Bromelain activates murine macrophages and natural killer cells in vitro.

The innate immune response is critical for effective immunity against most pathogens. In this study, we show that bromelain, a mixture of cysteine proteases, can enhance IFN-gamma-mediated nitric oxide and TNFalpha production by macrophages. Bromelain's effect was independent of endotoxin receptor activation and was not caused by direct modulation of IFN-gamma receptors. Instead, bromelain either enhanced or acted synergistically with IFN-gamma receptor-mediated signals. These effects were seen in both RAW 264.7, a macrophage cell line, and primary macrophage populations. Bromelain also increased IL-2- and IL-12-mediated IFN-gamma production by NK cells. These results indicate a potential role for bromelain in the activation of inflammatory responses in situations where they may be deficient, such as may occur in immunocompromised individuals.

Bromelain modulates T cell and B cell immune responses in vitro and in vivo.

The ability to modulate immune responses is a major aim of many vaccine and immunotherapeutic development programs. Bromelain, a mixture of cysteine proteases, modulates immunological responses and has been proposed to be of clinical use. However, the identity of the immune cells affected by bromelain and the specific cellular functions that are altered remain poorly understood. To address these shortcomings in our knowledge, we have used both in vitro and in vivo immunological assays to study the effects of bromelain. We found that bromelain enhanced T cell receptor (TCR) and anti-CD28-mediated T cell proliferation in splenocyte cultures by increasing the costimulatory activity of accessory cell populations. However, despite increased T cell proliferation, bromelain concomitantly decreased IL-2 production in splenocyte cultures. Additionally, bromelain did not affect TCR and CD28-induced proliferation of highly purified CD4+ T cells, but did inhibit IL-2 production by these cells. In vivo, bromelain enhanced T-cell-dependent, Ag-specific, B cell antibody responses. Again, bromelain induced a concomitant decrease in splenic IL-2 mRNA accumulation in immunized mice. Together, these data show that bromelain can simultaneously enhance and inhibit T cell responses in vitro and in vivo via a stimulatory action on accessory cells and a direct inhibitory action on T cells. This work provides important insights into the immunomodulatory activity of bromelain and has important implications for the use of exogenous cysteine proteases as vaccine adjuvants or immunomodulatory agents.

Bromelain, from pineapple stems, proteolytically blocks activation of extracellular regulated kinase-2 in T cells.

Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteases may act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis, apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimulatory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We show that bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via the TCR with anti-CD3epsilon mAb, or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain was dependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However, inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelain also inhibited PMA-induced IL-2, IFN-gamma, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNA production. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-induced cytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstream from ERK-2 in the Raf-1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cell signal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transduction pathways.

Bromelain protects piglets from diarrhoea caused by oral challenge with K88 positive enterotoxigenic Escherichia coli.

BACKGROUND: K88 positive enterotoxigenic Escherichia coli (K88+ ETEC) is an important cause of diarrhoea in young piglets. K88+ ETEC pathogenesis relies on attachment to specific glycoprotein receptors located on the intestinal mucosa. Proteolytic treatment of these receptors in vitro and in vivo prevents attachment of K88+ ETEC to piglet small intestines and may be of clinical use to prevent K88+ ETEC pathogenesis. AIMS: To determine whether bromelain, a proteolytic extract obtained from pineapple stems, would protect piglets against K88+ ETEC diarrhoea and to confirm and extend earlier findings on the effects of bromelain on K88+ ETEC receptors in vivo. METHODS: Bromelain (0, 12.5, or 125 mg) was orally administered to just weaned piglets for 10 days. One day following commencement of bromelain treatment, piglets were challenged with K88+ ETEC (5 x 10(10) K88ac:0149) for seven days. Intestinal contents from unchallenged piglets were obtained via an intestinal fistula, and tested for their ability to bind K88+ ETEC before and after bromelain treatment. RESULTS: Both doses of bromelain were successful in reducing the incidence of K88+ ETEC diarrhoea and protected piglets from life threatening disease. Bromelain treated pigs also had significantly increased weight gain compared with untreated pigs. Bromelain only temporarily inhibited K88+ ETEC receptor activity, with receptor activity being regenerated 30 hours following treatment, consistent with the regeneration of new enterocytes. CONCLUSION: Results show that bromelain can temporarily inactivate ETEC receptors in vivo and protect against ETEC induced diarrhoea. Bromelain may therefore be an effective prophylaxis against ETEC infection.

Bromelain prevents secretion caused by Vibrio cholerae and Escherichia coli enterotoxins in rabbit ileum in vitro.

BACKGROUND & AIMS: Diarrhea is a major cause of illness and death in children and young animals. The aim of this study was to investigate the possible therapeutic effect of bromelain, a proteolytic extract obtained from pineapple stems on bacterial toxin and second-messenger agonist-induced intestinal secretion. METHODS: The effect of bromelain pretreatment on short-circuit responses to Escherichia coli heat-labile enterotoxin, heat-stable enterotoxin, and Vibrio cholerae cholera toxin was evaluated in rabbit ileum mounted in Ussing chambers. RESULTS: Bromelain was 62% effective in preventing heat-labile enterotoxin-induced secretion, 51% effective against cholera toxin, and 35% effective against heat-stable enterotoxin [corrected]. Bromelain also prevented secretory changes caused by prostaglandin E2, theophylline, calcium-ionophore A23187, 8-bromoadenosine 3':5'-cyclic monophosphate, and 8-bromoguanosine 3':5'-cyclic monophosphate, well-known intracellular mediators of ion secretion. The efficacy of bromelain was not caused by reduced tissue viability resulting from its proteolytic effects on enterocytes, indicated by experiments measuring uptakes of nutrients into intestinal cells and experiments measuring short-circuit responses to glucose. CONCLUSIONS: Bromelain prevents intestinal fluid secretion mediated by secretagogues that act via adenosine 3':5'-cyclic monophosphate, guanosine 3':5'-cyclic monophosphate, and calcium-dependent signaling cascades. It may be clinically useful as an antidiarrheal drug.

Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine.

The virulence of enterotoxigenic Escherichia coli (ETEC) is attributed to their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. A novel approach to preventing ETEC induced diarrhoea would be to prevent attachment of ETEC to intestine by proteolytically modifying the receptor attachment sites. This study aimed to examine the effect of bromelain, a proteolytic extract obtained from pineapple stems, on ETEC receptor activity in porcine small intestine. Bromelain was administered orally to piglets and K88+ ETEC attachment to small intestine was measured at 50 cm intervals using an enzyme immunoassay. K88+ ETEC attachment to intestinal sections that were not treated with bromelain varied appreciably between sampling sites. Variability in receptor activity along the intestinal surface is though to be caused by the localised effects of endogenous proteases. Oral administration of exogenous protease inhibited K88+ ETEC attachment to pig small intestine in a dose dependent manner (p < 0.05). Attachment of K88+ ETEC was negligible after treatment, resembling the levels of attachment of K88 to piglets of the genetically determined non-adhesive phenotype, which are resistant to K88+ ETEC infection. Serum biochemical analysis and histopathological examination of treated piglets showed no adverse effects of the bromelain treatment. It is concluded that administration of bromelain can inhibit ETEC receptor activity in vivo and may therefore be useful for prevention of K88+ ETEC induced diarrhoea.

Detection of attachment of enterotoxigenic Escherichia coli (ETEC) to human small intestinal cells by enzyme immunoassay.

Simple immunoassays were developed to study the binding between enterocytes of the small intestine and other cell types, and enterotoxigenic Escherichia coli (ETEC). CFA/I or CFA/II pilus protein or CFA-positive E. coli bacteria were immobilised in wells of microtitre plates and incubated with vesicles or crude mucus prepared from human brush border enterocytes. Binding of the cell preparations was detected by adding specific rabbit anti-brush border IgG followed by urease-labelled goat anti-rabbit IgG and urea substrate. The binding of purified CFA/I to human or rabbit small intestine, human oral epithelial cells or Caco-2 cells was detected with specific anti-CFA/I IgG. Both human brush border and mucus-derived preparations were able to attach to ETEC. The binding was CFA-specific and strong enough to withstand several washings. In contrast, CFA/I did not bind to small intestinal cells of non-human small intestinal origin, indicating that there may be important differences in affinity between receptors present on human small intestinal cells and cells of non-human small intestinal origin. Antibodies directed against human small intestinal and non-small intestinal cells did not cross-react with either preparation, indicating that receptors between these different cell sources are different. The EIA proved useful during the identification of a newly-recognised 15 kDa bacterial surface component of ETEC strain H10407P, which may function as a putative attachment factor. The EIAs developed in this study were easy to perform and multiple tests could be performed on small samples, including biopsy samples obtained during endoscopy.

The distribution and stability of Escherichia coli K88 receptor in the gastrointestinal tract of the pig.

The levels of Escherichia coli K88 receptor were measured at various sites within the pig intestinal tract using an enzyme immunoassay. The amount of receptor in samples taken from K88-adhesive phenotype animals was found to vary widely along the length of the intestinal tract, but was usually highest in mucosal scrapings taken from the mid-small intestine. Receptor was evident in material collected near either end of the small intestine and was not apparent in material collected from the caecum or lower bowel. The ability of receptor-containing intestinal material to react with immobilized K88 adhesin was inhibited by exposure of the material to either trypsin or contents from the lower bowel, if the receptor-containing material was reacted with the immobilised K88 adhesin prior to exposure to trypsin or lower bowel contents, the bound material remained evident for 24 to 48 h. The possible implications of variable receptor activity in proteolytic environments in relation to pathogenesis and the determination of K88 phenotype in live pigs is discussed.

Efficacy of enteric-coated protease in preventing attachment of enterotoxigenic Escherichia coli and diarrheal disease in the RITARD model.

In this study, we report on a novel approach based on modification of the intestinal surface to prevent diarrhea caused by enterotoxigenic Escherichia coli (ETEC). The removable intestinal tie adult rabbit diarrhea (RITARD) model was used to test the efficacy of an enteric-coated protease preparation (Detach; Enzacor Technology Pty. Ltd.) in the prevention of bacterial attachment and diarrheal disease caused by colonization factor antigen I-positive (CFA/I+) E. coli H10407. Protease was administered orally to rabbits 18 h prior to challenge with 10(11) bacteria. Four groups of rabbits were inoculated with different ETEC strains which produced different combinations of adhesin and enterotoxin or with sterile phosphate-buffered saline. Occurrence of diarrhea during the subsequent 24-h incubation period was recorded. Oral administration of protease was successful in reducing diarrhea and diarrhea-induced death in six of seven (86%) rabbits infected with CFA/I+, heat-stable and heat-labile toxin-positive E. coli (H10407). Seven of eight (87%) rabbits not protected by protease treatment died or developed severe diarrhea. Quantitative analysis of bacterial cultures obtained from the small intestine of rabbits showed a significant (P less than 0.001) 2,000-fold reduction in CFU per centimeter of intestine following treatment with protease. The efficacy of protease treatment was 99.5%, with very wide confidence limits (greater than 0 to 99.9%). The data indicate that the use of protease to prevent ETEC diarrheal disease has considerable potential.