Platelet thrombus formation in eHUS is prevented by anti-MBL2.

E. coli associated Hemolytic Uremic Syndrome (epidemic hemolytic uremic syndrome, eHUS) caused by Shiga toxin-producing bacteria is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury that cause acute renal failure in up to 65% of affected patients. We hypothesized that the mannose-binding lectin (MBL) pathway of complement activation plays an important role in human eHUS, as we previously demonstrated that injection of Shiga Toxin-2 (Stx-2) led to fibrin deposition in mouse glomeruli that was blocked by co-injection of the anti-MBL-2 antibody 3F8. However, the markers of platelet thrombosis in affected mouse glomeruli were not delineated. To investigate the effect of 3F8 on markers of platelet thrombosis, we used kidney sections from our mouse model (MBL-2+/+ Mbl-A/C-/-; MBL2 KI mouse). Mice in the control group received PBS, while mice in a second group received Stx-2, and those in a third group received 3F8 and Stx-2. Using double immunofluorescence (IF) followed by digital image analysis, kidney sections were stained for fibrin(ogen) and CD41 (marker for platelets), von-Willebrand factor (marker for endothelial cells and platelets), and podocin (marker for podocytes). Electron microscopy (EM) was performed on ultrathin sections from mice and human with HUS. Injection of Stx-2 resulted in an increase of both fibrin and platelets in glomeruli, while administration of 3F8 with Stx-2 reduced both platelet and fibrin to control levels. EM studies confirmed that CD41-positive objects observed by IF were platelets. The increases in platelet number and fibrin levels by injection of Stx-2 are consistent with the generation of platelet-fibrin thrombi that were prevented by 3F8.

Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome.

BACKGROUND: The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. MATERIALS AND METHODS: We measured cell surface TF activity in response to tumor necrosis factor-alpha (TNF-alpha) (20 ng mL(-1), 2-144 h), Stx-1 (10(-11) mol L(-1), 4-144 h), or their combination (TNF-alpha 22 h and Stx-1 for the last 0.5-4 h of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). RESULTS AND CONCLUSIONS: We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/- 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx-1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-alpha markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS.

[Various routes of L-tryptophan transport in the small intestine of the chick]. / Razlichnye puti transporta L-triptofana v tonkoi kishke tsypliat.

Presence of two mechanisms in the accumulation of L-tryptophan by the mucosal cells of chicken small intestine was determined in vitro: sodium-independent and sodium-dependent those, the latter being of a major importance. Analysis of the accumulation kinetics in the linear stage showed that at the replacement of sodium with potassium ions in the incubation medium, a decrease of Kt (from 4.44 mM to 1.05 mM) as well as V (from 0.78 mM/min/cm to 0.12 mM/min/cm) occurs. High level of L-threonine, L-alpha-alanine, L-valine (100 mM) considerably inhibit the accumulation of L-tryptophan from 0.24 or 2.40 mM solution, the highest inhibition (78-96%) being related to the sodium-dependent transport mechanism. No considerable changes in Kt (4.0 mM) were observed in the saturated stage of transport whereas V showed a rapid decrease (0.22 mM/min/cm). Adding of modifier 0.64 mM L-alpha-alanine was followed by the increase both in Kt (5.71 mM) and V (0.37 mM/min/cm).

[The effect of sodium ions on amino acid and peptide assimilation in the small intestine of developing chicks]. / Vliianie ionov natriia na usvoenie aminokislot i peptidov v tonkoi kishke razvivaiushchikhsia tsypliat.

The absorption of free glycine and L-tryptophan as well as amino acids released in the hydrolysis of glycil-L-leucine and glycil-L-tryptophan was studied in vitro with accumulating preparations of intestinal mucosa of 1-65-day old chicken. Two ways of absorption of free and peptide amino acids were found: sodium-dependent and sodium-independent. Intensity of each of them depended on the age of chicken and was dissimilar for the compounds. The sodium-independent way is more important for glycine than for free amino acid. Absorption of glycil-L-tryptophan as well as its amino acids is mainly performed by the sodium-dependent way.

[Assimilation of free and peptide glycine in the chick small intestine]. / Usvoenie svobodnogo i peptidnogo glitsina tonkoi kishke tsypliat.

Absorption of free and peptide glycine released at the hydrolysis of glycyl-L-leucine, L-leucyl-glycine, glycyl-L-valine was investigated in experiments in vitro with accumulating mucosal preparations of the chick small intestine. An active accumulation of free amino acid occurred in the mucosa depending on sodium and oxygen presence in the incubation medium. The participation of sodium-dependent and sodium-independent components in the transport was revealed. Absorption of peptide glycine released at the hydrolysis of glycyl-L-leucine and glycyl-L-valine appeared to be a sodium-independent process and demanded the energy of aerobic metabolism. The data indicate the effect of various amino acids on the transport of free and peptide glycine. Activating effect of some of them on the absorption of glycine is supposed to be determined for this amino acid by a mechanism of exchange transport in the chick small intestine.

[Peptide interactions in the intestines of birds]. / Vzaimodeistvie peptidov v kishechnike ptits.

Interaction among various dipeptides was studied during their uptake in the mucosa of the small intestine of chicks. The effects observed were either inhibiting (L-valyl-L-valine inhibited the uptake of glycyl-L-alpha-alanine, glycyl-L-proline, L-leucyl-glycine, glycylglycine, and L-alpha-alanyl-L-alpha-alanine inhibited the uptake of glycyl-L-proline and L-leucylglycine) or stimulating (L-valyl-L-valine activated the uptake of glycyl-L-leucine), or neutral (L-alpha-alanyl-L-alpha-alanine exerted no significant effect on the absorption of glycyl-L-leucine and glycyl-L-valine). The amount and direction of the modifying effect depended on the agent concentration. Effects observed during the interaction of dipeptides and mixtures of free amino acids equimolar to them were identical. It is only possible if hydrolytic processes precede the transport and are closely related to them.

[Peptide transport and peptidase localization in the small intestine of chickens]. / Peptidnyi transport i lokalizatsiia peptidaz v tonkoi kishke tsypliat.

The mechanism of peptide transport was studied in vitro in the small intestine of chicks and accumulating preparations of mucosa using glycyl-1-tryptophan. A special criterion was proposed to differentiate the processes of intracellular and membrane hydrolysis of dipeptide basing on the analysis of transport intensity of amino acids released into the serosal solution after hydrolysis of dipeptide. 1-tryptophan released at hydrolysis was transported considerably more slowly than free amino acid. 1-tryptophan from the mixture with glycyne demonstrated the highest transport intensity in serosal solution as well as the highest accumulation in the mucosa cells from all forms of 1-tryptophan. At the same time the peptide form of glycyne was transported with the same intensity as that from the mixture with 1-tryptophan. According to the criterion applied it is supposed that unequal levels of 1-tryptophan and glycyne in the serosal solution prove the membrane hydrolysis of glycyne-1-tryptophan during ist transport in enterocytes.

[Membrane digestion and absorption in the bird]. / Membrannoe pishchevarenie i vsasyvanie u ptits.

Characteristics of membrane digestion in birds as well as its role in splitting up carbohydrates and proteins revealed the regulatory character of membrane digestion enzymes, their linkage to the enterocyte membrane, dependence on the age and the topography of the small intestine. The systems of active and exchange transport as well as its energetics are described. A close connection between membrane hydrolysis and absorption is shown as actualized by specialized digestive-transport complexes functioning in the enterocyte membrane. Fast and slow adaptation of membrane digestion and absorption are described as well as the ability of parasubstrate regulation of these systems.

[Intestinal dipeptidases and their regulation in chicks of different ages]. / Kishechnye dipeptidazy i ikh reguliatsiia u tsypliat raznogo vozrasta.

Studies have been made of changes in catalytic activity and regulatory properties of membrane and intracellular glycyl-valine dipeptidase from the small intestine of chicks ageing from 1 to 40 days. Gradual decrease in the activity of membrane glycyl-valine dipeptidase was found during postnatal life together with formation of proximodistal gradient in the distribution along the intestine with maximal activity in the distal part. The magnitude and direction of regulatory effects of modificators (tributyrine, sucrose, L-methionine) depend on the age of chicks. Intracellular enzyme does not undergo any significant changes in its regulatory properties and in the level of its activity along the intestine during postnatal development of chicks.

[Hydrolysis and absorption of lysozyme in the small intestine]. / Gidroliz i vsasyvanie lizotsima v tonkoi kishke.

Preparations of chicken small intestine were used in the experiment in vitro simulating processes of membranous digestion (inverted intestinal segments) and absorption (inverted intestinal myasis). It was established that lysozyme was hydrolyzed on the internal mucosa surface regardless of its concentration in the gastro-intestinal tract, and only insignificant quantity of lysozyme (0.027%) penetrates the intestinal wall. The method of lysozyme determination through its action on the cellular wall of Micrococcus lisodeicticus, and highly efficient liquid chromatography were used to study the transport process. The data presented have evidenced that lysozyme is well hydrolyzed under the action of intestinal peptide hydrolyses, and only insignificant amounts of non-splitted lysozyme can penetrate the blood.

Regulation of intestinal peptidases by nutrients in human fetuses and children.

Nutrient interactions during intestinal digestion has been established in animals. The present study investigated the effect of different nutrients on intestinal dipeptidase, tripeptidase, carboxypeptidase (EC 3.4.12.-) and aminopeptidase N (ApN) (EC 3.4.11.2) activities in human fetuses and children. The effect of nutrients on isolated porcine kidney ApN was also studied. Sucrose, lactose and tributyrin had no effect on di-, tri-, and carboxypeptidase activities of mucosal homogenates, but tributyrin significantly (20-50%) inhibited both fetal and postnatal ApN activity in the small intestine and colon. The pH-independent inhibition of ApN is specific for tributyrin and to the product of its hydrolysis, butyric acid. Glycerol, triolein, and natural oils did not affect ApN activity. The inhibition of ApN by tributyrin was dose and time dependent and occurred in enterocyte brush border membranes as well as in the purified enzyme from porcine kidney. The kinetics of the purified ApN showed that the tributyrin effect is primarily competitive and associated mainly with an increase in K(m). These observations demonstrate the possibility of intestinal and kidney ApN regulation by lipids and products of their hydrolysis. We speculate that nutrient interactions arose quite early in the evolution and represent a mechanism for the regulation of food digestion.