occurs in the CD45RO cells that support the involvement of these cells in allergy. Furthermore, results obtained in cells from immunized patients suggest that an AICD process may partly at least explain the mechanism of action of allergen immunotherapy.     

Denaturization of allergen P: effect on allergenicity, antigenicity and immunogenicity.

When allergen P was denatured by 8M urea, the modified molecule still reacted with IgE specific for the native allergen but not with hemagglutinating antibodies. Heating at 100 degrees C abolished the reaction in both cases. The results suggested differences between allergenic and antigenic capacities which may be based on structural differences of the antigenic determinants.     

Chronic intravenous injections of antigen induce and maintain tolerance in T cell receptor-transgenic mice

Antigen-specific T cell tolerance can be induced by systemic injection of high-dose antigen. In particular, a single intravenous (i.v.) injection of influenza virus hemagglutinin peptide in HNT-TCR transgenic mice induces T cell tolerance through thymocyte apoptosis as well as anergy and deletion of peripheral CD4⁺ T cells. We now show that this tolerance is reversed after 8 weeks probably due to the short in vivo half-life of the peptide. Since durable tolerance is required for this strategy to be of therapeutic value, we tested whether weekly i.v. injections of peptide (up to 12 weeks) could maintain the CD4⁺ T cell tolerance. Each injection induces a profound deletion of thymocytes, although their level recovers before the next injection. Therefore, during the treatment period, the thymus undergoes cycles of contraction/expansion. In the periphery, the number of CD4⁺ T cells is stably decreased and the persisting CD4⁺ T cells are hyporeactive both in vitro and in vivo. This tolerance is essentially peripheral since comparable results were obtained in thymectomized HNT-TCR mice injected weekly. Our data show that stable antigen-specific tolerance can be induced by repeated i.v. injections of antigen. These findings might have implications for the treatment of T cell-mediated autoimmune diseases.     

Analysis of the effect of repeated estrone injections on the mitotic activity of uterine epithelium in mice

Summary Estrone administration stimulates mitosis of the uterine epithelium in castrated mice, inhibiting it, however, in repated injections. Discontinuance of estrone administration at the time of intensive cell multiplication leads to reduction of mitotic activity of the tissue with its subsequent rise; as distinct from this, suspension of hormone administration against the background of low mitotic activity provokes its sharp rise already on the 3rd day.Folic acid and adenosintriphosphate exert no stimulating effect on cellular division during oestron therapy. Evidently, the inhibitory effect of repeated oestron injections is not connected with the disturbed DNA synthesis or with the exhaustion of the high energy phosphorus compounds stored in the cell.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 51,No. 3, pp. 102–105, March, 1961     

Enhanced reaginic antibody formation to ovalbumin in mice given repeated injections of concanavalin A.

Mice injected with 4 weekly doses of concanavalin A arabinogalactan complex (Con A-AG) give a secondary type of IgE response when they encounter ovalbumin (Ov) for the first time. The IgE-enhancing effect of Con A-AG immunization applies to Ov but not to various other antigens. However, no cross-reaction appears to exist at the humoral level between Con A and Ov. The apparent priming of mice to Ov by Con A can be adoptively transferred to syngeneic irradiated recipients by spleen cells.     

Immunomodulation with liposomes: the immune response elicited by liposomes with entrapped dichloromethylene-diphosphonate and surface-associated antigen or hapten

Macrophages in the murine spleen were eliminated by the intravenous administration of dichloromethylene-diphosphonate (DMDP) encapsulated in liposomes. The immune response against an antigen (bovine serum albumin, BSA) or hapten (dinitrophenyl, DNP) associated with the surface of the DMDP liposomes was studied. Significant effects of macrophage elimination on the primary (IgM), but not on the secondary (IgG), anti-BSA response were found. Dramatic effects on the response against liposome-associated DNP were observed in macrophage-depleted mice. The number of anti-DNP antibody-forming cells in the spleen decreased from 300 to 28 per section, and anti-DNP serum titres dropped to 12% of their normal values. Since a similar phenomenon was observed for TNP-Ficoll, a thymus-independent type-2 antigen (and not for thymus-dependent or thymus-independent type-1 antigens), we suggest that this response should be classified as thymus-independent type-2 on grounds of its in vivo behaviour. We conclude that adjuvant activity (and memory formation) of liposomes with antigen exposed on their surface occurs irrespective of the presence or absence of splenic macrophages, and that DMDP liposomes could be useful in drug targeting with antigenic liposomes.     

Differential immunogenicity and allergenicity of native and recombinant human lactoferrins: Role of glycosylation

Human native milk lactoferrin (LF) and recombinant forms of lactoferrin (rLF) are available with identical aa sequences, but different glycosylation patterns. Native lactoferrin (NLF) possesses the intrinsic ability to stimulate vigorous IgG and IgE antibody responses in BALB/c mice, whereas recombinant forms (Aspergillus or rice) are 40‐fold less immunogenic and 200‐fold less allergenic. Such differences are independent of endotoxin or iron content and the glycans do not contribute to epitope formation. A complex glycoprofile is observed for NLF, including sialic acid, fucose, mannose, and Lewis (Le)^x structures, whereas both rLF species display a simpler glycoprofile rich in mannose. Although Le^x type sugars play a Th2‐type adjuvant role, endogenous expression of Le^x on NLF did not completely account for the more vigorous IgE responses it provoked. Furthermore, coadminstration of rLF downregulated IgE and upregulated IgG2a antibody responses provoked by NLF, but was without effect on responses to unrelated peanut and chicken egg allergens. These results suggest glycans on rLF impact the induction phase to selectively inhibit IgE responses and that differential glycosylation patterns may impact on antigen uptake, processing and/or presentation, and the balance between Th1 and Th2 responses.     

Characterization of Leishmania donovani antigens encapsulated in liposomes that induce protective immunity in BALB/c mice

Leishmania donovani promastigote membrane antigens (LAg) encapsulated in positively charged liposomes have been found to induce very significant levels of protection against experimental visceral leishmaniasis. The protectively immunized animals exhibited profound delayed-type hypersensitivity and antibody responses. The extent of protection induced by the same antigens, however, varied depending on the charge of the vesicles, with maximum induction by positively charged liposomes, followed by neutral liposomes and last negatively charged liposomes. Characterization of LAg and LAg entrapped in liposomes of different charges by Western blot analysis revealed the immunodominance of gp63 in all three vaccine preparations. The strong reactivity of antigens in a restricted antigen profile that included, in addition to gp63, 72-, 52-, 48-, 45-, 39-, and 20-kDa components in neutral and positively charged liposomes contrasted with the reactivity of a greater number of LAg components in negatively charged liposomes. Resistance to visceral leishmaniasis appears to depend on the immunity induced by gp63 and a few select antigens in association with the right liposomes. A striking similarity between the immunogenic profile of partially purified soluble antigens and that of LAg in neutral and positively charged liposomes suggests the potentiality of these antigens in future vaccine studies of L. donovani.     

Intrahippocampal LPS injections reduce Aβ load in APP+PS1 transgenic mice

Multiple lines of evidence indicate that inflammatory processes are involved in the pathogenesis of Alzheimer’s disease. Lipopolysaccharide (LPS) is widely used to induce inflammation in experimental systems. Consequently we injected LPS or saline intrahippocampally in 11 and 16 months old APP+PS1 transgenic mice to induce brain inflammation, then used immunocytochemistry to examine amyloid pathology 7 days later. As expected, LPS activated microglia as indicated by a significant increase in the area covered by major histocompatibility complex-II (MHC-II) immunostaining in the mice injected with LPS compared to the saline injected. Simultaneously, Aβ immunostaining showed an unexpected reduction of the Aβ load in the mice injected with LPS compared to the saline injected. This effect of LPS on the Aβ load in APP+PS1 mice strengthens the hypothesis that moderate amounts of microglial activation may be beneficial in Alzheimer’s disease, by increasing the clearance of Aβ.     

Feeding in response to repeated protamine zinc insulin injections

Sustained overeating was induced in rats with repeated daily injections of protamine zinc insulin. The hyperphagia and hypophagia upon termination of insulin treatment were directly related to the amount of insulin that had been administered. The intake was regulatory since animals adjusted food consumption appropriately to compensate for caloric intake from glucose solutions, and they maintained stable caloric intake by reducing the weight of food ingested from a high fat diet. Overeating was unmodified by the use of an equicaloric high protein diet even though total weight gain was reduced. Injections of cycloheximide and dexamethasone prevented protamine zinc insulin induced overeating.     

Attempts to study the localization of liposomes and liposome entrapped antigen in the spleen.

Attempts were made to study the localization of intravenously injected liposomes and enclosed labelled antigens in the spleen of mice, using autoradiography. A more than hundred fold increased uptake of antigen by the spleen was obtained when liposome entrapped antigen was compared with free antigen or antigen-antibody complexes which have been used in earlier studies. No marked accumulation of the labelled antigen was found in the follicles of the spleen, although specific antibodies to the injected antigen have been injected simultaneously with the liposome entrapped antigen. It is argued that the bulk of the labelled antigen in the spleen 0.5 to 2 h after injection was still enclosed in the liposomes, whereas part of the label, arranged in patches in the red pulp and marginal zone, corresponds with labelled antigen in macrophages, released after digestion of the liposomal membranes. The present techniques did not enable the detection of the liposomes themselves.     

Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age.