Structural and immunoendocrine remodeling in gut, pancreas and thymus in weaning rats fed powdered milk diets rich in Maillard reactants.

Western diet is extending worldwide and suspected to be associated with various metabolic diseases. Many food products have skim milk powder added to it and, during processing, lactose reacts with milk proteins and Maillard reaction products (MRPs) are formed. Dietary MRPs are suggested risk factors for metabolic dysregulation, but the mechanisms behind are still enigmatic. Here we describe that weaning rats fed diets rich in MRPs are affected in both their immune and endocrine systems. Marked structural changes in pancreas, intestine and thymus are noted already after 1 week of exposure. The pancreatic islets become sparser, the intestinal mucosa is thinner, and thymus displays increased apoptosis and atrophy. Glucagon- like peptide-1 (GLP-1) seems to play a key role in that the number of GLP-1 expressing cells is up-regulated in endocrine pancreas but down-regulated in the intestinal mucosa. Further, intestinal GLP-1-immunoreactive cells are juxta positioned not only to nerve fibres and tuft cells, as previously described, but also to intraepithelial CD3 positive T cells, rendering them a strategic location in metabolic regulation. Our results suggest dietary MRPs to cause metabolic disorders, dysregulation of intestinal GLP-1- immunoreactive cells, arrest in pancreas development and thymus atrophy.

Skim milk powder with high content of Maillard reaction products affect weight gain, organ development and intestinal inflammation in early life in rats.

BACKGROUND: The intestinal tract is important for development of immune tolerance and disturbances are suggested to trigger autoimmune disorders. The aim of this study was to explore the effect of Maillard products in skim milk powder obtained after long storage, compared to fresh skim milk powder. METHODS: Young rats were weaned onto a diet based on skim milk powder with high concentration of Maillard products (HM-SM, n = 18) or low (C-SM, n = 18) for one week or four weeks. Weekly body weight and feed consumption were noted. At the end, organ weights, intestinal histology, permeability and inflammatory cytokines were evaluated. RESULTS: Rats fed with HM-SM had after one week, 15% less weight gain than controls, despite equal feed intake. After one week thymus and spleen were smaller, intestinal mucosa thickness was increased and acute inflammatory cytokines (IL-17, IL-1ß, MCP-1) were elevated. After four weeks, cytokines associated with chronic intestinal inflammation (fractalkine, IP-10, leptin, LIX, MIP-2, RANTES and VEGF) were increased in rats fed with HM-SM compared to C-SM. CONCLUSION: High content of Maillard products in stored milk powder caused an intestinal inflammation. Whether this is relevant for tolerance development and future autoimmune diseases remains to be explored.

Tetanus antigen modulates the gene expression profile of aluminum phosphate adjuvant in spleen lymphocytes in vivo.

Adjuvants play an important role in stimulation of the immune response to antigens. Very little is known about the molecular mechanisms of this stimulation. Here we address this issue by studying gene expression profiles from spleen lymphocytes after in vivo immunization of mice with a clinically relevant vaccine, tetanus toxoid formulated with aluminum phosphate as adjuvant (TT(ADJ)), or the adjuvant alone (ADJ). The Th1/Th2 response to TT(ADJ) was obtained from a combination of up- and downstream markers to conventional cytokines, which were in good agreement with cytokine protein levels. A clustering algorithm revealed that ADJ elicited expression of 47 genes active in cytotoxic lymphocytes, inflammation, oncogenesis, stress, toxicity and cell cycle regulation. In TT(ADJ) these adjuvant-elicited genes were expressed at lower levels and a compensatory onset of protective and inhibitory genes was observed. We conclude that the antigen, to a larger extent than previously recognized, modulates the molecular mechanism of the aluminum phosphate adjuvant and that the identified genes may serve as predictive biomarkers in the development of new adjuvants and vaccines.

Standardisation of allergens in Europe.

A general monograph for allergen products has now been adopted by the European Pharmacopoeia Commission. It is based on the use of an In-House Reference Preparation (IHRP), which shall be used in the control of every production batch after appropriate characterization. This characterization will depend on the intended use of the product. When possible, the biological potency should be established by skin testing and expressed in biological units. The monograph is the first mandatory regulation in Europe for allergen products and will guarantee the consistent composition and potency of the products on the market.

A study of some important vitamins and antioxidants in a blackcurrant jam with low sugar content and without additives.

Blackcurrant (Ribes nigrum) jam was manufactured with the aim of producing a jam with a low sugar content, and without any additives. Four temperatures were investigated, namely 60 degrees C, 76 degrees C, 92 degrees C and 97 degrees C. Processing time varied between 1-20 min. After processing, the highest content of ascorbic acid was found in the jam processed at 97 degrees C for 1 min, which contained 63.3 +/- 2.6 mg ascorbic acid/100 g jam. At all combinations investigated more than 60% of the original amount of ascorbic acid was retained after manufacturing and packaging. The jam made at 92 degrees C was stored in a shelf-life study for 13 months. The jam was then stored at 8 degrees C, ambient temperature and at 37 degrees C. At ambient temperature the jam was stored both in dark and in daylight, at 8 degrees C and at 37 degrees C the jam was stored in dark. After 13 months of storage, at 8 degrees C, 60% of the amount of ascorbic acid and 29% of the amount of anthocyanins were retained. In the jam stored at higher temperatures less of both was retained. The beta-carotene in the jam was found to be stable throughout the whole shelf-life study. Exposure to light did not have any effect on any of the components studied. The degradation of anthocyanins was best described by a second-order reaction and the activation energy was determined to be 90 kJ/mol. A jam of blackcurrant may be considered as a good source of vitamins and antioxidants after one year, if certain precautions concerning manufacture and storage conditions are taken.

Biodegradable microspheres. 16. Synthesis of primaquine-peptide spacers for lysosomal release from starch microparticles.

Classical procedures of peptide synthesis were applied to synthesize four groups of compounds, and analytical methods were developed for each of them. Two of the groups are tetrapeptide derivatives of the antileishmanial drug primaquine (PQ), with general structure NH2-X-Leu-Ala-Y-PQ. In the first group, Leu, Tyr, Lys, and Asp were used in the Y position, while X was Ala. In the second group, Ala, Tyr, Lys, and Asp were used in the X position, while Y was Leu. The derivatives are intended to be coupled, via their free alpha-amino group, to polyacryl starch microparticles, lysosomotropic drug carriers developed in our laboratory. Thus, a systematic study of the significance of the varying amino acid composition of the tetrapeptide spacer arm for the rate of lysosomal enzymatic release of PQ can be possible. A third group, comprising epsilon-aminocaproic acid-PQ derivatives which lack a free alpha-amino group, was synthesized. This was done to study the importance of enzymes, other than aminopeptidases, during lysosomal degradation of these derivatives. To allow HPLC analysis of the pattern of degradation of tetrapeptide-PQ derivatives, some shorter peptide-PQ derivatives (group four) were prepared as well.

Biodegradable microspheres. 17. Lysosomal degradation of primaquine-peptide spacer arms.

The pharmacological activity of drugs bound to lysosomotropic drug carriers will depend on the rate of release of the drugs from the drug-carrier complex. We have now studied the enzymatic release of primaquine (PQ) from two groups of peptide-PQ derivatives by their incubation with rat liver lysosomal fraction. The derivatives have the general structure NH2-X-Leu-Ala-Y-PQ and are intended to be coupled via their free alpha-amino group to starch microparticles. In the first group, Y was varied, being Leu, Tyr, Lys, or Asp, while X was Ala. In the second one, X was varied, being Ala, Tyr, Lys, or Asp, while Y was Leu. Thus, a systematic study of the significance of the varying amino acid composition of the tetrapeptides, which can serve as spacer arms in the microparticle-drug complexes, for the lysosomal release of PQ was possible. In addition, some epsilon-aminocaproic acid-PQ derivatives, which lack a free alpha-amino group, were incubated. This was done to study the importance of enzymes, other than aminopeptidases, during lysosomal degradation of these derivatives. The pattern and rate of degradation of all PQ derivatives was followed by HPLC analysis. The results obtained show that endopeptidases, as well as mono- and diaminopeptidases, degrade the derivatives. PQ cannot be cleaved directly from the derivatives by any carboxypeptidase-like enzyme. Asp peptides are digested slowly in the lysosomal fraction. The temporal aspects of reactions were quantitated using a kinetic model, in which first-order rate constants of all the steps of each peptide degradation sequence were estimated simultaneously.

Interferon-gamma in starch microparticles: nitric oxide-generating activity in vitro and antileishmanial effect in mice.

Recombinant mouse interferon-gamma (mu IFN-gamma) was covalently coupled to polyacryl starch microparticles, a lysosomotropic drug carrier. The microparticle-bound mu IFN-gamma was found to activate cultured macrophages for nitrite production and had an anti-leishmanial effect in mice. Low doses of mu IFN-gamma, which had no effect in the free form, when bound to microparticles significantly reduced the load of Leishmania donovani in infected mice. Further, inducement of nitrite production in cultured macrophages by microparticle-bound mu IFN-gamma required intact cell membrane receptors.

Biodegradable microspheres. XIII: Immune response to the DNP hapten conjugated to polyacryl starch microparticles.

Polyacryl starch microparticles are being investigated for use as drug carriers, especially in the treatment of intracellular parasitic diseases. The purpose of this work was to investigate the possible immune response to drugs coupled to the microparticles, using the dinitrophenyl group (DNP) as a model. The humoral immunogenicity of DNP hapten-conjugated polyacryl starch microparticles has been examined in mice. Microparticle:hapten complexes with different biodegradability were tested, as well as different dosages and routes of administration. Two DNP derivatives, Lys(DNP) and Leu-Ala-Lys(DNP), were coupled to microparticles. It was shown that Lys(DNP) was released from the particles by lysosomal enzymes only from the conjugate with the tripeptide DNP derivative. The DNP conjugated to the particles via the biodegradable Leu-Ala-Lys arm induced only a weak immune response without memory. No response was detected after injection of the Lys(DNP) microparticles. Thus, there should be no major immunological obstacles in using drug:microparticle complexes in the treatment of, for example, parasitic diseases.

Microparticles as potentially orally active immunological adjuvants.

A model microparticulate carrier system, capable of partially protecting an entrapped antigen against enzymatic degradation, has been successfully used to induce an enhanced secretory immune response to an incorporated model soluble antigen following oral administration to experimental rats. Ovalbumin was incorporated into polyacrylamide microparticles (2.55 microns diameter) and administered orally on four consecutive days to rats primed 14 days earlier by intraperitoneal injection. The memory secretory IgA antibody response (day 65) to the microparticle incorporated antigen group was significantly raised relative to a soluble antigen control group response (p less than 0.01), following administration of a booster dose of soluble antigen to both groups (day 61).

Biodegradable microspheres. XII: Properties of the crosslinking chains in polyacryl starch microparticles.

Polyacryl starch microparticles are under current investigation for use as lysosomotropic drug carriers. This paper describes some in vivo and in vitro properties of the crosslinking polymer chains in these particles. A radioactive label was introduced into the microparticle crosslinks by copolymerization of [14C]acrylamide. It was shown by gel permeation chromatography that the amount of tetramethylethylenediamine (TEMED) used in the microparticle polymerization influenced the molecular weight composition of the hydrocarbon chains. Increasing the TEMED concentration resulted in a higher proportion of shorter polymeric chains. After iv administration to mice, the microparticles were taken up mainly by the liver. Although presumably nonmetabolizable, a slow elimination (terminal half-life of 4-5 months) of the hydrocarbon chains from the liver was observed. This suggests that after exocytosis from the Kupffer cells or after their turnover, dissolved material is taken up by liver parenchymal cells and excreted into the bile.

Receptor-mediated uptake of starch and mannan microparticles by macrophages: relative contribution of receptors for complement, immunoglobulins and carbohydrates.

The contribution of different macrophage receptors to the uptake of polyacryl starch and polyacryl mannan microparticles by macrophages was studied. Both types of microparticle were taken up to a larger and similar extent when they had been pre-incubated with serum, indicating the importance of serum factors in the uptake process. These results were supported by organ distribution studies in mice, showing that the two microparticles were rapidly targeted to the liver and spleen after i.v. injection. The carbohydrate-specific mannose/fucose receptor was found to contribute significantly to the uptake of non-opsonized but not opsonized mannan microparticles. The two different microparticles were found to activate the alternative complement pathway and to adsorb immunoglobulin G, human serum albumin and fibronectin to their surface. Inhibition experiments provided evidence for the involvement of a complement receptor (CR3) and an Fc-receptor (FCR) for IgG in the uptake of opsonized microparticles.

Biodegradable microspheres. X: Some properties of polyacryl starch microparticles prepared from acrylic acid-esterified starch.

Acrylic acid-esterified starch was produced by reacting starch with acrylic acid chloride. This reaction was rapid and easy to control. Introduction of acrylic groups into starch reduced the enzymatic degradability of starch (e.g., with 12 acrylic groups/100 glucose residues, approximately 75% of the degradation products eluted before glucose on gel filtration). The degradability could be increased to a large extent by preincubation at pH 5.5 in vitro (e.g., after 16 weeks, the corresponding figure was approximately 15%). The acrylic acid-esterified starch was used to prepare polyacryl starch microparticles. These were rapidly eliminated from the circulation after iv injection in mice, mainly by uptake in the liver. The elimination of the microparticles from the liver, monitored with [14C]starch, displayed a half-life of approximately 3.5-4.5 months. After 5 and 6 months, approximately 30% of the initial radioactivity remained in the liver. This is equivalent to the amount anticipated from the enzymatic degradation of the monomer (acrylic acid-esterified starch) in vitro and the innate nondegradability of the 14C-marker. These results, taken together, indicate that the ester bond between starch and the hydrocarbon chain in polyacryl starch microparticles is hydrolyzed at lysosomal pH.

Biodegradable microspheres: polyacryl starch microparticles as a delivery system for the antileishmanial drug, sodium stibogluconate.

Liver parasite burdens of Leishmania donovani in the mouse have been determined after treatment with intravenous administration of sodium stibogluconate in the free or carrier form. The carrier form, in which the drug was covalently bound to polyacryl starch microparticles, was up to 100x more effective than the free form in this murine model of visceral leishmaniasis. Empty microparticles had no effect on liver parasite burdens and the enhanced in-vivo antileishmanial activity of the carrier form of the drug was apparently due to passive drug delivery to the infected liver.

Biodegradable microspheres. VI: Lysosomal release of covalently bound antiparasitic drugs from starch microparticles.

The possibilities of using polyacryl starch microparticles as a carrier for low molecular weight drugs have been investigated. Two drugs containing primary amino groups, primaquine and trimethoprim, have been covalently coupled to the starch microparticles via tri-, tetra-, and pentapeptide spacer arms. The drug-particle complexes were prepared by coupling different drug-peptide derivatives to the particles after activation of the starch with carbonyldiimidazole. The activation process with subsequent removal of activated groups did not change the degradability of the starch microparticles. The resulting drug-carrier complexes were stable in serum, while free drugs were released in a lysosome fraction. Thus, the microparticle-peptide-drug conjugates fulfill the basic requirements for a drug carrier used to target drugs to the lysosomes (e.g., for the treatment of lysosomal parasitic diseases).

Biodegradable microspheres. V: Stimulation of macrophages with microparticles made of various polysaccharides.

The interaction between four different microparticulate drug carriers and macrophages was investigated in vitro. The microparticles, consisting of crosslinked starch (1,4-alpha-D-glucan with 1,6-alpha-branches), dextran (1,6-alpha-D-glucan with 1,3-alpha-branches), lichenan (1,3-beta-D-glucan), or mannan (1,6-alpha-D-mannan with 1,2-alpha- and 1,3-alpha-branches), were investigated for their macrophage stimulatory properties. Macrophage stimulation was assayed by the uptake of [14C]glucosamine and stimulatory indices were calculated. Microparticles made of crosslinked lichenan were most stimulatory, followed by the biologically inert mannan and dextran microparticles. Biodegradable starch microparticles were less stimulatory to the macrophages than the other microparticles. All microparticles were phagocytosed to the same extent and stimulated the macrophages to release oxygen radicals. Lichenan, mannan, and dextran microparticles induced morphological changes in the macrophages when given in nontoxic doses. No morphological changes were observed when the macrophages were exposed to starch microparticles or soluble polysaccharides.