Anti-obesity activity of hen egg anti-lipase immunoglobulin yolk, a novel pancreatic lipase inhibitor.

BACKGROUND: There is completely no report about both hen egg anti-lipase immunoglobulin yolk (IgY) and its anti-obesity action. Thus, we tried to isolate and characterize a novel anti-lipase immunoglobulin from hen egg yolk. Moreover, we investigated whether hen egg yolk anti-lipase IgY inhibits pancreatic lipase activity in vitro, and examined its ability to prevent obesity in a murine high fat diet-induced obesity model. METHODS: We determined the inhibitory action of Anti-lipase IgY on lipase activity in vitro. We also focused our evaluation on the anti-obesity properties of Anti-lipase IgY in a murine high fat diet-induced obesity model. RESULTS: Anti-lipase IgY blocked porcine lipase activity with an IC50 of 0.49 µM. Supplementing the high fat diet with only 0.2% (w/w) of Anti-lipase IgY for 35 days significantly decreased the weights of intraperitoneal adipose tissues, epididymal, mesenteric, retroperitoneal and perirenal adipose tissues, and the amounts of hepatic total lipid, triglyceride, and cholesterol. This was accompanied by a significant increase in the fecal excretion of triglyceride in the absence of diarrhea. Furthermore, Anti-lipase IgY treatment restored body weight gain to levels similar to mice fed with Control IgY. CONCLUSIONS: This study provides the first report of the development of anti-lipase IgY and the direct evidence that inhibition of pancreatic lipase using Anti-lipase IgY is an effective anti-obesity treatment due to the associated increase in fecal excretion of triglyceride.

Oral passive IgY-based immunotherapeutics: a novel solution for prevention and treatment of alimentary tract diseases.

This commentary summarizes the laboratory investigations and clinical trials published recently involving per-oral application of IgY supplemented food for specific orogastrointestinal disease prevention and control purposes. The prolonged use and misuse of conventional antibacterial drugs has spawned antibiotic resistant microbes prompting scientists to search for other germ-killing options. In particular, the use of IgY as a novel mode of immunotherapy using oral chicken immunoglobulin (IgY) to confer passive immunity has gained much interest as an inexpensive non-antibiotic alternative for the prophylaxis and treatment of a wide variety of infectious diseases. The stability of IgY in the orogastrointestinal tract and its safety profile has been well-documented. IgY has been used in the treatment or prevention of dental caries, periodontitis and gingivitis, gastritis and gastric ulcer, oral thrush and infant rotavirus diarrhea. The recent clinical trials on IgY with encouraging results has catapulted into the market novel nutraceutical or health supplements for therapeutic or prophylactic intervention based on the consumption of mono-specific or mixed IgY formulations. With recent trends in consumer preference for natural materials to alleviate health concerns, the increasing healthcare costs and the recent advances in drug delivery systems, IgY is likely to shift from its mainly functional food status toward pharmaceuticalization in the foreseeable future.

Recombinant Shiga toxin B subunit can induce neutralizing immunoglobulin Y antibody.

Previously, we have shown that chickens immunized with Shiga toxin (Stx) produce Stx-neutralizing egg yolk immunoglobulin Y (IgY) antibody. The anti-Stx-1 IgY and anti-Stx-2 IgY exert their neutralizing activity through their antibody activity against the B subunit of the toxin but not the A subunit. In the present study, chickens were immunized with recombinant Stx-1 B subunit (rStx-1B) and recombinant Stx-2 B subunit (rStx-2B). Induced anti-rStx-1B and anti-rStx-2B IgY neutralized the toxicity of Stx-1 and Stx-2 against HeLa 229 cells. The neutralizing activity of anti-rStx-1B IgY on Stx-1 was almost 10 times stronger than that of anti-Stx-1 IgY, and that of anti-rStx-2B IgY was 2.6 times stronger than that of anti-Stx-2 IgY. Anti-rStx-1B and anti-rStx-2B IgY reacted with multimeric and monomeric forms of the B subunits in contrast to anti-Stx-1 and anti-Stx-2 IgY that reacted with only the multimeric form. These results indicated that recombinant B subunits were promising antigens for induction of neutralizing antibodies in chickens.

Randomized placebo-controlled clinical trial of immunoglobulin Y as adjunct to standard supportive therapy for rotavirus-associated diarrhea among pediatric patients.

This study aims to evaluate the effect of hyperimmune immunoglobulin Y (IgY) against human rotavirus (HRV) among pediatric patients receiving standard supportive treatment for rotavirus-associated diarrhea mostly with an enteric non-cholera co-pathogen in a hospital setting. Two natural HRV reassortant clinical strains ATCC VR 2273 and ATCC VR 2274 were used as mixed immunizing antigens in poultry hens to generate anti-HRV IgY (Rotamix IgY). The Rotamix IgY was used in laboratory and clinical studies against control or placebo IgY. The control or placebo IgY was prepared using tissue culture medium from mock-infected MA104 cell line as antigen for poultry immunization. In vitro, Rotamix IgY exhibited multi-serotypic cross neutralization activities along with synergistic effects against major global serotypes G1, G2, G3, G4 and other human or animal rotavirus strains when compared with mono-specific IgY. Suckling mice (ICR strain) pre-treated orally once with Rotamix IgY and then challenged with rotavirus 3h later showed a significant dose-dependent reduction in frequency (p<0.05) and duration (p<0.05) of diarrhea compared to placebo IgY-treated mice. Out of 114 children aged between 3 and 14 months and with diarrhea upon admission in a Myanmar hospital, 54 dehydrated and rotavirus-positive children were randomized into Rotamix IgY group and placebo IgY group. Of these, only 52 children had complete data with n=26 children per study group. Ninety-two percent of patients in each of these groups were positive for co-infecting enteric non-cholera pathogen and all patients received standard supportive therapy for diarrhea. The patients were monitored for volume and duration of oral rehydration fluid (ORF) and intravenous fluid (IVF) intake, daily stool frequency and overall duration of diarrhea, and frequency and duration of rotavirus shedding. Compared to placebo IgY group, the Rotamix IgY group had statistically significant reduction in mean ORF intake (p=0.004), mean duration of intravenous fluid administration (p=0.03), mean duration of diarrhea from day of admission (p<0.01) and mean duration of rotavirus clearance from stool from day of admission (p=0.05). Overall, our novel approach using oral Rotamix IgY for rotavirus-infected children mostly with non-cholera enteric pathogen co-infection appears to be a promising, safe and effective adjunct to management of acute diarrhea in pediatric patients.

The origin of IgG-containing cells in the bursa of Fabricius.

The bursa of Fabricius of the chicken is known as a primary lymphoid organ for B-cell development. Morphologically, the origin of IgG-containing cells in the bursa has not been clear until now, because abundant maternal IgG (MIgG) is transported to the chick embryo and distributed to the bursal tissue around hatching. Thus, it has been difficult to find out whether these cells themselves biosynthesize IgG or if they acquire MIgG via attachment to their surface. Our present study employing in situ hybridization clarified that IgG-containing cells in the medulla of bursal follicles did not biosynthesize IgG. To study the role of MIgG in the development of those IgG-containing cells, MIgG-free chicks were established from surgically bursectomized hen (SBx-hen). We found that, on the one hand, deprivation of MIgG from chicks completely inhibited the development of IgG-containing cells in the medulla after hatching. On the other hand, administration of MIgG to MIgG-free chicks recovered the emergence of those cells. In addition, we observed that those cells did not bear a B-cell marker and possessed dendrites with aggregated IgG. These results demonstrate that IgG-containing cells in the medulla are reticular cells that capture aggregated MIgG. Moreover, we show that the isolation of the bursa from environmental stimuli by bursal duct ligation (BDL) suppressed the development of IgG-containing cells after hatching. Thus, it is implied that environmental stimulations play a key role in MIgG aggregations and dendritic distributions of aggregated MIgG in the medulla after hatching.

Specific egg yolk immunoglobulin as a new preventive approach for Shiga-toxin-mediated diseases.

Shiga toxins (Stxs) are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC) infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY) from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection.

Passive oral immunization by egg yolk immunoglobulin (IgY) to Vibrio cholerae effectively prevents cholera.

In an attempt to prepare egg yolk immunoglobulin (IgY) to treat and prevent cholera, hens were immunized by a mixture of heat- or formalin-killed Vibrio cholerae O1 and O139 organisms, or by the recombinant cholera toxin B subunit (CTB). The IgYs were partially purified from egg yolk and orally administered to suckling mice before or after challenge with live O1 or O139 cells. The anti-O1 and O139 IgYs and the mixture of either IgY with anti-CTB IgY significantly protected the occurrence of cholera caused by both O1 and O139 infection. Since large amounts of IgY can be prepared very easily and at low cost, this seems to be a useful procedure for preventing and treating cholera.

In vitro and in vivo effectiveness of egg yolk antibody against Candida albicans (anti-CA IgY).

We prepared anti-Candida albicans antibody in chicken egg yolk (anti-CA IgY) and investigated its in vitro and in vivo effectiveness. Anti-CA IgY significantly reduced the adherence capacity of C. albicans to FaDu cells (human pharynx carcinoma cells) in a dose-dependent manner. The protective efficacy of anti-CA IgY was investigated in experimentally induced oral candidiasis in immunosuppressed mice. Oral administration of anti-CA IgY significantly reduced the number of C. albicans and the scores of the tongue lesions. Moreover, anti-CA IgY reduced the colonization of C. albicans in mice organs. These results indicate that anti-CA IgY has a protective effect against the oral candidiasis of experimentally infected mice and reduces the dissemination of C. albicans. Putting together, these results indicate that anti-CA IgY is effective against C. albicans. This effect might be due to the blocking of the binding of C. albicans to the host cells. Therefore, anti-CA IgY might be considered as a prophylactic immunotherapy or possibly an adjunctive antifungal therapy.

Effects of egg yolk antibody against Porphyromonas gingivalis gingipains in periodontitis patients.

Porphyromonas gingivalis gingipains is suspected to be one of the most important causative agents of periodontitis. We postulated that the inhibition of gingipains may reduce the pathogenic nature of P. gingivalis. Anti-P. gingivalis egg yolk antibody (IgY-GP) was isolated from the yolks of hens immunized with purified gingipains. We applied IgY-GP gel subgingivally in periodontitis patients who harbored P. gingivalis in their subgingival flora. Five pairs of contralateral anterior single-rooted teeth were selected. One tooth in each contralateral pair was randomly treated with IgY-GP and subgingival scaling and root planing, whereas the other tooth was treated with SRP alone. The number of P. gingivalis bacteria was assessed by real-time PCR. Bacterial levels were expressed as the percentage of total bacteria. The IgY-GP group had a significant reduction in probing depth. BOP significantly decreased in the IgY-GP group compared to the control group at week 4. The levels of P. gingivalis significantly increased in the control group at week 4, whereas the reduction in the levels of P. gingivalis was sustained in the IgY-GP group. Within the limitations of the present study, IgY-GP was shown to be an effective immunotherapeutic agent in the treatment of periodontitis.

Passive protection of dogs against clinical disease due to Canine parvovirus-2 by specific antibody from chicken egg yolk.

The protective effect of immunoglobulins derived from chicken egg yolk (IgY) against infection by Canine parvovirus 2 (CPV-2) was evaluated in 10 beagle dogs orally challenged with a strain of the virus. The 2-mo-old dogs were divided into 3 groups and treated with powders containing CPV-2 IgY or normal egg yolk for 7 d after the challenge. The 4 dogs receiving normal egg yolk (control group) demonstrated mild symptoms typical of CPV-2 infection, such as vomiting, diarrhea, and weight loss. No symptoms were observed by 16 d after challenge in the 3 dogs receiving 2 g of IgY powder. Of the 3 dogs receiving 0.5 g of IgY powder, 2 had clinical CPV-2 disease; however, the manifestations were less severe than in the control group. Furthermore, the IgY-treated groups had significantly greater weight gain and shorter duration of virus shedding than the control group. These results indicate that IgY is useful in protecting dogs from CPV-2-induced clinical disease.

Effect of egg yolk antibody on experimental Cryptosporidium parvum infection in scid mice.

In this study the effect of chicken egg yolk antibody (IgY) against Cryptosporidium parvum infection was examined. IgY sample was prepared from eggs of chickens immunized with C. parvum oocyst antigens. In vitro, antibody-treated sporozoites showed reduced binding to Caco-2 cells and lost vitality. These phenomena were not observed with a control IgY sample prepared from eggs of non-immunized chickens. Scid mice orally administered with the antibody demonstrated partial reduction in oocyst shedding after challenge with 10(3) oocysts. IgY, however, could not eliminate the infection after 17 days of continuous treatment. The potentials of using specific IgY for treatment and prevention of cryptosporidiosis were discussed.

Melanoidin, a food protein-derived advanced maillard reaction product, suppresses Helicobacter pylori in vitro and in vivo.

BACKGROUND: Extracellular urease proteins located on the surface of Helicobacter pylori are gastric mucin-targeted adhesins, which play an important role in infection and colonization to the host. In this study we have determined the inhibitory activity of a variety of melanoidins, protein-derived advanced Maillard reaction products, ubiquitously found in heat-treated foods, on urease-gastric mucin adhesion. In addition, we have determined the anticolonization effect of melanoidin I, prepared by the Maillard reaction between casein and lactose, in an animal model and in human subjects infected with this bacterium. METHODS: The inhibitory activity of each compound was determined by a competitive binding assay of labeled gastric mucin to plate-immobilized urease. Melanoidin I was used in an in vivo trial using euthymic hairless mice as an infection model. Melanoidin I was consumed for 8 weeks by subjects infected with H. pylori. The [(13)C] urease breath test and H. pylori-specific antigen in the stool (HpSA) test were performed on subjects at week 0 and week 8. RESULTS: A variety of food protein-derived melanoidins strongly inhibited urease-gastric mucin adhesion in the concentration range of 10 micro g/ml to 100 micro g/ml. In particular, melanoidin I significantly (p <.05) suppressed colonization of H. pylori in mice when given for 10 weeks via the diets. Eight weeks daily intake of 3 g melanoidin I significantly (p <.05) decreased the optical density of HpSA in subjects. CONCLUSION: Foods containing protein-derived melanoidins may be an alternative to antibiotic-based therapy to prevent H. pylori that combines safety, ease of administration and efficacy.

Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits.

In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits.

Enhancement of chicken resistance against Escherichia coli infection by oral administration of Bifidobacterium thermophilum preparations.

Three types of Bifidobacterium thermophilum extract were prepared and fed to 2-wk-old chickens to evaluate their usefulness in enhancing the defense activity of the chickens against pathogenic Escherichia coli. All three preparations resulted in significant reduction (P < 0.05) of E. coli numbers in the lungs of the treated chicken groups compared with the control nontreated group. Besides, improvement in the survival rate was observed in the treated chicken groups, especially the one administered the enzyme-digested B. thermophilum extract sample. Concanavalin A-stimulated lymphocytes from the latter group demonstrated significantly higher proliferation activity compared with those from the control group. These results suggest that oral administration of B. thermophilum preparations may be used to enhance the resistance of chickens against E. coli infection.