Effect of Tinospora cordifolia (Guduchi) on the phagocytic and pinocytic activity of murine macrophages in vitro.

Tinospora cordifolia (Guduchi) is a widely used herb in Ayurvedic system of medicine known to possess immunomodulatory properties. The present study was aimed to study the activation of macrophages after in vitro guduchi treatment. The aqueous extract of T. cordifolia was found to enhance phagocytosis and pinocytosis in vitro. The rate of pinocytosis by macrophages when measured by uptake of horseradish peroxidase was significantly increased after guduchi treatment as compared to medium alone. The macrophages demonstrated an increased phagocytosis to non-infective microorganisms (heat killed yeast) and live infective microorganisms (E. coli) after guduchi treatment. The results demonstrate that Guduchi enhances macrophage activation as analyzed by cytochemical parameters.

Intra- and trans-cellular delivery of enzymes by direct conjugation with non-multivalent anti-ICAM molecules.

Intercellular adhesion molecule 1 (ICAM-1) is a cell-surface protein overexpressed in many diseases and explored for endocytosis and transcytosis of drug delivery systems. All previous evidence demonstrating ICAM-1-mediated transport of therapeutics into or across cells was obtained using nanocarriers or conjugates coupled to multiple copies of anti-ICAM antibodies or peptides. Yet, transport of therapeutics linked to non-multivalent anti-ICAM ligands has never been shown, since multivalency was believed to be necessary to induce transport. Our goal was to explore whether non-multivalent binding to ICAM-1 could drive endocytosis and/or transcytosis of model cargo in different cell types. We found that anti-ICAM was specifically internalized by all tested ICAM-1-expressing cells, including epithelial, fibroblast and neuroblastoma cells, primary or established cell lines. Uptake was inhibited at 4°C and in the presence of an inhibitor of the ICAM-1-associated pathway, rather than inhibitors of the clathrin or caveolar routes. We observed minimal transport of anti-ICAM to lysosomes, yet prominent and specific transcytosis across epithelial monolayers. Finally, we coupled a model cargo (the enzyme horseradish peroxidase (HRP)) to anti-ICAM and separated a 1:2 antibody:enzyme conjugate for non-multivalent ICAM-1 targeting. Similar to anti-ICAM, anti-ICAM-HRP was specifically internalized and transported across cells, which rendered intra- and trans-cellular enzyme activity. Therefore, non-multivalent ICAM-1 targeting also provides transport of cargoes into and across cells, representing a new alternative for future therapeutic applications via this route.

DT(270-326) , a Truncated Diphtheria Toxin, Increases Blood-Tumor Barrier Permeability by Upregulating the Expression of Caveolin-1.

AIM: The nontoxic mutant of diphtheria toxin (DT) has been demonstrated to act as a receptor-specific carrier protein to delivery drug into brain. Recent research showed that the truncated "receptorless" DT was still capable of being internalized into cells. This study investigated the effects and potential mechanisms of DT(270-326) , a truncated "receptorless" DT, on the permeability of the blood-tumor barrier (BTB). METHODS: BTB and GECs were subjected to DT(270-326) treatment. HRP flux assays, immunofluorescent, co-immunoprecipitation, Western blot, CCK-8, and Flow cytometry analysis were used to evaluate the effects of DT(270-326) administration. RESULTS: Our results revealed that 5 µM of DT(270-326) significantly increased the permeability of BTBin vitro, which reached its peak at 6 h. The permeability was reduced by pretreatment with filipinIII. DT(270-326) co-localized and interacted with caveolin-1 via its caveolin-binding motif. The mRNA and protein expression levels of caveolin-1 were identical with the changes of BTB permeability. The upregulated expression of caveolin-1 was associated with Src kinase-dependent tyrosine phosphorylation of caveolin-1, which subsequently induced phosphorylation and inactivation of the transcription factor Egr-1. The combination of DT(270-326) with doxorubicin significantly enhanced the loss of cell viability and apoptosis of U87 glioma cells in contrast to doxorubicin alone. CONCLUSIONS: DT(270-326) might provide a novel strategy to increase the delivery of macromolecular therapeutic agents across the BTB.

The molecular mechanism and effect of cannabinoid-2 receptor agonist on the blood-spinal cord barrier permeability induced by ischemia-reperfusion injury.

Previous studies have shown that modulation of the receptor-mediated endocannabinoid system during ischemia injury can induce potent neuroprotective effects. However, little is known about whether cannabinoid-2 (CB2) receptor agonist would produce a protective effect on blood-spinal cord barrier (BSCB) during ischemia. Using an in vivo transient spinal cord ischemia model in rats, JWH-015 (1mg/kg, i.p.), a CB2 receptor selective agonist, or vehicles were injected 20 min before ischemia. The effects of JWH-015 on BSCB permeability, the major structural protein for the formation of caveolae, caveolin-1 (cav-1), tight junction (TJ) protein Occludin and zona occludens protein-1 (ZO-1) were examined at day 1, day 3 and day 7 of reperfusion after transient spinal cord ischemia in rats. Here we demonstrated that JWH-015 significantly down-regulated the expression of cav-1, up-regulated the expression of TJ proteins, and then decreased the permeability of BSCB compared with control group. In addition, using an in vitro BBB model, oxygen glucose deprivation (OGD) was applied to simulate spinal cord ischemia in vitro in Human brain microvascular endothelial cells (HBMECs). JWH-015 greatly increased the transepithelial electrical resistance (TEER) and changed the distribution of ZO-1 and Occludin. Moreover, JWH-015 induced the expression of p-PKB and p-FoxO1 protein and decreased the expression of cav-1, which were greatly reversed by ROS inhibitor or PI3K inhibitor. Taken together, all of these results suggested that JWH-015 might regulate the BSCB permeability and this effect could be related to paracellular and transcellular pathway. And pharmacological CB2R ligands offer a new strategy for BSCB protection during ischemic injury.

Minoxidil sulfate induced the increase in blood-brain tumor barrier permeability through ROS/RhoA/PI3K/PKB signaling pathway.

Adenosine 5'-triphosphate-sensitive potassium channel (KATP channel) activator, minoxidil sulfate (MS), can selectively increase the permeability of the blood-tumor barrier (BTB); however, the mechanism by which this occurs is still under investigation. Using a rat brain glioma (C6) model, we first examined the expression levels of occludin and claudin-5 at different time points after intracarotid infusion of MS (30 µg/kg/min) by western blotting. Compared to MS treatment for 0 min group, the protein expression levels of occludin and claudin-5 in brain tumor tissue of rats showed no changes within 1 h and began to decrease significantly after 2 h of MS infusion. Based on these findings, we then used an in vitro BTB model and selective inhibitors of diverse signaling pathways to investigate whether reactive oxygen species (ROS)/RhoA/PI3K/PKB pathway play a key role in the process of the increase of BTB permeability induced by MS. The inhibitor of ROS or RhoA or PI3K or PKB significantly attenuated the expression of tight junction (TJ) protein and the increase of the BTB permeability after 2 h of MS treatment. In addition, the significant increases in RhoA activity and PKB phosphorylation after MS administration were observed, which were partly inhibited by N-2-mercaptopropionyl glycine (MPG) or C3 exoenzyme or LY294002 pretreatment. The present study indicates that the activation of signaling cascades involving ROS/RhoA/PI3K/PKB in BTB was required for the increase of BTB permeability induced by MS. Taken together, all of these results suggested that MS might increase BTB permeability in a time-dependent manner by down-regulating TJ protein expression and this effect could be related to ROS/RhoA/PI3K/PKB signal pathway.

Paracellular versus transcellular intestinal permeability to gliadin peptides in active celiac disease.

The intestinal permeability of undegraded α9-gliadin peptide 31-49 (p31-49) and 33-mer gliadin peptides is increased in active celiac disease. Two distinct transport pathways have been proposed: paracellular leakage through epithelial tight junctions and protected transcellular transport. To analyze the relative contribution of these pathways, we compared mucosa-to-serosa permeability of small and large permeability markers [ionic conductance (G), mannitol, 182 Da; horseradish peroxidase, 40 kDa] and gliadin peptides [33-mer (p56-88, 3900 Da), 19-mer (p31-49, 2245 Da; and p202-220, 2127 Da), and 12-mer (p57-68, 1453 Da)] in duodenal biopsy specimens mounted in Ussing chambers. The permeability of intact peptides was much higher for p31-49 or 33-mer than for horseradish peroxidase, p202-220, and p57-68. A positive correlation was observed between G, an index of paracellular diffusion of ions, and mannitol permeability. The absence of correlation between G and permeability to intact 33-mer or p31-49 did not favor paracellular diffusion of the peptides. Immunofluorescence studies indicated that 33-mer enters the early endosome antigen 1-positive compartment but escapes the lysosomal-associated protein 2-positive compartment. The results underline that mannitol and ionic conductance G cannot be considered markers of permeability to gliadin peptides. In active celiac disease, increases in transcellular permeability to intact gliadin peptides might be considered in treatment strategies aimed at controlling epithelial permeability to gluten.

Delivering horseradish peroxidase as a respirable powder to the isolated, perfused, and ventilated lung of the rat: the pulmonary disposition of an inhaled model biopharmaceutical.

BACKGROUND: Our aim was to investigate the potential of the DustGun aerosol technology integrated with the isolated, perfused, and ventilated lung of the rat (IPL) to study the pulmonary disposition of an inhaled model biopharmaceutical, the 40-kDa protein horseradish peroxidase (HRP). METHOD: The DustGun aerosol technology was used to deliver respirable powder aerosols of HRP (the mass median aerodynamic diameter: 1.7 µm) as an 80-sec bolus to the IPL perfused in a single-pass mode. Lung perfusate was repeatedly sampled for 125 min after the HRP exposure. The amount of active HRP clearing with the perfusate or being retained in the lung was measured enzymatically. RESULTS AND CONCLUSIONS: The total amount of HRP deposited in the lungs was 335 ± 100 µg and 568 ± 47 µg for a low- and high-dose exposure, respectively. After inhalation, the initial appearance of HRP in the perfusate was rapid. However, the total amount of HRP that cleared with the perfusate remained below 0.5% of the deposited dose. The effect of opening the tight junctions between the alveolar epithelial cells on HRP absorption was studied by exposing the IPL to nebulized aerosols of either 0.02, 0.2, or 2% poly-L-Arginine (PLA) (MW 42.5 kDa) in phosphate-buffered saline (PBS) for 5 min, at 40 min after the HRP exposure. Subsequent exposure to 0.02% PLA did not affect HRP absorption. However, exposure to 0.2% PLA increased the absorption rate ninefold, and the total amount of HRP clearing with the perfusate increased to approximately 4% of the deposited dose. No further increase was obtained with 2% PLA, indicating a steep dose-response for the enhancer. It was concluded that the pulmonary absorption of HRP is quite slow, and absorption enhancers affecting tight junctions have a distinctive, yet limited efficiency. The presented inhalation technology can be very useful in studying the pulmonary absorption of biopharmaceuticals.

Intestinal epithelial barrier dysfunction and dendritic cell redistribution during early stages of inflammation in the rat: role for TLR-2 and -4 blockage.

BACKGROUND: Dendritic cell (DC) redistribution during early stages of enteritis may be related to ileal barrier dysfunction. We used a rat model of ileitis to examine this hypothesis. METHODS: Sprague-Dawley rats were injected with indomethacin or saline and euthanized 2, 6, 12, or 24 hours later. Ileal segments and mesenteric lymph nodes were obtained for morphological, bacterial, or functional studies. To determine the role of Toll-like receptor (TLR)-2 and -4 blockages, rats were pretreated with normal IgG, anti-TLR-2, or anti-TLR-4 antibodies prior to indomethacin or saline, and ileal segments were collected 24 hours later. RESULTS: In control rats, CD103+DC were mainly located in the lamina propria (LP) and some expressed TLR-2. TLR-4+ cells with different morphology and distribution from CD103+DC were also detected. In indomethacin-treated rats at 6-24 hours, inflammation was evident as was redistribution of CD103+DC from LP to Peyer's patches. We also observed TLR-2+ monocyte depletion and changes in TLR-4 distribution. At 2-6 hours we detected opened tight junctions as well as abnormal trans- and para-epithelial enteric bacterial infiltration, while macromolecular permeability was not significantly enhanced until 24 hours. In the absence of indomethacin, anti-TLR-2 blockage induced a significant increase of LP CD103+DC, while in the presence of indomethacin, anti-TLR-2 or -4 blockages significantly inhibited (P < 0.05) the reduction of LP CD103+DC. CONCLUSIONS: During the early stages of indomethacin-induced ileitis, epithelial barrier damage and abnormal bacterial infiltration into the mucosa occurred in conjunction with initial redistribution of CD103+DC. Furthermore, we showed that TLR-2 and -4 blockade regulates CD103+DC distribution during early phases in this experimental model.

Vascular endothelial growth factor and nitric oxide production in response to hypoxia in the choroid plexus in neonatal brain.

Damage to the choroid plexus in 1-day-old Wistar rats subjected to hypoxia was investigated. The mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal, inducible nitric oxide synthase (eNOS, nNOS, iNOS), and vascular endothelial growth factor (VEGF) along with nitric oxide (NO) production and VEGF concentration was up-regulated significantly in hypoxic rats. Ultrastructurally, the choroid plexus epithelial cells showed massive accumulation of glycogen. A striking feature was the extrusion of cytoplasmic fragments from the apical cell surfaces into the ventricular lumen following the hypoxic insult. Intraventricular macrophages showed increased expression of complement type 3 receptors, major histocompatibility complex class I and II antigens, and ED1 antigens. Following an intravenous injection of horseradish peroxidase (HRP), a large number of intraventricular macrophages were labeled suggesting enhanced leakage of the tracer from the blood vessels in the choroid plexus connective tissue stroma into the ventricular lumen. We suggest that increased production of NO in hypoxia is linked to the structural alteration of the choroid plexus, and along with VEGF, may lead to increased vascular permeability. Melatonin treatment reduced VEGF and NO levels as well as leakage of HRP suggesting its potential value in ameliorating damage in choroid plexus pathologies.

Increased epithelial uptake of protein antigens in the ileum of Crohn's disease mediated by tumour necrosis factor alpha.

BACKGROUND AND AIMS: The exact nature of the epithelial barrier defect in Crohn's disease remains to be elucidated. Previously we showed increased permeability to proteins in ileal Crohn's disease. Our aims were to study if this barrier defect (a) involves endocytotic uptake of antigens and (b) is related to low grade inflammation not detectable by histology. METHODS: Macroscopically normal segments of distal ileum of Crohn's disease patients (n = 10) were subgrouped into non-inflamed (histologically unaffected) and slightly inflamed tissues and studied in Ussing chambers, with normal ileal specimens from colon cancer patients (n = 9) as controls. Endocytotic uptake into enterocytes of the protein antigen horseradish peroxidase was assessed by measuring the area of horseradish peroxidase containing endosomes in electron photomicrographs. Mucosal tumour necrosis factor alpha (TNF-alpha) mRNA was quantified using real time polymerase chain reaction. For comparison, the effects of low doses of TNF-alpha on endosomal uptake of horseradish peroxidase were studied in cultured T84 cells grown on filter supports. RESULTS: The area of horseradish peroxidase containing endosomes was increased (p<0.001) in enterocytes of non-inflamed ileum of Crohn's disease (2.8 (0.7) mum(2)/300 mum(2)) compared with control ileum (0.6 (0.06)). In non-inflamed mucosa, a significant association between endosomal uptake and mucosal expression of TNF-alpha mRNA (p = 0.03) was found. Low concentrations of TNF-alpha (0.25-1.0 ng/ml) enhanced the endosomal uptake of horseradish peroxidase in polarised T84 cells, without affecting transepithelial electrical resistance. CONCLUSIONS: Our findings suggest increased endosomal uptake of antigens in ileal Crohn's disease that may be mediated by TNF-alpha. These data highlight the transcellular route of antigen uptake in barrier dysfunction and implicate the interaction between epithelial cells and the innate immune system in the development of mucosal inflammation.

N-methyl-d-aspartate-triggered neuronal death in organotypic hippocampal cultures is endocytic, autophagic and mediated by the c-Jun N-terminal kinase pathway.

Acute excitotoxic neuronal death was studied in rat organotypic hippocampal slices exposed to 100 micro mN-methyl-d-aspartate. Fulgurant death of pyramidal neurons occurred in the CA1 and CA3 regions and was already detectable within 2 h of the N-methyl-d-aspartate administration. Morphologically, the neuronal death was neither apoptotic nor necrotic but had the hallmarks of autophagic neuronal death, as shown by acid phosphatase histochemistry in both CA1 and CA3 and by electron microscopy in CA1. The dying neurons also manifested strong endocytosis of horseradish peroxidase or microperoxidase, occurring probably by a fluid phase mechanism, and followed, surprisingly, by nuclear entry. In addition to these autophagic and endocytic characteristics, there were indications that the c-Jun N-terminal kinase pathway was activated. Its target c-Jun was selectively phosphorylated in CA1, CA3 and the dentate gyrus and c-Fos, the transcription of which is under the positive control of c-Jun N-terminal kinase target Elk1, was selectively up-regulated in CA1 and CA3. All these effects, the neuronal death itself and the associated autophagy and endocytosis, were totally prevented by a cell-permeable inhibitor of the interaction between c-Jun N-terminal kinase and certain of its targets. These results show that pyramidal neurons undergoing excitotoxic death in this situation are autophagic and endocytic and that both the cell death and the associated autophagy and endocytosis are under the control of the c-Jun N-terminal kinase pathway.

The blood-brain barrier of the chick glycogen body (corpus gelatinosum) and its functional implications.

Among recent vertebrates only birds possess a glycogen body (corpus gelatinosum), located in the rhomboidal sinus of the lumbosacral region of the spinal cord and separated from the neural tissue proper. Because of the specific topographical situation of this circumventricular organ, the structure of its vascular system is of special interest with respect to the still unsolved functional problems. The existence of a blood-brain barrier is demonstrated by the exclusion of intravascularly injected tracer (horseradish peroxidase), and immunocytochemical demonstration of glucose transporter-1 as a functional marker and of neurothelin, occludin and ZO-1 as structural markers. Alkaline phosphatase and gamma-glutamyltransferase activities, two enzyme reactions frequently used for demonstration of an established blood-brain barrier in vitro, were localized histochemically on the plasmalemma of glycogen body cells and were absent from the endothelium. In addition, local enlargements of the intercellular space were observed by transmission and scanning electron microscopy. In accordance with the concept of a third circulation the cerebrospinal fluid may be the vehicle for distributing substances originating in the glycogen body to the CNS, while the vascular endothelium maintains the internal milieu by virtue of its dynamic barrier functions.

Reduced endocytosis and altered lysosome function in cisplatin-resistant cell lines.

We isolated human KB adenocarcinoma cisplatin-resistant (CP-r) cell lines with multidrug-resistance phenotypes because of reduced accumulation of cisplatin and other cytotoxic compounds such as methotrexate and heavy metals. The uptake of horseradish peroxidase (HRPO) and Texas Red dextran was decreased several-fold in KB-CP-r cells, indicating a general defect in fluid-phase endocytosis. In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptor-mediated endocytosis, was similar in sensitive and resistant cells. However, 40-60% of the (125)I-EGF released into the medium after uptake into lysosomes of KB-CP-r cells was TCA precipitable as compared to only 10% released by sensitive cells. These results indicate inefficient degradation of internalised (125)I-EGF in the lysosomes of KB-CP-r cells, consistent with slower processing of cathepsin L, a lysosomal cysteine protease. Treatment of KB cells by bafilomycin A(1), a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, (125)I-EGF, (14)C-carboplatin, and release of TCA precipitable (125)I-EGF. KB-CP-r cells also had less acidic lysosomes. KB-CP-r cells were crossresistant to Pseudomonas exotoxin, and Pseudomonas exotoxin-resistant KB cells were crossresistant to cisplatin. Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance.

Resistance of isolated mammalian spinal cord white matter to oxygen-glucose deprivation.

We found that isolated guinea pig spinal cord white matter is resistant to acute oxygen-glucose deprivation. Sixty minutes of oxygen-glucose deprivation resulted in a 60% reduction of compound action potential (CAP) conductance, and there was a near complete recovery after 60 min reperfusion. Corresponding horseradish peroxidase-exclusion assay showed little axonal membrane damage. To further deprive the axons of metabolic substrate, we added 2 mM sodium cyanide or 2 mM sodium azide, both mitochondrial suppressors, to the ischemic medium, which completely abolished CAP and resulted in a 15 to approximately 30% recovery postreperfusion. Both compounds preferentially reduced the conductance of large diameter axons. We suggest the residual ATP in our ischemic model can protect anatomic integrity and physiological functioning of spinal axons following ischemic insult. This further suggests that oxygen-glucose deprivation alone cannot be solely responsible for short-term functional and anatomic damage. The damaging effects of ischemia in vivo may be mediated by factors originating from the gray matter of the cord or other systemic factors; both were largely eliminated in our in vitro white matter preparation.

Increased endothelial vesicular transport correlates with increased blood-tumor barrier permeability induced by bradykinin and leukotriene C4.

Bradykinin and leukotriene C4 (LTC4) have been shown to increase molecular transport across the blood-tumor barrier (BTB). The aim of this study was to quantitatively assess whether an increase in vesicular transport or opening of tight junctions was responsible for this increase in permeability. Wistar rats bearing RG2 or C6 gliomas were infused with bradykinin or LTC4 through the right carotid artery for 15 min and then perfused to achieve fixation. Prepared specimens were observed using transmission electron microscopy. Infusion of either bradykinin or LTC4 resulted in significantly increased vesicular density in capillary endothelial cells of the BTB but not in normal brain capillaries. The opening of tight junctions, assessed by determining a cleft index, was found to be greater in tumor capillaries compared to normal controls. However, neither bradykinin nor LTC4 produce variations in the cleft index. A significant accumulation of horseradish peroxidase was seen in the intercellular peri-capillary spaces and in endothelial transport vesicles after infusion of bradykinin, demonstrating that the formation of vesicles was associated with macromolecular transcytosis. These findings suggest that pinocytotic vesicular transport is the primary means by which luminal to abluminal transport occurs in response to vasomodulation with bradykinin or LTC4.

Difference in penetration of horseradish peroxidase tracer as a foreign substance into the peri-implant or junctional epithelium of rat gingivae.

Horseradish peroxidase (HRP) tracer was applied to the gingival sulcus of implants or natural teeth at 5, 25, or 50 mg/ml to investigate the sealing capacities of the peri-implant epithelium (PIE) and junctional epithelium (JE); the extent of HRP penetration was observed under electron microscopy. A Ti-6Al-4V implant was inserted either immediately (immediate implantation) or 2 weeks (delayed implantation) after extraction of the maxillary left first molar of rats. The JE of the right molar was used as a control. Although the whole PIE of undecalcified sections appeared to be attached to the implant surface, electron microscopically, the internal basement lamina (IBL) and hemidesmosomes were deficient in the coronal-middle region of the PIE. There were extensive extracellular deposits of HRP in the intercellular spaces between PIE cells, and HRP was blocked to some extent by the lamina lucida and lamina densa of the external basal lamina and basal cell junction. HRP was detected in the connective tissue under the PIE, but was not found in the connective tissue under the JE. Intracellularly, HRP was found in the vesicles and granules of PIE cells and JE cells. These were fewer in number in PIE cells than in JE cells. There were no differences between the findings for immediate and delayed implantation. The results indicate that a deficiency in the IBL permitted penetration of HRP from the gingival sulcus into the connective tissue under the PIE, and suggest that the endocytotic capacity of PIE cells is inferior to that of JE cells.

Effect of fumonisin B1 on structure and function of macrophage plasma membrane.

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium moniliforme and related fungi, is nephrotoxic, neurotoxic, hepatotoxic, carcinogenic and immunosuppressive in animals and man. In this study we evaluate the modifications of fluidity, endocytosis and peroxidative damage of plasma membrane induced by FB1 in macrophage cell line J774A.1. In these immune cells FB1 (1-10 microM) enhances membrane fluidity and increases, time-dependently, the horseradish peroxidase (HRP) endocytosis. This effect is concentration-dependent, significant at 10 microM, and reverted by IFN-gamma (100 U/ml). Moreover, FB1 (1-10 microM) induces a membrane peroxidative damage as evident by the increase of malondialdehyde (MDA) production. All these mycotoxin effects provide additional insight into potential mechanism by which FB1, in macrophages, might enhance membrane damage and oxidative stress contributing to the pathogenesis of mycotoxin induced diseases.

Intestinal permeability and bacterial translocation are uncoupled after small bowel resection.

BACKGROUND/PURPOSE: Gut barrier failure and bacterial translocation have been proposed to cause infection and sepsis in patients with the short bowel syndrome. This study tested the hypothesis that permeability is increased in the adapting remnant ileum after massive small bowel resection (SBR). METHODS: Male ICR mice underwent a 50% proximal SBR or sham operation. At 3, 7, and 14 days, the ileum was mounted in an Ussing chamber. Mucosal-to-serosal flux of low (dextran) and high (horseradish peroxidase; HRP) molecular weight markers was determined. Additionally, bacterial translocation was measured by culturing blood, mesenteric lymph nodes, liver, and spleen at 3 and 14 days after SBR or sham operation. RESULTS: Permeability to dextran was reduced immediately after SBR but was no different at later time-points. HRP permeability was no different at any time-point. Translocation of Gram-negative bacteria to the mesenteric lymph nodes and liver was more frequent in the SBR animals 3 and 14 days postoperatively. CONCLUSIONS: Intestinal permeability to macromolecules is not increased after massive SBR, but the rate of translocation to the mesenteric lymph nodes and liver is elevated. This suggests that the mechanism for bacterial translocation after SBR does not involve alterations in gut permeability.

Distribution of intraventricularly administered antiamyloid-beta peptide (Abeta) antibody in the mouse brain.

There is considerable interest in utilizing the intracerebroventricular (icv) route of administration of antibodies in the brain for various studies and for the therapy of malignancies, but very little is known about the anatomic extent of distribution of the antibody in brain after injection into the third ventricle. To explore the potential for icv administration of antiamyloid-beta peptide (Abeta) in reducing Abeta toxicity in brain in Alzheimer's disease, we first mapped the time course and path of transit of horseradish peroxidase (HRP)-labeled antibody. The results show that, after a single injection into the mouse third venticle, the HRP-labeled antibody is localized within the microvasculature, first that of the corticohippocampal region close to the site of injection at 3 hr. By 24 hr, the antibody is distributed throughout the hippocampus and frontoparietal cortex close to the injection site, as well as in the deep and outer cerebral cortex and cerebellar cortex remote from the injection site. The injected antibody is almost entirely removed by 4 days. Therefore, the antibody had diffused throughout all the brain by 24 hr, showing the feasibility of small quantities of anti-Abeta antibody infused into the third ventricle to reach extracellular epitopes throughout the brain parenchyma rapidly.

Pancreatic acinar cell dysfunction in CFTR(-/-) mice is associated with impairments in luminal pH and endocytosis.

BACKGROUND & AIMS: We have previously shown that endocytosis at the apical plasma membrane in pancreatic acinar cells is coupled to ductal bicarbonate secretion into the lumen. We hypothesized that decreased bicarbonate secretion in cystic fibrosis (CF) inhibits apical endocytosis. The aim of this study was to determine in cftr(-/-) mice (1) if the pH of the pancreatic juice is acidic compared with wild-type (WT) controls, (2) if there is a selective block in endocytosis, and (3) if alkalinization of the luminal fluid reverses this defect. METHODS: Fluid secretion and pH of pancreatic juice were measured. Exocytosis, endocytosis, and morphology were compared in pancreatic lobules from cftr(-/-) and WT mice. RESULTS: Pancreatic juice pH was 8.12 +/- 0.06 in WT mice compared with 6.60 +/- 0.04 in cftr(-/-) mice. Although cholecystokinin-stimulated amylase secretion was not significantly different, endocytosis was markedly inhibited in cftr(-/-) compared with WT mice. Cleavage of GP2, a GPI-anchored protein tightly associated with activation of endocytosis, was also decreased. Incubation of lobules from cftr(-/-) mice at pH 8.3 reversed the luminal dilatation. CONCLUSIONS: These data indicate that apical endocytosis is selectively impaired in cftr(-/-) mice, which explains, in part, the luminal dilatation observed at the apical plasma membrane. In vitro alkalinization of luminal fluid led to reversal of defects in membrane dynamics, restored coupled exocytosis and endocytosis, and abolished the luminal dilatation in this animal model of CF. Acidic pH changes in luminal secretions may play a role in the pancreatic membrane dysfunction observed in CF.