Rafael Campos-Rodríguez

Entamoeba histolytica: immunohistochemical study of hepatic amoebiasis in mouse. Neutrophils and nitric oxide as possible factors of resistance.

Studies in mice have not rendered conclusive data on cell and humoral factors to support the resistance of this rodent to Entamoeba histolytica infection. In Balb/c and C3H/HeJ mice inoculated with live or fixed trophozoites, we studied the evolution of the hepatic lesion, the kinetics of inflammatory cells, and the participation of some humoral factors in the development of the hepatic amoebic lesion. From the first hour, amoebae were surrounded by neutrophils containing inducible nitric oxide synthase (iNOS); macrophages also expressing iNOS appeared lately, whereas NK cells were not part of the inflammatory infiltrates. On the fourth day, neutrophils, macrophages, T and B lymphocytes, plasma cells, and some NK cells limited the lesions and anti-amoeba antibodies appeared when most parasites had been eliminated. Therefore, the resistance of the mice to E. histolytica probably lies in non-specific immune responses, among which the activation of neutrophils and the production of nitric oxide (NO) may be important amoebicide factors.

Lactoferrin-lipopolysaccharide (LPS) binding as key to antibacterial and antiendotoxic effects

Lactoferrin (Lf), a multifunctional protein of the innate immune response, seems to act as a permeabilizing agent of Gram negative bacteria, apparently due to its interaction with enterobacterial lipopolysaccharide (LPS) on the bacterial surface. In both human and bovine Lf, a six residue sequence lying in an 18-loop region of the lactoferricin domain is key to Lf-LPS binding. There is much evidence that, by its action on LPS, Lf destabilizes the bacterial membrane and therefore increases bacterial permeability. By itself, Lf is not an effective antibacterial agent, but it permits the penetration of the bacterial membrane by some antibacterial substances whose hydrophobicity otherwise limits their efficacy. Additionally, Lf neutralizes free LPS by keeping the latter from forming complexes that activate TLR-4 signaling pathways. Such pathways, when over-activated, lead to the abundant production of pro-inflammatory mediators such as tumor necrosis factor (TNF) with fatal consequences to the host. The effect of Lf in reducing inflammation and destabilizing Gram negative bacteria has clinical implications in the control of sepsis, multiple organ dysfunction and bacterial invasion.

Effect of repeated restraint stress on the levels of intestinal IgA in mice

The effects of restraint stress on the intestinal humoral immune system, particularly those about intestinal IgA production, have not been explored in detail. Thus, the purpose of this study was to assess the effect of restraint stress on the production and secretion of intestinal IgA as well as on the number of IgA+ cells in the intestinal lamina propria. The involvement of glucocorticoids and catecholamines were also evaluated. Mice were exposed to 1 or 4 h restraint stress for 4 d. The intestinal IgA concentration was quantified by ELISA and the number of IgA containing cells in the lamina propria was determined by immunohistochemistry. The effects of restraint were also analyzed in mice submitted to different procedures: adrenalectomy, chemical sympathectomy, treatment with a glucocorticoid antagonist (RU486), dexamethasone and epinephrine. The main findings were that (1) chronic restraint-stress reduced the intestinal IgA concentration without changing the number of IgA+ cells in lamina propria; (2) adrenalectomy restored the production of IgA in stressed mice; (3) RU486 and chemical sympathectomy partially blocked the decrease in intestinal IgA in stressed mice; and (4) pharmacological doses of dexamethasone and epinephrine significantly reduced the intestinal IgA concentration and the number of IgA+ cells. The restraint stress probably reduced the intestinal IgA concentration through the effects of glucocorticoids and catecholamines.

Stress modulates intestinal secretory immunoglobulin A

Stress is a response of the central nervous system to environmental stimuli perceived as a threat to homeostasis. The stress response triggers the generation of neurotransmitters and hormones from the hypothalamus pituitary adrenal axis, sympathetic axis and brain gut axis, and in this way modulates the intestinal immune system. The effects of psychological stress on intestinal immunity have been investigated mostly with the restraint/immobilization rodent model, resulting in an up or down modulation of SIgA levels depending on the intensity and time of exposure to stress. SIgA is a protein complex formed by dimeric (dIgA) or polymeric IgA (pIgA) and the secretory component (SC), a peptide derived from the polymeric immunoglobulin receptor (pIgR). The latter receptor is a transmembrane protein expressed on the basolateral side of gut epithelial cells, where it uptakes dIgA or pIgA released by plasma cells in the lamina propria. As a result, the IgA-pIgR complex is formed and transported by vesicles to the apical side of epithelial cells. pIgR is then cleaved to release SIgA into the luminal secretions of gut. Down modulation of SIgA associated with stress can have negative repercussions on intestinal function and integrity. This can take the form of increased adhesion of pathogenic agents to the intestinal epithelium and/or an altered balance of inflammation leading to greater intestinal permeability. Most studies on the molecular and biochemical mechanisms involved in the stress response have focused on systemic immunity. The present review analyzes the impact of stress (mostly by restraint/immobilization, but also with mention of other models) on the generation of SIgA, pIgR and other humoral and cellular components involved in the intestinal immune response. Insights into these mechanisms could lead to better therapies for protecting against pathogenic agents and avoiding epithelial tissue damage by modulating intestinal inflammation.

Lactoferrin increases both resistance to Salmonella typhimurium infection and the production of antibodies in mice.

Lactoferrin (Lf) is a multifunctional iron-binding glycoprotein with antibacterial and immunomodulatory activities. The antibacterial influence of orally administered bovine Lf (bLf) against murine infection caused by Salmonella typhimurium (S. typhimurium) has scarcely been explored. In the current study, Balb/c mice were treated orally for 7 days with either 5 or 100mg of bovine lactoferrin (bLf). On day 7 of treatment, mice were intragastrically infected with a lethal or sublethal dose of colony forming units (CFU) of S. typhimurium. During treatment with bLf, feces from mice sublethally infected were harvested daily to prepare fecal suspensions, which were serially diluted and plated onto Salmonella Shigella agar to estimate CFU/g of feces. After sacrificing the animals on day 7, 14 or 21 post-infection, samples of intestinal fluid, Peyers patches (PP), liver and spleen were collected to count the number of CFU by plate dilution. Intestinal secretions were also employed, along with serum samples, to evaluate total IgA, IgG and IgM antibodies, and those against Salmonella surface proteins and bLf by ELISA assay. In lethally infected mice both bLf doses decreased mortality. In sublethally infected mice, both bLf doses decreased bacterial shedding in feces and intestinal fluid, and also reduced bacterial colonization at PP and bacterial translocation in the liver and spleen. Levels of total and those IgG and IgM in serum and IgA in intestinal secretions against Salmonella surface proteins and bLf were enhanced with both doses of bLf. These findings suggest that the effect of bLf against the infection by S. typhimurium in mice may be the result of an antimicrobial activity linked with its modulatory effect on immunocompetent cells (from intestinal and peripheral organs) involved in antibody production.

Regionalization of pIgR expression in the mucosa of mouse small intestine.

Few reports exist on the differences in cell populations or immunological functions between the proximal and distal segments of the small intestine (SI). In the current contribution we analyzed the expression of the polymeric immunoglobulin receptor (pIgR) and alpha chains as well as the density of IgA-producing cells from the proximal and distal intestinal segments from Balb/c mice. Furthermore, by using real-time RT-PCR we quantified the expression of cytokines (TNF-alpha, IFN-gamma, IL-4 and TGF-beta), Toll-like receptor-4 (TLR-4), and the glucocorticoid receptor (GR) involved in pIgR expression in intestinal epithelial cells (IEC). In this study, for the first time it has been demonstrated that the expression of the pIgR as well as alpha chain was greater in the proximal than the distal segment of the small intestine of normal mice. Moreover, we found striking differences in the expression of cytokines at the different intestinal compartments. Whereas the expression of TNF-alpha, IFN-gamma and TGF-beta was higher in lamina propria lymphocytes (LPL) of the distal than proximal segment, it was higher in IEC of the proximal than distal segment. In contrast, the expression of the gene for IL-4 was higher in the LPL of the proximal segment and the IEC of the distal segment. Although the overall expression of TNF-alpha, IL-4, IFN-gamma and TGF-beta was higher in the whole mucosa of the distal than proximal segment, we propose that cytokines produced by epithelial cells (TNF-alpha, IFN-gamma and TGF-beta) autocrinally up-regulate the expression of mRNA for the pIgR. Finally the expression of the GR was higher in the proximal segment, while the expression of the gene for TLR-4 was significantly higher in the IEC of the distal than proximal segment. The higher expression of pIgR found in the proximal segment is probably related to the effect on epithelial cells of the higher production of TNF-alpha, IFN-gamma and TGF-beta, as well as the higher expression of the glucocorticoid receptors. The increased expression of pIgR in the proximal segment appears primarily responsible for the increased secretory IgA levels in the small intestine of mice. These results confirm and extend previous findings supporting the compartmentalization of the intestinal immune system.

Hypophysectomy and neurointermediate pituitary lobectomy reduce serum immunoglobulin M (IgM) and IgG and intestinal IgA responses to Salmonella enterica serovar Typhimurium infection in rats.

ABSTRACT The influence of anterior pituitary hormones on the gastrointestinal tract of humans and animals has been reported. Hypophysectomy (HYPOX) in the rat causes atrophy of the intestinal mucosa, reduction of gastric secretion and intestinal absorption, and increased susceptibility to infections. To our knowledge, there are no studies on the humoral immune response of the gut-associated lymphoid tissue after HYPOX. We have reported that decreased secretion of vasopressin and oxytocin due to neurointermediate pituitary lobectomy (NIL) diminishes humoral and cell-mediated immune responses. However, no data have been published on whether NIL can affect intestinal immune responses. We analyzed the effects of HYPOX and NIL on bacterial colonization of the intestinal lumen, Peyers patches, and spleen as well as the serum immunoglobulin G (IgG) and IgM and specific intestinal IgA levels in response to Salmonella enterica serovar Typhimurium oral infection. Results showed the following: (i) Salmonella serovar Typhimurium was eliminated from the intestinal lumen at the same rate in rats that underwent a sham operation, HYPOX, and NIL; (ii) Salmonella serovar Typhimurium colonization of Peyers patches and spleen was significantly higher in both HYPOX and NIL rats than in sham-operated rats; (iii) serum IgG and IgM and intestinal IgA against surface proteins of Salmonella serovar Typhimurium were significantly lower in HYPOX and NIL rats than in sham-operated rats; and (iv) compared to NIL rats, higher Peyers patch and spleen bacterial colonization and decreased IgG, IgM, and IgA production were observed in HYPOX rats. We conclude that hormones from each pituitary lobe affect the systemic and gastrointestinal humoral immune responses through different mechanisms.

Repeated restraint stress increases IgA concentration in rat small intestine

The most abundant intestinal immunoglobulin and first line of specific immunological defense against environmental antigens is secretory immunoglobulin A. To better understand the effect of repeated stress on the secretion of intestinal IgA, the effects of restraint stress on IgA concentration and mRNA expression of the gene for the alpha-chain of IgA was assessed in both the duodenum and ileum of the rats. Restraint stress induced an increase in intestinal IgA, which was blocked by an adrenalectomy, suggesting a role of catecholamines and glucocorticoids. Whereas the blocking of glucocorticoid receptors by RU-486 did not affect the increased IgA concentration, it did reduce IgA alpha-chain mRNA expression in both segments, indicating a possible mediation on the part of glucocorticoids in IgA secretion by individual cells. Treatment with corticosterone significantly increased both the IgA concentration and IgA alpha-chain mRNA expression in ileum but not in duodenum, suggesting that glucocorticoids may act directly on IgA-antibody forming cells to increase IgA secretion in the former segment. A probable role by catecholamines was evidenced by the reduction in IgA concentration and IgA alpha-chain mRNA expression in both segments after a chemical sympathectomy with 6-hydroxydopamine (6-OHDA). Additionally, norepinephrine significantly reduced IgA alpha-chain mRNA levels but increased pIgR mRNA expression and IgA concentration in both intestinal segments. We propose that the increased intestinal IgA levels caused by repeated restraint stress is likely due to the effects of catecholamines on the transport of plgA across the epithelium.

Entamoeba histolytica: production of nitric oxide and in situ activity of NADPH diaphorase in amebic liver abscess of hamsters.

Entamoeba histolytica trophozoites were inoculated into the liver of hamsters and serum nitrate/nitrite levels [expressed as nitric oxide (NO) production] were determined at different times during amebic liver abscess (ALA) development. We also tested the effects of NO synthase (NOS) inhibitors such as NG-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, and dexamethasone during ALA production. Since NOS activity has been correlated with expression of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) in tissues, we performed histochemistry studies to determine the activity of the latter in livers infected with E. histolytica trophozoites. Production of NO in serum was directly proportional to the size of ALAs, and NOS inhibitors caused low levels of NO and smaller ALAs. Our data suggest that NO does not have any lytic effect on E. histolytica trophozoites and is therefore incapable of providing protection against the amebic liver infection. In addition, NADPHd activity was detected histochemically in hepatocytes and inflammatory cells associated with focal necrosis containing trophozoites. The positive reactivity observed in these parasites may be attributable to a close biochemical similarity of NADPHd to the NADPH:flavin oxidoreductase described in E. histolytica by other investigators.

Induction of Morphological and Electrophysiological Changes in Hamster Cornea after In Vitro Interaction with Trophozoites of Acanthamoeba spp.

ABSTRACT Acanthamoeba castellani and Acanthamoeba polyphaga are free-living amebae that cause keratitis and granulomatous encephalitis in humans. We have analyzed the early morphological and electrophysiological changes occurring during the in vitro interaction of cultured amebae with intact or physically damaged corneas obtained from hamsters. Both species of Acanthamoeba produced similar cytopathic changes, as seen by light microscopy and scanning electron microscopy. After adhesion to the epithelial surface, trophozoites formed clumps and migrated toward the cell borders, causing the separation of adjacent cells at 1 h of coculture. At later stages (2 to 4 h), some amebae were found under desquamating epithelial cells whereas others were seen associated with damaged cells or forming amebostome-like structures to ingest detached epithelial cells. Control corneas incubated in culture medium conditioned with amebae showed a cytoplasmic vacuolization and blurring of the epithelial-stromal junction. The early stages of corneal epithelial damage caused by amebae were also analyzed by measuring the transepithelial resistance changes in corneas mounted in Ussing chambers. Both species of Acanthamoeba caused a rapid decrease in electrical resistance. The present observations demonstrate that under in vitro conditions, Acanthamoeba trophozoites rapidly cause significant damage to the corneal epithelium. Furthermore, in our experimental model, previous physical damage to the corneas was not a prerequisite for the development of amebic corneal ulcerations.