Immune Modulatory Activities of Arginyl-Fructose (AF) and AF-Enriched Natural Products in In-Vitro and In-Vivo Animal Models.

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF (p < 0.01) or GF (p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM (p < 0.01), IgG (p < 0.05), IL-2 (p < 0.01), IL-4 (p < 0.01), IL-6 (p < 0.01), and IFN-γ (p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

Modulatory effect of Tagetes erecta flowers essential oils via Nrf2/HO-1/NF-κB/p65 axis mediated suppression of N-methyl-N'nitro-N-nitroguanidine (MNNG) induced gastric cancer in rats.

Protective effect of Tagetes erecta flowers essential oils was investigated on oxidative stress, immune response, inflammation, and apoptosis against N-methyl-N'nitro-N-nitroguanidine (MNNG) induced gastric cancer in rats. Essential oil were extracted from Tagetes erecta flowers and analyzed using gas chromatography-mass spectrometry (GC-MS). For observing a protective effect against MNNG induced gastric cancer, we divided rats into 4 groups (group A to D) having 10 rats in each group. Performed various experiments and measured a different parameters to investigate antioxidant activity, immune response, anti-inflammatory and anti-apoptotic activity. The levels of malondialdehyde were markedly increased in the presence of N-methyl-N'nitro-N-nitroguanidine, whereas, the antioxidant activities of superoxide dismutase, and catalase were lowered in the treated rats in contrast with the control. Intervention with TEEO to gastric cancer-induced rats upregulated the redox status and the activity of the immune system to decrease cancer risk. The proinflammatory cytokines (IL-6 and TNF-α) secretions that were induced by MNNG were markedly inhibited by TEEO. Administration of TEEO also significantly reduced terminal deoxynucleotidyl transferase dUTP nick end labeling positive gastric cancer cells, expression of mRNA of caspase-3, and Bax. Whereas, the expression of Bcl-2 was increased. Additionally, downregulation of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and IκBα degradation and the nuclear factor-κB (NF-κB) p65 expression in tissues of the stomach of MNNG-induced-rats were markedly elevated due to TEEO. This suggested possession of TEEO with a protective shield against MNNG induced gastric cancer by the exertion of antioxidative stress, anti-apoptotic response, the anti-inflammatory response through Nrf2/HO-1, and NF-κB signaling pathways.

Oleanane-type glycosides from the roots of Weigela florida "rumba" and evaluation of their antibody recognition.

Three triterpene glycosides were isolated from the roots of Weigela florida "rumba" (Bunge) A. DC.: two previously undescribed 3-O-ß-d-xylopyranosyl-(1→2)-[ß-d-xylopyranosyl-(1→4)]-ß-d-xylopyranosyl-(1→4)-ß-d-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyloleanolic acid (1) and 3-O-ß-d-xylopyranosyl-(1→2)-[ß-d-glucopyranosyl-(1→4)]-ß-d-xylopyranosyl-(1→4)-ß-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid (2), and one isolated for the first time from a natural source 3-O-ß-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyloleanolic acid (3). Their structures were elucidated mainly by 2D NMR spectroscopic analysis (COSY, TOCSY, NOESY, HSQC, HMBC) and mass spectrometry. Compounds 2 and 3 were further evaluated as antigens in enzyme-linked immunosorbent assay (ELISA) to recognize IgM antibodies in multiple sclerosis (MS) patients' sera.

Effects of formaldehyde inhalation on humoral immunity and protective effect of Nigella sativa oil: An experimental study.

AIM: This study was carried out to determine the effects of formaldehyde (FA) inhalation on the humoral immunity of rats and the protective effect of Nigella sativa (NS) oil. MATERIALS AND METHODS: The rats (n = 33) were divided into five groups, with five animals in the control group (FA-free air) and seven in the other four groups. Group FA1 was exposed to FA (5 ppm), group FA + NS1 was treated with NS and exposed to FA (5 ppm), group FA2 was exposed to FA (10 ppm), and group FA + NS2 was treated with NS and exposed to FA (10 ppm). At the end of a 4-week study period, blood samples were collected. Enzyme-linked immunosorbent assay was used to determine the levels of serum total immunoglobulin A (IgA), total immunoglobulin M (IgM), total immunoglobulin G (IgG), and complement 3 (C3). RESULTS: FA inhalation significantly increased serum IgA, IgM, and C3 levels and decreased serum IgG levels compared with the control group. NS administration decreased serum IgA, IgM, and C3 levels, which were induced by FA inhalation. CONCLUSION: FA inhalation significantly increased acute antibody responses and C3 levels in a dose-dependent manner compared with the control group. FA inhalation decreased the secondary immune response compared with the control group. Levels of acute antibody responses and complement following exposure to FA inhalation returned to normal following treatment with NS (immunoregulatory effect). However, NS did not affect the secondary immune response.

From goat colostrum to milk: physical, chemical, and immune evolution from partum to 90 days postpartum.

This study focused on the study of the changes originated in the milk from partum until d 90 of lactation. Ten multiparous Majorera goats, bred carefully under animal health standards, with a litter size of 2 kids (the average in this breed is 1.83 prolificacy) and similar gestation length (149 ± 1 d) were used. Goat kids were removed from their dams to avoid interferences with the study. Compositional content (fat, protein, and lactose) were measured, as well as some other properties, including pH, density, titratable acidity, ethanol stability, rennet clotting time, and somatic cell count. Moreover, immunity molecules (IgG, IgA, and IgM concentrations and chitotriosidase activity) received great attention. Fat and protein content were higher in the first days postpartum, whereas lactose content was lower. Density, titratable acidity, rennet clotting time, and somatic cell count decreased throughout the lactation period, whereas pH and ethanol stability increased. Relative to the immunological parameters, each measured parameter obtained its maximum level at d 0, showing the first milking as the choice to provide immunity to the newborn kids. On the other hand, this study might be used to establish what the best use is: processing or kid feeding.

Effect of social ranks and gestation housing systems on oxidative stress status, reproductive performance, and immune status of sows.

Ninety-six multiparous sows were randomly assigned into 2 different gestation housing systems on d 35 of gestation: individual gestational crates (n = 24) or small groups with 3 sows in gestational pens (n = 24). Sows were classified into 4 treatments based on gestation housing systems and social ranks within each gestational pen: sows housed in individual gestational crates were in control treatment (CON), and sows destined to high, middle, or low social ranks within each pen were classified into high social rank treatment (HR), middle social rank treatment (MR), and low social rank treatment (LR). The social rank of sows within a pen was determined by their winning percentage during aggressive interactions observed for a 4-d period after mixing on d 35 of gestation. Plasma samples collected from each sow on d 35, 60, 90, and 109 of gestation and d 1 and 18 of lactation were used to determine concentrations of malondialdehyde (MDA), protein carbonyls, 8-hydroxy-deoxyguanosine (8-OHdG), IgG, and IgM. Sows in HR had higher (P < 0.05) body weight during gestation and lactation, smallest (P < 0.05) litter weight at birth, increased (P < 0.05) number of stillborn than sows in MR and LR, and tended to have decreased (P = 0.073, P = 0.064) number of born alive compared with sows in CON and LR. Sows in LR had lower farrowing rate compared with sows in MR. Plasma concentration of protein carbonyl in HR was higher (P < 0.05) than that in MR on d 3 of lactation. Plasma concentrations of 8-OHdG in LR was greater (P < 0.05) than that in HR on d 90 of gestation, d 3 and 18 of lactation, and greater (P < 0.05) than CON on d 18 of lactation. The reproductive performance of sows from all of the social ranks was related to their oxidative stress status during gestation and lactation. Collectively, the reproductive performance, oxidative stress status, and immune status did not differ between sows housed in gestational crates (CON) and pens (HR + MR + LR). Sows in CON and MR did not show inferior reproductive performance during gestation and lactation. Sows in HR and LR had increased oxidative damage during late gestation and lactation which could contribute to the reduced litter size and litter weight in HR and lower farrowing rate in LR.

Intestinal IgA⁺ cell numbers as well as IgA, IgG, and IgM contents correlate with mucosal humoral immunity of broilers during supplementation with high fluorine in the diets.

Fluoride (F), a well-recognized harmful substance, is easily absorbed by the intestinal mucosa. The intestinal mucosal immune system is equipped with unique innate and adaptive defense mechanisms that provide a first line of protection against infectious agents. Meanwhile, immunoglobulins are the major secretory products of the adaptive immune system and their levels can be a strong indicator of a disease or condition. In this study, therefore, we investigated the effects of high dietary fluorine on the numbers of immunoglobulin A-positive (IgA(+)) cells in the lamina propria of intestines (duodenum, jejunum and ileum) by immunohistochemistry as well as on the contents of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in the mucosa of intestines (duodenum, jejunum, and ileum) by enzyme-linked immunosorbent assay (ELISA). A total of 280 1-day-old healthy avian broilers were randomly divided into four groups and fed on a corn-soybean basal diet as control diet (fluorine 22.6 mg/kg) or the same basal diet supplemented with 400, 800, and 1,200 mg/kg fluorine (high fluorine groups I, II, and III) in the form of sodium fluoride (NaF) for 42 days. The experimental data showed that the numbers of IgA(+) cells as well as the IgA, IgG, and IgM contents were significantly decreased (P < 0.01 or P < 0.05) in the high fluorine groups II and III when compared with those of the control group. It was concluded that dietary fluorine in the range of 800-1,200 mg/kg significantly reduced the numbers of the IgA(+) cells and the contents of aforementioned immunoglobulins in the intestines (duodenum, jejunum, and ileum) of broilers, which could finally impact the mucosal humoral immune function in the intestines by a way that reduces the lymphocyte population and/or lymphocyte activation.

Administration of RRR-α-tocopherol to pregnant mares stimulates maternal IgG and IgM production in colostrum and enhances vitamin E and IgM status in foals.

This study assessed the effect of a vitamin E supplement given to pregnant mares on immunoglobulins (Ig) levels in foals. In addition, the fatty acid (FA) content and composition of the mares' milk was assessed. Milk α-tocopherol concentrations were compared between pregnant Danish Warmblood mares (n = 17) given a daily oral supplement of 2500 international units (IU) RRR-α-tocopherol in the last 4 weeks of pregnancy and a group of unsupplemented mares (n = 17) receiving 170-320 IU vitamin E daily originating from the feed. Milk α-tocopherol was higher in supplemented mares (36.7, 12.4 and 9.8 µmol/l respectively) in relation to control mares (13.1, 6.4 and 5.8 µmol/l on days 1, 2 and 3 respectively; p < 0.001). Milk IgG was higher on days 2 and 3 post-partum (PP) in supplemented mares (1.03 and 0.73 mg/ml respectively) in relation to control mares (0.79 and 0.56 mg/ml respectively; p < 0.05). Milk IgM was higher on days 2 and 3 post-partum (PP) in supplemented mares (0.19 and 0.17 mg/ml) in relation to control mares (0.13 and 0.11 mg/ml respectively; p < 0.05). Plasma α-tocopherol in foals was higher from supplemented mares on days 1, 2 and 3 (5.7, 14.8 and 19.2 µmol/l respectively) in relation to foals from control mares (3.6, 6.1 and 7.6 respectively; p < 0.001). Foal plasma IgM was higher from supplemented mares on day 3 (0.50 mg/ml) in relation to foals from control mares (0.32 mg/ml; p < 0.001). The total FA content in milk was highest on day 1 (21.6 g FA/kg milk) in relation to days 2 and 3 (13.6 and 13.5 g FA/kg milk respectively; p < 0.001). In conclusion, a daily oral supplement of 2500 IU RRR-α-tocopherol increased α-tocopherol content in mare milk and foal plasma, IgG and IgM in mare milk and IgM in foal plasma.

Monoclonal antibody to fibulin-1 generated by genetic immunization.

Fibulin-1 (Fbln-1) is an extracellular matrix (ECM) and plasma glycoprotein. Considering the growing evidence indicating that Fbln-1 plays a role in cancer we sought to develop monospecific antibodies to better facilitate further studies of the function of Fbln-1 in breast cancer. Using a plasmid expression vector encoding full-length human Fbln-1D as an immunogen and CpG oligodeoxyribonucleotides as adjuvant a monoclonal antibody (MAb) against Fbln-1 was produced. This MAb, designated MEM-2 was of IgM isotype and reacted with bacterially expressed Fbln-1. Furthermore, MEM-2 reacted with Fbln-1 expressed in the ECM released by cultured human breast carcinoma SKBR-3 cells in ELISA, and also with Fbln-1 present in SKBR-3 cell extract in immunoprecipitation and Western blotting. MEM-2 also reacted with Fbln-1 in human breast carcinoma specimens. These findings illustrate the utility of genetic immunization as a means of generating monoclonal antibodies to tumor-related ECM proteins. MEM-2 represents a useful new tool for the study of Fbln-1 in breast cancer.

In vitro evaluation of DNA-DNA hybridization as a two-step approach in radioimmunotherapy of cancer.

The specific delivery of radioisotopes to a tumor at minimal radiation of normal tissue is the ultimate aim of radioimmunotherapy. In this respect a two-step pretargeting regimen generally leads to an improved tumor to normal tissue uptake ratio compared to direct administration of radioimmunoconjugates. In this paper, in vitro studies are described in which the specific hybridization of complementary DNA fragments is the recognition mechanism in a pretargeting regimen comprising tumor cell saturation with a monoclonal antibody (MoAb)-oligonucleotide conjugate, followed by administration of the radiolabeled complementary oligonucleotide. Complementary oligodeoxynucleotides (15-mers; melting temperature, 68 degrees C) were prepared on a DNA synthesizer. The 5'-end was derivatized with a functional group for labeling with iodine, and the 3'-end was substituted with an amino function suitable for conjugation to an antibody (or attachment of a biotin residue). Both terminal modifications ensure stability of the oligonucleotides against exonucleases because the unconjugated form is stable for 24 h and the conjugated form is stable for several days when incubated in human plasma at 37 degrees C. Antibody-DNA conjugates were prepared by introduction of sulfhydryl groups into the oligonucleotide, followed by conjugation to maleimide-substituted MoAbs. Typically, 3 oligonucleotides were conjugated to an IgG, and 4-6 were conjugated to an IgM with preservation of immunoreactivity. Histochemistry on fresh frozen sections of breast cancer tissue demonstrated qualitatively the specificity of our two-step procedure. In vitro experiments with human tumor cell lines and tumor-specific MoAbs showed that, after saturation with tumor-specific MoAb-DNA conjugates, quantitative hybridization of the tumor cell-bound oligonucleotides occurred at a 30-fold excess of the labeled complementary oligonucleotide: hybridization was complete after 30 min of incubation. No reaction was observed with an irrelevant MoAb-DNA conjugate. The oligonucleotide was neither taken up by tumor cells or endothelial cells nor hybridized to a significant extent with human genomic DNA. These data indicate the feasibility of this two-step approach in radioimmunotherapy.

Specific recognition of antibody-oligonucleotide conjugates by radiolabeled antisense nucleotides: a novel approach for two-step radioimmunotherapy of cancer.

One of the major challenges in radioimmunotherapy is the specific delivery of radioisotopes to tumor cells while minimizing normal tissue radiation. In this respect, the application of two-step pretargeting schemes generally leads to more favorable tumor to normal tissue uptake ratios than direct administration of radioimmunoconjugates. In this study, we present the specific hybridization of complementary DNA fragments as a novel recognition mechanism in pretargeting. Briefly, our strategy involves first administration of antibody-DNA conjugate, followed by targeting with radiolabeled complementary DNA (antisense DNA). Complementary oligodeoxynucleotides (14-mers, Tm = 57 degrees C), in which part of the phosphodiesters has been replaced by methylphosphonates (to ensure stability against nucleases), were prepared on a DNA synthesizer. The oligonucleotides were further derivatized via a uridine moiety at their 5'-end in such a way that radiolabeling or conjugation with antibodies could be accomplished. Both a murine IgG (anti-hCG) and the human anti-tumor IgM 16.88 were conjugated with one to three oligonucleotides via the heterobifunctional cross-linker SMCC. Incubation of these immunoconjugates with the radiolabeled antisense DNA revealed specific hybridization with the antibody-linked oligonucleotides. Antigen binding studies performed with antigen-coated matrices showed that the immunoreactivity of the antibody-DNA conjugate is preserved. Moreover, it is demonstrated that the radiolabeled DNA is still capable of hybridizing selectively with the oligonucleotides of the immunoconjugate, when the latter is bound to its antigen.

A lactogenic-immune-deficiency-syndrome in cows: unexplained phenomenon.

The majority of adult cows in a certain dairy herd, were found to have very low levels of immunoglobulins (Igs) in their colostrum. This phenomenon was defined by us as Lactogenic-Immune-Deficiency-Syndrome (LIDS). The mean IgG levels were 44.5 and 57.2 mg ml-1 respectively (on two different occasions) as compared to that of a control group which was 103.4 mg ml-1. The levels of Igs in the colostra of heifers from the same herd were found to be higher than those of adult cows. The degree of LIDS was found to be closely related to the age of cows in the herd. The low levels of Igs in the colostra were not directly linked to their concentrations in the sera of the affected cows. The relatively low amount of IgA in the affected colostra suggests that the local production in the lymph tissue associated with the mammary glands is impaired as well. In order to investigate the etiology of the phenomenon, tests were carried out to reveal whether bovine leucosis virus (BLV) infection or immune complexes were involved in the pathogenesis of LIDS. The results were negative. The etiology of LIDS remains for the time being unknown.

Physicochemical and immunochemical studies on bovine IgA and glycoprotein-a.

1. Bovine secretory IgA (SIgA) from colostrum (mol. wt. about 410,000) is composed of four alpha-chains (mol. wt. 61,000), four light chains (mol. wt. 23,000) and one molecule of glycoprotein-a (mol. wt. 70,000-86,000). The alpha-chains are antigenically and physicochemically distinct from the heavy chains of IgM, IgG1 and IgG2 while the light chains are identical to those occurring on other bovine immunoglobulins. Glycoprotein-a and bovine free secretory component are identical and the former name should be abolished. Much of this protein is covalently bonded to IgA. 2. The gel filtration behavior of serum IgA suggests it is a dimer. 3. The elution behavior of IgA and SIgA from ion-exchange columns and the solubility characteristic of SIgA in the presence of Zn2+ are similar to those of human and rabbit IgA. 4. The disc electrophoretic behavior of IgA and SIgA are distinct from IgM, dimeric IgG1, 7-S IgG and glycoprotein-a. Dimeric IgG1 (s20,w = 9.5) is abundant in colostrum and is similar in size to SIgA. 5. Bovine IgA shows physicochemical and immunochemical heterogeneity when studied by gel filtration, disc electrophoresis, immunoelectrophoresis and ultracentrifugational analyses. Lacrimal and nasal SIgA possess antigenic determinants absent on colostral SIgA.