Development of the equine gut microbiota.

Shortly after birth the mammalian gut is colonized, by a transient microbiota, highly susceptible to environment and diet, that eventually stabilizes and becomes the resident gut microbiota. In a window of opportunity during the colonization, oral tolerance is established towards resident bacteria. In this study, the development of the equine gut microbiota was investigated in ten foals from parturition until post weaning. We found great differences in the core species of the gut microbiota composition between time-matched samples on Day 7 and 20 post-partum. Between day 20 and Day 50 post-partum, we saw the gut microbiota became increasingly dominated by fiber fermenting species. After Day 50, no significant changes in species abundance were observed. Gene expression analysis of pro- and anti-inflammatory cytokines in the blood revealed no significant changes before and after weaning. In summary, relative stability of the gut microbiota was reached within 50 days post-partum and, weaning did not have a major impact on the microbial composition.

Expression of immune regulatory genes correlate with the abundance of specific Clostridiales and Verrucomicrobia species in the equine ileum and cecum.

Billions of bacteria inhabit the gastrointestinal tract. Immune-microbial cross talk is responsible for immunological homeostasis, and symbiotic microbial species induce regulatory immunity, which helps to control the inflammation levels. In this study we aimed to identify species within the equine intestinal microbiota with the potential to induce regulatory immunity. These could be future targets for preventing or treating low-grade chronic inflammation occurring as a result of intestinal microbial changes and disruption of the homeostasis. 16S rRNA gene amplicon sequencing was performed on samples of intestinal microbial content from ileum, cecum, and colon of 24 healthy horses obtained from an abattoir. Expression of genes coding for IL-6, IL-10, IL-12, IL-17, 18 s, TNFα, TGFß, and Foxp3 in the ileum and mesenteric lymph nodes was measured by qPCR. Intestinal microbiota composition was significantly different in the cecum and colon compared to the ileum, which contains large abundances of Proteobacteria. Especially members of the Clostridiales order correlated positively with the regulatory T-cell transcription factor Foxp3 and so did the phylum Verrucomicrobia. We conclude that Clostridiales and Verrucomicrobia have the potential to induce regulatory immunity and are possible targets for intestinal microbial interventions aiming at regulatory immunity improvement.

Probiotics and the immunological response to infant vaccinations; a double-blind randomized controlled trial.

OBJECTIVES: To examine the effect of a combination of probiotics on the antibody response to pneumococcal and pertussis vaccination in healthy Danish children, aged 8-14 months, at the time of starting day care. Moreover, the cytokine response to lipopolysaccharide of whole blood was assessed. METHODS: A total of 290 children were randomly allocated to receive a combination of Bifidobacterium animalis ssp. lactis and Lactobacillus rhamnosus GG daily for a 6-month intervention period, and blood samples were drawn at the start and end of the study. Specific antibody response towards Streptococcus pneumoniae serotypes and Bordetella pertussis toxin, as well as endotoxin-induced interleukin-6 (IL-6) and interferon-γ (IFN-γ) production in blood were analysed by Luminex and ELISA. RESULTS: There was no significant difference between the average individual changes from baseline to end of study in antibody concentrations for S. pneumoniae for both the probiotics (340.4% ± 11.2%) and the placebo group (382.9% ± 10.4%) (p 0.525), nor for B. pertussis toxin in the two groups (probiotics 190.1% ± 12.6% versus placebo 238.8% ± 1.1%, p 0.340). The average individual change in IL-6 concentration was significantly lower in the probiotics versus the placebo group (2.9% ± 10.3% versus 33.7% ± 9.0%, p 0.024), whereas there was no difference in IFN-γ concentration (0.0% ± 0.2% versus -0.2% ± 0.1%, p 0.279). CONCLUSIONS: The probiotic intervention did not affect the antibody response against S. pneumoniae and B. pertussis toxin in healthy Danish children.

Campylobacter jejuni carbon starvation protein A (CstA) is involved in peptide utilization, motility and agglutination, and has a role in stimulation of dendritic cells.

Campylobacter jejuni is the most frequent cause of severe gastroenteritis in the developed world. The major symptom of campylobacteriosis is inflammatory diarrhoea. The molecular mechanisms of this infection are poorly understood compared to those of less frequent disease-causing pathogens. In a previous study, we identified C. jejuni proteins that antibodies in human campylobacteriosis patients reacted with. One of the immunogenic proteins identified (Cj0917) displays homology to carbon starvation protein A (CstA) from Escherichia coli, where this protein is involved in the starvation response and peptide uptake. In contrast to many bacteria, C. jejuni relies on amino acids and organic acids for energy, but in vivo it is highly likely that peptides are also utilized, although their mechanisms of uptake are unknown. In this study, Biolog phenotype microarrays have been used to show that a ΔcstA mutant has a reduced ability to utilize a number of di- and tri-peptides as nitrogen sources. This phenotype was restored through genetic complementation, suggesting CstA is a peptide uptake system in C. jejuni. Furthermore, the ΔcstA mutant also displayed reduced motility and reduced agglutination compared to WT bacteria; these phenotypes were also restored through complementation. Murine dendritic cells exposed to UV-killed bacteria showed a reduced IL-12 production, but the same IL-10 response when encountering C. jejuni ΔcstA compared to the WT strain. The greater Th1 stimulation elicited by the WT as compared to ΔcstA mutant cells indicates an altered antigenic presentation on the surface, and thus an altered recognition of the mutant. Thus, we conclude that C. jejuni CstA is important not only for peptide utilization, but also it may influence host-pathogen interactions.

Establishment of tolerance to commensal bacteria requires a complex microbiota and is accompanied by decreased intestinal chemokine expression.

Intricate regulation of tolerance to the intestinal commensal microbiota acquired at birth is critical. We hypothesized that epithelial cell tolerance toward early gram-positive and gram-negative colonizing bacteria is established immediately after birth, as has previously been shown for endotoxin. Gene expression in the intestine of mouse pups born to dams that were either colonized with a conventional microbiota or monocolonized (Lactobacillus acidophilus or Eschericia coli) or germ free was examined on day 1 and day 6 after birth. Intestinal epithelial cells from all groups of pups were stimulated ex vivo with L. acidophilus and E. coli to assess tolerance establishment. Intestine from pups exposed to a conventional microbiota displayed lower expression of Ccl2, Ccl3, Cxcl1, Cxcl2, and Tslp than germ-free mice, whereas genes encoding proteins in Toll-like receptor signaling pathways and cytokines were upregulated. When comparing pups on day 1 and day 6 after birth, a specific change in gene expression pattern was evident in all groups of mice. Tolerance to ex vivo stimulation with E. coli was only established in conventional animals. Colonization of the intestine was reflected in the spleen displaying downregulation of Cxcl2 compared with germ-free animals on day 1 after birth. Colonization reduced the expression of genes involved in antigen presentation in the intestine-draining mesenteric lymph nodes, but not in the popliteal lymph nodes, as evidenced by gene expression on day 23 after birth. We propose that microbial detection systems in the intestine are upregulated by colonization with a diverse microbiota, whereas expression of proinflammatory chemokines is reduced to avoid excess recruitment of immune cells to the maturing intestine.

Maternal intake of fish oil but not of linseed oil reduces the antibody response in neonatal mice.

Dietary levels of n-3 PUFA are believed to influence the immune system. The importance of the source of n-3 PUFA is debated. This study addressed how the content and source of n-3 PUFA in the maternal diet influenced tissue FA composition and the immune response to ovalbumin (OVA) in mice pups. From the day of conception and throughout lactation, dams were fed diets containing 4% fat from linseed oil (LSO), fish oil (FO) or a n-3 PUFA-deficient diet (DEF). Pups were injected with OVA within 24 h of birth and sacrificed at weaning (day 21). Overall, the content of n-3 PUFA in milk, liver and spleen reflected the source and only minor differences were observed in brain phospholipid 22:6n-3. The source had only limited influence on the n-3 PUFA accretion in peripheral tissue, with most pronounced differences in the spleen. The marine PUFA-group had reduced levels of total OVA-specific antibodies and OVA-IgG1 titers in the pup blood, while the response in the LSO-group did not differ from that in the DEF-group. There were no statistical differences in the cytokine responses to OVA-stimulated splenocytes, but the decrease in IgG1 was paralleled by an increase in IFNγ-production and a decrease in IL-6-production. Our results indicate that maternal intake of FO, but not of LSO, changes the offspring's antigen-specific response and potentially increases Th1-polarization.

Dendritic cells from Peyer's patches and mesenteric lymph nodes differ from spleen dendritic cells in their response to commensal gut bacteria.

Commensal gut bacteria have potent effects on the immune system, which are partially mediated by intestinal dendritic cells (DC). Distinct commensals confer different properties to in vitro-generated DC. The aim of the present study was to reveal strain-dependent maturation patterns in primary DC. To this end, we compared the response of mouse Peyer's patch (PP) DC, mesenteric lymph node (MLN) DC and spleen DC to the commensal bacteria, Bifidobacterium longum Q46, Lactobacillus acidophilus X37 and Escherichia coli Nissle 1917. Bacterial maturation of DC occurred independently of tissue origin. Expression of CCR7 and CD103 on the surface of MLN DC, necessary for the induction of gut-homing regulatory T cells, increased with stimulation by Gram-positive commensals. Bacteria-dependent cytokine production (IL-6, IL-10 and TNF-alpha) was similar in spleen and MLN DC, and contaminant cells in these DC preparations produced IFN-gamma in response to L. acidophilus. In contrast, PP DC produced IL-6 only in response to E. coli, little IL-10 and no TNF-alpha, and this low cytokine production was not due to inhibition by IL-10 or TGF-beta. Bifidobacteria downregulate IL-6, TNF-alpha and IL-12 production induced in in vitro-generated DC by L. acidophilus. Similar inhibition was observed in splenic DC, but not in MLN DC. MLN cells responded to bacterial stimulation with higher IFN-gamma production than spleen cells, possibly due to the presence of more responsive natural killer cells. Commensal bacteria therefore play specific roles in the gut immune system distinguishable from the effect they would have if recognized by the systemic immune system.

Different kinetic in incorporation and depletion of n-3 fatty acids in erythrocytes and leukocytes of mice.

n-3 PUFA are well known for their anti-inflammatory effects. However, there has been only limited study on the kinetics of incorporation and depletion of n-3 PUFA in immune cells. In the present study we investigated the incorporation and depletion of n-3 PUFA in erythrocytes and leukocytes in mice during a 6-wk feeding period. Over the first 3-wk period (the incorporation period) the mice were fed a special diet with a high n-3/n-6 PUFA ratio. In the following 3-wk period (the depletion period) the mice were fed a standard chow diet. A linear increase of the concentration of EPA and DHA in erythrocyte membranes was observed during the incorporation period, whereas a stagnation was observed after the second week for leukocytes. The level of EPA did not fall to the background level after the depletion period, and the level of DHA was kept almost constant during the depletion period in the erythrocyte membranes. In leukocytes the concentration of both EPA and DHA decreased during the depletion period, but did not reach the background level after the 3-wk depletion. In conclusion, the kinetics of EPA and DHA in the different cells are different. The rate of incorporation is faster than that of depletion for n-3 PUFA. More n-3 PUFA can be incorporated into leukocytes in comparison with erythrocytes. The ratio of n-3/n-6 PUFA is more important than the amount of n-3 FA in changing the FA compositions of membrane lipids.

Immunogenicity of kappa-casein and glycomacropeptide.

Glycomacropeptide (GMP), arising from the cleavage of kappa-casein by chymosin or pepsin, has been correlated with a wide variety of biological activities including immunosuppression capacity, inhibition of pathogen invasion, and induction of satiety. Due to the interest in exploiting such potential of GMP, we aimed at characterizing the immunogenic properties of GMP as an indication of its potential allergenicity. Immunogenicity of kappa-casein and GMP were investigated using 2 animal models based on different routes of immunization: 1) mice immunized intraperitoneally or subcutaneously with either kappa-casein, polymerized GMP, GMP coupled to the immunogenic carrier ovalbumin, or GMP alone; 2) mice coadministered kappa-casein or GMP and cholera toxin. The specific antibody response to GMP was evaluated as well as the antigen-specific T-cell response. The results demonstrated that immunization or feeding with kappa-casein induced GMP-specific antibodies, whereas GMP per se lacked immunogenicity independently of the mode of presentation. The size of the presented form of GMP did not influence its immunogenicity. Because the results showed that GMP did not induce a specific T-cell response, we postulate that GMP lacks the ability to stimulate antigen-specific T cells.

Caseinomacropeptide self-association is dependent on whether the peptide is free or restricted in kappa-casein.

There is a general agreement that the experimentally determined molecular weight (MW) of caseinomacropeptide (CMP) is greater than the theoretical MW. Some studies suggest that this is due to a pH-dependent aggregation of monomeric CMP. How this aggregation is influenced by pH is not understood. This study was carried out to study the nature of CMP aggregates and to clarify which conditions affect aggregation of CMP. The apparent MW of CMP at different pH values was determined using size-exclusion chromatography. Caseinomacropeptide was further characterized by immunochemical analysis, sodium dodecyl sulfate-PAGE, N-terminal sequencing, and mass spectrometry. The hydrophobicity of CMP was studied by means of 1-anilino-naphthalene-8-sulfonic acid binding experiments. Four CMP products prepared by different methods were studied: CMP produced by enzymatic (chymosin or pepsin) hydrolysis of kappa-casein (CN), and 2 commercial CMP products. Both commercial products and CMP resulting from chymosin-hydrolysis of kappa-CN (at pH 6.6) had elution volumes with a MW corresponding to 35 kDA at pH 8.0 and 3.4. Caseinomacropeptide prepared from pepsin-hydrolysis of kappa-CN (at pH 2.5) eluted as multiple peaks with apparent MW of 35, 18, and 9 kDa, again independently of pH. Hydrolysis of kappa-CN with chymosin or pepsin at different pH values (pH 2.5, 3.4, and 6.6) produced differently sized aggregates of CMP, largely depending on the pH of the hydrolysis. These results indicate that, whereas CMP molecules are irreversibly associated, CMP in kappa-CN may associate reversibly in a pH-dependent manner. We suggest that interactions between para-kappa-CN parts of the kappa-CN molecules may be a requisite for the pH-dependent dissociation/association.

Milk-derived GM(3) and GD(3) differentially inhibit dendritic cell maturation and effector functionalities.

Gangliosides are complex glycosphingolipids, which exert immune-modulating effects on various cell types. Ganglioside GD(3) and GM(3) are the predominant gangliosides of human breast milk but during the early phase of lactation, the content of GD(3) decreases while GM(3) increases. The biological value of gangliosides in breast milk has yet to be elucidated but when milk is ingested, dietary gangliosides might conceptually affect immune cells, such as dendritic cells (DCs). In this study, we address the in vitro effect of GD(3) and GM(3) on DC effector functionalities. Treatment of bone marrow-derived DCs with GD(3) before lipopolysaccharide-induced maturation decreased the production of interleukin-6 (IL-6), IL-10, IL-12 and tumor necrosis factor-alpha as well as reduced the alloreactivity in mixed leucocyte reaction (MLR). In contrast, only IL-10 and IL-12 productions were significantly inhibited by GM(3,) and the potency of DCs to activate CD4(+) cells in MLR was unaffected by GM(3). However, both gangliosides suppressed expression of CD40, CD80, CD86 and major histocompatibility complex class II on DCs. Because GD(3) overall inhibits DC functionalities more than GM(3), the immune modulating effect of the ganglioside fraction of breast milk might be more prominent in the commencement of lactation during which the milk contains the most GD(3).

Induction of oral tolerance with micro-doses of ovomucoid depends on the length of the feeding period.

Oral administration of antigen induces antigen-specific immunologic tolerance, which is known to be dose-dependent. We studied the influence of continuous oral administration of nanogram and microgram doses of antigen on oral tolerance induction. Mice were continuously exposed to varying doses (1 ng-1 mg/day) of ovomucoid (OM) for a minimum of 30 days and a maximum of 100 days. It was possible to induce oral tolerance measured as reduced proliferation and antibody production (immunoglobulin (Ig)G1, IgG2a and total Igs) when mice were fed 1 mg of OM/day for 40 or 50 days. It was not possible to induce oral tolerance with daily doses of antigen of 10 microg or less. Feeding of 100 microg OM/day for 40 and 50 days and 1 mg OM/day for 30 days generated tolerization of Th2-dependent responses, but retained an intact response of Th1-dependent antibodies, whereas feeding of 1 mg OM/day for 40 and 50 days resulted in tolerization of both Th1- and Th2-antibody responses. The results presented here suggest that there is a threshold of microgram-doses below which oral tolerance cannot be induced, and that selective suppression of Th2 responses can be achieved by continuous microdose feeding, while an extension of the feeding dose or feeding period tolerizes both Th1- and Th2-dependent responses.

Protein modification by fermentation: effect of fermentation on the potential allergenicity of pea.

The effect of fermentation on components of potential significance for the allergenicity of pea was analyzed. Pea flour was fermented with three lactic acid bacteria, Pediococcus pentosaceus, Lactococcus raffinolactis, and Lactobacillus plantarum, and two fungi, Rhizopus microsporus, var. oligosporus and Geotrichum candidum. Residual antigenicity against antipea antibodies was reduced to 10% by the three lactic acid bacteria and R. microsporus. Reactions to anti-pea profilin and anti-Bet v 1 were still detectable after fermentation. The contents of lectin and pea protease inhibitor were not reduced by the microorganisms.

The use of antibodies for characterization and quantification of a recombinant protein.

In this study, we characterized proteinase A secreted by recombinant Saccharomyces cerevisiae bearing a multicopy plasmid containing the encoding gene (PEP4). Polyclonal and monoclonal antibodies were raised to study the product heterogeneity. Characterization of proteinase A was performed by immunoelectrophoresis and immunoblotting techniques. None of the monoclonal antibodies raised against proteinase A was found to react with the glycosyl side chains; thus cross-reaction with other glycosylated proteins (e.g. carboxypeptidase Y) was very low. This study allowed us to develop an ELISA method for the quantification of proteinase A in culture supernatants as well as the evaluation of monoclonal antibodies for their use in immunoaffinity chromatography.

Identification of IgE-binding egg white proteins: comparison of results obtained by different methods.

The binding of IgE to egg white proteins was investigated for 34 sera from adults with a positive case history and/or positive RAST towards egg, and the impact of experimental conditions on IgE binding in commonly used methods was studied. Radioimmunoblotting after SDS-PAGE of both reduced and unreduced egg white extracts showed complex reaction patterns. The results were confirmed by crossed radioimmunoelectrophoresis (CRIE). Radio dot immunobinding was used to investigate the effect of treatment of allergens for SDS-PAGE and to evaluate the other methods. As a conclusion, the use of combinations of at least two methods is recommended for the identification of IgE-binding egg white proteins. Of the 34 sera, 18 reacted with ovotransferrin, 13 with ovomucoid, 11 with ovalbumin and 5 with lysozyme. The amounts of IgE bound to ovalbumin and lysozyme were generally lower than the amounts bound to ovotransferrin and ovomucoid.

Gastric bicarbonate secretion and release of prostaglandin E2 are increased in duodenal ulcer patients but not in Helicobacter pylori-positive healthy subjects.

BACKGROUND: Duodenal ulcer (DU) patients have impaired proximal duodenal mucosal bicarbonate secretion at rest and in response to luminal acid with higher acid-stimulated mucosal release of prostaglandin (PG) E2 than healthy subjects. Our purpose was to determine whether this abnormality was present also in the stomach of DU patients. METHODS: Simultaneous determinations of gastric and duodenal bicarbonate secretion and luminal release of PGE2 were performed in 16 healthy volunteers (5 Helicobacter pylori-positive) and 8 inactive DU patients (all H. pylori-positive). RESULTS: In healthy volunteers the rates of gastroduodenal bicarbonate secretion and the release of PGE2 were not influenced by H. pylori status. In inactive DU patients the rates of basal (704 +/- 84 versus 356 +/- 40 mumol/h; mean +/- SEM) and vagally stimulated (modified sham feeding) (1724 +/- 376 versus 592 +/- 52 mumol/h) gastric bicarbonate secretion were higher (p < 0.05) than in the health, whereas the corresponding rates (339 +/- 42 versus 591 +/- 51 mumol/h and 543 +/- 99 versus 778 +/- 69 mumol/h) in duodenal bicarbonate secretion were lower (p < 0.05). In addition, inactive DU patients had higher basal (148 +/- 32 versus 53 +/- 5 ng/h) and stimulated (291 +/- 84 versus 131 +/- 25 ng/h) gastric release of PGE2, but only the basal release of PGE2 into the duodenum was significantly increased (20 +/- 3 versus 5 +/- 1 ng/h; p < 0.05). CONCLUSION: Increased mucosal production of PGE2 may be responsible for the abnormally high gastric secretion of bicarbonate in inactive DU patients. The defective duodenal secretion of bicarbonate observed in these patients may be a consequence of previous ulceration rather than the mere presence of H. pylori infection.

Optimization of hapten-protein conjugation by high-performance capillary electrophoresis.

HPCE has been found to be efficient for the optimization of conjugation procedures employed for coupling of the haptens p-nitrophenyl-alpha-D-galactopyranoside (PNPG) and soyasaponin I to a carrier protein, Kunitz soybean trypsin inhibitor (KSTI). The carbohydrate moieties of the haptens were oxidized with periodate followed by reaction with epsilon-amino groups of the carrier. For PNPG, the periodate oxidations (0.01-0.2 M NaIO4) were followed by HPCE and 0.1 M periodate was chosen as the optimum concentration. The coupling of PNPG to KSTI was found to proceed at a constant rate for more than 250 min. The reaction rate for the soyasaponin conjugation to KSTI declined after 80 min of incubation. The coupling of soyasaponin to KSTI was confirmed by enzyme-linked immunosorbent assay with monoclonal antibodies directed against soyasaponin I. The method was found to be particularly powerful for the investigation of conjugates with low epitope densities that are difficult to determine otherwise.

Purification of egg-white allergens.

Purification procedures for the four egg-white proteins ovomucoid, ovotransferrin, ovalbumin, and lysozyme are presented with reference to mechanistic studies at epitope levels of allergic reactions to these proteins. The applied procedures resulted in four preparations containing less than 0.1% contaminating proteins each. The purified protein preparations were characterized by SDS-PAGE and by crossed immunoelectrophoresis with polyclonal antibodies raised against an egg-white extract or the purified proteins. The necessity of these well-characterized proteins in studies on allergic reactions was shown by testing human sera in immunoblots of lysozyme, and by immunoblots of ovomucoid probing with antibodies against the proteins.

High-performance capillary electrophoresis for characterization of hapten-protein conjugates used for production of antibodies against soyasaponin I.

Micellar electrokinetic capillary chromatography using sodium cholate as the micellar phase has been investigated for characterization of hapten-protein conjugates. Special focus has been placed on the hapten soyasaponin I which is a quantitatively dominating glycoside in seeds of several legumes including pea (Pisum sativum L.) and soybean [Glycine max (L.) Merr.]. Soyasaponin I has been isolated from pea and used as hapten for production of anti-saponin specific polyclonal antibodies. Soyasaponin I was coupled to Kunitz soybean trypsin inhibitor (KSTI) and bovine serum albumin. The degree of coupling was determined by high-performance capillary electrophoresis (HPCE). Capillaries dynamically coated with zwitterions were found to be efficient for reduction of interaction between the silica capillary surface and the proteins. The applicability of HPCE for determination of coupling density was confirmed by investigation of a model hapten (p-nitrophenyl-alpha-D-galactoside; PNPG) coupled to KSTI. The PNPG-KSTI conjugates were examined by both HPCE and by spectrophotometric determination of the PNPG density on KSTI. The HPCE method was shown to be efficient in studies of the formation of hapten-protein conjugates and to be more specific than alternative techniques applied for determination of coupling densities.

High-performance capillary electrophoresis for the determination of trypsin and chymotrypsin inhibitors and their association with trypsin, chymotrypsin and monoclonal antibodies.

High-performance capillary electrophoresis (HPCE) was adapted for the determination of Kunitz soybean trypsin inhibitor, Bowman Birk inhibitor from soybean and protein-type proteinase inhibitors from pea (Pisum sativum L.). The method was developed for the determination and characterization of the inhibitors, the enzymes trypsin and chymotrypsin and the monoclonal antibodies (mAbs) raised against the inhibitors, and also the inhibitor-enzyme and inhibitor-mAb association complexes. The results from studies involving the use of various types of buffers revealed the advantages of having zwitterions such as trimethylammoniumpropyl sulphonate (AccuPure) or taurine included in the buffer. The use of capillaries dynamically coated with zwitterions efficiently reduced the interactions of the proteins with the silica capillary surface, which was important for the analyses for trypsin, chymotrypsin and mAbs and their association complexes with the inhibitors. The influence of temperature, voltage, pH and buffer type on migration times, resolution, peak areas and number of theoretical plates was investigated for the proteins studied. The proposed HPCE method is very suitable for studies of proteinase inhibitors compared with traditional inhibitor studies, and it gives efficient protein separations with the possibility of 245,000 plates/m.