Preservation of protective capacity of hyperimmune anti-Stx2 bovine colostrum against enterohemorrhagic Escherichia coli O157:H7 pathogenicity after pasteurization and spray-drying processes.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major etiologic agent that causes bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is the main virulence factor of EHEC responsible for the progression to HUS. Although many laboratories have made efforts to develop an effective treatment for Stx-mediated HUS, a specific therapy has not been found yet. Human consumption of bovine colostrum is known to have therapeutic effects against several gastrointestinal infections because of the peptide and proteins (including antibodies) with direct antimicrobial and endotoxin-neutralizing effects contained in this fluid. We have previously demonstrated that colostrum from Stx type 2 (Stx2)-immunized pregnant cows effectively prevents Stx2 cytotoxicity and EHEC O157:H7 pathogenicity. In this study we evaluated the preservation of the protective properties of hyperimmune colostrum against Stx2 (HIC-Stx2) after pasteurization and spray-drying processes by performing in vitro and in vivo assays. Our results showed that reconstituted HIC-Stx2 colostrum after pasteurization at 60°C for 60 min and spray-dried under optimized conditions preserved specific IgG that successfully neutralized Stx2 cytotoxicity on Vero cells. Furthermore, this pasteurized/dehydrated and reconstituted HIC-Stx2 preserved the protective capacity against EHEC infection in a weaned mice model. The consumption of hyperimmune HIC-Stx2 bovine colostrum could be effective for HUS prevention in humans as well as in EHEC control in calves. However, further studies need to be done to consider its use for controlling EHEC infections.

The oxidative stress induced in vivo by Shiga toxin-2 contributes to the pathogenicity of haemolytic uraemic syndrome.

Typical haemolytic uraemic syndrome (HUS) is caused by Shiga toxin (Stx)-producing Escherichia coli infections and is characterized by thrombotic microangiopathy that leads to haemolytic anaemia, thrombocytopenia and acute renal failure. Renal or neurological sequelae are consequences of irreversible tissue damage during the acute phase. Stx toxicity and the acute inflammatory response raised by the host determine the development of HUS. At present there is no specific therapy to control Stx damage. The pathogenic role of reactive oxygen species (ROS) on endothelial injury has been largely documented. In this study, we investigated the in-vivo effects of Stx on the oxidative balance and its contribution to the development of HUS in mice. In addition, we analysed the effect of anti-oxidant agents as therapeutic tools to counteract Stx toxicity. We demonstrated that Stx induced an oxidative imbalance, evidenced by renal glutathione depletion and increased lipid membrane peroxidation. The increased ROS production by neutrophils may be one of the major sources of oxidative stress during Stx intoxication. All these parameters were ameliorated by anti-oxidants reducing platelet activation, renal damage and increasing survival. To conclude, Stx generates a pro-oxidative state that contributes to kidney failure, and exogenous anti-oxidants could be beneficial to counteract this pathogenic pathway.

Tolerance to lipopolysaccharide promotes an enhanced neutrophil extracellular traps formation leading to a more efficient bacterial clearance in mice.

Tolerance to lipopolysaccharide (LPS) constitutes a stress adaptation, in which a primary contact with LPS results in a minimal response when a second exposure with the same stimulus occurs. However, active important defence mechanisms are mounted during the tolerant state. Our aim was to assess the contribution of polymorphonuclear neutrophils (PMN) in the clearance of bacterial infection in a mouse model of tolerance to LPS. After tolerance was developed, we investigated in vivo different mechanisms of bacterial clearance. The elimination of a locally induced polymicrobial challenge was more efficient in tolerant mice both in the presence or absence of local macrophages. This was related to a higher number of PMN migrating to the infectious site as a result of an increased number of PMN from the marginal pool with higher chemotactic capacity, not because of differences in their phagocytic activity or reactive species production. In vivo, neutrophils extracellular trap (NET) destruction by nuclease treatment abolished the observed increased clearance in tolerant but not in control mice. In line with this finding, in vitro NETs formation was higher in PMN from tolerant animals. These results indicate that the higher chemotactic response from an increased PMN marginal pool and the NETs enhanced forming capacity are the main mechanisms mediating bacterial clearance in tolerant mice. To sum up, far from being a lack of response, tolerance to LPS causes PMN priming effects which favour distant and local anti-infectious responses.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7.

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) produced by enterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

Renal damage and death in weaned mice after oral infection with Shiga toxin 2-producing Escherichia coli strains.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infections are considered a public health problem in both developed and developing countries because of their increasing incidence and the severity of clinical presentation. Approximately 10% of infected patients develop complications such as haemolytic uraemic syndrome (HUS) characterized by acute renal failure, thrombocytopenia and haemolytic anaemia. The precise sequence of events leading to HUS is still understood incompletely. Because of the lack of a reproducible small animal model for EHEC infections, in vivo studies examining EHEC-host early interactions are limited and insufficient. The aim of this study was to characterize the weaned BALB/c mouse as a model of E. coli O157:H7 infection. In this paper we report that human Shiga toxin 2 (Stx2)-producing EHEC strains can adhere to the intestinal epithelium of weaned BALB/c mice, and produce local damage which leads to systemic disease and death in a percentage of infected mice. The lethality of the EHEC strain is closely age-dependent, and is related to the bacterial ability to colonize intestine and to produce Stx2. It can be concluded that the weaned BALB/c mouse can be used as a small animal model to study host early responses, and the role of bacterial pathogenic factors in the induction of systemic disease, thus providing a useful tool for the evaluation of therapeutic or vaccine approaches.

Human antibody-dependent cellular cytotoxicity mediated by interferon gamma-activated neutrophils is impaired by vasoactive intestinal peptide.

The aim of the present study was to evaluate the effect of vasoactive intestinal peptide (VIP) on the expression and activity of receptors for the Fc portion of IgG (Fc gamma R) in human neutrophils. Cells were assayed under basal conditions and following in vitro stimulation with interferon gamma (IFN gamma). Antibody dependent-cellular cytotoxicity (ADCC) was chosen as a means of evaluating Fc gamma R activity. The results indicated that incubation with VIP (10(-6) M) during 18 h slightly diminished cytotoxicity of non stimulated neutrophils. In contrast, VIP exerted a marked inhibitory effect on neutrophils activated with IFN gamma. Similar results were obtained with forskolin, another agent that increases intracellular cAMP. Finally, using monoclonal antibodies and flow cytometry analysis, we found decreased membrane expression of Fc gamma R after VIP incubation. Taken together, these results show that VIP is able to act on human neutrophils, partially blocking IFN gamma-activation of Fc gamma R mediated functions. Modulation of neutrophil cytotoxic response by VIP may have an important role in limiting tissue injury during inflammation.

Different requirements for the induction of antibody-dependent and immune complexes triggered cytotoxicity mediated by monocytes.

We have previously shown that immune complexes triggered nonspecific cytotoxicity (NSC) towards nonsensitized target cells and antibody-dependent cellular cytotoxicity (ADCC), two functions mediated through monocyte Fc gamma receptors, employing different lytic mechanisms [Geffner, J. R., et al. (1986) Clin. Exp. Immunol. 67, 646]. In this report, we analyze some of the metabolic requirements involved in the induction of monocyte NSC and ADCC. The results showed NSC to be dependent on: (1) metabolic energy derived from glycolysis, (2) availability of external Ca2+, (3) calmodulin activity, (4) integrity of microtubules, but not the microfilament system, and (5) activation of phospholipase(s) and lipoxygenase. On the other hand, ADCC was not impaired by: (1) inhibition of glycolysis, (2) Ca2+ chelation, (3) disruption of microtubules, or (4) inhibition of calmodulin or lipoxygenase. It is concluded that monocyte NSC and ADCC are regulated by different endogenous signals.

Cyclophosphamide augments ADCC by increasing the expression of Fc-receptors.

Previous reports demonstrated that cyclophosphamide (Cy) enhances two Fc gamma receptor (Fc gamma R) mediated functions: antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis. In this paper we examine the mechanisms whereby Cy modifies the cytotoxic capacity of mouse splenocytes. The results indicate that the observed augmentation of ADCC could not be attributed to a higher proportion of macrophages and/or polymorphonuclear leukocytes (PMN), but rather to an enhanced activity per effector cell. Binding studies showed that this augmentation was associated with an increased number, but not an increased avidity of Fc gamma R sites. The possibility that the enhanced Fc gamma R expression by Cy may result in the alteration of other Fc gamma R-mediated functions is discussed.

Inhibition of lymphocyte and monocyte antibody-dependent cellular cytotoxicity by immune complexes: effect of normal human serum.

Antibody-dependent cellular cytotoxicity (ADCC) mediated by peripheral blood mononuclear cells (PBMC) and by isolated populations of lymphocytes and monocytes was compared for susceptibility to inhibition by soluble immune complexes (IC) and by heat-aggregated IgG (HAIgG). It was found that the decrease of ADCC was significantly higher in lymphocytes than in monocytes at all IC and HAIgG concentrations employed (P less than 0.001). The degree of inhibition of PBMC-mediated ADCC was similar to that observed in monocyte ADCC. In previous reports, we demonstrated that IC inhibition of PBMC-mediated ADCC could be reversed by normal human serum (NHS) used as a source of complement (C). In this paper, we study the effects of NHS on isolated populations of monocytes and lymphocytes. It was found that NHS was unable to modify the capacity of IC-blocked monocytes to mediate ADCC. On the contrary, NHS efficiently reversed the inhibition of both ADCC and Fc gamma R expression in IC-blocked lymphocytes. We propose that the regulation of Fc gamma R-IC interactions by C may constitute a physiological way to preserve Fc gamma R expression in lymphocytes.

[Interaction of immune complexes and their leukocyte receptors: regulation by the complement system and by cyclophosphamide]. / Interacción de los complejos inmunes y sus receptores leucocitarios: su regulación por el sistema complemento y la ciclofosfamida.

During the course of this investigation we have studied different aspects related to the interaction between immune complexes (CI) and their cellular receptors for the Fc-fragment of IgG (RFc gamma). Our first approach was to demonstrate that the alternative pathway of complement is the main one responsible for the CI-dissociation from their receptors of human peripheral mononuclear cells. This modulatory effect was studied throughout the functional restoration of antibody dependent cellular cytotoxicity (CCCDA), which is a mechanism susceptible to Cl-inhibition. The results suggest that the levels of circulating Cl do not necessarily correlate with the tissue damage they produce. Secondly, we have demonstrated that cyclophosphamide (Cy), which is a potent immunomodulating drug which has been used for the treatment of diseases characterized by high levels of Cl, enhances the capacity of the mononuclear phagocytic system to remove IgG-particulate complexes in mice. Finally, we have described a nonspecific cytotoxic system triggered by Cl against different target cells, through a mechanism that involves the reactive metabolites of O2.

Relevance of neutrophils in the murine model of haemolytic uraemic syndrome: mechanisms involved in Shiga toxin type 2-induced neutrophilia.

It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the haemolytic uraemic syndrome (HUS). However, the contribution of the inflammatory response in the pathogenesis of the disease has also been well established. Neutrophils (PMN) represent a central component of inflammation during infections, and patients with high peripheral PMN counts at presentation have a poor prognosis. The mouse model of HUS, by intravenous injection of pure Stx type 2 (Stx2), reproduces human neutrophilia and allows the study of early events in the course of Stx2-induced pathophysiological mechanisms. The aim of this study was to address the contribution of PMN on Stx2 toxicity in a murine model of HUS, by evaluating the survival and renal damage in mice in which the granulocytic population was depleted. We found that the absence of PMN reduced Stx2-induced lethal effects and renal damage. We also investigated the mechanisms underlying Stx2-induced neutrophilia, studying the influence of Stx2 on myelopoyesis, on the emergence of cells from the bone marrow and on the in vivo migration into tissues. Stx2 administration led to an accelerated release of bone marrow cells, which egress at an earlier stage of maturation, together with an increase in the proliferation of myeloid progenitors. Moreover, Stx2-treated mice exhibited a lower migratory capacity to a local inflammatory site. In conclusion, PMN are essential in the pathogenesis of HUS and neutrophilia is not merely an epiphenomenon, but contributes to Stx2-damaging mechanism by potentiating Stx2 toxicity.

Endogenous glucocorticoids modulate neutrophil function in a murine model of haemolytic uraemic syndrome.

Haemolytic uraemic syndrome (HUS) is caused by Shiga-toxin-producing Escherichia coli (STEC). Although, Shiga toxin type 2 (Stx2) is responsible for the renal pathogenesis observed in patients, the inflammatory response, including cytokines and polymorphonuclear neutrophils (PMN), plays a key role in the development of HUS. Previously, we demonstrated that Stx2 injection generates an anti-inflammatory reaction characterized by endogenous glucocorticoid (GC) secretion, which attenuates HUS severity in mice. Here, we analysed the effects of Stx2 on the pathogenic function of PMN and the potential role of endogenous GC to limit PMN activation during HUS development in a murine model. For this purpose we assessed the functional activity of isolated PMN after in vivo treatment with Stx2 alone or in simultaneous treatment with Ru486 (GC receptor antagonist). We found that Stx2 increased the generation of reactive oxygen intermediates (ROI) under phobol-myristate-acetate (PMA) stimulation and that the simultaneous treatment with Ru486 strengthened this effect. Conversely, both treatments significantly inhibited in vitro phagocytosis. Furthermore, Stx2 augmented in vitro PMN adhesion to fibrinogen (FGN) and bovine serum albumin (BSA) but not to collagen type I (CTI). Stx2 + Ru486 caused enhanced adhesion to BSA and CTI compared to Stx2. Whereas Stx2 significantly increased migration towards N-formyl-methionyl-leucyl-phenylalanine (fMLP), Stx2 + Ru486 treatment enhanced and accelerated this process. The percentage of apoptotic PMN from Stx2-treated mice was higher compared with controls, but equal to Stx2 + Ru486 treated mice. We conclude that Stx2 activates PMN and that the absence of endogenous GC enhances this activation suggesting that endogenous GC can, at least partially, counteract PMN inflammatory functions.

Melatonin-induced enhancement of antibody-dependent cellular cytotoxicity.

Antibody-dependent cellular cytotoxicity (ADCC) is a lytic mechanism in which a specific antibody acts cooperatively with leukocytic effector cells to induce target cell lysis. In this paper, the effect of exogenous melatonin on ADCC was examined. It was found that two evening intravenous injections of melatonin (1 mg/kg b.w.) was sufficient to enhance the capacity of splenocytes to mediate ADCC. This augmented activity returned to normal levels by day 6. Moreover, the opioid antagonist, naloxone, was unable to inhibit the ADCC enhancement, suggesting that melatonin did not operate through a naloxone-sensitive opiatergic mechanism. These results further support the modulatory action of melatonin on immune responses.

Effect of cyclophosphamide on the clearance of IgG-sensitized red cells in mice.

The effects of cyclophosphamide (Cy) on the clearance of IgG-sensitized erythrocytes (EA) were examined. The results clearly indicate that Cy treatment enhances the capacity of the mononuclear phagocytic system to remove antibody-coated cells from the circulation in normal and decomplemented mice. The enhanced rate of clearance is the consequence of an increased uptake of EA by the liver and spleen. We explored the possibility that the enhancement of Fc receptor-mediated clearance might be an important effect to be taken into account in the search for a more effective therapy of immune complexes diseases.

Enhancement of phagocytosis by different anti-neoplastic drugs.

The effect of various anti-neoplastic drugs on erythrophagocytosis was studied. All of them were able to induce a significant enhancement of phagocytosis when administered at single high doses to normal mice. The effect was exerted on antibody dependent phagocytosis and was not due to local inflammatory reactions since the subcutaneous route also induced increased phagocytic activity. Since the drugs tested are commonly used in cancer chemotherapy, their enhancing effect on phagocytosis may be relevant in the regulation of host defense mechanisms.

Seasonal variations in antibody-dependent cellular cytotoxicity regulation by melatonin.

Data collected over a period of 4 years show that melatonin (two daily i.v. injections of 0.1 mg/kg body wt. given at 16:00 h) was able to enhance antibody-dependent cellular cytotoxicity (ADCC) in summer, but not in winter. Dose-response curves carried out in January, May, July, and October suggest that the seasonal effects reported are related to differences in the sensitivity of mice to melatonin during the course of the year. These results show seasonal variations in the immune modulatory action of melatonin.

Impairment of the murine mononuclear phagocyte system function by antigenic stimulation.

This study examined the mononuclear phagocyte system (MPS) capacity to eliminate IgG-sensitized syngeneic erythrocytes (EA) after antigenic challenge. Survival data of EA in normal and preimmunized mice showed that a single dose of T-dependent antigen was able to delay Fc gamma R-dependent clearance. This impairment in EA elimination was dependent on the dose of antigen injected. On the other hand, T-independent antigens, B cell mitogen, and the inflammatory agent Freund's incomplete adjuvant were ineffective in modulating MPS function. As expected, the liver and the spleen were the main sites of EA trapping, but the spleen of immunized mice sequestered significantly less EA than that of control mice. Impaired clearance capacity was observed as soon as 24 hr after immunization and was persistent up to the seventh day after antigenic stimulation. Moreover, mice decomplementation by cobra venom factor treatment did not prevent the impairment of MPS by antigenic stimulation, suggesting that it is strictly Fc gamma R dependent. Our results indicate that stimulation of the immune system by T-dependent antigens can diminish the Fc gamma R-mediated clearance capacity of the MPS. The possible mechanisms involved in this regulation are discussed.

Immunomodulation exerted by cyclophosphamide is not interfered by N-acetyl cysteine.

Metabolism of cyclophosphamide (Cy) by liver enzymes results in cytostatic products and acrolein, which exerts urotoxicity. Experiments were designed to determine which metabolites are responsible for Cy-induced immunomodulation. For this purpose, mice were treated simultaneously with Cy and N-acetyl-cysteine (NAC), a thiol compound which reacts with acrolein, and different immunological functions were assayed. Results show that NAC did not interfere with Cy effects on antibody-dependent cellular cytotoxicity (ADCC), NK activity, delayed-type hypersensitivity (DTH) or antibody production, indicating that modulation of these functions by Cy is mediated by its cytostatic metabolites.

Fc gamma receptor-dependent clearance is enhanced following lipopolysaccharide in vivo treatment.

Lipopolysaccharides (LPS) occupy centre stage in the pathogenesis of gram-negative sepsis. Although LPS are potent stimulators of the mononuclear phagocyte system (MPS), their effects on immune complex (IC)-specific clearance have not yet been reported. In order to evaluate this issue, we examined the MPS function after LPS treatment by measuring intravascular removal rate of syngeneic erythrocytes sensitized with specific immunoglobulin G (IgG) (EA). Our findings showed that LPS, directly or through the release of endogenous cytokines, enhance Fc gamma receptor (Fc gamma R)-dependent clearance. The EA uptake by liver, spleen and bone marrow was significantly increased leading to an effective clearance of immune complexes. Splenic antibody-dependent cellular cytotoxicity (ADCC), an in vitro indicator of Fc gamma R functionality, was also increased after in vivo LPS treatment. However, cytometric studies showed that endotoxin did not modify Fc gamma R expression on splenocytes, but markedly enhanced the expression of CD11b/CD18 (Mac-1), an adhesion molecule closely related to Fc gamma R activity. We conclude that LPS enhance Fc gamma R-dependent effector functions and suggest that this effect is mediated through alterations in adhesion molecules.